A Novel Missense Variant of HOXD13 Caused Atypical Synpolydactyly by Impairing the Downstream Gene Expression and Literature Review for Genotype-Phenotype Correlations

Abstract

Synpolydactyly (SPD) is a hereditary congenital limb malformation with distinct syndactyly designated as SPD1, SPD2, and SPD3. SPD1 is caused by mutations of HOXD13, which is a homeobox transcription factor crucial for limb development. More than 143 SPD patients have been reported to carry HOXD13 mutations, but there is a lack of genotype-phenotype correlation. We report a novel missense mutation of c. 925A > T (p.I309F) in an individual with atypical synpolydactyly inherited from her father with mild clinodactyly and three other different alanine insertion mutations in HOXD13 identified by whole exome sequencing (WES) in 12 Chinese SPD families. Unlike polyalanine extension, which tends to form α-helix and causes protein aggregation in the cytoplasm as shown by molecular simulation and immunofluorescence, the c. 925A > T mutation impairs downstream transcription of EPHA7. We compiled literature findings and analyzed genotype-phenotype features in 173 SPD individuals of 53 families, including 12 newly identified families. Among the HOXD13-related individuals, mutations were distributed in three regions: polyalanine, homeobox, and non-homeobox. Polyalanine extension was the most common variant (45%), followed by missense mutations (32%) mostly in the homeobox compared with the loss-of-function (LOF) variants more likely in non-homeobox. Furthermore, a more severe degree and classic SPD were associated with polyalanine mutations although missense variants were associated with brachydactyly and syndactyly in hands and feet and LOF variants with clinodactyly in hands. Our study broadens the HOXD13 mutation spectrum and reveals the profile of three different variants and their severity of SPD, the genotype-phenotype correlation related to the HOXD13 mutation site provides clinical insight, including for genetic counseling.

Keywords: HoxD13; genotype and phenotype; polyalanine extension; synpolydactyly; transcription regulation.

FIGURE 1

Pedigrees and clinical phenotypes in SPD families with HOXD13 polyalanine extension. (A) The relationship charts of 10 families with synpolydactyly. (B) Clinical manifestations of the limbs in affected individuals, including synpolydactyly, syndactyly, syndactyly, clinodactyly, camptodactyly, brachydactyly.

FIGURE 2

Pedigrees and clinical phenotypes in SPD families with HOXD13 c.925A > T mutation. (A) The relationship chart of the family with atypical synpolydactyly. (B) Clinical manifestations of the limbs in affected individuals, including postaxial and preaxial polydactyly, camptodactyly.

FIGURE 3

The genotype–phenotype relationship in SPD patients with HOXD13 mutation. (A) The mutation sites in HOXD13. (B) The number of affected limbs with different phenotypes in different domains with HOXD13 mutation. (C) Heat map of the phenotype:genotype ratios in different HOXD13 mutation regions in hands and feet separately. (D) The scores of severity in different domains with HOXD13 mutation. (E) The scores of severity with different HOXD13 expansion sizes of polyalanine tract. (F) The scores of severity in the offsprings and parents who both have polyA mutations in the HOXD13 gene.

FIGURE 4

Subcellular location of HOXD13 protein. The blue color in the nucleus shows DAPI staining, and the green color shows HOXD13 staining. WT and I309F mutated HOXD13 protein are localized in nucleus only. Mutants with + 7A, + 8A, + 9A inserted in the polyalanine fragment partially entered the nucleus.

FIGURE 5

Molecular simulation of mutant HOXD13 and the transcriptional activity of I309F. (A) The simulated conformation of the polyalanine fragment dimer. Gray: 15 alanine (+ 0A), Red: 22 alanine (+ 7A). Blue: 23 alanine (+ 8A). Purple: 24 alanine (+ 9A). (B) The polymerization energy of alanine dimer. The PMF of 15, 22, 23, 24 alanine was 1.15, 11.71, 3.15, 4.58 kcal/mol, respectively. (C) I309 is located in the second α-helix of the homeobox domain. (D) The computed 3-D structure of the HOXD13 homeobox domain was generated by the I-TASSER online software. (E) Transactivation activity of the pGL3-EPHA renilla luciferase reporter plasmid by HOXD13 I309F mutants and WT. The results of the luciferase assay are presented as relative luciferase activity.