Functional screening of Alzheimer pathology genome-wide association signals in Drosophila.

We have leveraged a Drosophila model relevant to Alzheimer disease (AD) for functional screening of findings from a genome-wide scan for loci associated with a quantitative measure of AD pathology in humans. In six of the 15 genomic regions evaluated, we successfully identified a causal gene for the association, on the basis of in vivo interactions with the neurotoxicity of Tau, which forms neurofibrillary tangles in AD. Among the top results, rs10845990 within SLC2A14, encoding a glucose transporter, showed evidence of replication for association with AD pathology, and gain and loss of function in glut1, the Drosophila ortholog, was associated with suppression and enhancement of Tau toxicity, respectively. Our strategy of coupling genome-wide association in humans with functional screening in a model organism is likely to be a powerful approach for gene discovery in AD and other complex genetic disorders.

Infection-triggered familial or recurrent cases of acute necrotizing encephalopathy caused by mutations in a component of the nuclear pore, RANBP2.

Acute necrotizing encephalopathy (ANE) is a rapidly progressive encephalopathy that can occur in otherwise healthy children after common viral infections such as influenza and parainfluenza. Most ANE is sporadic and nonrecurrent (isolated ANE). However, we identified a 7 Mb interval containing a susceptibility locus (ANE1) in a family segregating recurrent ANE as an incompletely penetrant, autosomal-dominant trait. We now report that all affected individuals and obligate carriers in this family are heterozygous for a missense mutation (c.1880C-->T, p.Thr585Met) in the gene encoding the nuclear pore protein Ran Binding Protein 2 (RANBP2). To determine whether this mutation is the susceptibility allele, we screened controls and other patients with ANE who are unrelated to the index family. Patients from 9 of 15 additional kindreds with familial or recurrent ANE had the identical mutation. It arose de novo in two families and independently in several other families. Two other patients with familial ANE had different RANBP2 missense mutations that altered conserved residues. None of the three RANBP2 missense mutations were found in 19 patients with isolated ANE or in unaffected controls. We conclude that missense mutations in RANBP2 are susceptibility alleles for familial and recurrent cases of ANE.

CR1 is associated with amyloid plaque burden and age-related cognitive decline.

OBJECTIVE: Recently, genome-wide association studies have identified 3 new susceptibility loci for Alzheimer's disease (AD), CLU, CR1, and PICALM. We leveraged available neuropsychological and autopsy data from 2 cohort studies to investigate whether these loci are associated with cognitive decline and AD neuropathology. METHODS: The Religious Orders Study (ROS) and Rush Memory and Aging Project (MAP) are longitudinal studies that enroll nondemented subjects and include annual clinical evaluations and brain donation at death. We evaluated CR1 (rs6656401), CLU (rs11136000), and PICALM (rs7110631) in 1,666 subjects. We evaluated associations between genotypes and rate of change in cognitive function as well as AD-related pathology. Lastly, we used pathway analysis to determine whether relationships between single nucleotide polymorphisms and cognitive decline are mediated through AD pathology. RESULTS: Among our study cohort, the mean years of follow-up were 7.8 for ROS and 4.3 for MAP. Only the CR1 locus was associated with both global cognitive decline (p = 0.011) and global AD pathology (p = 0.025). More specifically, the locus affects the deposition of neuritic amyloid plaque (p = 0.009). In a mediation analysis, controlling for amyloid pathology strongly attenuated the effect of the CR1 locus on cognitive decline. INTERPRETATION: We found that common variation at the CR1 locus has a broad impact on cognition and that this effect is largely mediated by an individual's amyloid plaque burden. We therefore highlight 1 functional consequence of the CR1 susceptibility allele and generalize the role of this locus to cognitive aging in the general population.

IL2RA genetic heterogeneity in multiple sclerosis and type 1 diabetes susceptibility and soluble interleukin-2 receptor production.

Multiple sclerosis (MS) and type 1 diabetes (T1D) are organ-specific autoimmune disorders with significant heritability, part of which is conferred by shared alleles. For decades, the Human Leukocyte Antigen (HLA) complex was the only known susceptibility locus for both T1D and MS, but loci outside the HLA complex harboring risk alleles have been discovered and fully replicated. A genome-wide association scan for MS risk genes and candidate gene association studies have previously described the IL2RA gene region as a shared autoimmune locus. In order to investigate whether autoimmunity risk at IL2RA was due to distinct or shared alleles, we performed a genetic association study of three IL2RA variants in a DNA collection of up to 9,407 healthy controls, 2,420 MS, and 6,425 T1D subjects as well as 1,303 MS parent/child trios. Here, we report "allelic heterogeneity" at the IL2RA region between MS and T1D. We observe an allele associated with susceptibility to one disease and risk to the other, an allele that confers susceptibility to both diseases, and an allele that may only confer susceptibility to T1D. In addition, we tested the levels of soluble interleukin-2 receptor (sIL-2RA) in the serum from up to 69 healthy control subjects, 285 MS, and 1,317 T1D subjects. We demonstrate that multiple variants independently correlate with sIL-2RA levels.

The role of the CD58 locus in multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system associated with demyelination and axonal loss. A whole genome association scan suggested that allelic variants in the CD58 gene region, encoding the costimulatory molecule LFA-3, are associated with risk of developing MS. We now report additional genetic evidence, as well as resequencing and fine mapping of the CD58 locus in patients with MS and control subjects. These efforts identify a CD58 variant that provides further evidence of association with MS (P = 1.1 x 10(-6), OR 0.82) and the single protective effect within the CD58 locus is captured by the rs2300747(G) allele. This protective rs2300747(G) allele is associated with a dose-dependent increase in CD58 mRNA expression in lymphoblastic cell lines (P = 1.1 x 10(-10)) and in peripheral blood mononuclear cells from MS subjects (P = 0.0037). This protective effect of enhanced CD58 expression on circulating mononuclear cells in patients with MS is supported by finding that CD58 mRNA expression is higher in MS subjects during clinical remission. Functional investigations suggest a potential mechanism whereby increases in CD58 expression, mediated by the protective allele, up-regulate the expression of transcription factor FoxP3 through engagement of the CD58 receptor, CD2, leading to the enhanced function of CD4(+)CD25(high) regulatory T cells that are defective in subjects with MS.

Genetic variation in the IL7RA/IL7 pathway increases multiple sclerosis susceptibility.

Multiple sclerosis (MS) is characterized as an autoimmune demyelinating disease. Numerous family studies have confirmed a strong genetic component underlying its etiology. After several decades of frustrating research, the advent and application of affordable genotyping of dense SNP maps in large data sets has ushered in a new era in which rapid progress is being made in our understanding of the genetics underlying many complex traits. For MS, one of the first discoveries to emerge in this new era was the association with rs6897932[T244I] in the interleukin-7 receptor alpha chain (IL7RA) gene (Gregory et al. in Nat Genet 39(9):1083-1091, 2007; International Multiple Sclerosis Genetics Consortium in N Engl J Med 357(9):851-862, 2007; Lundmark in Nat Genet 39(9):1108-1113, 2007), a discovery that was accompanied by functional data that suggest this variant is likely to be causative rather than a surrogate proxy (Gregory et al. in Nat Genet 39(9):1083-1091, 2007). We hypothesized that variations in other genes functionally related to IL7RA might also influence MS. We investigated this hypothesis by examining genes in the extended biological pathway related to IL7RA to identify novel associations. We identified 73 genes with putative functional relationships to IL7RA and subsequently genotyped 7,865 SNPs in and around these genes using an Illumina Infinium BeadChip assay. Using 2,961 case-control data sets, two of the gene regions examined, IL7 and SOCS1, had significantly associated single-nucleotide polymorphisms (SNPs) that further replicated in an independent case-control data set (4,831 samples) with joint p values as high as 8.29 x 10(-6) and 3.48 x 10(-7), respectively, exceeding the threshold for experiment-wise significance. Our results also implicate two additional novel gene regions that are likely to be associated with MS: PRKCE with p values reaching 3.47 x 10(-4), and BCL2 with p values reaching 4.32 x 10(-4). The TYK2 gene, which also emerged in our analysis, has recently been associated with MS (Ban et al. 2009). These results help to further delineate the genetic architecture of MS and validate our pathway approach as an effective method to identify novel associations in a complex disease.

Fine-mapping the genetic basis of CRP regulation in African Americans: a Bayesian approach.

Basal levels of C-reactive protein (CRP) have been associated with disease, particularly future cardiovascular events. Twin studies estimate 50% CRP heritability, so the identification of genetic variants influencing CRP expression is important. Existing studies in populations of European ancestry have identified numerous cis-acting variants but leave significant ambiguity over the identity of the key functional polymorphisms. We addressed this issue by typing a dense map of CRP single-nucleotide polymorphisms (SNPs), and quantifying serum CRP in 594 unrelated African Americans. We used Bayesian model choice analysis to select the combination of SNPs best explaining basal CRP and found strong support for triallelic rs3091244 alone, with the T allele acting in an additive manner (Bayes factor > 100 vs. null model), with additional support for a model incorporating both rs3091244 and rs12728740. Admixture analysis suggested SNP rs12728740 segregated with haplotypes predicted to be of recent European origin. Using a cladistic approach we confirmed the importance of rs3091244(T) by demonstrating a significant partition of haplotype effect based on the rs3091244(C/T) mutation (F = 8.91, P = 0.006). We argue that weaker linkage disequilibrium across the African American CRP locus compared with Europeans has allowed us to establish an unambiguous functional role for rs3091244(T), while also recognising the potential for additional functional mutations present in the European genome.

Reproductive period and epigenetic modifications of the oxidative phosphorylation pathway in the human prefrontal cortex.

PURPOSE: Human females have a unique duration of post-reproductive longevity, during which sex-specific mechanisms ma influence later-life mechanisms of neuronal resilience and vulnerability. The maintenance of energy metabolism, through the oxidative phosphorylation (OXPHOS) apparatus, is essential for brain health. Given the known association between reproductive period (years from menarche to menopause) and cognitive aging, we examined the hypothesis that cumulative estrogen exposure across the lifetime may be associated with differential methylation of genes in the OXPHOS pathway. METHODS: Using DNA methylation patterns in the post-mortem dorsolateral prefrontal cortex (DLPFC) of 426 women prospectively followed until death in the Religious Orders Study and Rush Memory and Aging Project, we examined the relationship between reproductive period (subtracting age at menarche from age at menopause) and DNA methylation of a published set of autosomal OXPHOS genes previously implicated in stroke susceptibility. We then performed an unsupervised analysis of methylation levels across the Hallmark pathways from the Molecular Signatures Database. RESULTS: We observed a strong association between reproductive period and DNA methylation status across OXPHOS CpGs. We replicated this association between reproductive period and DNA methylation in a much larger set of OXPHOS genes in our unsupervised analysis. Here, reproductive period also showed associations with methylation in genes related to E2F, MYC and MTORC1 signaling, fatty acid metabolism and DNA repair. CONCLUSION: This study provides evidence from both a supervised and unsupervised analyses, that lifetime cumulative endogenous steroid exposures may play a role in maintenance of post-menopausal cellular balance, including in brain tissue.

Epigenomics of Alzheimer's disease.

Alzheimer's disease (AD) is a large and growing public health problem. It is characterized by the accumulation of amyloid ß peptides and abnormally phosphorylated tau proteins that are associated with cognitive decline and dementia. Much has been learned about the genomics of AD from linkage analyses and, more recently, genome-wide association studies. Several but not all aspects of the genomic landscape are involved in amyloid ß metabolism. The moderate concordance of disease among twins suggests other factors, potentially epigenomic factors, are related to AD. We are at the earliest stages of examining the relation of the epigenome to the clinical and pathologic phenotypes that characterize AD. Our literature review suggests that there is some evidence of age-related changes in human brain methylation. Unfortunately, studies of AD have been relatively small with limited coverage of methylation sites and microRNA, let alone other epigenomic marks. We are in the midst of 2 large studies of human brains including coverage of more than 420,000 autosomal cytosine-guanine dinucleotides with the Illumina Infinium HumanMethylation450 BeadArray, and histone acetylation with chromatin immunoprecipitation sequencing. We present descriptive data to help inform other researchers what to expect from these approaches to better design and power their studies. We then discuss future directions to inform on the epigenomic architecture of AD.

A genome-wide scan for common variants affecting the rate of age-related cognitive decline.

Age-related cognitive decline is likely promoted by accumulated brain injury due to chronic conditions of aging, including neurodegenerative and vascular disease. Because common neuronal mechanisms may mediate the adaptation to diverse cerebral insults, we hypothesized that susceptibility for age-related cognitive decline may be due in part to a shared genetic network. We have therefore performed a genome-wide association study using a quantitative measure of global cognitive decline slope, based on repeated measures of 17 cognitive tests in 749 subjects from the Religious Orders Study. Top results were evaluated in 3 independent replication cohorts, consisting of 2279 additional subjects with repeated cognitive testing. As expected, we find that the Alzheimer's disease (AD) susceptibility locus, APOE, is strongly associated with rate of cognitive decline (P(DISC) = 5.6 × 10(-9); P(JOINT)= 3.7 × 10(-27)). We additionally discover a variant, rs10808746, which shows consistent effects in the replication cohorts and modestly improved evidence of association in the joint analysis (P(DISC) = 6.7 × 10(-5); P(REP) = 9.4 × 10(-3); P(JOINT) = 2.3 × 10(-5)). This variant influences the expression of 2 adjacent genes, PDE7A and MTFR1, which are potential regulators of inflammation and oxidative injury, respectively. Using aggregate measures of genetic risk, we find that known susceptibility loci for cardiovascular disease, type 2 diabetes, and inflammatory diseases are not significantly associated with cognitive decline in our cohort. Our results suggest that intermediate phenotypes, when coupled with larger sample sizes, may be a useful tool to dissect susceptibility loci for age-related cognitive decline and uncover shared molecular pathways with a role in neuronal injury.

Intermediate phenotypes identify divergent pathways to Alzheimer's disease.

BACKGROUND: Recent genetic studies have identified a growing number of loci with suggestive evidence of association with susceptibility to Alzheimer's disease (AD). However, little is known of the role of these candidate genes in influencing intermediate phenotypes associated with a diagnosis of AD, including cognitive decline or AD neuropathologic burden. METHODS/PRINCIPAL FINDINGS: Thirty-two single nucleotide polymorphisms (SNPs) previously implicated in AD susceptibility were genotyped in 414 subjects with both annual clinical evaluation and completed brain autopsies from the Religious Orders Study and the Rush Memory and Aging Project. Regression analyses evaluated the relation of SNP genotypes to continuous measures of AD neuropathology and cognitive function proximate to death. A SNP in the zinc finger protein 224 gene (ZNF224, rs3746319) was associated with both global AD neuropathology (p = 0.009) and global cognition (p = 0.002); whereas, a SNP at the phosphoenolpyruvate carboxykinase locus (PCK1, rs8192708) was selectively associated with global cognition (p = 3.57 x 10(-4)). The association of ZNF224 with cognitive impairment was mediated by neurofibrillary tangles, whereas PCK1 largely influenced cognition independent of AD pathology, as well as Lewy bodies and infarcts. CONCLUSIONS/SIGNIFICANCE: The findings support the association of several loci with AD, and suggest how intermediate phenotypes can enhance analysis of susceptibility loci in this complex genetic disorder.

Integration of genetic risk factors into a clinical algorithm for multiple sclerosis susceptibility: a weighted genetic risk score.

BACKGROUND: Prediction of susceptibility to multiple sclerosis (MS) might have important clinical applications, either as part of a diagnostic algorithm or as a means to identify high-risk individuals for prospective studies. We investigated the usefulness of an aggregate measure of risk of MS that is based on genetic susceptibility loci. We also assessed the added effect of environmental risk factors that are associated with susceptibility for MS. METHODS: We created a weighted genetic risk score (wGRS) that includes 16 MS susceptibility loci. We tested our model with data from 2215 individuals with MS and 2189 controls (derivation samples), a validation set of 1340 individuals with MS and 1109 controls taken from several MS therapeutic trials (TT cohort), and a second validation set of 143 individuals with MS and 281 controls from the US Nurses' Health Studies I and II (NHS/NHS II), for whom we also have data on smoking and immune response to Epstein-Barr virus (EBV). FINDINGS: Individuals with a wGRS that was more than 1.25 SD from the mean had a significantly higher odds of MS in all datasets. In the derivation sample, the mean (SD) wGRS was 3.5 (0.7) for individuals with MS and 3.0 (0.6) for controls (p<0.0001); in the TT validation sample, the mean wGRS was 3.4 (0.7) for individuals with MS versus 3.1 (0.7) for controls (p<0.0001); and in the NHS/NHS II dataset, the mean wGRS was 3.4 (0.8) for individuals with MS versus 3.0 (0.7) for controls (p<0.0001). In the derivation cohort, the area under the receiver operating characteristic curve (C statistic; a measure of the ability of a model to discriminate between individuals with MS and controls) for the genetic-only model was 0.70 and for the genetics plus sex model was 0.74 (p<0.0001). In the TT and NHS cohorts, the C statistics for the genetic-only model were both 0.64; adding sex to the TT model increased the C statistic to 0.72 (p<0.0001), whereas adding smoking and immune response to EBV to the NHS model increased the C statistic to 0.68 (p=0.02). However, the wGRS does not seem to be correlated with the conversion of clinically isolated syndrome to MS. INTERPRETATION: The inclusion of 16 susceptibility alleles into a wGRS can modestly predict MS risk, shows consistent discriminatory ability in independent samples, and is enhanced by the inclusion of non-genetic risk factors into the algorithm. Future iterations of the wGRS might therefore make a contribution to algorithms that can predict a diagnosis of MS in a clinical or research setting.

Meta-analysis of genome scans and replication identify CD6, IRF8 and TNFRSF1A as new multiple sclerosis susceptibility loci.

We report the results of a meta-analysis of genome-wide association scans for multiple sclerosis (MS) susceptibility that includes 2,624 subjects with MS and 7,220 control subjects. Replication in an independent set of 2,215 subjects with MS and 2,116 control subjects validates new MS susceptibility loci at TNFRSF1A (combined P = 1.59 x 10(-11)), IRF8 (P = 3.73 x 10(-9)) and CD6 (P = 3.79 x 10(-9)). TNFRSF1A harbors two independent susceptibility alleles: rs1800693 is a common variant with modest effect (odds ratio = 1.2), whereas rs4149584 is a nonsynonymous coding polymorphism of low frequency but with stronger effect (allele frequency = 0.02; odds ratio = 1.6). We also report that the susceptibility allele near IRF8, which encodes a transcription factor known to function in type I interferon signaling, is associated with higher mRNA expression of interferon-response pathway genes in subjects with MS.

Long-term effects of dietary glycemic index on adiposity, energy metabolism, and physical activity in mice.

A high-glycemic index (GI) diet has been shown to increase adiposity in rodents; however, the long-term metabolic effects of a low- and high-GI diet have not been examined. In this study, a total of 48 male 129SvPas mice were fed diets high in either rapidly absorbed carbohydrate (RAC; high GI) or slowly absorbed carbohydrate (SAC; low GI) for up to 40 wk. Diets were controlled for macronutrient and micronutrient content, differing only in starch type. Body composition and insulin sensitivity were measured longitudinally by DEXA scan and oral glucose tolerance test, respectively. Food intake, respiratory quotient, physical activity, and energy expenditure were assessed using metabolic cages. Despite having similar mean body weights, mice fed the RAC diet had 40% greater body fat by the end of the study and a mean 2.2-fold greater insulin resistance compared with mice fed the SAC diet. Respiratory quotient was higher in the RAC group, indicating comparatively less fat oxidation. Although no differences in energy expenditure were observed throughout the study, total physical activity was 45% higher for the SAC-fed mice after 38 wk of feeding. We conclude that, in this animal model, 1) the effect of GI on body composition is mediated by changes in substrate oxidation, not energy intake; 2) a high-GI diet causes insulin resistance; and 3) dietary composition can affect physical activity level.