Identification And Functional Characterization Of The Disease-causing Mutation Of A Family With Nance-Horan Syndrome

Purpose:Nance-Horan syndrome (NHS) is a rare X-linked disorder characterized by congenital nuclear cataracts, dental anomalies, and craniofacial dysmorphisms. Mental retardation was present in about30%of the reported cases. The disease is caused by mutations in the NHS gene, which comprises10coding exons and encodes at least six isoforms as a result of alternative splicing. NHS-A and NHS-1A are two major isoforms of the NHS protein. The two isoforms are transcribed from exon1of the NHS gene, encoding a1630-amino-acid protein and a1651-amino-acid protein, respectively. Both of the two NHS protein isoforms contain a WASP family verprolin-homologous (WAVE) domain (WHD) in the N-terminus, functionally homologous to the WHD in WAVE proteins. The WHD can interact with members of the Abelson-interactor (Abi) protein family, such as Abil and Abi2, playing a regulatory role in actin remodelling and maintaining cell morphology. The purpose of this study was to investigate the clinical and molecular features of a four-generation Chinese family containing three affected males who presented with congenital cataract and microcornea. A combination of exome sequencing and Sanger sequencing was performed to identified the disease-causing mutation of the family. Functional analysis of the NHS gene was studied in vitro.Methods:1. Pedigree construction was conducted using Cyrillic software on the basis of the results of clinical examination and interview of the available members of the family. Inheritance mode of the family was analyzed by pedigree analysis. The ocular features and nonocular features of all male affects and female carriers were comprehensively examined. Genomic DNA was extracted from the blood.2. Whole exome sequencing analysis was performed on DNA from an affected male to scan for candidate mutations. Sanger sequencing was used to verify these candidate mutations in the whole family and50unrelated controls. Multiple sequence alignment of NHS protein sequences from seven different species was performed using CLC Sequence Viewer5.5(CLC bio A/S, http://www.clcbio.com).3. The mutant plasmid pEGFP-C2-NHS-c.322G>T (NHSE108X-EGFP) was construncted by site-directed mutagenesis on the basis of the wild plasmid pEGFP-C2-NHS (NHS-EGFP) expressing NHS-1A protein. NHS-EGFP, NHSE108X-EGFP and pEGFP-C2plasmids were transient transfected into Hek293T cell and SRA01/04cell. Subcellular localization of the GFP-fusion prqteins in SRA01/04cell was observed by inverted fluorescence microscopy. Quantitative real-time polymerase chain reaction (QRT-PCR) was performed to test the relative expression level of NHS mRNA in transfected Hek293T cell lines. Small inference RNA(siRNA) targeting NHS gene was transfected into human lens epitheliual cell line SRA01/04. The expressions level of Abi2protein was detected by western blot analysis. Transwell chamber test was used to observe the SRA01/04cell migration ability.Results:1. Seven members of a four-generation family participated in the study, including three affected males, two carrier females and two unaffected members. All affected males had received bilateral lensectomy at an early age to remove congenital nuclear cataracts. A male individual exhibited typical NHS dental anomalies. All presented with bilateral microcornea, nystagmus, and strabismus. All carrier females had bilateral lens opacity and bitemporal retraction. Pedigree analysis of the family revealed the mode of inheritance might be X-linked mode.2. A combination of exome sequencing and Sanger sequencing revealed a nonsense mutation c.322G>T (E108X) in exon1of the NHS gene, co-segregating with the disease in the family. The nonsense mutation led to the conversion of glutamic acid to a stop codon (E108X), resulting in truncation of the NHS protein. Multiple sequence alignments showed that codon108, where the mutation (c.322G>T) occurred, was located within a phylogenetically conserved region.3. The wild protein localize to the cytoplasm of SRA01/04cell, while the mutant protein was found in the nucleus and cytoplasm. QRT-PCR showed that the expression level of the mutated NHS was significantly decreased compared with that of the wild type (P<0.05). Western blot showed the expression level of Abi2protein in SRA01/04cell reduced after transfection of siRNA of NHS (P<0.05). Transwell assay results showed that knockdown of NHS expression reduced the migration ability of SRA01/04cell (P<0.05).Conclusions:1. We characterized a Chinese family with Nance-Horan syndrome. The phenotypic heterogeneity was found in the family.2. We identified a novel nonsense mutation (c.322G>T) in exon1of the NHS gene in a Chinese family. Multiple sequence alignments showed that codon108was located within a phylogenetically conserved region3. The noval mutation affected the subcellular localization of the NHS protein and the mRNA stability of the NHS gene. siRNA-NHS can reduce expression level of Abi2protein and the migration ability of human lens epithelial cell SRA01/04.