This article has Open Peer Review reports available.
8q24 sequence variants in relation to prostate cancer risk among men of African descent: A case-control study
© Benford et al; licensee BioMed Central Ltd. 2010
Received: 18 August 2009
Accepted: 28 June 2010
Published: 28 June 2010
Human chromosome 8q24 has been implicated in prostate tumorigenesis.
Consequently, we evaluated seven 8q24 sequence variants relative to prostate cancer (PCA) in a case-control study involving men of African descent. Genetic alterations were detected in germ-line DNA from 195 incident PCA cases and 531 controls using TaqMan polymerase chain reaction (PCR).
Inheritance of the 8q24 rs16901979 T allele corresponded to a 2.5-fold increase in the risk of developing PCA for our test group. These findings were validated using multifactor dimensionality reduction (MDR) and permutation testing (p = 0.038). The remaining 8q24 targets were not significantly related to PCA outcomes.
Although compelling evidence suggests that the 8q24 rs16901979 locus may serve as an effective PCA predictor, our findings require additional evaluation in larger studies.
Prostate cancer (PCA) accounts for 25% of all diagnosed cancer cases and was the second leading cause of cancer-related deaths in 2009 among U.S. men . Well-established risk factors of PCA include age, family history of PCA, and ethnicity. African American men carry a substantial portion of the disease burden and are 1.6- and 2.4-fold more likely to be diagnosed with PCA and die from disease, respectively, relative to their Caucasian counterparts . This disparity may be partially attributed to a combination of genetic predisposition and environmental exposures. Despite evidence supporting the role of genetics in PCA etiology, the search for common sequence variants as susceptibility markers has been largely unrewarding. However, recent genome-wide association studies (GWAS) indicate that chromosome 8q24 is a region of interest for further exploration of health disparity markers relative to PCA incidence and outcomes.
Several genetic variants or single nucleotide polymorphisms (SNPs) in the 8q24 region have been associated with PCA risk in Caucasians; however, similar studies on African Americans are only represented in a handful of published reports [3–8]. Amundattoir and co-workers (2006) demonstrated that the DG8S737- 8 microsatellite was significantly associated with PCA risk and disease progression in men of European and African descent . The association signal of DG8S737- 8 in Caucasians has been captured by rs1447295 or one of its linkage disequilibrium equivalents. This was not the case for African Americans, which suggests that the DG8S737- 8 microsatellite was either associated with increased PCA risk or is significantly correlated with other 8q24 sequence variants not detected in a GWAS scan or HapMap project. Two subsequent studies provide compelling evidence that two 8q24 sequence variants, rs6983561 and rs16901979, may be important PCA indicators for Caucasians and African Americans [3, 6]. Although the risk estimates for rs16901979 were similar between Caucasians and African Americans, this marker's higher prevalence among African Americans may partially explain the greater PCA incidence in this high-risk group. In fact, Gudmundson and co-workers (2007) suggested that the rs16901979 SNP alone could account for a large fraction of PCA risk among African Americans, as they had a considerably high population-attributable risk associated with this marker (24%) relative to Caucasians . Two other sequence variants, rs4242382 and rs4242384, were significantly related to organ-confined or aggressive disease when compared to disease-free men of European descent [9–11].
Unfortunately, the impact of these two loci and other 8q24 markers (e.g., rs6983561, rs1447295, rs11934905, rs16901979, and rs10090154) on PCA risk among men of African descent remains largely unknown. Although the 8q24 rs10090154T allele confers a 1.7-fold increase in PCA risk in a small case-control study set involving African Americans (85 cases and 149 disease-free men), this locus only reached borderline significance (OR = 1.69; 0.92-3.11).
To clarify the role of 8q24 sequence variants in PCA susceptibility among men of African descent, we sought to confirm previous reports and generate new data on the role of seven 8q24 sequence variants in PCA risk among 864 men of African descent (195 cases and 531 disease-free men). To overcome sample size issues and control for multiple comparisons, we used a multifactor dimensionality reduction (MDR) algorithm along with permutation testing. This approach will aid future studies that analyze different sequence variants within the 8q24 region to decode health disparities in high-risk subgroups, especially PCA risk in men of African descent.
Patient and Tumor Characteristics
No. of Participants, n
PSA in ng/ml
< 2.0, n (%)
Gleason Score n (%)
Global West African Ancestry
TaqMan allelic discrimination of 8q24 sequence variants
Polymorphisms detected in the 8q24 region were ascertained using TaqMan Polymerase Chain Reaction (PCR) allelic discrimination assays. The following seven alleles were detected: (1) rs6983561 (A > C); (2) rs1447295 (G > T); (3) rs4242384 (A > C); (4) rs4242382 (G > A); (5) rs11934905 (G > A); (6) rs16901979 (G > T); and (7) rs10090154 (G > A). Each allelic discrimination assay contained approximately 40 ng of germ-line DNA, 1× Universal Master Mix (Applied Biosystems, Foster City, CA), a 40× mixture containing 900 nM of each primer (forward and reverse), and 200 nM of each probe (FAM and VIC) to comprise a 5 μl reaction. To facilitate amplification of regions containing the aforementioned 8q24 SNPs, primers and probes were designed in our laboratory using sequences provided by NCBI  and placed into the FileBuilder software (Applied Biosystems). PCR reactions were carried out using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems). The thermocycling settings consisted of two holds at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec/cycle and 1 min at 60°C. The fluorescent intensity emitted from the probes was measured using the ABI 7900 sequence detection system and assigned genotypes with the SDS 2.2.1 software (Applied Biosystems). To minimize misclassification bias, laboratory technicians were blinded to the case status of study participants. Based on 24 non-DNA template controls per batch analysis, the percent cross-contamination during sample handling was less than 4.7%. Duplicate genotyping was performed on 72 randomly selected samples for quality control purposes, resulting in concordance rates of ≥ 97.5%. The genotype call rates ranged between 89.6 and 96.2% across the seven SNPs with a median value of 92.3%. Subjects (n = 62) with 4 or more missing SNP values across the 7 SNPs were removed from the final analysis.
One hundred ancestry autosomal markers were included to account for potential population stratification among our admixed population of self-reported African-Americans, West African Americans, East African Americans, and Afro-Caribbean Americans, as previously described . Study participants were grouped from lowest to highest genetic West African ancestry, with scores ranging from 0 to 100%. This marker was assembled using DNA from self-identified African-Americans (Coriell Institute for Medical Research, n = 96), Yoruban West Africans (HapMap, n = 60), West Africans (Bantu and Nilo Saharan speakers, n = 72), Europeans (New York City, n = 24), and Centre d'Etude du Polymorphisme Humain (CEPH) Europeans (HapMap Panel, n = 60). Individuals with a high degree of West African ancestry greater than or equal to 25% were included in the current study.
Screening for single gene markers predictive of PCA risk using conventional Logistic Regression (LR) Analysis
To assess whether inheritance of at least one variant 8q24 allele was associated with an elevated risk of developing PCA, we tested for significant differences in the distribution of seven 8q24 genotypes between 195 cases and 531 controls using the chi-square test of homogeneity. Associations between PCA risk and 8q24 sequence variants, expressed as odds ratios (ORs) and corresponding 95% confidence intervals (CIs), were estimated using unconditional multivariate LR models adjusted for potential confounders (age, PSA, and West African ancestry). All reported risk estimates and 95% CIs for the selected 8q24 loci used the following as reference genotypes: (1) rs6983561 (A/A); (2) rs1447295 (G/G); (3) rs4242384 (A/A); (4) rs4242382 (G/G); (5) rs11934905 (G/G); (6) rs16901979 (G/G); and (7) rs10090154 (G/G). For rs11934905, hetero- and homozygotes (AA + GA) were combined due to the low frequency of the minor allele. Test for trend included genotypes as ordinal variables. Statistical significance was assessed using a P-value < 0.05. All chi-square tests and LR analyses were conducted using the SAS 9.1.3 software (SAS Institute Inc., Cary, NC).
Validation of main effects using MDR
Multifactor dimensionality reduction (MDR) was used to evaluate and validate main effects associated with PCA risk. This algorithmic tool aids in the identification of high-risk markers using a cross validation strategy to estimate the classification and prediction accuracy of individual factor models [16, 17]. MDR is a data-mining platform that readily overcomes sample size limitations often encountered by parametric statistical methods (e.g., LR analysis) by collapsing high-dimensional genetic data into a single dimension. For this study, single factor loci were classified into high-risk and low-risk groups, which permitted the investigation of individual effects of variant 8q24 alleles in relation to PCA risk using a cross-validation and permutation testing scheme. MDR was utilized to generate a single model that maximized the number of individuals with the proper risk assignment. The best 8q24 sequence variant was selected among the seven loci that minimized the prediction error as well as maximized the cross validation consistency (CVC) and average testing accuracy (ATA). To evaluate the number of times the same single factor model was identified in each possible 9/10ths of the data, the average CVC (based on a scale from 0-100%) from the observed data was compared to the distribution of average consistencies under the null hypothesis of no association. Validation of models as effective predictors of PCA susceptibility was derived empirically from 10,000 permutations. This approach accounted for multiple testing issues as long as the entire model fitting procedure was repeated for each randomized dataset to provide an opportunity to identify false-positives. We considered MDR permutation results to be statistically significant at the 0.05 level.
Patient & Tumor Characteristics
The patient and tumor characteristics in the current study are summarized in Table 1. Cases were significantly older than controls and had higher PSA levels. There was significant difference in median West African genetic ancestry estimates when comparing cases and controls (P = 0.0273).
Prevalence of 8q24 variant alleles among men of African descent
Association between 8q24 Sequence Variants and Prostate Cancer Risk.
(95% CI) ††
1.35 (0.90 - 2.02)
1.78 (1.10 - 2.89)
1.70 (1.07 - 2.70)
1.99 (1.15 - 3.46)
AC + CC
1.46 (1.00 - 2.13)
1. 85 (1.18 - 2.91)
0.96 (0.67 - 1.37)
0.82 (0.55 - 1.26)
1.10 (0.66 - 1.85)
0.92 (0.49 - 1.70)
GT + TT
0.99 (0.71 - 1.39)
0.86 (0.45 - 1.61)
1.70 (1.18 - 2.45)
1.50 (0.99 - 2.28)
0.90 (0.35 - 2.27)
0.91 (0.32 - 2.53)
AC + CC
1.58 (1.11 - 2.25)
1.41 (0.95 - 2.11)
1.20 (0.84 - 1.72)
1.12 (0.74 - 1.69)
1.12 (0.68 - 1.84)
1.05 (0.60 - 1.86)
GA + AA
1.18 (0.85 - 1.65)
1.01 (0.75 - 1.62)
GA + AA
1.38 (0.64 - 2.98)
1.83 (0.54 - 6.24)
1.71 (1.14 - 2.56)
2.28 (1.42 - 3.69)
2.40 (1.49 - 3.87)
3.02 (1.72 - 5.31)
GT + TT
1.90 (1.30 - 2.77)
2.49 (1.58 - 3.92)
1.30 (0.90 - 1.88)
1.11 (0.72 - 1.69)
1.02 (0.39 - 2.64)
0.88 (0.30 - 2.57)
GA + AA
1.26 (0.89 - 1.81)
1.08 (0.72 - 1.63)
Association between 8q24 and PCA
Multifactor Dimensionality Reduction Models for 8q24 rs16901979 Sequence Variants
Cross Validation Consistency
Average Testing Accuracy
Permutation Testing p-value
Sequence variants localized to the 8q24 region have recently been associated with numerous cancer sites [4, 18, 21, 22]. In the current study, we evaluated the relationship between seven sequence variants and the risk of developing PCA and aggressive disease among 195 cases and 531 disease-free study participants. Our analyses revealed a statistically significant 2.5-fold increase in the risk of developing PCA among men of African descent who carry the rs16901979 T allele. This relationship remained important after adjusting for potential confounders and multiple comparisons. None of the other six sequence variants we analyzed were significantly related to PCA risk.
Our findings on the link between rs16901979 and PCA risk were validated using MDR, which remains effective with relatively small sample sizes. In addition, this observed correlation is further strengthened by two out of three other studies involving African American men. These two independent studies both revealed a 1.3- to 1.5-fold increase in the risk of developing PCA with 95% CIs that exclude unity [6, 7].
Several of the evaluated 8q24 SNPs may be more appropriate as PCA predictors among men of European- rather than African descent [3, 5, 6, 11, 23]. The rs4242384, rs4242382, rs1447295, and rs10090154 sequence variants were not significantly associated with PCA outcomes among men of African descent in the current study and/or published reports, but these markers appear to play a role in PCA risk or aggressive disease among men of European descent [3, 5–7, 9, 11, 19, 23–25]. Zheng and co-workers (2007) observed a 1.3- to 1.9-fold increase in PCA risk among European carriers of one or two rs4242382 A alleles (OR = 2.90; 95% CIs = 1.02 - 8.25 and OR = 2.90; 95% CIs = 1.02 - 8.25, respectively) [10, 11]. Moreover, Cussenot and co-workers (2008) also demonstrated a strong relationship between organ-confined (T1-T2, N0 M0) and advanced (T3-T4, N1M1) PCA among carriers of 8q24 rs4242382A and rs4242384C alleles . To our knowledge, there are no published reports on the relationship between the rs11934905 SNP localized to the 8q24 chromosome in relation to PCA among men of European or African descent.
We have considered the strengths and limitations of the current study. MDR controls for multiple comparisons and spurious risk estimates by using a cross validation and permutation testing scheme as a built-in feature. Misclassification by case status is also a potential limitation for this study. There is a possibility that some men who presented as disease-free following the initial diagnostic evaluation may eventually develop PCA. In an attempt to ease case-status misclassification, controls with at least one abnormal diagnostic test (i.e., PSA > 2.0 ng/ml or irregular DRE) underwent multiple core needle biopsies. Those with an abnormal biopsy were reclassified as cases. Men who received a normal biopsy test but had an abnormal PSA (> 4.0 ng/ml) and/or irregular DRE (n = 48) were excluded because we could not predict with any level of certainty whether they would eventually develop PCA. For similar reasons, we also excluded individuals (n = 65) who were diagnosed with BPH following biopsy. Notably, after close inspection of PCA tissue, it is feasible to overlook a microscopic nodule that can later develop into cancer ; however, this is an issue that plaques many cancer epidemiology studies. If controls in our study population were still misclassified after undergoing a PSA test, DRE, and/or multiple core needle biopsies, then we may expect our calculated risk estimates to slightly underestimate the relationship between the selected 8q24 loci and PCA susceptibility.
Another challenge for genetic epidemiology studies involving study participants of African descent is their unique population history of gene flow from divergent populations [27, 28]. The current study adjusted single locus models for population admixture using a West African ancestry score assigned to each study participant. Utilization of ancestry markers helps to eliminate misclassification of study participants based on confusion related to self-identified race/ethnicity (SIRE). Our findings suggest that adjusting our risk estimates for West African ancestry did not significantly change the risk estimates relative to unadjusted models; if anything, it makes them more precise to the nearest tenth.
Finally, it is feasible that the observed association is related to linkage disequilibrium of rs16901979 with unknown targets in this region. To address this issue, future studies will use next generation sequencing techniques to better consider the complete heterogeneity within this locus relative to PCA outcomes. Emphasis will be placed on sequence variants within region 3, as it is speculated that sequence variants may be related to a 2-fold increase in PCA risk [12, 13, 29]. Our findings demonstrating a significant relationship between rs16901979 and PCA risk have been submitted for further validation within pooled genetic analyses.
In summary, the 8q24 rs16901979 locus has a strong genetic linkage to PCA among men of African descent in the current investigation as well as other studies. Future multicenter collaborative efforts will facilitate the identification and validation of a reliable panel of genetic susceptibility biomarkers with the capacity to improve PCA detection and prognosis strategies, ultimately reducing PCA health disparities among all men.
The authors thank the men of African descent for their participation in the current study. This study was supported in part by the NIH R03 CA128028, the James Graham Brown Cancer Center, and the Bucks for Brains "Our Highest Potential" in Cancer Research Endowment. The authors appreciate access to the Center for Genetics and Molecular Medicine (CGeMM) DNA Core Facility at the UofL, directed by Dr. Ron Gregg, for use of their high-throughput genotyping facility. We also thank Andrew Marsh for editing this manuscript.
- American Cancer Society: Cancer Facts & Figures 2009. Atlanta, Amerian Cancer SocietyGoogle Scholar
- American Cancer Society: Cancer Facts & Figure for African Americans 2009-2010. Atlanta, Amerian Cancer SocietyGoogle Scholar
- Salinas CA, Kwon E, Carlson CS, Koopmeiners JS, Feng Z, Karyadi DM, Ostrander EA, Stanford JL: Multiple independent genetic variants in the 8q24 region are associated with prostate cancer risk. Cancer EpidemiolBiomarkers Prev. 2008, 17 (5): 1203-1213. 10.1158/1055-9965.EPI-07-2811.Google Scholar
- Xu J, Kibel AS, Hu JJ, Turner AR, Pruett K, Zheng SL, Sun J, Isaacs SD, Wiley KE, Kim ST, et al: Prostate cancer risk associated loci in African Americans. Cancer EpidemiolBiomarkers Prev. 2009, 18 (7): 2145-2149. 10.1158/1055-9965.EPI-09-0091.Google Scholar
- Amundadottir LT, Sulem P, Gudmundsson J, Helgason A, Baker A, Agnarsson BA, Sigurdsson A, Benediktsdottir KR, Cazier JB, Sainz J, et al: A common variant associated with prostate cancer in European and African populations. NatGenet. 2006, 38 (6): 652-658.Google Scholar
- Robbins C, Torres JB, Hooker S, Bonilla C, Hernandez W, Candreva A, Ahaghotu C, Kittles R, Carpten J: Confirmation study of prostate cancer risk variants at 8q24 in African Americans identifies a novel risk locus. Genome Res. 2007, 17 (12): 1717-1722. 10.1101/gr.6782707.View ArticlePubMedPubMed CentralGoogle Scholar
- Gudmundsson J, Sulem P, Manolescu A, Amundadottir LT, Gudbjartsson D, Helgason A, Rafnar T, Bergthorsson JT, Agnarsson BA, Baker A, et al: Genome-wide association study identifies a second prostate cancer susceptibility variant at 8q24. NatGenet. 2007, 39 (5): 631-637.Google Scholar
- Haiman CA, Patterson N, Freedman ML, Myers SR, Pike MC, Waliszewska A, Neubauer J, Tandon A, Schirmer C, McDonald GJ, et al: Multiple regions within 8q24 independently affect risk for prostate cancer. NatGenet. 2007, 39 (5): 638-644.Google Scholar
- Cussenot O, Azzouzi AR, Bantsimba-Malanda G, Gaffory C, Mangin P, Cormier L, Fournier G, Valeri A, Jouffe L, Roupret M, et al: Effect of genetic variability within 8q24 on aggressiveness patterns at diagnosis and familial status of prostate cancer. ClinCancer Res. 2008, 14 (17): 5635-5639. 10.1158/1078-0432.CCR-07-4999.Google Scholar
- Fitzgerald LM, Kwon EM, Koopmeiners JS, Salinas CA, Stanford JL, Ostrander EA: Analysis of recently identified prostate cancer susceptibility loci in a population-based study: associations with family history and clinical features. ClinCancer Res. 2009, 15 (9): 3231-3237. 10.1158/1078-0432.CCR-08-2190.Google Scholar
- Zheng SL, Sun J, Cheng Y, Li G, Hsu FC, Zhu Y, Chang BL, Liu W, Kim JW, Turner AR, et al: Association between two unlinked loci at 8q24 and prostate cancer risk among European Americans. JNatlCancer Inst. 2007, 99 (20): 1525-1533.View ArticleGoogle Scholar
- Raja J, Ramachandran N, Munneke G, Patel U: Current status of transrectal ultrasound-guided prostate biopsy in the diagnosis of prostate cancer. Clin Radiol. 2006, 61 (2): 142-153. 10.1016/j.crad.2005.10.002.View ArticlePubMedGoogle Scholar
- Gleason DF: Classification of prostatic carcinomas. Cancer Chemother Rep. 1966, 50 (3): 125-128.PubMedGoogle Scholar
- National Center for Biotechnology Information (NCBI) website. [http://www.ncbi.nlm.nih.gov/]
- Tian C, Hinds DA, Shigeta R, Kittles R, Ballinger DG, Seldin MF: A genomewide single-nucleotide-polymorphism panel with high ancestry information for African American admixture mapping. AmJHumGenet. 2006, 79 (4): 640-649.Google Scholar
- Hahn LW, Ritchie MD, Moore JH: Multifactor dimensionality reduction software for detecting gene-gene and gene-environment interactions. Bioinformatics. 2003, 19 (3): 376-382. 10.1093/bioinformatics/btf869.View ArticlePubMedGoogle Scholar
- Multi-factor dimensionality reduction. [http://www.epistasis.org]
- Park SL, Chang SC, Cai L, Cordon-Cardo C, Ding BG, Greenland S, Hussain SK, Jiang Q, Liu S, Lu ML, et al: Associations between variants of the 8q24 chromosome and nine smoking-related cancer sites. Cancer EpidemiolBiomarkers Prev. 2008, 17 (11): 3193-3202. 10.1158/1055-9965.EPI-08-0523.Google Scholar
- Cheng I, Plummer SJ, Jorgenson E, Liu X, Rybicki BA, Casey G, Witte JS: 8q24 and prostate cancer: association with advanced disease and meta-analysis. EurJHumGenet. 2008, 16 (4): 496-505.Google Scholar
- SNP500Cancer database. [http://snp500cancer.nci.nih.gov/]
- Schumacher FR, Feigelson HS, Cox DG, Haiman CA, Albanes D, Buring J, Calle EE, Chanock SJ, Colditz GA, Diver WR, et al: A common 8q24 variant in prostate and breast cancer from a large nested case-control study. Cancer Res. 2007, 67 (7): 2951-2956. 10.1158/0008-5472.CAN-06-3591.View ArticlePubMedGoogle Scholar
- Wokolorczyk D, Gliniewicz B, Sikorski A, Zlowocka E, Masojc B, Debniak T, Matyjasik J, Mierzejewski M, Medrek K, Oszutowska D, et al: A range of cancers is associated with the rs6983267 marker on chromosome 8. Cancer Res. 2008, 68 (23): 9982-9986. 10.1158/0008-5472.CAN-08-1838.View ArticlePubMedGoogle Scholar
- Severi G, Hayes VM, Padilla EJ, English DR, Southey MC, Sutherland RL, Hopper JL, Giles GG: The common variant rs1447295 on chromosome 8q24 and prostate cancer risk: results from an Australian population-based case-control study. Cancer EpidemiolBiomarkers Prev. 2007, 16 (3): 610-612. 10.1158/1055-9965.EPI-06-0872.Google Scholar
- Wang L, McDonnell SK, Slusser JP, Hebbring SJ, Cunningham JM, Jacobsen SJ, Cerhan JR, Blute ML, Schaid DJ, Thibodeau SN: Two common chromosome 8q24 variants are associated with increased risk for prostate cancer. Cancer Res. 2007, 67 (7): 2944-2950. 10.1158/0008-5472.CAN-06-3186.View ArticlePubMedGoogle Scholar
- Suuriniemi M, Agalliu I, Schaid DJ, Johanneson B, McDonnell SK, Iwasaki L, Stanford JL, Ostrander EA: Confirmation of a positive association between prostate cancer risk and a locus at chromosome 8q24. Cancer EpidemiolBiomarkers Prev. 2007, 16 (4): 809-814. 10.1158/1055-9965.EPI-06-1049.Google Scholar
- Loeb S, Catalona WJ: What is the prognostic impact of positive surgical margins in surgically treated prostate cancer?. Nat Clin Pract Urol. 2006, 3 (8): 418-419. 10.1038/ncpuro0549.View ArticlePubMedGoogle Scholar
- Shriver MD, Kittles RA: Genetic ancestry and the search for personalized genetic histories. NatRevGenet. 2004, 5 (8): 611-618.Google Scholar
- Kittles RA, Chen W, Panguluri RK, Ahaghotu C, Jackson A, Adebamowo CA, Griffin R, Williams T, Ukoli F, ms-Campbell L, et al: CYP3A4-V and prostate cancer in African Americans: causal or confounding association because of population stratification?. HumGenet. 2002, 110 (6): 553-560. 10.1007/s00439-002-0731-5.Google Scholar
- Gleason DF: Histologic grading of prostate cancer: a perspective. Hum Pathol. 1992, 23 (3): 273-279. 10.1016/0046-8177(92)90108-F.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/334/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.