Detection of mutations in COL4A5 gene by PCR SSCP analysis in Chinese Alport syndrome(AS) patients

Objective To detect mutations in COL4A5 gene encoding type Ⅳ collagen α 5 chain in Chinese AS patients Methods Genomic DNA from 18 unrelated patients (12 males and 6 females) with X linked AS were analyzed So far, 32 of 51 exons at the 3′ end portion of COL4A5 gene were amplified with the primers synthesized according to the published flanking intronic sequences The PCR products were further analyzed by agarose gel electrophoresis and single strand conformation polymorphism (SSCP) analysis The PCR products showing polymorphism by SSCP analysis were directly sequenced Results By PCR SSCP analysis, 4 PCR products were identified with different mobility of single strand DNA, exon 20, 39 and 46 in patient No 8; exon 26 in patient No 90 The PCR product of No 8 patient′s mother was revealed with a special pattern containing single strand DNA bands identical to those of the No 8 patient and the normal control DNA sequencing analyses revealed: (1) silent mutations in exon 46 and 39; (2) exon 20 was deleted with CC nucleotides and exon 26 was deleted with one C nucleotide , both of which induced nonsense mutations resulting in remarkably truncated α 5 chains of type Ⅳ collagen Conclusion For the first time, mutations in COL4A5 gene were identified in Chinese AS patients in the mainland of China Two small deletions were detected, one of which was accompanied by two silent mutations No major gene deletion or rearrangement was detected The heterozygote status of one patient′s mother was also demonstrated by PCR SSCP analysis