Head-and-neck squamous cell carcinoma risk in smokers: no association detected between phenotype and AHR, CYP1A1, CYP1A2, or CYP1B1 genotype
Jorge-Nebert et al. Human Genomics
Head-and-neck squamous cell carcinoma risk in smokers: no association detected between phenotype and AHR, CYP1A1, CYP1A2, or CYP1B1 genotype
Lucia F. Jorge-Nebert 0 3
Ge Zhang 0 1 3
Keith M. Wilson 2 3
Zhengwen Jiang 0 3 4
Randall Butler 3 5
Jack L. Gluckman 2 3
Susan M. Pinney 0 3
Daniel W. Nebert 0 3
0 Department of Environmental Health and Center for Environmental Genetics, University of Cincinnati Medical Center , Cincinnati, OH 45267-0056 , USA
1 Division of Human Genetics, Department of Pediatrics & Molecular
2 Department of Otolaryngology-Head and Neck Surgery, University of Cincinnati College of Medicine , Cincinnati, OH 45267-0528 , USA
3 Developmental Biology, Cincinnati Children's Hospital , Cincinnati, Ohio 45229-2899 , USA
4 Present address: Genesky Diagnostics , Suzhou , China
5 Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine , Cincinnati, OH 45267-0533 , USA
Background: Head-and-neck squamous cell carcinoma (HNSCC) differs between smokers and nonsmokers in etiology and clinical presentation. Because of demonstrated unequivocal involvement in smoking-induced cancer in laboratory animals, four candidate genes--AHR, CYP1A1, CYP1A2, and CYP1B1--were selected for a clinical genotype-phenotype association study of HNSCC risk in smokers. Thirty-six single-nucleotide variants (mostly tagSNPs) within and near these four genes [16 (AHR), 4 (CYP1A1), 4 (CYP1A2), and 12 (CYP1B1)] were chosen. Methods: Extreme discordant phenotype (EDP) method of analysis was used to increase statistical power. HNSCC patients--having smoked 1-40 cigarette pack-years--represented the “highly-sensitive” (HS) population; heavy smokers having smoked ≥80 cigarette-pack-years without any type of cancer comprised the “highly-resistant” (HR) group. The vast majority of smokers were intermediate and discarded from consideration. Statistical tests were performed on N = 112 HS and N = 99 HR DNA samples from whole blood. Conclusions: Among the four genes and flanking regions--one haploblock, ACTTGATC in the 5′ portion of CYP1B1, retained statistical significance after 100,000 permutations (P = 0.0042); among our study population, this haploblock was found in 36.4% of African-American, but only 1.49% of Caucasian, HNSCC chromosomes. Interestingly, in the 1000 Genomes Project database, frequency of this haplotype (in 1322 African and 1006 Caucasian chromosomes) is 0.356 and 0.003, respectively. This study represents an excellent example of “spurious association by population stratification”. Considering the cohort size, we therefore conclude that the variant alleles chosen for these four genes, alone or in combinations, are not statistically significantly associated with risk of cigarette-smoking-induced HNSCC.
AHR gene; CYP1A1; CYP1A2; CYP1B1 genes; Tag-SNPs (single nucleotide polymorphisms); Head-and-neck squamous cell carcinoma (HNSCC); Cigarette smoking; Extreme discordant phenotype method; Population stratification; Candidate-gene approach to genotype-phenotype association
Worldwide, head-and-neck squamous cell carcinoma
(HNSCC) is the sixth most common cancer. An
increased risk of HNSCC among cigarette smokers is well
known. In addition, high-risk types of human papilloma
virus (HPV) are associated with certain HNSCCs,
specifically a subset arising in the oropharynx. It also appears
very likely that there exists a genetic predisposition to
smoking-induced HNSCC risk. Clearly, “cancer”
represents a multifactorial trait involving hundreds, if not
thousands, of genes, plus epigenetic and environmental
effects. It remains possible, however, that—if a
candidate-gene approach that embraces a method having
sufficient statistical power is applied to a sufficiently
large cohort—then a genotype-phenotype association
might be demonstrated for one or more “small-effect”
genes. This study describes an attempt to establish such
Hundreds of polycyclic aromatic hydrocarbons (PAHs)
are present in cigarette smoke. Many laboratory animal
studies have demonstrated that aryl hydrocarbon
receptor (AHR)-regulated cytochrome P450 family-1 (CYP1)
enzymes (CYP1A1, CYP1A2, and CYP1B1) metabolize
PAHs to reactive oxygenated intermediates. When
cancer initiation occurs via “direct contact” with a
carcinogen, e.g., cigarette smoke, we believe that HNSCC will
more likely be associated with CYP1-mediated metabolic
activation [5, 27, 38], compared with a distal cancer site
such as kidney .
Description of the four candidate genes
AHR codes for a ligand-activated transcription factor
controlling numerous genes and critical cell pathways
, including up-regulation of CYP1A1, CYP1A2, and
CYP1B1 genes . AHR foreign ligands include
chemicals such as PAHs; polyhalogenated dibenzo-p-dioxins,
dibenzofurans and biphenyls; and benzoflavones found
especially in cruciferous plants . AHR endogenous
ligands include indoles and tryptophan-derived
moieties and an unknown number of the >150 members
of the lipid mediator second-messenger family [7, 32].
The highly conserved AHR exists in all vertebrates
and has also been reported to exist—without
ligandbinding properties—in mollusk, Caenorhabditis elegans,
and Drosophila .
CYP1A1 encodes the P450 monoxygenase that
metabolizes planar substrates, many of which are PAHs and
biphenyls. CYP1A1 metabolizes few, if any, drugs. Decades
of PAH-treated lab animal studies have shown strong
correlations of inducible CYP1A1 with various types of
cancer—in tissues in contact with the administered PAH
. Although basal CYP1A1 expression in animal and
human tissues is nearly always nil, inducible CYP1A1
activity is ubiquitous, located in virtually every tissue and
cell type of the body. For example, inducible CYP1A1
is found in white blood cells, endothelial cells of
blood vessels, lung, kidney, skin, and epithelial lining
of the head and neck and upper and lower
gastrointestinal (GI) tract. Inducible CYP1A1 also is seen
early in embryogenesis .
CYP1A2 codes for the CYP1A2 monooxygenase that
metabolizes about two dozen drugs—including caffeine
and theophylline—plus many environmental aromatic
amines. Substantial basal (constitutive) CYP1A2 activity
occurs in mammalian liver. Whereas >60-fold
differences in human hepatic CYP1A2 (mRNA, protein, and
activity) exist between individuals in any population
studied, etiology remains unknown. Human CYP1A2
gene expression is not detectable in embryo, fetus, or
kidney but is inducible by PAHs mostly in the liver, GI
tract, pancreas, nasal epithelium, brain, and lung .
CYP1B1 encodes the CYP1B1 monooxygenase which,
like CYP1A1, metabolizes numerous PAHs and
biphenyls, N-heterocyclic amines, arylamines and amino azo
dyes, and other carcinogenic and toxic environmental
chemicals. Also, like CYP1A1, CYP1B1 metabolizes few,
if any, drugs. Unlike CYP1A1, CYP1B1 often exhibits
substantial basal levels (e.g., endocrine tissues, tumors).
CYP1B1 expression is induced in vascular endothelial
cells, thymus/marrow and immune cells, breast,
prostate, uterus, epithelial lining of the head and neck and
upper GI tract, and various types of cancers .
Mouse Ahr knockouts and all three Cyp1 single-,
plus all three double- and the triple-knockout lines
are viable and able to reproduce––although serious
problems occur in Ahr(−/−) knockout [10, 20] and in
Cyp1a1/1a2/1b1(−/−) triple-knockout mice [8, 31].
Whereas no human “knockout” equivalent has been
found for AHR, CYP1A1, or CYP1A2, null mutations
in CYP1B1 are associated with primary congenital
glaucoma , suggesting that, during embryogenesis,
development of the eye’s anterior chamber requires
metabolism of a critical endogenous CYP1B1
substrate, most likely a lipid mediator .
Given any gradient for a multifactorial trait, if one
selects the two extremes of the phenotypic gradient 
and disregards intermediate responders in whom genes
contributing to phenotype are likely to overlap—then
statistical power can be increased . Among HNSCC
patients, we selected those with a history of 1–40
cigarette pack-years (Cig-Pk-Yrs) as “highly sensitive”
(HS). In the same clinic, we selected heavy smoker
volunteers with ≥80 Cig-Pk-Yrs having no types of cancer,
as “highly resistant” (HR) controls. All nonsmokers,
smokers intermediate between HS and HR criteria, and
other patients were excluded from our study.
SNP typing of the four genes
The human AHR gene is located at chromosome (Chr)
7p15 and CYP1B1 gene at Chr 2p22.2. The
CYP1A2_CYP1A1 locus, on Chr 15q24.1, contains the two genes
oriented head-to-head with a bidirectional promoter .
The purpose of this study was to search for single
single-nucleotide polymorphism (SNP) marker and
haplotype associations in these four selected genes that
might be statistically significantly correlated with greater
risk of HNSCC in HS cancer patients, compared with
HR heavy smokers having no cancer.
Clinical screening and patient/volunteer recruitment
HS patients were identified at the Barrett Cancer Center
(Department of Otolaryngology-Head and Neck Surgery,
University of Cincinnati College of Medicine) and the
Cincinnati Veterans’ Association (VA) Hospital.
Additional patients were identified within the Fernald
Community Project. HR volunteers were recruited at the
Barrett Cancer Center, Cincinnati VA Hospital, Fernald
Project, and throughout the community in response to
flyers. A questionnaire was used to identify HNSCC
patients—who qualified as HS because they had smoked
1–40 Cig-Pk-Yrs (e.g., “20 Cig-Pk-Yr” denotes someone
who might have smoked one pack per day for 20 years
or one-half pack per day for 40 years). Volunteers,
having no cancer of any kind (with exception of UV-caused
skin cancers), despite having smoked ≥80 Cig-Pk-Yrs,
qualified for the HR group and were also identified by
The Fernald Community blood samples originated
from an earlier independent study, as described . At
all times, we followed the clinical protocol—titled
“Human Cancer and AHR/CYP1A1/1A2/1B1 Gene
Polymorphisms” (#03-08-07-01)—annually approved for the
entire study, without any HIPAA issues or concerns, by
the University of Cincinnati Medical Center Institutional
Review Board (IRB).
Questions asked of all participants included self-identified
ethnicity, pipe or cigar smoking, second-hand smoke,
high-tar vs. low-tar cigarettes, alcohol consumption,
use of snuff/chewing tobacco, amount of coffee
consumed, occupational hazards, consumption and amount
of daily antioxidants, frequency of eating grilled meat,
and family history. Staff administering the
questionnaire ranked these eleven caveats as zero (little or
negligible contact), one (intermediate contact or exposure),
or two (high exposure). A copy of this questionnaire is
included as Additional file 1.
Whole blood for DNA isolation was collected from HS and
HR subjects via antecubital venipuncture. BD Vacutainer®
systems (Becton, Dickinson and Co.; Franklin Lakes, NJ)
were used. We collected two 13 × 100 mm Vacutainer®
K2EDTA tubes (lavender caps) for a total volume of 10 mL.
Freshly collected whole blood was refrigerated at 4 °C and
kept not longer than 24 h at that temperature. If genomic
DNA (gDNA) was not isolated from blood within 24 h of
collection, the blood was stored at −80 °C usually for not
longer than 8 weeks and then thawed the same day the
isolation procedure was performed.
For the 10 mL of blood—collected at the Barrett Cancer
Center and Cincinnati VA Hospital—gDNA was isolated
using the QIAamp® DNA Blood Maxi kit (QIAGEN,
Hilden, Germany) by applying the spin protocol.
Wholeblood samples from the Fernald Community cohort,
which had been stored at −70 °C, were thawed on the
day of isolation. Average volume of these samples was
2 mL; gDNA was therefore isolated using the QIAamp®
DNA Blood Midi kit (QIAGEN) and spin protocol. Both
QIAGEN kits employed special patented columns for
purification of gDNA present in blood pretreated with
proteases, in a highly denaturing medium containing
Highly purified gDNA isolated in this fashion was
quantified by UV absorption at 260 nm using a
minispectrometer (NanoDrop® ND-1000, Thermo Scientific;
Waltham, MA). Purity was evaluated using the 260/280
and 260/230 absorbance ratios.
Selection of common tag-SNPs
Tag-SNPs and other variants of interest were chosen in
order to study variation within each of the genetic loci
and to correlate these variations within each person in
the HS and HR cohorts. Tag-SNPs were selected by the
HaploView program , using a MAF cutoff of 10.0%
and an r2 threshold of 0.8. We used previous sequencing
data of the CYP1 locus (~40 kb) which includes the
23.3-kb bidirectional promoter [14, 15]. For AHR and
CYP1B1, we selected tag-SNPs from targeted
sequencing, including 10 kb of 5′ flanking regions in 24
Caucasian DNA samples (Z.J., data not shown). The ultimate
total was 36 tag-SNPs: 16 for AHR, 4 for CYP1A1, 4 for
CYP1A2, and 12 for CYP1B1.
These tag-SNPs are in linkage disequilibrium (LD)
with other variants in their vicinity. Thus, their selection
and genotyping were expected to simplify studies of the
four candidate genes—by reducing the number of
variants needed for a comprehensive study. Whole-genome
sequencing was not yet an option at the time of this
By site of collection
Genotyping of tag-SNPs
Tag-SNP genotypes for each HS and HR individual were
determined using the ABI Prism® SnaPshotTM Multiplex
system, which allows for the typing of up to ten SNPs
simultaneously. Chemistry of the applied kit is based on
the dideoxy single-base extension of unlabeled primer or
primers, catalyzed by AmpliTaq DNA polymerase, FS;
this permits incorporation of a single
fluorescentlylabeled ddNTP on the 3′-end of the primer.
Statistical analyses of genotype-phenotype association
Bioinformatics software HaploView (v. 4.2, Mark Daly’s
lab; MIT/Harvard Broad Institute) was used to
determine associations between the markers—variation in
SNPs—and phenotype in HS vs. HR samples. This
software also analyzes LD patterns, generating haploblocks,
and inferring haplotypes. Criteria applied to determine
haploblocks within genes was the “solid spine of LD”.
Associations between these haplotypes and phenotypes
were also calculated via the chi-square association test
of every haplotype (exhaustive haplotype test). In
addition, we applied the permutations test (results
randomized 100,000 times) to adjust for multiple tests of
multiple SNPs or haplotypes. Haplotype estimates for
each individual HS and HR were obtained through the
software program phase (v. 2.1.1, Matthew Stephens’s
lab; University of Chicago). Differences in frequencies of
haplotypes between populations were examined for
significance using Fisher’s exact test.
HNSCC in the present study refers exclusively to
squamous cell carcinoma of the oral cavity, larynx,
oropharynx, and hypopharynx. Tumors of the face, salivary
glands, nasopharynx, and brain were excluded. A
substantial number (~15%) of HNSCC patients have no
cigarette smoking history. Clinical and genetic features
of HNSCC in nonsmokers, former smokers, and current
smokers are known to be distinctly different .
Compared with malignancies in nonsmokers, smokers exhibit
more tumors of the larynx, hypopharynx, and floor of
the mouth; a much greater TP53 mutation rate; a
substantially higher percent of loss of heterozygosity at Chr
3p, 4q, and 11q13; and a greater overall average number
of chromosomal losses. In contrast, the percentage with
HPV infection was marginally, but not statistically, lower
in nonsmoker malignancies .
We excluded all nonsmokers (i.e., those having
smoked no more than one Cig-Pk-Yr, i.e., <7300
cigarettes, <365 packs, over a lifetime) because the
mechanism of HNSCC tumorigenesis is likely not to be relevant
to activation by cigarette smoke PAHs involving any of
the four genes under study. It should be noted that more
than 85% of all HNSCC smokers did not qualify because
they had smoked >40 Cig-Pk-Yrs and <80 Cig-Pk-Yrs;
this selective stringency creates a more robust statistical
power , but unfortunately fewer numbers of qualified
Over a 5-year period, we collected gDNA from a total
of 149 blood samples from the Barrett Cancer Center
and Cincinnati VA Hospital, plus 62 samples from the
Fernald Project. Of the 211 total samples, there were 94
and 18 HS patients from the Barrett Center/VA Hospital
and Fernald Project, respectively, and 55 and 44 HR
samples from the Barrett Center/VA Hospital and
Fernald Project, respectively. Total subjects having
highquality gDNA ultimately included 112 HS and 99 HR for
this study (Table 1).
Statistical analyses of covariates
With regard to the 11 covariates included in the
questionnaire (Appendix 1; described above), we specifically
focused on occupational history, amount of dietary
grilled meat, and family history of cancer (ranked as “0,”
“1,” or “2” for each individual); the other 8 parameters
did not have sufficient information for a substantial
number of participants. We used logistic regression and
random-forests models for statistical analysis of possible
associations between the selected best SNP markers and
these three covariates. No statistically significant
associations or trends of association (P < 0.05) were found
between any of these covariates and phenotype (data
not shown). The “self-identified ethnicity” was important
and is discussed in great detail below.
Genotype-phenotype association analysis
The four genes studied, with arrows displaying locations
of each selected tag-SNP, are illustrated in Fig. 1. It is
Table 1 Demographics of entire cohort studied
Fig. 1 Diagram of locations of all tag-SNPs and other SNPs in the four genes chosen for study. The AHR gene, having 11 exons, is located on
chromosome 7p15, and the transcribed region spans 47.53 kb; 16 tag-SNPs inside or near the gene were selected. CYP1A1 (spanning 6.07 kb) and
CYP1A2 (7.36 kb) on chromosome 15q24.1 each have seven exons and are arranged in tandem, head-to-head, with a 23.3-kb bidirectional promoter
between them; four tag-SNPs inside and near each gene were chosen. Human CYP1B1 on Chr 2p22.2 has three exons and spans 8.68 kb; 12 tag-SNPs
inside and near the gene were selected. Note that three SNPs (#2, 3 and 4) are located in CYP1B1 exon 3, within four nucleotides of one another. Closed
rectangles of the exons denote the translated region and open rectangles the 5′- and 3′-untranslated regions. Whereas translation of AHR is initiated at
the 3′ end of exon 1, all three CYP1 genes have noncoding first exons. Note that CYP1A1 and CYP1B1 are on reverse strand of gDNA, meaning that the
chromosomal location of each SNP for these two genes in the Genome Assembly is numbered from the 3′- to 5′-end, whereas AHR and CYP1A2 are
on the positive strand and therefore each SNP for these two genes is numbered from the 5′- to 3′-end.
noteworthy that two of the four genes are on the reverse
strand; by convention, all marker alleles were converted
to the positive strand. Moreover, CYP1A1 lies 5′-ward of
CYP1A2, with the two genes situated head-to-head and
a 23.3-kb bidirectional promoter between them; thus,
two tag-SNPs located 5′-ward of CYP1A1 and one
tagSNP 5′-ward of CYP1A2 exon 1 are located within the
bidirectional promoter—which has well-known
regulatory elements, i.e., AHR-binding sites .
Table 2 lists the 36 selected tag-SNPs, dbSNP rs
numbers, chromosomal positions, locations in or near each
gene, ancestral alleles (i.e., the “phylogenetic root” based
on sequence alignment of multiple (N = 6) primates),
reference/alternative alleles, alternative allele frequencies
[q(alt)] in Caucasian (EUR) samples from the 1000
Genomes Project, q(alt) in African (AFR) samples from the
1000 Genomes Project, and q(alt) found in the cohort
studied (HR and HS combined). We used Genome
Assembly GRCh38.p2 (Annotation Release 107) for this
analysis; data were calculated from the genomic sequences
of Ensembl transcripts AHR-002 (ENST00000242057),
CYP1A1-001 (ENST00000379727), CYP1A2-001 (ENST0
0000343932), and CYP1B1-001 (ENST00000610745).
Single-marker association study (allele test)
The entire cohort (HS and HR combined, N = 211)
included 196 self-identified Caucasian-Americans, 14
African-Americans, and 1 Latino (Table 1). Using the
chi-square test for allele-frequency difference of each
tag-SNP between HS and HR samples (Table 3), we
found three P values significant at the P < 0.05 level;
however, these associations did not retain statistical
significance in the 100,000 permutations test. This is a very
common observation and often not appreciated—when
comparing standard statistical tests with a permutation
test that is mandatory for association studies with regard
to multiple markers throughout all chromosomes .
Case-control association tests in Caucasians (N = 196)
were achieved by removing the 1 Latino and 14
AfricanAmerican subjects. Table 4 displays the HaploView
analysis—examining individual markers or SNPs. From
chisquare analysis, there is only one SNP that appears to
show a significant P value in the AHR gene: rs4719497
(P = 0.0134). All other SNPs have P values >0.05.
However, following permutation testing, rs4719497 did not
retain significance (P = 0.1048), indicating that this SNP did
not survive the mandatory correction for multi-testing.
Table 2 List of tag-SNPs, chromosomal location, SNP identifier number, chromosomal position, location in or near gene, ancestral
gene, reference/alternative alleles, alternative allele frequency q(alt) in Caucasian (EUR) and African (AFR) populations from the 1000
Genomes Project, and q(alt) in cohort studied
SNP # Gene Chr SNP ID Chromosomal Location in or Ancestral Reference q(alt) q(alt)
positiona near the geneb allelec /alternative allele EUR AFR
1 AHR 7 rs62444550 17290494 −8128C > A, promoter A C/A
2 AHR 7 rs77821156 17291826 −6796A > G, promoter A A/G
3 AHR 7 rs10250822 17293365 −5257 T > C, promoter T T/C
4 AHR 7 rs4719497 17295275 −3347 T > C, promoter T T/C
5 AHR 7 rs3757824 17296411 −2211 T > C, promoter T T/C
6 AHR 7 rs7796976 17298806 +185G > A, 5′-UTR, exon 1 G G/A
7 AHR 7 rs713150 17300533 +1912C > G, intron 1 C C/G
8 AHR 7 rs17722841 17303970 +5349G > A, intron 1 G G/A
9 AHR 7 rs2282885 17305990 +7369A > G, intron 1 A A/G
1 CYP1A1 15 (rev) rs4646903 74719300 CYP1A1*2A, Msp I RFLP Site, A A/G
2 CYP1A1 15 (rev) rs2606345 74724835 +776A > C, intron 1 C A/C
3 CYP1A1 15 (rev) rs3826042 74726564 −954C > T, promoter C C/T
4 CYP1A1 15 (rev) rs7495708 74727502 −1892 T > C, promoter C T/C
1 CYP1A2 15 rs2069514 74745879 CYP1A2*1C, −2965G > A, promoter G G/A
2 CYP1A2 15 rs762551 74749576 CYP1A2*1 F, +733A > C, intron 1 A A/C
3 CYP1A2 15 rs2470890 74755085 CYP1A2*1B, +6242 T > C, C T/C
4 CYP1A2 15 rs17861162 74756412 +7569C > G, downstream 3′ C C/G
1 CYP1B1 2 (rev) rs162562 38070372 +5810 T > G, 3′UTR T T/G
2 CYP1B1 2 (rev) rs1800440 38070996 CYP1B1*4, +5186 T > C, exon 3, N453S T T/C
3 CYP1B1 2 (rev) rs1056837 38071007 +5175G > A, exon 3, D449D (syn) G G/A
4 CYP1B1 2 (rev) rs1056836 38071060 CYP1B1*3, +5122C > G, exon 3, L432V C C/G
5 CYP1B1 2 (rev) rs1056827 38075034 CYP1B1*2, +1148C > A, exon 2, A119S A C/A
6 CYP1B1 2 (rev) rs10012 38075247 CYP1B1*2, +935G > C, exon 2, R48G C G/C
7 CYP1B1 2 (rev) rs162558 38076937 −756 T > C, promoter T T/C
8 CYP1B1 2 (rev) rs2855655 38077346 −1165C > T, promoter C C/T
9 CYP1B1 2 (rev) rs162557 38078309 −2128G > A, promoter G G/A
aGenome Assembly GRCh38.p2 (Annotation Release 107)
Haplotype association study
Figure 2 shows the LD heat map for CYP1B1 SNPs,
using r2 (correlation between pairs of loci) as a measure
of linkage disequilibrium. Haploblocks 1 (tag-SNPs #1, 2,
3, and 4) and 2 (tag-SNPs #5, 6, 7, 8, 9, 10, 11, and 12)
were determined by the program HaploView because
they showed the best continuous solid spine of LDs. We
defined haploblocks by applying this nonstringent
criterion—since no strong correlations were expected between
tag-SNP pairs, i.e., by definition, tag-SNPs are selected
because they should capture as much information as
possible about independent regions of the gene.
However, some of the SNPs selected for this study are in
very close proximity to one other and did show
substantially strong correlations (depicted as darkest squares in
the heat map, e.g., SNPs #5 and 6). We included these
SNPs in our study because of functional relevance in
cancer studies or known to alter enzyme activity levels.
LD heat maps and haploblocks were similarly generated
for the other three genes studied and were unremarkable
(data not shown).
Of the 38 haplotypes inferred in the six defined
haploblocks among our four genes in the entire cohort (Table 5,
N = 211), six were statistically significant (P < 0.05) by
chisquare analysis. After 100,000 permutations, however, only
one CYP1B1 haplotype in haploblock 2 (ACTTGATC)
Fig. 2 LD heat map of CYP1B1 SNPs from the entire cohort (N = 211), using r2 (correlation between pair of loci) as a measure of LD. Haploblock
2—which we had found initially as apparently the only haplotype (ACTTGATC) significantly associated with HNSCC risk in smokers—contains two
SNPs in the 5′ half of exon 2 plus six SNPs in the 5′-flanking region (reverse strand), extending as far 5′-ward as 4538 bp upstream of the transcription
initiation start-site. Shading denotes the following: white (r2 = 0), black (r2 = 1), and shades of gray (0 < r2 < 1), with deepening gray colors depicting
increasing r2 values (correlation) between SNP pairs. Numbers in each square indicate percentage of correlation (r2 × 100).
retained a statistically significant (P = 0.0042) association
with HNSCC risk in smokers.
The same analysis was applied to the Caucasian-only
sample (N = 196) and the results are shown in Table 6.
Only two haplotypes revealed significant P values: one in
AHR Block 1 (P = 0.0259) and one in CYP1B1 Block 2
(P = 0.0392). Following correction for multi-testing
(100K permutations), however, the appearance of
statistical significance was lost: P = 0.142 and 0.1666,
respectively. Note that the inferred haplotypes, and
order of their ranking by frequency (left-most columns),
differs between Table 5 (entire cohort) and Table 6
Further analysis of CYP1B1 haplotype ACTTGATC
In our initial approach, all HS and HR subjects (N = 211,
i.e., 422 chromosomes, including one self-identified
Latino) were included in the same association study of
haplotype ACTTGATC with HNSCC risk among
smokers, irrespective of racial origin (Table 5); and it
was initially exciting to find the ACTTGATC haplotype
(apparently) statistically significantly associated
(P = 0.0042) with cigarette smoking-induced risk of
HNSCC. When the Latino subject was excluded from
the analysis, leaving only African-American and
Caucasian-Americans (Table 7), Fisher’s exact test
indicated a significant difference in the frequency of this
haplotype between HS and HR (P = 0.0011).
However, when individuals carrying this haplotype
were identified by race (Table 8), it became apparent that
the haplotype was tracking extensively with African origin,
rather than HNSCC risk; in a total of 112 HS subjects
(224 chromosomes and excluding the one Latino), this
haplotype appeared 11 times—8 times in heterozygous
state in African-Americans and 3 times in heterozygous
state among Caucasian-Americans. This distribution is
disproportionately larger for African-Americans,
considering we had recruited only 11 African-American HS vs.
101 Caucasian-American HS patients. The Fisher exact
test demonstrated a significant difference (P < 0.0001) in
this haplotype frequency between African-American and
Caucasian-American HS chromosomes (Table 8).
From a total of 28 African-American chromosomes of
both HS and HR studied (Table 9), 8 carried the
ACTTGATC haplotype (allele frequency = 0.286). However,
when we compared this haplotype and HNSCC risk in
African-Americans, no statistically significant conclusion
could be reached (P = 0.141), perhaps because our study
is under-powered due to small sample sizes—especially a
control HR group comprising only six chromosomes.
Table 5 Comparison of haplotype frequencies (additive model), as tested by chi-square analysis and permutations test, HS vs. HR
samples, entire cohort (N = 211) (Continued)
AHR Block 1 SNPs 1, 2, 3, 4, 5, 7, AHR Block 2 SNPs 8, 9, 10, 11, 12, 13, 14, 15, 16, CYP1A1 SNPs 1, 2, 3, 4, CYP1A2 SNPs 1, 2, 3, CYP1B1 Block 1 SNPs 1, 2, 3, 4, CYP1B1
Block 2 SNPs 5, 6, 7, 8, 9, 10, 11, 12
*Statistically significant (P < 0.05)
**Statistically significant (P < 0.005)
To investigate the possibility that haplotype ACTTGATC
in our African-American cohort simply represented a
highfrequency haplotype among Africans, we turned to the
1000 Genomes Project and searched for average
frequencies of each SNP within CYP1B1 haploblock 2 among
African populations; we inferred the best reconstructed
haplotypes and estimated their frequencies (Additional file
2: Table S1). Interestingly, haplotype ACTTGATC was
found to be the most common (ranked #1)—among the
sum of all African genomes sequenced to date—with a
frequency of 0.356.
Regarding the majority of our HNSCC cohort who
were of self-identified Caucasian descent (N = 196,
i.e., 392 chromosomes), we failed to find an
association (P = 0.249) between haplotype ACTTGATC and
HNSCC risk (Table 10). Using data extracted from
the 1000 Genomes Project for European populations
(Additional file 2: Table S1) and analyzing it
similarly to what had been done for African populations,
we found that haplotype ACTTGATC has a
frequency of 0.003—ranking #9 among inferred
haplotypes for Europeans.
Taking into account the significant difference
(P < 0.0001) in frequency of haplotype ACTTGATC
between European and African populations represented in
the 1000 Genomes Project (Table 11), and the apparent
lack of significant association between this haplotype
and HNSCC risk when African-Americans (Table 9)
and Caucasian-Americans (Table 10) were analyzed
separately, we are able confidently to conclude that the
association found in our initial analysis was spurious and
caused by population stratification—a common artifact
that can lead many statisticians to reporting a false
positive (type I error) as a true positive.
The present study represents a candidate-gene analysis,
using the extreme discordant phenotype (EDP) method,
attempting to determine a genotype-phenotype
association—in which the trait being studied is HNSCC among
cigarette smokers. The four candidate genes included
AHR, CYP1A1, CYP1A2, and CYP1B1. Based on
innumerable laboratory animal studies, these four genes
were selected because they encode three CYP1 enzymes,
plus AHR which regulates the levels of these enzymes.
This gene battery  reflects one of the earliest
responses to cigarette smoke as an environmental signal.
Moreover, HNSCC is most likely to develop as the result
of “direct-contact” of head-and-neck epithelial cells with
chronic irritants in cigarette smoke. For example, if any
nonsmoker were to smoke even one cigarette—these
three CYP1 genes would become up-regulated by AHR
(to genetically varying degrees) within a few hours;
without further “stimulation” (i.e., no more cigarettes),
AHR-regulated CYP1 genes would quickly return to
baseline. In laboratory animals, there is ample evidence
that these four genes play an “up-front” role in
PAHinduced cancer [5, 27, 38].
Statistical power of EDP analysis
An accurate power calculation of EDP design—based on
exposure level [EDP design 2 of the  article [to a
known risk factor (e.g., cigarette smoking)] —would
require quantification of several features: (a) phenotypic
variance attributable to the environmental exposure, (b)
truncation selection areas (e.g., 1–40 cigarette
packyears for HS cases, >80 cigarette pack-years for HR
controls), and (c) disease prevalence. Given that these values
are not well known, it would be very difficult to provide
an accurate power calculation.
However, if we can roughly assume that cigarette
smoking explains >20% variance in liability of HNSCC
that the selection areas (1–40 vs. >80 cigarette
packyears) represent the bottom and the top 10% exposure
groups, respectively, and that the prevalence of HNSCC
in all smokers is ~1%, then our EDP design would have
similar power to a normal case/control study comprising
500~600 samples (Fig. 9 of the  article).
Previous genotype-phenotype association studies: HNSCC
and our four candidate genes
We found no studies in the literature concerning AHR
polymorphisms associated with HNSCC. With regard to
Table 8 Detailed studies of chromosomal frequencies of CYP1B1 haplotype ACTTGATC in haploblock 2
*Statistically significant (P < 0.0001) by Fisher’s exact test (two-tailed), comparing AFR with CAU (HS patients)
Table 7 Detailed studies of chromosomal frequencies of
CYP1B1 haplotype ACTTGATC in haploblock 2
Caucasian-Americans and African-Americans in present cohort,
combined (Latino-American excluded)
CYP1 polymorphisms and HNSCC risk, six of the
seemingly most relevant studies are detailed below.
(a) The Leu-432 allele of CYP1B1 has been associated
with a greater frequency [odds-ratio (O.R.) >4] of TP53
gene mutations and HNSCC in cigarette smokers ;
however, this “greater risk” is difficult to reconcile
experimentally—given the fact that the Leu-432 mutant
was shown in an E. coli expression assay in vitro to
metabolize PAHs only 1.2- to 1.5-fold better than the
Val-432 variant . (b) Four nonsynonymous coding
SNPs (p.R48G, p.A119S, p.L432V, and p.N453S) were
studied in 150 cases of HNSCC and 150 controls ;
when the four SNPs were analyzed as a haplotype, amino
acid changes p.A48G and p.A119S exhibited complete
LD in all cases and controls, and significant differences
(P < 0.05) were reported in the two haplotypes—GTCA
and GTGA—as being associated with increased risk of
HNSCC. (c) Another study, comparing 312 HNSCC
cases and 300 noncancer controls, looked at the impact
of 22 sequence variations in CYP1A1, CYP1B1, CYP2E1,
GSTM1, and six other genes encoding either
xenobioticmetabolizing or DNA repair enzymes ; using logit
regression and a Bayesian version of logit regression,
authors found significant (P < 0.05) associations of CYP1B1
p.L432V and CYP2E1 −70G > T (O.R. 10.84; 95% CI,
1.64–71.53), as well as CYP1B1 p.L432V and GSTM1
(0/0) null/null (OR 11.79; 95% CI, 2.18–63.77) with
HNSCC risk. (d) In another study of 153 HNSCC cases
and 145 controls with no current or previous diagnosis
of cancer , authors examined CYP1A1, CYP1A2,
CYP2E1, GSTM1, and GSTT1 SNPs as risk factors in
HNSCC; a significant difference (P < 0.001) was detected
for tobacco and alcohol consumption between cases and
controls; moreover, the CYP1A2*1D (OR 16.24) variant
and GSTM1(0/0) null alleles (OR 0.02) conferred
increased risk of HNSCC, and alcohol consumption in
HNSCC patients was associated with the CYP2E1*5B
variant allele (P < 0.0001, OR 190.6). (e) Performing
meta-analysis of six published case-control studies of
HNSCC—which included some studies that had
concluded no significant risk—authors  found that the
CYP1B1 p.L432V polymorphism was significantly related
with HNSCC risk (OR = 1.13, 95% CI = 1.03–1.25,
P = 0.014), whereas no significant association between
CYP1B1 p.N453S polymorphism and HNSCC risk was
found. (f ) Finally, a case-control study of 750 HNSCC
patients and equal number of healthy controls
investigated the association of polymorphisms in CYP1A1,
CYP1B1, CYP2E1, and GSTM1 with HNSCC ; cases
having variant haplotypes of both CYP1A1*2A and
CYP1A1*2C or CYP1B1*2 and CYP1B1*3 or CYP2E1*5B
and CYP2E1*6 were at significant (P < 0.05) risk of
developing HNSCC; statistical analysis revealed a more
than multiplicative interaction between combinations of
these variant CYP genotypes plus the GSTM1(0/0) null
genotype and between variant genotypes and tobacco
smoking or chewing or alcohol consumption—indicating
these genes contribute a “modest risk factor” for
developing HNSCC via gene-gene and gene-environment
interactions. To learn more about each CYP allele
described above, please refer to http://www.cypalleles.
What sets our study apart from all previous studies?
None of the studies mentioned above, however, had
distinguished between HNSCC in smokers and
nonsmokers. Also, in contrast to the present study that used
the EDP method of sample selection, all the studies
mentioned above used, as their control population—
nonsmokers, or any smoker not having cancer.
Furthermore, none of these studies employed any form of
multi-locus statistical testing; this is a very serious
recurring problem in the medical literature with regard to
studies attempting to discover associations between one
or more SNPs and a multifactorial trait [4, 36], which is
a type I (false-positive) error variously called “P < 0.05
studies”  and the “incidentalome” .
Table 9 Detailed studies of chromosomal frequencies of
CYP1B1 haplotype ACTTGATC in haploblock 2
A type I error (false-positive) likely resulting from
The present study, at least based on the size of our
cohort, rejected all four AHR, CYP1A1, CYP1A2, and
CYP1B1 genes as having any statistically significantly
detectable contribution to HNSCC in smokers; further, we
first found a false-positive observation—that the CYP1B1
haplotype ACTTGATC might be associated with
HNSCC risk in our cohort (Table 7). Instead, this finding
was realized to be extremely unlikely, due to haplotype
frequency differences of more than 100-fold between
African and Caucasian populations in the 1000 Genomes
Project (Additional file 2: Table S1); hence, our study is
an excellent teaching example of the consequences of a
population stratification artifact.
Increasing appreciation of CYP1-mediated detoxication,
as well as metabolic activation
One additional consideration is that we now know from
transgenic mouse studies that all three CYP1 enzymes
can participate in both metabolic activation and
detoxication—depending on dose, length of exposure, route of
administration, and target organ specificity [5, 35, 38].
Therefore, it seems clear that virtually all previous
epidemiological studies are ambiguous because such studies
have considered only the “metabolic activation”
component . Yet, when cancer initiation occurs via “direct
contact” of a carcinogen—such as cigarette smoke
exposure causing HNSCC—one would presume that
CYP1-mediated metabolic activation  would be a
much more likely determinant than detoxication .
However, in the population studied herein, we failed to
demonstrate a correlation between the genotype of AHR
and all three CYP1 genes and HNSCC in smokers. This
Table 10 Detailed studies of chromosomal frequencies of
CYP1B1 haplotype ACTTGATC in haploblock 2
Table 11 Detailed studies of chromosomal frequencies of
CYP1B1 haplotype ACTTGATC in haploblock 2
Caucasians (EUR) and Africans (AFR) in The 1000 Genomes Project
ACTTGATC* Other Total Haplotype
haplotypes chromosomes frequency
EUR 3 1003 1006 0.003
does not mean that—if a much larger cohort were
collected—an association between HNSCC and small-effect
contribution by one or another of these four genes
might not emerge. We found a lack of association
because our Caucasian and African cohorts were
apparently too small, especially the African population
(both HS and HR).
Environmental risk factors for HNSCC
Tobacco (smoking cigarettes, cigars or pipe,
secondhand smoke, chewing tobacco, and using snuff ) probably
represents the single greatest risk factor for developing
HNSCC. The vast majority (~85%) of HNSCC is
associated with tobacco usage. Heavy and frequent alcohol
consumption is the second highest risk factor. Often
intertwined with tobacco and heavy alcohol usage, poor
oral and dental hygiene, malnutrition, and cannabis
usage may enhance HNSCC risk. Men are two- to
threefold more likely than women to develop HNSCC, and
patients above age 40 are at higher risk. Exposure to
occupational inhalants (wood dust, paint fumes, and certain
chemicals) may increase a person’s HNSCC risk. Finally,
gastroesophageal-reflux disease (GERD),
laryngopharyngealreflux disease (LPRD), and a weakened immune system can
raise the risk of HNSCC. HPV is commonly seen in some
types of HNSCC, as detailed below.
Past and present concepts of the role of HPV in HNSCC
Over the past two decades, HPV-positive squamous cell
carcinoma has emerged as a distinct subset of HNSCC,
based on multiple lines of evidence. Such evidence for
an etiologic role of HPV includes (a) observations of
higher rate of anti-HPV antibody sero-positivity—even
after adjustment for smoking—among patients with
HNSCC, compared with that among cancer-free
individuals ; (b) preferential occurrence of HPV-positive
tumors in the oropharynx ; (c) unique demographic
features, including an increased number of lifetime
sexual partners and less exposure to tobacco and alcohol
; (d) lower rates of TP53 gene mutations, in
HPVpositive than HPV-negative oropharyngeal tumors ;
and (e) improved survival among HPV-positive
oropharynx cancers, compared to HPV-negative oropharynx
carcinomas in both retrospective  and prospective 
analyses of clinical trial data.
Given the clinical impact of HPV infection in
oropharyngeal SCCs, clinical testing for the presence of virus in
newly diagnosed tumors at this site is considered
mandatory and guides therapy—including enrollment in
clinical trials investigating de-intensified radiotherapy
regimens . Such testing may include
immunohistochemical staining for p16, the overexpression of which
occurs when HPV proteins E6 and E7 inactivate TP53
and RB1 proteins, thereby serving as a surrogate marker
of viral infection. Alternatively, HPV-specific methods
such as in situ hybridization staining of viral DNA or
polymerase chain reaction (PCR)-based detection of viral
DNA or RNA may be used. Many argue that p16
immunohistochemistry is sufficient for oropharyngeal
carcinomas  and that PCR-based tests may be overly
sensitive and detect latent HPV unrelated to
carcinogenesis in a given tumor .
Despite the well-established role of HPV in
oropharyngeal SCC and its attendant clinical implications, classic
carcinogenic effects of tobacco and alcohol appear to
abrogate the improved survival of these tumors.
Specifically, HPV infection may occur after significant exposure
to tobacco and/or alcohol and therefore may not be
associated with a statistically different outcome than
HPVnegative tumors . On the basis of these mitigating
effects of tobacco exposure in HPV-positive tumors, risk
stratification schemes that incorporate both smoking
history and tumor HPV status have been developed .
It should also be noted that the etiologic role and
clinical impact of HPV in nonoropharyngeal SCC has not
been clearly established; for example, the prevalence of
virus in a study of oral cavity SCCs was low, and clinical
features of patients with, vs. without, tumor-containing
HPV were similar .
Whereas our cohort did include 15-20% oropharyngeal
SCCs, we believe our results are still applicable to this
subset, although we did not analyze the tumors for HPV
infection. Specifically, it may be argued that—because
the carcinogenic effects of tobacco appear to supersede
the effects associated with HPV-driven oropharyngeal
SCC, and not all oropharyngeal tumors are etiologically
due to HPV—the lack of genotype-phenotype
correlation that we have clearly demonstrated in this study is
highly likely to hold true in the oropharynx, as well as in
other head-and-neck subsites.
With regard to our EDP method of rigorously selecting
cohorts of N = 112 highly-sensitive (HS) light smokers
with HNSCC and N = 99 highly resistant (HR) heavy
smokers with no cancer, we conclude that the carefully
chosen single-nucleotide variants located in and near
four genes—AHR, CYP1A1, CYP1A2, CYP1B1, alone, or
in combination—are not statistically significantly
associated with risk of cigarette-smoking-induced HNSCC.
One haploblock, ACTTGATC in the 5′ portion of
CYP1B1, retained statistical significance after 100,000
permutations, but this was discovered to be a type I
error (false positive) finding, due to spurious association
by population stratification.
AHR: Aryl hydrocarbon receptor protein; AHR: Gene encoding aryl
hydrocarbon receptor; Cig-Pk-Yr: Cigarette-pack-year (20 cigarettes smoked
per day for 1 year); CYP1A1: Gene encoding cytochrome P450 family 1
subfamily A member 1; CYP1A1: Name of enzyme; CYP1A2: Gene encoding
cytochrome P450 family 1 subfamily A member 2; CYP1A2: Name of enzyme;
CYP1B1: Gene encoding cytochrome P450 family 1 subfamily B member 1;
CYP1B1: Name of enzyme; EDP: Extreme discordant phenotype method of
population analysis; gDNA: Genomic DNA; HNSCC: Head-and-neck squamous
cell carcinoma; HPV: Human papilloma virus; LD: Linkage disequilibrium;
SNPs: Single nucleotide polymorphisms, variant alleles
We thank Drs. Michael Wolfe, Lyon Gleich, Reena Dhanda-Patil, and Li Jin for
their help early in this study. We are grateful to nurses Cathy Bailey, Nancy
Emrath, JaMyllah Payne, and especially Victoria Straughn for interfacing
tirelessly between the Department of Otolaryngology-Head and Neck Surgery
Outpatient Clinics (Barrett Cancer Center) and Cincinnati VA Hospital and our
laboratories. We thank colleagues for valuable discussions and careful
reading of this manuscript.
Grant support included National Institutes of Health Grants R01 DE016325 (to
D.W.N.), P30 ES006096 (to D.W.N. and S.M.P.), and a 2003–04 Pilot Program
Project sponsored by ES009066 (to D.W.N and J.L.G.).
Availability of data and materials
Data sharing not applicable to this article.
LFJ-N carried out the DNA isolation and molecular genetic studies. LFJ-N, GZ,
and ZJ performed statistical analyses. JLG and DWN originally designed the
study. LFJ-N, GZ, ZJ, RB, and DWN drafted the manuscript. JLG, KMW, and
DWN determined the clinical status for participation in the study. SMP
interfaced with nurses drawing bloods, coding of data, and assessing
questionnaires. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Clinical protocol—titled “Human Cancer and AHR/CYP1A1/1A2/1B1 Gene
Polymorphisms” (#03-08-07-01)—was approved annually, by the University of
Cincinnati Medical Center Institutional Review Board (IRB).
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