Mutation in collagen II alpha 1 isoforms delineates Stickler and Wagner syndrome phenotypes

Abstract

Purpose: Stickler syndrome is an arthro-ophthalmopathy with phenotypic overlap with Wagner syndrome. The common Stickler syndrome type I is inherited as an autosomal dominant trait, with causal mutations in collagen type II alpha 1 (COL2A1). Wagner syndrome is associated with mutations in versican (VCAN), which encodes for a chondroitin sulfate proteoglycan. A three-generation Caucasian family variably diagnosed with either syndrome was screened for sequence variants in the COL2A1 and VCAN genes.

Methods: Genomic DNA samples derived from saliva were collected from all family members (six affected and four unaffected individuals). Complete sequencing of COL2A1 and VCAN was performed on two affected individuals. Direct sequencing of remaining family members was conducted if the discovered variants followed segregation.

Results: A base-pair substitution (c.258C>A) in exon 2 of COL2A1 cosegregated with familial disease status. This known mutation occurs in a highly conserved site that causes a premature stop codon (p.C86X). The mutation was not seen in 1,142 ethnically matched control DNA samples.

Conclusions: Premature stop codons in COL2A1 exon 2 lead to a Stickler syndrome type I ocular-only phenotype with few or no systemic manifestations. Mutation screening of COL2A1 exon 2 in families with autosomal dominant vitreoretinopathy is important for accurate clinical diagnosis.

Figure 1

Study family pedigree. The family consisted of 14 individuals in three generations with six affected and four unaffected participants. Solid symbols indicate affected individuals. Asterisks indicate participating individuals for whom DNA was available for genomic analysis.

Figure 2

Sequence chromatogram of the Cys86X mutation in COL2A1 exon 2. The sequence chromatogram of COL2A1 exon 2 encompassing codon 86 demonstrates the c.258C>A mutation (GenBank NM_001844.4, +1 in cDNA numbering corresponding to the A of the methionine translation initiation codon) converting a cysteine codon to a stop codon in two affected individuals while the mutation is not present in two unaffected individuals. DNA analysis of all other affected family members cosegregated with this mutation while the mutation was not present in all unaffected family individuals.

Figure 3

COL2A1 cDNA structure and COL2A1 cDNA primer design. Exon 2 undergoes tissue-dependent alternative splicing. The COL2A1 type IIA isoform (A), expressed in the eye vitreous and in embryonic chondroprogenitor cells, includes exon 2 (Ex 2) whereas this exon is spliced in the COL2A1 type IIB isoform (B), which is expressed by adult differentiated chondrocytes. Primers were designed to amplify both cDNA isoforms: The COL2A1 cDNA primers span 303 bp when amplifying COL2A1 type IIB cDNA (excluding exon 2), and 510 bp when amplified COL2A1 type IIA cDNA (including exon 2). Ex 1, Ex 2, Ex 3, and Ex 8 depict exons 1, 2, 3, and 8.