Deletions in HOXD13 segregate with an identical,novel foot malformation in two unrelated families.

Synpolydactyly (SPD) is a dominantly inherited congenital limb malformation consisting of 3/4 syndactyly in the hands and 4/5 syndactyly in the feet, with digit duplication in the syndactylous web. The condition recently has been found to result from different-sized expansions of an amino-terminal polyalanine tract in HOXD13. We report a novel type of mutation in HOXD13, associated in some cases with features of classic SPD and in all cases with a novel foot phenotype. In two unrelated families, each with a different intragenic deletion in HOXD13, all mutation carriers have a rudimentary extra digit between the first and second metatarsals and often between the fourth and fifth metatarsals as well. This phenotype has not been reported in any mice with genetic modifications of the HoxD gene cluster. The two different deletions affect the first exon and the homeobox, respectively, in each case producing frameshifts followed by a long stretch of novel sequence and a premature stop codon. Although the affected genes may encode proteins that exert a dominant negative or novel effect, they are most likely to act as null alleles. Either possibility has interesting implications for the role of HOXD13 in human autopod development.     

HOXD13、FHL1和先天性马蹄内翻足的相关研究

为研究HOXD13、FHL1和先天性马蹄内翻足的关系, 应用变性梯度凝胶电泳技术检测HOXD13基因在先天性马蹄内翻足个体中的突变, 应用半定量RT-PCR及免疫组织化学技术检测HOXD13、FHL1在先天性马蹄内翻足患者肌肉组织中的表达; 并用软件预测FHL1基因上游HOXD13的结合位点, 凝胶阻滞试验(Electrophoretic mobility shift assay, EMSA)验证HOXD13和FHL1的相互作用。结果84例先天性马蹄内翻足患者中未发现HOXD13基因编码区突变存在。与同期正常足部肌肉组织相比HOXD13(33.3%), FHL1 (46.6%)在先天性马蹄内翻足患者肌肉组织中表达明显下调。EMSA结果表明, 当HOXD13存在时出现特异的DNA阻滞条带。上述结果说明：HOXD13的编码区突变可能不是先天性马蹄内翻足发生的原因, 而HOXD13和 FHL1表达水平的改变可能与马蹄内翻足畸形的发生有关, HOXD13可能通过直接调控FHL1发挥作用。     

The mouse Hoxd13(spdh) mutation, a polyalanine expansion similar to human type II synpolydactyly (SPD), disrupts the function but not the expression of other Hoxd genes.

Polyalanine expansion in the human HOXD13 gene induces synpolydactyly (SPD), an inherited congenital limb malformation. A mouse model was isolated, which showed a spontaneous alanine expansion due to a 21-bp duplication at the corresponding place in the mouse gene. This mutation (synpolydactyly homolog, spdh), when homozygous, causes malformations in mice similar to those seen in affected human patients. We have studied the genetics of this condition, by using several engineered Hoxd alleles, as well as by looking at the expression of Hox and other marker genes. We show that the mutated SPDH protein induces a gain-of-function phenotype, likely by behaving as a dominant negative over other Hox genes. The mutation, however, seems to act independently from Hoxa13 and doesn't appear to affect Hox gene expression, except for a slight reduction of the HOXD13 protein itself. Developmental studies indicate that the morphological effect is mostly due to a severe retardation in the growth and ossification of the bony elements, in agreement with a general impairment in the function of posterior Hoxd genes.     

A 117-kb Microdeletion Removing HOXD9–HOXD13 and EVX2 Causes Synpolydactyly

Studies in mouse and chick have shown that the 5' HoxD genes play major roles in the development of the limbs and genitalia. In humans, mutations in HOXD13 cause the dominantly inherited limb malformation synpolydactyly (SPD). Haploinsufficiency for the 5' HOXD genes has recently been proposed to underlie the monodactyly and penoscrotal hypoplasia in two children with chromosomal deletions encompassing the entire HOXD cluster. Similar deletions, however, have previously been associated with split-hand/foot malformation (SHFM), including monodactyly. Here we report a father and daughter with SPD who carry a 117-kb microdeletion at the 5' end of the HOXD cluster. By sequencing directly across the deletion breakpoint, we show that this microdeletion removes only HOXD9-HOXD13 and EVX2. We also report a girl with bilateral split foot and a chromosomal deletion that includes the entire HOXD cluster and extends approximately 5 Mb centromeric to it. Our findings indicate that haploinsufficiency for the 5' HOXD genes causes not SHFM but SPD and point to the presence of a novel locus for SHFM in the interval between EVX2 and D2S294. They also suggest that there is a regulatory region, upstream of the HOXD cluster, that is responsible for activating the cluster as a whole.     

Deletion of 2.7 kb near HOXD3 in an Arabian horse with occipitoatlantoaxial malformation

In the horse, the term occipitoatlantoaxial malformation (OAAM) is used to describe a developmental defect in which the first cervical vertebra (atlas) resembles the base of the skull (occiput) and the second cervical vertebra (axis) resembles the atlas. Affected individuals demonstrate an abnormal posture and varying degrees of ataxia. The homeobox (HOX) gene cluster is involved in the development of both the axial and appendicular skeleton. Hoxd3‐null mice demonstrate a strikingly similar phenotype to Arabian foals with OAAM. Whole‐genome sequencing was performed in an OAAM‐affected horse (OAAM1) and seven unaffected Arabian horses. Visual inspection of the raw reads within the region of HOXD3 identified a 2.7‐kb deletion located 4.4 kb downstream of the end of HOXD4 and 8.2 kb upstream of the start of HOXD3. A genotyping assay revealed that both parents of OAAM1 were heterozygous for the deletion. Additional genotyping identified two of 162 heterozygote Arabians, and the deletion was not present in 371 horses of other breeds. Comparative genomics studies have revealed that this region is highly conserved across species and that the entire genomic region between Hoxd4 and Hoxd3 is transcribed in mice. Two additional Arabian foals diagnosed with OAAM (OAAM 2 and 3) were genotyped and did not have the 2.7‐kb deletion. Closer examination of the phenotype in these cases revealed notable variation. OAAM3 also had facial malformations and a patent ductus arteriosus, and the actual malformation at the craniocervical junction differed. Genetic heterogeneity may exist across the HOXD locus in Arabian foals with OAAM.     

A group 13 homeodomain is neither necessary nor sufficient for posterior prevalence in the mouse limb

Posterior prevalence is the general property attributed to HOX proteins describing the dominant effect of more posterior HOX proteins over the function of anterior orthologs in common areas of expression. To explore the HOX group 13 protein domains required for this property, we used the mouse Prx-1 promoter to drive transgenic expression of Hox constructs throughout the entire limb bud during development. This system allowed us to conclusively demonstrate a hierarchy of Hox function in developing limbs. Furthermore, by substituting the HOXD11 or HOXA9 homeodomain for that of HOXD13, we show that a HOXD13 homeodomain is not necessary for posterior prevalence. Proximal expression of these chimeric proteins unexpectedly caused defects consistent with wild-type HOXD13 mediated posterior prevalence. Moreover, group 13 non-homeodomain residues appear to confer the property as proximal expression of HOXA9 containing the HOXD13 homeodomain did not result in limb reductions characteristic of HOXD13. These data are most compatible with models of posterior prevalence based on protein-protein interactions and support examination of the N-terminal non-homeodomain regions of Hox group 13 proteins as necessary agents for posterior prevalence.     

A novel stable polyalanine [poly(A)] expansion in the <Emphasis Type="Italic">HOXA13</Emphasis> gene associated with hand-foot-genital syndrome: proper function of poly(A)-harbouring transcription factors depends on a critical repeat length?

Hand-foot-genital syndrome (HFGS) is a dominantly inherited congenital malformation affecting the distal limbs and genitourinary tract. Here, we describe the phenotype and its molecular basis in a family that presented with HFGS. Genetic analysis revealed that the condition is caused by an 18-bp in-frame duplication within a cryptic trinucleotide repeat sequence encoding an 18-residue polyalanine tract in the homeoboxgene ( HOX) A13. This mutation expands the stretch with six extra alanine residues. Similar types of mutation (plus eight alanines) have recently been found in another HFGS family and also in the human HOXD13 gene (plus seven up to plus 14 residues) where it leads to synpolydactyly (SPD), a further congenital limb malformation rarely associated with genital abnormalities. As observed in our family, all the expanded tracts encoding polyalanine, either reported for HOXA13 or HOXD13, are quite stable when transmitted within affected families. Unlike disorders with unstable expansions of perfect trinucleotide repeats the molecular mechanism underlying these polyalanine expansions should be unequal crossing-over rather than replication slippage. The alanine tract elongation may prevent protein-protein interactions of the mutant HOXA13, thereby inducing a localized heterochrony in the sequence of distal limb and genitourinary development.     

Regulation of cell polarity in the cartilage growth plate and perichondrium of metacarpal elements by HOXD13 and WNT5A

The morphology of bones is genetically determined, but the molecular mechanisms that control shape, size and the overall gestalt of bones remain unclear. We previously showed that metacarpals in the synpolydactyly homolog (spdh) mouse, which carries a mutation in Hoxd13 similar to the human condition synpolydactyly (SPD), were transformed to carpal-like bones with cuboid shape that lack cortical bone and a perichondrium and are surrounded by a joint surface. Here we provide evidence that spdh metacarpal growth plates have a defect in cell polarization with a random instead of linear orientation. In parallel prospective perichondral cells failed to adopt the characteristic flattened cell shape. We observed a similar cell polarity defect in metacarpals of Wnt5a^−/− mice. Wnt5a and the closely related Wnt5b were downregulated in spdh handplates, and HOXD13 induced expression of both genes in vitro. Concomitant we observed mislocalization of core planar cell polarity (PCP) components DVL2 and PRICKLE1 in spdh metacarpals indicating a defect in the WNT/PCP pathway. Conversely the WNT/β-CATENIN pathway, a hallmark of joint cells lining carpal bones, was upregulated in the perichondral region. Finally, providing spdh limb explant cultures with cells expressing either HOXD13 or WNT5A led to a non-cell autonomous partial rescue of cell polarity the perichondral region and restored the expression of perichondral markers. This study provides a so far unrecognized link between HOX proteins and cell polarity in the perichondrium and the growth plate, a failure of which leads to transformation of metacarpals to carpal-like structures.     

四肢手足对称多指趾发育的基因特征与显微镜手术预后的相关性

目的:探讨四肢手足对称多指趾发育的基因特征与显微镜手术预后的相关性。方法:2016年2月至2020年1月选择在本院诊治的四肢手足对称多指趾患儿110例作为研究对象,所有患儿都给予显微手术治疗,记录预后情况。采用PCR方法检测HOXD13基因突变情况并进行相关性分析。结果:在110例患儿中,HOXD13基因突变45例,突变率为40.9%。突变组的性别、年龄、体重指数、血压、Wassel分型等与非突变组对比差异无统计学意义(P>0.05)。术后3个月突变组的疗效优良率为77.8%,显著低于非突变组的96.9%(P<0.05)。术后3个月突变组的肌腱粘连、切口感染、皮肤坏死、指趾间关节偏斜等并发症发生率为28.9%,显著高于非突变组的3.1%(P<0.05)。Pearson分析显示HOXD13基因突变与预后疗效、并发症等都存在相关性(P<0.05);多因素logistic回归分析分析显示HOXD13基因突变也是影响患儿预后疗效、并发症的主要因素(P<0.05)。结论:四肢手足对称多指趾患儿多伴随有HOXD13基因突变,显微手术具有很好的可行性,HOXD13基因突变与显微手术预后有显著相关性。     

Mutations in HOXD13 underlie syndactyly type V and a novel brachydactyly-syndactyly syndrome

HOXD13, the homeobox-containing gene located at the most 5' end of the HOXD cluster, plays a critical role in limb development. It has been shown that mutations in human HOXD13 can give rise to limb malformations, with variable expressivity and a wide spectrum of clinical manifestations. Polyalanine expansions in HOXD13 cause synpolydactyly, whereas amino acid substitutions in the homeodomain are associated with brachydactyly types D and E. We describe two large Han Chinese families with different limb malformations, one with syndactyly type V and the other with limb features overlapping brachydactyly types A4, D, and E and mild syndactyly of toes 2 and 3. Two-point linkage analysis showed LOD scores >3 (theta =0) for markers within and/or flanking the HOXD13 locus in both families. In the family with syndactyly type V, we identified a missense mutation in the HOXD13 homeodomain, c.950A-->G (p.Q317R), which leads to substitution of the highly conserved glutamine that is important for DNA-binding specificity and affinity. In the family with complex brachydactyly and syndactyly, we detected a deletion of 21 bp in the imperfect GCN (where N denotes A, C, G, or T) triplet-containing exon 1 of HOXD13, which results in a polyalanine contraction of seven residues. Moreover, we found that the mutant HOXD13 with the p.Q317R substitution was unable to transactivate the human EPHA7 promoter. Molecular modeling data supported these experimental results. The calculated interactions energies were in agreement with the measured changes of the activity. Our data established the link between HOXD13 and two additional limb phenotypes--syndactyly type V and brachydactyly type A4--and demonstrated that a polyalanine contraction in HOXD13, most likely, led to other digital anomalies but not to synpolydactyly. We suggest the term "HOXD13 limb morphopathies" for the spectrum of limb disorders caused by HOXD13 mutations.     

Monodactylous limbs and abnormal genitalia are associated with hemizygosity for the human 2q31 region that includes the HOXD cluster.

Vertebrates have four clusters of Hox genes (HoxA, HoxB, HoxC, and HoxD). A variety of expression and mutation studies indicate that posterior members of the HoxA and HoxD clusters play an important role in vertebrate limb development. In humans, mutations in HOXD13 have been associated with type II syndactyly or synpolydactyly, and, in HOXA13, with hand-foot-genital syndrome. We have investigated two unrelated children with a previously unreported pattern of severe developmental defects on the anterior-posterior (a-p) limb axis and in the genitalia, consisting of a single bone in the zeugopod, either monodactyly or oligodactyly in the autopod of all four limbs, and penoscrotal hypoplasia. Both children are heterozygous for a deletion that eliminates at least eight (HOXD3-HOXD13) of the nine genes in the HOXD cluster. We propose that the patients' phenotypes are due in part to haploinsufficiency for HOXD-cluster genes. This hypothesis is supported by the expression patterns of these genes in early vertebrate embryos. However, the involvement of additional genes in the region could explain the discordance, in severity, between these human phenotypes and the milder, non-polarized phenotypes present in mice hemizygous for HoxD cluster genes. These cases represent the first reported examples of deficiencies for an entire Hox cluster in vertebrates and suggest that the diploid dose of human HOXD genes is crucial for normal growth and patterning of the limbs along the anterior-posterior axis.     

Range of HOX/TALE superclass associations and protein domain requirements for HOXA13:MEIS interaction

AbdB-like HOX proteins form DNA-binding complexes with the TALE superclass proteins MEIS1A and MEIS1B, and trimeric complexes have been identified in nuclear extracts that include a second TALE protein, PBX. Thus, soluble DNA-independent protein-protein complexes exist in mammals. The extent of HOX/TALE superclass interactions, protein structural requirements, and sites of in vivo cooperative interaction have not been fully explored. We show that Hoxa13 and Hoxd13 expression does not overlap with that of Meis1-3 in the developing limb; however, coexpression occurs in the developing male and female reproductive tracts (FRTs). We demonstrate that both HOXA13 and HOXD13 associate with MEIS1B in mammalian and yeast cells, and that HOXA13 can interact with all MEIS proteins but not more diverged TALE superclass members. In addition, the C-terminal domains (CTDs) of MEIS1A (18 amino acids) and MEIS1B (93 amino acids) are necessary for HOXA13 interaction; for MEIS1B, this domain was also sufficient. We also show by yeast two-hybrid assay that MEIS proteins can interact with anterior HOX proteins, but for some, additional N-terminal MEIS sequences are required for interaction. Using deletion mutants of HOXA13 and HOXD13, we provide evidence for multiple HOX peptide domains interacting with MEIS proteins. These data suggest that HOX:MEIS interactions may extend to non-AbdB-like HOX proteins in solution and that differences may exist in the MEIS peptide domains utilized by different HOX groups. Finally, the capability of multiple HOX domains to interact with MEIS C-terminal sequences implies greater complexity of the HOX:MEIS protein-protein interactions and a larger role for variation of HOX amino-terminal sequences in specificity of function.