Further analysis of previously implicated linkage regions for Alzheimer's disease in affected relative pairs

Background Genome-wide linkage studies for Alzheimer's disease have implicated several chromosomal regions as potential loci for susceptibility genes. Methods In the present study, we have combined a selection of affected relative pairs (ARPs) from the UK and the USA included in a previous linkage study by Myers et al. (Am J Med Genet, 2002), with ARPs from Sweden and Washington University. In this total sample collection of 397 ARPs, we have analyzed linkage to chromosomes 1, 9, 10, 12, 19 and 21, implicated in the previous scan. Results The analysis revealed that linkage to chromosome 19q13 close to the APOE locus increased considerably as compared to the earlier scan. However, linkage to chromosome 10q21, which provided the strongest linkage in the previous scan could not be detected. Conclusion The present investigation provides yet further evidence that 19q13 is the only chromosomal region consistently linked to Alzheimer's disease

Canine Hereditary Ataxia in Old English Sheepdogs and Gordon Setters Is Associated with a Defect in the Autophagy Gene Encoding RAB24

Old English Sheepdogs and Gordon Setters suffer from a juvenile onset, autosomal recessive form of canine hereditary ataxia primarily affecting the Purkinje neuron of the cerebellar cortex. The clinical and histological characteristics are analogous to hereditary ataxias in humans. Linkage and genome-wide association studies on a cohort of related Old English Sheepdogs identified a region on CFA4 strongly associated with the disease phenotype. Targeted sequence capture and next generation sequencing of the region identified an A to C single nucleotide polymorphism (SNP) located at position 113 in exon 1 of an autophagy gene, RAB24, that segregated with the phenotype. Genotyping of six additional breeds of dogs affected with hereditary ataxia identified the same polymorphism in affected Gordon Setters that segregated perfectly with phenotype. The other breeds tested did not have the polymorphism. Genome-wide SNP genotyping of Gordon Setters identified a 1.9 MB region with an identical haplotype to affected Old English Sheepdogs. Histopathology, immunohistochemistry and ultrastructural evaluation of the brains of affected dogs from both breeds identified dramatic Purkinje neuron loss with axonal spheroids, accumulation of autophagosomes, ubiquitin positive inclusions and a diffuse increase in cytoplasmic neuronal ubiquitin staining. These findings recapitulate the changes reported in mice with induced neuron-specific autophagy defects. Taken together, our results suggest that a defect in RAB24, a gene associated with autophagy, is highly associated with and may contribute to canine hereditary ataxia in Old English Sheepdogs and Gordon Setters. This finding suggests that detailed investigation of autophagy pathways should be undertaken in human hereditary ataxia.American Kennel Club Canine Health Foundation (grant CHF 0407)American Kennel Club Canine Health Foundation (grant CHF 0925)Old English Sheepdog Club of AmericaTarTan Gordon Setter ClubEuropean Science Foundation (EURYI)Canine Health Information Center (DNA Repository

A genome-wide association study identifies protein quantitative trait loci (pQTLs)

There is considerable evidence that human genetic variation influences gene expression. Genome-wide studies have revealed that mRNA levels are associated with genetic variation in or close to the gene coding for those mRNA transcripts - cis effects, and elsewhere in the genome - trans effects. The role of genetic variation in determining protein levels has not been systematically assessed. Using a genome-wide association approach we show that common genetic variation influences levels of clinically relevant proteins in human serum and plasma. We evaluated the role of 496,032 polymorphisms on levels of 42 proteins measured in 1200 fasting individuals from the population based InCHIANTI study. Proteins included insulin, several interleukins, adipokines, chemokines, and liver function markers that are implicated in many common diseases including metabolic, inflammatory, and infectious conditions. We identified eight Cis effects, including variants in or near the IL6R (p = 1.8×10 -57), CCL4L1 (p = 3.9×10-21), IL18 (p = 6.8×10-13), LPA (p = 4.4×10-10), GGT1 (p = 1.5×10-7), SHBG (p = 3.1×10-7), CRP (p = 6.4×10-6) and IL1RN (p = 7.3×10-6) genes, all associated with their respective protein products with effect sizes ranging from 0.19 to 0.69 standard deviations per allele. Mechanisms implicated include altered rates of cleavage of bound to unbound soluble receptor (IL6R), altered secretion rates of different sized proteins (LPA), variation in gene copy number (CCL4L1) and altered transcription (GGT1). We identified one novel trans effect that was an association between ABO blood group and tumour necrosis factor alpha (TNF-alpha) levels (p = 6.8×10-40), but this finding was not present when TNF-alpha was measured using a different assay , or in a second study, suggesting an assay-specific association. Our results show that protein levels share some of the features of the genetics of gene expression. These include the presence of strong genetic effects in cis locations. The identification of protein quantitative trait loci (pQTLs) may be a powerful complementary method of improving our understanding of disease pathways. © 2008 Melzer et al

Integration of GWAS SNPs and tissue specific expression profiling reveal discrete eQTLs for human traits in blood and brain

Our knowledge of the transcriptome has become much more complex since the days of the central dogma of molecular biology. We now know that splicing takes place to create potentially thousands of isoforms from a single gene, and we know that RNA does not always faithfully recapitulate DNA if RNA editing occurs. Collectively, these observations show that the transcriptome is amazingly rich with intricate regulatory mechanisms for overall gene expression, splicing, and RNA editing. Genetic variability can play a role in controlling gene expression, which can be identified by examining expression quantitative trait loci (eQTLs). eQTLs are genomic regions where genetic variants, including single nucleotide polymorphisms (SNPs) show a statistical association with expression of mRNA transcripts. In humans, many SNPs are also associated with disease, and have been identified using genome wide association studies (GWAS) but the biological effects of those SNPs are usually not known. If SNPs found in GWAS are also found in eQTLs, then one could hypothesize that expression levels may contribute to disease risk. Performing eQTL analysis with GWAS SNPs in both blood and brain, specifically the frontal cortex and the cerebellum, we found both shared and tissue unique eQTLS. The identification of tissue-unique eQTLs supports the argument that choice of tissue type is important in eQTL studies (Paper I). Aging is a complex process with the mechanisms underlying aging still being poorly defined. There is evidence that the transcriptome changes with age, and hence we used the brain dataset from our first paper as a discovery set, with an additional replication dataset, to investigate any aging-gene expression associations. We found evidence that many genes were associated with aging. We further found that there were more statically significant expression changes in the frontal cortex versus the cerebellum, indicating that brain regions may age at different rates. As the brain is a heterogeneous tissue including both neurons and non-neuronal cells, we used LCM to capture Purkinje cells as a representative neuronal type and repeated the age analysis. Looking at the discovery, replication and Purkinje cell datasets we found five genes with strong, replicated evidence of age-expression associations (Paper II). Being able to capture and quantify the depth of the transcriptome has been a lengthy process starting with methods that could only measure a single gene to genome-wide techniques such as microarray. A recently developed technology, RNA-Seq, shows promise in its ability to capture expression, splicing, and editing and with its broad dynamic range quantification is accurate and reliable. RNA-Seq is, however, data intensive and a great deal of computational expertise is required to fully utilize the strengths of this method. We aimed to create a small, well-controlled, experiment in order to test the performance of this relatively new technology in the brain. We chose embryonic versus adult cerebral cortex, as mice are genetically homogenous and there are many known differences in gene expression related to brain development that we could use as benchmarks for analysis testing. We found a large number of differences in total gene expression between embryonic and adult brain. Rigorous technical and biological validation illustrated the accuracy and dynamic range of RNA-Seq. We were also able to interrogate differences in exon usage in the same dataset. Finally we were able to identify and quantify both well-known and novel A-to-I edit sites. Overall this project helped us develop the tools needed to build usable pipelines for RNA-Seq data processing (Paper III). Our studies in the developing brain (Paper III) illustrated that RNA-Seq was a useful unbiased method for investigating RNA editing. To extend this further, we utilized a genetically modified mouse model to study the transcriptomic role of the RNA editing enzyme ADAR2. We found that ADAR2 was important for editing of the coding region of mRNA as a large proportion of RNA editing sites in coding regions had a statistically significant decrease in editing percentages in Adar2 -/-Gria2 R/R mice versus controls. However, despite indications in the literature that ADAR2 may also be involved in splicing and expression regulatory machinery we found no changes in gene expression or exon utilization in Adar2 -/-Gria2 R/R mice as compared to their littermate controls (Paper IV). In our final study, based on the methods developed in Papers III and IV, we revisited the idea of age related gene expression associations from Paper II. We used a subset of human frontal cortices for RNA sequencing. Interestingly we found more gene expression changes with aging compared to the previous data using microarrays in Paper II. When the significant gene lists were analysed for gene ontology enrichment, we found that there was a large number of downregulated genes involved in synaptic function while those that were upregulated had enrichment in immune function. This dataset illustrates that the aging brain may be predisposed to the processes found in neurodegenerative diseases (Paper V)

Abundant Quantitative Trait Loci Exist for DNA Methylation and Gene Expression in Human Brain

A fundamental challenge in the post-genome era is to understand and annotate the consequences of genetic variation, particularly within the context of human tissues. We present a set of integrated experiments that investigate the effects of common genetic variability on DNA methylation and mRNA expression in four human brain regions each from 150 individuals (600 samples total). We find an abundance of genetic cis regulation of mRNA expression and show for the first time abundant quantitative trait loci for DNA CpG methylation across the genome. We show peak enrichment for cis expression QTLs to be approximately 68,000 bp away from individual transcription start sites; however, the peak enrichment for cis CpG methylation QTLs is located much closer, only 45 bp from the CpG site in question. We observe that the largest magnitude quantitative trait loci occur across distinct brain tissues. Our analyses reveal that CpG methylation quantitative trait loci are more likely to occur for CpG sites outside of islands. Lastly, we show that while we can observe individual QTLs that appear to affect both the level of a transcript and a physically close CpG methylation site, these are quite rare. We believe these data, which we have made publicly available, will provide a critical step toward understanding the biological effects of genetic variation

A genome-wide association study of myasthenia gravis

IMPORTANCE: Myasthenia gravis is a chronic, autoimmune, neuromuscular disease characterized by fluctuating weakness of voluntary muscle groups. Although genetic factors are known to play a role in this neuroimmunological condition, the genetic etiology underlying myasthenia gravis is not well understood. OBJECTIVE: To identify genetic variants that alter susceptibility to myasthenia gravis, we performed a genome-wide association study. DESIGN, SETTING, AND PARTICIPANTS: DNA was obtained from 1032 white individuals from North America diagnosed as having acetylcholine receptor antibody–positive myasthenia gravis and 1998 race/ethnicity-matched control individuals from January 2010 to January 2011. These samples were genotyped on Illumina OmniExpress single-nucleotide polymorphism arrays. An independent cohort of 423 Italian cases and 467 Italian control individuals were used for replication. MAIN OUTCOMES AND MEASURES: We calculated P values for association between 8114394 genotyped and imputed variants across the genome and risk for developing myasthenia gravis using logistic regression modeling. A threshold P value of 5.0 × 10(−8) was set for genome-wide significance after Bonferroni correction for multiple testing. RESULTS: In the over all case-control cohort, we identified association signals at CTLA4 (rs231770; P = 3.98 × 10(−8); odds ratio, 1.37; 95% CI, 1.25–1.49), HLA-DQA1 (rs9271871; P = 1.08 × 10(−8); odds ratio, 2.31; 95% CI, 2.02 – 2.60), and TNFRSF11A (rs4263037; P = 1.60 × 10(−9); odds ratio, 1.41; 95% CI, 1.29–1.53). These findings replicated for CTLA4 and HLA-DQA1 in an independent cohort of Italian cases and control individuals. Further analysis revealed distinct, but overlapping, disease-associated loci for early- and late-onset forms of myasthenia gravis. In the late-onset cases, we identified 2 association peaks: one was located in TNFRSF11A (rs4263037; P = 1.32 × 10(−12); odds ratio, 1.56; 95% CI, 1.44–1.68) and the other was detected in the major histocompatibility complex on chromosome 6p21 (HLA-DQA1; rs9271871; P = 7.02 × 10(−18); odds ratio, 4.27; 95% CI, 3.92–4.62). Association within the major histocompatibility complex region was also observed in early-onset cases (HLA-DQA1; rs601006; P = 2.52 × 10(−11); odds ratio, 4.0; 95% CI, 3.57–4.43), although the set of single-nucleotide polymorphisms was different from that implicated among late-onset cases. CONCLUSIONS AND RELEVANCE: Our genetic data provide insights into aberrant cellular mechanisms responsible for this prototypical autoimmune disorder. They also suggest that clinical trials of immunomodulatory drugs related to CTLA4 and that are already Food and Drug Administration approved as therapies for other autoimmune diseases could be considered for patients with refractory disease

A Genome-Wide Association Study of Myasthenia Gravis

Myasthenia gravis is a chronic, autoimmune, neuromuscular disease characterized by fluctuating weakness of voluntary muscle groups. Although genetic factors are known to play a role in this neuroimmunological condition, the genetic etiology underlying myasthenia gravis is not well understood

Genome-Wide Association Study of White Blood Cell Count in 16,388 African Americans: the Continental Origins and Genetic Epidemiology Network (COGENT)

Total white blood cell (WBC) and neutrophil counts are lower among individuals of African descent due to the common African-derived “null” variant of the Duffy Antigen Receptor for Chemokines (DARC) gene. Additional common genetic polymorphisms were recently associated with total WBC and WBC sub-type levels in European and Japanese populations. No additional loci that account for WBC variability have been identified in African Americans. In order to address this, we performed a large genome-wide association study (GWAS) of total WBC and cell subtype counts in 16,388 African-American participants from 7 population-based cohorts available in the Continental Origins and Genetic Epidemiology Network. In addition to the DARC locus on chromosome 1q23, we identified two other regions (chromosomes 4q13 and 16q22) associated with WBC in African Americans (P<2.5×10−8). The lead SNP (rs9131) on chromosome 4q13 is located in the CXCL2 gene, which encodes a chemotactic cytokine for polymorphonuclear leukocytes. Independent evidence of the novel CXCL2 association with WBC was present in 3,551 Hispanic Americans, 14,767 Japanese, and 19,509 European Americans. The index SNP (rs12149261) on chromosome 16q22 associated with WBC count is located in a large inter-chromosomal segmental duplication encompassing part of the hydrocephalus inducing homolog (HYDIN) gene. We demonstrate that the chromosome 16q22 association finding is most likely due to a genotyping artifact as a consequence of sequence similarity between duplicated regions on chromosomes 16q22 and 1q21. Among the WBC loci recently identified in European or Japanese populations, replication was observed in our African-American meta-analysis for rs445 of CDK6 on chromosome 7q21 and rs4065321 of PSMD3-CSF3 region on chromosome 17q21. In summary, the CXCL2, CDK6, and PSMD3-CSF3 regions are associated with WBC count in African American and other populations. We also demonstrate that large inter-chromosomal duplications can result in false positive associations in GWAS

Genome-Wide Association Study of White Blood Cell Count in 16,388 African Americans: the Continental Origins and Genetic Epidemiology Network (COGENT)

Total white blood cell (WBC) and neutrophil counts are lower among individuals of African descent due to the common African-derived “null” variant of the Duffy Antigen Receptor for Chemokines (DARC) gene. Additional common genetic polymorphisms were recently associated with total WBC and WBC sub-type levels in European and Japanese populations. No additional loci that account for WBC variability have been identified in African Americans. In order to address this, we performed a large genome-wide association study (GWAS) of total WBC and cell subtype counts in 16,388 African-American participants from 7 population-based cohorts available in the Continental Origins and Genetic Epidemiology Network. In addition to the DARC locus on chromosome 1q23, we identified two other regions (chromosomes 4q13 and 16q22) associated with WBC in African Americans (P<2.5×10−8). The lead SNP (rs9131) on chromosome 4q13 is located in the CXCL2 gene, which encodes a chemotactic cytokine for polymorphonuclear leukocytes. Independent evidence of the novel CXCL2 association with WBC was present in 3,551 Hispanic Americans, 14,767 Japanese, and 19,509 European Americans. The index SNP (rs12149261) on chromosome 16q22 associated with WBC count is located in a large inter-chromosomal segmental duplication encompassing part of the hydrocephalus inducing homolog (HYDIN) gene. We demonstrate that the chromosome 16q22 association finding is most likely due to a genotyping artifact as a consequence of sequence similarity between duplicated regions on chromosomes 16q22 and 1q21. Among the WBC loci recently identified in European or Japanese populations, replication was observed in our African-American meta-analysis for rs445 of CDK6 on chromosome 7q21 and rs4065321 of PSMD3-CSF3 region on chromosome 17q21. In summary, the CXCL2, CDK6, and PSMD3-CSF3 regions are associated with WBC count in African American and other populations. We also demonstrate that large inter-chromosomal duplications can result in false positive associations in GWAS

Resolving the polymorphism-in-probe problem is critical for correct interpretation of expression QTL studies.

Polymorphisms in the target mRNA sequence can greatly affect the binding affinity of microarray probe sequences, leading to false-positive and false-negative expression quantitative trait locus (QTL) signals with any other polymorphisms in linkage disequilibrium. We provide the most complete solution to this problem, by using the latest genome and exome sequence reference data to identify almost all common polymorphisms (frequency >1% in Europeans) in probe sequences for two commonly used microarray panels (the gene-based Illumina Human HT12 array, which uses 50-mer probes, and exon-based Affymetrix Human Exon 1.0 ST array, which uses 25-mer probes). We demonstrate the impact of this problem using cerebellum and frontal cortex tissues from 438 neuropathologically normal individuals. We find that although only a small proportion of the probes contain polymorphisms, they account for a large proportion of apparent expression QTL signals, and therefore result in many false signals being declared as real. We find that the polymorphism-in-probe problem is insufficiently controlled by previous protocols, and illustrate this using some notable false-positive and false-negative examples in MAPT and PRICKLE1 that can be found in many eQTL databases. We recommend that both new and existing eQTL data sets should be carefully checked in order to adequately address this issue