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Background: Idiopathic membranous nephropathy is a major cause of the nephrotic syndrome in adults, but its etiologic basis is not fully understood. We investigated the genetic basis of biopsy-proven cases of idiopathic membranous nephropathy in a white population.Methods: We performed independent genomewide association studies of single-nucleotide polymorphisms (SNPs) in patients with idiopathic membranous nephropathy from three populations of white ancestry (75 French, 146 Dutch, and 335 British patients). The patients were compared with racially matched control subjects; population stratification and quality controls were carried out according to standard criteria. Associations were calculated by means of a chi-square basic allele test; the threshold for significance was adjusted for multiple comparisons (with the Bonferroni method).Results: In a joint analysis of data from the 556 patients studied (398 men), we identified significant alleles at two genomic loci associated with idiopathic membranous nephropathy. Chromosome 2q24 contains the gene encoding M-type phospholipase A(2) receptor (PLA(2)R1) (SNP rs4664308, P=8.6×10(-29)), previously shown to be the target of an autoimmune response. Chromosome 6p21 contains the gene encoding HLA complex class II HLA-DQ alpha chain 1 (HLA-DQA1) (SNP rs2187668, P=8.0×10(-93)). The association with HLA-DQA1 was significant in all three populations (P=1.8×10(-9), P=5.6×10(-27), and P=5.2×10(-36) in the French, Dutch, and British groups, respectively). The odds ratio for idiopathic membranous nephropathy with homozygosity for both risk alleles was 78.5 (95% confidence interval, 34.6 to 178.2).Conclusions: An HLA-DQA1 allele on chromosome 6p21 is most closely associated with idiopathic membranous nephropathy in persons of white ancestry. This allele may facilitate an autoimmune response against targets such as variants of PLA2R1. Our findings suggest a basis for understanding this disease and illuminate how adaptive immunity is regulated by HLA. [ABSTRACT FROM AUTHOR]

BACKGROUNDCystic kidney disease (CyKD) is a predominantly familial disease in which gene discovery has been led by family-based and candidate gene studies, an approach that is susceptible to ascertainment and other biases.METHODSUsing whole-genome sequencing data from 1,209 cases and 26,096 ancestry-matched controls participating in the 100,000 Genomes Project, we adopted hypothesis-free approaches to generate quantitative estimates of disease risk for each genetic contributor to CyKD, across genes, variant types and allelic frequencies.RESULTSIn 82.3% of cases, a qualifying potentially disease-causing rare variant in an established gene was found. There was an enrichment of rare coding, splicing, and structural variants in known CyKD genes, with statistically significant gene-based signals in COL4A3 and (monoallelic) PKHD1. Quantification of disease risk for each gene (with replication in the separate UK Biobank study) revealed substantially lower risk associated with genes more recently associated with autosomal dominant polycystic kidney disease, with odds ratios for some below what might usually be regarded as necessary for classical Mendelian inheritance. Meta-analysis of common variants did not reveal significant associations, but suggested this category of variation contributes 3%-9% to the heritability of CyKD across European ancestries.CONCLUSIONBy providing unbiased quantification of risk effects per gene, this research suggests that not all rare variant genetic contributors to CyKD are equally likely to manifest as a Mendelian trait in families. This information may inform genetic testing and counseling in the clinic.

Class 2 HLA and PLA2R1 alleles are exceptionally strong genetic risk factors for membranous nephropathy (MN), leading, through an unknown mechanism, to a targeted autoimmune response. Introgressed archaic haplotypes (introduced from an archaic human genome into the modern human genome) might influence phenotypes through gene dysregulation. Here, we investigated the genomic region surrounding the PLA2R1 gene. We reconstructed the phylogeny of Neanderthal and modern haplotypes in this region and calculated the probability of the observed clustering being the result of introgression or common descent. We imputed variants for the participants in our previous genome-wide association study and we compared the distribution of Neanderthal variants between MN cases and controls. The region associated with the lead MN risk locus in the PLA2R1 gene was confirmed and showed that, within a 507 kb region enriched in introgressed sequence, a stringently defined 105 kb haplotype, intersecting the coding regions for PLA2R1 and ITGB6, is inherited from Neanderthals. Thus, introgressed Neanderthal haplotypes overlapping PLA2R1 are differentially represented in MN cases and controls, with enrichment In controls suggesting a protective effect., (Copyright © 2024 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Introduction: Steroid-sensitive nephrotic syndrome (SSNS) is the most common form of kidney disease in children worldwide. Genome-wide association studies (GWAS) have demonstrated the association of SSNS with genetic variation at HLA-DQ/DR and have identified several non- HLA loci that aid in further understanding of disease pathophysiology. We sought to identify additional genetic loci associated with SSNS in children of Sri Lankan and European ancestry., Methods: We conducted a GWAS in a cohort of Sri Lankan individuals comprising 420 pediatric patients with SSNS and 2339 genetic ancestry matched controls obtained from the UK Biobank. We then performed a transethnic meta-analysis with a previously reported European cohort of 422 pediatric patients and 5642 controls., Results: Our GWAS confirmed the previously reported association of SSNS with HLA-DR/DQ (rs9271602, P  = 1.12 × 10 -27 , odds ratio [OR] = 2.75). Transethnic meta-analysis replicated these findings and identified a novel association at AHI1 (rs2746432, P  = 2.79 × 10 -8 , OR = 1.37), which was also replicated in an independent South Asian cohort. AHI1 is implicated in ciliary protein transport and immune dysregulation, with rare variation in this gene contributing to Joubert syndrome type 3., Conclusions: Common variation in AHI1 confers risk of the development of SSNS in both Sri Lankan and European populations. The association with common variation in AHI1 further supports the role of immune dysregulation in the pathogenesis of SSNS and demonstrates that variation across the allele frequency spectrum in a gene can contribute to disparate monogenic and polygenic diseases., (© 2023 International Society of Nephrology. Published by Elsevier Inc.)

Introduction: Membranous nephropathy (MN) is the leading cause of nephrotic syndrome in adults and is characterized by detectable autoantibodies against glomerular antigens, most commonly phospholipase A2 receptor 1 (PLA2R1) and thrombospondin type-1 domain containing 7A (THSD7A). In Europeans, genetic variation in at least five loci, PLA2R1 , HLA-DRB1 , HLA-DQA1, IRF4, and NFKB1, affects the risk of disease. Here, we investigated the genetic risk differences between different autoantibody states., Methods: 1,409 MN individuals were genotyped genome-wide with a dense SNV array. The genetic risk score (GRS) was calculated utilizing the previously identified European MN loci, and results were compared with 4,929 healthy controls and 422 individuals with steroid-sensitive nephrotic syndrome., Results: GRS was calculated in the 759 MN individuals in whom antibody status was known. The GRS for MN was elevated in the anti-PLA2R1 antibody-positive ( N = 372) compared with both the unaffected control ( N = 4,929) and anti-THSD7A-positive ( N = 31) groups ( p < 0.0001 for both comparisons), suggesting that this GRS reflects anti-PLA2R1 MN. Among PLA2R1-positive patients, GRS was inversely correlated with age of disease onset ( p = 0.009). Further, the GRS in the dual antibody-negative group ( N = 355) was intermediate between controls and the PLA2R1-positive group ( p < 0.0001)., Conclusion: We demonstrate that the genetic risk factors for PLA2R1- and THSD7A-antibody-associated MN are different. A higher GRS is associated with younger age of onset of disease. Further, a proportion of antibody-negative MN cases have an elevated GRS similar to PLA2R1-positive disease. This suggests that in some individuals with negative serology the disease is driven by autoimmunity against PLA2R1., Competing Interests: The authors have no conflicts of interest to declare., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

Posterior urethral valves (PUV) are the commonest cause of end-stage renal disease in children, but the genetic architecture of this rare disorder remains unknown. We performed a sequencing-based genome-wide association study (seqGWAS) in 132 unrelated male PUV cases and 23,727 controls of diverse ancestry, identifying statistically significant associations with common variants at 12q24.21 (p=7.8 × 10 -12 ; OR 0.4) and rare variants at 6p21.1 (p=2.0 × 10 -8 ; OR 7.2), that were replicated in an independent European cohort of 395 cases and 4151 controls. Fine mapping and functional genomic data mapped these loci to the transcription factor TBX5 and planar cell polarity gene PTK7 , respectively, the encoded proteins of which were detected in the developing urinary tract of human embryos. We also observed enrichment of rare structural variation intersecting with candidate cis -regulatory elements, particularly inversions predicted to affect chromatin looping (p=3.1 × 10 -5 ). These findings represent the first robust genetic associations of PUV, providing novel insights into the underlying biology of this poorly understood disorder and demonstrate how a diverse ancestry seqGWAS can be used for disease locus discovery in a rare disease., Competing Interests: MC, OS, FL, AH, HS, CV, GB, WN, MZ, SW, YH, JC, DW, CM, AS, AK, RK, AW, DB, AL, DG No competing interests declared, (© 2022, Chan et al.)

Background: The endocytic reabsorption of proteins in the proximal tubule requires a complex machinery and defects can lead to tubular proteinuria. The precise mechanisms of endocytosis and processing of receptors and cargo are incompletely understood. EHD1 belongs to a family of proteins presumably involved in the scission of intracellular vesicles and in ciliogenesis. However, the relevance of EHD1 in human tissues, in particular in the kidney, was unknown., Methods: Genetic techniques were used in patients with tubular proteinuria and deafness to identify the disease-causing gene. Diagnostic and functional studies were performed in patients and disease models to investigate the pathophysiology., Results: We identified six individuals (5-33 years) with proteinuria and a high-frequency hearing deficit associated with the homozygous missense variant c.1192C>T (p.R398W) in EHD1 . Proteinuria (0.7-2.1 g/d) consisted predominantly of low molecular weight proteins, reflecting impaired renal proximal tubular endocytosis of filtered proteins. Ehd1 knockout and Ehd1 R398W/R398W knockin mice also showed a high-frequency hearing deficit and impaired receptor-mediated endocytosis in proximal tubules, and a zebrafish model showed impaired ability to reabsorb low molecular weight dextran. Interestingly, ciliogenesis appeared unaffected in patients and mouse models. In silico structural analysis predicted a destabilizing effect of the R398W variant and possible inference with nucleotide binding leading to impaired EHD1 oligomerization and membrane remodeling ability., Conclusions: A homozygous missense variant of EHD1 causes a previously unrecognized autosomal recessive disorder characterized by sensorineural deafness and tubular proteinuria. Recessive EHD1 variants should be considered in individuals with hearing impairment, especially if tubular proteinuria is noted., (Copyright © 2022 by the American Society of Nephrology.)

Membranous Nephropathy (MN) is a rare autoimmune cause of kidney failure. Here we report a genome-wide association study (GWAS) for primary MN in 3,782 cases and 9,038 controls of East Asian and European ancestries. We discover two previously unreported loci, NFKB1 (rs230540, OR = 1.25, P = 3.4 × 10 -12 ) and IRF4 (rs9405192, OR = 1.29, P = 1.4 × 10 -14 ), fine-map the PLA2R1 locus (rs17831251, OR = 2.25, P = 4.7 × 10 -103 ) and report ancestry-specific effects of three classical HLA alleles: DRB1*1501 in East Asians (OR = 3.81, P = 2.0 × 10 -49 ), DQA1*0501 in Europeans (OR = 2.88, P = 5.7 × 10 -93 ), and DRB1*0301 in both ethnicities (OR = 3.50, P = 9.2 × 10 -23 and OR = 3.39, P = 5.2 × 10 -82 , respectively). GWAS loci explain 32% of disease risk in East Asians and 25% in Europeans, and correctly re-classify 20-37% of the cases in validation cohorts that are antibody-negative by the serum anti-PLA2R ELISA diagnostic test. Our findings highlight an unusual genetic architecture of MN, with four loci and their interactions accounting for nearly one-third of the disease risk.

Background: Steroid-sensitive nephrotic syndrome (SSNS), the most common form of nephrotic syndrome in childhood, is considered an autoimmune disease with an established classic HLA association. However, the precise etiology of the disease is unclear. In other autoimmune diseases, the identification of loci outside the classic HLA region by genome-wide association studies (GWAS) has provided critical insights into disease pathogenesis. Previously conducted GWAS of SSNS have not identified non-HLA loci achieving genome-wide significance., Methods: In an attempt to identify additional loci associated with SSNS, we conducted a GWAS of a large cohort of European ancestry comprising 422 ethnically homogeneous pediatric patients and 5642 ethnically matched controls., Results: The GWAS found three loci that achieved genome-wide significance, which explain approximately 14% of the genetic risk for SSNS. It confirmed the previously reported association with the HLA-DR/DQ region (lead single-nucleotide polymorphism [SNP] rs9273542, P =1.59×10 -43 ; odds ratio [OR], 3.39; 95% confidence interval [95% CI], 2.86 to 4.03) and identified two additional loci outside the HLA region on chromosomes 4q13.3 and 6q22.1. The latter contains the calcium homeostasis modulator family member 6 gene CALHM6 (previously called FAM26F ). CALHM6 is implicated in immune response modulation; the lead SNP (rs2637678, P =1.27×10 -17 ; OR, 0.51; 95% CI, 0.44 to 0.60) exhibits strong expression quantitative trait loci effects, the risk allele being associated with lower lymphocytic expression of CALHM6 ., Conclusions: Because CALHM6 is implicated in regulating the immune response to infection, this may provide an explanation for the typical triggering of SSNS onset by infections. Our results suggest that a genetically conferred risk of immune dysregulation may be a key component in the pathogenesis of SSNS., (Copyright © 2019 by the American Society of Nephrology.)

Large-scale genome sequencing is poised to provide a substantial increase in the rate of discovery of disease-associated mutations, but the functional interpretation of such mutations remains challenging. Here we show that deletions of a sequence on human chromosome 16 that we term the intestine-critical region (ICR) cause intractable congenital diarrhoea in infants 1,2 . Reporter assays in transgenic mice show that the ICR contains a regulatory sequence that activates transcription during the development of the gastrointestinal system. Targeted deletion of the ICR in mice caused symptoms that recapitulated the human condition. Transcriptome analysis revealed that an unannotated open reading frame (Percc1) flanks the regulatory sequence, and the expression of this gene was lost in the developing gut of mice that lacked the ICR. Percc1-knockout mice displayed phenotypes similar to those observed upon ICR deletion in mice and patients, whereas an ICR-driven Percc1 transgene was sufficient to rescue the phenotypes found in mice that lacked the ICR. Together, our results identify a gene that is critical for intestinal function and underscore the need for targeted in vivo studies to interpret the growing number of clinical genetic findings that do not affect known protein-coding genes.

An HLA-DR3 association with membranous nephropathy (MN) was described in 1979 and additional evidence for a genetic component to MN was suggested in 1984 in reports of familial MN. In 2009, a pathogenic autoantibody was identified against the phospholipase A2 receptor 1 (PLA2R1). Here we discuss the genetic studies that have proven the association of human leucocyte antigen class II and PLA2R1 variants and disease in MN. The common variants in PLA2R1 form a haplotype that is associated with disease incidence. The combination of the variants in both genes significantly increases the risk of disease by 78.5-fold. There are important genetic ethnic differences in MN. Disease outcome is difficult to predict and attempts to correlate the genetic association to outcome have so far not been helpful in a reproducible manner. The role of genetic variants may not only extend beyond the risk of disease development, but can also help us understand the underlying molecular biology of the PLA2R1 and its resultant pathogenicity. The genetic variants identified thus far have an association with disease and could therefore become useful biomarkers to stratify disease risk, as well as possibly identifying novel drug targets in the near future.

Background For many patients with kidney failure, the cause and underlying defect remain unknown. Here, we describe a novel mechanism of a genetic order characterized by renal Fanconi syndrome and kidney failure. Methods We clinically and genetically characterized members of five families with autosomal dominant renal Fanconi syndrome and kidney failure. We performed genome-wide linkage analysis, sequencing, and expression studies in kidney biopsy specimens and renal cells along with knockout mouse studies and evaluations of mitochondrial morphology and function. Structural studies examined the effects of recognized mutations. Results The renal disease in these patients resulted from monoallelic mutations in the gene encoding glycine amidinotransferase (GATM), a renal proximal tubular enzyme in the creatine biosynthetic pathway that is otherwise associated with a recessive disorder of creatine deficiency. In silico analysis showed that the particular GATM mutations, identified in 28 members of the five families, create an additional interaction interface within the GATM protein and likely cause the linear aggregation of GATM observed in patient biopsy specimens and cultured proximal tubule cells. GATM aggregates-containing mitochondria were elongated and associated with increased ROS production, activation of the NLRP3 inflammasome, enhanced expression of the profibrotic cytokine IL-18, and increased cell death. Conclusions In this novel genetic disorder, fully penetrant heterozygous missense mutations in GATM trigger intramitochondrial fibrillary deposition of GATM and lead to elongated and abnormal mitochondria. We speculate that this renal proximal tubular mitochondrial pathology initiates a response from the inflammasome, with subsequent development of kidney fibrosis., (Copyright © 2018 by the American Society of Nephrology.)

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of CKD in the first three decades of life. However, for most patients with CAKUT, the causative mutation remains unknown. We identified a kindred with an autosomal dominant form of CAKUT. By whole-exome sequencing, we identified a heterozygous truncating mutation (c.279delG, p.Trp93fs*) of the nuclear receptor interacting protein 1 gene ( NRIP1 ) in all seven affected members. NRIP1 encodes a nuclear receptor transcriptional cofactor that directly interacts with the retinoic acid receptors (RARs) to modulate retinoic acid transcriptional activity. Unlike wild-type NRIP1, the altered NRIP1 protein did not translocate to the nucleus, did not interact with RAR α , and failed to inhibit retinoic acid-dependent transcriptional activity upon expression in HEK293 cells. Notably, we also showed that treatment with retinoic acid enhanced NRIP1 binding to RAR α RNA in situ hybridization confirmed Nrip1 expression in the developing urogenital system of the mouse. In explant cultures of embryonic kidney rudiments, retinoic acid stimulated Nrip1 expression, whereas a pan-RAR antagonist strongly reduced it. Furthermore, mice heterozygous for a null allele of Nrip1 showed a CAKUT-spectrum phenotype. Finally, expression and knockdown experiments in Xenopus laevis confirmed an evolutionarily conserved role for NRIP1 in renal development. These data indicate that dominant NRIP1 mutations can cause CAKUT by interference with retinoic acid transcriptional signaling, shedding light on the well documented association between abnormal vitamin A levels and renal malformations in humans, and suggest a possible gene-environment pathomechanism in this disease., (Copyright © 2017 by the American Society of Nephrology.)

Background: We describe molecular diagnosis in a complex consanguineous family: four offspring presented with combinations of three distinctive phenotypes; non-syndromic hearing loss (NSHL), an unusual skeletal phenotype comprising multiple fractures, cranial abnormalities and diaphyseal expansion, and significant developmental delay with microcephaly. We performed Chromosomal Microarray Analysis on the offspring with either the skeletal or developmental delay phenotypes, and linkage analysis and whole exome sequencing (WES) on all four children, parents and maternal aunt., Results: Chromosomal microarray and FISH analysis identified a de novo unbalanced translocation as a cause of the microcephaly and severe developmental delay. WES identified a NSHL-causing splice variant in an autosomal recessive deafness gene PDZD7 which resided in a linkage region and affected three of the children. In the two children diagnosed with an unusual skeletal phenotype, WES eventually disclosed a heterozygous COL1A1 variant which affects C-propetide cleavage site of COL1. The variant was inherited from an apparently unaffected mosaic father in an autosomal dominant fashion. After the discovery of the COL1A1 variant, the skeletal phenotype was diagnosed as a high bone mass form of osteogenesis imperfecta., Conclusions: Next generation sequencing offers an unbiased approach to molecular genetic diagnosis in highly heterogeneous and poorly characterised disorders and enables early diagnosis as well as detection of mosaicism.

Background: Membranous nephropathy (MN) is a common cause of nephrotic syndrome in adults. Previous genome-wide association studies (GWAS) of 300 000 genotyped variants identified MN-associated loci at HLA-DQA1 and PLA2R1., Methods: We used a combined approach of genotype imputation, GWAS, human leucocyte antigen (HLA) imputation and extension to other aetiologies of chronic kidney disease (CKD) to investigate genetic MN risk variants more comprehensively. GWAS using 9 million high-quality imputed genotypes and classical HLA alleles were conducted for 323 MN European-ancestry cases and 345 controls. Additionally, 4960 patients with different CKD aetiologies in the German Chronic Kidney Disease (GCKD) study were genotyped for risk variants at HLA-DQA1 and PLA2R1., Results: In GWAS, lead variants in known loci [rs9272729, HLA-DQA1, odds ratio (OR) = 7.3 per risk allele, P = 5.9 × 10 -27 and rs17830558, PLA2R1, OR = 2.2, P = 1.9 × 10 -8 ] were significantly associated with MN. No novel signals emerged in GWAS of X-chromosomal variants or in sex-specific analyses. Classical HLA alleles (DRB1*0301-DQA1*0501-DQB1*0201 haplotype) were associated with MN but provided little additional information beyond rs9272729. Associations were replicated in 137 GCKD patients with MN (HLA-DQA1: P = 6.4 × 10 -24 ; PLA2R1: P = 5.0 × 10 -4 ). MN risk increased steeply for patients with high-risk genotype combinations (OR > 79). While genetic variation in PLA2R1 exclusively associated with MN across 19 CKD aetiologies, the HLA-DQA1 risk allele was also associated with lupus nephritis (P = 2.8 × 10 -6 ), type 1 diabetic nephropathy (P = 6.9 × 10 -5 ) and focal segmental glomerulosclerosis (P = 5.1 × 10 -5 ), but not with immunoglobulin A nephropathy., Conclusions: PLA2R1 and HLA-DQA1 are the predominant risk loci for MN detected by GWAS. While HLA-DQA1 risk variants show an association with other CKD aetiologies, PLA2R1 variants are specific to MN., (© The Author 2016. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Background: Signaling through the T-cell receptor (TCR) is critical for T-cell development and function. Linker for activation of T cells (LAT) is a transmembrane adaptor signaling molecule that is part of the TCR complex and essential for T-cell development, as demonstrated by LAT-deficient mice, which show a complete lack of peripheral T cells., Objective: We describe a pedigree affected by a severe combined immunodeficiency phenotype with absent T cells and normal B-cell and natural killer cell numbers. A novel homozygous frameshift mutation in the gene encoding for LAT was identified in this kindred., Methods: Genetic, molecular, and functional analyses were used to identify and characterize the LAT defect. Clinical and immunologic analysis of patients was also performed and reported., Results: Homozygosity mapping was used to identify potential defective genes. Sanger sequencing of the LAT gene showed a mutation that resulted in a premature stop codon and protein truncation leading to complete loss of function and loss of expression of LAT in the affected family members. We also demonstrate loss of LAT expression and lack of TCR signaling restoration in LAT-deficient cell lines reconstituted with a synthetic LAT gene bearing this severe combined immunodeficiency mutation., Conclusion: For the first time, the results of this study show that inherited LAT deficiency should be considered in patients with combined immunodeficiency with T-cell abnormalities., (Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Objective: This study was undertaken to characterize the phenotype and response to treatment in patients with autosomal dominant FMF caused by MEFV p.M694del mutation and to use haplotype reconstruction to investigate the possibility of common ancestry., Methods: MEFV gene was analysed in 3500 subjects with suspected FMF referred to a single UK centre between 2002 and 2014. Patients with p.M694del underwent additional screening of the SAA1 gene as well as haplotype reconstruction of the MEFV locus., Results: The p.M694del variant was identified in 21 patients, sharing an identical disease haplotype that appears to have arisen about 550 years ago. The SAA1.1 allele was found in four patients, including two with AA amyloidosis. The clinical features comprised typical FMF symptoms with median age at onset of 18 years; three patients presented with AA amyloidosis, of whom two had had symptoms of FMF in retrospect. Fifteen patients had received colchicine treatment, all with excellent responses., Conclusion: The p.M694del variant is associated with autosomal dominantly inherited FMF in Northern European Caucasians. Symptoms may develop later in life than in classical recessive FMF but are otherwise similar, as is the response to colchicine treatment. The 14% incidence of AA amyloidosis may reflect delay in diagnosis associated with extreme rarity of FMF in this population. The common haplotype suggests a single founder living in about 1460., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The success of whole-exome sequencing to identify mutations causing single-gene disorders has been well documented. In contrast whole-exome sequencing has so far had limited success in the identification of variants causing more complex phenotypes that seem unlikely to be due to the disruption of a single gene. We describe a family where two male offspring of healthy first cousin parents present a complex phenotype consisting of peripheral neuropathy and bronchiectasis that has not been described previously in the literature. Due to the fact that both children had the same problems in the context of parental consanguinity we hypothesised illness resulted from either X-linked or autosomal recessive inheritance. Through the use of whole-exome sequencing we were able to simplify this complex phenotype and identified a causative mutation (p.R1070*) in the gene periaxin (PRX), a gene previously shown to cause peripheral neuropathy (Dejerine-Sottas syndrome) when this mutation is present. For the bronchiectasis phenotype we were unable to identify a causal single mutation or compound heterozygote, reflecting the heterogeneous nature of this phenotype. In conclusion, in this study we show that whole-exome sequencing has the power to disentangle complex phenotypes through the identification of causative genetic mutations for distinct clinical disorders that were previously masked.

Primary ovarian insufficiency (POI) is a distressing cause of infertility in young women. POI is heterogeneous with only a few causative genes having been discovered so far. Our objective was to determine the genetic cause of POI in a consanguineous Lebanese family with two affected sisters presenting with primary amenorrhoea and an absence of any pubertal development. Multipoint parametric linkage analysis was performed. Whole-exome sequencing was done on the proband. Linkage analysis identified a locus on chromosome 7 where exome sequencing successfully identified a homozygous two base pair duplication (c.1947&#95;48dupCT), leading to a truncated protein p.(Y650Sfs*22) in the STAG3 gene, confirming it as the cause of POI in this family. Exome sequencing combined with linkage analyses offers a powerful tool to efficiently find novel genetic causes of rare, heterogeneous disorders, even in small single families. This is only the second report of a STAG3 variant; the first STAG3 variant was recently described in a phenotypically similar family with extreme POI. Identification of an additional family highlights the importance of STAG3 in POI pathogenesis and suggests it should be evaluated in families affected with POI.

Myoclonus-dystonia (M-D) is a rare movement disorder characterized by a combination of non-epileptic myoclonic jerks and dystonia. SGCE mutations represent a major cause for familial M-D being responsible for 30%-50% of cases. After excluding SGCE mutations, we identified through a combination of linkage analysis and whole-exome sequencing KCTD17 c.434 G>A p.(Arg145His) as the only segregating variant in a dominant British pedigree with seven subjects affected by M-D. A subsequent screening in a cohort of M-D cases without mutations in SGCE revealed the same KCTD17 variant in a German family. The clinical presentation of the KCTD17-mutated cases was distinct from the phenotype usually observed in M-D due to SGCE mutations. All cases initially presented with mild myoclonus affecting the upper limbs. Dystonia showed a progressive course, with increasing severity of symptoms and spreading from the cranio-cervical region to other sites. KCTD17 is abundantly expressed in all brain regions with the highest expression in the putamen. Weighted gene co-expression network analysis, based on mRNA expression profile of brain samples from neuropathologically healthy individuals, showed that KCTD17 is part of a putamen gene network, which is significantly enriched for dystonia genes. Functional annotation of the network showed an over-representation of genes involved in post-synaptic dopaminergic transmission. Functional studies in mutation bearing fibroblasts demonstrated abnormalities in endoplasmic reticulum-dependent calcium signaling. In conclusion, we demonstrate that the KCTD17 c.434 G>A p.(Arg145His) mutation causes autosomal dominant M-D. Further functional studies are warranted to further characterize the nature of KCTD17 contribution to the molecular pathogenesis of M-D., (Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: In renal Fanconi's syndrome, dysfunction in proximal tubular cells leads to renal losses of water, electrolytes, and low-molecular-weight nutrients. For most types of isolated Fanconi's syndrome, the genetic cause and underlying defect remain unknown., Methods: We clinically and genetically characterized members of a five-generation black family with isolated autosomal dominant Fanconi's syndrome. We performed genomewide linkage analysis, gene sequencing, biochemical and cell-biologic investigations of renal proximal tubular cells, studies in knockout mice, and functional evaluations of mitochondria. Urine was studied with the use of proton nuclear magnetic resonance ((1)H-NMR) spectroscopy., Results: We linked the phenotype of this family's Fanconi's syndrome to a single locus on chromosome 3q27, where a heterozygous missense mutation in EHHADH segregated with the disease. The p.E3K mutation created a new mitochondrial targeting motif in the N-terminal portion of EHHADH, an enzyme that is involved in peroxisomal oxidation of fatty acids and is expressed in the proximal tubule. Immunocytofluorescence studies showed mistargeting of the mutant EHHADH to mitochondria. Studies of proximal tubular cells revealed impaired mitochondrial oxidative phosphorylation and defects in the transport of fluids and a glucose analogue across the epithelium. (1)H-NMR spectroscopy showed elevated levels of mitochondrial metabolites in urine from affected family members. Ehhadh knockout mice showed no abnormalities in renal tubular cells, a finding that indicates a dominant negative nature of the mutation rather than haploinsufficiency., Conclusions: Mistargeting of peroxisomal EHHADH disrupts mitochondrial metabolism and leads to renal Fanconi's syndrome; this indicates a central role of mitochondria in proximal tubular function. The dominant negative effect of the mistargeted protein adds to the spectrum of monogenic mechanisms of Fanconi's syndrome. (Funded by the European Commission Seventh Framework Programme and others.).

We aimed to develop and validate a reliable method for stable long-term recordings of EEG activity in zebrafish, which is less prone to artifacts than current invasive techniques. EEG activity was recorded with a blunt electrolyte-filled glass pipette placed on the zebrafish head mimicking surface EEG technology in man. In addition, paralysis of agarose-embedded fish using D-tubocurarine excluded movement artifacts associated with epileptic activity. This non-invasive recording technique allowed recordings for up to one hour and produced less artifacts than impaling the zebrafish optic tectum with a patch pipette. Paralyzed fish survived, and normal heartbeat could be monitored for over 1h. Our technique allowed the demonstration of specific epileptic activity in kcnj10a morphant fish (a model for EAST syndrome) closely resembling epileptic activity induced by pentylenetetrazol. This new method documented that seizures in the zebrafish EAST model were ameliorated by pentobarbitone, but not diazepam, validating its usefulness. In conclusion, non-invasive recordings in paralyzed EAST syndrome zebrafish proved stable, reliable and robust, showing qualitatively similar frequency spectra to those obtained from pentylenetetrazol-treated fish. This technique may prove particularly useful in zebrafish epilepsy models that show infrequent or conditional seizure activity.

Spinal muscular atrophies (SMAs) are a heterogeneous group of inherited disorders characterized by degeneration of anterior horn cells and progressive muscle weakness. In two unrelated families affected by a distinct form of autosomal-dominant distal SMA initially manifesting with calf weakness, we identified by genetic linkage analysis and exome sequencing a heterozygous missense mutation, c.616T>C (p.Cys206Arg), in F-box protein 38 (FBXO38). FBXO38 is a known coactivator of the transcription factor Krüppel-like factor 7 (KLF7), which regulates genes required for neuronal axon outgrowth and repair. The p.Cys206Arg substitution did not alter the subcellular localization of FBXO38 but did impair KLF7-mediated transactivation of a KLF7-responsive promoter construct and endogenous KLF7 target genes in both heterologously expressing human embryonic kidney 293T cells and fibroblasts derived from individuals with the FBXO38 missense mutation. This transcriptional dysregulation was associated with an impairment of neurite outgrowth in primary motor neurons. Together, these results suggest that a transcriptional regulatory pathway that has a well-established role in axonal development could also be critical for neuronal maintenance and highlight the importance of FBXO38 and KLF7 activity in motor neurons., (Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Recessive mutations in KCNJ10, which encodes an inwardly rectifying potassium channel, were recently identified as the cause of EAST syndrome, a severe and disabling multi-organ disorder consisting of epilepsy, ataxia, sensorineural deafness and tubulopathy that becomes clinically apparent with seizures in infancy. A Kcnj10 knockout mouse shows postnatal mortality and is therefore not suitable for drug discovery. Because zebrafish are ideal for in vivo screening for potential therapeutics, we tested whether kcnj10 knockdown in zebrafish would fill this need. We cloned zebrafish kcnj10 and demonstrated that its function is equivalent to that of human KCNJ10. We next injected splice- and translation-blocking kcnj10 antisense morpholino oligonucleotides and reproduced the cardinal symptoms of EAST syndrome - ataxia, epilepsy and renal tubular defects. Several of these phenotypes could be assayed in an automated manner. We could rescue the morphant phenotype with complementary RNA (cRNA) encoding human wild-type KCNJ10, but not with cRNA encoding a KCNJ10 mutation identified in individuals with EAST syndrome. Our results suggest that zebrafish will be a valuable tool to screen for compounds that are potentially therapeutic for EAST syndrome or its individual symptoms. Knockdown of kcnj10 represents the first zebrafish model of a salt-losing tubulopathy, which has relevance for blood pressure control.

Dystonia type 4 (DYT4) was first described in a large family from Heacham in Norfolk with an autosomal dominantly inherited whispering dysphonia, generalized dystonia, and a characteristic hobby horse ataxic gait. We carried out a genetic linkage analysis in the extended DYT4 family that spanned 7 generations from England and Australia, revealing a single LOD score peak of 6.33 on chromosome 19p13.12-13. Exome sequencing in 2 cousins identified a single cosegregating mutation (p.R2G) in the β-tubulin 4a (TUBB4a) gene that was absent in a large number of controls. The mutation is highly conserved in the β-tubulin autoregulatory MREI (methionine-arginine-glutamic acid-isoleucine) domain, highly expressed in the central nervous system, and extensive in vitro work has previously demonstrated that substitutions at residue 2, specifically R2G, disrupt the autoregulatory capability of the wild-type β-tubulin peptide, affirming the role of the cytoskeleton in dystonia pathogenesis., (Copyright © 2012 American Neurological Association.)

The M-type receptor for phospholipase A2 (PLA2R1) is the major target antigen in idiopathic membranous nephropathy (iMN). Our recent genome-wide association study showed that genetic variants in an HLA-DQA1 and phospholipase A2 receptor (PLA2R1) allele associate most significantly with biopsy-proven iMN, suggesting that rare genetic variants within the coding region of the PLA2R1 gene may contribute to antibody formation. Here, we sequenced PLA2R1 in a cohort of 95 white patients with biopsy-proven iMN and assessed all 30 exons of PLA2R1, including canonical (GT-AG) splice sites, by Sanger sequencing. Sixty patients had anti-PLA2R1 in serum or detectable PLA2R1 antigen in kidney tissue. We identified 18 sequence variants, comprising 2 not previously described, 7 reported as rare variants (<1%) in the Single Nucleotide Polymorphism Database or the 1000 Genomes project, and 9 known to be common polymorphisms. Although we confirmed significant associations among 6 of the identified common variants and iMN, only 9 patients had the private or rare variants, and only 4 of these patients were among the 60 who were PLA2R positive. In conclusion, rare variants in the coding sequence of PLA2R1, including splice sites, are unlikely to explain the pathogenesis of iMN.

Background/aims: Calcium homeostasis requires regulated cellular and interstitial systems interacting to modulate the activity and movement of this ion. Disruption of these systems in the kidney results in nephrocalcinosis and nephrolithiasis, important medical problems whose pathogenesis is incompletely understood., Methods: We investigated 25 patients from 16 families with unexplained nephrocalcinosis and characteristic dental defects (amelogenesis imperfecta, gingival hyperplasia, impaired tooth eruption). To identify the causative gene, we performed genome-wide linkage analysis, exome capture, next-generation sequencing, and Sanger sequencing., Results: All patients had bi-allelic FAM20A mutations segregating with the disease; 20 different mutations were identified., Conclusions: This autosomal recessive disorder, also known as enamel renal syndrome, of FAM20A causes nephrocalcinosis and amelogenesis imperfecta. We speculate that all individuals with biallelic FAM20A mutations will eventually show nephrocalcinosis., (Copyright © 2013 S. Karger AG, Basel.)

Renal fibromuscular dysplasia (FMD) can cause hypertension, and previous reports suggest that FMD is familial. We hypothesized that, in families containing an individual with proven FMD, relatives of index cases would have an increased risk of hypertension. ACTA2 mutations cause a spectrum of extra-renal arteriopathy, leading to our second hypothesis that mutations are implicated in FMD. The blood pressure of first-degree relatives was measured using standard devices and, when indicated, with 24-h ambulatory monitoring. Leucocyte DNA was obtained from FMD index cases and ACTA2 sequenced. Thirteen unrelated index cases, aged 2-32 (median 15) years, were recruited. Blood pressure was assessed in 40 first-degree relatives, comprising 22 parents aged 28-58 (median 44) years and 18 siblings aged 3-30 (median 13) years. Hypertension was evident in six (27%) parents but in none of the eight adult siblings. Of the ten screened siblings aged less than 18 years, one teenager was pre-hypertensive (90th-95th centile), the remainder being normotensive. No ACTA2 mutations were found in 13 index cases. Hypertension was evident in 20% of all assessed adult first-degree relatives and is therefore not increased relative to 25% of the adult population. Although hypertensive parents did not undergo angiography to assign FMD status, this observation, together with the lack of hypertension in 18 siblings, indicates that FMD is unlikely to confer an excess hypertension risk in first-degree relatives up to middle-age. Furthermore, in our cohort, FMD was not caused by ACTA2 mutations.

The K+ channel expressed by the KCNJ10 gene (Kir4.1) has previously demonstrated importance in retinal function in animal experiments. Recently, mutations in KCNJ10 were recognised as pathogenic in man, causing a constellation of symptoms, including epilepsy, ataxia, sensorineural deafness and a renal tubulopathy designated as EAST syndrome. We have studied the impact of KCNJ10 mutations on the human electroretinogram (ERG) in four unrelated patients with EAST syndrome. Corneal ganzfeld ERGs were elicited in response to flash stimuli of strengths of 0.001–10 phot cd s/m2 presented scotopically, and 0.3–10 phot cd s/m2 presented photopically. ERG waveforms from light-adapted retinae of all patients showed reduced amplitudes of the photopic negative response (PhNR) (P < 0.001). The photopic ERGs showed a delay in b-wave time to peak, but the photopic hill, i.e. the relative variation of time to peak and amplitude with luminance flash strength, was preserved. Scotopic ERGs to flash strengths 0.01 to 0.1 phot cd s/m2 showed a delay of up to 20 ms before the onset of the b-wave in two patients compared to controls. Stimulus–response functions were fitted by Michaelis–Menten equations and showed significantly lower retinal sensitivity in two patients than in controls (P < 0.001). Our study for the first time in the human ERG shows changes in association with KCNJ10 mutations affecting a Muller cell K+ channel. These data illustrate the role of KCNJ10 function in the physiology of proximal and possibly also the distal human retina.

Background: Arterial calcifications are associated with increased cardiovascular risk, but the genetic basis of this association is unclear., Methods: We performed clinical, radiographic, and genetic studies in three families with symptomatic arterial calcifications. Single-nucleotide-polymorphism analysis, targeted gene sequencing, quantitative polymerase-chain-reaction assays, Western blotting, enzyme measurements, transduction rescue experiments, and in vitro calcification assays were performed., Results: We identified nine persons with calcifications of the lower-extremity arteries and hand and foot joint capsules: all five siblings in one family, three siblings in another, and one patient in a third family. Serum calcium, phosphate, and vitamin D levels were normal. Affected members of Family 1 shared a single 22.4-Mb region of homozygosity on chromosome 6 and had a homozygous nonsense mutation (c.662C→A, p.S221X) in NT5E, encoding CD73, which converts AMP to adenosine. Affected members of Family 2 had a homozygous missense mutation (c.1073G→A, p.C358Y) in NT5E. The proband of Family 3 was a compound heterozygote for c.662C→A and c.1609dupA (p.V537fsX7). All mutations found in the three families result in nonfunctional CD73. Cultured fibroblasts from affected members of Family 1 showed markedly reduced expression of NT5E messenger RNA, CD73 protein, and enzyme activity, as well as increased alkaline phosphatase levels and accumulated calcium phosphate crystals. Genetic rescue experiments normalized the CD73 and alkaline phosphatase activity in patients' cells, and adenosine treatment reduced the levels of alkaline phosphatase and calcification., Conclusions: We identified mutations in NT5E in members of three families with symptomatic arterial and joint calcifications. This gene encodes CD73, which converts AMP to adenosine, supporting a role for this metabolic pathway in inhibiting ectopic tissue calcification. (Funded by the National Human Genome Research Institute and the National Heart, Lung, and Blood Institute of the National Institutes of Health.).

Charcot-Marie-Tooth disease type 2C (CMT2C) is an autosomal dominant neuropathy characterized by limb, diaphragm and laryngeal muscle weakness. Two unrelated families with CMT2C showed significant linkage to chromosome 12q24.11. We sequenced all genes in this region and identified two heterozygous missense mutations in the TRPV4 gene, C805T and G806A, resulting in the amino acid substitutions R269C and R269H. TRPV4 is a well-known member of the TRP superfamily of cation channels. In TRPV4-transfected cells, the CMT2C mutations caused marked cellular toxicity and increased constitutive and activated channel currents. Mutations in TRPV4 were previously associated with skeletal dysplasias. Our findings indicate that TRPV4 mutations can also cause a degenerative disorder of the peripheral nerves. The CMT2C-associated mutations lie in a distinct region of the TRPV4 ankyrin repeats, suggesting that this phenotypic variability may be due to differential effects on regulatory protein-protein interactions.

Background: Five children from two consanguineous families presented with epilepsy beginning in infancy and severe ataxia, moderate sensorineural deafness, and a renal salt-losing tubulopathy with normotensive hypokalemic metabolic alkalosis. We investigated the genetic basis of this autosomal recessive disease, which we call the EAST syndrome (the presence of epilepsy, ataxia, sensorineural deafness, and tubulopathy)., Methods: Whole-genome linkage analysis was performed in the four affected children in one of the families. Newly identified mutations in a potassium-channel gene were evaluated with the use of a heterologous expression system. Protein expression and function were further investigated in genetically modified mice., Results: Linkage analysis identified a single significant locus on chromosome 1q23.2 with a lod score of 4.98. This region contained the KCNJ10 gene, which encodes a potassium channel expressed in the brain, inner ear, and kidney. Sequencing of this candidate gene revealed homozygous missense mutations in affected persons in both families. These mutations, when expressed heterologously in xenopus oocytes, caused significant and specific decreases in potassium currents. Mice with Kcnj10 deletions became dehydrated, with definitive evidence of renal salt wasting., Conclusions: Mutations in KCNJ10 cause a specific disorder, consisting of epilepsy, ataxia, sensorineural deafness, and tubulopathy. Our findings indicate that KCNJ10 plays a major role in renal salt handling and, hence, possibly also in blood-pressure maintenance and its regulation., (2009 Massachusetts Medical Society)