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Vacuolar protein sorting 41 (VPS41) is as part of the Homotypic fusion and Protein Sorting (HOPS) complex required for lysosomal fusion events and, independent of HOPS, for regulated secretion. Here, we report three patients with compound heterozygous mutations in VPS41 (VPS41S285P and VPS41R662 * ; VPS41 c.1423‐2A>G and VPS41R662 *) displaying neurodegeneration with ataxia and dystonia. Cellular consequences were investigated in patient fibroblasts and VPS41‐depleted HeLa cells. All mutants prevented formation of a functional HOPS complex, causing delayed lysosomal delivery of endocytic and autophagic cargo. By contrast, VPS41S285P enabled regulated secretion. Strikingly, loss of VPS41 function caused a cytosolic redistribution of mTORC1, continuous nuclear localization of Transcription Factor E3 (TFE3), enhanced levels of LC3II, and a reduced autophagic response to nutrient starvation. Phosphorylation of mTORC1 substrates S6K1 and 4EBP1 was not affected. In a C. elegans model of Parkinson’s disease, co‐expression of VPS41S285P/VPS41R662 * abolished the neuroprotective function of VPS41 against α‐synuclein aggregates. We conclude that the VPS41 variants specifically abrogate HOPS function, which interferes with the TFEB/TFE3 axis of mTORC1 signaling, and cause a neurodegenerative disease., Compound heterozygous mutations in VPS41 were identified in patients with a neurodegenerative phenotype with dystonia and cerebellar atrophy. VPS41 variants obstruct the HOPS complex, leading to decreased endocytic and autophagy cargo transfer to lysosomes and inhibition of mTORC1 signaling.

The SET domain containing 2, histone lysine methyltransferase encoded by SETD2 is a dual-function methyltransferase for histones and microtubules and plays an important role for transcriptional regulation, genomic stability, and cytoskeletal functions. Specifically, SETD2 is associated with trimethylation of histone H3 at lysine 36 (H3K36me3) and methylation of α-tubulin at lysine 40. Heterozygous loss of function and missense variants have previously been described with Luscan-Lumish syndrome (LLS), which is characterized by overgrowth, neurodevelopmental features, and absence of overt congenital anomalies. We have identified 15 individuals with de novo variants in codon 1740 of SETD2 whose features differ from those with LLS. Group 1 consists of 12 individuals with heterozygous variant c.5218C>T p.(Arg1740Trp) and Group 2 consists of 3 individuals with heterozygous variant c.5219G>A p.(Arg1740Gln). The phenotype of Group 1 includes microcephaly, profound intellectual disability, congenital anomalies affecting several organ systems, and similar facial features. Individuals in Group 2 had moderate to severe intellectual disability, low normal head circumference, and absence of additional major congenital anomalies. While LLS is likely due to loss of function of SETD2, the clinical features seen in individuals with variants affecting codon 1740 are more severe suggesting an alternative mechanism, such as gain of function, effects on epigenetic regulation, or posttranslational modification of the cytoskeleton. Our report is a prime example of different mutations in the same gene causing diverging phenotypes and the features observed in Group 1 suggest a new clinically recognizable syndrome uniquely associated with the heterozygous variant c.5218C>T p.(Arg1740Trp) in SETD2.

BACKGROUND: Hyperkeratotic hand eczema (HHE) is a typical clinical hand eczema subtype with a largely unknown pathophysiology.OBJECTIVE: To investigate histopathology, expression of keratins (K), epidermal barrier proteins, and adhesion molecules in HHE.METHODS: Palmar skin biopsies (lesional and perilesional) were obtained from seven HHE patients and two healthy controls. Moreover, 135 candidate genes associated with palmoplantar keratoderma were screened for mutations.RESULTS: Immunofluorescence staining showed a significant reduction of K9 and K14 in lesional skin. Upregulation was found for K5, K6, K16, and K17 in lesional skin compared with perilesional and healthy palmar skin. Further, upregulation of involucrin and alternating loricrin staining, both in an extracellular staining pattern, was found. Filaggrin expression was similar in lesional, perilesional, and control skin. No monogenetic mutations were found.CONCLUSION: Currently, the phenotype of HHE is included in the hand eczema classification system; however, it can be argued whether this is justified. The evident expression of filaggrin and involucrin in lesional skin does not support a pathogenesis of atopic eczema. The upregulation of K6, K16, and K17 and reduction of K9 and K14 might contribute to the underlying pathogenesis. Unfortunately, comparison with hand eczema studies is not possible yet, because similar protein expression studies are lacking.

Epidermolysis bullosa is a group of genetic skin conditions characterized by abnormal skin (and mucosal) fragility caused by pathogenic variants in various genes. The disease severity ranges from early childhood mortality in the most severe types to occasional acral blistering in the mildest types. The subtype and severity of EB is linked to the gene involved and the specific variants in that gene, which also determine its mode of inheritance. Current treatment is mainly focused on symptomatic relief such as wound care and blister prevention, because truly curative treatment options are still at the preclinical stage. Given the current level of understanding, the broad spectrum of genes and variants underlying EB makes it impossible to develop a single treatment strategy for all patients. It is likely that many different variant-specific treatment strategies will be needed to ultimately treat all patients. Antisense-oligonucleotide (ASO)-mediated exon skipping aims to counteract pathogenic sequence variants by restoring the open reading frame through the removal of the mutant exon from the pre-messenger RNA. This should lead to the restored production of the protein absent in the affected skin and, consequently, improvement of the phenotype. Several preclinical studies have demonstrated that exon skipping can restore protein production in vitro, in skin equivalents, and in skin grafts derived from EB-patient skin cells, indicating that ASO-mediated exon skipping could be a viable strategy as a topical or systemic treatment. The potential value of exon skipping for EB is supported by a study showing reduced phenotypic severity in patients who carry variants that result in natural exon skipping. In this article, we review the substantial progress made on exon skipping for EB in the past 15 years and highlight the opportunities and current challenges of this RNA-based therapy approach. In addition, we present a prioritization strategy for the development of exon skipping based on genomic information of all EB-involved genes.

Spinal muscular atrophy is a severe autosomal recessive disease caused by disruptions in the SMN1 gene. The nearly identical SMN2 gene copy number is associated with disease severity. SMN1 duplication markers, such as c.*3+80T>G and c.*211_*212del, can assess residual carrier risk. An SMN2 disease modifier (c.859G>C) can help inform prognostic outcomes. The emergence of multiple precision gene therapies for spinal muscular atrophy requires accurate and rapid detection of SMN1 and SMN2 copy numbers to enable early treatment and optimal patient outcomes. We developed and evaluated a singletube PCR/capillary electrophoresis assay system that quantifies SMN1/2 copy numbers and genotypes three additional clinically relevant variants. Analytical validation was performed with human cell lines and whole blood representing varying SMN1/2 copies on four capillary electrophoresis instrument models. In addition, four independent laboratories used the assay to test 468 residual clinical genomic DNA samples. The results were >98.3% concordant with consensus SMN1/2 exon 7 copy numbers, determined using multiplex ligation-dependent probe amplification and droplet digital PCR, and were 100% concordant with Sanger sequencing for the three variants. Furthermore, copy number values were 98.6% (SMN1) and 97.1% (SMN2) concordant to each laboratory's own reference results. (J Mol Diagn

ARGONAUTE-2 and associated miRNAs form the RNA-induced silencing complex (RISC), which targets mRNAs for translational silencing and degradation as part of the RNA interference pathway. Despite the essential nature of this process for cellular function, there is little information on the role of RISC components in human development and organ function. We identify 13 heterozygous mutations in AGO2 in 21 patients affected by disturbances in neurological development. Each of the identified single amino acid mutations result in impaired shRNA-mediated silencing. We observe either impaired RISC formation or increased binding of AGO2 to mRNA targets as mutation specific functional consequences. The latter is supported by decreased phosphorylation of a C-terminal serine cluster involved in mRNA target release, increased formation of dendritic P-bodies in neurons and global transcriptome alterations in patient-derived primary fibroblasts. Our data emphasize the importance of gene expression regulation through the dynamic AGO2-RNA association for human neuronal development., AGO2 binds to miRNAs to repress expression of cognate target mRNAs. Here the authors report that heterozygous AGO2 mutations result in defects in neurological development and impair RNA interference.

Clinical severity and treatment response vary significantly between patients with spinal muscular atrophy. The approval of therapies and the emergence of neonatal screening programmes urgently require a more detailed understanding of the genetic variants that underlie this clinical heterogeneity. We systematically investigated genetic variation other than SMN2 copy number in the SMN locus. Data were collected through our single-centre, population-based study on spinal muscular atrophy in the Netherlands, including 286 children and adults with spinal muscular atrophy Types 1–4, including 56 patients from 25 families with multiple siblings with spinal muscular atrophy. We combined multiplex ligation-dependent probe amplification, Sanger sequencing, multiplexed targeted resequencing and digital droplet polymerase chain reaction to determine sequence and expression variation in the SMN locus. SMN1, SMN2 and NAIP gene copy number were determined by multiplex ligation-dependent probe amplification. SMN2 gene variant analysis was performed using Sanger sequencing and RNA expression analysis of SMN by droplet digital polymerase chain reaction. We identified SMN1–SMN2 hybrid genes in 10% of spinal muscular atrophy patients, including partial gene deletions, duplications or conversions within SMN1 and SMN2 genes. This indicates that SMN2 copies can vary structurally between patients, implicating an important novel level of genetic variability in spinal muscular atrophy. Sequence analysis revealed six exonic and four intronic SMN2 variants, which were associated with disease severity in individual cases. There are no indications that NAIP1 gene copy number or sequence variants add value in addition to SMN2 copies in predicting the clinical phenotype in individual patients with spinal muscular atrophy. Importantly, 95% of spinal muscular atrophy siblings in our study had equal SMN2 copy numbers and structural changes (e.g. hybrid genes), but 60% presented with a different spinal muscular atrophy type, indicating the likely presence of further inter- and intragenic variabilities inside as well as outside the SMN locus. SMN2 gene copies can be structurally different, resulting in inter- and intra-individual differences in the composition of SMN1 and SMN2 gene copies. This adds another layer of complexity to the genetics that underlie spinal muscular atrophy and should be considered in current genetic diagnosis and counselling practices., Clinical variability, variation in treatment response and the introduction of neonatal screening programmes urgently require a better understanding of the genetic background of spinal muscular atrophy. Here, we report that SMN2 copies are structurally different between and within patients and show that (partial) deletions, conversions and single-nucleotide variants correlate with clinical severity., Graphical Abstract Graphical Abstract

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Objective The clinical description of a novel TTR gene mutation characterized by a late onset amyloid cardiomyopathy. Methods and Results A 78-year-old man of Dutch origin with recent surgery for bilateral carpal tunnel syndrome (CTS) was admitted to our hospital because of heart failure with preserved ejection fraction (55%). Cardiac ultrasound showed thickened biventricular walls, and cardiac magnetic resonance imaging also showed late gadolinium enhancement. Early signs of a polyneuropathy were found by neurophysiological testing. A few months later, his 72-year-old sister was admitted to an affiliated hospital because of heart failure caused by a restrictive cardiomyopathy. In both patients, a subcutaneous abdominal fat aspirate was stained with Congo red and DNA was analyzed by direct sequencing of exons 1 to 4 of the transthyretin (TTR) gene. Both fat aspirates revealed transthyretin-derived (ATTR) amyloid. 99mTc-diphosphonate scintigraphy further confirmed cardiac ATTR amyloidosis in the male patient. DNA analysis of both patients showed a novel TTR mutation c.194C > G that encodes for the gene product TTR (p.A65G) ending up as the mature protein TTR A45G. The 56-year-old daughter of the male patient had the same TTR mutation. A full diagnostic workup did not reveal any signs of amyloidosis yet. Conclusions A novel amyloidogenic TTR mutation was found in a Dutch family. The clinical presentation of ATTR A45G amyloidosis in the affected family members was heart failure due to a late-onset cardiomyopathy. The systemic nature of this disease was reflected by bilateral CTS and by early signs of a polyneuropathy in the index patient.

Dominant mutations in the KLHL24 gene, encoding for kelch-like protein 24, have been implicated in the pathogenesis of epidermolysis bullosa simplex (EBS). So far, 26 patients from different ethnicities have been reported and all of them harboured a heterozygous KLHL24 start-codon mutation, with c.1A>G;p.Met1? being the most prevalent.1-3 Through this report, we aimed to expand the phenotypic spectrum by incorporating additional findings, in particular, dilated cardiomyopathy, seen in a Dutch family. This article is protected by copyright. All rights reserved.

Spinal muscular atrophy (SMA) is one of the leading genetic causes of infant mortality with an incidence of 1:10,000. The recently-introduced antisense oligonucleotide treatment improves the outcome of this disease, in particular when applied at an early stage of progression. The genetic cause of SMA is, in >95% of cases, a homozygous deletion of the survival motor neuron 1 (SMN1) gene, which makes the low-cost detection of SMA cases as part of newborn screening programs feasible. We developed and validated a new SALSA MC002 melting curve assay that detects the absence of the SMN1 exon 7 DNA sequence without detecting asymptomatic carriers and reliably discriminates SMN1 from its genetic homolog SMN2 using crude extracts from newborn screening cards. Melting curve analysis shows peaks specific for both the SMN1 gene and the disease modifying SMN2 homolog. The detection of the SMN2 homolog, of which the only clinically relevant difference from the SMN1 gene is a single nucleotide in exon 7, was only used to confirm a correct reaction in samples that lacked the SMN1 gene, and not for SMN2 quantification. We retrieved 47 DBS samples from children with genetically-confirmed SMA, after informed consent from parents, and 375 controls from the national archive of the Dutch National Institute for Public Health and the Environment (RIVM). The assay correctly identified all anonymized and randomized SMA and control samples (i.e., sensitivity and specificity of 100%), without the detection of carriers, on the three most commonly-used PCR platforms with melting curve analysis. This test’s concordance with the second-tier ‘golden standard’ P021 SMA MLPA test was 100%. Using the new P021–B1 version, crude extracts from DBS cards could also be used to determine the SMN2 copy number of SMA patients with a high level of accuracy. The MC002 test showed the feasibility and accuracy of SMA screening in a neonatal screening program.

Dystrophic epidermolysis bullosa (DEB) is a devastating blistering disease affecting skin and mucous membranes. It is caused by pathogenic variants in the COL7A1 gene encoding type VII collagen, and can be inherited dominantly or recessively. Recently, promising proof-of-principle has been shown for antisense oligonucleotide (AON)-mediated exon skipping as a therapeutic approach for DEB. However, the precise phenotypic effect to be anticipated from exon skipping, and which patient groups could benefit, is not yet clear. To answer these questions, we studied new clinical and molecular data on seven patients from the Dutch EB registry and reviewed the literature on COL7A1 exon skipping variants. We found that phenotypes associated with dominant exon skipping cannot be distinguished from phenotypes caused by other dominant DEB variants. Recessive exon skipping phenotypes are generally relatively mild in the spectrum of recessive DEB. Therefore, for dominant DEB, AON-mediated exon skipping is unlikely to ameliorate the phenotype. In contrast, the overall severity of phenotypes associated with recessive natural exon skipping pivots toward the milder end of the spectrum. Consequently, we anticipate AON-mediated exon skipping for recessive DEB caused by bi-allelic null variants should lead to a clinically relevant improvement of this devastating phenotype. Keywords: exon skipping, therapy, dystrophic epidermolysis bullosa, splicing, genotype-phenotype correlation

ObjectiveTo investigate mutations in genes that are potential modifiers of spinal muscular atrophy (SMA) severity.MethodsWe performed a hypothesis-based search into the presence of variants in fused in sarcoma (FUS), transactive response DNA-binding protein 43 (TDP-43), plastin 3 (PLS3), and profilin 2 (PFN2) in a cohort of 153 patients with SMA types 1–4, including 19 families. Variants were detected with targeted next-generation sequencing and confirmed with Sanger sequencing. Functional effects of the identified variants were analyzed in silico and for PLS3, by analyzing expression levels in peripheral blood.ResultsWe identified 2 exonic variants in FUS exons 5 and 6 (p.R216C and p.S135N) in 2 unrelated patients, but clinical effects were not evident. We identified 8 intronic variants in PLS3 in 33 patients. Five PLS3 variants (c.1511+82T>C; c.748+130 G>A; c.367+182C>T; c.891-25T>C (rs145269469); c.1355+17A>G (rs150802596)) potentially alter exonic splice silencer or exonic splice enhancer sites. The variant c.367+182C>T, but not RNA expression levels, corresponded with a more severe phenotype in 1 family. However, this variant or level of PLS3 expression did not consistently correspond with a milder or more severe phenotype in other families or the overall cohort. We found 3 heterozygous, intronic variants in PFN2 and TDP-43 with no correlation with clinical phenotype or effects on splicing.ConclusionsPLS3 and FUS sequence variants do not modify SMA severity at the population level. Specific variants in individual patients or families do not consistently correlate with disease severity.

Background and purposeNatural history studies in spinal muscular atrophy (SMA) have primarily focused on infants and children. Natural history studies encompassing all age groups and SMA types are important for the interpretation of treatment effects of recently introduced survival motor neuron gene-augmenting therapies.MethodsWe conducted a cross-sectional study to investigate muscle strength, Hammersmith Functional Motor Scale (Expanded) score and the patterns of muscle weakness in relation to age and SMA type.ResultsWe included 180 patients with SMA types 1-4 in the age range 1-77.5years with median disease duration of 18 (range 0-65.8)years. With the exception of the early phases of disease in which children with SMA types 2 and 3 may achieve new motor skills and show a temporary increase in muscle strength, cross-sectional data suggested that declining muscle strength and loss of motor skills over time are characteristic of all SMA types. Mean loss of strength was at least 1 point on the Medical Research Council score and 0.5 point on the Hammersmith Functional Motor Scale (Expanded) score per year. Trend lines compatible with deterioration of motor function and muscle strength started in childhood and continued into adulthood. The age at loss of specific motor skills was associated with disease severity. Triceps, deltoid, iliopsoas and quadriceps were the weakest muscles in all patients. Hierarchical cluster analysis did not show a segmental distribution of muscle weakness as suggested previously.ConclusionsProgressive muscle weakness and loss of motor function are characteristic of all SMA types and all ages.

PLEC, the gene encoding the cytolinker protein plectin, has eight tissue-specific isoforms in humans, arising by alternate splicing of the first exon. To date, all PLEC mutations that cause epidermolysis bullosa simplex (EBS) were found in exons common to all isoforms. Due to the ubiquitous presence of plectin in mammalian tissues, EBS from recessive plectin mutations is always associated with extracutaneous involvement including muscular dystrophy, pyloric atresia and cardiomyopathy. We studied a consanguineous family with sisters having isolated blistering suggesting EBS. Skin disease started with foot blisters at walking age and became generalized at puberty while sparing mucous membranes. DNA sequencing revealed a homozygous nonsense mutation (c.46C > T; p.Arg16X) in the first exon of the plectin variant encoding plectin isoform 1a (P1a). Immunofluorescence antigen mapping, transmission electron microscopy, western blot analysis and qRT-PCR were performed on patient skin and cultured keratinocytes, control myocardium and striated muscle samples. We found hypoplastic hemidesmosomes and intra-epidermal 'pseudo-junctional' cleavage fitting EBS. Screening for cardiomyopathy and muscle dystrophy showed no abnormalities. We report the first cases of autosomal-recessive EBS from P1a deficiency affecting skin, while mucous membranes, heart and muscle are spared. The dominant expression of the P1a isoform in epidermal basal cell layer and cultured keratinocytes suggests that mutations in the first exon of isoform 1a cause skin-only EBS without extracutaneous involvement. Our study characterizes yet another of the eight isoforms of plectin and adds a tissue-specific phenotype to the spectrum of 'plectinopathies' produced by mutations of unique first exons of this gene.

Proximal hereditary spinal muscular atrophy (SMA) is caused by homozygous deletion of the survival motor neuron (SMN) 1 gene and has a wide range of severity.1 Onset may occur prenatally up to the fourth decade of life, and motor deficits range from neonatal hypotonia to mild weakness in adulthood. The current classification of SMA distinguishes five SMA types (ie, type 0–4) based on the combination of age at onset and two acquired gross motor milestones.1 ,2 Distinction of additional subtypes based on differences in the age at onset, first for SMA type 3 (ie, 3a and 3b) and more recently for type 1 (ie, 1a–1c), has been proposed. This may help to further clarify differences in prognosis within SMA types and to balance baseline characteristics in clinical trials. We aimed to gain further insight into the added predictive value of motor milestones for the occurrence of SMA outcome or common complications, that is, death, respiratory insufficiency, scoliosis surgery and loss of ambulation. We included SMN2 copy number in our analysis, since this is the most important genetic biomarker of severity.3 We enrolled patients with SMA types 1–4 between September 2010 and August 2014. Methods are described in the online supplementary file. The clinical diagnosis of SMA was genetically confirmed in 200 patients. SMN2 copy number varied from 1 to 5 and overlapped between SMA types. SMN2 copy number correlated inversely with SMA type (p

The hereditary blistering disease junctional epidermolysis bullosa (JEB) is always accompanied by structural enamel abnormalities of primary and secondary dentition, characterized as amelogenesis imperfecta. Autosomal recessive mutations in LAMA3, LAMB3 and LAMC2 encoding the heterotrimer laminin 332 (LM-332) are among the genes causing JEB. While examining pedigrees of JEB patients with LAMA3 mutations, we observed that heterozygous carriers of functional null mutations displayed subtle enamel pitting in the absence of skin fragility or other JEB symptoms. Here, we report two new LAMA3 functional null mutations: nonsense c.2377C>T p.(Arg793Ter) and splice-site c.4684+1G>A mutation in heterozygous carriers exhibiting enamel pitting. Both parents had offspring affected with JEB and displayed subtle enamel pitting of secondary dentition without any sign of skin blistering. The reported enamel abnormality in LAMA3 mutation carriers could be attributed to a half dose effect of the laminin alpha 3 chain (haploinsufficiency).

IMPORTANCE Epidermolysis bullosa (EB) is a group of mechanobullous genodermatoses characterized by the fragility of skin and mucous membranes. Mutations in the ITGA6 and ITGB4 genes, encoding the hemidesmosomal protein alpha(6)beta(4)-integrin, have been involved in the pathogenesis of EB. To date, the inheritance of these particular genes is known to be exclusively autosomal recessive. Herein, we report a novel heterozygous missense mutation in the ITGB4 gene exerting a dominant negative effect that cosegregates with the EB phenotype in an extended family.OBSERVATIONS The clinical phenotype of affected individuals is primarily characterized by nail dystrophy and late onset of mild skin fragility and acral blistering. Some patients developed granulation tissue in the larynx, urethra, lacrimal duct, and external auditory canal. Sequencing the complete set of genes associated with EB revealed a heterozygous missense mutation in exon 5 of ITGB4: c.433G>T, p. Asp145Tyr. The mutation was found in the affected relatives and was not present in unaffected relatives and control DNA samples.CONCLUSIONS AND RELEVANCE This study highlights, for the first time to our knowledge, the possibility of a dominant mode of inheritance for a missense ITGB4 mutation in EB, thus expanding the mutational database and genotype-phenotype correlation for this rare disease.

Background Junctional epidermolysis bullosa, type Herlitz (JEB-H) is a lethal, autosomal recessive blistering disease caused by null mutations in the genes coding for the lamina lucida/densa adhesion protein laminin-332 (LAMB3, LAMA3 and LAMC2). Objectives To present the diagnostic features and molecular analyses of all 22 patients with JEB-H in the Dutch Epidermolysis Bullosa Registry between 1988 and 2011, and to calculate the disease incidence and carrier frequency in the Netherlands. Methods All patients were analysed with immunofluorescence antigen mapping (IF), electron microscopy (EM) and molecular analysis. Results The mean lifespan of our patients with JEB-H was 5 8 months (range 0.5-32.6). IF showed absent (91%) or strongly reduced (9%) staining for laminin332 with monoclonal antibody GB3. In EM the hemidesmosomes and sub-basal dense plates were hypoplastic or absent. We identified mutations in all 22 patients: in 19 we found LAMB3 mutations, in two LAMA3 mutations, and in one LAMC2 mutations. We found three novel splice site mutations in LAMB3: (i) c. 292A> G resulting in an out-of-frame skip of exon 3 and a premature termination codon (PTC); (ii) c. 1289-2_1296del10 leading to an out-of-frame skip of exon 12 and a PTC; and (iii) c. 3228+ 1G> T leading to an exon 21 skip. Conclusions All diagnostic tools should be evaluated to clarify the diagnosis of JEBH. We have identified 11 different mutations in 22 patients with JEB-H, three of them novel. In the Netherlands the incidence rate of JEB-H is 4.0 per one million live births. The carrier frequency of a JEB-H mutation in the Dutch population is 1 in 249.

Couples with recurrent miscarriage (RM) and men with poor semen quality may undergo genetic testing as part of the diagnostic work-up. This study explored their knowledge and perception of genetic testing, evaluated psychological wellbeing and identified associated variables. A prospective questionnaire study was conducted in seven clinical genetics centres and referring gynaecological departments in couples with RM or poor semen quality. Questionnaires were completed before disclosure of genetic test results. Main outcome measures were knowledge, perceived risk, anxiety and depression. Of 439 participants, 256 were not aware genetic testing was part of the diagnostic work-up. One-third (36% RM, 33% poor semen quality) indicated they had not received information about the genetic test from their doctor. Perceived risk of receiving an abnormal genetic test result was higher than objective risk. Anxiety was highly correlated with perceived risk. Women with RM were more anxious than women in the poor semen quality group or men (P

We describe the clinical phenotype of a novel de novo KNCA1 mutation, and functional characterization of the effects of the mutation on Kv1.1 channel function. HEK293 cells were transfected transiently with either wild-type or mutant channels. Representative currents were evoked after application of a series of square voltage steps from -80 mV to +50 mV in 200-ms intervals from Vh = -80 mV. Extracellular K(+) was added to evoke tail currents. Equal amounts of wild-type and Kv1.1(I262M) mutant DNA were transfected transiently in HEK293 cells to evaluate the influence of the mutation. We found that Kv1.1(I262M) leads to a defective voltage-gated potassium channel. Coexpression studies revealed a dominant-negative effect. We describe the phenotype of a novel KCNA1 mutation causing episodic ataxia. Patch-clamp studies confirm the pathogenicity of the mutation in vitro and suggest that it is dominant with respect to wild-type.

We reevaluated a unique family with two sibs who had a presumed autosomal recessively inherited syndrome characterized by mental retardation, microcephaly, short stature and absent phalanges. This family was originally described by Drayer et al. in 1977. Using modern molecular techniques, we demonstrated that the syndrome is caused by the recurrence of an apparently de novo 15qter deletion of 5.8 Mb. Analysis of polymorphic markers revealed that the deletion was of maternal origin in both cases, indicating germline mosaicism in the clinically unaffected mother. This study demonstrates the possibility of parental mosaicism and the risk of recurrence in sibs for terminal subtelomeric deletions.

Thirty-four children with genetically proven SMA type I (age at onset The median survival after diagnosis did not differ significantly between children diagnosed at birth (median survival 137 days, 95% CI 111-232 days) and those diagnosed later (median survival 159 days, 95% CI 141-256), implying that SMA I cases with different ages of onset show the same progression rate of the disease. The number of SMN2 copies was not clearly correlated with survival duration, possibly because of lack of statistical power due to the small number of cases with I or 3 SMN2 copies. The three cases alive at the end of the study had either three or an unknown number of SMN2 copies, which is in agreement with previously described cases showing longer survival with increasing number of SMN2 copies. All deceased children died of respiratory insufficiency and/or an intercurrent lung infection, indicating that the susceptibility of the child with SMA type I to respiratory infections plays an important role in determining the survival. (c) 2008 Elsevier B.V. All rights reserved.

Background Mutations in the gene COL17A1 coding for type XVII collagen cause non-Herlitz junctional epidermolysis bullosa (nH-JEB).Objectives Here we give an overview of the genotype-phenotype correlation in 12 patients from the Netherlands with type XVII collagen-deficient nH-JEB.Patient and methods Family and personal history and clinical presentation were recorded from each patient, and skin biopsies of intact and bullous skin were taken for immunofluorescence and electron microscopy. The mutations were identified by analysing the patient's DNA isolated from peripheral blood cells.Results DNA analysis identified five novel deletions: 1284delA, 1365delC, 3236delT, 3600-3601delCT and 4425delT. Interestingly, we identified a new patient, homozygous for 4425delT, with an exceptionally mild blistering phenotype. All together, three patients had more localized blistering confined to hands, lower legs and face, absent or very mild nail dystrophy, normal primary hair and sparse secondary hair. Nine patients had generalized blistering, nail dystrophy, sparse primary and absent secondary hair. All 12 patients had amelogenesis imperfecta (enamel pitting). Immunofluorescence (IF) antigen mapping with monoclonal antibodies 1A8C and 1D1 that bind to type XVII collagen, but not to its 97-kDa fragment was completely negative in patients with generalized blistering, whereas reduced in patients with localized blistering.Conclusions Our data reveal that in patients with COL17A1 mutations a localized nH-JEB phenotype can be differentiated from a generalized nH-JEB phenotype by IF antigen mapping. The data are important for genetic counselling at early age when the clinical phenotype is not yet clear.

Update to: European Journal of Human Genetics (2012) 20; doi:10.1038/ejhg.2012.62; published online 18 April 2012

Wij beschrijven een pasgeborene die zich presenteerde met asfyxie en een ernstige aangeboren hartafwijking. In het beloop bleek er daarnaast sprake te zijn van bewegingsarmoede en congenitale contracturen. Na het overlijden werd door middel van dna-diagnostiek de diagnose sma type 1, de ziekte van Werdnig-Hoffman, gesteld. De combinatie sma type 1 met een congenitale hartafwijking is bekend uit de medische literatuur, waarvan wij een overzicht geven. Kinderartsen dienen ook bij een kind met een congenitale hartafwijking alert te zijn op bijkomende (neurologische) verschijnselen. Het stellen van de diagnose sma type 1 heeft belangrijke genetische consequenties voor de ouders.

Mutation analysis was performed in four apparently unrelated Dutch families with pantothenate kinase-associated neurodegeneration, formerly known as Hallervorden-Spatz syndrome. A novel 3-bp deletion encompassing the nucleotides GAG at positions 1,142 to 1,144 of exon 5 of the PANK2 gene was found in all patients. One patient was compound heterozygous; she also carried a novel nonsense mutation (Ser68Stop). The other patients were homozygous for the 1142_1144delGAG mutation. The 1142_1144delGAG mutation was also found in a German patient of unknown descent. We used polymorphic microsatellite markers flanking the PANK2 gene (spanning a region of approximately 8 cM) for haplotype analyses in all these families. A conserved haplotype of 1.5 cM was found for the 1142_1144delGAG mutation carriers. All the Dutch families originated from the same geographical region within the Netherlands. The results indicate a founder effect and suggest that the 1142_1144delGAG mutation probably originated from one common ancestor. It was estimated that this mutation arose at the beginning of the ninth century, approximately 38 generations ago.

Item does not contain fulltext BACKGROUND: ALS is believed to be multifactorial in origin with modifying genes affecting its clinical expression. Childhood-onset spinal muscular atrophy (SMA) is an autosomal recessive disorder of motor neurons, caused by mutations of the survival motor neuron (SMN) gene. The SMN gene exists in two highly homologous variants: SMN1, the causative gene responsible for the production of the majority of functional SMN protein, and SMN2, responsible for the production of less protein but sufficient for modifying the SMA phenotype. OBJECTIVE: To test whether SMN genotypes are associated with susceptibility to and severity of sporadic ALS. METHODS: We performed competitive quantitative PCR analysis for both SMN1 and SMN2 genes in 242 clinically well-defined ALS patients and 175 controls. The combined determination of SMN1 and SMN2 copies also allowed for an estimation of the level of SMN for each patient (estimated SMN protein level = SMN1 copy number + 0.20 x SMN2 copy number). RESULTS: One copy of SMN1 was associated with an increased risk of developing ALS (odds ratio = 4.1, 95% CI = 1.2 to 14.2, p = 0.02) and ALS patients carried fewer SMN2 copy numbers (p < 0.001). Sixty-one percent of patients had an estimated protein SMN level < or = 2.2 vs only 36% of controls (p = 0.0000004). Multivariate Cox regression analyses showed that lower SMN2 copy numbers and lower levels of estimated SMN protein (hazard ratio = 1.3, 95% CI = 1.1 to 1.6, p = 0.03) were associated with an increased mortality rate. CONCLUSIONS: SMN genotypes producing less SMN protein increase susceptibility to and severity of ALS. 6 p.

In the present study, we aimed to elucidate the mechanism responsible for constitutive NF-kappaB DNA-binding activity in AML cells. Intervening in aberrant signaling pathway provides a rational approach for in vivo targeting of AML cells. Constitutive NF-kappaB DNA-binding activity was observed in 16 of 22 (73%) investigated AML cases and was, in general, associated with resistance to spontaneous apoptosis. Indeed, inhibition of NF-kappaB activity by the NF-kappaB inhibitor SN-50 peptide resulted in enhanced chemotherapy-induced apoptosis. In the majority of cases, constitutive NF-kappaB activity was mediated by a Ras/PI3 kinase (PI3-K)/protein kinase B (PKB)-mediated pathway. The PI3-K inhibitor Ly294002 and the Ras inhibitor L-744832 both inhibited PKB phosphorylation and NF-kappaB DNA-binding activity. The constitutive activation of Ras GTP-ase was caused by mutations in the gene encoding for N-Ras in 29% of the cases. The constitutive NF-kappaB activity could so far not be ascribed to the autocrine production of growth factors or to mutations in the Flt3 receptor, since anti-GM-CSF, -IL-1, -IL6, -TNFalpha or the tyrosine kinase inhibitor AG1296 did not affect the NF-kappaB DNA-binding activity. The present study demonstrates that Ras activation is an important pathway for triggering the NF-kappaB pathway in AML cells.

Spinal muscular atrophy (SMA) is caused by the homozygous deletion of the survival motor neuron (SMN)1 gene. The nearly identical SMN2 gene produces small amounts of full-length mRNA and functional SMN protein, due to a point mutation in a critical splicing site. Increasing SMN protein production by histone deacetylase inhibiting drugs such as valproic acid (VPA) is an experimental treatment strategy for SMA. To investigate whether an SMN-specific ELISA could detect changes in SMN protein expression in peripheral blood mononuclear cells (PBMCs) after treatment with VPA. The authors developed a sensitive SMN-specific ELISA. Six patients with SMA types 2 and 3 participated in the study. Recombinant SMN calibration curves were used to calculate SMN protein levels in PBMCs before and after 4&emsp14;months of VPA treatment. The SMN ELISA was able to detect small differences in SMN protein concentrations, and differences in SMN protein levels in Epstein-Barr virus immortalised lymphocyte cell lines from SMA type 1 and 2 patients, carriers and healthy individuals (p

We describe the clinical phenotype of a novel de novo KNCA1 mutation, and functional characterization of the effects of the mutation on Kv1.1 channel function. HEK293 cells were transfected transiently with either wild-type or mutant channels. Representative currents were evoked after application of a series of square voltage steps from -80 mV to +50 mV in 200-ms intervals from Vh = -80 mV. Extracellular K(+) was added to evoke tail currents. Equal amounts of wild-type and Kv1.1(I262M) mutant DNA were transfected transiently in HEK293 cells to evaluate the influence of the mutation. We found that Kv1.1(I262M) leads to a defective voltage-gated potassium channel. Coexpression studies revealed a dominant-negative effect. We describe the phenotype of a novel KCNA1 mutation causing episodic ataxia. Patch-clamp studies confirm the pathogenicity of the mutation in vitro and suggest that it is dominant with respect to wild-type.

In acute myelogenous leukemia (AML) and adult T-cell leukemia, it has been demonstrated that the transcription factor LIL-STAT is constitutively activated. To identify and characterize this unknown LIL-STAT protein, electrophoretic mobility shift assay (EMSA) and oligoprecipitation assays were performed by using lipopolysaccharide/interleukin-1 (IL-1)–responsive element (LILRE) oligonucleotide probes. EMSA demonstrated a significant increase in LIL-STAT binding to the LILRE oligonucleotides after interferon γ (IFN-γ) and IL-6 stimulation of THP-1 cells. In unstimulated THP-1 and AML cells, LILRE oligonucleotide probes bound only to STAT1 α and β isoforms. The LILRE element showed a significant increase in binding of both α and β isoforms of STAT1 and STAT3 upon IFN-γ and IL-6 stimulation. Similar results were observed with human monocytes upon IL-6 or IFN-γ stimulation. These studies indicate that LIL-STAT consists of STAT1 and STAT3 proteins that bind to the LILRE DNA consensus site in a stimulus-dependent way.

In the present study, we examined the underlying mechanism, which causes the constitutive tyrosine phosphorylation of signal transducer and activator of transcription 5 (STAT5) in acute myeloid leukemia (AML) blasts. Constitutive STAT5 phosphorylation was observed in 18 of 26 (69%) patients with AML. The constitutive STAT5 phosphorylation was caused by different mechanisms. In the majority of the investigated cases (71% (12 of 17)) constitutive STAT5 phosphorylation was associated with autophosphorylation of the type III receptor tyrosine kinase Flt3. In 47% (eight of 17) of these cases autophosphorylation of Flt3 coincided with tandem duplications of the Flt3 gene, resulting in constitutive phosphorylation of the receptor, while 24% (four of 17) of the cases demonstrated STAT5 phosphorylation and Flt3 autophosphorylation without mutations. In addition, a subset of AML cases (29% (five of 17)) had no autophosphorylation of the Flt3 receptor, but demonstrated constitutive STAT5 phosphorylation, which was partly due to autocrine growth factor production. All AML cases with high STAT5 and Flt3 phosphorylation demonstrated, in general, a lower percentage of spontaneous apoptosis, compared to AML blasts with no spontaneous STAT5 phosphorylation. Addition of the receptor tyrosine III kinase inhibitor AG1296 strongly inhibited STAT5 phosphorylation and enhanced the percentage of apoptotic cells without modulating the Bcl-xl protein levels. These data indicate that in the majority of AML cases the constitutive STAT5 phosphorylation is caused by Flt3 phosphorylation mostly due to mutations in the receptors and associated with a low degree of spontaneous apoptosis.

Epidermolysis bullosa simplex is an autosomal dominant inherited skin blistering disorder caused by mutations in the genes KRT5 or KRT14 coding for the basal epidermal keratins 5 and 14, respectively. We describe a novel heterozygous pathogenic missense mutation (KRT5:c.596A>T, p.Lys199Met) in a Hindoestan male with early onset localized epidermolysis bullosa simplex that segregated with the phenotype in the family. We also found a new heterozygous amino acid substitution polymorphism in the variable keratin 14 N-terminal head domain (KRT14:c.88C>T, p. Arg30Cys), that did not segregate with the phenotype.

guished according to phenotypic characteristics. These Background. Recently a locus for antenatal Bartter are: (i) the antenatal variant [3–5] also termed hypersyndrome associated with sensorineural deafness was prostaglandin E syndrome; (ii) the less severe ‘classical mapped to human chromosome 1p31 in a single con- variant’; and (iii) the Gitelman syndrome [6 ]. sanguineous Bedouin family (Brennan et al. Am J Hum The antenatal variant shows, in addition to the Genet 1998; 62: 355–361). above-described features, maternal polyhydramnios, Methods. By haplotype analysis we demonstrate link- premature delivery, severe polyuria, hypercalciuria, age to this locus in nine consanguineous families with nephrocalcinosis, and elevated urinary excretion of antenatal Bartter syndrome associated with sensorineu- prostaglandin E2. Patients fail to thrive and show ral deafness. developmental delay if not adequately treated by indoResults. The critical interval compatible with linkage methacin, and fluid and salt supplementation. Some of was refined to 4.0 cM by two novel recombinational the children show a typical facial appearance with events with markers D1S2661 and D1S475. prominent forehead, triangular faces, drooping mouth, Conclusion. We thereby confirmed this gene locus and and large eyes and pinnae. The antenatal variant is distinguished this clinical subtype from other variants caused either by mutations in the Na-K-2Cl cotransof Bartter syndrome as a new disease entity. porter gene (SLC12A1) [7,8] or by mutations in KCNJ1 encoding the inwardly rectifying K+ channel

Inherited hypokalemic renal tubulopathies are differentiated into at least three clinical subtypes: (1) the Gitelman variant of Bartter syndrome (GS); (2) hyperprostaglandin E syndrome, the antenatal variant of Bartter syndrome (HPS/aBS); and (3) the classic Bartter syndrome (cBS). Hypokalemic metabolic alkalosis and renal salt wasting are the common characteristics of all three subtypes. Hypocalciuria and hypomagnesemia are specific clinical features of Gitelman syndrome, while HPS/aBS is a life-threatening disorder of the newborn with polyhydramnios, premature delivery, hyposthenuria, and nephrocalcinosis. The Gitelman variant is uniformly caused by mutations in the gene for the thiazide-sensitive NaCl-cotransporter NCCT (SLC12A3) of the distal tubule, while HPS/aBS is caused by mutations in the gene for either the furosemide-sensitive NaK-2Cl-cotransporter NKCC2 (SLC12A1) or the inwardly rectifying potassium channel ROMK (KCNJ1). Recently, mutations in a basolateral chloride channel CLC-Kb (CLCNKB) have been described in a subset of patients with a Bartter-like phenotype typically lacking nephrocalcinosis. In this study, the screening for CLCNKB mutations showed 20 different mutations in the affected children from 30 families. The clinical characterization revealed a highly variable phenotype ranging from episodes of severe volume depletion and hypokalemia during the neonatal period to almost asymptomatic patients diagnosed during adolescence. This study adds 16 novel mutations to the nine already described, providing further evidence that mutations in the gene for the basolateral chloride channel CLC-Kb are the molecular basis of classic Bartter syndrome. Interestingly, the phenotype elicited by CLCNKB mutations occasionally includes HPS/aBS, as well as a Gitelman-like phenotype.

Identification of COL4A5 defects in Alport's syndrome by immunohistochemistry of skin. Background The COL4A3-COL4A4-COL4A5 network in the glomerular basement membrane is affected in the inherited renal disorder Alport's syndrome (AS). Approximately 85% of the AS patients are expected to carry a mutation in the X-chromosomal COL4A5 gene and 15% in the autosomal COL4A3 and COL4A4 genes. The COL4A5 chain is also present in the epidermal basement membrane (EBM). It is predicted that approximately 70% of the COL4A5 mutations prevent incorporation of this chain in basement membranes. Methods We investigated whether or not COL4A5 defects could be detected by immunohistochemical analysis of the EBM. Punch skin biopsies were obtained from 22 patients out of 17 families and two biopsy specimens from healthy males were used as controls. Results In four cases with the COL4A5 frameshift or missense mutations, the COL4A5 chain was either lacking from the EBM (male) or showed a focally negative pattern (female). In three other patients with a COL4A5 missense mutation, a COL4A3 and a COL4A4 mutation, respectively, the COL4A5 staining was normal. A (focally) negative EBM-COL4A5 staining was found in three patients of six families with a diagnosis of AS and in one family of a group of four families with possible AS. Conclusions The (focal) absence of COL4A5 in the EBM of skin biopsy specimens can be used for fast identification of COL4A5 defects. Combined with polymorphic COL4A5 markers, both postnatal and prenatal DNA diagnosis are possible in the family of the patient.

Background. Alport syndrome is a hereditary nephritis that may lead to end-stage renal disease (ESRD) in young adult life and is often associated with sensorineural deafness and/or ocular abnormalities. The majority of families are X-linked due to mutations in the COL4A5 gene at Xq22. Autosomal forms of the disease are also recognized with recessive disease, having been shown to be due to mutations in the COL4A3 and COL4A4 genes on chromosome 2. Familial benign haematuria has also been mapped to this region in some families. Subjects and methods. We describe a large family with autosomal dominant Alport syndrome in which males and females are equally severely affected and one member with a mild sensorineural deafness reached ESRD aged 35 years. Renal biopsy in four affected patients demonstrated characteristic thickened and split glomerular basement membranes on electron-microscopy. Results. Genetic linkage analysis using markers on chromosome 2q demonstrated co-segregation of the disease with the markers D2S351 and D2S401 with a maximum lod score of 3.4 at zero recombination. Linkage to the COL4A4 gene was confirmed using an intragenic COL4A4 polymorphism. Mutation analysis has revealed a missense Leu36Pro mutation in exon 5 of the adjacent COL4A3 gene in the unaffected mother, which may lead to a more severe phenotype in affected family members carrying this mutation. Conclusion. Mutations in the COL4A3 and COL4A4 genes can cause a spectrum of glomerular basement membrane disease ranging from autosomal recessive Alport syndrome to autosomal dominant Alport syndrome and familial benign haematuria.

Clinical manifestations of type IV collagen mutations can vary from the severe, clinically and genetically heterogeneous renal disorder, Alport syndrome, to autosomal dominant familial benign hematuria. The predominant form of Alport syndrome is X-linked; more than 160 different mutations have yet been identified in the type IV collagen alpha 5 chain (COL4A5) gene, located at Xq22-24 head to head to the COL4A6 gene. The autosomal recessive form of Alport syndrome is caused by mutations in the COL4A3 and COL4A4 genes, located at 2q35-37. Recently, the first mutation in the COL4A4 gene was identified in familial benign hematuria. This paper presents an overview of type IV collagen mutations, including eight novel COL4A5 mutations from our own group in patients with Alport syndrome. The spectrum of mutations is broad and provides insight into the clinical heterogeneity of Alport syndrome with respect to age at renal failure and accompanying features such as deafness, leiomyomatosis, and anti-GBM nephritis.

Spinal muscular atrophy with respiratory distress type 1 is an autosomal recessive disorder with early respiratory difficulties, distal muscle weakness, and contractures leading to foot deformities as the most striking clinical symptoms. Mutations of the gene encoding the immunoglobulin heavy chain p.-binding protein 2, mapped on chromosome 11q13, are the cause of the disease. We present the clinical and mutational characteristics of ten patients in the Netherlands who showed considerable clinical variability; they carried six novel mutations, including a deletion of exon 2. However, there were no clear phenotype genotype correlations. (C) 2013 Elsevier B.V. All rights reserved.

Gitelman syndrome is a mostly autosomal recessive disorder affecting the renal tubular function associated with hypokalemia and hypomagnesemia. Functional studies point to a defect in the distal renal tubule in the thiazide-sensitive, electroneutral sodium-chloride co-transporter (TSC). Based upon the localization of a 2.6 cDNA encoding the human TSC to chromosome 16q13, polymorphic markers spanning the region from 16p12 to 16q21 were tested for linkage to the Gitelman syndrome locus in three Dutch families with autosomal recessive inheritance of this disorder. Using two-point linkage analysis, a maximum LOD score (Zmax of 4.49 (at theta = 0.00) was found for the marker D16S408. One crucial recombination event places the Gitelman syndrome locus distal to D16S419 at 16q12-13. Subsequently we have tested our group of Gitelman patients for mutations in the human TSC gene. Two mutations were identified in three Gitelman families. Our study confirms that the human TSC gene is involved in Gitelman syndrome. Patients from three Gitelman families reveal two identical human TSC mutations, suggesting these families share a common ancestor.