Detection of Inherited Mutations in Brazilian Breast Cancer Patients Using Multi-Gene panel Testing


 Genetic diversity of germline variants in breast cancer (BC) predisposition genes, is unexplored in miscegenated people, such as Latin American populations. We evaluated 1,662 Brazilian BC patients, who underwent hereditary multi-gene panel testing (20–38 cancer susceptibility genes), to determine the spectrum and prevalence of (likely) pathogenic variants (P/LP) and variants of uncertain significance (VUS). In total, 161 (9.7%) participants carried germline P/LP variants in BRCA1/2 and 162 (9.7%) in other cancer predisposition genes. Overall, 341 distinctive P/LP variants were identified in 22 genes, including BRCA1(28%), BRCA2(19%), TP53(11%), MUTYH heterozygous (10%), ATM(9%), CHEK2(6%), and PALB2(5%). The Brazilian variant TP53 R337H (c.1010G > A, p.Arg337His), detected in 1.6% of BC patients and 0.09% of reference controls (RC), was strongly associated with odds of disease (OR = 17.67; 95%CI:9.21–34.76; p < 0.001). Heterozygous MUTYH c.1187G > A and MUTYH c.536A > G, detected in 0.78% (0.90% RC) and 0.48% (0.40% RC) of the patients, respectively, were not associated with the odds of BC, the former with OR = 0.87 (95%CI:0.49–1.53; p = 0.63) and the latter with OR = 1.20 (95%CI:0.58–2.49; p = 0.63). Besides, 766 individuals (46.1%) had 1 or more VUS. Concluding, the use of multi-gene panel testing doubled the identification of mutation carriers in Brazilian BC patients. Special attention should be given to TP53 mutations.


Introduction
Breast cancer (BC) is the most common cancer in women worldwide. In Brazil, an average of 66,280 women are diagnosed with carcinoma of the breast every year, accounting for 29.7% of all cancers in the female population 1 . Inherited pathogenic variants in highly penetrant predisposition genes are thought to be involved in about 10% of BC cases. Among the hereditary forms, the most frequent events are germline pathogenic variants in BRCA1/2 genes which predispose to hereditary breast and ovarian cancer syndrome (HBOC). The prevalence and spectrum of BRCA1/2 pathogenic variants vary among different populations and are responsible for only approximately 25-50% of the familial risk of BC [2][3][4] . As DNA sequencing technologies evolved, other cancer susceptibility genes have been discovered, including high-penetrant genes such as TP53, CDH1, STK11, PTEN and PALB2 (> 5 fold cancer relative risk), moderate-penetrant genes such as CHEK2 and ATM (2-5 fold cancer relative risk), and a number of common low-penetrant BC susceptibility loci identi ed through genome-wide association studies (1-2 fold cancer relative risk) [5][6][7] . The mutational spectrum of germline mutations in BC predisposition genes have been reported in single populations, with the majority of reports focused on Caucasians from Europe and North America. The population from Southern Hemisphere countries, except for Australia, are underrepresented and understudied in cancer genetic epidemiology research 2 .
The Brazilian population has unique ethnic characteristics. People miscegenation is a universal phenomenon, due to globalization and large waves of immigration. Brazil is considered an ethnic "melting pot", re ecting an admixture of European, Indigenous and Sub-Saharan African people, in addition to immigrants from a large number of European, Asian and Middle Eastern countries. Hence, Brazilian people offer a unique opportunity to advance the understanding of cancer genetic features in a miscegenated population 8 .
In Brazil, the majority of the inherited BC studies focused on the analyses of BRCA1/2 as well as TP53, given the relatively high population frequency of the TP53 R337H (also known as, c.1010G > A, p.Arg337His) founder mutation in people from the South and Southeast regions of Brazil 9 . However, the likelihood of carrying pathogenic mutation in other BC susceptibility genes among BRCA1/2 and TP53-negative patients is largely unexplored.
Recent advances in next generation sequencing (NGS) technology has reduced the cost of massively parallel sequencing, provided to physicians and patients the option of sequencing multiple genes simultaneously and broadened our understanding of the genetic etiology of inherited cancers. Multi-gene panel testing has proved useful as a diagnostic tool for disorders where similar phenotypes can be in uenced by multiple genes such as hereditary predisposition to BC, uncovering potentially actionable ndings that may be missed by traditional testing paradigms. Several laboratories have released commercial multi-gene panel testing ranging from six to > 100 genes 10 . Panels are cheaper, faster and increase the yield of genetic ndings, more than doubling the mutation detection rate in BRCA1/2-negative patients with suspected HBOC [11][12][13][14][15][16][17][18] . However, nding a mutation in a gene where the cancer risks and/or management strategies are not known, as well as the identi cation of higher numbers of variants of uncertain signi cance (VUS), can make the results cumbersome and challenging for a physician to interpret and guide treatment 10 .
These panels have been widely available in Brazil within the past 7 years, but no study has yet assessed the prevalence and mutational spectrum of germline variants in BC susceptibility genes other than BRCA1/2 and TP53 in a large cohort of individuals with BC, who were referred for genetic evaluation. Given the rapidly uptake of multi-gene panel testing in clinical practice, these data are urgently needed to inform genetic counseling. In this study, we report the results from 1,662 consecutive individuals with a history of BC who were referred for multi-gene panel testing.

Study population and prevalence of P and LP variants
This study involved a nationwide sample of 1,662 consecutive BC patients who underwent germline genetic testing with a multi-gene cancer panel between 2015 and 2017. Age at BC diagnosis, geographical region where the test was collected and prevalence of P/LP variants are listed in Table 1  Mean age at BC diagnosis was 42.9 ± 11.2 years. Age at diagnosis was signi cantly lower for BRCA1 mutation carriers (38.2 ± 9.3 years) than in the whole cohort (p < 0.001). Age at diagnosis was not associated with the presence of mutations in any other gene when compared to the whole cohort.
The majority of the tests were from patients who inhabited the Southeast region of Brazil (51.9%), which is the most populated in the country. Patients from all other regions were also well represented, except for patients from the North region (1.6%), which is the least densely populated, covered mostly by the Amazon Rainforest.
Both allelic heterogeneity and founder mutations played a role in inherited BC. Allelic heterogeneity among the patients was re ected in the appearance of 179 distinct mutations in 22 genes (Supplementary Table 3). Although the mutational pro le was heterogeneous, recurrent mutations (detected in three or more individuals) were found in 8 genes: APC, ATM, BARD1, BRCA1, BRCA2, CHEK2, MUTYH, and TP53 (Supplementary Table 1

Discussion
This is the largest nationwide cohort of Brazilian BC patients who underwent NGS muti-gene panel testing reported to date.
The most commonly mutated genes were BRCA1/2, which were identi ed in 10% of the entire cohort and accounted for almost 50% of all P/LP germline variants identi ed. In accordance with previous research from different countries, the use of a multi-gene panel test doubled the yield of P/LP variants detected, as well as increased in 12 times the chance of nding a VUS. Most signi cantly, this study differs from the others because it highlights the important contribution of Li-Fraumeni syndrome (LFS) to inherited BC burden in Brazil, due to the founder mutation TP53 R337H. It is worth emphasizing that the number of patients carrying this mutation is similar to the number of patients with BRCA1 c.5266dupC, which is the most prevalent BRCA1 pathogenic variant in our study.
The estimated frequency in the general population of P/LP BRCA1/2 mutations is 1:800-1:1000 per gene 21 [39][40][41][42][43] . The TP53 R337H variant confers a lifetime cancer risk by age 60 years of 80% in females and 47% in males. In comparison, in classic LFS, those with mutation located in typical DNAbinding domain, the cancer risk is 90% in women and 73% in men 44 . The reasons concerning the reduced penetrance of this variant is still controversial and usually associated with its location in the gene and biochemistry stability, which is pH dependent. A recent study showed that an extended haplotype cosegregating the TP53 R337H and XAF1 E134* alleles may lead to a more aggressive cancer phenotype than TP53 R337H alone, acting as a functional modi er by attenuating the transactivation of wild-type and hypomorphic TP53 variants, such as R337H. Carriers harboring the extended haplotype were more likely to be diagnosed with sarcomas and multiple tumors, nevertheless this association was not observed in BC patients. Further studies are needed to validate these ndings and evaluate their implications on genetic counseling and clinical management of TP53 R337H carriers 45 .
BC is the most common malignancy diagnosed in LFS. In Brazil, in high-risk BC patients, the prevalence of TP53 R337H ranged from 3.4-7.1% in the South/Southeast 46,47 and 0.9% in the Northeast region 30 (Table 3). In a cohort of 815 women affected by BC in southern Brazil who developed the disease before age 45 years, the prevalence of the TP53 R337H variant was 12.1% 47 . In our cohort, the prevalence of all P/LP TP53 variants was 2.2%, representing the third most commonly mutated gene among BC patients. The TP53 R337H variant was responsible for 70.3% all TP53 mutations identi ed. Removing the TP53 R337H variant, it becomes clear that the prevalence of other mutations in TP53 is low in the Brazilian population, following the same pattern as the worldwide prevalence.

Conclusion
In summary, the largest nationwide cohort of Brazilian BC patients who underwent NGS multi-gene panel testing identi ed that BRCA1/2 accounted for almost 50% of all P/LP germline variants. The third most frequently gene mutated was the TP53 due to the high number of TP53 R337H carriers in the South and Southeast region of Brazil. The high prevalence of this founder mutation has a signi cant impact in our screening and risk-reducing strategies. In addition, the use of multi-gene panel testing increased signi cantly the chance of nding a VUS.

Study population
Patients were eligible to participate if they were 18 years of age or older, had a personal diagnosis of BC, and were referred for a commercial multi-gene cancer panel testing at a College of American Pathology (CAP)-accredited laboratory (Mendelics Análise Genômica S.A., São Paulo, SP, Brazil). Informed consent for clinical testing and use of data for scienti c purposes was obtained and demographics/personal histories were collected from test requisition forms by the ordering physician. The protocol was approved by the Faculdade de Medicina da Universidade São Paulo (FMUSP) Institutional Review Board. All patient data were anonymized before analysis. The absence of personal information in medical requests were interpreted as "not provided".

Sequencing and variant interpretation
Genomic DNA was obtained from a buccal swab or peripheral blood sample using standard methods. DNA Sequencing was performed by high-end Illumina platforms (HiSeq 2500 and HiSeq 4000). Base calling was performed using original Illumina tools (bcl2fastq). Bioinformatics pipeline followed Broad Institute best practices (https://gatk.broadinstitute.org/hc/enus/sections/360007226651-Best-Practices-Work ows). After alignment to the reference genome GRCh37 / UCSC hg19, low quality and duplicate readings were removed, and variants (SNPs/indels) were detected with GATK HaplotypeCaller.
Enrichment and analysis concentrated on the coding sequences, anking intronic regions (± 20bp) and other speci c genomic regions previously identi ed that harbor causing variants. Promoters, untranslated regions and other non-coding regions were not analyzed. Exonic deletions and duplications (CNV) were identi ed using ExomeDepth, an R package that estimates the number of copies by comparing the reading depth for each target with the mean reading depth for the same target from samples genotyped from the same sequenced library. If a CNV was identi ed, multiplex ligation-dependent probe ampli cation (MLPA) assay was employed to con rm the nding. The variants were classi ed according to algorithms based on machine learning and described with a nomenclature compatible with the norms and guidelines of the American College of Medical Genetics and Genomics (ACMG)/Human Genome Variation Society (HGVS). Variants interpreted as pathogenic (P) and likely pathogenic (LP) were considered positive. All variants were evaluated by a medical geneticist or pathologist or certi ed oncologist.

Brazilian genomic database
Reference control data were obtained from the Mendelics Análise Genômica S.A. database, which contains panel and exome sequencing data from 18,919 Brazilian individuals, sequenced as part of various disease-speci c genetic tests, excluding samples from cancer cases. Associations of genes with BC were assessed by comparing frequency of mutations in genes of BC patients with those of reference controls.