The role of genetic variations on gene expression and splicing in control human brain: dissection of the aetiology of complex neurological diseases

Over the past two decades there has been a realization of the importance of understanding the underlying molecular mechanism of complex neurological diseases. GWAS studies confirmed a significant association between SNPs and complex neurologic and psychiatric diseases such as Parkinson’s disease and Alzheimer’s disease. In this project, the impact of genetic variations on gene expression and alternative splicing in control post-mortem human brain tissues from twelve different regions were assessed. These are disease associated regions and support different functional roles. They are: frontal cortex, temporal cortex, occipital cortex, white matter, hippocampus, thalamus, hypothalamus, putamen, substantia nigra, medulla, cerebellum and spinal cord. Based on 1231 RNA human exon arrays, genotyped and imputed DNA samples from 137 control human brain, brain transcriptome profiles, gene and exon expression quantitative trait loci (QTL) were identified in multiple brain regions. Significant region-specific exon and gene expression QTLs were reported. Cerebellum and white matter show more unique expression profiles and expression QTLs in comparison with other brain regions. Furthermore, alternative splicing patterns were in a specific group of regions such as the cortical regions. In addition, two neurodegenerative disease related genes were investigated in detail, namely LRRK2 and MAPT. Significant regional differences in expression at mRNA and protein levels were shown. Moreover, exon QTLs correlated with the expression of specific exons located in functional protein domains of LRRK2. Also, an exon QTL has been found that shows a protective effect against Parkinson’s disease with an increase in the inclusion of exon 3 in grey matter for MAPT. This study has yielded novel regional specific expression QTLs and novel insights into the expression, regulation and function of specific genes in different regions of control human brain that are related to neurological diseases. This reference dataset is a valuable resource to complement other datasets for research into the complex genetics of neurological diseases

The Friedreich ataxia GAA repeat expansion mutation induces comparable epigenetic changes in human and transgenic mouse brain and heart tissues

Friedreich ataxia (FRDA) is caused by a homozygous GAA repeat expansion mutation within intron 1 of the FXN gene, leading to reduced expression of frataxin protein. Evidence suggests that the mutation may induce epigenetic changes and heterochromatin formation, thereby impeding gene transcription. In particular, studies using FRDA patient blood and lymphoblastoid cell lines have detected increased DNA methylation of specific CpG sites upstream of the GAA repeat and histone modifications in regions flanking the GAA repeat. In this report we show that such epigenetic changes are also present in FRDA patient brain, cerebellum and heart tissues, the primary affected systems of the disorder. Bisulfite sequence analysis of the FXN flanking GAA regions reveals a shift in the FRDA DNA methylation profile, with upstream CpG sites becoming consistently hypermethylated and downstream CpG sites becoming consistently hypomethylated. We also identify differential DNA methylation at three specific CpG sites within the FXN promoter and one CpG site within exon 1. Furthermore, we show by chromatin immunoprecipitation (ChIP) analysis that there is overall decreased histone H3K9 acetylation together with increased H3K9 methylation of FRDA brain tissue. Further studies of brain, cerebellum and heart tissues from our GAA repeat expansion-containing FRDA YAC transgenic mice reveal comparable epigenetic changes to those detected in FRDA patient tissue. We have thus developed a mouse model that will be a valuable resource for future therapeutic studies targeting epigenetic modifications of the FXN gene to increase frataxin expression

Increased brain expression of GPNMB is associated with genome wide significant risk for Parkinson's disease on chromosome 7p15.3

Genome wide association studies (GWAS) for Parkinson's disease (PD) have previously revealed a significant association with a locus on chromosome 7p15.3, initially designated as the glycoprotein non-metastatic melanoma protein B (GPNMB) locus. In this study, the functional consequences of this association on expression were explored in depth by integrating different expression quantitative trait locus (eQTL) datasets (Braineac, CAGEseq, GTEx, and Phenotype-Genotype Integrator (PheGenI)). Top risk SNP rs199347 eQTLs demonstrated increased expressions of GPNMB, KLHL7, and NUPL2 with the major allele (AA) in brain, with most significant eQTLs in cortical regions, followed by putamen. In addition, decreased expression of the antisense RNA KLHL7-AS1 was observed in GTEx. Furthermore, rs199347 is an eQTL with long non-coding RNA (AC005082.12) in human tissues other than brain. Interestingly, transcript-specific eQTLs in immune-related tissues (spleen and lymphoblastoid cells) for NUPL2 and KLHL7-AS1 were observed, which suggests a complex functional role of this eQTL in specific tissues, cell types at specific time points. Significantly increased expression of GPNMB linked to rs199347 was consistent across all datasets, and taken in combination with the risk SNP being located within the GPNMB gene, these results suggest that increased expression of GPNMB is the causative link explaining the association of this locus with PD. However, other transcript eQTLs and subsequent functional roles cannot be excluded. This highlights the importance of further investigations to understand the functional interactions between the coding genes, antisense, and non-coding RNA species considering the tissue and cell-type specificity to understand the underlying biological mechanisms in PD

Widespread sex differences in gene expression and splicing in the adult human brain

There is strong evidence to show that men and women differ in terms of neurodevelopment, neurochemistry and susceptibility to neurodegenerative and neuropsychiatric disease. The molecular basis of these differences remains unclear. Progress in this field has been hampered by the lack of genome-wide information on sex differences in gene expression and in particular splicing in the human brain. Here we address this issue by using post-mortem adult human brain and spinal cord samples originating from 137 neuropathologically confirmed control individuals to study whole-genome gene expression and splicing in 12 CNS regions. We show that sex differences in gene expression and splicing are widespread in adult human brain, being detectable in all major brain regions and involving 2.5% of all expressed genes. We give examples of genes where sex-biased expression is both disease-relevant and likely to have functional consequences, and provide evidence suggesting that sex biases in expression may reflect sex-biased gene regulatory structures

Insights into the Influence of Specific Splicing Events on the Structural Organization of LRRK2

Leucine-rich repeat kinase 2 (LRRK2) is a large protein of unclear function. Rare mutations in the LRRK2 gene cause familial Parkinson's disease (PD) and inflammatory bowel disease. Genome-wide association studies (GWAS) have revealed significant association of the abovementioned diseases at the LRRK2 locus. Cell and systems biology research has led to potential roles that LRRK2 may have in PD pathogenesis, especially the kinase domain (KIN). Previous human expression studies showed evidence of mRNA expression and splicing patterns that may contribute to our understanding of the function of LRRK2. In this work, we investigate and identified significant regional differences in LRRK2 expression at the mRNA level, including a number of splicing events in the Ras of complex protein (Roc) and C-terminal of Roc domain (COR) of LRRK2, in the substantia nigra (SN) and occipital cortex (OCTX). Our findings indicate that the predominant form of LRRK2 mRNA is full length, with shorter isoforms present at a lower copy number. Our molecular modelling study suggests that splicing events in the ROC/COR domains will have major consequences on the enzymatic function and dimer formation of LRRK2. The implications of these are highly relevant to the broader effort to understand the biology and physiological functions of LRRK2, and to better characterize the role(s) of LRRK2 in the underlying mechanism leading to PD

Increased brain expression of GPNMB is associated with genome wide significant risk for Parkinson's disease on chromosome 7p15.3

Genome wide association studies (GWAS) for Parkinson's disease (PD) have previously revealed a significant association with a locus on chromosome 7p15.3, initially designated as the glycoprotein non-metastatic melanoma protein B (GPNMB) locus. In this study, the functional consequences of this association on expression were explored in depth by integrating different expression quantitative trait locus (eQTL) datasets (Braineac, CAGEseq, GTEx, and Phenotype-Genotype Integrator (PheGenI)). Top risk SNP rs199347 eQTLs demonstrated increased expressions of GPNMB, KLHL7, and NUPL2 with the major allele (AA) in brain, with most significant eQTLs in cortical regions, followed by putamen. In addition, decreased expression of the antisense RNA KLHL7-AS1 was observed in GTEx. Furthermore, rs199347 is an eQTL with long non-coding RNA (AC005082.12) in human tissues other than brain. Interestingly, transcript-specific eQTLs in immune-related tissues (spleen and lymphoblastoid cells) for NUPL2 and KLHL7-AS1 were observed, which suggests a complex functional role of this eQTL in specific tissues, cell types at specific time points. Significantly increased expression of GPNMB linked to rs199347 was consistent across all datasets, and taken in combination with the risk SNP being located within the GPNMB gene, these results suggest that increased expression of GPNMB is the causative link explaining the association of this locus with PD. However, other transcript eQTLs and subsequent functional roles cannot be excluded. This highlights the importance of further investigations to understand the functional interactions between the coding genes, antisense, and non-coding RNA species considering the tissue and cell-type specificity to understand the underlying biological mechanisms in PD

Mutations in the autoregulatory domain of β-tubulin 4a cause hereditary dystonia.

Dystonia type 4 (DYT4) was first described in a large family from Heacham in Norfolk with an autosomal dominantly inherited whispering dysphonia, generalized dystonia, and a characteristic hobby horse ataxic gait. We carried out a genetic linkage analysis in the extended DYT4 family that spanned 7 generations from England and Australia, revealing a single LOD score peak of 6.33 on chromosome 19p13.12-13. Exome sequencing in 2 cousins identified a single cosegregating mutation (p.R2G) in the β-tubulin 4a (TUBB4a) gene that was absent in a large number of controls. The mutation is highly conserved in the β-tubulin autoregulatory MREI (methionine-arginine-glutamic acid-isoleucine) domain, highly expressed in the central nervous system, and extensive in vitro work has previously demonstrated that substitutions at residue 2, specifically R2G, disrupt the autoregulatory capability of the wild-type β-tubulin peptide, affirming the role of the cytoskeleton in dystonia pathogenesis

Mutations in HPCA cause autosomal-recessive primary isolated dystonia

Reports of primary isolated dystonia inherited in an autosomal-recessive (AR) manner, often lumped together as "DYT2 dystonia," have appeared in the scientific literature for several decades, but no genetic cause has been identified to date. Using a combination of homozygosity mapping and whole-exome sequencing in a consanguineous kindred affected by AR isolated dystonia, we identified homozygous mutations in HPCA, a gene encoding a neuronal calcium sensor protein found almost exclusively in the brain and at particularly high levels in the striatum, as the cause of disease in this family. Subsequently, compound-heterozygous mutations in HPCA were also identified in a second independent kindred affected by AR isolated dystonia. Functional studies suggest that hippocalcin might play a role in regulating voltage-dependent calcium channels. The identification of mutations in HPCA as a cause of AR primary isolated dystonia paves the way for further studies to assess whether "DYT2 dystonia" is a genetically homogeneous condition or not

eQTL Catalogue 2023: New datasets, X chromosome QTLs, and improved detection and visualisation of transcript-level QTLs

The eQTL Catalogue is an open database of uniformly processed human molecular quantitative trait loci (QTLs). We are continuously updating the resource to further increase its utility for interpreting genetic associations with complex traits. Over the past two years, we have increased the number of uniformly processed studies from 21 to 31 and added X chromosome QTLs for 19 compatible studies. We have also implemented Leafcutter to directly identify splice-junction usage QTLs in all RNA sequencing datasets. Finally, to improve the interpretability of transcript-level QTLs, we have developed static QTL coverage plots that visualise the association between the genotype and average RNA sequencing read coverage in the region for all 1.7 million fine mapped associations. To illustrate the utility of these updates to the eQTL Catalogue, we performed colocalisation analysis between vitamin D levels in the UK Biobank and all molecular QTLs in the eQTL Catalogue. Although most GWAS loci colocalised both with eQTLs and transcript-level QTLs, we found that visual inspection could sometimes be used to distinguish primary splicing QTLs from those that appear to be secondary consequences of large-effect gene expression QTLs. While these visually confirmed primary splicing QTLs explain just 6/53 of the colocalising signals, they are significantly less pleiotropic than eQTLs and identify a prioritised causal gene in 4/6 cases

Genome-wide analyses for personality traits identify six genomic loci and show correlations with psychiatric disorders

Personality is influenced by genetic and environmental factors1 and associated with mental health. However, the underlying genetic determinants are largely unknown. We identified six genetic loci, including five novel loci2,3, significantly associated with personality traits in a meta-analysis of genome-wide association studies (N = 123,132–260,861). Of these genomewide significant loci, extraversion was associated with variants in WSCD2 and near PCDH15, and neuroticism with variants on chromosome 8p23.1 and in L3MBTL2. We performed a principal component analysis to extract major dimensions underlying genetic variations among five personality traits and six psychiatric disorders (N = 5,422–18,759). The first genetic dimension separated personality traits and psychiatric disorders, except that neuroticism and openness to experience were clustered with the disorders. High genetic correlations were found between extraversion and attention-deficit– hyperactivity disorder (ADHD) and between openness and schizophrenia and bipolar disorder. The second genetic dimension was closely aligned with extraversion–introversion and grouped neuroticism with internalizing psychopathology (e.g., depression or anxiety)