David Saffen

Skewed allele-specific expression of the NF1 gene in normal subjects: a possible mechanism for phenotypic variability in neurofibromatosis type 1.

Neurofibromatosis type 1 is an autosomal dominant disorder characterized by neurocutaneous abnormalities, learning disabilities, and attention-deficit disorder. Neurofibromatosis type 1 symptom severity can be highly variable even within families where all affected members carry the same mutation. We hypothesized that variation in the expression of the normal NF1 allele may be a mechanism that participates in producing variable phenotypes. We performed allelic expression imbalance assays on healthy control individuals to estimate the prevalence of skewed allelic expression of the NF1 gene. Approximately 30% of individuals in our sample population showed significant skewing of allelic expression away from the expected 50:50 ratio, indicating that differential regulation of the NF1 alleles occurs in a high proportion of individuals. Differences of up to 25% in allele-specific expression of the NF1 alleles were identified. In individuals with Neurofibromatosis type 1, who carry a mutant allele (haploinsufficient), this degree of expression skewing may be sufficient to modulate the phenotype.

Common Regulatory Variants of CYFIP1 Contribute to Susceptibility for Autism Spectrum Disorder (ASD) and Classical Autism

Based on the analysis of mRNA expression and genotype data from the “Brain Cloud” database, we identified seven SNPs within or near the autism candidate gene CYFIP1 that show nominally significant correlations between genotype and CYFIP1 mRNA expression in human dorsolateral prefrontal cortex. Analysis of transmission disequilibrium test (TDT) odds ratios (ORs) for these SNPs in a large Autism Genome Project (AGP) trio‐based association study revealed the high‐expression alleles of four of these SNPs (rs8028440, rs2289823, rs7403800 and rs3751566) to be susceptibility alleles. Correlations between the regression coefficients for mRNA expression and log10‐transformed TDT ORs were statistically significant [P = 0.008 (ASD); P = 0.002 (classical autism)]. Similarly, statistically significant correlations were obtained between levels of CYFIP1 mRNA expression predicted using the regression equations obtained from multiple linear regression analysis and log10‐transformed TDT ORs for specific combinations of genotypes for both ASD (rs2289823 + rs3751566: P = 0.008) and classical autism (rs2289823 + rs3751566: P = 0.008; rs2289823 + rs3751566 + rs765763: P = 0.0006) diagnoses. Together, these results support the hypothesis that high expression of CYFIP1 mRNA increases susceptibility for both ASD and classical autism.

Functional analysis reveals that RBM10 mutations contribute to lung adenocarcinoma pathogenesis by deregulating splicing.

RBM10 is an RNA splicing regulator that is frequently mutated in lung adenocarcinoma (LUAD) and has recently been proposed to be a cancer gene. How RBM10 mutations observed in LUAD affect its normal functions, however, remains largely unknown. Here integrative analysis of RBM10 mutation and RNA expression data revealed that LUAD-associated RBM10 mutations exhibit a mutational spectrum similar to that of tumor suppressor genes. In addition, this analysis showed that RBM10 mutations identified in LUAD patients lacking canonical oncogenes are associated with significantly reduced RBM10 expression. To systematically investigate RBM10 mutations, we developed an experimental pipeline for elucidating their functional effects. Among six representative LUAD-associated RBM10 mutations, one nonsense and one frameshift mutation caused loss-of-function as expected, whereas four missense mutations differentially affected RBM10-mediated splicing. Importantly, changes in proliferation rates of LUAD-derived cells caused by these RBM10 missense mutants correlated with alterations in RNA splicing of RBM10 target genes. Together, our data implies that RBM10 mutations contribute to LUAD pathogenesis, at least in large part, by deregulating splicing. The methods described in this study should be useful for analyzing mutations in additional cancer-associated RNA splicing regulators.

Evidence for contribution of common genetic variants within chromosome 8p21.2-8p21.1 to restricted and repetitive behaviors in autism spectrum disorders

BackgroundRestricted and Repetitive Behaviors (RRB), one of the core symptom categories for Autism Spectrum Disorders (ASD), comprises heterogeneous groups of behaviors. Previous research indicates that there are two or more factors (subcategories) within the RRB domain. In an effort to identify common variants associated with RRB, we have carried out a genome-wide association study (GWAS) using the Autism Genetic Resource Exchange (AGRE) dataset (n = 1,335, all ASD probands of European ancestry) for each identified RRB subcategory, while allowing for comparisons of associated single nucleotide polymorphisms (SNPs) with associated SNPs in the same set of probands analyzed using all the RRB subcategories as phenotypes in a multivariate linear mixed model. The top ranked SNPs were then explored in an independent dataset.ResultsUsing principal component analysis of item scores obtained from Autism Diagnostic Interview-Revised (ADI-R), two distinct subcategories within Restricted and Repetitive Behaviors were identified: Repetitive Sensory Motor (RSM) and Insistence on Sameness (IS). Quantitative RSM and IS scores were subsequently used as phenotypes in a GWAS using the AGRE ASD cohort. Although no associated SNPs with genome-wide significance (P < 5.0E-08) were detected when RSM or IS were analyzed independently, three SNPs approached genome-wide significance when RSM and IS were considered together using multivariate association analysis. These included the top IS-associated SNP, rs62503729 (P-value = 6.48E-08), which is located within chromosome 8p21.2-8p21.1, a locus previously linked to schizophrenia. Notably, all of the most significantly associated SNPs are located in close proximity to STMN4 and PTK2B, genes previously shown to function in neuron development. In addition, several of the top-ranked SNPs showed correlations with STMN4 mRNA expression in adult CEU (Caucasian and European descent) human prefrontal cortex. However, the association signals within chromosome 8p21.2-8p21.1 failed to replicate in an independent sample of 2,588 ASD probands; the insufficient sample size and between-study heterogeneity are possible explanations for the non-replication.ConclusionsOur analysis indicates that RRB in ASD can be represented by two distinct subcategories: RSM and IS. Subsequent univariate and multivariate genome-wide association studies of these RRB subcategories enabled the detection of associated SNPs at 8p21.2-8p21.1. Although these results did not replicate in an independent ASD dataset, genomic features of this region and pathway analysis suggest that common variants in 8p21.2-8p21.1 may contribute to RRB, particularly IS. Together, these observations warrant future studies to elucidate the possible contributions of common variants in 8p21.2-8p21.1 to the etiology of RSM and IS in ASD.

Autoregulation of RBM10 and cross-regulation of RBM10/RBM5 via alternative splicing-coupled nonsense-mediated decay

Abstract Mutations in the spliceosomal RNA binding protein RBM10 cause TARP syndrome and are frequently observed in lung adenocarcinoma (LUAD). We have previously shown that RBM10 enhances exon skipping of its target genes, including its paralog RBM5. Here, we report that RBM10 negatively regulates its own mRNA and protein expression and that of RBM5 by promoting alternative splicing-coupled nonsense-mediated mRNA decay (AS-NMD). Through computational analysis and experimental validation, we identified RBM10-promoted skipping of exon 6 or 12 in RBM10 and exon 6 or 16 in RBM5 as the underlying AS-NMD events. Importantly, we showed that LUAD-associated mutations affecting splice sites of RBM10 exons 6 or 12 abolished exon inclusion and correlated with reduced expression of RBM10 RNA. Together, our investigations have revealed novel molecular mechanisms underlying RBM10 autoregulation and cross-regulation of RBM5, thereby providing insights concerning the functions of RBM10 under various physiological and pathological conditions. Our combined computational and experimental approach should be useful for elucidating the role of AS-NMD in auto- and cross-regulation by other splicing regulators.

The schizophrenia/bipolar disorder candidate gene GNB1L is regulated in human temporal cortex by a cis-acting element located within the 3＇-region

Abstract22q11.2 deletion syndrome (DS) is a complex developmental disorder with a high incidence of psychiatric illnesses, including schizophrenia and mood disorders. Recent studies have identified Guanine Nucleotide Binding Protein (G protein) Beta Polypeptide 1-Like (GNB1L), located within the 1.5 Mbp 22q11.2 DS critical region, as a candidate liability gene for schizophrenia and bipolar disorder. In this study, we used mRNA expression measurements in Han Chinese postmortem temporal cortex and linkage disequilibrium (LD) analysis to show that GNB1L is regulated by a cis-acting genetic variant within the 3′-region of the gene. Significantly, this variant is located within an LD block that contains all of the common SNPs previously shown to associate with schizophrenia and bipolar disorder in Han Chinese and Caucasian populations. Contrary to our expectations, re-analysis of previously published case-control study data in light of our mRNA expression results implies that the GNB1L high-expression allele is the risk allele for schizophrenia and bipolar disorder in the Han Chinese population.

Evidence for genetic regulation of mRNA expression of the dosage-sensitive gene retinoic acid induced-1 (RAI1) in human brain.

RAI1 (retinoic acid induced-1) is a dosage-sensitive gene that causes Smith-Magenis syndrome (SMS) when mutated or deleted and Potocki-Lupski Syndrome (PTLS) when duplicated, with psychiatric features commonly observed in both syndromes. How common genetic variants regulate this gene, however, is unknown. In this study, we found that RAI1 mRNA expression in Chinese prefrontal and temporal cortex correlate with genotypes of common single nucleotide polymorphisms (SNPs) located in the RAI1 5′-upstream region. Using genotype imputation, “R2-Δ2” analysis, and data from the RegulomeDB database, we identified SNPs rs4925102 and rs9907986 as possible regulatory variants, accounting for approximately 30–40% of the variance in RAI1 mRNA expression in both brain regions. Specifically, rs4925102 and rs9907986 are predicted to disrupt the binding of retinoic acid RXR-RAR receptors and the transcription factor DEAF1 (Deformed epidermal autoregulatory factor-1), respectively. Consistent with these predictions, we observed binding of RXRα and RARα to the predicted RAI1 target in chromatin immunoprecipitation assays. Retinoic acid is crucial for early development of the central neural system, and DEAF1 is associated with intellectual disability. The observation that a significant portion of RAI1 mRNA expression is genetically controlled raises the possibility that common RAI1 5′-region regulatory variants contribute more generally to psychiatric disorders.

Psychiatric genetics in China: achievements and challenges.

To coordinate research efforts in psychiatric genetics in China, a group of Chinese and foreign investigators have established an annual “Summit on Chinese Psychiatric Genetics” to present their latest research and discuss the current state and future directions of this field. To date, two Summits have been held, the first in Changsha in April, 2014, and the second in Kunming in April, 2015. The consensus of roundtable discussions held at these meetings is that psychiatric genetics in China is in need of new policies to promote collaborations aimed at creating a framework for genetic research appropriate for the Chinese population: relying solely on Caucasian population-based studies may result in missed opportunities to diagnose and treat psychiatric disorders. In addition, participants agree on the importance of promoting collaborations and data sharing in areas where China has especially strong resources, such as advanced facilities for non-human primate studies and traditional Chinese medicine: areas that may also provide overseas investigators with unique research opportunities. In this paper, we present an overview of the current state of psychiatric genetics research in China, with emphasis on genome-level studies, and describe challenges and opportunities for future advances, particularly at the dawn of “precision medicine.” Together, we call on administrative bodies, funding agencies, the research community, and the public at large for increased support for research on the genetic basis of psychiatric disorders in the Chinese population. In our opinion, increased public awareness and effective collaborative research hold the keys to the future of psychiatric genetics in China.

The genetic architecture of autism spectrum disorders (ASDs) and the potential importance of common regulatory genetic variants

Currently, there is great interest in identifying genetic variants that contribute to the risk of developing autism spectrum disorders (ASDs), due in part to recent increases in the frequency of diagnosis of these disorders worldwide. While there is nearly universal agreement that ASDs are complex diseases, with multiple genetic and environmental contributing factors, there is less agreement concerning the relative importance of common vs rare genetic variants in ASD liability. Recent observations that rare mutations and copy number variants (CNVs) are frequently associated with ASDs, combined with reduced fecundity of individuals with these disorders, has led to the hypothesis that ASDs are caused primarily by de novo or rare genetic mutations. Based on this model, large-scale whole-genome DNA sequencing has been proposed as the most appropriate method for discovering ASD liability genes. While this approach will undoubtedly identify many novel candidate genes and produce important new insights concerning the genetic causes of these disorders, a full accounting of the genetics of ASDs will be incomplete absent an understanding of the contributions of common regulatory variants, which are likely to influence ASD liability by modifying the effects of rare variants or, by assuming unfavorable combinations, directly produce these disorders. Because it is not yet possible to identify regulatory genetic variants by examination of DNA sequences alone, their identification will require experimentation. In this essay, I discuss these issues and describe the advantages of measurements of allelic expression imbalance (AEI) of mRNA expression for identifying cis-acting regulatory variants that contribute to ASDs.

Exploring the role of common regulatory variants in the etiology of late-onset Alzheimer’s disease

Background Late-onset Alzheimer’s disease (LOAD) is a complex disorder with a significant genetic component (heritability ~ 0.8). It is distressingly common: in the United States, approximately 13% of individuals over the age of 65 and 43% of individuals over the age of 85 are estimated to have AD [2011 Alzheimer’s Disease Facts and Figures, Alzheimer’s Association]. Although specific mutations have been linked to familial forms of AD, mutations in AD candidate genes among LOAD cases are rare. For this reason there is currently great interest in identifying common genetic variants that contribute to this disorder. The goal of the current study is to quantify allele-specific differences in the expression of mRNAs encoding candidate LOAD susceptibility genes in human brain and elucidate the genetic basis for this differential expression. Haplotypes or combinations of genotypes that correlate with mRNA should be useful as markers in future genetic association studies aimed at identifying genes that contribute to AD risk and protection.