Association between hormonal genetic polymorphisms and ... - Nature

Prostate Cancer and Prostatic Diseases (2005) 8, 95–102 & 2005 Nature Publishing Group All rights reserved 1365-7852/05 $30.00 www.nature.com/pcan

Association between hormonal genetic polymorphisms and early-onset prostate cancer MS Forrest1,2, SM Edwards3, R Houlston3, Z Kote-Jarai3, T Key4, N Allen4, MA Knowles1, F Turner2, A Ardern-Jones5, A Murkin5, S Williams3, R Oram3, CR-UK/BPG UK prostate cancer study collaborators6, DT Bishop2,7 & RA Eeles3,5,7* 1

Cancer Research UK Cancer Medicine Research Division, University of Leeds, Leeds, UK; 2Cancer Research UK Genetic Epidemiology Division, University of Leeds, Leeds, UK; 3Institute of Cancer Research, Sutton, Surrey, UK; 4Cancer Research UK Epidemiology Unit, University of Oxford, Oxford, UK; and 5Royal Marsden NHS Trust, Sutton, Surrey, UK

We investigated the association between seven polymorphisms in four candidate genes involved in vitamin D and androgen metabolism with early-onset prostate cancer (CaP) risk. The polymorphisms were genotyped in 288 UK males who were diagnosed with CaP at the age of 55 y or younger and up to 700 population-based controls. An additional 50 cases (not selected for age) and 76 controls were also genotyped. Short (p22 repeats) AR (CAG)n repeats were associated with a significantly reduced risk of early onset CaP (OR 0.68, 95% CI 0.50–0.91) compared with men with long (422) repeats. Men homozygous for the leucine variant of SRD5A2 p.89V4L were also found to be at a significantly increased risk of CaP compared with men who were homozygous for the valine allele (OR 1.84, 95% CI 1.15–2.98). No associations were found with the AR (GGC)n, CYP17 Msp A1 I, VDR Taq I, SRD5A2 (TA)n and p.49A4T polymorphisms and CaP risk. These findings suggest that common polymorphisms in the AR and SRD5A2 genes may be associated with early-onset CaP in British men. Prostate Cancer and Prostatic Diseases (2005) 8, 95–102. doi:10.1038/sj.pcan.4500785 Published online 15 February 2005

Keywords: case–control study; genetic polymorphisms; early onset

Introduction After lung cancer, prostate cancer (CaP) is the most common cause of male cancer mortality in the United Kingdom, accounting for approximately 8973 cancer deaths in 2002 alone.1 The American Cancer Society estimates that there will be 230 110 new cases of CaP and 29 900 CaP deaths in the US in 2004.2 However, little is known about the causes of the disease, the only established risk factors being age,1 ethnicity/geographical

*Correspondence: RA Eeles, Translational Cancer Genetics Team, Institute of Cancer Research, Sutton, Surrey SM2 5PT, UK. E-mail: [email protected] 6 List available on request. 7 These authors contributed equally to this work. Received 1 September 2004; revised 17 December 2004; accepted 17 December 2004; published online 15 February 2005

location (eg, Japanese-Americans have a higher risk than native Japanese)3–5 and family history.6–8 Hormones are necessary for the growth and development of the prostate gland and have been implicated in the development of CaP. It has therefore been suggested that genetic polymorphisms that encode for enzymes involved in hormone biosynthesis and metabolism or receptor function may be related to cancer risk. Two such hormonal pathways include the vitamin D receptor pathway, which maintains calcium homeostasis and exerts antiproliferative and prodifferentiative effects on many cell types including the prostate, and the androgen receptor pathway, which acts to stimulate prostate cell growth directly (see Figure 1). The aim of this study is to investigate the association between several common polymorphisms in genes involved in these two pathways with early-onset CaP risk. The roles of the genes under investigation are illustrated in Figure 1 and include: CYP17 (cytochrome

Hormonal genetic polymorphisms and early-onset prostate cancer MS Forrest et al

96

Cholesterol

CYP11a1

Pregnenolone

Progesterone

HSD3B2

CYP17

CYP17

17α-Hydroxypregnenolone HSD3B2 CYP17

17α-Hydroxyprogesterone CYP17

Dehydroepiandrosterone HSD3B2 HSD17B1

∆5-Androstenediol

Androstenedione HSD17B1

HSD3B2

Testosterone SRD5A2

1,25 (OH)2D3

Dihydrotestosterone

Vitamin D Receptor

Androgen Receptor

Prostate Cell Division Decreased

Prostate Cell Division Increased

Figure 1 Hormonal genes involved in the regulation of prostate cell division. We investigated polymorphisms in CYP17, SRD5A2, the androgen receptor and the vitamin D receptor genes.

P-450 steroid 17a-hydroxylase/17,20 lyase), a gene that encodes an enzyme involved in testosterone synthesis within the testes;9 SRD5A2 (steroid 5a reductase type II), a gene that encodes for the enzyme which converts testosterone to dihydrotestosterone (DHT), its more active metabolite within the prostate;10,11 the AR (androgen receptor), which binds testosterone and DHT and upregulates prostate cell growth;12 and the VDR (vitamin D receptor), which binds vitamin D and downregulates prostate growth.13 Two AR gene polymorphisms have been extensively investigated with regard to CaP risk and include a polyglutamine (CAG) repeat and a polyglycine (GGC) repeat, both of which are found in the first exon encoding the N-terminal modulatory domain of the AR (the domain that binds to androgen response elements to regulate gene expression). Previous epidemiological studies have shown that men with relatively short AR polyglutamine (CAG)n repeats are at an increased risk of developing CaP or more aggressive CaP compared with men with longer Prostate Cancer and Prostatic Diseases

repeats.14–19 Two studies have also suggested a link to earlier onset CaP for smaller (CAG)n polymorphisms.20,21 The association between the (GGC)n polymorphism and CaP risk is more inconsistent, with both short and long repeats being found to predispose to CaP.15,17,22,23 A polymorphism (rs743572) has been identified in the CYP17 gene that contains a single base T4C transition in the 50 UTR of exon 1, which creates a novel Sp-1 type (CCACC) motif and a Msp A1 I restriction enzyme site (commonly denoted the A2 allele).24 Although this Sp-1 motif does not appear to influence Sp-1 binding,25 the variant allele might result in increased transcriptional activity, and hence increased androgen production. This polymorphism has been associated with an increased risk of polycystic ovaries and male premature pattern baldness,24 although the evidence that this polymorphism is related to breast cancer26,27 or CaP is inconsistent.28,29 Several SRD5A2 polymorphisms have been identified, the most studied of which include a TA dinucleotide repeat and the single nucleotide polymorphisms (SNPs) A49T and V89L. Davis and Russell30 originally reported three different alleles of the TA repeat in the 3’UTR, 0, 9 and 18 repeats, with 96% of alleles being assigned (TA)0.30 The observation that the rare (TA)18 allele was limited to African-American populations,30 who have a higher rate of CaP than Asian-American or Caucasian men, suggested that a long allele length may be associated with increased risk. However, case–control studies in other ethnic populations have not found the longer variant alleles to be associated with an increased CaP risk.31–34 A mis-sense substitution in SRD5A2 has been identified that substitutes alanine with threonine at codon 49 (A49T (rs9282858)). This polymorphism results in a variant protein whose in vitro enzymic activity is about five-fold higher than normal.35 Despite the low prevalence of this polymorphism (frequency of 2%), it has been associated with an increased risk of CaP in African-American and Hispanic men35 and also with advanced disease in a case series of prostate cancers.36 However, studies in Caucasian men have generally not found this polymorphism to be associated with an increased CaP risk in Caucasian men.33,37,38 A second mis-sense substitution in SRD5A2, V89L (rs523349), identified by Makridakis,39 substitutes amino-acid 89 from a valine to a leucine and is much more common than A49T at around 30% frequency.36,40,41 The variant L allele has been associated with reduced 5areductase type II enzyme activity in vitro41 and also with lower circulating A-diol-g levels, a marker of 5areductase type II activity and intraprostatic androgen activity in adult men.39–41 However, this polymorphism has not been found to be significantly associated with CaP risk among Caucasian men.28,40 The VDR gene encodes for the receptor that binds vitamin D (1,25-dihydroxyvitamin D3), a hormone involved in prostate cell growth. The TaqI polymorphism in codon 352 (rs731236) is silent, but has been shown to be associated with both circulating levels of osteocalcin, a VDR-regulated protein42 and bone density in osteoporotic women.43 The TaqI site is in linkage disequilibrium with a poly-A microsatellite polymorphism, with the t (presence of TaqI restriction site) and short poly-A (A14–A17, long poly-A ¼ A18–A22) almost always


coinciding.44,45 However, the epidemiological evidence that these polymorphisms are associated with a reduction in CaP risk is inconsistent.44–48 The aim of this study is to test the hypotheses that the CYP17 A2 allele, the SRD5A2 V89L LL genotype and the VDR tt genotype are associated with a reduction in CaP risk, and that shorter AR CAG and GGC repeats, and the SRD5A2 A49T T allele and (TA)9 allele are associated with an increased CaP risk in a unique population of early-onset (diagnosed aged 55 y or younger) CaP cases, and a smaller, unrestricted, pilot case–control population.

Materials and methods Subjects Case patients were identified from the Cancer Research UK/British Prostate Group UK Familial Prostate Cancer Study, as described elsewhere.49 DNA was available from 267 early-onset (aged 55 y or younger) CaP cases from across the UK who were predominantly identified by clinical symptoms rather than by screening. Of these, 28 cases were from CaP families (families containing one or more of: (a) at least three first-degree relatives with CaP; (b) three generations in either the proband’s paternal or maternal lineage; (c) two affected first-degree relatives where one of the relatives was age o55 y). The youngest age of diagnosis was 32 y, with a mean age of 51.1 y and a median age of 51.9 y. Population controls were taken from three previous studies. The first control set (set A) was comprised of 149 male and 135 female spouses of patients with colorectal cancer collected from Bristol, Wessex and the Thames area. These individuals were of Caucasian origin and had no personal or family history of cancer.50 The second set of population controls (set B) were 206 Caucasian females from the Oxford, Leeds and South London area, who were part of a breast cancer case–control study conducted by the UK National Case–Control Study Group (UK National Case–Control Study Group, 1989). All the women were cancer-free, randomly selected via general practice surgeries and aged up to 45 y. This set had previously been used as a control population for AR (CAG)n and (GGC)n repeat length polymorphisms and CaP risk.23 Controls (set C) for the SRD5A2 V89L polymorphism were derived from a previous study that investigated the association between this polymorphism and hormone levels as the RFLP test used was not successful for control sets A and B.51 This set of controls comprised over 600 Caucasian males selected from the Oxford UK component of the European Prospective Investigation into Cancer and Nutrition (EPIC) as described elsewhere51 and was not available for use with the other polymorphisms investigated. The three sets of controls provided a source of data for allele frequencies in the UK Caucasian population for comparison to our early-onset CaP cases. The use of female controls meant that we had data on up to 341 extra AR repeat alleles. Samples from a small case–control study of diet and CaP were also used as a pilot population for the

genotyping. Cases and controls were recruited from Oxfordshire, West Berkshire and Leeds as described elsewhere52 and DNA was available for 50 cases and 76 controls. Age of diagnosis ranged from 55 to 75 y with a median age of 69.0 y. One case had a father with CaP and one case had a brother with CaP.

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Genotyping of polymorphisms DNA was collected and extracted by standard methods, for example, Edwards et al.23 The AR CAG and GGC repeat polymorphisms of the cases and control set A samples were genotyped as described in Edwards et al.23 The CYP17 Msp A1 I polymorphism was genotyped as described in Feigelson et al26 in the cases and control sets A and B. The SRD5A2 TA repeats of the cases and control sets A and B were amplified using 25 ng of DNA with previously published primers (50 -GCTGATGAAAA CTGTCAAGCTGCTGA-30 and 50 -GCCAGCTGGCAGA ACGCCAGGAGAC-30 )30 at a concentration of 0.32 mM each in a total volume of 15 ml, with 0.5 units Amplitaq Gold (Applied Biosystems, Foster City, CA, USA) in 1 PE Buffer II (Applied Biosystems), 2 mM MgCl2 and 1 mM total dNTP. PCR conditions used were: 951C for 10 min followed by 35 cycles of 941C for 30 s, 641C for 1 min, 721C for 30 s, with a final extension step of 721C for 10 min. Products were run in 2% agarose gels and alleles called by length of PCR product (98 bp for 0 TA repeats, 116 bp for 9 TA repeats and 134 bp for 18 TA repeats). Minisequencing was used to genotype the SRD5A2 p.49 A4T polymorphism in the cases and control sets A and B. In all, 25 ng of genomic DNA were amplified in a total volume of 15 ml, with 0.45 U Amplitaq Gold (Applied Biosystems) in 1 PE Buffer II (Applied Biosystems), 2 mM MgCl2, 0.8 mM total dNTP and 0.12 mM each of primers 50 -AAGCACACGGAGATCCT and 50 -GGGCACCGC GAATCCGGCTGCTACC-30 GAAGGAAGGCCGCTCCTGCAGG-30 . PCR conditions used were: 951C for 10 min followed by 40 cycles of 941C for 30 s, 601C for 30 s, 721C for 20 s, with a final extension step of 721C for 10 min. PCR products were cleaned by sodium acetate precipitation and resuspended in 10 ml H2O. Minisequencing was carried out using the SNaPshot Ready Reaction Mix (Applied Biosystems) according to the manufacturer’s instructions and either primer 50 -CGGCGGCTACCCGCCTGCCA-30 (forward) or primer 50 -CAGGAACCAGGCGGCGCGGG-30 (reverse). Reactions using forward and reverse minisequencing primers were loaded in alternate lanes on ABI prism 377 DNA sequencers at 5 min intervals. As the interrogated base was different depending on the DNA strand investigated (G/A forward, C/T reverse) it was possible to run 2–3 samples per lane without the risk of lane miscalling. Variant samples were confirmed using both minisequencing primers. SRD5A2 V89L genotypes were determined in the cases and the EPIC controls as described by Lunn et al.28 VDR Taq 1 genotypes were determined in the cases and control sets A and B as described in Taylor et al.46 All PCR reactions were performed in Hybaid Touchdown and Applied Biosystems GeneAmp PCR System 9700 thermocyclers. Prostate Cancer and Prostatic Diseases


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Statistical methods

Results

Logistic regression was performed using STATA 6.0 (STATA Corporation) to provide odds ratios (OR), 95% confidence intervals (CI) and associated P-values for the risk of each polymorphism with CaP. The genotype frequencies for each polymorphism between cases and controls were tested using standard w2 tests. The heterogeneity of polymorphisms such as the AR CAG repeat means that the large number of alleles, often at low frequencies, greatly reduces the power of simple contingency table-based statistical tests such as w2. The CLUMP program,53 which uses a Monte Carlo approach to determine the significance of allele distributions (http://www.smd.qmul.ac.uk/statgen/dcurtis/software. html), was therefore used to investigate the association between any one genotype or group of genotypes and CaP risk.

The CYP 17 Msp A1 I, VDR Taq I and SRD5A2 V89L, A49 T and (TA)n polymorphisms were in Hardy–Weinberg equilibrium for all control groups both individually and combined and for both sexes of control set B. Since genotype frequencies were similar between the different control groups for all polymorphisms, the control groups were combined in subsequent analyses. The proportion of controls that possessed the variant alleles in the genes under investigation ranged from 8 to 48%. In all, 264/795 (33%) of control AR (CAG)n repeat lengths were greater than 22 repeats and 41% of AR (GGC)n repeat lengths were greater than 16 repeats (see Tables 1 and 2). For the VDR Taq I polymorphism, 50% possessed one variant t allele, and 17% possessed two variant alleles. For the SRD5A2 polymorphisms, 40% possessed one variant L allele, and 8% possessed two variant L alleles. For the

Table 1 Involvement of polymorphisms in the androgen and vitamin D receptors in early onset CaP risk Control set A (%)

Control set B (%)

Controls (%)

Cases (%)

OR (95% CI)

Pilot controls (%)

Pilot cases (%)

OR (95% CI)

AR (CAG)n p22 422

272 (67) 133 (33)

259 (66) 131 (34)

531 (67) 264 (33)

151 (58) 111 (42)

0.68 (0.50–0.91) 1.0

AR (CAG)n 21/22 o21/422

151 (37) 254 (63)

119 (31) 271 (69)

270 (34) 525 (66)

66 (25) 196 (75)

1.0 1.53 (1.10–2.13)

AR (GGC)n p16 416

244 (60) 165 (40)

168 (59) 116 (41)

412 (60) 282 (40)

158 (61) 102 (39)

1.06 (0.78–1.44) 1.0

47 (62) 29 (38)

32 (64) 18 (36)

1.10 (0.52–2.30) 1.0

VDR Taq I TT Tt tt

93 (35) 130 (49) 45 (17)

51 (29) 94 (53) 31 (18)

144 (32) 224 (50) 76 (17)

90 (34) 128 (49) 44 (17)

0.91 (0.65–1.28) 0.93 (0.59–1.46) 1.0

20 (30) 38 (57) 9 (13)

15 (33) 23 (50) 8 (17)

0.68 (0.23–2.01) 0.84 (0.26–2.70) 1.0

The AR (CAG)n polymorphism was shown to be associated with early onset CaP risk (data shown in bold).

Table 2 Involvement of polymorphisms in the steroid 5-alpha-reductase type II and cytochrome P450 17A1 in early-onset CaP risk Control set A (%)

Control set B (%)

Combined controls (%)

Cases (%)

OR (95% CI)

Pilot ctrls (%)

Pilot cases (%)

OR (95% CI)

CYP17 Msp A1 I A1/A1 A1/A2 A2/A2

112 (41) 130 (47) 32 (12)

73 (39) 92 (49) 23 (12)

185 (40) 222 (48) 55 (12)

98 (37) 132 (50) 32 (12)

1.0 1.12 (0.81–1.55) 1.10 (0.67–1.81)

23 (31) 43 (57) 9 (12)

20 (42) 20 (42) 8 (17)

1.0 0.53 (0.24–1.19) 1.02 (0.33–3.15)

SRD5A2 A49T AA AT TT

252 (93) 20 (7) 0

183 (95) 10 (5) 0

435 (94) 30 (6) 0

250 (94) 15 (6) 1 (0.4)

1.0 AT+TT:a 0.93 (0.50–1.74)

65 (90) 7 (10) 0

47 (100) 0 0

SRD5A2 (TA)n 0/0 0/9 9/9

222 (80) 51 (18) 3 (1)

154 (75) 45 (22) 5 (2)

376 (78) 96 (20) 8 (2)

195 (76) 57 (22) 3 (1)

1.0 0/9+9/9:a 1.11 (0.77–1.60)

59 (78) 15 (20) 2 (3)

39 (78) 10 (20) 1 (2)

1.0 0/9+9/9:a 0.98 (0.41–2.31)

EPIC Controls (%) 317 (52) 245 (40) 49 (8)

126 (48) 98 (38) 36 (14)

1.0 1.01 (0.74–1.38) 1.84 (1.15–2.98)

41 (55) 31 (41) 3 (4)

18 (37) 25 (51) 6 (12)

1.0 1.84 (0.86–3.95) 4.56 (1.02–20.27)

SRD5A2 V89L VV VL LL

There was a significant association between individuals homozygous for the SRD5A2 89L allele and early-onset CaP risk (data shown in bold). a Heterozygotes and homozygotes for rare alleles were combined for analyses.

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0.25

0.2

Frequency

p.A49A4T polymorphism, no controls and only one case was homozygous for the variant allele, and so these were grouped together with the heterozygous carriers, which comprised 6% of the population. Similarly, the prevalence of the (TA)n repeat polymorphism was low, with eight controls and three cases possessing the 9/9 genotype, and these individuals were also grouped with the heterozygotes for analysis, which comprised 22% of the population. For the CYP17 Msp A1 I polymorphism, 48% of the controls possessed one allele, and 12% possessed two copies of the variant allele. Since the average control AR CAG repeat number was 21.6, repeat lengths p22 were classified as ‘short’, and repeat lengths longer than 22 CAG repeats were classified as ‘long’. Compared with men with long (422) CAG repeats, men with short (p22) AR CAG repeats had a significantly reduced risk of early onset CaP (OR 0.68, 95% CI 0.50–0.91, P ¼ 0.007), as shown in Table 1. Each additional CAG repeat was associated with an increased risk of 4% (95% CI, 1 to 10%), but this was not statistically significant (P ¼ 0.09). The strongest association between (CAG) repeat length and CaP risk was found for men with (CAG)21 repeat lengths, as derived from CLUMP analysis, based on 1000 simulations (w2 of 6.6, which was achieved 87 times in 1000 simulations (P ¼ 0.088 after correction for multiple testing) and corresponded to an uncorrected P-value of 0.01). The most common control (CAG)n repeat lengths were 21 and 22 (see Figure 2), so it was decided to group these together and see if, as suggested by the CLUMP analysis, having a middling CAG repeat length had an effect on CaP risk. A w2 test of (CAG)o21/422 vs (CAG)21/ 22 gave an OR of 1.53 (95% CI 1.10–2.13, P ¼ 0.008), suggesting that both long and short AR CAG repeats predispose to early onset CaP. No association with early onset CaP risk was found for the AR (GGC)n repeat length polymorphism or the VDR polymorphism. Associations between the SRD5A2 p.89V4L, SRD5A2 A49T and CYP17 Msp A1 I polymorphisms and earlyonset CaP risk are shown in Table 2. Men homozygous for the SRD5A2 V89L leucine variant were found to be at almost twice the risk of developing early-onset CaP compared with men who were homozygous for the valine variant (OR 1.84, 95% CI 1.15–2.98, P ¼ 0.012). There was no significant difference between valine homozygotes and valine/leucine heterozygotes (OR 1.01, 95% CI 0.74–1.38, P ¼ 0.97). An increase in risk associated with the L allele was also observed in the pilot study population. Men who were homozygous for the leucine variant had a four-fold increased risk compared with men who were homozygous for the valine variant of SRD5A2 V89L (OR 4.56, 95% CI 1.02–20.27, P ¼ 0.046). Men who had a V/L genotype were at an intermediate risk (OR 1.84, 95% CI 0.86–3.95, P ¼ 0.12), but this difference was not statistically significant from that of the valine homozygotes. No significant associations with CaP risk were found for, SRD5A2 (TA)n, SRD5A2 A49T and CYP17 Msp A1 I (Table 2). One control individual was homozygous for the T variant allele and was therefore included in the AT genotype group for analysis. Excluding the 28 cases known to be from CaP families did not alter the findings for any of the polymorphisms investigated (see Supplemental Tables S1 and S2). In the pilot study, no cases

0.15

0.1

0.05

0 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 CAG repeat number

Figure 2 Frequencies of AR (CAG)n repeat polymorphisms in UK earlyonset CaP cases (white bars) and population controls (black bars). Logistic regression showed a significant decrease in early-onset CaP risk associated with p22 CAG repeats (OR 0.68, 95% CI 0.50–0.91). Linear regression analysis estimated a 4% increase in CaP risk for each extra CAG repeat (OR 1.04, s.e. 0.027, t ¼ 1.69, P4|t| 0.09, 95% CI 0.99–1.10). CLUMP analysis suggested that the strongest association with CaP risk was with (CAG)21 which gave a w2 of 6.6 (P ¼ 0.088, uncorrected P ¼ 0.01). A w2 test of (CAG)21/22 vs (CAG)o21/422 gave an OR of 1.53 (95% CI 1.10– 2.13, P ¼ 0.008), suggesting that both long and short AR CAG repeats predispose to early-onset CaP.

possessed an SRD5A2 49T allele, and a risk estimate was therefore not calculated for this population.

Discussion This is the largest study to investigate the association between polymorphisms in a range of candidate genes and early-onset CaP. This study has found that short (p22 repeats) AR (CAG)n was associated with a statistically significant 32% reduced risk of early onset CaP and that the L/L genotype of the SRD5A2 V89L polymorphism was associated with a statistically significant 85% increased risk of early-onset CaP. There was no evidence that other polymorphisms including the AR (GGC)n, CYP17 Msp A1 I, VDR taq I, SRD5A2 A49T or the SRD5A2 (TA)n were associated with early-onset CaP in this study population. Our finding that short (p22 repeats) AR (CAG)n was associated with a significant reduction in the risk of early-onset CaP are unexpected since previous case– control studies have found shorter CAG repeats to predispose towards CaP and in particular, aggressive CaP14–19 and earlier age at diagnosis.20,21 The precise role of the AR and other genes in prostate growth is not known. One explanation for our finding could be that a gene predisposing to CaP may interact with or be influenced by the AR gene. Men with mutations in this gene and longer CAG repeat lengths may be predisposed to early-onset CaP; in men without the mutation, shorter CAG repeats may be associated with an increased risk. This may explain why previous Prostate Cancer and Prostatic Diseases


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studies have found a consistent increase in CaP for short repeat lengths in men with a late age of onset. Indeed, the CLUMP analysis found both shorter CAG repeats and longer CAG repeats to be associated with an increased CaP, compared with average repeat lengths. Longer (X28) CAG repeats have also been associated with a lower age at diagnosis of breast cancer in women with germline BRCA1 mutations,54 although this was not substantiated in a study of early-onset breast cancer.55 Alternatively, the finding that shorter CAG repeats is associated with a reduced risk might be related to an as yet unknown negative feedback mechanism, whereby reduced growth stimuli caused by longer AR CAG repeats disrupt this loop causing cells to overcompensate, thus leading to earlier onset CaP. The finding that the L/L genotype of the SRD5A2 V89L polymorphism was associated with a statistically significant 85% increased risk of early-onset CaP was unexpected, although in line with our AR (CAG)n findings (as shown in Figure 1, these two genes effectively work in series to control prostate cell growth). The variant allele has been associated with lower levels of circulating A-diol-g, a serum marker of 5a-reductase type II activity and intraprostatic DHT production.39,51 One might therefore expect this polymorphism to be associated with a reduction in CaP risk, although previous studies have failed to find a significant association.28,36,40 Nevertheless, there is evidence that men with low testosterone levels have higher Gleason grades and worse outcomes than the prostate cancers that develop in men with normal testosterone.56–59 Unfortunately, we do not have clinical data for our cases, but it seems reasonable to assume that as they were predominantly detected by clinical symptoms, they had more advanced forms of CaP. Longer AR CAG repeats reduce the transactivation ability of the AR, and the leucine variant of SRD5A2 V89L decreases the amount of DHT available to bind to the AR (see Figure 1), suggesting that there could be a common mechanism for early onset CaP predisposition involving reduced transcription of genes that are regulated by the AR. The fact that not just one, but two polymorphisms examined gave the opposite, yet complementary, results to those expected suggests that our findings are not anomalous— one contrary result could be due to chance, but two is unlikely. We found no evidence to suggest that the AR (GGC)n, VDR taq I, CYP17 Msp A1 I A2 or the SRD5A2 A49T polymorphisms were associated with early-onset CaP. Previous studies on the association between CaP risk and the AR GGC repeat polymorphism,15,17,23 the VDR taq I polymorphism,44–48 the CYP17 Msp A1 I polymorphism28,29,60,61 and the SRD5A2 A49T polymorphism33,35,37,38 have been highly inconsistent. However, the low prevalence of some of these variant alleles (particularly the SRD5A2 A49 T T allele) limits the power to detect a significant association, especially in studies with relatively few cases. There may be several explanations for the inconsistencies between the results from this study compared with previous studies. Firstly, this study population consisted of men diagnosed with CaP at the age of 55 or younger, an age group which accounts for only one percent of UK CaP cases.62 No other published population has selected as stringently for age of onset and most


did not account for age at all. Secondly, rates of CaP vary between different countries5 and differences in genetic, dietary and/or environmental factors between populations might account for our findings. However, few risk factors for CaP have been established, making it unlikely that adjustment for other factors would have largely altered the results. Thirdly, the lack of widespread PSA testing in the UK means that the majority of early-onset cases in this study population presented with clinical symptoms. In general, PSA testing, which is widespread in the US, has led to an earlier age at diagnosis, with some estimating that this test lowers the age at diagnosis by X5 years on average.63 The present study population may therefore be clinically different to that of other CaP populations that have been studied thus far. The finding that the AR CAG repeat polymorphism and the SRD5A2 V89L polymorphism were associated with a reduced risk of early-onset CaP raises a number of interesting questions about the nature of early-onset CaP. It is interesting that a recent clinical trial using Finasteride, the SRD5A2 inhibitor, as a chemopreventative agent for CaP, found that while CaP was less likely in those treated, there was a significantly higher prevalence of high grade (Gleason 7–10) disease (P ¼ 0.005) than in untreated men.58 If inhibiting SRD5A2 has this effect, then perhaps low levels of activity of SRD5A2 and AR over a longer period of time will produce similar effects, with fewer, but more aggressive cases of CaP resulting which present at an earlier age. Our results could thus be due to ascertainment bias. More work is required to determine if early-onset CaP is a different disease to CaP detected at a later age and hence different polymorphisms are involved in the disease, or if the same polymorphisms play different roles.

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Supplementary Information accompanies the paper on Prostate Cancer website (http://www.nature.com/PCAN).
