The Investigation into CYP2E1 in Relation to the Levelof Response to Alcohol Through a Combination of Linkage and Association Analysis2
Amy Webb, Penelope A. Lind, Jelger Kalmijn, Heidi S. Feiler, Tom L. Smith,Marc A. Schuckit, and Kirk Wilhelmsen
Background:
A low level of response to alcohol during an individual’s early experience withalcohol is associated with an increase risk of alcoholism. A family-based genome-wide linkageanalysis using sibling pairs that underwent an alcohol challenge where the level of response toalcohol was measured with the Subjective High Assessment Scale (SHAS) implicated the 10q ter-minal (10qter) region.
CYP2E1
, a gene known for its involvement with ethanol metabolism, mapsto this region.
Methods:
Variance component multipoint linkage analysis was performed on a combined mapof single-nucleotide polymorphism (SNP) and microsatellite data. To account for the heterogene-ity evident in the dataset, a calculation assuming locus heterogeneity was made using the Hetero-geneity Log of Odds (HLOD) score. Association between SNP marker allele counts and copynumber and SHAS scores were evaluated using a logistic regression model.
Results:
Linkage analysis detected significant linkage to
CYP2E1,
which was diminishedbecause of apparent locus heterogeneity traced to a single family with extreme phenotypes. Inretrospect, circumstances recorded during testing for this family suggest that their phenotype dataare likely to be unreliable. Significant allelic associations were detected for several
CYP2E1
poly-morphisms and the SHAS score. DNA sequencing from families that contributed the greatest evi-dence for linkage did not detect any changes directly affecting the primary amino acid sequence.With the removal of a single family, combined evidence from microsatellites and SNPs offerssignificant linkage between the level of response to alcohol and the region on the end of chromo-some 10.
Conclusion:
Combined linkage and association indicate that sequence changes in or near
CYP2E1
affect the level of response to alcohol providing a predictor of risk of alcoholism. Theabsence of coding sequence changes indicates that regulatory sequences are responsible. Implicat-ing
CYP2E1
in the level of response to alcohol allows inferences to be made about how the brainperceives alcohol.
Key Words:
Level of Response to Alcohol, Linkage Analysis, Association Analysis, CombinedLinkage and Association, CYP2E1, Locus Heterogeneity.
W
HILE A NUMBER of phenotypic factors can affectthe risk of alcoholism, one of the most studied end-ophenotypes is an individual’s level of response to alcoholduring their early experience with alcohol (Heath et al., 1999).The level of response to alcohol can be reliably measured withthe Subjective High Assessment Scale (SHAS) during an alco-hol challenge or by the Self-Rating of the Effects of Alcohol,which uses recall to establish the number of drinks requiredto reach an effect. Children of alcoholics have a greater riskof alcoholism when they have a lower level of response(Pollock, 1992; Schuckit and Smith, 1996; Schuckit et al.,2000). A low level of response established early in an indivi-dual’s drinking career can lead to higher future drinking levels(Ehlers et al., 1999; Heath et al., 1999; Volavka et al., 1996).Populations at historically higher risk of alcoholism, such asNative American or Korean, need to consume larger amountsof alcohol to become intoxicated (Garcia-Andrade et al.,1997; Luczak et al., 2002; Wall et al., 1999) compared tothose with lower risk (Monteiro et al., 1991) who exhibit amore intense level of response to alcohol. Several studies have
A C E R 1 3 1 7
B
Dispatch: 1.9.10 Journal: ACER
CE: Nathiya
Journal Name Manuscript No.
Author Received: No. of pages: 9 PE: Amal
From the Department of Biomedical Engineering (AW), Univer-sity of North Carolina at Chapel Hill, Chapel Hill, North Carolina;Genetic Epidemiology (PAL), Queensland Institute of Medical Research, Brisbane, Queensland, Australia; Department of Psychiatry(JK, TLS, MAS), University of California, San Diego, and the Vet-erans Affairs San Diego Healthcare System, San Diego, California;Life Sciences Division (HSF), Lawrence Berkeley National Labora-tory, Berkeley, California; and Department of Genetics and Neurology(KW), University of North Carolina at Chapel Hill, Chapel Hill,North Carolina.Received for publication November 12, 2009; accepted June 21, 2010.Reprint requests: Kirk C. Wilhelmsen, MD,PhD, UNC-CH Department of Neurology and Genetics, 120 Mason Farm Road,Campus Box #7264, Chapel Hill, North Carolina 27599-7264; Tel.:(919) 966-1373; Fax: (919) 966-3630; E-mail: kirk@med.unc.eduCopyright
Ó
2010 by the Research Society on Alcoholism.
DOI: 10.1111/j.1530-0277.2010.01317.x
Alcoholism: Clinical and Experimental Research
Vol. 35, No. 1January 2011
Alcohol Clin Exp Res,
Vol 35, No 1, 2011: pp 1–9 1
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implicated genes showing association with the level of response to alcohol (
GABA
,
5-HT
, and
KCNMA1
) (Barret al., 2003; Dick et al., 2006; Schuckit et al., 2005). The evi-dence proving association for these genes is weak by currentstandards that have been developed as a consequence fromthe technological advances enabling genome-wide associationstudies. Even though these genes may affect the level of response to alcohol to some degree, it is possible that thesereported associations reflect the typical reporting bias seen incandidate gene studies.Initially, data were collected from 139 sibling pairs (Wil-helmsen et al., 2003). Variance component analysis found asignificant Log of Odds (LOD) score peak of 3.2 for theSHAS score at the 10qter region. Of the genes located at10qter
, CYP2E1
has a known involvement with ethanolmetabolism. The CYP2E1 enzyme metabolizes ethanol andacetaminophen, as well as many toxicologic and carcinogeniccompounds and can be induced by ethanol and nicotine(Tanaka et al., 2000). In the second stage of the study, when99 newly collected sibling pairs were added (Schuckit et al.,2005), the peak at 10qter was significantly diminished. As willbe described in this paper, it was initially assumed that thediminishment was because of locus heterogeneity, but ulti-mately the reduced evidence for linkage was explained by asingle family with extreme and unreliable phenotypes.Most of the ethanol that is consumed is oxidized by theliver using alcohol dehydrogenase (ADH). At the high con-centrations associated with chronic alcohol consumption,metabolism of ethanol to acetaldehyde increases while thesubsequent conversion into acetate is decreased, leading toeven higher levels of acetaldehyde. It was shown in rats thatchronic consumption reduced the oxidation of acetaldehydein the liver, thus providing an explanation for the high bloodacetaldehyde levels measured after chronic use in human sub- jects (Lieber, 1999). Acetaldehyde is toxic and highly reactive(Zakhari, 2006), binding to nearby proteins thus creating anantibody response, decreased DNA repair, and glutathionedepletion ultimately reducing the ability of the liver to clearfree radicals (Lieber, 1997). As a result from the oxidation,NAD
+
is reduced with the addition of an electron to formNADH (Zakhari, 2006) used by mitochondria for ATP syn-thesis. At high concentrations, ethanol is oxidized by ADH ata higher rate leading to an increase in the NADH
⁄
NAD
+
ratio (Zakhari, 2006).
CYP2E1
is part of the microsomal ethanol oxidizing sys-tem (MEOS) accounting for up to 10% of ethanol oxidationin the liver (Tanaka et al., 2000). Once the ADH pathwaybecomes saturated because of high ethanol concentrations,the MEOS pathway activity increases (Lieber, 1997). By theMEOS pathway, CYP2E1 metabolizes ethanol and other sub-strates into toxic metabolites creating free radicals in the formof reactive oxygen (O
2
) intermediates creating oxidative stressleading to liver damage. CYP2E1 uses O
2
to oxidize ethanolto aldehyde and NADPH to NADP
+
. While generally usedbiosynthetically, NADPH can be regenerated from NADP
+
with the conversion of NADH to NAD
+
. In the absence of NADPH, oxidation of ethanol to aldehyde by CYP2E1results in superoxides (Zakhari, 2006). An excess of reducedNADH in addition to the increased activity of hydrogen shut-tles in mitochondria results in an increased intake of electronsleading to an increase in superoxide anions (Seitz and Stickel,2007). The increased creation of reactive oxygen species, orROS as a result from the shift in cellular redox state, coupledwith the reduced ability to clear these free radicals, because of the increase in acetaldehyde, is thought to be a major drivingforce in the development of alcohol-related liver disease.The catalase pathway can oxidize ethanol in conjunctionwith hydrogen peroxide–generating systems, such as NADPHoxidase (Zakhari, 2006). The catalase pathways play a largerrole in the oxidation of ethanol in the brain, where little ADHoxidation occurs (Zakhari, 2006). In a study by Vasiliouand colleagues (2006), it was found that animals with aknockout of either catalase or CYP2E1 were more sensitiveto the sedative effects of ethanol than control, wild-typeanimals. The study found that CYP2E1 did not contributesignificantly to ethanol clearance in the brain, but wasinstead involved with ethanol processing in the brain affectingsensitivity.High ethanol concentrations can interfere with the abilityof CYP2E1 to metabolize other substrates because of compe-tition from the shared oxidation pathway leading to reduceddrug clearance and elevated drug concentrations (Tanakaet al., 2000). The interaction of certain drugs with alcohol willlead to a long-lasting, enhanced drug effect, often leading tooverdose. A similar relationship is thought to exist with nico-tine. It has been shown that smokers have a more rapid etha-nol clearance than nonsmokers, suggesting a biological basisfor the correlation of tobacco and alcohol consumption seenin alcoholics (Schoedel and Tyndale, 2003).A number of polymorphisms in
CYP2E1
have been testedin relation to alcoholism and a number of related disorders,including many types of cancer, with varying, often conflict-ing, results. Carriers of the c2 allele of CYP2E1*5B haveincreased risk of alcoholic liver disease and are more likely toconsume excessive amounts of alcohol possibly because of thehigher transcriptional levels of CYP2E1 seen with this allele(Grove et al., 1998; Pirmohamed et al., 1995; Watanabeet al., 1994). Variants in the gene have been implicated in theincreased risk of different types of cancer relating to the respi-ratory and digestive systems (Bouchardy et al., 2000; Liuet al., 2001; Yu et al., 1995).Because of the previously described relationship between
CYP2E1
and the metabolism of ethanol and positive linkageresults concerning the level of response to alcohol in relationto alcoholism, a number of single-nucleotide polymorphisms(SNP) were genotyped in the
CYP2E1
to further elucidate thegene’s role in alcohol response. Both genotype and copy num-ber were tested for association with the level of response toalcohol as measured by the SHAS questionnaire. Combinedlinkage and association analysis was performed to determinewhether a single marker or haplotype could account for thelinkage signal seen at 10qter.
2
WEBB ET AL.
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METHODS
Alcohol Challenge
The data collection protocol was approved by the Human SubjectsProtection Committee at the University of California in San Diegoand used written, informed consent. The design for the alcohol chal-lenge is fully described in the initial report by Wilhelmsen and col-leagues (2003). Male and female subjects ranging in age from 18 to29 years old were recruited from a population of college students.Chosen sibling pairs reported having an alcohol-dependent parent,but were not alcohol dependent themselves. The siblings included43.7% men and 56.3% women. They had an average age of 22.4 and14.2 years of education. Caucasians constituted 72.2%, 20.0% wereHispanic, and 7.8% were African-American. For 85.0% of the sub- jects, the alcohol-dependent parent was the father, whereas for 4.4%it was the mother, in 4.0% it was both parents, and in 6.6% the moreintensive interview revealed that neither parent met full criteria fordependence.To measure each participant’s response to alcohol, the SHASquestionnaire was administered. For the challenge, each subject wasgiven 8 minutes to consume a 20% by volume solution of 95% etha-nol, at 0.75 ml
⁄
kg for women and 0.9 ml
⁄
kg for men. Baseline levelsfor each score (SHAS, body sway, and breath alcohol level) weremeasured prior to the challenge and then were measured at multipleset time intervals throughout the 3 hours challenge. Ultimately, thechanges in SHAS score and body sway at 1 hour after the challengewere used as phenotypes for the genome-wide genetic analysis. Geno-typing was performed on 811 microsatellite markers across thegenome.
Taqman Genotyping
Genomic DNA was extracted from whole peripheral bloodsamples. Genotyping was performed on 10 SNPs with Taqmangenotyping assays with locus-specific PCR primers and fluorescentallele-specific probes designed by Applied Biosystems
3
. StandardTaqman protocol was followed and endpoint amplification intensitywas measured by the 7900 ABI Sequence Detector
4
. The position of the genotyped markers in relation to
CYP2E1
can be found inFig. 1. The HapMap Consortium reported 3 major haplotypes in theCaucasian population, as seen in Fig. 1, which could be distinguishedby the initial 2 SNPs that were genotyped. Table 1 lists the namesand positions of the genotyped SNPs.
Copy Number Analysis
The copy number of
CYP2E1
was determined for each sample inquadruplicate through the amplification of both a probe specific to
CYP2E1
and a standard probe by real-time PCR using the standardGene Dosage protocol provided by Applied Biosystems (Livak andSchmittgen, 2001). Preliminary amplification showed the 2 probesused for analysis had different efficiencies of amplification, whichwas corrected by a standard dilution curve added to each plate. Thefold increase after
n
number of cycles was calculated by (efficiency)
n
,and the ratio of this increase between the target and reference genesprovided copy number. Standard copy number quantificationassumes equal amplification efficiencies, but this is not always avalid assumption. Correcting for even small differences in amplifica-tion efficiencies leads to less variability among the quadruplicate
Fig. 1.
11
Location of genotyped single-nucleotide polymorphisms (SNPs) in relation to
CYP2E1
on chromosome 10. The top of the figure shows the posi-tion on chromosome 10 with each SNP location indicated by triangles. The middle part of the figure shows the position of
CYP2E1
with exons representedas yellow rectangles and introns as the lines between. At the bottom, phased haplotypes derived from the HapMap Caucasian (CEU) population are shown.Each vertical block represents a SNP genotyped in HapMap. Not all of these markers were genotyped in the study, so vertical black lines through the haplo-type figure indicate actual genotyped SNPs.
Table 1.
Translation of Identification Valuesfor Genotyped Single-Nucleotide Polymorphism (SNPs) and Position
10
rs ID ABI assay ID Build 129 position (bp) Other namesrs10776687 hCV2431881 135184332rs9418990 hCV2431878 135187956rs2070673 hCV2431871 135190557 CYP2E1*7_-333T>A– hCV30633979 135192024 CYP2E1*2,g.1132G>Ars943975 hCV7468406 135192250rs6413421 hCV25594214 135195801rs915909 hCV7468401 135197387 CYP2E1_6498C>T(I321I)rs2515641 hCV16026002 135201352 CYP2E1_10463T>C(F421F)rs2480258 hCV2431850 135202090rs2249695 hCV2431848 135202158A listing of the various identification names for the SNPs genotyped in the study based on the Applied Biosystems ID. Included under ‘‘othernames’’ are names commonly used for specific markers.
L O W R E S O L U T I O N F I G
THE INVESTIGATION INTO CYP2E1
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Scientists have discovered a gene variant that may protect against alcoholism.
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