Whole exome sequencing identified a novel DAG1 mutation in a patient with rare,mild and late age of onset muscular dystrophy‐dystroglycanopathy

Muscular dystrophy‐dystroglycanopathy (limb‐girdle), type C, 9 (MDDGC9) is the rarest type of autosomal recessive muscular dystrophies. MDDGC9 is manifested with an early onset in childhood. Patients with MDDGC9 usually identified with defective glycosylation of DAG1, hence it is known as “dystroglycanopathies”. Here, we report a Chinese pedigree presented with mild MDDGC9. The proband is a 64 years old Chinese man. In this family, both the proband and proband's younger brother have been suffering from mild and late onset MDDGC9. Muscle biopsy showed that the left deltoid muscle with an advanced stage of dystrophic change. Immunohistochemistry staining of dystrophin, α‐sarcoglycan, β‐sarcoglycan and dysferlin are normal. Molecular genetic analysis of the proband has been done with whole exome sequencing. A homozygous novel missense mutation (c.2326C>T; p.R776C) in the exon 3 of the DAG1 gene has been identified in the proband. Sanger sequencing revealed that this missense mutation is co‐segregated well among the affected and unaffected (carrier) family members. This mutation is not detected in 200 normal healthy control individuals. This novel homozygous missense mutation (c.2326C>T) causes substitution of arginine by cystine at the position of 776 (p.R776C) which is evolutionarily highly conserved. Immunoblotting studies revealed that a significant reduction of α‐dystroglycan expression in the muscle tissue. The novelty of our study is that it is a first report of DAG1 associated muscular dystrophy‐dystroglycanopathy (limb‐girdle), type C, 9 (MDDGC9) with mild and late age of onset. In Chinese population this is the first report of DAG1 associated MDDGC9.     

Identification of two novel mutations in POU4F3 gene associated with autosomal dominant hearing loss in Chinese families

Autosomal dominant non‐syndromic hearing loss is genetically heterogeneous with 47 genes identified to date, including POU4F3. In this study, by using a next‐generation sequencing panel targeting 127 deafness genes, we identified a pathogenic frameshift mutation c.704_705del and a missense mutation c.593G>A in two three‐generation Chinese families with late‐onset progressive ADNSHL, respectively. The novel mutations of POU4F3 co‐segregated with the deafness phenotype in these two families. c.704_705del caused a frameshift p.T235fs and c.593G>A caused an amino acid substitution of p.R198H. Both mutations led to an abnormal and incomplete protein structure. POU4F3 with either of the two mutations was transiently transfected into HEI‐OC1 and HEK 293 cell lines and immunofluorescence assay was performed to investigate the subcellular localization of mutated protein. The results indicated that both c.704_705del (p.T235fs) and c.593G>A (p.R198H) could impair the nuclear localization function of POU4F3. The p.R198H POU4F3 protein was detected as a weak band of the correct molecular weight, indicating that the stability of p.R198H POU4F3 differed from that of the wild‐type protein. While, the p.T235fs POU4F3 protein was expressed with a smaller molecular weight, implying this mutation result in a frameshift and premature termination of the POU4F3 protein. In summary, we report two novel mutations of POU4F3 associated with progressive ADNSHL and explored their effects on POU4F3 nuclear localization. These findings expanded the mutation spectrum of POU4F3 and provided new knowledge for the pathogenesis of POU4F3 in hearing loss.     

New mutation in the <Emphasis Type="Italic">MYOC</Emphasis> gene and its association with primary open-angle glaucoma in a Chinese family

Myocilin (MYOC) gene is expressed in many ocular tissues, including the trabecular meshwork, a specialized eye tissue essential in regulating intraocular pressure. Many mutations in MYOC have been detected in primary open-angle glaucoma (POAG). We investigated whether MYOC mutations contributed to the susceptibility to POAG in a Chinese family. In a four-generation family affected with POAG, ocular examinations were performed on all members of the pedigree to determine their disease status, and 200 healthy matched controls were recruited. PCR–restriction fragment length polymorphism (PCR–RFLP) analysis and DNA sequencing were used to determine the mutations in MYOC. Biological software was used to analyze the corresponding proteins for missense mutations. The c.1084G>− was found, for the first time, in four of eight affected patients and in one of two patients with suspected POAG. The c.1006C>T mutation was found in two of eight patients and in one of 19 subjects who were asymptomatic. The frequencies of c.1084G>− and c.1006C>T were 12.82 and 7.69%, respectively, in patients but not in the controls. These data provide additional clues to the pathogenesis of POAG because no other mutation was detected in either group. Our results suggest that the MYOC c.1084G>− may contribute to a genetic predisposition to POAG.     

A missense variant in the NSDHL gene in a Chihuahua with a congenital cornification disorder resembling inflammatory linear verrucous epidermal nevi

Congenital hemidysplasia with ichthyosiform nevus and limb defects syndrome in humans is a genodermatosis characterized by inflammatory linear verrucous epidermal nevi (ILVEN), often showing a striking lateralization pattern. It is caused by variants in the NSDHL gene encoding a 3β‐hydroxysteroid dehydrogenase involved in the cholesterol biosynthesis pathway. In the present study, we investigated a female Chihuahua, which showed clinical and histological signs of ILVEN. We performed a candidate gene analysis in the affected animal. This analysis revealed a single missense variant in the NSDHL gene in the affected dog (XM_014111859.2:c.700G>A). The variant is predicted to cause a non‐conservative amino acid change from glycine to arginine, XP_013967334.1:p.(Gly234Arg). The mutant allele was absent from WGS data of 594 genetically diverse dogs and eight wolves. Sanger sequencing confirmed that the variant was heterozygous in the affected dog and absent from 22 control Chihuahuas. Based on the knowledge about the functional impact of NSDHL variants in dogs and other species, c.700G>A is probably pathogenic and a convincing candidate causative variant for the observed skin lesions in the affected Chihuahua.     

The caveolin‐3 P104L mutation in LGMD‐1C patients inhibits non‐insulin‐stimulated glucose metabolism and growth but promotes myocyte proliferation

The caveolin‐3 (CAV3) protein is known to be specifically expressed in various myocytes, and skeletal muscle consumes most of the blood glucose as an energy source to maintain normal cell metabolism and function. The P104L mutation in the coding sequence of the human CAV3 gene leads to autosomal dominant disease limb‐girdle muscular dystrophy type 1C (LGMD‐1C). We previously reported that C2C12 cells transiently transfected with the P104L CAV3 mutant exhibited decreased glucose uptake and glycogen synthesis after insulin stimulation. The present study aimed to examine whether the P104L mutation affects C2C12 cell glucose metabolism, growth, and proliferation without insulin stimulation. C2C12 cells stably transfected with CAV3‐P104L were established, and biochemical assays, western blot analysis and confocal microscopy were used to observe glucose metabolism as well as cell growth and proliferation and to determine the effect of the P104L mutation on the PI3K/Akt signaling pathway. Without insulin stimulation, C2C12 cells stably transfected with the P104L CAV3 mutant exhibited decreased glucose uptake and glycogen synthesis, decreased CAV3 expression and reduced localization of CAV3 and GLUT4 on the cell membrane. The P104L mutant significantly reduced the cell diameters, but accelerated cell proliferation. Akt phosphorylation was inhibited, and protein expression of GLUT4, p‐GSK3β, and p‐p70s6K, which are molecules downstream of Akt, was significantly decreased. The CAV3‐P104L mutation inhibits glycometabolism and cell growth but accelerates C2C12 cell proliferation by reducing CAV3 protein expression and cell membrane localization, which may contribute to the pathogenesis of LGMD‐1C.     

Recessive TRAPPC11 Mutations Cause a Disease Spectrum of Limb Girdle Muscular Dystrophy and Myopathy with Movement Disorder and Intellectual Disability

Myopathies are a clinically and etiologically heterogeneous group of disorders that can range from limb girdle muscular dystrophy (LGMD) to syndromic forms with associated features including intellectual disability. Here, we report the identification of mutations in transport protein particle complex 11 (TRAPPC11) in three individuals of a consanguineous Syrian family presenting with LGMD and in five individuals of Hutterite descent presenting with myopathy, infantile hyperkinetic movements, ataxia, and intellectual disability. By using a combination of whole-exome or genome sequencing with homozygosity mapping, we identified the homozygous c.2938G>A (p.Gly980Arg) missense mutation within the gryzun domain of TRAPPC11 in the Syrian LGMD family and the homozygous c.1287+5G>A splice-site mutation resulting in a 58 amino acid in-frame deletion (p.Ala372_Ser429del) in the foie gras domain of TRAPPC11 in the Hutterite families. TRAPPC11 encodes a component of the multiprotein TRAPP complex involved in membrane trafficking. We demonstrate that both mutations impair the binding ability of TRAPPC11 to other TRAPP complex components and disrupt the Golgi apparatus architecture. Marker trafficking experiments for the p.Ala372_Ser429del deletion indicated normal ER-to-Golgi trafficking but dramatically delayed exit from the Golgi to the cell surface. Moreover, we observed alterations of the lysosomal membrane glycoproteins lysosome-associated membrane protein 1 (LAMP1) and LAMP2 as a consequence of TRAPPC11 dysfunction supporting a defect in the transport of secretory proteins as the underlying pathomechanism.     

Oculocutaneous Albinism Type 3 (OCA3): Analysis of Two Novel Mutations in <Emphasis Type="Italic">TYRP1</Emphasis> Gene in Two Chinese Patients

Oculocutaneous albinism (OCA) is a genetic disease characterized by the reduction or deficiency of melanin in eyes, skin, and hair. OCA exhibits genetic heterogeneity. Presently, there are four types of OCA named as OCA1, OCA2, OCA3, and OCA4. OCA3 is more common in African born blacks but rarely found in other ethnic populations. Our recent genotyping of patients with OCA of Chinese descent has identified two patients who were not OCA1, OCA2, or OCA4. Examination and analysis of the TYRP1 gene identified them to be having OCA3. PCR and DNA sequencing analysis found that the mutant TYPR1 alleles were present in each of the two patients, c.780-791del/c.1067G>A (p.R356Q) and c.625G>TT (p.G209LfsX1)/c.643C>T (p.H215Y). The c.780-791del and c.1067G>A mutations have been already reported. However, the c.625G>TT and c.643C>T mutations have not been previously reported and were found to be maternal and paternal mutations, respectively. Moreover, population screening and bioinformatic analysis were carried out to determine the effects of these two mutations which revealed that both the mutation were pathogenic. Based on the similar mild phenotype of these two patients, we suggest that OCA3 might be prevalent within the Chinese population.     

Novel mutation in the human HPRT1 gene and the Lesch-Nyhan disease

Lesch-Nyhan disease (LND) is a rare X-linked inherited neurogenetic disorder of purine metabolism in which the enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt) is defective. The authors report a novel point mutation that led to HGprt-related neurological dysfunction (HND) in a family in which there was a missense mutation in exon 6 of the coding region of the HPRT1 gene: g.34938G>T, c.403G>T, p.D135Y. Molecular diagnosis is consistent with the genetic heterogeneity of the HPRT1 gene responsible for HGprt deficiency. It allows fast, accurate carrier detection and genetic counseling.     

Two missense mutations in exon 9 of caprine PRLR gene were associated with litter size

Guanzhong (n = 321) and Boer (n = 191) goat breeds were used to detect single nucleotide polymorphisms (SNPs) in the coding regions of the prolactin receptor (PRLR) gene by DNA sequencing and PCR‐RFLP. Two SNPs (c.1457G>A and c.1645G>A) were identified that caused amino acid variations p.Ser485Asn and p.Val548Met respectively. Statistical results indicated that the c.1457G>A and c.1645G>A SNPs were significantly associated with litter size in Boer and Guanzhong goat breeds. Further analysis revealed that combined genotype C4 (GGGG) and haplotype G‐G were better than the others for litter size in both goat breeds. These results might contribute to goat genetic resources and breeding.     

Identification of a novel RPGRIP1 mutation in an Iranian family with leber congenital amaurosis by exome sequencing

Leber congenital amaurosis (LCA) is a heterogeneous, early‐onset inherited retinal dystrophy, which is associated with severe visual impairment. We aimed to determine the disease‐causing variants in Iranian LCA and evaluate the clinical implications. Clinically, a possible LCA disease was found through diagnostic imaging, such as fundus photography, autofluorescence and optical coherence tomography. All affected patients showed typical eye symptoms associated with LCA including narrow arterioles, blindness, pigmentary changes and nystagmus. Target exome sequencing was performed to analyse the proband DNA. A homozygous novel c. 2889delT  (p.P963 fs) mutation in the RPGRIP1 gene was identified, which was likely the deleterious and pathogenic mutation in the proband. Structurally, this mutation lost a retinitis pigmentosa GTPase regulator (RPGR)‐interacting domain at the C‐terminus which most likely impaired stability in the RPGRIP1 with the distribution of polarised proteins in the cilium connecting process. Sanger sequencing showed complete co‐segregation  in this pedigree. This study provides compelling evidence that the c. 2889delT  (p.P963 fs) mutation in the RPGRIP1 gene works as a pathogenic mutation that contributes to the progression of LCA.     

Detection of c.139G&gt;A (D47N) mutation in <Emphasis Type="BoldItalic">GJA8</Emphasis> gene in an extended family with inheritance of autosomal dominant zonular cataract without pulverulent opacities by exome sequencing

The aim of this study was to identify the gene causing bilateral autosomal dominant zonular congenital cataract (ADZCC) without pulverulent opacities in an extended Muslim family by exome sequencing and subsequent analysis. An extended family of 37 members (14 affected and 23 unaffected) who belong to different nuclear families was screened for causative gene. Proband and her unaffected son were screened for causative variant by exome sequencing followed by Sanger sequencing of the proband’s entire nuclear family. The rest of the members were further screened for variants detected, by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS PCR). Review of exome sequencing data of the proband and her unaffected son for 40 known genes causing congenital nonsyndromic cataracts revealed two variants, namely c.139G>A (p.Asp47Asn; D47N) in the GJA8 gene and c.2036C>T in the FYCO1 gene to be potentially pathogenic. Further, validation of these two variants in the entire family showed cosegregation of c.139G>A variant in GJA8 with ADZCC without pulverulent opacities. Variation of c.2036C>T in FYCO1 was not associated with disease in the family. The mutation c.139G>A in the GJA8 gene detected in the present study was also previously reported in Caucasian and Chinese families but with different phenotypes, i.e. nuclear and nuclear pulverulent cataracts. Thus, the mutation c.139G>A in GJA8 appears to exhibit marked interfamilial phenotypic variability.     

汉族马凡综合征(MFS)患者FBN1基因两种新发突变分析

为调查马凡综合征(Marfan syndrome, MFS)患者的原纤维蛋白-1(Fibrillin-1, FBN1)基因突变情况, 应用聚合酶链反应(PCR)和变性高效液相色谱法(Denaturing high-performance liquid chromatography, DHPLC)对MFS患者的FBN1基因进行突变筛查, 对DHPLC初筛异常的DNA片段进行测序分析。结果在两个MFS家系中发现FBN1基因两种新的突变: 一种为复合突变包含第55号外显子的缺失突变c.6862_6871delGGCTGTGTAG (p.Gly2288MetfsX109)、同义突变c.6861A>G和内含子的突变c.[6871+1_6871+11delGTAAGAGGATC; 6871+34dupCATCAGAAGTGACAGTGGACA]; 另一种为第20号外显子的错义突变c.2462G>A(p.Cys821Tyr)。研究表明, FBN1基因的缺失突变c.[6862_6871delGGCTGTGTAG; 6871+1_6871+11delGTAAGAGGATC] (p.Gly2288MetfsX109)和错义突变c.2462G>A(p.Cys821Tyr)可能分别是这两个家系患者的致病原因。     

A novel heterozygous COL4A4 missense mutation in a Chinese family with focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is the most common glomerular histological lesion associated with high‐grade proteinuria and end‐stage renal disease. Histologically, FSGS is characterized by focal segmental sclerosis with foot process effacement. The aim of this study was to identify the disease‐causing mutation in a four‐generation Chinese family with FSGS. A novel missense mutation, c.1856G>A (p.Gly619Asp), in the collagen type IV alpha‐4 gene (COL4A4) was identified in six patients and it co‐segregated with the disease in this family. The variant is predicted to be disease‐causing and results in collagen IV abnormalities. Our finding broadens mutation spectrum of the COL4A4 gene and extends the phenotypic spectrum of collagen IV nephropathies. Our study suggests that exome sequencing is a cost‐effective and efficient approach for identification of disease‐causing mutations in phenotypically complex or equivocal disorders. Timely screening for COL4A3/COL4A4 mutations in patients with familial FSGS may help both accurately diagnose and treat these patients.     

Reducing blood glucose level in TIDM mice by orally administering the silk glands of transgenic hIGF-I silkworms

Protein O-mannosyltransferase 1 (POMT1) and its homolog, POMT2, are responsible for the catalysis of the first step in O-mannosyl glycan synthesis. Mutations in their genes are associated with a type of congenital muscular dystrophy called Walker-Warburg syndrome. Arg⁶⁴, Glu⁷⁸ and Arg¹³⁸ in the N-terminus region of ScPmt1p, a POMT homolog in Saccharomyces cerevisiae, are important for transferase activity. Arg¹³⁸ is also essential for complex formation with ScPmt2p. Here we examined the effects of replacing the corresponding residues in human POMT1 and POMT2 with Ala on complex formation and enzymatic activity. The human POMT1 mutants lost almost all transferase activity while the POMT2 mutants retained enzymatic activity. Neither mutant lost its ability to form complexes with the native counter component. These results indicate that ScPmtps and human POMTs have different mechanisms of complex formation. They also suggest that human POMT1 and POMT2 have discrete functions since the effect of amino acid substitutions on enzymatic activity are different.     

Identification of a novel COL4A5 mutation in a Chinese family with X-linked Alport syndrome using exome sequencing

Alport syndrome (AS) is an inherited disorder and clinically characterized by glomerulonephritis and end-stage kidney disease (ESRD). The aim of this study was to identify the gene responsible for glomerulopathy in a 4-generation Chinese pedigree. Exome sequencing was conducted in four patients of the family, and then direct sequencing was performed in other members of the pedigree. A novel missense mutation c.368G>A (p.Gly123Glu) in the collagen type IV alpha-5 gene (COL4A5) was found to be the genetic cause. The p.Gly123Glu mutation occurs prior to Gly-X-Y repeats in the alpha-5 chain of type IV collagen. Neither sensorineural hearing loss nor ocular abnormalities were present in patients of this family. Other clinical features, such as age of onset, age of ESRD, disease severity and complications, varied among patients of this family. Our finding may provide new insights into the cause and diagnosis of AS, and also have implications for genetic counseling.     

Heterozygous mutations in ZP1 and ZP3 cause formation disorder of ZP and female infertility in human

The human zona pellucida (ZP) is a highly organized glycoprotein matrix that encircles oocytes and plays an essential role in successful reproduction. Previous studies have reported that mutations in human ZP1, ZP2 and ZP3 influence their functions and result in a lack of ZP or in an abnormal oocytes and empty follicle syndrome, which leads to female infertility. Here, we performed whole‐exome sequencing in two probands with primary infertility whose oocytes lacked a ZP, and we identified a heterozygous mutation in ZP1 (NM_207341:c.326G>A p.Arg109His), which is situated in the N‐terminus, and a heterozygous mutation in ZP3 (NM_001110354:c.400G>A p.Ala134Thr), which is situated in the ZP domain. The effects of the mutations were investigated through structure prediction and in vitro studies in HeLa cells. The results, which were in line with the phenotype, suggested that these mutations might impede the function of cross‐linking and secretion of ZP proteins. Our study showed that the two mutations in ZP1 and ZP3 influenced the formation of the ZP, causing female infertility. Meanwhile, these data highlight the importance of the ZP1 N‐terminus in addition to the conserved domains for ZP1 function and ZP formation. Additionally, the patient with the ZP1 mutation delivered a baby following intracytoplasmic sperm injection (ICSI); thus, we suggest the targeted genetic diagnosis of ZP genes to choose appropriate fertilization methods and improve the success rate of assisted reproductive technology (ART) treatments.     

Mutation Screening of Retinal Dystrophy Patients by Targeted Capture from Tagged Pooled DNAs and Next Generation Sequencing

Purpose

Retinal dystrophies are genetically heterogeneous, resulting from mutations in over 200 genes. Prior to the development of massively parallel sequencing, comprehensive genetic screening was unobtainable for most patients. Identifying the causative genetic mutation facilitates genetic counselling, carrier testing and prenatal/pre-implantation diagnosis, and often leads to a clearer prognosis. In addition, in a proportion of cases, when the mutation is known treatment can be optimised and patients are eligible for enrolment into clinical trials for gene-specific therapies.

Methods

Patient genomic DNA was sheared, tagged and pooled in batches of four samples, prior to targeted capture and next generation sequencing. The enrichment reagent was designed against genes listed on the RetNet database (July 2010). Sequence data were aligned to the human genome and variants were filtered to identify potential pathogenic mutations. These were confirmed by Sanger sequencing.

Results

Molecular analysis of 20 DNAs from retinal dystrophy patients identified likely pathogenic mutations in 12 cases, many of them known and/or confirmed by segregation. These included previously described mutations in ABCA4 (c.6088C>T,p.R2030*; c.5882G>A,p.G1961E), BBS2 (c.1895G>C,p.R632P), GUCY2D (c.2512C>T,p.R838C), PROM1 (c.1117C>T,p.R373C), RDH12 (c.601T>C,p.C201R; c.506G>A,p.R169Q), RPGRIP1 (c.3565C>T,p.R1189*) and SPATA7 (c.253C>T,p.R85*) and new mutations in ABCA4 (c.3328+1G>C), CRB1 (c.2832_2842+23del), RP2 (c.884-1G>T) and USH2A (c.12874A>G,p.N4292D).

Conclusions

Tagging and pooling DNA prior to targeted capture of known retinal dystrophy genes identified mutations in 60% of cases. This relatively high success rate may reflect enrichment for consanguineous cases in the local Yorkshire population, and the use of multiplex families. Nevertheless this is a promising high throughput approach to retinal dystrophy diagnostics.     

Genetic analysis of hereditary multiple exostoses in Tunisian families: a novel frame-shift mutation in the <Emphasis Type="Italic">EXT1</Emphasis> gene

Hereditary multiple exostoses (HME) is an autosomal dominant orthopaedic disorder most frequently caused by mutations in the EXT1 gene. The aim of the present study is to determine the underlying molecular defect of HME in two multigenerational Tunisian families with 21 affected members and to examine the degree of intrafamilial variability. Linkage analysis was performed using three microsatellite markers encompassing the EXT1 locus and mutation screening was carried out by direct sequencing. In family 1, evidence for linkage to EXT1 was obtained on the basis of a maximum LOD score of 4.26 at θ = 0.00 with D8S1694 marker. Sequencing of the EXT1 revealed a heterozygous G > T transversion (c.1019G>T) in exon 2, leading to a missense mutation at the codon 340 (p.Arg340Leu). In family 2 we identified a novel heterozygous 1 bp deletion in the exon 1 (c.529_531delA) leading to a premature codon stop and truncated EXT1 protein expression (p.Lys177LysfsX15). This mutation was associated with the evidence of an intrafamilial clinical variability and considered to be a novel disease-causing mutation in the EXT1 gene. These findings provide additional support for the involvement of EXT1 gene in the HME disease.