Deep Sequencing of 71 Candidate Genes to Characterize Variation Associated with Alcohol Dependence.

paper Cited Public

Authors: Clark, Shaunna L; McClay, Joseph L; Adkins, Daniel E; Kumar, Gaurav; Aberg, Karolina A; Nerella, Srilaxmi; Xie, Linying; Collins, Ann L; Crowley, James J; Quackenbush, Corey R; Hilliard, Christopher E; Shabalin, Andrey A; Vrieze, Scott I; Peterson, Roseann E; Copeland, William E; Silberg, Judy L; McGue, Matt; Maes, Hermine; Iacono, William G; Sullivan, Patrick F; Costello, Elizabeth J; van den Oord, Edwin J
Year: 2017
Journal: Alcoholism, clinical and experimental research
PMID: 28196272
DOI: 10.1111/acer.13352
PMCID: PMC5378639

BACKGROUND: Previous genomewide association studies (GWASs) have identified a number of putative risk loci for alcohol dependence (AD). However, only a few loci have replicated and these replicated variants only explain a small proportion of AD risk. Using an innovative approach, the goal of this study was to generate hypotheses about potentially causal variants for AD that can be explored further through functional studies. METHODS: We employed targeted capture of 71 candidate loci and flanking regions followed by next-generation deep sequencing (mean coverage 78X) in 806 European Americans. Regions included in our targeted capture library were genes identified through published GWAS of alcohol, all human alcohol and aldehyde dehydrogenases, reward system genes including dopaminergic and opioid receptors, prioritized candidate genes based on previous associations, and genes involved in the absorption, distribution, metabolism, and excretion of drugs. We performed single-locus tests to determine if any single variant was associated with AD symptom count. Sets of variants that overlapped with biologically meaningful annotations were tested for association in aggregate. RESULTS: No single, common variant was significantly associated with AD in our study. We did, however, find evidence for association with several variant sets. Two variant sets were significant at the q-value <0.10 level: a genic enhancer for ADHFE1 (p = 1.47 × 10 ; q = 0.019), an alcohol dehydrogenase, and ADORA1 (p = 5.29 × 10 ; q = 0.035), an adenosine receptor that belongs to a G-protein-coupled receptor gene family. CONCLUSIONS: To our knowledge, this is the first sequencing study of AD to examine variants in entire genes, including flanking and regulatory regions. We found that in addition to protein coding variant sets, regulatory variant sets may play a role in AD. From these findings, we have generated initial functional hypotheses about how these sets may influence AD.

#	Section	Preview
0	INTRODUCTION	Alcohol dependence (AD) is a disorder characterized by compulsive and uncontrolled consumption of…
1	INTRODUCTION	One explanation is that instead of following the common-disease common variant hypothesis, which…
2	INTRODUCTION	Exome sequencing studies (Ng et al., 2009), which interrogate the protein coding portion of the…
3	INTRODUCTION	The goal of this paper was to generate hypotheses about potentially causal variants for AD that can…
4	INTRODUCTION	To achieve our goal, we employ targeted capture (Gnirke et al., 2009) of 71 AD candidate genes in…
5	INTRODUCTION	As there may be a large number of causal risk variants that have small effects (Manolio et al.,…
6	MATERIALS & METHODS — Samples	The sequenced sample comprises 363 subjects from the Virginia Twin Study on Adolescent Behavioral…
7	MATERIALS & METHODS — Measures	The phenotype considered in this study is a count of the number of alcohol dependence (AD) symptoms…
8	MATERIALS & METHODS — Measures	Descriptive data for each sample are shown in Table 1. The samples had comparable rates of…
9	MATERIALS & METHODS — Sequencing	We used the solution-based hybridization targeted capture technology (SureSelectXT, Agilent) to…
10	MATERIALS & METHODS — Sequencing	The loci included in our targeted capture library were as follows: genes identified through…
11	MATERIALS & METHODS — Sequencing	The libraries were paired-end sequenced (75bp + 35bp reads) on the SOLiD 5500xl (Life Technologies).…
12	MATERIALS & METHODS — Variant Calling and Annotation	The variants were called and filtered using GATK (McKenna et al., 2010) according to best practices…
13	MATERIALS & METHODS — Variant Calling and Annotation	All variants passing quality control (QC) were annotated to examine if variants overlapped with…
14	MATERIALS & METHODS — Statistical Analyses — Individual Variants	To determine if AD symptom count was associated with any single, common variant, we performed single…
15	MATERIALS & METHODS — Statistical Analyses — Sets of Variants	Variant set-based association tests were performed using SKAT (Lee et al., 2012b) to identify sets…
16	MATERIALS & METHODS — Statistical Analyses — Sets of Variants	The VTSABD and MTFS were analyzed together as a mega-analysis to increase power and ensure that the…
17	MATERIALS & METHODS — Statistical Analyses — Sets of Variants	Previous studies have shown that under certain conditions SKAT can be biased when calculating…
18	RESULTS — Variant Calling	In the 71 loci investigated, we identified 25,922 variants of which 18,442 (71.4%) were rare and…
19	RESULTS — Individual Variants	The Quantile-Quantile (QQ) plot of the individual, common variant meta-analysis results (Figure 1)…

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Deep Sequencing of 71 Candidate Genes to Characterize Variation Associated with Alcohol Dependence.

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