Detection of mutations in the COL4A5 gene by analyzing cDNA of skin fibroblasts

• Published in: Kidney international Vol. 67; no. 4; pp. 1268 - 1274
• Main Authors: Wang, Fang, Wang, Yunfeng, Ding, Jie, Yang, Jiyun
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Inc 01.04.2005; Nature Publishing; Elsevier Limited
• Subjects: Biological and medical sciences; Chromosomes, Human, X; collagen type IV; Collagen Type IV - genetics; fibroblast; Fibroblasts - cytology; Fibroblasts - physiology; hereditary; Humans; Medical sciences; messenger; mutation; nephritis; Nephritis, Hereditary - genetics; Nephrology. Urinary tract diseases; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger - genetics; Skin - cytology; Skin Physiological Phenomena; transcription; nephritis; mutation; fibroblast; transcription; RNA; messenger; hereditary; collagen type IV; Kidney disease; Nephrology; Urinary system disease; Transcription; Collagen type IV; Hereditary; Urology; Genetic disease; Nephropathy; Complementary DNA; Gene; Genetics; Skin; Diagnosis; Mutation; Fibroblast
• ISSN: 0085-2538; 1523-1755
• DOI: 10.1111/j.1523-1755.2005.00204.x

Detection of mutations in the COL4A5 gene by analyzing cDNA of skin fibroblasts. Alport syndrome is a progressive hereditary glomerulonephritis that is characterized by hematuria, sensorineural deafness, ocular lesions, and progressive renal failure. The majority of cases (about 85%) are caused by mutations in the COL4A5 gene on the X chromosome which encodes the type IV collagen α5 chain (X-linked Alport syndrome). In this study we performed a systematic analysis of the entire coding region of COL4A5 mRNA in 31 unrelated Chinese X-linked Alport syndrome patients and four controls by using reverse transcription-polymerase chain reaction (RT-PCR) and direct sequencing methods. The mRNA analyzed was isolated from cultured skin fibroblasts of Alport syndrome patients. The entire sequences of mRNA of the controls corresponded exactly to the published sequence. There were 28 variants detected by analyzing mRNA of COL4A5 in 28/31 patients. Of those, a total of 25 functionally significant COL4A5 mutations was confirmed in 25/31 patients by using RT-PCR method and subsequently confirmed at genomic DNA level, which included seven different mutations described in previous reports, and 18 novel mutations. The mutation detection rate was 80.6% (25/31), which is comparable with the highest previous detection sensitivity of COL4A5 mutations in evident X-linked Alport syndrome using genomic DNA. Furthermore, three splicing mutations that occurred at the cryptic splice sites and would be overlooked or simply considered as intronic sequence variations by solely analyzing genomic DNA were identified in this study. RT-PCR and direct sequencing using cultured skin fibroblasts RNA is a practical approach with high sensitivity for genetic analysis in X-linked Alport syndrome patients.