A high frequency of LOH (61%) has been detected in a series of breast tumours at a site at a 17p13.3 suggesting the presence of a tumour suppressor gene involved in breast cancer on chromosome 17p (Mackay <I>et al., </I>1988a). Following on from this work the incidence of LOH in primary breast tumours was determined at other loci on chromosome 17p and served a two fold purpose. First, the pattern of LOH at different loci on chromosome 17 in individual breast tumours was determined in order to define a shortest region of overlap (SRO). Identification of such a region, commonly lost in all tumours showing LOH, could be used to pinpoint the position of the putative tumour suppressor gene. Secondly, as two of the probes used to investigate LOH detect loci close to the known tumour suppressor gene p53, a potential role of the p53 gene in breast cancer development could be investigated. Two tumour samples showed LOH at the p53 locus and not at the more distal 17p13.3 site, suggesting the involvement of the p53 gene in breast tumorigenesis. Furthermore LOH at the p53 gene locus was detected in 49% of primary breast tumour samples. Exons 5-9 of the p53 gene, which contain known mutation hotspots, were examined for mutation in 78 primary breast tumours using the HOT (amplification and mismatch detection) technique. Twenty four (31%) of the breast tumours were found to possess a somatically acquired p52 mutation, mostly single base substitutions resulting in missense mutations. In tumours with data on both LOH and p53 mutation, the majority of p53 mutations were found to be accompanied by LOH, indicating the importance of the removal of the tumour suppressive function of the gene.

In the first part of the study complete ascertainment of facial clefts born in the West of Scotland between 01/01/80 and 31/12/84 was attempted. 286 cases were identified giving a birth prevalence of 1.53 per 1000 total births. Of these 51/286 had cleft lip only, 147/286 had cleft palate only and 88/286 had both. Of the 139 patients with cleft lip with or without cleft palate (CL(P)), the lip defect was unilateral in 86/139, midline in 2/139, bilateral in 41/139 and 10/139 could not be classified. There was significant excess of left-sided cleft lip (55/86) among the unilateral defects (x<SUP>2 =</SUP> 6.7 p< 0.05). In the CL(P) group there was an excess of males with a male:female ratio of 1.84:1 (YM x<SUP>2 =</SUP> 8.36 p< 0.01). 48/139 of the CL(P) group has congenital anomalies associated with the facial cleft. In the cleft palate only group (CP) the male:female ratio was 0.81:1, this female excess was not statistically significant. 86 of the 147 CP cases had congenital anomalies associated with their facial cleft. The nature of the malformations associated with the facial clefts were recorded in both groups. Data from this study are in broad agreement with previous studies from European populations. There does, however, appear to be an excess of CP cases in this and other Scottish studies. This study provides an unbiased cohort of patients that can be used in future haplotype association studies and to calculate empiric recurrence risks for use in counselling parents of affected children. In the second part of the study the <i>in situ</i> hybridisation technique was employed to study the role of transforming growth factor type beta (TGF beta) isoforms in murine palatogenesis.

Prior to this study a 360 kb YAC was isolated, Y263, which was found to contain the proximal and distal limits of the RP3 region and, therefore, should contain at least part of the gene. In this study, Y263 was used to improve the physical and genetic characterisation of the RP3 region. Y263 was successfully targeted, with a vector containing a neomycin resistance gene, into the CYBB gene which defines the distal limit of RP3. The neomycin resistance gene provides the selection required to insert the YAC into a mammalian cell line. This derivative YAC has also been mapped using rare cutting restriction enzymes and PFGE. Insertion of the vector increased the size of Y263 and thus enabled it to be separated from yeast chromosome III by PFGE. The ability to isolate Y263 by PFGE is useful for many applications. Y263 has also facilitated genetic mapping of the RP3 region. A novel technique using biotin and streptavidin selection has been used to clone a new microsatellite repeat (JL152) from the YAC. JL152 is highly polymorphic, 8 alleles having been identified to date, and it has a heterozygosity value of 0.76. Sequence analysis has shown that JL152 is located in intron 5 of the CYBB gene. JL152 was shown to be tightly linked to RP3 giving a maximum likelihood lod score of 7.8 at a recombination fraction of zero. This new marker provides additional information for presymptomatic diagnosis and has proved useful in classifying X-linked RP families at RP3 or RP2 (the other X-linked RP locus localised to the short arm of the X chromosome).

A sample of 35 families with ADPKD from central Scotland, previously typed with two markers from the PKD1 region [3'HVR. However, there is one recombinant with CMM65 (D16S84), and one with 26-6 (D16S125), which localises PKD1 to between these markers. In order to obtain cloned DNA from this genetically defined region of interest, spanning approximately 750 kb of DNA, yeast artificial chromosomes (YACs) were isolated from available libraries.

Androgen insensitivity is an X-linked hereditary disorder characterised by failure of virilisation during <I>in utero</I> and pubertal development. The genetic basis of the disorder is now well defined with the finding that deletions or point mutations of the androgen receptor (AR) gene are present in many affected patients. The AR is an intracellular steroid receptor and acts by binding to DNA to cause transactivation of target genes. The receptor protein possesses different structural domains which are essential to effect hormone binding, DNA binding and transactivating functions. The identification of mutations within the AR gene of affected individuals has allowed the elucidation of certain key amino acids essential for receptor function. The mutations which have been identified are located throughout the gene and cause a spectrum of functional impairment from abolition of binding to subtle effects on conformational stability of the receptor/ligand complex. In the light of this it has been suggested that androgen receptor gene mutations may be responsible for infertility in otherwise normal men. DNA was extracted from whole blood obtained from six patients with complete androgen insensitivity and from nine patients with features of partial androgen insensitivity and the individual exons of the AR gene were amplified by polymerase chain reaction. Direct sequencing of the exons was performed to detect the presence of point mutations. Of the fifteen patients, point mutations were detected in three with complete androgen insensitivity and in four with partial insensitivity. Another of the patients showed an amplification of the glutamine homopolymeric repeat region of the receptor; this abnormality is known to be associated with spinal and bulbar muscular atrophy (Kennedy's disease) and a review of the case history suggested that he had an atypical presentation of this disease.

This thesis describes a) the isolation and mapping of new markers from human chromosome 11q12-13 and b) the mutational analysis of a gene from this region as a candidate for an inherited deafness syndrome. A number of markers were concentrated in a sub region of 11q13 which had been linked to several important disorders, and these were used to isolate YACs. Thus the formation of a cloned DNA map of the region was initiated. In parallel I examined the Olfactory Marker Protein (OMP) gene as a candidate for Usher Syndrome Type IB, by DNA sequencing and mutational analysis of affected individuals. Usher syndrome is characterised by deafness and retinitis pigmentosa. In Type I, vestibular dysfunction is also a feature of the disorder. The disorder is genetically heterogeneous and for each subtype several genetic loci have been linked in individual populations: Type IB has been linked to 11q13. A stereociliary defect was proposed, and the mapping of OMP to the linked region, its expression in olfactory nasal cilia and the CNS, and the mapping of the mouse OMP gene to the synthetic region on mouse chromosome 7 very close to <I>Shaker-1, </I>a possible mouse homologue of Usher syndrome type I, justified close examination of OMP as a candidate for both disorders. A human genomic clone was therefore obtained and sequenced, several different methods of mutation detection compared, and the value of the chemical mismatch cleavage technique demonstrated. At the same time, comparative mapping was carried out by mapping candidates from the <I>Shaker-1 </I>nonrecombinant region on the somatic cell hybrid panel. Variants of the OMP gene were identified, but were formally excluded as candidates when a gene located very close to OMP was shown by others to be mutated in Shaker-1 mice and USHIB patients.

No description available.

No description available.

The aim of this project was to identify loci for recessive forms of RP by genetic linkage analysis, using patients with arRP and BBS, and to test for mutations in any resultant candidate genes. Pedigrees with arRP from south-central Sardinia, an ethnic outlier with a higher prevalence of recessive disease, were studied initially. Linkage analysis was carried out using genome-wide microsatellite markers, and genetic heterogeneity was identified among the 11 families studied. Examination of the data revealed potential linkage to D14S80, on chromosome 14q11, in a subset of families. Fine mapping with further markers in this region identified a region of homozygosity in one consanguineous family, suggesting identity-by-descent. A strong candidate gene for RP, neural retina specific leucine zipper (NRL), was found to be located with the region of homozygosity. NRL codes for an evolutionarily conserved protein which is expressed in all layers of the neural retina, and is thought to have a role in the transcriptional regulation of retinal genes, including rhodopsin. The NRL gene was studied in the consanguineous family by direct sequencing and mutation analysis, but no mutations were identified within the coding region. A total of 28 BBS pedigrees collected worldwide were studied using markers in regions where linkage was established during the course of the study. These loci are located on chromosomes 11q13, 16q21, 3p13-p12 and 15q22.3-q23. The results revealed significant genetic heterogeneity, with most families showing linkage to 11q13, and others being consistent with linkage to the 16q or 15q chromosomal regions. Some of the larger pedigrees could be assigned to specific loci, but many of the smaller families were too small for a definite assignment. The results of multipoint linkage analyses in these three linked chromosomal regions will help narrow down the region of search for candidate genes.

Genomic DNA from two unrelated patients suffering from isolated central hypothyroidism was screened for germline inactivating mutations. Patient 1 appeared to retain normal TRH-R sequence whereas, Patient 2 was found to have inherited a mutant TRH-R allele from each of his unaffected parents. Neither of the mutant TRH-Rs (M-STOP from the mother; F-TM3 from the father), when expressed <I>in vitro, </I>displayed TRH binding or therefore, second messenger function, explaining this patient's unresponsiveness to TRH administration. This is the first report of a naturally occurring mutation in a PLC- linked GPCR, and represents a novel cause of isolated central hypothyroidism. The affected patient appears to represent a naturally occurring TRH-R knock-out, a condition which due to the postulated various and widespread actions of TRH was presumed to be a lethal genetic condition. Generation of a transgenic mouse TRH-R knock-out model will allow more detailed analysis of the functions of TRH during embryogenesis and subsequent postnatal development, both in the brain and in extraneural locations. The results of the work described in this thesis indicate that the possession of inactivated TRH-Rs results in central hypothyroidism in one out of two randomly selected patients. It remains to be determined how prevalent such mutations may be in patients with this rare disorder and indeed in other congruous diseases. Patients presenting with other TSH-deficient conditions may reveal additional naturally occurring inactivating mutations. Site-directed mutagenesis of the hTRH-R has shown that substitution of a single amino acid within the receptor protein (Thr265 at the intron/exon boundary and within a site recognised as important for G-protein coupling) transforms it to a more active state. It is therefore feasible that such activating mutations of the hTHR-R may remain to be identified as underlying other abnormalities of the pituitary-thyroid axis.