Deep Sequencing of Three Loci Implicated in Large-Scale Genome-Wide Association Study Smoking Meta-Analyses.
BACKGROUND
Genome-wide association study meta-analyses have robustly implicated three loci that affect susceptibility for smoking: CHRNA5\CHRNA3\CHRNB4, CHRNB3\CHRNA6 and EGLN2\CYP2A6. Functional follow-up studies of these loci are needed to provide insight into biological mechanisms. However, these efforts have been hampered by a lack of knowledge about the specific causal variant(s) involved. In this study, we prioritized variants in terms of the likelihood they account for the reported associations.


METHODS
We employed targeted capture of the CHRNA5\CHRNA3\CHRNB4, CHRNB3\CHRNA6, and EGLN2\CYP2A6 loci and flanking regions followed by next-generation deep sequencing (mean coverage 78×) to capture genomic variation in 363 individuals. We performed single locus tests to determine if any single variant accounts for the association, and examined if sets of (rare) variants that overlapped with biologically meaningful annotations account for the associations.


RESULTS
In total, we investigated 963 variants, of which 71.1% were rare (minor allele frequency < 0.01), 6.02% were insertion/deletions, and 51.7% were catalogued in dbSNP141. The single variant results showed that no variant fully accounts for the association in any region. In the variant set results, CHRNB4 accounts for most of the signal with significant sets consisting of directly damaging variants. CHRNA6 explains most of the signal in the CHRNB3\CHRNA6 locus with significant sets indicating a regulatory role for CHRNA6. Significant sets in CYP2A6 involved directly damaging variants while the significant variant sets suggested a regulatory role for EGLN2.


CONCLUSIONS
We found that multiple variants implicating multiple processes explain the signal. Some variants can be prioritized for functional follow-up.
Center for Biomarker Research and Precision Medicine, School of Pharmacy, Virginia Commonwealth University, Richmond, VA; slclark2@vcu.edu.
Shaunna L
SL
Clark
Center for Biomarker Research and Precision Medicine, School of Pharmacy, Virginia Commonwealth University, Richmond, VA;
Joseph L
JL
McClay
Center for Biomarker Research and Precision Medicine, School of Pharmacy, Virginia Commonwealth University, Richmond, VA;
Daniel E
DE
Adkins
Center for Biomarker Research and Precision Medicine, School of Pharmacy, Virginia Commonwealth University, Richmond, VA;
Karolina A
KA
Aberg
Center for Biomarker Research and Precision Medicine, School of Pharmacy, Virginia Commonwealth University, Richmond, VA;
Gaurav
G
Kumar
Center for Biomarker Research and Precision Medicine, School of Pharmacy, Virginia Commonwealth University, Richmond, VA;
Sri
S
Nerella
Center for Biomarker Research and Precision Medicine, School of Pharmacy, Virginia Commonwealth University, Richmond, VA;
Linying
L
Xie
Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC;
Ann L
AL
Collins
Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC;
James J
JJ
Crowley
Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC;
Corey R
CR
Quakenbush
Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC;
Christopher E
CE
Hillard
Department of Biostatistics, School of Medicine, Virginia Commonwealth University, Richmond, VA;
Guimin
G
Gao
Center for Biomarker Research and Precision Medicine, School of Pharmacy, Virginia Commonwealth University, Richmond, VA;
Andrey A
AA
Shabalin
Virginia Institute for Psychiatric and Behavioral Genetics, Virginia Commonwealth University, Richmond, VA;
Roseann E
RE
Peterson
Department of Psychiatry and Behavioral Sciences, Duke University Medical Center, Durham, NC;
William E
WE
Copeland
Virginia Institute for Psychiatric and Behavioral Genetics, Virginia Commonwealth University, Richmond, VA;
Judy L
JL
Silberg
Virginia Institute for Psychiatric and Behavioral Genetics, Virginia Commonwealth University, Richmond, VA;
Hermine
H
Maes
Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC; Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden.
Patrick F
PF
Sullivan
Department of Psychiatry and Behavioral Sciences, Duke University Medical Center, Durham, NC;
Elizabeth J
EJ
Costello
Center for Biomarker Research and Precision Medicine, School of Pharmacy, Virginia Commonwealth University, Richmond, VA;
Edwin J
EJ
van den Oord
D000328:Adult; D005260:Female; D005787:Gene Frequency; D020022:Genetic Predisposition to Disease; D014644:Genetic Variation; D055106:Genome-Wide Association Study; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D012907:Smoking / Q000235:genetics*; D014029:Tobacco Use Disorder / Q000235:genetics