A loss of function mutation in the COL9A2 gene causes autosomal recessive Stickler syndrome

Stickler syndrome is characterized by ocular, auditory, skeletal, and orofacial abnormalities. We describe a family with autosomal recessive Stickler syndrome. The main clinical findings consisted of high myopia, vitreoretinal degeneration, retinal detachment, hearing loss, and short stature. Affected family members were found to have a homozygous loss‐of‐function mutation in COL9A2, c.843_c.846 + 4del8. A family with autosomal recessive Stickler syndrome was previously described and found to have a homozygous loss‐of‐function mutation in COL9A1. COL9A1, COL9A2, and COL9A3 code for collagen IX. All three collagen IX α chains, α1, α2, and α3, are needed for formation of functional collagen IX molecule. In dogs, two causative loci have been identified in autosomal recessive oculoskeletal dysplasia. This dysplasia resembles Stickler syndrome. Recently, homozygous loss‐of‐function mutations in COL9A2 and COL9A3 were found to co‐segregate with the loci. Together the data from the present study and the previous studies suggest that loss‐of‐function mutations in any of the collagen IX genes can cause autosomal recessive Stickler syndrome. © 2011 Wiley‐Liss, Inc.     

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.     

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.     

LOXL3 novel mutation causing a rare form of autosomal recessive Stickler syndrome

Stickler syndrome is a collagenopathy that is typically inherited as autosomal dominant disease caused by monoallelic mutations in COL2A1, COL11A2, and COL11A1. Rarely, biallelic mutations in COL9A1, COL9A2, and COL9A3 cause an autosomal recessive Stickler syndrome. One previous report described two siblings with Stickler syndrome and a homozygous mutation in LOXL3, suggesting that biallelic mutations in LOXL3 can also cause autosomal recessive Stickler syndrome. LOXL3 is a member of the lysyl oxidase family of genes which encode enzymes oxidizing the side chain of peptidyl lysine permitting the covalent crosslinking of collagen and elastin chains. Therefore, LOXL3 deficiency is expected to result in collagen defect. Furthermore, Loxl3 deficient mouse model demonstrated features overlapping with Stickler syndrome. In this report, we describe a child and his father who had clinical features consistent with Stickler syndrome and found to have a homozygous novel mutation c.1036C>T (p.Arg346Trp) in LOXL3. This report not only supports that biallelic LOXL3 mutations cause autosomal recessive Stickler syndrome, but also further delineates the phenotype associated with LOXL3 mutations. In addition, the family described here shows an interesting example for pseudodominance, which can be observed in recessive diseases when one parent is affected and the other is heterozygous carrier.     

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.     

Novel COL9A3 mutation in a family with multiple epiphyseal dysplasia

Multiple epiphyseal dysplasia (MED) is a common skeletal dysplasia characterized by mild to moderate short stature, early-onset of osteoarthritis (OA) mainly in the hip and knee joints, and abnormally small and/or irregular epiphyses. MED is clinically and genetically heterogeneous. Six causative genes of MED have been reported, including type IX collagen genes (COL9A1, COL9A2, COL9A3). All the type IX collagen mutations previously reported cause exon skipping that loses the COL3 domain. Here we have identified a novel COL9A3 mutation co-segregating in a three-generation family with MED. The mutation (IVS3 + 5G > A) was speculated to lose the COL3 domain by skipping of exon 3, which was confirmed by in vitro analysis. The patients were of normal height and had minimal complaints with phenotypes being more severe in male patients. The radiographic phenotypes of the patients were relatively milder than those of previously reported cases, and were indistinguishable to common, idiopathic OA.     

Pseudoachondroplasia and multiple epiphyseal dysplasia: a 7-year comprehensive analysis of the known disease genes identify novel and recurrent mutations and provides an accurate assessment of their relative contribution

Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are relatively common skeletal dysplasias resulting in short-limbed dwarfism, joint pain, and stiffness. PSACH and the largest proportion of autosomal dominant MED (AD-MED) results from mutations in cartilage oligomeric matrix protein (COMP); however, AD-MED is genetically heterogenous and can also result from mutations in matrilin-3 (MATN3) and type IX collagen (COL9A1, COL9A2, and COL9A3). In contrast, autosomal recessive MED (rMED) appears to result exclusively from mutations in sulphate transporter solute carrier family 26 (SLC26A2). The diagnosis of PSACH and MED can be difficult for the nonexpert due to various complications and similarities with other related diseases and often mutation analysis is requested to either confirm or exclude the diagnosis. Since 2003, the European Skeletal Dysplasia Network (ESDN) has used an on-line review system to efficiently diagnose cases referred to the network prior to mutation analysis. In this study, we present the molecular findings in 130 patients referred to ESDN, which includes the identification of novel and recurrent mutations in over 100 patients. Furthermore, this study provides the first indication of the relative contribution of each gene and confirms that they account for the majority of PSACH and MED.     

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.     

Splicing mutations in the COL3 domain of collagen IX cause multiple epiphyseal dysplasia

We report on a three-generation family with multiple epiphyseal dysplasia (MED). The propositus had typical MED findings of knees, ankles, elbows, and hands in childhood. The 2 other affected relatives were adults. The main clinical findings consisted of osteochondritis dissecans and osteoarthritis of the knees. DNA of the propositus was screened for mutations by conformation sensitive gel electrophoresis in all known candidate genes for MED, cartilage oligomeric matrix protein, and the COL9A1, COL9A2, and COL9A3 genes coding for the alpha1, alpha2, and alpha3 chains of collagen IX. The screening identified a unique change in PCR products of exon 3 of the COL9A3 gene. Sequencing indicated a G to A mutation in the acceptor splice site (G(-1)IVS2-->A) of intron 2 in all affected relatives, but not in unaffected relatives. Analysis of RNA from the propositus indicated a skipping of exon 3, and thus, a deletion of 12 amino acid residues as a consequence of the mutation. All four other collagen IX mutations previously described in MED have consequences identical to that characterized here, thus it seems likely that this type of mutation in collagen IX plays an important role in the pathogenesis of MED.     

Alport syndrome: impact of digenic inheritance in patients management

Alport syndrome (ATS) is a genetically heterogeneous nephropathy with considerable phenotypic variability and different transmission patterns, including monogenic (X‐linked/autosomal) and digenic inheritance (DI). Here we present a new series of families with DI and we discuss the consequences for genetic counseling and risk assessment. Out of five families harboring variants in more than one COL4 gene detected by next generation sequencing (NGS), minigene‐splicing assay allowed us to identify four as true digenic. Two families showed COL4A3/A4 mutations in cis, mimicking an autosomal dominant inheritance with a more severe phenotype and one showed COL4A3/A4 mutations in trans, mimicking an autosomal recessive inheritance with a less severe phenotype. In a fourth family, a de novo mutation (COL4A5) combined with an inherited mutation (COL4A3) triggered a more severe phenotype. A fifth family, predicted digenic on the basis of silico tools, rather showed monogenic X‐linked inheritance due to a hypomorphic mutation, in accordance with a milder phenotype. In conclusion, this study highlights the impact of DI in ATS and explains the associated atypical presentations. More complex inheritance should be therefore considered when reviewing prognosis and recurrence risks. On the other side, these findings emphasize the importance to accompany NGS with splicing assays in order to avoid erroneous identification of at risk members.     

Prenatal course of metaphyseal anadysplasia associated with homozygous mutation in MMP9 identified by exome sequencing

Metaphyseal anadysplasia (MANDP) is a rare autosomal recessive form of skeletal dysplasia characterized by normal length at birth and transitory bowing of the legs. Although several families with MANDP have been reported, homozygous mutations in the matrix metalloproteinase type 9 (MMP9) gene have been described in only one consanguineous family, and thus the pre and postnatal phenotypic spectrum is still obscure. A clinically similar but more severe type is caused by autosomal‐dominant inheritance and is caused by mutations in matrix metalloproteinase type 13 gene (MMP13). Here, we report the prenatal and early postnatal course of two affected sib fetuses with early sonographic evidence of long bone shortening and postnatally no metaphyseal changes. Whole‐exome sequencing revealed homozygous mutation in MMP9 in both fetuses suggesting a diagnosis of MANDP. We propose that MANDP should be considered in pregnancies with early prenatal shortening of the long bones without associated finding of lethal skeletal dysplasias. In addition, the finding of homozygous mutation in non‐consanguineous parents of Jewish‐Caucasus ancestry may suggest unawareness of such relation or the occurrence of a founder mutation in this gene.     

Parental somatogonadal COL2A1 mosaicism contributes to intrafamilial recurrence in a family with type 2 collagenopathy

The COL2A1 gene encodes the alpha‐1 chain of procollagen type 2. Pathogenic variants in the COL2A1 gene are associated with several different types of skeletal dysplasia collectively known as type 2 collagenopathies. Type 2 collagenopathies have an autosomal dominant inheritance. Some germline or somatogonadal mosaicism cases have been reported. We investigated whether somatogonadal mosaicism occurred in a family with two children suspected of type 2 collagenopathies or related diseases. First, we detected a pathogenic variant in the COL2A1 gene in the two affected children by whole exome sequencing (WES). Next, we performed targeted deep sequencing to their parents without the variant by WES. A low level of COL2A1 mosaicism was revealed in the mother's tissues. We concluded that the mother had somatogonadal mosaicism with the COL2A1 mutation arose in the epiblast, and that the intrafamilial recurrence rate of the disease by the somatogonadal mosaicism was higher than by the germline mosaicism. This report suggests that parental low‐level mosaicism should be evaluated in those parents with children carrying de novo germline mutations and the targeted deep sequencing is useful to detect them.     

Vertical transmission of a frontonasal phenotype caused by a novel ALX4 mutation

Frontonasal dysplasias (FND) comprise a spectrum of disorders caused by abnormal median facial development. Its etiology is still poorly understood but recently frontonasal dysplasia phenotypes were linked to loss‐of‐function mutations in the ALX homeobox gene family, which comprises the ALX1, ALX3, and ALX4 genes. All ALX‐related frontonasal phenotypes till date had been compatible with an autosomal recessive mode of inheritance. In contrast, heterozygous loss‐of‐function mutations in ALX4 had been only associated with isolated symmetrical parietal ossification defects at the intersection of the sagittal and lambdoid sutures, known as enlarged parietal foramina. We report a family with vertical transmission from mother to son of mild frontonasal dysplasia phenotype caused by a novel ALX4 gene mutation (c.1080‐1089_delGACCCGGTGCinsCTAAGATCTCAACAGAGATGGCAACT, p.Asp326fsX21).This is the first report of a frontonasal phenotype related to a heterozygous mutation in ALX4. This mutation is predicted to cause the loss of the aristaless domain in the C‐terminal region of the protein and preserves the homeodomain. We speculate that a different mechanism, a dominant‐negative effect, is responsible for the distinct phenotype in this family. © 2013 Wiley Periodicals, Inc.     

Multiple SLC26A2 mutations occurring in a three-generational family

Multiple epiphyseal dysplasias (MED) are a group of heterogeneous skeletal dysplasias, which share a common phenotype: short stature, skeletal deformities, joint pain and early onset osteoarthritis. Mutations in COMP account for approximately half of autosomal dominant MED cases whilst SLC26A2 mutations account for ～25% of the recessive cases in the Caucasian population. We present here an interesting family, which was thought to initially have an autosomal dominant skeletal dysplasia. Using a targeted sequencing skeletal dysplasia panel, the proband was found to be a compound heterozygote for two mutations in SLC26A2, one novel mutation, p.Ser522Phe and the other, the common mutation, p.Arg279Trp. In addition to the classical characteristics of MED, she presented with an atypical feature, bilateral synostoses between the 2nd and 3rd metatarsals. The parents were confirmed to be heterozygous for the two mutations but interestingly, the maternal grandfather, who had MED, was found to be homozygous for the common SLC26A2 mutation.     

Mutations in the known genes are not the major cause of MED; distinctive phenotypic entities among patients with no identified mutations

Multiple epiphyseal dysplasia (MED) is a clinically and genetically heterogeneous chondrodysplasia. Mutations in six genes (COMP, COL9A1, COL9A2, COL9A3, MATN3 and DTDST) have been reported, but the genotype-phenotype correlations and the proportions of cases due to mutations in these genes are still not well characterized. We performed a clinical, radiological and molecular analysis of known MED genes on 29 consecutive MED patients. The mutation analysis resulted in identification of the DTDST mutation in four patients (14%), the COMP mutation in three (10%) and the MATN3 mutation in three (10%). Thus, a disease-causing mutation was identified in 10 patients altogether (34%). The phenotypic features observed in the patients with mutations were in accordance with previously described phenotypes, but two new distinct phenotypic entities were identified in patients in whom no mutation was found. One of them was characterized by severe, early-onset dysplasia of the proximal femurs with almost complete absence of the secondary ossification centres and abnormal development of the femoral necks. The other phenotype was characterized by 'mini-epiphyses', resulting in severe dysplasia of the proximal femoral heads. The findings suggest that mutations in the known genes are not the major cause of MED and are responsible for less than half of the cases. The existence of additional MED loci is supported by the exclusion of known loci by mutation analysis and finding of specific subgroups among these patients.     

Genotypic and phenotypic characterization of Chinese patients with osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a rare hereditary skeletal dysplasia, characterized by recurrent fractures and bone deformity. This study presents a clinical characterization and mutation analysis of 668 patients, aiming to establish the mutation spectrum and to elucidate genotype–phenotype correlations in Chinese OI patients. We identified 274 sequence variants (230 in type I collagen encoding genes and 44 in noncollagen genes), including 102 novel variants, in 340 probands with a detection rate of 90%. Compared with 47 loss‐of‐function variants detected in COL1A1, neither nonsense nor frameshift variants were found in COL1A2 (p < 0.0001). The major cause of autosomal recessive OI was biallelic variants in WNT1 (56%, 20/36). It is noteworthy that three genomic rearrangements, including one gross deletion and one gross duplication in COL1A1 as well as one gross deletion in FKBP10, were detected in this study. Of ten individuals with glycine substitutions that lie towards the N‐terminal end of the triple‐helical region of the α1(I) chain, none exhibited hearing loss, suggesting a potential genotype–phenotype correlation. The findings in this study expanded the mutation spectrum and identified novel correlations between genotype and phenotype in Chinese OI patients.