Improving mutation screening in familial hematuric nephropathies through next generation sequencing.
ABSTRACT: 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: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: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 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:BACKGROUND: Autosomal forms of Alport syndrome represent 20% of all patients (15% recessive and 5% dominant). They are caused by mutations in the COL4A3 and COL4A4 genes, which encode a-3 and a-4 collagen IV chains of the glomerular basement membrane, cochlea and eye. Thin basement membrane nephropathy may affect up to 1% of the population. The pattern of inheritance in the 40% of cases is the same as autosomal dominant Alport syndrome: heterozygous mutations in these genes. The aim of this study is to detect new pathogenic mutations in the COL4A4 gene in the patients previously diagnosed with autosomal Alport syndrome and thin basement membrane nephropathy in our hospital. METHODS: We conducted a clinical and genetic study in eleven patients belonging to six unrelated families with aforementioned clinical symptoms and a negative study of COL4A3 gene. The molecular study was made by conformation of sensitive gel electrophoresis (CSGE) and direct sequencing of the fragments that show an altered electrophoretic migration pattern. RESULTS: We found two pathogenic mutations, not yet described: IVS3 + 1G > C is a replacement of Guanine to Cytosine in position +1 of intron 3, in the splicing region, which leads to a pathogenic mutation. c.4267C > T; p.P1423S is a missense mutation, also considered pathogenic. We also found seven new polymorphisms. CONCLUSIONS: We describe two new pathogenic mutations, responsible for autosomal dominant Alport syndrome. The other families of the study were undiagnosed owing to problems in the method employed and the possibility of mutations in other genes, giving rise to other diseases with similar symptoms.
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 (AS) is a hereditary disease caused by mutations in COL4A3-5 genes. Recently, comprehensive genetic analysis has become the first-line diagnostic tool for AS. However, no reports comparing mutation identification rates between conventional sequencing and comprehensive screening have been published. METHODS:In this study, 441 patients clinically suspected of having AS were divided into two groups and compared. The initial mutational analysis method involved targeted exome sequencing using next-generation sequencing (NGS) (n = 147, NGS group) or Sanger sequencing for COL4A3/COL4A4/COL4A5 (n = 294, Sanger group). RESULTS:In the NGS group, 126 patients (86%) were diagnosed with AS by NGS, while two had pathogenic mutations in other genes, NPHS1 and EYA1. Further, 239 patients (81%) were diagnosed with AS by initial analysis in the Sanger group. Thirteen patients who were negative for mutation detection in the Sanger group were analyzed by NGS; three were diagnosed with AS. Two had mutations in CLCN5 or LAMB2. The final variant detection rate was 90%. DISCUSSION:Our results reveal that Sanger sequencing and targeted exome sequencing have high diagnostic ability. NGS also has the advantage of detecting other inherited kidney diseases and pathogenic mutations missed by Sanger sequencing.
Project description:Alport syndrome is an inherited disease characterized by hematuria, progressive renal failure, hearing loss, and ocular abnormalities. Autosomal recessive Alport syndrome is suspected in consanguineous families and when female patients develop renal failure. Fifteen percent of patients with Alport syndrome have autosomal recessive inheritance caused by two pathogenic mutations in either COL4A3 or COL4A4. Here, we describe the mutations and clinical features in 40 individuals including 9 children and 21 female individuals (53%) with autosomal recessive inheritance indicated by the detection of two mutations. The median age was 31 years (range, 6-54 years). The median age at end stage renal failure was 22.5 years (range, 10-38 years), but renal function was normal in nine adults (29%). Hearing loss and ocular abnormalities were common (23 of 35 patients [66%] and 10 of 18 patients [56%], respectively). Twenty mutation pairs (50%) affected COL4A3 and 20 pairs affected COL4A4. Of the 68 variants identified, 39 were novel, 12 were homozygous changes, and 9 were present in multiple individuals, including c.2906C>G (p.(Ser969*)) in COL4A4, which was found in 23% of the patients. Thirty-six variants (53%) resulted directly or indirectly in a stop codon, and all 17 individuals with early onset renal failure had at least one such mutation, whereas these mutations were less common in patients with normal renal function or late-onset renal failure. In conclusion, patient phenotypes may vary depending on the underlying mutations, and genetic testing should be considered for the routine diagnosis of autosomal recessive Alport syndrome.
Project description:Autosomal recessive Alport syndrome is a progressive hematuric glomerulonephritis characterized by glomerular basement membrane abnormalities and associated with mutations in either the COL4A3 or the COL4A4 gene, which encode the alpha3 and alpha4 type IV collagen chains, respectively. To date, mutation screening in the two genes has been hampered by the lack of genomic structure information. We report here the complete characterization of the 48 exons of the COL4A4 gene, a comprehensive gene screen, and the subsequent detection of 10 novel mutations in eight patients diagnosed with autosomal recessive Alport syndrome. Furthermore, we identified a glycine to alanine substitution in the collagenous domain that is apparently silent in the heterozygous carriers, in 11.5% of all control individuals, and in one control individual homozygous for this glycine substitution. There has been no previous finding of a glycine substitution that is not associated with any obvious phenotype in homozygous individuals.
Project description:Alport syndrome (AS) is a genetically heterogeneous disorder that is characterized by hematuria, progressive renal failure typically resulting in end-stage renal disease, sensorineural hearing loss, and variable ocular abnormalities. Only 15% of cases with AS are autosomal recessive and are caused by mutations in the COL4A3 or COL4A4 genes, encoding type IV collagen.Clinical data in a large consanguineous family with four affected members were reviewed, and genomic DNA was extracted. For mapping, 15 microsatellite markers flanking COL4A3, COL4A4, and COL4A5 in 16 family members were typed. For mutation screening, all coding exons of COL4A3 were polymerase chain reaction- amplified and Sanger-sequenced from genomic DNA.The disease locus was mapped to chromosome 2q36.3, where COL4A3 and COL4A4 reside. Sanger sequencing revealed a novel mis-sense mutation (c.2T>C; p.M1T) in exon 1 of COL4A3. The identified nucleotide change was not found in 100 healthy ethnicity-matched controls via Sanger sequencing.We present a large consanguineous Turkish family with AS that was found to have a COL4A3 mutation as the cause of the disease. Although the relationship between the various genotypes and phenotypes in AS has not been fully elucidated, detailed clinical and molecular analyses are helpful for providing data to be used in genetic counseling. It is important to identify new mutations to clarify their clinical importance, to assess the prognosis of the disease, and to avoid renal biopsy for final diagnosis.