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Keywords:

  • ADH7;
  • genetic variant;
  • genetic susceptibility;
  • head and neck cancer;
  • molecular epidemiology

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES

BACKGROUND:

The authors conducted a hospital-based study of 1110 patients with squamous cell carcinoma of the head and neck (SCCHN) and a control group of 1129 patients to replicate the associations reported by a recent, large European study between 2 potentially functional single nucleotide polymorphisms (SNPs) of the alcohol dehydrogenase (ADH) genes, a substitution in ADH1B at amino acid 48 from arginine to histidine (R48H) (reference SNP number [rs]1229984; guanine to adenine [G[RIGHTWARDS ARROW]A]) and a substitution in ADH7 at amino acid 92 from alanine to glycine (A92G) (rs1573496; cytosine to guanine [C[RIGHTWARDS ARROW]G]), and the risk of squamous cell carcinoma of the head and neck (SCCHN).

METHODS:

Multivariate logistic regression was used to calculate adjusted odds ratios (ORs) and 95% confidence intervals (CIs). False-positive report probabilities (FPRPs) also were calculated for significant findings.

RESULTS:

The ADH7 A92G GG and combined CG + GG genotypes were associated with a decreased risk of SCCHN (GG: adjusted OR, 0.32; 95% CI, 0.13-0.82; CG + GG: adjusted OR, 0.74; 95% CI, 0.59-0.94; FPRP, .098) compared with the CC genotype. This association was also evident in subgroups of older patients (aged >57 years), men, former smokers, patients with oral cancer, and patients with N) lymph node status (P < .05 for all); however, such associations were not observed for the ADH1B R48H SNP.

CONCLUSIONS:

The current results support the ADH7 A92G SNP as a marker for the risk of SCCHN in non-Hispanic white populations. Cancer 2010. © 2010 American Cancer Society.

Head and neck cancer, which includes cancers that arise in the oral cavity, pharynx, and larynx, is the sixth most common type of cancer. It is estimated that there are about 650,000 new cases and 350,000 new deaths worldwide every year.1 All head and neck cancers are characteristic of squamous cell carcinoma of the head and neck (SCCHN).2 Although smoking and alcohol consumption are known as major risk factors for SCCHN, only few smokers and drinkers develop SCCHN, suggesting that there is genetic susceptibility to this disease in the general population.

Acetaldehyde, as the first product of ethanol metabolism, is a main carcinogen involved in the etiology of alcohol-related cancer.3 Although studies have indicated that ethanol can be oxidized to acetaldehyde by several pathways in humans, it is believed that alcohol dehydrogenase (ADH) plays a major role in this metabolic process.4 There are 7 isozymes of human ADH that are coded by different genes. Because of high similarities in structure and kinetic properties, these ADH isozymes are classified into 5 classes (ie, Class I: ADH1A, ADH1B, ADH1C; Class II: ADH4; Class III: ADH5; Class IV: ADH7; and Class V: ADH6).5 There are several hundred genetic variants reported in these 7 ADH genes,6 and cumulative evidence indicates that some of these genetic polymorphisms may play an important role in the etiology of SCCHN.

For example, studies in Asian populations have indicated that carriers of the guanine-guanine (GG) genotype (formerly named ADH1B *1/*1) of the single nucleotide polymorphism (SNP) arginine-to-histidine substitution at amino acid 48 (R48H) (reference SNP number [rs]1229984; guanine to adenine [G[RIGHTWARDS ARROW]A]) may have a higher risk than carriers of other genotypes of developing cancers of the upper aerodigestive tract (UADT) (including cancers of the oral cavity, pharynx, and esophagus) among moderate or heavy drinkers compared with lighter drinkers.7-9 Moreover, studies in European ethnic-origin populations have indicated that the A allele of the ADH1B R48H may be associated with a decreased risk of UADT cancers regardless of drinking status.10 In addition, the GG genotype of the ADH1C isoleucine-to-valine substitution at amino acid 350 (I350V) (rs698; A[RIGHTWARDS ARROW]G) also was associated with an increased risk of SCCHN, because it modified the biologically effective dose of alcohol in a US case-control study11; however, a study in European populations suggested an opposite effect.10 To date, most published studies of the associations between ADH polymorphisms and the risk of SCCHN have dealt with ADH1B and ADH1C, but the results were not conclusive.12-14

All ADH genes are located in a cluster (5′-ADH7-ADH1C-ADH1B-ADH1A-ADH6-ADH4-ADH5-3′) of approximately 370 kb in the long arm of chromosome 4.15 Studies already have indicated that there is strong linkage disequilibrium (LD) among variants of the ADH1B and ADH1C genes.16, 17 Moreover, LD analysis for SNPs that cover this region from HapMap data also indicated strong LD among 7 ADH genes.18 The strong LD over most of the region that covers these ADH genes makes it difficult to determine the exact contributions of individual variants. To date, most published studies of associations between ADH genes and cancers, as well as other alcohol-related diseases, failed to address this issue, which may explain the inconsistent results in published studies.19 Because there is high LD among SNPs in these ADH genes, we believed it would be ideal to systemically assess the associations between all genetic variants of these ADH genes and the risk of SCCHN.

Recently, having analyzed the LD pattern of SNPs in all ADH genes, Hashibe and colleagues selected 6 representative missense SNPs in ADH genes to assess the impact of genetic variants of ADH genes on the risk of UADT cancers in a large, multicenter European study. Consequently, 2 significant SNPs, ADH1B R48H (rs1229984; G[RIGHTWARDS ARROW]A) and ADH7 the alanine-to-glycine substitution at amino acid 92 (A92G) (rs1573496; C[RIGHTWARDS ARROW]G), were associated with risk of the UADT cancers in 3800 cases and 5200 controls.18 Because SCCHN is 1 of the UADT cancers, we used DNA samples from our ongoing SCCHN study for a replication study to test the hypothesis that these 2 functional SNPs, ADH1B R48H (rs1229984; G[RIGHTWARDS ARROW]A) and ADH7 A92G (rs1573496; C[RIGHTWARDS ARROW]G), identified as important SNPs in UADT cancers, are associated with the risk of SCCHN in non-Hispanic white individuals in a Texas population.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES

Study Patients

We studied patients who were recruited from our ongoing investigations into the molecular epidemiology of SCCHN, for which the recruitment of patients has been described elsewhere.20, 21 In brief, all patients with newly diagnosed, untreated SCCHN had their disease confirmed histopathologically at The University of Texas M. D. Anderson Cancer Center between May 1995 and December 2007. Patients with second SCCHN primary tumors, primary tumors of the nasopharynx or sinonasal tract, primary tumors outside the UADT, cervical metastases of unknown origin, or any histopathologic diagnosis other than SCCHN were excluded. All eligible patients were approached for their consent to participate, and the response rate was approximately 90%. Consequently, the current case-control study included 1110 patients (cases) with primary tumors of the oral cavity (n = 327; 29.4%), the pharynx (n = 609; 54.9%, including 566 tumors of the oropharynx and 43 tumors of the hypopharynx), or the larynx (n = 174; 15.7%).

According to the American Joint Committee on Cancer,22 regional lymph node involvement of SCCHN was defined as follows: N0, no regional lymph node metastasis; N1, metastasis in a single ipsilateral lymph node that measured ≤3 cm in greatest dimension; N2, metastasis in a single ipsilateral lymph node that measured >3 cm but <6 cm in greatest dimension or in multiple ipsilateral lymph nodes, none of which measured ≥6 cm in greatest dimension, or in any bilateral or contralateral lymph node that measured <6 cm in greatest dimension; and N3, metastasis in any lymph node that measured ≥6 cm in greatest dimension. The extent of the primary SCCHN was defined as follows: T1, tumor that measured ≤2 cm at greatest dimension; T2, tumor that measured >2 cm but <4 cm in greatest dimension; T3, tumor that measured ≥4 cm in greatest dimension; and T4, tumor that invaded adjacent structures.

Self-reported, cancer-free individuals were recruited from M. D. Anderson Cancer Center visitors during the same period (the control group). These individuals were not related genetically to the enrolled cases or to each other and accompanied patients to the clinics but were not seeking medical care. We first surveyed potential controls at the clinics by using a short questionnaire to determine their willingness to participate in research studies and to obtain demographic information for frequency matching to the cases by age (±5 years) and sex. For the eligible controls, the response rate was approximately 90%.

After providing written informed consent, each participant completed a questionnaire to provide additional information about demographics and risk factors, such as age, sex, ethnicity, tobacco smoking, and alcohol use. Participants who had smoked <100 cigarettes in their lifetime were considered “never smokers,” and all others were considered “ever smokers.” For ever smokers, those who had quit smoking for more than 1 year before recruitment were considered “former smokers,” and the remaining participants were considered “current smokers.” Participants who had used alcoholic beverages at least once a week for >1 year were considered “ever drinkers,” and all others were considered “nondrinkers.” Among ever drinkers, those who had quit drinking for more than 1 year before recruitment were considered “former drinkers,” and the others were considered “current drinkers.” Each participant also provided a 1-time, 30-mL blood sample for further biomarker assays. Because genotype frequencies may vary between ethnic groups, and few minority patients were recruited, we only included non-Hispanic white individuals in the current analysis. The research protocol was approved by the M. D. Anderson Cancer Center institutional review board.

Genotyping

Genomic DNA was extracted from the buffy-coat fraction of the blood samples by using a blood DNA mini kit (Qiagen Inc., Valencia, Calif) according to the manufacturer's instructions. Restriction fragment length polymorphism-polymerase chain reaction (PCR) was used to identify the genotypes of the ADH1B R48H (rs1229984, G[RIGHTWARDS ARROW]A) and ADH7 A92G (rs1573496, C[RIGHTWARDS ARROW]G) polymorphisms. The PCR mixture included approximately 20 ng of genomic DNA, 0.1 mM deoxynucleotide triphosphate, and 1 × PCR buffer (50 mM KCl, 10 mM Tris HCl, and 0.1% Triton X-100); 1.5 mM MgCl2, 0.5 U Taq polymerase (Denville Scientific Inc.; Metuchen, NJ); and 2 pmol of each primer. The genomic DNA samples were amplified with 2 primers (mismatch bases are in italics): 5′-AGAAACACAATTTCAGGAATTTGGGT-3′ (forward) and 5′-ACTAACCACGTGGTCATCTGCG-3′ (reverse) for ADH1B R48H and 5′-TTGTGGTTTGA CACCTGCAT-3′ (forward) and 5′-ATTTTGGCCA CAGGAATCTG-3′ (reverse) for ADH7 A92G. There were 132-base pair (bp) and 163-bp PCR products for ADH1B R48H and ADH7 A92G, respectively. The final PCR reaction products were digested with the restriction enzymes HhaI for ADH1B R48H and BtsI for ADH7 A92G (New England Biolabs, Beverly, Mass) and were separated in 3% and 2% gels, respectively. The PCR assays were conducted, and the results were evaluated without knowledge of the individual's case or control status. Greater than 10% of samples were selected randomly for repeated assays, and the results from both sets of analyses were 100% concordant.

Statistical Analysis

The chi-square test was used to compare the differences in frequency distributions of demographic variables, other known risk factors, and alleles and genotypes of the ADH1B R48H and ADH7 A92G SNPs between cases and controls. In addition, the associations between the SNPs and the risk of SCCHN were estimated by computing odds ratio (ORs) and 95% confidence intervals (CIs) with and without adjustment for age, sex, smoking status, and drinking status in unconditional multivariate logistic regression analyses. Stratified analyses were performed to explore possible interactions between each SNP and stratified variables using similar unconditional multivariate logistic regression models. The ALLELE and HAPLOTYPE procedures in the SAS software (version 9.13; SAS Institute, Cary, NC) were used to calculate LD and to conclude haplotype frequencies based on the observed genotypes.

We also calculated the false-positive report probability (FPRP),23 which depends on the previously calculated probability that the SNP is associated with SCCHN, the power of the current study, and the observed P value. We set .2 as an FPRP threshold (ie, the probability of a false-positive result is <20%). For all significant results in the current study, we assigned a prior probability of .01 to detect an OR of 1.56 (for a risk effect) or 0.67 (for a protective effect) for an association with genotypes and haplotypes of each SNP. Only significant results with an FPRP value >.2 were considered a noteworthy association. All statistical tests were 2-sided, a P value of .05 was considered significant, and all tests were performed using the SAS software package.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES

Characteristics of Study Patients

Characteristics of the study population are listed in Table 1. Both cases and controls had similar frequency distributions of age (P = .482) and sex (P = .708), suggesting that the frequency matching was adequate. However, there were more current smokers and drinkers among cases than among controls (P < .001 for both), and those variables were controlled for in later multivariate logistic regression analyses.

Table 1. Frequency Distributions of Selected Variables in Patients With Squamous Cell Carcinoma of the Head and Neck and in Cancer-Free Controls
VariableNo. of Subjects (%)Pa
Cases, n=1110Controls, n=1129
  • SD indicates standard deviation.

  • a

    Two-sided chi-square tests were used to calculate differences in the distribution between cases and controls.

  • b

    One case was missing information regarding primary tumor classification and lymph node metastasis.

Age, y   
 Mean±SD57.2 ± 11.156.8 ± 11.0.414
 ≤50302 (27.2)317 (28.1).482
 51-57290 (26.1)270 (23.9) 
 >57518 (46.7)542 (48) 
Sex   
 Women273 (24.6)270 (23.9).708
 Men837 (75.4)859 (76.1) 
Smoking status   
 Never308 (27.8)552 (48.9)<.001
 Former380 (34.2)412 (36.5) 
 Current422 (38)165 (14.6) 
Alcohol use   
 Never304 (27.4)494 (43.8)<.001
 Former242 (21.8)182 (16.1) 
 Current564 (50.8)453 (40.1) 
Primary tumor classificationb   
 T1282 (25.4)  
 T2401 (36.1)  
 T3222 (20)  
 T4204 (18.5)  
Lymph node metastasisb   
 N0404 (36.4)  
 N1160 (14.4)  
 N2502 (45.3)  
 N343 (3.9)  
Tumor site   
 Oral cavity327 (29.4)  
 Pharynx609 (54.9)  
 Larynx174 (15.7)  

Association Between ADH1B and ADH7 Genotypes and SCCHN Risk

The genotype and allele distributions of the 2 selected SNPs among cases and controls are summarized in Table 2. The observed genotype frequencies of ADH1B R48H in controls were in Hardy-Weinberg equilibrium (P = .108), but those of ADH7 A92G were not (P = .025). There was no statistically significant difference in the frequencies of both ADH1B R48H genotypes and alleles between cases and controls (P = .374 and P = .689, respectively). However, for ADH7 A92G, cases had a significant lower frequencies of CG and GG genotypes (P < .001) and of the G allele (P < .001) compared with controls. With adjustment for age, sex, smoking status, and drinking status, logistic regression analysis suggested a significantly lower risk of SCCHN associated with the ADH7 GG genotype (adjusted OR, 0.32; 95% CI, 0.13-0.82) and the combined CG + GG genotypes (adjusted OR, 0.74; 95% CI, 0.59-0.94). However, these associations were not observed for ADH1B R48H.

Table 2. Genotype Frequencies of Alcohol Dehydrogenase (ADH) Variant ADH1B R48H and ADH7 A92G Polymorphisms in Patients With Squamous Cell Carcinoma of the Head and Neck and in Cancer-Free Controls and Associations With the Risk of Squamous Cell Carcinoma of the Head and Neck
VariableNo. (%)PaOR [95% CI]
Cases, n=1110Controls, n=1129CrudeAdjustedb
  • OR indicates odds ratio; CI, confidence interval; R48H, substitution from arginine to histidine in amino acid 48; G[RIGHTWARDS ARROW]A, guanine to adenine; A92G, substitution from alanine to glycine in amino acid 92; C[RIGHTWARDS ARROW]G, cytosine to guanine; rs, reference single nucleotide polymorphism; NA, not applicable.

  • a

    Two-sided chi-square tests were used to calculate differences in the frequency distribution of genotypes, combined genotypes, or alleles between cases and controls.

  • b

    Adjusted for age, sex, smoking, and drinking status.

ADH1B R48H rs1229984: G[RIGHTWARDS ARROW]A       
 GG105995.4107595.2.3741.001.00
 AG514.6524.6 1.00 [0.67-1.48]1.33 [0.88-2.02]
 AA00.020.2 NANA
 AG and AA514.6544.8.8230.96 [0.65-1.42]1.29 [0.86-1.94]
 A allele frequency0.0230.025.689    
ADH7 A92G rs1573496: C[RIGHTWARDS ARROW]G       
 CC94885.490279.9.0001.001.00
 CG15614.120618.2 0.72 [0.57-0.90]0.79 [0.62-1.00]
 GG60.5211.9 0.27 [0.11-0.68]0.32 [0.13-0.82]
 CG and GG16214.622720.1.0000.68 [0.54-0.85]0.74 [0.59-0.94]
 G allele frequency0.0760.110<.001    

We further stratified the observed associations of ADH1B R48H and ADH7 A92G genotypes with risk for SCCHN stratified by age, sex, smoking and drinking status, lymph node metastasis, primary tumor status, and tumor site. Table 3 demonstrates that, for ADH7 A92G, the decreased risk of SCCHN associated with the combined ADH7 CG + GG genotypes was significant for subgroups of older individuals (aged >57 years; adjusted OR, 0.67; 95% CI, 0.48-0.94), men (adjusted OR, 0.70; 0.54-0.91), former smokers (adjusted OR, 0.68; 0.46-0.99), those with N0 lymph node metastasis (adjusted OR, 0.64; 95% CI, 0.45-0.90), and those with oral cavity disease (adjusted OR, 0.63; 95% CI, 0.43-0.91); and the decreased risk was marginal for those with T1 (adjusted OR, 0.69; 0.48-1.01) and T2 (adjusted OR, 0.72; 95% CI, 0.52-1.00) primary tumors. For ADH1B R48H, although no overall risk was observed, a significantly increased risk of SCCHN was associated with the combined ADH1B AG + AA genotypes in subgroups of younger individuals (aged ≤50 years; adjusted OR, 2.39; 95% CI, 1.12-5.10) and in those with N2 lymph node metastasis (adjusted OR, 1.62; 95% CI, 1.00-2.63). Because the findings in fewer subgroups were significant, the results for ADH1B R48H probably were caused by chance. Furthermore, there were no associations modulated by subgroups of alcohol use, although the 2 genes are involved in alcohol metabolism.

Table 3. Stratification Analysis of the Associations of Genotypes of Alcohol Dehydrogenase (ADH) Variants ADH1B R48H and ADH7 A92G With the Risk of Squamous Cell Carcinoma of the Head and Neck
Stratified VariableNo. of Cases/ ControlsADH1B R48H, rs1229984: G[RIGHTWARDS ARROW]AADH7 A92G, rs1573496: C[RIGHTWARDS ARROW]G
GG: Cases/ Controls, %GG vs AG+ AA: Adjusted OR (95% CI)aPbCC: Cases/ Controls, %CC vs CG+ GG: Adjusted OR (95% CI)aPb
  • R48H indicates substitution from arginine to histidine in amino acid 48; rs, reference single nucleotide polymorphism; G[RIGHTWARDS ARROW]A, guanine to adenine; A92G, substitution from alanine to glycine in amino acid 92; C[RIGHTWARDS ARROW]G, cytosine to guanine; OR, odds ratio; CI, confidence interval.

  • a

    Adjusted by age, sex, and smoking and drinking status.

  • b

    Two-sided chi-square tests were obtained from multivariate logistic regression models that were adjusted for age, sex, and smoking and drinking status. The sum of patients in some strata was less than the total number because of missing information.

  • c

    Statistically significant in multivariate logistic regression analysis.

  • d

    Included 565 cases with disease of the oropharynx and 44 cases with disease of the hypopharynx.

Age, y       
 ≤50302/31793.4/96.22.39 (1.12-5.10).02585.5/80.40.74 (0.48-1.14).171
 51-57290/27094.8/931.00 (0.47-2.10).99684.5/80.70.90 (0.57-1.44).664
 ≥57518/54296.9/95.80.90 (0.45-1.79).75785.7/79.20.67 (0.48-0.94)c.022
Sex       
 Men837/85995.3/95.21.26 (0.79-2.02).33385.9/79.90.70 (0.54-0.91)c.008
 Women273/27095.6/95.21.34 (0.57-3.14).50183.9/80.00.90 (0.56-1.44).654
Smoking status       
 Never308/55291.6/94.41.59 (0.90-2.67).11881.2/79.40.90 (0.63-1.28).547
 Former380/41296.3/94.90.77 (0.38-1.54).45385.8/79.90.68 (0.46-0.99)c.043
 Current422/16597.4/98.82.14 (0.46-10.00).33488.2/81.80.61 (0.36-1.01).055
Alcohol use       
 Never304/49491.8/93.31.39 (0.80-2.40).24083.6/78.70.74 (0.51-1.07).110
 Former242/18297.9/96.70.77 (0.22-2.68).68285.1/76.90.67 (0.40-1.13).134
 Current564/45396.3/96.71.35 (0.66-2.76).40886.5/82.30.80 (0.56-1.14).219
Primary tumor classification       
 T1282/112995.0/95.21.24 (0.67-2.30).50085.8/79.90.69 (0.48-1.01).055
 T2401/112994.5/95.21.53 (0.90-2.61).11385.8/79.90.72 (0.52-1.00).047
 T3222/112996.4/95.21.05 (0.48-2.31).89584.2/79.90.78 (0.52-1.17).226
 T4204/112996.6/95.21.09 (0.47-2.55).82885.3/79.90.76 (0.49-1.18).222
Lymph node metastasis       
 N0404/112996.5/95.20.93 (0.50-1.75).82687.6/79.90.64 (0.45-0.90)c.010
 N1160/112995.6/95.21.24 (0.54-2.86).61186.9/79.90.63 (0.39-1.04).069
 N2502/112994.0/95.21.62 (1.00-2.63).04883.7/79.90.81 (0.60-1.07).140
 N343/1129100.0/95.279.1/79.91.15 (0.54-2.47).720
Tumor site       
 Oral cavity327/112994.5/95.21.48 (0.82-2.66).18987.5/79.90.63 (0.43-0.91)c.014
 Pharynxd609/112995.2/95.21.20 (0.74-1.94).46683.9/79.90.80 (0.61-1.04).093
 Larynx174/112997.7/95.20.92 (0.31-2.73).87786.8/79.90.68 (0.41-1.13).141

Association Between ADH1B and ADH7 Haplotypes and SCCHN Risk

The LD analysis indicated that the 2 SNPs were in incomplete LD in our study population (D′ = 0.386; r2 = 0.031; P < .001), and 4 possible haplotypes were inferred based on the observed genotype data (Table 4). The global test for haplotype distribution between cases and controls was statistically significant (P < .001). When the most common haplotype “GC” (ie, ADH1B G + ADH7 C) was used as the reference, the haplotype “GG” (ie, ADH1B G + ADH7 G) was associated with a significant protection against the risk of SCCHN (adjusted OR, 0.74; 95% CI, 0.59-0.92), suggesting a major role of the ADH7 G allele, although the ADH1B G allele and the ADH7 G allele had opposite effects. In contrast, the haplotype “AC” (ie, ADH1B A + ADH7 C) was associated with a significantly increased risk of SCCHN (adjusted OR, 1.71; 95% CI, 1.02-2.87), suggesting a major role of the ADH7 C allele. Although the effect of the combined haplotype “AG” did not reach significance (adjusted OR, 0.70; 95% CI, 0.36-1.34), the nonsignificant protective effect of this haplotype further supports a protective role of the ADH7 G allele.

Table 4. The Frequency of Inferred Haplotypes of the Alcohol Dehydrogenase (ADH) Variants ADH1B R48H and ADH7 A92G Based on Observed Genotypes and Their Association With the Risk of Squamous Cell Carcinoma of the Head and Neck
ADH1B R48H, rs1229984: G[RIGHTWARDS ARROW]AADH7 A92G, rs1573496: C[RIGHTWARDS ARROW]GNo. of Chromosomes (%)a,bOR [95% CI]
CasesControlsCrudeAdjustedc
  • OR indicates odds ratio; CI, confidence interval; R48H, substitution from arginine to histidine in amino acid 48; rs, reference single nucleotide polymorphism; G[RIGHTWARDS ARROW]A, guanine to adenine; A92G, substitution from alanine to glycine in amino acid 92; C[RIGHTWARDS ARROW]G, cytosine to guanine.

  • a

    Data are presented as the number (%) of total chromosomes for cases (total chromosomes, N=2220) and controls (total chromosomes, N=2258).

  • b

    Two-sided chi-square tests were used to calculate haplotype frequency distributions between cases and controls (16.93 with 3 degrees of freedom; P = .001).

  • c

    Adjusted for age, sex, smoking, and drinking status.

GC2016 (90.8)1982 (87.78)1.001.00
GG153 (6.9)220 (9.74)0.68 [0.55-0.85]0.74 [0.59-0.92]
AC36 (1.6)28 (1.24)1.26 [0.77-2.08]1.71 [1.02-2.87]
AG15 (0.7)28 (1.24)0.53 [0.28-0.99]0.70 [0.36-1.34]

Finally, the FPRP values at different prior probability levels for all significant findings are summarized in Table 5. For a prior probability of .01, assuming that the OR for specific genotype was 0.67 (protection) or 1.56 (risk), with statistical power of .548 and .421, the FPRP values were .098 and .190, respectively, for an association of the combined ADH7 CG + GG genotypes with a reduced risk of SCCHN in all individuals and in men. The FPRP value for an association between the haplotype “GG” and the risk of SCCHN was .103 with a power of .601. These 3 significant associations were considered noteworthy findings in the current study, because the probability of a false-positive result was <20%. In contrast, greater FPRP values were observed for the other significant associations between ADH variants and SCCHN, suggesting some possible bias in the findings.

Table 5. False-Positive Report Probability Values for Associations Between the Risk of Squamous Cell Carcinoma of the Head and Neck and the Frequency of Genotypes and Haplotypes of Alcohol Dehydrogenase (ADH) Variants ADH1B R48H and ADH7 A92G
Genotype/HaplotypePositive OR (95% CI)aPbStatistical PowercPrior Probability
.25.1.01.001.0001
  • OR indicates odds ratio; 95% CI, 95% confidence interval; A92G, substitution from alanine to glycine in amino acid 92; rs, reference single nucleotide polymorphism; C[RIGHTWARDS ARROW]G, cytosine to guanine; N, lymph node status; A-C, adenine-cytosine.

  • a

    Crude OR.

  • b

    The omnibus chi-square test was used to calculate the genotype and haplotype frequency distributions.

  • c

    Statistical power was calculated using the number of observations in the subgroup and the OR and P values in this table.

  • d

    Statistically significant in false-positive report probability analysis.

  • e

    Haplotypes were constructed in the order of ADH1B R48H and ADH7 A92G.

ADH7 A92G, rs1573496: C[RIGHTWARDS ARROW]G        
 GG vs CC        
  All patients0.27 (0.11-0.68).000.007.115.281.812.978.998
 GG+CG vs CC        
  All patients0.68 (0.54-0.85).001.548.003.010.098d.522.916
  Age ≥57 y0.63 (0.46-0.87).005.373.039.110.575.932.993
  Men0.65 (0.50-0.84).001.421.007.021.190d.703.960
  Former smoking0.66 (0.45-0.96).027.461.151.349.855.983.998
  N00.56 (0.40-0.78).001.152.010.029.246.767.971
  Oral cavity site0.57 (0.40-0.82).002.197.028.080.489.906.990
 Haplotypee        
  A-C vs G-C0.53 (0.28-0.99).001.020.095.240.776.972.997
  G-G vs G-C0.68 (0.55-0.85).001.601.003.010.103d.538.921

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES

In this hospital-based case-control study, we investigated the associations between 2 reportedly important SNPs, ADH1B R48H (rs1229984; G[RIGHTWARDS ARROW]A) and ADH7 A92G (rs1573496; C[RIGHTWARDS ARROW]G), and the risk of SCCHN in a non-Hispanic white population. We observed that an overall reduced risk of SCCHN was associated with the GG genotype or the combined CG + GG genotypes of the ADH7 A92G SNP, and this reduced risk also was evident in subgroups of older individuals, men, former smokers, those who had cancers of the oral cavity, and those who had tumors without metastasis. These results are consistent with what was reported previously in a large European study of UADT cancers.18 Although we did not observe an overall association between the ADH1B R48H SNP and the risk of SCCHN, the “GG” haplotype of these 2 SNPs appeared to be associated with a significantly lower SCCHN risk (mostly the effect of the ADH7 G allele); however, the “AC” haplotype was associated with a significantly higher SCCHN risk (mostly the effect of the ADH1B A allele) compared with the most common “GC” haplotype.

The ADH1B R48H AA genotype encodes an enzyme in which the homodimers are about 40 times more active than the enzymes encoded by the AG and GG genotype4; however, the AA genotype is common in Asian populations but rare in European populations,15, 24 and most studies on the effects of ADH1B polymorphisms on cancer have been in Asian populations. For example, the risk of esophageal squamous cell carcinoma increased as the numbers of ADH1B R48H G alleles increased in Taiwanese,7 and the GG genotype was associated with a >5-fold increased risk of SCCHN among Japanese men who were moderate-to-heavy drinkers.25 Nevertheless, several later studies in European populations also reported that the ADH1B GG genotype was associated with an increased risk of UADT cancers and an interaction with alcohol consumption,10, 18 although no association was reported between the GG genotype and the risk of laryngeal cancer, even taking into account alcohol consumption, in a German population.26

Our study did not observe an overall association between ADH1B R48H genotypes and the risk of SCCHN or between different subgroups of alcohol use. This discrepancy may have been resulted from several factors. First, the ADH1B R48H A allele frequency was rare in our study (0.023 for cases and 0.025 for controls). Second, the effect of ADH1B variants on SCCHN was striking only in the moderate and high levels of alcohol consumption,27 but there were more never drinkers in our study than in other studies.28 It is conceivable that the rare ADH1B R48H A allele with low alcohol consumption did not allow us to detect the potential effect of this SNP on the risk of SCCHN. However, the current study did validate the association between the more frequent ADH7 A92G SNP (the G allele frequency was 0.076 in cases and 0.110 in controls) and the risk of SCCHN in our study population, consistent with the results from a large, multicenter, European case-control study.18 Studies have indicated that the ADH7 SNPs were associated with alcoholism,29 which appears to be independent from a causative factor in ADH1B.30 Nevertheless, we have not observed any associations that were modulated by alcohol use status, although the effect of ADH7 A92G reportedly was more striking in moderate and heavy alcohol drinkers.18 It is likely that the former and current drinkers in our study may have included both light and heavy drinkers, which may weaken the association between ADH7 A92G and alcohol use.

The ADH7 gene is expressed only in the epithelial tissues of the upper gastrointestinal tract, down to the stomach, and the eyes; whereas other ADH genes are expressed mainly in the liver,31, 32 and ADH7 has the highest maximal activity for ethanol among the ADHs.33 These findings suggest that ADH7 is probably the first of the ADHs in metabolizing the ingested alcohol before alcohol has been absorbed into the blood. A recent in vivo study also identified alleles of selected ADH7 SNPs that influenced the early stages of alcohol metabolism.34 Because the upper gastrointestinal tract is the first organ to ingest ethanol, a causal association would be expected between variants in ADH7 and the risk of SCCHN.

There are 2 main polarized haplotype blocks that have been identified in the ADH7 gene region that include sequences flanking the 5′ and 3′ ends of the ADH7 transcription unit.35 The SNP A92G locus is located in the 5′ haplotype block of the ADH7 gene. Therefore, our findings suggest that the effect of this SNP on the risk of SCCHN also may caused by other SNPs in high LD with it. Because LD between ADH7 and other ADH genes was low,36 it is likely that there are other potential causal polymorphisms in the ADH7 gene, especially in the 5′ haplotype block of ADH7.

There are several limitations in our study. First, although our sample size was relatively large, it still was not large enough to identify the possible effect of alcohol consumption pattern on the association. Second, this was a hospital-based case-control study, and the genotype frequency in the control group may not represent the true frequency in the general population because of potential selection bias. This may have led to the deviation from Hardy-Weinberg equilibrium in the control group by the genotype frequency of ADH7 A92G, which may have weaken any possible interaction between variants in ADH and smoking and alcohol use. These limitations should be overcome in the future by large, well designed, prospective studies. In summary, we have confirmed that the G allele of the ADH7 A92G (rs1573496; C[RIGHTWARDS ARROW]G) SNP is associated with a reduced risk of SCCHN in non-Hispanic whites. More studies with multiethnic groups are warranted to explore the role of ADH7 SNPs in the etiology of SCCHN.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES

We thank Margaret Lung and Kathryn Patterson for their assistance in recruiting the subjects and gathering the questionnaire information and Yawei Qiao, Jianzhong He, Kejing Xu, and Min Zhao for laboratory assistance.

CONFLICT OF INTEREST DISCLOSURES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES

Supported by National Institutes of Health grants R01 ES11740-07 (Qingyi Wei, principal investigator), R01 CA131274-01 (Qingyi Wei, principal investigator), P50 CA97007-07 (Scott Lippman, principal investigator), and P30 CA16672 (The University of Texas M. D. Anderson Cancer Center).

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. CONFLICT OF INTEREST DISCLOSURES
  8. REFERENCES