Characterization of Expression Quantitative Trait Loci in Pedigrees from Colombia and Costa Rica Ascertained for Bipolar Disorder

Variants regulating gene expression (expression quantitative trait loci, eQTL) are at a high frequency among SNPs associated with complex traits. Genome-wide characterization of gene expression is an important tool in genetic mapping studies of complex disorders, including many psychiatric disorders. Further, implicating eQTL to specific tissue types is key to understanding functional variation in disease development. Our group, in collaboration with Chiara Sabatti (Statistics, Stanford) and Nelson B. Freimer (David Geffen School of Medicine, UCLA), developed a novel approach for analyzing eQTL and applied the method to a dataset from a bipolar disorder study.

Current approaches to implicating eQTL specific to tissues lack sufficient power in large-scale studies of human brain related traits, such as bipolar disorder. Together with the University of California San Francisco, Universidad de Costa Rica, Universidad de Antioquia, Medellín, Colombia, and Tel Aviv University, our group adopted a novel approach to assess the heritability and genetic regulation of gene expression related to bipolar disorder in populations from Costa Rica and Colombia.

This project examines 786 genotyped subjects originally recruited in a study of bipolar disorder, all related within 26 extended families. While the subjects in this study were originally recruited as part of an investigation for severe bipolar disorder (BP1), we found no relationship between the observed gene expression data and BP1. Instead, we use this unique Latin American population to explore the architecture of genetic regulation. Specifically, we estimate heritability, evaluate the relative importance of local vs. distal genomic variation, identify variants with regulatory effects, and analyze the role of multiple associated SNPs in the same region.

Our group adopted a novel hierarchical testing procedure that leads to the analysis of eQTL data in a stage-wise manner with increasing levels of detail. This design allows us to compare estimates of the heritability of gene expression obtained using both traditional and genotype-based methods. First, we apply a multiscale testing strategy to identify SNPs that have regulatory effects (eSNPs) on BP1. Second, we investigate which specific probes are influenced by these eSNPs. This hierarchical testing procedure effectively controls error rates and leverages the heterogeneity across genetic variants to preserve computational power.

We use this approach to measure gene expression in lymphoblastoid cell lines (LCLs) in subjects from extended families, segregating for BP1. Our results suggest that variation in expression values is heritable and that, at least in samples including related individuals, relying on theoretical kinship coefficients or on realized genotype correlation for estimation of heritability leads to similar results.

Expression heritability and proportion of genetic variance due to local effects. For more information, see our paper. For more information, see our paper.

Variance decomposition approaches suggest that on average 30% of the genetic variance is due to local regulation. In the majority of probes under local regulation in our sample, more than one typed SNP is required to account for expression variation. This finding can be interpreted as the result of heterogeneity, but also could reflect un-typed causal variants that are tracked by more than one typed SNP.

The knowledge we acquired by studying the genetic regulatory network within these pedigrees, instead, can be used to inform our mapping studies: eSNPs might receive a higher prior probability of association, or be assigned a larger portion of the allowed global error rate when using a weighted approach to testing. We will report elsewhere on the results of these investigations.

For more information, see our paper, which is available for download through PLoS Genetics: http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1006046.

The full citation to our paper is: 

Peterson, C.B., Jasinska, A.J., Gao, F., Zelaya, I., Teshiba, T.M., Bearden, C.E., Cantor, R.M., Reus, V.I., Macaya, G., López-Jaramillo, C. and Bogomolov, M., 2016. Characterization of Expression Quantitative Trait Loci in Pedigrees from Colombia and Costa Rica Ascertained for Bipolar Disorder. PLoS Genet, 12(5), p.e1006046.

 

Simultaneous modeling of disease status and clinical phenotypes to increase power in GWAS

Michael Bilow and Eleazar Eskin, together with Fernando Crespo, Zhicheng Pan, and Susana Eyheramendy, recently released a novel method for accurate joint modeling of clinical phenotype and disease status. This approach incorporates a clinical phenotype into case/control studies under the assumption that the genetic variant can affect both.

Genetic case-control association studies have found thousands of associations between genetic variants and disease. Most studies collect data from individuals with and without disease, and they often search for variants with different frequencies between the groups. Jointly modelling clinical phenotype and disease status is a promising way to increase power to detect true associations between genetics and disease. In particular, this method increases potential for discovering genetic variants that are associated with both a clinical phenotype and a disease.

However, standard multivariate techniques fail to effectively solve this problem because their case-control status is discrete and not continuous. Standard approaches to estimate model parameters are biased due to the ascertainment in case/control studies. We present a novel method that resolves both of these issues for simultaneous association testing of genetic variants that have both case status and a clinical covariate.

In our paper, we show the utility of our method using data from the North Finland Birth Cohort (NFBC) dataset. NFBC enrolled almost everyone born in 1966 in Finland’s two most northern provinces. The NFBC dataset consists of 10 phenotypes and genotypes at 331,476 genetic variants measured in 5,327 individuals. We focus our study on the LDL cholesterol and triglyceride levels phenotypes.

Our evaluation strategy analyzes a subset of the NFBC data and compares what we discover here to what was discovered in the full NFBC dataset—which we treat as the gold standard. We compare the performance of our novel approach to three other methods: (1) the single univariate test applied to the disease status, (2) the multivariate approach applied to the disease status and the clinical phenotype modeled as a multivariate normal distribution, and (3) the liability threshold model treating the clinical phenotype as a covariate.

Using the univariate approach, the p-values are much weaker in comparison to those observed in the full NFBC dataset. Running the multivariate approaches, incorporating the triglyceride levels phenotypes, increased power (i.e., more significant p-values than SNPs).

Our method has the highest power in all scenarios. The advantage of our method is greater when there are substantial amounts of selection bias compared to lower amounts of selection bias. Our method is even more powerful when the correlation between the clinical covariate and the disease liability is lower, because we explicitly estimate the underlying liability using all of the data.

For more information, see our paper in Genetics: http://www.genetics.org/content/early/2017/01/27/genetics.116.198473

The software implementing the methods described in this paper was developed by Fernando Crespo and is available at: http://genetics.cs.ucla.edu/multipheno/ and
https://github.com/facrespo/BivariateProbitContinueEM

An illustration of the distribution of liability in a case-control study under selection bias. For more information, read our paper.

The full citation to our paper is:
Bilow, M., Crespo, F., Pan, Z., Eskin, E. and Eyheramendy, S., 2017. Simultaneous Modeling of Disease Status and Clinical Phenotypes to Increase Power in GWAS. Genetics, pp.genetics-116.

 

Profiling adaptive immune repertoires across multiple human tissues by RNA Sequencing

In a project led by Serghei Mangul, members of our lab recently developed and tested a novel computational method that uses regular RNA-Seq data to rapidly and accurately profile the human immune system. Mangul and his collaborators, including UCLA graduate student Harry (Taegyun) Yang and 2016 B. I. G. Summer undergraduate participants Jeremy Rotman, Benjamin Statz, and Will Van Der Wey, recently published their results in a paper on bioRxiv.

Discoveries in human immunology and advancements in development of treatments for many common human diseases depend on detailed reconstructions of the adaptive immune repertoire. The “adaptive” immune repertoire recognizes pathogens and toxins that the “innate” defense system misses. Assay-based genetic studies provide a detailed view of these adaptive systems by profiling the genetic expression and repertoires of B and T cell receptors. Assay-based approaches have accurately characterized the immune repertoire of peripheral blood.

However, these methods are expensive and smaller in scale when compared to standard RNA sequencing (RNA-seq). Characterizing the immunological repertoires of other tissues, including barrier tissues like skin and mucosae, requires large-scale study. RNA-Seq can capture the entire cellular population of a sample, including B and T cell and their receptors.

ImReP is the first method to efficiently extract B and T cell receptor derived reads from RNA-Seq data, accurately assemble CDR3 sequences, the most variable regions of these receptors, and determine their antigen specificity. Mangul and his team used simulated data to test the feasibility of using RNA-Seq to study the adaptive immune repertoire. ImReP is able to identify 99% CDR3-derived reads from the RNA-Seq mixture, suggesting it is a powerful tool for profiling RNA-Seq samples of immune-related tissues.

They also compared methods and investigated the sequencing depth and read length required to reliably assemble B and T cell receptor sequences from RNA-Seq data. ImReP consistently outperformed existing methods in both recall and precision rates for the majority of simulated parameters. Notably, ImReP was the only method with acceptable performance at 50bp read length, reconstructing with higher precision rate significantly more CDR3 clonotypes.

Mangul and his team applied ImReP to 8,555 samples across 544 individuals from 53 tissues obtained from Genotype-Tissue Expression study (GTEx v6). The data was derived from 38 solid organ tissues, 11 brain subregions, whole blood, and three cell lines. ImRep identified over 26 million reads overlapping 3.8 million distinct CDR3 sequences that originate from diverse human tissues.

Using ImReP, they created a systematic atlas of immunological sequences for B and T cell repertoires across a broad range of tissue types, most of which were not previously studied for B and T cell repertoires. They also examined the compositional similarities of clonal populations between tissues to track the flow of B and T clonotypes across immune-related tissues, including secondary lymphoid and organs encompassing mucosal, exocrine, and endocrine sites.

Advantages of using RNA-Seq to study immune repertoires include the ability to simultaneously capture both B and T cell clonotype populations during a single run, simultaneously detect overall transcriptional responses of the adaptive immune system, and scaling up the atlas of B and T cell receptors that will provide valuable insights into immune responses across various autoimmune diseases, allergies, and cancers.

Read more about ImReP in the full article, which is available for download on bioRxivhttp://biorxiv.org/content/early/2016/11/22/089235.article-metrics

ImReP was created by Igor Mandric and Serghei Mangul. ImReP is freely available at: https://sergheimangul.wordpress.com/imrep/

The atlas of T and B cell receptors, the largest collection of CDR3 sequences and tissue types, is freely available at https://sergheimangul.wordpress.com/atlas-immune-repertoires/. This resource has potential to enhance future studies in areas such as immunology and advance development of therapies for human diseases.

The full citation to our paper is:

Mangul, S., Mandric, I., Yang, H.T., Strauli, N., Montoya, D., Rotman, J., Van Der Wey, W., Ronas, J.R., Statz, B., Zelikovsky, A. and Spreafico, R., 2016. Profiling adaptive immune repertoires across multiple human tissues by RNA Sequencing. bioRxiv, p.089235.

 

Figure 1. Overview of ImReP.

Figure 1. Overview of ImReP. (See full paper for details.)

 

Figure 6. Flow of T and B cell clonotypes across diverse human tissues.

Figure 6. Flow of T and B cell clonotypes across diverse human tissues. (See full paper for details.)