Allissa Dillman

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.

Genome-wide Screen Identifies rs646776 near Sortilin as a Regulator of Progranulin Levels in Human Plasma

Recent studies suggest progranulin (GRN) is a neurotrophic factor. Loss-of-function mutations in the progranulin gene (GRN) cause frontotemporal lobar degeneration (FTLD), a progressive neurodegenerative disease affecting ∼10% of early-onset dementia patients. Using an enzyme-linked immunosorbent assay, we previously showed that GRN is detectable in human plasma and can be used to predict GRN mutation status. This study also showed a wide range in plasma GRN levels in non-GRN mutation carriers, including controls. We have now performed a genome-wide association study of 313,504 single-nucleotide polymorphisms (SNPs) in 533 control samples and identified on chromosome 1p13.3 two SNPs with genome-wide significant association with plasma GRN levels (top SNP rs646776; p = 1.7 × 10⁻³⁰). The association of rs646776 with plasma GRN levels was replicated in two independent series of 508 controls (p = 1.9 × 10⁻¹⁹) and 197 FTLD patients (p = 6.4 × 10⁻¹²). Overall, each copy of the minor C allele decreased GRN levels by ∼15%. SNP rs646776 is located near sortilin (SORT1), and the minor C allele of rs646776 was previously associated with increased SORT1 mRNA levels. Supporting these findings, overexpression of SORT1 in cultured HeLa cells dramatically reduced GRN levels in the conditioned media, whereas knockdown of SORT1 increased extracellular GRN levels. In summary, we identified significant association of a locus on chromosome 1p13.3 with plasma GRN levels through an unbiased genome-wide screening approach and implicated SORT1 as an important regulator of GRN levels. This finding opens avenues for future research into GRN biology and the pathophysiology of neurodegenerative diseases.

MAPT expression and splicing is differentially regulated by brain region: relation to genotype and implication for tauopathies

The MAPT (microtubule-associated protein tau) locus is one of the most remarkable in neurogenetics due not only to its involvement in multiple neurodegenerative disorders, including progressive supranuclear palsy, corticobasal degeneration, Parksinsons disease and possibly Alzheimers disease, but also due its genetic evolution and complex alternative splicing features which are, to some extent, linked and so all the more intriguing. Therefore, obtaining robust information regarding the expression, splicing and genetic regulation of this gene within the human brain is of immense importance. In this study, we used 2011 brain samples originating from 439 individuals to provide the most reliable and coherent information on the regional expression, splicing and regulation of MAPT available to date. We found significant regional variation in mRNA expression and splicing of MAPT within the human brain. Furthermore, at the gene level, the regional distribution of mRNA expression and total tau protein expression levels were largely in agreement, appearing to be highly correlated. Finally and most importantly, we show that while the reported H1/H2 association with gene level expression is likely to be due to a technical artefact, this polymorphism is associated with the expression of exon 3-containing isoforms in human brain. These findings would suggest that contrary to the prevailing view, genetic risk factors for neurodegenerative diseases at the MAPT locus are likely to operate by changing mRNA splicing in different brain regions, as opposed to the overall expression of the MAPT gene.

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.

mRNA expression, splicing and editing in the embryonic and adult mouse cerebral cortex

The complexity of the adult brain is a result of both developmental processes and experience-dependent circuit formation. One way to look at the differences between embryonic and adult brain is to examine gene expression. Previous studies have used microarrays to address this in a global manner. However, the transcriptome is more complex than gene expression levels alone, as alternative splicing and RNA editing generate a diverse set of mature transcripts. Here we report a high-resolution transcriptome data set of mouse cerebral cortex at embryonic and adult stages using RNA sequencing (RNA-Seq). We found many differences in gene expression, splicing and RNA editing between embryonic and adult cerebral cortex. Each data set was validated technically and biologically, and in each case we found our RNA-Seq observations to have predictive validity. We provide this data set and analysis as a resource for understanding gene expression in the embryonic and adult cerebral cortex.

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

Genome-wide association studies have nominated many genetic variants for common human traits, including diseases, but in many cases the underlying biological reason for a trait association is unknown. Subsets of genetic polymorphisms show a statistical association with transcript expression levels, and have therefore been nominated as expression quantitative trait loci (eQTL). However, many tissue and cell types have specific gene expression patterns and so it is not clear how frequently eQTLs found in one tissue type will be replicated in others. In the present study we used two appropriately powered sample series to examine the genetic control of gene expression in blood and brain. We find that while many eQTLs associated with human traits are shared between these two tissues, there are also examples where blood and brain differ, either by restricted gene expression patterns in one tissue or because of differences in how genetic variants are associated with transcript levels. These observations suggest that design of eQTL mapping experiments should consider tissue of interest for the disease or other traits studied.

FUS mutations in sporadic amyotrophic lateral sclerosis.

Mutations in the FUS gene have recently been described as a cause of familial amyotrophic lateral sclerosis (ALS), but their role in the pathogenesis of sporadic ALS is unclear. We undertook mutational screening of all coding exons of FUS in 228 sporadic ALS cases, and, as previous reports suggest that exon 15 represents a mutational hotspot, we sequenced this exon in an additional 1295 sporadic cases. Six variants in six different cases were found, indicating that FUS mutations can underlie apparently sporadic ALS, but account for less than 1% of this form of disease.

Initial Assessment of the Pathogenic Mechanisms of the Recently Identified Alzheimer Risk Loci

Recent genome wide association studies have identified CLU, CR1, ABCA7 BIN1, PICALM and MS4A6A/MS4A6E in addition to the long established APOE, as loci for Alzheimers disease. We have systematically examined each of these loci to assess whether common coding variability contributes to the risk of disease. We have also assessed the regional expression of all the genes in the brain and whether there is evidence of an eQTL explaining the risk. In agreement with other studies we find that coding variability may explain the ABCA7 association, but common coding variability does not explain any of the other loci. We were not able to show that any of the loci had eQTLs within the power of this study. Furthermore the regional expression of each of the loci did not match the pattern of brain regional distribution in Alzheimer pathology. Although these results are mainly negative, they allow us to start defining more realistic alternative approaches to determine the role of all the genetic loci involved in Alzheimers disease.

Variation in tau isoform expression in different brain regions and disease states

Progressive supranuclear palsy (PSP) is the most common atypical parkinsonian disorder. Abnormal tau inclusions, in selected regions of the brain, are a hallmark of the disease and the H1 haplotype of MAPT, the gene encoding tau, is the major risk factor in PSP. A 3-repeat and 4-repeat (4R) tau isoform ratio imbalance has been strongly implicated as a cause of disease. Thus, understanding tau isoform regional expression in disease and pathology-free states is crucial to elucidating the mechanisms involved in PSP and other tauopathies. We used a tau isoform-specific fluorescent assay to investigate relative 4R-tau expression in 6 different brain regions in PSP cases and healthy control samples. We identified a marked difference in 4R-tau relative expression, across brain regions and between MAPT haplotypes. Highest 4R-tau expression levels were identified in the globus pallidus compared with pons, cerebellum, and frontal cortex. 4R-tau expression levels were related to the MAPT H1 and H1c haplotypes. Similar regional variation was seen in PSP case and in control samples.

Age-associated changes in gene expression in human brain and isolated neurons

Previous studies have suggested that there are genes whose expression levels are associated with chronological age. However, which genes show consistent age association across studies, and which are specific to a given organism or tissue remains unresolved. Here, we reassessed this question using 2 independently ascertained series of human brain samples from 2 anatomic regions, the frontal lobe of the cerebral cortex and cerebellum. Using microarrays to estimate gene expression, we found 60 associations between expression and chronological age that were statistically significant and were replicated in both series in at least 1 tissue. There were a greater number of significant associations in the frontal cortex compared with the cerebellum. We then repeated the analysis in a subset of samples using laser capture microdissection to isolate Purkinje neurons from the cerebellum. We were able to replicate 5 gene associations from either frontal cortex or cerebellum in the Purkinje cell dataset, suggesting that there is a subset of genes which have robust changes with aging. Of these, the most consistent and strongest association was with expression of RHBDL3, a rhomboid protease family member. We confirmed several hits using an independent technique (quantitative reverse transcriptase polymerase chain reaction) and in an independent published sample series that used a different array platform. We also interrogated larger patterns of age-related gene expression using weighted gene correlation network analysis. We found several modules that showed significant associations with chronological age and, of these, several that showed negative associations were enriched for genes encoding components of mitochondria. Overall, our results show that there is a distinct and reproducible gene signature for aging in the human brain.