Pseudoachondroplasia and multiple epiphyseal dysplasia: New etiologic developments

Pseudoachondroplasia (PSACH) (OMIM#177170) and multiple epiphyseal dysplasia (MED) are separate but overlapping osteochondrodysplasias. PSACH is a dominantly inherited disorder characterized by short-limb short stature, loose joints, and early-onset osteoarthropathy. The diagnosis is based on characteristic clinical and radiographic findings. Only mutations in the cartilage oligomeric matrix protein (COMP) gene have been reported in PSACH, and all family studies have been consistent with linkage to the COMP locus on chromosome 19. Multiple epiphyseal dysplasia (MED) is a relatively mild chondrodysplasia but like PSACH, MED causes early-onset joint degeneration, particularly of the large weight-bearing joints. Given the clinical similarity between PSACH and MED, it was not surprising that the first MED locus identified was the COMP gene (EDM1). Mutations causing MED have now been identified in five other genes (COL9A1, COL9A2, COL9A3, DTDST, and MATN3), making MED one of the most genetically heterogeneous disorders. This article reviews the clinical features of PSACH and MED, the known mutations, and the pathogenetic effect of COMP mutations on the cartilage extracellular matrix.     

Finer linkage mapping of a primary hip osteoarthritis susceptibility locus on chromosome 6

Primary osteoarthritis (OA) is a common late-onset disease that exhibits complex genetic transmittance. A previous genome-wide linkage scan of OA affected sibling pair families (ascertained by total joint replacement surgery) identified a region of suggestive linkage on chromosome 6, with a maximum multipoint-LOD score (MLS) of 2.9 in 194 families containing sibling pairs concordant for total hip replacement (THR-families). However, up to 50 cM of the chromosome had a multipoint-LOD score >2.0, indicating that the susceptibility locus was poorly mapped. We have now genotyped chromosome 6 to a higher density in an expanded cohort of 378 THR-families. We obtained an MLS of 2.8 to an 11.4 cM interval defined by markers D6S452 and 509-8B2, which map between 70.5 to 81.9 cM from the 6p-telomere. Stratification by gender revealed that this linkage was completely accounted for by female THR-families (n=146), with an MLS of 4.0 and with the highest two-point LOD score being 4.6 for marker D6S1573 (75.9 cM). The 11.4 cM interval just encompasses the candidate gene COL9A1 (81.9 cM). We identified and then genotyped twenty common single nucleotide polymorphisms (SNPs) from within COL9A1 in the 146 probands from our female THR-families and in 215 age-matched female controls. No SNP allele, genotype or haplotype demonstrated association to disease. Overall, we have narrowed the chromosome 6 OA susceptibility locus to a point at which linkage disequilibrium/association analysis is feasible, we have demonstrated that this locus is female specific, and found no evidence that COL9A1 encodes for the susceptibility.     

Clinical and radiographic features of multiple epiphyseal dysplasia not linked to the COMP or type IX collagen genes

Multiple epiphyseal dysplasia (MED) is a mild chondrodysplasia affecting the structural integrity of cartilage and causing early-onset osteoarthrosis in adulthood. The condition is genetically heterogeneous. Mutations in the COMP gene and in two genes (COL9A2; COL9A3), coding respectively for the alpha2(IX) and alpha3(IX) chains of type IX collagen, can cause the autosomal dominant forms of MED. Mutations in the DTDST gene have recently been identified in a recessive form of MED. However, for the majority of MED cases, the genetic defect still remains undetermined. We report a three-generation family with an autosomal dominant form of MED, characterised by normal stature, joint pain in childhood and early-onset osteoarthrosis, affecting mainly the hips and knees. Based on discordant inheritance among affected individuals linkage of the phenotype to the COMP, COL9A1, COL9A2, COL9A3 genes was excluded. Our study provides evidence that at least another locus, distinct from COL9A1, is involved in autosomal dominant MED.     

Correlation of linkage data with phenotype in eight families with Stickler syndrome

The clinical findings of eight families with Stickler syndrome were analyzed and compared with the results of linkage studies using a marker for the type II collagen gene (COL2A1). In six families, there was linkage of the phenotype to COL2A1. The manifestations of the affected individuals were similar to those of the original Stickler syndrome family [Stickler et al., Mayo. Clin. Proc. 40:433–455, 1965] and resembled the phenotype of the previously reported individuals or families with Stickler syndrome in which a dominant mutation in the COL2A1 gene has been identified. Linkage to COL2A1 was excluded in the two remaining families. The most striking difference between these two types of families was the absence of severe myopia and retinal detachment in the two unliked families. In the COL2A1 unlinked families, linkage of the phenotype to genes (COL11A1 and COL11A2) that encode proα chains of type XI collagen, a minor cartilage-specific collagen, was also excluded. Since Stickler syndrome can be produced by mutations in COL2A1, COL11A1, and COL11A2, our data suggest that there is at least a fourth locus for Stickler syndrome. Am. J. Med. Genet. 80:121–127, 1998. © 1998 Wiley-Liss, Inc.     

Linkage of epidermolysis bullosa simplex to keratin gene loci.

Epidermolysis bullosa simplex (EBS) is an autosomal dominant disorder characterised by intraepidermal blistering of the skin. Two families with Weber-Cockayne EBS have been analysed for linkage to keratin gene loci. In the first family, linkage was found to chromosome 17 markers flanking the keratin 14 gene (D17S74: Zmax = +2.45, theta = 0.10; COL1A1: Zmax = +0.97, theta = 0.00) and markers near the keratin 5 gene on chromosome 12 were excluded (D12S17: Z less than -2.0, theta = 0.08; COL2A1: Z less than -2.0, theta = 0.13). In contrast, the second family showed linkage to the region containing the keratin 5 gene (D12S17: Zmax = +1.37, theta = 0.08; COL2A1: Zmax = +0.33, theta = 0.15) and was not linked to the keratin 14 gene (D17S74: Z less than -2.0, theta = 0.14). The Weber-Cockayne form of EBS is genetically heterogeneous with linkage to different keratin gene loci.     

Linkage analysis of five fibrillar collagen loci in a large French Marfan syndrome family.

Marfan syndrome consists of a group of dominantly inherited disorders of connective tissue with wide clinical variability. Using the candidate gene approach, we have attempted to map the gene defect in a large French Marfan syndrome family with no ocular manifestations. We performed linkage studies with polymorphic probes for five structural procollagen genes. The data obtained exclude linkage of Marfan syndrome to the two major fibrillar collagen (COL1A1, COL1A2, and COL2A1) genes. These results confirm previously published data obtained from smaller pedigrees. A small positive lod score (Z = 0.99, theta = 0.00) was obtained for the COL3A1-COL5A2 gene cluster located on chromosome 2.     

The molecular basis of classic Ehlers-Danlos syndrome: a comprehensive study of biochemical and molecular findings in 48 unrelated patients

Classic Ehlers-Danlos syndrome (EDS) is characterized by fragile and hyperextensible skin, atrophic scarring, and joint hypermobility. Mutations in the COL5A1 and the COL5A2 gene encoding the alpha1(V) and the alpha2(V) chains, respectively, of type V collagen have been shown to cause the disorder, but it is unknown what proportion of classic EDS patients carries a mutation in these genes. We studied fibroblast cultures from 48 patients with classic EDS by SDS-PAGE for the presence of type V collagen defects. An abnormal collagen pattern was detected in only 2 out of 48 cell lines, making this a poor method for routine diagnostic evaluation. A total of 42 out of 48 (88%) patients were heterozygous for an expressed polymorphic variant in COL5A1. cDNA from 18 (43%) of them expressed only one COL5A1 allele. In 37 patients, the COL5A1/A2 genes were then analyzed by SSCP and conformation sensitive gel electrophoresis (CSGE). A total of 26 patients that were mutation-negative after SSCP/CSGE screening were reanalyzed by dHPLC. In addition, 11 other patients were analyzed by dHPLC only. In total, 17 mutations leading to a premature stop codon and five structural mutations were identified in the COL5A1 and the COL5A2 genes. In three patients with a positive COL5A1 null-allele test, no causal mutation was found. Overall, in 25 out of 48 patients (52%) with classic EDS, an abnormality in type V collagen was confirmed. Variability in severity of the phenotype was observed, but no significant genotype-phenotype correlations emerged. The relatively low mutation detection rate suggests that other genes are involved in classic EDS. We excluded the COL1A1, COL1A2, and DCN gene as major candidate genes for classic EDS, since no causal mutation in these genes was found in a number of patients who tested negative for COL5A1 and COL5A2.     

Association between COL1A1 gene polymorphisms and bone size in Caucasians

Bone size is an important determinant of bone strength and a risk factor of osteoporotic fracture. Several studies indicate that bone size has a high heritability. Thus, a better understanding of genetic factors regulating bone size might have important clinical implications. In the present study, we examined the relationship between the collagen type I alpha 1 (COL1A1) gene and bone size at the spine, hip and wrist in a sample of 1873 subjects of Caucasian origin from 405 nuclear families. Three single-nucleotide polymorphisms (SNPs) in the COL1A1 gene were analyzed. The minor allele frequencies were 15.4, 18.8, and 1.9% for SNP1, SNP2, and SNP3, respectively. Haplotypes were reconstructed based on the family information as well as marker genotypes using the program Genehunter. We did not find evidence of population stratification, within-family association, or linkage for either single SNPs or haplotypes at any skeletal site. Suggestive evidence of total association was observed for the wrist size at SNP2 (P=0.011). After adjusting age, sex, height, and weight, subjects with the T allele of SNP2 had, on average, 3.05% smaller wrist size than noncarriers. When the subjects were divided into families with only female offspring and families with male offspring only, similar total associations were found at the wrist size for SNP2 with P-values of 0.011 and 0.010, respectively. In conclusion, the COL1A1 gene may have some effects on bone size variation at the wrist, but not at the spine or hip in our Caucasian nuclear families.     

Stickler syndrome: further mutations in COL11A1 and evidence for additional locus heterogeneity

Stickler syndrome (hereditary arthro-ophthalmopathy) is a dominantly inherited connective tissue disorder with ocular, oro-facial, auditory and skeletal manifestations. It is genetically and phenotypically heterogeneous with the majority of families having mutations in the gene encoding type II collagen (COL2A1) and exhibiting a characteristic 'membranous' or type 1 vitreous phenotype. More recently a novel mutation in the gene encoding the alpha1 chain of type XI collagen (COL11A1) was reported in a Stickler syndrome pedigree with a different 'beaded' or type 2 vitreous phenotype. In the present study five more families with the type 2 vitreous phenotype were examined for linkage to four candidate genes: COL2A1, COL5A2, COL11A1 and COL11A2. Two families were linked to COL11A1 and sequencing identified mutations resulting in shortened alphal(XI) collagen chains, one via exon skipping and the other via a multiexon deletion. One of the families showed weak linkage to COL5A2 but sequencing the open reading frame failed to identify a mutation. In the remaining two families all four loci were excluded by linkage analysis. These data confirm that mutations in COL11A1 cause Stickler syndrome with the type2 vitreous phenotype and also reveal further locus heterogeneity.     

Segregation analysis of dominant osteogenesis imperfecta in Italy.

We have performed linkage analysis in seven Italian families, in which mild osteogenesis imperfecta (OI) segregated as a dominant trait, by means of six DNA restriction fragment length polymorphisms (RFLPs) of type I collagen genes. OI type I was linked to the alpha 1(I) gene (COL1A1) in two families, and to the alpha 2(I) gene (COL1A2) in one family. OI type IV segregated with COL1A2 in two families. In two OI type I families, the molecular genetic data were insufficient for exclusion of one gene. Four DNA polymorphisms were particularly informative for cosegregation analysis of OI in Italian kindreds.     

Missense mutation in a von Willebrand factor type A domain of the alpha 3(VI) collagen gene (COL6A3) in a family with Bethlem myopathy

The Bethlem myopathy is a rare autosomal dominant proximal myopathy characterized by early childhood onset and joint contractures. Evidence for linkage and genetic heterogeneity has been established, with the majority of families linked to 21q22.3 and one large family linked to 2q37, implicating the three type VI collagen subunit genes, COL6A1 (chromosome 21), COL6A2 (chromosome 21) and COL6A3 (chromosome 2) as candidate genes. Mutations of the invariant glycine residues in the triple-helical domain-coding region of COL6A1 and COL6A2 have been reported previously in the chromosome 21-linked families. We report here the identification of a G-->A mutation in the N-terminal globular domain-coding region of COL6A3 in a large American pedigree (19 affected, 12 unaffected), leading to the substitution of glycine by glutamic acid in the N2 motif, which is homologous to the type A domains of the von Willebrand factor. This mutation segregated to all affected family members, to no unaffected family members, and was not identified in 338 unrelated Caucasian control chromosomes. Thus mutations in either the triple-helical domain or the globular domain of type VI collagen appear to cause Bethlem myopathy.      

Mutations of COL10A1 in Schmid metaphyseal chondrodysplasia

Schmid metaphyseal chondrodysplasia (SMCD) is a dominantly inherited cartilage disorder caused by mutations in the gene for the hypertrophic cartilage extracellular matrix structural protein, collagen X (COL10A1). Thirty heterozygous mutations have been described, about equally divided into two mutation types, missense mutations, and mutations that introduce premature termination signals. The COL10A1 mutations are clustered (33/36) in the 3' region of exon 3, which codes for the C-terminal NC1 trimerization domain. The effect of COL10A1 missense mutations have been examined by in vitro expression and assembly assays and cell transfection studies, which suggest that a common consequence is the disruption of collagen X trimerization and secretion, with consequent intracellular degradation. The effect of COL10A1 nonsense mutations in cartilage tissue has been examined in two patients, demonstrating that the mutant mRNA is completely removed by nonsense mediated mRNA decay. Thus for both classes of mutations, functional haploinsufficiency is the most probable cause of the clinical phenotype in SMCD.     

Autosomal dominant spondylarthropathy due to a type II procollagen gene(COL2A1) point mutation

Osteoarthrosis represents a very common disease with heterogeneous etiology. In some pedigrees linkage of the condition with the type II collagen gene (COL2A1) has been established, but information on the underlying gene defect is still incomplete as only one mutation causing this phenotype has been identified. We analyzed the COL2A1 gene in a 27-year-old woman and her 47-year-old mother presenting with severe premature osteoarthrosis and X-ray signs compatible with mild spondyloepiphyseal dysplasia. Examination of the complete gene in both patients was done by amplification of all 54 exons, screening of the PCR products by SSCP-analysis, and subsequent sequencing. In mother and daughter a G to A transition at the 5′-end of exon 21 was detected, leading to a substitution of serine for glycine at position 274 of the triple helical domain. The mutation was not present in unaffected family members or in healthy control individuals. The autosomal dominant spondylarthropathies may represent the less severe entities of the clinical spectrum of type II collagenopathies. © 1994 Wiley-Liss, Inc.     

Linkage analysis in dominantly inherited osteogenesis imperfecta

The only serious attempts at linkage in osteogenesis imperfecta (OI) have shown that the disease is linked to type 1 collagen genes in all families studied in which it segregrates as a clear mendelian dominant trait. For prenatal diagnosis the probability that a new family is linked can be taken as greater than 0.95 and this figure is augmented as more meioses are studied. Some phenotype correlations, notably between the OI type IV phenotype and linkage to COL1A2 and between presenile hearing loss in OI type I and linkage to COL1A1, can be used to improve risk estimates substantially in families where there are no segregation data to distinguish whether COL1A1 or COL1A2 is the mutant locus.     

Use of type VII collagen gene (COL7A1) markers in prenatal diagnosis of recessive dystrophic epidermolysis bullosa.

Generalised recessive dystrophic epidermolysis bullosa (EB) is a severe inherited disease in which patients suffer from blistering and scarring of the skin and mucous membranes after minor mechanical trauma. Tight genetic linkage has been established to the type VII collagen gene (COL7A1) at 3p21, with no evidence of locus heterogeneity. Several COL7A1 mutations have now been identified in recessive dystrophic EB patients. Prenatal diagnosis has been performed by examination of a fetal skin biopsy taken at about 16 weeks' gestation, and relies on identification of characteristic ultrastructural and immunohistochemical changes. We have now achieved a first trimester prenatal diagnosis using intragenic and flanking COL7A1 markers in a pregnancy at risk for recessive dystrophic EB. Segregation of the informative markers predicted the baby would be an unaffected carrier. The pregnancy continued to term and a healthy baby was born, confirming this result.     

Epistatic Interactions between Genomic Regions Containing the COL1A1 Gene and Genes Regulating Osteoclast Differentiation may Influence Femoral Neck Bone Mineral Density

Bone mineral density (BMD) is a primary risk indicator of osteoporotic fractures, which are largely determined by the actions of multiple genes. Genetic linkage studies have seldom explored epistatic interaction of genes for BMD. To evaluate potential genetic interactions for BMD at the femoral neck (FN) we conducted a variance component linkage analysis, to test epistatic effects between the genomic region containing the COL1A1 (collagen type I alpha 1) gene and the genomic regions containing genes regulating osteoclast differentiation (e.g. TNFRSF11A encoding RANK (receptor for activation of nuclear factor kappa B), TNFSF11 encoding RANKL (RANK ligand), IL1A (interleukin-1 alpha), IL6 (interleukin-6), etc) in 3998 Caucasian subjects from 434 pedigrees. We detected significant epistatic interactions between the regions containing COL1A1 with IL6 (p = 0.004) and TNFRSF1B encoding TNFR2 (tumor necrosis factor receptor 2) (p = 0.003), respectively. In summary, we identified the epistatic effects on BMD between regions containing several prominent candidate genes. Our results suggested that the IL6 and TNFRSF1B genes may regulate FN BMD variation through interactions with the COL1A1 gene, which should be substantiated by other, or population-based, association studies.     

成骨不全一家系的COL1A1基因突变分析

目的探讨一个成骨不全家系的COL1A1基因的突变位点及其与临床特征的关系。方法收集一个成骨不全家系的临床资料，采用聚合酶链反应以及直接测序法对家系内成员进行COL1A1基因突变位点检测，同时对50名无血缘关系健康对照者的该位点进行限制性内切酶分析。结果该家系中成骨不全患者均存在COL1A1基因的第2461位点G→A突变（17．821S），但其临床特征不一致。而在家系内非患者及正常对照者中均未发现该突变。结论COL1A1基因突变是中国人群中成骨不全致病原因之一。成骨不全的表型不仅与基因型有关，还与遗传背景有关。     

Population haplotype analysis and evolutionary relations of the COL2A1 gene

We have determined the allele frequencies and pairwise linkage disequilibria of restriction fragment length polymorphisms (RFLPs) distributed over the entire COL2A1 gene (spanning 23.6 kb) in a population of unrelated Dutch Caucasians. Pairwise linkage disequilibrium analysis of RFLP sites between exon 5B and 51 indicated a high degree of partly positive (the rare alleles of both loci are associated) and partly negative (the rare allele is associated with the common allele) linkage disequilibrium.
The high degree of linkage disequilibrium enabled the assignment of 13 out of 128 possible haplotypes with 7 RFLPs. An evolutionary tree of these haplotypes was derived using a minimum spanning tree approach, indicating at least two ancestral haplotypes. Our data indicate that disease related population studies involving the COL2A1 gene should include a minimum of 4 RFLPs (D9, A9, H33, P51) to obtain 98% of possible haplotypes occurring.     

A family with Classical Ehlers-Danlos Syndrome (cEDS), mild bone fragility and without vascular complications,caused by the p.Arg312Cys mutation in COL1A1

The Ehlers-Danlos syndromes (EDS) are heritable disorders of connective tissue (HDCT) with joint hypermobility, skin hyperextensibility and tissue fragility, which were recently re-classified (2017 International Classification). Most patients (>90%) with Classical Ehlers-Danlos syndrome (cEDS) have a mutation in the COL5A1 or COL5A2 genes encoding type V procollagen. A small number of patients with the p.Arg312Cys mutation in COL1A1 have been reported with overlapping features of both cEDS and vascular EDS (vEDS). In this report, we describe two patients from a large family with this mutation and clinical features consistent with cEDS without vascular complications. The proband presented with congenital hip dislocation (previously reported in one patient), the mother of the proband with multiple fractures in childhood, and dental defects (novel findings). The small number of patients reported with this mutation and proportion with vascular complications suggests that vascular surveillance should still be recommended.     

Exclusion of the COL2A1 gene as the mutation site in diastrophic dysplasia.

The involvement of the cartilage specific type II collagen gene (COL2A1) was studied in nine patients with diastrophic dysplasia in the Finnish population, where the prevalence of this chondrodystrophy clearly exceeds that reported for other populations. COL2A1 was chosen as the candidate gene based on previous morphological and chemical studies which suggested abnormal structure of type II collagen in diastrophic dysplasia. Southern analysis of the patients' DNA showed no disease related differences in any of the restriction fragments covering the 30 kb COL2A1 gene. As a second approach, the nine patients and their 74 relatives were studied for the inheritance of the type II collagen gene. Three of the patients with diastrophic dysplasia were not homozygous for the intragenic RFLP markers, which suggests that the disease is not linked to the type II collagen gene. Multipoint linkage analysis gave a lod score of -2.95, which conclusively excluded the COL2A1 gene as the mutation site in diastrophic dysplasia in these families.