THIRD INTERNATIONAL CONFERENCE ON ALZHEIMER’S DISEASE S69

sample has been completely ascertained. Model fitting approaches can ~imuitsneously estimate the importance d age. genetlc variance. shared environment, and environments unique to the individual. Specifk putative risk factors will be examined using rslathre risk and logistk regression technlquee.

269 A PHYSICAL MAP OF THE 21421.2421.3 REGION INCLUDING THE p-AMYLOID PRECURSOR PROTEIN GENE. W. Van Hul, M. Cruts, L. Stuyver, C. Brahe and C. Van Broeckhoven. Neurogenetics Laboratory, Born Bunge Foundation, University of Antwerp (IJIA) and Innogenelics Inc., Belgium; Institute di Genetica Umana, Universita Cattolica de1 Sacro Cuore, Roma, Italy.

The Pamyloid protein (PAP) is a major constituent of the pathological brain lesions found in patients with Alzheimer disease (AD). The PAP is a 4 kD proteolysis product of the !3-amyloid precursor protein &4PP). The PAPP gene was located in 21q21.2-q21.3, a region linked to familial AD. Therefore, the PAPP gene has been considered as a candidate gene for AD. The recent finding of single base mutations in the PAPI’ gene in some AD families confirmed that the PAPP plays a keyrole in the AD pathology. Nevertheless, in other AD families recombinants between AD and the @API’ gene were reported suggesting that in these families the PAPP gene is not the primary mutation site. However, the possibility remained that the exclusion of the PAPP gene was the result of intragenic recombination events. To test for the latter possibility we estimated the physical size of the PAPP gene by pulsed field gel electrophoresis. Clones recognizing the promotor sequence and different parts of the PAPP cDNA were used. Our results indicated a minimal size for the PAPI’ gene of 280 kb and a maximal size of 440 kb. The relatively small size of the PAPP gene suggested that intragenic recombinations are not to be expected with a high frequency.

Information on the possible occurence of intragenic recombinants can also be gained by combining data of the PAPI’ gene with data obtained with closely linked flanking markers. We mapped four markers pMC1.28d (D21S147), pMC1.4la, pLSB8 (D21S179) and pLSB29 (D21S180) between the breakpoints of the cell hybrids (1;21) and Acem and thus in 21q21.2q21.3, the same chromosomal region as the PAPP gene. Their exact position relative to the PAPP gene was determined by pulsed field gel electrophoresis. Polymorphisms detected with these markers can be helpful in the linkage analysis of the PAPP gene in less informative AD families.

270 DETECTION OF POLYMORPHISMS IN CANDIDATE GENES IN ALZHEIMER’S DISEASE, S.E. Poduslo and P. Decker. Department of Neurology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430.

Alzheimer’s Disease is a progressive neurodegenerative disorder characterized by loss of memory, reasoning, perception, and orientation. The pathological changes in brain involve a loss of neurons from particular areas, the formation of intracellular neurofibrillary tangles and extracellular amyloid-containing neuritic plaques. While the cause of the disease may be multiiactorial, there are undoubtedly genetic components. A gene for the early onset formmayhaveitslocusonchromosome21whilethelateonsetform may be linked to chromosome 19. Our strategy is to analyze candidate genes (e.g. neurotransmitter receptors, growth factor receptors) whose proteins may be affected by the disease. The technology that we are using is exquisitely sensitive and can detect polymorphisms with single base changes (Poduslo, Dean, Kolch, and O’Brien, “Detecting high-resolution polymorphisms in human coding loci by combining PCR and single strand polymorphism (SSCP) analysis,” Amer. J. Human Genetics B:loS, 1991). Sequences of interest are ampliied and simultaneously radio-labeled by the polymerase chain reaction. The samples are then denatured and subjected to electrophoresis for SSCP analysis. In addition, if restriction enzymes that cut frequently (with four base pair recognition sequences) are used in digests of the amplified DNA, multiple genetic variations can be resolved after electrophoresis. Using this strategy we have detected a polymorphism in the dopamine D2 receptor gene located on chromosome llq22-q23. While this polymorphism occurs in 10% of the CEPH parents, it

occurs in over 30% of the Alzheimer’s patients that we have. We are sequencing this polymorphism in patients and controls and determining whether there are associations with subtypes of the disease. With this technology and using this approach the genetic basis of this disease may be more quickly delineated. (Supported by the Alzheimer’s Institute.)

271 TWO FAMILIES AFFECTED WITH FATAL FAMILIAL INSOMNIA HAVE A MUTATION.,AT CODON 178 IN.THE PRION PROTEIN GENE. Rr Medori , H.J. Tritschler , E. Lugaresi , P. Montagna , A. -Blanc, .F. Villare, L. Autilio Gambetti, P. Gambetti. Clinica Neurologica dell' Universita' di Bologna, Bologna Italy; Division of Neuropathology, Case Western Reserve University, Cleveland, USA. We studied the biochemical and genetic abnormalities . two unrelated pedigrees kimilial insomnia (FFI)

affected with fatal , a disease we first described

in 1986. FFI is characterized by progressive and untreatable insomnia, autonomic dysfunctions and different degrees of motor disturbances such as myoclonus and ataxia, with a mean duration of 13 months. The second Italian, unrelated family presented with an identical condition. The neuropathology from eight affected members showed neuronal loss and reactive astrogliosis of the anterior and dorsal-medial nuclear groups of the thalami, and mild spotty gliosis. Spongiform degeneration was observed only in two brains. Western and dot blotting of brain tissue to reveal the presence of protease-resistant Prion protein (PrP). Direct sequencing and/or ASP I digestion of the PCR amplified PrP coding region showed a G->A mutation in the codon 178 of the PrP coding region resulting in the substitution of aspartate with asparagine. ASP I digestion of the amplified coding region showed the same mutation in the other family. Linkage analysis showed that the mutation segregated with the disease. We conclude that a distinct phenotype is associated with the PrP codon 178 mutation.

272 LINKAGE CALCULATIONS FOR ALZHEIMER'S FAMILIES, D.F. Andrews, M.E. Percy, and M. Pintillie. Department of Preventive Medicine and Biostatistics, University of Toronto, Toronto M5S lAl, Canada, and Surrey Place Centre, Toronto M5S X2, Canada. Linkage analysis for Alzheimer's disease is complicated by difficulty of diagnosis and aoe dependence of incidence. The incorporation of these complications into a statistical model requires, in particular, specification of the probability that a person of age x will develop symptoms. The calculation of the likelihoods used in analysis is exceed- ingly complex for large pedigrees. We have developed a new procedure based on the theory of Konq for the calculation of likelihoods in the analysis of Alzheimer's families. Efficient simulation procedures have been developed for these calculations. These developments permit the evaluation of the effect of both linkage and penetrance parameters. The farm 4

rocedures developed are beinq a plied to the study of a y with late onset dementia of c: t e Alzheimer type that may

be associated with an abnormality on chromosome 22 (Percy et al, 1991). Several other families with early onset Alzheimer disease and a mutation in the beta-amyloid precursor protein locus on chromosome 21 will also serve as useful model systems for evalua- tion of the effectiveness of our new linkage program in identify- ing the location of disease genes. Supported by the Alzheimer's Association/Ana M. Buchanan Memorial Pilot Research Grant (Alzheimer's Disease and Related Disorders Assoc. Inc. PRG-90-123).

References Kong, A. (1989). Efficient methods for computing linkaae likeli- hoods of recessive diseases in inbred pediorees. Technical Report University of Chicago. Percy, M.E., Markovic, V.D., Crapper McLachlan, D.R., Berg, J.M., Hummel, J.T., Laing, M.E., Dearie, T.G., Andrews, D.F. (1991). Family with a 22-derived marker chromosome and late-onset demen- tia of the Alzheimer type: I. Application of a new model for estimation of the risk of disease associated with the marker. Am. J. Med. Genet. 39: 307-313.