Exome Sequencing of Extended Families with Alzheimer's Disease Identifies Novel Genes Implicated in Cell Immunity and Neuronal Function.

OBJECTIVE: Alzheimer's disease (AD) is a neurodegenerative disorder for which more than 20 genetic loci have been implicated to date. However, studies demonstrate not all genetic factors have been identified. Therefore, in this study we seek to identify additional rare variants and novel genes potentially contributing to AD. METHODS: Whole exome sequencing was performed on 23 multi-generational families with an average of eight affected subjects. Exome sequencing was filtered for rare, nonsynonymous and loss-of-function variants. Alterations predicted to have a functional consequence and located within either a previously reported AD gene, a linkage peak (LOD>2), or clustering in the same gene across multiple families, were prioritized. RESULTS: Rare variants were found in known AD risk genes including AKAP9, CD33, CR1, EPHA1, INPP5D, NME8, PSEN1, SORL1, TREM2 and UNC5C. Three families had five variants of interest in linkage regions with LOD>2. Genes with segregating alterations in these peaks include CD163L1 and CLECL1, two genes that have both been implicated in immunity, CTNNA1, which encodes a catenin in the cerebral cortex and MIEF1, a gene that may induce mitochondrial dysfunction and has the potential to damage neurons. Four genes were identified with alterations in more than one family include PLEKHG5, a gene that causes Charcot-Marie-Tooth disease and THBS2, which promotes synaptogenesis. CONCLUSION: Utilizing large families with a heavy burden of disease allowed for the identification of rare variants co-segregating with disease. Variants were identified in both known AD risk genes and in novel genes.

Targeted sequencing of ABCA7 identifies splicing, stop-gain and intronic risk variants for Alzheimer disease.

Several variants in the gene ABCA7 have been identified as potential causal variants for late-onset Alzheimer's disease (LOAD). In order to replicate these findings, and search for novel causal variants, we performed targeted sequencing of this gene in cohorts of non-Hispanic White (NHW) and African-American (AA) LOAD cases and controls. We sequenced the gene ABCA7 in 291 NHW LOAD cases and 103 controls. Variants were prioritized for rare, damaging variants and previously reported variants associated with LOAD, and were follow-up genotyped in 4076 NHW and 1157 AA cases and controls. We confirm three previously associated ABCA7 risk variants and extend two of these associations to other populations, an intronic variant in NHW (P=3.0×10-3) (originally reported in a Belgian population), and a splice variant originally associated in the Icelandic population, which was significantly associated in the NHW cohort (P=1.2×10-6) and nominally associated in the AA cohort (P=0.017). We also identify a 3'-UTR splice variant that segregates in four siblings of one family and is nominally associated with LOAD (P=0.040). Multiple variants in ABCA7 contribute to LOAD risk.

Utility of blood pressure genetic risk score in admixed Hispanic samples.

Hypertension is strongly influenced by genetic factors. Although hypertension prevalence in some Hispanic sub-populations is greater than in non-Hispanic whites, genetic studies on hypertension have focused primarily on samples of European descent. A recent meta-analysis of 200 000 individuals of European descent identified 29 common genetic variants that influence blood pressure, and a genetic risk score derived from the 29 variants has been proposed. We sought to evaluate the utility of this genetic risk score in Hispanics. The sample set consists of 1994 Hispanics from 2 cohorts: the Northern Manhattan Study (primarily Dominican/Puerto Rican) and the Miami Cardiovascular Registry (primarily Cuban/South American). Risk scores for systolic and diastolic blood pressure were computed as a weighted sum of the risk alleles, with the regression coefficients reported in the European meta-analysis used as weights. Association of risk score with blood pressure was tested within each cohort, adjusting for age, age2, sex and body mass index. Results were combined using an inverse-variance meta-analysis. The risk score was significantly associated with blood pressure in our combined sample (P=5.65 × 10-4 for systolic and P=1.65 × 10-3 for diastolic) but the magnitude of the effect sizes varied by degree of European, African and Native American admixture. Further studies among other Hispanic sub-populations are needed to elucidate the role of these 29 variants and identify additional genetic and environmental factors contributing to blood pressure variability in Hispanics.

GWAS analysis implicates NF-κB-mediated induction of inflammatory T cells in multiple sclerosis.

To identify genes and biologically relevant pathways associated with risk to develop multiple sclerosis (MS), the Genome-Wide Association Studies noise reduction method (GWAS-NR) was applied to MS genotyping data. Regions of association were defined based on the significance of linkage disequilibrium blocks. Candidate genes were cross-referenced based on a review of current literature, with attention to molecular function and directly interacting proteins. Supplementary annotations and pathway enrichment scores were generated using The Database for Annotation, Visualization and Integrated Discovery. The candidate set of 220 MS susceptibility genes prioritized by GWAS-NR was highly enriched with genes involved in biological pathways related to positive regulation of cell, lymphocyte and leukocyte activation (P=6.1E-15, 1.2E-14 and 5.0E-14, respectively). Novel gene candidates include key regulators of NF-κB signaling and CD4+ T helper type 1 (Th1) and T helper type 17 (Th17) lineages. A large subset of MS candidate genes prioritized by GWAS-NR were found to interact in a tractable pathway regulating the NF-κB-mediated induction and infiltration of pro-inflammatory Th1/Th17 T-cell lineages, and maintenance of immune tolerance by T-regulatory cells. This mechanism provides a biological context that potentially links clinical observations in MS to the underlying genetic landscape that may confer susceptibility.

Genetic overlap between Alzheimer's disease and Parkinson's disease at the MAPT locus.

We investigated the genetic overlap between Alzheimer's disease (AD) and Parkinson's disease (PD). Using summary statistics (P-values) from large recent genome-wide association studies (GWAS) (total n=89 904 individuals), we sought to identify single nucleotide polymorphisms (SNPs) associating with both AD and PD. We found and replicated association of both AD and PD with the A allele of rs393152 within the extended MAPT region on chromosome 17 (meta analysis P-value across five independent AD cohorts=1.65 × 10(-7)). In independent datasets, we found a dose-dependent effect of the A allele of rs393152 on intra-cerebral MAPT transcript levels and volume loss within the entorhinal cortex and hippocampus. Our findings identify the tau-associated MAPT locus as a site of genetic overlap between AD and PD, and extending prior work, we show that the MAPT region increases risk of Alzheimer's neurodegeneration.

Vascular tone pathway polymorphisms in relation to primary open-angle glaucoma.

AIMS: Vascular perfusion may be impaired in primary open-angle glaucoma (POAG); thus, we evaluated a panel of markers in vascular tone-regulating genes in relation to POAG. METHODS: We used Illumina 660W-Quad array genotype data and pooled P-values from 3108 POAG cases and 3430 controls from the combined National Eye Institute Glaucoma Human Genetics Collaboration consortium and Glaucoma Genes and Environment studies. Using information from previous literature and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, we compiled single-nucleotide polymorphisms (SNPs) in 186 vascular tone-regulating genes. We used the 'Pathway Analysis by Randomization Incorporating Structure' analysis software, which performed 1000 permutations to compare the overall pathway and selected genes with comparable randomly generated pathways and genes in their association with POAG. RESULTS: The vascular tone pathway was not associated with POAG overall or POAG subtypes, defined by the type of visual field loss (early paracentral loss (n=224 cases) or only peripheral loss (n=993 cases)) (permuted P≥0.20). In gene-based analyses, eight were associated with POAG overall at permuted P<0.001: PRKAA1, CAV1, ITPR3, EDNRB, GNB2, DNM2, HFE, and MYL9. Notably, six of these eight (the first six listed) code for factors involved in the endothelial nitric oxide synthase activity, and three of these six (CAV1, ITPR3, and EDNRB) were also associated with early paracentral loss at P<0.001, whereas none of the six genes reached P<0.001 for peripheral loss only. DISCUSSION: Although the assembled vascular tone SNP set was not associated with POAG, genes that code for local factors involved in setting vascular tone were associated with POAG.

Follow-up of loci from the International Genomics of Alzheimer's Disease Project identifies TRIP4 as a novel susceptibility gene.

To follow-up loci discovered by the International Genomics of Alzheimer's Disease Project, we attempted independent replication of 19 single nucleotide polymorphisms (SNPs) in a large Spanish sample (Fundació ACE data set; 1808 patients and 2564 controls). Our results corroborate association with four SNPs located in the genes INPP5D, MEF2C, ZCWPW1 and FERMT2, respectively. Of these, ZCWPW1 was the only SNP to withstand correction for multiple testing (P=0.000655). Furthermore, we identify TRIP4 (rs74615166) as a novel genome-wide significant locus for Alzheimer's disease risk (odds ratio=1.31; confidence interval 95% (1.19-1.44); P=9.74 × 10(-)(9)).

Genetically meaningful phenotypic subgroups in autism spectrum disorders.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder with strong evidence for genetic susceptibility. However, the effect sizes for implicated chromosomal loci are small, hard to replicate and current evidence does not explain the majority of the estimated heritability. Phenotypic heterogeneity could be one phenomenon complicating identification of genetic factors. We used data from the Autism Diagnostic Interview-Revised, Autism Diagnostic Observation Schedule, Vineland Adaptive Behavior Scales, head circumferences, and ages at exams as classifying variables to identify more clinically similar subgroups of individuals with ASD. We identified two distinct subgroups of cases within the Autism Genetic Resource Exchange dataset, primarily defined by the overall severity of evaluated traits. In addition, there was significant familial clustering within subgroups (odds ratio, OR ≈ 1.38-1.42, P < 0.00001), and genotypes were more similar within subgroups compared to the unsubgrouped dataset (Fst = 0.17 ± 0.0.0009). These results suggest that the subgroups recapitulate genetic etiology. Using the same approach in an independent dataset from the Autism Genome Project, we similarly identified two distinct subgroups of cases and confirmed this severity-based dichotomy. We also observed evidence for genetic contributions to subgroups identified in the replication dataset. Our results provide more effective methods of phenotype definition that should increase power to detect genetic factors influencing risk for ASD.

Parkinson disease loci in the mid-western Amish.

Previous evidence has shown that Parkinson disease (PD) has a heritable component, but only a small proportion of the total genetic contribution to PD has been identified. Genetic heterogeneity complicates the verification of proposed PD genes and the identification of new PD susceptibility genes. Our approach to overcome the problem of heterogeneity is to study a population isolate, the mid-western Amish communities of Indiana and Ohio. We performed genome-wide association and linkage analyses on 798 individuals (31 with PD), who are part of a 4,998 member pedigree. Through these analyses, we identified a region on chromosome 5q31.3 that shows evidence of association (p value < 1 × 10(-4)) and linkage (multipoint HLOD = 3.77). We also found further evidence of linkage on chromosomes 6 and 10 (multipoint HLOD 4.02 and 4.35 respectively). These data suggest that locus heterogeneity, even within the Amish, may be more extensive than previously appreciated.

Independent and epistatic effects of variants in VPS10-d receptors on Alzheimer disease risk and processing of the amyloid precursor protein (APP).

Genetic variants in the sortilin-related receptor (SORL1) and the sortilin-related vacuolar protein sorting 10 (VPS10) domain-containing receptor 1 (SORCS1) are associated with increased risk of Alzheimer's disease (AD), declining cognitive function and altered amyloid precursor protein (APP) processing. We explored whether other members of the (VPS10) domain-containing receptor protein family (the sortilin-related VPS10 domain-containing receptors 2 and 3 (SORCS2 and SORCS3) and sortilin (SORT1)) would have similar effects either independently or together. We conducted the analyses in a large Caucasian case control data set (n=11,840 cases, 10,931 controls) to determine the associations between single nucleotide polymorphisms (SNPs) in all the five homologous genes and AD risk. Evidence for interactions between SNPs in the five VPS10 domain receptor family genes was determined in epistatic statistical models. We also compared expression levels of SORCS2, SORCS3 and SORT1 in AD and control brains using microarray gene expression analyses and assessed the effects of these genes on γ-secretase processing of APP. Several SNPs in SORL1, SORCS1, SORCS2 and SORCS3 were associated with AD. In addition, four specific linkage disequilibrium blocks in SORCS1, SORCS2 and SORCS3 showed additive epistatic effects on the risk of AD (P≤0.0006). SORCS3, but not SORCS2 or SORT1, showed reduced expression in AD compared with control brains, but knockdown of all the three genes using short hairpin RNAs in HEK293 cells caused a significant threefold increase in APP processing (from P<0.001 to P<0.05). These findings indicate that in addition to SORL1 and SORCS1, variants in other members of the VPS10 domain receptor family (that is, SORCS1, SORCS2, SORCS3) are associated with AD risk and alter APP processing. More importantly, the results indicate that variants within these genes have epistatic effects on AD risk.

Genome-wide association study of Alzheimer's disease.

In addition to apolipoprotein E (APOE), recent large genome-wide association studies (GWASs) have identified nine other genes/loci (CR1, BIN1, CLU, PICALM, MS4A4/MS4A6E, CD2AP, CD33, EPHA1 and ABCA7) for late-onset Alzheimer's disease (LOAD). However, the genetic effect attributable to known loci is about 50%, indicating that additional risk genes for LOAD remain to be identified. In this study, we have used a new GWAS data set from the University of Pittsburgh (1291 cases and 938 controls) to examine in detail the recently implicated nine new regions with Alzheimer's disease (AD) risk, and also performed a meta-analysis utilizing the top 1% GWAS single-nucleotide polymorphisms (SNPs) with P<0.01 along with four independent data sets (2727 cases and 3336 controls) for these SNPs in an effort to identify new AD loci. The new GWAS data were generated on the Illumina Omni1-Quad chip and imputed at ~2.5 million markers. As expected, several markers in the APOE regions showed genome-wide significant associations in the Pittsburg sample. While we observed nominal significant associations (P<0.05) either within or adjacent to five genes (PICALM, BIN1, ABCA7, MS4A4/MS4A6E and EPHA1), significant signals were observed 69-180 kb outside of the remaining four genes (CD33, CLU, CD2AP and CR1). Meta-analysis on the top 1% SNPs revealed a suggestive novel association in the PPP1R3B gene (top SNP rs3848140 with P = 3.05E-07). The association of this SNP with AD risk was consistent in all five samples with a meta-analysis odds ratio of 2.43. This is a potential candidate gene for AD as this is expressed in the brain and is involved in lipid metabolism. These findings need to be confirmed in additional samples.

Mutation screening of spastin, atlastin, and REEP1 in hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) comprises a group of clinically and genetically heterogeneous diseases that affect the upper motor neurons and their axonal projections. Over 40 chromosomal loci have been identified for autosomal dominant, recessive, and X-linked HSP. Mutations in the genes atlastin, spastin and REEP1 are estimated to account for up to 50% of autosomal-dominant HSP and currently guide the molecular diagnosis of HSP. Here, we report the mutation screening results of 120 HSP patients from North America for spastin, atlastin, and REEP1, with the latter one partially reported previously. We identified mutations in 36.7% of all tested HSP patients and describe 20 novel changes in spastin and atlastin. Our results add to a growing number of HSP disease-associated variants and confirm the high prevalence of atlastin, spastin, and REEP1 mutations in the HSP patient population.

Variants in several genomic regions associated with asperger disorder.

Asperger disorder (ASP) is one of the autism spectrum disorders (ASD) and is differentiated from autism largely on the absence of clinically significant cognitive and language delays. Analysis of a homogenous subset of families with ASP may help to address the corresponding effect of genetic heterogeneity on identifying ASD genetic risk factors. To examine the hypothesis that common variation is important in ASD, we performed a genome-wide association study (GWAS) in 124 ASP families in a discovery data set and 110 ASP families in a validation data set. We prioritized the top 100 association results from both cohorts by employing a ranking strategy. Novel regions on 5q21.1 (P = 9.7 × 10(-7) ) and 15q22.1-q22.2 (P = 7.3 × 10(-6) ) were our most significant findings in the combined data set. Three chromosomal regions showing association, 3p14.2 (P = 3.6 × 10(-6) ), 3q25-26 (P = 6.0 × 10(-5) ) and 3p23 (P = 3.3 × 10(-4) ) overlapped linkage regions reported in Finnish ASP families, and eight association regions overlapped ASD linkage areas. Our findings suggest that ASP shares both ASD-related genetic risk factors, as well as has genetic risk factors unique to the ASP phenotype.

A non-synonymous SNP within membrane metalloendopeptidase-like 1 (MMEL1) is associated with multiple sclerosis.

Several single-nucleotide polymorphism (SNP) genome-wide association studies (GWASs) have been completed in multiple sclerosis (MS). Follow-up studies of the variants with the most promising rankings, especially when supplemented by informed candidate gene selection, have proven to be extremely successful. In this study we report the results of a multi-stage replication analysis of the putatively associated SNPs identified in the Wellcome Trust Case Control Consortium non-synonymous SNP (nsSNP) screen. In total, the replication sample consisted of 3444 patients and 2595 controls. A combined analysis of the nsSNP screen and replication data provides evidence implicating a novel additional locus, rs3748816 in membrane metalloendopeptidase-like 1 (MMEL1; odds ratio=1.16, P=3.54 × 10⁻6) in MS susceptibility.

Association and gene-gene interaction of SLC6A4 and ITGB3 in autism.

Autism is a heritable neurodevelopmental disorder with substantial genetic heterogeneity. Studies point to possible links between autism and two serotonin related genes: SLC6A4 and ITGB3 with a sex-specific genetic effect and interaction between the genes. Despite positive findings, inconsistent results have complicated interpretation. This study seeks to validate and clarify previous findings in an independent dataset taking into account sex, family-history (FH) and gene-gene effects. Family-based association analysis was performed within each gene. Gene-gene interactions were tested using extended multifactor dimensionality reduction (EMDR) and MDR-phenomics (MDR-P) using sex of affecteds and FH as covariates. No significant associations with individual SNPs were found in the datasets stratified by sex, but associations did emerge when we stratified by family history. While not significant in the overall dataset, nominally significant association was identified at RS2066713 (P = 0.006) within SLC6A4 in family-history negative (FH-) families, at RS2066713 (P = 0.038) in family-history positive (FH+) families but with the opposite risk allele as in the FH- families. For ITGB3, nominally significant association was identified at RS3809865 overall (P = 0.040) and within FH+ families (P = 0.031). However, none of the associations survived the multiple testing correction. MDR-P confirmed gene-gene effects using sex of affecteds (P = 0.023) and family history (P = 0.014, survived the multiple testing corrections) as covariates. Our results indicate the extensive heterogeneity within these two genes among families. The potential interaction between SLC6A4 and ITGB3 may be clarified using family history as an indicator of genetic architecture, illustrating the importance of covariates as markers of heterogeneity in genetic analyses.

Genetics and pathogenesis of multiple sclerosis.

Multiple sclerosis (MS) is an idiopathic autoimmune neurodegenerative disease. Like many common diseases, MS has a genetic component; however, as with most complex diseases, the genetic architecture may be influenced by heterogeneity, incomplete penetrance, polygenic inheritance, and environmental factors. This clinically complex disease has provided great challenges for geneticists over the years. Although the first consistent genetic association to MS (with HLA-DR*1501) was discovered more than 30 years ago, lack of consistently replicated genetic results has plagued the scientific community. New study design methods (particularly genome-wide associations studies [GWAS]) along with genome project data and larger datasets have allowed several additional MS genes to be identified and consistently replicated. Thus, after many years of frustration, the strong genetic component associated with MS is finally beginning to be characterized.

Examination of tetrahydrobiopterin pathway genes in autism.

Autism is a complex disorder with a high degree of heritability and significant phenotypic and genotypic heterogeneity. Although candidate gene studies and genome-wide screens have failed to identify major causal loci associated with autism, numerous studies have proposed association with several variations in genes in the dopaminergic and serotonergic pathways. Because tetrahydrobiopterin (BH4) is the essential cofactor in the synthesis of these two neurotransmitters, we genotyped 25 SNPs in nine genes of the BH4 pathway in a total of 403 families. Significant nominal association was detected in the gene for 6-pyruvoyl-tetrahydropterin synthase, PTS (chromosome 11), with P = 0.009; this result was not restricted to an affected male-only subset. Multilocus interaction was detected in the BH4 pathway alone, but not across the serotonin, dopamine and BH4 pathways.

Follow-up examination of linkage and association to chromosome 1q43 in multiple sclerosis.

Multiple sclerosis (MS) is a debilitating neuroimmunological and neurodegenerative disease affecting >4,00,000 individuals in the United States. Population and family-based studies have suggested that there is a strong genetic component. Numerous genomic linkage screens have identified regions of interest for MS loci. Our own second-generation genome-wide linkage study identified a handful of non-major histocompatibility complex regions with suggestive linkage. Several of these regions were further examined using single-nucleotide polymorphisms (SNPs) with average spacing between SNPs of approximately 1.0 Mb in a dataset of 173 multiplex families. The results of that study provided further evidence for the involvement of the chromosome 1q43 region. This region is of particular interest given linkage evidence in studies of other autoimmune and inflammatory diseases including rheumatoid arthritis and systemic lupus erythematosus. In this follow-up study, we saturated the region with approximately 700 SNPs (average spacing of 10 kb per SNP) in search of disease-associated variation within this region. We found preliminary evidence to suggest that common variation within the RGS7 locus may be involved in disease susceptibility.

Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal degenerative motor neuron disorder. Ten percent of cases are inherited; most involve unidentified genes. We report here 13 mutations in the fused in sarcoma/translated in liposarcoma (FUS/TLS) gene on chromosome 16 that were specific for familial ALS. The FUS/TLS protein binds to RNA, functions in diverse processes, and is normally located predominantly in the nucleus. In contrast, the mutant forms of FUS/TLS accumulated in the cytoplasm of neurons, a pathology that is similar to that of the gene TAR DNA-binding protein 43 (TDP43), whose mutations also cause ALS. Neuronal cytoplasmic protein aggregation and defective RNA metabolism thus appear to be common pathogenic mechanisms involved in ALS and possibly in other neurodegenerative disorders.

Examination of association of genes in the serotonin system to autism.

Autism is characterized as one of the pervasive developmental disorders, a spectrum of often severe behavioral and cognitive disturbances of early development. The high heritability of autism has driven multiple efforts to identify genetic variation that increases autism susceptibility. Numerous studies have suggested that variation in peripheral and central metabolism of serotonin (5-hydroxytryptamine) may play a role in the pathophysiology of autism. We screened 403 autism families for 45 single nucleotide polymorphisms in ten serotonin pathway candidate genes. Although genome-wide linkage scans in autism have provided support for linkage to various loci located within the serotonin pathway, our study does not provide strong evidence for linkage to any specific gene within the pathway. The most significant association (p = 0.0002; p = 0.02 after correcting for multiple comparisons) was found at rs1150220 (HTR3A) located on chromosome 11 ( approximately 113 Mb). To test specifically for multilocus effects, multifactor dimensionality reduction was employed, and a significant two-way interaction (p value = 0.01) was found between rs10830962, near MTNR1B (chromosome11; 92,338,075 bp), and rs1007631, near SLC7A5 (chromosome16; 86,413,596 bp). These data suggest that variation within genes on the serotonin pathway, particularly HTR3A, may have modest effects on autism risk.