METHODS

Subjects

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.

Mutation analysis

Collagen 9 gene analysis

Sequences corresponding to exons 8 to 10 of COL9A1,²⁰ exons 2 to 4 of COL9A2,²⁰ and COL9A3²¹ 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 μl that contained 20 to 40 ng genomic DNA, 5 to 10 pmol of the PCR primers, 1.5 mM of MgCl₂, 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, USA) instead of ethidium bromide. All samples were also analysed by direct sequencing of PCR products with fluorescence labelled dideoxy nucleotides and on-line fluorescence detection using ABI PRISM^TM377 or 3100 sequencer apparatus and the BigDye terminator cycle sequencing kit (Applied Biosystems). In family 2, all 32 exons and the boundaries of COL9A2 and COL9A3, and all 38 exons and the boundaries of COL9A1 were screened by CSGE from the affected individuals.¹¹

Sequences corresponding to all 19 exons and exon boundaries of COMP²⁴ were amplified by PCR to obtain products of 259 to 401 bp (table 1). These PCR products were analysed by CSGE followed by direct sequencing as indicated above.

RESULTS

Clinical findings

Family 1

The eldest boy in this sibship (fig 1, subject III-1) was noted by his paediatrician to have mild muscular weakness around the age of three years. At five years, he was referred for neuropaediatric consultation and was found to have a moderately raised plasma creatine kinase (between 440 and 1647 U/l in six different blood samples; normal for age, <195 U/l). Clinical examination confirmed mild muscular weakness without other major findings. Subsequently he complained of joint pain and was referred to a rheumatologist for investigation of possible myositis or other rheumatological disorder. Skeletal radiographs were taken and epiphyseal changes at the knees were noted, prompting a review of the findings of the skeletal dysplasia group. A diagnosis of epiphyseal dysplasia with mild myopathy was made and molecular investigation of collagen IX genes was recommended. His clinical course was mild, with occasional joint complaints but no significant limitation or handicap in his daily life.

The younger siblings of III-1 were fraternal twins—a girl and a boy (III-2 and III-3 in fig 1). The boy had motor features similar to those of his elder brother and he was included in the neuropaediatric consultation. He was also found to have raised creatine kinase (411 and 315 U/l on two occasions; normal, <195 U/l) and, following identification of epiphyseal dysplasia in his elder brother, he was found to have similar radiographic changes (figs 2 and 3).

The other twin, a girl (III-2 in fig 1), was clinically healthy. Neither her paediatrician nor her parents considered her to have muscular or other clinical features like those of her brothers. When mutation analysis revealed that she was also carrying the COMP mutation, x rays of her hand and knee were done. These showed the unequivocal presence of epiphyseal dysplasia (fig 3). Her plasma creatine kinase on that occasion was 208 U/l (normal for her age, <171 U/l).

The height of all three children was in the upper range of normal. Family history revealed that the parents originated from the Balkans. The father (subject II-1) had precocious osteoarthritis of both hip joints. He had been a construction worker but had to be placed on disability leave in his early thirties because of pain and disability. He required bilateral hip joint replacement at age 33 years. His mother (grandmother of the affected siblings; subject I-1 in fig 1) had a similar history of osteoarthritis and had had bilateral hip replacement surgery in her forties. Radiographic data were not available; blood could be obtained from the father but not from the grandmother, who lived abroad. Plasma creatine kinase in the father was 222 U/l (normal for age, 180 U/l).

Family 2

A 12 year old girl (individual III-5 in fig 1) was referred for recurrent joint pain. Her mother (II-3) also gave a history of frequent joint pain, and her older brother (III-4) mentioned occasional knee pain. Radiographs were obtained. Knee x rays showed changes suggestive of MED in the girl (fig 3, panel D), and magnetic resonance imaging (MRI) was done (fig 4). Knee radiographs of the mother and brother showed only mild changes (fig 3, panels E and F). The family members consented to donate blood for molecular analysis but considered their knee affliction to be of little significance and they where lost to follow up; their plasma creatine kinase could not be obtained. The radiographic pattern at the knee, with the clefts on the lateral sides of the distal femoral and proximal tibial epiphyses (“Gletscherspalte”, glacier crevice; fig 3, panel D) was considered similar to that described in published reports in association with collagen IX mutations. However, no mutations were identified and investigations were stopped. Only a few years later, upon identification of family 1 (above), the DNA samples were resubmitted to COMP mutation analysis which revealed the presence of the same mutation (see below) in the index case, in her mother, and also in her brother, who had mild radiographic changes.

Radiological findings

The radiographic features associated with the R718W mutation are shown in figs 2 to 4. The hands showed some delay in epiphyseal maturation with slightly irregular contours of the carpal elements and a small radial distal epiphysis. The proximal femoral epiphyses in case III-3 (at age seven years) have low-normal size and regular contours, while in case III-5 (at age 14 years), they are small and have an irregular contour. The knees show the most significant changes. A lateral view of the knee of subject III-1 at age 11½ years shows a moderately small patella with an accessory cranial ossification centre and markedly irregular femoral epiphyses with frayed contours and the possible presence of a loose body. Figure 3 delineates the sequence of radiographic changes at the knee. At ages six and seven years there are moderately small epiphyses with slightly irregular contours. Between the ages of 11 and 13 years (probably coinciding with the pubertal growth spurt and osseous maturation), an additional ossification centre appears at the medial condyle and extends to form a ring at the periphery of the epiphysis; it is at this stage that the aspect is characteristic (resembling a “crevice” in a glacier) and can be seen both in the distal femoral and the proximal tibial epiphyses. At later ages, the “crevice” sign is no longer visible but the epiphyses are flatter than normal.

Mutation analysis

Because the phenotype of the affected individuals studied here was similar to that caused by collagen IX gene mutations, these genes were analysed for mutations. All reported mutations of collagen IX genes are clustered in the splice sites of exon 3 of COL9A2 or COL9A3, or the splice acceptor site of exon 8 of COL9A1.^1,2 Therefore the candidate exons were analysed by CSGE and sequencing from the index case of family I (fig 1), his affected father, and his unaffected mother. Analysis of the candidate exons of COL9A1, COL9A2, and COL9A3 genes identified five sequence variations. Two of them were in COL9A1 (IVS8⁺¹³C>T, IVS10⁺⁴⁵G>A), two in COL9A2 (IVS2–⁴²T>C, IVS4⁺³⁶C>A), and one in COL9A3 (e2⁺¹⁵C>A; P29P). They were not disease causing because no co-segregation with the variation and phenotype was observed or they were also present in control samples (data not shown). The analysis did not reveal any putative disease causing variations.

Because no mutations were found in the candidate exons of collagen IX genes in this family in spite of an unequivocable MED phenotype, mutation screening was then extended to COMP. COMP mutations are among the most common causes of the dominant form of MED. All 19 exons and the boundaries of COMP were analysed by CSGE from three individuals in family 1 (fig 1, II:1, II:2, and III:1). In addition, all exons and the boundaries were sequenced from the proband. A unique CSGE pattern in exon 18 of COMP was present in the affected boy (III:1) and his affected father (II:1). Sequencing of the product identified a c.2152 C>T transition converting the codon CGG for R718 (arginine) to TGG for W (tryptophan) (fig 5). A previously reported polymorphism, IVS18⁺⁵³ T>C,¹¹ was also present in the same PCR product in the affected family members and seen on CSGE analysis as well (fig 5). CSGE analysis of the other exons did not reveal any other heteroduplexes, and no other sequence variations were found in sequencing. After identifying the R718W mutation in two affected family members (II:1, III:1), the analysis of the twins showed that they had also inherited the R718W mutation and the IVS18⁺⁵³T>C polymorphism from their father. The unaffected mother did not have the R718W mutation or the polymorphism. The fact that the polymorphism was seen in one family only, and that the mutation itself occurs at a CpG dinucleotide, suggests independent occurrence.

Initially, all exons and the boundaries of COL9A1, COL9A2, and COL9A3 were analysed from the three affected individuals from family 2. The analysis failed to identify any disease causing mutations. When the R718W mutation was identified in family 1 because the phenotype was similar to family 2, the three affected members of family 2 were analysed for the presence of R718W mutation in COMP by CSGE and sequencing. The same R718W mutation was identified in all three individuals. Subsequent CSGE analysis of the other 18 exons and the boundaries of COMP did not indicate any other sequence variations in these family members. Sequencing of the remaining 18 COMP exons from the proband’s sample did not reveal any other sequence variations.

DISCUSSION

We identified seven individuals in two families who were heterozygous for an amino acid substitution in the C-terminal globular domain of COMP. In spite of its relatively large size, mutations in the C-terminal domain producing a skeletal phenotype seem to be relatively rare and may tend to cluster around sensitive microdomains. According to the review of genotype–phenotype correlations by Briggs and Chapman,¹ mutation of threonine-585 has been observed in two unrelated individuals with MED, and two closely situated mutations, glutamate-583 and histidine-587 have been observed in patients with PSACH. Recently, three novel mutations were reported in the C-terminal domain of COMP: a nucleotide insertion leading to premature termination at codon 742 (giving MED); a substitution of glutamate 719 with aspartate (resulting in severe PSACH); and substitution of arginine 718 with tryptophan, which is the same mutation we found in our study subjects.^12,25 Apparently, arginine 718 and glutamate 719 define a second functionally important microdomain in the C-terminal domain of COMP.

Mabuchi et al observed R718W segregating in a family, but little clinical detail or radiographic data were given, except for height at –3 SD and a diagnosis of “Fairbank” type MED.¹² While saturating the mutation map of the molecule contributes to the genotype–phenotype database and delineates functional regions of the molecule, the clinical and radiographic findings in the two families reported here provide novel information, such as the association of this mutation with myopathy, the surprising overlap between radiographic findings in this family with those reported for collagen IX associated MED, and the dynamic changes at the distal femoral epiphyses that delineate a diagnostic time window.

Muscle weakness was the presenting clinical sign in the two index cases in family 1, and clear elevation of plasma creatine kinase in both confirmed the presence of myopathy. Creatine kinase levels were also slightly raised in their younger sister and their father. Myopathy has previously been reported in association with MED and COL9A3 mutation¹³; interestingly, in that family also two boys had clinically relevant myopathy, while female mutation carriers were affected subclinically. In that family, elevation of plasma creatine kinase was less marked than in the two boys studied here. In addition, the proband in another family with a COL9A3 mutation, an 11 year old boy, was initially evaluated by a neuropaediatrician because of stiffness in the knees, clumsiness, and muscle weakness in the legs.¹⁴ It is not unusual for children who are later diagnosed as having PSACH to present before the onset of short stature with neuromuscular symptoms, most often gait disturbances. In view of the molecular interaction between COMP and collagen IX, it may be that a common supramolecular complex in muscle (possibly at the junction between myocytes and adjacent matrix structures) is affected. Little is known so far about a possible function of collagen IX and COMP in muscle and this aspect remains to be investigated.

A review of the radiographic features of a series of patients with dominant MED suggested a distinct pattern, allowing differentiation between forms caused by mutations in the collagen IX genes and forms caused by mutations in COMP.¹⁹ The knee “crevice” sign can be recognised in other report of MED—for example, in Hatori et al²⁶ (case 6 in fig 5 in that paper), and, significantly, in collagen IX associated MED.^14,16 The accessory cranial ossification centre of the patella has also been observed in collagen IX-MED¹⁴ (compare fig 3 in that report with the findings presented here). This was what prompted us to investigate collagen IX before COMP. Apparently neither sign is specific for collagen IX associated MED as it can also be seen in COMP associated MED. Thus although these observations do not refute the distinction proposed by Unger et al that patient groups may tend to show differences,¹⁹ there is considerable overlap. It is true that two affected individuals (I-1 and II-1) developed severe hip arthritis in adulthood, which is more typical of COMP-MED. At present there is not enough follow up information on adult patients with collagen IX-MED to determine whether severe hip involvement in adult life is a clear distinguishing criterion between the two forms of MED.

The lack of specificity of the radiographic signs during childhood and adolescence, and the presence of myopathy, points to a common molecular pathogenesis. Conceivably, the function of a supramolecular complex is disrupted in dominant MED, with similar consequences irrespective of whether the primary defect is in one component (COMP) or the other (collagen IX). From a diagnostic perspective, we suggest that it may be more practical to investigate the COMP gene first and the collagen IX genes only after COMP mutation screening has proven negative.