RNase L Variants Do Not Appear to Impact on Clinical Features of Sporadic Prostate Cancer Patients

doi:10.4236/oju.2013.37054

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Open Journal of Urology, 2013, 3, 287-292

Published Online November 2013 (http://www.scirp.org/journal/oju)

http://dx.doi.org/10.4236/oju.2013.37054

Open Access OJU

RNase L Variants Do Not Appear to Impact on Clinical

Features of Sporadic Prostate Cancer Patients*

Frank T. D’Arcy1,2, Ruth Foley1, Thomas H. Lynch2

1Institute of Molecular Medicine, Trinity College, Dublin, Ireland

2Department of Urology, St. James Hospital, Dublin, Ireland

Email: [email protected]

Received November 4, 2013; revised November 23, 2013; accepted November 28, 2013

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

Introduction: Prostate cancer is the most common non-cutaneous male cancers, contributing to significant mortality

rates globally. Mutations of RNase L, an enzyme involved in inflammatory and immunological pathways, have been

speculated to predispose to cancer. This study assesses three different mutations of the RNase L gene in Irish prostate

cancer patients, including one linked with general cancer susceptibility never investigated before in prostate cancer

(rs3738579), and reports on links with aggressive cancer. Methods: 134 patients had their RNase L mutation status

determined by polymerase chain reaction (PCR) of serum DNA. Complementary clinical details for each patient are

statistically analysed. Results: No link to age of diagnosis, high grade disease or prostate specific antigen (PSA) level at

diagnosis was demonstrated with any of the studied single nucleotide polymorphisms (SNP). The SNP variation was

consistent with that of published international series. Conclusion: SNP genotypic frequencies in Ireland are consistent

with international findings. The studied RNase L mutations including rs3738579 do not appear to have a significant

impact on our patient population.

Keywords: RNase L; R462Q; D541E; Prostate Cancer

1. Introduction

RNase L is a gene found on the hereditary prostate can-

cer (HPC) locus of chromosome 1 that codes for a latent

endoribonuclease. This enzyme participates in an interferon

inducible RNA decay pathway that has a role in inflam-

mation and cellular immunity against viral infection. Sus-

tained activation will lead to apoptosis. The gene is 741

amino acids long with 8 exons and is roughly 13 kilo-

bases. It is converted from an inactive monomeric form

to a potent dimeric structure by the action of a series of

2’ to 5’ linked oligodenylates, commonly known as 2 -

5 A.

RNase L has been a candidate gene for prostate cancer

researchers for a number of years and many variants in-

cluding R462Q [1], E265X [1], M1I [2] and 471ΔAAAG [3]

have been described.

The R462Q RNase L mis-sense mutation (rs486927)

arises when a substitution of the “G” to “A” base along

exon 1 at mRNA position 1552 gives an amino acid

change of argenine to glutamine at amino acid position

462. The consequence of this is a variant of the gene that

produces an enzyme functioning at a third of the wild-

type enzyme’s efficiency [4]. It has been of interest to

researchers since it was linked to prostate cancer in 2002

[2]. Subsequent studies have been mixed with some show-

ing a link with this variant and familial prostate cancer

[5,6], whilst others have not [7]. A meta-analysis of 7

papers has failed to show a significant link between this

mutation and prostate cancer regardless of ethnicity or

family history [8].

D541E (rs627928) comes about following a substitution

of the “T” base at mRNA position 1790 with “G”, caus-

ing a change in the protein at amino acid position 541

from aspartate to glutamic acid. This area of the gene

codes for a region within the protein kinase domain of

the enzyme, an area with an important role in dimeriza-

tion of the protein into its active state. Despite the muta-

tion, this enzyme has comparable catalytic activity with

the wildtype variant [4]. Its significance is the source of

some debate within the literature. Studies have linked

*All funding for described work came via Trinity College Dublin and

The Irish Cancer Society.

F. T. D’ARCY ET AL.

288

this change to an increased risk of familial prostate can-

cer [7] and sporadic, metastatic disease [9], but in a Japa-

nese study [10] the wildtype variant was actually linked

to an increased risk of familial prostate cancer. Other

investigators have found no significant associations with

it [1,5,11].

An undescribed mutation (rs3738579) located in the 5’

untranscribed region (5’-UTR) has been identified as a

general indicator of increased cancer susceptibility [12].

The exact role of this variant on expression is unclear but

mutations are seen disproportionally in cervical as well

as head and neck squamous cell cancers.

Work in the fields of cervical cancer (human papillo-

mavirus) [13], gastric cancer (helicobacter pylori) [14],

B-cell lymphomas (Epstein-Barr virus) [15] and osteo-

genic sarcomas (chronic osteomyelitis) [16] have all found

definite links between chronic infection and neoplasia.

Whilst no such links have yet been established in prostate

cancer, there are inconsistent reports in the literature of

positive associations of the disease with sexually trans-

mitted infections (STIs) [17-19], and one study finding

men with 25 or more sexual partners being 2.8 times

more likely to be diagnosed with prostate cancer than a

man with 5 or less partners [20]. If inflammation or viral

infection plays a role in prostate cancer development, it

is speculated that polymorphisms in genes involved in

the inflammatory and infectious disease pathways like

RNase L could be important.

This study aims to sequence RNase L single nucleo-

tide polymorphisms (SNPs) in men with known cases of

sporadic prostate cancer and correlate to the clinical as-

pects of the cancer.

2. Methods and Materials

Patient samples were obtained from the Prostate Cancer

Research Consortium (PCRC) bio-repository. This is a

collaboration of three University institutions affiliated

with five major hospitals. Complementary clinical details

relating to each patient are also logged into a password

protected, secure database. All volunteers were pre-op-

erative radical prostatectomy patients with localised pros-

tate cancer, enrolled with full consent in line with ethical

advice. All cases of prostate cancer analysed were spo-

radic.

Using the database of known SNPs published online at

the National Centre for Biotechnology Information (NCBI),

the sequence of code surrounding the SNPs of interest

were obtained.

Primers were designed using Primer Express (Applied

Biosystems, USA). Details of the actual sequence of in-

terest and the primer set are given in Table 1. Primers

were supplied by MWG and were purchased at 0.01

mol scale, HPS purification.

DNA was extracted from peripheral blood DNA by the

Autopure automated system (Qiagen, USA) which uses

puregene chemistry. The product was purified (Qiaquick

PCR purification kit from Qiagen, USA) and sequencing

reactions with a fluorescent dye terminator (BigDye v3.1,

ABI) were performed. Results were interpreted with Se-

quencing Analysis v5.1 from Applied Biosystems.

Polymerase chain reaction (PCR) was carried out with

the product initially being exposed to 94˚C for thirty

seconds, followed by 35 cycles where the temperature

alternated from 94˚C for 30 more seconds, then [53.1˚C

(R462Q)/53˚C (D541E)/55˚C (5’-UTR)] for 30 seconds

followed by a minute at 72˚C. The reaction culminated in

30 minutes exposure to 72˚C.

3. Results

Table 2 illustrates the frequencies of each RNase L SNP

studied in the prostate cancer population. These figures

are in keeping with those published on the NCBI database.

R462Q

9% of patients had the homozygous AA genotype

(R462Q), which corresponds to the less efficient variant of

the RNase L enzyme, while 52.2% were heterozygous

for this SNP. Table 3 outlines R462Q status versus histo-

logical disease aggression. On initial inspection it would

appear that a disproportionate amount of men with the

AA (i.e. the R462Q) genotype have tumours that are of

Gleason score 7 or greater (7 out of 11). This association,

whilst a trend did not demonstrate statistical significance

(p = 0.258). No association was seen between this SNP

and either percentage gland involvement by tumour (p =

0.57) or number of affected first-degree relatives (p =

0.69). Furthermore, anova 1-way p values for age and

prostate specific antigen (PSA) levels at diagnosis also

failed to demonstrate any statistical significance (p =

Table 1. Description of primers selected for each SNP studied.

DbSNP ID Base Amino acid Primer Sequence

Forward 5’-TGGAAGCGTGTTTGGATGTG-3’

rs486907 G/A R462Q Reverse 5’-TGCAGATCCTGGTGGGTGTA -3’

Forward 5’-TTGATTTATGGCTTTTGTGCAGG-3’

rs627928 T/G D541E Reverse 5’-TGAGGTCCTTAGTTTCCTCATCT-3’

Forward 5’-GTGGAAT GTCAGAAGAC TGAGAAC-3’

rs3738579 C/T - Reverse 5’-AATGCCACCTGCTACCACTT-3’

Open Access OJU

F. T. D’ARCY ET AL. 289

Table 2. Summary of sequencing results for each SNP in

men with prostate cancer.

SNP Genotype Frequency (%)

AA (Mutation) 12/134 (9%)

AG 70/134 (52.2%)

R462Q

GG (Wildtype) 52/134 (38.8%)

GG (Mutation) 35/91 (38.5%)

GT 38/91 (41.7%) D541E

TT (Wildtype) 18/91 (19.8%)

CC (Mutation) 18/136 (13.2%)

CT 55/136 (40.4%) 5’-UTR (rs3738579)

TT (Wildtype) 63/136 (46.4%)

Table 3. Gleason score and SNP status at the exon 1/R462Q

SNP.

Gleason Score AA AG GGTotal numbers P value

4 - 6 4 39 23 66

7 - 9 7 26 23 56

Total 11 65 46 122 p = 0.258

0.60 and 0.44 respectively).

D541E

38.5% of prostate cancer patients in the study were

shown to carry the D541E mutation. Analysis of comple-

mentary clinical data revealed no association with either

percentage gland involvement (p = 0.58), number of

first-degree relatives affected (p = 0.058) or age at diag-

nosis (p = 0.68). Analysis of PSA at diagnosis (on Table

4) revealed that men with the mutation had a much

higher PSA at diagnosis however this fell just short of

statistical significance (p = 0.06).

Analysis of Gleason score of tumour and SNP status

failed to show any statistically significant association (p

= 0.68).

5’-UTR

Statistical analysis of Gleason score (p = 0.24), per-

centage gland involvement (p = 0.5), family history (p =

0.49), age (p = 0.29) or PSA at diagnosis (p = 0.77)

failed to reveal any association with this SNP (see Table

5).

4. Discussion and Review of the Literature

To our knowledge this is the first study of the RNase L

SNP rs3738579 in the context of prostate cancer and also

adds to the debate about the significance of D541E and

R462Q.

There are a number of studies in the literature exam-

ining prostate cancer and the R462Q mutation. The results

of these are summarised in Table 6 and a comparison of

the frequency that the mutation is detected across these

series are displayed in the Forest plot given in Figure 1.

This shows our findings to be consistent with interna-

tionally published series.

Table 4. Breakdown of PSA values (given as ng/ml) versus

genotype at D541E SNP.

Genotype Mean ± SDMedian Anova 1-way p value

GG (D541E)8.42 ± 3.138.25

GT 7.08 ± 3.266.25

TT 6.84 ± 2.076.6

0.0634

Table 5. Analysis of low and high grade tumours versus

5’-UTR locus SNP status.

Gleason score CCCTTT Total numbersp value

4 - 6 8 29 29 66

7 - 9 8 22 26 56

Total 16 51 55 122 0.24

0.000.05 0.100.15 0.20 0.25

系列1

Rokman

Wa ng

Wiklund

Meier

D’Arcy

AUTHORS

Comparison

of AA

mutation

representation

on Exon 1

462

throughout

major

published

series

AA Proportion

Figure 1. Expression of mutated form of Exon 1, AA (R 462Q),

as a proportion of total numbers examined for all major

studies, expressed on a Forest plot. Y-axis has study’s au-

thor’s name whilst X-axis contains proportion of AA geno-

type given with 95% confidence limits.

D541E mutation has been the focus of multiple studies.

We fail to demonstrate a significant clinical association

with this mutation. Results summary and comparison of

frequency of mutation are given in Table 7 and Figure 2

respectively.

Rokman’s study focused on 492 patients with prostate

cancer, 47 of whom reported a positive family history.

They were consecutive cases diagnosed at the University

Hospital, Tampere, Finland, over a three-year period.

This population would have differed from the one we are

reporting on by including men with both organ confined

and metastatic disease. Control samples came from 566

healthy male blood donors. This study failed to show any

significant difference in D541E status between prostate

cancer, BPH and normal control groups and concluded

that this mutation doesn’t have an important role in pros-

tate cancer within their population. It did however dem-

onstrate an association between R462Q status and heredi-

tary prostate cancer.

Wang’s study came from Minnesota in the United

States and included a total of 825 patients with prostate

Open Access OJU

F. T. D’ARCY ET AL.

290

Table 6. Comparison of results of analysis of R462Q mutation (“AA” genotype) throughout different studies within the litera-

ture.

Study Author Population Patient group AA (Mut) GenotypeAG Genotype GG Genotype Total

Prostate cancer 39 (16.7%) 106 (45.5%) 88 (37.8%) 233

Rokman [1] Finnish Control 23 (13.1%) 84 (47.7%) 69 (39.2%) 176

Prostate cancer 102 (11.1%) 427 (46.5%) 389 (42.4%) 918

Wang [5] Hispanic and White. USA Control 67 (13.5%) 233 (47.3%) 193 (39.1%) 493

Prostate cancer 247 (15.2%) 778 (48.0%) 597 (36.8%) 1622

Wiklund [7] Swedish Control 115 (14.4%) 384 (48.2%) 297 (37.4%) 796

Prostate cancer 59 (16.3%) 171 (47.1%) 133 (36.6%) 363

Maier [11] German Control 37 (17.9%) 97 (46.9%) 73 (35.2%) 207

D’Arcy Irish Prostate cancer 12 (9.0%) 70 (52.2%) 52 (38.8%) 134

Table 7. Comparison of results of analysis of D541E mutation (“GG” genotype) throughout different studies within the litera-

ture.

Study Author Population Patient group GG (Mut) Genotype GT Genotype TT Genotype Total

Prostate cancer 78 (33.5%) 126 (54.1%) 29 (12.4%) 233

Rokman [1] Finnish Control 56 (31.8%) 91 (51.7%) 29 (16.5%) 176

Prostate cancer 181 (19.5%) 476 (51.2%) 272 (29.3%) 929

Wang [5] Hispanic and White. USA Control 107 (21.1%) 228 (44.9%) 173 (34.0%) 508

Prostate cancer 462 (33.9%) 668 (49.0%) 233 (17.1%) 1363

Wiklund [7] Swedish Control 257 (32.5%) 372 (47.0%) 162 (20.5%) 791

Prostate cancer 125 (34.4%) 176 (48.5%) 62 (17.1%) 363

Maier [11] German Control 69 (33.3%) 97 (46.9%) 41 (19.8%) 207

Prostate cancer 55 (36.7%) 73 (48.7%) 22 (14.6%) 150

Noonan-Wheeler [9] European-Americans Control 44 (25.7%) 94 (55.0%) 33 (19.3%) 171

Prostate cancer 51 (50.5%) 32 (31.7%) 18 (17.8%) 101

Nakazato [10] Japanese Control 59 (56.2%) 43 (40.9%) 3 (2.9%) 105

D’Arcy Irish Prostate cancer 35 (38.5%) 38 (41.7%) 18 (19.8%) 91

0.000.20 0.400.60 0.80

系列

Wan g

Wiklund

Meier

N-Wheeler

Na ka za to

Rokma n

Comparison

of GG

mutation

representa tion

onExon 3

throughout

major

published

series

AUT HO RS

GG Proportion

D’Arcy

Figure 2. Expression of mutate d form of Exo n 3, GG ( D 541E),

as a proportion of total numbers examined for all major

studies, expressed on a Forest plot. Y-axis has study’s au-

thor’s name whilst X-axis contains proportion of GG geno-

type given with 95% confidence limits.

cancer, made up of 326 familial prostate cancer patients

and 499 cases of sporadic prostate cancer, all of the latter

group comprising of organ confined disease. Control group

came from men of a similar age from the local popula-

tion (Rochester Epidemiology Project), who were invited

to enrol and subsequently had a careful history, physical

examination and if necessary a TRUS biopsy. Once pros-

tate cancer had been ruled out, they were incorporated as

a control group. In total there were 510 such men. This

study also linked R462Q status with hereditary prostate

cancer and in such men disease was associated with an

earlier age of onset. Like Rokman’s work, this study also

failed to demonstrate a correlation between the D541E

mutation and prostate cancer risk.

Wiklund’s series came from patients registered on

Cancer Prostate, a Swedish nationwide database of pros-

tate cancer patients younger than 79 diagnosed between

July 1st 2001 and 30th September 2002. Patients were

invited to take part in the study and a blood sample for

analysis was taken. In total, 1636 prostate cancer patients

with a full spectrum of disease from early to advanced

were included. Control samples were matched to similar

people from the Swedish Population registry, who were

invited to take part. This involved filling a questionnaire

and giving a blood sample. In total 801 controls were

analysed. No significant association between R462Q status

and prostate cancer risk was demonstrated in this study.

It did reveal a significant link (p = 0.03) between familial

Open Access OJU

F. T. D’ARCY ET AL. 291

prostate cancer risk and D541E mutation, however no link

between this variant and either age at diagnosis or tu-

mour aggressiveness was found.

Maier’s patients were recruited from the Prostate Can-

cer Genetics Project. This is a database made up of men

from predominately from the South of Germany, the ma-

jority of whom would have undergone radical prostatec-

tomies. Patients are encouraged to enrol in this study by

their urologist and there are no selection criteria. At risk

families are identified by interview of the patient. There

were a total of 303 of such patients. 227 sporadic cases

were also sequenced as well as 207 control samples.

These samples came from healthy, elderly men with no

history of prostate cancer and negative DRE and/or nor-

mal PSA levels. D541E or R462Q failed to demonstrate any

significant association with prostate cancer in this study.

Noonan-Wheeler’s study originated in Missouri, USA.

It examined RNase L in men with aggressive, metastatic

cancer and healthy controls. Patients were recruited from

the outpatient department and were required to have a

PSA over 50 ng/ml or radiological/pathological evidence

of metastatic disease. The control group consisted of men

older than 75 with normal PSA levels and rectal exami-

nation with no background of prostate cancer. There

were a total of 150 patients and 171 controls examined. It

was shown that D541E was over-represented (p = 0.045)

in patients with metastatic disease, and concluded that

such patients were at an increased risk for sporadic, me-

tastatic disease (OR = 1.68).

Nakazato’s study came from Japan and examined

RNase L in familial prostate cancer cases and healthy

controls. It comprised 101 patients with a positive family

history of cancer (29 of whom had 3 or more affected

family members) and 105 controls. Prostate cancer pa-

tients ranged in age from 40 to 88 years, encompassed

organ confined and metastatic disease and had 76 men

with a Gleason score of 7 or greater and the remaining 26

with a Gleason score of 6 or less. Control cases were

recruited from the outpatients department and were of

similar age. Patients with an elevated PSA or abnormal

DRE were excluded from this group. Interestingly this

study demonstrated a significant link between the wild-

type DD variant and familial prostate cancer (p = 0.0004,

OR = 7.37), possibly an observation unique to the Japa-

nese population.

5. Conclusion

In conclusion, no statistically significant correlation was

proven in the Irish population between Gleason score,

percentage gland involvement, patient age, PSA or fam-

ily history with any of the studied SNPs. In particular the

SNP rs3738579 failed to highlight men with significant

clinical cancer.

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