Screening of BRCA1 variants c.190T>C, 1307delT, g.5331G>A and c.2612C>T in breast cancer patients from North India

Abstract

The polymorphic variants of BRCA1, which lead to amino acid substitutions, have a known pathogenic role in breast cancer. The present study investigated in North Indian breast cancer patients the association of risk with four reported pathogenic variants of BRCA1: c.190T>C (p.Cys64Arg), 1307delT, g.5331G>A (p.G1738R) and c.2612C>T (p.Pro871Leu). Genotyping was done by PCR-RFLP method in 255 clinically confirmed breast cancer patients and 255 age and gender matched healthy individuals. For c.190T>C, 1307delT and g.5331G>A, all the patients and controls had the wild-type genotype indicating no association with breast cancer risk. For c.2612C>T polymorphism, the frequency of the CC, CT, and TT genotypes was 14.5 vs 15.7%, 59.6 vs 53.7% and 25.9 vs 30.6% in breast cancer patients and controls respectively. The frequency of heterozygotes (CT genotype) was higher in cases than controls but the difference was not statistically significant. Genetic model analysis showed no association of the four analyzed BRCA1 variants with breast cancer risk with any model. The studied variants were not associated with the risk of breast cancer in Punjab, North west India, suggesting a need for further screening of other BRCA1 variants. It is the first reported study on these 4 variants from India.

Keywords
BRCA1; variants; breast cancer; North India

Introduction

Breast cancer is the most common cancer in the world (Ghoncheh et al., 2016). In India, according to the National Cancer Registry Programme (NCRP 2012-2014), incidence of breast cancer ranks amongst the top in the following individual registries across India: Delhi (41.0%), Chennai (37.9%), Bangalore (34.4%) and Thiruvananthapuram district (33.7%). In the state of Punjab, the highest incidence rate of breast cancer has been reported in districts of Bathinda (37.3%), followed by Mohali (34.3%), Patiala (32.5%) and Faridkot (31.5%) (NCRP 2012-2013), with increasing frequency of sporadic breast cancer being reported from Amritsar (Batra et al., 2010).

One of the well-studied genes that has been associated with breast cancer, BRCA1 (MIM 113705), is located at the chromosomal position 17q21 and contains 24 exons (Miki et al., 1994). It encodes a multi-domain protein BRCA1, which has four major domains: a RING domain, the BRCA1 serine domain and two BRCA1 C terminus (BRCT) domains (Clark et al., 2012). BRCA1 interacts with a variety of other proteins to carry out multiple functions at the cellular level, like controlling the cell cycle, DNA damage repair, regulation of transcription, replication, recombination and chromatin hierarchical control (Parvin, 2004).

Mutations in BRCA1 and BRCA2 genes are the most susceptible causes amongst the genetic risk factors that play a crucial role in familial breast cancer (Naga, 2011). A frequency of 2.9-24% of BRCA1/2 mutations has been reported in familial breast cancer patients of South India (Vaidyanathan et al., 2009). However, the reported frequency of mutations in BRCA2 is remarkably lower than in BRCA1 in India (Kim and Choi, 2013).

Populations of Caucasian and Indoscythian mixed racial ancestry inhabit the state of Punjab in North India (Bhasin et al., 1992). Therefore, the BRCA1 variants were chosen for the study considering their prevalence in similar Caucasian and South Asian populations in other parts of the world. The four variants of BRCA1 [c.190T>C (p.Cys64Arg), 1307delT, g.5331G>A (p.G1738R) and c.2612C>T (p.Pro871Leu)] were investigated in the current study. The variant c.2612C>T (p.Pro871Leu) is a non-synonymous polymorphism of BRCA1 that leads to the substitution of proline by leucine at position 871, a region where recombinase RAD51 interacts with BRCA1 to aid homologous recombination (Miao et al., 2017). The T allele has been reported to show an association with breast cancer risk in Chinese populations (Xu et al., 2018) though some studies have reported no significant association between the p.Pro871Leu variant of BRCA1 and breast cancer risk. The 1307delT variant of BRCA1, present in codon 396 of exon 11, causes a frameshift mutation that introduces a premature stop codon at amino acid residue 409 and leads to protein truncation. It was reported in the South Indian population (Gajalakshmi et al., 2007). The c.1907T>C variant of BRCA1 is a missense mutation present in exon 5 (Cys64Arg). Molecular modelling indicated that the substitution of cysteine with an arginine probably disturbs the structure of the BRCA1 RING finger domain, which is responsible for the interaction with BARD1 and essential for the tumor-suppressor activity of the BRCA1-BARD1 complex (Karami and Mehdipour, 2013). g.5331G>A (p.G1738R) is a missense variant of BRCA1 that affects the binding affinity of the protein by destabilizing it (Williams et al., 2003; Glover, 2006). It has been reported as a pathogenic mutation (Chenevix-Trench et al., 2006; Karchin et al., 2007).

Although a few common variants of BRCA1 have been repeatedly reported from South India, the dissimilarity between the populations of North India: Caucasians, Scythians and Indo-Europeans (Bhasin et al., 1992; Reich et al., 2009) and South India: Dravidian (Reich et al., 2009), led us to consider studying the variants in North India. Thus, the current study aimed to screen the Punjabi population from Amritsar and adjoining regions of Punjab, North India, for the four variants of BRCA1 [c.190T>C (p.Cys64Arg), 1307delT, g.5331G>A (p.G1738R) and c.2612C>T (p.Pro871Leu)] to investigate their association with breast cancer risk. Identifying BRCA1 polymorphisms that are associated with breast cancer risk can also be further explored for involvement in the therapy response of patients. BRCA1 c.190T>C (p.Cys64Arg) and g.5331G>A (p.G1738R) have never been reported from India in any of the studies so far. Also, these four variants together have not been investigated in Indian patients in any previous study.

Subjects and Methods

Study subjects

In this case-control study, patients were selected from a tertiary care hospital, Sri Guru Ram Das Institute of Medical Sciences and Research, Vallah, Amritsar, Punjab. The majority of the patients (81%) belonged to the Majha region of Punjab, i.e., to the Amritsar district (34%) and its adjoining districts of Gurdaspur (32%) and Tarn Taran (15%). The rest of the patients belonged to the districts of Jalandhar (5%), Kapurthala (5%), Hoshiarpur (4%), Pathankot (3%), Ferozpur (2%) and Mansa (1%). Three patients, each belonging to the states of Jammu and Kashmir, Himachal Pradesh and Uttar Pradesh, were also included. A total of 255 (5 males and 250 females) clinically confirmed breast cancer patients and 255 age and gender matched normal healthy individuals were recruited in the present study between January 2013 and March 2017. Patients who had received chemotherapy, radiotherapy or blood transfusion before surger, or had previous history of any malignancy were excluded from the study. Controls were biologically unrelated to cancer patients and were from the same geographical region as the patients. Individuals in the control group had no family history of any cancer or any other chronic disease. This study was undertaken after approval by the institutional ethics committee of Guru Nanak Dev University, Amritsar, Punjab, India. After informed consent, as per tenets of the Declaration of Helsinki,a5 mL peripheral venous blood sample was collected in an EDTA vial from each subject.

DNA isolation and screening of BRCA1 variants

Genomic DNA was extracted from peripheral blood lymphocytes by a standard phenol-chloroform method (Adeli and Ogbonna, 1990). Four variants of BRCA1, c.190T>C (p.Cys64Arg), 1307delT, g.5331G>A (p.G1738R) and c.2612C>T (p.Pro871Leu) were screened by a PCR-RFLP method using published primer sequences (Gajalakshmi et al., 2007; Karami and Mehdipour, 2013; Xu et al., 2018).

The PCR assays were set up in a 15 μL reaction volume containing 50 ng of DNA, 1X Taq buffer with 1.5 mM MgCl2, 0.3 μL dNTPs mixture (Bangalore GeNei), 6 pmol of each primer (Sigma, St. Louis, MO, USA), 1U Taq DNA Polymerase (Bangalore GeNei). The PCR conditions were: an initial denaturation at 95 °C for 5 min, followed by 35 cycles with denaturation at 95 °C for 45 s, annealing at 58 °C for 30 s and extension at 72 °C for 45 s, and a final extension step at 72 °C for 10 min, run in a Mastercycler gradient (Eppendorf, Germany) machine. A negative control without template DNA was run in each reaction to check for contamination. The PCR products were analysed on 2% ethidium bromide-stained agarose gels. Amplified products were digested with the respective restriction enzymes following the manufacturer’s instructions (New England Biolabs, Beverly, MA), details of which are shown in Table 1. The restriction digestion reaction products were also analysed on 2.5% ethidium bromide-stained agarose gels. The genotype was assigned to each sample according to the size of the fragments obtained after digestion (Table 1). Genotyping was performed without knowing the status of the subjects.

Table 1
Details of analyzed BRCA1 variants and restriction conditions used for genotyping.

S. No.	Variant	PCR product size (bp)	Restriction enzyme used	Digestion temperature	Fragment size (bp) after digestion	Alleles
1.	c.190T>C (p.Cys64Arg)	193 bp	SnaBI	37 °C	193	T
					124 and 69	C
2.	g. 5331G>A (p.G1738R)	233 bp	BsaXI	37 °C	112, 91 and 30	G
					233	A
3.	1307delT	151 bp	DdeI	37 °C	151	WT
					96 and 55	MUT
4.	c.2612C>T (p.Pro871Leu)	125 bp	HpaII	37 °C	99 and 26	C
					125	T

• WT - wild type; MUT - mutant.

Statistical analysis

A chi-square test was used to test for Hardy-Weinberg Equilibrium (HWE) and difference of genotype and allele frequencies between the breast cancer patients and normal controls. For determining the association between BRCA1 variants with breast cancer risk, the odds ratio (OR) with 95% confidence interval (CI) was calculated. A genetic model analysis was done to check for an association of the BRCA1 variants with breast cancer risk. A value of p ≤ 0.05 was considered statistically significant. Statistical analysis was performed by using SPSS (Version 20.0; SPSS, Inc).

Results

The present study recruited 255 clinically and histopathologically confirmed breast cancer patients and 255 healthy controls. Out of the 255 patients, 250 (98%) were females and 5 (2%) were males. The mean age of the patients was 50.7 ± 11.43 years (range 27-85 years) and that of controls was 48.56 ± 14.26 (range 17-90 years). The frequency of patients diagnosed with stage I, stage II, stage III and stage IV breast cancer were 14.9%, 46.8%, 25.5% and 12.8%, respectively (Table 2). Out of these, 40% of the patients were triple negative for the receptor status. The majority of the patients had sporadic breast cancer (78.8%), while 7.5% reported a history of breast cancer in their family (Table 3). Another 13.7% of the patients had a history of cancer of other sites. Hence, 21.2% of the patients had a family history of any type of cancer.

Table 2
Clinical profile of breast cancer patients.

Parameter	Total n (%)	Females n (%)	Males n (%)
Stage
I	35(14.9)	35(15.2)	0(0)
II	110(46.8)	105(46.7)	5(100)
III	60(25.5	60(26.1)	0(0)
IV	30(12.8)	30(13)	0(0)
Breast side
Left	116(47.2)	113(46.9)	3(60)
Right	121(49.2)	120(49.8)	1(20)
Bilateral	9(3.6)	8(3.3)	1(20)
Histological Type
DCIS	6(2.7)	6(2.8)	0(0)
DCIS-Papilary	3(1.3)	3(1.4)	0(00
IDC	205(91.9)	200(91.7)	5(100)
IDC-Mucinous	5(2.2)	5(2.2)	0(0)
IDC-Medulllary	1(0.5)	1(0.5)	0(0)
IDC-Pagets	1(0.5)	1(0.5)	0(0)
Invasive Lobular Carcinoma	2(0.9)	2(0.9)	0(0)

• n: number of subjects

Table 3
Breast cancer patients with family history of cancer.

Category	Cases	% of Total Cases
Sporadic cases	201	78.8
Familial cases	54	21.2
History of breast cancer
1st Relatives	11	4.3
2nd Relatives	5	2.0
3rd Relatives	3	1.2
History of other cancer
1st Relatives	23	9.0
2nd Relatives	12	4.7
3rd Relatives	0	0

The genotype and allele frequencies of the four analysed BRCA1 variants in cases and controls are shown in Table 4. For the 3 variants c.190T>C (p.Cys64Arg), 1307delT and c.5331G>A (p.G1738R) all the patients and controls had the wild-type genotype (^{Figures S1}-^S4). The observed genotype frequencies for the c.2612C>T polymorphism were in HWE in the controls (p=0.11). The frequency of the CC, CT and TT genotypes of c.2612C>T polymorphism were 14.5% vs. 15.7%, 59.6% vs. 53.7% and 25.9% vs 30.6%, respectively, in breast cancer patients and control individuals. No association was observed between the breast cancer risk and the CT (OR = 1.19, 95% CI: 0.73-1.98, p=0.48) and TT (OR = 0.91, 95% CI:0.53-1.59, p=0.75) genotypes. The c.2612 C>T polymorphism did not show an association with breast cancer in any of the genetic models (Table 4). The T allele frequency was higher than that of the C allele in both patients and controls, but the difference between patients and controls was not significant (p=0.57). The difference in the frequency distribution of the C and T alleles in breast cancer patients with involvement of left, right or bilateral sides was statistically non-significant (p=0.55). This difference was also not significant (p=0.60) amongst patients at different stages of breast cancer.

Table 4
Association analyses of BRCA1 variants with breast cancer risk.

Variant	Genotype	Patients n (%)	Controls n (%)	OR (95%CI)	p-value
c.190T>C (p.Cys64Arg)	TT	255(100)	255(100)	-	NC
	TC	0	0
	CC	0	0
1307delT	WT/WT	255(100)	255(100)	-	NC
	WT/MUT	0	0
	MUT/MUT	0	0
c.5331G>A (p.G1738R)	GG	255(100)	255(100)	-	NC
	GA	0	0
	AA	0	0
c.2612 C>T (p.Pro871Leu)	CC	37(14.5)	40(15.7)	1 (ref)
	CT	152(59.6)	137(53.7)	1.19(0.73-1.98)	0.48
	TT	66(25.9)	78(30.6)	0.91(0.53-1.59)	0.75
	Allele
	C	226(44.3)	217(42.5)	1 (ref)
	T	284(55.7)	293(57.5)	0.93(0.73-1.19)	0.57
Genetic Models
Dominant Model CT/TT vs CC				1.10(0.67-1.78)	0.71
Co-dominant Model TT vs CT =CT vs CC				0.92(0.70-1.20)	0.54
Recessive Model TT vs CC/CT				0.79(0.54-1.17)	0.24

• n: number of subjects; OR: odds ratio; CI: confidence intervals; NC: not calculated.

To evaluate the relation between the parameters like age at diagnosis, menopausal history, habitat, diet and BMI with the BRCA1 p.Pro871Leu variant, the cases and controls were stratified according to the aforementioned parameters. We found no significant difference in the genotypic frequencies among patients and controls. However, there was a significant difference in C and T allele frequencies among cases and controls for parameters like age at diagnosis in the group 36-50 years (OR = 0.47, 95% CI = 0.32-0.70, p=0.00), frequency of premenopausal women (OR = 0.56, 95% CI = 0.36-0.87, p=0.01), frequency of post-menopausal women (OR = 0.65, 95% CI = 0.46-0.90, p=0.01), rural habitat (OR = 0.51, 95% CI = 0.37-0.87, p=0.000), vegetarian diet (OR = 0.61, 95% CI = 0.44-0.85, p=0.003) and obesity (BMI ≥ 23 kg/m2) (OR = 0.62, 95% CI = 0.46-0.84, p=0.002) (Table 5).

Table 5
Correlation of BRCA1 p.Pro871Leu variant with demographic parameters in subjects.

Parameter	Genotypes				OR (95% CI)	p-value	Allele				OR (95% CI)	p-value
	Cases		Controls				Cases		Controls
	CC N (%)	CT+TT N (%)	CC N (%)	CT+TT N (%)			C N (%)	T N (%)	C N (%)	T N (%)
Age at diagnosis (years)
≤ 35	5(13.9)	26(10.9)	3(11.1)	45(20)	0.35(0.08-1.57)	0.17	26(11.7)	36(12.7)	34(19.9)	62(18.6)	0.76(0.39-1.46)	0.41
36-50	15(41.7)	118(49.6)	8(29.6)	96(42.7)	0.66(0.27-1.61)	0.36	106(47.5)	118(41.7)	62(36.3)	146(43.9)	0.47(0.32-0.70)	0.00
51-65	13(36.1)	77(32.4)	6(22.2)	66(29.3)	0.54(0.19-1.5)	0.24	73(32.7)	107(37.8)	45(26.3)	99(29.7)	0.67(0.42-1.06)	0.08
≥ 66	3(8.3)	17(7.1)	10(37.1)	18(8.0)	3.15(0.74-13.45)	0.12	18(8.1)	22(7.8)	30(17.5)	26(7.8)	1.41(0.62-3.19)	0.41
Menstrual history
premenopausal	12(35.3)	66(32.0)	6(26.1)	88(42.3)	0.38(0.13-1.05)	0.06	71(33.3)	85(31.8)	60(39.5)	128(41.3)	0.56(0.36-0.87)	0.01
postmenopausal	22(64.7)	140(68.0)	17(73.9)	120(57.7)	1.46(0.56-3.18)	0.76	142(66.7)	182(68.2)	92(60.5)	182(58.7)	0.65(0.46-0.90)	0.01
Habitat
rural	27(77.1)	163(76.9)	16(61.5)	162(73)	0.60(0.31-1.15)	0.12	167(77.0)	213(76.9)	101(60.8)	255(77.3)	0.51(0.37-0.69)	0.000
urban	8(22.9)	49(23.1)	10(38.5)	60(27)	1.02(0.37-2.78)	0.97	50(23.0)	64(23.1)	65(39.2)	75(22.7)	0.68(0.67-1.82)	0.68
Diet
vegetarian	24(68.6)	136(64.5)	16(61.5)	134(61.5)	0.68(0.34-1.33)	0.26	146(67.3)	174(63.3)	102(61.8)	198(61.3)	0.61(0.44-0.85)	0.003
non-vegetarian	11(31.4)	75(35.5)	10(38.5)	84(38.5)	0.81(0.33-2.02)	0.65	71(32.7)	101(36.7)	63(38.2)	125(38.7)	0.72(0.47-1.10)	0.13
BMI
non-obese	7(20.6)	60(29.9)	5(19.2)	42(19.0)	1.02(0.30-3.43)	0.97	56(26.9)	78(29.8)	30(18.1)	64(19.5)	0.65(0.38-1.14)	0.13
obese	27(79.4)	141(70.1)	21(80.8)	179(81.0)	0.61(0.33-1.23)	0.12	152(73.1)	184(70.2)	136(81.9)	264(80.5)	0.62(0.46-0.84)	0.002

• n: number of subjects; OR: odds ratio; CI: confidence intervals; NC: not calculated; p<0.05 (significance).

Discussion

In the present case-control study, we assessed the relationship of c.190T>C (p.Cys64Arg), 1307delT, g.5331G>A (p.G1738R) and c.2612C>T variants of BRCA1 with breast cancer risk. The variants p.Cys64Arg and p.G1738R have never been reported from any Indian study so far, and also c.2612C>T had not yet been reported from India at the time when the present study was undertaken. It has been only recently reported from a population of West India (Gujarat), where 32 polymorphisms and two novel mutations in BRCA1 were found in 35 breast cancer patients by an NGS method (Shah et al., 2018). Among these, c.2612C>T was found in six out of the 35 patients.

The c.190T>C variant has been reported as a pathological mutation in families with HBOC (history of breast and ovarian cancer) in Polish (Jakubowska et al., 2001), Italian (Willems et al., 2009; Caleca et al., 2014), Brazilian (Fernandes et al., 2016), French (Azzollini et al., 2017) and Japanese (Arai et al., 2018) populations. A few in-vitro studies have also revealed its deleterious effects (Cochran et al., 2015; Anantha et al., 2017). In the present study, only the wild type genotype (TT) of c. 190T>C was found in all the patients and controls from Amritsar.

The 1307delT variant was first time reported in a South Indian family with Dravidian ancestry having breast cancer (Gajalakshmi et al., 2007). The South Indian population represents a racial compositon different from that of North India. In the present study all the patients and controls, which were from the Northwest part of India, with Caucasian and Indoscythian ancestry (Reich et al., 2009), had the wild type genotype for this variant.

The g.5331G>A (p.G1738R) missense mutation has been repeatedly found in Greek families with breast cancer (Belogianni et al., 2004; Armaou et al., 2009; Koumpis et al., 2011), ovarian cancer (Stavropoulou et al., 2013) or both (Konstantopoulou et al., 2008; Karami and Mehdipour, 2013). It is also reported to be a deleterious mutation in in-vitro assays (Carvalho et al., 2007; Chenevix-Trench et al., 2016). None of the patients or controls in the present study carried this mutation.

The c.2612C>T (p.Pro871Leu) non-synonymous SNP causes an amino acid change from proline to leucine at position 871 in the BRCA1 protein. The previous published studies give contradictory reports on the role of p.P871L and the risk for various types of cancers. The wild type genotype (CC) was associated with increased risk of gastric cancer (Wang et al., 2015), esophageal squamous cell carcinoma (ESCC) (Zhang et al., 2013) and non-Hodgkin Lymphoma (NHL) (Chen et al., 2013), but not associated with thyroid carcinoma (Xu et al., 2012) and ovarian cancer (Wenham et al., 2003; Auranen et al., 2005). In a recent meta-analysis (Xu et al., 2018), the p.Pro871Leu polymorphism was linked to decreased risk of various cancers like ESCC, cervical cancer, gastric cancer and NHL among Asians. But some studies from Chinese populations have reported significant associations between the BRCA1 p.Pro871Leu variant and breast cancer, where the CT genotype provides increased risk to breast cancer in these populations (Huo et al., 2009; Wang et al., 2009). The homozygous variant TT genotype has also been reported to be associated with reduction in glioblastoma risk in Caucasians (Chang et al., 2008) and NHL risk in Korea (Kim et al., 2014). The T allele of the p.Pro871Leu polymorphism has been shown to be linked with miR-628 dependent BRCA1 reduction, indicating that cells with the T allele would express higher BRCA1 as compared to those with the C allele (Nicoloso et al., 2010). In the present study, none of the genotypes of this variant were found to be associated with breast cancer risk. Similar findings have been reported in a Czech population (Soucek et al., 2007), where no association was reported. Meta-analyses carried out in 2014 (Qin et al., 2014) and 2017 (Miao et al., 2017) reported no association of the p.Pro871Leu polymorphism and breast cancer risk. Contradictory results have also been reported for the BRCA1 871L allele, as significantly associated with increased risk of breast cancer in a Polish population (Smolarz et al., 2017). An association of the TT genotype with reduced breast cancer risk in Chinese has also been reported (Zhou et al., 2009).

When assessed for response to therapy, p.Pro871Leu showed no association with either advanced phases of CML or treatment response (Gutierrez-Malacatt et al., 2016). The CT/TT genotypes have been reported to be associated with ovarian cancer progression in non-African American patients, but not with progression-free survival (PFS) and overall survival (OS) in patients treated with cisplatin and paclitaxel (Tian et al., 2013). The TT genotype has been reported to not only contribute to increased breast cancer risk associated with combined estrogen monotherapy in postmenopausal women in Germany (Abbas et al., 2010), but also with recurrence of TNBC after radiotherapy in Han Chinese (Shi et al., 2017).

Understanding of the biological mechanisms underlying breast cancer predisposition by genomic profiling can help to define molecular targets for chemoprevention and biomarkers of breast cancer risk. India is the second most populous country in the world, but not much information on BRCA1 is available as of now. The 185delAG variant has been found to be the most common mutation in BRCA1 in India, as revealed by a few reported studies of limited sample sizes (range 19 - 204). It has been found in variable frequencies in the studied groups from North India: 7.14% (Kumar et al., 2002), 0.49% (Saxena et al., 2006) and South India: 7 out of 19 members in a single family study (Kadalmani et al., 2007), 54.5% (Vinodkumar et al., 2007) and 16.4% (Vaidyanathan et al., 2009). The present study is the first report providing baseline data on four variants of BRCA1 from the Amritsar and adjoining regions of Punjab, Northwest India. The three mutations [c.190T>C (p.Cys64Arg), 1307delT and g.5331G>A (p.G1738R)], which have been widely known as pathogenic in Caucasians, have no mutant genotype in the studied population. For the c.2612C>T polymorphism also, the T allele frequency was slightly higher than that of the C allele, and there was a preponderance of the CT genotype, but no association with the breast cancer risk. A marked variation in the distribution of p.Pro871Leu in the Northwest Indian population from the previously reported populations of South India has been observed. The present study indicates the need of screening Indian populations for other mutations/variants in BRCA1 gene for identifying breast cancer risk and possible targets for therapy.

Acknowledgments

We are thankful to all the subjects who participated in the present study. Financial assistance came from an UGC vide project grant F.No. 41-824/2012/(SR) to Dr. Vasudha Sambyal, UGC-UPE and UGC-CPEPA to Dr. Vasudha Sambyal and Dr. Kamlesh Guleria. The fellowship from UGC-UPE and CSIR vide F.No. 09/254(0258)/2016-EMR-I to Akeen Kour is also highly acknowledged. We would also like to thank Dr. Geeta Sharma, former Principal and Dr. Manjit Singh Uppal, Principal, Sri Guru Ram Das Institute of Medical Sciences and Research, Vallah,Amritsar, Punjab, for allowing access to patients and other facilities helpful in carrying out the present study.

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Internet resources

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Supplementary material

The following online material is available for this article:

Figure S1 - Agarose gel picture for BRCA1 c.190T>C (p.Cys64Arg).

Figure S2 - Agarose gel picture for BRCA1 1307delT.

Figure S3 - Agarose gel picture for BRCA1 c.5331G>A (p.G1738R).

Figure S4 - Agarose gel picture for BRCA1 c.2612 C>T (p.Pro871Leu).

Edited by

Associate editor:

Mara Hutz

Publication Dates

• Publication in this collection
  20 May 2020
• Date of issue
  2020

History

• Received
  16 Jan 2019
• Accepted
  22 June 2019