Molecular analysis of collagen XVIII reveals novel mutations, presence of a third isoform, and possible genetic heterogeneity in Knobloch syndrome

Abstract

Knobloch syndrome (KS) is a rare disease characterized by severe ocular alterations, including vitreoretinal degeneration associated with retinal detachment and occipital scalp defect. The responsible gene, COL18A1, has been mapped to 21q22.3, and, on the basis of the analysis of one family, we have demonstrated that a mutation affecting only one of the three COL18A1 isoforms causes this phenotype. We report here the results of the screening of both the entire coding region and the exon-intron boundaries of the COL18A1 gene (which includes 43 exons), in eight unrelated patients with KS. Besides 20 polymorphic changes, we identified 6 different pathogenic changes in both alleles of five unrelated patients with KS (three compound heterozygotes and two homozygotes). All are truncating mutations leading to deficiency of one or all collagen XVIII isoforms and endostatin. We have verified that, in exon 41, the deletion c3514-3515delCT, found in three unrelated alleles, is embedded in different haplotypes, suggesting that this mutation has occurred more than once. In addition, our results provide evidence of nonallelic genetic heterogeneity in KS. We also show that the longest human isoform (NC11-728) is expressed in several tissues (including the human eye) and that lack of either the short variant or all of the collagen XVIII isoforms causes similar phenotypes but that those patients who lack all forms present more-severe ocular alterations. Despite the small sample size, we found low endostatin plasma levels in those patients with mutations leading to deficiency of all isoforms; in addition, it seems that absence of all collagen XVIII isoforms causes predisposition to epilepsy.

Figure 1

Diagram of the N-terminal region of COL18A1, showing the two distinct promoters and the alternate transcription start sites

Figure 2

Haplotype for families with KS. a, Patients from three unrelated families (KS3, KS4, and KS5), sharing the allele c3514-3515delCT. Asterisks denote the haplotypes harboring the allele c3514-3515delCT. b, Patients from two unrelated families, homozygous for IVS1-2A→T. c, Familial cases in which no pathogenic change was detected in the patients.

Figure 3

RT-PCR using primers for the human NC11-728 COL18A1 variant mRNA (208 bp) in different human tissues. Lane a, NC11-728. Lane b, β-actin (250 bp), used as positive control. Lane c, NC11-728 together with β-actin.