Association of Androgen Receptor,Prostate-Specific Antigen,and CYP19 Gene Polymorphisms with Prostate Carcinoma and Benign Prostatic Hyperplasia in a North Indian Population

Abstract

The genes involved in androgen pathway and metabolism have been reported to contribute considerably to prostate carcinoma (CaP) risk. The present study investigated the association of androgen receptor (AR), prostate-specific antigen (PSA or KLK3), and cytochrome P450 (CYP19) gene polymorphisms in CaP (n=105) and benign prostatic hyperplasia (BPH) (n=120) in comparison to normal healthy controls (n=106) in an Indian population. We also evaluated the functional consequences of these gene variants on AR and PSA mRNA expression. Significant association of short AR CAG repeats (≤24) with risk of CaP (odds ratios [OR]=2.98, p<0.001) and BPH (OR=1.96, p=0.01) was observed; however, CYP19 gene polymorphism was not found to be associated with disease phenotype (p>0.05). PSA G-158A SNP was found to be significantly associated with risk of CaP (AA: OR=2.68, p=0.016 and GA: OR=2.07, p=0.018) p-trend 0.031 and BPH (AA: OR=3.46, p<0.001 and GA: OR=2.47, p=0.03) p-trend 0.009, respectively. PSA G-158A genotype independently increased the risk of developing BPH (OR=16.37, p<0.001), irrespective of AR CAG repeat length. Using quantitative real-time polymerase chain reaction, we found a significant upregulation of AR and PSA mRNA expression in CaP comparison to BPH. While short AR CAG (≤24) repeats were associated with higher AR mRNA expression in CaP (p=0.002), the PSA SNP did not correlate with its mRNA expression. Interestingly, significantly higher risk estimates for CaP were observed for the combined analysis of short AR CAG and CYP19 genotypes (A2A2) (OR=7.18, p<0.001) or A2A3 (OR=7.60, p=0.004). Our results suggest significant association of androgen signaling gene polymorphisms with risk of CaP and BPH and provide evidence for a putative functional role of AR CAG repeat in regulating its mRNA expression and warrant the need of larger studies in the Indian population to confirm our results.

Introduction

Prostate carcinoma (CaP) is the second most frequently diagnosed cancer and the sixth leading cause of cancer death in men worldwide (Jemal et al., 2011). In India, CaP is the fifth most common cancer among men (Sinha et al., 2003). Although the incidence of CaP in India is lower as compared with the Western population, the last two decades have witnessed a substantial increase in its incidence (Satyanarayana and Asthana, 2008).

Androgen receptor (AR) is considered a critical effector for development and progression of CaP (Buchanan et al., 2001). The first exon of the AR gene contains a polymorphic CAG repeat tract. Progressive expansion of the CAG repeat has been associated with a linear decrease in the AR transactivation (Santos et al., 2003). Several studies have shown the association of short AR CAG polymorphism with the risk of CaP (Modugno et al., 2001; Balic et al., 2002; Rodriguez-Gonzalez et al., 2009) while other studies failed to document a similar association (Latil et al., 2001; Gsur et al., 2002; Salinas et al., 2005). However, the functional role of AR CAG repeat polymorphism on its mRNA expression has not been studied in clinical samples of CaP.

Prostate-specific antigen (PSA or KLK3) is the best studied AR target gene and is thought to contribute to CaP progression through its protease activity and its ability to induce epithelial-mesenchymal transition and cell migration (Jariwala et al., 2007). The serum PSA (sPSA) level is associated with the pathologic stage and grade of CaP and is considered indispensable in the posttreatment monitoring of patients for progression and recurrence (Erbersdobler et al., 2010). An important PSA G-158A SNP located within ARE I (Lai et al., 2007) has been identified to be associated with CaP risk using genome-wide scan for CaP susceptibility loci with SNPs in the KLK3 gene (Guy et al., 2009).

Androgen deprivation and administration of estrogens are an established mode of therapy for CaP and it is suspected that early exposure to estrogens initiates carcinogenesis among different tissues, including the prostate gland, and that decrease of the androgen/estrogen ratio with aging could be responsible in part for CaP (Cussenot et al., 2007). Aromatase encoded by CYP19 is a key enzyme that converts testosterone into estrogen in men, and is suggested to play an important role in the development of benign prostatic hyperplasia (BPH) and CaP (Tsuchiya et al., 2006). CYP19 has a tetranucleotide (TTTA) repeat, which is reported to be associated with CaP, breast cancer, and postmenopausal bone metabolism (Modugno et al., 2001; Ellem et al., 2004).

The present study had been initiated to investigate the role of androgen signaling (AR and PSA) and metabolism pathway (CYP19) gene variants in CaP and BPH. In addition, we report for the first time the functional impact of AR CAG repeats on its gene expression in Indian CaP patients.

Materials and Methods

Study subjects

Two hundred and seventy-nine cases (279) of prostate enlargement were registered for the study. After a thorough clinical examination, all cases underwent uroflowmetry, digital rectal examination, and sPSA levels. In 245 patients the sPSA levels were found to be > 4 ng/mL and were subjected to sextant or sextant plus site specific or 12-core transrectal ultrasonography-guided prostate biopsy. One hundred and five cases were diagnosed with CaP and 120 cases were diagnosed with BPH. Twenty BPH cases were excluded from the study as 4 cases had malignancy in the urinary bladder and 16 BPH cases could not be age matched with CaP cases. In addition, 106 age-matched healthy control individuals were also enrolled for the study. Controls were ruled out for previous history of any malignancy or urological disorders.

Tissue samples for AR and PSA mRNA expression analysis were collected from 32 CaP and 30 BPH patients.

Informed consent was obtained from all the participating patients and healthy controls and the study was carried out with the approval of Ethical Review Committee of Safdarjung Hospital, New Delhi.

Polymerase chain reaction-based GeneScan analysis: AR CAG and CYP19 TTTA repeat analysis

The AR and CYP19 repeat polymorphisms were analyzed using fragment analysis (Tsuchiya et al., 2006; dos Santos et al., 2008). Repeat number for both AR CAG and CYP19 TTTA repeats was confirmed by direct sequencing on a 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA).

Polymerase chain reaction-restriction fragment length polymorphism assay: PSA G-158A polymorphism

The PSA G-158A polymorphism was determined by the polymerase chain reaction (PCR)-restriction fragment length polymorphism as described previously (Rao et al., 2003). Genotyping results were validated by direct sequencing.

DNA and RNA extraction and quantitative real time-polymerase chain reaction

Genomic DNA was extracted from blood leucocytes using a standard phenol-chloroform extraction method (Blin and Stafford, 1976). Total cellular RNA was extracted from fresh-frozen tissue sections using Trizol (Molecular Research Center, Cincinnati, OH). AR and PSA mRNA expression was determined by TaqMan quantitative real-time polymerase chain reaction according to manufacturer's protocol. The assay ID numbers for AR and PSA (KLK3) were Hs00171172_m1 and Hs03063374_m1, respectively.

Statistical analysis

Hardy-Weinberg equilibrium was calculated using the χ² test in the CaP, BPH, and controls (d.f.=1). The multinomial logistic regression analysis was performed for risk assessment of polymorphisms. The AR CAG repeats were categorized dichotomously as short (≤24 repeat units) and long (>24 repeat units) taking into consideration the results of AR CAG repeats as continuous variable wherein the analysis showed the maximum relative risk with repeat length of 24. The Dunn-Sidak correction was applied for the multiple testing, the adjusted p-value was obtained by the equation as (1−[1−α]^1/3), where α=0.05 and 3 accounted for three independent hypothesis (AR, PSA, and CYP19) being tested.

The multiple endogenous control strategy using GAPDH, TBP, and 18S rRNA genes was used for normalization as described previously (Soni et al., 2011). Data analysis for AR and PSA mRNA expression was performed using the Integromics RealTime StatMiner package. Fold changes were calculated on filtered and quantile normalized data using the DDCT method. The Wilcoxon Rank-Sum test was performed for testing the significant difference for mean mRNA expression in CaP and BPH. Correlation of the genotypes and mRNA expression was performed using the Mann-Whitney or the Kruskal-Wallis test. A two-sided p≤0.05 was considered statistically significant. All statistical analysis was performed using the SPSS 17.0 software package.

On the basis of an earlier study in Indian population (Mittal et al., 2007), AR CAG short allele frequency among cases of CaP (n=135) studied was estimated as 78.5%, while for controls (n=142) it was found to be 52.1% and the odds ratios (OR) was estimated as 3.36, which was the maximum value among all studied genotypes. Taking these two percentages and the OR at 5% level of significance and 90% power, the minimum sample size that should be studied to test for this significant difference was found to be 75.

Results

The mean age (mean±SD) of CaP (68.63±9.83), BPH (67.12±9.10), and controls (62±10.62) was not significantly different (Table 1).

Table 1.

Clinico-Pathological Characteristics of the Study Subjects

	For gene expression study		For polymorphism study
Clinico-pathologic characteristics	CaP, n=32 (%)	BPH, n=30 (%)	CaP, n=105 (%)	BPH, n=120 (%)	Controls n=106 (%)
Age
Mean±SD	61.90±8.50	64.87±9.34	68.63±9.83	67.12±9.10	62±10.62
Median	61.5	62	70	66	62
PSA (ng/mL), median	41.5	6.8	45	6	<1
Prostate volume (cm³), mean±SD	43.13±19.88	24.13±6.72	41.53±17.00	23.33±6.21
Cores procured n (%)
Sextant	6 (19.0)	10 (33.0)	10 (10.6)	18 (15.0)
Sextant plus site specific	12 (37.5)	3 (10.0)	22 (23.4)	34 (28.3)
Greater than sextant	4 (12.5)	3 (10.0)	15 (16.0)	12 (10.0)
TURP n (%)	10 (31.0)	14 (47.0)	47 (50.0)	56 (46.7)
Gleason score n (%)
<7 (5-6)	11 (34.4)		66 (70.0)
≥7 (7-9)	21 (65.6)		28 (29.8)

BPH, benign prostatic hyperplasia; CaP, prostate carcinoma; PSA, prostate-specific antigens; TURP, trans urethral resection of prostate.

The frequency distribution of AR CAG, PSA G-158A, and CYP19 TTTA genotypes in CaP, BPH, and controls is shown in Table 2. A statistically significant association of short AR CAG repeats (≤24) was found with CaP risk (odds ratios [OR]=2.98 [95% confidence interval (CI)=1.69-5.24, p<0.001]). Results were observed in the similar direction when BPH cases were compared with controls, however, with lower risk estimates (OR=1.96 [95% CI=1.15-3.32, p=0.01]).

Table 2.

Genotype Frequencies and Odds Ratios of Polymorphisms Associated with Prostate Cancer Risk Among Different Groups

					OR [95% CI], p^a
Gene	Genotype	CaP, n=105 (%)	Controls, n=106 (%)	BPH, n=120 (%)	CaP vs. controls	BPH vs. controls
AR	>24^b	32 (30.5)	46 (43.4)	48 (40.0)	1	1
	≤24^b	73 (69.5)	60 (56.6)	72 (60.0)	2.98 [1.69-5.24], <0.001	1.96 [1.15-03.32], 0.01
PSA	GG	36 (34.3)	57 (53.8)	32 (26.7)	1	1
	GA	47 (44.8)	36 (34.0)	70 (59.8)	2.07 [1.13-3.77], 0.018	2.47 [1.07-5.68], 0.03
	AA	22 (21.0)	13 (12.3)	18 (15.0)	2.68 [1.20-5.98], 0.016	3.46 [1.92-6.25], <0.001
CYP19	A1A1 (7/7)	10 (9.5)	10 (9.5)	10 (8.3)	1	1
	A1A2 (7/8)	17 (16.2)	24 (22.6)	24 (20.0)	0.71 [0.24-2.07], 0.53	1.00 [0.35-2.84], 1.00
	A1A3^c (7/9)	11 (10.5)	17 (16.0)	22 (18.3)	0.65 [0.20-2.06], 0.46	1.29 [0.44-3.82], 0.64
	A2A2 (8/8)	25 (23.8)	15 (14.2)	15 (12.5)	1.67 [0.56-4.93], 0.36	1.00 [0.32-3.10], 1.00
	A2A3 (8/9)	6 (5.7)	4 (3.8)	12 (10.0)	1.50 [0.32-6.99], 0.61	3.00 [0.72-12.55], 0.13
	A2A6^c (8/12)	22 (21.0)	20 (18.9)	21 (17.5)	1.10 [0.38-3.19], 0.86	1.00 [0.36-3.06], 0.93
	A3A3^c (9/9)	8 (7.6)	2 (2.8)	9 (7.5)	2.67 [0.54-13.08], 0.23	3.00 [0.62-14.47], 0.17
	A6A6^c (12/12)	6 (5.7)	13 (12.3)	7 (5.8)	0.46 [0.13-1.70], 0.25	0.54 [0.15-1.92], 0.34

Significant p-values after Dunn-Sidak correction are given in bold.

Number of repeats.

Risk assessment of the less common genotypes was derived by merging with other categories.

AR, androgen receptor; OR, odds ratios; CI, confidence interval.

For PSA G-158A SNP, genotype frequencies among controls were found in agreement with the expected HWE frequencies (p=0.66, 0.14, and 0.18, respectively, for CaP, BPH, and controls). Significant association of the A allele has been found with risk of CaP both in heterozygous (GA) and homozygous (AA) conditions, the risk of the AA genotype to CaP (OR=2.68 [95% CI=1.20-5.98, p=0.016]) and BPH (OR=3.46 [95% CI=1.92-6.25, p<0.001]) being higher than that of the GA genotype to both CaP (OR=2.07 [95% CI=1.13-3.77, p=0.018]) and BPH (OR=2.47 [95% CI=1.07-5.68, p=0.03]). A significant increasing trend (p<0.001) has been observed in risk for both CaP and BPH from the GA to AA genotypes. According to the HapMap project data, in the Asian population, 69.7% of individuals were GG homozygotes, 29.2% were GA heterozygotes, and 1.1% were AA homozygotes for PSA G-158A polymorphism. The frequencies for control individuals found in our data were 53.8% for GG genotype and 34.0% for GA genotype, while 12.3% were AA genotype.

Seven different alleles (designated A1 to A7) of CYP19 TTTA repeat polymorphism were identified in our study population. A2 allele (eight repeats) was found to contribute significantly toward the risk of BPH (OR=2.76 [95% CI=1.01-7.54, p=0.047]) and moderately to CaP (OR=2.78 [95% CI=0.97-7.97, p=0.06]) (Table 3). However, none of the CYP19 genotypes was found to confer significant risk for CaP or BPH in the study population (Table 2).

Table 3.

Allelic Distribution of CYP19 TTTA Repeats in CaP, BPH, and Controls

Allele ^a	CaP, n=105 (%)	BPH, n=120 (%)	Controls, n=106 (%)	CaP vs. controls OR [95% CI], p	BPH vs. controls OR [95% CI], p
A1	38 (23.60)	56 (28.14)	48 (28.57)	1	1
A2	70 (43.48)	72 (36.18)	63 (37.50)	2.78 [0.97-7.97], 0.057	2.76 [1.01-7.54], 0.047
A3	25 (15.53)	43 (21.60)	24 (14.29)	2.35 [0.84-6.58], 0.10	1.89 [0.70-5.07], 0.21
A6	28 (17.39)	28 (14.07)	33 (19.64)	2.56 [0.92-7.11], 0.07	2.20 [0.77-6.30], 0.14

The allelic frequencies were derived from the primary genotype distribution (A1: by combining the frequencies of A1A1, A1A2, and A1A3 genotypes; A2: by combining A1A2, A2A2, A2A3, and A2A6; A3: by combining A1A3, A2A3, and A3A3; and A6: by combining frequencies of A2A6 and A6A6).

TTTA, tetranucleotide.

Analysis of combined effects of AR CAG repeats and PSA G-158A genotypes on CaP susceptibility showed that patients with short AR CAG (≤24) and GA (OR=14.99 [95% CI=2.37-13.81, p<0.001]) and AA genotypes (OR=8.87 [95% CI=1.76-15.51, p=0.003]) of PSA gene had a significant risk for CaP compared with other combinations. In BPH patients, significant increase in risk was also found in patients with short AR CAG repeats (≤24) and GA (OR=21.07 [95% CI=3.97-31.03, p<0.001]) and AA genotypes (OR=11.61 [95% CI=2.48-29.05, p=0.003]) of PSA (Table 4a). Interestingly, significant risk was also found in BPH patients having longer AR CAG repeats (>24) with GA genotype of PSA gene (OR=16.37 [95% CI=2.82-19.83, p<0.001]), suggesting that the PSA G-158A genotype might be an independent risk factor for BPH irrespective of AR CAG repeat status.

Table 4.

Combined Effects of AR CAG Repeats with PSA and CYP19 Genotypes on the Risk of CaP and BPH

(a) AR	PSA	CaP	BPH	Control	CaP vs. controls OR [95% CI]	p^a	BPH vs. controls OR [95% CI]	p^a
>24	GG	32 (30.2)	7 (5.8)	22 (21.0)	1	1	1	1
	GA	22 (20.8)	36 (30.0)	35 (33.3)	0.21 [0.49-3.20]	0.65	16.37 [2.82-19.83]	<0.001
	AA	6 (5.7)	5 (4.2)	16 (15.2)	1.57 [0.63-8.34]	0.21	3.31 [0.90-16.10]	0.069
≤24	GG	25 (23.6)	25 (20.8)	14 (13.3)	2.60 [0.86-4.71]	0.11	9.09 [1.70-12.28]	0.003
	GA	14 (13.2)	34 (28.3)	12 (11.4)	14.99 [2.37-13.81]	<0.001	21.07 [3.97-31.03]	<0.001
	AA	7 (6.6)	13 (10.8)	6 (5.7)	8.87 [1.76-15.51]	0.003	11.61 [2.48-29.05]	0.001

(b) AR	CYP19				CaP vs. controls OR [95% CI]	p^a	BPH vs. controls OR [95% CI]	p^a
>24	A1A1	8 (7.6)	3 (2.5)	6 (5.7)	1	1	1	1
	A1A2	11 (10.5)	9 (7.5)	10 (9.4)	1.00 [0.53-18.92]	1.00	2.67 [0.28-25.64]	0.40
	A1A6	7 (6.7)	8 (6.7)	10 (9.4)	1.33 [0.20-9.08]	0.77	0.89 [0.16-5.08]	0.89
	A2A2	19 (18.1)	6 (5.0)	5 (4.7)	1.50 [0.16-14.42]	0.73	3.00 [0.45-20.15]	0.26
	A2A3	3 (2.9)	9 (7.5)	0 (0)	1.20 [0.17-14.42]	0.73	0.80 [0.13-4.87]	0.81
	A2A6	14 (13.3)	8 (6.7)	8 (7.5)	1.14 [0.14-9.29]	0.90	1.52 [0.25-9.30]	0.67
	A3A3	7 (6.7)	4 (3.3)	1 (0.9)	0.86 [0.12-6.14]	0.88	8.57 [0.15-4.76]	0.86
	A6A6	4 (3.8)	1 (0.8)	6 (5.7)	0.57 [0.06-5.57]	0.64	0.19 [0.02-2.50]	0.21
≤24	A1A1	2 (1.9)	7 (5.8)	4 (3.8)	2.67 [0.36-19.71]	0.34	1.56 [0.24-9.91]	0.64
	A1A2	6 (5.7)	15 (12.5)	14 (13.2)	14.00 [0.94-207.60]	0.055	1.6 [0.38-7.02]	0.52
	A1A3^b	4 (3.8)	14 (11.7)	7 (6.6)	3.50 [0.52-23.56]	0.20	3.29 [0.67-16.26]	0.14
	A2A2	6 (5.7)	9 (7.5)	10 (9.4)	7.18 [6.51-7.92]	<0.001	2.13 [0.41-11.06]	0.37
	A2A3	3 (2.9)	3 (2.5)	4 (3.8)	7.60 [1.07-54.09]	0.04	2.40 [0.38-15.32]	0.36
	A2A6^b	8 (7.6)	13 (10.8)	12 (11.3)	1.40 [0.20-9.87]	0.74	1.87 [0.34-10.25]	0.48
	A3A3^b	1 (1.0)	5 (4.2)	2 (1.9)	2.20 [0.33-14.72]	0.42	2.00 [0.37-10.92]	0.42
	A6A6^b	2 (1.9)	6 (5.0)	7 (6.6)	1.33 [0.161-11.08]	0.79	1.33 [0.20-8.71]	0.76

Significant p-values after Dunn-Sidak correction are given in bold.

Risk assessment of the less common genotypes was derived by merging with other categories.

Combination of short AR CAG repeats (≤24) with the most common genotypes A2A2 (OR=7.18 [95% CI=6.51-7.92, p<0.001]) or A2A3 (OR=7.60 [95% CI=1.07-54.09, p=0.004]) of CYP19 TTTA repeats revealed a significantly increased risk for CaP (Table 4b). No significant risk was observed for any combinations of AR CAG and CYP19 TTTA repeats for BPH. The combined effect of PSA and CYP19 genotypes was not evaluated due to inadequate number of samples in each group.

No significant association was observed between the AR, PSA, and CYP19 gene variants and median levels of sPSA, prostate volume, or Gleason scores (p>0.05).

A statistically significant (p=0.004) difference in the mean (mean±SD) mRNA expression of AR (0.756±4.591) in CaP was observed compared with BPH cases (−4.042±3.569). Similar significant (p=0.049) difference in mean PSA mRNA expression in CaP (−6.653±4.990) was found compared with BPH (−9.402±5.390). Both AR and PSA genes showed nearly threefold increased mRNA expression in CaP as compared with BPH. Increased mRNA expression of AR gene was found in cases of CaP with short AR (CAG) repeats (p=0.002) while no significant association was observed in PSA mRNA expression and PSA genotypes (GG, GA, or AA) in CaP patients (p>0.005). No significant difference in the expression of either AR or PSA was found among different genotypes in BPH cases (Table 5).

Table 5.

Mean Gene Expression of AR and PSA Across Different Genotypes

		Mean±SD
Gene	Genotypes	CaP	BPH	p Value
AR	>24	−3.09±3.15	−3.43±3.21	0.002
	≤24	0.33±4.69	−4.55±3.86
PSA	GG	−9.48±1.00	−10.52±5.89	>0.05
	GA	−5.66±5.34	−8.41±5.55
	AA	−9.06±4.02	−9.98±1.08

Discussion

Global ethnic variation explains the substantial differences in the incidence of CaP existing throughout the world, reported highest in Western countries and lowest in developing countries (Zeegers et al., 2004). The association between the AR CAG repeats and CaP susceptibility has been studied across populations. Recently, a meta-analysis conducted on AR CAG repeats and CaP revealed that after classifying studies by geographic areas, carriers of AR CAG repeats of ≥20 had 11% decreased risk of CaP in populations from United States, 53% from Europe, and 20% from Asia (p>0.05), whereas comparison of ≥23 repeats with others generated a significant prediction in European populations (Gu et al., 2012). In the present study, we have demonstrated a significant association between the short AR CAG repeats (≤24) and risk of CaP and BPH in the Indian population; however, mean repeat length and the cutoff values for CaP were found to be different from previously reported studies (Krishnaswamy et al., 2006; Mittal et al., 2007), which might be attributed to population heterogeneity. The only other study, with 57 Indian BPH patients, showed no significant association of BPH with short AR CAG repeat length (Mishra et al., 2005). These controversial results can be explained by the low power of the study previously conducted in Indian BPH patients.

Previous studies have suggested association of PSA G-158A polymorphism with risk and disease progression of CaP in Caucasian populations; however, the allele associated with risk was found to be inconsistent (Chiang et al., 2004; Schatzl et al., 2005). The present study on PSA G-158A polymorphism demonstrated that men with GA or AA genotype have a significantly increased risk for developing CaP and BPH. The present study did not show any significant alteration in median sPSA levels when cross-classified by AR, PSA, and CYP19 genotypes.

An altered ratio of testosterone and estrogen has been related to the onset of both CaP and BPH (Ellem and Risbridger, 2007). Haiman et al. (2000) demonstrated that the presence of at least one allele with more than seven (TTTA) repeats of the CYP19 polymorphism was associated with a lower level of plasma androstenedione, a higher level of estrogens, and a higher estrone-to-androstenedione ratio. This polymorphism influences the activity of aromatase enzyme both in vivo and in vitro (Hammoud et al., 2010). However, the present study did not find any significant association of CYP19 genotypes with the risk of CaP or BPH. While the A2 allele has found to contribute significantly toward the risk of BPH, it moderately influences the risk of CaP. However, this finding needs further replication in larger cohorts.

Since the allelic variability at a single locus cannot explain the etiology of CaP, therefore, combined effects of polymorphisms in the genes under study were evaluated for their synergistic effects. The present study demonstrated that combination of short AR CAG repeats (≤24) with GA/AA genotypes of PSA gene further increased the risk for both CaP and BPH; the similar increased risk has been reported previously (Xue et al., 2000; Gsur et al., 2002). Interestingly, the present study has also shown increased risk for BPH when long AR CAG repeats (>24) were combined with PSA AA or GA genotypes. The current study also demonstrated that the combination of short AR CAG genotype (≤24) with the most common CYP19 TTTA genotypes (A2A2) or (A2A3) significantly increases the risk for CaP but not for BPH. This finding is interesting as the two genes are essential for homeostasis and the balance in the two hormones might be a decisive factor in prostate carcinogenesis.

Altered expression of the AR has been demonstrated both in human tissues and animal model systems of CaP (Scher et al., 2004). In the present study increased mRNA expression of both AR and PSA genes was observed in CaP compared with BPH. Likewise, it has been suggested that shorter AR CAG repeats impose a higher transactivation activity on the receptor and have increased binding activity for androgens (Feldman, 1997). Our observations complement the study, as a significantly higher AR mRNA expression was observed in CaP cases with short AR CAG repeats (≤24).

The present study does have its own limitations and strengths. The major limitation of the present study is the small sample size. Even with low sample size, the study had 90% power to test for significant difference between AR CAG repeat polymorphism in CaP and controls as explained in the Materials and Methods section. The strength of the current study is the investigation of complex combined effects among AR, PSA, and CYP19 genes in prostate carcinogenesis and effects of AR CAG repeat length on AR mRNA expression in CaP patients. These findings warrant replication as their interpretation is limited somewhat by small sample size. One solution to overcome some of these limitations is to increase statistical power. Therefore, resolution may ultimately require the meta-analysis in the Indian population and comparison with other populations.

Conclusion

In summary, the results in the present study suggest significant association of AR CAG repeats and PSA G-158A polymorphism with the risk of CaP and BPH independently as well as in combination. We provided evidence that short AR CAG repeats (≤24) exceedingly transactivate AR mRNA expression in CaP. Our results could serve as a potential platform for future genetic research on CaP in the Indian population using studies with large sample size.

Footnotes

Acknowledgments

The authors sincerely thank patients, their family members, and volunteers for their kind support. The authors thank Department of Science and Technology (DST) for providing necessary funds to support this study. J.B. is supported by NHMRC early career fellowship.

Author Disclosure Statement

No competing financial interests exist.

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