Mutations in DYNC2H1, the cytoplasmic dynein 2, heavy chain 1 motor protein gene, cause short-rib polydactyly type I, Saldino-Noonan type.

The short-rib polydactyly syndromes (SRPS) are autosomal recessively inherited, genetically heterogeneous skeletal ciliopathies. SRPS phenotypes were historically categorized as types I-IV, with type I first delineated by Saldino and Noonan in 1972. Characteristic findings among all forms of SRP include short horizontal ribs, short limbs and polydactyly. The SRP type I phenotype is characterized by a very small thorax, extreme micromelia, very short, poorly mineralized long bones, and multiple organ system anomalies. To date, the molecular basis of this most severe type of SRP, also known as Saldino-Noonan syndrome, has not been determined. We identified three SRP cases that fit the original phenotypic description of SRP type I. In all three cases, exome sequence analysis revealed compound heterozygosity for mutations in DYNC2H1, which encodes the main component of the retrograde IFT A motor, cytoplasmic dynein 2 heavy chain 1. Thus SRP type I, II, III and asphyxiating thoracic dystrophy (ATD), which also result from DYNC2H1 mutations. Herein we describe the phenotypic features, radiographic findings, and molecular basis of SRP type I.

Next-generation sequencing for disorders of low and high bone mineral density.

UNLABELLED: To achieve an efficient molecular diagnosis of osteogenesis imperfecta (OI), Ehlers-Danlos syndrome (EDS), and osteopetrosis (OPT), we designed a next-generation sequencing (NGS) platform to sequence 34 genes. We validated this platform on known cases and have successfully identified the causative mutation in most patients without a prior molecular diagnosis. INTRODUCTION: Osteogenesis imperfecta, Ehlers-Danlos syndrome, and osteopetrosis are collectively common inherited skeletal diseases. Evaluation of subjects with these conditions often includes molecular testing which has important counseling and therapeutic and sometimes legal implications. Since several different genes have been implicated in these conditions, Sanger sequencing of each gene can be a prohibitively expensive and time-consuming way to reach a molecular diagnosis. METHODS: In order to circumvent these problems, we have designed and tested a NGS platform that would allow simultaneous sequencing on a single diagnostic platform of different genes implicated in OI, OPT, EDS, and other inherited conditions, leading to low or high bone mineral density. We used a liquid-phase probe library that captures 602 exons (~100 kb) of 34 selected genes and have applied it to test clinical samples from patients with bone disorders. RESULTS: NGS of the captured exons by Illumina HiSeq 2000 resulted in an average coverage of over 900X. The platform was successfully validated by identifying mutations in six patients with known mutations. Moreover, in four patients with OI or OPT without a prior molecular diagnosis, the assay was able to detect the causative mutations. CONCLUSIONS: In conclusion, our NGS panel provides a fast and accurate method to arrive at a molecular diagnosis in most patients with inherited high or low bone mineral density disorders.

Identification of loss-of-function mutations of SLC35D1 in patients with Schneckenbecken dysplasia, but not with other severe spondylodysplastic dysplasias group diseases.

BACKGROUND: Schneckenbecken dysplasia (SBD) is an autosomal recessive lethal skeletal dysplasia that is classified into the severe spondylodysplastic dysplasias (SSDD) group in the international nosology for skeletal dysplasias. The radiological hallmark of SBD is the snail-like configuration of the hypoplastic iliac bone. SLC35D1 (solute carrier-35D1) is a nucleotide-sugar transporter involved in proteoglycan synthesis. Recently, based on human and mouse genetic studies, we showed that loss-of-function mutations of the SLC35D1 gene (SLC35D1) cause SBD. OBJECT: To explore further the range of SLC35D1 mutations in SBD and elucidate whether SLC35D1 mutations cause other skeletal dysplasias that belong to the SSDD group. METHODS AND RESULTS: We searched for SLC35D1 mutations in five families with SBD and 15 patients with other SSDD group diseases, including achodrogenesis type 1A, spondylometaphyseal dysplasia Sedaghatian type and fibrochondrogenesis. We identified four novel mutations, c.319C>T (p.R107X), IVS4+3A>G, a 4959-bp deletion causing the removal of exon 7 (p.R178fsX15), and c.193A>C (p. T65P), in three SBD families. Exon trapping assay showed IVS4+3A>G caused skipping of exon 4 and a frameshift (p.L109fsX18). Yeast complementation assay showed the T65P mutant protein lost the transporter activity of nucleotide sugars. Therefore, all these mutations result in loss of function. No SLC35D1 mutations were identified in all patients with other SSDD group diseases. CONCLUSION: Our findings suggest that SLC35D1 loss-of-function mutations result consistently in SBD and are exclusive to SBD.

Mutations in FLNB cause boomerang dysplasia.

Boomerang dysplasia (BD) is a perinatal lethal osteochondrodysplasia, characterised by absence or underossification of the limb bones and vertebrae. The BD phenotype is similar to a group of disorders including atelosteogenesis I, atelosteogenesis III, and dominantly inherited Larsen syndrome that we have recently shown to be associated with mutations in FLNB, the gene encoding the actin binding cytoskeletal protein, filamin B. We report the identification of mutations in FLNB in two unrelated individuals with boomerang dysplasia. The resultant substitutions, L171R and S235P, lie within the calponin homology 2 region of the actin binding domain of filamin B and occur at sites that are evolutionarily well conserved. These findings expand the phenotypic spectrum resulting from mutations in FLNB and underline the central role this protein plays during skeletogenesis in humans.

Fine mapping of the X-linked split-hand/split-foot malformation (SHFM2) locus to a 5.1-Mb region on Xq26.3 and analysis of candidate genes.

Split-hand/split-foot malformation (SHFM) is a genetically heterogeneous disorder, with five known loci, that causes a lack of median digital rays, syndactyly, and aplasia or hypoplasia of the phalanges, metacarpals, and metatarsals. In the only known SHFM2 family, affected males and homozygous females exhibit monodactyly or bidactyly of the hands and lobster-claw feet. This family (1) was revisited to include additional subjects and genealogical data. All 39 affected males and three females fully expressed the SHFM, while 13 carrier females examined exhibited partial expression of SHFM. We narrowed the previously linked 22-Mb genetic interval on Xq24-q26 (2), by analyzing additional family members and typing additional markers. The results define a 5.1-Mb region with a new centromeric boundary at DXS1114 and a telomeric boundary at DXS1192. We did not identify mutations in the exons and exon/intron boundaries of 19 candidate genes. These data suggest that the mutation may lie in a regulatory region of one of these candidate genes or in another gene within the SHFM2 region with unclear role in limb development.

Novel mutations in the EXT1 gene in two consanguineous families affected with multiple hereditary exostoses (familial osteochondromatosis).

Multiple hereditary exostoses (HME) is an autosomal dominant developmental disorder exhibiting multiple osteocartilaginous bone tumors that generally arise near the ends of growing long bones. Here, we report two large consanguineous families from Pakistan, who display the typical features of HME. Affected individuals also show a previously unreported feature--bilateral overriding of single toes. Analysis using microsatellite markers for each of the known EXT loci, EXT1, EXT2, and EXT3 showed linkage to EXT1. In the first family, mutation analysis of the EXT1 gene revealed that affected individuals were heterozygous for an in-frame G-to-C transversion at the conserved splice donor site in intron 1. This mutation is predicted to disrupt splicing of the first intron and produce a frameshift that leads to a premature termination codon. In the second family, an insertion of an A in exon 8 is predicted to produce a frameshift at codon 555 followed by a premature termination, a further 10 codons downstream. In both families, an increased number of affected male subjects were observed. In affected females in family 2, phenotypic variability and incomplete penetrance were noted.

A locus for spondylocarpotarsal synostosis syndrome at chromosome 3p14.

Spondylocarpotarsal synostosis syndrome is a rare autosomal recessive disorder characterised by vertebral fusions, frequently manifesting as an unsegmented vertebral bar, as well as fusions of the carpal and tarsal bones. In a study of three consanguineous families and one non-consanguineous family, linkage analysis was used to establish the chromosomal location of the disease gene. Linkage analysis localised the disease gene to chromosome 3p14. A maximum lod score of 6.49 (q = 0) was obtained for the marker at locus D3S3532 on chromosome 3p. Recombination mapping narrowed the linked region to the 5.7 cM genetic interval between the markers at loci D3S3724 and D3S1300. A common region of homozygosity was found between the markers at loci D3S3724 and D3S1300, defining a physical interval of approximately 4 million base pairs likely to contain the disease gene. Identification of the gene responsible for this disorder will provide insight into the genes that play a role in the formation of the vertebral column and joints.

Mutation in the cartilage-derived morphogenetic protein-1 (CDMP1) gene in a kindred affected with fibular hypoplasia and complex brachydactyly (DuPan syndrome).

The present authors have previously described a consanguineous Pakistani family with fibular hypoplasia and complex brachydactyly (DuPan syndrome) inherited as an autosomal recessive trait. All affected individuals showed either reductions or absence of bones in the limbs, and appendicular bone dysmorphogenesis with unaffected axial bones. Obligate heterozygote parents were phenotypically normal. Mutations in the cartilage-derived morphogenetic protein 1 (CDMP1) gene have been reported in two acromesomelic chondrodysplasias (i.e. Hunter-Thompson type and Grebe type) which are phenotypically related to DuPan syndrome. CDMP1, a member of the transforming growth factor beta super-family of secreted signalling molecules, has been reported to regulate limb patterning and distal bone growth. Therefore, the present authors examined genomic DNA from the family with DuPan syndrome for mutations in the CDMP1 gene. Affected individuals were homozygous for a missense mutation, T1322C, in the coding region of the CDMP1 gene. This mutation was not found in 44 control subjects of Pakistani origin. The T1322C change predicts a leu441pro substitution in the mature domain of the CDMP1 protein. This is likely to cause a conformational change in the CDMP1 protein that influences the expression of genes which are required for normal bone development. This finding extends the spectrum of phenotypes produced by defects in the CDMP1 gene.

Identification of human FEM1A, the ortholog of a C. elegans sex-differentiation gene.

We report the isolation, genomic structure, chromosomal location, and expression pattern of the FEM1A gene, the human ortholog of the Caenorhabditis elegans fem-1 and mouse Fem1a genes. The coding sequence is 1851 bp and encodes a 617 amino acid protein. The human FEM1A protein has 65% identity with the mouse Fem1a protein and 34% identity with the C. elegans fem-1 protein, indicating conservation of this protein. The N-terminal region of the encoded protein contains six ankyrin repeat elements, a motif found in signaling and transcriptional regulatory molecules such as Notch and glp1. The gene was highly expressed in human kidney and cardiac tissue, and was expressed at lower levels in multiple tissues, including cartilage. FEM1A was localized to chromosome 5q23.1, a region of conserved synteny with a portion of mouse chromosome 17 that contains Fem1a. In C. elegans, fem-1 is involved in a pathway necessary for sex determination. The identification of a human homolog of this conserved gene suggests a potential role for this sex-determining molecule in humans.

Double heterozygosity for pseudoachondroplasia and spondyloepiphyseal dysplasia congenita.

Pseudoachondroplasia (PSACH) and spondyloepiphyseal dysplasia congenita (SEDC) are autosomal dominant forms of short-limb short stature caused by mutations in genes that encode structural components of the cartilage extracellular matrix. PSACH results from mutations in the cartilage oligomeric matrix protein (COMP) gene, while SEDC is caused by mutations in the gene for type II procollagen (COL2A1). We report a child with a distinct skeletal dysplasia due to the combined phenotypes of PSACH and SEDC. The proband's mother had PSACH and his father had SEDC. The child was suspected of having both phenotypes on the basis of the severity of his clinical and radiographic findings, and this was confirmed by molecular analysis. The COMP gene mutation (C348R), while not previously published, is typical of those in PSACH patients, whereas the COL2A1 mutation (T1370M) is somewhat atypical, as it predicts an amino acid change within the carboxyl-terminal region of the protein. Both mutations segregated with their respective phenotypes within this family. The description and natural history of the double heterozygote phenotype may be useful in counseling families regarding risk and prognosis.

Procollagen II amino propeptide processing by ADAMTS-3. Insights on dermatosparaxis.

The amino and carboxyl propeptides of procollagens I and II are removed by specific enzymes as a prerequisite for fibril assembly. Null mutations in procollagen I N-propeptidase (ADAMTS-2) cause dermatosparaxis in cattle and the Ehlers-Danlos syndrome (dermatosparactic type) in humans by preventing proteolytic excision of the N-propeptide of procollagen I. We have found that procollagen II is processed normally in dermatosparactic nasal cartilage, suggesting the existence of another N-propeptidase(s). We investigated such a role for ADAMTS-3 in Swarm rat chondrosarcoma RCS-LTC cells, which fail to process the procollagen II N-propeptide. Stable transfection of RCS-LTC cells with bovine ADAMTS-2 or human ADAMTS-3 partially rescued the processing defect, suggesting that ADAMTS-3 has procollagen II N-propeptidase activity. Human skin and skin fibroblasts showed 30-fold higher mRNA levels of ADAMTS-2 than ADAMTS-3, whereas ADAMTS-3 mRNA was 5-fold higher than ADAMTS-2 mRNA in human cartilage. We propose that both ADAMTS-2 and ADAMTS-3 process procollagen II, but ADAMTS-3 is physiologically more relevant, given its preferred expression in cartilage. The findings provide an explanation for the sparing of cartilage in dermatosparaxis and, perhaps, for the relative sparing of some procollagen I-containing tissues.

Defects in extracellular matrix structural proteins in the osteochondrodysplasias.

Mutations in the genes that encode structural proteins of the extracellular matrix affect one or more steps in the diverse set of coordinated events necessary for ordered skeletal development. Depending on the role of the gene product and the severity of the defect, disruption of endochondral ossification and linear growth, the structural integrity and stability of articular cartilage, and/or mineralization can occur. Several themes have emerged from the molecular dissection of these disorders; most of the osteochondrodysplasias that result from defects in structural proteins are inherited in an autosomal dominant fashion; a spectrum of related clinical phenotypes can be produced by distinct mutations in the same gene; haploinsufficiency for the gene product usually produces a milder clinical phenotype than do mutations resulting in synthesis of structurally abnormal proteins. For structural defects, a dominant-negative effect resulting from presence of the abnormal protein in the matrix appears to be the primary determinant of phenotype. Secondary effects on extracellular matrix protein structure can result from defects in post-translational maturation, including hydroxylation, sulfation and proteolytic cleavage, and produce distinct osteochondrodysplasias. Overall, the inherited disorders of skeletogenesis have revealed the exquisite sensitivity of the architecture of the extracellular matrix to the quantity and quality of matrix molecules.

Multiple epiphyseal dysplasia: radiographic abnormalities correlated with genotype.

Multiple epiphyseal dysplasia (MED) is an osteochondrodysplasia characterized clinically by mild short stature and early-onset degenerative joint disease and radiographically by epiphyseal hypoplasia/dysplasia. MED is genetically heterogeneous, with autosomal dominant cases resulting from mutations in at least three genes: the cartilage oligomeric matrix protein (COMP) gene (EDM1) and the COL9A2 (EDM2) and COL9A3 (EDM3) genes of type IX procollagen. We present here a comparison of the radiographic phenotypes of MED patients with type IX collagen gene mutations and those with COMP gene mutations. We reviewed radiographs from two patients with MED produced by COMP mutations, two families with COL9A2 mutations, and one family with a mutation in COL9A3. The data demonstrated that the patients with type IX collagen defects had more severe joint involvement at the knees and relative hip sparing, while the patients with COMP mutations had significant involvement at the capital femoral epiphyses and irregular acetabuli. This pattern of joint involvement was consistent regardless of overall degree of severity of the phenotype.

Rapid determination of COL2A1 mutations in individuals with Stickler syndrome: analysis of potential premature termination codons.

Stickler syndrome is one of the milder phenotypes resulting from mutations in the gene that encodes type-II collagen, COL2A1. All COL2A1 mutations known to cause Stickler syndrome result in the formation of a premature termination codon within the type-II collagen gene. COL2A1 has 10 in-frame CGA codons, which can mutate to TGA STOP codons via a methylation-deamination mechanism. We have analyzed these sites in genomic DNA from a panel of 40 Stickler syndrome patients to test the hypothesis that mutations that cause Stickler syndrome preferentially occur at these bases. Polymerase chain reaction (PCR) amplification of genomic DNA containing each of the in-frame CGA codons was done by one of two methods: either using primers that amplify DNA that includes the CGA codon, or using allele-specific primers that either amplify normal sequence containing a CGA codon or amplify a mutant sequence containing a TGA codon. Analysis of PCR products by restriction endonuclease digestion or sequencing demonstrated the presence of a normal or mutated codon. TGA mutations were identified in eight patients, at five of the 10 in-frame CGA codons. The identification of these mutations in eight of 40 patients demonstrates that these sites are common sites for mutations in individuals with Stickler syndrome and, we propose, should be analyzed as a first step in the search for mutations that result in this disorder.

A locus for an autosomal dominant form of progressive renal failure and hypertension at chromosome 1q21.

Linkage studies were performed in a large family with an autosomal dominant phenotype characterized by nephropathy and hypertension. In this family of Iraqi Jewish origin, the nephropathy develops into progressive renal failure. By performing a genomewide linkage search, we localized the disease gene to chromosome 1q21; the highest LOD score was obtained for the marker at locus D1S305, which yielded a maximum LOD score of 4.71 at a recombination fraction of 0. Recombination mapping defined an interval of approximately 11.6 cM, between the markers at loci D1S2696 and D1S2635, that contains the disease gene. Localization of the disease-causing gene in this family represents a necessary step toward isolation of the defective gene and toward a deeper understanding of the mechanisms of hypertension and progressive renal failure.

Exclusion of the Ellis-van Creveld region on chromosome 4p16 in some families with asphyxiating thoracic dystrophy and short-rib polydactyly syndromes.

Ellis-van Creveld syndrome (EVC) is a relatively rare, usually non-lethal, autosomal recessive skeletal dysplasia characterized by short stature, polydactyly, cardiac and renal anomalies. Linkage analysis has localized the disease gene to chromosome 4p16, with the markers at loci D4S827 and D4S3135 defining the centromeric and telomeric limits of the linked interval, respectively. There has been long-term speculation that asphyxiating thoracic dystrophy (ATD) and the short-rib polydactyly syndromes (SRP) represent the severe end of the EVC disease spectrum. We performed linkage analysis using markers from the EVC region in seven families manifesting either ATD or SRP type III. In two of the families, one segregating ATD and one SRP kindred, linkage of the phenotype to the EVC region was excluded. In the other five families linkage of the phenotype to the EVC region could not be excluded, but the families were too small for linkage to the region to be established. The exclusion of the EVC region in ATD and SRP III families suggests that locus heterogeneity exists within the short-rib dysplasia (with and without polydactyly) group of disorders.

Exon skipping mutation in the COL9A2 gene in a family with multiple epiphyseal dysplasia.

Previous linkage analysis (Briggs, M.D., Choi, H.-C., Warman, M.L. et al., 1994. Genetic mapping of a locus for multiple epiphyseal dysplasia (EDM 2) to a region of chromosome 1 containing a type IX collagen gene. Am. J. Hum. Genet. 55, 678-684) in a large English family with multiple epiphyseal dysplasia established the EDM2 locus, a region of chromosome 1 containing the COL9A2 collagen gene. We now report that affected members of this family are heterozygous for a single base transversion (T-->G) at the sixth position of the intron 3 splice donor of COL9A2. The mutation leads to skipping of exon 3 during splicing, and results in a 36-nucleotide deletion in COL9A2 transcripts derived from the mutant allele. Skipping of exon 3 predicts an in-frame deletion of 12 amino acid residues within the COL3 domain of the alpha2(IX) chain. This is the fifth instance of an exon 3 deletion within the COL3 region of collagen IX heterotrimers causing the MED phenotype, as yet the only type IX collagen defect identified in this disorder. Electron microscopy (EM) of chondrocytes obtained from articular cartilage of one affected individual in the family demonstrated normal appearing rough endoplasmic reticulum (RER). In addition, the articular cartilage matrix did not show any gross abnormalities in the quantity or caliber of collagen fibrils.

Widely distributed mutations in the COL2A1 gene produce achondrogenesis type II/hypochondrogenesis.

The COL2A1 gene was assayed for mutations in genomic DNA from 12 patients with achondrogenesis type II/hypochondrogenesis. The exons and flanking sequences of the 54 exons in the COL2A1 gene were amplified by a series of specific primers using PCR. The PCR products were scanned for mutations by conformation sensitive gel electrophoresis, and PCR products that generated heteroduplex bands were then sequenced. Mutations in the COL2A1 gene were found in all 12 patients. Ten of the mutations were single base substitutions that converted a codon for an obligate glycine to a codon for an amino acid with a bulkier side chain. One of the mutations was a change in a consensus RNA splice site. Another was an 18-base pair deletion of coding sequences. The results confirmed previous indications that conformation sensitive gel electrophoresis is highly sensitive for detection of mutations in large and complex genes. They also demonstrate that most, if not all, patients with achondrogenesis type II/hypochondrogenesis have mutations in the COL2A1 gene.

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.