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J Med Genet 2003;40:942-948
Multiple epiphyseal dysplasia (MED) is clinically and genetically a heterogeneous disorder that affects growth centres and results in delayed and irregular mineralisation of the ossification centres. (1) (2) Recessively inherited MED (rMED; MIM 226900) accounts for a significant proportion of MED cases and is associated with mutations in the sulphate transporter gene, DTDST/SLC26A2. (3)(4) More often, MED is inherited as a dominant trait. Thus far, five different genes have been implicated in dominantly inherited MED: the gene for cartilage oligomeric matrix protein, COMP (MIM 600310); the genes for the [alpha]1, [alpha]2, and [alpha]3 chains of collagen IX, COL9A1 (MIM 120165), COL9A2 (MIM 120260), and COL9A3 (MIM 120270); and the gene for matrilin-3, MATN3 (MIM 602109). Patients with the severe forms of MED have short stature and major disability because of joint pain and stiffness. In the milder forms, height can be normal and joint complaints minimal.
Mutations in COMP typically lead to the severe forms of dominant MED (MIM 132400) and can also cause a related but more severe disorder--pseudoachondroplasia (PSACH, MIM 177170). COMP is a pentameric extracellular glycoprotein that belongs to the thrombospondin protein family. (5-7) It consists of a coiled coil N-terminal domain responsible for pentamerisation, four epidermal growth factor (EGF)-like repeats, eight thrombospondin type 3 (T3) repeats, and a large C-terminal globular domain. Mutations in COMP that cause MED are located in the T3 repeats. (1) (2) Mutations in these repeats alter the conformation of the protein and affect its ability to bind calcium. (8-10) No mutations have been reported in the N-terminal domain or the EGF-like domains in MED. Only four mutations causing MED have been found in the C-terminal domain--two (T585R and T585M) in patients with unclassified MED, (1) (11) and the other two (R718W and N742fsX743) in patients with "severe MED" and "ribbing type MED", respectively. (12)
Altogether eight mutations have been identified in the collagen IX genes in MED patients. (11) (13-18) All reported mutations are clustered in the splice donor or acceptor site of exon 3 of COL9A2 or COL9A3 or in the splice acceptor site of exon 8 of COL9A1. The consequence of these mutations is skipping of exon 3 within the COL3 domain, leading to an in-frame 12 amino acid deletion from either the [alpha]2(IX) or [alpha]3(IX) chain, respectively; or in the case of the [alpha]1(IX) chain, skipping of exon 8 and/or exon 10, leading to an in-frame 25, 21, or 49 amino acid deletion within the COL3 domain. Patients with collagen IX mutation are typically of normal >to near normal height. Dysplastic changes are mainly seen in the knees, and the hips are relatively spared. (1) (19) In contrast, the presence of dysplastic capital femoral epiphyses and severely irregular acetabuli is suggestive of COMP mutations. (1) (19)
We undertook a clinical and molecular study of two families with an MED phenotype very similar to those individuals previously reported with mutations in the collagen IX genes. Surprisingly, affected members in both families had a mutation in COMP, R718W, suggesting that this mutation and mutations in collagen IX may share the same molecular pathogenesis.
We studied two MED families, family 1 and family 2 (fig 1). Family 1 was a three generation family in which two children presented with muscular weakness, moderately raised creatine kinase, and joint pain. Family 2 was a two generation family in which a 12 year old girl presented with joint, and especially knee, pain. All subjects were examined clinically and radiographically. Affected and unaffected family members were informed about the nature of the molecular investigations aimed at determining the cause of the muscle and joint disease in some family members, and gave consent to venepuncture and molecular analysis. Genomic DNA was extracted using standard methods.
Collagen 9 gene analysis
Sequences corresponding to exons 8 to 10 of COL9A1, (20) exons 2 to 4 of COL9A2, (20) and COL9A3 (21) and the corresponding intronic flanking sequences were amplified by polymerase chain reaction (PCR) to obtain products of 252 to 396 base pairs (bp) to be used for mutation screening by conformation sensitive gel electrophoresis (CSGE) (table 1). The amplifications were carried out in a volume of 20 [micro]1 that contained 20 to 40 ng genomic DNA, 5 to 10 pmol of the PCR primers, 1.5 mM of MgC[l.sub.2], 0.2 mM of deoxynucleotide triphosphates (dNTPs), and one unit of AmpliTaq Gold DNA polymerase (Applied Biosystems). The thermocycling and the CSGE analysis conditions were the same as described earlier (22) (23) with the exception that the CSGE gels were stained with SYBR Gold nucleic acid gel stain (Eugene, …