BRCA1 and BRCA2 gene mutation spectrum and high frequency of BRCA1 185delAG among breast and ovarian cancer patients from Southern India.

doi:10.21203/rs.3.rs-3129325/v1

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BRCA1 and BRCA2 gene mutation spectrum and high frequency of BRCA1 185delAG among breast and ovarian cancer patients from Southern India.

https://doi.org/10.21203/rs.3.rs-3129325/v1

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In a developing country like India, genomic data sets for even the most clinically relevant genes like BRCA1 and BRCA2 are relatively scarce. There is also a need to identify and screen population specific BRCA hotspot mutations to pave the way for affordable genetic testing strategies in clinical practice. We have carried out an ambispective study to evaluate Next-generations Sequencing (NGS) based approach to identify pathogenic variants in BRCA1 and BRCA2 genes among 772 breast and ovarian cancer patients. The target enrichment was carried out using the in-house designed Multiplex-PCR for BRCA1 and BRCA2, followed by targeted NGS on Ion Torrent Personal Genome Machine. Additionally, allele-specific PCR (ASPCR) based genotyping of BRCA1 c.68_69delAG also known as 185delAG, was carried out in 149 patients. We identified 181 BRCA1 and BRCA2 variants, and based on ACMG 2015 guidelines, these variants were classified as 111 pathogenic or likely pathogenic and 70 VUS (Variant with uncertain significance). The 185delAG was identified as a recurrent mutation in the Southern Indian population accounting for 25.21% of the pathogenic variants. In addition, a family history of cancers of the breast, ovary, pancreas, or prostate (BOPP) was found to be associated with a higher risk of identifying a deleterious BRCA1/2 variant [OR=2.15 (95%CI 1.46-3.2) p≤0.0001]. These results suggest that Multiplex PCR coupled NGS is a sensitive and specific strategy for BRCA testing. However, ASPCR-based genotyping of 185delAG followed by targeted NGS would be cost-effective in South Indian patients.

Biological sciences/Genetics/Sequencing/Next-generation sequencing

Health sciences/Medical research/Genetics research

BRCA genes

NGS

allele-specific PCR

recurrent mutation

BOPP

Breast and ovarian malignancies are the most prevalent and leading cause of cancer related deaths in Indian women(1, 2). Data from cancer registries suggest that up to one-third (3–24%) of these cancers may be hereditary(3, 4). Even though most of the highly penetrant mutations have been reported in BRCA1 and BRCA2 genes(5), routine screening for these causative variants in a clinical setting is still in a primitive stage in India.

Next-generation sequencing (NGS) based multigene panel testing is increasingly adopted as a rapid technique for identifying pathogenic BRCA mutations with an accuracy comparable to traditional methods(6). The increasing adoption of parallel multigene testing, and coverage of multiple genes, has improved the identification of patients with pathogenic mutations (7). Additionally, confirmation of these results with traditional Sanger sequencing ensures the specificity of these test results (8, 9).

In this study, we have established and utilized targeted NGS of BRCA1 and BRCA2 genes to screen 772 patients with breast or/and ovarian cancers, including subjects with strong family history of breast, ovarian, pancreatic, or prostate cancers (BOPP). In addition, we aimed to identify clinico-pathological factors that are predictive for identifying pathogenic BRCA variants and recurrent population-specific hotspot mutations.

This study was conducted at a tertiary care hospital with patients predominantly from southern and eastern India and Bangladesh. Overall, the wide geographical distribution of patients provides a reasonable snapshot of the distribution of BRCA mutations among a genetically heterogeneous population.

Patients

The indications for testing were in accordance with the NCCN guidelines for BRCA mutation testing(10). Patients were eligible if they fulfilled any one of the following criteria: a) young onset breast cancer (age <45 years), b) young onset triple-negative breast cancer (age <60 years), (c) Breast cancer at any age with >1 close relative (first, second or third-degree) diagnosed to have breast cancer at age <50 years or carcinoma ovary, pancreas, or prostate at any age. (d) Epithelial ovarian cancer (including fallopian tube or peritoneal cancer) (e) Male breast cancer (e) Exocrine pancreatic cancer at any age (f) High grade carcinoma prostate (Gleasons score >7) with >1 close relative (first, second or third degree relative) with carcinoma breast (g) first- or second-degree relative meeting the criteria listed above (f) family history of BRCA mutation.

In this study, 772 eligible patients were evaluated, and patients who did not have complete clinical details regarding cancer stage, reasons for testing, or treatment were excluded. Patient screening and recruitment for germline BRCA mutations was carried out in Medical Oncology, Endocrine Surgery, Radiation Oncology, and Gynaecological Oncology departments, at Christian Medical College, Vellore, India. The genotyping and sequencing experiments were carried out in the Molecular Endocrinology Laboratory, Department of Endocrinology, Christian Medical College, Vellore, India. This study was approved by the institutional review board IRB no 13205.

Sequencing and Bioinformatics

DNA extraction: Whole blood samples were utilized for DNA extraction using the Promega Maxwell® RSC DNA extraction kit, and the samples were stored at -20⁰ C.

Target enrichment: We have developed a novel multiplex PCR-based enrichment for BRCA1 and BRCA2 genes using a QIAGEN® Multiplex PCR kit, followed by sequencing on Ion Torrent™ PGM. The primers include 25 bp upstream and downstream of each exon to capture the splice junctions and the 5´ UTR and 3´UTR regions. Primers were designed in-house (Supplementary material; Table1), and the multiplex PCRs were carried out (Figure.1a-1d).

Library preparation: The NGS library preparation was carried out as per the published protocol(11). In short, the library preparation for the pooled PCR amplicons, adaptor ligation, nick repair, and amplification were performed using the NEBNext® Fast DNA Library Prep Set for Ion Torrent™. Before pooling, the sample multiplexing was carried out using the Ion Xpress™ barcode adapters (Ion Torrent, Life Technologies). The adaptor ligated samples were size-selected on E-Gel® Size Select™ 2% pre-cast agarose gels. These libraries were amplified for 4-6 cycles of PCR, and the concentration for equimolar pooling of the samples was done using KAPA Library Quantitation Kit (Roche).

Template preparation: The clonal amplification (template preparation) was performed using Ion OneTouch OT2 Emulsion PCR using the Ion OneTouch™ Hi-Q Template Kit (Ion Torrent, Life Technologies). Further, the streptavidin-coupled Dynabeads®-based enrichment was carried out on Ion OneTouch Enrichment System (Ion Torrent, Life Technologies) before sequencing.

Sequencing and bioinformatic analysis: Targeted Sequencing was performed on Ion Torrent PGM using Ion PGM™ 200 Sequencing Kit (Ion Torrent, Life Technologies), utilizing 316 chips and 318 chips. The generated sequencing data was mapped to the human genome reference hg19 using Torrent Mapping Alignment Program (TMAP). The Torrent suite software with v5.0 was used for analysis. The coverage analysis was carried out using Torrent Coverage Analysis (Supplementary material Figure 1), and variants were identified using the Torrent Variant Caller. In addition, we utilized DNA Star software (DNASTAR, Madison, WI, USA) for annotation, variant calling, and further data analysis.

The aligned reads with TMAP and the VCF files generated with Ion torrent suit software were further analyzed using the wANNOVAR, a free web-based tool for variant calling and annotation(12). The variants identified in the homopolymer regions were also manually checked using Integrative Genomics Viewer (IGV v.2.9.4). All the variants identified in the homopolymer regions and also the pathogenic/likely pathogenic/variant of uncertain significance were further confirmed by Sanger sequencing.

Allele-specific PCR (ASPCR) for BRCA1 185delAG

Allele-specific PCR (ASPCR) based genotyping for the BRCA1:c.68_69delAG (185delAG) hotspot mutation has been established in-house. The PCR protocol was standardized with appropriate positive and negative controls using EmeraldAmp® Max PCR master mix (TaKaRa Bio), and the validation of the positive results was done using Sanger sequencing (ABI 3130 genetic analyzer, Applied Biosystems).

Statistical analysis

Baseline clinical characteristics were described using appropriate measures of central tendency. Recurrent mutations were identified, and the geographical origins of the patient were traced to look for population-specific patterns. Associations between patient characteristics and the presence of pathogenic BRCA1/BRCA2 mutations were tested by the Chi-square test. Multivariate logistic regression was done to eliminate confounding effects. A significance level of p≤0.05 was used in the analysis. All analysis were performed using SPSS.

A total of 772 patients with breast and/or ovarian cancers were included in this study and additional 149 patients were screened only for the 185delAG hotspot mutation. The characteristics of the study subjects are presented in Table 1. We identified 181 (23.4%) patients with clinically significant variants and based on ACMG 2015 guidelines, these variants were classified: 111 pathogenic or likely pathogenic and 70 VUS. The overall diagnostic yield (mutation positivity), including all cases, was 23%. There was a higher yield seen in patients with family history (34.9%) than those without family history (20.4%), (Table 2). The BRCA1 mutation load in this study was comparatively higher with 118 variants, while 68 variants were identified in the BRCA2 gene. We identified five novel variants (two pathogenic, two likely pathogenic, and one VUS). A list of the identified pathogenic and likely pathogenic variants is given in Table 3.

Multiplex PCR based target enrichments followed by sequencing yielded a mean coverage of >500X with more than 98% of the bases covered at a minimum coverage of 20X (yielding coverage of coding regions and splice site regions without any gaps). Sanger sequencing validation results were consistent with the NGS readouts. However, the challenge with the Ion Torrent sequencing technology is the presence of homopolymer errors(false calls at the repeats of the same nucleotide) (31). Therefore, the variants identified in the homopolymer regions were filtered by cross-comparing similar allele frequency false calls across the samples. Also, the unique variant calls in the homopolymer region specific to a sample, even with < 40% allele frequency calls, were further confirmed by Sanger sequencing. With these strategies, we were able to identify and validate variants even in the homopolymers regions in BRCA1 and BRCA2 genes.

In this study, out of 772 patients, 181 patients were identified with clinically significant variants, which consisted of 63 frameshift (34.8%), 26 nonsense (14.4%) and 64 missense (35.4%) mutations. Splice site variants were identified in 23 (12.7%) patients (Figure 3).

Description of Novel Variants

In this study, we have identified five novel BRCA variants.

Subject BC294 was found to be positive for a heterozygous BRCA1 deletion c.5334_5335del (p.Asp1778GlufsTer72) that results in a frameshift at codon 1778, which creates a premature translational stop signal at codon 1849 instead of 1885. The patient had bilateral metachronous breast cancer with a strong family history of first- and second-degree relatives with breast and ovarian cancers.

Subject BC219, with serous cystadenocarcinoma of the ovary, was positive for a novel heterozygous BRCA1 nonsense variant c.1369G>T (p. Glu457Ter), resulting in premature termination. Also, the patient had recurrent metastatic disease and has been counselled to use PARP inhibitors. She had no first or second-degree family members with HBOC (Hereditary Breast and Ovarian Cancer). According to ACMG 2015 guidelines, these novel BRCA1 loss-of-function variants are likely pathogenic. The families of both these patients have been referred for genetic counselling and are on regular monitoring.

The subject BC704 diagnosed with ovarian cancer at 57 years of age, was found to be positive for a novel frameshift BRCA2(NM_000059.4):c.7219del (p.Val2407PhefsTer60) variant, resulting in premature truncation of BRCA2 protein at 2466 instead of 3419. There was no family history of cancers.

Another novel likely pathogenic frameshift BRCA2(NM_000059.4):c.521del (p.Arg174LeufsTer11) variant was identified in the subject BC724 who was diagnosed with estrogen-receptor positive breast cancer and the age of onset was 29. The patient, paternal grandfather’s, brother had prostate cancer.

Subject BC152, is a 13 years old girl from Bangladesh who underwent BRCA testing because of a strong family history with her mother, who was diagnosed with carcinoma breast at the age of 33 years. The proband was found to be positive for a novel BRCA2 variant c.6661A>G (p.Asn2221Asp). The identified BRCA2 variant is not reported in GnomAD exomes or 1000genome databases. However, the mutation status of other family members is unknown and based on ACMG 2015 guidelines this variant has been classified as a variant with uncertain significance.

These results were further validated with sanger sequencing and NGS (Supplementary material Figure 2a, 2b, 3 and 4).

Recurrent Mutations

We identified six recurrent variants in the BRCA1 gene-BRCA1(NM_007300.4):c.68_69del, c.3995G>T, c.5098del, c.5429del, c.55C>T and c.4738G>C and three recurrent variants in the BRCA2 gene- BRCA2(NM_000059.4):c.2593G>C, c.4570_4573del and c.5604_5605del. The recurrent variants with ACMG classification have been listed in Table 4.

The BRCA1(NM_007300.4):c.68_69delAG on the BRCA1 gene, also known as the 185delAG, was identified 29 times out of 115 deleterious BRCA1/2 variants (25.2%). Of these 29 patients, 11 were diagnosed with carcinoma breast and 13 with ovary. Interestingly, out of 67 patients screened due to strong family history, five were positive for 185delAG mutation. This mutation accounted for 25.2% of all pathogenic variants in our cohort.

There were four patients (BC086, BC094, BC116 and BC757 ) in our cohort positive for BRCA1(NM_007300.4):c.3995G>T (p.Gly1332Val) variant in exon 10 of BRCA1 gene; BC086 diagnosed with breast cancer at the age of 26 whose mother had breast cancer and BC094 and BC757 had ovarian cancer at the age of 68 and 70 respectively. BC094with subsequent recurrence at 70 years of age and BC757 had a strong family history of cancers. This missense variant results in the substitution of amino acid Glycine to Valine at position 1332. The majority of the in-silico predictions suggest a damaging outcome and also the minor allele frequency (0.0000653) of 1 in 15306 (in South Asians) and only two heterozygote alleles have been reported in gnomAD (13,14). This variant has not been previously described in affected individuals(15,16). However, in this study, four positive alleles among a cohort of 772 patients suggest that the variant may have a likely pathogenic role in Breast and Ovarian cancer and require further investigations.

There were two patients (BC012 and BC175) positive for BRCA1(NM_007300.4):c.5098del (p.Leu1700Ter) nonsense mutation, which results in the deletion of a single nucleotide from exon 16 causing a translational premature stop signal at codon 1700. Both these patients were diagnosed with early onset (age 36 and 37 respectively) triple negative breast cancer with a strong family history of cancer.

Two patients (BC169 and BC001) were positive for a pathogenic BRCA1(NM_007300.4):c.5429del (p.Ala1810ValfsTer4) mutation in exon 22. BC169 was diagnosed with epithelial ovarian cancer at 44 while BC001 had triple negative breast cancer at 37 . No family history of breast, ovarian, pancreatic or prostate cancer is known in these families.

The pathogenic variant BRCA1(NM_007300.4):c.55C>T (p.Gln19Ter) in exon 2 was identified in two patients (BC023 and BC328) diagnosed with triple negative breast cancer at ages 52 and 37 respectively. The patient BC023 had recurrent breast cancer after ten years and her daughter expired due to bilateral breast cancer. The patient BC328, as per the records has no family history.

Two patients (BC120 & BC172) tested positive for BRCA2(NM_000059.4):c.2593G>C (Glu865Gln) variant in exon 11 with uncertain significance. BC120 (age 38 years) was screened due to a family history of breast cancer, whereas BC172 was diagnosed with estrogen positive breast cancer at 37 years, however, the family history (as per the record) is not known.

The pathogenic variant BRCA1:c.4738G>C was identified in two patients (Subject ID: BC569 and BC587). The patient BC587 was diagnosed with ovarian cancer at the age of 62 years and had a strong family history of cancer. BC569 also had ovarian cancer at 49 and had a family history of cancers.

Another recurrent variant in our cohort was BRCA2:c.4570_4573delTTTC (p.Phe1524IlefsTer18), identified in patients with subject ID BC502 and BC579. BC 502 was diagnosed with Ca ovary at 33 and has no family history known to date while the BC579 has a family history of cancer and is diagnosed with Ca ovary at 60.

Two patients, BC484 and BC571 were positive for a pathogenic BRCA2:c.5604_5605del (p.Asp1868GlufsTer4) frameshift mutation leading to protein truncation. The patient BC484 was diagnosed with Ca ovary at 54 with a history of cancer in the family and BC571 had estrogen-positive breast cancer at 31 with a family history of cancer.

Factors predicting deleterious BRCA1/2 Variants and 185delAG mutation:

We performed a univariate analysis of the factors predictive of the presence of BRCA1 or BRCA2 mutations. Triple negative breast cancer, hormone positive breast cancer, and a family history of BOPP malignancy were significantly associated with higher risks of detecting BRCA1 or BRCA2 mutations. After multivariate binary logistic regression, only a positive family history of BOPP malignancy remained significantly associated with being BRCA mutation positive (Out of 181 positive patients, 58 had a family history of BOPP cancers and 123 did not have any family history (OR 2.15 [95% C.I 1.46 to 3.16] p= <0.0001).

In a logistic regression model for factors predictive of a 185delAG variant, being of South Indian origin was the only significant factor identified (OR 4.93 [95% C.I 95%=1.86 to 13.04, p=0.0013). Out of 29 delAG positive patients, 24 are from south India and 5 are from northern India.

Characteristics of patients positive for variants

There were 111 patients identified with pathogenic/likely pathogenic mutations and the mean age of these patients was (41.2 ±14) years. Out of these 111 patients, 51 were diagnosed with breast cancer while 54 with ovarian cancer, and six were considered for genetic screening due to a strong family history of BOPP cancer.

Interestingly, 89/111 (80%) pathogenic/likely pathogenic mutations were found in the BRCA1 gene and remaining 22/111 (20%) in BRCA2 gene (Table 4). Twenty-seven patients were positive for mutations in exon 2 (3 missense, 22 185delAG mutation and other dels) and 37 patients with mutations in exon 10 of BRCA1 gene in the study cohort. Therefore, BRCA1 exons 2 and 10 can be considered as hotspot exons and by screening these exons alone can yield, identification of 57% of pathogenic/likely pathogenic mutations.

There were 70 patients identified to have a VUS, requiring further investigation to understand the role of these variants in HBOC. The clinical features of patients positive for VUS variants varied with age and family history (Supplementary Material Table 2).

BRCA1 and BRCA2 are tumor suppressor genes well-known for their causative role in HBOC when mutated (17,18). Although other genes such as PALB2, RAD51, BARD1, ATM, CHEK2, and CDH1 are also implicated in patients with breast cancer, the strongest association remains with mutations in BRCA1 and BRCA2(19). The prevalence of BRCA1 and BRCA2 mutations varies in different populations (20,21). Decision making for the test is important for patient-centred medical care and risk-reducing treatments (22,23).

NGS based testing and its advantages

In the current study, we have identified 181 BRCA variants resulting in a positivity rate of 23.4%. Other Indian studies have found mutation frequencies varying from 2.9% to 28% depending on the technique used(4,24). NGS-based investigations have reported a diagnostic rate ranging from 25-30%, similar to that seen in our study[5, 15, 25, 26].

Indians have a higher incidence of BRCA1 than BRCA2 compared to other Asian populations

In this study, we identified a higher proportion of BRCA1 mutations (15.2%) than BRCA2 mutations (8.2%). Previous studies have demonstrated that India and Pakistan have a higher proportion of BRCA1 mutations than BRCA2 (4,5,15,24), which contrasts with the pattern seen in other Asian populations (26). Since BRCA1 is known to have an earlier onset of HBOC, this may partially explain the high incidence of young onset breast cancer in the subcontinent (27).

185delAG is a recurrent mutation in South India

There were 29 patients tested positive for 185delAG using NGS or allele-specific PCR (Figure 2). Twenty four out of these 29 patients were from South India, leading to an estimated odds ratio of 4.93 (95% CI=1.86 to 13.04, p=0.0013) for a South Indian HBOC patient having this mutation compared to those from other parts of India. Also, this mutation occurred frequently and was identified 29 times accounting for 32.6% of all the pathogenic BRCA1 variants in our cohort.

The BRCA1(NM_007300.4):c.68_69del otherwise known as the 185delAG is a known hotspot mutation described as a founder mutation in the Ashkenazi Jew population (28). Previous studies have demonstrated a prevalence ranging from 10.6% to 16.4% among the South Indian population (4,29). A study from eastern India of 231 HBOC patients did not find any patients with this mutation (30) further corroborating the regional specificity of this finding.

The ASPCR established in this study has shown to be highly specific and sensitive in identifying the 185delAG. The sensitivity and specificity of this assay was validated using Sanger and NGS (Figure 3a, 3b). Using ASPCR, we have additionally screened 149 patients (100 South Indians and 49 Non-South Indians) for 185delAG. Seven subjects were found to be positive for 185delAG. Overall, 29/772 (NGS +ASPCR) patients were positive for this mutation, and 24 were from South India. These results further corroborate the need for testing BRCA1 185delAG among the high-risk individuals from South India. These findings strongly argue that 185delAG may have an independent founder effect in the South Indian population.

NGS based testing would enable comprehensive testing strategies and also aid in identifying and establishing population specific hotspot mutations screening strategies for mass screening. Therefore, based on our findings, we recommend patients from South India with a lack of access and financial constraints can be offered screening for the 185delAG mutation as a first line testing in a clinical setting.

What factors affected chances of pathogenic/VUS BRCA variant detection

In this study, patient selection was predominantly based on the NCCN guidelines, which identify those at the highest risk of hereditary cancer syndrome. However, an analysis of these clinco-pathological criteria revealed that only a family history of BOPP malignancies was significantly predictive of a deleterious BRCA mutation. This is similar to other Indian studies, which also recognize the age of diagnosis and family history as the most predictive factors in our population(15). Studies from other countries have questioned the applicability of NCCN guideline-based testing in their populations(31). Therefore, there is a need for large cohort studies in India to help identify additional predictive factors relevant to our population.

Our study limitation was that it was hospital-based and included an ‘enriched’ population of cancer patients who fulfilled ‘high-risk’ criteria for testing. This is, therefore, not reflective of the prevalence of BRCA mutations in the general population. Also, the patients were recruited retrospectively with clinical information obtained from the medical records therefore, all the inherent drawbacks of a retrospective cohort study are applicable. Another limitation includes the lack of family screening for patients who tested positive for novel variants.

The strengths of our study include establishment and utilization of in-house NGS based testing which has shown to be a cost-effective, sensitive and specific method for detecting BRCA1 and BRCA2 variants. Additionally, BRCA1 and BRCA2 genes were evaluated as a single assay, and the targeted NGS panel has room for expansion. As 185delAG mutation in the BRCA1 gene is a mutational hotspot in the South Indian population with a possible founder effect, the development of the 185delAG screening tool for South Indian patients has also shown to be a rapid, cost-effective and sensitive method for initial screening of suspected patients. A positive test will preclude the need for further evaluation of the entire BRCA1 and BRCA2 genes however a negative test will mandate an evaluation of the targeted panel of genes.

Data Availability: The datasets generated during and/or analysed during the current study are available with corresponding author and will be available on request.

Acknowledgement

We would like to thank all our patients and their families for their consent and participation in the study. We acknowledge Mrs. Manika Varshini and Ms. Judith Evangeline for their involvement in the initial stages of the study.

Author Contributions:

Study conception and design: Dr. Aaron Chapla and Dr. Ashish Singh. NGS and ASPCR design and standardization: Dr. Aaron Chapla, Mrs. Deny Varghese, Mr. Parthiban R. Material preparation, data collection, analysis: Dr. Ajoy Oommen John, Dr. Ashish Singh, Dr. Anjana Joel, Ms. Pratibha Yadav, Dr. Divya Bala Thumaty, Dr. Fibi Ninan K, Dr. Josh Thomas Georgy, Dr. Anish Jacob Cherian, Dr. Shawn Thomas, Dr. Anitha Thomas, Dr. Vinotha Thomas, Dr. Abraham Peedicayil, Ms. Lavanya Ravichandran, Mr. Jabasteen Johnson, Dr. Bijesh Yadav, Dr. Patricia S, Dr. Selvamani B, Dr. Deepak Abraham, Dr. MJ Paul, Dr. Raju Titus Chacko, Dr. Nihal Thomas, Dr. Ashish Singh, Dr. Aaron Chapla. First draft of the manuscript was written by Dr. Ajoy Oommen John, Dr. Aaron Chapla, Dr. Ashish Singh and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Ethical Approval:

This study was approved by the institutional review board IRB no 13205 (retro) dated 22.07.2020.

Competing Interests: The authors have no competing interest.

Consent for participation: Informed consent was obtained from all individual participants included in the study.

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Alemar B, Gregório C, Herzog J, Bittar CM, Netto CBO, Artigalas O, et al. BRCA1 and BRCA2 mutational profile and prevalence in hereditary breast and ovarian cancer (HBOC) probands from Southern Brazil: Are international testing criteria appropriate for this specific population? PLOS ONE. 2017 Nov 21;12(11):e0187630.

Table 1: Baseline disease characteristics:

	Breast Cancer (N=464)	Ovarian Cancer (N=241)	Screening (N=67)
Age in years (mean±SD)	42.1±10	48.4±11	NA
Non NCCN guideline compliant testing (%)	27 (9.8%)	6 (3.9%)	8 (27.5%)
ER and HER2 Positive breast cancer (%)	36 (7.8%)	-	-
ER Positive breast cancer (%)	145 (31.3%)
Her2 Positive breast cancer (%)	28 (6%)
Triple-negative breast cancer (%)	227 (49%)

Table 2: Diagnostic yield based on the family history of cancers, age at onset and type of cancer.

Cancer type versus family history	Diagnostic yield			Overall diagnostic yield
	With Family history		Without Family history	Overall diagnostic yield
All cases	(34.9%) 58/166		(20.4%) 123/604	(23.44%) 181/772
Hormone receptor positive breast cancer	(27.8%)10/36		(13.1%) 16/122	(16.5%)26/158
Triple negative breast cancer	(34.3%) 12/35		(22.2%) 44/198	(23%)56/233
Ovarian cancer	(50%) 26/52		(25.4%) 48/189	(30.7%) 74/241
Age at diagnosis versus family history	Diagnostic yield			Overall diagnostic yield
	With family history		Without family history	Overall diagnostic yield
<40 years 40-50 years >50 years	(36.5%) 15/41		(21.1%) 42/199	(23.7%)57/240
	(40%) 20/50		(21.7%) 41/189	(25.4%) 61/240
	(34.7%) 17/49		(19.9%) 35/176	(23.1%) 52/225
Cancer type versus age at diagnosis	Diagnostic yield			Overall diagnostic yield
	<40 years	40-50 years	>50 years
TOTAL CASES	(23.75%) 57/240	(25.4%) 61/240	(23.1%) 52/225	(23.44%) 181/772
Hormone Receptor positive breast cancer	(19.3%) 11/57	(14.9%) 10/67	(14.7%) 5/34	(16.5%) 26/158
Hormone Receptor negative HER2 positive breast cancer	(23%) 3/13	(28.5%) 2/7	(11.1%) 1/9	(20.7%) 6/29
Triple negative breast cancer	(24.8%) 25/101	(23.7%) 18/76	(23.6%) 13/55	(24%) 56/233
Ovarian cancer	(29.6%) 16/54	(38%) 27/71	(26.7%) 31/116	(30.7%) 74/241

Table3: List of pathogenic and likely pathogenic variants identified in BRCA1 & BRCA2 genes.

S.No	Gene Variant	Protein Change	ACMG Classification	Frequency
1	BRCA1(NM_007300.4):c.68_69del	(p.Glu23ValfsTer17)	P	14
2	BRCA1(NM_007300.4):c.5429del	(p.Ala1810ValfsTer4)	P	2
3	BRCA1(NM_007300.4):c.5217G>C	(p.Trp1739Cys)	P	1
4	BRCA2(NM_000059.4):c.9100C>T	(p.Gln3034Ter)	P	1
5	BRCA1(NM_007300.4):c.5098del	(p.Leu1700Ter)	P	2
6	BRCA1(NM_007300.4):c.55C>T	(p.Gln19Ter)	P	2
7	BRCA2(NM_000059.4):c.9276T>G	(p.Tyr3092Ter)	P	1
8	BRCA1(NM_007300.4):c.5278G>T	(p.Asp1760Tyr)	LP	1
9	BRCA2(NM_000059.4):c.3187C>T	(p.Gln1063Ter)	P	1
10	BRCA1(NM_007300.4):c.5334_5335del	(p.Asp1778GlufsTer72)	LP*	2
11	BRCA1(NM_007300.4):c.34C>T	(p.Gln12Ter)	P	1
12	BRCA2(NM_000059.4):c.5621_5628del	(p.Ile1874LysfsTer5)	P	1
13	BRCA2(NM_000059.4):c.92G>A	(p.Trp31Ter)	P	1
14	BRCA1(NM_007294.4):c.4389C>A	(p.Tyr1463Ter)	P	1
15	BRCA1(NM_007300.4):c.5314C>T	(p.Arg1772Ter)	P	1
16	BRCA1(NM_007294.4):c.1450G>T	(p.Gly484Ter)	P	1
17	BRCA1(NM_007300.4):c.1327A>T	(p.Lys443Ter)	P	1
18	BRCA1(NM_007300.4):c.5367del	(p.Tyr1790MetfsTer24)	P	1
19	BRCA1(NM_007300.4):c.315T>G	(p.Tyr105Ter)	LP	1
20	BRCA2(NM_000059.4):c.4003G>T	(p.Glu1335Ter)	P	1
21	BRCA1(NM_007300.4):c.1188del	(p.Asp396GlufsTer14)	P	2
22	BRCA2(NM_000059.4):c.9222_9223insTT	(p.Ile3075LeufsTer2)	P	1
23	BRCA2(NM_000059.4):c.5073dup	(p.Trp1692MetfsTer3)	LP	1
24	BRCA1(NM_007300.4):c.220C>T	(p.Gln74Ter)	P	1
25	BRCA1(NM_007300.4):c.4780del	(p.Asp1594MetfsTer28)	P	1
26	BRCA1(NM_007300.4):c.5135C>T	(p.Thr1712Ile)	LP	1
27	BRCA1(NM_007300.4):c.5572T>C	(p.Trp1858Arg)	LP	1
28	BRCA1(NM_007300.4):c.66dup	(p.Glu23ArgfsTer18)	P	1
29	BRCA1(NM_007294.4):c.5363G>T	(p.Gly1788Val)	LP	1
30	BRCA1(NM_007300.4):c.1360del	(p.Ser454ValfsTer21)	P	1
31	BRCA1(NM_007300.4):c.850del	(p.Gln284SerfsTer14)	P	1
32	BRCA1(NM_007300.4):c.5599C>T	(p.Gln1867Ter)	P	1
33	BRCA2(NM_000059.4):c.4415_4418del	(p.Lys1472ThrfsTer6)	P	1
34	BRCA1(NM_007300.4):c.53T>G	(p.Met18Arg)	LP	1
35	BRCA1(NM_007300.4):c.1749_1755del	(p.Lys583AsnfsTer3)	P	1
36	BRCA2(NM_000059.4):c.4478_4481del	(p.Glu1493ValfsTer10)	P	1
37	BRCA1(NM_007300.4):c.1369G>T	(p.Glu457Ter)	LP*	1
38	BRCA1(NM_007300.4):c.3995G>T	p.Gly1332Val	LP	4
39	BRCA1(NM_007294.4):c.5251C>T	(p.Arg1751Ter)	P	1
40	BRCA1(NM_007294.4):c.5509T>C	(p.Trp1837Arg)	P	1
41	BRCA2(NM_000059.4):c.4074dup	p.Thr1359TyrfsTer2	LP	1
42	BRCA1(NM_007300.4):c.5090T>G	p.Leu1697Ser	P	1
43	BRCA1(NM_007300.4):c.1450G>T	p.Gly484Ter	P	1
44	BRCA1(NM_007300.4):c.4717del	p.Tyr1573ThrfsTer7	P	1
45	BRCA2(NM_000059.4):c.5604_5605del	p.Asp1868GlufsTer4	P	2
46	BRCA2(NM_000059.4):c.7219_7220del	p.Val2407SerfsTer4	P	1
47	BRCA1(NM_007300.4):c.547+1G>T		P	1
48	BRCA1(NM_007300.4):c.5469+2T>G		P	1
49	BRCA2(NM_000059.4):c.4570_4573del	p.Phe1524IlefsTer18	P	2
50	BRCA1(NM_007300.4):c.3607C>T	p.Arg1203Ter	P	1
51	BRCA1(NM_007300.4):c.4738G>C	p.Glu1580Gln	P	1
52	BRCA1(NM_007300.4):c.4681del	p.Glu1561LysfsTer8		1
53	BRCA2(NM_000059.4):c.521del	p.Arg174LeufsTer11	P	1
54	BRCA1(NM_007300.4):c.1116G>A	p.Trp372Ter	P	1
55	BRCA1(NM_007300.4):c.5497_5512del	p.Pro1833ArgfsTer17	P	1
56	BRCA1(NM_007300.4):c.4433C>G	p.Ser1478Ter	P	1
57	BRCA1(NM_007300.4):c.2215_2216insT	p.Lys739IlefsTer4	P	1
58	BRCA1(NM_007300.4):c.3008_3009del	p.Phe1003Ter	P	1
59	BRCA2(NM_000059.4):c.6645C>G	p.Tyr2215Ter	P	1
60	BRCA1(NM_007300.4):c.4185+1G>A		P	1
61	BRCA1(NM_007300.4):c.2362del	p.Val788LeufsTer4	P	1
62	BRCA2(NM_000059.4):c.9106C>T	p.Gln3036Ter	P	1
63	BRCA1(NM_007300.4):c.3328_3329del	p.Lys1110AlafsTer4	P	1
64	BRCA1(NM_007300.4):c.3358_3359del	p.Val1120Ter	P	1
65	BRCA1(NM_007300.4):c.1631_1632del	p.Gln544ArgfsTer6	P	1
66	BRCA1(NM_007300.4):c.3880_3881insT	p.Ser1294MetfsTer9	P	1
67	BRCA1(NM_007300.4):c.2311_2317del	p.Pro773LeufsTer17	P	1
68	BRCA1(NM_007300.4):c.3706_3713del	p.Asn1236PhefsTer5	P	1
69	BRCA1(NM_007300.4):c.4672C>T	p.Gln1558Ter	P	1
70	BRCA2(NM_000059.4):c.8487+1G>A		P	1
71	BRCA1(NM_007300.4):c.4752C>A	p.Tyr1584Ter	P	1
72	BRCA1(NM_007300.4):c.5507G>A	p.Trp1836Ter	P	1
73	BRCA2(NM_000059.4):c.7008-2A>G		P	1
74	BRCA1(NM_007300.4):c.2214dup	p.Lys739Ter	P	1
75	BRCA1(NM_007300.4):c.726del	p.Ser242ArgfsTer5	P	1
76	BRCA1(NM_007300.4):c.2217del	p.Val740CysfsTer13	P	1
77	BRCA1(NM_007300.4):c.1881_1884del	p.Ser628GlufsTer3	P	1

*The above variants have been submitted to ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/submitters/508251/)

P:Pathogenic, LP: Likely Pathogenic

Table 4: Distribution of BRCA1 and BRCA2 variants in our cohort

Diagnosis	No. of patients with BRCA1 variants	No. of patients with BRCA2 variants
Ca ovary	50 (27.6%)	24 (13.3%)
ER+ Ca breast	12 (6.6%)	13 (7.2%)
HER2+ Ca breast	3 (1.7%)	3 (1.7%)
Triple Negative Ca breast	40 (22.1%)	15 (8.3%)
Screened due to family history	8 (4.4%)	3 (1.7%)

Table 4: BRCA1 and BRCA2 mutations in patients with different types of cancer. *ER: Estrogen receptor, HER2= Human epidermal factor, TNBC= Triple Negative Breast Cancer

There is no duality of interest

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BRCA1 and BRCA2 gene mutation spectrum and high frequency of BRCA1 185delAG among breast and ovarian cancer patients from Southern India.

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