Gerard D. Schellenberg, PhD - US grants

Alzheimer's Disease (AD) is a common neurodegenerative disease for which no treatment or preventative measure exist. Affected individuals eventually require institutional care and invariably die from disease-related conditions. Over 3 million individuals are affected in the US at an annual cost of over $50 billion/year. Although onset can occur as early as the 3rd decade of life, AD is a disease of the elderly; as many as 10% of those over 65 yrs and 50% of those over 85 yr have AD. AD may be related to athe normal aging; autopsies of cognitively normal elderly persons show a low density of amyloid plaques, neurofibrillary tangles, features characteristic of AD. Therefore, studies of AD are important for both solving the disease and for better understanding normal aging. Defective genes are responsible for AD in some families. One AD gene is the amyloid precursor gene (APP) on chromosome 21. APP mutations cause autosomal dominant familial AD (FAD) in some early-onset kindreds. However, in the majority of early-onset families (90-95%) and in late-onset families, FAD is not caused by APP mutations. We used linkage analysis to identify an early-onset FAD locus on chromosome 14. This locus accounts for FAD in most of the early-onset kindreds which do not have APP mutations. We propose to identifying and clone the chromosome 14 gene using positional cloning techniques. Polymorphic markers will be identified for this region and mapped using the CEPH panel. These markers will be used to refine the position of the chromosome 14 FAD locus by linkage analysis. We will generate a radiation hybrid cell panel and a pulse field gel electrophoresis physical map of the FAD region for fine- scale mapping. Once the location has been refined, the corresponding DNA will be cloned, primarily using YAC clones, and a contig assembled. Genes in this contig will be identified and screened by DNA sequence analysis for FAD mutations. Once identified, the gene's function will be sought to understand its role in AD pathogenesis and possibly normal aging. Identification of the chromosome 14 gene may also help resolve the role of genes in late-onset AD.

Werner's syndrome (WS) is an autosomal dominant recessive disorder which is characterized by the premature occurrence of are-related diseases and the premature appearance of some of the physical features associated with normal aging. WS subjects develop a striking array of age-related degenerative and proliferative disorders early in life. These include some of the most significant age-related diseases of man such as arteriosclerosis, (atherosclerosis, arteriolosclerosis, medial calcinosis, heart valve calcification), a variety of benign and malignant neoplasms, diabetes mellitus, osteoporosis and ocular cataracts. Other features include cutaneous atrophy, short stature, graying or loss of hair, hypogonadism, altered skin pigmentation, high-pitched voice, hyperkeratosis, tight skin, a bird-like facies, cutaneous ulcers of the legs, telangiectasia, and a shortened life expectancy. Onset of at least some of the symptoms is usually noted after adolescence although shortened stature may indicate some undefined alteration in development. Recently, linkage analysis was used to assign the locus for WS (designated WRN) to 8p21. This proposal outlines an approach for identifying the WRN gene by positional cloning techniques. Preliminary work has identified 3 markers which are in linkage disequilibrium with the WRN mutation(s). This observation suggests that the these markers are with O.5-1Mb of the WRN gene. This region has been cloned in yeast artificial chromosome (YAC) clones. New polymorphic markers will be identified so that the region of disequilibrium can be further refined. Genes in the region will be identified by hybridizing cDNA libraries to YACs and by exon trapping. These genes will be sequenced as candidate genes to detect mutations. Once the WRN gene is identified, the long-range goal 'will be to find the function of the gene product and to understand its role in the aging process and its role in major human diseases including arteriosclerosis, neoplasms, diabetes mellitus, and osteoporosis.

Genetic studies have demonstrated three genetic loci for Alzheimer's disease; the amyloid precursor protein (APP) gene, a chromosome 14 locus, and the apolipoprotein E (ApoE) locus/region of chromosome 19. While the first 2 are probably relatively rare causes of Alzheimer's disease (AD), ApoE is a significant risk factor for AD in late-onset AD. In addition to these known loci, other AD genes remain to be identified. One may be at the HLA A locus. Others remain to be mapped. ApoE genotype has become a covariable for many studies of AD, including epidemiologic, genetic, clinical, and neuropathologic studies. In many of these efforts, the ApoE genotype is being used to attempt to correlate phenotypic features with genetic subtypes. Other studies are using ApoE genotypes to attempt to identify the mechanism by which the ApoE epsilon4 allele serves as a risk factor for AD, and the mechanism by which the epsilon2 allele may be protective. The Molecular Genetic Analysis will provide services to projects in the University of Washington Alzheimer's Disease Research Center (UW-ADRC), the University of Kansas Alzheimer's Disease Research Center, the Southern California Alzheimer's Disease Research Center, and to funded projects outside of these Alzheimer's Disease Research Centers. The following services will be provided: 1) DNA will be prepared and banked from AD patients and appropriate controls from a variety of studies. When possible, serum and plasma will also be banked. Each individual study will supply blood samples for preparation of DNA, plasma and serum. 2) All patients and controls will be genotyped for the ApoE polymorphism consisting of the epsilon2/epsilon3/epsilon4 haplotype system. 3) A new ApoE genotyping method will be developed which will be based on the oligo- ligation assay (OLA) methodology. The assay will be easily automated for high-throughput genotyping. 4) The Core will provide the capacity to genotype new genetic markers for AD and other closely related disorders as these markers become available (e.g., the chromosome 14 locus and other LOAD loci). One potential marker is the HLA-A locus. Other early onset and late-onset markers are expected to be developed as new AD loci are identified. Also, as non-DNA markers in serum or plasma are developed, this Core will assay for these new markers.

A unique disease known as amyotrophic lateral sclerosis (ALS) and Parkinsonism Dementia Complex (PDC) or ALS/PDC is observed in the Chamorro people of Guam. The clinical features of ALS observed are similar if not identical to ALS observed in other parts of the world. However, Guam ALS is neuropathologically unique in that neurofibrillary tangles, are observed in the cortex and spinal cord, a finding atypical of classical ALS. PDC is Parkinsonism features observed with dementia, again, the neuropathologic features, which are essentially identical to Guam ALS, distinguish PDC from Parkinson's disease. Guam ALS and PDC are considered different clinical. The goal of this proposal is to identify the genetic susceptibility factors involved in ALS/PDC. To accomplish this goal, the mode of inheritance of ALS/PDC will be examined in 1st degree relatives of a case-control Registry established in 1958-1963. Analysis of segregation patterns should provide information to generate a genetic model of ALS/PDC. Since environmental factors, possibly unique to Gum, also probably play a role in susceptibility, environmental risk factors will be incorporated into the genetic model. To actually identify genetic factors, linkage analysis of ALS/PDC pedigrees will be performed to map the susceptibility gene(s). If needed, non-parametric methods will be used for mapping loci. Results from genetic modeling will be used to design the analysis methods.

Autism is one of the most common developmental disorders, with an occurrence rate of approximately 2-5 per 10,000 in the general population, although true prevalence rates may be as high as 1 per 1000. It is a severe disease with a life long course, and is evident as early as one year of age. Characteristic impairments include difficulty processing social and emotional information, language abnormalities, and repetitive or stereotyped behaviors. Additionally, 75 percent of autistic persons function in the mentally retarded range. The etiology of this disorder is unknown, but evidence for genetic influences is storng. Twin studies shown that monozygotic twins are frequently both affected while dizygotic twins have a lower rate of concordance. Studies of families of autism probands show that there is an elevated risk in sibs of probands relative to the general population. Data from both twin and family studies indicates that the genetic component of autism is the result of multiple genes which interact (epistatic multigenic inheritance). We propose to identify the genetic loci involved in autism. A cohort of 200-250 families with 2 or more affected sibs affected with autism will be ascertained and characterized. The typical family will be 2 affected sibs and both parents. The autistic subjects will be rigorously evaluated to establish the diagnosis of autism. They will also be evaluated with a battery neurocognitive test to potential evaluate sbtypes of autism which may provide markers for genetic heterogeneity. Blood samples will be obtained from members (Affected subjects and their parents and DNA prepared for genetic analysis. In cases where a limited amount of blood can be obtained, permanent cell lines will be prepared. DNA from the family subjects will be genotyped for markers scattered on all chromosomes and the data analyzed by sib-pair methods. The ultimate goal is to identify the chromosomal location of all of the genetic loci contributing to autism with the eventual goal of cloning all of these genes. Unraveling th genetics of autism should provide molecular tools for understanding the pathogenesis of autism.

Alzheimer's disease (AD) is a common neurodegenerative disorder affecting more than 3 million people in the United States. AD etiology is only partially understood. Four genes have been implicated in the inheritance of AD and additional genes remain to be identified. Recently, we and others studying frontotemporal dementia (FTD), found causative mutations in the gene encoding tau. Tau is the main protein of neurofibrillary tangles (NFT's) that are found in both AD and FTD. Other variants of tau pathology are also found in FTD (e.g glial fibrillary tangles). The FTD mutations demonstrate that genetic alterations in tau cause both neurodegeneration and tau pathology, that in some cases closely parallels the changes observed in AD. Thus, tau appears to be an active participant in neurodegenerative events and tau pathology is not just a secondary consequence of AD changes. Different tau mutations cause FTD by different mechanisms; in some cases, the biochemical properties of tau are altered while for other mutations, splicing of exon 10 appears to be altered. Mutations that affect splicing appear to act by 3 mechanisms; some affect the 5'splice site of exon 10, others alter exon splicing enhancer (ESE) regulatory elements, and at least one alters an exon splicing silencer. One goal of this proposal is to understand how the tau gene is regulated in normal and disease cells. First, the genomic sequence of the mouse and human tau gene will be determined and compared to identify conserved non-repeat sequences. Conserved regions are potential cis-acting regulatory elements. Second, the role of these conserved sequences in the regulation of normal splicing will be determined. Third, the mechanism by which polymorphisms and FTD mutations affect this splicing will be determined. We have identified over 30 tau polymorphisms in normal subjects and many of these are in potential regulatory sequences. A fourth goal is to evaluate the role of tau variants in AD. AD subjects will be screened for causative mutations as well as mutations that affect progression. A fifth goal is to refine the location of additional AD loci using Monte Carlo simulation methods for analysis of family data.

DESCRIPTION (Adapted from the application): Alzheimer's disease (AD) is a common neurodegenerative disorder affecting more than three million people in the United States. AD etiology is only partially understood. Recently, the applicants and others studying frontotemporal dementia (FTD), found causative mutations in the gene encoding tau. Tau is the main protein of neurofibrillary tangles (NFT's) that are found in both AD and FTD. Other variants of tau pathology are also found in FTD (e.g., glial fibrillary tangles). The FTD mutations show that genetic alterations in tau cause both neurodegeneration and tau pathology, that in some cases closely parallels the changes observed in AD. Different tau mutations cause FTD by different mechanisms; in some cases, the biochemical properties of tau are altered while in others, splicing of exon 10 (E10) is altered. Mutations affecting splicing act by altering at least four different regulatory mechanisms: 1) the strength of the 5?splice site of E10, 2) an exon splicing enhancer regulatory element in E10, 3) an exon splicing silencer in E10, and 4) an inhibitory sequence directly adjacent to the 3? end of E10. FTD mutations either increase or decrease E10 incorporation into tau mRNA. In some families that show linkage to chromosome 17, no mutations in the open reading frame of tau or in sequences directly flanking exons have been found. Also, for progressive supranuclear palsy (PSP), association studies demonstrate that tau genetic variability is a risk factor for PSP, yet no mutations/risk factors are present in the open reading frame of tau. Thus, additional regulatory mutations in intronic sequences not directly adjacent to exons remain to be found. The different mechanisms affected by the different mutations are responsible for the diverse phenotype observed in different FTD kindreds. The following will be performed: 1) additional FTD families will be screened for tau mutations, 2) sporadic FTD subjects will be screened for mutations, 3) the functional consequences of each mutation on RNA splicing and tau protein faction will be determined, 4) a P1 artificial chromosome (PAC) clone containing the complete tau gene will be characterized for use in generating transgenic animals, 5) tau mutations will be introduced into the PAC, 6) the normal and mutant PACs will be used to generate transgenic animals. This work will determine how tau mutations function in vivo and generate transgenic animals with tau pathology.

DESCRIPTION (Adapted from the application): Genetic studies have demonstrated four genetic loci for Alzheimer's disease; the amyloid precursor protein (APP) gene, presenilin 1 (PS 1) on chromosome 14, presenilin 2 (PS2) on chromosome 1, and the apolipoprotein E (ApoE) locus/region of chromosome 19. While the first 3 are rare causes of Alzheimer s disease (AD), ApoE is a significant risk factor for late-onset AD. In addition to these known loci, other AD genes remain to be identified. Candidates for additional AD loci include the HLA A gene on chromosome 6, and the alpha-2-macroglobulin and lipoprotein receptor protein genes on chromosome 12. Others remain to be mapped. ApoE genotype has become a covariate for many studies of AD, including epidemiologic, genetic, clinical, and neuropathologic studies. In many of these efforts, the ApoE genotype is being used to attempt to correlate phenotypic features with genetic subtypes. Other studies are using ApoE genotypes to attempt to identify the mechanism by which the ApoE e4 allele serves as a risk factor for AD, and the mechanism by which the e2 allele may be protective. The Molecular Genetic Analysis core (Core G) will provide services to projects in the University of Washington Alzheimer s Disease Research Center (UW-ADRC), other AD and related projects at the University of Washington, the University of Southern California ADRC, and other researchers at the University of Southern California. The following services will be provided: 1) DNA will be prepared and banked from AD patents and appropriate controls from a variety of studies. When possible, serum and plasma will also be banked. Each individual study will supply blood samples for preparation of DNA, plasma and serum. 2) All patients and controls will be genotyped for the ApoE polymorphism consisting of the e2/e3/e4 haplotype system. Assays will also be developed for the ApoE promoter region polymorphisms. 3) A new ApoE genotyping method will be developed which will be based on real-time quantitative PCR methods. The assay will be easily automated for high-throughput genotyping. 4) The core will provide the capacity to genotype new genetic markers for AD and other closely related disorders as these markers become available. 5) The core will screen early-onset familial subjects for APP, PS1 and PS2 mutations.

Alzheimer's disease (AD) is the mostcommoncause of dementia inthe elderly. Inthe U.S., this disease affects approximately 3-4 million persons, costing the U.S. economy more than $50 billion peryear. The cause(s) of this debilitating neurodegenerative disease is (are) presently unknown. However,a large body of evidence indicates that at least some, if not all, AD cases are due to genetic factors. Genetic analysis of families with multiple casesof early-onset AD has shownthat 3 autosomal dominantgenesare responsible for atleastsome occurrences of the disease. Inthesefamilies, offspring of affected personsare at least at 50% risk of inheriting a familial ad (FAD)gene and developing AD. Late-onset FAD(LOFAD) appearsto involve othergenes andisa more complex disease. Using linkage analysis, other sophisticated statistical genetic methods, and positional cloning approaches, the long-range goal of this project is to identifythe underlying causes of AD by identifying the genes responsible for genetic forms of late-onset AD. Using genetic linkage analysis based on Monte Carlo MarkovChain methods, we identified a quantitative trait locus on chromosome 19p13.2 that affectsAD risk. This locus wasidentifiedasaquantitative trait thataffects age-of-onset. The 19p locus targeted by this project is distinct from ApoE, another LOFADgene located at 19q13. To identify this new LOFADgene by positional cloning, the following steps will be performed. First, a physical, sequence, and gene map of 19p13.2 spanning the region indicated by linkage analysis will be generated. Second, genes in this region will be screened for polymorphic sites bydatabase analysis and DMA sequence analysis. Third, polymorphisms spanning the region will beusedto testfor linkage disequilibrium in the region. Polymorphic sites tested will include short tandem repeatpolymorphic sites and single nudeotide polymorphism (SNP) sites. Fourth, SNP's in genes in the region will betested as pathogenic sites in multiple familial and case-control samples to identify the true pathogenic allele. Fifth, when the gene and pathogenic alleles are found, functional assayswill bedevised to determine the mechanism of pathogenesis leading toAD. Identification of additional LOFADgenes should greatly enhance our understanding ofADandpotentially leadto new tvDes of therapies. PERFORMANCE SfTEtS) (organization, city, states) ; . Veterans Affairs Puget Sound Health CareSystem,Seattle Division; 1660S. Columbian Way, Seattle, WA98108 (an affiliate hospital of the University of Washington): Phone (206) 764-2701; FAX (206) 764-2569 University of Washington, Seattle, WA KEY PERSONNEL Seeinstructionson Page 11. Usecontinuation pagesasneededto provide the reo^i^ irrfbrmatwn inthefomtf shown below. Name Organization Role on Project C i/Schellenberg, Gerard D., Ph.D. CVBird, Thomas D., M.D. CVWijsman, E. M., Ph.D. [unreadable] l/Yu,Chang-En, Ph.D. VeteransAffairs Puget Sound Health Care System P.I. VeteransAffairs Puget SoundHealth Care System co-P.I. University of Washington co-P.I. Veterans Affairs Puget Sound Health Care System -co-P.I. PHS398 (Rev. 4/98) Page 2 BB Number pages consecutively atthe bottom throughout the application.Dono*usesuffixes such as3a,3b. CC Prin^^Pivestigator/Program Director (Last, first, middle): Sc^^Pnberg, Gerard David Type the name of the principal investigator/program director at the top of each printed page and each continuation page. (For type specifications, see instructions on pages.) RESEARCHGRANT TABLE OF CONTENTS Page Numbers Face Page _ .-. '. _....'. _ 1 Description,

DESCRIPTION (provided by applicant): A unique disease known as amyotrophic lateral sclerosis (ALS) and Parkinsonism Dementia Complex (PDC) or ALS/PDC is observed in the Chamorro people of Guam. The clinical features of ALS observed are similar if not identical to ALS observed in other parts of the world. However, Guam ALS is neuropathologically unique in that neurofibfillary tangles, are observed in the cortex and spinal cord, a finding atypical of classical ALS. PDC is Parkinsonism features observed with dementia; again, the neuropathologic features, which are essentially identical to Guam ALS, distinguish PDC from Parkinson's disease. Guam ALS and PDC are considered different clinical manifestations of the same disease. Guam dementia is another disease entity that is clinically indistinguishable from Alzheimer's disease (AD) and may be an additional form of ALS/PDC. Clustering of ALS/PDC in families and a high incidence in specific villages on Guam indicates that genetic factors are important in disease susceptibility. Also, the increase in age-of-onset of ALS and PDC, and the decrease in the incidence of ALS indicate that environmental factors, possibly unique to Guam, are also involved. The goal of this proposal is to identify the genetic susceptibility factors involved in ALS/PDC. Previous work indicated that tau is a susceptibility factor for ALS/PDC but is not the major gene responsible for this group of diseases. This proposal will focus on: 1) identifying additional susceptibility factors for ALS/PDC; 2) identifying the tau alleles the confer susceptibility to ALS/PDC; 3) characterize Guam dementia in terms of genetic markers for AD such as ApoE promoter polymorphisms and ALS/PDC such as tau.

Abnormally aggregates of the protein tau in the form of neurofibrillary tangles (NFT's) and glial tangles are found in a number of neurodegenerative diseases including Alzheimer's disease (AD) and frontotemporal dementia with parkinsonism - chromosome 17 type (FTDP-17). For most of these diseases, the role tau plays in disease initiation and progression is not understood. However, for the autosomal-dominant disorder FTDP-17, mutations in MAPT, the gene that encodes tau protein, cause the disease. The clinical and neuropathologic phenotype produced by different MAPT mutations is highly variable. For some mutations, the initial clinical feature is limited to executive function deterioration. For others, severe behavioral disturbances such as disinhibition or psychosis are the initial symptoms. Different neuropathologic phenotypes are observed. For some mutations, aggregated tau is only seen in neurons as NFT's. For others, both neuronal and glial aggregates are found (glial fibdllary tangles, GFT's). The regional distribution of pathology is also varied. In some cases, pathology is seen broadly distributed in the frontotemporal lobes while in other cases, only brain stem regions are involved as seen in progressive supra nuclear palsy (PSP). Two types of FTDP-17 mutations are known. One type is missense mutations that act at the protein level. The second type of mutations, which is the focus of this application, alter the regulation of the alternative splicing of tau exon 10 (El0). Also, susceptibility to PSP is caused by an unidentified allele(s) at a MAPT polymorphic site(s) that influences MAPTsplicing. For FTDP-17 mutations, phenotypic variability is potentially dependent on differential expression patterns of trans-acting splicing regulatory factors in different subpopulations of neurons and glial cells. Differential phosphorylation of trans-acting factors in different cell types may also be involved in phenotypic vadabUity. In this proposal, we will identify the cis-acting sequence elements in tau that affect the regulation of alternative splicing. We will also identify the trans-factors that interact with the cis-acting elements. Identification of c/s-elements may lead to therapeutic approaches based on evolving strategies for manipulating RNA in vivo. Likewise, identification of trans-factors may lead to protein targets such as kinases that are also therapeutic targets.

The overall goal is to identify genes that modify the age-at-onset of Alzheimer's disease (AD). There are four known AD loci: 1) the amyloid precursor protein (APP) gene on chromosome 21; 2) the PSEN1 gene on chromosome 13; 3) the PSEN2 gene on chromosome 1; 4) the chromsome 19 apolipoprotein (APOE) gene. Mutations in APP and PSEN1 exhibit autosomal dominant inheritance of AD. Penetrance of disease is age-dependent and complete by 65 years (early-onset AD). PSEN2 mutations are also inherited by an autosomal dominant mechanism, but penetrance is extended over a much longer age-span. In PSEN2 carriers, AD onset ranges from 43 to >89 years. APOE is a susceptibility gene for AD. Inheritance of the APOE epsilon4 allele increases the risk of developing AD, but this allele is neither necessary nor sufficient to cause AD. The epsilon4 allele modifies the onset-age of PSEN1- and PSEN2-induced AD. Numerous other candidate regions and genes have also been suggested, though none has been unambiguously confirmed. The general hypotheses are: 1) The onset age of PSEN1- and PSEN2-induced AD is variable and determined at least in part by other inherited modifier loci. These modifier genes include APOE and other as yet unidentified loci; 2) The location of these modifier genes can be identified by linkage analysis of PSEN1 and PSEN2 AD families. The actual genes responsible for onset modification can be identified by positional cloning methods. We will perform linkage analysis of PSEN1 and PSEN2 families using APOE as a covariant, and age-of-onset as a quantitative trait. Markov chain Monte Carlo analysis methods will be used to detect modifier loci. The families to be studied include the Volga German families (PSEN2N141-1 mutation), a large Columbian family (PSEN1 E280A mutation), and a group of PSEN1 mutation families assembled by the University of Washington Alzheimer's Disease Research Center. In addition, APOE haplotypes constructed from a panel of 21 single nucleotide polymorphism sites will be examined to determine APOE sites other than the epsilon2/epsilon3/epsilon4 polymorphisms contribute to the modification of the age of AD onset.

DESCRIPTION (provided by applicant): This collaborative application is submitted in response to RFA MH-09-171. The root causes of autism remain unknown, limiting efforts to understand disease heterogeneity, diagnose cases, and prevent and treat disease. Epidemiological findings have repeatedly and unequivocally determined that heritable variation in DNA plays a substantial role in the etiology of autism and autism spectrum disorders, yet traditional efforts to identify the genetic basis of this striking heritability have met with very limited success to date and have therefore provided limited insight into disease biology. We propose here an unprecedented partnership between expert large- scale sequencing centers (at the Baylor College of Medicine and the Broad Institute of MIT and Harvard) and a collaborative network of research labs focused on the genetics of autism (brought together by the Autism Genome Project and the Autism Consortium). These groups will work together to utilize dramatic new advances in DNA sequencing technology to reveal the genetic architecture of autism, first through a detailed examination of 1000 genes implicated by previous genetic studies or postulated to be functionally relevant, and later, as the technology continues to advance, through unbiased whole-genome sequencing. The goal is to conclusively identify which genes harbor individual or collections of rare DNA variants that predispose to autism, and thus translate the abstract heritability into solid biological clues to disease pathogenesis that can be studied molecularly and approached therapeutically. These efforts and their follow-up, which will be performed on thousands of autism families collected by the autism research groups and being provided with phenotype data to NIMH repositories, will form the cornerstone of autism genetic research going forward. PUBLIC HEALTH RELEVANCE: We propose a partnership between expert large-scale sequencing centers and a collaborative network of research labs focused on the genetics of autism to utilize novel high-throughput genome sequencing to discover specific genes underlying the significant heritability of autism. Without knowledge of the specific genes and DNA variants that predispose to disease, we lack the basic starting point with which we might understand the biological processes of disease. By combining large, well-characterized patient samples, experienced DNA sequencing teams and a collaborative, expert analysis and follow-up network, this study will provide novel insight into disease biology and will expose genes and pathways that constitute high priority targets for therapeutic development.

DESCRIPTION (provided by applicant): Genome-wide association (GWA) methods have now been successfully used to detect disease-related susceptibility genes numerous genetically complex disorders. In these studies, a critical element for success is that the sample size be large enough so that there is adequate power to detect genes with small effect sizes at a genome-wide significance level. As an example, type 2 diabetes, susceptibility genes with odds ratios of ~1.3 were detected with cohorts of ~15,000 cases and a comparable number of controls. For AD, preliminary GWA studies utilized samples of 1,000 cases or less have been performed. These studies have the power to detect modest effect loci but not the small effect genes typically found in the analysis of other complex disorders. The primary goals of this application are to: 1) Assemble enough AD cases and comparable elderly normal controls to detect small effect loci;2) Perform GWA studies using a high- density genotyping platform to detect small effect loci that contribute to AD susceptibility;3) Analyze a large familial AD collection using the same genotyping platform so that family-based methods can be applied to AD gene discovery;4) Provide a DNA, phenotype, and genotype resource of well- characterized AD cases, mild cognitive impaired (MCI) subjects, and controls for the genetics community to analyze. The cases, MCI subjects, and controls will be the approximately 16,500 subjects currently being studied by the 29 NIA-funded Alzheimer's Disease Centers (ADC's). Extensive phenotype data as a Uniform Data Set (UDS) for these subjects is in a centralized database (National Alzheimer Coordinating Center or NACC). DNA from these subjects will be deposited in the National Cell Repository for Alzheimer's Disease (NCRAD), an existing repository. A second cohort will be multiplex families with extensive phenotype data and DNA currently available from the NIMH Genetics Initiative repository. This study will make use of the infrastructure of the existing Alzheimer's Disease Genetics Consortium (ADGC) to coordinate that data from multiple GWA studies for subsequent meta and combined analysis of multiple AD GWA studies, and to provide investigators with AD GWA data for subsequent analysis. PUBLIC HEALTH RELEVANCE: Alzheimer's disease affects over 5 million people in the US costing the Federal Government over $100 billion dollars/year. Because our population is aging, in 2050, if the disease remains untreatable, there will be 16 million people in the US with AD costing $1 trillion dollars/year. Presently there are no methods of preventing AD or halting progression. Thus, more fundamental knowledge on disease mechanism is needed.

? DESCRIPTION (provided by applicant): This proposal describes future work of the Alzheimer's Disease Genetics Consortium (ADGC). The goal is to deconstruct the complete genetic architecture of Alzheimer's disease (AD), and to determine how all inherited factors contribute to the AD phenotype. To this end we will identify, annotate, replicate, and validate all DNA variants that increase risk or protect against AD, determine what genes are connected to these variants, and evaluate the contribution of each to total AD risk. The rationale for the following genetics/genomics project is to: 1) Predict who will develop AD. 2) Fully reveal all AD genetics in all ethnic groups. 3) Understand the pathogenesis of AD. 4) Identify novel therapeutic targets for AD. Therefore, we will identify new genes/therapeutic targets for AD using methods that make use of data from not only genotyping arrays but also massively parallel DNA sequence approaches. Because much of what we know about AD genetics comes from Caucasians AD studies, we will focus future analysis on not only Caucasians but also on African Americans, Latinos, and Asians. To resolve AD genetics, we will: in AIM 1, use functional genomics to identify AD risk and protective variants in cis- acting regulatory elements, and identifying the genes affected by these CREs; in AIM 2, we will use in silico systems biology approaches to integrate information from all AD genes to identify interaction networks and pathways relevant to AD; in AIM 3, we will identify additional AD rare-variant genes using gene-based (including CREs) analyses. All ethnic groups will be analyzed by these methods; in AIM 4, we will perform whole exome sequencing on African American subjects to generalize findings made on Caucasians, to refine gene localization, to identify novel variants, and to identify novel genes found only in other ethnic groups. This will be followed up by targeted sequencing in African Americans and Latinos; in AIM 5 we will assemble and harmonize phenotypes available in multiple cohorts to identify subtypes of AD and genes associated with variants associated with those subtypes.

PROJECT SUMMARY: The project goal is to identify genes that modify tau pathogenicity. Two approaches will be used, both based on unbiased screens, and thus both can potentially reveal new features of taumediated toxicity. The first approach is to use human genetics to identify genes that contribute to risk for frontotemporal lobar dementia (FTLD). Previously we performed a genome-wide association study (GWAS) using progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) cases, both of which are FTLDs. Five genes were identified: MAPT, NELL2, the FAM76B/translokin/MTMR2 gene cluster, MOBP, and STX6. We will follow up on these loci, performing further human genetics studies and functional analysis. The second approach for identifying modifying loci is to use C. elegans as a tauopathy model. We will identify genes that suppress the toxic effects of tau in C. elegans and translate the finding into a mammalian model system. The Specific Aims are: 1) Follow-up the PSP/CBD GWAS, by collecting additional PSP/CBD subjects, performing dense SNP mapping of the susceptibility genes, examine the expression of the PSP/CBD susceptibility genes with respect to genotype, and test these susceptibility genes in our C. elegans model. 2) Sequence genes involved in FTLD related neurodegenerative diseases. Subjects to be sequenced will primarily be FTLD cases. These experiments will identify rare variants that cause FTLD. 3) Identify new tau toxicity modifiers in our C. elegans model. 4) Generate a mouse knockout of an orthologue of a previously identified C. elegans suppressor gene (SUT2). These mice, null for the mammalian gene (mSUT2) will be crossed with a tau transgenic mouse PSI9 that develops a tauopathy-related phenotype. These experiments will determine if loss of mSUT2 can suppress tauopathy as SUT2 does in C. elegans. This project will identify genes that cause/modify tau toxicity, which is the key to undestanding FTLD. These findings will also be important to Alzheimer's disease, a disorder that also has prominent tau pathology.

DESCRIPTION (provided by applicant): Our unique resource of extended pedigrees with autism spectrum disorder (ASD) will allow us to make important contributions to genetic studies of ASD. We will sequence family members from the most informative pedigrees to study genetic variation contributing to ASD and related phenotypes. We will work to discover new variation, and also use the resource to characterize variants in conjunction with existing whole exome data available through our collaborations. We will test findings in up to 10 other families available from the Autism Genome Project (AGP) network of collaborators. We will also make the resource available to the broader scientific community. Extended families offer an excellent opportunity to identify and study genetic variation, giving a complementary approach to ongoing studies of simplex and small multiplex families. The current collection of families represents some of the largest pedigrees with ASD in the world. We have already detected significant linkage evidence in some of these families with clinical diagnosis and also with related phenotypes, including gender; Full Scale IQ; discrepancy between verbal and nonverbal IQ; language delay, Insistence on Sameness, Repetitive Sensory-Motor Actions (RSMA), overall clinical severity, and regressive onset (all derived from the ADI); head circumference; and the Broader Autism Phenotype. Sequence data in these extended families will result in highly accurate and extensive genetic information. We will identify familial variation in these data, and predict potentially deleterious variants using new informatics approaches. We will refine information about risk by comparing to ongoing sequence projects. We will also use the ongoing sequence projects to help prioritize the familial variant discovery, and choose the best for replication efforts in other AGP families. Finally, we will investigate sequence variants found by simplex/small family sequencing to determine specificity and penetrance in our extended families. Our proposed project will benefit from the continued collaboration of excellent molecular, analytic, and clinical expertise in the Autism Genome Project to enable the most effective use of this unique resource.

DESCRIPTION (provided by applicant): This proposal, entitled the Consortium for Alzheimer's Sequence Analysis (CASA) describes plans to analyze whole exome and whole genome sequence data generated from subjects with Alzheimer's disease (AD) and elderly normal controls. These data will be generated by the National Human Genome Institute Large-Scale Sequence Program. The goal of the planned analyses is to identify genes that have alleles that protect against or increase susceptibility to AD. This is a multiple PI proposal, a collaboration between five senior AD genetics investigators (Farrer, Haines, Mayeux, Pericak-Vance, Schellenberg). CASA has 4 cores and 3 projects. Core A is the Administrative core that will coordinate all aspects of CASA. Core B is the Analysis Statistics and Innovation core that will design and assist analysis by other cores/projects and devise novel methods of statistical analysis. Core C is that Data Management and Information Transfer Core that will implement analyses designed by Core B and the projects. Core C will provide high-performance computing for CASA. These three cores are mandated by FOA PAR-12-183. Core D is the In Silico Functional Genomics Core that will annotate AD-associated variants and perform pathway and interaction analyses. Project 1 will evaluate variants detected in the sequence data for association with AD to identify protective and susceptibility alleles. Project 2 will evaluate sequence data from multiplex AD families to identify variants associated with AD risk and protection, and evaluate variant co-segregation with AD. Project 3 will focus on structural variants (insertion-deletions, copy number variants, and chromosomal rearrangements). The project will use existing methods and develop and implement novel approaches for detecting structural variants. The projects and cores are highly interdependent. For example, structural variants identified by Project 3 will be integrated with single nucleotide AD-associated variants identified by Projects 1 and 2. Likewise variants identified by Project 1 will be tested in the family-based data sets. Core B will assist all projects in designing analyses and Core C will implement Project analyses. Core D will annotate and help interpret results from all projects.

The role of the RFA-mandated Biostatistics and Data Analysis Core (Core C) in the Coordinating Center for Genetics and Genomics of Alzheimer's Disease (CGAD) is to design and implement all aspects of the analysis of Alzheimer's Disease Sequence Project (ADSP) and non-ADSP data (Combined Discovery, Replication and Extended Replication Phases). This analysis design will occur in collaboration with all ADSP/RFA-AG-16002 UO1 and other AD genetics investigators with Core C leading this interaction. To this end, Core C personnel will work with Core A to establish mechanisms for CGAD and other ADSP investigators to work together. The mission of the Core is to design analyses that make use of all available Alzheimer disease (AD)-relevant genetics data including whole genome sequence (WGS) and whole exome sequence (WES) from the ADSP Discovery Phase, targeted genome sequence (TGS) from the Replication Phases, non-ADSP sequence data, and genome-wide single nucleotide variant (SNV) data from large cohorts. Core C will design harmonization protocols for genetic data. Core B will execute these protocols to prepare analysis-ready files for Core C analysis. Special consideration will be given to family-based analyses because the multiplex kindreds being used by the ADSP and non-ADSP groups for sequencing have additional subjects that are not being sequenced but with genome-wide SNV data available. Special consideration will also be given to analyzing African American and Caribbean Hispanic data. Core C tasks will be: 1) re-evaluate the Discovery Phase analyses for additional findings that may be considered in the Replication Phase; 2) design and conduct Replication Phase analyses; 3) design and conduct comprehensive analyses of the aggregate Discovery and Replication Phase data; 4) design and conduct analyses to incorporate genetic data generated outside the ADSP for subjects and families used in the ADSP discovery and Replication Phases; 5) design and conduct the Extended Replication Phase analyses to incorporate genetic data from subjects and cohorts not directly involved in the ADSP project; 6) design post analyses; 7) develop novel analyses approaches.

? DESCRIPTION (provided by applicant): The goal of Alzheimer's disease (AD) genetics research is to identify genetic variants that cause, influence risk, or protect against this disordr, and to identify the underlying genes affected by these variants. These genes are then potential therapeutic targets. The goal of the NIA Coordinating Center for Genetics and Genomics of Alzheimer's Disease (CGAD, this application) is to facilitate AD gene discovery by coordinating analysis of all AD-relevant data. As mandated by RFA AG16001, there are 3 cores and an Overall Component. The mandated cores are: 1) Administrative (Core A); 2) Data Management, Harmonization, and Information Transfer Core (Core B); and 3) Biostatistics and Data Analysis Core (Core C). As mandated by RFA AG16001, CGAD will assemble all data (Cores A and B) generated by the Alzheimer's Disease Sequence Project (ADSP) from both the Discovery Phase and the Replication Phase, and all data from non-ADSP sources (Core A) including that generated by grants funded under RFA AG16002. CGAD will; 1) create and support a collaborative network of all CGAD, ADSP, RFA AG16002, and other AD genetics investigators (Core A); 2) harmonize all genetic and phenotype data and fully annotate all variants (Core B); 2) design all harmonization and annotation protocols (Core C, implemented by Core B); 3) design analysis protocols for all data (Core C); 4) implement analyses plans (Core C, except for computationally intensive protocols that will be executed by Core B); 5) broadly distribute primary data, harmonized annotated analysis-ready files, and analyses results including depositing appropriate data into qualified access databases [National Institute on Aging Genetics of Alzheimer's Disease Storage site (NIAGADS) and database of Genotypes and Phenotypes (dbGaP)] (Core B). As mandated by RFA AG16001, all harmonization protocols and analyses plans will be refined in collaboration with all ADSP, RFA AG16002, and other AD genetics investigators. The Overall Component describes in detail the proposed activities and analyses to be executed by the cores. We will analyze Replication Phase data using single and gene-based analyses. Both single nucleotide variants (SNVs) and structural variants (SVs) will be analyzed. We will perform a combined analysis of Replication and Discovery Phase data. We will also perform an Extended Replication Phase using non- ADSP data. We will analyze data from all phases in a pathway network analysis, an interaction analysis, and a polygenic risk score. These gene-discovery activities will lead to potential targets for developing therapeutics.

The role of the National Institute on Aging (NIA) Coordinating Center for Genetics and Genomics of Alzheimer's Disease (CGAD) is to coordinate the integration and meta-analysis of all available Alzheimer's disease (AD) relevant genetic data with the goal of identifying AD risk/causative/protective genetic variants and eventual therapeutic targets. To this end, CGAD will: 1) Harmonize all AD-relevant genetic data and phenotype data to be compatible for use in subsequent analysis. This will include sequence data generated by the Alzheimer's Disease Sequence Project (ADSP) and data from other sources; 2) Analyze the Alzheimer's Disease Sequence Project (ADSP) Replication Phase data for AD risk/causative/protective genetic variants; 3) Provide a combined analysis of the ADSP Discovery and Replication data for AD risk/causative/protective genetic variants; and 4) Extend the Replication analyses by meta-analyzing ADSP and non-ADSP genetics data for AD risk/causative/protective genetic variants. As mandated by NIA, the CGAD will broadly disseminate all results and derivative data [e.g. imputed genotypes, variant call format (VCF) files recalled using ADSP protocols, etc.]. All CGAD results and derivative data will be distributed to ADSP and RFA-AG- 16002 UO1 and other AD investigators, particularly those working on functional analysis of AD-associated genetic variants. The role of Administrative Core (Core A) is to coordinate all the above aspects CGAD activities carried out by Cores B, C, and the Overall Component. Core A will also coordinate all CGAD activities with the activities of other ADSP/RFA-AG-16002 grant investigators, and other AD genetics investigators. The goal is to enhance ADSP activities and increase the efficiency of AD genetics research. Specific tasks of the Administrative Core are: 1) Support and foster collaboration between ADSP and AD genetics investigators, and RFA-AG-16002 investigators; 2) Coordinate the collection of genetic and phenotype data from ADSP Discovery and Replication phase, and all relevant non-ADSP data, and transfer the data to Core B; 3) Coordinate data harmonization of all AD-relevant genetic and phenotype data and analysis for AD risk and protective variants; 4) Distribute analysis-ready files and analysis results to ADSP and all AD genetics investigators. Files will be fully annotated. Analysis files will be results from joint and meta- analysis results generated by CGAD, the ADSP, and RFA-AG-16002; 5) Disseminate data/findings to a diverse group of investigators including ADSP investigators and Replication phase collaborators, AD genetics investigators including those funded by NIA through other mechanisms (e.g. RO1's), and the broader scientific community through data sharing via National Institute on Aging Genetics of Alzheimer's Disease storage site (NIAGADS) and database of Genotypes and Phenotypes (dbGaP); 6) Lead Research Round table where genetic results are shared with AD investigators working on molecular mechanisms related to pathogenesis.

PROJECT SUMMARY/ABSTRACT Progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) are Parkinsonian disorders with predominant tau pathology at autopsy. A common non-recombining haplotype at the microtubule associated protein tau gene (MAPT) locus on chromosome 17q21 increases the risk of PSP and CBD and recently our genome-wide association efforts identified variation in additional genes/loci (STX6, EIF2AK3, SOS1, KIF13B, and MOBP/Appoptosin). In addition, other genes, as yet undetected, likely contribute to susceptibility to PSP and CBD. This study aims to resolve the disease-associated genetic variation within the exome and whole genome sequence data from PSP and CBD patients, to determine the pathological consequences and mechanisms underlying these complex neurodegenerative diseases characterized by tau pathology, thus identifying potential therapeutic targets. For Aim 1, we will analyze a unique dataset consisting of over 600 exomes and 2400 whole genome sequences derived from pathology-confirmed PSP patients and 350 exomes from CBD patients. These data will be compared to the 5000 control exomes available through the Alzheimer's sequencing project for single nucleotide variant (SNV) analysis. In Aim 2, we will expand our analysis of this cohort and specifically analyze structural variants (SV) and copy number variations (CNV) that contribute to PSP and CBD using a range of analytical software programs which we have extensively tested to achieve optimal sensitivity for each type of variation. For Aim 3, we will use the available exome and whole genome sequence data to assess the association of genetic variation on tau toxicity and pathology with Core C, by assessing tau burden in different brain regions and microgliosis as a surrogate of neuronal cell loss. On-going efforts have shown that using quantitative pathologic measures can help distinguish subtypes of PSP. For Aim 4, we will determine the effect of significantly associated variants/genes identified. We will examine mRNA expression, RNA-Seq data, in vitro functional studies to characterize the effect of the observed mutation on tau aggregation and microtubule assembly, and the effect of these genes/variants on tau protein levels and isoforms in postmortem brain tissue. In summary, the combination of whole exome and whole genome sequence data from pathologically-confirmed and highly-phenotyped patients with an in-depth analytical plan focusing on SNV, CNV, SV and quantitative traits, provides a unique opportunity for novel gene discovery. Identifying novel genes for tauopathies is a critical step towards a better understanding of the pathomechanisms underlying this group of disorders and may help identify prognostic biomarkers for these devastating disorders.

PROJECT 2: Project Summary/Abstract Frontotemporal lobar dementia (FTLD) is the second most common cause of dementia in people over 65 years of age. In a subset of FTLD cases, aggregated tau is the major neuropathological feature (FTLD-Tau). FTLD- Tau includes progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD), which are the focus of this project. In both disorders, aggregated tau is found in both neurons and glial cells. PSP and CBD belong to a larger group of neurodegenerative diseases called tauopathies, disorders with aggregated tau as part of the neuropathologic signature of each disease. The most common tauopathy that has the largest economic, medical, and personal costs is Alzheimer's disease (AD). All tauopathies are presently essentially untreatable. The goal of this project is to deconstruct the genetic and genomic architecture of tauopathies with a focus on PSP and CBD. The rationale for the project is to: 1) Predict who will develop tauopathies. When prevention therapies become available, genetics will contribute to predicting who should be treated and when. 2) Understand tauopathy pathogenesis. Gene discovery can identify pathways not presently implicated in these disorders. Pathways potentially involved in FTLD-Tau include all aspects of tau spreading including initial aggregate formation, release form cells, uptake by adjacent cells, release in to the cytoplasm, formation of seeded aggregates, and response to either a toxic tau-related entity, or response to tau depletion. 3) Identify therapeutic targets. Genetic studies leading to gene discovery are essential for finding new therapeutic targets. We will use a multi-pronged approach to tauopathy genetics. We will: 1) Identify genes where loss- of-function variants prevent tauopathies (protective genes) using a C. elegans tauopathy model; 2) Identify novel PSP and CBD causative/risk genes using whole exome sequence analysis to identify causative variants. Genes identified in both phases will then be tested in both C. elegans and mammalian tauopathy models; 3) identify causative variants in MAPT region regulatory elements that affect expression of MAPT and other nearby genes. This work has the potential to identify therapeutic targets for not only PSP and CBD, but also to AD, the most prevalent tauopathy.

Progressive supranuclear palsy (PSP) is the most common frontotemporal lobar degeneration associated with tau pathology. While rare pathogenic variants, common risk factors, and ? more recently ? rare risk-associated variants have been identified in PSP, a significant proportion of the heritability for neurodegenerative tauopathies and other frontotemporal lobar degenerations remains unexplained, strongly suggesting that additional genetic risk factors await discovery. In this application, we propose to identify novel genetic variation associated with PSP using a multi-stage strategy. First, we will detect variants through whole-genome sequencing of neuropathologically characterized PSP. Second, we will prioritize pathological brain tissue samples for a multidimensional screen that includes transcriptional, proteomics, and epigenetic assays. Through recursive application of a prioritization algorithm, regions and variants most likely to have a high impact on disease risk will be identified. Finally, we will follow up on these variants using a high-throughput functional screen. This project taps unprecedented pathologic resources of PSP, leverages a pathologic and genetic infrastructure created with support from private foundations, and offers to transform our understanding of the genetic architecture of PSP and to advance towards the biology and downstream effects of this prototypical tauopathy downstream effects of this prototypical tauopathy.

Overall Project Summary Alzheimer?s disease (AD) affects 5.8 million people in the United States, and is an immense burden on patients, caregivers and on the economy. No disease-modifying treatments or preventions exist, and we need better understanding of the disease and new therapeutic strategies. Genetic discoveries are one source of candidate therapeutic targets. One source of genetic targets is the Alzheimer?s Disease Sequencing Project (ADSP), a National Institute on Aging (NIA) initiative since 2012 to sequence genomes and exomes of AD subjects and cognitively normal elderly controls. The Genome Center for Alzheimer?s Disease (GCAD) is the analysis coordinating center for the ADSP. In the previous grant cycle, GCAD processed all AD-relevant sequencing data producing harmonized genetic data for AD research. This renewal responds to the increase in the amount and complexity of ADSP sequence data, the collection of new types of data, and an expansion of the types of analysis being performed. In addition to sequence data, GCAD will harmonize functional genomics data. GCAD will provide fully quality-controlled and annotated genetic and functional genomics data that is analysis ready. In addition to AD, GCAD will also work with data for AD related disorders (ADRD). These include frontotemporal dementias (FTDs), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Lewy body dementia (LBD), and Parkinson?s disease with dementia (PD-d). In the Y6-10 funding period, GCAD will assemble and harmonize whole-genome/whole-exome sequencing data, and provide it to ADSP investigators and the general scientific community. GCAD will work with US and non-US groups to analyze data by fostering a collaborative environment, and providing infrastructure support. GCAD will also assemble and harmonize functional genomics data which will be integral to identifying genes as candidate drug targets. The research plan will lead to a high quality, comprehensive, harmonized collection of genetic and functional data with detailed supporting resources including documentation and optimized computer codes. This resource will be invaluable for the entire AD research community.

Core A Project Summary/Abstract Core A (Administrative Core) will oversee, facilitate and coordinate all GCAD activities carried out by the other two cores. Core A will also coordinate analyses by investigators funded to analyze Alzheimer?s Disease Sequencing Project (ADSP) data. Core A Specific Aims are: (1) Coordinate acquisition of ADRD genetic, FG, and phenotype data; (2) Coordinate data processing, harmonization and analyses; (3) Support/foster collaboration between ADSP investigators and related projects; (4) Disseminate data and findings to a diverse group of ADRD researchers; (5) Provide administrative support to GCAD including governance, Institution Review Board (IRB) protocol review, and data sharing compliance.