Spondyloepiphyseal dysplasia in a Cape Town family: linkage with the gene for type II collagen (COL2A1).

A moderately severe form of autosomal dominant (AD) spondyloepiphyseal dysplasia (SED) has been documented in 14 individuals in 3 generations of a family in Cape Town, South Africa. Affected persons had a short trunk; radiographic investigations indicated that skeletal involvement was worst in the hips and spine. Linkage studies with restriction fragment length polymorphisms (RFLPs) associated with the COL2A1 gene and the phenotype yielded a maximal LOD score of 4.51 at theta = 0.00. This result suggests that the structural locus for type II collagen is primarily involved in the pathogenesis of this form of SED.     

Spondyloepiphyseal dysplasia in a cape town family: Linkage with the gene for type II collagen (COL2A1)

A moderately severe form of autosomal dominant (AD) spondyloepiphyseal dysplasia (SED) has been documented in 14 individuals in 3 generations of a family in Cape Town, South Africa. Affected persons had a short trunk; radiographic investigations indicated that skeletal involvement was worst in the hips and spine. Linkage studies with restriction fragment length polymorphisms (RFLPs) associated with the COL2A1 gene and the phenotype yielded a maximal LOD score of 4.51 at theta = 0.00. This result suggests that the structural locus for type II collagen is primarily involved in the pathogenesis of this form of SED. © 1992 Wiley-Liss, Inc.     

Autosomal dominant (Beukes) premature degenerative osteoarthropathy of the hip joint unlinked to COL2A1

Molecular investigations have been undertaken in several separate large South African families with autosomal dominant skeletal dysplasias in which premature degenerative osteoarthropathy of the hip joint was the major manifestation. There are sometimes additional minor changes in the spine and these conditions fall into the general spondyloepiphyseal dysplasia (SED) nosological category. In some kindreds, linkage between phenotype and the type II collagen gene (COL2A1) has been established, while in others there is no linkage. We have now completed molecular linkage investigations in an Afrikaner family named Beukes, in which 47 members in 6 generations have premature osteoarthropathy of the hip joint. A LOD score of minus infinity indicates that this condition is not the result of a defect of the COL2A1 gene. © 1994 Wiley-Liss, Inc.     

Stickler-like syndrome due to a dominant negative mutation in the COL2A1 gene

The type II collagenopathies include a wide spectrum of phenotypes ranging from mild spondylo epiphyseal dysplasia (SED) to severe achondrogenesis/ hypochondrogenesis. Several attempts have been made at providing phenotype-genotype correlations in this group of disorders. In this report we discuss a South African family in which four members have a phenotype resembling Stickler syndrome type 1. Ocular problems and conductive deafness predominate, while skeletal changes resemble those of a mild form of multiple epiphyseal dysplasia (MED). In distinction to the classical form of Stickler syndrome, the affected persons have stubby digits. DNA analysis of the exons of the COL2A1 gene documented a C-T transversion in exon 39, resulting in an Arg704Cys substitution in the triple helical domain of the type II collagen peptide; this nontermination mutation may be indicative of further heterogeneity in the Stickler group of disorders or of a new syndrome amongst the type II collagenopathies. Am. J. Med. Genet. 80:6–11, 1998. © 1998 Wiley-Liss, Inc.     

Ehlers-Danlos syndrome and type III collagen abnormalities: a variable clinical

Ehlers-Danlos syndrome (EDS) comprises ten types. EDS IV is the most severe type because of its often lethal complications, such as arterial rupture. EDS IV is caused by an abnormality of collagen type III as a result of mutations in the corresponding gene COL3A1. A collagen type III abnormality is also seen in patients with EDS without the classical severe EDS IV phenotype. We report on 11 patients with type III collagen abnormality and normal collagen V in whom clinically EDS II, III, and IV were diagnosed. There is no correlation between the type of collagen III anomaly and the clinical phenotype. It is concluded that type III collagen abnormality may lead to a phenotypic spectrum and that it does not predict the severity and course of the disease.     

Vitreoretinopathy with phalangeal epiphyseal dysplasia,a type II collagenopathy resulting from a novel mutation in the C-propeptide region of the molecule

A large family with dominantly inherited rhegmatogenous retinal detachment, premature arthropathy, and development of phalangeal epiphyseal dysplasia, resulting in brachydactyly was linked to COL2A1, the gene encoding proalpha1(II) collagen. Mutational analysis of the gene by exon sequencing identified a novel mutation in the C-propeptide region of the molecule. The glycine to aspartic acid change occurred in a region that is highly conserved in all fibrillar collagen molecules. The resulting phenotype does not fit easily into pre-existing subgroups of the type II collagenopathies, which includes spondyloepiphyseal dysplasia, and the Kniest, Strudwick, and Stickler dysplasias.     

A family with Stickler syndrome type 2 has a mutation in the COL11A1 gene resulting in the substitution of glycine 97 by valine in alpha 1 (XI) collagen

Stickler syndrome (hereditary arthro-ophthalmopathy) is the commonest inherited cause of retinal detachment and one of the commonest autosomal dominant connective tissue dysplasias. There is clinical and locus heterogeneity with about two thirds of families linked to the gene encoding type II procollagen (COL2A1). Families with Sticklers syndrome type 1 have a characteristic congenital vitreous anomaly and are linked without recombination to markers at the COL2A1 locus. In contrast families with the type 2 variety have a different vitreo- retinal phenotype and are not linked to the COL2A1 gene. Type XI collagen is a quantitatively minor fibrillar collagen related to type V collagen and associated with the more abundant type II collagen fibrils. A mutation in COL11A2, the gene for alpha 2 (XI) procollagen, has recently been found in a family described as having Stickler syndrome, although there was no ocular involvement. Here we show for the first time that a family with the full Type 2 Stickler syndrome including vitreous and retinal abnormalities is linked to the COL11A1 gene and characterise the mutation as a Glycine to Valine substitution at position 97 of the triple helical domain caused by a single base G-- >T mutation. These results are the first to provide confirmation that type XI collagen is an important structural component of human vitreous. They also support previous work suggesting that mutations in the genes encoding collagen XI can give rise to some manifestations of Stickler syndrome, but of these, only mutations in COL11A1 will give the full syndrome including the vitreo-retinal features.      

Type II collagen screening in the human chondrodysplasias

Abnormalities of type II collagen have been considered strong candidates for causing human condrodysplasias. We have employed peptide mapping to screen for several types of type II colagen abnormalities in cartilage samples from 66 patients with 20 separate disorders. Except for achondrogenesis type II (Langer-Saldino) and spondyloepiphyseal dysplasia (SED) congenita in which abnormalities have been described and diastrophic dysplasia in which the changes were probably secondary, no abnormalities were detected. Within the limitations of the screening technique, the results combined with other data from the literature suggest that abnormalities of this molecule are not common causes of chondrodysplasias outside of the achondrogenesis type II-SED congenita family of disorders.     

Identification of a locus for a form of spondyloepiphyseal dysplasia on chromosome 15q26.1: exclusion of aggrecan as a candidate gene

We have investigated a family with an autosomal dominant form of spondyloepiphyseal dysplasia (SED) characterised by short stature and severe premature degenerative arthropathy. Previous studies have excluded linkage between this condition and the locus for the type II collagen gene. Here we report the identification of linkage between this disorder and a locus on the long arm of chromosome 15 between markers D15S979 and D15S1004. According to current linkage maps and sequence data, this locus includes that of the aggrecan gene (AGC1). Our linkage data from the SED family show, however, that AGC1 maps to a locus that is proximal to D15S979. This proximal location for AGC1 is further supported by linkage data from a second family with an autosomal recessive form of multiple epiphyseal dysplasia that also maps to the SED locus. In both families AGC1 is therefore excluded as a candidate gene.     

Cells expressing partially unfolded R789C/p.R989C type II procollagen mutant associated with spondyloepiphyseal dysplasia undergo apoptosis

We investigated the effects of the presence of R75C (p.R275C), R519C (p.719C), R789C (p.R989C), and G853E (p.G1053E) type II collagen (COL2A1) mutants, associated with distinct forms of spondyloepiphyseal dysplasia (SED), on the biological processes occurring in chondrocytic cells harboring those mutants. Mutant-specific biological responses of cells were initiated by activating tetracycline (Tet)-dependent expression of type II collagen mutants. Employing microscopic and biochemical assays, we determined that cells expressing the thermolabile R789C (p.R989C) type II collagen mutant undergo apoptosis. In contrast, in cells expressing the thermostable R75C (p.R275C), R519C (p.719C), and G853E (p.G1053E) mutants, apoptotic markers were not apparent. We also demonstrated that the R789C (p.R989C) mutant formed atypical complexes with endoplasmic reticulum (ER)-resident chaperones, thereby indicating an "unfolded protein response" (UPR) of cells harboring this specific mutant. Apoptotic changes were also demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and cleaved caspase 3 assays in the growth plates of mice harboring the R992C (p.R1147C) substitution in type II collagen. Based on these results, we propose that the intracellular presence of structurally altered type II collagen mutants could activate an apoptotic response, thereby limiting cell survival. By analyzing the response of cells to the altered structure of collagen mutants, our study contributes to better understanding the molecular basis of the pathological changes seen in vivo at the tissue level.     

An α1 II Gly913 to Cys substitution prevents the matrix incorporation of type II collagen which is replaced with type I and III collagens in cartilage from a patient with hypochondrogenesis

A heterozygous mutation in the COL2A1 gene was identified in a patient with hypochondrogenesis. The mutation was a single nucleotide transition of G3285T that resulted in an amino acid substitution of Cys for Gly⁹¹³ in the α1(II) chain of type II collagen. This amino acid change disrupted the obligatory Gly-X-Y triplet motif required for the normal formation of a stable collagen triple helix and prevented the deposition of type II collagen into the proposita's cartilage, which contained predominantly type I and III collagens and minor amounts of type XI collagen. Biosynthetic analysis of collagens produced and secreted by the patient's chondrocytes cultured in alginate beads was consistent with the in vivo matrix composition, demonstrating that the main products were type I and III collagens, along with type XI collagen. The synthesis of the cartilage-specific type XI collagen at similar levels to controls indicated that the isolated cartilage cells had re-differentiated to the chondrocyte phenotype. The chondrocytes also produced small amounts of type II collagen, but this was post-translationally overmodified and not secreted. These data further delineate the biochemical and phenotypic consequences of mutations in the COL2A1 gene and suggest that cartilage formation and bone development can take place in the absence of type II collagen. © 1996 Wiley-Liss, Inc.     

Exclusion of type II and type VI procollagen gene mutations in a five-generation family with multiple epiphyseal dysplasia

We have studied a family with an autosomal dominant form of multiple epiphyseal dysplasia (MED) inherited through at least 5 generations. Bilateral deformity of the hips with subsequent degenerative arthritis was the most common and most severe change observed in the affected relatives. Abnormalities of the knees, ankles, and shoulders were also noted in some affected individuals. Radiological examination showed changes in affected joints consistent with epiphyseal dysplasia. In early stages, the articular surfaces appeared flattened or irregular in shape. In advanced stages, epiphyseal fragmentation, joint surface erosion, and extensive remodeling were observed. The abnormalities of the epiphyses suggested that the primary defect might be in a structural component of the epiphyseal cartilage matrix. The gene encoding type II collagen (COL2A1) was tested for genetic linkage to MED in this family by restriction fragment length polymorphism (RFLP) analysis. Recombination between COL2A1 and MED was observed, ruling out COL2A1 as the site of the mutation. The genes encoding the 3 chains of type VI collagen were also excluded on the basis of discordant inheritance. The disease in this family is therefore not the result of mutations in the genes encoding type II or type VI collagen. © 1993 Wiley-Liss, Inc.     

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.     

Mutation and polymorphism spectrum in osteogenesis imperfecta type II: implications for genotype-phenotype relationships

Osteogenesis imperfecta (OI), also known as brittle bone disease, is a clinically and genetically heterogeneous disorder primarily characterized by susceptibility to fracture. Although OI generally results from mutations in the type I collagen genes, COL1A1 and COL1A2, the relationship between genotype and phenotype is not yet well understood. To provide additional data for genotype-phenotype analyses and to determine the proportion of mutations in the type I collagen genes among subjects with lethal forms of OI, we sequenced the coding and exon-flanking regions of COL1A1 and COL1A2 in a cohort of 63 subjects with OI type II, the perinatal lethal form of the disease. We identified 61 distinct heterozygous mutations in type I collagen, including five non-synonymous rare variants of unknown significance, of which 43 had not been seen previously. In addition, we found 60 SNPs in COL1A1, of which 17 were not reported previously, and 82 in COL1A2, of which 18 are novel. In three samples without collagen mutations, we found inactivating mutations in CRTAP and LEPRE1, suggesting a frequency of these recessive mutations of approximately 5% in OI type II. A computational model that predicts the outcome of substitutions for glycine within the triple helical domain of collagen alpha1(I) chains predicted lethality with approximately 90% accuracy. The results contribute to the understanding of the etiology of OI by providing data to evaluate and refine current models relating genotype to phenotype and by providing an unbiased indication of the relative frequency of mutations in OI-associated genes.     

A single base mutation in COL5A2 causes Ehlers-Danlos syndrome type II.

Ehlers-Danlos syndrome (EDS) is a heterogeneous group of connective tissue disorders. Recently mutations have been found in the genes for type V collagen in a small number of people with the most common forms of EDS, types I and II. Here we characterise a COL5A2 mutation in an EDS II family. Cultured dermal fibroblasts obtained from an affected subject synthesised abnormal type V collagen. Haplotype analysis excluded COL5A1 but was concordant with COL5A2 as the disease locus. The entire open reading frame of the COL5A2 cDNA was directly sequenced and a single base mutation detected. It substituted a glycine residue within the triple helical domain (G934R) of alpha2(V) collagen, typical of the dominant negative changes in other collagens, which cause various other inherited connective tissue disorders. All three affected family members possessed the single base change, which was absent in 50 normal chromosomes.     

High efficiency of mutation detection in type 1 stickler syndrome using a two-stage approach: vitreoretinal assessment coupled with exon sequencing for screening COL2A1

Stickler syndrome is a genetically heterogeneous disorder that affects the ocular, skeletal, and auditory systems. To date three genes, COL2A1, COL11A1, and COL11A2, encoding the heterotypic type II/XI collagen fibrils present in vitreous and cartilage have been shown to have mutations that result in Stickler syndrome. As systemic features in this disorder are variable we have used an ophthalmic examination to differentiate those patients with a membranous vitreous phenotype associated with mutations in COL2A1, from other patients who may have mutations in other genes. Gene amplification and exon sequencing was used to screen 50 families or sporadic cases with this membranous phenotype, for mutations in COL2A1. Mutations were detected in 47 (94%) cases consisting of 166 affected and 78 unaffected individuals. We also demonstrate that the predominantly ocular form of type 1 Stickler syndrome is not confined to mutations in the alternatively spliced exon 2. Using splicing reporter constructs we demonstrate that a mutant GC donor splice site in intron 51 can be spliced normally; this contributed to the predominantly ocular phenotype in the family in which it occurred.     

The immune response of guinea-pigs to type II collagen: poor cross-reactivity with homologous type II collagen accounts for resistance to collagen-induced arthritis.

In order to determine the susceptibility of guinea-pigs to collagen-induced arthritis (CIA), Hartley and Strain 13 guinea-pigs were immunized with heterologous or homologous type II collagen. None of the animals developed CIA. Because immunity to type II collagen plays a critical role in CIA, we characterized the guinea-pig's immune response to determine the basis for this resistance. Guinea-pigs develop cellular and humoral reactivity to heterologous type II collagen similar to that of CIA-susceptible rats. The reactions distinguish type I from type II collagen but not among several heterologous type II collagens. The cell-mediated immune (CMI) response was specific for determinants on the primary amino acid structure of collagen, whether native or denatured collagen was used for immunization; however, the humoral response was specific for the form of the molecule used for immunization. Guinea-pigs differ from CIA-susceptible rats in that immunization with homologous or heterologous type II collagen fails to induce significant cross-reactive immunity with the homologous antigen. A transient arthritis could be induced in animals immunized with heterologous type II collagen by injecting them intra-articularly with heterologous but not with homologous type II collagen. Our results show that the disparity between immunity to type II collagen and the susceptibility to develop CIA in guinea-pigs is due to their poor cross-reactive immune response to autologous type II collagen.     

A specific collagen type II gene (COL2A1) mutation presenting as spondyloperipheral dysplasia

We report on a patient with a skeletal dysplasia characterized by short stature, spondylo-epiphyseal involvement, and brachydactyly E-like changes. This condition has been described as spondyloperipheral dysplasia and the few published cases suggest autosomal dominant inheritance with considerable clinical variability. We found our sporadic case to be due to a collagen type II defect resulting from a specific COL2A1 mutation. This mutation is the first to be located at the C-terminal outside the helical domain of COL2A1. A frameshift as consequence of a 5 bp duplication in exon 51 leads to a stop codon. The resulting truncated C-propeptide region seems to affect helix formation and produces changes of chondrocyte morphology, collagen type II fibril structure and cartilage matrix composition. Our case with its distinct phenotype adds another chondrodysplasia to the clinical spectrum of type II collagenopathies. © 1996 Wiley-Liss, Inc.     

Tissue and cell studies of the growth plate in the chondrodysplasias

As the morphologic expression of the chondrocytic differentiation pathway responsible for bone development and growth, the growth plate has been investigated extensively in the chondrodysplasias. Unique morphologic abnormalities identified in many disorders have provided insight into pathogenetic mechanisms and have been useful diagnostically and nosologically. Biochemical studies have detected evidence of type II collagen defects in patients having disorders in the achondrogenesis type II-spondyloepiphyseal dysplasias (SED) congenita family of chondrodysplasias. Most promising may be the cell culture systems now being developed for human chondrocytes. Preliminary results suggest that they will allow the cellular and molecular biology of the dysplastic growth plate to be directly analyzed.     

Dominant negative mutations in the C-propeptide of COL2A1 cause platyspondylic lethal skeletal dysplasia, torrance type, and define a novel subfamily within the type 2 collagenopathies

Platyspondylic lethal skeletal dysplasia (PLSD) Torrance type (PLSD-T) is a rare skeletal dysplasia characterized by platyspondyly, brachydactyly, and metaphyseal changes. Generally a perinatally lethal disease, a few long-term survivors have been reported. Recently, mutations in the carboxy-propeptide of type II collagen have been identified in two patients with PLSD-T, indicating that PLSD-T is a type 2 collagen-associated disorder. We studied eight additional cases of PLSD-T and found that all had mutations in the C-propeptide domain of COL2A1. The mutational spectrum includes missense, stop codon and frameshift mutations. All non-sense mutations were located in the last exon, where they would escape non-sense-mediated RNA-decay. We conclude that PLSD-T is caused by mutations in the C-propeptide domain of COL2A1, which lead to biosynthesis of an altered collagen chain (as opposed to a null allele). Similar mutations have recently been found to be the cause of spondyloperipheral dysplasia, a non-lethal dominant disorder whose clinical and radiographical features overlap those of the rare long-term survivors with PLSD-T. Thus, spondyloperipheral dysplasia and PLSD-T constitute a novel subfamily within the type II collagenopathies, associated with specific mutations in the C-propeptide domain and characterized by distinctive radiological features including metaphyseal changes and brachydactyly that set them apart from other type 2 collagenopathies associated with mutations in the triple-helical domain of COL2A1. The specific phenotype of C-propeptide mutations could result from a combination of diminished collagen fibril formation, toxic effects through the accumulation of unfolded collagen chains inside the chondrocytes, and alteration of a putative signaling function of the carboxy-propeptide of type 2 collagen.