Comprehensive analysis of germline mutations in northern Brazil: a panel of 16 genes for hereditary cancer-predisposing syndrome investigation

Next generation sequencing (NGS) has been a handy tool in clinical practice, mainly due to its efficiency and cost-effectiveness. It has been widely used in genetic diagnosis of several inherited diseases, and, in clinical oncology, it may enhance the discovery of new susceptibility genes and enable individualized care of cancer patients. In this context, we explored a pan-cancer panel in the investigation of germline variants in Brazilian patients presenting clinical criteria for hereditary cancer syndromes or familial history. Seventy-one individuals diagnosed or with familial history of hereditary cancer syndromes were submitted to custom pan-cancer panel including 16 high and moderate penetrance genes previously associated with hereditary cancer syndromes (APC, BRCA1, BRCA2, CDH1, CDKN2A, CHEK2, MSH2, MSH6, MUTYH, PTEN, RB1, RET, TP53, VHL, XPA and XPC). All pathogenic variants were validated by Sanger sequencing. We identified a total of eight pathogenic variants among 12 of 71 individuals (16.9%). Among the mutation-positive subjects, 50% were diagnosed with breast cancer and had mutations in BRCA1, CDH1 and MUTYH. Notably, 33.3% were individuals diagnosed with polyposis or who had family cases and harbored pathogenic mutations in APC and MUTYH. The remaining individuals (16.7%) were gastric cancer patients with pathogenic variants in CDH1 and MSH2. Overall, 54 (76.05%) individuals presented at least one variant uncertain significance (VUS), totalizing 81 VUS. Of these, seven were predicted to have disease-causing potential. Overall, analysis of all these genes in NGS-panel allowed the identification not only of pathogenic variants related to hereditary cancer syndromes but also of some VUS that need further clinical and molecular investigations. The results obtained in this study had a significant impact on patients and their relatives since it allowed genetic counselling and personalized management decisions.

(Continued from previous page) Conclusion: Overall, analysis of all these genes in NGS-panel allowed the identification not only of pathogenic variants related to hereditary cancer syndromes but also of some VUS that need further clinical and molecular investigations. The results obtained in this study had a significant impact on patients and their relatives since it allowed genetic counselling and personalized management decisions.
Keywords: Hereditary cancer, Next generation sequencing, Pan-cancer panel, Pathogenic variant

Background
Hereditary cancer syndrome is a genetic predisposition to several types of cancer caused by pathogenic germline variants in one or more genes [1]. It corresponds to 5-10% of all cancers and have peculiar clinical aspects, such as the onset at an early age, high lifetime risk for multiple primary tumors, and cancer occurring in successive generations of the family [2,3].
Most hereditary cancer syndromes display autosomal dominant inheritance involving genes that are mainly controlling cell cycle or DNA repair [4]. For example, mutations in BRCA1 and BRCA2 confer 40-80% lifetime risk of developing breast cancer, and 11-50% of developing ovarian cancer [5]; germline variants in at least one of the DNA mismatch repair genes (eg. MSH2, MLH1, MSH6, and PMS1) affect in up to 80% of the Lynch syndrome individuals [6]; and mutations in CDH1 gene are detected in 30 to 40% of families with hereditary diffuse gastric cancer (HDGC) [7].
For a long time, single-gene analyses were used for detection of the genetic cause of hereditary cancers. However, the recent advances in DNA sequencing techniques have allowed the application of next generation sequencing (NGS) in clinical practice, including in genetic diagnosis of inheritance disease. In addition to its rapid, efficient and cost-effective approach for testing multiple cancer susceptibility genes, it may enhance the discovery of new susceptibility genes and enable individualized care of cancer patients [8,9].

Pathogenic variants validation
Pathogenic variants presence were validated by Sanger sequencing in ABI 3130 (Applied Biosystems) as follows. The location of interest was amplified by PCR using primers shown in Supplementary Table S1. Sanger Sequencing was carried out with 1 μL of purified PCR product of each exon, 0.5 μL of the forward/ reverse specific primer, 0.5 μL of ABI Prism Bid Dye Terminator Cycle Sequencing v3.1 Kit (Applied Biosystems, USA), 3 μL of SaveMoney buffer, and 10 μL of water to a final volume of 15 μL. The thermocycling reaction proceeded as follows: 96°C for 1 min, followed by 35 cycles of 96°C for 15 s, 50°C for 15 s and 60°C for 4 min. The sequence information was interpreted by ABI Analysis Software™. The electropherograms were analyzed using the Chromas 2.6.6 software and compared with the reference sequence obtained from NCBI.
Almost all probands with positive results for pathogenic variants had only a single mutation (n = 11), with the exception of one proband affected with colonic  (Fig. 1). Among all individuals tested, 54 (76.05%) presented at least one VUS and 14 individuals had a negative result for both pathogenic variants and VUS.
Most VUS were in intronic, intragenic or untranslated regions (UTR) and, therefore, it was not possible to predict their pathogenicity (Supplementary Table S2). Only 12 variants were submitted to the prediction (Supplementary Table S3), among these seven were predicted to be deleterious by at least five predictors (six missense variants and one structural interaction variant), suggesting their disease-causing potential.

Family screening
The pan-cancer panel allowed us to identify familial mutation in affected patients, directing screening on family members and, consequently, to provide a personalized genetic counselling and management decisions. Examples of families that were benefited from the pan-cancer panel results are depicted in Fig. 2 and Fig. 3.
Family A (Fig. 2) proband was submitted to genetic testing due to colonic polyposis diagnosis. Two pathogenic variants in MUTYH gene were identified, c.1147delC and c.1187G > A. Nine unaffected family members were investigatedof these, three men and two women presented c.1147delC, two women presented c.1187G > A, while one man was compound heterozygous for these variants. Family B (Fig. 3) proband was submitted to genetic testing since they met the clinical criteria for HBOC. This individual presented BRCA1 c.1961delA. Sixteen family members were tested to this variant, being seven mutation-positive (four men and three women).
Therefore, as pathogenic variants were identified in unaffected individuals with an affected family member, it allowed the choosing of an appropriate surveillance approach and management decisions for the mutationnegative subjects.

Discussion
Advances in NGS technologies and their costeffectiveness have made multigene panels a useful diagnostic tool in oncology clinic, particularly for offering an increase in mutation detection rate, which benefits individuals without family history information or atypical phenotype [30]. In the present study, a custom pancancer panel was developed and applied for the detection of hereditary cancer-related pathogenic mutations.
Variant analysis revealed the presence of at least one pathogenic variant in 16.9% of the analyzed individuals.  Most of the individuals were breast cancer patients, being BRCA1 mutations responsible for 50% of the pathogenic variants in these patients. In the example of a family which was benefited from the multigene panel analysis, BRCA1 c.1961delA was also reported in men, demonstrating the importance of BRCA1 screening not only in women, since men with pathogenic variants in this gene have a slightly higher risk for prostate cancer and male breast cancer [31,32]. Germline mutations in BRCA1 and BRCA2 are very relevant to the development of HBOC, however, they are responsible for only 15-25% of such cases, which demonstrates the significant contribution of other genes [33,34]. In this study, the remaining pathogenic variants in breast cancer occurred in CDH1 and MUTYH.
CDH1 encodes E-cadherin, a protein responsible for calcium-dependent cell-to-cell adhesion. Germline mutations in this gene have been reported to cause hereditary diffuse gastric cancer (HDGC), a disorder that leads to an increased risk for diffuse gastric cancer and lobular breast cancer (LBC) [35]. However, substantial evidences have demonstrated an increased risk for LBC among CDH1 mutations carriers regarding their familial history for diffuse gastric cancer (DGC) [36]. In this study, CDH1 c.1003 C > T was reported in a breast cancer patient without familial history for DGC. MUTYH mutations are the cause of MUTYHassociated polyposis (MAP), a syndrome that predisposes to colorectal polyposis and colorectal cancer [37]. Here, MUTYH c.1187G > A was reported in two unrelated individuals with breast cancer. This variant is the most frequent of all MUTYH mutations in various populations [38], but the association between this variant and breast cancer remains controversial. In Northern Israel, monoallelic inheritance was associated with an elevated risk of breast cancer [39], while in Non-Hispanic individuals of European ancestry there was no positive association [40]. Thus, our preliminary finding in the Brazilian population reinforces the need for further studies and for genetic counseling for families that have this variant segregating.
Here, one individual with colonic polyposis showed compound heterozygous mutations in MUTYH (c.1147delC and c.1187G > A). Genetic counselling was essential to identify other biallelic carriers in the family, in addition to monoallelic carriers (potential increased breast cancer risk), since these individuals may have a 2fold increased risk for colorectal cancer and other cancers when compared to the general population [41].
Among the individuals with gastric cancer, one presented a mutation in CDH1 and, another, in MSH2. CDH1 alterations underlie HDGC, conferring risk of 56-70% higher [42]. Gastric cancer occurrence has a frequency of 1-6% in individuals with a Lynch syndromeassociated mutation, and this risk increases by 9% for those that present germline MSH2 mutations [43,44]. In this study, a mutation in this gene was identified in a gastric cancer patient (c.388_389delCA)until now, this variant had been reported only in Brazilian Lynch syndrome patients [45,46].
Multigene pan-cancer panels that include a variety of cancer types might contribute to better understanding an individual's risk for cancer, but it also raises new challenges in genetic counselling [9]. These challenges are even more complex in populations with a broad lack of information, such as the Brazilian one. Our population has several particular genomic features resulting from a high degree of admixture [47]. Most of the available genomic databases contain data from ancestry populations that do not entirely represent our genetic composition. It may explain the low number of pathogenic variants found in this study − it is possible that among the VUS and the "not described" variants there are some pathogenic variants that have not been associated with clinical data yet due to the lack of studies and validation [48]. Among the 81 VUS identified, seven were predicted with deleterious potential by at least five different predictors, reinforcing the need for functional studies that validate their pathogenicity.
Moreover, it is important to comprehend that most studies on cancer genetics were carried out in North America and Western Europe, thus reflecting the mutation burden in subjects of European ancestry. It is known that people of distinct ethnicities inherit a different pattern of pathogenic mutations from their ancestors. For instance, BRCA1 and BRCA2 show significant global variations according to contribution in regional cancer incidence and to mutation spectrum [49]. Only a minor part of the heritability of cancer risk has been elucidated so far, and further whole exome sequencing studies are needed to significantly increase the identification of hereditary cancer genes [8].
Despite the small sample size, which may not fully represent the Brazilian population, our results suggest the existence of a unique genetic background which needs to be more explored. Considering Brazil as a continental country submitted to different colonization processes, further studies should include samples from the different regions of the country.

Conclusions
In conclusion, our findings contributes to the description of pathogenic variants background in Northern Brazil, as well as demonstrated the potential of a multigene panel in identifying pathogenic variants in genes not typically tested in hereditary cancer specific cases. The results obtained in this study observed 81 VUS, seven predicted with deleterious potential, reinforcing the need to identify pathogenicity of these variants in our population. These results had a great impact on the patients and their relatives since it allowed genetic counselling and personalized management decisions.