Epidemiologic studies have reported the association of X-ray repair cross-complementary group 1 (XRCC1) Arg399Gln polymorphisms with susceptibility to squamous cell carcinoma of the head and neck (HNSCC). However, the results were conflictive rather than conclusive. The purpose of this study was to clarify the association of XRCC1 Arg399Gln variants with HNSCC risk.
Systematic searches were performed through the search engines of PubMed, Elsevier, Science Direct, CNKI and Chinese Biomedical Literature Database. Summary odds ratio (OR) with 95% confidence intervals (CI) was computed to estimate the strength association.
Overall, we did not observe any association of XRCC1 Arg399Gln polymorphisms with HNSCC risk in total population (OR = 0.95, 95% CI: 0.76–1.19 for Gln/Gln vs. Arg/Arg, OR = 1.05, 95% CI: 0.92–1.20 for Arg/Gln vs. Arg/Arg, and OR = 1.03, 95% CI: 0.90–1.18 for Gln/Gln+Arg/Gln vs. Arg/Arg) based on 18 studies including 3917 cases and 4560 controls. In subgroup analyses, we observed an increased risk of XRCC1 399 Arg/Gln genotype for HNSCC in Caucasians (OR = 1.20, 95% CI: 1.00–1.44) and Gln/Gln genotype for larynx squamous cell carcinoma (OR = 1.63, 95% CI: 1.10–2.40). We did not observe any association between XRCC1 Arg399Gln variants and HNSCC risk in additional subgroup analyses.
Citation: Wang Y, Chu X, Meng X, Zou F (2013) Association of X-ray Repair Cross Complementing Group 1 Arg399Gln Polymorphisms with the Risk of Squamous Cell Carcinoma of the Head and Neck: Evidence from an Updated Meta-Analysis. PLoS ONE 8(10): e77898. doi:10.1371/journal.pone.0077898
Editor: Michael Scheurer, Baylor College of Medicine, United States of America
Received: June 17, 2013; Accepted: September 5, 2013; Published: October 30, 2013
Copyright: © 2013 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by National Basic Research Program of China (No. 2012CB518200) and National Nature Science Foundation of China (No. 81272180). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The global incidence of squamous cell carcinoma of the head and neck (HNSCC) is 540,000 per year, with an annual mortality rate of 271,000. HNSCC is a malignant disease of the upper aerodigestive tract, including oral, pharyngeal and laryngeal regions. HNSCC occurs most commonly in men between the sixth and seventh decade of life. Epidemiologic data have shown that tobacco smoking and alcohol consumptions are the main etiological factors in the carcinogenesis of squamous cell carcinoma of the head and neck .
Although a majority of people are exposed to such risk factors, only a small fraction of them develop HNSCC . This indicates that an individual’s susceptibility might play a certain role in HNSCC carcinogenesis. Recently, Increasing evidence has been accumulated to support the hypothesis that common genetic polymorphisms in genes involved in DNA repair capacity may be of importance in determining an individual’s susceptibility to develop HNSCC –.
XRCC1 is a major DNA repair gene involved in base excision repair (BER) and single-strand breaks (SSBs) repair. XRCC1 interacts strongly with Poly [ADP-ribose] polymerase 1 (PARP1), which recognizes SSBs, and LIGIII that seals SSBs and BER intermediates . Several single nucleotide polymorphisms (SNP) have been identified in the XRCC1 gene, three of which (Arg194Trp, Arg280His and Arg399Gln) occur within conserved sequences, as described by Shen et al . A polymorphism Arg399Gln (rs25487) leads to amino acid substitutions (exon 10, G-A). This mutation could alter XRCC1 function, diminish repair kinetics, and influence susceptibility to adverse health effect, such as cancer. To date, a number of studies have investigated the association between XRCC1 Arg399Gln polymorphisms and HNSCC risk , , , –. However, the results from epidemiologic studies have been inconsistent. Moreover, Li et al  conducted a meta-analysis on the association of XRCC1 Arg399Gln polymorphisms with HNSCC risk in 2007 and did not observe any significant association based on 7 published studies. Ever since, several new studies have provided additional data on the association between XRCC1 Arg399Gln polymorphisms and HNSCC risk. In order to address a more precise estimation of this relationship, a meta-analysis including a total of 18 studies was performed, which may provide the more comprehensive evidence for the association of XRCC1 Arg399Gln variants with HNSCC risk.
Materials and Methods
Literature and Methods
Systematic searches in Pubmed/Medline, Elsevier, Science Direct, Chinese National Knowledge Infrastructure (CNKI) and Chinese Biomedical Literature Database were performed using the following search terms: “head and neck cancer” or “oral cancer” or “larynx cancer” or “pharynx cancer” and “polymorphism” and “XRCC1”. Additional studies were identified using the reference lists of the selected papers. The ending date of publication search was March 1, 2013.
The following criteria were used for study selection: (1) The papers should evaluate the association between XRCC1 Arg399Gln polymorphisms and HNSCC risk; (2) Case-control studies or cohort studies; (3) Sufficient data were used for estimating OR with 95% CI; (4) When more than one article was identified for the same study population, we included the publication containing more information. The exclusion criteria were (1) studies on nasopharyngeal cancer; (2) studies that could not offer the number of cases and controls or other essential information; (3) reviews or studies with overlapping patient populations; (4) studies without histologically confirmed information for HNSCC.
In total, 30 published articles were identified with the association between XRCC1 Arg399Gln polymorphisms and head and neck cancer risk. We reviewed all papers in accordance with the criteria listed, above; seven studies without histologically confirmed information for HNSCC, three overlapping studies and two reviews were excluded. At last, 18 original articles that reported the association between XRCC1 Arg399Gln polymorphisms and HNSCC risk were determined to be eligible to enter our study (Figure S1 Flow diagram).
Data were carefully extracted and tabulated by two of the authors independently, and then inputted into an electronic database. The following information was subtracted from each study: author’s name, publishing date, country, ethnicity of subjects, source of control, total number of cases and controls and number of every genotype. If the study provided stratum information, the data coming from similar stratum were added up to make full use of the data. Characteristics of selected studies were summarized in Table 1.
Quantitative Data Synthesis
The strength of association between XRCC1 Arg399Gln polymorphisms and HNSCC risk was measured by OR with 95% CI. Data were combined using both a fixed-effects model and a random-effects model . Heterogeneity was assessed by the Cochrane Q statistics test. The fixed-effects model is applied when the effects are assumed to be homogenous, while the random-effects model is applied when they are heterogeneous. The potential publication bias was firstly estimated by visual inspection of the funnel plot. An asymmetric plot suggests a possible publication bias. The funnel plot asymmetry was assessed by the methods of Egger’s test and Begg’s test , . We tested whether genotype frequencies of controls were in Hardy-Weinberg equilibrium (HWE) using the χ2 test.
All of the statistical tests used in this meta-analysis were performed with Review Manager (Version 184.108.40.206, the Cochrane Collaboration) and STATA10.0 software package (Stata Corporation, College Station, Texas). All the tests were two-sided, a P value of less than 0.05 for any test was considered to be statistically significant.
A database was established according to the extracted information from all eligible articles. Essential information was listed in Table 1, which indicated the first author’s name, year of publication, source of control, country, ethnicity of subjects, the number of case and control and P value of HWE. There were a total of 18 studies with 3917 cases and 4560 controls concerning the XRCC1 Arg399Gln polymorphisms related to HNSCC risk.
Test of Heterogeneity
The heterogeneity of XRCC1 codon 399 Gln/Gln vs. Arg/Arg, Arg/Gln vs. Arg/Arg and Gln/Gln+Arg/Gln vs. Arg/Arg was analyzed for 18 identified studies. The results showed that XRCC1 codon 399 Gln/Gln vs. Arg/Arg, Arg/Gln vs. Arg/Arg and Gln/Gln+Arg/Gln vs. Arg/Arg for Caucasians and oral squamous cell carcinoma, Gln/Gln vs. Arg/Arg for healthy population-based control and larynx squamous cell carcinoma, and Arg/Gln vs. Arg/Arg for hospital-based control and healthy population-based control had no heterogeneity with a P value ≥0.05, therefore, we analyzed the summary odds ratios for them with a fixed-effects model. Random-effects model was used to analyze the summary ORs for the rest.
Quantitative Data Synthesis
Table 2 listed the summary ORs of XRCC1 Arg399Gln polymorphisms related to HNSCC risk on the basis of 3917 cases and 4560 controls. We did not observe any association of XRCC1 Arg399Gln polymorphisms with HNSCC risk in the total population (OR = 0.95, 95% CI: 0.76–1.19 for Gln/Gln vs. Arg/Arg (Fig. 1), OR = 1.05, 95% CI: 0.92–1.20 for Arg/Gln vs. Arg/Arg (Fig. 2), and OR = 1.03, 95% CI: 0.90–1.18 for Gln/Gln+Arg/Gln vs. Arg/Arg (Fig. 3)) based on 18 identified studies including 3917 cases and 4560 controls. In subgroup analysis, we observed an increased risk of XRCC1 codon 399 Arg/Gln genotype for HNSCC in Caucasians (OR = 1.20, 95% CI: 1.00–1.44) and Gln/Gln genotype for larynx squamous cell carcinoma (OR = 1.63, 95% CI: 1.10–2.40). We did not observe any association between XRCC1 Arg399Gln variants and HNSCC risk in additional subgroup analyses (Table 2).
Publication bias was assessed by funnel plot, the shape of the funnel plot seemed to be approximately symmetrical (Fig. 4, 5, 6). The results from Egger’s test and Begg’s test indicated that there were no obvious publication biases in our current meta-analysis (Table 2).
Sensitivity analyses were conducted to determine the influence of the individual dataset on the summary ORs by sequential removal of each eligible study. The overall effects were not altered when the studies were homogenous for Gln/Gln vs. Arg/Arg, Arg/Gln vs. Arg/Arg and Gln/Gln+Arg/Gln vs. Arg/Arg among total population by removing some eligible studies (Fig. S2, S3, S4).
XRCC1 is an important component of the base excision repair system, which is the predominant DNA repair pathway for small base lesions resulting from oxidation and alkylation damage . The gene is located on chromosome 19q13.2–13.3, which has 32,354 base pairs and consists of 17 exons and 16 introns. It encodes a 70-kDa scaffolding protein consisting of 633 amino acids, which coordinates numerous protein-protein interactions, including with DNA ligase III and DNA polymerase at the site of damage , . More than 300 validated single nucleotide polymorphisms in the XRCC1 gene were reported in the dbSNP database (http://www.ncbi.nlm.nih.gov/SNP), among them, rs25487 in XRCC1 codon 399 of exon 10 was the most extensively studied polymorphic site. Recently, meta-analysis studies have reported that XRCC1 Arg399Gln variant was associated with the risk of certain cancers, such as breast cancer , nasopharyngeal carcinoma , cervical carcinoma  and glioma . In this paper, we performed a systematic literature review to comprehensively evaluate the association between sequence variants in XRCC1 Arg399Gln and the risk of squamous cell carcinoma of the head and neck. We also estimated the possible effect modifications by source of control, ethnicity of subjects, tumor site and HWE in control. In summary, we did not observe an association of XRCC1 Arg399Gln polymorphisms with HNSCC risk in the total population. Our findings are consistent with the previous meta-analysis study .
HNSCC includes tumors from different sites including oral, pharynx and larynx region, risk factors for these cancers may be different. For example, oral cavity and laryngeal cancers are mainly associated with tobacco use and alcohol consumption, while oropharyngeal cancers are principally related to viral infection, such as human papillomavirus (HPV) . Unlike the HPV-negative oropharyngeal cancers, the HPV-positive subset is not related to tobacco or alcohol use, but with certain types of sexual behaviours. The HPV 16 subtype is present in up to 90% of HPV-related oropharyngeal cancers, while HPVs 18, 31 and 33 have been identified in the remainder. Recently, HPV has been recognized as a good prognostic factor in head and neck cancer, which has been attributed to several mechanisms, including absence of field cancerisation and increased sensitivity to chemoradiation therapy . When subgroup analyses were conducted by tumor site, the subjects carrying Gln/Gln genotype had an increased risk in larynx squamous cell carcinoma subgroup. There was no association of XRCC1 Arg399Gln polymorphisms with oral squamous cell carcinoma. We did not perform subgroup analyses on other tumor sites, owing to unavailable data.
Considering that the frequency of the XRCC1 399 Gln allele variant is significantly different among different ethnic population. Allele frequency patterns of XRCC1 Arg399Gln polymorphism vary greatly among major ethnic groups. The frequency of Gln allele was more than 0.3 in Caucasians , , but less than 0.2 in Asian population , . When stratified by ethnicity, we observed an association of XRCC1 Arg399Gln polymorphisms with HNSCC risk among Caucasians, but not among Asian population. Different ethnicities cancer susceptibility associated with the XRCC1 Arg399Gln polymorphisms was also observed in previous meta-analyses of lung cancer and breast cancer , . This discrepancy among different ethnicities may be due to the different genetic backgrounds in these populations, subsequently leading to different genetic susceptibility to the same disease.
It is widely acknowledged that deviation from HWE may point to methodological weaknesses, such as biased selection of subjects or genotyping errors. The results of genetic association studies might be spurious if the distribution frequency of genotypes in the control groups was not in agreement with HWE . To address this problem, subgroup analysis was conducted in this study by HWE in controls. When the study  that significantly deviated from HWE was excluded from this present analysis, we did not observe a substantial modification of the results, suggesting that this factor might not have effects on the overall estimates in the current meta-analysis.
This meta-analysis should be interpreted within the context of its limitations. First, only published articles were included in this study. Therefore, publication bias is very likely to occur. To address this issue, Egger’s test and Begg’s test were conducted. Our results indicated that the likelihood of key publication bias might not be present in this meta-analysis. Secondly, each study had different eligibility criteria for subjects and different source of controls, which should be taken into account while interpreting the combined estimates. When subgroup analysis was performed by source of control, we observed any association between XRCC1 Arg399Gln polymorphisms and HNSCC risk neither in hospital-based control nor in healthy population-based control. Thirdly, the summary odds ratios were based on individual unadjusted estimates, while a more precise analysis might be performed if detailed individual data were available, which could allow for an adjusted estimation by sex, age, tobacco use, alcohol consumption and environment factors. In addition, the study number was limited and the sample size was relatively small in subgroup analyses, as a consequence, our estimates of the association in subgroup analyses might have occurred by chance. Thus, the results must be interpreted with caution.
In conclusion, this systematic review demonstrates that XRCC1 Arg399Gln variants appear not to be a risk factor of HNSCC in the total population. However, XRCC1 Arg399Gln variants might be a potential risk factor for HNSCC among Caucasians and for larynx squamous cell carcinoma. Large scale studies with the pooling of individual study data should be taken into account in the future studies to verify the results from this current meta-analysis.
Sensitivity analysis for Gln/Gln vs. Arg/Arg.
Sensitivity analysis for Arg/Gln vs. Arg/Arg.
Sensitivity analysis for Gln/Gln+Arg/Gln vs. Arg/Arg.
PRISMA 2009 Checklist.
We would like to thank Isioma Enwerem’s kindly helps for editing the English language of this manuscript.
Conceived and designed the experiments: YDW FZ. Performed the experiments: YDW XWC. Analyzed the data: YDW XWC. Contributed reagents/materials/analysis tools: XWC XJM. Wrote the paper: YDW.
- 1. Kostrzewska-Poczekaj M, Gawecki W, Illmer J, Rydzanicz M, Gajecka M, et al. (2013) Polymorphisms of DNA repair genes and risk of squamous cell carcinoma of the head and neck in young adults. Eur Arch Otorhinolaryngol 270: 271–276.
- 2. Foulkes WD, Brunet JS, Sieh W, Black MJ, Shenouda G, et al. (1996) Familial risks of squamous cell carcinoma of the head and neck: retrospective case-control study. BMJ 313: 716–721.
- 3. Gugatschka M, Dehchamani D, Wascher TC, Friedrich G, Renner W (2011) DNA repair gene ERCC2 polymorphisms and risk of squamous cell carcinoma of the head and neck. Exp Mol Pathol 91: 331–334.
- 4. Khlifi R, Rebai A, Hamza-Chaffai A (2012) Polymorphisms in human DNA repair genes and head and neck squamous cell carcinoma. J Genet 91: 375–384.
- 5. Li C, Hu Z, Lu J, Liu Z, Wang LE, et al. (2007) Genetic polymorphisms in DNA base-excision repair genes ADPRT, XRCC1, and APE1 and the risk of squamous cell carcinoma of the head and neck. Cancer 110: 867–875.
- 6. Yu H, Liu Z, Huang YJ, Yin M, Wang LE, et al. (2012) Association between single nucleotide polymorphisms in ERCC4 and risk of squamous cell carcinoma of the head and neck. PLoS One 7: e41853.
- 7. Vidal AE, Boiteux S, Hickson ID, Radicella JP (2001) XRCC1 coordinates the initial and late stages of DNA abasic site repair through protein-protein interactions. EMBO J 20: 6530–6539.
- 8. Shen MR, Jones IM, Mohrenweiser H (1998) Nonconservative amino acid substitution variants exist at polymorphic frequency in DNA repair genes in healthy humans. Cancer Res 58: 604–608.
- 9. Applebaum KM, McClean MD, Nelson HH, Marsit CJ, Christensen BC, et al. (2009) Smoking modifies the relationship between XRCC1 haplotypes and HPV16-negative head and neck squamous cell carcinoma. Int J Cancer 124: 2690–2696.
- 10. Bogela A, Yalikun Y, Ayihen Q (2011) Study on polymorphism and genetic susceptibility of laryngeal of Arg399Gln XRCC1 gene in different ethnic groups in Xinjiang. Journal of Xinjiang Medical University 34: 1247–1250.
- 11. Csejtei A, Tibold A, Koltai K, Varga Z, Szanyi I, et al. (2009) Association between XRCC1 polymorphisms and head and neck cancer in a Hungarian population. Anticancer Res 29: 4169–4173.
- 12. Demokan S, Demir D, Suoglu Y, Kiyak E, Akar U, et al. (2005) Polymorphisms of the XRCC1 DNA repair gene in head and neck cancer. Pathol Oncol Res 11: 22–25.
- 13. Dos Reis MB, Losi-Guembarovski R, de Souza Fonseca Ribeiro EM, Cavalli IJ, Morita MC, et al. (2013) Allelic variants of XRCC1 and XRCC3 repair genes and susceptibility of oral cancer in Brazilian patients. J Oral Pathol Med 42: 180–185.
- 14. Harth V, Schafer M, Abel J, Maintz L, Neuhaus T, et al. (2008) Head and neck squamous-cell cancer and its association with polymorphic enzymes of xenobiotic metabolism and repair. J Toxicol Environ Health A 71: 887–897.
- 15. Kietthubthew S, Sriplung H, Au WW, Ishida T (2006) Polymorphism in DNA repair genes and oral squamous cell carcinoma in Thailand. Int J Hyg Environ Health 209: 21–29.
- 16. Kowalski M, Przybylowska K, Rusin P, Olszewski J, Morawiec-Sztandera A, et al. (2009) Genetic polymorphisms in DNA base excision repair gene XRCC1 and the risk of squamous cell carcinoma of the head and neck. J Exp Clin Cancer Res 28: 37.
- 17. Krupa R, Kasznicki J, Gajecka M, Rydzanicz M, Kiwerska K, et al. (2011) Polymorphisms of the DNA repair genes XRCC1 and ERCC4 are not associated with smoking- and drinking-dependent larynx cancer in a Polish population. Exp Oncol 33: 55–56.
- 18. Kumar A, Pant MC, Singh HS, Khandelwal S (2012) Associated risk of XRCC1 and XPD cross talk and life style factors in progression of head and neck cancer in north Indian population. Mutat Res 729: 24–34.
- 19. Olshan AF, Watson MA, Weissler MC, Bell DA (2002) XRCC1 polymorphisms and head and neck cancer. Cancer Lett 178: 181–186.
- 20. Tae K, Lee HS, Park BJ, Park CW, Kim KR, et al. (2004) Association of DNA repair gene XRCC1 polymorphisms with head and neck cancer in Korean population. Int J Cancer 111: 805–808.
- 21. Varzim G, Monteiro E, Silva RA, Fernandes J, Lopes C (2003) CYP1A1 and XRCC1 gene polymorphisms in SCC of the larynx. Eur J Cancer Prev 12: 495–499.
- 22. Yang Y, Tian H, Zhang Z, He S, Hu C (2008) Association of the XRCC1 and hOGG1 polymorphism with the risk of laryngeal carcinoma. Chin J Med Genet 25: 211–213.
- 23. Majumder M, Sikdar N, Ghosh S, Roy B (2007) Polymorphisms at XPD and XRCC1 DNA repair loci and increased risk of oral leukoplakia and cancer among NAT2 slow acetylators. Int J Cancer 120: 2148–2156.
- 24. DerSimonian R, Laird N (1986) Meta-analysis in clinical trials. Control Clin Trials 7: 177–188.
- 25. Egger M, Davey Smith G, Schneider M, Minder C (1997) Bias in meta-analysis detected by a simple, graphical test. Bmj 315: 629–634.
- 26. Begg CB, Mazumdar M (1994) Operating characteristics of a rank correlation test for publication bias. Biometrics 50: 1088–1101.
- 27. Almeida KH, Sobol RW (2007) A unified view of base excision repair: lesion-dependent protein complexes regulated by post-translational modification. DNA Repair (Amst) 6: 695–711.
- 28. Kubota Y, Nash RA, Klungland A, Schar P, Barnes DE, et al. (1996) Reconstitution of DNA base excision-repair with purified human proteins: interaction between DNA polymerase beta and the XRCC1 protein. EMBO J 15: 6662–6670.
- 29. Li H, Ha TC, Tai BC (2009) XRCC1 gene polymorphisms and breast cancer risk in different populations: a meta-analysis. Breast 18: 183–191.
- 30. Huang GL, Guo HQ, Yu CY, Liu XY, Li BB, et al. (2011) XRCC1 polymorphisms and risk of nasopharyngeal carcinoma: a meta-analysis. Asian Pac J Cancer Prev 12: 2329–2333.
- 31. Liu YT, Shi JP, Fu LY, Zhou B, Wang HL, et al. (2013) Gene polymorphism of XRCC1 Arg399Gln and cervical carcinoma susceptibility in Asians: a meta-analysis based on 1,759 cases and 2,497 controls. Asian Pac J Cancer Prev 14: 189–193.
- 32. Jiang L, Fang X, Bao Y, Zhou JY, Shen XY, et al. (2013) Association between the XRCC1 polymorphisms and glioma risk: a meta-analysis of case-control studies. PLoS One 8: e55597.
- 33. Zhang C, Song X, Zhu M, Shi S, Li M, et al. (2013) Association between MMP1–1607 1G>2G polymorphism and head and neck cancer risk: a meta-analysis. PLoS One 8: e56294.
- 34. Thibaudeau E, Fortin B, Coutlee F, Nguyen-Tan P, Weng X, et al. (2013) HPV Prevalence and Prognostic Value in a Prospective Cohort of 255 Patients with Locally Advanced HNSCC: A Single-Centre Experience. Int J Otolaryngol 2013: 437815.
- 35. Zhang Y, Newcomb PA, Egan KM, Titus-Ernstoff L, Chanock S, et al. (2006) Genetic polymorphisms in base-excision repair pathway genes and risk of breast cancer. Cancer Epidemiol Biomarkers Prev 15: 353–358.
- 36. Zienolddiny S, Campa D, Lind H, Ryberg D, Skaug V, et al. (2006) Polymorphisms of DNA repair genes and risk of non-small cell lung cancer. Carcinogenesis 27: 560–567.
- 37. Long X, Ma Y, Wei Y, Deng Z (2004) POLYMORPHISM OF DNA REPAIR GENE XRCC1 AND RISK OF HEPATOCELLULAR CARCINOMA. JOURNAL OF GUANGXI MEDICAL UNIVERSITY 21: 313–315.
- 38. Yin J, Vogel U, Ma Y, Qi R, Sun Z, et al. (2007) The DNA repair gene XRCC1 and genetic susceptibility of lung cancer in a northeastern Chinese population. Lung Cancer 56: 153–160.
- 39. Kiyohara C, Takayama K, Nakanishi Y (2006) Association of genetic polymorphisms in the base excision repair pathway with lung cancer risk: a meta-analysis. Lung Cancer 54: 267–283.
- 40. Salanti G, Amountza G, Ntzani EE, Ioannidis JP (2005) Hardy-Weinberg equilibrium in genetic association studies: an empirical evaluation of reporting, deviations, and power. Eur J Hum Genet 13: 840–848.