Novel point mutations in GDF5 associated with two distinct limb malformations in Chinese: brachydactyly type C and proximal symphalangism

Growth/differentiation factor 5 (GDF5) is a secreted growth factor that plays a key regulatory role in embryonic skeletal and joint development. Mutations in the GDF5 gene can cause different types of skeletal dysplasia, including brachydactyly type C (BDC) and proximal symphalangism (SYM1). We report two novel mutations in the GDF5 gene in Chinese families with distinct limb malformations. In one family affected with BDC, we identified a novel nonsense mutation, c.1461T > G (p.Y487X), which is predicted to truncate the GDF5 precursor protein by deleting 15 amino acids at its C-terminus. In one family with SYM1, we found a novel missense mutation, c.1118T > G (p.L373R), which changes a highly conserved amino acid in the prodomain of GDF5. We transfected COS-7 cells with retroviral constructs to express human wild-type or mutant GDF5 cDNAs. The mature GDF5 protein was detected, as in the wild-type, in supernatant derived from the p.L373R mutant GDF5 transfected cells, but not in the supernatant from the p.Y487X mutant transfected cells, indicating that the two mutations led to different fates of the mutant GDF5 proteins, thereby producing distinct limb phenotypes. Wei Yang and Lihua Cao contributed equally to the work.     

The mutational spectrum of brachydactyly type C

Growth/differentiation factor-5 (GDF5), also known as cartilage-derived morphogenetic protein-1 (CDMP-1), is a secreted signaling molecule that participates in skeletal morphogenesis. Heterozygous mutations in GDF5, which maps to human chromosome 20, occur in individuals with autosomal dominant brachydactyly type C (BDC). Here we show that BDC is locus homogeneous by reporting a GDF5 frameshift mutation segregating with the phenotype in a family whose trait was initially thought to map to human chromosome 12. We also describe heterozygous mutations in nine additional probands/families with BDC and show nonpenetrance in a mutation carrier. Finally, we show that mutant GDF5 polypeptides containing missense mutations in their active domains do not efficiently form disulfide-linked dimers when expressed in vitro. These data support the hypothesis that BDC results from functional haploinsufficiency for GDF5.     

Two novel disease-causing variants in BMPR1B are associated with brachydactyly type A1

Brachydactyly type A1 is an autosomal dominant disorder primarily characterized by hypoplasia/aplasia of the middle phalanges of digits 2–5. Human and mouse genetic perturbations in the BMP-SMAD signaling pathway have been associated with many brachymesophalangies, including BDA1, as causative mutations in IHH and GDF5 have been previously identified. GDF5 interacts directly as the preferred ligand for the BMP type-1 receptor BMPR1B and is important for both chondrogenesis and digit formation. We report pathogenic variants in BMPR1B that are associated with complex BDA1. A c.975A>C (p.(Lys325Asn)) was identified in the first patient displaying absent middle phalanges and shortened distal phalanges of the toes in addition to the significant shortening of middle phalanges in digits 2, 3 and 5 of the hands. The second patient displayed a combination of brachydactyly and arachnodactyly. The sequencing of BMPR1B in this individual revealed a novel c.447-1G>A at a canonical acceptor splice site of exon 8, which is predicted to create a novel acceptor site, thus leading to a translational reading frameshift. Both mutations are most likely to act in a dominant-negative manner, similar to the effects observed in BMPR1B mutations that cause BDA2. These findings demonstrate that BMPR1B is another gene involved with the pathogenesis of BDA1 and illustrates the continuum of phenotypes between BDA1 and BDA2.     

A mutation in the receptor binding site of GDF5 causes Mohr-Wriedt brachydactyly type A2

Background

Brachydactyly type A2 (OMIM 112600) is characterised by hypoplasia/aplasia of the second middle phalanx of the index finger and sometimes the little finger. BDA2 was first described by Mohr and Wriedt in a large Danish/Norwegian kindred and mutations in BMPR1B were recently demonstrated in two affected families.

Methods

We found and reviewed Mohr and Wriedt''s original unpublished annotations, updated the family pedigree, and examined 37 family members clinically, and radiologically by constructing the metacarpo‐phalangeal profile (MCPP) pattern in nine affected subjects. Molecular analyses included sequencing of BMPR1B, linkage analysis for STS markers flanking GDF5, sequencing of GDF5, confirmation of the mutation by a restriction enzyme assay, and localisation of the mutation inferred from the very recently reported GDF5 crystal structure, and by superimposing the GDF5 protein sequence onto the crystal structure of BMP2 bound to Bmpr1a.

Results

A short middle phalanx of the index finger was found in all affected individuals, but other fingers were occasionally involved. The fourth finger was characteristically spared. This distinguishes Mohr‐Wriedt type BDA2 from BDA2 caused by mutations in BMPR1B. An MCPP analysis most efficiently detected mutation carrier status. We identified a missense mutation, c.1322T>C, causing substitution of a leucine with a proline at amino acid residue 441 within the active signalling domain of GDF5. The mutation was predicted to reside in the binding site for BMP type 1 receptors.

Conclusion

GDF5 is a novel BDA2 causing gene. It is suggested that impaired activity of BMPR1B is the molecular mechanism responsible for the BDA2 phenotype.     

Modulation of GDF5/BRI-b signalling through interaction with the tyrosine kinase receptor Ror2

The brachydactylies are a group of inherited disorders of the hands characterized by shortened digits. Mutations in the tyrosine kinase receptor Ror2 cause brachydactyly type B (BDB). Mutations in GDF5, a member of the BMP/TGF-beta ligand family, cause brachydactyly type C (BDC) whereas mutations in the receptor for GDF5, BRI-b, cause brachydactyly type A2 (BDA2). There is considerable degree of phenotypic overlap between the subtypes BDB, BDC and BDA2. Here we demonstrate that all three components are involved in GDF5 induced regulation of chondrogenesis. We show that Ror2 (tyrosine kinase receptor) and BRI-b (serine/threonine kinase receptor) form a ligand independent heteromeric complex. The frizzled-like-CRD domain of Ror2 is required for this complex. Within that complex Ror2 gets transphosphorylated by BRI-b. We show that Ror2 modulates GDF5 signalling by inhibition of Smad1/5 signalling and by activating a Smad-independent pathway. Both pathways however, are needed for chondrogenic differentiation as demonstrated in ATDC5 cells. The functional interaction of Ror2 with GDF5 and BRI-b was genetically confirmed by the presence of epistatic effects in crosses of Ror2, BRI-b and Gdf5 deficient mice. These results indicate for the first time a direct interaction of Ser/Thr- and Tyr-Kinase receptors and provide evidence for modulation of the Smad-pathway and GDF5 triggered chondrogenesis.     

Brachydactyly type A2 associated with a defect in proGDF5 processing

We investigated a family with a brachydactyly type A2 and identified a heterozygous arginine to glutamine (R380Q) substitution in the growth/differentiation factor 5 (GDF5) in all affected individuals. The observed mutation is located at the processing site of the protein, at which the GDF5 precursor is thought to be cleaved releasing the mature molecule from the prodomain. In order to test the effect of the mutation, we generated the GDF5-R380Q mutant and a cleavage-resistant proGDF5 mutant (R380A/R381A) in vitro. Both mutants were secreted from chicken micromass cultures, but showed diminished biological activity. Western blot analyses showed that wt GDF5 was processed by the chicken micromass cells, whereas the mutants were not, indicating that the mutations interfere with processing and that this leads to a strong reduction of biological activity. To test the requirements for GDF5 processing in vitro we produced recombinant human (rh) proGDF5 wild-type protein in Escherichia coli. The results show that unprocessed (rh) proGDF5 is virtually inactive but can be proteolytically activated by different enzymes such as trypsin, furin, and MMP3. (rh) proGDF5 could thus be used as a locally administered depot form with retarded release of activity. In contrast to mature rhGDF5, (rh) proGDF5 shows a high solubility at physiological pH, a characteristic that might be useful for therapeutic applications.     

Homozygous missense and nonsense mutations in BMPR1B cause acromesomelic chondrodysplasia-type Grebe

Acromesomelic chondrodysplasias (ACDs) are characterized by disproportionate shortening of the appendicular skeleton, predominantly affecting the middle (forearms and forelegs) and distal segments (hands and feet). Here, we present two consanguineous families with missense (c.157T>C, p.(C53R)) or nonsense (c.657G>A, p.(W219*)) mutations in BMPR1B. Homozygous affected individuals show clinical and radiographic findings consistent with ACD-type Grebe. Functional analysis of the missense mutation C53R revealed that the mutated receptor was partially located at the cell membrane. In contrast to the wild-type receptor, C53R mutation hindered the activation of the receptor by its ligand GDF5, as shown by reporter gene assay. Further, overexpression of the C53R mutation in an in vitro chondrogenesis assay showed no effect on cell differentiation, indicating a loss of function. The nonsense mutation (c.657G>A, p.(W219*)) introduces a premature stop codon, which is predicted to be subject to nonsense-mediated mRNA decay, causing reduced protein translation of the mutant allele. A loss-of-function effect of both mutations causing recessive ACD-type Grebe is further supported by the mild brachydactyly or even non-penetrance of these mutations observed in the heterozygous parents. In contrast, dominant-negative BMPR1B mutations described previously are associated with autosomal-dominant brachydactyly-type A2.     

Mutations in GDF5 presenting as semidominant brachydactyly A1

Brachydactyly A1 (BDA1) is an autosomal dominant disorder characterized by shortness of all middle phalanges of the hands and toes, shortness of the proximal phalanges of the first digit, and short stature. Missense mutations in the Indian Hedgehog gene (IHH) are known to cause BDA1, and a second locus has been mapped to chromosome 5p. In a consanguineous French Canadian kindred with BDA1, both IHH and the 5p locus were excluded. Microsatellites flanking GDF5 on chromosome 20q were found to cosegregate with the disease. Sequencing of the GDF5 coding region revealed that a mildly affected individual in the family was heterozygous, and that all of the severely affected individuals were homozygous for a novel missense c.1195C>T mutation that predicts a p.Arg399Cys substitution at a highly conserved amino acid. Functional analysis demonstrated that although the p.Arg399Cys mutant is able to stimulate chondrogenesis, it is much less effective than wild‐type GDF5. This data confirms genetic heterogeneity in BDA1, demonstrates that mutations upstream of IHH can result in BDA1, and shows that BDA1 can result from semidominant mutations in GDF5. Hum Mutat 31:1–8, 2010. © 2010 Wiley‐Liss, Inc.     

A homozygous BMPR1B mutation causes a new subtype of acromesomelic chondrodysplasia with genital anomalies

We present a patient with acromesomelic chondrodysplasia and genital anomalies caused by a novel homozygous mutation in BMPR1B, the gene coding for bone morphogenetic protein receptor 1B. The 16 year old girl, the offspring of a multiconsanguinous family, showed a severe form of limb malformation consisting of aplasia of the fibula, severe brachydactyly, ulnar deviation of the hands, and fusion of carpal/tarsal bones. In addition, she presented with hypoplasia of the uterus and ovarian dysfunction resulting in hypergonadotrophic hypogonadism. Mutation analysis of BMPR1B revealed a homozygous 8 bp deletion (del359–366). This mutation is expected to result in a loss of function and is thus different from the heterozygous missense mutations in BMPR1B recently shown to cause brachydactyly type A2 through a dominant negative effect. The patient's skeletal phenotype shows an overlap with the clinical spectrum of the acromesomelic chondrodysplasias of the Grebe, Hunter-Thompson, and DuPan types caused by homozygous mutations in the gene coding for growth differentiation factor 5 (GDF5) which is a high-affinity ligand to BMPR1B. However, the phenotype described here differs from GDF5 associated chondrodysplasias because of the additional presence of genital anomalies and the distinct limb phenotype.     

A novel mutation in GDF5 causes autosomal dominant symphalangism in two Chinese families

Proximal symphalangism (SYM1) is an autosomal dominant disorder characterized by ankylosis of the proximal interphalangeal joints and fusion of carpal and tarsal bones. We identified and characterized two five-generation Chinese families with SYM1. The two families share some similarities (e.g., osseous fusion of interphalangeal joints of the 2-4 fingers) with SYM1 families with mutations in the NOG gene or the family with mutation R438L recently reported in the GDF5 gene (encoding a bone morphogenetic protein family member). However, they show some unique features including the absence of cuboid bone, the lack of shortness of the first and fifth metacarpal bones, and manifestation of flat feet. Genome-wide linkage analysis of the two families mapped the disease gene to marker D20S112 with a combined LOD score of 4.32. Mutational analysis revealed a novel E491K mutation in the GDF5 gene in both families. The mutation occurs at a highly conserved residue in the TGF-beta domain of GDF5 and represents the second GDF5 mutation identified for SYM1 to date. The E491K mutation co-segregated with the affected individuals in the two families, and did not exist in unaffected family members or 200 normal controls. These results indicate that defects in GDF5 can cause SYM1 in the Chinese population, and expand the spectrum of clinical phenotypes associated with mutant GDF5.     

A novel mutation in CDMP1 causes brachydactyly type C with “angel-shaped phalanx”. A genotype–phenotype correlation in the mutational spectrum

Brachydactyly type C (BDC), a well-recognized autosomal dominant hand malformation, displays brachymesophalangy of the second, third, and fifth fingers, a short first metacarpal, hyperphalangy, and ulnar deviation of the index finger. An “angel-shaped phalanx” is a distinctive radiological sign that can be found in BDC and other skeletal dysplasias, such as angel-shaped phalango-epiphyseal dysplasia (ASPED), an autosomal dominant skeletal abnormality characterized by a typical angel-shaped phalanx, brachydactyly, specific radiological findings, abnormal dentition, hip dysplasia, and delayed bone age. BDC and ASPED result from mutations in the CDMP1 gene. We report here a Mexican patient with BDC and clinical features of ASPED who carries a novel mutation in CDMP1, confirming that BDC and ASPED are part of the CDMP1 mutational spectrum. Based on the large number of clinical features in common, we suggest that both anomalies are part of the same clinical spectrum. Supported by an extensive review of the literature, a possible genotype–phenotype correlation in the mutational spectrum of this gene is proposed.     

一个短指家系临床特征调查及其致病基因的定位

目的 通过对山东省一个A1型短指(brachydactyly type A1,BDA1)家系的临床特征及致病基因分析,确定该病的遗传类型及其发生机制.方法 经家系调查及临床检查确定疾病类型;通过致病基因微卫星多态位点进行连锁分析;采用修饰引物产生引入酶切位点的方法来区分突变基因.结果 该家系的短指症为A1型,常染色体显性遗传;发病原因为位于染色体2q35-2q36的IHH基因(indian hedgehog gene)发生了G298A(D100N)错义突变.结论 中国山东A1型短指家系的发病机理是IHH基因发生了G298A(D100N)错义突变所致.     

一个短指家系临床特征调查及其致病基因的定位

目的 通过对山东省一个A1型短指(brachydactyly type A1,BDA1)家系的临床特征及致病基因分析,确定该病的遗传类型及其发生机制.方法 经家系调查及临床检查确定疾病类型;通过致病基因微卫星多态位点进行连锁分析;采用修饰引物产生引入酶切位点的方法来区分突变基因.结果 该家系的短指症为A1型,常染色体显性遗传;发病原因为位于染色体2q35-2q36的IHH基因(indian hedgehog gene)发生了G298A(D100N)错义突变.结论 中国山东A1型短指家系的发病机理是IHH基因发生了G298A(D100N)错义突变所致.     

一个短指家系临床特征调查及其致病基因的定位

目的 通过对山东省一个A1型短指(brachydactyly type A1,BDA1)家系的临床特征及致病基因分析,确定该病的遗传类型及其发生机制.方法 经家系调查及临床检查确定疾病类型;通过致病基因微卫星多态位点进行连锁分析;采用修饰引物产生引入酶切位点的方法来区分突变基因.结果 该家系的短指症为A1型,常染色体显性遗传;发病原因为位于染色体2q35-2q36的IHH基因(indian hedgehog gene)发生了G298A(D100N)错义突变.结论 中国山东A1型短指家系的发病机理是IHH基因发生了G298A(D100N)错义突变所致.     

一个短指家系临床特征调查及其致病基因的定位

目的 通过对山东省一个A1型短指(brachydactyly type A1,BDA1)家系的临床特征及致病基因分析,确定该病的遗传类型及其发生机制.方法 经家系调查及临床检查确定疾病类型;通过致病基因微卫星多态位点进行连锁分析;采用修饰引物产生引入酶切位点的方法来区分突变基因.结果 该家系的短指症为A1型,常染色体显性遗传;发病原因为位于染色体2q35-2q36的IHH基因(indian hedgehog gene)发生了G298A(D100N)错义突变.结论 中国山东A1型短指家系的发病机理是IHH基因发生了G298A(D100N)错义突变所致.     

一个短指家系临床特征调查及其致病基因的定位

目的 通过对山东省一个A1型短指(brachydactyly type A1,BDA1)家系的临床特征及致病基因分析,确定该病的遗传类型及其发生机制.方法 经家系调查及临床检查确定疾病类型;通过致病基因微卫星多态位点进行连锁分析;采用修饰引物产生引入酶切位点的方法来区分突变基因.结果 该家系的短指症为A1型,常染色体显性遗传;发病原因为位于染色体2q35-2q36的IHH基因(indian hedgehog gene)发生了G298A(D100N)错义突变.结论 中国山东A1型短指家系的发病机理是IHH基因发生了G298A(D100N)错义突变所致.     

一个短指家系临床特征调查及其致病基因的定位

目的 通过对山东省一个A1型短指(brachydactyly type A1,BDA1)家系的临床特征及致病基因分析,确定该病的遗传类型及其发生机制.方法 经家系调查及临床检查确定疾病类型;通过致病基因微卫星多态位点进行连锁分析;采用修饰引物产生引入酶切位点的方法来区分突变基因.结果 该家系的短指症为A1型,常染色体显性遗传;发病原因为位于染色体2q35-2q36的IHH基因(indian hedgehog gene)发生了G298A(D100N)错义突变.结论 中国山东A1型短指家系的发病机理是IHH基因发生了G298A(D100N)错义突变所致.     

一个短指家系临床特征调查及其致病基因的定位

目的 通过对山东省一个A1型短指(brachydactyly type A1,BDA1)家系的临床特征及致病基因分析,确定该病的遗传类型及其发生机制.方法 经家系调查及临床检查确定疾病类型;通过致病基因微卫星多态位点进行连锁分析;采用修饰引物产生引入酶切位点的方法来区分突变基因.结果 该家系的短指症为A1型,常染色体显性遗传;发病原因为位于染色体2q35-2q36的IHH基因(indian hedgehog gene)发生了G298A(D100N)错义突变.结论 中国山东A1型短指家系的发病机理是IHH基因发生了G298A(D100N)错义突变所致.     

一个短指家系临床特征调查及其致病基因的定位

目的 通过对山东省一个A1型短指(brachydactyly type A1,BDA1)家系的临床特征及致病基因分析,确定该病的遗传类型及其发生机制.方法 经家系调查及临床检查确定疾病类型;通过致病基因微卫星多态位点进行连锁分析;采用修饰引物产生引入酶切位点的方法来区分突变基因.结果 该家系的短指症为A1型,常染色体显性遗传;发病原因为位于染色体2q35-2q36的IHH基因(indian hedgehog gene)发生了G298A(D100N)错义突变.结论 中国山东A1型短指家系的发病机理是IHH基因发生了G298A(D100N)错义突变所致.     

一个短指家系临床特征调查及其致病基因的定位

目的 通过对山东省一个A1型短指(brachydactyly type A1,BDA1)家系的临床特征及致病基因分析,确定该病的遗传类型及其发生机制.方法 经家系调查及临床检查确定疾病类型;通过致病基因微卫星多态位点进行连锁分析;采用修饰引物产生引入酶切位点的方法来区分突变基因.结果 该家系的短指症为A1型,常染色体显性遗传;发病原因为位于染色体2q35-2q36的IHH基因(indian hedgehog gene)发生了G298A(D100N)错义突变.结论 中国山东A1型短指家系的发病机理是IHH基因发生了G298A(D100N)错义突变所致.