Detection of mutations in the COL4A5 gene in over 90% of male patients with X-linked Alport's syndrome by RT-PCR and direct sequencing.

X-linked Alport's syndrome is caused by mutations in the COL4A5 gene encoding the type IV collagen alpha5 chain (alpha5[IV]). Polymerase chain reaction-single-str and conformation polymorphism (PCR-SSCP) on genomic DNA has previously been used to screen for mutations in the COL4A5 gene, but this method was relatively insensitive, with mutations detected in less than 50% of patients. Here, we report a systematic analysis of the entire coding region of the COL4A5 gene, using nested reverse-transcription-polymerase chain reaction (RT-PCR) and the direct sequence method using leukocytes. This study examines twenty-two unrelated Japanese patients with X-linked Alport's syndrome showing abnormal expression of alpha5(IV) in the glomerular or epidermal basement membranes. Mutations that were predicted to be pathogenic were identified in 12 of the 13 male patients (92%) and five of the nine female patients (56%). Six patients had missense mutations, four had out-of-frame deletion mutations, three had nonsense mutations, and three had mutations causing exon loss of the transcript. The current study shows that nested RT-PCR and the direct sequence method using leukocytes are highly sensitive and offer a useful approach for systematic gene analysis in patients with X-linked Alport's syndrome.