A mutation in the alpha 3 chain of type IX collagen causes autosomal dominant multiple epiphyseal dysplasia with mild myopathy

Abstract

Multiple epiphyseal dysplasia (MED) is a degenerative cartilage condition shown in some cases to be caused by mutations in genes encoding cartilage oligomeric matrix protein or type IX collagen. We studied a family with autosomal dominant MED affecting predominantly the knee joints and a mild proximal myopathy. Genetic linkage to the COL9A3 locus on chromosome 20q13.3 was established with a peak log(10) odds ratio for linkage score of 3.87 for markers D20S93 and D20S164. Reverse transcription-PCR performed on the muscle biopsy revealed aberrant mRNA lacking exon 3, which predicted a protein lacking 12 amino acids from the COL3 domain of alpha3(IX) collagen. Direct sequencing of genomic DNA confirmed the presence of a splice acceptor mutation in intron 2 of the COL9A3 gene (intervening sequence 2, G-A, -1) only in affected family members. By electron microscopy, chondrocytes from epiphyseal cartilage exhibited dilated rough endoplasmic reticulum containing linear lamellae of alternating electron-dense and electron-lucent material, reflecting abnormal processing of mutant protein. Type IX collagen chains appeared normal in size and quantity but showed defective cross-linking by Western blotting. The novel phenotype of MED and mild myopathy is likely caused by a dominant-negative effect of the exon 3-skipping mutation in the COL9A3 gene. Patients with MED and a waddling gait but minimal radiographic hip involvement should be evaluated for a primary myopathy and a mutation in type IX collagen.

Figure 1

Pedigree of MED with mild myopathy genotyped at markers from chromosome 20q13.3. The disease haplotype is boxed. All affected family members share a common allele of markers D20S173, D20S93, and D20S164. D20S93* lies within an intron of the COL9A3 gene (34). Circles, female; squares, male; shaded symbols, affected; open symbols, unaffected.

Figure 2

(a--d) Radiographs from the proband (individual IV-5) at 10 yr of age. (a) Anteroposterior view of both knees. Epiphyses are abnormally shaped especially at subchondral bone regions. (b) Abnormal ankle radiograph with irregular shaping of distal tibial epiphysis and a rounded talar surface. (c) Anteroposterior radiograph centered at the wrist shows irregular carpals and a mildly shortened distal ulna. (d) Normal hip and pelvic radiograph. Knee radiographs showing progressive degenerative osteoarthritis in (e) individual III-9 at 38 yr and her mother II-3 at 52 yr (f) and 63 yr (g).

Figure 3

Histogram of quadriceps muscle biopsy from the proband showing mild variability in fiber diameter compared with an age-matched control.

Figure 4

Electron micrographs of epiphyseal chondrocytes with dilated RER containing (a) linear lamellar material in longitudinal section (×8,000) and (b) hexagonal arrays of punctate material in cross-section (×12,970). Fat inclusions are seen in both the cytoplasm and RER.

Figure 5

(a) Reverse transcription–PCR from skeletal muscle cDNA using primers flanking the mutation. Lane M, molecular size markers in bp; lane 1, patient sample showing a normal upper band and an abnormal lower band lacking the 36-bp exon 3; lane 2, normal control containing a single upper band; lane 3, negative control without DNA. (b) Chromatogram of genomic DNA sequence from the proband. The G-to-A mutation is present in a heterozygous state at position −1 with respect to the exon (). (c) Schematic of the splice defect relative to the domain structure of COL9A3. COL1–3, collagenous domains 1–3; and NC1–4, noncollagenous domains 1–4.

Figure 6

Collagen IX protein analysis. (a) Western blot analysis of denaturant-extracted intact collagen type IX chains showing no apparent quantitative difference between control (CNTL) and patient (MED) samples. (b) Western blot analysis of CNBr-extracted, cross-linked collagen IX fragments showing a clear deficiency in cross-linked α3CB6 fragments in patient (MED) compared with control (CNTL) samples.