A genome is constantly damaged by endogenous and exogenous factors (Liu and Huang, 2014; Bian et al., 2019). Effectively repairing the damaged DNA is vital to all life (Jeggo et al., 2016). The DNA damage repair (DDR) system is composed of multiple functional pathways and is universally preserved in living organisms. Each pathway consists of multiple genes that repair special types of DNA damage: BER (base excision repair) pathway repairs the lesions with small bases (Willey et al., 2014); DR (direct reversal) pathway repairs the DNA damaged by ubiquitous alkylating agents (Volkert, 1988); FA (Fanconi anemia) pathway repairs the damages of strand cross-link errors (Ceccaldi et al., 2016); HR (homologous recombination) (Liang et al., 2005) and NHEJ (nonhomologous end joining) pathways (Budman and Chu, 2005) repair double strand DNA breaks; MMR (mismatch repair) pathway repairs mismatched errors (Lynch et al., 2015); NER (nucleotide excision repair) pathway repairs the DNA damaged by helix-distorting DNA errors (Reardon and Sancer, 2006).

Genetic variation of insertion, deletion, duplication, indel, single nucleotide change etc. is common in DDR genes (Seplyarskiy and Sunyaev, 2021). While most genetic variants in DDR genes are benign or neutral, a portion of the variants are pathogenic that they damage the function of the affected DDR genes, causing genome instability and high risk of diseases. In medical terminology, a genetic variant can be classified into pathogenic, likely pathogenic, variant of uncertain significance, likely benign, or benign. Pathogenic and likely pathogenic variants (PVs) have direct clinical significance in disease diagnosis, treatment and prognosis but benign and likely benign variants (BVs) don’t, whereas the function of uncertain significance variants (VUS) remains unclear (Richards et al., 2015).

The PVs in DDR genes are well determined as the genetic predisposition for high disease risk, in particular, cancer (Lynch, 2010; Xue, et al., 2012; Fu, et al., 2014; Simons, et al., 2014). For example, the tumor suppressor BRCA1 in the homologous DNA damage repair pathway plays essential roles in repairing double-strand DNA breaks. PVs in BRCA1 lead to deficiency of the homologous DNA damage repair pathway, increasing the risk of breast and ovarian cancer (Welcsh and King, 2001; Narod and Foulkes, 2004). One of the distinct features of modern humans from many other mammals is the high susceptibility to cancer (David and Zimmerman, 2010). This could also be attributed to specific genetic factors in humans, to which the DDR PVs can be directly relevant. Determining the evolutionary origin and arising time of DDR PVs in modern humans can provide deep in-sight into the evolutionary basis of human diseases and develop strategies to control disease risk. Our recent studies in the human DDR genes including BRCA1, BRCA2, TP53, MUTHY, and PALB2 etc. revealed that the DDR PVs were not transmitted from nonhuman species through cross-species conservation but arose in humans themselves (Li, et al., 2022; Chian, et al., 2023; Kou, et al., 2023; Xiao, et al., 2023). The results promoted us to question whether the result observed in these genes could be universal that the PVs in all human DDR genes were originated from humans themselves. We expanded the study by testing the origins of PVs in all 169 human DDR genes. The results confirmed that nearly all PVs in the 169 DDR genes were originated in humans themselves (Zhao, et al., 2024). However, human evolution has lasted for over 6 million-years since the separation from Chimp. While our study determined that the PVs in human DDR genes is basically originated from humans themselves, the precise time point for when the PVs arose along human evolutionary history remain unknown. One of the most significant events in human evolution history is the latest out-of-Africa migration, which played fundamental roles in the formation of modern humans. Our previous study (Zhao et al., 2024) used the PV data from ancient humans determine that the origin of human DDR PVs was from humans themselves. However, the precise arising timing of the DDR PVs along human evolution process remains to be determined.

Modern non-African humans (hereafter: non-African) were the direct descendants of the latest out-of-Africa human migrants that occurred 50,000–60,000 years ago (Hublin, et al., 2017) staying largely in Persian Plateau and spread to the world 45,000 years ago (Vallini et al., 2024). The non-African migrants were more homogeneous than the ancestral African due to the founder effects and the bottleneck effects of small population size when out-of-Africa migration occurred (Tishkoff, et al., 2009). Although out-of-Africa humans could inherit certain genetic variants from their African ancestors, novel genetic variation should arise and be selected in the decedent out-of-Africa humans following their adaptation to new environments and increased population size. These novel variants should be largely absent in the ancestors of the out-of-Africa humans and the descendants of ancient African (hereafter: African) except these occurred coincidentally. Rapid progress in archaeological genomics has led to the identification of rich genetic variation data including the PVs from ancient humans, which were mostly from the descendants of out-of-Africa migrants composed of the ancient non-African living in Eurasia continent within the last 10,000 years (Kou, et al., 2023). The main goal of our current study is to narrow down the arising time of human DDR PVs by referring to the out-of-Africa migration as the cutting point. We consider that the assumptions “zoom-in” from the million-year human evolution history to the Out-Of-Africa period of 60,000 years ago, which played one of the most significant roles in the formation of modern humans, to determine the arising time of human DDR PVs. The results from the much simplified analytic process can open a door to further study the complexities of DDR PV origins. Thus, we hypothesized that comparison of the DDR PVs among African, non-African and ancient humans should allow to determine the arising time by referring out-of-Africa migration as the cutting point. The PVs absence in African but sharing between ancient humans and non-African would imply that the PVs were originated after the out-of-Africa migration; the PVs shared in African, ancient humans and non-African would imply that the PVs were originated in African; and the PVs present only in African but not in ancient humans and non-African would imply that the PVs were originated in African.

To test our hypotheses, we identified the DDR PVs in 169 human DDR genes originated in 28,872 African, 222,342 non-African, and 5,031 ancient humans. We made direct comparison of DDR PVs between African and non-African. We also used the DDR PVs of ancient humans as the intermediate to further compare the DDR PVs between African and non-African. We further validated the results by comparing the PVs in BRCA and TP53 in the three cohorts. The results reveal that DDR PVs in modern humans mostly arose after the latest human out-of-Africa migration.

We designed the strategy for our study: 1. Direct comparing the DDR PVs between African and non-African populations to estimate the similarity and difference of DDR PVs between the two populations after their separation since out-of-Africa migration; 2. Using the DDR PVs derived from ancient humans as the reference to further test the similarity and difference of DDR PVs between the two populations; 3. Using the PVs in BRCA and TP53 as the examples to validate the results from the comparisons. Figure 1 outlines the working flow of analytic process.

Determination of the evolution origin and arising time of PVs in human DDR genes is important, as it will provide information to address the following fundamental questions in human genetics and human diseases: 1. Understand the genetic basis of human cancer. A fundamental question in biology is “where we come, where we go.” The same applies for genetic variation. Only after knowing the origin and arising time, we can better understand the genetic basis of human cancer risk. The germline PVs in human DDR genes provides an ideal system to address the issue; 2. Understand the origin of human disease susceptibility. The PVs in human DDR genes are well determined as the genetic predisposition in causing high risk of cancer by functioning as the first hit in initiating the oncogenic process. This is best represented by many “founder mutation” in human DDR genes, such as the 185delAG (c.68_69delAG) founder mutation in Ashkenazi Jews that causing high risk of breast cancer in Ashkenazi Jews population. Determination of the origin and timing of the mutation greatly enhances cancer prevention in the population; and 3. Understand why modern humans are at much higher cancer risk than many other mammals such as elephant, whales, and chimpanzee.

By systematically comparing DDR PVs among modern African, modern non-African, and ancient humans, our study reveals that most of the DDR PVs in modern humans originated after the latest out-of-Africa migration.

Our current study aims to understand the arising time of disease-causing DDR PVs risk in modern humans by referring the well-established Out-of-Africa model (Hublin, et al., 2017). The key points of the model include that 1). African is the most genetically diversified among global ethnic human populations; 2). Modern humans were originated from African; 3). Only part of genetic variants in the descendants of out-of-Africa migrants was inherited from African ancestors; 4). de novo genetic variation could arise in the descendants of out-of-Africa migrants by adaptation, selection and population expansion. The de novo genetic variants were absent in their African ancestors.

The increased population size since the latest out-of-Africa migration can be an important factor contributing to the de novo variants, as the increased population size can greatly increase the number of DDR PVs in the human population considering the consistent mutation rate in the human genome (Cabrera, 2021). It is determined that only a few hundred to thousands of individuals moved out of Africa 60,000 years ago (Zhivotovsky, et al., 2003). Since then, the size of human population had increased slowly until the end of the last glacial period approximately 10,000 years ago (Henn et al., 2012). Following the agricultural revolution occurred 6,000 years ago (Gignoux, et al., 2011) and the industrial revolution centuries ago, the size of human population has been expanding significantly till the current size of modern humans. Although the same DDR pathways and DDR genes were affected by DDR PVs in both African and non-African, the spectrum of DDR PVs between African and non-African differs substantially after their separation by the latest out-of-Africa migration. After about 2,000 generations since the latest out-of-Africa migration, de novo DDR PVs can arise constantly following the expansion of the human population (Hudjashov, et al., 2007). The high PV sharing rate between ancient humans and non-African but low between ancient humans and African is due to the relationship of ancient humans closer to non-African (within 10,000 years) than African. These findings also explain the high ethnic specificity of DDR PVs in modern humans (Bhaskaran et al., 2021; Qin et al., 2022; Qin et al., 2023). The fact that most human DDR PVs have only a few thousand years of history also implies that human DDR PVs could be too young to allow evolutionary selection to function effectively. Whether the PVs would be eliminated or integrated into human genome re-mains to be determined with longer time.

For the following reasons, TP53 and BRCA were selected from all DDR genes as the examples for detailed analysis. As the most mutated tumor suppressor gene, TP53 is mutated in more than half of human cancer types (Narod and Foulkes 2004). The presence of a group of hot-spot PVs in African makes TP53 a valuable tool to test the origin of DDR PVs (Olivier et al., 2010; Monti et al., 2020). The finding that only 2 of the 19 ancient TP53 PVs were shared with African is consistent with the observation that TP53 variation has lower prevalence in African than in non-African (0.07% in African, 0.12% in South Asian, 0.15% in East Asian, 0.16% in Latin American, and 0.24%–0.28% in European) (de Andrade et al., 2017), whereas TP53 hotspot PVs are also highly prevalent in modern humans (Freed-pastor and Prives, 2012; Baugh et al., 2018; Kou et al., 2023). As represented by the four TP53 hotspot PVs, those shared between African and non-African were likely originated from African and inherited by non-African after the out-of-Africa migration. BRCA is under strong positive selection in humans (Huttley, et al., 2000; Fleming, et al., 2003; Abkevich, et al., 2004; Pavlicek, et al., 2004; Burk-Herrick, et al., 2006) for its new roles gained in regulating immunity (O’Connell, 2010), gene expression and metabolism (Lou, et al., 2014), neural development (Rosen, et al., 2006), and reproduction (Smith et al., 2013; Pao, et al., 2014). BRCA variation is also highly prevalent in non-African and African with high ethnic-specificity (Qin et al., 2022). These features make BRCA PVs as ideal markers to test the relationship of DDR PVs between African and non-African before and after out-of-Africa migration. Our previous studies revealed that the PVs in TP53 and BRCA were originated from humans themselves but not from non-human species (Li et al., 2022; Kou et al., 2023). Furthermore, rich PV data in TP53 and BRCA are available and widely applied in guiding clinical cancer diagnosis, treatment and prognosis (Cline et al., 2018; de Andrade et al., 2022). These features made TP53 and BRCA as ideal models to validate the observed DDR PVs between African and non-African populations.

Direct comparison of the DDR PVs between African and non-African showed substantial differences (Figure 3). However, the results could be biased due to the different population sizes of African and non-African included in the study. We further used the DDR PVs from ancient humans as the intermediate for the comparison. The ancient humans used in the study were mostly dated within the last 10,000 years, therefore, closer to non-African than the African. Using the ancient DDR PVs for the comparison eased the uncertainty attributed by different population sizes and enhanced the reliability of the results.

The results from our study have an immediate impact on the characterization of human DDR PVs. Many genetic variants have been identified, but their pathogenicity remain to be determined. For example, 89.7% of the 72,330 BRCA variants identified in modern humans remain uncharacterized (https://brcaexchange.org/factsheet, accessed 8 December 2023). To ease the situation, in silico computational programs are often applied to predict pathogenicity for the unknown variants. Certain computational programs are designed using the concept of evolutionary conservation. However, our studies showed that DDR PVs did not originate from nonhuman species through evolutionary conservation but arose from humans themselves (Li, et al., 2022; Chian, et al., 2023; Kou, et al., 2023; Xiao, et al., 2023), and our current study further reveals that most human DDR PVs arose after the latest human out-of-Africa migration. While evolutionary conservation-based approaches can be powerful in analyzing the benign variants in DDR genes highly conserved between human and nonhuman species, they are not suitable in identifying the PVs in human DDR genes as they are basically absent in non-human species. Instead, focusing on the humans themselves via non-conservation-based approaches, such as those based on the impact of variants on protein structural stability, will be necessary to define human DDR PVs (Tam, et al., 2020).

There are limitations in our study. Although the study included 28,872 modern African individuals, the number is smaller than the ones of non-African. The unbalanced population size between African and non-African could influence the results of the PV comparison. It is well determined that African population has the most diversified genetics among global ethnic populations. However, current human genetic study is heavily biased towards European and descendent populations that contributed most of the genetic variation data currently available. This is reflected by the limited BRCA PV data derived from African population. The lack of genomic data from ancient African can also limit the results from the comparison. This is also the reason that we used two sets of African BRCA PV data in our analysis in order to compensate for the weakness. In addition, ancient human genomes are often not fully covered by sequences because of the damaged ancient DNA samples. Therefore, the actual DDR PVs in the ancient humans could be higher than identified.

In summary, by tracing the PV data in modern African, modern non-African, and ancient humans, our study demonstrates that the DDR PVs in modern humans arose mostly after the latest human out-of-Africa migration. The information provides a foundation to understand the genetic basis for disease susceptibility including cancer in modern humans.