Pooled analysis indicates that the GSTT1 deletion, GSTM1 deletion, and GSTP1 Ile105Val polymorphisms do not modify breast cancer risk in BRCA1 and BRCA2 mutation carriers

Abstract

The GSTP1, GSTM1, and GSTT1 detoxification genes all have functional polymorphisms that are common in the general population. A single study of 320 BRCA1/2 carriers previously assessed their effect in BRCA1 or BRCA2 mutation carriers. This study showed no evidence for altered risk of breast cancer for individuals with the GSTT1 and GSTM1 deletion variants, but did report that the GSTP1 Ile105Val (rs1695) variant was associated with increased breast cancer risk in carriers. We investigated the association between these three GST polymorphisms and breast cancer risk using existing data from 718 women BRCA1 and BRCA2 mutation carriers from Australia, the UK, Canada, and the USA. Data were analyzed within a proportional hazards framework using Cox regression. There was no evidence to show that any of the polymorphisms modified disease risk for BRCA1 or BRCA2 carriers, and there was no evidence for heterogeneity between sites. These results support the need for replication studies to confirm or refute hypothesis-generating studies.

Introduction

Breast cancer risk is greatly increased in female carriers of germline mutations in the BRCA1 or BRCA2 genes compared with the general population. However, the estimated penetrance of deleterious mutations does vary according to study ascertainment, being somewhat lower in carriers recruited via population-based studies compared to those identified in studies of multiple case families, and risks have been found to vary depending on the cancer site of the first individual that led to the family ascertainment [1–3]. It is generally accepted that genetic modifiers explain some of this difference in penetrance and, indeed, recent studies by the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) have provided evidence showing that genetic variation outside of the BRCA1 and BRCA2 genes can modify risk of breast cancer in carriers of pathogenic BRCA1 and BRCA2 mutations [4–6].

Candidate breast cancer modifier genes include those that are involved in the metabolism of carcinogens. The phase II glutathione S-transferase genes catalyze the glutathione-mediated reduction of exogenous and endogenous electrophiles with broad and overlapping substrate specificity [7, 8], generally producing readily excreted water-soluble compounds. Thus, allelic variants associated with altered detoxification rates of potential carcinogens have long been postulated to confer an increased susceptibility to cancer [9]. The GSTP1, GSTM1, and GSTT1 genes have functional polymorphisms that are frequently present in the general population. Inherited homozygous deletions of the GSTT1 and GSTM1 gene lead to the complete absence of enzyme activity [10–12], whereas the GSTP1 A313G substitution results in an isoleucine to valine amino acid substitution at position 104, which has altered specific activity and decreased heat stability [13–15].

There is conflicting evidence for a role for GST functional polymorphisms with risk of breast and bladder cancer, and other smoking-related cancers [16–19]. However, their effects on breast cancer risk in carriers of BRCA1 or BRCA2 mutations have been reported in only a single study [20]. Their analysis of 320 BRCA1/2 carriers showed no evidence for altered risk of breast cancer for individuals with the GSTT1 and GSTM1 deletion variants, but did report that the GSTP1 Ile105Val variant was associated with increased breast cancer risk (HR 1.36 (p = 0.04) for heterozygotes, and HR 2.00 (P = 0.01) for homozygotes). After stratification by gene, the effect of the GSTP1 variant remained significant among BRCA2 carriers only (Hazard Ratio = 3.20 (95% CI 1.26–8.09, P = 0.01)).

We undertook a study to collate existing data for these polymorphisms from CIMBA consortium members to assess the association of GST polymorphisms with risk of breast cancer in a larger sample set.

Subjects

The characteristics of the study samples are shown in Table 1. A total of 473 BRCA1 and 245 BRCA2 carriers, from four sites within CIMBA, had been genotyped for the GSTT1 and GSTM1 deletion polymorphisms, and the GSTP1 Ile105Val polymorphism. All sites provided information for the GSTT1 and GSTM1 polymorphisms, and all except the MAGIC site provided data for the GSTP1 missense polymorphism. The ascertainment of carriers to these four sites is described in detail elsewhere [4]. Only carriers of pathogenic mutations were included. Ethical approvals for recruitment and genotyping were obtained from the institutional review boards or ethics committees at all the sites. Written informed consent was obtained from each participant.

Table 1.

Characteristics of Study Subjects

	BRCA1		BRCA2		Group TOTAL
	n	(% of total)	n	(% of total)
EMBRACE	236	(49.9)	90	(36.7)	326
KconFaB	93	(19.7)	73	(29.8)	166
Inherit	73	(15.4)	82	(33.5)	155
Magic*	71	(15.0)	0	(0.0)	71
Total	473		245		718
Affected with breast cancera	251	(53.1)	136	(55.5)
Affected with ovarian cancera	42	(8.9)	13	(5.3)
Unaffectedb	180	(38.1)	96	(39.2)

^*GSTP1 genotypes were not available for this sample set

^aRefers to first cancer for individuals reporting both breast and ovarian cancer. Individuals with ovarian cancer were censored as unaffected at the age of diagnosis for analysis

^bIncludes unaffected individuals censored at the age of prior bilateral mastectomy

Molecular methods

Genotyping for the MAGIC [21], and kConFab, EMBRACE and INHERIT samples [22], was as described previously. In brief, PCR-agarose methodology was used to detect the homozygous wildtype and GSTT1 and GSTM1 deletion variants; ABI Prism 7700 Sequence Detection System (SDS) methodology was used for genotyping the GSTP1 A to G Ile105Val variant (rs1695).

Statistical methods

Individuals with a first primary invasive breast cancer diagnosis were considered to be affected, while individuals with no reported breast or ovarian cancer were censored at the age of interview, or at the age of prior bilateral prophylactic mastectomy. Individuals with a first primary ovarian cancer diagnosis were censored as unaffected at the age at onset of ovarian cancer. Analyses of association between genotype and breast cancer risk were performed using Cox regression with time to breast cancer onset as the end point. Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated separately for BRCA1 and BRCA2 carriers, and in each, study group and year of birth (categorized into subgroups 1910–1939, 1940–1949, 1950–1959, and 1960+) were included as covariates in the analysis. Secondary analyses also adjusted for ethnicity (Caucasian, other). Adjustment for other potential confounders was not considered due to the relatively small sample size of this study, which included only a limited number of CIMBA subjects with existing genotyping data. Confidence limits for the rate ratio were calculated using a robust variance approach to allow for the dependence among individuals in the same family [23]. In order to address the problem of non-random sampling of mutation carriers with respect to the disease phenotype, we analyzed using the weighted Cox regression approach [24], where individuals were weighted such that observed breast cancer incidence rates in the study sample are consistent with established breast cancer risk estimates for BRCA1 and BRCA2 mutation carriers [1]. R version 2.7.0 was used for statistical analyses.

Results and Discussion

The estimated hazard ratios associated with the GST polymorphisms are shown in Table 2. There was no evidence for an association between any of the GST polymorphisms and risk in BRCA1 or BRCA2 mutation carriers. This included the GSTP1 Ile105Val variant: the HR (95% CI) for this ValVal homozygote genotype was 0.89 (0.44–1.49) for BRCA1 carriers, and 0.81 (0.40–1.65) for BRCA2 carriers. There was no evidence for heterogeneity in the hazard ratios between studies for any of the three polymorphisms analyzed (P ≥ 0.3). The overall findings were little different when analyses were adjusted additionally for ethnicity. For example, the HR (95% CI) for the GSTP1 ValVal genotype was 0.92 (0.46–1.84) for BRCA1 carriers, and 0.80 (0.39–1.65) for BRCA2 carriers.

Table 2.

Association of GSTT1, GSTM1, and GSTP1 genotypes with breast cancer risk in BRCA1 and BRCA2 carriers

Gene	Genotype	BRCA1				BRCA2
		Genotype frequency	Adjusted group and year of birth			Genotype frequency	Adjusted group and year of birth
			P	HR	(95% CI)		P	HR	(95% CI)
GSTT1	Null	0.20	0.60	0.90	(0.59–1.36)	0.14	0.71	1.13	(0.59–2.19)
GSTM1	Null	0.52	0.3	1.19	(0.84–1.67)	0.48	0.5	0.84	(0.53–1.33)
GSTP1	AG	0.47	0.82	1.05	(0.70–1.57)	0.42	0.86	1.05	(0.62–1.77)
	GG	0.10	0.74	0.89	(0.44–1.79)	0.16	0.56	0.81	(0.40–1.65)

Although our genotyping method for GSTM1 and GSTT1 does not distinguish heterozygotes from wild-type homozygotes [25], our data provide no evidence for association between breast cancer risk for carriers and three GST common polymorphisms. Notably, the GSTP1 105Val variant, which was previously reported to be associated with increased risk of breast cancer in a small study of 90 BRCA2 carriers [20], was associated with non-significant reduced risk for BRCA2 carriers in our larger sample of 245 women with BRCA2 mutations. Although the sample size of this study is still relatively small, there is a statistically significant difference (P = 0.02) between the HR for BRCA2 in this study and that reported by Kadouri et al [20]. These results support the need for replication studies to confirm or refute hypothesis-generating studies, including analyses that use existing unpublished data collated by consortia.

kConFab—The Kathleen Cuningham Consortium for Research into Familial Breast Cancer

We wish to thank Heather Thorne, Eveline Niedermayr, all the kConFab research nurses and staff, the heads and staff of the Family Cancer Clinics, and the Clinical Follow Up Study (funded by NHMRC grants 145684, 288704, and 454508) for their contributions to this resource, and the many families who contribute to kConFab. The kConFab is supported by grants from the National Breast Cancer Foundation, the National Health and Medical Research Council (NHMRC), and by the Queensland Cancer Fund, the Cancer Councils of New South Wales, Victoria, Tasmania and South Australia, and the Cancer Foundation of Western Australia. ABS is an NHMRC Senior Research Fellow, and GC-T is an NHMRC Senior Principal Research Fellow.

Epidemiological study of BRCA1 and BRCA2 mutation carriers (EMBRACE)

DE is the PI of the study. DE, SP, and MC are funded by Cancer Research, UK Grants C1287/A10118 and C1287/A8874. EMBRACE Collaborating Centers are: Coordinating Centre, Cambridge: Susan Peock, Margaret Cook, Clare Oliver, Debra Frost; North of Scotland Regional Genetics Service, Aberdeen: Helen Gregory, Zosia Miedzybrodzka; Northern Ireland Regional Genetics Service, Belfast: Patrick Morrison; West Midlands Regional Clinical Genetics Service, Birmingham: Trevor Cole, Carole McKeown, Amy Taylor; South West Regional Genetics Service, Bristol: Alan Donaldson; East Anglian Regional Genetics Service, Cambridge: Joan Paterson; Medical Genetics Services for Wales, Cardiff: Alexandra Murray, Mark Rogers, Emma McCann; St James’s Hospital, Dublin & National Centre for Medical Genetics, Dublin: John Kennedy, David Barton; South East of Scotland Regional Genetics Service, Edinburgh: Mary Porteous; Peninsula Clinical Genetics Service. Exeter: Carole Brewer, Emma Kivuva, Anne Searle, Selina Goodman; West of Scotland Regional Genetics Service, Glasgow: Rosemarie Davidson, Victoria Murday, Nicola Bradshaw, Lesley Snadden, Mark Longmuir, Catherine Watt; South East Thames Regional Genetics Service, Guys Hospital London: Louise Izatt, Gabriella Pichert, Caroline Langman. North West Thames Regional Genetics Service. Harrow: Huw Dorkins; Leicestershire Clinical Genetics Service; Leicester: Julian Barwell; Yorkshire Regional Genetics Service, Leeds: Carol Chu, Tim Bishop, Julie Miller; Merseyside & Cheshire Clinical Genetics Service. Liverpool: Ian Ellis; Manchester Regional Genetics Service, Manchester: D Gareth Evans, Fiona Lalloo, Felicity Holt; North East Thames Regional Genetics Service, NE Thames: Alison Male, Anne Robinson. Nottingham Centre for Medical Genetics, Nottingham: Carol Gardiner; Northern Clinical Genetics Service, Newcastle: Fiona Douglas, Oonagh Claber; Oxford Regional Genetics Service, Oxford: Lisa Walker, Diane McLeod; The Institute of Cancer Research and Royal Marsden NHS Foundation Trust: Ros Eeles, Susan Shanley, Nazneen Rahman, Richard Houlston, Elizabeth Bancroft, Lucia D’Mello, Elizabeth Page, Audrey Ardern-Jones, Anita Mitra; North Trent Clinical Genetics Service, Sheffield: Jackie Cook, Oliver Quarrell, Cathryn Bardsley; South West Thames Regional Genetics Service, London: Shirley Hodgson, Sheila Goff, Glen Brice, Lizzie Winchester; Wessex Clinical Genetics Service; Princess Anne Hospital, Southampton: Diana Eccles, Anneke Lucassen, Gillian Crawford, Emma Tyler, Donna McBride. ACA is a Cancer Research UK Senior Cancer Research Fellow. CIMBA data management is supported by Cancer Research UK. The kConFab and EMBRACE genotyping was supported by an NHMRC Programme grant to GCT. MAGIC data collection and analysis was supported by R01-CA102776 to TRR.

Interdisciplinary Health Research International Team on Breast Cancer Susceptibility (INHERIT BRCAs)

Jacques Simard, Francine Durocher, Rachel Laframboise, Marie Plante, Centre Hospitalier Universitaire de Québec & Laval University, Québec, Canada; Peter Bridge, Jilian Parboosingh, Molecular Diagnostic Laboratory, Alberta Children’s Hospital, Calgary, Canada; Jocelyne Chiquette, Hôpital du Saint-Sacrement, Québec, Canada; Bernard Lespérance, Hôpital du Sacré-Cœur de Montréal, Montréal, Canada. Jacques Simard- J.S. is Chairholder of the Canada Research Chair in Oncogenetics. This study was supported by the Canadian Institutes of Health Research for the “CIHR Team in Familial Risks of Breast Cancer” program.

Modifiers and Genetics in Cancer (MAGIC)

This work was supported by the NIH grants R01-CA102776 and R01-CA083855 (to TRR). The MAGIC Consortium includes the following centers and individuals: Baylor- Charles A. Sammons Cancer Center (Joanne L, Blum, M.D. Ph.D.; Becky Althaus, R.N., C.G.C.; Gaby Ethington), Baylor College of Medicine (Claire Noll; Sharon Plon, M.D., Ph.D.), Beth Israel Deaconess Medical Center (Nadine Tung, M.D.), City of Hope National Medical Center (Sharon Sand; Jeffrey N. Weitzel, M.D.), Creighton University (Carrie Snyder, B.A.; Henry T. Lynch, M.D.; Patrice Watson, Ph.D.), Dana-Farber Cancer Institute (Kathryn Stoeckert; Judy E. Garber, M.D., M.P.H.), Duke University (Sydnee Crankshaw; Joellen Schildkraut, Ph.D.), Evanston Northwestern Healthcare Center for Medical Genetics (Suzanne M. O’Neill, Ph.D.; Christina Selkirk; Wendy S. Rubinstein, M.D., Ph.D.), Fox Chase Cancer Center (Mary B. Daly, M.D., Ph.D.; Andrew Godwin, Ph.D.), Queensland Institute of Medical Research (Georgia Chenevix-Trench), Georgetown University (Claudine Isaacs, M.D.), Jonsson Comprehensive Cancer Center at the University of California-Los Angeles (Joyce Seldon; Patricia A. Ganz, M.D.), Mayo Clinic College of Medicine (Linda Wadum; Fergus Couch, Ph.D.), University of Chicago (Shelly Cummings; Olufunmilayo Olopade, M.D.), University of California-Irvine (Susan L. Neuhausen, Ph.D.; Linda Steele), University of Pennsylvania Health System (Susan Domchek, M.D.; Katherine Nathanson M.D.; Tara Friebel, M.P.H.; Timothy Rebbeck, Ph.D.), University of Texas Southwestern (Gail Tomlinson, M.D.), University of Vienna (Christian Singer, M.D.), and Women’s College Hospital (Steven A. Narod, M.D.).