Identification of nine novel mutations in cartilage oligomeric matrix protein in patients with pseudoachondroplasia and multiple epiphyseal dysplasia

Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1) are allelic disorders caused by mutations in the gene encoding cartilage oligomeric matrix protein (COMP). PSACH is a dominant condition characterized by disproportionate short stature, joint laxity, and early-onset osteoarthritis. EDM1 is a less severe skeletal dysplasia associated with average to mild short stature, joint pain, and early-onset osteoarthritis. COMP is an extracellular matrix protein present in cartilage, ligament, and tendon tissues. Here, we report on nine novel mutations in COMP causing PSACH and EDM1. Four of these mutations are in exons 13C and 14 where no previous mutations had been reported. One of those mutations was identified in two separate EDM1 families. In addition, we have identified the first case of PSACH resulting from an expansion of the five aspartates in exon 17B. We are also reporting a mutation in a third PSACH family with somatic/germline mosaicism. Therefore, this report increases the range of mutations that cause PSACH and EDM1 and provides additional regions to target for mutational analysis. Am. J. Med. Genet. 85:486–490, 1999. © 1999 Wiley-Liss, Inc.     

Pseudoachondroplasia and multiple epiphyseal dysplasia: mutation review, molecular interactions, and genotype to phenotype correlations

Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) constitute a bone dysplasia family, which is both genetically and phenotypically heterogeneous. The disease spectrum ranges from mild MED, which manifests with pain and stiffness in the joints and delayed and irregular ossification of the epiphyses, to the more severe PSACH, which is characterized by marked short stature, deformity of the legs, and ligamentous laxity. PSACH is almost exclusively caused by mutations in cartilage oligomeric matrix protein (COMP) whereas various forms of MED are caused by mutations in the genes encoding COMP, type IX collagen (COL9A1, COL9A2, and COL9A3), matrilin-3 (MATN3), and solute carrier member 26, member 2 gene (SLC26A2). In this review we discuss specific disease-causing mutations and the clustering of these mutations in functionally and structurally important regions of the respective gene products, genotype to phenotype correlations, and the diagnostic relevance of mutation screening in these osteochondrodysplasias.     

Revisit of multiple epiphyseal dysplasia: ethnic difference in genotypes and comparison of radiographic features linked to the COMP and MATN3 genes

Multiple epiphyseal dysplasia (MED) is a genetically heterogeneous group of diseases characterized by variable degrees of epiphyseal abnormality primarily involving the hip and knee joints. The purpose of this study was to investigate the frequency of mutations in individuals with a clinical and radiographic diagnosis of MED and to test the hypothesis that characteristic radiological findings may be helpful in predicting the gene responsible. The radiographs of 74 Korean patients were evaluated by a panel of skeletal dysplasia experts. Six genes known to be associated with MED (COMP, MATN3, COL9A1, COL9A2, COL9A3, and DTDST) were screened by sequencing. Mutations were found in 55 of the 63 patients (87%). MATN3 mutations were found in 30 patients (55%), followed by COMP mutations in 23 (41%), and COL9A2 and DTDST mutations in one patient (2%) each. Comparisons of radiographic findings in patients with COMP and MATN3 mutations showed that albeit marked abnormalities in hip and knee joints were observed in both groups, the degree of involvement and the morphology of dysplastic epiphyses differed markedly. The contour of the pelvic acetabulum, the presence of metaphyseal vertical striations, and/or the brachydactyly of the hand were also found to be highly correlated with the genotypes. The study confirms that MATN3 and COMP are the genes most frequently responsible for MED and that subtle radiographic signs may give precious indications on which gene(s) should be prioritized for mutational screening in a given individual.     

Exome sequencing reveals a novel COL2A1 mutation implicated in multiple epiphyseal dysplasia

Mutations in the COMP, COL9A1, COL9A2, COL9A3, MATN3, and SLC26A2 genes cause approximately 70% of multiple epiphyseal dysplasia (MED) cases. The genetic changes involved in the etiology of the remaining cases are still unknown, suggesting that other genes contribute to MED development. Our goal was to identify a mutation causing an autosomal dominant form of MED in a large multigenerational family. Initially, we excluded all genes known to be associated with autosomal dominant MED by using microsatellite and SNP markers. Follow‐up with whole‐exome sequencing analysis revealed a mutation c.2032G>A (p.Gly678Arg) in the COL2A1 gene (NCBI Reference Sequence: NM_001844.4), which co‐segregated with the disease phenotype in this family, manifested by severe hip dysplasia and osteoarthritis. One of the affected family members had a double‐layered patella, which is frequently seen in patients with autosomal recessive MED caused by DTDST mutations and sporadically in the dominant form of MED caused by COL9A2 defect.     

A compound heterozygote of novel and recurrent DTDST mutations results in a novel intermediate phenotype of Desbuquois dysplasia, diastrophic dysplasia, and recessive form of multiple epiphyseal dysplasia

Diastrophic dysplasia sulfate transporter (DTDST) is required for synthesis of sulfated proteoglycans in cartilage, and its loss-of-function mutations result in recessively inherited chondrodysplasias. The 40 or so DTDST mutations reported to date cause a group of disorders termed the diastrophic dysplasia (DTD) group. The group ranges from the mildest recessive form of multiple epiphyseal dysplasia (r-MED) through the most common DTD to perinatally lethal atelosteogenesis type II and achondrogenesis 1B. Furthermore, the relationship between DTDST mutations, their sulfate transport function, and disease phenotypes has been described. Here we report a girl with DTDST mutations: a compound heterozygote of a novel p.T266I mutation and a recurrent p.DeltaV340 mutation commonly found in severe phenotypes of the DTD group. In infancy, the girl presented with skeletal manifestations reminiscent of Desbuquois dysplasia, another recessively inherited chondrodysplasia, the mutations of which have never been identified. Her phenotype evolved with age into an intermediate phenotype between r-MED and DTD. Considering her clinical phenotypes and known phenotypes of p.DeltaV340, p.T266I was predicted to be responsible for mild phenotypes of the DTD group. Our results further extend the phenotypic spectrum of DTDST mutations, adding Desbuquois dysplasia to the list of differential diagnosis of the DTD group.     

Novel and recurrent mutations clustered in the von Willebrand factor A domain of MATN3 in multiple epiphyseal dysplasia

Multiple epiphyseal dysplasia (MED) is a common skeletal dysplasia characterized by joint pain and stiffness, delayed and irregular ossification of epiphyses, and early-onset osteoarthritis. Six genes responsible for MED have been identified, including COMP, COL9A1, COL9A2, COL9A3, DSTDT and MATN3. MATN3 encodes matrilin-3, a cartilage-specific extracellular matrix protein. To date, seven different MATN3 mutations have been identified; all are located within the beta-sheet regions of the von Willebrand factor type A (vWFA) domain, which is encoded by exon 2. We examined MATN3 mutations in27 Japanese MED patients who were possibly autosomal dominant inheritance and had been excluded for COMP mutations. Ten of them had a positive family history. We examined all eight exons of MATN3 by PCR and direct sequencing from genomic DNA. We have identified four missense mutations in eight unrelated families; two are novel, and two have been characterized previously. Like previously characterized MATN3 mutations, those identified in this study are clustered within exon 2, specifically in and around the 2nd beta-sheet region of the vWFA domain (aa. 120-127). Contrary to the previous assumption that the MATN3 mutation in MED is confined to the beta-sheet regions, one novel mutation (p.F105S) is located outside the beta-sheet region, within an alpha-helix region.     

SLC26A2 disease spectrum in Sweden – high frequency of recessive multiple epiphyseal dysplasia (rMED)

Diastrophic dysplasia (DTD) is an autosomal recessive skeletal dysplasia caused by SLC26A2 mutations. Clinical features include short stature, joint contractures, spinal deformities, and cleft palate. SLC26A2 mutations also result in other skeletal dysplasias, including the milder recessive multiple epiphyseal dysplasia (rMED). DTD is overrepresented in Finland and we speculated that this may have influenced the prevalence and spectrum of SLC26A2‐related skeletal conditions also in Sweden. We reviewed the patient registry at Department of Clinical Genetics, Karolinska University Hospital, Stockholm to identify subjects with SLC26A2 mutations. Seven patients from six families were identified; clinical data were available for six patients. All but one patient had one or two copies of the Finnish SLC26A2 founder mutation IVS1+2T>C. Arg279Trp mutation was present in compound heterozygous form in five patients with phenotypes consistent with rMED. Their heights ranged from ?2.6 to ?1.4 standard deviation units below normal mean and radiographic features included generalised epiphyseal dysplasia and double‐layered patellae. Two rMED patients had hypoplastic C2 and cervical kyphosis, a severe manifestation previously described only in DTD. Our study confirms a high prevalence of rMED in Sweden and expands the phenotypic manifestations of rMED.     

Clinical and molecular characterization of Diastrophic Dysplasia in the Portuguese population

SLC26A2-related dysplasias encompass a spectrum of diseases: from lethal achondrogenesis type 1B (ACG1B; MIM #600972) and atelosteogenesis type 2 (AO2; MIM #256050) to classical diastrophic dysplasia (cDTD; MIM #222600) and recessive multiple epiphyseal dysplasia (rMED; MIM #226900). This study aimed at characterizing clinically, radiologically and molecularly 14 patients affected by non-lethal SLC26A2-related dysplasias and at evaluating genotype-phenotype correlation. Phenotypically, eight patients were classified as cDTD, four patients as rMED and two patients had an intermediate phenotype (mild DTD - mDTD, previously 'DTD variant'). The Arg279Trp mutation was present in all patients, either in homozygosity (resulting in rMED) or in compound heterozygosity with the known severe alleles Arg178Ter or Asn425Asp (resulting in DTD) or with the mutation c.727-1G>C (causing mDTD). The 'Finnish mutation', c.-26+2T>C, and the p.Cys653Ser, both frequent mutations in non-Portuguese populations, were not identified in any of the patients of our cohort and are probably very rare in the Portuguese population. A targeted mutation analysis for p.Arg279Trp and p.Arg178Ter in the Portuguese population allows the identification of approximately 90% of the pathogenic alleles.     

Mutations in the diastrophic dysplasia sulfate transporter (DTDST) gene (SLC26A2): 22 novel mutations, mutation review, associated skeletal phenotypes, and diagnostic relevance

Mutations in the DTDST gene can result in a family of skeletal dysplasia conditions which comprise two lethal disorders, achondrogenesis type 1B (ACG1B) and atelosteogenesis type 2 (AO2); and two non-lethal disorders, diastrophic dysplasia (DTD) and recessive multiple epiphyseal dysplasia (rMED). The gene product is a sulfate-chloride exchanger of the cell membrane. Inactivation of the sulfate exchanger leads to intracellular sulfate depletion and to the synthesis of undersulfated proteoglycans in susceptible cells such as chondrocytes and fibroblasts. Genotype-phenotype correlations are recognizable, with mutations predicting a truncated protein or a non-conservative amino acid substitution in a transmembrane domain giving the severe phenotypes, and non-transmembrane amino acid substitutions and splice site mutations giving the milder phenotypes. The clinical phenotype is modulated strictly by the degree of residual activity. Over 30 mutations have been observed, including 22 novel mutations reported here. The most frequent mutation, 862C>T (R279W), is a mild mutation giving the rMED phenotype when homozygous and mostly DTD when compounded; occurrence at a CpG dinucleotide and its panethnic distribution suggest independent recurrence. Mutation IVS1+2T>C is the second most common mutation, but is very frequent in Finland. It produces low levels of correctly spliced mRNA, and results in DTD when homozygous. Two other mutations, 1045-1047delGTT (V340del) and 558C>T (R178X), are associated with severe phenotypes and have been observed in multiple patients. Most other mutations are rare. Heterozygotes are clinically unaffected. When clinical samples are screened for radiologic and histologic features compatible with the ACG1B/AO2/DTD/rMED spectrum prior to analysis, the mutation detection rate is high (over 90% of alleles), and appropriate genetic counseling can be given. The sulfate uptake or sulfate incorporation assays in cultured fibroblasts have largely been replaced by mutation analysis, but may still be useful in cases where mutation analysis is not informative. Although supplementation of patients' cultured cells with thiols may bypass the transporter defect and enhance sulfation of proteoglycans, therapeutic approaches are not yet available. Mouse models for this and other disorders of sulfate metabolism are being developed to help in developing therapeutic treatments.     

Report of five novel and one recurrent COL2A1 mutations with analysis of genotype-phenotype correlation in patients with a lethal type II collagen disorder

Achondrogenesis II-hypochondrogenesis and severe spondyloepiphyseal dysplasia congenita (SEDC) are lethal forms of dwarfism caused by dominant mutations in the type II collagen gene (COL2A1). To identify the underlying defect in seven cases with this group of conditions, we used the combined strategy of cartilage protein analysis and COL2A1 mutation analysis. Overmodified type II collagen and the presence of type I collagen was found in the cartilage matrix of all seven cases. Five patients were heterozygous for a nucleotide change that predicted a glycine substitution in the triple helical domain (G313S, G517V, G571A, G910C, G943S). In all five cases, analysis of cartilage type II collagen suggested incorporation of the abnormal alpha1(II) chain in the extracellular collagen trimers. The G943S mutation has been reported previously in another unrelated patient with a strikingly similar phenotype, illustrating the possible specific effect of the mutation. The radiographically less severely affected patient was heterozygous for a 4 bp deletion in the splice donor site of intron 35, likely to result in aberrant splicing. One case was shown to be heterozygous for a single nucleotide change predicted to result in a T1191N substitution in the carboxy-propeptide of the proalpha1(II) collagen chain. Study of the clinical, radiographic, and morphological features of the seven cases supports evidence for a phenotypic continuum between achondrogenesis II-hypochondrogenesis and lethal SEDC and suggests a relationship between the amount of type I collagen in the cartilage and the severity of the phenotype.     

Identification of sequence polymorphisms in two sulfation-related genes, PAPSS2 and SLC26A2, and an association analysis with knee osteoarthritis

Osteoarthritis (OA) is one of the most common musculoskeletal disorders and is characterized by degeneration of articular cartilage. Sulfation of extracellular ma-trix proteins in articular cartilage is an important step in maintaining normal cartilage metabolism. Two sulfation-related genes have been reported as the causal genes of severe chondrodysplasias: mutations in PAPSS2 (3′-phosphoadenosine 5′-phosphosulfate synthase 2) cause spondylo-epimetaphyseal dysplasia (SEMD), and mutations in SLC26A2 (solute carrier family 26, member 2) cause diastrophic dysplasia. Given their critical roles in cartilage metabolism and the severe phenotypes that result from mutations in these genes, we examined PAPSS2 and SLC26A2 as candidate susceptibility loci for OA. We identified sequence polymorphisms in the coding and core promoter regions of these genes and analyzed their potential association with knee OA within the Japanese population. Ten sequence polymorphisms were detected in PAPSS2 and five in SLC26A2. An association analysis showed suggestive association of one minor polymorphism in the promoter region of SLC26A2. This 4-bp adenine deletion allele, del4A, was over-represented in knee OA (P = 0.043, odds ratio = 3.43) and is thought to confer a minor susceptibility to knee OA within the Japanese population. Haplotype analysis showed no evidence of association with the two genes, however, excluding them as major susceptibility loci for knee OA. Received: May 7, 2001 / Accepted: June 21, 2001     

Screening of SLC25A13 mutations in early and late onset patients with citrin deficiency and in the Japanese population: Identification of two novel mutations and establishment of multiple DNA diagnosis methods for nine mutations

We have recently identified SLC25A13 on chromosome 7q21.3 as the gene responsible for adult‐onset type II citrullinemia (CTLN2) and found seven mutations in the SLC25A13 gene of CTLN2 patients. Most recently, the SLC25A13 mutations have been detected in neonatal/infantile patients with a type of neonatal hepatitis associated with cholestasis (NICCD). In the present study, we identified a novel mutation, E601X, in the SLC25A13 gene and established multiple DNA diagnosis methods for eight mutations by using a genetic analyzer with GeneScan and the single primer extension procedure (SNaPshot). An additional novel missense mutation (variation), E601K, was detected by SNaPshot analysis and was indistinguishable from the mutation E601X detected by the PCR/RFLP method. Multiple DNA diagnoses for the nine mutations revealed that 100 (male/female: 70/30) out of 115 CTLN2 and 38 (14/24) out of 45 NICCD patients tested were homozygotes or compound heterozygotes. The frequency of homozygotes carrying SLC25A13 mutations in both alleles is estimated to be minimally 1 in 21,000 from carrier detection (18 in 1,315 individuals tested) in the Japanese population. The differences in the gender ratio and in mutation types between CTLN2 and NICCD patients are significant. It is, however, unknown whether all homozygotes with mutated SLC25A13 in both alleles suffer from NICCD, CTLN2, both, or neither. Hum Mutat 19:122–130, 2002. © 2002 Wiley‐Liss, Inc.