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.
| Identifer | oai:union.ndltd.org:oulo.fi/oai:oulu.fi:isbn951-42-5686-7 |
| Date | 07 June 2000 |
| Creators | Martin, P. (Paula) |
| Publisher | University of Oulu |
| Source Sets | University of Oulu |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis, info:eu-repo/semantics/publishedVersion |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess, © University of Oulu, 2000 |
| Relation | info:eu-repo/semantics/altIdentifier/pissn/0355-3221, info:eu-repo/semantics/altIdentifier/eissn/1796-2234 |
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