| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Molecular Biology and Genetics |
Departments of Laboratory Medicine and Pathology [L. W., D. A. E., E. C., A. F. M., J. M. C., S. N. T.], Health Sciences Research [S. K. M., S. L. S., B. J. P., S. J. J., J. R. C., D. J. S.], and Urology [D. A. E., M. L. B.], Mayo Clinic and Foundation, Rochester, Minnesota 55905
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In addition to possible germ-line mutations, two common polymorphisms
(Ser217Leu and Ala541Thr) in HPC2/ELAC2 have been reported
to increase the risk for PC (15
, 16)
. These variants have
been estimated to be responsible for 
5% of PC in the general
population.
To confirm whether alterations of HPC2/ELAC2 are associated with HPC, we screened 300 PC patients (2 affected members/family) from 150 families with HPC (14) for potential germ-line mutation. We also examined the frequency of two common polymorphisms (Ser217Leu and Ala541Thr) in a sample set consisting of 446 HPC patients and 502 controls.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
200 high-risk families
were identified. More detailed family histories were obtained over the
telephone, and three to four generation pedigrees were constructed.
Families having a minimum of 3 affected men with PC were enrolled for
additional study. For the purposes of this study, we have defined HPC
as those families having a minimum of 3 affected men with PC. Blood was
collected by a number of methods from as many family members as
possible, resulting in a total of 473 affected men from 181 families.
For 164 of these families, DNA was available on multiple living
affected men. For the remaining 17 families, DNA was available on only
a single affected individual. All men who contributed a blood specimen
and who had PC had their cancers verified by review of medical records
and pathological confirmation. One family has Hispanic ancestry; the
remainder are Caucasian. For our mutation study, 2 affected members (the proband and 1 randomly selected affected male) from each of 150 HPC families were selected for additional analysis (total 300 patients). For our association study, we used all affected men from the same generation (i.e., siblings and cousins) to avoid large differences in ages and secular trends according to year of diagnosis. Thus, 446 HPC cases, consisting of singletons, siblings, and cousins, were used for our association study. The research protocol and informed consent forms were approved by the Mayo Clinic Institutional Review Board.
Population Controls for Association Study.
The Olmsted County Study of Urinary Symptoms and Health Status among
men cohort was initiated in 1989–1990 and has been established and
maintained by our research team over the past 10 years (17
, 18)
. The initial cohort was drawn from the population of Olmsted
County, which serves as the laboratory for the Rochester Epidemiology
Project (19)
. The initial cohort was randomly selected
from an age- and residence (City of Rochester versus balance
of Olmsted County)-stratified sampling frame constructed from the
Rochester Epidemiology Project. Of the 2115 men from the initial
cohort, 475 were selected for a clinical urological examination
(in-clinic cohort; Ref. 20
). This examination included:
DRE and TRUS of the prostate, abdominal ultrasound for postvoid
residual urine volume, serum PSA and creatinine measurement, focused
urological physical examination, and cryopreservation of serum for
subsequent sex hormone assays. Any patient with an abnormal DRE,
elevated serum PSA level, or suspicious lesion on TRUS was evaluated
for prostatic malignancy. If the DRE and TRUS were unremarkable and the
serum PSA level was elevated (>4 ng/ml), a sextant
biopsy (three cores from each side) of the prostate was performed. An
abnormal DRE or TRUS result, regardless of the serum PSA level,
prompted a biopsy of the area in question. In addition, a sextant
biopsy of the remaining prostate was performed. Those men who were
found to be without PC based on this extensive work-up at baseline or
at any of the follow-up exams through 1994, with augmentation with
random samples from the population accrued over that time, were used as
the control population for this study (n = 502; Ref. 21
).
Control Population for Mutation Screening.
DNA was also available from 200 healthy blood bank donors. These
specimens were used to determine the frequency of variant alleles
identified through mutation screening.
PCR Primers.
On the basis of published sequences (GenBank accession no. AF304370 for
cDNA and AC005277 for genomic DNA), we designed 21 pairs of primers for
amplifying 23 of the 24 exons containing coding sequences. The primers
for mutational screening were generally selected to cover 
50 bp on
either side of the coding sequence. The sequences of these primers are
listed on Table 1
.
|
Genotyping.
Two polymorphisms (Ser217Leu and Ala541Thr) in the
HPC2/ELAC2 gene were genotyped in 446 cases with HPC and 502
population controls. The primer pair Exon7-TaqI/BfaI (Table 1)
was used to amplify a 227-bp region containing the Ser217Leu
variant. The primer pair Exon17-Fnu4HI was used to amplify a 197-bp
fragment containing the Ala541Thr variant. All PCR reactions were
carried out in a 12.5-µl reaction volume consisting of 1 x AmpliGold buffer II, 2 mM
MgCl2, 100 µM each
deoxynucleotide triphosphate, 6.25 pmol of each primers, 0.5 unit of
TaqAmpliGold DNA polymerase, and 50 ng of template DNA. PCR was
performed using a Tetrad thermal cycler (MJ Research, Cambridge, MA)
with the following conditions: initial denaturation at 94°C for 12
min, followed by 35 cycles at 94°C for 20 s, 60°C for 30 s, and 72°C for 1 min. Five µl of the PCR product was digested with
the appropriate restriction enzyme (TaqIa for Exon 7 and Fnu4HI for
Exon 17; New England Biolabs), according to the manufacturer’s
recommendation. Fragments were resolved on a 3% agarose gel and
recorded on a Gel Documentation System (Bio-Rad).
All genotyping results were confirmed by a second technique:
pyrosequencing (26)
. The PCR primers used for pyrosequencing were
identical to the those used for the RFLP analysis except that one of
the primer was biotin labeled to capture single-stranded molecules for
subsequent sequencing (Table1). The PCR products were mixed with
magnetic beads (Dynal Biotech, Oslo, Norway) and incubated at
65°C for 15 min. The immobilized strand was then separated in 0.5
M NaOH and transferred to annealing buffer (20
mM Tris-Acetate and 5 mM
MgCl2) containing 18 pmol of sequencing primer (Table 1)
.
Pyrosequencing was performed on a PSQ96 instrument (Pyrosequencing AB,
Uppsala, Sweden), according to the manufacturer’s instructions.
Statistical Analysis.
The association of each of the two polymorphisms (Ser217Leu and
Ala541Thr) with HPC was evaluated by two statistical approaches. The
first was a comparison of the genotype frequencies between cases and
controls using a test for trends in the number of variant alleles,
analogous to Armitage’s test for trends in proportions
(27)
, yet with the appropriate variance to account for the
correlated family data (28)
. The second method was logistic regression,
used to evaluate the main effects of the variants (coded as 0,1,2
according to the number of variants in the genotype) but adjusted for
the potential confounding factors of age and BMI. For these analyses,
age was defined as age at diagnosis for cases and age at blood draw for
the controls. BMI (at the time of recruitment for both cases and
controls) was calculated as weight in kg divided by height in meters,
squared. For the regression analyses, age was categorized using
quartiles of the combined distribution of cases and controls (four
quartiles: 42–52, 53–62, 63–69, and 70+), and BMI was dichotomized
(28 versus >28). To account for correlations among cases
from the same family, generalized estimating equations
(29)
were used, assuming an exchangeable working
correlation matrix. All reported Ps are two sided.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. Among these variants, 13 were intronic, and 10 were exonic. Of the 10
exonic changes, 9 were located in protein-coding sequence and included
5 missense, 3 silent, and 1 nonsense alteration. The single nonsense
mutation in exon 7, Glu216Stop, was identified in pedigree 59. Because
this mutation created a restriction site (CGAG > CTAG)
recognized by Bfa I, we genotyped all available samples in this family
using a Bfa I-based PCR assay (Fig. 1)
. Sequence analysis confirmed that 2 of the 3 affected men were
carriers of this nonsense mutation. This alteration was not present in
the one female available for study.
|
|
For the intronic variants (Table 2)
, we identified a 17-bp duplication
(CCCACACATCTTCACTA) within intron 5, 44 bp upstream of exon 6, in 13 of
148 mixed HPC samples (mixed samples refer to simultaneous CSGE
analysis of 2 patient specimens in a single PCR reaction; see
"Materials and Methods"). Subsequent analysis demonstrated this
duplication in 9 of 100 mixed normal blood bank controls. We also
identified a common 6-bp deletion/insertion polymorphism within intron
10, 182 bp upstream of exon 11. This deletion was found in 113 of 150
mixed cases and 71 of 100 mixed normal blood bank controls. A
mononucleotide repeat (A)10–13 was found in
intron 1, 88 bp upstream of exon 2. The remaining variants were single
nucleotide substitution (Table 2)
.
We also analyzed all variant sequences using a splice site predictor program4 but did not find any indication that any of these alterations affected splicing.
Gene Association Studies.
Characteristics of the hereditary cases and the population-based
controls used for the gene association studies are presented in Table 3
. Hereditary cases were significantly older than controls (median 66
years versus 55 years respectively, P < 0.0001). Nonetheless, there was substantial overlap in the age
distribution between cases and controls. When subjects were grouped
according to age quartiles, the respective percentage of controls
versus cases in the four age groups were: 44.4
versus 3.8%, age 53 years; 26.1
versus 28.5%, age 53–63 years; 12.0 versus
37.8%, age 63–69 years; and 17.5 versus 29.9%,
age > 69 years. The cases also had a significantly
lower BMI than controls (median 26.6 versus 27.8
respectively, P < 0.0001). Because of these
differences, age and BMI were included in all logistic regression
models to statistically adjust for potential confounding effects.
|
, and the results for Ala541Thr are presented in Table 5
. The Leu217 and Thr541 allele frequencies in 446
hereditary cases were 32.3 and 5.4%, respectively. These frequencies
did not differ statistically from those found in the unaffected
population-based control subjects, 31.8 and 5.2%, respectively
(Tables 4
and 5)
. The Thr541 variant was observed only in the
presence of Leu217 alleles, consistent with the findings by
Tavtigian et al. and Rebbeck et al. (15
, 16)
. Neither of these variants alone was associated with HPC
when all cases were compared with the controls or when subsets of
cases, stratified on nodal status, stage, and grade, were compared with
controls. The upper confidence limits for the ORs suggest that the
relative risk associated with the Ser217Leu variant allele
is <1.23 and that for the variant of Ala541Thr is <1.60. For Tables 4
and 5
, the allele covariates in the logistic regression models were
included as 0, 1, 2 (i.e., counts of the variant allele in
the genotypes), which is appropriate for a log-additive
(multiplicative) effect of the variant alleles. To avoid this
assumption, we reanalyzed the data using a simple indicator variable
for carriers of the variants (i.e., grouping homozygous and
heterozygous carriers into a single group); the adjusted ORs and CIs
for these analyses were 1.03 (0.80, 1.33) for Ser217Leu and 0.98 (0.61,
1.56) for Ala541Thr, similar to those reported in Tables 4
and 5
.
|
|
.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. Remarkably, eight of the nine siblings developed malignancies. Both
parents were also diagnosed with cancer. In all, 10 of 11 members of
this nuclear family suffered from cancer in their lifetimes. The
prevalence of malignancy in this family, and the rarity of the tumor
types (other than prostate and breast), suggests a genetic contribution
in this lineage. Mutational analysis demonstrated that 2 of the 3 available affected men in this family carried the nonsense mutation. The 1 unaffected male was not a carrier. Unfortunately, we could not analyze the other 2 affected men because they were deceased, and specimens were not available. Because this mutation is predicted to cause a truncated protein, this alteration is likely to be a causative germ-line change in this family. However, labeling this missense mutation as a causative alteration assumes an inactivating genetic mechanism, for which there is currently no independent evidence. One possible explanation for the lack of a germ-line mutation in the 1 affected male is the presence of a phenocopy. This nonsense alteration was also not detected in the one female available for testing. Because other affected family members were unavailable for testing, it was not possible to additionally explore the role of the Glu216Stop mutation in cancer formation for this family.
In addition to the one nonsense mutation, three novel missense mutations were also identified: Arg211Gln, Gly487Arg, and Gly806Arg. However, only the Gly487Arg variant was found in normal blood bank donors, suggesting that this allele may be a rare polymorphism. The Arg211Gln alteration was found in only a single individual and in none of the 200 normal blood bank donors. Unfortunately, these data provide no evidence that any of these variants are important in susceptibility to PC. Although germ-line mutation of HPC2/ELAC2 does not appear to be a common cause of HPC in the present study, identification of a nonsense mutation in 1 family with multiple cancers suggests a limited role of this gene in HPC and, possibly, in other types of cancers as well. Overall, the low frequency of mutations observed in the study is similar to that reported by Tavtigian et al. (15) , having found a causative mutation in only 2 of their 42 kindreds with evidence for linkage at 17p. In a study recently published by Xu et al. (30) , however, there was no evidence of linkage to chromosome 17 in the total sample nor in any of the subgroups tested, and there were no novel mutations in the coding region of HPC2/ELAC2 in 93 probands with HPC.
In addition to examining the HPC2/ELAC2 gene for the
presence of specific mutations, we also examined two common
polymorphisms for their association with PC risk. Despite the recent
report by Tavtigian et al. and Rebbeck et al.
(15
, 16)
suggesting that the Leu217/Thr541 genotypes were
significantly more common among hereditary and unselected PC cases
compared with control subjects, we failed to detect any statistically
significant increased risk of these genotypes in our HPC cases. Both
Tavtigian et al. and Rebbeck et al. (15
, 16)
found the largest association when considering joint effect
of both variants. However a similar analysis for our data resulted in
no significant association with an OR of 1.04 (95% CI, 0.57–1.89;
Table 6
), with an upper confidence limit less than the OR of 2.37
reported by Rebbeck et al. (16)
. For our data,
we have 98% power to detect an OR of 2.37, with our observed
frequency of 10.4% of the controls who carried variants at both sites
and an effective sample size of 269 cases (to account for relationships
among our 446 HPC cases). A similar result was also reported recently
by Xu et al. (30)
. In their association
studies, both family-based and population-based tests failed to reveal
any statistically significant differences in the allele frequency of
these two polymorphisms between patients with PC and control subjects.
However, although not statistically significant, Xu et al.
(30)
did note a trend toward higher Leu 217 homozygous
carrier rates in patients (9.4%) than in the controls (7.7%; odds
radio, 1.3). The analysis of a larger set of samples may be necessary
to adequately address this question.
A potential source of bias in our study is that the controls tended to
be younger than the cases by 10 years on average. It is possible
that some of our controls will have PC in later years. However, our
regression adjustment for age differences failed to suggest that either
Leu217 or Thr541 were associated with PC. In addition to using age
quartiles to adjust for the effect of age, we added age and the square
of age to the regression models to have more refined adjustments for
age. All of these analyses were quite close to the results presented in
Tables 4
5
6
, suggesting that the imbalance of age is not a major source
of bias. Given the similar allele frequencies between our cases and
controls, it is very unlikely that our conclusions would differ if some
controls developed PC later in life. In fact, we would expect the ORs
to be even closer to unity if some of the controls are misclassified.
Similarly, the imbalance of BMI levels between cases and controls did
not seem to be a source of bias, because the estimated ORs were similar
whether or not BMI was included in the regression model.
The genetic complexity of PC, the presence of phenocopies within high-risk pedigrees, and the late age at diagnosis all contribute to the difficulty of identifying PC susceptibility genes. In this study, we detected a nonsense mutation in the HPC2/ELAC2 gene, confirming its potential role in genetic susceptibility to PC. However, our data also suggests that germ-line mutations of the HPC2/ELAC2 are rare in HPC and that the variants Leu217 and Thr541 do not appear to influence the risk of HPC. In conclusion, our data suggested that the HPC2/ELAC2 gene plays a limited role in genetic susceptibility to HPC.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported by Grants CA 72818, AR30582, and
DK58859 from the Public Health Service, NIH. 
2 To whom requests for reprints should be
addressed, at Laboratory Genetics/HI 970, Mayo Clinic Rochester, 200
First Street SW, Rochester, MN 55905. Phone: (507) 284-4696; Fax:
(507) 284-0043; E-mail: sthibodeau{at}mayo.edu
3 The abbreviations used are: PC, prostate cancer;
HPC, hereditary prostate cancer; DRE, digital rectal examination; TRUS,
transrectal ultrasound; PSA, prostate-specific antigen; CSGE,
conformation-sensitive gel electrophoresis; BMI, body mass index; OR,
odds ratio; CI, confidence interval.
4 Internet address:
http://www.fruitfly.org/seq_tools/splice.html.
Received 3/12/01. Accepted 7/11/01.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  Y.-C. Chen, E. Giovannucci, P. Kraft, and D. J.Hunter Sequence variants of elaC homolog 2 (Escherichia coli) (ELAC2) gene and susceptibility to prostate cancer in the Health Professionals Follow-Up Study Carcinogenesis, May 1, 2008; 29(5): 999 - 1004. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Rennert, C. M. Zeigler-Johnson, K. Addya, M. J. Finley, A. H. Walker, E. Spangler, D. G.B. Leonard, A. Wein, S. B. Malkowicz, and T. R. Rebbeck Association of Susceptibility Alleles in ELAC2/HPC2, RNASEL/HPC1, and MSR1 with Prostate Cancer Severity in European American and African American Men Cancer Epidemiol. Biomarkers Prev., April 1, 2005; 14(4): 949 - 957. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Narla, A. DiFeo, H. L. Reeves, D. J. Schaid, J. Hirshfeld, E. Hod, A. Katz, W. B. Isaacs, S. Hebbring, A. Komiya, et al. A Germline DNA Polymorphism Enhances Alternative Splicing of the KLF6 Tumor Suppressor Gene and Is Associated with Increased Prostate Cancer Risk Cancer Res., February 15, 2005; 65(4): 1213 - 1222. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Cybulski, T. Huzarski, B. Gorski, B. Masojc, M. Mierzejewski, T. Debniak, B. Gliniewicz, J. Matyjasik, E. Zlowocka, G. Kurzawski, et al. A Novel Founder CHEK2 Mutation is Associated with Increased Prostate Cancer Risk Cancer Res., April 15, 2004; 64(8): 2677 - 2679. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. J. Schaid The complex genetic epidemiology of prostate cancer Hum. Mol. Genet., April 1, 2004; 13(90001): R103 - 121. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Cybulski, B. Gorski, T. Debniak, B. Gliniewicz, M. Mierzejewski, B. Masojc, A. Jakubowska, J. Matyjasik, E. Zlowocka, A. Sikorski, et al. NBS1 Is a Prostate Cancer Susceptibility Gene Cancer Res., February 15, 2004; 64(4): 1215 - 1219. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. H. Seppala, T. Ikonen, V. Autio, A. Rokman, N. Mononen, M. P. Matikainen, T. L. J. Tammela, and J. Schleutker Germ-Line Alterations in MSR1 Gene and Prostate Cancer Risk Clin. Cancer Res., November 1, 2003; 9(14): 5252 - 5256. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Stanford, L. P. Sabacan, E. A. Noonan, L. Iwasaki, J. Shu, Z. Feng, and E. A. Ostrander Association of HPC2/ELAC2 Polymorphisms with Risk of Prostate Cancer in a Population-based Study Cancer Epidemiol. Biomarkers Prev., September 1, 2003; 12(9): 876 - 881. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. K. Nam, W. W. Zhang, J. Trachtenberg, E. Diamandis, A. Toi, M. Emami, M. Ho, J. Sweet, A. Evans, M. A.S. Jewett, et al. Single Nucleotide Polymorphism of the Human Kallikrein-2 Gene Highly Correlates With Serum Human Kallikrein-2 Levels and in Combination Enhances Prostate Cancer Detection J. Clin. Oncol., June 15, 2003; 21(12): 2312 - 2319. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Severi, G. G. Giles, M. C. Southey, A. Tesoriero, W. Tilley, P. Neufing, H. Morris, D. R. English, M. R. E. McCredie, P. Boyle, et al. ELAC2/HPC2 Polymorphisms, Prostate-Specific Antigen Levels, and Prostate Cancer J Natl Cancer Inst, June 4, 2003; 95(11): 818 - 824. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Takaku, A. Minagawa, M. Takagi, and M. Nashimoto A candidate prostate cancer susceptibility gene encodes tRNA 3' processing endoribonuclease Nucleic Acids Res., May 1, 2003; 31(9): 2272 - 2278. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Hemminki and K. Czene Attributable Risks of Familial Cancer from the Family-Cancer Database Cancer Epidemiol. Biomarkers Prev., December 1, 2002; 11(12): 1638 - 1644. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. R. Rebbeck Inherited Genotype and Prostate Cancer Outcomes Cancer Epidemiol. Biomarkers Prev., October 1, 2002; 11(10): 945 - 952. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Simard, M. Dumont, P. Soucy, and F. Labrie Perspective: Prostate Cancer Susceptibility Genes Endocrinology, June 1, 2002; 143(6): 2029 - 2040. [Full Text] [PDF]
|
|
|
|
|
|
Y.-Q. Wu, H. Chen, M. A. Rubin, K. J. Wojno, and K. A. Cooney Loss of Heterozygosity of the Putative Prostate Cancer Susceptibility Gene HPC2/ELAC2 Is Uncommon in Sporadic and Familial Prostate Cancer Cancer Res., December 1, 2001; 61(24): 8651 - 8653. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
