Variation in the vitreous phenotype of Stickler syndrome can be caused by different amino acid substitutions in the X position of the type II collagen Gly-X-Y triple helix

Abstract

Stickler syndrome is a dominantly inherited disorder characterized by arthropathy, midline clefting, hearing loss, midfacial hypoplasia, myopia, and retinal detachment. These features are highly variable both between and within families. Mutations causing the disorder have been found in the COL2A1 and COL11A1 genes. Premature termination codons in COL2A1 that result in haploinsufficiency of type II collagen are a common finding. These produce a characteristic congenital "membranous" anomaly of the vitreous of all affected individuals. Experience has shown that vitreous slit-lamp biomicroscopy can distinguish between patients with COL2A1 mutations and those with dominant negative mutations in COL11A1, who produce a different "beaded" vitreous phenotype. Here we characterize novel dominant negative mutations in COL2A1 that result in Stickler syndrome. Both alter amino acids in the X position of the Gly-X-Y triple-helical region. A recurrent R365C mutation occurred in two unrelated sporadic cases and resulted in the membranous vitreous anomaly associated with haploinsufficiency. In a large family with linkage to COL2A1, with a LOD score of 2.8, a unique L467F mutation produced a novel "afibrillar" vitreous gel devoid of all normal lamella structure. These data extend the mutation spectrum of the COL2A1 gene and help explain the basis for the different vitreous phenotypes seen in Stickler syndrome.

Figure 1

Clinical phenotypes. A–C, MS12, age 17 years, showing mild nasal-root hypolasia and moderate midfacial hypoplasia and slender digits. D–F, MS16, age 14 years. Note anteverted nares and nasal-root hyoplasia and joint laxity. G–I, III-7, MS25, age 29 years. Note well-developed nasal bridge and root, moderate midfacial hypoplasia, and slender digits.

Figure 2

Vitreous phenotypes in Stickler syndrome. A, Membranous congenital vitreous anomaly seen in MS12 and MS16. Note vestigial gel occupying retrolental space and bordered by a distinct folded membrane (arrows). B, Beaded congenital vitreous anomaly: COL11A1 mutation (see Martin et al. 1999). Note irregularly thickened fiber bundles, giving a string-of-pearls appearance (arrows). C, Afibrillar congenital vitreous anomaly seen in MS25. Note complete absence of visible fiber bundles. D, Normal vitreous appearance. Note healthy compact homogenous fibrillar array.

Figure 3

COL2A1 genomic sequencing. The heterozygous (arrows) mutation-containing sequences obtained from affected individuals in MS12, MS16, and MS25 are shown under the normal sequence (N). Intron sequences are in lowercase, and coding sequences are in uppercase.

Figure 4

DNA analysis of families. A, DNA containing exon 26, which was amplified from MS16 (1) and MS12 (2–5) and digested with BbvI. Extra bands in samples from affected individuals, visualized after electrophoresis, are marked with arrows. Undigested DNA (lane U) and standard size markers (bp) were included on the gel. B, Results of [³³P]dideoxynucleotide sequencing reaction, which was used to detect the mutation in MS25. Samples correspond to the pedigree shown above the gel. Family members not included in the analysis are situated on either side of the gel. Nucleotide numbers correspond to the complete COL2A1 gene sequence (Genome Database accession number L10347).

Figure 5

COL2A1 cDNA sequencing. Sequencing of cDNA amplified from an individual (MS20 C) with a nonsense mutation in exon 42 showed loss of heterozygosity when compared with the genomic sequence (MS20 G). Amplified cDNA from MS16 and MS25 showed heterozygosity for the mutations seen in genomic DNA (fig. 1).

Figure 6

Position of Arg→Cys substitutions in fibrillar collagens. The position of cysteine substitutions in the α1(I), α1(II), and α2(XI) collagens are illustrated. X-position changes are indicated below the line representing each molecule, whereas Y-position changes are indicated above the lines. Numbers represent the amino acid position within the helix. The resulting phenotypes are osteoarthritis (OA), spondyloepipheseal dysplasia (SED), SED congenita (SEDC), Stickler syndrome (SS), mild chondrodysplasia (MC), Stickler-like syndrome (SLS), classical Ehlers-Danlos syndrome (CEDS), and nonsyndromic deafness (DFN).