John R. Gilbert, PhD - US grants

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder with a high mutation rate which affects numerous organ systems. TS is characterized by hamartomas and harmartias and symptoms can vary from benign skin macules to mental retardation with epilepsy to premature death. The estimated prevalence of TSC is 1 per 10,000. TSC is a heterogeneous disorder. In 1987 genetic linkage was demonstrated between TSC and the ABO blood group on chromosome 9q34. Approximately one third of TSC families seem to be linked to the 9q loci. More recently this laboratory, using families clearly unlinked to the 9q loci, has shown linkage between the D16S283 marker, located at chromosome 16p13, and a large group of TSC families. Progress in isolating the 9q34 TSC gene has proceeded relatively slowly primarily because of family heterogeneity. Recent identification of a second TSC loci at 16p13, which includes the majority of the TSC families, allows the clarification of these issues and should greatly accelerate progress in isolating the 9q TSC gene. Although TSC was first linked to 9q six years ago all that could be said until recently is that the disease loci lay in a region of approximately 20 cM distal to D9S125 in 9q34. Recent analyses have, however, localized the chromosome 9 TSC loci to a region of approximately 2 cM flanked proximally by DBH and distally by D9S114. Efforts to further refine the TSC loci by developing new markers are presently underway. With the isolation of close flanking markers the chromosome 9 TSC region may be mapped using pulse field electrophoresis and Yeast Artificial Chromosomes (YAC)'s isolated from the new CEPH megabase library and existing YAC libraries. The TSC region will be physically clone using Yacs and a cosmic contig encompassing the TSC locus. New markers will be generated, genes contained within the region will be isolated and tested as TSC candidate genes.

Alzheimer's disease *AD) is a devastating neurological disorder characterized by a progressive dementia which affects 5-10% of the elderly over age 65. Genetic factors have clearly been implicated in the early and late-onset familial forms of the linked disease. A form of late-onset AD has been show to be linked to the proximal long arm of chromosome 19 and subsequently associated with Apolipoprotein E (ApoE). Apo E is a plasma apolipoprotein that is involved in lipid transport and metabolism. Apo E is produced in brain and found in three major isoforms in the human population which have been designated E2. E3. E4. These alleles occur at a frequency of approximately 8, 76, and 16 per cent, respectively. Recently, an association between the ApoE4 allele and an increased risk of late-onset AD has been established. Subsequent studies have demonstrated that Apo E2 has a protective effect or delays the age of onset of Ad. The exact mechanism by which the different isoforms exert their effects are as yet unknown. To study the mechanisms by which Apo E exerts its effects and the role which Apo E plays in brain molecular biology, physiology, and metabolism awe are creating Apo E 2. 3. and 4 transgenic mice. Cosmid libraries from human AD patients with the appropriate Apo E genotype were prepared in the cosmid vectors pWE15 and SuperCos 1. Apo E containing cosmid clones were isolated and restriction mapped. Clones of the proper size containing the brain expression control regions for Apo E were produced and purified. Experiments to produce Apo E transgenic mice using microinjection procedures will be carried out using ice which lack a functional Apo E gene (Apo E "knockout" mice). Mice, lacking a functional murine Apo E, will be produced which are homozygous and heterozygous for the human Apo E2, 3 and 4 alleles. These mice will serve as a model system for the study of the role of Apo E in brain function and metabolism and its potential role in Alzheimer's disease.

DESCRIPTION: The purpose of this proposal is to isolate genes involved in autistic disorder (AD). The gene hunt will focus on chromosome 15q11-q13 because of recent evidence, some of it from the investigator's laboratory, that this region is linked to AD. The first set of aims is to develop markers from genomic contigs to genetically fine map the region in AD families in order to define regions of high recombination as closely as possible and determine the most likely location of the AD locus. The families will also be examined for methylation abnormalities and chromosomal duplications, insertion, deletions, or inversions. The second set of aims revolves around the isolation and analysis of AD candidate genes for direct identification of causative mutations.

Essential Tremor (ET) is a heterogenous tremor disorder characterized by a core group of features. The tremor syndrome is characterized by postural and kinetic tremor affecting the arms and hands, although the head, voice, and legs may also be affected. Although frequently described as a benign disorder, this is not true; many patients are socially and physically handicapped, with some patients being totally disabled. The differential diagnosis list for ET is extensive including dystonia, Parkinsonism, myoclonus, peripheral neuropathy, and other conditions. Prevalence estimates range widely, depending upon methodology and diagnostic criteria, from 0.003 to as high as 2% in the general population, with as much as 5% of the population affected over the age of 65. There are no known biological or diagnostic neuropathological markers for ET. The estimates of ET cases presenting with a positive family history range from 17.4% to 100%. Recent studies indicate that up to 96% of ET may be dominantly inherited. Clinical and genetic heterogeneity have slowed linkage studies. To date three loci associated with ET have been linked: 1) Familial Essential Tremor 1 (FET1) has been mapped in a series of Icelandic families on chromosome 3q13; (2) ETM mapped, in four unrelated US families, to chromosome 2p22-p25; and (3) a third locus maps, in a family that segregates both Parkinson's disease and postural tremor consistent with ET, to Chromosome 4p. We have, to date, ascertained, twelve ET and ET/PD linkage quality families. The largest pure ET kindred (DUK13001) have been excluded from known ET loci. The aims of this proposal are to ascertain and sample large families with ET, carry out a complete ET genome scan to establish linkage for these and additional ET families, identify new ET disease loci, and isolate and characterize ET genes, beginning with DUK13001 ET family.