Yonago Acta medica 2006;49:1–7

HRAS1 Variable Number of Tandem Repeats Polymorphismin Japanese Patients with Colorectal Adenoma and Cancer
Masahiko Miura, Naoto Maeda and Yoshikazu Murawaki
Division of Medicine and Clinical Science, Department of Multidisciplinary Internal Medicine,
School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8504 JapanThe highly polymorphic HRAS1 variable number of tandem repeats (VNTR) has been
described as an inherited predisposing factor in various human cancers. The aim of thepresent study was to evaluate the association between the presence of rare HRAS1 VNTR
alleles and colorectal adenoma and cancer. A total of 165 Japanese patients underwent
total colonoscopy with informed consent, and were divided into 2 groups: colorectal
neoplastic and non-neoplastic patients. Two hundred and sixteen HRAS1 VNTR alleles
from 108 colorectal neoplastic patients (67 adenomas and 41 cancers) and 114 alleles
from 57 non-neoplastic patients were genotyped using PCR-based long-agarose gel electrophoresis assay of peripheral blood leukocyte DNA. Rare alleles were differentiatedfrom 4 types of common allele (a1, a2, a3 and a4) by shifts in electrophoretic mobility.
The prevalence of rare HRAS1 VNTR alleles was higher in colorectal neoplastic patients
than in non-neoplastic patients (25.4% and 34.1% versus 8.8%). The adjusted odds ratio with at least one rare allele was 8.65 (95% conﬁdence interval = 2.93–25.53, P < 0.0001)
in colorectal neoplastic patients. The presence of rare HRAS1 VNTR alleles could be a
genetic predisposing factor for risk of colorectal neoplasm in Japanese people.
Key words: colorectal neoplasm; HRAS1 variable number of tandem repeats; polymorphism

The multifarious molecular changes in cancer
development frequently involve alterations on
minisatellites or variable number of tandem repeats (VNTR) as well as allelic deletions or lossof heterozygosity. Since the HRAS1 VNTR allelicpolymorphism in normal individuals and cancer
patients was surveyed by Krontiris et al. in 1985,
this polymorphism has been reported to be an
inherited predisposing factor in various cancers,
including colorectal, ovarian and lung cancers,
so far (Krontiris et al., 1993; Lindstedt et al.,
1999; Weitzel et al., 2000). The proto-oncogene
HRAS1, located on the short arm of chromosome
11, encompasses 4 exons ﬂanked by a VNTR region at the 3' end which consists of a basic 28 bp

Recently, the rate of colorectal cancer has been
rapidly increasing in Japan, and colorectal cancer
is now the main cause of death from malignant
disease as in many other countries (Yoshimi andSobue, 2004). In fact, age-standardized incident
rates are similar to rates in US populations of
Caucasians (Yiu et al., 2004). The reason has
generally been ascribed to the westernized diet,characterized by a high intake of fat and meat especially after World War II (Kono, 2004), whereas the relevance of genetic predispositions has notbeen sufﬁciently analyzed.
Colorectal cancer is a multifactorial disease,with dietary factors, lifestyle habits and genetic
predispositions contributing to its development.

Abbreviations: CI, conﬁdence interval; OR, odds ratio; PCR, polymerase chain reaction; VNTR, variable number of tandem repeats1M. Miura et al.

consensus sequence. The HRAS1 VNTR consistsof four common progenitor alleles, which werepreviously identiﬁed as a1, a2, a3 and a4 having
repeats of 30, 46, 68 and 84 units, respectively.
In addition, several rare variants are thought toderive from germ-line mutations of the nearest
common alleles (Kasperczyk et al., 1990). These
common alleles represent 94% in Caucasian populations (Krontiris et al., 1985). Minisatellite alleles frequently vary not only in the number of repeat copies but also in the interspersion pattern of
the repeat sequence along the VNTR (Conway etal., 1996; Ding et al., 1999). Although the actualrole of specific HRAS1 VNTR alleles in cancer
susceptibility is not yet known, it has been shownthat the HRAS1 VNTR binds at least 4 membersof the rel NF-κB family of transcriptional regulatory factors, suggesting that the VNTR could affect cancer susceptibility by transcriptional modulation of HRAS1 or other nearby genes (Trepicchio
and Krontiris, 1992; Green and Krontiris, 1993;
Mukhopadhyay et al., 1995). The aim of the present study is to examine whether Japanese individuals with rare HRAS1 VNTR alleles are at anincreased risk for developing colorectal neoplasm.

was obtained from all patients and non-neoplasm
subjects. This study was approved by the Institutional Review Boards of Tottori University Faculty of Medicine (approval number: G 16).
HRAS1 VNTR genotyping
Genomic DNA was extracted from peripheral
blood samples using DnaQuick II DNA extraction
kits (Dainippon Pharmaceutical, Osaka, Japan)
as recommended by the supplier. Using the extracted DNA as a template, the HRAS1 VNTR region was ampliﬁed by polymerase chain reaction(PCR) using a commercially available kit, Expand
High Fidelity PCR System (Roche Diagnostics,
Mannheim, Germany), according to the manufacturer's instructions. The sequences of the primers
were: the forward primer, 5'-ATC TAC AGT CCC
CCT TGC CG -3'; the reverse primer, 5'-GCAACT GAC CGT GCA AGT CA -3'. Reactions
were carried out using the following conditions: 2
min at 95°C and then ampliﬁed for 10 cycles consisting of 15 s at 94°C, 30 s at 62°C and 2 min at
72°C, followed by 20 cycles of 15 s at 94°C, 30 s
at 62°C and 2 min plus cycle elongation of 5 s foreach cycle at 72°C. A ﬁnal extension step at 72°
C for 7 min was added to terminate the ampliﬁcation.
Subsequently, the PCR product was electrophoresed on a 1% agarose gel 30 cm in length
using TBE buffer (45 mM Tris-borate, pH 8.3
and 2 mM EDTA) to conﬁrm the size. Molecularsize marker IV (Nippon Gene, Tokyo, Japan) anda 100 bp ladder marker (Bio-Rad Lab., Hercules,CA) were loaded in approximately every 7th and8th lane on all gels to minimize artifacts due togel distortion. Gels were stained with ethidium
bromide and destained in water to allow direct
visualization of the alleles (Fig. 1). The 4 mostcommon HRAS1 VNTR alleles were labeled a1,
a2, a3 and a4. The other alleles were labeled interms of the difference in number of 28 bp repeat
units from that of “the common allele” closest in
size. For example, “a1+” refers to the allele with
28 bp repeat unit more than a1, “a1-” refers to theMaterials and Methods
Subjects
A total of 165 Japanese subjects (78 women and
87 men; age range, 19–90 years; median, 67
years) were studied between August 2003 andMarch 2005. All of the subjects had undergone
total colonoscopy just before the enrollment. For
extraction of DNA, 5 mL of peripheral blood
was obtained from each of the 108 patients withhistologically conﬁrmed colorectal neoplasm (41cancers and 67 adenomas) and 57 non-neoplasm(neoplasm-free) subjects. Subjects were interviewed for medical history, family history of neoplastic disease and health habits such as alcohol
intake and cigarette smoking. Before enrollment
and blood sampling, written informed consent
2HRAS1 polymorphism and colorectal neoplasm

Fig. 1. Representative examples of the HRAS1 VNTR allele. Rare alleles are indicated by the addition (+) or deletion(–) of 28 bp motifs from common alleles. Lanes M4 and L: molecular weight standards.

allele with 28 bp repeat unit less than a1; these arereferred to as “rare alleles”.

Results

Statistical analysis

Characteristics of research subjects

As in other published studies, alleles were categorized into two sets: common and rare. The association between rare HRAS1 VNTR alleles andcolorectal neoplasm was assessed by comparing
the proportions of patients and non-neoplasm subjects with one or more rare alleles. The statisticalsigniﬁcance of the different means or proportions
was measured using analysis of variance or chisquare test. To evaluate increased risk of colorectal neoplasm associated with the presence of rare
HRAS1 alleles, odds ratios (ORs) and 95% conﬁdence intervals (CIs) were computed and adjusted
by logistic regression for several covariates potentially associated with neoplastic risk such as age,sex, family history of neoplasm, alcohol intake
and smoking status. The statistical analyses were
performed using the software package SPSS II for
Windows (version 11.0 J, SPSS Japan, Tokyo).

The characteristics of participants are summarized in Table 1. Adenoma cases were more likely
than non-neoplasm cases to be male and to havea family history (all of malignancy), and cancer
cases were more likely to be older and to smoke
habitually. No differences between neoplastic andnon-neoplastic cases were observed in other clinicopathological variables.
HRAS1 VNTR allelic patterns
The HRAS1 VNTR allelic patterns are shown in
Table 2. We genotyped 216 HRAS1 VNTR alleles
from 108 patients with colorectal neoplasm (41cancers and 67 adenomas) and 114 alleles from 57
non-neoplasm subjects. Regarding the distribution of common alleles in our study, most subjects
had a1 alleles, and none had a3 or a4 alleles.

3M. Miura et al.

Table 1. Characteristics of research subjects
Variables

Non-neoplasm [57]
NumberAdenoma [67]
Number (%)(%)

Age (year)
60.6 ± 16.6
Sex (male/female)
23/34
Family history
All of malignancies
18/50 (36.0)
Only colorectal cancer
7/50(14.0)
Alcohol*(60 mg/day)
9/53(17.0)
Smoking*(habitually)
15/52 (28.8)

Colorectal neoplasm
P†
Cancer [41]
Number
(%)

65.4 ± 9.4
41/26
32/53
3/53
12/53
22/53

(60.4)( 5.7)(22.6)(41.5)

0.11
0.02

68.4 ± 10.9
23/18

0.01
0.15
0.46
0.17

19/41
8/41
7/41
21/41

P†
0.01
0.12

(46.3)(19.5)(17.1)(51.2)

0.32
0.48
0.99
0.03

Continuous variables expressed as mean ± SD.
[ ], number of subjects.
* Assessed only in available patients.
† P values were calculated versus non-neoplasm. Statistical signiﬁcance was deﬁned as P < 0.05.

Association between HRAS1 VNTR andcolorectal neoplasm

the OR was 13.16 (95% CI = 3.69–47.00, P <0.0001). The OR in adenoma was calculated at
6.59 (95% CI = 2.07–21.02, P < 0.001).

To evaluate increased risk of colorectal neoplasm
associated with the presence of rare HRAS1 alleles, odds ratios (ORs) and 95% CIs were computed and adjusted by logistic regression forseveral covariates potentially associated withneoplastic risk such as age, sex, family historyof neoplasm, alcohol intake and smoking status(Table 3). The adjusted OR for colorectal neoplasm was 8.65 (95% CI = 2.93–25.53, P < 0.0001).
Repeating the analyses among cancer cases only,

Discussion
In addition to many environmental factors, genetic polymorphisms are thought to inﬂuence the
carcinogenesis of colorectal cancer. Although
there have been many candidate genes associated with colorectal carcinogenesis, only 2 genes,
HRAS1 VNTR and APC-1 1307K, were foundTable 2. HRAS1 VNTR allelic patterns
Non-neoplasm

Common alleles

a1
a2
a3
a4
Subtotal

Rare alleles~a1a1+~a2a2+~a3a3+~a4a4+~
Subtotal

Total

Number(%)

Colorectal neoplasm
AdenomaCancerNumber(%)
Number
(%)

103
1
0
0
104

(90.3)
(0.9)
(0)
(0)
(91.2)

100
0
0
0
100

9
(7.9)
1
(0.9)
0
(0)
0
(0)
0
(0)
10
(8.8)
114
[57 individuals](74.6)(0)(0)(0)(74.6)

19(14.2)
15(11.2)
0
(0)
0
(0)
0
(0)
34
(25.4)
134[67 individuals]

4

54
0
0
0
54

(65.9)(0)(0)(0)(65.9)

17(20.7)
11(13.4)
0
(0)
0
(0)
0
(0)
28
(34.1)
82[41 individuals]

HRAS1 polymorphism and colorectal neoplasm

Table 3. Association between HRAS1 VNTR and colorectal neoplasm
Genotypes

Non-neoplasm [57] Neoplasm [108]Common/common
Common/rare or rare/rare
Odds ratio
95% conﬁdence interval
P

50
7
1.00
2.93–25.53
Not signiﬁcant

60
48
8.65
2.07–21.02< 0.0001

Type of tumor
Adenoma [67]Cancer [41]
42
25
6.59
3.69–47.00< 0.001

18
23
13.16
< 0.0001[ ], number of subjects.
P: less than 0.05 is signiﬁcant.

least one rare allele was 8.65 (P < 0.0001), showing the close association of HRAS1 VNTR polymorphisms with colorectal neoplasms in Japanese. Furthermore, the adjusted OR of colorectal
cancer was markedly higher. The recent increasein colorectal cancer patients in Japanese may be
due to additional external factors such as post-war
dietary changes in individuals with genetic predispositions.
This is the ﬁrst report to clarify the signiﬁcant relationship of HRAS1 VNTR and colorectal
adenoma. Previous studies reported only somecomparison with colorectal cancer, and did not
discuss adenoma. Generally, neoplasms develop
in the adenoma-carcinoma sequence (Vogelstein
et al., 1988) in most colorectal cancers. It has
been suggested that HRAS1 VNTR polymorphisms are involved in the early stage of tumorigenesis as a genetic predisposing factor.
We also found the difference in OR between
adenoma (OR; 6.59) and cancer (OR; 13.16) patients. Although the reason for the increase in OR
in colorectal cancer patients is not clear, many
hereditary predispositions may be involved in the
carcinogenesis in a complex manner.
Since Krontiris et al. (1985) reported on theassociation of HRAS1 VNTR with carcinogenesis,
other associations with various other cancers besides colorectal cancer have been reported mainlyin Europe and America. However, some reports
presented negative views of a causal relationshipin recent cases of breast cancer and urinary bladder cancer (Firgaira et al., 1999; van Gils et al.,to have a polymorphism that increases colorectal carcinogenesis in meta-analysis reported by
Houlston and Tomlinson (2001). De Jong et al.
(2002) reviewed 20 genes (30 gene polymorphisms) discussed in several reports, and speculated that HRAS1 VNTR, GSTT1, NAT2 (phenotype), ALDH2 and TNF-α (α2 allele) increasecarcinogenetic risk. They summarized that
HRAS1 VNTR was involved in colon carcinogenesis with an OR of 2.50 to 2.67. However, thesestudies of the association between HRAS1 VNTR
polymorphisms and colorectal cancer were performed on Caucasians, while there has been no
study performed on Japanese. Racial differencesin the HRAS1 VNTR allele distribution have beenreported (Weston et al., 1991). In our subjects,
the most common alleles were a1, and no larger
alleles (a3 and a4 alleles) were noted. In a previously reported HRAS1 VNTR allele distribution
in Japanese people (Ishikawa et al., 1987; Honda
et al., 1988; Pierce et al., 2000), a1 accounted fora high ratio, and the frequencies of a2, a3 and a4
alleles were less than 10% in each allele. The absence of a3 and a4 in our study suggests the presence of regional differences, in addition to racialdifferences, but the details are not clear becauseof the small number of patients.
In this study, in agreement with previous reports on Caucasians (Klingel et al., 1991; Krontiris et al., 1993; Gosse-Brun et al., 1998; Vega etal., 2001), the frequency of the rare alleles was
significantly higher in the neoplasm group andthe adjusted OR of colorectal neoplasm with at5

M. Miura et al.

2002; Tamimi et al., 2003). This may have been
due to advances in molecular-biological techniques that have allowed the resolution of singlerepeat differences (particularly in larger sizes),
leading to a more precise classification of common and rare alleles than classiﬁcation by Southern blot. In recent reports of colorectal cancer
using the PCR method (Vogelstein et al., 1988;
Vega et al., 2001), a significant correlation wasnoted between HRAS1 VNTR and colorectal cancer. In this study, we also used the PCR method,and found significant differences between nonneoplasm and neoplasm patients.
In conclusion, there is a strong association
between rare alleles of the HRAS1 VNTR andcolorectal neoplasm in Japan. If these results are
conﬁrmed, then from the standpoint of preventive
medicine, HRAS1 VNTR polymorphisms may
be useful in identifying risk groups for colorectal
neoplasms. Further investigation of differencesin HRAS1 VNTR polymorphisms in development
sites, histologic types, and prognosis of colon cancer may be necessary.

Hum Mol Genet 1999;8:515–521.
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GG, McCredie MR, et al. HRAS1 rare minisatellite
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6 Green M, Krontiris TG. Allelic variation of reporter
gene activation by the HRAS1 minisatellite. Genomics 1993;17:429–434.
7 Honda K, Ishizaki K, Ikenaga M, Toguchida J, Inamoto T, Tanaka K, et al. Increased frequency ofspeciﬁc alleles of the c-Ha-ras gene in Japanese cancer patients. Hum Genet 1988;79:297–300.
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and colorectal tumor r isk. Gastroenterology
2001;121:282–301.
9 Ishikawa J, Maeda S, Takahashi R, Kamidono S,
Sugiyama T. Lack of correlation between rare Haras alleles and urothelial cancer in Japan. Int J Cancer 1987;40:474–478.
10 Kasperczyk A, DiMa r tino NA, K rontiris TG.
Minisatellite allele diversification: the origin ofrare alleles at the HRAS1 locus. Am J Hum Genet
1990;47:854–859.
11 Klingel R, Mittelstaedt P, Dippold WG, Meyer zum
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12 Kono S. Secular trend of colon cancer incidence andmortality in relation to fat and meat intake in Japan.
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13 Krontiris TG, DiMartino NA, Colb M, Parkinson
DR. Unique allelic restriction fragments of the human Ha-ras locus in leukocyte and tumour DNAs ofcancer patients. Nature 1985;313:369–374.
14 Krontiris TG, Devlin B, Karp DD, Robert NJ, RischN. An association between the risk of cancer andmutations in the HRAS1 minisatellite locus. N Engl
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15 Lindstedt BA, Ryberg D, Zienolddiny S, Khan H,
Haugen A. Hras1 VNTR alleles as susceptibility markers for lung cancer: relationship to microsatellite instability in tumors. Anticancer Res
1999;19:5523–5527.
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17 Pierce LM, Sivaraman L, Chang W, Lum A, Donlon
T, Seifried A, et al. Relationships of TP53 codon 72
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Ackowledgments: The authors are indebted to Prof. Eiji
Nanba and Mrs. Kaori Adachi of the Gene ResearchCenter, Tottori University Faculty of Medicine, for technical support.
This study has been awarded prizes at United European Gastroenterology Week 2004 in Prague, the World
Conference of Gastroenterology 2005 in Montreal, andDigestive Disease Week-Japan 2005 in Kobe.
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Received December 9, 2005; accepted December 21, 2005
Corresponding author: Masahiko Miura7