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The Prostate 33:9–12 (1997)
Genetic Variation of 3b-Hydroxysteroid
Dehydrogenase Type II in Three Racial/Ethnic
Groups: Implications for Prostate Cancer Risk
Sunita A. Devgan,1 Brian E. Henderson,2 Mimi C. Yu,2 Chen-Yang Shi,3
Malcolm C. Pike,2 Ronald K. Ross,2 and Juergen K.V. Reichardt1,4*
1
Department of Biochemistry and Molecular Biology, University of Southern California
School of Medicine, USC/Norris Comprehensive Cancer Center, Los Angeles, California
2
Department of Preventive Medicine, University of Southern California School of Medicine,
USC/Norris Comprehensive Cancer Center, Los Angeles, California
3
Department of Community, Occupational and Family Medicine, National University of
Singapore, Singapore
4
Institute for Genetic Medicine, University of Southern California School of Medicine,
USC/Norris Comprehensive Cancer Center, Los Angeles, California
BACKGROUND. Elevated prostatic dihydrotestosterone (DHT) has been suggested to increase the risk of prostate cancer. The HSD3B2 gene encodes the type II 3b-hydroxysteroid
dehydrogenase: one of two enzymes that initiate the inactivation of DHT. Thus, the HSD3B2
gene is a candidate gene for predisposition to prostate cancer.
METHODS. We have determined the distribution of a complex dinucleotide repeat in the
HSD3B2 gene in high-risk African-Americans, intermediate-risk Euro-Americans, and lowrisk Asians. Genomic DNA from 312 individuals was amplified by polymerase chain reaction
(PCR) and analyzed by electrophoresis on denaturing polyacrylamide gels.
RESULTS. We have found that certain alleles are either unique to or much more common in
either African-Americans, Asians, or Euro-Americans. Our data also substantially expand the
number of alleles reported for the complex dinucleotide repeat polymorphism in the HSD3B2
gene.
CONCLUSIONS. Our report demonstrates substantial genetic variation in the HSD3B2 gene.
We hypothesize that allelic variants of the HSD3B2 gene may play a role in predisposition to
prostate cancer, and in explaining the substantial racial/ethnic variation in risk. Prostate
33:9–12, 1997. © 1997 Wiley-Liss, Inc.
KEY WORDS:
androgen metabolism; PCR; complex dinucleotide repeat polymorphism; racial/ethnic variation of prostate cancer risk
INTRODUCTION
Prostate cancer in the United States will be diagnosed in some 317,000 men in 1996 and over 41,000
will die of this disease [1]. This cancer is characterized
by marked variations in incidence among U.S. population groups: African-American men have a 70% increase in risk when compared to Euro-American (i.e.,
non-Hispanic White) males, while Asian-American
men (of either Chinese or Japanese ancestry) have an
approximately 60% decrease in risk when compared
© 1997 Wiley-Liss, Inc.
to males of European ancestry [2]. We have previously
suggested that elevated testosterone (T) levels and, in
particular, intraprostatic dihydrotestosterone (DHT)
Contract Grant sponsor: National Institutes of Health; Contract
Grant numbers: CA17054, CA53890, CA54281, and CA68581.
*Correspondence to: Juergen K.V. Reichardt, Institute for Genetic Medicine, USC/Norris Comprehensive Cancer Center, HMR413, 2011 Zonal
Ave., Los Angeles, CA 90033.
E-mail: reichard@zygote.hsc.usc.edu
Received 29 April 1996; Accepted 17 June 1996
10
Devgan et al.
levels may be responsible for some of this variation in
risk [3]. Molecular studies thus far have revealed few
mutations responsible for predisposition to prostate
cancer and early tumor progression [4,5].
T is converted to the more active intracellular metabolite, DHT, by steroid 5a-reductase [6]. DHT then
binds to the androgen receptor (AR) and the DHT-AR
complex transactivates a number of genes with ARresponsive elements [6]. This results in a variety of
physiologic effects including cell division. DHT is inactivated by either 3a- or 3b-hydroxysteroid dehydrogenase, further modified and ultimately excreted [6].
Thus, the 3-hydroxysteroid dehydrogenases are involved in the regulation of DHT levels which modulate prostatic cell division.
Two isozyme forms of 3b-hydroxysteroid dehydrogenase have been reported in humans. The type I enzyme is encoded by the HSD3B1 gene and is expressed
mostly in the breast, placenta, and skin [7]. The type II
enzyme is encoded by the HSD3B2 gene and is primarily expressed in adrenals, testis, and ovary [7]. The
human HSD3B1 and HSD3B2 genes have been shown
to be closely linked and mapped to a single band on
the short arm of chromosome 1 [1p13; 7,8]. Both genes
have been cloned and shown to be very similar spanning about 8 kb of genomic DNA in four exons each
[7]. A number of mutations in the HSD3B2 gene have
been found to cause a rare human disorder, congenital
adrenal hyperplasia [9]. These (and other) mutations,
however, do not appear to be involved in prostatic
diseases in adults.
Verreault et al. [10] reported a complex (TG)n (TA)n
(CA) n dinucleotide repeat polymorphism in the
HSD3B2 gene which can be detected by polymerase
chain reaction (PCR). The variable region, located
within the third intron of the gene and reported to
consist of eight alleles, was typed in 52 Caucasian individuals [10].
We have initiated systematic molecular investigations into the role of androgen-metabolic genes in
the racial/ethnic variation of prostate cancer risk. We
recently reported genetic variation of the SRD5A2
gene encoding steroid 5a-reductase which synthesizes
DHT from T [6,11]. The high-risk African-American
sample contained alleles that were absent from intermediate-risk Euro-Americans and low-risk Asians
[11]. Here, we examined the hypothesis that HSD3B2
is also a candidate gene for prostate cancer predisposition by genotyping males from three population
groups at very different risks for prostate cancer: highrisk African-American, intermediate-risk EuroAmerican, and low-risk Asian men. We report significant polymorphism in the HSD3B2 gene with some
alleles restricted to high-risk African-Americans. Several other alleles are characterized by significant dif-
ferences in their racial distribution. These data are
consistent with our hypothesis that androgenmetabolic genes such as HSD3B2 and SRD5A2 play a
role in predisposition to prostate cancer and may explain, at least in part, the substantial racial/ethnic
variation in risk.
MATERIALS AND METHODS
HSD3B2 Genotyping
Our study subjects consisted of multiethnic,
male residents of Los Angeles County (of African,
European, Chinese, and Japanese ancestry) and Chinese male residents of Singapore. The Los Angeles
component has been described previously [11,12]
and Chinese subjects in Singapore are part of an
ongoing cohort study similar in both design and
goals to the multiethnic cohort study in Los Angeles
[11,12].
Genomic DNA of study subjects of AfricanAmerican, Asian, and Euro-American men [11] was
PCR amplified using the primer pair of Verreault et al.
[10]. Syntheses of primers were performed in 30 nM
scales on a Beckman Oligo1000 (Beckman Instruments, Fullerton, CA). One primer was radiolabeled
by kinasing with g[32P]ATP (6,000 Ci/mmol; NEN,
Boston, MA) and T4 polynucleotide kinase (Promega,
Madison, WI; 13). PCR products were obtained in a
TwinBlock thermal cycler (Ericomp, San Diego, CA)
by repeating the following cycle 30 times: denaturation at 92°C for 2 min, annealing for 1 min at 62°C,
and extension at 72°C for 2 min. PCR reactions were
denatured and then fractionated on 4.5% denaturing
polyacrylamide gels (‘‘sequencing gels’’; 13) in parallel with DNA sequencing reactions obtained with Sequenase 2.0 (USB, Cleveland, OH). Gels were dried
and exposed overnight to Kodak BIOMAX autoradiography film (Rochester, NY).
Statistical Analysis
The ‘‘exact’’ method of Mehta and Patel [14] was
used to compare the overall prevalence of alleles
among the three racial/ethnic groups and between
each pair of groups. All tests were statistically highly
significant (two sided, P < 0.000001). Since the overall
tests were highly significant, we then tested individual
allele comparison using Fisher’s exact test (comparing
each allele against all others combined) between each
set of two racial/ethnic groups. Significant differences
in these results are quoted as one-sided P values in the
Results section [15].
HSD3B2 Polymorphism and Prostate Cancer Risk
TABLE I. Distribution of HSD3B2 Alleles in
Three Populations*
Allele (bp)
271–273
275
281
283–287
289
291
293–295
302–334
338
340
342
344–372
AfricanAmerican
(n = 256)
Euro-American
(n = 248)
Asian
(n = 120)
0
0.055
0.016
0.004
0.336
0.254
0.051
0.066
0.039
0.137
0.023
0.020
0.008
0
0.020
0.020
0.516
0.105
0.012
0.024
0.032
0.222
0.028
0.016
0.033
0
0.175
0.008
0.367
0.150
0.033
0.125
0.008
0.075
0.017
0.008
*Allele frequency figures were rounded as necessary and, therefore, may not add up to 1.0 (or 100% in the text).
RESULTS
We have further investigated the published
HSD3B2 genotyping system which involves a complex
(TG)n (TA)n (CA)n dinucleotide repeat and was reported to consist of eight alleles [10]. Thus far, we have
identified a total of 25 alleles. Table I presents allele
frequencies for the HSD3B2 gene by race in 312 control
subjects of African-American, Asian, and European
heritage (624 alleles). Some alleles were combined into
‘‘families’’ to condense the data for easier analysis (cf.
Table I). All alleles are identified by their size in base
pairs (bp).
The 289 bp allele is the most common allele in all
three population groups examined (Table I) in agreement with the previously published data on Caucasians alone [10]. This marker is significantly more
common among Euro-Americans (allele frequency =
51.6%) than among African-Americans (allele frequency = 33.6%; P = 0.00003) or among Asians (allele
frequency = 36.7%; P = 0.005). Several other allelic
markers show statistically significant differences in allele distribution in the three populations examined.
The second most common allele in all populations is
291 bp in size but its frequency differs dramatically
between African- (allele frequency = 25.4%) and EuroAmericans (allele frequency = 10.5%; P = 0.000009;
Table I). The same allele is intermediate in frequency
among Asians (15.0%; P = 0.015 vs. AfricanAmericans). The 275 bp allele has thus far been found
only in African-American men with a frequency of
5.5% (P = 0.00064 vs. Euro-Americans; P = 0.0041 vs.
Asians; Table I). The 293–295 bp family of alleles appears to be more common in African-Americans (allele
11
frequency = 5.1%; Table I) than it is among EuroAmericans (allele frequency = 1.2%; P = 0.011; Table I).
Furthermore, alleles ranging from 302 to 334 bp in size
are most common among people of Asian ancestry
(allele frequency = 12.5%). This allele frequency is
much lower among Euro-Americans (allele frequency
= 2.4%; P = 0.0002; Table I). These alleles are, however,
intermediate among African-American men when
compared to Asians (allele frequency = 6.6%; P =
0.047). The 281 bp allele is much more common among
Asians (allele frequency = 17.5%) than among African(allele frequency = 1.6%) or Euro-Americans (allele
frequency = 2%; Table I). Finally, the 340 bp allele is
more prominent among people of European ancestry
(allele frequency = 22.2%; Table I) than among those of
African (allele frequency = 13.7%; P = 0.009) or Asian
descent (allele frequency = 7.5%; P = 0.0002).
DISCUSSION
We report here extensive genotypic data on three
populations at markedly different risk for prostate
cancer: high-risk African-Americans, intermediaterisk Euro-Americans, and low-risk Asians. We PCR
typed the previously reported complex (TG)n (TA)n
(CA)n dinucleotide repeat polymorphism in the human HSD3B2 gene [10]. Our data indicate 1) that this
gene is more polymorphic than previously reported,
and 2) that certain polymorphisms are distributed differently among the three racial groups examined
(Table I). Thus far, we have identified 25 different alleles which expand substantially the original report of
8 alleles [10]. For ease of analysis, we have grouped
these 25 alleles into 12 families (Table I). In agreement
with the data on Caucasians reported earlier [10], the
289 bp allele is the most prominent allele in all three
populations examined (Table I). We also found one
allele that is unique to the African-American men
sampled (275 bp; Table I). Furthermore, we identified
two families of alleles—291 and 293–295 bp—that are
significantly more common among AfricanAmericans than they are among either EuroAmericans or Asians (Table I). The 340 bp allele is
significantly more common among people of European ancestry than among the two other groups we
genotyped in this study. Finally, we have identified
two families of alleles, 281 and 302–334 bp, that are
more common in Asian men (Table I).
We have previously proposed that the difference
in the racial/ethnic incidence of prostate cancer [2]
may be determined in part by different levels of T
and more specifically its intraprostatic metabolite
DHT [3]. The present study provides additional molecular genetic support for our hypothesis. The statistically significant finding of HSD3B2 repeat alleles that
12
Devgan et al.
are present only or more prominently so in high-risk
African-Americans or low-risk Asians (Table I) supports our initial study (3). We hypothesize here based
on our polymorphic data (Table I) that certain 3bhydroxysteroid dehydrogenase enzyme variants encoded by mutant HSD3B2 genes may result in altered
enzyme activity. Such mutations could result in
slower than normal degradation of DHT through decreased enzyme activity. Intraprostatic DHT levels
would rise accordingly, resulting in an increased risk
for developing prostate cancer. Conversely, other mutations that increase HSD3B2 activity might reduce the
risk for developing prostate cancer by increasing the
rate of DHT degradation. These hypothetical alleles
that may influence prostate cancer risk could be
marked by particular repeat alleles. The complex dinucleotide repeat polymorphism we typed in this
study is intronic [10] and, therefore, unlikely to be of
functional significance in this context. However, this
polymorphic repeat is a useful marker for molecular
investigations into prostate cancer risk.
The present report complements our previous work
on another androgen metabolic gene, SRD5A2, encoding steroid 5a-reductase type II [11]. In that communication we characterized substantial racial/ethnic
variation at this locus that correlated with risk. These
two investigations into genetic variability of two candidate genes support a role for genes encoding androgen-metabolic enzymes in explaining the racial/ethnic
differences in prostate cancer risk.
ACKNOWLEDGMENTS
This work was supported by NIH grants CA17054,
CA53890, CA54281, and CA68581. We thank Kristine
Monroe and Peggy Wan for help with data recording
and statistical computation.
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