Clinical and genetic analysis in Chinese families with synpolydactyly, and cellular localization of HOXD13 with different length of polyalanine tract

Abstract

Synpolydactyly (SPD) is caused by mutations in the transcription factor gene HOXD13. Such mutations include polyalanine expansion (PAE), but further study is required for the phenotypic spectrum characteristics of HOXD13 PAE. We investigated four unrelated Chinese families with significant limb malformations. Three PAEs were found in the HOXD13 polyalanine coding region: c.172_192dup (p.Ala58_Ala64dup) in Family 1, c.169_192dup (p.Ala57_Ala64dup) in Family 2, and c.183_210dup (p.Ala62_Ala70dup) in Family 3 and Family 4. Interestingly, we identified a new manifestation of preaxial polydactyly in both hands in a pediatric patient with an expansion of seven alanines, a phenotype not previously noted in SPD patients. Comparing with the wild-type cells and mutant cells with polyalanine contractions (PACs), the HOXD13 protein with a PAE of nine-alanine or more was difficult to enter the nucleus, and easy to form inclusion bodies in the cytoplasm, and with the increase of PAE, the more inclusion bodies were formed. This study not only expanded the phenotypic spectrum of SPD, but also enriched our understanding of its pathogenic mechanisms.

Keywords: HOXD13; polyalanine expansion; preaxial polydactyly; synpolydactyly; variant.

FIGURE 1

Pedigrees of four unrelated families with the synpolydactyly. The arrows indicate the proband. The asterisks represent the individuals in the families who provided peripheral blood samples.

FIGURE 2

Clinical manifestation of patients with synpolydactyly. (A) Bilateral preaxial polydactyly of hands and postaxial synpolydactyly of feet found in a pediatric patient. (B) Bilateral or unilateral synpolydactyly in 3/4 fingers of the affected individuals in SPD families. (C) Congenital syndactyly between the 3/4 fingers. (D) There were syndactyly in the proband’s 3/4 fingers. The arrows indicate the patients’ fingers.

FIGURE 3

Polyalanine expansions of HOXD13 identified in the families with synpolydactyly. (A) Expansion of seven-alanine, c.172_192dup (p.Ala58_Ala64dup), in Family 1. (B) Expansion of eight-alanine, c.169_192dup (p.Ala57_Ala64dup), in Family 2. (C) Expansion of nine-alanine, c.183_210dup (p.Ala62_Ala70dup), in Family 3 and Family 4. (D) Take Family 4 as an example, pattern diagram of different codons in HOXD13 polyalanine tract of the normal allele and mutant allele with expansion of nine-alanine. (E) Two bands (182bp and 161bp) were separated on 8% neutral polyacrylamide gel. The result showed that all patients had the duplication.

FIGURE 4

Cellular localization of the fusion protein of GFP-HOXD13 in COS-7 cells. (A) Expression of eight vectors: GFP, pEGFP-C3 vector; WT, the pEGFP-C3 with wild type HOXD13; three HOXD13 poly-Ala mutant plasmids with polyalanine contractions (PACs) (−13A, −11A and -7A); three HOXD13 poly-Ala mutant plasmids with polyalanine expansions (PAEs) (+9A, +14A, and +17A). The wild type and the PAC mutant of HOXD13 did not affect the cellular localization of the fusion protein, but the PAE of HOXD13 caused the aggregation of fusion proteins in the cytoplasm. Moreover, with the increase of PAE, the inclusion body of fusion protein in cytoplasm increased. (B) Schematic structure of HOXD13, the polyalanine tract located in the first exon and the homeobox domain located in the second exon.