De novo mutations in COL4A5 identified by whole exome sequencing in 2 girls with Alport syndrome in Korea.
ABSTRACT: Alport syndrome (ATS) is an inherited glomerular disease caused by mutations in one of the type IV collagen novel chains (?3, ?4, and ?5). ATS is characterized by persistent microscopic hematuria that starts during infancy, eventually leading to either progressive nephritis or end-stage renal disease. There are 3 known genetic forms of ATS, namely X-linked ATS, autosomal recessive ATS, and autosomal dominant ATS. About 80% of patients with ATS have X-linked ATS, which is caused by mutations in the type IV collagen ?5 chain gene, COL4A5. Although an 80% mutation detection rate is observed in men with X-linked ATS, some difficulties do exist in the genetic diagnosis of ATS. Most mutations are point mutations without hotspots in the COL4A3, COL4A4, and COL4A5 genes. Further, there are insufficient data on the detection of COL4A3 and COL4A4 mutations for their comparison between patients with autosomal recessive or dominant ATS. Therefore, diagnosis of ATS in female patients with no apparent family history can be challenging. Therefore, in this study, we used whole-exome sequencing (WES) to identify mutations in type IV collagen in 2 girls with glomerular basement membrane structural changes suspected to be associated with ATS; these patients had no relevant family history. Our results revealed de novo c.4688G>A (p.Arg1563Gln) and c.2714G>A (p.Gly905Asp) mutations in COL4A5. Therefore, we suggest that WES is an effective approach to obtain genetic information in ATS, particularly in female patients without a relevant family history, to detect unexpected DNA variations.
Project description:<h4>Background</h4>Collagen IV-related nephropathies, including thin basement membrane nephropathy and Alport Syndrome (AS), are caused by defects in the genes COL4A3, COL4A4 and COL4A5. Diagnosis of these conditions can be hindered by variable penetrance and the presence of non-specific clinical or pathological features.<h4>Methods</h4>Three families with unexplained inherited kidney disease were recruited from Shanghai, China. Whole exome sequencing (WES) was performed in the index case from each family and co-segregation of candidate pathogenic mutations was tested by Sanger sequencing.<h4>Results</h4>We identified COL4A4 missense variants [c.G2636A (p.Gly879Glu) and c.C4715T (p.Pro1572Leu)] in the 21-year-old male proband from family 1, who had been diagnosed with mesangial proliferative nephropathy at age 14. COL4A4 c.G2636A, a novel variant, co-segregated with renal disease among maternal relatives. COL4A4 c.C4715T has previously been associated with autosomal recessive AS and was inherited from his clinically unaffected father. In family 2, a novel COL4A3 missense mutation c.G2290A (p.Gly997Glu) was identified in a 45-year-old male diagnosed with focal segmental glomerulosclerosis and was present in all his affected family members, who exhibited disease ranging from isolated microscopic hematuria to end stage renal disease (ESRD). In family 3, ESRD occurred in both male and females who were found to harbor a known AS-causing COL4A5 donor splice site mutation (c.687+1G>A). None of these variants were detected among 100 healthy Chinese individuals.<h4>Conclusion</h4>WES identified 2 novel and 2 known pathogenic COL4A3/COL4A4/COL4A5 mutations in 3 families with previously unexplained inherited kidney disease. These findings highlight the clinical range of collagen IV-related nephropathies and resolved diagnostic confusion arising from atypical or incomplete clinical/histological findings, allowing appropriate counselling and treatment advice to be given.
Project description:Alport syndrome (ATS) is a hereditary nephropathy often associated with sensorineural hypoacusis and ocular abnormalities. Mutations in the COL4A5 gene cause X-linked ATS. Mutations in COL4A4 and COL4A3 genes have been reported in both autosomal recessive and autosomal dominant ATS. The conventional mutation screening, performed by DHPLC and/or Sanger sequencing, is time-consuming and has relatively high costs because of the absence of hot spots and to the high number of exons per gene: 51 (COL4A5), 48 (COL4A4) and 52 (COL4A3). Several months are usually necessary to complete the diagnosis, especially in cases with less informative pedigrees. To overcome these limitations, we designed a next-generation sequencing (NGS) protocol enabling simultaneous detection of all possible variants in the three genes. We used a method coupling selective amplification to the 454 Roche DNA sequencing platform (Genome Sequencer junior). The application of this technology allowed us to identify the second mutation in two ATS patients (p.Ser1147Phe in COL4A3 and p.Arg1682Trp in COL4A4) and to reconsider the diagnosis of ATS in a third patient. This study, therefore, illustrates the successful application of NGS to mutation screening of Mendelian disorders with locus heterogeneity.
Project description:Alport syndrome (AS) is a clinically and genetically heterogeneous, progressive nephropathy caused by mutations in COL4A3, COL4A4, and COL4A5, which encode type IV collagen. The large sizes of these genes and the absence of mutation hot spots have complicated mutational analysis by routine polymerase chain reaction (PCR)-based approaches. Here, in order to design a rapid and effective method for the genetic diagnosis of AS, we developed a strategy by utilizing targeted capture associated with next-generation sequencing (NGS) to analyze COL4A3, COL4A4, and COL4A5 simultaneously in 20 AS patients. All the coding exons and flanking sequences of COL4A3, COL4A4, and COL4A5 from the probands were captured followed by HiSeq 2500 sequencing. Candidate mutations were validated by classic Sanger sequencing and quantitative (q)PCR. Sixteen patients (16/20, 75%) showed X-linked inheritance, and four patients (4/20, 20%) showed autosomal recessive inheritance. None of the individuals had autosomal-dominant AS. Fifteen novel mutations, 6 known mutations, and 2 novel fragment deletions were detected by targeted capture and NGS. Of these novel mutations, 12, 3, and 2 mutations were detected in COL4A5, COL4A4, and COL4A3, respectively. A comparison of the clinical manifestations caused by different types of mutations in COL4A5 suggested that nonsense mutations and glycine substitution by an acidic amino acid are more severe than the other missense mutations. Pathogenic mutations were detected in 20 patients. These novel mutations can expand the genotypic spectrum of AS. Our results demonstrated that targeted capture and NGS technology are effective in the genetic diagnosis of AS.
Project description:Alport syndrome is an inherited renal disorder characterized by glomerular basement membrane lesions with hematuria, proteinuria and frequent hearing defects and ocular abnormalities. The disease is associated with mutations in genes encoding ?3, ?4, or ?5 chains of type IV collagen, namely COL4A3 and COL4A4 in chromosome 2 and COL4A5 in chromosome X. In contrast to the well-known X-linked and autosomal recessive phenotypes, there is very little information about the autosomal dominant. In view of the wide spectrum of phenotypes, an exact diagnosis is sometimes difficult to achieve.We investigated a Spanish family with variable phenotype of autosomal dominant Alport syndrome using clinical, histological, and genetic analysis.Mutational analysis of COL4A3 and COL4A4 genes showed a novel heterozygous mutation (c. 998G > A; p.G333E) in exon 18 of the COL4A3 gene. Among relatives carrying the novel mutation, the clinical phenotype was variable. Two additional COL4A3 mutations were found, a Pro-Leu substitution in exon 48 (p.P1461L) and a Ser-Cys substitution in exon 49 (p.S1492C), non-pathogenics alone.Carriers of p.G333E and p.P1461L or p.S1492C mutations in COL4A3 gene appear to be more severely affected than carriers of only p.G333E mutation, and the clinical findings has an earlier onset. In this way, we could speculate on a synergistic effect of compound heterozygosity that could explain the different phenotype observed in this family.
Project description:BACKGROUND:Alport syndrome is an inherited renal disease caused by mutations in COL4A3, COL4A4, or COL4A5 genes. Coexisting mutations in either two of the three genes in Alport patients have been reported recently. However, the effect of heterozygous mutations in COL4A3 or COL4A4 genes in X-linked Alport syndrome (XLAS) patients is unclear. METHODS:Using targeted next-generation sequencing, six unrelated Chinese children were identified to have a combination of a pathogenic variant in COL4A5 and a heterozygous mutation in COL4A3 or COL4A4. They were three males and three females. Another three XLAS males each with only one pathogenic variant in COL4A5 were included. The clinical data were analyzed and compared between the males in two groups (group 1, males with a pathogenic variant in COL4A5 and a heterozygous pathogenic variant in COL4A3 or COL4A4; group 2, males with only one pathogenic variant in COL4A5). RESULTS:Patients with XLAS who also had heterozygous pathogenic COL4A3 or COL4A4 variants accounted for 1% of Alport syndrome. In this study, three children showed coexisting pathogenic variants in COL4A5 and COL4A3. Two children showed pathogenic variants in COL4A5 and COL4A4. One child had pathogenic variants in the three COL4A3-5 genes, in which the pathogenic variant in COL4A5 was de novo and the pathogenic variants in COL4A4 and COL4A3 were inherited independently (in trans). The site and type of mutations in COL4A5 were similar between the two groups. It was revealed that males in group 1 presented more severe proteinuria than males in group 2 (p < 0.05). CONCLUSION:The present study provides further evidence for complicated genotype in Alport syndrome. For the first time, we reported a case with three pathogenic variants in COL4A5, COL4A3, and COL4A4 genes. Moreover, we found that heterozygous pathogenic COL4A3 or COL4A4 variants are likely to make XLAS disease more serious.
Project description:Alport syndrome is an inherited nephropathy associated with mutations in genes encoding type IV collagen chains present in the glomerular basement membrane. COL4A5 mutations are associated with the major X-linked form of the disease, and COL4A3 and COL4A4 mutations are associated with autosomal recessive and dominant forms (thought to be involved in 15% and 1%-5% of the families, respectively) and benign familial hematuria. Mutation screening of these three large genes is time-consuming and expensive. Here, we carried out a combination of multiplex PCR, amplicon quantification, and next generation sequencing (NGS) analysis of three genes in 101 unrelated patients. We identified 88 mutations and 6 variations of unknown significance on 116 alleles in 83 patients. Two additional indel mutations were found only by secondary Sanger sequencing, but they were easily identified retrospectively with the web-based sequence visualization tool Integrative Genomics Viewer. Altogether, 75 mutations were novel. Sequencing the three genes simultaneously was particularly advantageous as the mode of inheritance could not be determined with certainty in many instances. The proportion of mutations in COL4A3 and COL4A4 was notably high, and the autosomal dominant forms of Alport syndrome appear more frequently than reported previously. Finally, this approach allowed the identification of large COL4A3 and COL4A4 rearrangements not described previously. We conclude that NGS is efficient, reduces screening time and cost, and facilitates the provision of appropriate genetic counseling in Alport syndrome.
Project description:BACKGROUND:About 40-50% of patients with familial microscopic hematuria (FMH) caused by thin basement membrane nephropathy (TBMN) inherit heterozygous mutations in collagen IV genes (COL4A3, COL4A4). On long follow-up, the full phenotypic spectrum of these patients varies a lot, ranging from isolated MH or MH plus low-grade proteinuria to chronic renal failure of variable degree, including end-stage renal disease (ESRD). METHODS:Here, we performed Whole Exome Sequencing (WES) in patients of six families, presenting with autosomal dominant FMH, with or without progression to proteinuria and loss of renal function, all previously found negative for severe collagen IV mutations. Hierarchical filtering of the WES data was performed, followed by mutation prediction analysis, Sanger sequencing and genetic segregation analysis. RESULTS:In one family with four patients, we found evidence for the contribution of two co-inherited variants in two crucial genes expressed in the glomerular basement membrane (GBM); LAMA5-p.Pro1243Leu and COL4A5-p.Asp654Tyr. Mutations in COL4A5 cause classical X-linked Alport Syndrome, while rare mutations in the LAMA5 have been reported in patients with focal segmental glomerulosclerosis. The phenotypic spectrum of the patients includes hematuria, proteinuria, focal segmental glomerulosclerosis, loss of kidney function and renal cortical cysts. CONCLUSIONS:A modifier role of LAMA5 on the background of a hypomorphic Alport syndrome causing mutation is a possible explanation of our findings. Digenic inheritance is another scenario, following the concept that mutations at both loci more accurately explain the spectrum of symptoms, but further investigation is needed under this concept. This is the third report linking a LAMA5 variant with human renal disease and expanding the spectrum of genes involved in glomerular pathologies accompanied by familial hematurias. The cystic phenotype overlaps with that of a mouse model, which carried a Lama5 hypomorphic mutation that caused severely reduced Lama5 protein levels and produced kidney cysts.
Project description:Introduction:Alport syndrome (AS) is caused by mutations in ?3/?4/?5 (IV) collagen genes, the severity of which determine the progression of AS. Posttransplantation outcome is good, although anti-glomerular basement membrane (anti-GBM) glomerulonephritis occurs in 3% to 5% of recipients, clustering in patients with a severe mutation. We assessed whether the severity of the underlying AS mutation affects graft and patients outcome after transplantation, including the occurrence of anti-GBM nephritis. Methods:We included 73 AS patients with an identified mutation (COL4A5, 57 patients; COL4A3, 9 patients; COL4A4, 6 patients; heterozygous composite COL4A3 and A4, 1 patient) who underwent transplantation between 1971 and 2014 and who had received a total of 93 kidney grafts. Results:In all, 41 patients had a severe mutation (COL4A5, 30 patients; COL4A3, 6 patients; COL4A4, 5 patients), and 32 had a nonsevere mutation (COL4A5, 27 patients; COL4A3, 4 patients; COL4A4, 1 patient). Patient survival was similar in patients with severe and nonsevere mutations (89% vs. 84% at 5 years, 83% vs. 75% at 10, 15, and 20 years; P = 0.46). Graft survival was not affected by the severity of mutation (77% vs. 63% at 5 years, 60% vs. 55% at 10 years, 55% vs. 55% at 15 years, and 55% vs. 50% at 20 years; P = 0.65). Clinically significant anti-GBM glomerulonephritis occurred in 1 male patient with severe COL4A5 mutation 6 years after transplantation recurred in a subsequent graft, leading twice to graft loss. Conclusion:Although severe mutations affect the severity of AS, they do not have an impact on patient and graft survival after transplantation. De novo anti-GBM nephritis after transplantation was less frequent than previously reported, occurring in only 1.4% of AS patients, and in 2% of males with COL4A5 mutation.
Project description:BACKGROUND:Alport syndrome (AS) is an inherited progressive renal disease caused by mutations in COL4A3, COL4A4, and COL4A5 genes. The large sizes of these genes and the absence of mutation hot spots have complicated mutational analysis by routine PCR-based approaches. In recent years, the development of next-generation sequencing (NGS) has made possible the time- and cost-effective and accurate analysis of the three genes in a single step. METHODS:Here, we analyze COL4A3, COL4A4, and COL4A5 simultaneously in 29 AS patients using NGS. Candidate mutations were validated by classic Sanger sequencing and Real-time PCR. RESULTS:Twenty two new mutations and 10 known mutations were detected. Of those novel mutations, 18, 3, and 1 mutations were detected in COL4A5, COL4A4, and COL4A3, respectively. Twenty six patients showed X-linked inheritance, one showed autosomal recessive inheritance and two showed digenic inheritance (DI). CONCLUSION:A comparison of the clinical manifestations caused by different types of mutations in COL4A5 suggested that large fragment mutations are relatively more severe than the other missense mutations and AS by some mutations may show inter- and intra-familial phenotypic variability. It is important to consider these transmission patterns in the clinical evaluation according to the results of genetic testing, especially for DI. Twenty two new mutations can expand the genotypic spectrum of AS.
Project description:BACKGROUND:Alport Syndrome (AS) is a progressive hereditary glomerular disease. It is often accompanied by sensorineural hearing loss and ocular abnormalities and can sometimes develop into end stage renal disease (ESRD), which is caused by mutations in the genes encoding the collagen type IV family of proteins. METHODS:This study analyzed the association between the clinical data of seven AS families and genes and the disease progression of different mutation types, including COL4A3 (OMIM 120070)?COL4A4 (OMIM 120131), and COL4A5 (OMIM303630). RESULTS:A total of six new pathogenic mutation sites, one complex heterozygous mutation at COL4A3, and a combined mutation of COL4A5 and INF2 (OMIM 610982) were identified in this study. It was revealed that the clinical manifestations of X-linked AS caused by mutations in the COL4A5 gene were more severe in males than in females. In addition, the difference in patient phenotype can be attributed to the location of gene mutations affecting the protein domain or functional domain. Our data suggested that the gene deletion and nonsense mutations had a high risk for progression to ESRD. CONCLUSION:Our results revealed the spectrum of type IV collagen genes, which contribute to the enrichment of database resources and has important implications in the diagnosis, prognosis, and guiding treatment of AS.