Bacteriophage gene targeting vectors generated by transplacement.

A rate-determining step in gene targeting is the generation of the targeting vector. We have developed bacteriophage gene targeting vectorology, which shortens the timeline of targeting vector construction. Using retro-recombination screening, we can rapidly isolate targeting vectors from an embryonic stem cell genomic library via integrative and excisive recombination. We have demonstrated that recombination can be used to introduce specific point mutations or unique restriction sites into gene targeting vectors via transplacement. Using the choline/ethanolamine kinase alpha and beta genes as models, we demonstrate that transplacement can also be used to introduce specifically a neo resistance cassette into a gene targeting phage. In our experience, the lambdaTK gene targeting system offers considerable flexibility and efficiency in TV construction, which makes generating multiple vectors in one week's time possible.

Clinical and molecular genetic characterisation of a family segregating autosomal dominant retinitis pigmentosa and sensorineural deafness.

AIMS/BACKGROUND: To characterise clinically a large kindred segregating retinitis pigmentosa and sensorineural hearing impairment in an autosomal dominant pattern and perform genetic linkage studies in this family. Extensive linkage analysis in this family had previously excluded the majority of loci shown to be involved in the aetiologies of RP, some other forms of inherited retinal degeneration, and inherited deafness. METHODS: Members of the family were subjected to detailed ophthalmic and audiological assessment. In addition, some family members underwent skeletal muscle biopsy, electromyography, and electrocardiography. Linkage analysis using anonymous microsatellite markers was performed on DNA samples from all living members of the pedigree. RESULTS: Patients in this kindred have a retinopathy typical of retinitis pigmentosa in addition to a hearing impairment. Those members of the pedigree examined demonstrated a subclinical myopathy, as evidence by abnormal skeletal muscle histology, electromyography, and electrocardiography. LOD scores of Zmax = 3.75 (theta = 0.10), Zmax = 3.41 (theta = 0.10), and Zmax = 3.25 (theta = 0.15) respectively were obtained with the markers D9S118, D9S121, and ASS, located on chromosome 9q34-qter, suggesting that the causative gene in this family may lie on the long arm (q) of chromosome 9. CONCLUSIONS: These data indicate that the gene responsible for the phenotype in this kindred is located on chromosome 9 q. These data, together with evidence that a murine deafness gene is located in a syntenic area of the mouse genome, should direct the research community to consider this area as a candidate region for retinopathy and/or deafness genes.

Neurons from stem cells: implications for understanding nervous system development and repair.

Neurodegenerative diseases cost the economies of the developed world billions of dollars per annum. Given ageing population profiles and the increasing extent of this problem, there has been a surge of interest in neural stem cells and in neural differentiation protocols that yield neural cells for therapeutic transplantation. Due to the oncogenic potential of stem cells a better characterisation of neural differentiation, including the identification of new neurotrophic factors, is required. Stem cell cultures undergoing synchronous in vitro neural differentiation provide a valuable resource for gene discovery. Novel tools such as microarrays promise to yield information regarding gene expression in stem cells. With the completion of the yeast, C. elegans, Drosophila, human, and mouse genome projects, the functional characterisation of genes using genetic and bioinformatic tools will aid in the identification of important regulators of neural differentiation.

Exclusion of the involvement of all known retinitis pigmentosa loci in the disease present in a family of Irish origin provides evidence for a sixth autosomal dominant locus (RP8).

Retinitis Pigmentosa (RP) is the most prevalent degenerative retinal disease of mendelian origin, currently affecting approximately 1.5 million people worldwide. To date it has been established that a minimum of five different genes maybe involved in the pathogenesis of autosomal dominant forms of RP (adRP). The genes encoding two retinal specific proteins, rhodopsin and peripherin/RDS, have been implicated in causing adRP due to the observation of many different mutations in these genes in patients suffering from RP. The three remaining adRP genes have been mapped to specific regions of human chromosomes but as yet are uncharacterized. We have investigated if there is evidence for the presence of another locus in the genome which when mutated causes adRP. We have utilised polymorphic genetic markers which have previously been mapped to each of the regions known to harbour adRP genes, to test for the exclusion or linkage of the disease gene segregating in a pedigree of Irish origin and find no evidence for linkage. Hence we provide definitive evidence for the involvement of yet another locus. The implications of high levels of genetic heterogeneity inherent in adRP are discussed in relation to diagnosis, prognosis and future therapies.

Novel mutations in the TIGR gene in early and late onset open angle glaucoma.

A gene for juvenile onset, open angle glaucoma (JOAG) has been localized to chromosome 1q21-31 in several families. Mutations in the trabecular meshwork-induced glucocorticoid response protein (TIGR) gene, which maps to this region, recently have been found in families segregating both JOAG and a later onset form of primary open angle glaucoma (POAG). We have analysed the TIGR gene in two families; one Spanish family segregating autosomal dominant JOAG and an Irish family with a later onset form of autosomal dominant POAG. We have found a G-T transversion in the first base of codon 426 in all affected members of the Spanish family, which results in a valine to phenylalanine amino acid substitution. We have also found a G-A transition at the first base of codon 367 that segregates through all but one branch of the Irish family and results in a glycine to arginine amino acid substitution. Members of this family that carry the Gly367Arg change also share a common haplotype that is neither present in any of the unaffected members of the family, nor in the branch that does not segregate the mutation. Identification of further mutations in the TIGR gene increases its importance in the etiology of open angle glaucoma.

A novel Asp380Ala mutation in the GLC1A/myocilin gene in a family with juvenile onset primary open angle glaucoma.

Glaucoma describes a clinically and genetically heterogeneous group of diseases that result in optic neuropathy and progressive loss of visual fields. A gene for juvenile onset primary open angle glaucoma JOAG) has recently been mapped to 1q21-31. Mutations in the trabecular meshwork induced glucocorticoid response gene (TIGR, also known as myocilin or the GLC1A locus) have been found to cause both juvenile and later onset primary open angle glaucoma. Family TCD-POAG1 is a Spanish kindred, which segregates JOAG in an autosomal dominant fashion. This family was found to be linked to the previously identified GLC1A locus on chromosome 1q. Direct sequencing of the TIGR/myocilin gene showed a heterozygous A to C transition in codon 380, resulting in the substitution of alanine for aspartic acid (Asp380Ala). This substitution created a StyI restriction site, which segregated with the JOAG phenotype and permitted rapid screening of all members of the family. This restriction site was not present in 60 controls.

Retinitis pigmentosa and progressive sensorineural hearing loss caused by a C12258A mutation in the mitochondrial MTTS2 gene.

Family ZMK is a large Irish kindred that segregates progressive sensorineural hearing loss and retinitis pigmentosa. The symptoms in the family are almost identical to those observed in Usher syndrome type III. Unlike that in Usher syndrome type III, the inheritance pattern in this family is compatible with dominant, X-linked dominant, or maternal inheritance. Prior linkage studies had resulted in exclusion of most candidate loci and >90% of the genome. A tentative location for a causative nuclear gene had been established on 9q; however, it is notable that no markers were found at zero recombination with respect to the disease gene. The marked variability in symptoms, together with the observation of subclinical muscle abnormalities in a single muscle biopsy, stimulated sequencing of the entire mtDNA in affected and unaffected individuals. This revealed a number of previously reported polymorphisms and/or silent substitutions. However, a C-->A transversion at position 12258 in the gene encoding the second mitochondrial serine tRNA, MTTS2, was heteroplasmic and was found in family members only. This sequence change was not present in 270 normal individuals from the same ethnic background. The consensus C at this position is highly conserved and is present in species as divergent from Homo sapiens as vulture and platypus. The mutation probably disrupts the amino acid-acceptor stem of the tRNA molecule, affecting aminoacylation of the tRNA and thereby reducing the efficiency and accuracy of mitochondrial translation. In summary, the data presented provide substantial evidence that the C12258A mtDNA mutation is causative of the disease phenotype in family ZMK.

RED: the analysis, management and dissemination of expressed sequence tags.

The Rancourt EST Database (RED) is a web-based system for the analysis, management, and dissemination of expressed sequence tags (ESTs). RED represents a flexible template DNA sequence database that can be easily manipulated to suit the needs of other laboratories undertaking mid-size sequencing projects.

A mutation (Met-->Arg) in the type I keratin (K14) gene responsible for autosomal dominant epidermolysis bullosa simplex.

We have identified a single base change in exon 4 of the type I keratin gene which results in the replacement of a methionine for an arginine residue at codon 272 in an Irish family displaying an autosomal dominant simplex (Koebner) form of epidermolysis bullosa (EB). This family had previously provided tentative evidence for linkage to genetic markers on chromosome 1q. The mutation cosegregates with the disease, producing a lod score of 4.8 at theta = 0.

Evidence for genetic heterogeneity in Best's vitelliform macular dystrophy.

Best's vitelliform macular dystrophy is an early onset, autosomal dominant macular degeneration. Linkage analysis has previously mapped a disease locus in this disorder to the pericentromeric region of chromosome 11. We examined two families, one of German and one of Irish origin, both affected with this disorder. The Irish family (BTMD1) showed strong evidence for linkage to the previously reported locus on chromosome 11. Linkage of the disease locus to the same region of chromosome 11 has been significantly excluded in the German family (Fam E), thereby providing evidence of locus heterogeneity in this clinically unique condition.

Strategems in vitro for gene therapies directed to dominant mutations.

A major difficulty associated with the design of gene therapies for autosomal dominant diseases is the immense intragenic heterogeneity often encountered in such conditions. In order to overcome such difficulties we have designed, and evaluated in vitro, three strategies which avoid a requirement to target individual mutations for genetic suppression. In the first, normal and mutant alleles are suppressed by targeting sequences in transcribed but untranslated regions of transcript (UTRs), enabling introduction of a replacement gene with the correct coding sequencing but altered UTRs to prevent suppression. A second approach involves suppression in coding sequence and concurrent introduction of a replacement gene by exploiting the degeneracy of the genetic code. A third strategy utilises intragenic polymorphism to suppress the disease allele specifically, the advantage being that a proportion of patients with different disease mutations have the same polymorphism. These approaches provide a wider choice of target sequence than those directed to single disease mutations and are appropriate for many mutations within a given gene. General methods for suppression may be directed towards the primary defect or a secondary effect associated with the disease process, such as apoptosis. Three general methods targeting the primary defect which circumvent problems of allelic genetic heterogeneity are explored in vitro using hammerhead ribozymes designed to target transcripts from the rhodopsin, peripherin and collagen 1A1 and 1A2 genes, extensive genetic heterogeneity being a feature of associated disease pathologies.

Three keratin gene mutations account for the majority of dominant simplex epidermolysis bullosa cases within the population of Ireland.

We have located three extended families in Ireland (population 3.5 million) with autosomal dominant simplex forms of Epidermolysis Bullosa (EBS). A mutation within the keratin type I (K14) gene (Met-->272-->Arg) in one family suffering from the generalized simplex (Koebner) form of the disease has been previously described (Humphries et al., Hum Mutat 2:37-42, 1993). Here we report on the identification of mutations within the remaining two families, both of whom suffer from the Weber-Cockayne form of the disease. These mutations, within the type II keratin (K5) gene, are Asn-->193-->Lys and Met-->327-->Thr. They have been shown in each case to co-segregate with the disease and are not present in the normal population. Within the three families, a total of 44 living persons with such mutations have been identified, providing a minimum prevalence estimate for the disease in the Irish population of approximately 1 in 80,000, compared to an overall estimated global incidence at birth for all forms of EB of 1 in 50,000. Therefore, these three mutations probably account for the majority of cases of EBS within this population.

ES cell neural differentiation reveals a substantial number of novel ESTs.

We have used a method for synchronously differentiating murine embryonic stem (ES) cells into functional neurons and glia in culture. Using subtractive hybridization we isolated approximately 1200 cDNA clones from ES cell cultures at the neural precursor stage of neural differentiation. Pilot studies indicated that this library is a good source of novel neuro-embryonic cDNA clones. We therefore screened the entire library by single-pass sequencing. Characterization of 604 non-redundant cDNA clones by BLAST revealed 96 novel expressed sequence tags (ESTs) and an additional 197 matching uncharacterized ESTs or genomic clones derived from genome sequencing projects. With the exception of a handful of genes, whose functions are still unclear, most of the 311 known genes identified in this screen are expressed in embryonic development and/or the nervous system. At least 80 of these genes are implicated in disorders of differentiation, neural development and/or neural function. This study provides an initial snapshot of gene expression during early neural differentiation of ES cell cultures. Given the recent identification of human ES cells, further characterization of these novel and uncharacterized ESTs has the potential to identify genes that may be important in nervous system development, physiology and disease.