How accurate can genetic predictions be?
 Jonathan M Dreyfuss†^{1, 2}Email author,
 Daniel Levner†^{3, 4},
 James E Galagan^{5, 6},
 George M Church^{3, 4} and
 Marco F Ramoni^^{1, 7, 8}
DOI: 10.1186/1471216413340
© Dreyfuss et al.; licensee BioMed Central Ltd. 2012
Received: 4 November 2011
Accepted: 1 July 2012
Published: 24 July 2012
Abstract
Background
Presymptomatic prediction of disease and drug response based on genetic testing is a critical component of personalized medicine. Previous work has demonstrated that the predictive capacity of genetic testing is constrained by the heritability and prevalence of the tested trait, although these constraints have only been approximated under the assumption of a normally distributed genetic risk distribution.
Results
Here, we mathematically derive the absolute limits that these factors impose on test accuracy in the absence of any distributional assumptions on risk. We present these limits in terms of the bestcase receiveroperating characteristic (ROC) curve, consisting of the bestcase test sensitivities and specificities, and the AUC (area under the curve) measure of accuracy. We apply our method to genetic prediction of type 2 diabetes and breast cancer, and we additionally show the best possible accuracy that can be obtained from integrated predictors, which can incorporate nongenetic features.
Conclusion
Knowledge of such limits is valuable in understanding the implications of genetic testing even before additional associations are identified.
Background
Accurate presymptomatic prediction of disease and drug response is a vital component of personalized medicine, which could allow for improved clinical decisionmaking and targeted prevention strategies, easing both the burden and costs of disease [1]. Already, several companies offer consumers personalized risk assessments, lifestyle recommendations, and 'nutraceuticals' based on their genetic profiles [2]. Unfortunately, most genetic factors associated with common traits explain only a small portion of the phenotypic variance (the “missing heritability” problem [3]), making genetic prediction currently difficult [4]. Investment into studies that assay rare variants [5] and the use of informative polymorphisms that do not individually pass stringent statistical tests of association [6] can improve the accuracy of predictions, but the extent to which predictions can be improved is unclear. Thus, identifying the bounds on the accuracy of predictive genetic testing based on readilyknown disease parameters (such as prevalence and heritability) can be an invaluable planning tool.
Although the accuracy of a medical test can be measured in many ways, the concepts of sensitivity and specificity are paramount [7]. Frequently, the test result is continuous (e.g. the individual’s predicted risk), while the clinical decision and true outcome are binary (e.g. either the person will get sick or not), so that different thresholds of the test result yield different pairs of sensitivity and specificity. The receiver operator characteristic (ROC) curve depicts this tradeoff between sensitivity and specificity across all possible thresholds, and the area under this curve (AUC) is the most widely used metric to summarize the accuracy of a test. An AUC of 1 indicates perfect prediction while an AUC of 0.5 represents random guessing.
Evidence that a bound on maximum predictive accuracy exists can be found in heritability. The heritability of a trait (in the broadsense) is the proportion of phenotypic variation in the population that can be attributed to genetic variation; that is, it reflects the contribution of genetic factors relative to environmental ones. Narrowsense heritability measures the corresponding quantity for additive genetic variance only, which excludes genetic effects such as dominance and epistasis. The heritability of binary phenotypes can be computed directly on the observed binary scale. However, it may also be calculated on a liability scale, where it is assumed that an individual has the binary trait if their risk exceeds a threshold. Both types of heritability can be estimated using familybased studies, such as twin studies [8], and the two scales can be mapped to each other [9].
The impact of heritability on genetic test accuracy can be seen by examining its two extremes: a trait that has 100% heritability, such as a Mendelian trait, can be predicted with certainty from the genotype; in contrast, a trait with 0% heritability is not influenced by genetic factors, and thus genetic tests cannot produce any useful information. Previous groundbreaking works have investigated the bounds prevalence and heritability impose on predictive accuracy using simulations [10], analytical results utilizing genotype relative risks and their frequencies [11], and analytical approximations under the assumption of a normally distributed liability [12, 13]. Here, we mathematically elucidate the absolute bounds on the specificities, sensitivities, and AUC for genetic testing given any values of heritability and prevalence of the tested trait, without making any assumptions about the risk distribution.
Results
Common complex traits are typically the combined effect of genetic and environmental factors. Since no practical predictor can account for all factors and their interactions, clinical prediction can at best assign probabilistic risks rather than deterministic outcomes. Viewed on the population level, these risk assignments can be seen as comprising a risk distribution, which is an estimate of the population’s true risk distribution. Maximal predictive accuracy occurs when the estimated risk matches the true risk.
where i = 1,…,n indexes people, n is the sample size, risk_{ i } is individual i’s genetic risk (i.e. the conditional probability of the trait given genes), and $\overline{risk}$is the average genetic risk, which equals the average population risk (see Methods). The meaning of risk depends on the context: for instance, when the phenotype is current disease status, the average risk in the population is its prevalence, whereas in prediction of lifetime illness, risk is the lifetime risk of disease. (When possible, we nonetheless opt for the term prevalence.) Equation 1 mathematically expresses that heritability is the proportion of phenotypic variance explained by the genetic risk distribution.
Breast cancer has the same maximal AUC as T2D, albeit with a distinct ROC curve from T2D. Breast cancer was found to have a prevalence of 4% [19], and we calculated its heritability on the binary scale to be 11% (see Methods), which yields a maximum AUC of 89%. Although this is the same maximum AUC as for T2D, the sensitivity/specificity pairs for breast cancer (Figure 3) are not identical to those for T2D, owing to the different disease parameters. For example, to reach a specificity of 99%, sensitivity cannot exceed 24%, which is substantially lower than the corresponding maximal sensitivity of T2D when specificity is 99%. The divergence of these two ROC curves as specificity approaches 100% illustrates the importance of identifying the maximal ROC curve, rather than relying on the maximal AUC alone.
Heritability is the proportion of phenotypic variance explained by all genetic factors, but our analytic approach can treat the proportion of phenotypic variance explained by any particular set of factors. If the proportion of phenotypic variance explained by a particular set of genes is known, that proportion of variance explained could be substituted for heritability in our model. For instance, if a subset of genes could explain 50% of the genetic variance of T2D (i.e. explain 13% of phenotypic variance), then the maximum achievable AUC of this subset would be 80%.
Our method can also be applied in elucidating the maximum accuracy of predictors that integrate features such as gene expression, de novo mutation, body mass index, and lifestyle (which are not fully inherited). The proportion of variance explained by such an integrated predictor can then be greater than heritability. When there are no geneenvironment interactions, this difference is the proportion of phenotypic variation that these features explain independently of genes. For example, weekly physical activity can explain 4% of phenotypic variance of T2D (see Methods), is moderately heritable [20], and was found to not interact with wellknown gene variants in T2D [21]. Accordingly, the proportion of variance explained by the integrated predictor comprised of genomic profile and physical activity does not increment by the full 4% beyond the heritability of T2D. If the proportion of T2D variance that physical activity explains independently of genes was known to be only 3%, say, then the integrated predictor’s maximum AUC would be calculated based on a proportion of variance explained of 29% (sum of 26% and 3%), which yields a maximum AUC of 90%. If, however, we did not have an estimate for the proportion of T2D variance that physical activity explains independently of genes, then we could conservatively use 4% in the previous calculation, yielding a similar AUC. This analysis applies to predictors based on nongenetic features that are supplemented by genetics. In general, the estimation of the proportion of variance explained by integrated predictors is complicated by the interaction of genetic and nongenetic features; our method can nonetheless be applied when the interaction can be estimated or bounded. Note that genetic testing alone can still accurately predict outcome for some small, extreme risk groups (such as those with highly penetrant variants), but such a test will not benefit the general population without both a high sensitivity and specificity [22].
Discussion
Our results are general and apply to any binary trait, and they rely on only two commonly estimated parameters. Although the quality of the results is only as good as the estimates of prevalence and heritability for the population in question, our method allows for ranges of prevalences and heritabilities to be considered, which can provide important insight into predictive accuracies. Nonetheless, care must be taken when applying these statistics, as different estimates apply in different situations. For example, in assessing limits to the prediction of lifelong risk, lifelong risk estimates should be used in place of prevalence estimates. In particular, the ballooning lifelong risk of T2D in the USA [23] implies genetic prediction of lifetime T2D will become more difficult.
Using this approach, one may evaluate a proposed GWAS based on parameters such as sample size and the number of loci sampled.
Heritability estimates for any binary trait can be used by our method. Broadsense heritability estimates are needed to cap predictive accuracy, since genetic predictors can exploit dominance and epistatic interactions not measured by narrowsense heritability estimates. However, if a genetic predictor is constructed as an additive model in line with the assumptions of narrowsense heritability, then its maximum accuracy can be calculated using narrowsense heritability; thus, these estimates can also be used, albeit with a slightly different interpretation. Heritability estimates on the normal liability scale can be used after they are transformed to the observed binary scale, e.g. using the method proposed by Dempster and Lerner [8, 9]. Heritability on the binary scale can be sensitive to prevalence [26], but its use avoids the assumption of normallydistributed liability, which requires that the trait be affected by many genes, all with small effect (normallydistributed liability effectively requires a purely unimodal genetic risk distribution). In fact, when variants with particularly large effects do exist—such as APOE in Alzheimer’s disease [27], BRCA1 and BRCA2 in breast and ovarian cancer [28], and LRRK2 in Parkinson’s disease [29]—previous authors have suggested simulations in lieu of their analytical approximation [13]. Moreover, because liability cannot be measured, the distributional assumptions on liability are virtually untestable [30].
Our maximal ROC curves (Figure 3) can be substantially higher than those given by the beta distribution, which is an accurate proxy for multiple previous reports [10, 12, 13], indicating that the maximal accuracies of genetic prediction may be substantially higher than previously thought. This difference highlights the importance that the risk distribution can have in the power of genetic prediction. Furthermore, as we are only now beginning to uncover the risk distributions of common complex diseases, it seems important to understand the absolute, distributionindependent limits on genetictest accuracy, which we present here.
Conclusion
We have given exact limits on genetic prediction for any binary trait imposed by the epidemiological parameters of prevalence and heritability. Knowledge of these limits can help delineate the maximal benefits associated with genetic testing, which can allow for costbenefit analyses, regulation, and clinical guidelines regarding genetic testing even before additional associations are identified. We have also illustrated how these limits can help us prioritize the allocation of research resources, by showing how they can assist in the prioritization and design of future association studies. The calculations presented in this paper could further be used to mitigate the possibility of investing in the development of a genetic test which could never be accurate enough to be of clinical relevance.
Methods
To optimize over the set of risk distributions subject to the disease parameters of average risk and proportion of variance explained (PVE), we modeled a categorical distribution (which resembles a histogram) with b + 1 bins located at 0, 1/b, 2/b, … , 1 representing risk groups, so i/b represents the conditional probability of disease given a set of factors for individuals in risk group i (e.g. people in the 1/b group have risk 1/b). An example of such a distribution is depicted in Figure 1. The probability that someone falls into bin i is p_{ i }, where the p_{ i }‘s (for i = 0,…,b) sum to one. We restrict the average risk (e.g. prevalence) and PVE (e.g. heritability) using two observations. (1) By the law of total probability, the unconditional probability of disease is simply the mean of the conditional risk distribution, i.e. it is equal to the average risk. (2) The PVE relates to the risk distribution through Equation 1. (Equation 1 can be understood as the R^{2} from the regression: binary phenotype = risk + error, where risk is a probability.)
Here, n_{ j } individuals have risk j/b, i.e. they are assigned to risk group (histogram bin) j, and p_{ j } = n_{ j }/n is the probability that a random individual is assigned to risk group j.
With this model of the risk distribution and constraints, we can identify the bestcase AUC and optimal sensitivity/specificity pairs using the procedures detailed below. Because these procedures associate a single genetic risk distribution with the bestcase AUC and a potentially different risk distribution with each optimal sensitivity/specificity pair, it is possible that only some of these sensitivity/specificity pairs may be realizable for a single trait in practice. Consequently, these sensitivity/specificity pairs cannot be used directly to derive the maximal AUC.
Area under ROC curve
The numerator of this expression can be conveniently represented as p^{ T }Qp + b^{ 2 }k, where Q is a symmetric matrix whose entry at row i and column j is j(b + i)/2 for i ≥ j.
where the sum of the p_{ i }‘s (for i = 1,…,b) must not exceed 1, and each p_{ i } is bounded between 0 and 1.
The parameters k PVE, and b are predefined constants. Note that for b = 100, as well as for all the values of b we examined, Q is negative definite, so that this is a convex program. Hence, there are efficient solution methods to identify the global maximum. Using the quadprog package [32] in the R software [14], we solved this program for values of k and PVE with b = 100. When b = 1000, all maximal AUCs shown in Figure 2 change by less than 0.01%. In fact, using b = 10 does not change any of these maximal AUCs more than 1% from that calculated with b = 1000. Note also that given an estimated risk distribution vector p, a researcher can directly calculate the AUC from the objective function. To calculate the AUC of the beta distribution for given levels of k and PVE, we discretized the beta distribution with parameters a = k(1/PVE1) and b = (1k)(1/PVE1), which uniquely satisfy the constraints.
Sensitivity/specificity pairs
We optimized sensitivity for any given value of specificity, average risk, proportion of variance explained, and threshold using a linear programming model. This was implemented in the lpSolve package in R [14] using 1000 bins. We then optimized the sensitivities over the thresholds to obtain the maximal sensitivity for every specificity, average risk, and proportion of variance explained.
Calculations for examples
To calculate the proportion of T2D variance explained by physical activity we used Equation 1, where the risk distribution was defined by the prevalence and the relative risks of exercise [33]. To calculate the heritability of breast cancer on the binary scale we used twice the difference in correlation between monozygotic and dizygotic twin pairs, where correlations were computed on binary outcomes from 44,788 pairs of Nordic twins [34].
Author’s contribution
JMD designed the study, carried out the analysis, and drafted the manuscript. DL designed the study and drafted the manuscript. JEG provided computing resources and helped direct the study. GMC helped direct the study. MFR designed the study and critically revised the manuscript. All authors read and approved the manuscript.
Notes
Abbreviations
 ROC:

receiveroperating characteristic
 AUC:

area under ROC curve
 T2D:

type 2 diabetes
 GWAS:

genomewide association study
 PVE:

proportion of variance explained.
Declarations
Acknowledgements
We dedicate this to Marco Ramoni, who tragically passed away in June 2010.
Authors’ Affiliations
References
 Collins FS, Green ED, Guttmacher AE, Guyer MS: A vision for the future of genomics research. Nature. 2003, 422 (6934): 835847. 10.1038/nature01626.View ArticlePubMedGoogle Scholar
 Janssens AC, Gwinn M, Bradley LA, Oostra BA, van Duijn CM, Khoury MJ: A critical appraisal of the scientific basis of commercial genomic profiles used to assess health risks and personalize health interventions. Am J Hum Genet. 2008, 82 (3): 593599. 10.1016/j.ajhg.2007.12.020.PubMed CentralView ArticlePubMedGoogle Scholar
 Manolio TA, Collins FS, Cox NJ, Goldstein DB, Hindorff LA, Hunter DJ, McCarthy MI, Ramos EM, Cardon LR, Chakravarti A, Cho JH, Guttmacher AE, Kong A, Kruglyak L, Mardis E, Rotimi CN, Slatkin M, Valle D, Whittemore AS, Boehnke M, Clark AG, Eichler EE, Gibson G, Haines JL, Mackay TF, McCarroll SA, Visscher PM: Finding the missing heritability of complex diseases. Nature. 2009, 461 (7265): 747753. 10.1038/nature08494.PubMed CentralView ArticlePubMedGoogle Scholar
 Kraft P, Hunter D: Genetic Risk Prediction — Are We There Yet?. N Engl J Med. 2009, 360 (17): 17011703. 10.1056/NEJMp0810107.View ArticlePubMedGoogle Scholar
 Goldstein DB: Common genetic variation and human traits. N Engl J Med. 2009, 360 (17): 16961698. 10.1056/NEJMp0806284.View ArticlePubMedGoogle Scholar
 Yang J, Benyamin B, McEvoy BP, Gordon S, Henders AK, Nyholt DR, Madden PA, Heath AC, Martin NG, Montgomery GW, Goddard ME, Visscher PM: Common SNPs explain a large proportion of the heritability for human height. Nat Genet. 2010, 42 (7): 565569. 10.1038/ng.608.PubMed CentralView ArticlePubMedGoogle Scholar
 Pepe MS: The statistical evaluation of medical tests for classification and prediction. 2003, Oxford: Oxford University PressGoogle Scholar
 Visscher PM, Hill WG, Wray NR: Heritability in the genomics era–concepts and misconceptions. Nat Rev Genet. 2008, 9 (4): 255266.View ArticlePubMedGoogle Scholar
 Dempster ER, Lerner IM: Heritability of Threshold Characters. Genetics. 1950, 35 (2): 212236.PubMed CentralPubMedGoogle Scholar
 Janssens AC, Aulchenko YS, Elefante S, Borsboom GJ, Steyerberg EW, van Duijn CM: Predictive testing for complex diseases using multiple genes: fact or fiction?. Genet Med. 2006, 8 (7): 395400. 10.1097/01.gim.0000229689.18263.f4.View ArticlePubMedGoogle Scholar
 Moonesinghe R, Liu T, Khoury MJ: Evaluation of the discriminative accuracy of genomic profiling in the prediction of common complex diseases. Eur J Hum Genet. 2010, 18 (4): 485489. 10.1038/ejhg.2009.209.PubMed CentralView ArticlePubMedGoogle Scholar
 Wray NR, Yang J, Goddard ME, Visscher PM: The genetic interpretation of area under the ROC curve in genomic profiling. PLoS Genet. 2010, 6 (2): e100086410.1371/journal.pgen.1000864.PubMed CentralView ArticlePubMedGoogle Scholar
 So HC, Sham PC: A unifying framework for evaluating the predictive power of genetic variants based on the level of heritability explained. PLoS Genet. 2010, 6 (12): e100123010.1371/journal.pgen.1001230.PubMed CentralView ArticlePubMedGoogle Scholar
 Team RDC: R: A language and environment for statistical computing. 2008, Vienna: R Foundation for Statistical ComputingGoogle Scholar
 Tuomilehto J, Lindström J, Eriksson JG, Valle TT, Hämäläinen H, IlanneParikka P, KeinänenKiukaanniemi S, Laakso M, Louheranta A, Rastas M, Salminen V, Uusitupa M: Prevention of type 2 diabetes mellitus by changes in lifestyle among subjects with impaired glucose tolerance. N Engl J Med. 2001, 344 (18): 13431350. 10.1056/NEJM200105033441801.View ArticlePubMedGoogle Scholar
 Janssens AC, van Duijn CM: Genomebased prediction of common diseases: advances and prospects. Hum Mol Genet. 2008, 17 (R2): R166173. 10.1093/hmg/ddn250.View ArticlePubMedGoogle Scholar
 Talmud PJ, Hingorani AD, Cooper JA, Marmot MG, Brunner EJ, Kumari M, Kivimaki M, Humphries SE: Utility of genetic and nongenetic risk factors in prediction of type 2 diabetes: Whitehall II prospective cohort study. BMJ. 2010, 340: b483810.1136/bmj.b4838.PubMed CentralView ArticlePubMedGoogle Scholar
 Poulsen P, Kyvik KO, Vaag A, BeckNielsen H: Heritability of type II (noninsulindependent) diabetes mellitus and abnormal glucose tolerance–a populationbased twin study. Diabetologia. 1999, 42 (2): 139145. 10.1007/s001250051131.View ArticlePubMedGoogle Scholar
 Risch N: The genetic epidemiology of cancer: interpreting family and twin studies and their implications for molecular genetic approaches. Cancer Epidemiol Biomarkers Prev. 2001, 10 (7): 733741.PubMedGoogle Scholar
 Lauderdale DS, Fabsitz R, Meyer JM, Sholinsky P, Ramakrishnan V, Goldberg J: Familial determinants of moderate and intense physical activity: a twin study. Med Sci Sports Exerc. 1997, 29 (8): 10621068. 10.1097/0000576819970800000012.View ArticlePubMedGoogle Scholar
 Scott RA, Chu AY, Grarup N, Manning AK, Hivert MF, Shungin D, Tonjes A, Yesupriya A, Barnes D, BouatiaNaji N, Glazer NL, Jackson AU, Kutalik Z, Lagou V, Marek D, RasmussenTorvik LJ, Stringham HM, Tanaka T, Aadahl M, Arking DE, Bergmann S, Boerwinkle E, Bonnycastle LL, Bornstein SR, Brunner E, Bumpstead SJ, Brage S, Carlson OD, Chen H, Chen YD: No Interactions Between Previously Associated 2Hour Glucose Gene Variants and Physical Activity or BMI on 2Hour Glucose Levels. Diabetes. 2012, 61 (5): 12911296. 10.2337/db110973.PubMed CentralView ArticlePubMedGoogle Scholar
 Janssens AC, Pardo MC, Steyerberg EW, van Duijn CM: Revisiting the clinical validity of multiplex genetic testing in complex diseases. Am J Hum Genet. 2004, 74 (3): 585588. 10.1086/382052. author reply 588589PubMed CentralView ArticlePubMedGoogle Scholar
 Narayan KM, Boyle JP, Thompson TJ, Sorensen SW, Williamson DF: Lifetime risk for diabetes mellitus in the United States. JAMA. 2003, 290 (14): 18841890. 10.1001/jama.290.14.1884.View ArticlePubMedGoogle Scholar
 Wray NR, Goddard ME, Visscher PM: Prediction of individual genetic risk of complex disease. Curr Opin Genet Dev. 2008, 18 (3): 257263. 10.1016/j.gde.2008.07.006.View ArticlePubMedGoogle Scholar
 Daetwyler HD, Villanueva B, Woolliams JA: Accuracy of predicting the genetic risk of disease using a genomewide approach. PLoS One. 2008, 3 (10): e339510.1371/journal.pone.0003395.PubMed CentralView ArticlePubMedGoogle Scholar
 Robertson A, Lerner IM: The Heritability of AllorNone Traits: Viability of Poultry. Genetics. 1949, 34 (4): 395411.PubMed CentralPubMedGoogle Scholar
 Farrer LA, Cupples LA, Haines JL, Hyman B, Kukull WA, Mayeux R, Myers RH, PericakVance MA, Risch N, van Duijn CM: Effects of age, sex, and ethnicity on the association between apolipoprotein E genotype and Alzheimer disease. A metaanalysis. APOE and Alzheimer Disease Meta Analysis Consortium. JAMA. 1997, 278 (16): 13491356. 10.1001/jama.1997.03550160069041.View ArticlePubMedGoogle Scholar
 Fu R, Harris EL, Helfand M, Nelson HD: Estimating risk of breast cancer in carriers of BRCA1 and BRCA2 mutations: a metaanalytic approach. Stat Med. 2007, 26 (8): 17751787. 10.1002/sim.2811.View ArticlePubMedGoogle Scholar
 Healy DG, Falchi M, O'Sullivan SS, Bonifati V, Durr A, Bressman S, Brice A, Aasly J, Zabetian CP, Goldwurm S, Ferreira JJ, Tolosa E, Kay DM, Klein C, Williams DR, Marras C, Lang AE, Wszolek ZK, Berciano J, Schapira AH, Lynch T, Bhatia KP, Gasser T, Lees AJ, Wood NW, Consortium IL: Phenotype, genotype, and worldwide genetic penetrance of LRRK2associated Parkinson's disease: a casecontrol study. Lancet Neurol. 2008, 7 (7): 583590. 10.1016/S14744422(08)701170.PubMed CentralView ArticlePubMedGoogle Scholar
 Falconer DS: Introduction to Quantitative Genetics. 1981, New York: Longman, 2Google Scholar
 Hanley JA, McNeil BJ: The meaning and use of the area under a receiver operating characteristic (ROC) curve. Radiology. 1982, 143 (1): 2936.View ArticlePubMedGoogle Scholar
 Goldfarb D, Idnani A: A numerically stable dual method for solving strictly convex quadratic programs. Math Program. 1983, 27 (1): 33View ArticleGoogle Scholar
 Hu FB, Sigal RJ, RichEdwards JW, Colditz GA, Solomon CG, Willett WC, Speizer FE, Manson JE: Walking compared with vigorous physical activity and risk of type 2 diabetes in women: a prospective study. JAMA. 1999, 282 (15): 14331439. 10.1001/jama.282.15.1433.View ArticlePubMedGoogle Scholar
 Lichtenstein P, Holm NV, Verkasalo PK, Iliadou A, Kaprio J, Koskenvuo M, Pukkala E, Skytthe A, Hemminki K: Environmental and heritable factors in the causation of cancer–analyses of cohorts of twins from Sweden, Denmark, and Finland. New Engl J Med. 2000, 343 (2): 7885. 10.1056/NEJM200007133430201.View ArticlePubMedGoogle Scholar
Copyright
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.