X-linked cataract and Nance-Horan syndrome are allelic disorders

Abstract

Nance-Horan syndrome (NHS) is an X-linked developmental disorder characterized by congenital cataract, dental anomalies, facial dysmorphism and, in some cases, mental retardation. Protein truncation mutations in a novel gene (NHS) have been identified in patients with this syndrome. We previously mapped X-linked congenital cataract (CXN) in one family to an interval on chromosome Xp22.13 which encompasses the NHS locus; however, no mutations were identified in the NHS gene. In this study, we show that NHS and X-linked cataract are allelic diseases. Two CXN families, which were negative for mutations in the NHS gene, were further analysed using array comparative genomic hybridization. CXN was found to be caused by novel copy number variations: a complex duplication-triplication re-arrangement and an intragenic deletion, predicted to result in altered transcriptional regulation of the NHS gene. Furthermore, we also describe the clinical and molecular analysis of seven families diagnosed with NHS, identifying four novel protein truncation mutations and a novel large deletion encompassing the majority of the NHS gene, all leading to no functional protein. We therefore show that different mechanisms, aberrant transcription of the NHS gene or no functional NHS protein, lead to different diseases. Our data highlight the importance of copy number variation and non-recurrent re-arrangements leading to different severity of disease and describe the potential mechanisms involved.

Figure 1.

Pedigree structures. Families A, B, C, D, E, F and G were diagnosed with NHS. Families H and I were diagnosed with X-linked cataract. Black boxes denote affected males; dotted circles, carrier females; clear boxes and circles, unaffected individuals; and clear circles with a question mark, females with unknown clinical status.

Figure 2.

Patient phenotypes. (A) Carrier cataract phenotype (I:1) of Family A. (B) Family B dental phenotype (II:3) showing screwdriver-shaped incisors and broad diastema mediale with hyperplastic frenulum mediale and (C) hypoplastic enamel. (D) Family C proband (II:1) with dental anomalies and (E) sutural cataract phenotype of carrier mother (I:1). (F) Family E proband (III:2) with screwdriver-shaped incisors. (G) Relatively mild cataract phenotype of affected male (II:1) in Family F. (H) Typical features of NHS in affected male II:1 from Family G.

Figure 3.

Patient mutations identified in the NHS gene. (A) Schematic representation of the X-chromosomal region surrounding the NHS gene (16.80–18.30 Mb). Arrows in red indicate position and direction of genes. Genomic structure of the NHS gene is shown with mutations identified to date. (B) Sequence chromatograms showing protein truncation mutations identified in Families A–F. Mutations detected in affected male sequence are shown below the unaffected control male chromatograms. (C) Agarose gels showing amplification of NHS exon 1a and SCML2 exons 1 and 14 and absence of NHS exon 8, SCML1 exons 1 and 8, RAI2 exon 3, CXorf20 exons 1 and 14 for the affected male in NHS Family G (II:1) compared with a control male (+). Corresponding amplicon sizes are indicated.

Figure 4.

Sequence analysis of chromosome re-arrangements. Sequence analysis of breakpoint junctions of chromosome re-arrangements in Families F, H and I, and conserved regions in Families H and I. Normal distal genomic sequence is shown in orange on the top line; patient genomic breakpoint sequence is shown on the middle line; normal proximal genomic sequence is shown in blue on the bottom line. Chromosome positions are indicated along the sequences. (A) Chromosomal re-arrangement in Family F. A 17 bp fragment (green) between the distal and the proximal reference sequence is derived from the reverse strand in the intergenic region between NHS and SCML1 genes. Regions of homology at the distal and proximal breakpoint sequences are boxed. (B) Breakpoint 4 of the complex duplication–triplication copy number variation in Family H. An insertion of 8 bp at the distal breakpoint (green) and an 18 bp deletion at the proximal breakpoint (blue dash) were detected. ‘Mirrored’ sequences at the distal and proximal breakpoint regions are boxed. (C) Deletion breakpoint in Family I shows identical nucleotides between the proximal and distal flanking sequence. A region of complete homology is boxed. (D) Breakpoint 4 in Family H occurs within a 240 bp sequence conserved across species. (E) Sequence conservation across species of a 340 bp region within the deletion in Family I.

Figure 5.

Copy number variations identified in CXN families. (A) Array CGH results from patient IV:4 in Family H. A complex duplication–triplication was found between ChX:17105121 and ChX:17926943. Position of the NHS gene and neighbouring genes are shown, and affected genes are highlighted in red. Genes present at normal copy number are shown in green. A schematic representation of the duplication–triplication region is shown below depicting breakpoint regions 1–4. (B) Relative quantification of qPCR data showing R_o values averaged between affected samples IV:1 and IV:4 normalized to R_o values averaged between WT samples III:2 and V:3. (C) Sequence derived from breakpoint 3 is shown with ChrX:17800261 joined to sequence of unknown origin, and sequence derived from breakpoint 4 is shown with ChrX:17926944 sequence joined to ChrX:17105038 sequence. (D) Genetic analysis of other relatives in the family demonstrated segregation of this complex re-arrangement in Family H with all affected and carriers individuals. (E) Array CGH results from patient III:1 in Family I. A deletion of a 4.8 kb region is located between ChrX:17461256 and ChrX:17466720. This deleted region is located within intron 1 of the NHS gene and is shown in red above the CGH data. (F) Agarose gel showing that PCR across the deletion breakpoint in affected male III:1 (CXN Family I) produces a 913 bp fragment compared with a control (+) fragment of 5724 bp. Fragment sizes are indicated. (G) Patient chromatogram showing the breakpoint sequence, which occurs at ChrX:17461765.