Abstract
The nucleotide sequence of the entire COL9A3 gene, coding
for the human α3(IX) chain, was determined. The gene was
approximately 23 kb in length and consisted of 32 exons. The polymerase chain
reaction (PCR)-based procedure of conformation-sensitive gel electrophoresis
(CSGE) was used to screen the gene for sequence variations and mutations
in 83 unrelated patients with generalized primary osteoarthritis
(OA), 31 with rheumatoid arthritis (RA), 171 with intervertebral disc
disease (IDD), and 50 with various osteochondrodysplasias. The frequencies
of certain sequence variations in healthy individuals were also
determined.
The COL9A3 gene was analyzed for mutations in two unrelated
families with multiple epiphyseal dysplasia (MED). The analysis
revealed a splice site mutation leading to skipping of exon 3 and
an in-frame loss of 12 amino acid residues in the COL3 domain, the
first diseasecausing mutation to be identified in the COL9A3 gene.
Sequencing also indicated a 9 bp deletion in one allele in the second MED
family that removed a Gly-Pro-Pro triplet. Surprisingly, the deletion
did not co-segregate with the MED phenotype in the family. A similar
9 bp deletion, was also found in an unrelated family with no obvious
phenotype, suggesting that the two 9 bp deletions represent neutral
sequence variants. A construct with the deletion was then made in
order to produce a recombinant protein, and the mutant type IX collagen
was analyzed under reducing conditions by SDS-PAGE. The results
indicated that the recombinant type IX collagen proteins consisted
of three α chains, α1(IX), α2(JX), α3(IX),
in a 1:1:1 ratio. To study the triple helix stability, pepsin treatment
followed by SDS-PAGE was performed on normally folded and denatured
recombinant type IX collagen samples. The results demonstrated that
the recombinant type IX collagen containing the Gly-X-Y deletion
in the a3(IX) chain is secreted as a correctly folded triple-helical
molecule.
CSGE analysis of exon 5 of the COL9A3 gene identified two
nucleotide variations in the same codon, and thus three alleles:
CGG (Arg), CAG (Gln), and TGG (Trp). The frequency of the Trp for Arg
substitution, the Trp3 allele, was 0.244 among the probands with
the IDD, while its overall frequency in the combined group of all
non-IDD cases was 0.093. This difference was significant, with a
p-value of 0.000013. The Trp3 allele increases the relative risk
of IDD by a factor of 2.6 (95 percent confidence interval, 1.6 to
4.3).
COL9A3 mutations are shown to be associated with mild cartilage
and intervertebral disc diseases.
| Identifer | oai:union.ndltd.org:oulo.fi/oai:oulu.fi:isbn951-42-5456-2 |
| Date | 15 November 1999 |
| Creators | Paassilta, P. (Petteri) |
| 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, 1999 |
| Relation | info:eu-repo/semantics/altIdentifier/pissn/0355-3221, info:eu-repo/semantics/altIdentifier/eissn/1796-2234 |
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