- Email: [email protected]
WITHOUT AN AD-ASSOCIATED TREM2 VARIANT
P644
Poster Presentations: Monday, July 17, 2017
Figure 1. Pedigree of the family investigated in this study. The index case has sporadic AD and no other causes of cognitive impairment. All members of generation II were confirmed normal on assessment. Information on generation I was by family report. The sib-pair are c.2279A>C EPHA1 heterozygotes.
maging Initiative. Variants not found in any cases were annotated using the Ensembl Variant Effect Predictor toolset. Results: Analysis disclosed the presence of the variant NM_005232.4: c.2279A>C (p.Asn760Thr) on exon 14 of the EPHA1 gene in the mother and maternal uncle. The variant was not found in the 1000 Genomes, ExAC, ALS, or Geno2MP databases. Polyphen2, an in silico prediction tool, suggests that the effect of the variant is likely benign (0.099). Conclusions: The presence of this EPHA1 variant in a healthy older APOE4 homozygote and APOE4 heterozgyote sib-pair combined with its absence in the related index case and in nearly 6,000 AD cases, suggests that it may be a private, protective variant. A common, protective polymorphism on EPHA1 has been reproducibly identified in large genome-wide association studies. Additional screening of this family is underway and functional assays will be undertaken on neurons derived from fibroblasts donated by the mother and affected daughter.
P2-097
WITHDRAWN
P2-098
TSPO IMMUNOSTAINING IN AD CASES WITH/WITHOUT AN AD-ASSOCIATED TREM2 VARIANT
Angela Hodges1,2, Yau Mun Lim3, 1Maurice Wohl Clinical Neuroscience Institute James Black Centre Institute of Psychiatry, Psychology & Neuroscience (IoPPN) King’s College London, London, United Kingdom; 2King’s College London, London, United Kingdom; 3King’s College London, Institute of Psychiatry, Psychology & Neuroscience, London, United Kingdom. Contact e-mail: [email protected]
Figure 2. Reported cerebrospinal fluid levels of Ab42 peptide, total tau (T-tau), and phospho-tau (P-tau) protein in the index case are consistent with the diagnosis of AD. (A) Technical results reported from Athena Diagnostics. (B) Subject data is plotted on the graph to illustrate the position of the individual’s results relative to recognized reference points for diagnostic cutoff values.
index case exhibited a typical amnestic presentation of AD and underwent spinal fluid analysis for AD biomarkers, which strongly supported the diagnosis. The mother underwent research clinical and neuropsychological evaluations confirming her to be cognitively healthy. The uncle underwent a phone interview with a CDR of 0. Whole exome sequencing was performed on all family members on the Illumina HiSeq2000 platform. The raw data was analyzed with BWA (read mapping) and GATK (variant calling) software packages. Variants were filtered to select mutations present in only the mother and uncle, but not the daughter. These variants were then screened against 5,656 AD cases from the Alzheimer’s Disease Sequencing Project, and 128 AD cases with whole genome sequencing from the Alzheimer’s Disease Neuroi-
Background: The Peripheral-Type Benzodiazepine Receptor (TSPO) is a well-established target for the Positron Emission Tomography (PET) ligands [11C]-PK11195 and [11C]-PBR28. TSPO is believed to translocate cholesterol across the mitochondrial intermembrane space and is highly expressed in activated glia. TSPO PET ligands thus enable sensitive in vivo monitoring of neuroinflammation, a phenomenon closely associated with neuronal cell death in Alzheimer’s disease (AD). AD patients have recently been identified with rare disease-associated variants in the TREM2 gene (1, 2). Disease vulnerability in these people appears to be linked to inappropriate microglial function and immune response. TREM2 is a plasma membrane receptor highly expressed in brain microglia (3) where it is believed to regulate phagocytic and inflammatory microglial responses to brain pathology. We therefore sought to establish if TSPO levels are impaired in AD patients who have a disease-associated TREM2 variant and establish which glial cells impairment is associated. Methods: Hippocampal sections (5-7mM) from age and gender matched AD (N¼37) and control (N¼18) donors were analysed by immunohistochemistry using published TSPO antibodies (4, 5) (PAB7095 and NP155). The AD cases consisted of Braak-matched cases (IV) with TREM2+ (N¼16) and without TREM2-(N¼21) disease-associated variants in TREM2. Sections were also immunostained for p-tau (AT8), Ab (4G8) and by co-immunoflurescence with markers of microglia (Iba-1, CD68 and HLA-DR,DP,DQ) and astrocytes (GFAP) to ascertain cell types expressing TSPO. Results: We will report results of
Poster Presentations: Monday, July 17, 2017
immunohistochemistry analyses using two previously reported TSPO antibodies in human post mortem brain sections. As reported previously, these antibodies stain macrophages, astrocytes and microglia in AD brains with more limited staining in non-AD cases. We used astrocyte and microglia specific markers to determine which cells were differentially stained between TREM2+ and TREM2- AD cases and determine differences in cells adjacent to amyloid or Tau which stain for TSPO. Conclusions: The biomarker TSPO has been used extensively to explore immune activation in vivo in the brain. Levels are increased in AD, particularly associated with the emergence of abnormal Tau and neuronal cell loss. Our results will facilitate interpretation of future PET imaging in this group of patients using the TSPO tracers [11C]-PK11195 or [11C]-PBR28.
P2-099
INVESTIGATING THE IMPACT OF NONCODING ALZHEIMER’S DISEASEASSOCIATED VARIANTS ON BIN1 REGULATION
Anna N. Barrett, Julie Williams, Nick Bray, Matt Hill, Cardiff University, Cardiff, United Kingdom. Contact e-mail: [email protected] Background: In 2013 the International Genomics of Alzheimer’s
disease Project performed the largest genome wide association study on late onset Alzheimer’s disease (LOAD) to date. This study identified a single nucleotide polymorphism at the BIN1 locus as the second most significant region associated with LOAD. The genomic region of association lies approximately 30Kb upstream of BIN1. Rs6733839, a cytosine to thymine change, is the most significant SNP within the BIN1 region associated with LOAD, having a p value of 6.9x10-44. As this region of association is outside of the protein-coding region it is possible that this locus may have a regulatory function and dysregulation of BIN1 expression may be a pathological mechanism increasing susceptibility for LOAD. Methods: Allele specific expression assays are able to measure cis-regulatory effects of DNA variants on gene expression. This assay was used to investigate differential expression of BIN1alleles in relation to BIN1 genotype in adult post-mortem prefrontal cortex tissue. Data from the Roadmap Epigenomics Project show evidence of epigenetic modifications indicative of DNA enhancer activity at the BIN1index SNP in monocytes. Using gene reporter assays we quantified the enhancer activity of this region in a number of cell lines to investigate allele specific enhancer activity. Results: Preliminary data indicate the presence of differential BIN1 allelic expression in prefrontal cortex tissue, indicating that BIN1 expression is subject to control by cis-regulatory variation. Data discussing the association between allele expression levels and genotype will be presented at AAIC 2017. Initial gene reporter assays in HEK293 cells indicated this locus has enhancer activity in an orientation specific manner however rs6733839 genotype showed no effect on enhancer function. This assay was performed in more disease relevant immune cell lines to elucidate the impact of rs6733839 genotype on enhancer activity. This data will be presented at AAIC 2017. Conclusions: Despite identifying variants associated with disease, the functional significance and cellular context of the effect of these variants remains unknown. Our observations lead to the hypothesis that BIN1 regulation is implicated in LOAD and rs6733839, located within a regulatory region, may impact on enhancer function.
P2-100
P645
CLINICAL AND GENETIC STUDY OF A JAPANESE FAMILY WITH COMPLICATED HEREDITARY SPASTIC PARAPLEGIA AND ALZHEIMER’S DISEASE
Celeste Montecchiani1,2, Laura D’Onofrio1, Marzia Mearini1, Fabrizio Gaudiello1, Marialuisa Miele1, Carlo Caltagirone1,3, Toshitaka Kawarai4, Antonio Orlacchio1,2, 1IRCCS Santa Lucia, Rome, Italy; 2University of Perugia, Perugia, Italy; 3University of Rome “Tor Vergata,” Rome, Italy; 4University of Tokushima, Tokushima, Japan. Contact e-mail: [email protected] Background: Mutations in the SPG4/SPAST gene are the major cause
of hereditary spastic paraplegias (HSPs), a group of genetic disorders leading to progressive spasticity and weakness of the lower limbs. Alzheimer’s disease (AD) is a degenerative disorder of the central nervous system. Early-onset familial AD (EOFAD) accounts for 35% of all AD cases and denotes families in which onset is reliably before age 60 to 65 years and sometimes before age 55 years. Two SPG4/SPAST Italian families with pure HSP and AD were previously described by our research team. Methods: Whole exome sequencing (WES), Sanger sequencing, Multiplex Ligation dependent Probe Amplification (MLPA) analysis, bioinformatics, neurological evaluation, diagnostic imaging, and pathological assessment. Results: One Japanese woman, belonging to a kindred emigrated in Italy, was diagnosed as certainly affected by slowly progressive autosomal dominant HSP, according to the Harding criteria. The age at onset of the first motor symptoms was 21 years. At 62 years, the phenotype was complicated by cognitive impairment; a diagnosis of EOFAD based on the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA) criteria was formulated. The case was definite Alzheimer’s disease (autopsy proven): the brain pathology showed plaques with a congophilic core and neuritic pathology. Additional six affected subjects revealed the same initial symptoms of dementia and HSP was diagnosed. Additional clinical signs, such as scoliosis and pes cavus, were present in the affected individuals. WES revealed in all patients a new heterozygous change in SPG4/SPAST. MLPA analysis excluded the presence of deletions/ duplications in SPG4/SPAST. Bioinformatic analyses and population study confirm a pathogenetic role of such mutation. Screening of PS1, PS2, and bAPP genes did not reveal any coding mutation. No affected subject carried out APOEgenotype ε4/ε3 or ε4/ε4. Conclusions: To our knowledge, this work describes the first Japanese family with a new SPG4/SPAST mutation and association of a complicated form of HSP and EOFAD.
P2-101
CROSS STUDY INTEGRATIVE NETWORK ANALYSIS IDENTIFICATION OF AD DISEASE ETIOLOGY
Benjamin A. Logsdon, Thanneer M. Perumal, Solveig K. Sieberts, Larsson Omberg, Lara M. Mangravite, Sage Bionetworks, Seattle, WA, USA. Contact e-mail: ben.logsdon@ sagebase.org Background: Alzheimer’s disease is a devastating illness with no
known efficacious pharmacological intervention. Identification and characterization of novel aspects of AD biology is essential to develop treatments for this complex disease. We use a systems biology approach for this task, with an integrative network analysis of five publically available expression data sets from patients with Alzheimer’s Disease including expression data from the ROSMAP study, the MSSM study, the Mayo RNAseq study, the MCADGS
Poster Presentations: Monday, July 17, 2017
Figure 1. Pedigree of the family investigated in this study. The index case has sporadic AD and no other causes of cognitive impairment. All members of generation II were confirmed normal on assessment. Information on generation I was by family report. The sib-pair are c.2279A>C EPHA1 heterozygotes.
maging Initiative. Variants not found in any cases were annotated using the Ensembl Variant Effect Predictor toolset. Results: Analysis disclosed the presence of the variant NM_005232.4: c.2279A>C (p.Asn760Thr) on exon 14 of the EPHA1 gene in the mother and maternal uncle. The variant was not found in the 1000 Genomes, ExAC, ALS, or Geno2MP databases. Polyphen2, an in silico prediction tool, suggests that the effect of the variant is likely benign (0.099). Conclusions: The presence of this EPHA1 variant in a healthy older APOE4 homozygote and APOE4 heterozgyote sib-pair combined with its absence in the related index case and in nearly 6,000 AD cases, suggests that it may be a private, protective variant. A common, protective polymorphism on EPHA1 has been reproducibly identified in large genome-wide association studies. Additional screening of this family is underway and functional assays will be undertaken on neurons derived from fibroblasts donated by the mother and affected daughter.
P2-097
WITHDRAWN
P2-098
TSPO IMMUNOSTAINING IN AD CASES WITH/WITHOUT AN AD-ASSOCIATED TREM2 VARIANT
Angela Hodges1,2, Yau Mun Lim3, 1Maurice Wohl Clinical Neuroscience Institute James Black Centre Institute of Psychiatry, Psychology & Neuroscience (IoPPN) King’s College London, London, United Kingdom; 2King’s College London, London, United Kingdom; 3King’s College London, Institute of Psychiatry, Psychology & Neuroscience, London, United Kingdom. Contact e-mail: [email protected]
Figure 2. Reported cerebrospinal fluid levels of Ab42 peptide, total tau (T-tau), and phospho-tau (P-tau) protein in the index case are consistent with the diagnosis of AD. (A) Technical results reported from Athena Diagnostics. (B) Subject data is plotted on the graph to illustrate the position of the individual’s results relative to recognized reference points for diagnostic cutoff values.
index case exhibited a typical amnestic presentation of AD and underwent spinal fluid analysis for AD biomarkers, which strongly supported the diagnosis. The mother underwent research clinical and neuropsychological evaluations confirming her to be cognitively healthy. The uncle underwent a phone interview with a CDR of 0. Whole exome sequencing was performed on all family members on the Illumina HiSeq2000 platform. The raw data was analyzed with BWA (read mapping) and GATK (variant calling) software packages. Variants were filtered to select mutations present in only the mother and uncle, but not the daughter. These variants were then screened against 5,656 AD cases from the Alzheimer’s Disease Sequencing Project, and 128 AD cases with whole genome sequencing from the Alzheimer’s Disease Neuroi-
Background: The Peripheral-Type Benzodiazepine Receptor (TSPO) is a well-established target for the Positron Emission Tomography (PET) ligands [11C]-PK11195 and [11C]-PBR28. TSPO is believed to translocate cholesterol across the mitochondrial intermembrane space and is highly expressed in activated glia. TSPO PET ligands thus enable sensitive in vivo monitoring of neuroinflammation, a phenomenon closely associated with neuronal cell death in Alzheimer’s disease (AD). AD patients have recently been identified with rare disease-associated variants in the TREM2 gene (1, 2). Disease vulnerability in these people appears to be linked to inappropriate microglial function and immune response. TREM2 is a plasma membrane receptor highly expressed in brain microglia (3) where it is believed to regulate phagocytic and inflammatory microglial responses to brain pathology. We therefore sought to establish if TSPO levels are impaired in AD patients who have a disease-associated TREM2 variant and establish which glial cells impairment is associated. Methods: Hippocampal sections (5-7mM) from age and gender matched AD (N¼37) and control (N¼18) donors were analysed by immunohistochemistry using published TSPO antibodies (4, 5) (PAB7095 and NP155). The AD cases consisted of Braak-matched cases (IV) with TREM2+ (N¼16) and without TREM2-(N¼21) disease-associated variants in TREM2. Sections were also immunostained for p-tau (AT8), Ab (4G8) and by co-immunoflurescence with markers of microglia (Iba-1, CD68 and HLA-DR,DP,DQ) and astrocytes (GFAP) to ascertain cell types expressing TSPO. Results: We will report results of
Poster Presentations: Monday, July 17, 2017
immunohistochemistry analyses using two previously reported TSPO antibodies in human post mortem brain sections. As reported previously, these antibodies stain macrophages, astrocytes and microglia in AD brains with more limited staining in non-AD cases. We used astrocyte and microglia specific markers to determine which cells were differentially stained between TREM2+ and TREM2- AD cases and determine differences in cells adjacent to amyloid or Tau which stain for TSPO. Conclusions: The biomarker TSPO has been used extensively to explore immune activation in vivo in the brain. Levels are increased in AD, particularly associated with the emergence of abnormal Tau and neuronal cell loss. Our results will facilitate interpretation of future PET imaging in this group of patients using the TSPO tracers [11C]-PK11195 or [11C]-PBR28.
P2-099
INVESTIGATING THE IMPACT OF NONCODING ALZHEIMER’S DISEASEASSOCIATED VARIANTS ON BIN1 REGULATION
Anna N. Barrett, Julie Williams, Nick Bray, Matt Hill, Cardiff University, Cardiff, United Kingdom. Contact e-mail: [email protected] Background: In 2013 the International Genomics of Alzheimer’s
disease Project performed the largest genome wide association study on late onset Alzheimer’s disease (LOAD) to date. This study identified a single nucleotide polymorphism at the BIN1 locus as the second most significant region associated with LOAD. The genomic region of association lies approximately 30Kb upstream of BIN1. Rs6733839, a cytosine to thymine change, is the most significant SNP within the BIN1 region associated with LOAD, having a p value of 6.9x10-44. As this region of association is outside of the protein-coding region it is possible that this locus may have a regulatory function and dysregulation of BIN1 expression may be a pathological mechanism increasing susceptibility for LOAD. Methods: Allele specific expression assays are able to measure cis-regulatory effects of DNA variants on gene expression. This assay was used to investigate differential expression of BIN1alleles in relation to BIN1 genotype in adult post-mortem prefrontal cortex tissue. Data from the Roadmap Epigenomics Project show evidence of epigenetic modifications indicative of DNA enhancer activity at the BIN1index SNP in monocytes. Using gene reporter assays we quantified the enhancer activity of this region in a number of cell lines to investigate allele specific enhancer activity. Results: Preliminary data indicate the presence of differential BIN1 allelic expression in prefrontal cortex tissue, indicating that BIN1 expression is subject to control by cis-regulatory variation. Data discussing the association between allele expression levels and genotype will be presented at AAIC 2017. Initial gene reporter assays in HEK293 cells indicated this locus has enhancer activity in an orientation specific manner however rs6733839 genotype showed no effect on enhancer function. This assay was performed in more disease relevant immune cell lines to elucidate the impact of rs6733839 genotype on enhancer activity. This data will be presented at AAIC 2017. Conclusions: Despite identifying variants associated with disease, the functional significance and cellular context of the effect of these variants remains unknown. Our observations lead to the hypothesis that BIN1 regulation is implicated in LOAD and rs6733839, located within a regulatory region, may impact on enhancer function.
P2-100
P645
CLINICAL AND GENETIC STUDY OF A JAPANESE FAMILY WITH COMPLICATED HEREDITARY SPASTIC PARAPLEGIA AND ALZHEIMER’S DISEASE
Celeste Montecchiani1,2, Laura D’Onofrio1, Marzia Mearini1, Fabrizio Gaudiello1, Marialuisa Miele1, Carlo Caltagirone1,3, Toshitaka Kawarai4, Antonio Orlacchio1,2, 1IRCCS Santa Lucia, Rome, Italy; 2University of Perugia, Perugia, Italy; 3University of Rome “Tor Vergata,” Rome, Italy; 4University of Tokushima, Tokushima, Japan. Contact e-mail: [email protected] Background: Mutations in the SPG4/SPAST gene are the major cause
of hereditary spastic paraplegias (HSPs), a group of genetic disorders leading to progressive spasticity and weakness of the lower limbs. Alzheimer’s disease (AD) is a degenerative disorder of the central nervous system. Early-onset familial AD (EOFAD) accounts for 35% of all AD cases and denotes families in which onset is reliably before age 60 to 65 years and sometimes before age 55 years. Two SPG4/SPAST Italian families with pure HSP and AD were previously described by our research team. Methods: Whole exome sequencing (WES), Sanger sequencing, Multiplex Ligation dependent Probe Amplification (MLPA) analysis, bioinformatics, neurological evaluation, diagnostic imaging, and pathological assessment. Results: One Japanese woman, belonging to a kindred emigrated in Italy, was diagnosed as certainly affected by slowly progressive autosomal dominant HSP, according to the Harding criteria. The age at onset of the first motor symptoms was 21 years. At 62 years, the phenotype was complicated by cognitive impairment; a diagnosis of EOFAD based on the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA) criteria was formulated. The case was definite Alzheimer’s disease (autopsy proven): the brain pathology showed plaques with a congophilic core and neuritic pathology. Additional six affected subjects revealed the same initial symptoms of dementia and HSP was diagnosed. Additional clinical signs, such as scoliosis and pes cavus, were present in the affected individuals. WES revealed in all patients a new heterozygous change in SPG4/SPAST. MLPA analysis excluded the presence of deletions/ duplications in SPG4/SPAST. Bioinformatic analyses and population study confirm a pathogenetic role of such mutation. Screening of PS1, PS2, and bAPP genes did not reveal any coding mutation. No affected subject carried out APOEgenotype ε4/ε3 or ε4/ε4. Conclusions: To our knowledge, this work describes the first Japanese family with a new SPG4/SPAST mutation and association of a complicated form of HSP and EOFAD.
P2-101
CROSS STUDY INTEGRATIVE NETWORK ANALYSIS IDENTIFICATION OF AD DISEASE ETIOLOGY
Benjamin A. Logsdon, Thanneer M. Perumal, Solveig K. Sieberts, Larsson Omberg, Lara M. Mangravite, Sage Bionetworks, Seattle, WA, USA. Contact e-mail: ben.logsdon@ sagebase.org Background: Alzheimer’s disease is a devastating illness with no
known efficacious pharmacological intervention. Identification and characterization of novel aspects of AD biology is essential to develop treatments for this complex disease. We use a systems biology approach for this task, with an integrative network analysis of five publically available expression data sets from patients with Alzheimer’s Disease including expression data from the ROSMAP study, the MSSM study, the Mayo RNAseq study, the MCADGS