Two novel missense substitutions in the VSX1 gene: clinical and genetic analysis of families with Keratoconus from India

Abstract

Background: Visual system homeobox gene (VSX1) plays a major role in the early development of craniofacial and ocular tissues including cone opsin gene in the human retina. To date, few disease-causing mutations of VSX1 have been linked to familial and sporadic keratoconus (KC) in humans. In this study, we describe the clinical features and screening for VSX1 gene in families with KC from India.

Methods: Clinical data and genomic DNA were collected from patients with clinically diagnosed KC and their family members. The study was conducted on 20 subjects of eight families from India. The coding exons of VSX1 gene were amplified using PCR and amplicons were analyzed by direct sequencing. Predictive effect of the mutations was performed using Polyphen-2, SIFT and mutation assessor algorithms. Additionally, haplotypes of VSX1 gene were constructed for affected and unaffected individuals using SNPs.

Results: In the coding region of VSX1, one novel missense heterozygous change (p.Leu268His) was identified in five KC patients from two unrelated families. Another family of three members had a novel heterozygous change (p.Ser251Thr). These variants co-segregated with the disease phenotype in all affected individuals but not in the unaffected family members and 105 normal controls. In silico analysis suggested that p.Leu268His could have a deleterious effect on the protein coded by VSX1, while p.Ser251Thr has a neutral effect on the functional properties of VSX1. Haplotype examination revealed common SNPs around the missense change (p.Leu268His) in two unrelated KC families.

Conclusions: In this study, we add p.Leu268His, a novel missense variation in the coding region of VSX1 to the existing repertoire of VSX1 coding variations observed in Indian patients with the characteristic phenotype of KC. The variant p.Ser251Thr might be a benign polymorphism, but further biophysical studies are necessary to evaluate its molecular mechanism. The shared haplotype by two families with the same variant suggests the possibility of a founder effect, which requires further elucidation. We suggest that p.Leu268His might be involved in the pathogenesis of KC, which may help in the genetic counselling of patients and their family.

Fig. 1

Pedigrees of the KC families with novel coding variants in VSX1 Legend: A, B, C denotes three unrelated families. Squares and circles indicate males and females, respectively. Black symbols indicate affected members and open symbols indicate unaffected individuals. The black arrow indicates the proband, the sign ‘+’ represents the wild type and the mutations identified are p. Leu268His, p. Ser251Thr

Fig. 2

Novel VSX1 coding variants in KC families A, B: Comparison of DNA sequence chromatogram of an unaffected individual (top) with an affected (bottom). A: Patients DNA from KC–01, KC–02 revealed heterozygous T-to-A (c.803 T > A) transition (black arrow) in exon 4 of VSX1, resulting in a leucine 268 (CTC) to histidine (CAC) change (p. Leu268His). B: Affected individuals from family KC–03 shows a heterozygous T > A (c.751 T > A) nucleotide change in exon 4 of VSX1, which altered the serine 251 (TCC) to theronine (ACC) amino acid change (p. Ser251Thr). C: Amino acid sequence alignment of the human VSX1 protein (amino acids from 234–274) with other species. The Ser 251 and Leu 268 are shown in red

Fig. 3

Corneal topography of the KC probands with VSX1 coding variants. A. Pentacam image of patient II:2 from KC–01, both eyes show an area of inferior steepening on the sagittal curvature map with gross inferior-superior asymmetry, more in the right eye. This area of steepening corresponds to areas of abnormal elevation on both the anterior and posterior elevation maps with values suggestive of Keratoconus (KC) with an inferior cone. B. In the second family (KC–02), patient II:1 has an area of central steepening on the sagittal curvature map. This area of steepening corresponds to areas of abnormal elevation on both the anterior and posterior elevation maps with values suggestive of KC with a central cone in the left eye (OS); right eye (OD) showing the posterior elevation, suggestive of early KC. C. The sagittal curvature on Scheimpflug imaging of patient II:2 from the third family (KC–03) with the left eye showing (OS) an area of central steepening. The anterior and posterior curvature maps show areas of abnormal elevation with values suggestive of KC with a central cone. The corneal thickness map also shows an area of central thinning which is corresponding to the areas of abnormal elevation. The right eye (OD) shows a fairly central area of steepening with features suggestive of advanced KC. There is gross posterior elevation with significant corneal thinning (thinnest pachymetry of 370 μm) in the central 3 mm zone

Fig. 4

Pedigrees and haplotype analysis of VSX1 containing L268H and S251T variants in KC families. Legend: A,B,C The haplotype segregating with L268H and S251T are presented on white and gray backgrounds, respectively. Sharing of three common SNPs markers (KC–01,KC–02) families were shown in red