Table 1.

Published studies on BRCA1 promoter methylation in breast and/or ovarian cancer.

#	Reference	Methodology	Regiona	DNA source	Study groups	Age (years)	Findings
1	Chen et al. (2006)44	Bisulfite cloning Sanger sequencing	−591 to +66	Whole blood	Affected BRCA1 and BRCA2 mutation-negative women with a strong family history (n = 41) Unaffected women (n = 19)	20–50	No significant difference in BRCA1 methylation levels of individual CpGs between affected and unaffected women
2	Snell et al. (2008)69	MethyLight Real-time PCR	+14 to +16	Whole blood	Women diagnosed with BRCA1-like BC (n = 7)	35–51	One women was methylated at the BRCA1 promoter in her DNA derived from blood and buccal mucosa (10% and 5%, respectively). Two women had ~1% BRCA1 methylation
		MS-HRM Digital MS-HRM Sanger sequencing	−55 to +44	Buccal mucosa Breast tumour	Corresponding buccal mucosa DNA (n = 1) Tumour DNA (n = 5)		Tumour-derived DNAs from all three women had high levels of BRCA1 methylation (61–100%)
3	Kontorovich et al. (2009)39	MS-qPCR Sanger sequencing	−728 to −1052	Whole blood	Affected BRCA1/2 mutation carriers (n = 48) Unaffected BRCA1/2 mutation carriers (n = 41) Affected non-mutation carriers (n = 52) Healthy controls (n = 89)	26–62	Promoter methylation only detected in BRCA1 (5.6–7.3% in each study group) BRCA1 promoter methylation detected in ~5% of the Israeli Jewish women regardless of the BRCA1/2 status Proportion with BRCA1 promoter methylation according to age (all groups): <40 years: 9/118 (7.6%) 41–50 years: 4/68 (5.9%) 51–60 years: 1/27 (3.7%) ≥61 years: 0/17 (0%)
4	Cho et al. (2010)80	MethyLight Real-time PCR	+8 to +27	Breast tumour Adjacent normal tissue White blood cells	BC patients (n = 40) Healthy controls (n = 40)	< 40–> 60	Most patients with methylation at BRCA1, HIN1, RASSF1A and CDH1 in their blood-derived DNA were also methylated in their corresponding tumour-derived DNA Gene-specific methylation in blood-derived DNA the same in cases and controls
5	Wong et al. (2011)21	MS-HRM MethyLight	−55 to +44 +14 to +16	Whole-blood Breast tumour	Group 1: Young women diagnosed with ‘BRCA1-like’ BC (n = 52) Group 2: Young women diagnosed with non ‘BRCA1-like’ BC (n = 164) Unaffected women (n = 169)	< 40	BRCA1 promoter methylation detected in blood-derived DNA of 30% Group 1 women compared with Group 2 (P = 0.000002) and unaffected women (P = 0.004). BRCA1 promoter methylation associated with rs11655505 C > T (P = 0.035) and rs799906 A > G (P = 0.017). BRCA1 promoter methylation in blood-derived DNA was associated with a 3.5-fold increased risk of developing ‘BRCA1-like’ BC (95% CI: 1.4, 10.5).
6	Al-Moghrabi et al. (2011)64	MSP High-resolution sodium bisulfite sequencing	−37 to +44 –567 to +44	Breast tumour Whole blood	Tumour-derived DNA from affected Arab women (n = 47) Blood-derived DNA from other women diagnosed with BC (n = 7) Healthy controls (n = 73)	22–80	Strong association between BRCA1 methylation and young age (≤40 years) at diagnosis and high-grade tumours (67%) (P = 0.0038) BRCA1 promoter methylated in blood-derived DNA of 8/73 healthy controls (10.9%) and 2/7 BC patients (28%) BRCA1 CpG island highly methylated in blood-derived DNA in both cases and controls Sporadic BRCA1 methylation detected in the core promoter region (−218 to +1)
7	Wojdacz et al. (2011)67	MS-HRM	−21 to +44	Whole blood	Affected women (n = 180) Unaffected women (n = 108)	> 50	No significant difference in frequency of BRCA1 methylation between cases (20%) and controls (20%)
8	Iwamoto et al. (2011)20	qMSP	+8 to +27	Whole blood	Affected women (n = 200) Unaffected women (n = 200)	30–69	BRCA1 promoter methylation detected in blood-derived DNA of 21.5% (43/200) affected women and 13.5% (27/200) unaffected women ORadj for BC is 1.73; 95% CI: 1.01, 2.96; P = 0.045)
9	Hansmann et al. (2012)22	Pyrosequencing Bisulfite plasmid sequencing Promoter sequencing	−116 to +116	Whole blood	Affected women negative for BRCA1 and BRCA2 mutations (n = 641)	< 40–>60	1.4% (9/641) patients had BRCA1 methylation (6–20%). BRCA1 epimutations were  (i) Significantly more frequent in index patients with OC than BC (P = 0.016) (ii) More frequent in families with BC and OC history than with BC alone (P = 0.01) (iii) Significantly enriched in women with early-onset BC without a family history of cancer, compared with women with familial BC after the age of 36 years (P = 0.047)
10	Bosviel et al. (2012)40	QAMA	−662 to −678	Whole blood	Mutation-negative affected women (n = 902) Unaffected women (n = 990)	26–89	BRCA1 promoter methylation rate was 47.1% (95% CI: 46.1, 48.1) in affected women and 45.9% (95% CI: 45.0, 46.8) in unaffected women (P = 0.08) In blood-derived DNA of affected women, BRCA1 promoter methylation was associated with  (i) Age (P = 0.017)  (ii) Post-menopausal status (P = 0.013)  (iii) BMI < 20 (P = 0.0095)  (iv) WHR (≤76.8) (P = 0.0027) BRCA1 promoter methylation was not associated with Scarff-Bloom-Richardson tumour grade and size, histological type and lymph node metastasis
11	Al-Moghrabi et al. (2014)66	MSP	−156 to +34	White blood cell Breast tumour	Affected women (n = 155) Unaffected women (n = 143) Tumour tissues from 22 methylation-positive patients (n = 19)	23–73	14.2% (22/155) affected women (20 were <50 years old) and 9.1% (13/143) unaffected women (11 were <40 years old) had BRCA1 promoter methylation in their blood DNA 66.7% affected women had BRCA1 promoter methylation in blood-derived and tumour-derived DNA BRCA1 methylation was higher in cases than controls (corresponding lower mRNA expression)
12	Gupta et al. (2014)65	MS-HRM	−21 to +44	Whole blood	Affected BRCA1-mutation carriers (n = 30) Affected non carriers (n = 36) Unaffected women (n = 36)	> 60 (mean age)	BRCA1 promoter methylation present in 41.7% (15/36) affected mutation-negative women but not affected mutation carriers (P < 0.01) BRCA1 methylation-positive women tend to be younger (average 42.5 years) OR for BC in affected mutation-negative women with detectable BRCA1 methylation was 12.1 (95% CI: 2.5, 59.0) BRCA1 methylation more common in TNBC [40.9% (9/22)], medullary BC [45.4% (10/22)] and both triple-negative and medullary BC [50% (4/8)]
13	Cho et al. (2015)68	MethyLight Real-time PCR	−55 to +16	WBC Breast tumour	Population-based cases (n = 1021) Population-based controls (n = 1036) Tumour tissue (n = 569)	< 40–> 75	BRCA1 hypermethylation in WBC associated with an increased risk of BC (≥0.1% methylation (OR 1.31; 95% CI: 0.98, 1.75)) Lack of concordance between tumor tissue and paired WBC DNA methylation Limited evidence for BRCA1 methylation in WBC DNA and BC risk
14	Evans et al. (2018)45	Pyrosequencing Bisulfite sequencing	−55 to +44	Lymphocyte Blood Buccal mucosa Hair follicle Breast tumour	Lymphocyte: Unrelated individuals from high-risk BC and OC families (n = 49) Blood: Family 1 (n = 10); Family 2 (n = 4) Buccal mucosa: Family 1 (n = 9); Family 2 (n = 4) Hair follicle: Family 1 (n = 9); Family 2 (n = 4) Breast tumour: Family 1 (n = 1); Family 2 (n = 0)	31–85	BRCA1 promoter methylation detected in:  (i) 2/49 women diagnosed with early-onset BC (lymphocyte and blood) (ii) Six family members in Family 1. 2/6 diagnosed with early-onset BC (iii) Four family members in Family 2. 1/4 diagnosed with early-onset OC (iv) associated with a 5’UTR dominantly inherited c.−107A > T variant No correlation between methylation levels and clinical phenotype The epimutation accounts for at least 1.25% BRCA1 pathogenic variants in high-risk BC and OC families

The methodology, region interrogated, DNA source, age of participants, study groups and findings for each study are as listed. # denotes the study number corresponding to Fig. 1

BC breast cancer, MethyLight MethyLight Real-time PCR, MS-HRM methylation-specific High Resolution Melt analysis, MSP methylation-specific PCR, OC ovarian cancer, QAMA Quantitative Analysis of Methylated Alleles, WBC white blood cell

^aRelative to BRCA1 transcription start site