- Email: [email protected]
An Angiotensin-Converting Enzyme (ACE) polymorphism is associated with intracerebral hemorrhage recurrences in Cerebral Amyloid Angiopathy patients
Poster Presentations P4 132 of these having either probable or possible dementia. Quality control procedures indicate that nearly 800,000 SNPs are available for analysis. Initial studies of a smaller subset of this dataset revealed potential linkage to regions on chromosomes 1q, 3q22, 5q, 18q, and 21q. Our simulation studies suggest that statistical analyses are sensitive to the methods used to reduce complex pedigrees to more computationally tractable sizes. Conclusions: Detailed simulation, linkage, and association studies are ongoing and will be presented. Initial results suggest several regions of linkage and that analyses are sensitive to preservation of pedigree structure. P4-115
GENETIC VARIANTS IN PRESENILINS AND CORRELATION TO AMYLOID-BETA 42 LEVELS IN CEREBROSPINAL FLUID AND DIAGNOSIS OF ALZHEIMER’S DISEASE
Christine A. F. von Arnim1, Elena Lebedeva1, Dietmar Thal1, ¨ zer1, Bjo¨rn von Einem1, Estifanos Ghebremedhin2, Joachim Strauss1, Esra O 1 1 Hayrettin Tumani , Markus Otto , Matthias W. Riepe1, Albert C. Ludolph1, Julia Kirchheiner1, 1Ulm University, 89081 Ulm, Germany; 2Frankfurt University, Frankfurt, Germany. Contact e-mail: [email protected] Background: Aim of this study was to assess genetic variability in human presenilin 1 and 2 (PSEN) genes in Alzheimer’s disease (AD) patients in relation to b-amyloid 42 (Ab42) levels in cerebrospinal fluid (CSF) and clinical symptoms. We hypothesized that by specific phenotyping through biomarkers the chance of identifying novel disease-modifying genetic variations might be increased. Methods: We therefore analyzed 314 subjects defined by their clinical diagnosis and Ab42 levels (cut-off 600 pg/ml). To confirm this data we employed an independent control group of 109 autopsy cases characterized for levels of amyloid plaque deposition. In all patients with early-onset AD, all exons of PSEN1 and PSEN2 were sequenced. In the whole sample, haplotype tagging SNPs in PSEN1 and PSEN2 were analyzed. Results: Three heterozygous amino acid exchange mutations were found (one novel in PSEN1, and one novel, one known in PSEN2) in the subgroup of EO-AD patients. Haplotype analysis revealed one distinct haploblock in PSEN2. The most frequent haplotypes were detected by analysis of 4 tagging SNPs. In PSEN1 no haploblocks were detected. CSF Ab42 increased with the number of PSEN2 haplotypes 1 TCTG (p¼0.009). A similar trend for haplotype 2 CCCG in the late-onset group (p¼0.033) was observed. Homozygous carriers of haplotype 4 defined by rs11405 were underrepresented in the late-onset AD group compared to the control group (p¼0.026). Genotyping of the autopsy brain samples revealed that 4 out of 5 carriers of haplotype 4/4 did not have any b-amyloid deposits in the medial temporal lobe. Conclusions: While non-synonymous mutations of PSEN1 and PSEN2 were found only sporadically, haplotype analysis of PSEN2 revealed associations with the level of Ab42 in early-onset and late-onset AD patients. The underrepresentation of haplotype 4/4 among AD patients and lack of b-amyloid deposits in brain autopsy samples from 4/4 carriers might point to a potentially protective effect of this haplotype on amyloid plaques formation. P4-116
METHYLATION STUDIES IN BRAINS FROM PATIENTS WITH FAMILIAL ALZHEIMER’S DISEASE
Jesper Brohede, Maria Ahlkvist, Bengt Winblad, Caroline Graff, Karolinska Institutet, Huddinge, Sweden. Contact e-mail: [email protected] Background: CpG-islands are stretches of DNA with high density of CG (which is the motif where the C has potential to be methylated) and are often located in the vicinity of a gene. These CpG-islands are thought to regulate the expression of the associated gene in a tissue and time specific manner. Neuropathology in AD differs with various regions of the brain where, for instance, the plaque formation starts in the cortical areas whereas cerebellum is rarely affected until very late stages of the disease. These differences may be due to regional methylation differences which can modify the expression levels of genes that are involved in the plaque formation. In order to investigate if this may be the case we undertook a pilot study where we determined the methylation levels in the CpG-islands of amyloid-b precursor protein
P465
(APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2) in DNA extracted from different brain regions of a limited number of AD patients. Methods: DNA from frozen brain tissue was extracted from six individuals diagnosed with familial AD. Four of the individuals carried the APPswe mutation and the two remaining samples had familial AD with unknown mutation. For each patient we collected tissue from cerebellum, frontal cortex, parietal cortex and temporal cortex. The DNA samples were bisulfite converted and parts of the CpG-islands of APP, PSEN1 and PSEN2 were PCR amplified using primers specific to the bisufite converted sequence. Resulting amplicons were subsequently sequenced using Sanger sequencing and capillary electrophoresis. The bisulfite treatment will convert all unmethylated cytosines into uracils which will be changed into thymine after PCR amplification. Results: For the CpG-island associated with APP we bisulfite sequenced a 238bp fragment, including 16 CpG sites, and for CpG island associated with PSEN1 we bisulfite sequenced a 228bp fragment, including 17 CpG sites. No high quality sequences could be obtained for the CpG island of PSEN2. No sequences showed any detectable methylation in the any of the brain regions studied. Conclusions: It appears as if the studied parts of the CpG-islands of APP and PSEN1 are unmethylated in cerebellum, frontal cortex, parietal cortex, and temporal cortex. P4-117
AN ANGIOTENSIN-CONVERTING ENZYME (ACE) POLYMORPHISM IS ASSOCIATED WITH INTRACEREBRAL HEMORRHAGE RECURRENCES IN CEREBRAL AMYLOID ANGIOPATHY PATIENTS
Mar Hernandez Guillamon1, Sophie Domingues1, Merce` Boada2,3, Mireia Pare´s1, Israel Ferna´ndez Cadenas1, Isabel Herna´ndez2, Pablo Martı´nez-Lage2, Pilar Delgado1, Joan Montaner1,3, 1Intitut Recerca, Hospital Vall d’Hebron, Barcelona, Spain; 2Fundacio´ ACE, Barcelona, Spain; 3Servei Neurologia, Hospital Vall d’Hebron, Barcelona, Spain. Contact e-mail: [email protected] Background: Cerebral Amyloid Angiopathy (CAA) is characterized by the deposition of the beta-amyloid protein within brain arteries and is a cause of lobar intracerebral hemorrhage (ICH). CAA is also a common pathological finding in Alzheimer disease (AD) patients with a frequency of up to 98% in autopsy series. We have no clinical data to identify those CAA or AD patients prone to new brain bleedings. Therefore, we aimed to study predictive value of single nucleotide polymorphisms (SNPs) and their function in association to higher rates of ICH recurrence in a cohort of CAA patients. Methods: Patients with a CAA-related ICH were included in the study. MRI data, leukoaraiosis degree and microbleed count, was collected for each patient. 19 SNPs located in 10 candidate genes related to brain hemorrhages were analyzed, including 11 Tag SNPs located in the ACE gene. Statistical analysis was performed through survival curves and Cox regression. Serum ACE and beta-amyloid (1-40) levels were then determined by ELISA. Results: 77 patients were included in the study, with a mean age of 75 years. On baseline MRI, 50% of patients had at least 2 microbleeds and mild to moderate leukoaraiosis degree. During the follow-up (36 months), 24 patients presented a recurrent ICH. Five polymorphisms were related to the presence of recurrent ICH, including four in the ACE gene, although only 2 SNPs remained significantly associated to ICH recurrence after Cox regression analysis adjusting for microbleeds number and leukoaraiosis degree, the e2 allele of the ApoE gene (p¼0.028) and the T allele of the rs4311 SNP of the ACE gene (p¼0.005). Serum ACE levels were higher in T allele carriers (T carriers: 267.6 6 41.2; CC: 203.1 6 51.4 mg/mL; p¼0.013) and ACE and beta-amyloid levels were strongly correlated (p¼0.004). Conclusions: An association between the T allele of the ACE rs4311 polymorphism and ICH recurrence was observed in our CAA cohort. ACE protein levels were significantly higher in carriers of the T allele and were strongly correlated to beta-amyloid (1-40) circulating levels. This data suggests that ACE rs4311 genotypes information might add valuable prognostic information and open therapeutic expectations in CAA patients that merits further investigation.
GENETIC VARIANTS IN PRESENILINS AND CORRELATION TO AMYLOID-BETA 42 LEVELS IN CEREBROSPINAL FLUID AND DIAGNOSIS OF ALZHEIMER’S DISEASE
Christine A. F. von Arnim1, Elena Lebedeva1, Dietmar Thal1, ¨ zer1, Bjo¨rn von Einem1, Estifanos Ghebremedhin2, Joachim Strauss1, Esra O 1 1 Hayrettin Tumani , Markus Otto , Matthias W. Riepe1, Albert C. Ludolph1, Julia Kirchheiner1, 1Ulm University, 89081 Ulm, Germany; 2Frankfurt University, Frankfurt, Germany. Contact e-mail: [email protected] Background: Aim of this study was to assess genetic variability in human presenilin 1 and 2 (PSEN) genes in Alzheimer’s disease (AD) patients in relation to b-amyloid 42 (Ab42) levels in cerebrospinal fluid (CSF) and clinical symptoms. We hypothesized that by specific phenotyping through biomarkers the chance of identifying novel disease-modifying genetic variations might be increased. Methods: We therefore analyzed 314 subjects defined by their clinical diagnosis and Ab42 levels (cut-off 600 pg/ml). To confirm this data we employed an independent control group of 109 autopsy cases characterized for levels of amyloid plaque deposition. In all patients with early-onset AD, all exons of PSEN1 and PSEN2 were sequenced. In the whole sample, haplotype tagging SNPs in PSEN1 and PSEN2 were analyzed. Results: Three heterozygous amino acid exchange mutations were found (one novel in PSEN1, and one novel, one known in PSEN2) in the subgroup of EO-AD patients. Haplotype analysis revealed one distinct haploblock in PSEN2. The most frequent haplotypes were detected by analysis of 4 tagging SNPs. In PSEN1 no haploblocks were detected. CSF Ab42 increased with the number of PSEN2 haplotypes 1 TCTG (p¼0.009). A similar trend for haplotype 2 CCCG in the late-onset group (p¼0.033) was observed. Homozygous carriers of haplotype 4 defined by rs11405 were underrepresented in the late-onset AD group compared to the control group (p¼0.026). Genotyping of the autopsy brain samples revealed that 4 out of 5 carriers of haplotype 4/4 did not have any b-amyloid deposits in the medial temporal lobe. Conclusions: While non-synonymous mutations of PSEN1 and PSEN2 were found only sporadically, haplotype analysis of PSEN2 revealed associations with the level of Ab42 in early-onset and late-onset AD patients. The underrepresentation of haplotype 4/4 among AD patients and lack of b-amyloid deposits in brain autopsy samples from 4/4 carriers might point to a potentially protective effect of this haplotype on amyloid plaques formation. P4-116
METHYLATION STUDIES IN BRAINS FROM PATIENTS WITH FAMILIAL ALZHEIMER’S DISEASE
Jesper Brohede, Maria Ahlkvist, Bengt Winblad, Caroline Graff, Karolinska Institutet, Huddinge, Sweden. Contact e-mail: [email protected] Background: CpG-islands are stretches of DNA with high density of CG (which is the motif where the C has potential to be methylated) and are often located in the vicinity of a gene. These CpG-islands are thought to regulate the expression of the associated gene in a tissue and time specific manner. Neuropathology in AD differs with various regions of the brain where, for instance, the plaque formation starts in the cortical areas whereas cerebellum is rarely affected until very late stages of the disease. These differences may be due to regional methylation differences which can modify the expression levels of genes that are involved in the plaque formation. In order to investigate if this may be the case we undertook a pilot study where we determined the methylation levels in the CpG-islands of amyloid-b precursor protein
P465
(APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2) in DNA extracted from different brain regions of a limited number of AD patients. Methods: DNA from frozen brain tissue was extracted from six individuals diagnosed with familial AD. Four of the individuals carried the APPswe mutation and the two remaining samples had familial AD with unknown mutation. For each patient we collected tissue from cerebellum, frontal cortex, parietal cortex and temporal cortex. The DNA samples were bisulfite converted and parts of the CpG-islands of APP, PSEN1 and PSEN2 were PCR amplified using primers specific to the bisufite converted sequence. Resulting amplicons were subsequently sequenced using Sanger sequencing and capillary electrophoresis. The bisulfite treatment will convert all unmethylated cytosines into uracils which will be changed into thymine after PCR amplification. Results: For the CpG-island associated with APP we bisulfite sequenced a 238bp fragment, including 16 CpG sites, and for CpG island associated with PSEN1 we bisulfite sequenced a 228bp fragment, including 17 CpG sites. No high quality sequences could be obtained for the CpG island of PSEN2. No sequences showed any detectable methylation in the any of the brain regions studied. Conclusions: It appears as if the studied parts of the CpG-islands of APP and PSEN1 are unmethylated in cerebellum, frontal cortex, parietal cortex, and temporal cortex. P4-117
AN ANGIOTENSIN-CONVERTING ENZYME (ACE) POLYMORPHISM IS ASSOCIATED WITH INTRACEREBRAL HEMORRHAGE RECURRENCES IN CEREBRAL AMYLOID ANGIOPATHY PATIENTS
Mar Hernandez Guillamon1, Sophie Domingues1, Merce` Boada2,3, Mireia Pare´s1, Israel Ferna´ndez Cadenas1, Isabel Herna´ndez2, Pablo Martı´nez-Lage2, Pilar Delgado1, Joan Montaner1,3, 1Intitut Recerca, Hospital Vall d’Hebron, Barcelona, Spain; 2Fundacio´ ACE, Barcelona, Spain; 3Servei Neurologia, Hospital Vall d’Hebron, Barcelona, Spain. Contact e-mail: [email protected] Background: Cerebral Amyloid Angiopathy (CAA) is characterized by the deposition of the beta-amyloid protein within brain arteries and is a cause of lobar intracerebral hemorrhage (ICH). CAA is also a common pathological finding in Alzheimer disease (AD) patients with a frequency of up to 98% in autopsy series. We have no clinical data to identify those CAA or AD patients prone to new brain bleedings. Therefore, we aimed to study predictive value of single nucleotide polymorphisms (SNPs) and their function in association to higher rates of ICH recurrence in a cohort of CAA patients. Methods: Patients with a CAA-related ICH were included in the study. MRI data, leukoaraiosis degree and microbleed count, was collected for each patient. 19 SNPs located in 10 candidate genes related to brain hemorrhages were analyzed, including 11 Tag SNPs located in the ACE gene. Statistical analysis was performed through survival curves and Cox regression. Serum ACE and beta-amyloid (1-40) levels were then determined by ELISA. Results: 77 patients were included in the study, with a mean age of 75 years. On baseline MRI, 50% of patients had at least 2 microbleeds and mild to moderate leukoaraiosis degree. During the follow-up (36 months), 24 patients presented a recurrent ICH. Five polymorphisms were related to the presence of recurrent ICH, including four in the ACE gene, although only 2 SNPs remained significantly associated to ICH recurrence after Cox regression analysis adjusting for microbleeds number and leukoaraiosis degree, the e2 allele of the ApoE gene (p¼0.028) and the T allele of the rs4311 SNP of the ACE gene (p¼0.005). Serum ACE levels were higher in T allele carriers (T carriers: 267.6 6 41.2; CC: 203.1 6 51.4 mg/mL; p¼0.013) and ACE and beta-amyloid levels were strongly correlated (p¼0.004). Conclusions: An association between the T allele of the ACE rs4311 polymorphism and ICH recurrence was observed in our CAA cohort. ACE protein levels were significantly higher in carriers of the T allele and were strongly correlated to beta-amyloid (1-40) circulating levels. This data suggests that ACE rs4311 genotypes information might add valuable prognostic information and open therapeutic expectations in CAA patients that merits further investigation.