Our study sought to adapt well-established international eligibility guidelines to perform a pilot of clinical germline genetics testing among patients with breast and prostate cancers in Rwanda. We adapted NCCN guidelines and developed context-specific genetic counseling and testing protocols to facilitate the ascertainment of PV and VUS in this population and contribute to the characterization of inherited cancers in individuals of African descent. First, while our breast cancer study cohort was similar in age and disease characteristics to previous studies of germline genetic testing conducted from SSA countries, our study represents a large cohort of patients from a previously undertested SSA population region who underwent multi-gene panel testing12,13,19,20,21,22. In our study, young BC patients with triple negative disease were the most likely to harbor PV. Second, we demonstrate that applying standard guidelines in patient selection for genetic testing with a curated multi-gene panel testing resulted in a high yield of PV across BC patients which helps to strengthen the case for more routine implementation of germline genetic testing in cancer care in SSA. Finally, our study shows a clear pattern of inheritability of the common highly penetrant genes between regions of Africa and the diaspora. Whereas BRCA2 was more prevalent in our BC cohort and many other in Eastern Africa, BRCA1 has been reported to be more common in Western Africa, Caribbean and African American women with BC12,15,23,24,25.

The majority of PV identified in BC study patients (94%) were consistent with established BC risk, including ATM, BRCA1, BRCA2, CHEK2, and TP53. A high prevalence of BRCA1 or BRCA2 PV have been previously reported in unselected breast cancer cases from Nigeria, Cameroon, and Uganda12,15,19 with BRCA1 prevalence of 6-7% and BRCA2 prevalence of 4-5%. We observed a prevalence of 6.5% for BRCA1 PV, 8.9% for BRCA2 PV, and an aggregate BRCA1/2 rate of 15.4% in the present study. Thus, our estimate of BRCA1 PV is similar to that reported in other African populations, but our prevalence of BRCA2 is somewhat higher than previously reported. In contrast, PV prevalence in unselected breast cancer case series in largely European descent populations has been estimated to be approximately 6% for BRCA1/2 in women diagnosed before age 3620, 11.2% in women with triple negative breast cancer21, 4.7% BRCA1/2 PV prevalence in patients ages 35–64 years22. African Americans have been reported to have lower prevalence of BRCA1/2 PV compared to European Americans in the range of 4% compared to 5% in European Americans and a 1.3% BRCA1 PV prevalence in African Americans compared to 2.2% among Non-Hispanic whites26. Larger African studies are needed to further establish the PV prevalence.

Two PV were observed in genes that had not been previously associated with breast cancer. One breast cancer case carried a PV in RET, a proto-oncogene that confers risks of Hirschsprung Disease 1 and Multiple Endocrine Neoplasia, Type 2 A. FGFR1, an established low penetrance breast cancer susceptibility locus, is a paralog of RET. RET exhibits elevated expression and survival in some breast tumors27,28,29. A single PV in WRN was also identified in one breast cancer case. WRN is a RecQ-like helicase associated with (autosomal recessive) Werner and Bloom syndromes that are involved in DNA damage repair. However, no association of WRN and breast cancer have been previously reported although Werner Syndrome confers risk of sarcomas, melanoma, and thyroid cancers30. Thus, while germline variation in RET and WRN are not breast cancer susceptibility genes, they each could be involved in breast cancer etiology. However, based on the pedigrees, these incidental findings do not provide evidence that the PV identified at these genes is causative. While these PV are uncommon, it is likely that their occurrence is unrelated to the cause of these malignancies.

We also observed three PV in a single breast cancer case diagnosed at age 38 with triple negative breast cancer: TP53 c.1010 G > A (p.Arg337His), BRCA2 c.3720_3723del (p.Phe1241-Valfs*17), and ATM c.7913 G > A (Table 2). ATM c.7913 G > A has been previously reported in Cameroon8. TP53 (p.Arg337His) has been observed in Sudan and is a founder mutation in Brazil31,32. This PV has been reported to occur at a relatively high frequency in Li-Fraumeni Syndrome families and confers early onset breast cancer risk in Brazil33,34,35,36,37,38. This PV is a founder mutation based on its occurrence on a common haplotype39. The observation of this PV in Eastern Africa raises the possibility of an alternate origin of this “founder” mutation reported commonly in Brazil.

Of note, multiple identical PV were identified in fBC cases that were not known to be biological relatives. These included three occurrences of c.7913 G > A (p.Trp2638*) at ATM, seven occurrences of c.4065_4068del (p.Asn1355Lysfs*10) at BRCA1, three occurrences of c.1053del (p.Lys351Asnfs*16) at BRCA1, three occurrences each of c.3720_3723del (p.Phe1241Valfs*17) and c.5633dup (p.Asn1878Lysfs*4) at BRCA2, and two occurrences of a large deletion involving exons 1-4 at BRCA2. BRCA1 c.4065_4068del has been reported in numerous populations worldwide40,41. This variant was identified in an earlier Rwandan cohort16 and has been uniformly reported in most East African countries and Northern Africa in patients with similar disease phenotype11,12,23. The same variant was also reported in a large cancer genetics clinic in Norway that studied seven distinct families whose ancestries were unclear25. Studies are underway to determine whether this PV reflects a distinct haplotype in Rwanda from those previously described. Similarly, c.5633dup has been previously reported in a study of Cameroonian and Ugandan breast cancer cases, and c.1053del has been reported in Uganda12. BRCA2 c.3720_3723del has been previously reported in a set of early onset Rwandan breast cancer cases16. These observations suggest either hot spots for mutation in these genes or common (founder) PV that are relatively common in Africa. Other variants observed in this study have also been reported elsewhere. For example, BRCA1 c.1504_1508 is commonly found in Eastern and Northern Africa, but also identified in a population of unlikely African origin11,14.

One man with breast cancer was found to be a BRCA2 carrier. While the sample size of men with breast cancer was low, the rate of PV in our cohort (20%) is within range of the prevalences seen in other populations42,43.

BRCA2 was the most commonly reported PV among men of African descent diagnosed with PC with a frequency of 1.75%30. However, we observed that Rwandan PC cases have a lower prevalence of PV than reported in other populations30,44,45,46,47,48. We did not observe any PV in HOXB13, TP53, or the Lynch Syndrome genes, which have been proposed as PC susceptibility genes in other populations48,49.

In contrast to breast cancer, the majority of PV observed in PC cases (71%) occurred in genes that are not currently thought to be PC susceptibility genes, including BRIP1, RAD50, RAD51D, and RECQL4 (Supplementary Table 2). BRIP1, RAD50, and RAD51D interact with BRCA1 and/or BRCA2 in DNA damage repair and related functions. Thus, while not known to be PC susceptibility loci, these genes are plausibly associated with PC and may warrant further evaluation in African and other populations. PV at RECQL450,51,52 were observed in two PC cases (c.1048_1049del and c.3416del). RECQL4 is associated with (autosomal recessive) Rothmund-Thomson syndrome, RAPADILINO syndrome, and Baller-Gerold Syndrome52. While this gene is located at chromosome 8q24, it is found approximately 15 Mb away from the PC susceptibility locus thought to regulate MYC in conferring PC susceptibility53, and thus it is unlikely that these PV would explain long-range regulation of MYC or the association of prostate tumors at this locus.

The disparities in genomic testing led to many variants found in the population of African descent in SSA to be classified as variants of uncertain significance (VUS)54. 115 patients (33.6%) with breast cancer and 109 patients (31.8%) with PC were observed to carry VUS. Similar prevalences have been reported in other SSA populations, congruent with findings in generally under-represented populations, highlighting the need for further larger population studies and analyses12,19. In addition to giving a thorough explanation of non-actionable findings, we presented the VUS rates in this selected population with a suggestive phenotype in order to lay the groundwork for future studies on variant reclassification.

Our study had some limitations. First, as a cancer case series, there is limited possibility of generalizing the findings across patient populations in Rwanda and beyond. We were also unable to establish a cause-effect relationship between the variants and patient’s cancers due to the lack of a population-based non-cancer control group. Second, family history was taken during the time of encounter (pre-genetic testing counseling session) with the study participants, which was limited by recall bias and relatively poor access of the overall population to cancer diagnostic services. Third, given a regional history of migration, we could not ascertain the origins of the unrelated probands found with recurrent variants. Finally, there were practical challenges with many samples requiring retesting and a high number of participation refusals for repeat testing; this may point to potentially inadequate education during the pre-genetic testing counseling and underappreciation of burdens related to patient participation in the study. Subsequent studies will focus on cascade testing of at-risk relatives.

Our study shows a high yield of PV among high-risk individuals, when ascertained using standard guidelines. Although not validated in African populations, the NCCN guidelines provide a flexible framework to identify the population requiring cancer genetic testing based on demographic and clinical factors, even in the absence of reported family history. Further confirmatory studies are needed for validation purposes, including to determine if NCCN guidelines need to be adapted to the African setting and incidence of pathogenic variants in patients who do not meet NCCN guidelines. This work is planned in subsequent larger studies.

The World Health Organization recognizes human genomics as a means to prevent, diagnose and predict, manage, monitor, and treat genetic conditions55. A balance needs to be maintained between approaching cancers through familial programs and evidence-based universal testing for eligible individuals and cancers, which could both prove to be innovative ways to address and curb cancer mortality in Africa. Their impact has a wide reach, in that it improves healthcare systems through human and infrastructural capacity building, while emphasizing on a precise nature of cancer prevention and treatment54,56,57. The purpose of genetic testing is to provide individuals and systems with potentially actionable information regarding cancer. Our approach of identifying patients based on standard eligibility criteria was mostly dependent on age and disease characteristics, and less with family history, which was incomplete in most cases. In the example of Rwanda, and as illustrated on our pedigrees, familial structures and knowledge of pertinent medical history are limited. This is a common theme in most SSA-based cancer genetic studies, usually compounded with lack of follow-up, resulting in missed opportunities for familial identification, cascade testing and the implementation of intensified screening and/or risk-reducing interventions. Approaching families can help identify individuals at high-risk of cancers, and this could complement cancer screening efforts that are ongoing on the continent. Population-based screening for cancer-susceptibility genes has an advantage of identifying all carriers regardless of family history and is a proactive option that solves the failure of prevention resulting from eligible patient genetic testing. However, the adoption of this screening in low-resource settings requires a careful consideration of several factors, including the population-specific cancer risk conferred by these genes, the cancer screening infrastructural availability after genetic testing, and would be warranted in communities where a greater acceptance of cancer genetic testing exists, and a prevalence of mutational prevalence has been established58. Our study used multi-gene panel testing, which is now the standard procedure for cancer genetics services, owing to its cost-effectiveness, speed, and efficiency, when compared to whole genome sequencing. For low resource settings however, a precise curation of genes for different disease conditions is needed, to limit the risk of incidental findings beyond the cancer scope, that these developing settings might not be able to manage.

The critical next steps will consist in understanding the study and result disclosure experience of the probands through qualitative surveys, providing a structure for disclosure of the genetic results from the identified probands to at-risk relatives, and subsequent cascade genetic testing58,59. Effective risk communication and models of disclosure that would perform well in SSA have not been explored at length, and low health literacy could impact the probands’ intent to communicate, the information content and the motivation to undergo genetic testing58,59,60,61. However, a shift from traditional familial disclosure and letter sharing to individualized, culturally sensitive methods has resulted in positive outcomes with regard to genetic risk assessment in women of African descent and their family members and could be tested in our population. SSA health systems differ, and methods of familial approach (physician or proband-led) could vary from a setting to another. Understanding personal values and the health-seeking behaviors of the at-risk relatives are important in establishing a relationship with families, and to facilitate the risk communication and genetic testing. Finally, health insurance schemes in Rwanda (both public and private) cover access to screening, prevention and essential cancer treatment5, but some mitigating strategies, including access to PARP inhibitors are not widely available (for the public insurance scheme). The current study provides a basis for future interventions, including clinical trials to test efficacy of these medications in an African population.

By using adapted eligibility guidelines, our study findings contribute to bridge the gap in the diversity of reported PV and their relationship with breast and prostate cancer susceptibility in the Rwandan population. As screening, prevention, and targeted treatment solutions emerge for individuals who carry PV, there is need for familial cancer registries for an early ascertainment, better cancer risk assessment, tailored clinical-grade laboratory solutions and diversified treatment modalities in low-resource settings.