Brit-Maren M. Schjeide

Comprehensive research synopsis and systematic meta-analyses in Parkinson's disease genetics : The PDGene database

More than 800 published genetic association studies have implicated dozens of potential risk loci in Parkinsons disease (PD). To facilitate the interpretation of these findings, we have created a dedicated online resource, PDGene, that comprehensively collects and meta-analyzes all published studies in the field. A systematic literature screen of ∼27,000 articles yielded 828 eligible articles from which relevant data were extracted. In addition, individual-level data from three publicly available genome-wide association studies (GWAS) were obtained and subjected to genotype imputation and analysis. Overall, we performed meta-analyses on more than seven million polymorphisms originating either from GWAS datasets and/or from smaller scale PD association studies. Meta-analyses on 147 SNPs were supplemented by unpublished GWAS data from up to 16,452 PD cases and 48,810 controls. Eleven loci showed genome-wide significant (P<5×10−8) association with disease risk: BST1, CCDC62/HIP1R, DGKQ/GAK, GBA, LRRK2, MAPT, MCCC1/LAMP3, PARK16, SNCA, STK39, and SYT11/RAB25. In addition, we identified novel evidence for genome-wide significant association with a polymorphism in ITGA8 (rs7077361, OR 0.88, P = 1.3×10−8). All meta-analysis results are freely available on a dedicated online database (www.pdgene.org), which is cross-linked with a customized track on the UCSC Genome Browser. Our study provides an exhaustive and up-to-date summary of the status of PD genetics research that can be readily scaled to include the results of future large-scale genetics projects, including next-generation sequencing studies.

The Role of Clusterin, Complement Receptor 1, and Phosphatidylinositol Binding Clathrin Assembly Protein in Alzheimer Disease Risk and Cerebrospinal Fluid Biomarker Levels

CONTEXT Two recent and simultaneously published genome-wide association studies independently implicated clusterin (CLU), complement receptor 1 (CR1), and phosphatidylinositol binding clathrin assembly protein (PICALM) as putative novel Alzheimer disease (AD) risk loci. Despite their strong statistical support, all 3 signals emerged from heterogeneous case-control populations and lack replication in different settings. OBJECTIVE To determine whether genetic variants in CLU, CR1, and PICALM confer risk for AD in independent data sets (n = 4254) and to test the impact of these markers on cerebrospinal fluid (CSF)-Aβ42 and total-tau protein levels (n = 425). DESIGN Genetic association study using family-based and case-control designs. SETTING Ambulatory or hospitalized care. PARTICIPANTS Family samples originate from mostly multiplex pedigrees recruited at different centers in the United States (1245 families, 2654 individuals with AD, and 1175 unaffected relatives). Unrelated case-control subjects originate from 1 clinical center in Germany (214 individuals with AD and 211 controls). All subjects were of European descent. MAIN OUTCOME MEASURES The association between 5 genetic variants in CLU, CR1, and PICALM and risk for AD, and the correlation between these 5 genetic variants and CSF-Aβ42 and tau levels. RESULTS All 3 investigated loci showed significant associations between risk for AD (1-tailed P values ranging from <.001 to .02) and consistent effect sizes and direction. For each locus, the overall evidence of association was substantially strengthened on meta-analysis of all available data (2-tailed P values ranging from 1.1 × 10(-16) to 4.1 × 10⁻⁷). Of all markers tested, only rs541458 in PICALM was shown to have an effect on CSF protein levels, suggesting that the AD risk allele is associated with decreased CSF Aβ42 levels (2-tailed P = .002). CONCLUSIONS This study provides compelling independent evidence that genetic variants in CLU, CR1, and PICALM are genetically associated with risk for AD. Furthermore, the CSF biomarker analyses provide a first insight into the potentially predominant pathogenetic mechanism(s) underlying the association between AD risk and PICALM.

Assessment of Alzheimer’s disease case–control associations using family-based methods

The genetics of Alzheimer’s disease (AD) is heterogeneous and remains only ill-defined. We have recently created a freely available and continuously updated online database (AlzGene; http://www.alzgene.org) for which we collect all published genetic association studies in AD and perform systematic meta-analyses on all polymorphisms with sufficient genotype data. In this study, we tested 27 genes (ACE, BDNF, CH25H, CHRNB2, CST3, CTSD, DAPK1, GALP, hCG2039140, IL1B, LMNA, LOC439999, LOC651924, MAPT, MTHFR, MYH13, PCK1, PGBD1, PRNP, PSEN1, SORCS1, SORL1, TF, TFAM, TNK1, GWA_14q32.13, and GWA_7p15.2), all showing significant association with AD risk in the AlzGene meta-analyses, in a large collection of family-based samples comprised of 4,180 subjects from over 1,300 pedigrees. Overall, we observe significant association with risk for AD and polymorphisms in ACE, CHRNB2, TF, and an as yet uncharacterized locus on chromosome 7p15.2 [rs1859849]. For all four loci, the association was observed with the same alleles as in the AlzGene meta-analyses. The convergence of case–control and family-based findings suggests that these loci currently represent the most promising AD gene candidates. Further fine-mapping and functional analyses are warranted to elucidate the potential biochemical mechanisms and epidemiological relevance of these genes.

No Association between CALHM1 and Alzheimer's Disease Risk

In a recent paper in Cell, Dreses-Werringloer et al. (2008) reported the identification and functional characterization of a new Alzheimer’s disease (AD) gene, CALHM1, encoding calcium homeostasis modulator 1. CALHM1 (formerly annotated as FAM26C) represents a compelling candidate gene for late-onset AD as it is located on chromosome 10q24, a consistently replicated AD linkage region (Bertram et al., 2007), is highly expressed in the hippocampus, which is severely affected by AD-related pathology, and is involved in calcium ion homeostasis, which may be disrupted in AD. In view of these converging leads, Dreses-Werringloer et al. (2008) sequenced the open reading frame of CALHM1 in a small sample of AD patients and healthy controls and identified a nonsynonymous polymorphism (Pro86Leu, rs2986017) whose minor leucine allele showed a higher frequency in the AD patients. Followup analyses in four additional independent samples of ~3400 DNAs revealed a consistent overrepresentation of the same allele in AD cases compared to controls in each dataset. From the combined analyses the authors estimated that inheritance of the leucine allele modestly, but significantly, elevated the risk for AD by ~40% (p value = 2 × 10−10). Their genetics findings were supported by data generated in a large number of functional genomics and biochemical experiments showing evidence that the risk-associated leucine allele leads to a loss of protein function, including attenuated permeability to calcium ions and reduced cytosolic calcium ion levels, which, in turn, were associated with an increase in the pathogenic peptide, amyloid-β (Aβ). Here, we present an independent assessment of the potential association between AD and the Pro-86Leu single-nucleotide polymorphism (SNP) in CALHM1 in more than 8100 subjects from several independent datasets comprised of AD families—including those in which the original chromosome 10q24 linkage signal was identified (Bertram et al., 2000)—as well as unrelated cases and controls, and we find no evidence of a genetic association in these samples. The family-based datasets (CAG, NIA, NIMH, NCRAD) tested in this project are of self-reported European (Caucasian) ancestry collected in the US for the study of genetic factors in AD (see Table S1, available online, for a summary of sample characteristics). All samples were primarily sibships and lacked parental genotypes. With the exception of the CAG sample, the majority of pedigrees analyzed here were nuclear families ascertained on the basis of multiple affected individuals. In addition to containing at least one affected relative pair, many pedigrees also had DNA available from additional affected or unaffected individuals (mostly siblings). The diagnosis of definite, probable, or possible AD was made according to NINCDS/ADRDA criteria for affected individuals in all four samples (McKhann et al., 1984). In addition to the family samples, we genotyped ~1300 unrelated AD cases and controls, which were collected at two sites in Northern Europe, Sweden, and Finland (Table S1). All subjects were Caucasian, and AD patients fulfilled NINCDS/ADRDA criteria for probable AD. The Swedish AD patients were ascertained at the Memory Disorder Unit at Uppsala University Hospital. Healthy control subjects were recruited from the same geographic region following advertisements in local newspapers and displayed no signs of dementia upon neuropsychological testing. The Finnish subjects were gathered from Eastern Finland and were examined in the Department of Neurology of Kuopio University Hospital. Control subjects had no signs of dementia following neuropsychological testing. Finally, we assessed the CALHM1 locus in two previously published high-density genome-wide association studies (GWAS) (Li et al., 2008; Reiman et al., 2007) for which genotype data are publicly available. Together, these studies investigated 2900 unrelated AD cases and controls. Given that the rs2986017 (Pro86Leu) variant was not tested directly in either of the GWAS, we tested two SNPs showing strong and significant linkage disequilibrium with the rs2986017 (Pro86Leu) variant (rs2986030 [D′ = 0.89, r2 = 0.71] and rs1555823 [D′ = 0.89, r2 = 0.64]). Note that the power to detect the effect sizes described in the original report (Dreses-Werringloer et al., 2008) was still very high (>90%) due to the strong linkage disequilibrium between these variants (Table S2). Genotyping of the rs2986017 (Pro86Leu) variant in the US family samples and the Northern European case-control datasets was based on an individually optimized single-base extension reaction detected by high-efficiency fluorescent polarization (HEFP; described in Bertram et al., 2005; protocols available on request). Overall, genotyping efficiency was 97.6%, and the average error rate was below 0.2%. Resequencing of the Pro86Leu variant in 20 individuals revealed 100% concordance with the HEFP genotype results. None of the markers deviated significantly from Hardy-Weinberg equilibrium. To test for association with AD risk in the family-based samples we used PBAT (http://www.biostat.harvard.edu/~clange/default.htm) applying an additive model. Odds ratios (ORs) for the family samples were calculated by fitting a conditional logistic regression model to each dataset, where family defines the stratum. To test for association in the case-control samples (including the markers extracted from the GWAS) we calculated allele-based study-specific crude ORs, 95% confidence intervals, and p values for each marker (Bertram et al., 2007). To combine the effect size estimates obtained in this study with those estimated in the original publication, summary ORs across all samples were calculated using the DerSimonian and Laird random effects model, in line with the analyses routinely performed for the AlzGene database (Bertram et al., 2007). Power calculations (performed in PBAT) suggested that we had sufficient (i.e., 70% or greater) power to detect the genetic effect size estimated in the original study in each of the samples, with the exception of the CAG dataset (Table S2). As shown in Figure S1 and Table S2, none of the eight samples we investigated showed evidence for significant association between the rs2986017 (Pro86Leu) variant in CALHM1 and AD risk (p values ranging from 0.15 to 0.84). Stratification by age of AD onset (using 65 years as cutoff) or APOE e4-genotype did not appreciably change these results (data not shown; stratified analyses were not possible in the GWAS samples as no onset age or APOE e4 data were supplied). Effect size estimates indicated insignificant ORs that were opposite in direction to those reported by Dreses-Werringloer et al. (2008) in six of the eight samples (see Figure S1). Accordingly, summary ORs calculated across the newly genotyped samples in our study (labeled “This study” in Figure S1) were insignificant (OR = 0.94 [95% CI: 0.83–1.07], p = 0.4) and tended toward null when combined with the published GWAS genotype data (“All follow-up”; OR = 0.99 [95% CI: 0.91–1.09], p = 0.9). Upon combining these data with the results of the original study (“All studies”), that is, generating a meta-analysis on all ~11,700 currently available subjects, the overall summary OR became insignificant as well (OR = 1.13 [95% CI: 0.99–1.27], p = 0.06). Using rs1555823 (instead of rs2986030) as proxy for the rs2986017 (Pro86Leu) variant in the GWAS samples revealed even less pronounced and less significant overall effects (data not shown). Thus, we have independently assessed the potential association between AD risk and the rs2986017 (Pro86Leu) variant in the CALHM1 gene in a large number of independent datasets, including AD families in which the original chromosome 10q24 linkage signal was identified (Bertram et al., 2000). Despite good to excellent power to detect genetic effect sizes on the order described by Dreses-Werringloer et al. (2008), no association between CALHM1 and AD was observed, either in the individual samples or in the combined analyses of more than 8100 subjects. Based on these negative data, it is doubtful that CALHM1 represents more than a minor genetic determinant of AD risk.

MANBA, CXCR5, SOX8, RPS6KB1 and ZBTB46 are genetic risk loci for multiple sclerosis

A recent genome-wide association study reported five loci for which there was strong, but sub-genome-wide significant evidence for association with multiple sclerosis risk. The aim of this study was to evaluate the role of these potential risk loci in a large and independent data set of ≈ 20,000 subjects. We tested five single nucleotide polymorphisms rs228614 (MANBA), rs630923 (CXCR5), rs2744148 (SOX8), rs180515 (RPS6KB1), and rs6062314 (ZBTB46) for association with multiple sclerosis risk in a total of 8499 cases with multiple sclerosis, 8765 unrelated control subjects and 958 trios of European descent. In addition, we assessed the overall evidence for association by combining these newly generated data with the results from the original genome-wide association study by meta-analysis. All five tested single nucleotide polymorphisms showed consistent and statistically significant evidence for association with multiple sclerosis in our validation data sets (rs228614: odds ratio = 0.91, P = 2.4 × 10(-6); rs630923: odds ratio = 0.89, P = 1.2 × 10(-4); rs2744148: odds ratio = 1.14, P = 1.8 × 10(-6); rs180515: odds ratio = 1.12, P = 5.2 × 10(-7); rs6062314: odds ratio = 0.90, P = 4.3 × 10(-3)). Combining our data with results from the previous genome-wide association study by meta-analysis, the evidence for association was strengthened further, surpassing the threshold for genome-wide significance (P < 5 × 10(-8)) in each case. Our study provides compelling evidence that these five loci are genuine multiple sclerosis susceptibility loci. These results may eventually lead to a better understanding of the underlying disease pathophysiology.

GAB2 as an Alzheimer Disease Susceptibility Gene: Follow-up of Genomewide Association Results

BACKGROUND Genomewide association (GWA) studies have recently implicated 4 novel Alzheimer disease (AD) susceptibility loci (GAB2, GOLM1, and 2 uncharacterized loci to date on chromosomes 9p and 15q). To our knowledge, these findings have not been independently replicated. OBJECTIVE To assess these GWA findings in 4 large data sets of families affected by AD. DESIGN Follow-up of genetic association findings in previous studies. SETTING Academic research. PARTICIPANTS More than 4000 DNA samples from almost 1300 families affected with AD. MAIN OUTCOME MEASURES Genetic association analysis testing of 4 GWA signals (rs7101429 [GAB2], rs7019241 [GOLM1], rs10519262 [chromosome 15q], and rs9886784 [chromosome 9p]) using family-based methods. RESULTS In the combined analyses, only rs7101429 in GAB2 yielded significant evidence of association with the same allele as in the original GWA study (P =.002). The results are in agreement with recent meta-analyses of this and other GAB2 polymorphisms suggesting approximately a 30% decrease in risk for AD among carriers of the minor alleles. None of the other 3 tested loci showed consistent evidence for association with AD across the investigated data sets. CONCLUSIONS GAB2 contains genetic variants that may lead to a modest change in the risk for AD. Despite these promising results, more data from independent samples are needed to better evaluate the potential contribution of GAB2 to AD risk in the general population.

Regulation of growth hormone expression by Delta-like protein 1 (Dlk1)

Delta-like protein 1 (Dlk1) is a transmembrane protein characterized by epidermal growth factor (EGF)-like repeats. Dlk1, which is also known as preadipocyte factor 1 (pref-1) because of its ability to inhibit preadipocyte differentiation, regulates the differentiation of several other cell types through unknown mechanisms. To elucidate Dlk1 functions, identification of Dlk1-regulated target genes is critical. The observation that Dlk1 is expressed in many endocrine tissues suggests that Dlk1 may have endocrine-related functions. Because Dlk1 is expressed in GH producing cells, we hypothesize that one function of Dlk1 is to regulate GH expression. We found that GH mRNA, protein, and secretion were significantly decreased in GH3 pituitary cell clones that stably express Dlk1. In contrast, Dlk1 expression was unable to alter prolactin expression. Co-transfection of GH3 cells with a GH promoter-regulated reporter gene showed that Dlk1 repressed GH promoter activity. Deletion and mutation analysis of the GH promoter indicated that Pit-1 binding sites in the GH promoter are required for Dlk1-mediated repression. Furthermore, Dlk1 expression represses Pit-1-mediated transcription when both proteins are co-expressed in MCF-7 cells. Deletion analysis of Dlk1 revealed that the ability of Dlk1 to regulate GH promoter activity is independent of both its EGF-like repeats and its ability to modulate MAP kinase activity. The observation that Dlk1 regulates GH expression identifies the first endocrine function of Dlk1, establishes GH as a Dlk1-regulated target gene, and provides a model system to facilitate studies of Dlk1-mediated signaling.

MicroRNA-138 is a potential regulator of memory performance in humans

Genetic factors underlie a substantial proportion of individual differences in cognitive functions in humans, including processes related to episodic and working memory. While genetic association studies have proposed several candidate “memory genes,” these currently explain only a minor fraction of the phenotypic variance. Here, we performed genome-wide screening on 13 episodic and working memory phenotypes in 1318 participants of the Berlin Aging Study II aged 60 years or older. The analyses highlight a number of novel single nucleotide polymorphisms (SNPs) associated with memory performance, including one located in a putative regulatory region of microRNA (miRNA) hsa-mir-138-5p (rs9882688, P-value = 7.8 × 10−9). Expression quantitative trait locus analyses on next-generation RNA-sequencing data revealed that rs9882688 genotypes show a significant correlation with the expression levels of this miRNA in 309 human lymphoblastoid cell lines (P-value = 5 × 10−4). In silico modeling of other top-ranking GWAS signals identified an additional memory-associated SNP in the 3′ untranslated region (3′ UTR) of DCP1B, a gene encoding a core component of the mRNA decapping complex in humans, predicted to interfere with hsa-mir-138-5p binding. This prediction was confirmed in vitro by luciferase assays showing differential binding of hsa-mir-138-5p to 3′ UTR reporter constructs in two human cell lines (HEK293: P-value = 0.0470; SH-SY5Y: P-value = 0.0866). Finally, expression profiling of hsa-mir-138-5p and DCP1B mRNA in human post-mortem brain tissue revealed that both molecules are expressed simultaneously in frontal cortex and hippocampus, suggesting that the proposed interaction between hsa-mir-138-5p and DCP1B may also take place in vivo. In summary, by combining unbiased genome-wide screening with extensive in silico modeling, in vitro functional assays, and gene expression profiling, our study identified miRNA-138 as a potential molecular regulator of human memory function.

Aging and KIBRA/WWC1 genotype affect spatial memory processes in a virtual navigation task

Spatial navigation relies on multiple mnemonic mechanisms and previous work in younger adults has described two separate types of spatial memory. One type uses directional as well as boundary‐related information for spatial memory and mainly implicates the hippocampal formation. The other type has been linked to directional and landmark‐related information and primarily involves the striatum. Using a virtual reality navigation paradigm, we studied the impacts of aging and a single nucleotide polymorphism (SNP rs17070145) of the KIBRA gene (official name: WWC1) on these memory forms. Our data showed that older adults spatial learning was preferentially related to processing of landmark information, whereas processing of boundary information played a more prominent role in younger adults. Moreover, among older adults T‐allele carriers of the examined KIBRA polymorphism showed better spatial learning compared to C homozygotes. Together these findings provide the first evidence for an effect of the KIBRA rs17070145 polymorphism on spatial memory in humans and age differences in the reliance on landmark and boundary‐related spatial information.

Closing the case of APOE in multiple sclerosis: no association with disease risk in over 29 000 subjects

Background Single nucleotide polymorphisms (SNPs) rs429358 (ε4) and rs7412 (ε2), both invoking changes in the amino-acid sequence of the apolipoprotein E (APOE) gene, have previously been tested for association with multiple sclerosis (MS) risk. However, none of these studies was sufficiently powered to detect modest effect sizes at acceptable type-I error rates. As both SNPs are only imperfectly captured on commonly used microarray genotyping platforms, their evaluation in the context of genome-wide association studies has been hindered until recently. Methods We genotyped 12 740 subjects hitherto not studied for their APOE status, imputed raw genotype data from 8739 subjects from five independent genome-wide association studies datasets using the most recent high-resolution reference panels, and extracted genotype data for 8265 subjects from previous candidate gene assessments. Results Despite sufficient power to detect associations at genome-wide significance thresholds across a range of ORs, our analyses did not support a role of rs429358 or rs7412 on MS susceptibility. This included meta-analyses of the combined data across 13 913 MS cases and 15 831 controls (OR=0.95, p=0.259, and OR 1.07, p=0.0569, for rs429358 and rs7412, respectively). Conclusion Given the large sample size of our analyses, it is unlikely that the two APOE missense SNPs studied here exert any relevant effects on MS susceptibility.