DNA sequence of the cystatin C gene on a Japanese amyloid angiopathy patient

FOURTH INTERNATIONAL

CONFERENCE

ON ALZHEIMER’S

subjects the onset of the disease has been estimated around 60-65 years with progressive intellectual deficit, development of myoclonic jerks and epileptic seizures in the advanced stages, and exitus 6 to 8 years after the onset for infectious disease in a state of profound dementia.

530 PROBABLE AP-2 BINDING SEQUENCE WITHIN EXON-1 OF HUMAN AND PORCINE CHOLINE ACETYLTRANSFERASE GENES REGULATES TRANSCRIPTION IN NEURAL CELLS. F.Baskin, Y.-P.Li, R.N.Rosenberg and R.M.Davis. Dept.of Neurology, IJ.T.Southwestern Medical Center, Dallas Texas 75235 USA. Although choline acetyltransferase (ChAT), one rate limiting enzyme in the synthesis of is induced by NGF and other neuroacetylcholine, regtropic factors, its precise transcriptional ulation is only now being determined. Previous work determined that a 263 nucleotide sequence within the first coding exon of the porcine ChAT gene increased the expression of luciferase and CAT reporter genes when included in DNA constructs transfected into either SK-N-BE2-Ml7 (adrenergic) or SK-MC-IXC (cholinergic) neuroblastoma cells. Deletion experiments indicate that that this lo-20 fold increase in gene expression resides within a 5' 16 nucleotide segment. Mutation experiments indicate that CCGCGGGG or CCTCGGGG sequences within the human and porcine segments, respectively, are necessary and sufficient for this activbut not ity. Constructs containing these sequences, inactive sequences containing 2-nucleotide tandem substitutions, demonstrate specific binding to proteins from adrenergic and cholinergic cells in gel retardation experiments. Both the human and porcine sequences are in agreement with a consensus binding sequence for AP-2, a nuclear transcription factor expressed only in neural crest and skin cells. Additional experiments, now in progress, utilizing synthetic AP-2 should confirm that it is an AP-2 binding sequence within the first coding exon of the human and porcine ChAT genes which allows its transcriptional enhancement in neural cells. AP-2 analogs or inducers may be useful in future tissue specific ameliorations of AD and other cholinergic deficits.

S129

DISEASE

products were precipitated with isopropanol in the presence of ammonium acetate. DNA sequencing was carried out by the dideoxy chain termination method. No mutations were detected in the sequences of exons 2 and 3. The Leu-68-Gln mutation of the HCCAA-I type was not observed in exon 2. We are now on the way to sequence exon 1 and promoter region.

532 MOLECULAR

ANALYSIS

CHROMOSOME ALZHEIMER’S

OF THE REGION ON

14 ASSOCIATED

WITH EARLY-ONSET

DISEASE

Michael L Hutton and Richard Crook Suncoast Alzheimer’s Disease Laboratory, MDC14, University of South Florida, Tampa, Fl33613 Early-onset

Alzheimer’s disease is a heterogeneous

disorder for which two genetic loci, on chromosomes 14 and 21, are known to exist. The gene on chromosome 21 has been identified as the amyloid precursor protein’

(APP) while

the

gene on chromosome 14 has not yet been isolated. Detailed linkage analysis of the chromosome 14 region2

(14q24 3) that is associated with the majority of cases of earlyonset Alzheimer’s disease has led to the isolation of a series of YACs spanning the region that contains the affected gene (C van Broeckhoven and A. M begun

to

trappin&DNA

analyze

this

Goate, unpublished) YAC

contig

using

Our initial

selection approach

We have an

exon-

studies are

focusing on that part of the contig that is known to contain the c-fos gene as a means of assessing the efficiency expressed sequence isolation protocols

of our

Data on the progress

of this study will be presented

References 1 Goate, A.M et al (I 991) Nature 349, 704-706 2 Schellenberg, G.D et al (I 992)

Science 258,668-671.

531 DNA sequence of the cystatin C gene on a Japanese Amyloid Angiopathy Patient A. Nagai, S. Kobayashi, K. Shimode, S. Fujihara, and K. Imaoka. Third division of Internal Medicine, Shimane Medical University, Lzumo, Japan In hereditary cystatin C amyloid angiopathy in Iceland (HCCAA-I), a Leu-68-Gin mutation of the cystatin gene has been detected and may play an important role in the pathophysiology of deposition of amyloid fibrils in cerebral blood vessels and brain hemorrhage. On the other hand, it has also been suggested that cystatin C is not an intrinsic component of amyloid fibrils in a case of non-hereditary cerebral amyloid angiopathy (CAA) showing dual immunohistochemical reactivity with antibodies to both P-protein and cystatin C. We investigated whether mutations exist in a case of possibly familial CAA in Japanese (CCAA-J) on whose cerebral blood vessels both b-protein and cystatin C was confirmed by immunostaining. We attempted to sequence exons 1, 2, 3 and promoter region of the cystatin C from the genomic DNA of a CCAA-J patient. Genomic DNA from fresh frozen brain of a CCAA-J patient was prepared by the SDS-proteinase K method and phenol/chloroform extraction. Oligonucleotide primers were set for PCR ampIification of 467, 217, 202 and 220 bp segments in exons 1, 2, 3 and promoter region (Promoter region was amplified by the method of Balbin et al.) and bands with the respective sizes were confirmed by agarose gel electrophoresis. For generating predominantly single-strand products, asymmetric PCR was performed on each region and

533 OF A STS BASED YAC CONTIG MAP OF THE EARLY-ONSET ALZHEIMER’S DISEASE REGION ON CHROMOSOME 14C124.3.M. Cruts’, H. Backhovens’, D. Le Paslie?, J. Weissenbach’, J.-J. Martin’, C. Van Broeckhoven’, ‘Born-Bunge Foundation, Laboratory of Neurogenetics, Universiteitsplein I, B-2610 Antwerpen, Belgium, ‘C.E.P.H., 27 rue Juliette Dodu, F-75010 Paris, France, ’GBnBthon, 1 rue de I’lntemationale BP60, 91002 Evry, France. initial linkage analysis studies have demonstrated that a locus for familial early-onset Alrheimefs disease (AD) is located on chromosome 14q24.3 in a region of 22 CM between the markers D14S52 and D14S53. We were able to reduce the candidate region to 6 CM between the markets D148277 and Dl4S61 based on linkage studies in the 2 early-onset AD families AD/A and AD/B. Using the CEPH YAC libraries we have constructed a STS based YAC contig map of the candidate region, ranging from D14S63 at the proximal side to D14S53 at the distal side with only 3 remaining gaps between Dl4S71 and D14S77, Dl4S277 and D14S269, and Dl4S256 and Dl4S57. From the YAC contig map, we can estimate that the candidate region between Dl4S277 and D14S61 corresponds to a minimal size of 2.7 Mb. The maximal size could not be estimated since the YAC contig is not contiguous between Dl4S77 and Dt4S71. Three expressed sequences were localised on the YAC contig map: the celullar oncogene FOS, the transforming growth factor 63 (TGFB3) and D14SlOl E, a brain-expressed EST. Both FOS and TFGB3 are located within the AD candidate region. CONSTRUCTION