We use cookies and similar technologies to recognize your repeat visits and preferences, as well as to measure the effectiveness of campaigns and analyze traffic. To learn more about cookies, including how to disable them, view our Privacy Policy.
By clicking "I accept" on this banner or using our site, you consent to the use of cookies unless you have disabled them.

 View our Privacy Policy I accept

Buy generic levitra from canada

António Guterres will on Wednesday launch the #PledgetoPause campaign as part of wider efforts to create what the UN hopes will be “a levitra 20mg cena new social media norm” to help buy generic levitra from canada combat the impact of viral misinformation. The campaign is based on research which indicates that taking a brief pause before sharing information can significantly lessen the inclination to share shocking or emotive material, and slow the spread of misinformation. Deadly consequences “During buy generic levitra from canada the erectile dysfunction treatment levitra, the wrong information can be deadly. Take the pledge to pause and help stop the spread of misinformation”, the UN chief said in a video message recorded for the campaign. The message, which begins with a five-second pause, will be shared on his Twitter and Instagram accounts, and is designed to be replicated by other leaders, influencers and members of the public.

#PledgetoPause aims buy generic levitra from canada to increase media literacy so that social media users can spot misinformation and stop themselves from passing it on. Break the chain of misinformation The new campaign is part of Verified, a UN initiative launched in May to share science-backed health information and stories of global solidarity around erectile dysfunction treatment. The UN describes it as the first global behaviour-change campaign on misinformation, to mobilise experts and researchers, governments, influencers, civil society, businesses, regulators and the media, under a single message. #PledgetoPause. “erectile dysfunction treatment is not just a health crisis, but a communications emergency as well.

When misinformation spreads, the public loses trust and too often makes decisions that hamper the public response and even their own lives”, said Melissa Fleming, head of the UN’s Department for Global Communications. “It is increasingly clear that we cannot successfully tackle the levitra without also addressing online misinformation. Each and every one of us can help break the chain of misinformation by pausing before we share.” Ms. Fleming added that the UN is also working with social media platforms to recommend changes. Civil society support The #PledgetoPause campaign aims to reach a global audience of one billion, online and through partnerships, by the end of December.Civil society collaborators supporting Pause include organizations from around the world that are fighting misinformation, such as Chequeado, Newschecker.in and First Draft.

African media organizations such as MultiChoice and Yuvaa also are helping to distribute campaign messaging..

Is levitra better than viagra

Levitra
Viagra super active
Sildalis
Cialis sublingual
Average age to take
Ask your Doctor
You need consultation
Yes
Ask your Doctor
Buy with Paypal
Online
Online
Yes
Yes
Free pills
Indian Pharmacy
Online Drugstore
On the market
RX pharmacy
Take with alcohol
20mg 120 tablet $269.95
100mg 120 capsule $191.95
100 + 20mg 180 tablet $249.95
20mg 360 tablet $719.95
Generic
Yes
No
No
No
Side effects
Ask your Doctor
Consultation
100 + 20mg
Consultation
Female dosage
Canadian pharmacy only
100mg
In online pharmacy
20mg

IntroductionThe mammalian kinesin superfamily proteins (KIFs) are microtubule and ATP-dependent molecular motors, which is levitra better than viagra were first identified Where to buy norvasc pills in 1985 as axonal transporters in squid and bovine brains.1 Forty-five different kinesin family member (KIF) genes were identified in the mouse genome so far, 44 of which are present in the human genome. Phylogenetic analysis based on sequence homology between the human and the mouse genome led to the classification of KIF genes into 16 families, from kinesin-1 to kinesin-14B (figure 1).2 The first kinesins discovered belong to the kinesin-1 family (KIF5A, KIF5B and KIF5C), and they form a heterotetramer of two heavy chains and two light chains (KLC1-4).2 KIF genes encode KIFs, a specific class of motor proteins generating intracellular motility by driving directional transport of various cargoes such as organelles, protein complexes and mRNAs along the microtubule system.2 Studies using knockout mouse models by Hirokawa and colleagues significantly contributed to elucidate the roles of kinesins in mammalian physiology. Their role in transport is fundamental to cellular logistics and performance, and the molecular motors are not only effectors of signal transduction cascades but also transport and/or bind to important signal transduction molecules to actively modulate their function.3Phylogenetic tree of mammalian kinesin superfamily genes identified in the human (and mouse) genome and classified in 16 subfamilies (from kinesin 1 to 14B) (adapted from Hirokawa et al 3)." data-icon-position data-hide-link-title="0">Figure 1 Phylogenetic tree of mammalian kinesin superfamily genes identified in the human (and mouse) genome and classified in 16 subfamilies (from kinesin 1 to 14B) (adapted from Hirokawa et al3).The first kinesins were observed in the context of axonal transport in neurons, and a novel disease entity of ‘motor–proteinopathy’ was proposed for the pathogenesis of axonal neuropathies in 2001.4 is levitra better than viagra Due to their role in cellular membrane trafficking, however, kinesins are essential for the functioning of many polar cell types, such as neurons, epithelial cells, sperm cells or stem cells during organogenesis. Kinesins also play a fundamental role in cell-cycle dynamics, both during mitotic and meiotic processes.

They regulate chromosomal condensation and alignment, spindle formation, cytokinesis and cell-cycle progression.5 It is estimated that about a dozen kinesins are is levitra better than viagra involved in the cell cycle. Among these, there is a specific subclass of chromokinesins (kinesin 4 and kinesin 10 family) which are able to bind chromosomes.6 Recently, KIFs were discovered to act as microtubule stabilisers (KIF26A and KIF21A) and depolymerisers (KIF2A and KIF2C), activities which are important for both cellular morphogenesis and mammalian development, playing a role in neuronal and axonal morphology and ciliogenesis.7Alterations in motor kinesins are leading to human disease by various pathological mechanisms, including cancer and multifactorial and monogenic disorders. Variants in 18 out of the 44 human KIF genes were identified to cause monogenic disorders, following different modes of Mendelian inheritance and associated with a wide is levitra better than viagra spectrum of clinical signs. These range from lethal and multiple to isolated congenital anomalies—including birth defects potentially detectable in the foetal period by current prenatal imaging studies—to postnatally apparent neurodevelopmental disorders, intellectual disability and neurological conditions.We will review the current state of knowledge of the biological processes kinesins are involved in and discuss their emerging role in human disease, particularly in birth defects and congenital anomaly syndromes.

Birth defects remain a leading cause of perinatal lethality in industrialised countries.8 Structural anomalies are recognised with increasing reliability during early pregnancy by the use of high-resolution ultrasound technologies, thus raising questions about diagnosis, aetiology, prognosis and recurrence risk, particularly in the presence of more than one anomaly, which most likely indicates a genetic aetiology. We identify recurrent phenotype patterns caused by alterations in KIF genes, and we outline the complexity of phenotype–genotype correlations mirroring the processes of intracellular microtubule-mediated is levitra better than viagra transport and movement, in which kinesins play a fundamental role. There are likely many more relationships between the clinical signs and the genetic variants to be identified in the future, and the functional network of kinesins and their role in human disease need to be further elucidated. We propose is levitra better than viagra to introduce the term ‘kinesinopathies’ for this group of conditions, which are phenotypically and genetically overlapping and characterised by the functional impairment of a specific group of molecular motors.

We hope that their systematic approach leads to a better recognition in clinical practice, as well as in genome-wide sequencing for diagnosis and research, and opens strategies for the future development of molecular therapies.KIF structureAll KIFs have a phylogenetically well-conserved motor domain head, consisting of an ATP-binding motif and a microtubule-binding domain. Depending on the position of the motor domain, kinesins can be subdivided into N-kinesins (amino-terminal motor domain), M-kinesins (middle-region motor domain) and C-kinesins (carboxy-terminal motor domain).2 Most kinesins belong to the N-kinesin subgroup, but members of the kinesin 13A family (figure 1) belong to the M-kinesin subtype, while KIF1C, KIF2C and KIF3C belong to the C- kinesin subfamily.3 Both N-kinesins and C-kinesins are responsible for plus end and minus end-directed motility, M-kinesins for depolymerisation of microtubules in is levitra better than viagra tubulin molecules. However, there are a few exceptions to this categorisation.9 The motor domain head attaches to the neck, the coiled coil stalk and the tail. The kinesins’ neck is family-specific and responsible for the direction of motility or regulation of activity.

The coiled coil stalk and tail are involved in is levitra better than viagra kinesin dimerisation and/or interactions with cargoes. Kinesins typically use scaffold proteins and adaptor proteins to bind their cargoes but can sometimes bind the cargo directly. Scaffolds and adaptors might also have regulatory roles in kinesin-driven intracellular transport, that is, recognising specific cargoes and regulating their loading and is levitra better than viagra unloading.3Role of KIFs in physiology and diseaseThe application of genome-wide sequencing for gene identification in research or for clinical diagnostic purposes significantly contributes to the identification of KIF candidate genes. Genotype–phenotype correlations in KIF gene-related disorders, together with functional and animal studies, continue to elucidate the complex involvement of KIFs in human developmental pathways and disease.

Table 1 summarises the monogenic conditions caused by variants affecting the function of KIF genes.View this table:Table 1 Specific monogenic disorders caused by variants affecting the function of KIF genesView this table:Table 2 Summary of phenotypes and genotypes of KIF149 26 30 31The kinesins’ functions in physiological processes, however, are complex and still incompletely understood, but their role in is levitra better than viagra cell-cycle progression and regulation, including both meiosis and mitosis, in intracellular trafficking, axonal transport, microtubule activity and ciliogenesis, is increasingly studied. Figure 2 summarises the clustering of KIF genes according to their functional roles and the phenotypical consequences as identified to date in 32 out of the 44 human kinesin genes.Assignment and clustering of KIF genes to various functions and relation to birth defect or monogenic phenotype groups. Detailed phenotypes are shown in tables 1 and 3 is levitra better than viagra. Cancer and multifactorial conditions are not included.

CNS, central nervous system." data-icon-position data-hide-link-title="0">Figure 2 Assignment and clustering of KIF genes to various functions and relation to birth defect or monogenic phenotype groups. Detailed phenotypes are shown in tables 1 and 3 is levitra better than viagra. Cancer and multifactorial conditions are not included. CNS, central nervous is levitra better than viagra system.Kinesins play a pivotal role during early development and organogenesis.

Microcephaly is one of the most frequently associated clinical signs, mirroring a defect in the regulation of the final number of neurons during development.10KIF4A is a motor protein that translocates PRC1, a cytokinesis protein, to the ends of the spindle microtubules during mitosis, regulates the PARP1 activity in brain development and the survival of neurons, and is a member of the L1CAM recycling pathway. Variants in L1CAM cause X-linked isolated is levitra better than viagra and syndromic hydrocephalus. KIF4A was recently proposed as a candidate gene for hydrocephalus.11KIFs are involved in neuronal branching, and microtubule depolarisation, operated by KIF2A M-kinesin, was suggested to suppress collateral branch extension during brain development, leading to anomalies of cortical development, including agyria and pachygyria, subcortical band heterotopia and corpus callosum anomalies.12Functional disruption of KIF genes in knockout mice often results in embryonic lethality, for example, for Kif18A, Kif10, Kif3A, Kif3B and Kif5B,13–17 highlighting the importance of kinesins in embryonic and foetal development. A study on KIF16B demonstrated that microtubule-based trafficking is responsible for early development and stem cell survival.18 KIF26B is essential in kidney development, contributing to the adhesion of mesenchymal cells to the ureteric bud.3 KIF26A was suggested to play a role in enteric nervous system development, because knockout mice develop a megacolon and enteric nerve hypoplasia,19 and to negatively regulate nociceptive sensation.20A significant number of KIFs play a prominent role in ciliogenesis and cilia function.

They regulate cilia length, ciliary assembly/disassembly and can have motile cilia-specific functions.21 Some KIFs, specifically found in primary cilia (PC), regulate the length of the axoneme and its disassembly when re-entering the cell cycle.KIF7, also a key component of the Hedgehog signalling is levitra better than viagra pathway, is responsible for cilia length regulation through suppression of microtubule polymerisation.7 KIF7 variants cause hydrolethalus, acrocallosal, and Joubert and Al-Gazali-Bakalinova syndromes.22 Kif2A knockout mice have severe brain defects, and KIF2A variants in humans lead to microcephaly because of cell-cycle delay in cellular progenitors resulting from cilia disassembly defects. KIF24, belonging to the same kinesin 13 family, plays a role in both microtubule depolymerising activity and regulation of the early steps of ciliogenesis. Other PC-related KIFs recently identified are KIF5B, KIF1C and KIF13B, and a potential role in cilia was hypothesised for KIF11 and KIF14.KIF3 protein complex (KIF3A-KIF3B-KAP3 heterotetramer) is a molecular motor necessary for intraflagellar transport (IFT) but is also involved in ciliogenesis of motile is levitra better than viagra cilia. Kif3a-knockout or Kif3b-knockout mice are prenatally lethal, exhibiting anomalies similar to ciliopathy phenotypes, including the disturbance of left–right body determination.3KIF19A is localised at the tip of motile cilia and performs motor and microtubule-depolymerising activities during IFT.

Kif19a-knockout mice present with hydrocephalus and female infertility, common signs in ciliary defects, due to abnormally elongated cilia with altered motility, not able to generate proper fluid flow.9Further KIFs, which may have specific roles in motile is levitra better than viagra cilia, are KIF27, KIF9, KIF6 and KIF18B. Regarding the involvement of numerous KIFs in cilia-related processes, it is not surprising that many disorders caused by variants affecting KIF gene function are presenting with anomalies reminiscent of ciliopathies.Kinesin motors have a fundamental role in neuronal function, as they are responsible for the transport of synaptic vesicle precursors and transmitter receptors along axons and dendrites from the neuron body.3 Molecular motor activity as for KIF1A, KIF5 and KIF17 is important for higher brain functions, such as learning and memory through regulation of synaptic transmission.5 Dysfunction can be associated with intellectual disability and global developmental delay (table 1).Impaired function can also result in peripheral neuropathies (KIF5A, KLC2, KIF1A and KIF1B) and ocular motility disorders (KLC2 and KIF21A)23 24 when axon elongation in the peripheral nervous system and optic nerve is affected. KIF5A variants are associated with epileptic phenotypes both in humans and mice25 because the transport of neurotransmitter receptors is disturbed and inhibitory regulation is altered.Due to their role in cell-cycle regulation, kinesins are important in male spermatogenesis and female oogenesis. They are involved in all steps of spermatogenesis 26 and, based on previous animal is levitra better than viagra studies, they may represent a potential target to treat male infertility.

In female meiosis, 13 KIF genes were studied in animal models. There is some evidence that kinesin expression is vulnerable to maternal ageing and is levitra better than viagra environmental factors, such as oocyte cryopreservation and alcohol consumption. It may be promising to expand research in this field in order to clarify the mechanisms and factors contributing to oocyte quality decline.27Many kinesins were extensively studied in the fields of cancer development, progression and therapy. Deregulation of the mitotic kinesins by both overexpression and decreased expression causes cancer progression or can be a prognostic marker in various tumours.28 The cell-permeable small-molecule mitotic is levitra better than viagra inhibitor monastrol was discovered in 199929 and was shown to arrest cells in mitosis by specifically inhibiting KIF11, a kinesin important for spindle bipolarity.

The bipolar mitotic spindle is replaced by a monoastral microtubule array surrounded by a ring of chromosomes, which gave the inhibitor its name. The mitotic spindle is now is levitra better than viagra a well-known target of chemotherapy, and inhibitors of the mitotic kinesins KIF11, KIF10 and KIF1C are being studied for this purpose.28 30 The redundancy of some kinesins allows them to escape pharmacological inhibition. For example, in the absence of KIF10, KIF15 is able to replace all of its essential functions in spindle assembly. Cilia-related KIF7, KIF13B and KIF27 are involved in SHh signalling and may be a future target in cancer research.28Some kinesins confer susceptibility to a range of multifactorial, metabolic and neurodegenerative conditions.

KIF13B contributes to the enhancement of endocytosis of low-density lipoprotein (LDL) receptor-related protein 1 that is levitra better than viagra is involved in the recognition and internalisation of LDL and factor VIII. Kif13b-knockout mice have hypercholesterolaemia and higher factor VIII serum levels.5 KIF12 is implicated in the pathogenesis of type 2 diabetes, protecting pancreatic β cells from the oxidative stress caused by nutritional excess.5 Variants in KIF1B or KIF21B confer susceptibility to multiple sclerosis (OMIM %612596, #126200).31 32 KIF5A was associated with Amyotrophic lateral sclerosis (OMIM #617921).33 KIF3 complex and KIF17 were recently uncovered to be involved in schizophrenia.34 35 Further studies, however, are needed to clarify the precise role of KIFs in neurodegenerative processes and psychiatric conditions.KIF14 -related birth defects. Lessons learntAdvances is levitra better than viagra in next-generation sequencing technologies have revolutionised our understanding of Mendelian disorders, including birth defect phenotypes, by sequencing the coding genome (exome) or entire genome at an unprecedented resolution in a comparably short time span. The technology has been extensively used for gene identification approaches in research for many years, enabling unparalleled genotype–phenotype correlations and the definition of novel pathways of related genes and disorders at an accelerated pace, traditionally focusing on postnatal disorders.

Filges and Friedman36 postulated that a number of novel is levitra better than viagra disease genes causing birth defects could be identifiable through the investigation of lethal foetal phenotypes since they would represent the extreme end of allelic milder and viable postnatal phenotypes with less specific or recognisable anomaly patterns. Based on embryonically or perinatally lethal mouse models (www.informatics.jax.org and www.dmdd.org.uk), it is estimated that knockout variants in about 30% of human protein coding genes may present with a phenotype of early lethality. The identification of KIF14 loss of function variants in fetuses with a lethal multiple congenital anomaly syndrome and the subsequent description of the allelic postnatal viable phenotype and further functional characterisation of KIF14 in developmental processes are recent examples of how to study those embryonic lethal phenotypes in order to understand the role of genes for which little to nothing is known.Filges et al identified autosomal recessive compound heterozygous loss of function variants in KIF14 using family-based exome sequencing in a recurrent severe lethal phenotype (OMIM #616258). It was the first human phenotype reported due to is levitra better than viagra variants in the human KIF14 gene (figure 3).37 The two affected siblings presented with intrauterine growth retardation (IUGR), oligohydramnios, severe microcephaly, renal cystic dysplasia or agenesis, genital tract malformations (uterine hypoplasia and vaginal atresia), as well as cerebral and cerebellar hypoplasias with partial or total agenesis of the vermis, arhinencephaly, agenesis of occipital lobes/corpus callosum at second trimester ultrasound scan.

Cross-species comparison to the laggard spontaneous mice mutant, characterised by homozygous variants of the Kif14 gene,38 confirmed a phenotypical overlap. An increased number of binucleated cells in the tissue histology of the two fetuses were in concordance with the key role of KIF14 during mitosis participating in chromosomes’ congression and alignment, as well as is levitra better than viagra in cytokinesis39 and the observation of binucleated cells as a consequence of failed cytokinesis in mammalian KIF14 knockdown cells. During cytokinesis, PRC1 localises KIF14 at the central spindle and midbody, which in turn recruits citron rho-interacting kinase (CIT) to the midbody. CIT, in turn, acts is levitra better than viagra as a negative regulator of KIF14 activity.

Knockdown of KIF14 in mammalian cells results in impaired localisation of CIT during mitosis.40Structure of KIF14 and summary of all published KIF14 variants affecting function.10 37 41 42 The N-terminal region (aa 1–356) is important for its interactions with PRC1 and the protein’s localisation at the central spindle and midbody. The kinesin motor domain (aa 358–701) is responsible for the microtubule-dependent ATPase activity. The FHA domain is levitra better than viagra (aa 825–891). Stalk and tail region (aa 891–1648) are necessary for the interaction with the protein CRIK (aa 901–1189, red diagonal lines).

There are four additional coiled-coil domains is levitra better than viagra (light blue-coloured areas).61 FHA, forkhead associated. Aa, amino acid." data-icon-position data-hide-link-title="0">Figure 3 Structure of KIF14 and summary of all published KIF14 variants affecting function.10 37 41 42 The N-terminal region (aa 1–356) is important for its interactions with PRC1 and the protein’s localisation at the central spindle and midbody. The kinesin motor domain (aa is levitra better than viagra 358–701) is responsible for the microtubule-dependent ATPase activity. The FHA domain (aa 825–891).

Stalk and tail region (aa 891–1648) are necessary for the interaction with the protein CRIK (aa 901–1189, red diagonal is levitra better than viagra lines). There are four additional coiled-coil domains (light blue-coloured areas).61 FHA, forkhead associated. Aa, amino acid.Filges et al pointed out that KIF14 should be considered a candidate gene for viable postnatal phenotypes, including isolated microcephaly.34 Additional individuals with autosomal recessive variants in KIF14 and isolated primary microcephaly were then described9 41 42 (table 2).Impaired cytokinesis, increased apoptosis and reduced cell motility were confirmed in cells from the described patients, pointing to a new cellular pathway in the pathogenesis of microcephaly.43 Apart from one case with small kidneys with increased echogenicity, none of these 18 patients had associated kidney anomalies. However, a targeted exome sequencing study in 204 unrelated patients with congenital anomalies of the kidney and urinary tract (CAKUT) reported two more cases of renal anomalies, bilateral hypoplasia or agenesis, caused by KIF14 variants.44 Further nine cases had an associated renal phenotype, which ranged is levitra better than viagra from bilateral renal agenesis to cystic or non-cystic renal hypodysplasia.42 Table 2 and figure 3 summarise KIF14 variants and the associated phenotypes.

Loss of function variants more likely lead to multiple congenital anomalies, while hypomorphic variants result in a milder phenotype without renal involvement, although phenotype–genotype correlations remain preliminary for the time being.The phenotypical spectrum ranging from isolated primary microcephaly to congenital anomalies reminiscent of ciliopathy phenotypes suggested a complex role for KIF14 in developmental processes and raised a number of questions about the relationship between its established role in cell division and its possible function in ciliary pathways. Functional studies of absent KIF14 protein in the development of human foetal tissues and mutant zebrafish provided evidence for similarities and differences between mitotic events occurring during proliferation in the development of both brain and kidney.42 The observation that KIF14-stained midbodies accumulate within the lumen of the branch tips of ureteric buds in human foetal kidneys provided a key clue to better understand the mechanism is levitra better than viagra through which the loss of KIF14 affects both brain and kidney development in humans. It was previously demonstrated that the secretion and accumulation of midbody remnants in the cerebrospinal fluid in mice during the early stages of brain development correspond to the amplification of neural progenitors.45 Kif14 mutant zebrafish phenotypes supported the hypothesis of a potential role for KIF14 in cilia. In vitro and in vivo analyses suggested that loss of kif14 causes ciliary anomalies through an accumulation is levitra better than viagra of mitotic cells in ciliated tissues but failed to establish a direct functional link.21 42 Further mechanisms remain to be elucidated.

Overexpression of KIF14 in various types of tumours was suggested to be a possible prognostic marker and a potential target for therapeutic purposes.46Kinesinopathies in birth defect phenotypes. Recurrent themesIn the last few years, an increasing number of variants in KIF genes were described to cause isolated as well as multiple congenital anomalies. There is a huge variability of phenotypes caused by variants is levitra better than viagra even within the same gene. However, we can identify recurrent clinical signs that should alert the clinician to suspect a KIF gene-related disorder and the molecular geneticist to include KIF genes in multigene-panel and genome-wide sequencing approaches.

This will become particularly relevant in prenatal and perinatal medicine, which focuses on the is levitra better than viagra detection of structural anomalies in the fetus and the newborn by using ultrasound and MRI or autopsy when the outcome is lethal. We have summarised the predominant and recurrent structural anomalies in kinesinopathies reported so far that would likely become apparent during the foetal period in table 3 and the syndromic disorders in table 1.View this table:Table 3 KIF gene-related structural congenital anomalies recurrently described in prenatal phenotypesSupplemental materialConsistent with the kinesins’ role in the development of the central nervous system (CNS), brain anomalies of various degrees are a frequent clinical sign, particularly microcephaly, but include lissencephaly, polymicrogyria, thinned or agenesis of the corpus callosum, arhinencephaly, cerebral hypoplasia or atrophy, cerebellar hypoplasia or atrophy, brainstem hypoplasia and a molar tooth sign on brain imaging.12 22 37 44 47–51Primary microcephaly can be detected prenatally or at birth12 22 47 48 50 51 and can present as an isolated or syndromic condition as, for example, caused by variants in KIF149 or in KIF11 (microcephaly with or without chorioretinopathy, lymphoedema or mental retardation. OMIM #152950).48KIF7 variants were related to macrocephaly in the presence is levitra better than viagra of congenital hydrocephalus (hydrolethalus syndrome LS2, OMIM # 614120). Isolated hydrocephalus was reported for KIF4A in a single case.11Foetal akinesia and arthrogryposis (KIF5C12, KIF1434 and KIF26B50) are likely secondary to the neurological compromise of the fetus but can also appear as an early sign of abnormal CNS development, which should prompt specialist CNS sonographic and MRI evaluation of the fetus.Further anomalies of the limbs include camptodactyly (KIF26B50), clubfoot (KIF1A51), rocker-bottom feet (KIF26B50) and congenital lymphoedema of the limbs (dorsa of feet, lower extremities and, rarely, hands) in cases with KIF11 gene mutations.48 In particular, KIF7 gene variants have been related to various anomalies of the hands (tapered fingers, fifth finger clinodactyly, brachydactyly, preaxial or postaxial polydactyly, bifid terminal phalanges of the thumbs, spindle-shaped fingers, clinodactyly and soft tissue webbing) and feet (toe syndactyly, preaxial or postaxial polydactyly, and duplicated halluces).22CAKUT and genital anomalies are reported in various kinesinopathies including renal agenesis or hypoplasia (KIF1437 and KIF1252), ureteral hypoplasia (KIF1437), congenital megabladder (KIF1252) and vesicoureteral reflux (KIF1252), uterine hypoplasia and vaginal atresia (KIF1437) and hypospadias and chordae (KIF16B49).IUGR is recurrently detected (KIF5C12, KIF1437, KIF1053, KIF1554 and KIF2A12) and is particularly relevant when occurring simultaneously with one of the other recurrent clinical signs, indicating a potential syndromic KIF-related disorder.

Oligohydramnios or polyhydramnios is most likely secondary to a primary organ anomaly.There are a few kinesinopathy syndromes that have been specifically reported to be lethal, such as the ciliary phenotype (OMIM #616258), caused by variants in KIF1434, and hydrolethalus syndrome (OMIM #614120), caused by variants in KIF7.22 However, lethality is usually closely related to the specific major anomalies, and it can be hypothesised that such a lethal phenotype will exist for all KIF gene-related disorders.Developmental delay, intellectual disability, seizures, and sensory and motor disturbances of the peripheral nervous system, as well as eye anomalies, such as microphthalmy, optic nerve pallor, fibrosis of extraocular muscles and chorioretinopathy, will escape detection in the foetal period but are reported in postnatal patients.Kinesin pathways in birth defectsFunctional studies of kinesins in birth defects are still sparse, and little is known about their networks and pathways. In order to improve our understanding, we used the Ingenuity Pathway Analysis (IPA Qiagen, Redwood City, California, USA) to visualise and analyse the connections between the 13 kinesin motor proteins associated with structural congenital anomalies (KIF5C, KIF1A, KIF1BP, KIF14, KIF16B, KIF7, KIF4A, KIF11, KIF10, KIF26B, KIF12, KIF15 and KIF2A) and in up to 10 of is levitra better than viagra each of their most significant downstream proteins. The connections are defined as protein–protein interactions, activation, regulation of binding, expression, localisation, phosphorylation, protein–RNA interactions, molecular cleavage, ubiquitination, protein–DNA interactions, inhibition, translocation and transcription. Figure 3 displays the results is levitra better than viagra.

We used the software Gephy55 to look for all possible interactions between all proteins of the network and also used the IPA data to retrieve the canonical pathways involved. Figure 4 and online supplementary material, table is levitra better than viagra 4, summarise the results. KIF7, KIF14 and KIF12 are located within the same network, and because of multiple connections between themselves and their downstream proteins, it is not surprising that they are all involved in kidney anomalies. IPA data is levitra better than viagra are based on current publications and are therefore subject to bias because proteins that are most interconnected are also most probably those that have been more extensively studied.

However, we consider the KIF genes coding for proteins seeming less important within the network to be strong candidates for future studies of human developmental disorders.IPA of the 13 kinesins known to be involved in birth defects with respect to their position in the cell. Proteins displayed on the right side of the figure, below the tag ‘other’, are those for which no subcellular location is known. Birth defect-related kinesins and their connection with each other are highlighted in green is levitra better than viagra. Light blue-coloured downstream proteins are those which are known to cause birth defects when altered.

Yellow-coloured proteins are is levitra better than viagra those involved in neurological disorders overlapping with the clinical features of kinesinopathies. The legend of the biological function associated with every molecule is displayed on the right. Path Designer is levitra better than viagra by IPA was used for the figure design. IPA, Ingenuity Pathway Analysis." data-icon-position data-hide-link-title="0">Figure 4 IPA of the 13 kinesins known to be involved in birth defects with respect to their position in the cell.

Proteins displayed on the right side of the figure, below the tag ‘other’, are those for which no subcellular location is known. Birth defect-related kinesins and their connection with each is levitra better than viagra other are highlighted in green. Light blue-coloured downstream proteins are those which are known to cause birth defects when altered. Yellow-coloured proteins are those involved in is levitra better than viagra neurological disorders overlapping with the clinical features of kinesinopathies.

The legend of the biological function associated with every molecule is displayed on the right. Path Designer by is levitra better than viagra IPA was used for the figure design. IPA, Ingenuity Pathway Analysis.Closing remarks and future perspectivesNovel KIF genes are increasingly identified, and there is a growing body of literature demonstrating the impact of kinesin dysfunction in human disease. We propose to introduce the term kinesinopathies for conditions caused by variants in KIF genes, since recurrent and common functional and phenotypical is levitra better than viagra themes can be observed.

In analogy to ciliopathies56 and rasopathies,57 the delineation of the clinical, genetic and functional hallmarks of kinesinopathies will be important to better recognise these conditions, to understand the pathomechanisms and to ultimately improve the clinical management of the patients. Previously, the unified view of the phenotype characteristics of ciliary dysfunction allowed a tremendous increase in awareness, both in clinic and research, and the further identification of yet unrecognised ciliary disorders and the genes and proteins involved in their pathogenesis.56Remarkable progress was achieved in assigning function to kinesins through their study in isolated and multiple congenital anomaly phenotypes. They are one large superfamily of molecular motors out of three (kinesins, dyneins and myosins), which is of key importance in several fundamental cellular processes using microtubules as rails for directional anterograde intracellular transport, including its regulation and modulating signal transduction.5 is levitra better than viagra Kinesin motors are most important for the movement of chromosomes along the spindles during chromosome segregation, regulation of spindle formation, cell division and cytokinesis. These essential and broad cellular functions are critical for many physiological processes such as neuronal function and survival, some ciliary functions and ciliogenesis, determination of the left/right asymmetry of our body and regulation of organogenesis, thus explaining the impact and emerging recognition of kinesins in embryonic and foetal development.

Defects can result in neuropathies, is levitra better than viagra higher brain functions and structural brain anomalies. Multiple congenital anomalies, including the kidney and urinary tract and limb anomalies, are repeatedly reported. Microcephaly, which is usually not associated with genes implicated in specific ciliary mechanisms, and CNS is levitra better than viagra anomalies are the most recurrent clinical signs in both the prenatal and postnatal phenotypes described so far. The discovery of the implication of KIF14 in microcephaly further suggested a possible novel role of other microcephaly proteins in cytokinesis.

A number of syndromic kinesinopathies present, however, with phenotype patterns reminiscent of ciliopathies. So far, however, a direct functional impact was confirmed in only a few and could not be demonstrated, for example, for KIF14, despite an overlapping clinical is levitra better than viagra pattern. In turn, ciliopathies are a clinically and genetically heterogeneous group of conditions themselves. Studying tissue and cell type-specific function and expression may help to further define the specific defects related to the individual aberrant kinesin.The pleiotropic nature of human kinesinopathies, however, is just emerging, but is levitra better than viagra their study promises to provide important insights into human developmental pathways.

Seemingly unrelated clinical entities are highlighting a common theme. In a relatively short time span, monogenic KIF-related disorders were identified to present with often severe and lethal antenatal anomalies, with multiple or isolated congenital anomalies, neurodevelopmental and is levitra better than viagra neurological disorders, or an increased susceptibility to multifactorial conditions. We focused on the emerging role of kinesins in structural congenital anomalies because, as illustrated for the KIF14 gene, great potential to decipher allelic viable phenotypes and developmental pathways lies in the study of these human knockout phenotypes at the severe end of the phenotypical spectrum. Knockout variants in about 30% of human protein coding genes in our genome may present with a phenotype of early lethality, and KIF genes seem to play an important role in such fundamental processes of human development.

Identifying and characterising the variants, genes and phenotypes will extend our knowledge on early human development and pathomechanisms, and will ultimately also is levitra better than viagra improve the clinical utility of genome-wide sequencing approaches for prenatal and postnatal application by our increased ability to interpret loss of function and hypomorphic variants alike. Furthermore, kinesins were extensively studied in cancer research and therapeutic strategies targeting their specific functions, such as the example of monastrol and other inhibitors of the mitotic kinesins may be adopted in the future. There are likely many more kinesinopathies to be is levitra better than viagra unravelled in the field of birth defects because of their pivotal role in cellular logistics, but their recognition in clinics and research will depend on our ability to identify and characterise the common clinical, molecular and functional themes of these disorders and to use them to improve our understanding of their disease mechanisms.IntroductionIntellectual disability (ID) affects about 3% of individuals worldwide and raises significant issues in terms of diagnostic, management and genetic counselling. The presence of pigmentation anomalies in a patient with ID represents helpful clinical clues in order to narrow the range of aetiological hypothesis.

Hypomelanosis of Ito (HMI, MIM #300337) is an unspecific term encompassing a heterogeneous group of disorders characterised by cutaneous hypopigmented whorls and streaks along Blaschko’s lines is levitra better than viagra and variable extracutaneous features affecting the musculoskeletal and nervous systems.1 The cutaneous pattern therefore represents a non-specific hallmark of mosaicism in these neurocutaneous conditions. Genetic mosaicism is due to postzygotic mutations, either chromosomal rearrangements or point mutations, whereas random X inactivation in females leads to functional mosaicism.2 Unravelling the molecular basis of pigmentary mosaicism (PM) is still a challenge due to clinical and genetic heterogeneity, technical difficulties in detecting mosaic mutations by classical sequencing approaches and the complexities of obtaining affected tissue. As part of a collaborative group, we recently reported de novo mutations in exons 3 and 4 of transcription factor E3 (TFE3) as the cause for HMI in four unrelated individuals, including one male, as well as syndromic ID without pigmentary disorders in a female.3TFE3 belongs to the MITF family of mammalian basic helix–loop–helix zipper transcription factors, together is levitra better than viagra with TFEB and TFEC. All four can form homodimers or heterodimers with each other.4 Embryonic expression of TFE3 orthologues Tfe3a and Tfe3b was demonstrated in the zebrafish in a wide range of tissues.5 TFE3 subcellular localisation plays a crucial role in the regulation of cellular homeostasis and embryonic stem cell (ESC) differentiation.

The phosphorylated TFE3 is retained in the cytoplasm, whereas dephosphorylated protein translocates to the nucleus to promote the transcription of target genes involved in lysosomal biogenesis and autophagy.6 TFE3 relocalisation to the nucleus is driven on various stressors, such as starvation,7 8 DNA damage,9 mitochondrial damage,10 Golgi stress11 and pathogens12 in an mTORC1-dependent manner, and oxidative stress13 or cadmium exposition14 in an mTORC1-independent manner. Moreover, lysosomal signalling-induced nucleocytoplasmic redistribution of TFE3 is essential to the regulation of ESC renewal.3 15 By restricting nuclear localisation is levitra better than viagra and activity of Tfe3, lysosome activity, the tumour suppressor protein folliculin and the Ragulator protein complex enable the exit from pluripotency and therefore drive differentiation. Conversely, enforced nuclear Tfe3 enables ESCs to withstand differentiation.15 In humans, TFE3 mutations have long been known in cancer. Gene fusions by translocations or other chromosomal rearragements involving TFE3 and five partner genes have indeed been reported to occur in a subset of renal cell carcinomas (RCCs), referred to as ‘TFE-fusion RCC’, and, more rarely, to lung sarcoma and perivascular epithelioid cell tumours.16 Beyond these data on TFE3 function, by the report of a series of 17 individuals harbouring de novo mutations is levitra better than viagra in exons 3 and 4 of TFE3, we emphasise their phenotype and bring additional clinical insight toward the recognition of this novel developmental disorder.ResultsWe describe a series of 17 patients carrying a de novo mutation in TFE3, 5 of them being previously published with limited clinical information.3 Twelve were females and five were males.

Their age ranged from 12 months to 22 years. Five were referred for HMI, five is levitra better than viagra for syndromic ID and five for suspicion of storage disorder.Clinical dataThe clinical features are summarised in table 1. Additional information can be found in online supplementary data 1.Supplemental materialView this table:Table 1 Clinical and molecular features of the 17 patients with an TFE3 mutationNeonatal course was remarkable for nine patients. History of jaundice, hepatomegaly or feeding difficulties was reported for three patients each, hypoglycaemia for two and cholestasis for one.

All these features were transient.Developmental delay, usually severe is levitra better than viagra and noticeable from the first months of life, was a constant feature in all the individuals. Only 6 patients were able to walk at the time of the study, whereas 11 were still unable to walk. All patients were is levitra better than viagra non-verbal, except for two older patients who could speak a few words. Neurological examination was abnormal in 12 individuals and consisted in truncal hypotonia, associated with lower limb spasticity (6 individuals) or ataxia (2 adults).

Behavioural issues is levitra better than viagra such as autistic features and sleeping disturbance were noted for 11 patients. Eleven patients developed epilepsy, onset in the first decade and characterised as intractable in three of them. Brain MRI was normal in 10 individuals and abnormal in 6 patients (hydrocephaly, short corpus callosum, Dandy-Walker malformation, arachnoid cyst, periventricular white matter lesions, delayed myelination and cerebral atrophy). The sensory anomaly was congenital hearing loss (5 patients), and ophtalmological anomalies (10 patients) consisted of strabismus, hyperopia, retinal degeneration, depigmented macule on the iris, oculomotor apraxia or impaired vision of cortical origin.Facial dysmorphism shared among the patients consisted in coarseness, flat nasal bridge, short nose with anteverted nares, widely spaced eyes, almond-shaped is levitra better than viagra eyes, thick lips, facial hypertrichosis, fleshy earlobe, and full and pink cheeks (figure 1).

Twelve patients had pigmentation anomalies, located on Blaschko’s lines for 10 of them (figure 2). One was diagnosed at 4 years old with is levitra better than viagra histiocytofibroma. Moderate to severe postnatal growth retardation affected 10 patients, who had a length between −2.0 and −4.5 SD. Obesity affected is levitra better than viagra 13 individuals.

Skeletal anomalies were frequent (11 individuals) and consisted of flat or clubfeet, hyperlordosis, scoliosis, hip dislocation, limitation of elbow extension and genu valgum. Recurrent s of the is levitra better than viagra upper airways were noted in five patients. One had a documented neutropenia. Early-onset chronic interstitial lung disease was reported in two patients.

Nail clubbing was noted in is levitra better than viagra two individuals. Visceral malformations consisted of congenital heart defect (left ventricle dilatation, aortic insufficiency and patent ductus arteriosus) in three patients, umbilical hernia in three individuals, lateral semicircular canal dysplasia, posterior plagiocephaly, sleep apnoea syndrome, anteriorly displaced anus and hypospadias in one individual each.Facial phenotypes of seven patients. (A–C) Patient 5, aged is levitra better than viagra 6 months (A,B) and 3 years (C). (D) Patient 8, aged 5 years.

(E,F) Patient 2, aged 5 and 20 years is levitra better than viagra. (G–I) Patient 3, aged 1 year (G) and 3 years (H,I). (J,K) Patient 13, aged 22 years. (L–O) Patient 6, aged 8 months (L), 20 months (M) and is levitra better than viagra 3 years (N,O).

(P,Q) Patient 10 aged 22 years." data-icon-position data-hide-link-title="0">Figure 1 Facial phenotypes of seven patients. (A–C) Patient 5, is levitra better than viagra aged 6 months (A,B) and 3 years (C). (D) Patient 8, aged 5 years. (E,F) Patient is levitra better than viagra 2, aged 5 and 20 years.

(G–I) Patient 3, aged 1 year (G) and 3 years (H,I). (J,K) Patient 13, aged 22 years. (L–O) Patient 6, aged 8 months (L), 20 months (M) and 3 is levitra better than viagra years (N,O). (P,Q) Patient 10 aged 22 years.Cutaneous features.

(A) Patient 3 is levitra better than viagra. Umbilical hernia, widely spaced nipples and hypopigmentation on the left side of the abdomen. (B) Patient is levitra better than viagra 17. Hypotonia, umbilical hernia and widely spaced nipples.

(C) Patient is levitra better than viagra 8. Blaschko’s lines on the back. (D) Patient 6. Hypopigmentation on the left side of is levitra better than viagra the abdomen.

(E) Patient 7. Blaschko’s lines is levitra better than viagra on the abdomen and right side of the trunk. (F) Patient 13. Hand.

Note clubbing of thumbnail and loose skin. (G) Patient 17. Blaschko’s lines on the left side of the abdomen. (H) Patient 17.

Hand. Note tapering fingers and wrinkled skin. (I) Patient 1. Linear hypopigmentation on the back.

(J) Patient 6. Blaschko’s lines on the back. (K) Patient 3. Blachko’s lines on the right lower limb.

(L) Patient 6. Blaschko’s lines on the right lower limb. (M,N) Patient 7. Linear hyperpigmentation on the lower limbs.

(O) Patient 11. Blaschko’s lines on the back." data-icon-position data-hide-link-title="0">Figure 2 Cutaneous features. (A) Patient 3. Umbilical hernia, widely spaced nipples and hypopigmentation on the left side of the abdomen.

(B) Patient 17. Hypotonia, umbilical hernia and widely spaced nipples. (C) Patient 8. Blaschko’s lines on the back.

(D) Patient 6. Hypopigmentation on the left side of the abdomen. (E) Patient 7. Blaschko’s lines on the abdomen and right side of the trunk.

(F) Patient 13. Hand. Note clubbing of thumbnail and loose skin. (G) Patient 17.

Blaschko’s lines on the left side of the abdomen. (H) Patient 17. Hand. Note tapering fingers and wrinkled skin.

(I) Patient 1. Linear hypopigmentation on the back. (J) Patient 6. Blaschko’s lines on the back.

(K) Patient 3. Blachko’s lines on the right lower limb. (L) Patient 6. Blaschko’s lines on the right lower limb.

(M,N) Patient 7. Linear hyperpigmentation on the lower limbs. (O) Patient 11. Blaschko’s lines on the back.Molecular resultsThe characteristics of the 13 different de novo TFE3 variants identified in the 17 unrelated individuals are summarised in table 2.

All but one were missense variants, affecting nine different aminoacids. One was a splice donor mutation. This mutation was reported a few weeks ago in a patient with a similar phenotype.21 Two variants were localised in exon 3 and 11 in exon 4 (figure 3). All were absent from public databases and were predicted to be pathogenic by prediction softwares.

TFE3 protein and localisation of the missense variants identified. In bold are variants identified in two patients. In bold and underlined is the variant identified in three patients. In green is the intronic variant." data-icon-position data-hide-link-title="0">Figure 3 TFE3 protein and localisation of the missense variants identified.

In bold are variants identified in two patients. In bold and underlined is the variant identified in three patients. In green is the intronic variant.View this table:Table 2 Molecular data of the 13 de novo TFE3 mutations. Characteristics, inheritance, frequency in the public database GnomAD, prediction scores regarding pathogenicity (Polyphen, Grantham and CADD (Combined Annotation Dependant Depletion) scores).

The transcript is NM_006521.5The putative mosaicism was assessed through X inactivation studies in females and analysis of the exome sequencing data in males, by checking the total number of reads covering the variant, as well as the number of reads supporting the presence of the variant (table 1). Allele frequencies in females were always consistent with a constitutional heterozygous mutation. X inactivation was skewed in blood of the female patients 1, 3 and 7 and in fibroblasts of Patient 2. X inactivation was random in fibroblasts of Patient 1 and 3.

Regarding the male patients, the mutation was identified in 65% of the reads for Patient 17% and 88% of the reads for Patient 15 (106/120). No mosaicism was detected in the blood of Patient 13, 14 and 16 despite the presence of pigmentary manifestations in Patient 16.DiscussionTFE3 functions in signalling of the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1). The PIK3-AKT-mTOR pathway plays a role in the regulation of cellular growth, proliferation, survival and metabolism. Overactivation of the mTOR signalling is responsible for neurocutaneous disorders and cancers.22 Somatic mutations in TSC1, TSC2, AKT3, PIK3CA and MTOR are responsible for focal cortical dysplasia type II (MIM607341),23–25 hemimegalencephaly26 and megalencephaly.27 The phenotype ascribed to germline TSC1/TSC2, PTEN, MTOR and PK3R2/AKT3/CCND2 mutations – respectively in tuberous sclerosis (TS, MIM 191100), Cowden syndrome (CS, MIM 158350), Smith-Kingsmore syndrome (MIM 616638) and Megalencephaly, Polymicrogyria, Polydactyly and Hydrocephalus syndrome 1, 2 and 3 (MPPH1/2/3, MIM 603387/615939/615938) - is characterised by the association of ID, epilepsy, brain malformations and skin tumours.

Similarly, all the individuals harbouring a de novo TFE3 mutation reported in the series presented with a severe neurodevelopmental disorder. Delayed psychomotor development was constant. The youngest patient to acquire independent walking was 30 months old, and more than half of the patients aged over 18 months (57%), did not acquire walk at the last examination. Conversely to patients with MTOR, AKT3 or PTEN mutation, none of the patients described in this series had macrocephaly.

Brain imaging was abnormal in 35% of the patients. Hydrocephaly and corpus callosum dysgenesis, identified in respectively three and one individual, were previously reported in patients with mosaic gain of function MTOR mutations.28 29 One patient had surgery to remove an early-onset histiocytofibroma. However, no other skin tumour was reported, either in this patient or in any other from the series.Pigmentation anomalies, along Blaschko’s lines or, for one patient, as a large hyperpigmented area, were present in a majority of the individuals (71%) in the series, including 40% of the males and 83% of the females. PM along Blaschko’s lines is highly suggestive of genetic mosaicism.30 Genomic mosaicism is defined by the presence of at least two cell populations with different genotypes in an individual originating from one zygote and mainly occurs through post-zygotic event, whereas females can present with functional (epigenetics) mosaicism due to X inactivation.2 PM is a classical feature of X-linked male-lethal genodermatosis, such as incontinentia pigmenti (IP, MIM #308300), focal dermal hypoplasia (FDH, MIM #305600), chondrodysplasia punctuata type 2 (Conradi-Hunermann-Happle syndrome, CDPX2, MIM #302960) and linear skin defects with multiple congenital anomalies (LSDMCA1, MIM #309801).

In these conditions, the overwhelming predominance of affected females is a consequence of the male lethality, and the PM a manifestation of the functional mosaicism occurring in females. Similarly, the majority of individuals with de novo TFE3 variants in our cohort were females (sex ratio female:male was 12:5 (2.4)). The study of X-inactivation on non-cultured fibroblasts was consistent with functional mosaicism in two affected females with PM who harboured random X-inactivation, whereas a third female without PM had skewed X-inactivation. In IP, FDH and CDPX2, most hemizygous males die in utero.

However, there have been reports of surviving males 31–33 with an estimated prevalence around 10% in FDH and IP.32 34 The majority of them are explained by post-zygotic mutations or chromosomal anomalies (Klinefelter syndrome). Non-mosaic males have also been reported in FDH and IP – respectively about 17% and 45% of the affected males harbour a non-mosaic variant.32 35 In our series, males represented 29% of the patients with a de novo TFE3 variant. A mosaic variant was identified in blood for half of them. None had Klinefelter syndrome.

Interestingly, mosaicism was detected in only one out of the two males with PM, and one male with a mosaic variant had no pigmentation anomalies noted on examination. It is still possible that subtle pigmentation anomalies were missed on examination. Moreover, somatic mosaicism can be difficult to detect. Recent studies have shown that a large proportion of de novo mutations presumed to be germline had in fact occurred as post-zygotic event.36 In the males of this cohort, WES was performed on leucocytes-derived DNA and no other tissue was studied.

Therefore, it is possible that a low mosaicism was not detected. Finally, it is probable than TFE3 mutations account for a significant proportion of patients with HMI. Indeed, in this population, the high frequency of ID, epilepsy, coarse facial features has long been emphasised in the literature.37By its ability to bind the coordinated lysosomal enhancement and regulation (CLEAR) sites in the promotor region of target genes, TFE3 is involved in the control lysosomal biogenesis, autophagy and endocytosis.8 Several patients of the series indeed had clinical and biochemical features that pointed toward an inborn error of metabolism. Lysosomal storage disorder was suspected due to the variable association of coarse facial features (88%), skeletal anomalies (65%) –flat or clubfeet, hyperlordosis, hip dislocation, limitation of elbow extension, genu valgum, scoliosis–, postnatal growth retardation (59%), history of speech or developmental regression (29%) congenital hearing loss (29%), recurrent upper airways s (29%) neonatal liver anomalies such as hepatomegaly and cholestasis (18%), upper airways s (24%), umbilical hernia (18%), sleep apnoea syndrome (6%) and aortic insufficiency (6%).

Other metabolic anomalies observed in the series were obesity, defined in children by body mass index (BMI) (weight/height2) above the WHO curve, present in the oldest patients (76%), neonatal transient hypoglycaemia (12%), and hyperlactataemia (6%). Dysregulation of lipid metabolism, via suppression of thermogenesis and decreased lipolysis, thus leading to increased adipose tissue, was previously observed in adipose-specific TFE3 transgenic mice.38 Similarly to lysosomes, mitochondrias have a key role in cellular metabolism, including autophagy. Recent data demonstrate that mitochondrial and lysosomal metabolisms are interrelated.39 Muscle biopsy, performed in two individuals from this cohort, showed fat and glycogen accumulation, muscular fibre size irregularity, without evidence of mitochondrial dysfunction. Interestingly in the more recent data, evidences showing that TFE3 plays a role in the regulation of the circadian oscillations of the expression of genes involved in autophagy and lipid metabolism, and that Tfe3 knock-out mice had abnormal circadian behaviour.40 Indeed, in our series, five patients (29%) were noted to have sleep disturbance.

This could be due to circadian rhythms alteration. Finally, TFE3 has been shown to be involved in the regulation of innate immune response in macrophages via the FLCN-AMPK signalling axis,41 and of B-lymphocytes activation.42 Along these lines, four patients of the series (24%) had a history of recurrent s, associated with documented neutropenia in one of them. As shown in table 3, a summary of the frequency of the features observed in the cohort, facial dysmorphism was constant and strikingly similar among the patients. More than two-third had anteverted nares, broad flat nasal bridge, almond-shaped and widely spaced eyes, puffy cheeks and coarse facial features (thick lips and fleshy earlobes).

More than half had facial hypertrichosis. All individuals presented with at least four of the above features. One patient had an extreme facial phenotype of hypertelorism, bifid nose and bilateral cleft lip and palate. Whether these frontonasal dysplasia features may be associated with the TFE3 mutation remains unclear.

No other mutation in known genes was found in Patient 1’s exome sequencing data.View this table:Table 3 Frequency of the clinical features observed in the seriesTFE3 is a highly conserved protein, intolerant to loss of function as supported by data from the GnomAD browser43 (probability of being loss-of-function intolerant (pLI) evaluated at 0.98, observed:expected ratio=0.06) and to missense variants (Z=2.15). Moreover, TFE3 does not, or only in a few tissues, escape X inactivation, suggesting that TFE3 gene dosage is crucial to cell function.44 45 In vitro, Villegas et al recently showed that the absence of either TFE3 exon 3 or 4 resulted in a nuclear gain-of-function Tfe3 allele in ESCs, indicating that both exons 3 and 4 are required for Tfe3 inactivation.3 Nuclear localisation and resistance to differentiation were proved in Tfe3 knock-out (K.O.) ESCs expressing murine Tfe3 alleles (Gln118Pro and Pro185Leu, corresponding to mutations Gln119Pro and Pro186Leu identified in individuals referred to as patients 1 and 2 in this series). Based on the analysis of TFE3 secondary structure,46 indicating that residues 110–215 are predicted to form a domain of two stable alpha helices that might be disrupted by mutations in exons 3 and 4, and the observation of a similar phenotype in patients harbouring mutations in exons 3 and 4, it was suggested that Tfe3 exons 3 and 4 form a Rag binding fold whose structural integrity is indispensable for lysosome-mediated cytoplasmic Tfe3 inactivation.3 In this series, the recurrent mutations Arg117Gln, Leu191Pro and Thr187Met were present in respectively two, two and three patients. The aminoacid in position 187 was mutated in five patients.

In addition, 13 of the described mutations were localised between positions p.184 and p.201. This proximity could account for the absence of obvious genotype–phenotype correlation. The canonical splice site variant in intron 4 identified in patient 9 might lead to in-frame exon skipping of exon 4. The clinical picture of the patient with this splice site variant perfectly fits with the syndrome described here.

As a consequence, we raise the hypothesis of a gain-of-function effect of this variant.In conclusion, de novo mutations in exons 3 and 4 of the X-linked gene TFE3 are responsible for a neurocutaneous disorder with specific and recognisable facial dysmorphism, lysosomal storage disorder-like features and PM. This series unravels TFE3 as a major gene responsible for HMI and for a rare cause of syndromic ID. Furthermore, we provide clinical and molecular data on a previously unidentified lysosomal storage disorder, in which new insights, especially biochemical features, will probably be investigated further, together with the description of more patients. Further delineation of this phenotype will indeed allow a better understanding of the link between lysosomal signalling and development.

Finally, the evidence for mosaicism in this recently described disorder highlights the importance of considering mosaic variants on next-generation sequencing reports in diagnostic, including for patients without suggestive phenotype..

IntroductionThe mammalian kinesin superfamily proteins (KIFs) are microtubule and ATP-dependent molecular motors, which were first identified in 1985 as axonal transporters in squid and bovine brains.1 Forty-five different kinesin family member (KIF) genes were identified buy generic levitra from canada in the mouse genome so far, 44 of which are present in the human genome. Phylogenetic analysis based on sequence homology between the human and the mouse genome led to the classification of KIF genes into 16 families, from kinesin-1 to kinesin-14B (figure 1).2 The first kinesins discovered belong to the kinesin-1 family (KIF5A, KIF5B and KIF5C), and they form a heterotetramer of two heavy chains and two light chains (KLC1-4).2 KIF genes encode KIFs, a specific class of motor proteins generating intracellular motility by driving directional transport of various cargoes such as organelles, protein complexes and mRNAs along the microtubule system.2 Studies using knockout mouse models by Hirokawa and colleagues significantly contributed to elucidate the roles of kinesins in mammalian physiology. Their role in transport is fundamental to cellular logistics and performance, and the molecular motors are not only effectors of signal transduction cascades but also transport and/or bind to important signal transduction molecules to actively modulate their function.3Phylogenetic tree of mammalian kinesin superfamily genes identified in the human (and mouse) genome and classified in 16 subfamilies (from kinesin 1 to 14B) (adapted from Hirokawa et al 3)." data-icon-position data-hide-link-title="0">Figure 1 Phylogenetic tree of mammalian kinesin superfamily genes identified in the human (and mouse) genome and classified in 16 subfamilies (from kinesin 1 to 14B) (adapted from Hirokawa et al3).The first kinesins were observed in the context of axonal transport in neurons, and a novel disease entity of ‘motor–proteinopathy’ was proposed for the pathogenesis of axonal neuropathies in 2001.4 Due to their role in cellular membrane trafficking, however, kinesins are essential for the functioning of many polar cell types, buy generic levitra from canada such as neurons, epithelial cells, sperm cells or stem cells during organogenesis. Kinesins also play a fundamental role in cell-cycle dynamics, both during mitotic and meiotic processes.

They regulate chromosomal condensation and alignment, spindle formation, cytokinesis and buy generic levitra from canada cell-cycle progression.5 It is estimated that about a dozen kinesins are involved in the cell cycle. Among these, there is a specific subclass of chromokinesins (kinesin 4 and kinesin 10 family) which are able to bind chromosomes.6 Recently, KIFs were discovered to act as microtubule stabilisers (KIF26A and KIF21A) and depolymerisers (KIF2A and KIF2C), activities which are important for both cellular morphogenesis and mammalian development, playing a role in neuronal and axonal morphology and ciliogenesis.7Alterations in motor kinesins are leading to human disease by various pathological mechanisms, including cancer and multifactorial and monogenic disorders. Variants in 18 out of the 44 human KIF genes were identified to cause monogenic disorders, following different modes of Mendelian inheritance and associated with a buy generic levitra from canada wide spectrum of clinical signs. These range from lethal and multiple to isolated congenital anomalies—including birth defects potentially detectable in the foetal period by current prenatal imaging studies—to postnatally apparent neurodevelopmental disorders, intellectual disability and neurological conditions.We will review the current state of knowledge of the biological processes kinesins are involved in and discuss their emerging role in human disease, particularly in birth defects and congenital anomaly syndromes.

Birth defects remain a leading cause of perinatal lethality in industrialised countries.8 Structural anomalies are recognised with increasing reliability during early pregnancy by the use of high-resolution ultrasound technologies, thus raising questions about diagnosis, aetiology, prognosis and recurrence risk, particularly in the presence of more than one anomaly, which most likely indicates a genetic aetiology. We identify recurrent phenotype patterns caused by alterations in KIF genes, and we outline the complexity of buy generic levitra from canada phenotype–genotype correlations mirroring the processes of intracellular microtubule-mediated transport and movement, in which kinesins play a fundamental role. There are likely many more relationships between the clinical signs and the genetic variants to be identified in the future, and the functional network of kinesins and their role in human disease need to be further elucidated. We propose to introduce the term ‘kinesinopathies’ for this group of conditions, which are phenotypically and genetically overlapping and characterised by the functional impairment of a specific buy generic levitra from canada group of molecular motors.

We hope that their systematic approach leads to a better recognition in clinical practice, as well as in genome-wide sequencing for diagnosis and research, and opens strategies for the future development of molecular therapies.KIF structureAll KIFs have a phylogenetically well-conserved motor domain head, consisting of an ATP-binding motif and a microtubule-binding domain. Depending on the position of the motor domain, kinesins can be subdivided into N-kinesins (amino-terminal motor domain), M-kinesins (middle-region motor domain) and C-kinesins (carboxy-terminal motor domain).2 Most kinesins belong to the N-kinesin subgroup, but members of the kinesin 13A family (figure 1) belong to the M-kinesin subtype, while KIF1C, KIF2C and KIF3C belong to the C- kinesin subfamily.3 Both N-kinesins and C-kinesins are responsible for plus end and minus end-directed motility, M-kinesins buy generic levitra from canada for depolymerisation of microtubules in tubulin molecules. However, there are a few exceptions to this categorisation.9 The motor domain head attaches to the neck, the coiled coil stalk and the tail. The kinesins’ neck is family-specific and responsible for the direction of motility or regulation of activity.

The coiled coil stalk and tail are involved in kinesin buy generic levitra from canada dimerisation and/or interactions with cargoes. Kinesins typically use scaffold proteins and adaptor proteins to bind their cargoes but can sometimes bind the cargo directly. Scaffolds and adaptors might also have regulatory roles in kinesin-driven intracellular transport, that is, recognising specific cargoes and regulating their loading and unloading.3Role of KIFs in physiology and diseaseThe application of genome-wide sequencing for gene identification in research or for clinical diagnostic purposes significantly contributes to the identification of KIF candidate genes buy generic levitra from canada. Genotype–phenotype correlations in KIF gene-related disorders, together with functional and animal studies, continue to elucidate the complex involvement of KIFs in human developmental pathways and disease.

Table 1 summarises the monogenic conditions caused by variants affecting the function of KIF genes.View this table:Table 1 Specific monogenic disorders caused by variants affecting the function buy generic levitra from canada of KIF genesView this table:Table 2 Summary of phenotypes and genotypes of KIF149 26 30 31The kinesins’ functions in physiological processes, however, are complex and still incompletely understood, but their role in cell-cycle progression and regulation, including both meiosis and mitosis, in intracellular trafficking, axonal transport, microtubule activity and ciliogenesis, is increasingly studied. Figure 2 summarises the clustering of KIF genes according to their functional roles and the phenotypical consequences as identified to date in 32 out of the 44 human kinesin genes.Assignment and clustering of KIF genes to various functions and relation to birth defect or monogenic phenotype groups. Detailed phenotypes buy generic levitra from canada are shown in tables 1 and 3. Cancer and multifactorial conditions are not included.

CNS, central nervous system." data-icon-position data-hide-link-title="0">Figure 2 Assignment and clustering of KIF genes to various functions and relation to birth defect or monogenic phenotype groups. Detailed phenotypes buy generic levitra from canada are shown in tables 1 and 3. Cancer and multifactorial conditions are not included. CNS, central nervous system.Kinesins play a pivotal buy generic levitra from canada role during early development and organogenesis.

Microcephaly is one of the most frequently associated clinical signs, mirroring a defect in the regulation of the final number of neurons during development.10KIF4A is a motor protein that translocates PRC1, a cytokinesis protein, to the ends of the spindle microtubules during mitosis, regulates the PARP1 activity in brain development and the survival of neurons, and is a member of the L1CAM recycling pathway. Variants in buy generic levitra from canada L1CAM cause X-linked isolated and syndromic hydrocephalus. KIF4A was recently proposed as a candidate gene for hydrocephalus.11KIFs are involved in neuronal branching, and microtubule depolarisation, operated by KIF2A M-kinesin, was suggested to suppress collateral branch extension during brain development, leading to anomalies of cortical development, including agyria and pachygyria, subcortical band heterotopia and corpus callosum anomalies.12Functional disruption of KIF genes in knockout mice often results in embryonic lethality, for example, for Kif18A, Kif10, Kif3A, Kif3B and Kif5B,13–17 highlighting the importance of kinesins in embryonic and foetal development. A study on KIF16B demonstrated that microtubule-based trafficking is responsible for early development and stem cell survival.18 KIF26B is essential in kidney development, contributing to the adhesion of mesenchymal cells to the ureteric bud.3 KIF26A was suggested to play a role in enteric nervous system development, because knockout mice develop a megacolon and enteric nerve hypoplasia,19 and to negatively regulate nociceptive sensation.20A significant number of KIFs play a prominent role in ciliogenesis and cilia function.

They regulate cilia length, ciliary assembly/disassembly and can have motile cilia-specific functions.21 Some KIFs, specifically found in primary cilia (PC), regulate the length of the axoneme and its disassembly when re-entering the cell cycle.KIF7, also a key component of the Hedgehog signalling pathway, is responsible for cilia length regulation through suppression of microtubule polymerisation.7 KIF7 variants cause hydrolethalus, acrocallosal, and buy generic levitra from canada Joubert and Al-Gazali-Bakalinova syndromes.22 Kif2A knockout mice have severe brain defects, and KIF2A variants in humans lead to microcephaly because of cell-cycle delay in cellular progenitors resulting from cilia disassembly defects. KIF24, belonging to the same kinesin 13 family, plays a role in both microtubule depolymerising activity and regulation of the early steps of ciliogenesis. Other PC-related buy generic levitra from canada KIFs recently identified are KIF5B, KIF1C and KIF13B, and a potential role in cilia was hypothesised for KIF11 and KIF14.KIF3 protein complex (KIF3A-KIF3B-KAP3 heterotetramer) is a molecular motor necessary for intraflagellar transport (IFT) but is also involved in ciliogenesis of motile cilia. Kif3a-knockout or Kif3b-knockout mice are prenatally lethal, exhibiting anomalies similar to ciliopathy phenotypes, including the disturbance of left–right body determination.3KIF19A is localised at the tip of motile cilia and performs motor and microtubule-depolymerising activities during IFT.

Kif19a-knockout mice present with hydrocephalus and female infertility, common signs in ciliary defects, due to abnormally elongated cilia with altered motility, not able to generate proper fluid flow.9Further KIFs, which may have specific roles in motile cilia, buy generic levitra from canada are KIF27, KIF9, KIF6 and KIF18B. Regarding the involvement of numerous KIFs in cilia-related processes, it is not surprising that many disorders caused by variants affecting KIF gene function are presenting with anomalies reminiscent of ciliopathies.Kinesin motors have a fundamental role in neuronal function, as they are responsible for the transport of synaptic vesicle precursors and transmitter receptors along axons and dendrites from the neuron body.3 Molecular motor activity as for KIF1A, KIF5 and KIF17 is important for higher brain functions, such as learning and memory through regulation of synaptic transmission.5 Dysfunction can be associated with intellectual disability and global developmental delay (table 1).Impaired function can also result in peripheral neuropathies (KIF5A, KLC2, KIF1A and KIF1B) and ocular motility disorders (KLC2 and KIF21A)23 24 when axon elongation in the peripheral nervous system and optic nerve is affected. KIF5A variants are associated with epileptic phenotypes both in humans and mice25 because the transport of neurotransmitter receptors is disturbed and inhibitory regulation is altered.Due to their role in cell-cycle regulation, kinesins are important in male spermatogenesis and female oogenesis. They are involved in all steps of spermatogenesis 26 and, based on previous animal studies, they may represent buy generic levitra from canada a potential target to treat male infertility.

In female meiosis, 13 KIF genes were studied in animal models. There is buy generic levitra from canada some evidence that kinesin expression is vulnerable to maternal ageing and environmental factors, such as oocyte cryopreservation and alcohol consumption. It may be promising to expand research in this field in order to clarify the mechanisms and factors contributing to oocyte quality decline.27Many kinesins were extensively studied in the fields of cancer development, progression and therapy. Deregulation of the mitotic kinesins by both overexpression and buy generic levitra from canada decreased expression causes cancer progression or can be a prognostic marker in various tumours.28 The cell-permeable small-molecule mitotic inhibitor monastrol was discovered in 199929 and was shown to arrest cells in mitosis by specifically inhibiting KIF11, a kinesin important for spindle bipolarity.

The bipolar mitotic spindle is replaced by a monoastral microtubule array surrounded by a ring of chromosomes, which gave the inhibitor its name. The mitotic spindle is now a well-known target of chemotherapy, and inhibitors of the mitotic kinesins KIF11, KIF10 and KIF1C are being studied for this purpose.28 30 buy generic levitra from canada The redundancy of some kinesins allows them to escape pharmacological inhibition. For example, in the absence of KIF10, KIF15 is able to replace all of its essential functions in spindle assembly. Cilia-related KIF7, KIF13B and KIF27 are involved in SHh signalling and may be a future target in cancer research.28Some kinesins confer susceptibility to a range of multifactorial, metabolic and neurodegenerative conditions.

KIF13B contributes to the enhancement of endocytosis of low-density lipoprotein (LDL) buy generic levitra from canada receptor-related protein 1 that is involved in the recognition and internalisation of LDL and factor VIII. Kif13b-knockout mice have hypercholesterolaemia and higher factor VIII serum levels.5 KIF12 is implicated in the pathogenesis of type 2 diabetes, protecting pancreatic β cells from the oxidative stress caused by nutritional excess.5 Variants in KIF1B or KIF21B confer susceptibility to multiple sclerosis (OMIM %612596, #126200).31 32 KIF5A was associated with Amyotrophic lateral sclerosis (OMIM #617921).33 KIF3 complex and KIF17 were recently uncovered to be involved in schizophrenia.34 35 Further studies, however, are needed to clarify the precise role of KIFs in neurodegenerative processes and psychiatric conditions.KIF14 -related birth defects. Lessons learntAdvances in next-generation sequencing technologies have buy generic levitra from canada revolutionised our understanding of Mendelian disorders, including birth defect phenotypes, by sequencing the coding genome (exome) or entire genome at an unprecedented resolution in a comparably short time span. The technology has been extensively used for gene identification approaches in research for many years, enabling unparalleled genotype–phenotype correlations and the definition of novel pathways of related genes and disorders at an accelerated pace, traditionally focusing on postnatal disorders.

Filges and Friedman36 postulated that buy generic levitra from canada a number of novel disease genes causing birth defects could be identifiable through the investigation of lethal foetal phenotypes since they would represent the extreme end of allelic milder and viable postnatal phenotypes with less specific or recognisable anomaly patterns. Based on embryonically or perinatally lethal mouse models (www.informatics.jax.org and www.dmdd.org.uk), it is estimated that knockout variants in about 30% of human protein coding genes may present with a phenotype of early lethality. The identification of KIF14 loss of function variants in fetuses with a lethal multiple congenital anomaly syndrome and the subsequent description of the allelic postnatal viable phenotype and further functional characterisation of KIF14 in developmental processes are recent examples of how to study those embryonic lethal phenotypes in order to understand the role of genes for which little to nothing is known.Filges et al identified autosomal recessive compound heterozygous loss of function variants in KIF14 using family-based exome sequencing in a recurrent severe lethal phenotype (OMIM #616258). It was buy generic levitra from canada the first human phenotype reported due to variants in the human KIF14 gene (figure 3).37 The two affected siblings presented with intrauterine growth retardation (IUGR), oligohydramnios, severe microcephaly, renal cystic dysplasia or agenesis, genital tract malformations (uterine hypoplasia and vaginal atresia), as well as cerebral and cerebellar hypoplasias with partial or total agenesis of the vermis, arhinencephaly, agenesis of occipital lobes/corpus callosum at second trimester ultrasound scan.

Cross-species comparison to the laggard spontaneous mice mutant, characterised by homozygous variants of the Kif14 gene,38 confirmed a phenotypical overlap. An increased number of binucleated cells in the tissue histology of the two fetuses were in concordance with the key role of KIF14 during mitosis participating in chromosomes’ congression and alignment, as well as in cytokinesis39 and the observation of binucleated cells as a consequence of failed cytokinesis buy generic levitra from canada in mammalian KIF14 knockdown cells. During cytokinesis, PRC1 localises KIF14 at the central spindle and midbody, which in turn recruits citron rho-interacting kinase (CIT) to the midbody. CIT, in turn, acts as a negative buy generic levitra from canada regulator of KIF14 activity.

Knockdown of KIF14 in mammalian cells results in impaired localisation of CIT during mitosis.40Structure of KIF14 and summary of all published KIF14 variants affecting function.10 37 41 42 The N-terminal region (aa 1–356) is important for its interactions with PRC1 and the protein’s localisation at the central spindle and midbody. The kinesin motor domain (aa 358–701) is responsible for the microtubule-dependent ATPase activity. The FHA domain buy generic levitra from canada (aa 825–891). Stalk and tail region (aa 891–1648) are necessary for the interaction with the protein CRIK (aa 901–1189, red diagonal lines).

There are four buy generic levitra from canada additional coiled-coil domains (light blue-coloured areas).61 FHA, forkhead associated. Aa, amino acid." data-icon-position data-hide-link-title="0">Figure 3 Structure of KIF14 and summary of all published KIF14 variants affecting function.10 37 41 42 The N-terminal region (aa 1–356) is important for its interactions with PRC1 and the protein’s localisation at the central spindle and midbody. The kinesin motor domain (aa 358–701) is buy generic levitra from canada responsible for the microtubule-dependent ATPase activity. The FHA domain (aa 825–891).

Stalk and tail region (aa 891–1648) are necessary for the interaction with the protein CRIK buy generic levitra from canada (aa 901–1189, red diagonal lines). There are four additional coiled-coil domains (light blue-coloured areas).61 FHA, forkhead associated. Aa, amino acid.Filges et al pointed out that KIF14 should be considered a candidate gene for viable postnatal phenotypes, including isolated microcephaly.34 Additional individuals with autosomal recessive variants in KIF14 and isolated primary microcephaly were then described9 41 42 (table 2).Impaired cytokinesis, increased apoptosis and reduced cell motility were confirmed in cells from the described patients, pointing to a new cellular pathway in the pathogenesis of microcephaly.43 Apart from one case with small kidneys with increased echogenicity, none of these 18 patients had associated kidney anomalies. However, a targeted exome sequencing study in 204 unrelated patients with congenital anomalies of the kidney and urinary tract (CAKUT) reported two more cases of renal anomalies, bilateral hypoplasia or agenesis, caused by KIF14 variants.44 Further nine cases had an associated renal phenotype, which ranged from bilateral renal agenesis to cystic or non-cystic renal hypodysplasia.42 Table 2 and figure 3 summarise buy generic levitra from canada KIF14 variants and the associated phenotypes.

Loss of function variants more likely lead to multiple congenital anomalies, while hypomorphic variants result in a milder phenotype without renal involvement, although phenotype–genotype correlations remain preliminary for the time being.The phenotypical spectrum ranging from isolated primary microcephaly to congenital anomalies reminiscent of ciliopathy phenotypes suggested a complex role for KIF14 in developmental processes and raised a number of questions about the relationship between its established role in cell division and its possible function in ciliary pathways. Functional studies of buy generic levitra from canada absent KIF14 protein in the development of human foetal tissues and mutant zebrafish provided evidence for similarities and differences between mitotic events occurring during proliferation in the development of both brain and kidney.42 The observation that KIF14-stained midbodies accumulate within the lumen of the branch tips of ureteric buds in human foetal kidneys provided a key clue to better understand the mechanism through which the loss of KIF14 affects both brain and kidney development in humans. It was previously demonstrated that the secretion and accumulation of midbody remnants in the cerebrospinal fluid in mice during the early stages of brain development correspond to the amplification of neural progenitors.45 Kif14 mutant zebrafish phenotypes supported the hypothesis of a potential role for KIF14 in cilia. In vitro and in vivo analyses suggested that loss of kif14 causes ciliary anomalies through an accumulation of mitotic cells in ciliated tissues but failed to establish a direct functional link.21 42 Further buy generic levitra from canada mechanisms remain to be elucidated.

Overexpression of KIF14 in various types of tumours was suggested to be a possible prognostic marker and a potential target for therapeutic purposes.46Kinesinopathies in birth defect phenotypes. Recurrent themesIn the last few years, an increasing number of variants in KIF genes were described to cause isolated as well as multiple congenital anomalies. There is a huge variability of phenotypes caused by variants even within the same buy generic levitra from canada gene. However, we can identify recurrent clinical signs that should alert the clinician to suspect a KIF gene-related disorder and the molecular geneticist to include KIF genes in multigene-panel and genome-wide sequencing approaches.

This will become particularly relevant in prenatal and perinatal medicine, which focuses on the detection of structural anomalies in the fetus and the newborn by using ultrasound and buy generic levitra from canada MRI or autopsy when the outcome is lethal. We have summarised the predominant and recurrent structural anomalies in kinesinopathies reported so far that would likely become apparent during the foetal period in table 3 and the syndromic disorders in table 1.View this table:Table 3 KIF gene-related structural congenital anomalies recurrently described in prenatal phenotypesSupplemental materialConsistent with the kinesins’ role in the development of the central nervous system (CNS), brain anomalies of various degrees are a frequent clinical sign, particularly microcephaly, but include lissencephaly, polymicrogyria, thinned or agenesis of the corpus callosum, arhinencephaly, cerebral hypoplasia or atrophy, cerebellar hypoplasia or atrophy, brainstem hypoplasia and a molar tooth sign on brain imaging.12 22 37 44 47–51Primary microcephaly can be detected prenatally or at birth12 22 47 48 50 51 and can present as an isolated or syndromic condition as, for example, caused by variants in KIF149 or in KIF11 (microcephaly with or without chorioretinopathy, lymphoedema or mental retardation. OMIM #152950).48KIF7 variants were related to macrocephaly in the presence of buy generic levitra from canada congenital hydrocephalus (hydrolethalus syndrome LS2, OMIM # 614120). Isolated hydrocephalus was reported for KIF4A in a single case.11Foetal akinesia and arthrogryposis (KIF5C12, KIF1434 and KIF26B50) are likely secondary to the neurological compromise of the fetus but can also appear as an early sign of abnormal CNS development, which should prompt specialist CNS sonographic and MRI evaluation of the fetus.Further anomalies of the limbs include camptodactyly (KIF26B50), clubfoot (KIF1A51), rocker-bottom feet (KIF26B50) and congenital lymphoedema of the limbs (dorsa of feet, lower extremities and, rarely, hands) in cases with KIF11 gene mutations.48 In particular, KIF7 gene variants have been related to various anomalies of the hands (tapered fingers, fifth finger clinodactyly, brachydactyly, preaxial or postaxial polydactyly, bifid terminal phalanges of the thumbs, spindle-shaped fingers, clinodactyly and soft tissue webbing) and feet (toe syndactyly, preaxial or postaxial polydactyly, and duplicated halluces).22CAKUT and genital anomalies are reported in various kinesinopathies including renal agenesis or hypoplasia (KIF1437 and KIF1252), ureteral hypoplasia (KIF1437), congenital megabladder (KIF1252) and vesicoureteral reflux (KIF1252), uterine hypoplasia and vaginal atresia (KIF1437) and hypospadias and chordae (KIF16B49).IUGR is recurrently detected (KIF5C12, KIF1437, KIF1053, KIF1554 and KIF2A12) and is particularly relevant when occurring simultaneously with one of the other recurrent clinical signs, indicating a potential syndromic KIF-related disorder.

Oligohydramnios or polyhydramnios is most likely secondary to a primary organ anomaly.There are a few kinesinopathy syndromes that have been specifically reported to be lethal, such as the ciliary phenotype (OMIM #616258), caused by variants in KIF1434, and hydrolethalus syndrome (OMIM #614120), caused by variants in KIF7.22 However, lethality is usually closely related to the specific major anomalies, and it can be hypothesised that such a lethal phenotype will exist for all KIF gene-related disorders.Developmental delay, intellectual disability, seizures, and sensory and motor disturbances of the peripheral nervous system, as well as eye anomalies, such as microphthalmy, optic nerve pallor, fibrosis of extraocular muscles and chorioretinopathy, will escape detection in the foetal period but are reported in postnatal patients.Kinesin pathways in birth defectsFunctional studies of kinesins in birth defects are still sparse, and little is known about their networks and pathways. In order to improve our understanding, we used the Ingenuity Pathway Analysis (IPA Qiagen, Redwood City, California, USA) to visualise and analyse the connections between the 13 kinesin motor proteins associated with structural congenital anomalies (KIF5C, KIF1A, KIF1BP, KIF14, KIF16B, KIF7, KIF4A, KIF11, KIF10, KIF26B, KIF12, KIF15 and buy generic levitra from canada KIF2A) and in up to 10 of each of their most significant downstream proteins. The connections are defined as protein–protein interactions, activation, regulation of binding, expression, localisation, phosphorylation, protein–RNA interactions, molecular cleavage, ubiquitination, protein–DNA interactions, inhibition, translocation and transcription. Figure 3 displays the results buy generic levitra from canada.

We used the software Gephy55 to look for all possible interactions between all proteins of the network and also used the IPA data to retrieve the canonical pathways involved. Figure 4 and online supplementary buy generic levitra from canada material, table 4, summarise the results. KIF7, KIF14 and KIF12 are located within the same network, and because of multiple connections between themselves and their downstream proteins, it is not surprising that they are all involved in kidney anomalies. IPA data are based on current publications and are therefore subject to bias because proteins buy generic levitra from canada that are most interconnected are also most probably those that have been more extensively studied.

However, we consider the KIF genes coding for proteins seeming less important within the network to be strong candidates for future studies of human developmental disorders.IPA of the 13 kinesins known to be involved in birth defects with respect to their position in the cell. Proteins displayed on the right side of the figure, below the tag ‘other’, are those for which no subcellular location is known. Birth defect-related buy generic levitra from canada kinesins and their connection with each other are highlighted in green. Light blue-coloured downstream proteins are those which are known to cause birth defects when altered.

Yellow-coloured proteins are those buy generic levitra from canada involved in neurological disorders overlapping with the clinical features of kinesinopathies. The legend of the biological function associated with every molecule is displayed on the right. Path Designer by IPA was used for the buy generic levitra from canada figure design. IPA, Ingenuity Pathway Analysis." data-icon-position data-hide-link-title="0">Figure 4 IPA of the 13 kinesins known to be involved in birth defects with respect to their position in the cell.

Proteins displayed on the right side of the figure, below the tag ‘other’, are those for which no subcellular location is known. Birth defect-related kinesins buy generic levitra from canada and their connection with each other are highlighted in green. Light blue-coloured downstream proteins are those which are known to cause birth defects when altered. Yellow-coloured proteins are those involved in neurological disorders overlapping with buy generic levitra from canada the clinical features of kinesinopathies.

The legend of the biological function associated with every molecule is displayed on the right. Path Designer by buy generic levitra from canada IPA was used for the figure design. IPA, Ingenuity Pathway Analysis.Closing remarks and future perspectivesNovel KIF genes are increasingly identified, and there is a growing body of literature demonstrating the impact of kinesin dysfunction in human disease. We propose to introduce the term kinesinopathies for conditions caused by variants in KIF genes, since recurrent and common functional and phenotypical themes can buy generic levitra from canada be observed.

In analogy to ciliopathies56 and rasopathies,57 the delineation of the clinical, genetic and functional hallmarks of kinesinopathies will be important to better recognise these conditions, to understand the pathomechanisms and to ultimately improve the clinical management of the patients. Previously, the unified view of the phenotype characteristics of ciliary dysfunction allowed a tremendous increase in awareness, both in clinic and research, and the further identification of yet unrecognised ciliary disorders and the genes and proteins involved in their pathogenesis.56Remarkable progress was achieved in assigning function to kinesins through their study in isolated and multiple congenital anomaly phenotypes. They are one large superfamily of molecular motors out of three (kinesins, dyneins and myosins), which is of key importance in several fundamental cellular processes using microtubules as rails for directional anterograde intracellular transport, including its regulation and modulating signal transduction.5 Kinesin motors are most important for the movement of chromosomes along the spindles during chromosome segregation, regulation of spindle formation, cell division and buy generic levitra from canada cytokinesis. These essential and broad cellular functions are critical for many physiological processes such as neuronal function and survival, some ciliary functions and ciliogenesis, determination of the left/right asymmetry of our body and regulation of organogenesis, thus explaining the impact and emerging recognition of kinesins in embryonic and foetal development.

Defects can buy generic levitra from canada result in neuropathies, higher brain functions and structural brain anomalies. Multiple congenital anomalies, including the kidney and urinary tract and limb anomalies, are repeatedly reported. Microcephaly, which is usually not associated with genes implicated in specific ciliary mechanisms, and CNS anomalies are the most buy generic levitra from canada recurrent clinical signs in both the prenatal and postnatal phenotypes described so far. The discovery of the implication of KIF14 in microcephaly further suggested a possible novel role of other microcephaly proteins in cytokinesis.

A number of syndromic kinesinopathies present, however, with phenotype patterns reminiscent of ciliopathies. So far, however, a direct buy generic levitra from canada functional impact was confirmed in only a few and could not be demonstrated, for example, for KIF14, despite an overlapping clinical pattern. In turn, ciliopathies are a clinically and genetically heterogeneous group of conditions themselves. Studying tissue and cell type-specific function and expression may help to further define the specific defects related to the individual aberrant kinesin.The pleiotropic nature of human kinesinopathies, however, is just emerging, but their study promises to provide important insights into buy generic levitra from canada human developmental pathways.

Seemingly unrelated clinical entities are highlighting a common theme. In a relatively short time span, monogenic KIF-related disorders were identified to present with often severe and lethal antenatal anomalies, with multiple or isolated congenital anomalies, neurodevelopmental and neurological disorders, buy generic levitra from canada or an increased susceptibility to multifactorial conditions. We focused on the emerging role of kinesins in structural congenital anomalies because, as illustrated for the KIF14 gene, great potential to decipher allelic viable phenotypes and developmental pathways lies in the study of these human knockout phenotypes at the severe end of the phenotypical spectrum. Knockout variants in about 30% of human protein coding genes in our genome may present with a phenotype of early lethality, and KIF genes seem to play an important role in such fundamental processes of human development.

Identifying and characterising the variants, genes and phenotypes will extend our knowledge on buy generic levitra from canada early human development and pathomechanisms, and will ultimately also improve the clinical utility of genome-wide sequencing approaches for prenatal and postnatal application by our increased ability to interpret loss of function and hypomorphic variants alike. Furthermore, kinesins were extensively studied in cancer research and therapeutic strategies targeting their specific functions, such as the example of monastrol and other inhibitors of the mitotic kinesins may be adopted in the future. There are likely many more kinesinopathies to be unravelled in the field of birth defects because of their pivotal role in cellular logistics, but their recognition in clinics and research will depend on our ability to identify and characterise the common clinical, molecular and functional themes of these disorders and to use them to improve our understanding of their disease mechanisms.IntroductionIntellectual disability (ID) affects about 3% of individuals worldwide and raises significant issues in terms of diagnostic, management buy generic levitra from canada and genetic counselling. The presence of pigmentation anomalies in a patient with ID represents helpful clinical clues in order to narrow the range of aetiological hypothesis.

Hypomelanosis of Ito (HMI, MIM #300337) is an unspecific term encompassing a heterogeneous group of disorders characterised by cutaneous hypopigmented whorls and streaks along Blaschko’s lines and variable extracutaneous features affecting the musculoskeletal and nervous systems.1 buy generic levitra from canada The cutaneous pattern therefore represents a non-specific hallmark of mosaicism in these neurocutaneous conditions. Genetic mosaicism is due to postzygotic mutations, either chromosomal rearrangements or point mutations, whereas random X inactivation in females leads to functional mosaicism.2 Unravelling the molecular basis of pigmentary mosaicism (PM) is still a challenge due to clinical and genetic heterogeneity, technical difficulties in detecting mosaic mutations by classical sequencing approaches and the complexities of obtaining affected tissue. As part of a collaborative group, we recently reported de novo mutations in exons 3 and 4 of transcription factor E3 (TFE3) as the cause for HMI in four unrelated individuals, including one male, as well as syndromic ID without pigmentary disorders in a female.3TFE3 belongs to the MITF family of buy generic levitra from canada mammalian basic helix–loop–helix zipper transcription factors, together with TFEB and TFEC. All four can form homodimers or heterodimers with each other.4 Embryonic expression of TFE3 orthologues Tfe3a and Tfe3b was demonstrated in the zebrafish in a wide range of tissues.5 TFE3 subcellular localisation plays a crucial role in the regulation of cellular homeostasis and embryonic stem cell (ESC) differentiation.

The phosphorylated TFE3 is retained in the cytoplasm, whereas dephosphorylated protein translocates to the nucleus to promote the transcription of target genes involved in lysosomal biogenesis and autophagy.6 TFE3 relocalisation to the nucleus is driven on various stressors, such as starvation,7 8 DNA damage,9 mitochondrial damage,10 Golgi stress11 and pathogens12 in an mTORC1-dependent manner, and oxidative stress13 or cadmium exposition14 in an mTORC1-independent manner. Moreover, lysosomal signalling-induced nucleocytoplasmic redistribution of TFE3 is essential to the regulation of ESC renewal.3 15 By restricting nuclear localisation and activity of buy generic levitra from canada Tfe3, lysosome activity, the tumour suppressor protein folliculin and the Ragulator protein complex enable the exit from pluripotency and therefore drive differentiation. Conversely, enforced nuclear Tfe3 enables ESCs to withstand differentiation.15 In humans, TFE3 mutations have long been known in cancer. Gene fusions by translocations or other chromosomal rearragements involving TFE3 and five partner genes have indeed been reported to occur in a subset of renal cell carcinomas (RCCs), referred to as ‘TFE-fusion RCC’, and, more rarely, to lung sarcoma and perivascular epithelioid cell tumours.16 Beyond these data on TFE3 function, by the report of a series of 17 individuals harbouring de novo mutations in exons 3 and 4 of TFE3, we emphasise their phenotype and bring additional clinical insight toward the recognition of this novel developmental disorder.ResultsWe describe a series of 17 patients carrying a de novo mutation in TFE3, 5 of them being previously published with limited clinical information.3 Twelve buy generic levitra from canada were females and five were males.

Their age ranged from 12 months to 22 years. Five were referred for HMI, five for buy generic levitra from canada syndromic ID and five for suspicion of storage disorder.Clinical dataThe clinical features are summarised in table 1. Additional information can be found in online supplementary data 1.Supplemental materialView this table:Table 1 Clinical and molecular features of the 17 patients with an TFE3 mutationNeonatal course was remarkable for nine patients. History of jaundice, hepatomegaly or feeding difficulties was reported for three patients each, hypoglycaemia for two and cholestasis for one.

All these features were transient.Developmental delay, buy generic levitra from canada usually severe and noticeable from the first months of life, was a constant feature in all the individuals. Only 6 patients were able to walk at the time of the study, whereas 11 were still unable to walk. All patients were non-verbal, except for two older patients who could speak a buy generic levitra from canada few words. Neurological examination was abnormal in 12 individuals and consisted in truncal hypotonia, associated with lower limb spasticity (6 individuals) or ataxia (2 adults).

Behavioural issues such as autistic buy generic levitra from canada features and sleeping disturbance were noted for 11 patients. Eleven patients developed epilepsy, onset in the first decade and characterised as intractable in three of them. Brain MRI was normal in 10 individuals and abnormal in 6 patients (hydrocephaly, short corpus callosum, Dandy-Walker malformation, arachnoid cyst, periventricular white matter lesions, delayed myelination and cerebral atrophy). The sensory anomaly was congenital hearing loss (5 buy generic levitra from canada patients), and ophtalmological anomalies (10 patients) consisted of strabismus, hyperopia, retinal degeneration, depigmented macule on the iris, oculomotor apraxia or impaired vision of cortical origin.Facial dysmorphism shared among the patients consisted in coarseness, flat nasal bridge, short nose with anteverted nares, widely spaced eyes, almond-shaped eyes, thick lips, facial hypertrichosis, fleshy earlobe, and full and pink cheeks (figure 1).

Twelve patients had pigmentation anomalies, located on Blaschko’s lines for 10 of them (figure 2). One was diagnosed at 4 years old buy generic levitra from canada with histiocytofibroma. Moderate to severe postnatal growth retardation affected 10 patients, who had a length between −2.0 and −4.5 SD. Obesity affected buy generic levitra from canada 13 individuals.

Skeletal anomalies were frequent (11 individuals) and consisted of flat or clubfeet, hyperlordosis, scoliosis, hip dislocation, limitation of elbow extension and genu valgum. Recurrent s of the upper airways were noted buy generic levitra from canada in five patients. One had a documented neutropenia. Early-onset chronic interstitial lung disease was reported in two patients.

Nail clubbing was noted buy generic levitra from canada in two individuals. Visceral malformations consisted of congenital heart defect (left ventricle dilatation, aortic insufficiency and patent ductus arteriosus) in three patients, umbilical hernia in three individuals, lateral semicircular canal dysplasia, posterior plagiocephaly, sleep apnoea syndrome, anteriorly displaced anus and hypospadias in one individual each.Facial phenotypes of seven patients. (A–C) Patient buy generic levitra from canada 5, aged 6 months (A,B) and 3 years (C). (D) Patient 8, aged 5 years.

(E,F) Patient buy generic levitra from canada 2, aged 5 and 20 years. (G–I) Patient 3, aged 1 year (G) and 3 years (H,I). (J,K) Patient 13, aged 22 years. (L–O) Patient buy generic levitra from canada 6, aged 8 months (L), 20 months (M) and 3 years (N,O).

(P,Q) Patient 10 aged 22 years." data-icon-position data-hide-link-title="0">Figure 1 Facial phenotypes of seven patients. (A–C) Patient buy generic levitra from canada 5, aged 6 months (A,B) and 3 years (C). (D) Patient 8, aged 5 years. (E,F) Patient 2, buy generic levitra from canada aged 5 and 20 years.

(G–I) Patient 3, aged 1 year (G) and 3 years (H,I). (J,K) Patient 13, aged 22 years. (L–O) Patient 6, aged 8 months (L), 20 months (M) and buy generic levitra from canada 3 years (N,O). (P,Q) Patient 10 aged 22 years.Cutaneous features.

(A) Patient buy generic levitra from canada 3. Umbilical hernia, widely spaced nipples and hypopigmentation on the left side of the abdomen. (B) Patient 17 buy generic levitra from canada. Hypotonia, umbilical hernia and widely spaced nipples.

(C) Patient 8 buy generic levitra from canada. Blaschko’s lines on the back. (D) Patient 6. Hypopigmentation on buy generic levitra from canada the left side of the abdomen.

(E) Patient 7. Blaschko’s lines on the abdomen and right side of the trunk buy generic levitra from canada. (F) Patient 13. Hand.

Note clubbing of thumbnail and loose skin. (G) Patient 17. Blaschko’s lines on the left side of the abdomen. (H) Patient 17.

Hand. Note tapering fingers and wrinkled skin. (I) Patient 1. Linear hypopigmentation on the back.

(J) Patient 6. Blaschko’s lines on the back. (K) Patient 3. Blachko’s lines on the right lower limb.

(L) Patient 6. Blaschko’s lines on the right lower limb. (M,N) Patient 7. Linear hyperpigmentation on the lower limbs.

(O) Patient 11. Blaschko’s lines on the back." data-icon-position data-hide-link-title="0">Figure 2 Cutaneous features. (A) Patient 3. Umbilical hernia, widely spaced nipples and hypopigmentation on the left side of the abdomen.

(B) Patient 17. Hypotonia, umbilical hernia and widely spaced nipples. (C) Patient 8. Blaschko’s lines on the back.

(D) Patient 6. Hypopigmentation on the left side of the abdomen. (E) Patient 7. Blaschko’s lines on the abdomen and right side of the trunk.

(F) Patient 13. Hand. Note clubbing of thumbnail and loose skin. (G) Patient 17.

Blaschko’s lines on the left side of the abdomen. (H) Patient 17. Hand. Note tapering fingers and wrinkled skin.

(I) Patient 1. Linear hypopigmentation on the back. (J) Patient 6. Blaschko’s lines on the back.

(K) Patient 3. Blachko’s lines on the right lower limb. (L) Patient 6. Blaschko’s lines on the right lower limb.

(M,N) Patient 7. Linear hyperpigmentation on the lower limbs. (O) Patient 11. Blaschko’s lines on the back.Molecular resultsThe characteristics of the 13 different de novo TFE3 variants identified in the 17 unrelated individuals are summarised in table 2.

All but one were missense variants, affecting nine different aminoacids. One was a splice donor mutation. This mutation was reported a few weeks ago in a patient with a similar phenotype.21 Two variants were localised in exon 3 and 11 in exon 4 (figure 3). All were absent from public databases and were predicted to be pathogenic by prediction softwares.

TFE3 protein and localisation of the missense variants identified. In bold are variants identified in two patients. In bold and underlined is the variant identified in three patients. In green is the intronic variant." data-icon-position data-hide-link-title="0">Figure 3 TFE3 protein and localisation of the missense variants identified.

In bold are variants identified in two patients. In bold and underlined is the variant identified in three patients. In green is the intronic variant.View this table:Table 2 Molecular data of the 13 de novo TFE3 mutations. Characteristics, inheritance, frequency in the public database GnomAD, prediction scores regarding pathogenicity (Polyphen, Grantham and CADD (Combined Annotation Dependant Depletion) scores).

The transcript is NM_006521.5The putative mosaicism was assessed through X inactivation studies in females and analysis of the exome sequencing data in males, by checking the total number of reads covering the variant, as well as the number of reads supporting the presence of the variant (table 1). Allele frequencies in females were always consistent with a constitutional heterozygous mutation. X inactivation was skewed in blood of the female patients 1, 3 and 7 and in fibroblasts of Patient 2. X inactivation was random in fibroblasts of Patient 1 and 3.

Regarding the male patients, the mutation was identified in 65% of the reads for Patient 17% and 88% of the reads for Patient 15 (106/120). No mosaicism was detected in the blood of Patient 13, 14 and 16 despite the presence of pigmentary manifestations in Patient 16.DiscussionTFE3 functions in signalling of the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1). The PIK3-AKT-mTOR pathway plays a role in the regulation of cellular growth, proliferation, survival and metabolism. Overactivation of the mTOR signalling is responsible for neurocutaneous disorders and cancers.22 Somatic mutations in TSC1, TSC2, AKT3, PIK3CA and MTOR are responsible for focal cortical dysplasia type II (MIM607341),23–25 hemimegalencephaly26 and megalencephaly.27 The phenotype ascribed to germline TSC1/TSC2, PTEN, MTOR and PK3R2/AKT3/CCND2 mutations – respectively in tuberous sclerosis (TS, MIM 191100), Cowden syndrome (CS, MIM 158350), Smith-Kingsmore syndrome (MIM 616638) and Megalencephaly, Polymicrogyria, Polydactyly and Hydrocephalus syndrome 1, 2 and 3 (MPPH1/2/3, MIM 603387/615939/615938) - is characterised by the association of ID, epilepsy, brain malformations and skin tumours.

Similarly, all the individuals harbouring a de novo TFE3 mutation reported in the series presented with a severe neurodevelopmental disorder. Delayed psychomotor development was constant. The youngest patient to acquire independent walking was 30 months old, and more than half of the patients aged over 18 months (57%), did not acquire walk at the last examination. Conversely to patients with MTOR, AKT3 or PTEN mutation, none of the patients described in this series had macrocephaly.

Brain imaging was abnormal in 35% of the patients. Hydrocephaly and corpus callosum dysgenesis, identified in respectively three and one individual, were previously reported in patients with mosaic gain of function MTOR mutations.28 29 One patient had surgery to remove an early-onset histiocytofibroma. However, no other skin tumour was reported, either in this patient or in any other from the series.Pigmentation anomalies, along Blaschko’s lines or, for one patient, as a large hyperpigmented area, were present in a majority of the individuals (71%) in the series, including 40% of the males and 83% of the females. PM along Blaschko’s lines is highly suggestive of genetic mosaicism.30 Genomic mosaicism is defined by the presence of at least two cell populations with different genotypes in an individual originating from one zygote and mainly occurs through post-zygotic event, whereas females can present with functional (epigenetics) mosaicism due to X inactivation.2 PM is a classical feature of X-linked male-lethal genodermatosis, such as incontinentia pigmenti (IP, MIM #308300), focal dermal hypoplasia (FDH, MIM #305600), chondrodysplasia punctuata type 2 (Conradi-Hunermann-Happle syndrome, CDPX2, MIM #302960) and linear skin defects with multiple congenital anomalies (LSDMCA1, MIM #309801).

In these conditions, the overwhelming predominance of affected females is a consequence of the male lethality, and the PM a manifestation of the functional mosaicism occurring in females. Similarly, the majority of individuals with de novo TFE3 variants in our cohort were females (sex ratio female:male was 12:5 (2.4)). The study of X-inactivation on non-cultured fibroblasts was consistent with functional mosaicism in two affected females with PM who harboured random X-inactivation, whereas a third female without PM had skewed X-inactivation. In IP, FDH and CDPX2, most hemizygous males die in utero.

However, there have been reports of surviving males 31–33 with an estimated prevalence around 10% in FDH and IP.32 34 The majority of them are explained by post-zygotic mutations or chromosomal anomalies (Klinefelter syndrome). Non-mosaic males have also been reported in FDH and IP – respectively about 17% and 45% of the affected males harbour a non-mosaic variant.32 35 In our series, males represented 29% of the patients with a de novo TFE3 variant. A mosaic variant was identified in blood for half of them. None had Klinefelter syndrome.

Interestingly, mosaicism was detected in only one out of the two males with PM, and one male with a mosaic variant had no pigmentation anomalies noted on examination. It is still possible that subtle pigmentation anomalies were missed on examination. Moreover, somatic mosaicism can be difficult to detect. Recent studies have shown that a large proportion of de novo mutations presumed to be germline had in fact occurred as post-zygotic event.36 In the males of this cohort, WES was performed on leucocytes-derived DNA and no other tissue was studied.

Therefore, it is possible that a low mosaicism was not detected. Finally, it is probable than TFE3 mutations account for a significant proportion of patients with HMI. Indeed, in this population, the high frequency of ID, epilepsy, coarse facial features has long been emphasised in the literature.37By its ability to bind the coordinated lysosomal enhancement and regulation (CLEAR) sites in the promotor region of target genes, TFE3 is involved in the control lysosomal biogenesis, autophagy and endocytosis.8 Several patients of the series indeed had clinical and biochemical features that pointed toward an inborn error of metabolism. Lysosomal storage disorder was suspected due to the variable association of coarse facial features (88%), skeletal anomalies (65%) –flat or clubfeet, hyperlordosis, hip dislocation, limitation of elbow extension, genu valgum, scoliosis–, postnatal growth retardation (59%), history of speech or developmental regression (29%) congenital hearing loss (29%), recurrent upper airways s (29%) neonatal liver anomalies such as hepatomegaly and cholestasis (18%), upper airways s (24%), umbilical hernia (18%), sleep apnoea syndrome (6%) and aortic insufficiency (6%).

Other metabolic anomalies observed in the series were obesity, defined in children by body mass index (BMI) (weight/height2) above the WHO curve, present in the oldest patients (76%), neonatal transient hypoglycaemia (12%), and hyperlactataemia (6%). Dysregulation of lipid metabolism, via suppression of thermogenesis and decreased lipolysis, thus leading to increased adipose tissue, was previously observed in adipose-specific TFE3 transgenic mice.38 Similarly to lysosomes, mitochondrias have a key role in cellular metabolism, including autophagy. Recent data demonstrate that mitochondrial and lysosomal metabolisms are interrelated.39 Muscle biopsy, performed in two individuals from this cohort, showed fat and glycogen accumulation, muscular fibre size irregularity, without evidence of mitochondrial dysfunction. Interestingly in the more recent data, evidences showing that TFE3 plays a role in the regulation of the circadian oscillations of the expression of genes involved in autophagy and lipid metabolism, and that Tfe3 knock-out mice had abnormal circadian behaviour.40 Indeed, in our series, five patients (29%) were noted to have sleep disturbance.

This could be due to circadian rhythms alteration. Finally, TFE3 has been shown to be involved in the regulation of innate immune response in macrophages via the FLCN-AMPK signalling axis,41 and of B-lymphocytes activation.42 Along these lines, four patients of the series (24%) had a history of recurrent s, associated with documented neutropenia in one of them. As shown in table 3, a summary of the frequency of the features observed in the cohort, facial dysmorphism was constant and strikingly similar among the patients. More than two-third had anteverted nares, broad flat nasal bridge, almond-shaped and widely spaced eyes, puffy cheeks and coarse facial features (thick lips and fleshy earlobes).

More than half had facial hypertrichosis. All individuals presented with at least four of the above features. One patient had an extreme facial phenotype of hypertelorism, bifid nose and bilateral cleft lip and palate. Whether these frontonasal dysplasia features may be associated with the TFE3 mutation remains unclear.

No other mutation in known genes was found in Patient 1’s exome sequencing data.View this table:Table 3 Frequency of the clinical features observed in the seriesTFE3 is a highly conserved protein, intolerant to loss of function as supported by data from the GnomAD browser43 (probability of being loss-of-function intolerant (pLI) evaluated at 0.98, observed:expected ratio=0.06) and to missense variants (Z=2.15). Moreover, TFE3 does not, or only in a few tissues, escape X inactivation, suggesting that TFE3 gene dosage is crucial to cell function.44 45 In vitro, Villegas et al recently showed that the absence of either TFE3 exon 3 or 4 resulted in a nuclear gain-of-function Tfe3 allele in ESCs, indicating that both exons 3 and 4 are required for Tfe3 inactivation.3 Nuclear localisation and resistance to differentiation were proved in Tfe3 knock-out (K.O.) ESCs expressing murine Tfe3 alleles (Gln118Pro and Pro185Leu, corresponding to mutations Gln119Pro and Pro186Leu identified in individuals referred to as patients 1 and 2 in this series). Based on the analysis of TFE3 secondary structure,46 indicating that residues 110–215 are predicted to form a domain of two stable alpha helices that might be disrupted by mutations in exons 3 and 4, and the observation of a similar phenotype in patients harbouring mutations in exons 3 and 4, it was suggested that Tfe3 exons 3 and 4 form a Rag binding fold whose structural integrity is indispensable for lysosome-mediated cytoplasmic Tfe3 inactivation.3 In this series, the recurrent mutations Arg117Gln, Leu191Pro and Thr187Met were present in respectively two, two and three patients. The aminoacid in position 187 was mutated in five patients.

In addition, 13 of the described mutations were localised between positions p.184 and p.201. This proximity could account for the absence of obvious genotype–phenotype correlation. The canonical splice site variant in intron 4 identified in patient 9 might lead to in-frame exon skipping of exon 4. The clinical picture of the patient with this splice site variant perfectly fits with the syndrome described here.

As a consequence, we raise the hypothesis of a gain-of-function effect of this variant.In conclusion, de novo mutations in exons 3 and 4 of the X-linked gene TFE3 are responsible for a neurocutaneous disorder with specific and recognisable facial dysmorphism, lysosomal storage disorder-like features and PM. This series unravels TFE3 as a major gene responsible for HMI and for a rare cause of syndromic ID. Furthermore, we provide clinical and molecular data on a previously unidentified lysosomal storage disorder, in which new insights, especially biochemical features, will probably be investigated further, together with the description of more patients. Further delineation of this phenotype will indeed allow a better understanding of the link between lysosomal signalling and development.

Finally, the evidence for mosaicism in this recently described disorder highlights the importance of considering mosaic variants on next-generation sequencing reports in diagnostic, including for patients without suggestive phenotype..

Where can I keep Levitra?

Keep out of the reach of children. Store at room temperature between 15 and 30 degrees C (59 and 86 degrees F). Throw away any unused medicine after the expiration date.

How to take levitra

Shutterstock Former Surgeon General Jerome Adams and the National Action Alliance for Suicide Prevention recently issued a document urging a national call to action for suicide prevention.In 1999, the surgeon general issued the first call to action, and the National Strategy for Suicide Prevention was updated in 2012 to identify 13 goals and 60 objectives.More than 47,000 people died by suicide in 2019, and millions struggled with serious thoughts of suicide.Suicide prevention is a complex issue how to take levitra requiring comprehensive solutions, according to the strategy report. There is how to take levitra no one-size-fits-all solution. Certain groups have disproportionately high rates of suicide. These groups include indigenous communities, veterans, military service members, and ethnic, racial, sexual, and gender minorities.Prevention efforts must identify and support how to take levitra individuals and groups at risk, support those affected by suicide, provide effective crisis response and care for suicide risk, and promote resilience and wellness.People who have experience with suicide and suicide prevention should guide the efforts, the National Action Alliance said.

€œWe know how to take levitra that the erectile dysfunction disease-2019 (erectile dysfunction treatment) levitra is taking a tremendous toll on Americans emotional and economic well-being,” the alliance said. €œWhile no one is immune from the stress and anxiety resulting from this crisis, these effects are magnified in households that already faced systemic disparities before the levitra began.”.

Shutterstock Former Surgeon General Jerome Adams and buy generic levitra from canada the National Action Alliance for Suicide Prevention recently issued a document urging a national call to action for suicide prevention.In 1999, the surgeon general issued the first call to action, and the National Strategy for Suicide Prevention was updated in 2012 to identify 13 goals and 60 objectives.More than 47,000 blog people died by suicide in 2019, and millions struggled with serious thoughts of suicide.Suicide prevention is a complex issue requiring comprehensive solutions, according to the strategy report. There is no one-size-fits-all solution buy generic levitra from canada. Certain groups have disproportionately high rates of suicide. These groups include indigenous communities, veterans, military service members, and ethnic, racial, sexual, and gender minorities.Prevention efforts must identify and support individuals and groups at risk, support those affected by suicide, provide effective crisis response and care for suicide risk, and promote resilience and wellness.People who have experience with suicide and suicide prevention should guide the efforts, the National Action Alliance said. €œWe know that the erectile dysfunction disease-2019 (erectile dysfunction treatment) levitra is taking a tremendous toll on Americans emotional and economic well-being,” the alliance said.

€œWhile no one is immune from the stress and anxiety resulting from this crisis, these effects are magnified in households that already faced systemic disparities before the levitra began.”.

Free levitra trial offer

Adam Woodrum was out for a bike ride with his wife levitra 20mg price australia and free levitra trial offer kids on July 19 when his then 9-year-old son, Robert, crashed. €œHe cut himself pretty bad, and I could tell right away he needed stitches,” said Woodrum. Because they were on bikes, he called the fire department free levitra trial offer in Carson City, Nevada. €œThey were great,” said Woodrum. €œThey took him free levitra trial offer on a stretcher to the ER.” Robert received stitches and anesthesia at Carson Tahoe Regional Medical Center.

He’s since recovered nicely. Then the denial free levitra trial offer letter came. The Patient. Robert Woodrum, covered under free levitra trial offer his mother’s health insurance plan from the Nevada Public Employees’ Benefits Program Total Bill. $18,933.44, billed by the hospital Service Provider.

Carson Tahoe free levitra trial offer Regional Medical Center, part of not-for-profit Carson Tahoe Health Medical Service. Stitches and anesthesia during an emergency department visit What Gives. The Aug free levitra trial offer. 4 explanation of benefits (EOB) document said the Woodrum’s claim had been rejected and their patient responsibility would be the entire sum of $18,933.44. This case involves an all-too-frequent dance between different types of insurers about which one should pay a patient’s bill free levitra trial offer if an accident is involved.

All sides do their best to avoid paying. And, no surprise to Bill of the Month free levitra trial offer followers. When insurers can’t agree, who gets a scary bill?. The patient free levitra trial offer. The legal name for the process of determining which type of insurance is primarily responsible is subrogation.

Could another policy — say, auto or home coverage or workers’ compensation — be obligated free levitra trial offer to pay if someone was at fault for the accident?. Subrogation is an area of law that allows an insurer to recoup expenses should a third party be found responsible for the injury or damage in question. Health insurers say subrogation helps hold down premiums by reimbursing them for their medical costs. About two weeks after the accident, Robert’s parents — both lawyers — got the EOB informing free levitra trial offer them of the insurer’s decision. The note also directed questions to Luper Neidenthal &.

Logan, a law firm in Columbus, Ohio, that specializes free levitra trial offer in helping insurers recover medical costs from “third parties,” meaning people found at fault for causing injuries. The firm’s website boasts that “we collect over 98% of recoverable dollars for the State of Nevada.” Another letter also dated Aug. 4 soon arrived from HealthScope Benefits, a large administrative firm that processes claims for free levitra trial offer health plans. The claim, it said, included billing codes for care “commonly used to treat injuries” related to vehicle crashes, slip-and-fall accidents or workplace hazards. Underlined for emphasis, one sentence warned that the denied claim would not be free levitra trial offer reconsidered until an enclosed accident questionnaire was filled out.

Adam Woodrum, who happens to be a personal injury attorney, runs into subrogation all the time representing his clients, many of whom have been in car accidents. But it still came as a shock, he said, to have his health insurer deny payment because there was no third party responsible for their free levitra trial offer son’s ordinary bike accident. And the denial came before the insurer got information about whether someone else was at fault. €œIt’s like deny now free levitra trial offer and pay later,” he said. €œYou have insurance and pay for years, then they say, ‘This is denied across the board.

Here’s your $18,000 bill.’” Although Adam Woodrum is a personal injury attorney, he says it still free levitra trial offer came as a shock to have his health insurer deny the claim after his son, Robert, got stitches in July following a bike crash. (Maggie Starbard for KHN) Woodrum and his son, Robert, get ready to bike near their home in Carson City, Nevada, on Nov. 7. (Maggie Starbard for KHN) When contacted, the Public Employees’ Benefits Program in Nevada would not comment specifically on Woodrum’s situation, but a spokesperson sent information from its health plan documents. She referred questions to HealthScope Benefits about whether the program’s policy is to deny claims first, then seek more information.

The Little Rock, Arkansas-based firm did not return emails asking for comment. The Nevada health plan’s documents say state legislation allows the program to recover “any and all payments made by the Plan” for the injury “from the other person or from any judgment, verdict or settlement obtained by the participant in relation to the injury.” Attorney Matthew Anderson at the law firm that handles subrogation for the Nevada health plan said he could not speak on behalf of the state of Nevada, nor could he comment directly on Woodrum’s situation. However, he said his insurance industry clients use subrogation to recoup payments from other insurers “as a cost-saving measure,” because “they don’t want to pass on high premiums to members.” Despite consumers’ unfamiliarity with the term, subrogation is common in the health insurance industry, said Leslie Wiernik, CEO of the National Association of Subrogation Professionals, the industry’s trade association. “Let’s say a young person falls off a bike,” she said, “but the insurer was thinking, ‘Did someone run him off the road, or did he hit a pothole the city didn’t fill?. €™â€ Statistics on how much money health insurers recover through passing the buck to other insurers are hard to find.

A 2013 Deloitte consulting firm study, commissioned by the Department of Labor, estimated that subrogation helped private health plans recover between $1.7 billion and $2.5 billion in 2010 — a tiny slice of the $849 billion they spent that year. Medical providers may have reason to hope that bills will be sent through auto or homeowner’s coverage, rather than health insurance, as they’re likely to get paid more. That’s because auto insurers “are going to pay billed charges, which are highly inflated,” said attorney Ryan Woody, who specializes in subrogation. Health insurers, by contrast, have networks of doctors and hospitals with whom they negotiate lower payment rates. Resolution.

Because of his experience as an attorney, Woodrum felt confident it would eventually all work out. But the average patient wouldn’t understand the legal quagmire and might not know how to fight back. €œI hear the horror stories every day from people who don’t know what it is, are confused by it and don’t take appropriate action,” Woodrum said. €œThen they’re a year out with no payment on their bills.” Or, fearing for their credit, they pay the bills. After receiving the accident questionnaire, Woodrum filled it out and sent it back.

There was no liable third party, he said. No driver was at fault. His child just fell off his bicycle. HealthScope Benefits reconsidered the claim. It was paid in September, two months after the accident.

The hospital received less than half of what it originally billed, based on rates negotiated through his health plan. The insurer paid $7,414.76 of the cost, and the Woodrums owed $1,853.45, which represented their share of the deductibles and copays. Adam Woodrum and his son, Robert, bike near their home in Carson City, Nevada, on Nov. 7.(Maggie Starbard for KHN) The Takeaway. The mantra of Bill of the Month is don’t just write the check.

But also don’t ignore scary bills from insurers or hospitals. It’s not uncommon for insured patients to be questioned on whether their injury or medical condition might have been related to an accident. On some claim forms, there is even a box for the patient to check if it was an accident. But in the Woodrums’ case, as in others, it was an automatic process. The insurer denied the claim based solely on the medical code indicating a possible accident.

If an insurer denies all payment for all medical care related to an injury, suspect that some type of subrogation is at work. Don’t panic. If you get an accident questionnaire, “fill it out, be honest about what happened,” said Sean Domnick, secretary of the American Association for Justice, an organization of plaintiffs lawyers. Inform your insurer and all other parties of the actual circumstances of the injury. And do so promptly.

That’s because the clock starts ticking the day the medical care is provided and policyholders may face a statutory or contractual requirement that medical bills be submitted within a specific time frame, which can vary. €œDo not ignore it,” said Domnick. €œTime and delay can be your enemy.” Bill of the Month is a crowdsourced investigation by KHN and NPR that dissects and explains medical bills. Do you have an interesting medical bill you want to share with us?. Tell us about it!.

Julie Appleby. jappleby@kff.org, @Julie_Appleby Related Topics Contact Us Submit a Story TipBrandon Hudgins works the main floor at Fleet Feet, a running-shoe store chain, for more than 30 hours a week. He chats with customers, measuring their feet and dashing in and out of the storage area to locate right-sized shoes. Sometimes, clients drag their masks down while speaking. Others refuse to wear masks at all.So he worries about erectile dysfunction treatment.

And with good reason. Across the U.S., erectile dysfunction treatment hospitalizations and deaths are hitting record-shattering new heights. The nation saw 198,633 new cases on Friday alone.Unlike in the early days of the levitra, though, many stores nationwide aren’t closing. And regular erectile dysfunction treatment testing of those working remains patchy at best.“I’ve asked, what if someone on staff gets symptoms?. ‘You have to stay home,’” said Hudgins, 33, who works in High Point, North Carolina.

But as an hourly employee, staying home means not getting paid. €œIt’s stressful, especially without regular testing. Our store isn’t very big, and you’re in there all day long.”To the store’s credit, Hudgins said the manager has instituted a locked-door policy, where employees determine which customers can enter. They sanitize the seating area between customers and administer regular employee temperature checks. Still, there’s no talk of testing employees for erectile dysfunction treatment.

Fleet Feet did not respond to multiple requests to talk about its testing policies.The federal Centers for Disease Control and Prevention issued guidance to employers to include erectile dysfunction treatment testing, and it advised that people working in close quarters be tested periodically. However, the federal government does not require employers to offer those tests.But the board overseeing the California Division of Occupational Safety and Health, known as Cal/OSHA, on Thursday approved emergency safety rules that are soon likely to require the state’s employers to provide erectile dysfunction treatment testing to all workers exposed to an outbreak on the job at no cost to the employees. Testing must be repeated a week later, followed by periodic testing.California would be the first state to mandate this, though the regulation doesn’t apply to routine testing of employees. That is up to individual businesses.Across the nation, workplaces have been the source of major erectile dysfunction outbreaks. Meat-processing plants, grocery stores, farms, schools, Amazon warehouses — largely among the so-called essential workers who bear the brunt of erectile dysfunction treatment s and deaths.The U.S.

Occupational Safety and Health Administration inspects workplaces based on workers’ complaints — over 40,000 of which related to erectile dysfunction treatment have been filed with the agency levitra online overnight at the state and federal levels.Workers “have every right to be concerned,” said Dr. Peter Chin-Hong, an epidemiologist at the University of California-San Francisco. €œThey are operating in a fog. There is little economic incentive for corporations to figure out who has erectile dysfunction treatment at what sites.”Waiting for symptoms to emerge before testing is ill-considered, Chin-Hong noted. People can exhibit no symptoms while spreading the levitra.

A CDC report found that, among people with active s, 44% reported no symptoms. Email Sign-Up Subscribe to KHN’s free Morning Briefing. Yet testing alone cannot protect employees. While workplaces can vary dramatically, Chin-Hong emphasized the importance of enforcing safety guidelines like social distancing and wearing face masks, as well as being transparent with workers when someone gets sick.Molly White, who works for the Missouri state government, was required to return to the office once a week starting in July. But White, who is on drugs to suppress her immune system, feared her employer’s “cavalier attitude toward erectile dysfunction treatment and casual risk taking.” Masks are encouraged for employees but are not mandatory, and there’s no testing policy or even guidance on where to get tested, she said.

White filed for and received an Americans With Disabilities Act exception, which lasts through the end of the year, to avoid coming into the office.After a cluster of 39 erectile dysfunction treatment cases emerged in September in the building where she normally works, White was relieved to at least get an email notifying her of the outbreak. A few days later, Gov. Mike Parson visited the building, and he tested positive for erectile dysfunction treatment soon after.Following pressure from labor groups, Amazon reported in a blog post last month that almost 20,000 employees had tested positive or been presumed positive for erectile dysfunction treatment since the levitra began. To help curb future outbreaks, the online retailing giant, which also owns Whole Foods, built its own testing facilities, hired lab technicians and said it planned to conduct 50,000 daily tests across 650 sites by this month.The National Football League tests players and other essential workers daily. An NFL spokesperson said the league conducts 40,000 to 45,000 tests a week through New Jersey-based BioReference Laboratories, though both organizations declined to share a price tag.

Reports over the summer estimated the season’s testing program would cost about $75 million.Not all companies, particularly those not in the limelight, have the interest — or the money — to regularly test workers.“It depends on the company how much they care,” said Gary Glader, president of Horton Safety Consultants in Orland Park, Illinois. Horton works with dozens of companies in the manufacturing, construction and transportation industries to write exposure control plans to limit the risk of erectile dysfunction treatment outbreaks and avoid OSHA citations. €œSome companies could care less about their people, never have.”IGeneX, a diagnostic testing company in Milpitas, California, gets around 15 calls each day from companies across the country inquiring about its employer testing program. The lab works with about 100 employers — from 10-person outfits to two pro sports teams — mainly in the Bay Area. IGeneX tests its own workers every other week.One client is Tarana Wireless, a nearby telecommunications company that needs about 30 employees in the office at a time to operate equipment.

In addition to monthly erectile dysfunction treatment tests, the building also gets cleaned every two hours, and masks are mandatory.“It’s definitely a burden,” said Amy Beck, the company’s director of human resources. €œWe are venture-backed and have taken pay cuts to make our money extend longer. But we do this to make everyone feel safe. We don’t have unlimited resources.”IGeneX offers three prices, depending on how fast a company wants the results. $135 for a polymerase chain reaction (PCR) test with a 36- to 48-hour turnaround — down to around $100 a test for some higher-volume clients.

One-day testing costs $250, and it’s $400 for a six-hour turnaround.In some cases, IGeneX is able to bill the companies’ health insurance plan.“Absolutely, it’s expensive,” said IGeneX spokesperson Joe Sullivan. €œI don’t blame anyone for wanting to pay as little as possible. It’s not ‘one and done,’ which companies are factoring in.”Plus, cheaper, rapid options like Abbott’s antigen test, touted by the Trump administration, have come under fire for being inaccurate.For those going into work, Chin-Hong recommends that companies test their employees once a week with PCR tests, or twice a week with the less sensitive antigen tests.Ideally, Chin-Hong said, public health departments would work directly with employers to administer erectile dysfunction treatment testing and quash potential outbreaks. But, as KHN has reported extensively, these local agencies are chronically underfunded and overworked. Free community testing sites can sometimes take days to weeks to return results, bogged down by extreme demand at commercial labs like Quest Diagnostics and LabCorp and supply chain problems.Hudgins, who receives his health insurance through North Carolina’s state exchange, tries to get a monthly erectile dysfunction treatment test at CVS on his own time.

But occasionally, his insurance — which requires certain criteria to qualify — has declined to pay for it, he said.“Being in the service industry in a state where numbers are ridiculously high,” he said in an email, “I see volumes of people every day, and I think getting tested is the smart and considerate thing to do.” Hannah Norman. hannahn@kff.org, @hnorms Related Topics Public Health States erectile dysfunction treatmentCalifornia Gov. Gavin Newsom’s maskless dinner with medical industry lobbyists and others at a Napa County restaurant where meals cost a minimum of $350 per head was just about the last straw for some beleaguered California small-business owners.With their livelihoods on the line, a growing number of them are openly defying the latest orders to shut down as erectile dysfunction treatment cases skyrocket in California — and pointing to Newsom’s bad behavior.“We are definitely not complying. We have enough information to make an educated decision. The data do not back another shutdown,” said Miguel Aguilar, founder and owner of Self Made Training Facility, based in Temecula, California, which leases space to physical trainers and nutrition advisers and has 40 locations across 11 states, including 15 in California.The news of Newsom’s Nov.

6 dinner at the French Laundry in Yountville only strengthened Aguilar’s resolve. €œYes, we all make mistakes, but his apology was pathetic,” Aguilar said. €œHe told us he was outdoors, but then the photos surfaced. He can attend in-person gatherings, but we can’t?. There’s absolutely no trust there.” Email Sign-Up Subscribe to KHN’s free Morning Briefing.

New erectile dysfunction treatment cases and hospitalizations have surged at an alarming rate in California, with a seven-day average of over 11,500 cases Saturday, more than triple the number of a month earlier. Hospitalizations have doubled over the same period, according to the Los Angeles Times, part of a national trend that has pushed total erectile dysfunction treatment s in the U.S. Above 12 million.In most California counties, restaurants, fitness clubs, yoga studios, churches, movie theaters and museums that have already been through two previous shutdowns and reopenings since March are once again required to cease indoor operations — just as winter hits. Some are laying off workers for the third time this year.Add to that the failure of Congress to pass another stimulus package and, in many cases, a preexisting mistrust of government mandates. It all amounts to more disgruntled entrepreneurs.Larry McNamer, owner of Major’s Diner in the tiny San Diego County community of Pine Valley, said he is continuing to serve people indoors, even though the county closed indoor dining on Nov.

14 in accordance with state regulations. He doesn’t believe the government has the right to impose such an ordinance on him. And, he said, Newsom’s dinner fiasco helped him make his decision to stay open.“We’re having to deal with all of the lying, the hypocrisy — you’ve got a governor that’s running around ignoring his own mandates,” McNamer said.McNamer knows the levitra is real, he said. He is seating only a quarter of his normal indoor capacity and has added distance between tables. But after closing the restaurant from March 15 to May 23, laying off half his employees and falling $200,000 behind on rent and other bills, McNamer isn’t sure how much more his business can take.Last Wednesday, he was hit with a cease-and-desist order from the county, threatening him with a fine of $1,000 for each offense.

San Diego County law enforcement officers are aggressively pursuing violations of public health orders, and the county has issued at least 83 citations to businesses since Nov. 16.In many other counties, including Riverside, Orange, San Bernardino and Placer, sheriffs and police departments have rejected the erectile dysfunction treatment ordinances or expressed reluctance to enforce them.Last week, Newsom announced that 41 of California’s 58 counties — representing 94% of the population — were in the state’s “purple” tier — the most severe of four color-coded risk levels that impose increasingly restrictive limits on business activities. That was up from 13 purple counties the week before.A few days later, the governor ordered a curfew, requiring people in the purple counties to stay at home between 10 p.m. And 5 a.m. Unless they’re performing essential activities, including certain jobs, grocery shopping or going to the doctor.Los Angeles County went a step further Sunday, banning outdoor dining for at least three weeks.

Unlike earlier in the year when that measure was ordered, now no federal financial aid is available to restaurants or their employees. Indoor dining has been shut down in the county for months.Despite plunging revenue, mounting debt and the frustrating uncertainty of shifting goal posts, many small-business owners are not defying the latest public health restrictions, either out of a sense of responsibility or fear of enforcement actions — or of contracting the levitra themselves.Those who do flout public health ordinances are doing so for a variety of reasons, with economics topping the list.“There are people who are protecting their employment, protecting their income,” said Vickie Mays, a clinical psychologist and professor of health policy and management at UCLA’s Fielding School of Public Health. €œThere are no stimulus checks coming. There’s no alternative.”Many people who own their own businesses “have taken other risks in their lives, and the risks they have taken have paid off, so there’s a belief that despite this risk, you’re not going to get infected,” Mays said.Many business owners, whether they comply with the health orders or not, believe their industries are being unfairly targeted and that the risk of viral spread in their establishments is not as great as officials say.Scott Slater, who owns two restaurants in San Diego’s seaside community of La Jolla, said he was frustrated by the public health focus on restaurants when a lot of erectile dysfunction treatment transmission is happening in private home gatherings.“We’re a perfect scapegoat,” Slater said. €œThey can control us, but they can’t control someone’s own home.” He called Newsom’s dinner “a slap in the face” but said he and his wife are complying with the new restrictions, scraping by on catering, takeout and delivery — though he estimates they are $200,000 behind on rent.Francesca Schuler, CEO of Stockton, California-based In-Shape Health Clubs, which has more than 60 fitness centers and just laid off most of its staff for the third time this year, said gyms should be viewed as part of the solution, not the problem.“I look at people who are dying of erectile dysfunction treatment, and it’s people who are overweight, who have high blood pressure or diabetes,” said Schuler, who is respecting the closure orders despite her objection to them.

€œThere are a lot of people who are trying to exercise to stay healthy, yet they shut down gyms while people can still go to tattoo parlors, to McDonald’s and to liquor stores. I just don’t get it.”Mays, however, said gyms are considered high-risk because “people are breathing hard. They are expelling air further.”And there are multiple ways people can stay fit without going to a gym, though outdoor exercise can be difficult sometimes because of heat and wildfire smoke, or in high-crime areas.In many cases, the levitra restrictions are crushing enterprises small-business owners have struggled to build over a lifetime. They’ve invested their savings, time, sweat and dreams in building something from the ground up, and now it’s threatened.Aguilar, who owns the training facility company, said he comes from a broken family, was homeless and penniless at age 16 and later got his start giving physical training lessons out of his garage. From that, he built his coast-to-coast chain.“At this point,” he said, “if I’m going to lose it all, I might as well go down fighting.” This KHN story first published on California Healthline, a service of the California Health Care Foundation.

Bernard J. Wolfson. bwolfson@kff.org, @bjwolfson Anna Almendrala. aalmendrala@kff.org, @annaalmendrala Related Topics Public Health erectile dysfunction treatmentJournalists from KHN and the Guardian have identified 1,413 workers who reportedly died of complications from erectile dysfunction treatment they contracted on the job. Reporters are working to confirm the cause of death and workplace conditions in each case.

They are also writing about the people behind the statistics — their personalities, passions and quirks — and telling the story of every life lost.Explore the new interactive tool tracking those health worker deaths. More From This Series. Related Topics Health Industry erectile dysfunction treatment Doctors Investigation Lost On The Frontline Nursing HomesThis story is part of a partnership that includes KCUR, NPR and Kaiser Health News. This story can be republished for free (details). Registered nurse Pascaline Muhindura has spent the past eight months treating erectile dysfunction treatment patients at Research Medical Center in Kansas City, Missouri.But when she returns home to her small town of Spring Hill, Kansas, she’s often stunned by what she sees, like on a recent stop for carryout food.“No one in the entire restaurant was wearing a mask,” Muhindura said. €œAnd there’s no social distancing.

I had to get out, because I almost had a panic attack. I was like, ‘What is going on with people?. Why are we still doing this?. '”Many rural communities across the U.S. Have resisted masks and calls for social distancing during the erectile dysfunction levitra, but now rural counties are experiencing record-high and death rates.Critically ill rural patients are often sent to city hospitals for high-level treatment and, as their numbers grow, some urban hospitals are buckling under the added strain.

Kansas City has a mask mandate, but in many smaller communities nearby, masks aren’t required — or masking orders are routinely ignored. In the past few months, rural counties in both Kansas and Missouri have seen some of the highest rates of erectile dysfunction treatment in the country.At the same time, according to an analysis by KHN, about 3 in 4 counties in Kansas and Missouri don’t have a single intensive care unit bed, so when people from these places get critically ill, they’re sent to city hospitals.A recent patient count at St. Luke’s Health System in Kansas City showed a quarter of erectile dysfunction treatment patients had come from outside the metro area.Two-thirds of the patients coming from rural areas need intensive care and stay in the hospital for an average of two weeks, said Dr. Marc Larsen, who leads erectile dysfunction treatment at St. Luke’s.“Not only are we seeing an uptick in those patients in our hospital from the rural community, they are sicker when we get them because [doctors in smaller communities] are able to handle the less sick patients,” said Larsen.

€œWe get the sickest of the sick.” Email Sign-Up Subscribe to KHN’s free Morning Briefing. Dr. Rex Archer, head of Kansas City’s health department, warns that capacity at the city’s 33 hospitals is being put at risk by the influx of rural patients.“We’ve had this huge swing that’s occurred because they’re not wearing masks, and yes, that’s putting pressure on our hospitals, which is unfair to our residents that might be denied an ICU bed,” Archer said.A study newly released by the Centers for Disease Control and Prevention showed that Kansas counties that mandated masks in early July saw decreases in new erectile dysfunction treatment cases, while counties without mask mandates recorded increases.Hospital leaders have continued to plead with Missouri Republican Gov. Mike Parson, and with Kansas’ conservative legislature, to implement stringent, statewide mask requirements but without success.Parson won the Missouri gubernatorial election on Nov. 3 by nearly 17 percentage points.

Two days later at a erectile dysfunction treatment briefing, he accused critics of “making the mask a political issue.” He said county leaders should decide whether to close businesses or mandate masks.“We’re going to encourage them to take some sort of action,” Parson said Thursday. €œThe holidays are coming and I, as governor of the state of Missouri, am not going to mandate who goes in your front door.”In an email, Dave Dillon, a spokesperson for the Missouri Hospital Association, agreed that rural patients might be contributing to hospital crowding in cities but argued that the strain on hospitals is a statewide problem.The reasons for the rural erectile dysfunction treatment crisis involve far more than the refusal to mandate or wear masks, according to health care experts.Both Kansas and Missouri have seen rural hospitals close year after year, and public health spending in both states, as in many largely rural states, is far below national averages.Rural populations also tend to be older and to suffer from higher rates of chronic health conditions, including heart disease, obesity and diabetes. Those conditions can make them more susceptible to severe illness when they contract erectile dysfunction treatment.Rural areas have been grappling with health problems for a long time, but the erectile dysfunction has been a sort of tipping point, and those rural health issues are now spilling over into cities, explained Shannon Monnat, a rural health researcher at Syracuse University.“It’s not just the rural health care infrastructure that becomes overwhelmed when there aren’t enough hospital beds, it’s also the surrounding neighborhoods, the suburbs, the urban hospital infrastructure starts to become overwhelmed as well,” Monnat said.Unlike many parts of the U.S., where erectile dysfunction treatment trend lines have risen and fallen over the course of the year, Kansas, Missouri and several other Midwestern states never significantly bent their statewide curve.Individual cities, such as Kansas City and St. Louis, have managed to slow cases, but the continual emergence of rural hot spots across Missouri has driven a slow and steady increase in overall new case numbers — and put an unrelenting strain on the states’ hospital systems.The months of slow but continuous growth in cases created a high baseline of cases as autumn began, which then set the stage for the sudden escalation of numbers in the recent surge.“It’s sort of the nature of epidemics that things often look like they’re relatively under control, and then very quickly ramp up to seem that they are out of hand,” said Justin Lessler, an epidemiologist at Johns Hopkins Bloomberg School of Public Health.Now, a recent local case spike in the Kansas City metro area is adding to the statewide surge in Missouri, with an average of 190 erectile dysfunction treatment patients per day being admitted to the metro region’s hospitals. The number of people hospitalized throughout Missouri increased by more than 50% in the past two weeks.Some Kansas City hospitals have had to divert patients for periods of time, and some are now delaying elective procedures, according to the University of Kansas Health system’s chief medical officer, Dr.

Steven Stites.But bed space isn’t the only hospital resource that’s running out. Half of the hospitals in the Kansas City area are now reporting “critical” staffing shortages. Pascaline Muhindura, the nurse who works in Kansas City, said that hospital workers are struggling with anxiety and depression.“The hospitals are not fine, because people taking care of patients are on the brink,” Muhindura said. €œWe are tired.”This story is from a reporting partnership that includes KCUR, NPR and KHN. Alex Smith, KCUR.

@AlexSmithKCUR Related Topics Health Industry Multimedia Public Health States Audio erectile dysfunction treatment Hospitals Kansas Missouri Rural Medicine.

Adam Woodrum was out for a bike ride with his wife and levitra cheapest price kids on July 19 when his buy generic levitra from canada then 9-year-old son, Robert, crashed. €œHe cut himself pretty bad, and I could tell right away he needed stitches,” said Woodrum. Because they were buy generic levitra from canada on bikes, he called the fire department in Carson City, Nevada.

€œThey were great,” said Woodrum. €œThey took him on a stretcher to the ER.” Robert received stitches and anesthesia at Carson Tahoe Regional Medical buy generic levitra from canada Center. He’s since recovered nicely.

Then the buy generic levitra from canada denial letter came. The Patient. Robert Woodrum, covered under his mother’s health insurance plan from the Nevada Public Employees’ Benefits Program Total buy generic levitra from canada Bill.

$18,933.44, billed by the hospital Service Provider. Carson Tahoe Regional Medical Center, part of not-for-profit buy generic levitra from canada Carson Tahoe Health Medical Service. Stitches and anesthesia during an emergency department visit What Gives.

The Aug buy generic levitra from canada. 4 explanation of benefits (EOB) document said the Woodrum’s claim had been rejected and their patient responsibility would be the entire sum of $18,933.44. This case buy generic levitra from canada involves an all-too-frequent dance between different types of insurers about which one should pay a patient’s bill if an accident is involved.

All sides do their best to avoid paying. And, no surprise to Bill of buy generic levitra from canada the Month followers. When insurers can’t agree, who gets a scary bill?.

The patient buy generic levitra from canada. The legal name for the process of determining which type of insurance is primarily responsible is subrogation. Could another policy — say, auto or home coverage or workers’ compensation — buy generic levitra from canada be obligated to pay if someone was at fault for the accident?.

Subrogation is an area of law that allows an insurer to recoup expenses should a third party be found responsible for the injury or damage in question. Health insurers say subrogation helps hold down premiums by reimbursing them for their medical costs. About two weeks after the accident, Robert’s buy generic levitra from canada parents — both lawyers — got the EOB informing them of the insurer’s decision.

The note also directed questions to Luper Neidenthal &. Logan, a law firm in Columbus, Ohio, that specializes in helping insurers recover medical costs from “third parties,” meaning people found at buy generic levitra from canada fault for causing injuries. The firm’s website boasts that “we collect over 98% of recoverable dollars for the State of Nevada.” Another letter also dated Aug.

4 soon arrived from HealthScope Benefits, a large administrative firm that processes claims for health buy generic levitra from canada plans. The claim, it said, included billing codes for care “commonly used to treat injuries” related to vehicle crashes, slip-and-fall accidents or workplace hazards. Underlined for emphasis, one buy generic levitra from canada sentence warned that the denied claim would not be reconsidered until an enclosed accident questionnaire was filled out.

Adam Woodrum, who happens to be a personal injury attorney, runs into subrogation all the time representing his clients, many of whom have been in car accidents. But it still came as a shock, he said, to buy generic levitra from canada have his health insurer deny payment because there was no third party responsible for their son’s ordinary bike accident. And the denial came before the insurer got information about whether someone else was at fault.

€œIt’s like deny now and pay buy generic levitra from canada later,” he said. €œYou have insurance and pay for years, then they say, ‘This is denied across the board. Here’s your $18,000 bill.’” Although Adam Woodrum is a personal injury attorney, he says it still came as a shock to have his health insurer buy generic levitra from canada deny the claim after his son, Robert, got stitches in July following a bike crash.

(Maggie Starbard for KHN) Woodrum and his son, Robert, get ready to bike near their home in Carson City, Nevada, on Nov. 7. (Maggie Starbard for KHN) When contacted, the Public Employees’ Benefits Program in Nevada would not comment specifically on Woodrum’s situation, but a spokesperson sent information from its health plan documents.

She referred questions to HealthScope Benefits about whether the program’s policy is to deny claims first, then seek more information. The Little Rock, Arkansas-based firm did not return emails asking for comment. The Nevada health plan’s documents say state legislation allows the program to recover “any and all payments made by the Plan” for the injury “from the other person or from any judgment, verdict or settlement obtained by the participant in relation to the injury.” Attorney Matthew Anderson at the law firm that handles subrogation for the Nevada health plan said he could not speak on behalf of the state of Nevada, nor could he comment directly on Woodrum’s situation.

However, he said his insurance industry clients use subrogation to recoup payments from other insurers “as a cost-saving measure,” because “they don’t want to pass on high premiums to members.” Despite consumers’ unfamiliarity with the term, subrogation is common in the health insurance industry, said Leslie Wiernik, CEO of the National Association of Subrogation Professionals, the industry’s trade association. “Let’s say a young person falls off a bike,” she said, “but the insurer was thinking, ‘Did someone run him off the road, or did he hit a pothole the city didn’t fill?. €™â€ Statistics on how much money health insurers recover through passing the buck to other insurers are hard to find.

A 2013 Deloitte consulting firm study, commissioned by the Department of Labor, estimated that subrogation helped private health plans recover between $1.7 billion and $2.5 billion in 2010 — a tiny slice of the $849 billion they spent that year. Medical providers may have reason to hope that bills will be sent through auto or homeowner’s coverage, rather than health insurance, as they’re likely to get paid more. That’s because auto insurers “are going to pay billed charges, which are highly inflated,” said attorney Ryan Woody, who specializes in subrogation.

Health insurers, by contrast, have networks of doctors and hospitals with whom they negotiate lower payment rates. Resolution. Because of his experience as an attorney, Woodrum felt confident it would eventually all work out.

But the average patient wouldn’t understand the legal quagmire and might not know how to fight back. €œI hear the horror stories every day from people who don’t know what it is, are confused by it and don’t take appropriate action,” Woodrum said. €œThen they’re a year out with no payment on their bills.” Or, fearing for their credit, they pay the bills.

After receiving the accident questionnaire, Woodrum filled it out and sent it back. There was no liable third party, he said. No driver was at fault.

His child just fell off his bicycle. HealthScope Benefits reconsidered the claim. It was paid in September, two months after the accident.

The hospital received less than half of what it originally billed, based on rates negotiated through his health plan. The insurer paid $7,414.76 of the cost, and the Woodrums owed $1,853.45, which represented their share of the deductibles and copays. Adam Woodrum and his son, Robert, bike near their home in Carson City, Nevada, on Nov.

7.(Maggie Starbard for KHN) The Takeaway. The mantra of Bill of the Month is don’t just write the check. But also don’t ignore scary bills from insurers or hospitals.

It’s not uncommon for insured patients to be questioned on whether their injury or medical condition might have been related to an accident. On some claim forms, there is even a box for the patient to check if it was an accident. But in the Woodrums’ case, as in others, it was an automatic process.

The insurer denied the claim based solely on the medical code indicating a possible accident. If an insurer denies all payment for all medical care related to an injury, suspect that some type of subrogation is at work. Don’t panic.

If you get an accident questionnaire, “fill it out, be honest about what happened,” said Sean Domnick, secretary of the American Association for Justice, an organization of plaintiffs lawyers. Inform your insurer and all other parties of the actual circumstances of the injury. And do so promptly.

That’s because the clock starts ticking the day the medical care is provided and policyholders may face a statutory or contractual requirement that medical bills be submitted within a specific time frame, which can vary. €œDo not ignore it,” said Domnick. €œTime and delay can be your enemy.” Bill of the Month is a crowdsourced investigation by KHN and NPR that dissects and explains medical bills.

Do you have an interesting medical bill you want to share with us?. Tell us about it!. Julie Appleby.

jappleby@kff.org, @Julie_Appleby Related Topics Contact Us Submit a Story TipBrandon Hudgins works the main floor at Fleet Feet, a running-shoe store chain, for more than 30 hours a week. He chats with customers, measuring their feet and dashing in and out of the storage area to locate right-sized shoes. Sometimes, clients drag their masks down while speaking.

Others refuse to wear masks at all.So he worries about erectile dysfunction treatment. And with good reason. Across the U.S., erectile dysfunction treatment hospitalizations and deaths are hitting record-shattering new heights.

The nation saw 198,633 new cases on Friday alone.Unlike in the early days of the levitra, though, many stores nationwide aren’t closing. And regular erectile dysfunction treatment testing of those working remains patchy at best.“I’ve asked, what if someone on staff gets symptoms?. ‘You have to stay home,’” said Hudgins, 33, who works in High Point, North Carolina.

But as an hourly employee, staying home means not getting paid. €œIt’s stressful, especially without regular testing. Our store isn’t very big, and you’re in there all day long.”To the store’s credit, Hudgins said the manager has instituted a locked-door policy, where employees determine which customers can enter.

They sanitize the seating area between customers and administer regular employee temperature checks. Still, there’s no talk of testing employees for erectile dysfunction treatment. Fleet Feet did not respond to multiple requests to talk about its testing policies.The federal Centers for Disease Control and Prevention issued guidance to employers to include erectile dysfunction treatment testing, and it advised that people working in close quarters be tested periodically.

However, the federal government does not require employers to offer those tests.But the board overseeing the California Division of Occupational Safety and Health, known as Cal/OSHA, on Thursday approved emergency safety rules that are soon likely to require the state’s employers to provide erectile dysfunction treatment testing to all workers exposed to an outbreak on the job at no cost to the employees. Testing must be repeated a week later, followed by periodic testing.California would be the first state to mandate this, though the regulation doesn’t apply to routine testing of employees. That is up to individual businesses.Across the nation, workplaces have been the source of major erectile dysfunction outbreaks.

Meat-processing plants, grocery stores, farms, schools, Amazon warehouses — largely among the so-called essential workers who bear the brunt of erectile dysfunction treatment s and deaths.The U.S. Occupational Safety and Health Administration inspects workplaces based on workers’ complaints — over 40,000 of which related to erectile dysfunction treatment have been filed with the agency at the state and federal levels.Workers “have every right to be concerned,” said Dr. Peter Chin-Hong, an epidemiologist at the University of California-San Francisco.

€œThey are operating in a fog. There is little economic incentive for corporations to figure out who has erectile dysfunction treatment at what sites.”Waiting for symptoms to emerge before testing is ill-considered, Chin-Hong noted. People can exhibit no symptoms while spreading the levitra.

A CDC report found that, among people with active s, 44% reported no symptoms. Email Sign-Up Subscribe to KHN’s free Morning Briefing. Yet testing alone cannot protect employees.

While workplaces can vary dramatically, Chin-Hong emphasized the importance of enforcing safety guidelines like social distancing and wearing face masks, as well as being transparent with workers when someone gets sick.Molly White, who works for the Missouri state government, was required to return to the office once a week starting in July. But White, who is on drugs to suppress her immune system, feared her employer’s “cavalier attitude toward erectile dysfunction treatment and casual risk taking.” Masks are encouraged for employees but are not mandatory, and there’s no testing policy or even guidance on where to get tested, she said. White filed for and received an Americans With Disabilities Act exception, which lasts through the end of the year, to avoid coming into the office.After a cluster of 39 erectile dysfunction treatment cases emerged in September in the building where she normally works, White was relieved to at least get an email notifying her of the outbreak.

A few days later, Gov. Mike Parson visited the building, and he tested positive for erectile dysfunction treatment soon after.Following pressure from labor groups, Amazon reported in a blog post last month that almost 20,000 employees had tested positive or been presumed positive for erectile dysfunction treatment since the levitra began. To help curb future outbreaks, the online retailing giant, which also owns Whole Foods, built its own testing facilities, hired lab technicians and said it planned to conduct 50,000 daily tests across 650 sites by this month.The National Football League tests players and other essential workers daily.

An NFL spokesperson said the league conducts 40,000 to 45,000 tests a week through New Jersey-based BioReference Laboratories, though both organizations declined to share a price tag. Reports over the summer estimated the season’s testing program would cost about $75 million.Not all companies, particularly those not in the limelight, have the interest — or the money — to regularly test workers.“It depends on the company how much they care,” said Gary Glader, president of Horton Safety Consultants in Orland Park, Illinois. Horton works with dozens of companies in the manufacturing, construction and transportation industries to write exposure control plans to limit the risk of erectile dysfunction treatment outbreaks and avoid OSHA citations.

€œSome companies could care less about their people, never have.”IGeneX, a diagnostic testing company in Milpitas, California, gets around 15 calls each day from companies across the country inquiring about its employer testing program. The lab works with about 100 employers — from 10-person outfits to two pro sports teams — mainly in the Bay Area. IGeneX tests its own workers every other week.One client is Tarana Wireless, a nearby telecommunications company that needs about 30 employees in the office at a time to operate equipment.

In addition to monthly erectile dysfunction treatment tests, the building also gets cleaned every two hours, and masks are mandatory.“It’s definitely a burden,” said Amy Beck, the company’s director of human resources. €œWe are venture-backed and have taken pay cuts to make our money extend longer. But we do this to make everyone feel safe.

We don’t have unlimited resources.”IGeneX offers three prices, depending on how fast a company wants the results. $135 for a polymerase chain reaction (PCR) test with a 36- to 48-hour turnaround — down to around $100 a test for some higher-volume clients. One-day testing costs $250, and it’s $400 for a six-hour turnaround.In some cases, IGeneX is able to bill the companies’ health insurance plan.“Absolutely, it’s expensive,” said IGeneX spokesperson Joe Sullivan.

€œI don’t blame anyone for wanting to pay as little as possible. It’s not ‘one and done,’ which companies are factoring in.”Plus, cheaper, rapid options like Abbott’s antigen test, touted by the Trump administration, have come under fire for being inaccurate.For those going into work, Chin-Hong recommends that companies test their employees once a week with PCR tests, or twice a week with the less sensitive antigen tests.Ideally, Chin-Hong said, public health departments would work directly with employers to administer erectile dysfunction treatment testing and quash potential outbreaks. But, as KHN has reported extensively, these local agencies are chronically underfunded and overworked.

Free community testing sites can sometimes take days to weeks to return results, bogged down by extreme demand at commercial labs like Quest Diagnostics and LabCorp and supply chain problems.Hudgins, who receives his health insurance through North Carolina’s state exchange, tries to get a monthly erectile dysfunction treatment test at CVS on his own time. But occasionally, his insurance — which requires certain criteria to qualify — has declined to pay for it, he said.“Being in the service industry in a state where numbers are ridiculously high,” he said in an email, “I see volumes of people every day, and I think getting tested is the smart and considerate thing to do.” Hannah Norman. hannahn@kff.org, @hnorms Related Topics Public Health States erectile dysfunction treatmentCalifornia Gov.

Gavin Newsom’s maskless dinner with medical industry lobbyists and others at a Napa County restaurant where meals cost a minimum of $350 per head was just about the last straw for some beleaguered California small-business owners.With their livelihoods on the line, a growing number of them are openly defying the latest orders to shut down as erectile dysfunction treatment cases skyrocket in California — and pointing to Newsom’s bad behavior.“We are definitely not complying. We have enough information to make an educated decision. The data do not back another shutdown,” said Miguel Aguilar, founder and owner of Self Made Training Facility, based in Temecula, California, which leases space to physical trainers and nutrition advisers and has 40 locations across 11 states, including 15 in California.The news of Newsom’s Nov.

6 dinner at the French Laundry in Yountville only strengthened Aguilar’s resolve. €œYes, we all make mistakes, but his apology was pathetic,” Aguilar said. €œHe told us he was outdoors, but then the photos surfaced.

He can attend in-person gatherings, but we can’t?. There’s absolutely no trust there.” Email Sign-Up Subscribe to KHN’s free Morning Briefing. New erectile dysfunction treatment cases and hospitalizations have surged at an alarming rate in California, with a seven-day average of over 11,500 cases Saturday, more than triple the number of a month earlier.

Hospitalizations have doubled over the same period, according to the Los Angeles Times, part of a national trend that has pushed total erectile dysfunction treatment s in the U.S. Above 12 million.In most California counties, restaurants, fitness clubs, yoga studios, churches, movie theaters and museums that have already been through two previous shutdowns and reopenings since March are once again required to cease indoor operations — just as winter hits. Some are laying off workers for the third time this year.Add to that the failure of Congress to pass another stimulus package and, in many cases, a preexisting mistrust of government mandates.

It all amounts to more disgruntled entrepreneurs.Larry McNamer, owner of Major’s Diner in the tiny San Diego County community of Pine Valley, said he is continuing to serve people indoors, even though the county closed indoor dining on Nov. 14 in accordance with state regulations. He doesn’t believe the government has the right to impose such an ordinance on him.

And, he said, Newsom’s dinner fiasco helped him make his decision to stay open.“We’re having to deal with all of the lying, the hypocrisy — you’ve got a governor that’s running around ignoring his own mandates,” McNamer said.McNamer knows the levitra is real, he said. He is seating only a quarter of his normal indoor capacity and has added distance between tables. But after closing the restaurant from March 15 to May 23, laying off half his employees and falling $200,000 behind on rent and other bills, McNamer isn’t sure how much more his business can take.Last Wednesday, he was hit with a cease-and-desist order from the county, threatening him with a fine of $1,000 for each offense.

San Diego County law enforcement officers are aggressively pursuing violations of public health orders, and the county has issued at least 83 citations to businesses since Nov. 16.In many other counties, including Riverside, Orange, San Bernardino and Placer, sheriffs and police departments have rejected the erectile dysfunction treatment ordinances or expressed reluctance to enforce them.Last week, Newsom announced that 41 of California’s 58 counties — representing 94% of the population — were in the state’s “purple” tier — the most severe of four color-coded risk levels that impose increasingly restrictive limits on business activities. That was up from 13 purple counties the week before.A few days later, the governor ordered a curfew, requiring people in the purple counties to stay at home between 10 p.m.

And 5 a.m. Unless they’re performing essential activities, including certain jobs, grocery shopping or going to the doctor.Los Angeles County went a step further Sunday, banning outdoor dining for at least three weeks. Unlike earlier in the year when that measure was ordered, now no federal financial aid is available to restaurants or their employees.

Indoor dining has been shut down in the county for months.Despite plunging revenue, mounting debt and the frustrating uncertainty of shifting goal posts, many small-business owners are not defying the latest public health restrictions, either out of a sense of responsibility or fear of enforcement actions — or of contracting the levitra themselves.Those who do flout public health ordinances are doing so for a variety of reasons, with economics topping the list.“There are people who are protecting their employment, protecting their income,” said Vickie Mays, a clinical psychologist and professor of health policy and management at UCLA’s Fielding School of Public Health. €œThere are no stimulus checks coming. There’s no alternative.”Many people who own their own businesses “have taken other risks in their lives, and the risks they have taken have paid off, so there’s a belief that despite this risk, you’re not going to get infected,” Mays said.Many business owners, whether they comply with the health orders or not, believe their industries are being unfairly targeted and that the risk of viral spread in their establishments is not as great as officials say.Scott Slater, who owns two restaurants in San Diego’s seaside community of La Jolla, said he was frustrated by the public health focus on restaurants when a lot of erectile dysfunction treatment transmission is happening in private home gatherings.“We’re a perfect scapegoat,” Slater said.

€œThey can control us, but they can’t control someone’s own home.” He called Newsom’s dinner “a slap in the face” but said he and his wife are complying with the new restrictions, scraping by on catering, takeout and delivery — though he estimates they are $200,000 behind on rent.Francesca Schuler, CEO of Stockton, California-based In-Shape Health Clubs, which has more than 60 fitness centers and just laid off most of its staff for the third time this year, said gyms should be viewed as part of the solution, not the problem.“I look at people who are dying of erectile dysfunction treatment, and it’s people who are overweight, who have high blood pressure or diabetes,” said Schuler, who is respecting the closure orders despite her objection to them. €œThere are a lot of people who are trying to exercise to stay healthy, yet they shut down gyms while people can still go to tattoo parlors, to McDonald’s and to liquor stores. I just don’t get it.”Mays, however, said gyms are considered high-risk because “people are breathing hard.

They are expelling air further.”And there are multiple ways people can stay fit without going to a gym, though outdoor exercise can be difficult sometimes because of heat and wildfire smoke, or in high-crime areas.In many cases, the levitra restrictions are crushing enterprises small-business owners have struggled to build over a lifetime. They’ve invested their savings, time, sweat and dreams in building something from the ground up, and now it’s threatened.Aguilar, who owns the training facility company, said he comes from a broken family, was homeless and penniless at age 16 and later got his start giving physical training lessons out of his garage. From that, he built his coast-to-coast chain.“At this point,” he said, “if I’m going to lose it all, I might as well go down fighting.” This KHN story first published on California Healthline, a service of the California Health Care Foundation.

Bernard J. Wolfson. bwolfson@kff.org, @bjwolfson Anna Almendrala.

aalmendrala@kff.org, @annaalmendrala Related Topics Public Health erectile dysfunction treatmentJournalists from KHN and the Guardian have identified 1,413 workers who reportedly died of complications from erectile dysfunction treatment they contracted on the job. Reporters are working to confirm the cause of death and workplace conditions in each case. They are also writing about the people behind the statistics — their personalities, passions and quirks — and telling the story of every life lost.Explore the new interactive tool tracking those health worker deaths.

More From This Series. Related Topics Health Industry erectile dysfunction treatment Doctors Investigation Lost On The Frontline Nursing HomesThis story is part of a partnership that includes KCUR, NPR and Kaiser Health News. This story can be republished for free (details). Registered nurse Pascaline Muhindura has spent the past eight months treating erectile dysfunction treatment patients at Research Medical Center in Kansas City, Missouri.But when she returns home to her small town of Spring Hill, Kansas, she’s often stunned by what she sees, like on a recent stop for carryout food.“No one in the entire restaurant was wearing a mask,” Muhindura said.

€œAnd there’s no social distancing. I had to get out, because I almost had a panic attack. I was like, ‘What is going on with people?.

Why are we still doing this?. '”Many rural communities across the U.S. Have resisted masks and calls for social distancing during the erectile dysfunction levitra, but now rural counties are experiencing record-high and death rates.Critically ill rural patients are often sent to city hospitals for high-level treatment and, as their numbers grow, some urban hospitals are buckling under the added strain.

Kansas City has a mask mandate, but in many smaller communities nearby, masks aren’t required — or masking orders are routinely ignored. In the past few months, rural counties in both Kansas and Missouri have seen some of the highest rates of erectile dysfunction treatment in the country.At the same time, according to an analysis by KHN, about 3 in 4 counties in Kansas and Missouri don’t have a single intensive care unit bed, so when people from these places get critically ill, they’re sent to city hospitals.A recent patient count at St. Luke’s Health System in Kansas City showed a quarter of erectile dysfunction treatment patients had come from outside the metro area.Two-thirds of the patients coming from rural areas need intensive care and stay in the hospital for an average of two weeks, said Dr.

Marc Larsen, who leads erectile dysfunction treatment at St. Luke’s.“Not only are we seeing an uptick in those patients in our hospital from the rural community, they are sicker when we get them because [doctors in smaller communities] are able to handle the less sick patients,” said Larsen. €œWe get the sickest of the sick.” Email Sign-Up Subscribe to KHN’s free Morning Briefing.

Dr. Rex Archer, head of Kansas City’s health department, warns that capacity at the city’s 33 hospitals is being put at risk by the influx of rural patients.“We’ve had this huge swing that’s occurred because they’re not wearing masks, and yes, that’s putting pressure on our hospitals, which is unfair to our residents that might be denied an ICU bed,” Archer said.A study newly released by the Centers for Disease Control and Prevention showed that Kansas counties that mandated masks in early July saw decreases in new erectile dysfunction treatment cases, while counties without mask mandates recorded increases.Hospital leaders have continued to plead with Missouri Republican Gov. Mike Parson, and with Kansas’ conservative legislature, to implement stringent, statewide mask requirements but without success.Parson won the Missouri gubernatorial election on Nov.

3 by nearly 17 percentage points. Two days later at a erectile dysfunction treatment briefing, he accused critics of “making the mask a political issue.” He said county leaders should decide whether to close businesses or mandate masks.“We’re going to encourage them to take some sort of action,” Parson said Thursday. €œThe holidays are coming and I, as governor of the state of Missouri, am not going to mandate who goes in your front door.”In an email, Dave Dillon, a spokesperson for the Missouri Hospital Association, agreed that rural patients might be contributing to hospital crowding in cities but argued that the strain on hospitals is a statewide problem.The reasons for the rural erectile dysfunction treatment crisis involve far more than the refusal to mandate or wear masks, according to health care experts.Both Kansas and Missouri have seen rural hospitals close year after year, and public health spending in both states, as in many largely rural states, is far below national averages.Rural populations also tend to be older and to suffer from higher rates of chronic health conditions, including heart disease, obesity and diabetes.

Those conditions can make them more susceptible to severe illness when they contract erectile dysfunction treatment.Rural areas have been grappling with health problems for a long time, but the erectile dysfunction has been a sort of tipping point, and those rural health issues are now spilling over into cities, explained Shannon Monnat, a rural health researcher at Syracuse University.“It’s not just the rural health care infrastructure that becomes overwhelmed when there aren’t enough hospital beds, it’s also the surrounding neighborhoods, the suburbs, the urban hospital infrastructure starts to become overwhelmed as well,” Monnat said.Unlike many parts of the U.S., where erectile dysfunction treatment trend lines have risen and fallen over the course of the year, Kansas, Missouri and several other Midwestern states never significantly bent their statewide curve.Individual cities, such as Kansas City and St. Louis, have managed to slow cases, but the continual emergence of rural hot spots across Missouri has driven a slow and steady increase in overall new case numbers — and put an unrelenting strain on the states’ hospital systems.The months of slow but continuous growth in cases created a high baseline of cases as autumn began, which then set the stage for the sudden escalation of numbers in the recent surge.“It’s sort of the nature of epidemics that things often look like they’re relatively under control, and then very quickly ramp up to seem that they are out of hand,” said Justin Lessler, an epidemiologist at Johns Hopkins Bloomberg School of Public Health.Now, a recent local case spike in the Kansas City metro area is adding to the statewide surge in Missouri, with an average of 190 erectile dysfunction treatment patients per day being admitted to the metro region’s hospitals. The number of people hospitalized throughout Missouri increased by more than 50% in the past two weeks.Some Kansas City hospitals have had to divert patients for periods of time, and some are now delaying elective procedures, according to the University of Kansas Health system’s chief medical officer, Dr.

Steven Stites.But bed space isn’t the only hospital resource that’s running out. Half of the hospitals in the Kansas City area are now reporting “critical” staffing shortages. Pascaline Muhindura, the nurse who works in Kansas City, said that hospital workers are struggling with anxiety and depression.“The hospitals are not fine, because people taking care of patients are on the brink,” Muhindura said.

€œWe are tired.”This story is from a reporting partnership that includes KCUR, NPR and KHN. Alex Smith, KCUR. @AlexSmithKCUR Related Topics Health Industry Multimedia Public Health States Audio erectile dysfunction treatment Hospitals Kansas Missouri Rural Medicine.