Screening of AD-patients for mutations in the APP-gene
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THIRD INTERNATIONAL CONFERENCE ON ALZHEIMER’S DISEASE s71 271 ABSElKE OF 717 APP MDTATION IN ITALIAN FAWILIES WITR EARLY ORSET FAMILIAL ALZREIMRR'S DISRASE. B. Nacmias, S. Sorbi, G.Tesco, M. Mortilla, P. Forleo, S. Latorraca, P. Piersanti, S. Piacentini, L. Amaducci. Department of Neurological and Psychiatric Sciences, University of Florence, Florence, Italy. A missense mutation (Val-->Ile) in exon 17 of amyloid precursor protein gene has been reported in five early onset Familial Alzheimer's disease (FAD) pedigrees (Goate et al, Nature 1991, Nakuse et al, Lancet 1991, Lucotte et al Nature 1991) but not in other patients with familial and sporadic Alzheimer's disease (Van Dujin et al. Lancet 1991), suggesting genetic heterogeneity. We have studied affected and non affected members from 7 large Italian families with early onset autosomal dominant FAD coseg- regating in 4 to 10 generations. Mean age at onset was 45.0 (SD 5.46) years and mean disease duration was 4.42 (SD 2.32) years. The disease was previollsly shown to be linked to markers mapping on chromosome 21 in one of these families. One pg of DNA, obtained from peripheral blood, lymphoblasts, fibroblasts and in two cases from frozen and fixed brain, extracted by phenol-chloroform procedure, was amplified by polymerase chain reaction (PCR). PCR products were digested with Bcl I for 4 hours at 5OoC and then electrophoresed on 3% agarose. None of the patients have shown the Bcl I restriction fragment length polymorphism (RFLP) in exon 17 carrying the Val-->Ile mutation. These results suggest that the 117 APP mutation occurs only in some FAD families which may represent a subtype or a variant of FAD. 278 SCREENING OF AD-PATIENTS FOR MUTATIONS IN THE APP-GENE, T. Hartmannl, G. W. Rebeck2, U. Monningl, G. Konigl. C. L. Masters3 and K. Beyreutherlv Center for Molecular Biology Heidelberg, University Heidelberg, D-6900 Heidelberg. Germanyl; Dept. of Neurology, Massachusetts General Hospital, Boston, MA 02114, USA2; Dept. of Pathology, University of Melbourne, Parkville, Victoria 3052, Australia3 Recently mutations in exon 17 of the APP gene have been identified in some cases of familial AD. To identify further’mutations in the APP gene which could cause AD, we have also examined additional regions of the APP gene from AD and control patients. RNA of stimulated lymphocytes from patients was isolated and selected regions throughout the APP-gene were amplified by PCR of cDNA from these samples. The PCR-products were examined either by Temperature Gradient Gel-Electrophoresis (TGGE) or Single- Stranded Conformation Analysis (SSCA). Both methods detect mutations indirectly by changes in the secondary structure of the DNA due to changes in the nucleotide sequence with a sensitivity that allows the detection of most, hut not all single base pair changes. This allowed us to analyse regions up to 300-400bp (SSCA) or more than 1OOObp (TGGE) in a single experiment spanning several exons. As controls we used 4 APP constructs with different exon 17 mutations. In our ongoing survey so far we have not found any mutations in the selected regions of the APP gene of the over 50 patients we have examined. The method applied is fast and inexpensive allowing a rapid sreening of the entire APP gene. 279 SCREENING AND IDENTIFICATION OF MUTATIONS IN THE ARYMID GENE AND ALEHEIURR'S DISEASE. R.-C. Chartier- Iiarlin, St. For the Alaheimer’s Disease Research Group. Wary's Hospital Medical School, London W2 lPG, United Kingdom. Norfolk Place, During the last ten years linkage analysis have show" the genetic heterogeneity of Alzheimer'sdisease (An), We have concentrated our work on a large early-onsetfamily linked to chromosome 21. In this family we discovered a mutation in exe" 17 of the APP gene. This mutation, which led to a Val>Ile sub- stitution at codon 717, is only present in the affected members and not found in the general population. This mutation creates a Bcl I restriction site allowing a" easy screening and we identi- fied a second early onset family carrying this mutation. Se- quencing of exe" 16 and 17 (encoding for the B-amyloid peptide) in 14 earlv Onset families has not revealed other mutations.To select femiiies linked to the APP gene, we analysed the inheri - tance of this gene in AD families using an highly polymorphic marker D21S210 which is located close to the APP eene. This method allowed us to select a" other early onset pedigree. Direct sequencing of exe" 17 identified a second mutation at the same codon 717 changing the valine in a glycine. At the same period a" other mutation again at codon 717 inducing a phenyla- lanine has been described. The occurrence of 3 mutations at the same codon strongly support their pathogenic role To know if other mutations occur in the APP gene in the other early onset pedigrees where linkage failed to give a definitive answer we scre&d the APP gene by SSCP analysis. This method detects the 3 mutations APP717. We analvse exe" 1 to 18 of the APP eene and a region of the APP promo&. An other mutation APP717 (?al>Ile) was identified but no other mutation anywhere else. Due to the importance of the codon 717 in AD, we have also developed a quicker technic, specific oligo hybridisation, to analyse the 6 possible changes at this codon. The 3 mutations are detectable with this method, but so far no other mutations in sporadic cases or late onset families have been discovered. Worldwide 9 early onset families carry the mutations APP717. It seems now clear that these mutations occur at a crucial position in APP. and are responsible for the disease in a" small propor- tion of early onset families. Nevertheless they point out the major role of APP in AD, and place it as the central event in the pathogenesis of AD. 280 IDENTIFICATION OF AN AMYLOID PRECURSOR-LIKE PROTEIN MCALIZED ON CHROMOSOME 19 W. Wasco, B. Hyman, and R. Taxi, Dept. Neurology, MGH. Boston, MA. We have isolated a cDNA from a mouse brain library that encodes a protein whose predicted amino acid sequence is 42X identical to the amyloid fiprotein precursor (APP). This 653 amino acid amyloid precursor-like protein (APLP) is similar to APP in overall structure as well as amino acid sequence. The amino acid homologies are particularly strong in three distinct regions of the two proteins where the identities are 47X. 54% and 56%. All three reeions are also strongly conserved in the drosophila APP-like gene APPL. Notably, 11 cysteine residues are conserved in the cysteine-rich region, as are a" extracellular glycosylation site and the cytoplasmic clathrin-binding domain. The cytoplasmic domain is also conserved in the partial cDNA reported to encode an APP-like gene irl testes. These data sugesst that APP is part of a highly conserved gene family. The APLP cDNA hybridizes to two messages of approximately 2.4 and 1.6 kb that are present in mouse brain and neuroblastoma cells. APLP has been mapped to human chromosome 19 in the same general region as a locus that has bee" reported to be linked to a form of late-onset familial Alzheimer disease. I" situ hybridization studies show APP and -- APLP to be expressed in similar human brain regions and cell populatio"s. We are currently testing APLP as a candidate gene for late-onset familial Alzheimer disease. Additionally, we are screening the human genome for other members of what appears to be a conserved family of APP-like genes. 281 THE EXPRESSIION OF THE AMYLOID PRECURSOR PROTEIN (APP) IS REGULATED BY lW0 G&ELEMENTS IN THE PROMOTER, P. Pollwein, R. Pollwein, C. L Masters* and K Ekqreuther. Center for Molecular Biology Heidelbarg , Uniwrsityof Heidelberg,D-6gOO Heidelberg, Germany. *Department of Pathology, University of Melboume.Parkville, Victoria 3052, Australia Because chronic dii such as AMaimer% disease (AD) are expected to be relatiwty resistant towards therapy, cur aim is to postpone the progressionot amybid depositionin AD interfering with the regulation of the APP gene. This gene is known to be highly expressed in neuronal and to a lesser extent in peripheral cells. lt was also reported, that the APP gene can be induced in viva and in vitro.
Transcript of Screening of AD-patients for mutations in the APP-gene
THIRD INTERNATIONAL CONFERENCE ON ALZHEIMER’S DISEASE s71
271 ABSElKE OF 717 APP MDTATION IN ITALIAN FAWILIES WITR EARLY ORSET FAMILIAL ALZREIMRR'S DISRASE. B. Nacmias, S. Sorbi, G.Tesco, M. Mortilla, P. Forleo, S. Latorraca, P. Piersanti, S. Piacentini, L. Amaducci. Department of Neurological and Psychiatric Sciences, University of Florence, Florence, Italy. A missense mutation (Val-->Ile) in exon 17 of amyloid precursor protein gene has been reported in five early onset Familial Alzheimer's disease (FAD) pedigrees (Goate et al, Nature 1991, Nakuse et al, Lancet 1991, Lucotte et al Nature 1991) but not in other patients with familial and sporadic Alzheimer's disease (Van Dujin et al. Lancet 1991), suggesting genetic heterogeneity. We have studied affected and non affected members from 7 large Italian families with early onset autosomal dominant FAD coseg- regating in 4 to 10 generations. Mean age at onset was 45.0 (SD 5.46) years and mean disease duration was 4.42 (SD 2.32) years. The disease was previollsly shown to be linked to markers mapping on chromosome 21 in one of these families. One pg of DNA, obtained from peripheral blood, lymphoblasts, fibroblasts and in two cases from frozen and fixed brain, extracted by phenol-chloroform procedure, was amplified by polymerase chain reaction (PCR). PCR products were digested with Bcl I for 4 hours at 5OoC and then electrophoresed on 3% agarose. None of the patients have shown the Bcl I restriction fragment length polymorphism (RFLP) in exon 17 carrying the Val-->Ile mutation. These results suggest that the 117 APP mutation occurs only in some FAD families which may represent a subtype or a variant of FAD.
278 SCREENING OF AD-PATIENTS FOR MUTATIONS IN THE APP-GENE, T. Hartmannl, G. W. Rebeck2, U. Monningl, G. Konigl. C. L. Masters3 and K. Beyreutherlv Center for Molecular Biology Heidelberg, University Heidelberg, D-6900 Heidelberg. Germanyl; Dept. of Neurology, Massachusetts General Hospital, Boston, MA 02114, USA2; Dept. of Pathology, University of Melbourne, Parkville, Victoria 3052, Australia3 Recently mutations in exon 17 of the APP gene have been identified in some cases of familial AD. To identify further’mutations in the APP gene which could cause AD, we have also examined additional regions of the APP gene from AD and control patients. RNA of stimulated lymphocytes from patients was isolated and selected regions throughout the APP-gene were amplified by PCR of cDNA from these samples. The PCR-products were examined either by Temperature Gradient Gel-Electrophoresis (TGGE) or Single- Stranded Conformation Analysis (SSCA). Both methods detect mutations indirectly by changes in the secondary structure of the DNA due to changes in the nucleotide sequence with a sensitivity that allows the detection of most, hut not all single base pair changes. This allowed us to analyse regions up to 300-400bp (SSCA) or more than 1OOObp (TGGE) in a single experiment spanning several exons. As controls we used 4 APP constructs with different exon 17 mutations. In our ongoing survey so far we have not found any mutations in the selected regions of the APP gene of the over 50 patients we have examined. The method applied is fast and inexpensive allowing a rapid sreening of the entire APP gene.
279 SCREENING AND IDENTIFICATION OF MUTATIONS IN THE ARYMID GENE AND ALEHEIURR'S DISEASE. R.-C. Chartier- Iiarlin, St.
For the Alaheimer’s Disease Research Group. Wary's Hospital Medical School,
London W2 lPG, United Kingdom. Norfolk Place,
During the last ten years linkage analysis have show" the genetic heterogeneity of Alzheimer's disease (An), We have concentrated our work on a large early-onset family linked to chromosome 21. In this family we discovered a mutation in exe" 17 of the APP gene. This mutation, which led to a Val>Ile sub- stitution at codon 717, is only present in the affected members
and not found in the general population. This mutation creates a Bcl I restriction site allowing a" easy screening and we identi- fied a second early onset family carrying this mutation. Se- quencing of exe" 16 and 17 (encoding for the B-amyloid peptide) in 14 earlv Onset families has not revealed other mutations. To select femiiies linked to the APP gene, we analysed the inheri - tance of this gene in AD families using an highly polymorphic marker D21S210 which is located close to the APP eene. This method allowed us to select a" other early onset pedigree. Direct sequencing of exe" 17 identified a second mutation at the same codon 717 changing the valine in a glycine. At the same period a" other mutation again at codon 717 inducing a phenyla- lanine has been described. The occurrence of 3 mutations at the same codon strongly support their pathogenic role To know if other mutations occur in the APP gene in the other early onset pedigrees where linkage failed to give a definitive answer we scre&d the APP gene by SSCP analysis. This method detects the 3 mutations APP717. We analvse exe" 1 to 18 of the APP eene and a region of the APP promo&. An other mutation APP717 (?al>Ile) was identified but no other mutation anywhere else. Due to the importance of the codon 717 in AD, we have also developed a quicker technic, specific oligo hybridisation, to analyse the 6 possible changes at this codon. The 3 mutations are detectable with this method, but so far no other mutations in sporadic cases or late onset families have been discovered. Worldwide 9 early onset families carry the mutations APP717. It seems now clear that these mutations occur at a crucial position in APP. and are responsible for the disease in a" small propor- tion of early onset families. Nevertheless they point out the major role of APP in AD, and place it as the central event in the pathogenesis of AD.
280 IDENTIFICATION OF AN AMYLOID PRECURSOR-LIKE PROTEIN MCALIZED ON CHROMOSOME 19 W. Wasco, B. Hyman, and R. Taxi, Dept. Neurology, MGH. Boston, MA.
We have isolated a cDNA from a mouse brain library that encodes a protein whose predicted amino acid sequence is 42X identical to the amyloid fi protein precursor (APP). This 653 amino acid amyloid precursor-like protein (APLP) is similar to APP in overall structure as well as amino acid sequence. The amino acid homologies are particularly strong in three distinct regions of the two proteins where the identities are 47X. 54% and 56%. All three reeions are also strongly conserved in the drosophila APP-like gene APPL. Notably, 11 cysteine residues are conserved in the cysteine-rich region, as are a" extracellular glycosylation site and the cytoplasmic clathrin-binding domain. The cytoplasmic domain is also conserved in the partial cDNA reported to encode an APP-like gene irl testes. These data sugesst that APP is part of a highly conserved gene family. The APLP cDNA hybridizes to two messages of approximately 2.4 and 1.6 kb that are present in mouse brain and neuroblastoma cells. APLP has been mapped to human chromosome 19 in the same general region as a locus that has bee" reported to be linked to a form of late-onset familial Alzheimer disease. I" situ hybridization studies show APP and -- APLP to be expressed in similar human brain regions and cell populatio"s. We are currently testing APLP as a candidate gene for late-onset familial Alzheimer disease. Additionally, we are screening the human genome for other members of what appears to be a conserved family of APP-like genes.
281 THE EXPRESSIION OF THE AMYLOID PRECURSOR PROTEIN (APP) IS REGULATED BY lW0 G&ELEMENTS IN THE PROMOTER, P. Pollwein, R. Pollwein, C. L Masters* and K Ekqreuther. Center for Molecular Biology Heidelbarg , Uniwrsity of Heidelberg, D-6gOO Heidelberg, Germany. *Department of Pathology, University of Melboume. Parkville, Victoria 3052, Australia
Because chronic dii such as AMaimer% disease (AD) are expected to be relatiwty resistant towards therapy, cur aim is to postpone the progression ot amybid deposition in AD interfering with the regulation of the APP gene. This gene is known to be highly expressed in neuronal and to a lesser extent in peripheral cells. lt was also reported, that the APP gene can be induced in viva and in vitro.
