Annette Payne

Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12)

Retinitis pigmentosa (RP) comprises a clinically and genetically heterogeneous group of diseases that afflicts approximately 1.5 million people worldwide. Affected individuals suffer from a progressive degeneration of the photoreceptors, eventually resulting in severe visual impairment. To isolate candidate genes for chorioretinal diseases, we cloned cDNAs specifically or preferentially expressed in the human retina and the retinal pigment epithelium (RPE) through a novel suppression subtractive hybridization (SSH) method. One of these cDNAs (RET3C11) mapped to chromosome 1q31–q32.1, a region harbouring a gene involved in a severe form of autosomal recessive RP characterized by a typical preservation of the para-arteriolar RPE (RP12; ref. 3). The full-length cDNA encodes an extracellular protein with 19 EGF-like domains, 3 laminin A G-like domains and a C-type lectin domain. This protein is homologous to the Drosophila melanogaster protein crumbs (CRB), and denoted CRB1 (crumbs homologue 1). In ten unrelated RP patients with preserved para-arteriolar RPE, we identified a homozygous AluY insertion disrupting the ORF, five homozygous missense mutations and four compound heterozygous mutations in CRB1. The similarity to CRB suggests a role for CRB1 in cell-cell interaction and possibly in the maintenance of cell polarity in the retina. The distinct RPE abnormalities observed in RP12 patients suggest that CRB1 mutations trigger a novel mechanism of photoreceptor degeneration.

Chromosomal duplication involving the forkhead transcription factor gene FOXC1 causes iris hypoplasia and glaucoma

The forkhead transcription factor gene FOXC1 (formerly FKHL7) is responsible for a number of glaucoma phenotypes in families in which the disease maps to 6p25, although mutations have not been found in all families in which the disease maps to this region. In a large pedigree with iris hypoplasia and glaucoma mapping to 6p25 (peak LOD score 6.20 [recombination fraction 0] at D6S967), no FOXC1 mutations were detected by direct sequencing. However, genotyping with microsatellite repeat markers suggested the presence of a chromosomal duplication that segregated with the disease phenotype. The duplication was confirmed in affected individuals by FISH with markers encompassing FOXC1. These results provide evidence of gene duplication causing developmental disease in humans, with increased gene dosage of either FOXC1 or other, as yet unknown genes within the duplicated segment being the probable mechanism responsible for the phenotype.

GCAP1(Y99C) mutant is constitutively active in autosomal dominant cone dystrophy

GCAP1 stimulates photoreceptor guanylate cyclase (GC) in bleached vertebrate photoreceptors when [Ca2+]free decreases but is inactivated when cytoplasmic [Ca2+]free increase after dark adaptation. A Y99C mutation in GCAP1 has recently been found to be associated with autosomal dominant cone dystrophy. We show that the GCAP1(Y99C) mutant and native GCAP1 are highly effective in stimulation of photoreceptor GC1. The Ca2+ sensitivity of the mutant GCAP1, however, is markedly altered, causing reduced but persistent stimulation of GC1 under physiological dark conditions. These results are consistent with a model in which enhanced GC activity in dark-adapted cones leads to elevated levels of cytoplasmic cGMP. Alterations in physiological cGMP levels are also associated with other retinal degenerations, including Lebers congenital amaurosis.

Clustering and frequency of mutations in the retinal guanylate cyclase (GUCY2D) gene in patients with dominant cone-rod dystrophies

Editor—Guanylate cyclase (retGC-1) is a key enzyme in the recovery phase of phototransduction in both cone and rod photoreceptor cells.1 Upon excitation by a photon of light, an enzymatic cascade of events occurs which leads to the hydrolysis of cGMP and the closure of the cGMP gated cation channels. This results in hyperpolarisation of the plasma membrane and the generation of a signal higher up in the visual pathway. Upon closure of the ion channels, the cytosolic levels of Ca2+ decrease because export by the Na+, K+, Ca2+exchanger continues. This reduced Ca2+ concentration results in the activation of retGC by activating proteins (GCAPs) and the increased conversion of GTP to cGMP, thus restoring the level of cGMP in the photoreceptors to their dark level. Mutations in GUCY2D, the gene encoding retGC-1 , are a cause of Leber congenital amaurosis (LCA1), a recessive condition which manifests itself either at birth or during the first few months of life as total or near total blindness.2 3 Recently, we identified mutations in GUCY2D in four British families with autosomal dominant cone-rod dystrophy (ADCORD).4Subsequent to this, mutations in this gene were shown to be responsible for ADCORD in a French,5 a Swiss,6 and a Norwegian7 family. In all seven families, the mutations are either in the same or in adjacent codons in a highly conserved region of the protein. In our four families and in the Swiss and Norwegian families, mutations were found in either codon 837 or 838,4 6 7 whereas codons 837-839 each encode for an amino acid substitution in the French family.5 In order to determine whether ADCORD arising from mutations in GUCY2D are restricted to these codons and how important these …

A Locus for Autosomal Recessive Congenital Microphthalmia Maps to Chromosome 14q32

Congenital microphthalmia (CMIC) (OMIM 309700) may occur in isolation or in association with a variety of systemic malformations. Isolated CMIC may be inherited as an autosomal dominant, an autosomal recessive, or an X-linked trait. On the basis of a whole-genome linkage analysis, we have mapped the first locus for isolated CMIC, in a five-generation consanguineous family with autosomal recessive inheritance, to chromosome 14q32. All affected individuals in this family have bilateral CMIC. Linkage analysis gave a maximum two-point LOD score of 3.55 for the marker D14S65. Surrounding this marker is a region of homozygosity of 7.3 cM, between the markers D14S987 and D14S267, within which the disease gene is predicted to lie. The genes for several eye-specific transcription factors are located on human chromosome 14q and in the syntenic region of mouse chromosome 12. However, both CHX10 (14q24.3), mutations of which give rise to CMIC in mouse models, and OTX2 (14q21-22) can be excluded as candidates for autosomal recessive congenital microphthalmia (arCMIC), since they map outside the critical disease region defined by recombination events. This suggests that arCMIC is caused by defects in a novel developmental gene that may be important or even essential in eye development.

Genetic analysis of the guanylate cyclase activator 1B (GUCA1B) gene in patients with autosomal dominant retinal dystrophies

The guanylate cyclase activator proteins (GCAP1 and GCAP2) are calcium binding proteins which by activating Ret-GC1 play a key role in the recovery phase of phototransduction. Recently a mutation in theGUCA1A gene (coding for GCAP1) mapping to the 6p21.1 region was described as causing cone dystrophy in a British family. In addition mutations in Ret-GC1have been shown to cause Leber congenital amaurosis and cone-rod dystrophy. To determine whether GCAP2 is involved in dominant retinal degenerative diseases, the GCAP2 gene was screened in 400 unrelated subjects with autosomal dominant central and peripheral retinal dystrophies. A number of changes involving the intronic as well as the coding sequence were observed. In exon 1 a T to C nucleotide change was observed leaving the tyrosine residue 57 unchanged. In exon 3 a 1 bp intronic insertion, a single nucleotide substitution G to A in the intron 3′ of this exon, and a GAG to GAT change at codon 155 were observed. This latter change results in a conservative change of glutamic acid to aspartic acid. In exon 4 a 7 bp intronic insertion, a single nucleotide A to G substitution in the intron 5′ of this exon, and a single base pair change C to G in the intron 3′ of exon 4 were seen. None of these changes would be expected to affect correct splicing of this gene. All these changes were observed in controls. The results of this study do not show any evidence so far that GCAP2 is involved in the pathogenesis of autosomal dominant retinal degeneration in this group of patients. All the changes detected were found to be sequence variations or polymorphisms and not disease causing.

Epigenetic Inactivation Implies Independent Functions for Insulin-like Growth Factor Binding Protein (IGFBP)-Related Protein 1 and the Related IGFBPL1 in Inhibiting Breast Cancer Phenotypes

Purpose: To analyze epigenetic regulation of two related genes, insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) and IGFBPL1, and its significance as a determinant of clinical phenotypes in human breast cancer. Experimental Design: We have investigated the expression and epigenetic regulation of IGFBP-rP1 and IGFBPL1 in human breast cancer cell lines and primary and metastatic carcinomas. Results: Expression of IGFBP-rP1 and IGFBPL1 is down-regulated in breast cancer cell lines. Aberrant methylation in the CpG islands of each gene correlates well with loss of expression at the mRNA level. Analysis of methylation in DNA isolated from human primary breast tumors showed that methylation in either gene was associated with a worse overall survival (OS; P = 0.008) and disease-free survival (DFS) following surgery (P = 0.04) and worse DFS following adjuvant chemotherapy (P = 0.01). Methylation of IGFBP-rP1 alone was associated with a trend toward decreased OS (P = 0.10) and decreased DFS (P = 0.25). Methylation in IGFBPL1 was clearly associated with worse OS (P = 0.001) and DFS (P < 0.0001). Methylation in either IGFBP-rP1 or IGFBPL1 was significantly associated with nodal disease (P < 0.001). Conclusions: Expression of IGFBP-rP1 and IGFBPL1 is regulated by aberrant hypermethylation in breast cancer, implying that inactivation of these genes is involved in the pathogenesis of this malignancy. Analysis of methylation of these genes may have utility in prediction of clinical phenotypes, such as nodal disease and response to chemotherapy.

The use of an e-learning constructivist solution in workplace learning

Motivation -- To investigate whether an e-learning approach which uses constructivist principles can be successfully applied to train employees in a highly specialised skill thought to require expert individuals and extensive prolonged training. Research approach -- The approach involved the development of an e-learning package which included simulations and interactivity, then experimental testing in the workplace environment with the collection of both quantitative and qualitative data to assess the effectiveness of the package. Findings -- Our study shows that this e-learning strategy improved the skills of the inexperienced operator significantly. We therefore propose that such programmes could be used as a work based training aids and used as a model system for the training of employees in complex skilled tasks in the workplace. Research Implications -- This research illustrates that the e-learning can be applied outside the traditional learning environment to train unskilled employees to undertake complex practical tasks which traditionally would involve prohibitively expensive instruction. This work also illustrates that simulations and interactivity are a powerful tools in the design of successful e-learning packages in preparing learners for real world practical situations. We would suggest that workplace learners can be better served by e-learning environments as they allow asynchronous learning and private study which are valued by employees who have other demands on their time and are more comfortable receiving tuition privately. Originality/Value -- This research uniquely demonstrates the harnessing of simulations and interactivity in an e-learning environment for high skill level workplace training. Take away message -- In conclusion we present an e-learning solution to the teaching of a complex task which at present is seen as too costly and specialised to be worth sending employees on a traditional training course to learn. The use of constructivism principles to aid its design, along with acknowledged e-learning best practice demonstrates that this medium can be used successfully in the training of highly specialised and skilled tasks required in the modern workplace.

Severe autosomal dominant retinitis pigmentosa caused by a novel rhodopsin mutation (Ter349Glu)

Mutations in the rhodopsin gene are reported to be responsible for approximately 25% of all cases of autosomal dominant Retinitis pigmentosa (adRP). Affected individuals from a large family with an unusually severe form of adRP were screened for mutations in the rhodopsin gene. Direct sequencing of exon 5 revealed a TAA to GAA transversion at nucleotide 5276 / codon 349, which was confirmed by Dde 1 restriction digest analysis. This change would replace the normal termination codon with a glutamic acid residue (Ter‐349‐Glu, or X349E). The next predicted termination codon (TAA) lies 153bp downstream at nucleotides 5429 to 5431. Termination of transcription at this point would add an additional 51 amino‐acid residues to the carboxy terminus of the rhodopsin molecule. This mutation is unique in producing a mutant rhodopsin in which all of the normal 348 amino‐acid residues remain intact. It produces one of the most severe adRP phenotypes ever observed in a family with a rhodopsin mutation. In view of this the Ter‐349‐Glu mutation is worthy of further investigation to determine how the presence of this particular mutant opsin leads to rod photoreceptor degeneration.

Phenotype of autosomal recessive congenital microphthalmia mapping to chromosome 14q32

BACKGROUND Congenital microphthalmia (OMIM: 309700) may occur in isolation or in association with a variety of systemic malformations. Isolated microphthalmia may be inherited as an autosomal dominant, an autosomal recessive, or an X linked trait. METHODS Based on a whole genome linkage analysis, in a six generation consanguineous family with autosomal recessive inheritance, the first locus for isolated microphthalmia was mapped to chromosome 14q32. Eight members of this family underwent clinical examination to determine the nature of the microphthalmia phenotype associated with this locus. RESULTS All affected individuals in this family suffered from bilateral microphthalmia in association with anterior segment abnormalities, and the best visual acuity achieved was “perception of light”. Corneal changes included partial or complete congenital sclerocornea, and the later development of corneal vascularisation and anterior staphyloma. Intraocular pressure, as measured by Schiotz tonometry, was greatly elevated in many cases. CONCLUSIONS This combination of ocular defects suggests an embryological disorder involving tissues derived from both the neuroectoderm and neural crest. Other families with defects in the microphthalmia gene located on 14q32 may have a similar ocular phenotype aiding their identification.