The X-linked form of Alport syndrome (AS) is caused by mutations in the COL4A5 gene encoding the alpha5 chain of type IV collagen. Most COL4A5 mutations are individual, and mutation analysis is complicated by the size of the gene and the number of exons. Larger structural rearrangements account for 10-15% of mutations. We have established a method for mutation analysis of COL4A5 based on reverse transcriptase-polymerase chain reaction analysis of mRNA from cultured skin fibroblasts and multiplex ligation-dependent probe amplification (MLPA) on genomic DNA. One advantage of using skin biopsies for the mRNA analysis is the possibility of immunohistochemical staining for the alpha5(IV) chain on skin sections to support a diagnosis of X-linked AS. A mutation was detected in all five cases included. One patient presenting with AS and diffuse leiomyomatosis was found to have a COL4A5 deletion extending into and comprising COL4A6 exons 1, 1', and 2. We have evaluated the MLPA assay on samples from 67 previously tested AS patients (45 males and 22 females) and 20 controls. We found that the combination of cDNA and MLPA analysis improves the mutation detection rate in COL4A5 and that MLPA should be the first step in genetic testing for X-linked AS.