Brooks, S.P.; (2006) Molecular genetic and functional analyses of X-linked congenital cataract. Doctoral thesis , University of London.

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Abstract

Nance-Horan Syndrome (NHS) is an X-linked developmental syndrome characterised by congenital cataract, dental anomalies, and dysmorphological features often associated with mental retardation. The NHS locus on Xp22.13 is encompassed by the disease locus for X- linked congenital cataract (CXN). Analysis of microsatellites within the CXN family resulted in refinement of the CXN disease interval, reducing the region of overlap between the CXN and NHS disease loci. Candidate genes in the overlapping intervals were identified bioinformatically and their genomic structures evaluated. Patient DNA was screened by direct sequencing, resulting in the identification of mutations within a novel gene in four British families with NHS, but not the CXN family. This novel gene, named NHS, is encoded by at least 10 exons transcribed into at least five mRNA isoforms A, B, C, D, and E (encoding a putative 1,630 a.a., 1,335 a.a., 1,474 a.a., 1,453 a.a., and 1,473 a.a. protein, respectively). All mutations identified are truncating and three mutations have been identified in exon 1, which are only expressed in isoform A. This implies that mutations in isoform A are sufficient to cause disease in families with NHS. Functional clues for the NHS protein were investigated resulting in identification of three new genes with significant homology to NHS {lcub}NHS-Like 1 (NHSL1), NHSL2 and NHSL3). All four genes share a conserved genomic structure. Fetal expression analysis of NHS, NHSL1 and NHSL2 suggests that NHSL1 and NHSL2 are more ubiquitous than NHS. Analysis of the NHS family of proteins revealed significant homology to members of the WASP family, which consists of WASP, N-WASP and WAVE 1-3. The WASP protein family play a crucial role in regulating actin dynamics, directly linking small GTPase signalling to actin polymerisation through activation of the Arp2/3 complex. An anti-peptide antibody to the C-terminus of NHS, completely conserved across species, was raised and characterised. A major NHS isoform (approximately 170 kDa) was detected in several cell lines. Subcellular localisation studies in MTLn3 cells showed localization of endogenous NHS to the leading edge of lamellipodia, a localisation pattern reminiscent of the Arp2/3 complex. Endogenous NHS also localised to some actin stress fibres. Homology to the WASP protein family and localisation of endogenous NHS to the leading edge of lamellipods strongly supports a role for NHS in actin cytoskeletal dynamics during development.

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