Abstract

Type IV collagen is only found in basement membranes, where it is the major structural component, providing a framework for the binding of other basement membrane components and a substratum for cells. The type IV collagen molecule is triple-helical and composed of three a chains which exist as six distinct forms (α1–α6). Abnormalities in this basement membrane collagen structure and function are connected to both inherited and acquired diseases.

Alport syndrome is a hereditary kidney disease associated with extrarenal complications, such as sensorineural deafness and eye abnormalities. The disease is caused by mutations in the COL4A3, COL4A4 and COL4A5 genes, coding for the type IV collagen α3, α4 and α5 chain genes, respectively. About 85% of the Alport syndrome cases are X-linked dominant, caused by mutations in the COL4A5 gene. In order to develop a basis for automated mutation analysis of the COL4A5 gene, previously unknown intron sequences flanking exons 2 and 37 were determined. Intron sequences flanking the other 49 exons were expanded from 35 to 190, and additionally, two novel 9 bp exons (exons 41A and 41B) were characterized in the large intron 41. In addition to optimization of the PCR amplification and sequencing conditions for all 51 exons and exon flanking sequences, optimization for the 820 bp promoter region and for the two novel exons was performed as well. Mutations were found in 79 unrelated patients of the 107 studied. This gives a high mutation detection rate of almost 75% in comparison with 50%, at its best, in other extensive mutation analyses of the COL4A5 gene using SSCP analysis. None of the mutations involved the promoter region or exons 41A and 41B.

Circulating antibodies against basement membrane components have been recognized in some autoimmune diseases. Goodpasture syndrome is a rare autoimmune disease characterized by progressive glomerulonephritis and pulmonary hemorrhage. The target of the antibodies in this disease has been shown to be the noncollagenous NC1 domain of type IV collagen α3 chain. For unknown reasons, a minority of Alport syndrome patients also develops antibodies against α3 and α5 chains after renal transplantation with manifestation of severe anti-GBM disease. In order to investigate the antibodies both in Goodpasture and Alport syndrome, the NC1 domains of all six type IV collagen chains were produced as recombinant proteins in bacterial and mammalian expression systems, and an ELISA method was developed for antibody detection. Antibodies were found in both syndromes, interestingly also in Alport syndrome patients without the anti-GBM disease.

The results of this work have a significant clinical value by providing for the first time complete, effective DNA-based analysis of all exon/intron and promoter regions of the COL4A5 gene in Alport syndrome.