BRCA genes as candidates for colorectal cancer genetic testing panel: systematic review and meta-analysis

Background Breast cancer susceptibility gene (BRCA) mutation carriers are at an increased risk for breast, ovarian, prostate and pancreatic cancers. However, the role of BRCA is unclear in colorectal cancer; the results regarding the association between BRCA gene mutations and colorectal cancer risk are inconsistent and even controversial. This study aimed to investigate whether BRCA1 and BRCA2 gene mutations are associated with colorectal cancer risk. Methods In this systematic review, we searched PubMed/MEDLINE, Embase and Cochrane Library databases, adhering to PRISMA guidelines. Study quality was assessed using the Newcastle–Ottawa Scale (NOS). Unadjusted odds ratios (ORs) were used to estimate the probability of Breast Cancer Type 1 Susceptibility gene (BRCA1) and Breast Cancer Type 2 Susceptibility gene (BRCA2) mutations in colorectal cancer patients. The associations were evaluated using fixed effect models. Results Fourteen studies were included in the systematic review. Twelve studies, including seven case–control and five cohort studies, were included in the meta-analysis. A significant increase in the frequency of BRCA1 and BRCA2 mutations was observed in patients with colorectal cancer [OR = 1.34, 95% confidence interval (CI) = 1.02–1.76, P = 0.04]. In subgroup analysis, colorectal cancer patients had an increased odds of BRCA1 (OR = 1.48, 95% CI = 1.10–2.01, P = 0.01) and BRCA2 (OR = 1.56, 95% CI = 1.06–2.30, P = 0.02) mutations. Conclusions BRCA genes are one of the genes that may increase the risk of developing colorectal cancer. Thus, BRCA genes could be potential candidates that may be included in the colorectal cancer genetic testing panel. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-023-11328-w.


Introduction
Colorectal cancer (CRC) ranks third among the most common malignancies in men and women and is among the leading causes of cancer-related deaths worldwide [1].In the past 20 years, the age of onset of CRC tends to be younger [2].CRC is characterised by higher incidence, younger onset age and genetic susceptibility, but the data on the molecular characteristics of CRC is relatively limited [3,4].Therefore, in the context of precision medicine, a broader molecular tumour map should be explored to update the biomarkers and therapeutic targets in CRC patients.
The breast cancer type 1 susceptibility gene (BRCA1) and breast cancer type 2 susceptibility gene (BRCA2) were first mapped to chromosome arms 17q and 13q in the 1990s [5,6].BRCA1 and BRCA2 as tumour suppression genes are crucial in the DNA repair process by homologous recombination, which plays an essential role in chromosome integrity [7].The additional functions of BRCA genes, such as chromatin remodelling and transcriptional control, also contributed to tumour suppression [8,9].BRCA1 or BRCA2 mutation carriers show a lifetime risk of up to approximately 85% and 20%-40% for breast and ovarian cancers, respectively [10][11][12].BRCA mutations are also known to be risk factors of pancreatic and prostate cancers [13,14].In a large case-control study comparing 3030 patients with pancreatic cancer with reference controls, significant associations were observed between pancreatic cancer and BRCA2 [1.9% of cases and 0.3% of controls; odds ratio (OR), 6.20; 95% confidence interval (CI), 4.62-8.17]and BRCA1 (0.6% of cases and 0.2% of controls; OR, 2.58; 95% CI, 1.54-4.05)mutations [15].
Studies have indicated recently that BRCA mutation was associated with the development of CRC [16,17].Allelic losses at the BRCA1 locus have been detected in almost 50% of sporadic CRC cases [18].A retrospective cohort study in North America and Western Europe investigated families with ovarian or breast cancer and found the relative risk of CRC in BRCA1 mutation carriers to be 4.11 (95% CI, 2.36-7.15)[19].Another study analysed the coding regions of 27 cancer-predisposing genes in 12,503 unselected Japanese CRC patients and 23,705 controls by using target sequencing and a genome-wide SNP chip, which identified that the pathogenic variants of BRCA1 (OR, 2.6) and BRCA2 (OR, 1.9) were significantly associated with CRC development [20].Contrarily, a retrospective study in five countries from Canada, the United States or Europe reported that the incidence of CRC in BRCA1 (standardised incidence ratio (SIR), 0.92; 95% CI, 0.54-1.40,P = 0.7) and BRCA2 (SIR, 0.82; 95% CI, 0.30-1.81,P = 0.7) mutation carriers was not greater as compared to that of the general population [16].Thus, the association between BRCA gene mutations and CRC risk remains controversial [21,22].The present study aimed to investigate whether the probability of BRCA gene mutations is increased in patients with CRC.

Methods
The present study adhered to the PRISMA guidelines [23] and has been registered at PROSPERO (ID: CRD42022366024).

Search strategy
An electronic search was conducted using the following bibliographic databases: PubMed/MEDLINE, Embase and Cochrane Library.The search was executed by two investigators and included a combination of indexing (MeSH terms in PubMed and EMTREE terms in Embase) and entry terms, including 'Genes, BRCA1' and 'Genes, BRCA2' , and 'Colorectal Neoplasms' , 'Colonic Neoplasms' , and 'Rectal Neoplasms' , respectively, and translated for each database.A preliminary selection was made for all titles and appropriate abstracts were reviewed.We also performed a manual check of the reference list of key articles to identify recent relevant publications.The last date of search was 10 October 2022, and no language restrictions were applied.

Study selection
Participants, interventions, comparators, outcomes and research methods (PICOs) guided the eligibility screening for inclusion in our study, which were as follows: 1) participants: human adults (age > 18 years) identified as BRCA gene mutation carriers or diagnosed with CRC; 2) intervention: not applicable; 3) comparisons: colorectal cancer incidence or probability of BRCA gene mutations in the general population; 4) outcome: incidence of colorectal neoplasms or probability of BRCA gene mutations; and 5) studies: cohort or case-control studies.The exclusion criteria were as follows: subjects had no confirmed BRCA1 or BRCA2 mutation; only family or kinship analysis was reported; and commentaries, editorials, letters or review papers.

Summary measures
To quantify the probability of BRCA gene mutations in CRC, we used unadjusted ORs as a generic metric.Those studies that were included reported ORs, SIRs, and hazard ratios (HRs), or provided sufficient information to calculate the ORs.Unadjusted ORs for each study were calculated from a 2 × 2 contingency table created for each study.The OR values calculated herein were used in all subgroup meta-analyses.Individual studies have reported the effects of age, sex and pathological outcomes.

Data extraction
Two authors screened the title and abstract independently.Disagreements were resolved through a discussion and consensus, and outstanding issues were decided by a third party.Each reviewer extracted the following information from each study: title, journal, publication date, study population, type of CRC and control.

Study quality
The quality of the included studies was assessed using the Newcastle-Ottawa Scale (NOS).

Statistical analysis
Meta-analysis was performed using Review Manager (RevMan [Computer program].Version 5.4.1,The Cochrane Collaboration, 2020).The results are expressed as ORs.The OR and 95% CI were estimated using the inverse variance method.The fixed effects models were used to assess ORs for differences.An OR > 1.00 indicated a higher risk of CRC with BRCA1 or BRCA2 mutations.If the 95% CI included 1.00, the OR was not statistically significant.
The Cochran Q statistic measured heterogeneity by a weighted sum of squares and the I 2 statistic to quantify the total percentage of variations due to heterogeneity in each study.A P value for the Cochran Q test was < 0.05 and I 2 exceeded 50%, which was used as the cut-off value indicating a statistically significant heterogeneity.Publication bias was presented by using a contour-enhanced funnel plot of standard error against the effect estimate.

Results
Initially, we retrieved 1023 relevant abstracts from Pub-Med, 1125 from Embase and 35 from Cochrane Library using predetermined search terms (Fig. 1).The other three records were obtained by searching the references of related studies.A total of 1132 records were obtained after removal of duplicates.Altogether, 1103 articles were excluded based on the review of abstracts, leaving 29 articles assessed in full text.Among them, 14 articles met the requirements.The statistical information of two articles was insufficient, and the remaining 12 studies, including seven case-control and five cohort studies, were included in the meta-analysis.All studies were published between 1994 and 2021.These studies are summarised in Table 1.
Table 2 summarises the genome sequencing techniques used in the included literature.Dobbins et al. [26] and Akcay et al. [24] applied the exome sequencing to analyse cancer susceptibility genes.Other studies used firstgeneration sequencing to detect the BRCA1 and BRCA2 genes.
Various research methods were reported.Seven case-control studies examined patients with CRC and explored the probability of BRCA gene mutations.Five cohort studies calculated the risk of CRC in BRCA gene

Records identified through database searching N=2183
Additional records identified through other sources N=3 Records screened after removal of duplicates N=1132 Full-text articles assessed for eligibility N=29
To generalize to a wider population, we performed a subgroup analysis of patients with non-Ashkenazi Jewish inheritance, which showed that BRCA mutation frequencies were statistically higher in non-Ashkenazi CRC cases, with no heterogeneity (OR = 1.61, 95% CI = 1.26-2.07,P = 0.0002) (Fig. 3).
The funnel plots were symmetric, indicating that there was no evidence of publication bias in the studies included in the meta-analysis (Fig. 4).Applying the leaveone-out sensitivity analysis did not significantly alter the pooled estimates of the association between BRCA mutation and CRC risk, except in one study [20].The results of the sensitivity analysis were shown in Fig. 5.

Discussion
This meta-analysis involved a total of seven case-control and five cohort studies and showed the proportion of BRCA1 and BRCA2 gene mutations is increased in patients with CRC.Several published studies support this conclusion.Yurgelun et al. [33] reported 1% BRCA1/2 mutations in a series of 1058 CRC patients, which was greater than the expected prevalence of BRCA1/2 mutations (0.25%;1:400).Akcay et al. [24] reported three BRCA1/2 pathogenic mutations in 189 CRC patients (1.6%) compared to two in 490 cancer-free elderly controls (0.4%).Fujita et al. [20] studied 12503 unselected CRC patients and concluded that BRCA1 (OR = 2.6, P = 0.034) and BRCA2 (OR = 1.9, P = 0.0041) mutations were significantly associated with CRC.
In this meta-analysis, we were surprised to find a potential protective effect of BRCA gene mutations against colorectal cancer in three studies that did not distinguish between BRCA1 and BRCA2 (Fig. 2 1.1.3).This finding was more pronounced in two studies that were published earlier [25,28].Both studies detected only three common founder mutations in BRCA1 and BRCA2.In contrast, Akcay et al. [24] used nextgeneration sequence-based multigene panel assays BRCA1 has vital roles in multiple DNA repair pathways (particularly homologous recombination, nonhomologous end-joining and single-strand annealing) and checkpoint regulation [34,35].The primary function of BRCA2 is in homologous recombination.BRCA2 mediates the Fig. 4 Funnel plot of standard error by effect estimate for overall meta-analysis of the association between BRCA mutation and colorectal cancer Fig. 5 Sensitivity analysis of meta-analysis of the association between BRCA gene mutations and colorectal cancer risk recruitment of the recombinase to DNA double strand breaks, which is not only essential for homologous recombination but is also responsible for the tumour's suppressive function of this repair process [36].Loss of BRCA1 or BRCA2 function in normal cells leads to growth defects that, combined with a subsequent loss of other DDR mediators, promote tumour development.
BRCA1 and BRCA2 are detected in at least 5% of unselected patients with breast cancer and in approximately 30% of patients with a positive family history of breast or ovarian cancer [37,38].The presence of these mutations is associated with younger age at cancer diagnosis and higher risk of cancer recurrence [39,40].Currently, effective biomarker targeted oral medications, namely poly (ADP-ribose) polymerase (PARP) inhibitors, have been approved by the U.S. Food and Drug Administration (FDA) and by the European Medicines Agency (EMA).PARP inhibitors exploit and exacerbate these tumour vulnerabilities by inducing DNA damage, preventing DNA repair and amassing unresolved replication intermediates that instigate replication and mitotic catastrophe [41].Olaparib, a PARP inhibitor, was approved for human epidermal growth factor receptor type 2 (HER2)negative locally advanced or metastatic breast cancer with germline BRCA1/2 mutations, and as maintenance therapy for platinum-sensitive advanced ovarian cancer with germline mutations in DNA repair genes BRCA1/2 [42,43].A meta-analysis showed that breast or ovarian cancer patients carrying BRCA gene mutations significantly benefited progression free survival (breast cancer: HR, 0.64, 95% CI, 0.55-0.75,P < 0.001; ovarian cancer: HR, 0.33, 95% CI, 0.27-0.42,P < 0.001) by the addition of PARP inhibitors to conventional therapy [44].In addition, multiple clinical trials have shown the efficacy of PARP inhibitors in BRCA-mutated prostate cancer, pancreatic cancer, and small-cell lung cancer (SCLC) [45][46][47].The efficacy of PARP inhibitors in BRCA-mutated colorectal cancer is worthy of expectation.
The incidence of colorectal cancer is rising at an alarming rate among young adults aged between 18 and 50 years [48].The proportion of BRCA1 and BRCA2 mutation carriers is high among patients with youngonset (aged 18-40 years) invasive breast cancer cancer [49].Similar findings have been reported in young-onset colorectal cancer.Suchy et al. [31] found an excess of BRCA1 mutations in 851 patients who were diagnosed with colorectal cancer at age 60 or earlier compared with 4,570 population controls.Phelan et al. [16] followed about 7000 BRCA1/2 mutation carriers and showed a nearly five-fold risk for CRC in female carriers of the BRCA1 gene mutation who were below the age of 50 compared with general population.Unfortunately, we were unable to assess the association between BRCA1 or BRCA2 mutations and risk for young-onset CRC in this meta-analysis, since authors have different definitions of 'young-onset' and set different standards, which interferes with the analysis.Some studies have reached different conclusions.Oh et al. [22] included 14 studies prior to 2017 for meta-analysis and concluded that the risk of CRC is moderately elevated in BRCA1 but not in BRCA2 mutation carriers.But this meta-analysis included pedigree studies and putative BRCA mutation carriers as many of the included patients did not undergo formal genetic testing.Cullinane et al. [21] conducted a meta-analysis of seven cohort and four case-control studies and reported no significant increase in CRC risk in BRCA1/2 mutation carriers.Both of the systematic reviews included studies prior to 2018.Our meta-analysis updated two recent studies [20,24] and drew interesting conclusions that BRCA1/2 gene mutations were increased in patients with CRC.The Fujita et al. [20] 2020 study had a high relative weight in both the BRCA1 and BRCA2 subgroup analyses, which may be one reason why the results of this study differ from those of other studies.Besides, the development of genome sequencing technology may be another vital reason.
Most studies used Sanger sequencing, named as first-generation sequencing technology.This technique can only obtain one sequence per reaction and the sequencing throughput is very low [50].The BRCA1 gene contains 22 exons spanning approximately 110 kb of DNA; it is difficult to cover the complete sequence through the Sanger sequencing.Thus, based on Sanger sequencing, the early cohort research concentrated on only fewer mutation sites of a gene.For example, Niell et al. [30] only investigated the association between the mutations of BRCA1 187delAG and 5385insC and BRCA2 6174delT and increased risk of CRC in 1422 cases and 1566 controls.Genome sequencing became even more far reaching along with the introduction of next-generation sequencing (NGS) methods in 2005.The biggest advantage of second-generation sequencing over first-generation sequencing is its high throughput and low cost per base, which greatly promotes the popularization of gene sequencing [51].Two studies( Dobbins et al. and Akcay et al.) [24,26], which used second-generation sequencing, were included in the present meta-analysis.These studies provided more intact data and made our conclusions more significant.However, no technique is absolutely perfect and the technological limitations of NGS remain.Short reads, assembly of a large number of short fragments and PCR amplification make it easy to cause information loss and reduce the accuracy of sequencing when detecting complex repetitive sequences or long copy number variants.The third-generation single-molecule sequencing technology [52,53] performs de novo sequencing on a single long sequence and can be used to find long-fragment variants on the human genome, creating accurate human genome maps.While the technology is currently limited to the laboratory setting, it is believed that third-generation sequencing could be used in the clinic to identify potential long copy number genetic mutations in the near future.
The present meta-analysis has several limitations.First, we must acknowledge the presence of heterogeneity in the study design of the literature.Several articles included only Ashkenazi Jews, who had higher odds of BRCA mutations.A subgroup analysis was performed to account for this heterogeneity, which suggested that the rate of BRCA mutation remains high in non-Ashkenazi Jews patients with CRC.Besides, Some of the included studies did not distinguish between BRCA1 and BRCA2, and some did not distinguish between colon and rectal cancer [32].Some studies examined multiple genes, but in the case of the BRCA1 and BRCA2 genes, only the three most common founder mutations were included in the analysis [25,28,31].The low mutation rates of BRCA1 and BRCA2 in some studies may be due to the detection of only three founder mutations or limited testing technology.We performed subgroup analyses to reduce heterogeneity in study design, but we were unable to perform subgroup analyses according to cancer type, age, and sex, because of the lack of significant statistical data.Second, most of the included studies were retrospective, and only study was prospective, which may lead to a selection bias.We could not control for the effect of other risk factors on the study results, such as diet, smoking and family history.Each study used different effect measures, including OR, SIR and RR, which must be transformed into OR for meta-analysis.
In conclusion, in this meta-analysis, BRCA genes are one of the genes that may increase the risk of developing colorectal cancer.We, thus, suggest that BRCA genes could be potential candidates which may be included in the colorectal cancer genetic testing panel.
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Table 1
Characteristics of included studies

Table 2
detection methods of included studies Forest plot of the association between overall BRCA mutation and colorectal cancer risk expressed as unadjusted odds ratio.1.2.1 Forest plots of the associations of BRCA1 mutations with colorectal cancer risk.1.2.2Forest plots of the associations of BRCA2 mutations with colorectal cancer risk.1.2.3 Forest plots of the associations of BRCA1/2 mutations with colorectal cancer risk 3ig.3Forest plot of the relationship between BRCA mutations and colorectal cancer risk, expressed as unadjusted odds ratios, among non-Ashkenazic Jews and multiple ligation-dependent probe amplification to detect BRCA1/2 genes and did not reach similar conclusions.BRCA1 is a versatile protein that links DNA damage sensing and DNA damage response (DDR) effectors.