Identification of three novel mutations in the COL2A1 gene in four unrelated Chinese families with spondyloepiphyseal dysplasia congenita

Introduction

Spondyloepiphyseal dysplasia congenita (SEDC) is an autosomal dominant skeletal dysplasia characterized by short stature, abnormal epiphyses, and flattened vertebral bodies. The condition occurs through a mutation in the COL2A1 gene that encodes the type II procollagen alpha1 chain (proalpha1 (II)).

Method and Results

We investigated nine affected individuals from four unrelated Chinese families with SEDC. We screened for COL2A1 gene mutations, and identified found four missense mutations (G447A, G456A, R789C and G1152D). The G447A, G456A and G1152D mutations are novel and the R789C mutation has been reported previously in several other studies with a strikingly similar phenotype.

Conclusions

Our study extends the mutation spectrum of SEDC and is helpful in early molecular diagnoses of SEDC.     

Novel and recurrent COMP (cartilage oligomeric matrix protein) mutations in pseudoachondroplasia and multiple epiphyseal dysplasia

Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are common skeletal dysplasias with impaired enchondral ossification and premature degenerative joint disease. The two disorders were in the past considered to be distinct clinical entities; however, recent studies have proven that both diseases can result from mutations of the gene encoding cartilage oligomeric matrix protein (COMP). To characterize further COMP mutations and investigate phenotype-genotype relationships, we screened this gene in 15 patients with PSACH or MED by directly sequencing polymerase chain reaction products from genomic DNA. We identified ten mutations involving conserved residues among the eight calmodulin-like repeats of the gene product: seven were novel missense mutations in exons 9, 10, 11, 13 or 14, and the other three resulted from deletion of one of the five GAC repeats in exon 13. We have found that the GAC repeats in the 7th calmodulin-like repeat in exon 13 represent a hot-spot for mutation, and that mutations in the 7th calmodulin-like repeat produce severe PSACH phenotypes while mutations elsewhere in the gene exhibit mild PSACH or MED phenotypes. These genotype-phenotype correlations may facilitate molecular diagnosis and classification of PSACH and MED, and provide insight into the relationship between structure and function of the COMP gene product. Received: 1 June 1998 / Accepted: 22 October 1998     

Identification and molecular characterization of two novel mutations in COL1A2 in two Chinese families with osteogenesis imperfecta

Osteogenesis imperfecta (OI, also known as brittle bone disease) is caused mostly by mutations in two type Ⅰ collagen genes, COL1A1 and COL1A2 encoding the pro-α1 (Ⅰ) and pro-α2 (Ⅰ) chains of type Ⅰ collagen, respectively. Two Chinese families with autosomal dominant OI were identified and characterized. Linkage analysis revealed linkage of both families to COL1A2 on chromosome 7q21.3-q22.1. Mutational analysis was carried out using direct DNA sequence analysis. Two novel missense mutations, c.3350A＞G and c.3305G＞C, were identified in exon 49 of COL1A2 in the two families, respectively. The c.3305G＞C mutation resulted in substitution of a glycine residue (G) by an alanine residue (A) at codon 1102 (p.G1102A), which was found to be mutated into serine (S), argine (R), aspartic acid (D), or valine (V) in other families. The c.3350A＞G variant may be a de novo mutation resulting in p.Y1117C. Both mutations co-segregated with OI in respective families, and were not found in 100 normal controls. The G1102 and Y1117 residues were evolutionarily highly conserved from zebrafish to humans. Mutational analysis did not identify any mutation in the COX-2 gene (a modifier gene of OI). This study identifies two novel mutations p.G1102A and p.Y1117C that cause OI, significantly expands the spectrum of COL1A2 mutations causing OI, and has a significant implication in prenatal diagnosis of OI.     

Analysis of site-directed mutations in human pro-alpha 2(I) collagen which block cleavage by the C-proteinase

We have used site-directed mutagenesis to obtain human pro alpha 2(I) cDNAs containing novel mutations designed to inhibit cleavage at the C-proteinase site. Deletion of six relatively conserved amino acids which surround the cleavage site did not interfere with assembly of the triple helix in transfected rat cells, but blocked cleavage of the constituent mutated chains by endogenous C-proteinase. Substitution for a conserved Asp, which forms part of the Ala-Asp bond cleaved by C-proteinase, also blocked cleavage by endogenous C-proteinase. The conserved Asp is, therefore, a necessary component of the C-proteinase cleavage site. Incubation in vitro with a purified mouse C-proteinase, confirmed both mutations to be resistant to cleavage by high concentrations of the physiologically relevant enzyme. Mutant pro alpha 2(I) chains, resistant to cleavage by C-proteinase in culture media, were processed in cell layers by a different protease which cleaved telopeptide domains. Naturally occurring mutations at the C-proteinase site have not been described in human patients. The mutations characterized here, further define the C-proteinase cleavage site and provide reagents which may be informative when introduced into transgenic mice.     

Identification of two novel mutations in POU4F3 gene associated with autosomal dominant hearing loss in Chinese families

Autosomal dominant non‐syndromic hearing loss is genetically heterogeneous with 47 genes identified to date, including POU4F3. In this study, by using a next‐generation sequencing panel targeting 127 deafness genes, we identified a pathogenic frameshift mutation c.704_705del and a missense mutation c.593G>A in two three‐generation Chinese families with late‐onset progressive ADNSHL, respectively. The novel mutations of POU4F3 co‐segregated with the deafness phenotype in these two families. c.704_705del caused a frameshift p.T235fs and c.593G>A caused an amino acid substitution of p.R198H. Both mutations led to an abnormal and incomplete protein structure. POU4F3 with either of the two mutations was transiently transfected into HEI‐OC1 and HEK 293 cell lines and immunofluorescence assay was performed to investigate the subcellular localization of mutated protein. The results indicated that both c.704_705del (p.T235fs) and c.593G>A (p.R198H) could impair the nuclear localization function of POU4F3. The p.R198H POU4F3 protein was detected as a weak band of the correct molecular weight, indicating that the stability of p.R198H POU4F3 differed from that of the wild‐type protein. While, the p.T235fs POU4F3 protein was expressed with a smaller molecular weight, implying this mutation result in a frameshift and premature termination of the POU4F3 protein. In summary, we report two novel mutations of POU4F3 associated with progressive ADNSHL and explored their effects on POU4F3 nuclear localization. These findings expanded the mutation spectrum of POU4F3 and provided new knowledge for the pathogenesis of POU4F3 in hearing loss.     

Identification of mutations in two families with sporadic hemophilia A

Summary Direct sequencing of segments of the factor VIII gene in 30 hemophiliacs with sporadic disease (32+ kb of sequence in total) revealed two missense transitions: glutamate 1704 to lysine (E¹⁷⁰⁴K) in a patient with severe hemophilia A and proline 2300 to serine (P²³⁰⁰S) in a patient with mild hemophilia. Both transitions are likely to be causative mutations because the amino acids affected were evolutionarily conserved. Haplotype and sequence analysis of the mother and grandparents of patient HA12 (E¹⁷⁰⁴K) indicate that the mutation arose in the grandfather who was 27 years old when his daughter was conceived. The origin of mutation in patient HA39 (P²³⁰⁰S) could not be determined. As mutations that cause mild disease can be found in seemingly unrelated families, 96 unrelated hemophiliacs were screened rapidly for the P²³⁰⁰S mutation with polymerase chain reaction (PCR) amplification of specific alleles (PASA). None of these patients had the mutation. PASA was also used to conveniently assess a polymorphic site in intron 7. The polymorphism is estimated to be informative in 13% of Korean females and in 23% of Western European females.     

Phenotypic variability in two families with novel splice-site and frameshift NF2 mutations

Neurofibromatosis 2 (NF2) is a clinically variable autosomal dominant disorder, caused by mutations in the NF2 tumor suppressor gene on chromosome 22q12, that predisposes to nervous system tumors and ocular abnormalities. To assess intrafamilial phenotypic variability, we performed mutation analysis and clinical assessment on two multigeneration NF2 families with five patients and seven asymptomatic first-degree relatives of patients. One family had a point mutation of agCC→ggCC at position 1447–2 at the exon 13/14 boundary predicted to lead to an altered splice acceptor sequence and exon deletion. The other family had an insertion of 2 base pairs (TC) at position 761 in exon 8, leading to a frameshift. Both mild and severe phenotypes occurred in each family, indicating that phenotypic variability in NF2 can be caused by factors other than NF2 mutations. Genetic counseling of NF2 families should include the possibility that presymptomatic NF2 mutation carriers can develop a different phenotype than previously diagnosed patients. Received: 4 January 1996 / Revised: 26 March 1996     

High levels of expression of full length human pro-alpha 2(V) collagen cDNA in pro-alpha 2(V)-deficient hamster cells

A full length cDNA encoding human pro-alpha 2(V) collagen was constructed. Partial sequencing of the cDNA and primer extension analysis of mRNA from fibroblasts found that pro-alpha 2(V) mRNA differs from the mRNAs of other fibrillar collagens in the increased length of its 5'-untranslated region. The pro-alpha 2(V) cDNA was placed downstream of the human cytomegalovirus immediate early promoter/regulatory sequences for expression studies in cultured Chinese hamster lung cells. These cells have been shown previously to synthesize large quantities of pro-alpha 1(V) homotrimers as their only collagenous product. Transfection resulted in a number of clonal cell lines that express human alpha 2(V) RNA at levels comparable to, and in some cases greater than, levels found in normal human skin fibroblasts. Pro-alpha 2(V) chains produced in the majority of clonal lines were of sufficient quantity to complex all available endogenous pro-alpha 1(V) chains. Chimeric heterotrimers, composed of hamster alpha 1(V) and human alpha 2(V) chains in a 2:1 ratio, were stable to pepsin digestion and were found predominantly associated with the cell layer. Surprisingly, pro-alpha 2(V) chains, in excess to pro-alpha 1(V) chains, were found in the extracellular matrix and, in much greater abundance, in media. These chains were pepsin sensitive, indicating that pro-alpha 2(V) chains can be secreted as nonstable homotrimers or as free chains.     

Novel types of COMP mutations and genotype-phenotype association in pseudoachondroplasia and multiple epiphyseal dysplasia

Mutations in the gene encoding cartilage oligomeric matrix protein ( COMP) cause two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). More than 40 mutations have been identified; however, genotype-phenotype relationships are not well delineated. Further, mutations other than in-frame insertion/deletions and substitutions have not been found, and currently known mutations are clustered within relatively small regions. Here we report the identification of nine novel and three recurrent COMP mutations in PSACH and MED patients. These include two novel types of mutations; the first, a gross deletion spanning an exon-intron junction, causes an exon deletion. The second, a frameshift mutation that results in a truncation of the C-terminal domain, is the first known truncating mutation in the COMP gene. The remaining mutations, other than a novel exon 18 mutation, affected highly conserved aspartate or cysteine residues in the calmodulin-like repeat (CLR) region. Genotype-phenotype analysis revealed a correlation between the position and type of mutations and the severity of short stature. Mutations in the seventh CLR produced more severe short stature compared with mutations elsewhere in the CLRs ( P=0.0003) and elsewhere in the COMP gene ( P=0.0007). Patients carrying mutations within the five-aspartates repeat (aa 469-473) in the seventh CLR were extremely short (below -6 SD). Patients with deletion mutations were significantly shorter than those with substitution mutations ( P=0.0024). These findings expand the mutation spectrum of the COMP gene and highlight genotype-phenotype relationships, facilitating improved genetic diagnosis and analysis of COMP function in humans.     

A novel mutation in the EXT2 gene identified in two unrelated Chinese families with hereditary multiple exostoses

Hereditary multiple exostoses (HME) is an autosomal dominant disorder characterized by benign bone tumors. In this report, we describe two unrelated Chinese families with HME. Linkage analysis and mutation detection was performed. Clinical analysis was also performed for some affected individual in both families. Linkage with the EXT2 was established in both families. A novel mutation, c505 G > T, was identified in both families. Further allelic heterogeneity of EXT2 was demonstrated by the intrafamilial and interfamilial variability in clinical phenotype.     

Mutations in cartilage oligomeric matrix protein causing pseudoachondroplasia and multiple epiphyseal dysplasia affect binding of calcium and collagen I, II, and IX

Mutations in type 3 repeats of cartilage oligomeric matrix protein (COMP) cause two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). We expressed recombinant wild-type COMP that showed structural and functional properties identical to COMP isolated from cartilage. A fragment encompassing the eight type 3 repeats binds 14 calcium ions with moderate affinity and high cooperativity and presumably forms one large disulfide-bonded folding unit. A recombinant PSACH mutant COMP in which Asp-469 was deleted (D469 Delta) and a MED mutant COMP in which Asp-361 was substituted by Tyr (D361Y) were both secreted into the cell culture medium of human cells. Circular dichroism spectroscopy revealed only small changes in the secondary structures of D469 Delta and D361Y, demonstrating that the mutations do not dramatically affect the folding and stability of COMP. However, the local conformations of the type 3 repeats were disturbed, and the number of bound calcium ions was reduced to 10 and 8, respectively. In addition to collagen I and II, collagen IX also binds to COMP with high affinity. The PSACH and MED mutations reduce the binding to collagens I, II, and IX and result in an altered zinc dependence. These interactions may contribute to the development of the patient phenotypes and may explain why MED can also be caused by mutations in collagen IX genes.     

Identification of the type IX collagen polypeptide chains. The alpha 2(IX) polypeptide carries the chondroitin sulfate chain(s)

Type IX collagen has recently been shown to contain glycosaminoglycan chain(s) and furthermore to be immunologically identical with proteoglycan Lt (Vaughan, L., Winterhalter, K. H., and Bruckner, P. (1985) J. Biol. Chem. 260, 4758-4763). Here we demonstrate that the chondroitin sulfate carrying 115-kDa polypeptide of type IX collagen corresponds to the alpha 2(IX) chain. In addition the 84- and 68-kDa polypeptides were identified as the alpha 1(IX) and the alpha 3(IX) chains, respectively. This conclusion is based on a comparison of the tryptic fingerprints of the 84-, 115-, and 68-kDa chains of type IX collagen on high performance liquid chromatography with the similarly treated C2, C3, and C5 chains of the peptic fragment HMW. In addition, we provide evidence that both the C3 and C4 components of HMW are derived from the alpha 2(IX) chain.     

Biosynthetic processing of the pro-alpha 1(V)2pro-alpha 2(V) collagen heterotrimer by bone morphogenetic protein-1 and furin-like proprotein convertases.

The low abundance fibrillar collagen type V is incorporated into and regulates the diameters of type I collagen fibrils. Bone morphogenetic protein-1 (BMP-1) is a metalloprotease that plays key roles in regulating formation of vertebrate extracellular matrix; it cleaves the C-propeptides of the major fibrillar procollagens I-III and processes precursors to produce the mature forms of the cross-linking enzyme prolysyl oxidase, the proteoglycan biglycan, and the basement membrane protein laminin 5. Here we have successfully produced recombinant pro-alpha1(V)(2)pro-alpha2(V) heterotrimers, and we have used these to characterize biosynthetic processing of the most prevalent in vivo form of type V procollagen. In addition, we have compared the processing of endogenous pro-alpha1(V) chains by wild type mouse embryo fibroblasts and by fibroblasts derived from embryos doubly homozygous null for the Bmp-1 gene and for a gene encoding the closely related metalloprotease mammalian Tolloid-like 1. Together, results presented herein indicate that within pro-alpha1(V)(2)pro-alpha2(V) heterotrimers, pro-alpha1(V) N-propeptides and pro-alpha2(V) C-propeptides are processed by BMP-1-like enzymes, and pro-alpha1(V) C-propeptides are processed by furin-like proprotein convertases in vivo.