Abstract
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.
Highlights
Alport Syndrome (AS) is a type of inherited nephropathy characterized by hematuria, proteinuria, and progressive renal failure, often associated with extrarenal manifestations such as sensorineural hypoacusis and ocular abnormalities [1]
A total of 272 variants and 2 fragment deletions were identified in COL4A3, COL4A4, and COL4A5 from the 20 patients diagnosed with AS using targeted capture and next-generation sequencing (NGS)
We identified 16 missense mutations, one nonsense mutation, three frameshift mutations, and one 50-untranslated region (UTR) mutation
Summary
Alport Syndrome (AS) is a type of inherited nephropathy characterized by hematuria, proteinuria, and progressive renal failure, often associated with extrarenal manifestations such as sensorineural hypoacusis and ocular abnormalities [1]. The pathogenesis of AS is genetically heterogeneous and is caused by mutations in the genes encoding type IV collagen [2, 3]. 104200) and autosomal dominant inheritance (ADAS OMIM no.203780) caused by mutations in COL4A3 or COL4A4 located in 2q36.3 [7,8,9,10,11]. Individuals homozygous for COL4A3 and COL4A4 genes resulting in ARAS have similar clinical symptoms and prognosis as males showing XLAS, reaching ESRD in the first or second decade of life [17]. The clinical phenotypes of heterozygous mutations in COL4A3 and COL4A4 are heterogeneous, and can cause autosomal dominant AS (ADAS), thin basement membrane nephropathy (TBMN), focal segmental glomerulosclerosis (FSGS) and benign familial hematuria (BFH) [18, 19]. In this study, we developed a strategy to analyze COL4A3, COL4A4, and COL4A5 simultaneously using targeted capture and NGS in 20 patients with AS
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