Co-occurrence of orofacial clefts and clubfoot phenotypes in a sub-Saharan African cohort: Whole-exome sequencing implicates multiple syndromes and genes

Abstract

Background: Orofacial clefts (OFCs) are congenital malformations of the face and palate, with an incidence of 1 per 700 live births. Clubfoot or congenital talipes equinovarus (CTEV) is a three-dimensional abnormality of the leg, ankle, and feet that leads to the anomalous positioning of foot and ankle joints and has an incidence of 1 per 1000 live births. OFCs and CTEV may occur together or separately in certain genetic syndromes in addition to other congenital abnormalities. Here, we sought to decipher the genetic etiology of OFC and CTEV that occurred together in six probands.

Methods: At the time of recruitment, the most clinically obvious congenital anomalies in these individuals were the OFC and CTEV. We carried out whole-exome sequencing (WES) on DNA samples from probands and available parents employing the Agilent SureSelect XT kit and Illumina HiSeq2500 platform, followed by bioinformatics analyses. WES variants were validated by clinical Sanger Sequencing.

Results: Of the six probands, we observed probable pathogenic genetic variants in four. In three probands with probable pathogenic genetic variants, each individual had variants in three different genes, whereas one proband had probable pathogenic variant in just one gene. In one proband, we observed variants in DIS3L2, a gene associated with Perlman syndrome. A second proband had variants in EPG5 (associated with Vici Syndrome), BARX1 and MKI67, while another proband had potentially etiologic variants in FRAS1 (associated with Fraser Syndrome 1), TCOF1 (associated with Treacher Collins Syndrome 1) and MKI67. The last proband had variants in FRAS1, PRDM16 (associated with Cardiomyopathy, dilated, 1LL/Left ventricular noncompaction 8) and CHD7 (associated with CHARGE syndrome/Hypogonadotropic hypogonadism 5 with or without anosmia).

Conclusion: Our results suggest that clubfoot and OFCs are two congenital abnormalities that can co-occur in certain individuals with varying genetic causes and expressivity, warranting the need for deep phenotyping.

Keywords: clubfoot; genetic syndromes; orofacial clefts; whole-exome sequencing.

FIGURE 1

Phenotypes observed in the proband of Family 5. (a) Bilateral cleft lip and palate. (b) Bilateral clubfoot phenotype with one foot slightly superimposed on the other to depict the severity of the defect. (c) Two feet standing in isolation to depict the syndactyly on left foot but lack of same in the right foot. (d and e) syndactyly of third, fourth, and fifth toes of the left foot

FIGURE 2

Electropherogram and simulation of the effect of mutations on protein structure by HOPE for the novel variants. (a) electropherogram for c.1594C>T variant in EPG5 (NM_020964.3), with a white vertical line indicating the position of the variant—top electropherogram shows the heterozygous variant in proband and bottom one is for the mother who lacked the variant. (b) electropherogram for c.4248delA in CHD7 (NM_017780.4), with a white vertical line indicating the position of the variant—top electropherogram indicates the heterozygous deletion in the proband whereas the bottom is for the mother who did not have the variant. (c) Schematic simulation of mutant protein for p.Pro293His in DIS3L2 (NM_001257281.2) by HOPE: proline is more hydrophobic and smaller than histidine. (d) Ribbon simulation of mutant protein for p.Pro293His in DIS3L2 by HOPE: green and red indicate wild‐type and mutant residues, respectively. (e) Schematic representation of p.His532Tyr variant in EPG5: the mutant residue, (tyrosine) is bigger and more hydrophobic than the wild‐type residue (histidine)