An acceptor splice site mutation in HOXD13 results in variable hand,but consistent foot malformations

HOXD13 is the most 5' of the HOXD cluster of homeobox genes in chromosome band 2q31.1. Heterozygous expansions of a polyalanine tract in HOXD13 are typically associated with synpolydactyly characterized by insertional digit duplication associated with syndactyly. We screened for mutations of HOXD13 in patients with a variety of limb malformations and identified a novel heterozygous mutation (758-2delA) in a three-generation family without the typical synpolydactyly phenotype in the hands, but with bilateral partial duplication of the 2nd metatarsals within the first web space of the feet. This mutation locates in the acceptor splice site of exon 2 and is predicted to cause failure of normal splicing of HOXD13. The foot abnormality in this family is similar to that described in two families by Goodman et al. [1998: Am. J. Hum. Genet. 63: 992-1000] in which different deletions of HOXD13 were reported. These findings together lend support to a distinct phenotype resulting from haploinsufficiency of HOXD13.     

A large Turkish kindred with syndactyly type II (synpolydactyly). 2. Homozygous phenotype?

Syndactyly type II (synpolydactyly (SPD)) is an autosomal dominant condition with typical abnormalities of the distal parts of both upper and lower limbs. We report here a previously undescribed phenotypic feature of people with severe hand and foot deformities who were born to two affected parents. This is the first example of SPD subjects manifesting a very distinctive phenotype, suggesting that they must be homozygous for this condition. The typical characteristic clinical features in these subjects are as follows: (1) short hands with wrinkled fatty skin and short feet; (2) complete soft tissue syndactyly involving all four limbs; (3) polydactyly of the preaxial, mesoaxial, and postaxial digits of the hands; (4) loss of the normal tubular shape of the carpal, metacarpal, and phalangeal bones, so as to give polygonal structures; (5) loss of the typical structure of the cuboid and all three cuneiform bones while the talus calcaneus and navicular bones remain intact; (6) large bony islands instead of metatarsals, most probably because of cuboid-metatarsal and cuneiform-metatarsal fusions; and (7) severe middle phalangeal hypoplasia/aplasia as well as fusion of some phalangeal structures that are associated with the loss of normal phalangeal pattern. We report seven subjects with this phenotype from three different branches of a very large SPD pedigree exhibiting the same phenotype with minimal variation. In mice, the Polysyndactyly (Ps) mutation shows a pattern of synpolydactyly very similar to that of human SPD, suggesting that they may well be homologous mutations. A molecular genetic study is currently under way to determine the chromosomal location of the SPD locus in humans and to identify the corresponding homologous region in mice.     

A nonsense mutation in the HOXD13 gene underlies synpolydactyly with incomplete penetrance

Synpolydactyly 1 (SPD1; OMIM 186000), also known as type II syndactyly, is a dominantly inherited limb malformation that is characterized by an increased number of digits. SPD1 is most commonly caused by polyalanine repeat expansions in the coding region of the HOXD13 gene, which are believed to show a dominant-negative effect. In addition, missense and out-of-frame deletion mutations in the HOXD13 gene are also known to cause SPD, and the mechanism responsible for the phenotype appears to be haploinsufficiency. Here, we analyzed a large consanguineous family from Pakistan with SPD showing a wide variation in phenotype among affected individuals. We performed genetic linkage analysis, which identified a region on chromosome 2 containing the HOXD13 gene. Haplotype analysis with microsatellite markers suggested segregation of the phenotype with HOXD13 gene with incomplete penetrance. Direct sequencing analysis of HOXD13 gene revealed a nonsense mutation, designated as Q248X. All affected individuals with the severe SPD phenotype are homozygous for the mutation, whereas those with the mild SPD phenotype are heterozygous for the mutation. Furthermore, some unaffected individuals also carry the mutation in the heterozygous state, showing incomplete penetrance. Our results show the first nonsense mutation in the HOXD13 gene underlying a severe form of SPD in the homozygous state, and a milder form of SPD with ～50% penetrance in the heterozygous state, most likely because of the production of 50% of protein compared with normal individuals.     

Synpolydactyly and HOXD13 polyalanine repeat: addition of 2 alanine residues is without clinical consequences

Background  

Type II syndactyly or synpolydactyly (SPD) is clinically very heterogeneous, and genetically three distinct SPD conditions are known and have been designated as SPD1, SPD2 and SPD3, respectively. SPD1 type is associated with expansion mutations in HOXD13, resulting in an addition of ≥ 7 alanine residues to the polyalanine repeat. It has been suggested that expansions ≤ 6 alanine residues go without medical attention, as no such expansion has ever been reported with the SPD1 phenotype.     

Polyalanine repeat expansion mutation of the HOXD13 gene in a Chinese family with unusual clinical manifestations of synpolydactyly

Synpolydactyly (SPD) is an autosomal dominant limb malformation caused by mutations in the gene HOXD13. We investigated a Chinese family in which three individuals across three generations were affected with distinctive limb malformations. We extracted genomic DNA from the affected and three unaffected individuals from this family as well as 100 unrelated controls, for mutation detection by DNA sequencing. The family was characterized by camptodactyly and symphalangism of fingers two to five, transverse phalanx and osseous fusion of the third metacarpal with the proximal phalanx, as well as the coexistence of mild and more severe bilateral phenotypes. We identified a duplication mutation, c. 186-212dup, in exon 1 of the HOXD13 gene in the affected individuals from this family; it was not present in the unaffected individuals or the 100 unrelated individuals. And we also did not find polymorphism among the controls. This study has expanded the phenotypic spectrum of known HOXD13 polyalanine repeat mutations and provided more information about the polymorphic nature of the polyalanine repeat. In addition, new clinical manifestations have been added to the spectrum of possible synpolydactyly phenotypes.     

Genetic heterogeneity of synpolydactyly: a novel locus SPD3 maps to chromosome 14q11.2-q12

Syndactyly type II or synpolydactyly (SPD) is the second most frequent syndactyly type and is inherited in an autosomal dominant fashion. The cardinal features of this malformation are the cutaneous or bony fusion of third and fourth fingers, and fourth and fifth toes associated with additional digital elements within the web. It shows incomplete penetrance and high inter- and intrafamilial phenotypic variability. Two loci are known for SPD (MIM 186000, MIM 608180) associated with mutations in HOXD13 and FBLN1, respectively. Here, we report further genetic heterogeneity for SDP. Employing a whole genomic screen, we demonstrate, in a large Pakistani kindred, that the classical phenotype of SPD maps on a new locus at chromosome 14q11.2-q12. The highest LOD score (Z(max) = 4.06) was obtained with microsatellite marker D14S264, and the multipoint LOD score reached a maximum of 5.01. Haplotype analysis revealed that the disease interval is flanked by microsatellite markers D14S283 and D14S1060, encompassing a physical distance of 10.72 Mb. We propose to allocate to this locus the symbol SPD3 (synpolydactyly 3), and to name the loci associated with HOXD13 or FBLN1 mutations SPD1 and SPD2, respectively.     

HOXD13 polyalanine tract expansion in classical synpolydactyly type Vordingborg

In 1927, Oluf Thomsen, in a classic paper, described a seven-generation family with autosomal dominant axial synpolydactyly (SPD)--the Vordingborgtyp of axis duplication and dysostosis. Expansion of a polyalanine tract in the HOXD13 gene is known to cause synpolydactyly. We have rediscovered part of the family described by Thomsen, and detected a 9 triplet polyalanine expansion within HOXD13segregating with the disorder. The phenotypic spectrum in mutation carriers ranged from severe to inapparent bone malformations. In the latter case, only dermatoglyphics revealed the genetic status.     

HOXD13-associated synpolydactyly: Extending and validating the genotypic and phenotypic spectrum with 38 new and 49 published families

PurposeHOXD13 is an important regulator of limb development. Pathogenic variants in HOXD13 cause synpolydactyly type 1 (SPD1). How different types and positions of HOXD13 variants contribute to genotype-phenotype correlations, penetrance, and expressivity of SPD1 remains elusive. Here, we present a novel cohort and a literature review to elucidate HOXD13 phenotype-genotype correlations.MethodsPatients with limb anomalies suggestive of SPD1 were selected for analysis of HOXD13 by Sanger sequencing, repeat length analysis, and next-generation sequencing. Literature was reviewed for HOXD13 heterozygotes. Variants were annotated for phenotypic data. Severity was calculated, and cluster and decision-tree analyses were performed.ResultsWe identified 98 affected members of 38 families featuring 11 different (likely) causative variants and 4 variants of uncertain significance. The most frequent (25/38) were alanine repeat expansions. Phenotypes ranged from unaffected heterozygotes to severe osseous synpolydactyly, with intra- and inter-familial heterogeneity and asymmetry. A literature review provided 160 evaluable affected members of 49 families with SPD1. Computer-aided analysis only corroborated a positive correlation between alanine repeat length and phenotype severity.ConclusionOur findings support that HOXD13-protein condensation in addition to haploinsufficiency is the molecular pathomechanism of SPD1. Our data may, also, facilitate the interpretation of synpolydactyly radiographs by future automated tools.     

A large Turkish kindred with syndactyly type II (synpolydactyly). 1. Field investigation, clinical and pedigree data.

A very large Turkish family with syndactyly type II (synpolydactyly (SPD)) is described, which originated from and is mainly concentrated in the village of Derbent, Afyon. The kindred consists of 425 subjects over seven generations, of whom 182 are affected. It appears that a founder effect in this village has led to this extensive kindred. This condition is inherited as an autosomal dominant trait with variable expressivity and an estimated penetrance of 96%. Penetrance is different between the upper (96%) and lower (69.5%) extremities. No excess of affected males or females or other associated features were documented in this condition. Variations in the involvement of one or both hands, upper or lower extremities, bone and soft tissue, as well as variation in the affected subjects of two successive generations were documented. We also noted that metacarpal and metatarsal involvement and middle phalangeal hypoplasia of the feet are the consistent features of SPD and, therefore, should be considered as characteristic of this phenotype. We observed four different phenotypes in various branches of the Derbent kindred: (1) subjects presenting typical features of SPD; (2) subjects exhibiting both pre- and post-axial polydactyly simultaneously; (3) persons manifesting postaxial polydactyly type A; and (4) subjects born to two affected parents with severe hand and foot deformities that have not been previously described in any other SPD families (that is, homozygotes). A total of 27 affected offspring were born to two such affected parents, of whom seven are expected to be homozygous for the SPD gene. This group is presented in an accompanying paper in this issue of the Journal. A molecular study is currently under way to identify the chromosomal location of the defective gene.     

Clinical phenotype associated with homozygosity for a HOXD13 7-residue polyalanine tract expansion

Synpolydactyly (SPD) is an autosomal dominant malformation of the distal limbs caused by mutations in the homeobox gene HOXD13 located on chromosome 2q31. We detail the clinical findings in a consanguineous Pakistani family segregating a HOXD13 7-residue polyalanine tract expansion. Three members of this pedigree were heterozygotes with features typical of SPD. Two further members demonstrate a more severe phenotype consistent with homozygosity for the familial mutation. We also report a child from a consanguineous Somali family homozygous for the same molecular lesion. Characteristic changes include a complex central polydactyly in the hands, abnormal modelling of the metacarpals and metatarsals, an increased number of carpal bones with abnormal shapes, hypoplasia or absence of the fifth digital rays in the feet, hypoplasia of the middle phalanges and abnormally long proximal phalanges in hands and feet. These cases illustrate the distinct phenotype associated with homozygosity for a HOXD13 mutation and also highlight the importance of considering homozygosity for a dominant mutation in consanguineous pedigrees.     

An N-terminal G11A mutation in HOXD13 causes synpolydactyly and interferes with Gli3R function during limb pre-patterning

Synpolydactyly (SPD) is a distal limb anomaly characterized by incomplete digit separation and the presence of supernumerary digits in the syndactylous web. This phenotype has been associated with mutations in the homeodomain or polyalanine tract of the HOXD13 gene. We identified a novel mutation (G11A) in HOXD13 that is located outside the previously known domains and affects the intracellular half life of the protein. Misexpression of HOXD13(G11A) in the developing chick limb phenocopied the human SPD phenotype. Finally, we demonstrated through in vitro studies that this mutation has a destabilizing effect on GLI3R uncovering an unappreciated mechanism by which HOXD13 determines the patterning of the limb.     

Type II familial synpolydactyly: report on two families with an emphasis on variations of expression

Type II familial synpolydactyly is rare and is known to have variable expression. However, no previous papers have attempted to review these variations. The aim of this paper was to review these variations and show several of these variable expressions in two families. The classic features of type II familial synpolydactyly are bilateral synpolydactyly of the third web spaces of the hands and bilateral synpolydactyly of the fourth web spaces of the feet. Several members of the two families reported in this paper showed the following variations: the third web spaces of the hands showing syndactyly without the polydactyly, normal feet, concurrent polydactyly of the little finger, concurrent clinodactyly of the little finger and the ‘homozygous'' phenotype. It was concluded that variable expressions of type II familial synpolydactyly are common and awareness of such variations is important to clinicians.     

Syndactyly and preaxial synpolydactyly in the single Sfrp2 deleted mutant mice

Secreted Frizzled-related protein 2 (Sfrp2) or Stromal Cell Derived Factor-5 (SDF-5) is highly expressed in the developing limbs. Here we showed the single Sfrp2 inactivation in mice resulted in syndactyly and preaxial synpolydactyly, predominantly in the hindlimbs. Tails were often kinked. A penetrance of the syndactyly was highest in 129/SvJ or CBA/N x 129/SvJ background and the phenotype was haploinsufficient. Preaxial synpolydactyly was seen in homozygous mutants in C57BL/6 x 129/SvJ. Of note, syndactyly showed retarded apoptosis of the second and the third interdigital spaces; concomitantly, mesodermal Msx2 expression was down-regulated. Impaired digital anlagen maturation was also noticeable in the same position. Preaxial synpolydactyly of the Sfrp2 mutants was a non-mirror image type and Shh independent. Although joint formation was not disrupted, chondrocyte maturation was preaxially disturbed. Our results suggest that the Sfrp2 deleted mice can be a useful animal model to study human syndactyly/preaxial synpolydactyly defects.     

中国人并多指(趾)畸形家系中HOXD13基因突变及产前诊断

目的对中国山东一个并多指(趾)，又称Ⅱ型并指(趾)，畸形大家系进行致病基因突变的鉴定．确定中国人并多指(趾)畸形家系中是否存在HOXD13基因突变；通过检测突变HOXD13基因对高危胎儿进行产前基因诊断。方法根据家族史、临床体征和手足X线检查进行临床诊断；采集家系成员外周血标本及受检孕妇羊水和绒毛标本，常规提取基因组DNA；设计并合成1对特异引物，通过PCR扩增HOXD13基因第1外显子内多聚丙氨酸链编码序列；PCR扩增片段经琼脂糖凝胶电泳检测，异常扩增片段经TA克隆后测序鉴定；产前诊断中，通过PCR扩增、变性聚丙烯酰胺凝胶电泳和银染检查HOXD13基因内及基因两侧共3个微卫星多态标记进行单体型分析。结果本家系4代54人，患者16人(男6人，女10人)；手足共同表现为：3／4完全并指伴软组织蹼内多指，4／5并趾伴软组织蹼内多趾．外显率为100％，表现度变异明显。上述表现符合典型常染色体显性并多指(趾)的表型特征。对家系中18人(患者9人)进行HOXD13基因分析，结果显示：全部患者多聚丙氨酸链中丙氨酸残基数由正常的15个延长为24个。通过HOXD13基因多聚丙氨酸链延展突变检测和单体型分析，对家系中1女性患者两次怀孕进行产前诊断，发现胎儿均携带突变HOXD13基因。结论首次在中国人典型并多指(趾)大家系中发现HOXD13基因多聚丙氨酸链延展突变；联合HOXD13基因多聚丙氨酸链延展突变检测和单体型分析，首次完成2例并多指(趾)胎儿的产前基因诊断。     

Point Mutation of Hoxd12 in Mice

Purpose

Genes of the HoxD cluster play a major role in vertebrate limb development, and changes that modify the Hoxd12 locus affect other genes also, suggesting that HoxD function is coordinated by a control mechanism involving multiple genes during limb morphogenesis. In this study, mutant phenotypes were produced by treatment of mice with a chemical mutagen, N-ethyl-N-nitrosourea (ENU). We analyzed mutant mice exhibiting the specific microdactyly phenotype and examined the genes affected.

Materials and Methods

We focused on phenotype characteristics including size, bone formation, and digit morphology of ENU-induced microdactyly mice. The expressions of several molecules were analyzed by genome-wide screening and quantitative real-time PCR to define the affected genes.

Results

We report on limb phenotypes of an ENU-induced A-to-C mutation in the Hoxd12 gene, resulting in alanine-to-serine conversion. Microdactyly mice exhibited growth defects in the zeugopod and autopod, shortening of digits, a missing tip of digit I, limb growth affected, and dramatic increases in the expressions of Fgf4 and Lmx1b. However, the expression level of Shh was not changed in Hoxd12 point mutated mice.

Conclusion

These results suggest that point mutation rather than the entire deletion of Hoxd12, such as in knockout and transgenic mice, causes the abnormal limb phenotype in microdactyly mice. The precise nature of the spectrum of differences requires further investigation.     

Synpolydactyly: clinical and molecular advances

Synpolydactyly (SPD) is a rare limb deformity showing a distinctive combination of syndactyly and polydactyly. Of the nine non-syndromic syndactylies, it is clinically and genetically one of the most heterogeneous malformation. SPD families may show clinical features consistent with the Temtamy and McKusick criteria as well as additional phenotypic variants, which vary from case to case. In certain instances, these variants predominate in a given family, while the typical SPD features remain less explicit. We have reviewed all the clinical variants occurring in well-documented SPD families. We conclude that typical SPD features can be delineated from minor clinical variants. Then, we propose to lump all the phenotypic variants, manifesting themselves in SPD families into three categories: (i) typical SPD features, (ii) minor variants, and (iii) unusual phenotypes. Next, we discuss the likely reasons for the occurrence of minor variants and the obvious lack of penetrance in SPD families. Finally, we show that for the SPD phenotype associated with HOXD13 mutations, a straightforward genotype–phenotype correlation is weak. Our lumping and splitting scheme for SPD phenotypic variants could be useful for the understanding of this interesting malformation.     

A large duplication involving the IHH locus mimics acrocallosal syndrome

Indian hedgehog (Ihh) signaling is a major determinant of various processes during embryonic development and has a pivotal role in embryonic skeletal development. A specific spatial and temporal expression of Ihh within the developing limb buds is essential for accurate digit outgrowth and correct digit number. Although missense mutations in IHH cause brachydactyly type A1, small tandem duplications involving the IHH locus have recently been described in patients with mild syndactyly and craniosynostosis. In contrast, a ～600-kb deletion 5' of IHH in the doublefoot mouse mutant (Dbf) leads to severe polydactyly without craniosynostosis, but with craniofacial dysmorphism. We now present a patient resembling acrocallosal syndrome (ACS) with extensive polysyndactyly of the hands and feet, craniofacial abnormalities including macrocephaly, agenesis of the corpus callosum, dysplastic and low-set ears, severe hypertelorism and profound psychomotor delay. Single-nucleotide polymorphism (SNP) array copy number analysis identified a ～900-kb duplication of the IHH locus, which was confirmed by an independent quantitative method. A fetus from a second pregnancy of the mother by a different spouse showed similar craniofacial and limb malformations and the same duplication of the IHH-locus. We defined the exact breakpoints and showed that the duplications are identical tandem duplications in both sibs. No copy number changes were observed in the healthy mother. To our knowledge, this is the first report of a human phenotype similar to the Dbf mutant and strikingly overlapping with ACS that is caused by a copy number variation involving the IHH locus on chromosome 2q35.