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DETECTION OF COL4A5 AND COL4A5/COL4A6 MUTATIONS IN CHINESE ALPORT SYNDROME (AS) PATIENTS

Wang F, Ding J, Guo S-H, Yang J-Y

Department of Pediatrics, First Hospital, Peking University, Beijing, China

 

Objective: To detect mutations in either the COL4A5 or the COL4A5/COL4A6 genes encoding the α5 and the α6 chains of type collagen in Chinese AS patients.

Methods: Genomic DNA from 36 unrelated patients with x-linked AS were analyzed. So far, all 51 exons of the COL4A5 gene and exon 1-4of the COL4A6 gene were amplified with the primers synthesized according to the published flanking intronic sequences. The PCR products were further analyzed by single strand conformation polymorphism (SSCP) analysis or directly sequenced. The genomic DNA with the absence of exons in the COL4A5/COL4A6 genes was further analyzed by Southern hybridization to identify the breakpoint.

Results: PCR-SSCR analysis of the COL4A5 gene revealed:(1) exon 20, 39 and 46 in patient No.3, exon 17 in patient No.5, exon 4in patient No.8, exon 9 in patient No.12, and exon 26 in patient No.14 have a different mobility of the single strand DNA; (2) No.3 patient’s mother and sister, No.8 patient’s grandmother and No.12 patient’s mother have a special pattern containing two types of the single strand DNA band, in which one band was identical to that of the corresponding male proband, and the other band to the normal control. DNA sequencing analyses revealed: (1) silent mutations in exon 39 and 46; (2) exon 17 and exon 26 were deleted with one C nucleotide and exon 20 was deleted with CC nucleotides all of which induced nonsense mutations resulting in remarkably truncated α5 chains of type collagen; (3) a CA substitution in one strand of exon 4 and a normal sequence in another strand. PCR amplification of exons showed that in patient No.22 there were no exon 1 in COL4A5 and exons 1-3 in COL4A6.

Conclusion: Three small deletions were detected, one of which was companied by two silent mutations. For the first time, a large deletion of the paired COL4A5 and COL4A6 genes was detected in Chinese AS patients in the mainland of China. The heterozygous status of one patient and four x-linked AS carrier women was also demonstrated by analyzing the COL4A5 gene.