A novel NHS mutation causes Nance-Horan Syndrome in a Chinese family
The study presented here describes the clinical and molecular genetic analysis of a Chinese family exhibiting Nance-Horan Syndrome (NHS). Mutations in the NHS gene have been reported from various populations throughout the world, including Turkish, Indian, Tunisian, Australian, Taiwanese, European and Chinese populations. Clinical evaluation of the available affected male subjects revealed typical NHS ocular and nonocular features that are more severe than in obligate carriers, in concordance with previous reports [8, 11, 17]. Both affected male subjects experienced profound vision loss, and one (II: 5) underwent cataract surgery. Mild mental retardation was exhibited by an affected male (III: 1) in this family. Mental retardation in NHS is highly variable and has been reported in 20–30% of affected subjects, which suggests it is a major component of the NHS syndrome [5].
NHS carrier females manifest mild and variable phenotypes. In one study, Y-sutural posterior lens opacities associated with microcornea and dental anomalies was observed in 100% of carrier females [18, 19], though carriers presenting clear to total lens opacities with small cornea, strabismus and nystagmus have also reported [11]. In our carrier females (II: 2; III: 7), cataract was no longer observed due to cataract surgery prior to the study. Although both carriers were of normal intellect, with normal facial features, microcornea, nystagmus, high myopia and strabismus were found.
Whole-exome sequencing followed by Sanger sequencing of variants identified a novel splice site mutation (c.1045?+?2T??A) in the NHS gene, mapping to the short arm of chromosome Xp22.13.
Splice site mutations represent a significant proportion of gene alterations leading to Mendelian disorders [20]. Such mutations influencing precursor mRNA splicing either result in complete skipping of one or more exons, retention of introns, creation of a pseudo-exon or activation of a cryptic splice site with in an exon or an intron [21]. Activation of cryptic splice site is the second most frequent consequence of splicing defect after exon skipping [22]. The identified sequence variant (c.1045?+?2T??A) in the NHS gene, located just after exon 4 at the 5? consensus donor site, disrupts the natural splicing of exons 4-5.
The presence of aberrant splicing in the NHS gene caused by the NM_198270; c.1045?+?2T??A mutation was experimentally assessed by RT-PCR. Gel analysis showed and confirmed the presence of amplicons of two different lengths (421 bp, 837 bp) in a carrier female compared to a shorter amplicon (421 bp) in a control and a longer amplicon (837 bp) in an affected male. Sanger sequencing of cDNA confirmed the addition of 416 nucleotides to the mutated sequence.
The female fetus was at the 22nd week of gestation at the time of sample collection for DNA extraction. Sequencing of the NHS gene revealed the splice site mutation in a heterozygous state (m/+). Sonographic scan of the fetus (data not shown) and follow-up ophthalmological examination after birth confirmed the presence of congenital cataract, in concordance with the molecular genetic analyses.
The NHS protein has multiple isoforms, and cell type-dependent differential expression is observed. The two major isoforms, NHS-A and NHS -1A, which are implicated in the pathogenesis of NHS, contain a functional WAVE homology domain at the N-terminus. This domain interacts with members of the Abelson-interactor (Abi) family and contributes to actin remodeling regulation and maintenance of cell morphology [9]. NHS-A, the neuronal and epithelial-specific isoform, is mainly expressed in the human eye lens, and the encoded protein associates with the peripheral cell membrane in the lens epithelium [23]. The identified splice site substitution in intron 4 induces a putative premature stop codon 7 codons downstream (p.G349-IVLVFSX), which is likely to result in a truncated NHS protein. Truncating mutations may lead to nonsense mediated decay (NMD), which results in complete loss of mutated proteins. However, it has been reported that some mRNAs harboring premature termination codons located in some specific regions can escape the NMD pathway, leading to an abnormal protein product [24]. Previously described RT-PCR analysis for a truncating mutation in exon 1(c.115C??T; Q39X) of the NHS gene in an Indian NHS family found no evidence of NMD pathway involvement [17]. In the present study, RT-PCR analysis depicting the presence of an aberrant mRNA transcript in the affected and carrier subjects also suggested escape from the NMD pathway. As a possible consequence, a truncated NHS protein composed of 355 amino acids with a damaging effect would be produced in the cells of these individuals, causing the disease phenotype. However, additional work is required to understand the exact pathogenic mechanism underlying NHS.