Pathogenic germline variants in BRCA1 and TP53 increase lung cancer risk in Chinese

Abstract Backgroud Multiple studies have identified pathogenic germline variants in cancer susceptibility genes (CSGs) in Chinese lung cancer patients; however, accurate assessment of these variants' contributions to cancer predisposition is always hampered by the absence of data on the prevalence of these variants in the general population. It is necessary to conduct a large‐scale case–control study to identify CSGs that significantly increase the risk of lung cancer. Materials and methods We performed targeted sequencing of a CSGs panel in 1117 lung cancer patients and 16,327 controls from the general Chinese population. Results In comparison to controls, lung cancer patients had a considerably higher prevalence of pathogenic and likely pathogenic (P/LP) variations. Among lung cancer patients, 72% of P/LP variants carriers did not have a family cancer history, who would be ignored if germline testing was only provided for patients meeting family history‐based criteria. Furthermore, compared to individuals with late‐onset lung cancer, patients with early‐onset lung cancer had a considerably higher prevalence of P/LP variations. With odds ratios (ORs) ranging from 4‐fold (BRCA1: OR, 4.193; 95%CI, 1.382–10.768) to 29‐fold (TP53: OR, 29.281; 95%CI, 1.523–1705.506), P/LP variants in the BRCA1 and TP53 genes were discovered to be strongly related to increased lung cancer risk. Additionally, with ORs ranging from 7.322‐fold to infinity, we discovered 23 variations previously categorized as non‐P/LP variants were highly enriched in lung cancer patients. Conclusion Our findings indicated that P/LP variants in BRCA1 and TP53 conferred increased risk of lung cancer in Chinese.


| INTRODUCTION
][4] Additionally, substantial evidence showed that genetic factors were also a significant contributor to the risk of lung cancer.Through a large prospective twin-based investigation, it was estimated that the overall heritability of lung cancer was 18%. 5 Previous studies identified multiple risk loci of lung cancer through genome-wide association studies (GWAS), while GWAS-derived polygenic risk score (PRS) was proved to be an effective tool to predict lung cancer risk. 6,7Notably, the incidence of lung cancer among light smokers with high PRS was higher than that among heavy smokers with low PRS, indicating that genetic factors were also important lung cancer predictors and might be used in population screening programs. 7These germline-derived genetic biomarkers may be especially helpful in screening because they do not alter throughout a person's life despite changes in other risk variables.
][10][11][12][13] Hu et al 11 demonstrated that there were 64 (1.03%) carriers of BRCA1 and BRCA2 pathogenic germline variants in 6220 Chinese non-small cell lung cancer (NSCLC) patients, with BRCA2 variants being the most predominant.Similarly, pathogenic germline variants in BRCA1 and BRCA2 were detected in 6 (0.34%) and 16 (0.79%)participants, respectively, in another study which involved 1764 Chinese lung cancer patients. 13When expanding to multiple cancer susceptibility genes (CSGs) panel, the prevalence of pathogenic germline variants in Chinese lung cancer patients increased to 3.8%. 13,14n the TCGA cohort, 5.5% of 1087 lung cancer patients were found to carry pathogenic germline variants using a 152-gene panel. 15It was suggested that multigene panel testing would improve the discovery of heritable variants over targeted testing based on guidelines. 16dentification of a genetic predisposition would substantially assist the carrier in selecting risk-reducing interventions before developing cancer, as well as in making appropriate treatment options once diagnosed with cancer.Previous investigations on the role of CSGs germline variants in lung cancer were mainly focused on patients, while information on the prevalence of germline variants in the general population was always absent which impeded the precise evaluation of their contributions to cancer risk.In this study, we aimed to investigate the risk of CSGs germline variants to lung cancer by assessing their prevalence in both unselected lung cancer patients and the general population in China.

| Participants
Patients who had a current or previous diagnosis of lung cancer were recruited from Henan Cancer Hospital.Patients were unselected for histopathology, stage of disease, and family history of cancer.Individuals from the general population who underwent routine health checkup and denied personal cancer history were recruited as controls.Blood or saliva samples were collected from participants to extract genomic DNA after anonymization.Clinical information including sex, age, histopathology, smoking, and family cancer history were collected from lung cancer patients, while only sex and age were available from controls.The study was performed in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Henan Cancer Hospital and Shanghai Ethics Committee for Clinical Research.Written informed consent was obtained from all subjects.

| Targeted sequencing of CSGs
Genomic DNA was extracted from whole blood or saliva using MagMAX™ DNA Multi-Sample Ultra 2.0 Kit (Thermo Fisher Scientific, Waltham, MA, USA).An amplification-based panel designed by Fluidigm D3 Assay Design program (Fluidigm, San Francisco, CA, USA) was applied to amplify exons and exon/intron boundaries of APC, ATM, BRCA1, BRCA2, CDH1, CHEK2, MLH1, MSH2, MSH6, PALB2, PTEN, STK11, and TP53 genes.PCR amplification of targeted sequences were performed using targeted DNA Seq Library Preparation reagents on Fluidigm LP 192.24 chip (Fluidigm).PCR products were collected and then purified using Agencourt AMPure XP (Beckman Coulter, Brea, CA, USA) before being sequenced on HiSeq X Ten instrument (Illumina, San Diego, CA, USA).The sequencing coverage and quality statistics for each sample are summarized in Table S1.

| Germline variant calling and classification
Germline variant calling was carried out as described previously. 17Briefly, sequencing reads were mapped to human reference genome (hg19) and germline variants were called using GATK.NM_000038.5   18 variants were classified as pathogenic (P), likely pathogenic (LP), variant of uncertain significance (VUS), likely benign (LB), and benign (B).All variants classified as P/LP were further validated by Sanger sequencing.

| Statistics analysis
Comparisons between different groups were performed using Mann-Whitney test, chi-square test, or Fisher exact test as appropriate.p value was calculated using a 2-sided hypothesis test, and p < 0.05 was considered statistically significant.The Benjamini-Hochberg method was applied for multiple hypothesis testing.All statistical analysis was performed using R package (version 3.5.3).

| Cohort characteristics
A total of 1117 unselected lung cancer patients including 691 males and 426 females were enrolled in the study.The characteristics of patients including sex, age, histopathology, and smoking status are shown in Table 1.In addition, 16,327 controls from the general population who denied personal cancer history were also involved in the study.The distribution of sex and age of controls are listed in Table 1.The mean age of the controls (39.7 ± 11.3 for males and 38.1 ± 8.8 for females) was significantly younger than that of patients (59.8 ± 9.79 for males and 58.1 ± 10.9 for females) (p < 0.01).0][21][22] Among the 1117 patients, 704 (63%) had adenocarcinoma lung cancer, 198 (17.8%) had squamous cell lung cancer, 199 (17.8%) had small cell lung cancer, and 16 (1.4%)had lung cancer of other histopathological categories.Squamous cell lung cancer was more common in males (26.9%) than in females (2.8%), while adenocarcinoma lung cancer was more common in females (83.3%) than in males (50.5%) (p < 0.01).The majority of male patients (72.6%)S2.

| Germline variants in 1117 lung cancer patients
A multigene panel was applied in targeted sequencing, and germline variants were identified in both lung cancer patients and controls.We identified 1257 unique variants in 1117 lung cancer patients, with pathogenic and likely pathogenic variants (P/LP) accounting for 2.0%, benign and likely benign variants (B/LB) accounting for 30.8%,variants of uncertain significance (VUS) accounting for 14.2%, variants with conflicting interpretations of pathogenicity accounting for 8.2%, and unclassified variants (variants that are not recorded in ClinVar) accounting for 44.8% (Table 2).Integration of epidemiological, genetic, and/or histopathological data, as well as in silico prediction and/or in vitro functional analysis, was suggested to be the best way to interpret the role and mechanism of unclassified variants in cancer risk, which always required collaboration with other groups. 23Therefore, in the future, we will collaborate with other research teams to categorize these unclassified variants unambiguously.A total of 15 P/LP variants were found in 691 male patients, while 10 P/LP variants were found in 426 female patients (Table 3 and Table S3).The carrier frequency of P/LP variants was quite similar in male (15 out of 691, 2.2%) and female (10 out of 426, 2.3%) patients.Among the 25 carriers, 15 had adenocarcinoma lung cancer, six had squamous cell lung cancer, three had small cell lung cancer, and one had lung cancer of other histopathological categories.Only seven carriers (28%) self-reported having a family history of various malignancies including lung cancer, implying that 72% of the carriers would be missed if germline testing was only provided for patients who met family history-based criteria (Table S3).Notably, the prevalence of P/LP variants was 5.9% in patients with early-onset lung cancer (age < 50 years old), while it was significantly lower (1.5%) in patients with late-onset cancer (age ≥ 50 years old), suggesting that P/LP variants might play a more importantly role in younger patients (Figure 1).

| Germline variants in 16,327 controls
A total of 7505 unique germline variants were found in 16,327 controls, of which 150 (2%) were P/LP variants, 1217 (16.2%) were B/LB variants, 1336 (17.8%) were VUS, 432 (5.8%) were variants with conflicting interpretations of pathogenicity, and 4370 (58.2%) were unclassified variants (Table 2).The majority of P/LP variants (129 out of 150) were found only once, whereas 21 of them were recurrent in two or three individuals.Notably, one person had two P/LP variants (one in TP53 and one in PALB2) simultaneously (Table 3 and Table S4).Totally, there were 180 carriers carrying 181 P/LP variants, and no difference in the carrier frequency was found between males (11 out of 965, 1.1%) and females (169 out of 15,362, 1.1%).Besides, five P/LP variants (one in ATM, two in BRCA2, and two in PALB2) were also found in 1117 lung cancer patients (Table S4).
In addition to P/LP variants, we found 23 variants previously classified as B/LB, VUS, or unclassified variants in ClinVar database might also be associated with increased risk for lung cancer, with adjusted p values <0.05 and ORs varying from 7.322 to infinity (Table 4 and Table S5).These 23 variants were found in exons, introns, or untranslated regions (UTRs) of 11 genes, including APC, ATM, BRCA1, BRCA2, CDH1, CHEK2, MSH2, MSH6, PTEN, STK11, and TP53.Eight of the 23 variants were nonsynonymous, two were synonymous, while 10 and six were situated in introns and UTRs, respectively.The majority of the 23 variants had no allele frequency information in the 1000 Genome, ExAC, and gnomAD databases, whereas only a few were reported with the maximum allele frequency at 0.1% in East Asian population (Table S5).Our findings indicated P/LP variants of the BRCA1 and TP53 genes, as well as 23 rare variants, contributed significantly to lung cancer susceptibility.

| DISCUSSION
The potential significance of germline pathogenic variants in lung cancer susceptibility has received a lot of attention in recent years.5][26] However, it has been proposed that the profile of germline variants in Chinese lung cancer patients may differ ethnically from that of western patients. 27Case-control studies are needed to uncover ethnic-specific germline variants that increase the risk of lung cancer in the Chinese population.
Lung cancer, colorectal cancer, and breast cancer were reported to be the first, second, and fourth most frequent malignancies in China in 2020. 28P/LP germline variants in ATM, BRCA1, BRCA2, CDH1, CHEK2, PALB2, PTEN, STK11, and TP53 genes have been linked to an elevated risk of breast cancer, whereas APC, CHEK2, MLH1, MSH2, MSH6, PTEN, and STK11 have been identified as causal genes in colorectal cancer. 29,30n a recent study, P/LP germline variants in ATM, BRCA1, BRCA2, CHEK2, MSH6, PALB2, and TP53 were reported to be enriched in lung cancer patients. 27Our targeted sequencing panel, which includes the13 genes mentioned above (APC, ATM, BRCA1, BRCA2, CDH1, CHEK2, MLH1, MSH2, MSH6, PALB2, PTEN, STK11,  and TP53), may be capable of evaluating germline predisposition for lung cancer, colorectal cancer, and breast cancer simultaneously (Figure S1).Using the 13-genes panel, we attempted to identify germline variants that significantly increase risk to lung cancer in this study.Due to the restricted number of genes in this panel, it is inevitable that other genes that contribute to lung cancer predisposition may be ignored.However, given that there were over 10,000 samples to be investigated in our study, using the 13-genes panel would significantly reduce the experimental cost compared to a more comprehensive panel.
In this case-control study, we found that germline P/LP variants were considerably more prevalent in lung cancer patients than in the controls (2.24% vs. 1.11%, p < 0.01).Our findings demonstrated that germline P/LP variants in the BRCA1 and TP53 genes increased the risk of lung cancer in Chinese people.We also discovered 23 rare variants in the APC, ATM, BRCA1, BRCA2, CDH1, CHEK2, MSH2, MSH6, PTEN, STK11, and TP53 genes that may confer increased risk for lung cancer.However, in-depth mechanistic studies on these rare variants are needed to substantiate their pathogenic roles in lung cancer.
The prevalence of germline BRCA1 and BRCA2 P/ LP variants was 0.24% and 0.79%, respectively, according to a large survey encompassing 6220 Chinese patients with advanced NSCLC. 11Carriers of BRCA1 and BRCA2 P/LP variants accounted for 0.79% and 0.60% of 1017 lung cancer patients with adenocarcinoma or squamous cell carcinoma, respectively, in the TCGA cohort. 15We found that 1.17% of 1117 unselected Chinese lung cancer patients carried BRCA1 (0.54%) and BRCA2 (0.63%) P/LP variants.These findings showed that the proportion of BRCA1 and BRCA2 carriers differed between cohorts, with more BRCA2 carriers discovered in Chinese patients and more BRCA1 carriers detected in TCGA (non-Chinese) lung cancer patients.However, this did not imply that BRCA2 played a more important role in Chinese lung cancer patients than BRCA1.In fact, when their prevalence in the controls was taken into account, P/LP variants of BRCA1 but not BRCA2 were significantly related with an elevated risk of lung cancer, as demonstrated by our study.Compared to the controls, enrichment of BRCA1 variants in lung cancer patients was statistically significant, with an OR of 4.193 (95% CI 1.382-10.768,p = 0.006), but it was not significant for BRCA2.Our findings suggested that, in addition to breast and ovarian cancer, P/LP variants of BRCA1 also conferred increased risk for lung cancer in Chinese people.
Previous studies revealed that the prevalence of ATM, CHEK2, PALB2, and TP53 germline P/LP variants ranged from 0.11% to 0.29%, 0.39% to 0.40%, 0.11% to 0.29%, and 0.10% to 0.34%, respectively, in two independent Chinese cohorts of lung cancer patients. 13,14However, due to a paucity of data on the prevalence of variants in the controls, it was unclear how much these genes contributed to the risk of lung cancer.Taking the advantage of enrolling large number of controls, we found that despite the fact that P/LP variants of ATM, CHEK2, PALB2, and TP53 were enriched in lung cancer patients, only TP53 exhibited statistical significance with an OR of 29.281 (95% CI, 1.523-1705.506,p = 0.012).In addition, 23 rare variants in the exonic, intronic, or UTR regions of the APC, ATM, BRCA1, BRCA2, CDH1, CHEK2, MSH2, MSH6, PTEN, STK11, and TP53 genes were also found to be significantly enriched in lung cancer patients, implying that these variants may also predispose risks for lung cancer, which needs to be confirmed in other datasets.Furthermore, while pathogenic germline variants of MLH1 have been reported in lung cancer patients in Western countries, 31 we did not discover P/LP variants in the MLH1 gene in the 1117 Chinese patients with lung cancer, indicating ethnic disparities in CSGs may exist.
Lung cancer diagnosed before the age of 50 was always referred to as early-onset lung cancer.3][34] Polymorphisms in genes involved in xenobiotic metabolism have been identified as hereditary risk factors for early-onset lung cancer. 33ur results showed that the prevalence of germline P/ LP variants in patients with early-onset lung cancer was significantly higher than in individuals with late-onset lung cancer (5.9% vs. 1.5%, p < 0.01).Similar observation was also reported in a study involving 2984 patients with various types of cancer. 16Thus, germline P/LP variants might impose a larger risk in younger patients than in elder ones.
According to a recent study, 1019 out of 2984 patients (34.1%) with various malignancies had a family cancer history in first-degree relatives, implying that genetic factors might play an important role in multiple types of cancer. 16Consistently, 218 out of the 1117 unselected lung cancer patients (19.5%) involved in our study self-reported a family cancer history in one or more first-degree relatives.However, P/LP variants were only found in seven of the 218 patients (3.2%), and the majority patients with a family cancer history lacked P/LP variants in the targeted genes we examined.There might be more deleterious variants or risk loci in other genes that contributed to the heritability of lung cancer, and an extended gene panel would improve the possibility of detecting more heritable variants. 16However, 18 of the 25 P/LP variant carriers (72%) in our study did not have a family cancer history, which would have been missed if germline testing was offered only on family history-based criteria.It has been recommended that germline genetic testing be integrated into somatic genomic testing to identify additional clinically actionable variants. 19,35his study has several limitations.First, the age and sex of the controls did not match that of the lung cancer patients.The majority of individuals in the control group (94.1%) were female and much younger than lung cancer patients.Consistently, according to a survey carried out in the United States with ~34,000 responders, 94.8% of those who had taken genetic testing were females. 22It is likely that young ladies were more willing to undergo routine health checks as well as genetic testing.However, because a germline variant is transferred straight from a parent to a child at conception, age, or sex would not introduce bias into its prevalence.Second, this was a retrospective study.A long-term follow-up study for the control group would be of great importance in determining the incidence of malignancies in P/LP variant carriers.Third, the number of CSGs investigated in this study is restricted, and the amplicon-based NGS approach we applied was incapable of detecting large deletions, duplications, and copy number variations in target genes.Whole exome sequencing could address these issues, but the cost would be much higher when investigating 10,000 samples.Fourth, there was no family cancer history in the controls, and genetic testing was not performed on the family members of P/LP variant carriers, which might limit the generalizability of our findings.
In summary, our results indicated that P/LP variants in the BRCA1 and TP53 genes significantly elevated the risk of lung cancer in Chinese people.In addition, we revealed that 23 variants previously classified as B/LB, VUS, or unclassified variants were significantly enriched in lung cancer patients compared to controls.Our findings highlight the need for a case-control study to evaluate the contribution of CSGs to cancer risk.

F I G U R E 1
Prevalence of P/LP variants in lung cancer patients and controls.(A) Prevalence of P/LP variants in 1117 lung cancer patients and 16,327 controls.A total of 25 and 181 P/LP variants were identified in patients and controls respectively, and the prevalence of P/LP variants was significantly higher in patients (25/1117, 2.24%) than in controls (181/16327, 1.11%).(B) Prevalence of P/LP variants in early-onset and late-onset lung cancer patients.A total of 11 P/LP variants were identified in 186 early-onset patients, and 14 P/LP variants were identified in 931 late-onset patients.Compared to late-onset patients, the prevalence of P/LP variants was significantly higher in earlyonset patients (5.9% vs. 1.5%).** represents p value <0.01.T A B L E 4Variants associated with increased risk for lung cancer.
T A B L E 1self-reported being smokers, while only two female patients (0.5%) were smokers.A total of 218 patients (19.5%) self-reported having at least one first-degree relative with cancer.Detailed information on family cancer history is provided in Table Prevalence of P/LP variants in lung cancer patients and controls.
T A B L E 3