Research ArticleOriginal Article

The mutation landscape of multiple cancer predisposition genes in Chinese familial/hereditary breast cancer families

Li Dong, Hailian Zhang, Huan Zhang, Yingnan Ye, Yanan Cheng, Lijuan Li, Lijuan Wei, Lei Han, Yandong Cao, Shixia Li, Xishan Hao, Juntian Liu and Jinpu Yu
Cancer Biology & Medicine June 2022, 19 (6) 850-870; DOI: https://doi.org/10.20892/j.issn.2095-3941.2021.0011

Abstract

Objective: Approximately 5%–10% of breast cancer (BC) patients display familial traits that are genetically inherited among the members of a family. The purpose of this study was to profile the germline mutations in 43 genes with different penetration rates and their correlations with phenotypic traits in Chinese familial BC families.

Methods: Ion Torrent S5™-based next generation sequencing was conducted on 116 subjects from 27 Chinese familial BC families.

Results: Eighty-one germline mutations in 27 BC predisposition genes were identified in 82.8% (96/116) of the cases. Among these, 80.8% of the mutated genes were related to DNA damage repair. Fourteen possible disease-causing variants were identified in 13 of 27 BC families. Only 25.9% (7/27) of the BC families exhibited hereditary deficiency in BRCA1/2 genes, while 22.2% of the BC families exhibited defects in non-BRCA genes. In all, 41.7% (40/96) of the mutation carriers had BRCA mutations, 88.5% (85/96) had non-BRCA mutations, and 30.2% (29/96) had both BRCA and non-BRCA mutations. The BC patients with BRCA mutations had a higher risk of axillary lymph node metastases than those without mutations (P < 0.05). However, the BC patients with non-BRCA mutations frequently had a higher occurrence of benign breast diseases than those without mutations (P < 0.05).

Conclusions: In addition to BRCA1/2, genetic variants in non-BRCA DNA repair genes might play significant roles in the development of familial/hereditary BC. Therefore, profiling of multiple BC predisposition genes should be more valuable for screening potential pathogenic germline mutations in Chinese familial/hereditary BC.

keywords

Introduction

For Chinese women, breast cancer (BC) has become the most common malignant tumor and the fifth most common cause of cancer death. Approximately 5%–10% of BC patients display familial traits that are genetically inherited among the members of a family1, and are significantly regulated by varied genetic factors. Among these genetic factors, driver genes directly stimulate BC carcinogenesis, while predisposition genes generally increase the hereditary genetic risk of BC and are the most important causes of familial clustering in BC. However, no study focusing on the germline genetic profiling of multiple BC predisposition genes has been reported in Chinese hereditary BC families. BRCA1 and BRCA2 are 2 well-known high penetration BC predisposition genes in hereditary BC2. BRCA1/2 mutations are characteristic of an increased lifetime risk for hereditary breast and ovarian cancer syndrome3. The cumulative risk of BC in women with BRCA mutations is as high as 80% by the age of 704,5. Clinical studies have shown that patients with BRCA mutations have a higher incidence of early-onset BC, bilateral BC, triple-negative BC, lymph node metastasis, and ipsilateral and contralateral BC recurrence6–9. Patients with BRCA-related BC are also at high risk for other cancers, such as pancreatic cancers, gastrointestinal malignancies, and melanomas10. Identification of germline mutations in BRCA1/2 will not only help to identify high risk hereditary BC patients, but will also change screening, cancer risk management, and therapeutic strategies for their family members.

However, only 20%–40% of familial hereditary BC are caused by BRCA1/2 mutations11. There is a large percentage of familial BC not associated with BRCA1/2 mutations. Currently, more BC predisposition genes have been identified, including genes with high penetration (TP53, CDH1, PTEN, and STK11), moderate penetration (PALB2, CHEK2, ATM, NBN, etc.), and low penetration (MLH1, MSH2, MSH6, PMS2, MEN1, etc.)12–14. Most BC predisposition genes are DNA damage repair (DDR)-related genes. DDR is an important part of the mammalian cell defense mechanism and includes 5 different but functionally interrelated pathways: base excision repair (BER), nucleotide excision repair, mismatch repair (MMR), homologous recombination repair (HR), and nonhomologous end joining15. DDR genes recover the DNA damage caused by various factors in vivo and in vitro, thus maximizing the stability of genetic material. The decline or lack of DDR ability can lead to genome instability and the occurrence of cancer16.

It has been reported that genomic instability caused by DDR gene deficiency is one of the most important reasons for the occurrence of BC17–19. Comprehensive screening of genetic variants of DDR genes would therefore help to precisely evaluate hereditary susceptibilities to BC in high risk families. Next-generation sequencing (NGS) has recently enabled massive parallel sequencing at low cost, which makes high-throughput gene testing commercially available for hereditary BC susceptibility assessment with high accuracy and high efficiency20.

In this study, a total of 116 subjects from 27 Chinese hereditary BC families were enrolled, including both BC patients and their relatives. Ion Torrent S5™-based NGS was conducted to detect multiple types of germline variants in 43 genes and compare their correlations with phenotypic traits. We found that 80.8% of the mutated genes were related to DDR. Only 25.9% of BC families exhibited hereditary deficiency in BRCA1/2 genes, while 22.2% of the BC families exhibited defects in non-BRCA genes. The BRCA mutation patients had a higher occurrence of axillary lymph node metastases, while the non-BRCA mutation patients frequently had a higher occurrence of benign breast diseases than those without mutations. Genetic variants in non-BRCA DDR genes might therefore play significant roles in the development of Chinese familial/hereditary BC, and more extensive BC predisposition genes should be considered to evaluate hereditary BC susceptibilities in high risk families.

Materials and methods

Sample collection

A total of 27 hereditary BC families in China were enrolled and admitted to the Second Department of Breast Cancer of Tianjin Medical University Cancer Institute and Hospital (TMUCIH) from January 2017 to January 2019. The criterion for the collection of hereditary BC families was that the families should include ≥ 2 patients with breast and/or ovarian cancer among first- and second-degree relatives. This study was approved by the Ethics Committee of Tianjin Medical University (Approval No. Ek2018050). Written consent was obtained from all patients.

We selected at least 1 BC patient and 1 family member from each hereditary BC family. Finally, a total of 116 subjects from 27 families were collected, which included 45 patients (42 BC, 2 ovarian cancer, and 1 endometrial cancer) and 71 healthy family members. All subjects were Chinese. Among the 42 BC patients, 36 were initially treated at TMUCIH. Clinical characteristics for these 36 patients were collected, including the age of onset, unilateral/bilateral, primary tumor diameter size, regional lymph node status, clinical stage, tumor grade, histological type, luminal type, benign breast disease, and recurrence or metastasis. When the clinical characteristics of the 36 patients were analyzed, the data of the additional 47 BC patients with a family history of BC were also included. These 47 familial BC patients were all females and were also treated at TMUCIH. The age of the patients ranged from 26–76 years. The median age was 51 years and the average age was 50.1 years. Of them, 31.9% were younger than 45 years, 36.1% had lymph node metastasis, 17.0% were triple negative BC, 29.7% were in stage III, and 21.2% were histological grade III (details are shown in Supplementary Table S1). All 47 familial BC patients were sequenced by the same assay panel as the 27 pedigree samples.

This study was approved by the ethics committee of TMUCIH, and all included subjects signed informed consent forms.

NGS panel

In this study, the 43 genes selected were as follows: AKT1, APC, ATM, ATR, BAP1, BARD1, BLM, BRAF, BRCA1, BRCA2, BRIP1, CCND1, CDH1, CDK4, CHEK2, CYP1B1, EGFR, EPCAM, ERBB2, ERBB4, ERCC1, FANCD2, FANCI, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, NOTCH1, PALB2, PIK3CA, PMS2, PTEN, RAD50, RAD51C, RAD51D, RET, SMAD4, STK11, TP53, XPC, and XRCC1 (Table 1). Most of the 43 genes, such as APC, ATM, ATR, BAP1, BARD1, BLM, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, CYP1B1, EPCAM, ERCC1, FANCD2, FANCI, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, STK11, XPC, and XRCC1 were DDR genes. The panel was composed of the whole coding sequence and splicing region (exonic boundaries ± 10 bp) of each gene. The target region size of the panel was 114 kb, and 99% of the target region was covered with 1,352 amplicons (Analyses, Beijing, China).

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Table 1

Gene list

NGS and data processing

Genomic DNA was extracted from peripheral blood samples (2–5 mL) using a QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The DNA library was constructed using GO prep kits (Analyses), in which every library from different samples was marked with varied indices. The prepared libraries were sequenced by Ion S5 (TMO, Shanghai, China). Qualified reads were aligned to the human reference genome hg19 by TMAP (v.5.10). The target regions were sequenced at a depth > 200 times. Germline mutations (SNV/small InDel < 22 bp) were detected using TVC software (v.5.10). Then, Ensembl Variant Effect Predictor software (http://grch37.ensembl.org/info/docs/tools/vep/index.html) was used for variant interpretation, such as HGVS notation, Population Allele Frequencies from GnomAD (http://gnomad.broadinstitute.org/), 1K Genomes Project (http://www.1000genomes.org), Clinical Significance States assigned by HGMD (http://www.hgmd.cf.ac.uk/), ClinVar (http://www.ncbi.nlm.nih.gov/clinvar/), BRCA Exchange (https://brcaexchange.org/), and BIC (https://research.nhgri.nih.gov/bic/). In addition, all significant mutations, including pathogenic variants, likely pathogenic variants, and variants of uncertain significance (VUS) were confirmed by Sanger sequencing.

Variant classification

All mutations were classified according to the American College of Medical Genetics (ACMG) professional practice and guidelines21. Following the principles of the ACMG, all germline mutations were classified as pathogenic (P), likely pathogenic (LP), uncertain significance (US), likely benign (LB), or benign (B). Each pathogenic criterion was weighted as very strong (PVS1: nonsense, frameshift, canonical ± 1 or 2 splice sites, initiation codon), strong (PS1–4); moderate (PM1–6), or supporting (PP1–5), and each benign criterion was weighted as stand-alone (BA1), strong (BS1–4), or supporting (BP1–6). The other mutations were classified as variants of uncertain significance (VUS).

Statistical analysis

The correlations between genetic variants and clinicopathological characteristics of patients were determined using the Student’s t-test, the chi-square test, or Fisher’s precise test. P-values less than 0.05 were considered to be statistically significant.

Results

Quality assessment of sequencing data

More than 26.7 GB of sequencing data were generated from 116 clinical samples. An average of 1.0 million reads for each sample was obtained. The depth of each variant was mainly 200–1,500×, and the average depth was more than 800× (Figure 1A). On average, 98.9% of all reads could be mapped back to the hg19 genome (Figure 1B) and 96.0% of all reads were mapped to targeted regions (Figure 1C). After variant calling, the allele fraction plots of all the variants demonstrated a clear bimodal distribution pattern peaking at 0.5 and 1.0, which indicated that a typical distribution pattern of germline mutations was achieved (Figure 1D).

Figure 1
Figure 1

Quality assessment of the sequencing data. (A) The sequencing depth of variants. (B) Percentage of all mapped reads for samples. (C) Percentage of reads mapped to target regions for samples. (D) The distribution of allele fractions across all identified variants.

Identification of germline mutations

We detected 37,009 variants among 43 genes in 116 subjects from 27 families. After variant filtering (Figure 2), 81 germline mutations in 26 genes were identified in 96 subjects (Figure 3A; more details are available in Supporting Information Supplementary Table S2). The genes with ≥ 5 mutations were BRCA1, BRCA2, ATM, BLM, BRIP1, MSH6, and RAD50. Of the mutated genes, 80.8% (21/26) were DDR genes (ATM, BRCA1, BRCA2, BAP1, BARD1, BRIP1, BLM, CHEK2, FANCD2, FANCI, MRE11A, NBN, PALB2, RAD50, RAD51C, MLH1, MSH2, MSH6, EPCAM, PMS2, and MUTYH), and 19.2% (5/26) were driver genes (APC, CDH1, RET, STK11, and TP53). Among these DDR genes, 71.4% (15/21) were involved in HR, 23.8% (5/21) were involved in MMR, and 4.8% (1/21) were involved in BER. Of the 81 mutations, 67.9% were found in HR genes, 19.8% in MMR genes, 2.5% in BER genes, and 9.9% in driver genes (Figure 3B). More than 90% of the mutations occurred in DDR genes. Of these mutations, 10 (12.3%) were pathogenic or likely pathogenic (P/LP), and 71 (88.7%) were VUS. There were 7 P/LP mutations detected in BRCA1/2 genes and 3 P/LP mutations detected in non-BRCA genes. Four VUS were considered high risk based on software predictions and literature reports. In this article, P/LP mutations and high risk VUS were defined as possible disease-causing mutations22.

Figure 2
Figure 2

Filtering steps of mutations. *Predicted as damaging by multiple software programs or reported in cancer patients.

Figure 3
Figure 3

Distribution of the mutated genes. (A) Number of mutations in the mutated genes. (B) Percentage of mutations in different gene types. HR: homologous recombination repair, MMR: mismatch repair, BER: base excision repair.

The correlation between genetic mutations and hereditary BC families

Among the 27 familial BC families, 48.1% (13/27) had possible disease-causing mutations in known BC predisposition genes, including BRCA1, BRCA2, BLM, BRIP1, MSH2, MSH6, RAD51C, and RET. The cause of hereditary BC in 51.9% (14/27) of the families was unknown. Hereditary BC in 25.9% (7/27) of the families was associated with BRCA1/2 genes, while that in 22.2% (6/27) was associated with non-BRCA genes (Table 2). This showed that testing the non-BRCA genes increased the detection of hereditary BC by 22.2%.

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Table 2

The correlation between mutation genes and cancer types of hereditary breast cancer families

Of these 27 families, 11 (40.7%) were characterized by BC only, 4 (14.8%) by both BC and ovarian cancer, and 12 (44.5%) by other cancer types besides BC (and ovarian cancer), such as lung cancer, stomach cancer, esophageal cancer, colorectal cancer, and endometrial cancer (Table 3). Of the families with BC only, 27.3% (3/11) were related with BRCA genes and 18.2% (2/11) were related with non-BRCA genes. However, of the families with cancer types other than BC (and ovarian cancer), more were related with non-BRCA genes than BRCA genes (33.3% vs. 16.7%) (Table 2).

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Table 3

The corresponding variants and the cancer types of each family

The distribution of possible disease-causing mutations in BRCA1/2 genes

Among the mutation carriers, 24.0% (23/96) carried possible disease-causing mutations in BRCA genes. Of them, 47.8% (11/23) were carriers of the BRCA1 gene, and 52.2% (12/23) were carriers of the BRCA2 gene. Therefore, mutation carriers of the BRCA2 gene occurred 1.09 times more frequently than those of the BRCA1 gene in these 27 Chinese hereditary BC families.

Four possible disease-causing mutations in the BRCA1 gene were found in 27 familial BC families. There were 3 mutations located in exon 10 and 1 located in exon 23 (Figure 4A). BRCA1 p.Glu1836fs was located in the BRCT2 domain of BRCA1. The BRCT domain is found in a large variety of proteins involved in DNA repair, recombination, and cell cycle control, and functions as a protein-protein interaction module23,24. BRCA1 p.Thr327fs was located upstream of the serine-rich domain associated with BRCT and found in a family with 2 BC patients, 1 of whom was diagnosed with BC at 32 years of age and died of BC. The healthy member with this mutation is taking tamoxifen orally to prevent BC under the guidance of her physician. BRCA1 p.Asp1362fs and p.Leu1306fs were not located in the functional domain of BRCA1. Both were found in a family with BC and ovarian cancer. Four possible disease-causing mutations in the BRCA1 gene previously found in 146 sporadic BC patients were located in exons 4, 10, 23, and all in the functional domain22. There was no significant difference in the location of these mutations in the BRCA1 gene between pedigrees and sporadic patients. Of the 8 possible disease-causing mutations in the BRCA1 gene, 7 (87.5%) were frameshift mutations, and 5 (62.5%) were located in the functional domain of the BRCA1 gene, especially BRCT2 (Figure 4A). The patients with BRCA1 p.Ile1824fs and p.Leu1306fs were diagnosed with BC at the age of ≤ 45 years and had lymph node metastases. The tumor-node-metastasis (TNM) stage of BRCA1 p.Ile1824fs mutation carriers was stage III. The patient with BRCA1 p.Leu481fs was diagnosed with BC at the age of > 45 years but had lymph node metastasis and was in stage III. The BRCA1 p.Asp1362fs mutation carriers were ≥ 45 years of age, in stage I, and had no lymph node metastasis.

Three possible disease-causing mutations in the BRCA2 gene were found in 27 familial BC families and located in exons 3, 15, and 23 (Figure 4B). BRCA2 p.Arg2520Term was located in the helical domain of BRCA2. The region interacts with the DSS1 (deleted in split hand/split foot) protein in mammalian cells, which is required for normal cell growth25. BRCA2 p.Glu97Term and p.Ser2984Term were not located in the functional domain of BRCA2. BRCA2 p.Glu97Term was found in a family with 2 BC patients. The onset age of the patients was over 50 years. BRCA2 p.Ser2984Term was found in a family with 3 BC patients. Among them, 1 patient developed BC at the age of 35 years, and 1 patient experienced contralateral BC after she was diagnosed with BC at 42 years of age. Six possible disease-causing mutations in the BRCA2 gene found previously in 146 sporadic BC patients were located in exons 3, 11, 19, and 23. The distribution of possible disease-causing mutations found in sporadic patients may be more dispersed in the BRCA2 gene22. Of the 9 possible disease-causing mutations of the BRCA2 gene, 6 (66.7%) were nonsense mutations, and mutations within exon 11 of BRCA2 were the most common. We found that most of the mutations carried by patients with an onset age of ≤ 45 years were located in the region of exon 15 or behind exon 15, and 80.0% of the patients with BRCA2 possible disease-causing mutations had lymph node metastases. The patients with BRCA2 p.Glu38Lys, p.Val2050fs, p.Arg2520Term, p.Ser2984Term, and p.Trp2990Term were diagnosed with BC at the age of ≤ 45 years and had lymph node metastases. The TNM stage of BRCA2 p.Glu38Lys, p.Val2050fs, and p.Trp2990Term mutation carriers was stage III. The BRCA2 p.Ser1404Term mutation carriers were more than 45 years of age, were in stage I, and had no lymph node metastasis.

Figure 4
Figure 4

The distribution of BRCA1/2 mutations in databases (HGMD, ClinVar, and Gnomad) and the disease-causing mutations in BRCA1/2 genes and its effects on the BRCA protein. (A) The disease-causing mutations in the BRCA1 gene. (B) The disease-causing mutations in the BRCA2 gene.

Possible disease-causing mutations in non-BRCA genes

Seven possible disease-causing mutations in non-BRCA genes were found in 27 familial BC families including 3 likely pathogenic mutations in the BLM, BRIP1, and MSH6 genes, and 4 high risk VUSs in the BLM, MSH2, RAD50C, and RET genes. BLM p.Asp1116fs was not present in population databases such as ExAC, gnomAD, and 1,000 Genomes. This sequence change duplicated 1 nucleotide from exon 17 of the BLM mRNA (c.3349dupA), causing a frameshift variant at codon 1,116. Loss-of-function variants in the BLM gene are known to be the pathogenic mechanism for Bloom syndrome26. The sequence change of BRIP1 p.Lys222Term replaced A with T from exon 7 of the BRIP1 mRNA (c.664A>T), causing a nonsense mutation at codon 222. It was also not present in population databases such as ExAC, gnomAD, and 1,000 Genomes and was recorded as pathogenic in the ClinVar database (SCV001187697). MSH6 p.Arg841fs was not present in population databases such as ExAC, gnomAD, and 1,000 Genomes. This sequence change deleted 1 nucleotide from exon 4 of the MSH6 mRNA (c.2522delG), causing frameshift variants at codon 841. Loss-of-function variants in the MSH6 gene are known to be the pathogenic mechanism for Lynch syndrome27,28. According to the guidelines of the ACMG, these 3 mutations were classified as likely pathogenic.

Four VUS were considered high risk in this study, namely, BLM p.Leu60Ile, MSH2 p.Met688Ile, RAD50C p.Arg370Term, and RET p.Glu632Lys. BLM p.Leu60Ile, MSH2 p.Met688Ile, and RET p.Glu632Lys were predicted to be damaging by multiple software programs. BLM p.Leu60Ile was recorded as having conflicting interpretations of pathogenicity in the ClinVar database without clinical information. MSH2 p.Met688Ile was recorded with uncertain significance in the ClinVar database and reported in colorectal cancer, endometrial cancer, and Lynch syndrome29–32. RET p.Glu632Lys was recorded with uncertain significance in the ClinVar database and reported in medullary thyroid carcinoma33,34, Hirschsprung’s disease35, esophageal cancer36, colorectal cancer37 and sporadic pheochromocytoma38. The nonsense mutation RAD50C p.Arg370Term exhibited the termination codon at 370 amino acids. It was not clear whether RAD50C p.Arg370Term would lead to nonsense mutation-mediated mRNA decay because it was located in the last exon. It was also recorded with uncertain significance in the ClinVar database. Overall, the studies have shown that deletion of the terminal 11 amino acid residues led to cell localization errors of the RAD51C protein39.

Comutation of BRCA and non-BRCA genes in the BC pedigree

In this study, 82.76% (96/116) of all subjects were found to carry at least 1 gene mutation. Only 34.4% (33/96) of the mutation carriers had 1 mutation, while 65.6% (63/96) had ≥ 2 mutations simultaneously. It is common that 1 person carries > 1 mutation, which was detected either in BRCA1/2 genes or non-BRCA genes (Figure 5). Twenty-nine (25.0%, 29/116) subjects carried both BRCA and non-BRCA mutations, namely, 12 patients and 17 healthy members. These non-BRCA mutations occurred in the ATM, BAP1, BLM, BRIP1, CHEK2, EPCAM, MLH1, MSH2, MSH6, MUTYH, PMS2, RAD50, RAD51C, and TP53 genes, 92.9% of which were related to DDR genes. Among the comutation samples of BRCA and non-BRCA genes, there were 19 carriers with possible disease-causing mutations in BRCA genes and 5 carriers with possible disease-causing mutations in non-BRCA genes. Two carriers had possible disease-causing mutations in both BRCA genes and non-BRCA genes. Both were from 1 family with 1 BC patient and 1 ovarian cancer patient.

Figure 5
Figure 5

The distribution of mutations and clinical features across all subjects.

The clinicopathological characteristics of hereditary BC patients with varied genetic mutations

According to the mutation status (P, LP, VUS), 36 BC patients were divided into 3 groups: the BRCA mutation group (n = 9), non-BRCA mutation group (n = 20), and nonmutation group (n = 7) (Table 4). The BRCA mutation group had a comparably higher risk of axillary lymph node metastasis than the nonmutation group (77.8% vs. 28.6%, P = 0.049). There was no significant difference in age, unilateral or bilateral, tumor size, TNM stage, tumor grade, histological type, luminal type, history of benign breast diseases, recurrence, or metastasis between the BRCA mutation group and the nonmutation group. In contrast, the non-BRCA mutation group had a significantly higher occurrence of benign breast disease before BC than the nonmutation group (70.0% vs. 14.3%, P = 0.021). However, there was no significant difference in other parameters.

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Table 4

Clinicopathological characteristics among 36 patients with different mutation status

The average onset age of BC in the BRCA mutation group was younger than that in the nonmutation group (44.8 ± 9.5 vs. 51.3 ± 6.8) (Table 4). However, the difference was not statistically significant. The small sample size might be an important factor. We therefore included an additional 47 BC patients with family histories of BC. Among these 83 familial BC patients, 20 had mutations in BRCA1/2 genes, 25 had non-BRCA mutations, and 38 had no mutations. We compared the correlations between BRCA mutation status and the clinical and pathological features of patients (Table 5). The results showed that the average onset age in the BRCA mutation group was significantly younger than that in the nonmutation group (45.7 ± 9.6 vs. 51.9 ± 8.7, P = 0.015). Furthermore, the percentages of young BC (55.6% vs. 28.6%, P = 0.023), lymph node metastatic (70.0% vs. 28.9%, P = 0.005), clinical stage III (35.0% vs. 18.4%, P = 0.011), and triple-negative BC (40.0% vs. 7.9%, P = 0.002) were higher in the BRCA mutation group than in the nonmutation group. In contrast, no significant difference was detected when comparing the above clinicopathological features between the non-BRCA mutation group and the non-mutation group.

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Table 5

Clinicopathological characteristics among 83 patients with different mutation status

Classical pedigree analysis of hereditary BC families

Figure 6 shows several representative families enrolled in this study. As shown in Figure 6A, the family included 4 BC patients, of whom 1 died and 1 suffered bilateral BC. We collected samples from other patients. The sequencing results showed that all 3 patients carried BRCA2 p.Arg2520Term and CHEK2 p.Ala480Thr mutations. According to the ACMG classification, CHEK2 p.Ala480Thr is a VUS and BRCA2 p.Arg2520Term is a pathogenic mutation. Therefore, the BRCA2 p.Arg2520Term mutation may be the genetic pathogenic mutation of this family. The earliest onset age of BC patients in this family was 30 years of age, and the oldest was 40 years of age. The onset age of BC caused by the BRCA2 p.Arg2520Term mutation may be earlier. In the fourth generation, 2 young family members, 26 and 22 years of age, respectively, also carried BRCA2 p.Arg2520Term. Although they are still healthy, it was recommended that prevention and follow-up should be strengthened.

Figure 6
Figure 6

The family tree of classical pedigrees in this study.

As shown in Figure 6B, the pathogenic mutation BRCA1 Asp1362fs was found in 1 BC patient, 1 ovarian cancer patient, and 2 healthy family members. The BC patient was diagnosed at the age of 58 years, and the ovarian cancer patient was diagnosed at the age of 63 years. BC or ovarian cancer caused by BRCA1 Asp1362fs may develop later. It is noteworthy that their mother was not a cancer patient, while 1 of their maternal aunts (their mother’s sister) suffered from BC. Genetic testing for these patients was not available because they had died. In addition to BC and ovarian cancer, this family also had 1 esophageal cancer patient. However, there was no definite link between the occurrence of esophageal cancer and the BRCA1 Asp1362fs mutation. The 2 healthy family members with BRCA1 Asp1362fs were 32 and 29 years of age, respectively. They may have not reached the age of onset. However, early prevention and regular physical examination were recommended.

Figure 6C shows a family with 2 BC patients. Patient III.1 was diagnosed with BC at the age of 57 years. Her sister (Patient III.2) was diagnosed with BC at the age of 52 years. They both carried BLM p.Asp1116fs and BRCA2 p.Ile1929Val. According to the ACMG classification, BLM p.Asp1116fs is a likely pathogenic mutation and BRCA2 p.Ile1929Val is a VUS. The BLM gene encodes the DNA helicase RecQ protein on chromosome 15q26, which unwinds a variety of DNA substrates including Holliday junctions, and is involved in several pathways contributing to the maintenance of genome stability40. BLM p.Asp1116fs was presumably the main genetic cause of BC in this family. One of the healthy family members was also detected with BLM p.Asp1116fs at the age of 37 years. Because she might have a high risk of BC, we suggested that she have regular physical examinations for the possible early prevention of BC.

In Figure 6D, we collected 5 samples from this family. BC patient II.1 was detected with BRCA1 p.Glu1836fs and BRIP1 p.Lys222Term. These 2 mutations are likely pathogenic according to the ACMG guidelines. Her mother had ovarian cancer. Because she had already died, her sample could not be collected. One healthy family member was detected with p.Glu1836fs and BRIP1 p.Lys222Term and another with BRCA1 p.Glu1836fs. BRCA1 p.Glu1836fs has been reported in a Chinese BC patient41 and recorded as pathogenic in the LOVD database (https://brcaexchange.org/variant/451386). It was indicated that mutation of BRCA1 p.Glu1836fs can lead to the occurrence of BC without the mutation of BRIP1 p.Lys222Term. The protein encoded by BRIP1 interacts with the BRCT repeats of BRCA1 protein. The bound complex is important in the normal double-strand break repair function of BRCA142. In this family, healthy carriers of likely pathogenic mutations were also recommended to seek close follow-up and early prevention of BC.

Annual breast magnetic resonance imaging or mammography screening is recommended for younger pathogenic mutation carriers. For healthy women with BRCA pathogenic mutations who have no fertility requirements, chemoprevention or risk-reduction surgery is recommended to reduce the risk of BC occurrence43.

Discussion

We performed NGS for 116 subjects from 27 familial BC families based on the Ion Torrent S5 platform. The average sequencing depth was more than 800×. The percentage of hereditary BC in families caused by mutations in known genetic predisposition genes was approximately 48.1%, which was higher than that of other reports of familial BC44–46. This was probably because of the high sensitivity of amplicon-based NGS. In addition, the range of genes tested was increased in this study.

In this study, 43 genes were used to detect the hereditary risk of familial BC and other BC-related inherited syndromes. Most of them, such as AKT1, APC, ATR, ATM, BAP1, BARD1, BLM, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, ERBB2, ERCC1, FANCI, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PALB2, PIK3CA, PMS2, PTEN, RAD50, RAD51C, RAD51D, RET, STK11, and TP53 have been reported to be associated with BC susceptibilities14,47–51. In addition, there are still some cancer predisposition genes reported in other BC-related inherited syndromes, such as CYP1B1, CCND1, CDK4, ERBB4, FANCD2, NOTCH1, SMAD4, XPC, and XRCC152–55, most of which belong to DDR-related genes.

BRCA1 and BRCA2 are 2 well-known high penetration predisposition genes in hereditary BC. Hereditary BC with BRCA mutations is more invasive than that without BRCA mutations9,22,56. In this study, comparative analyses of clinicopathological features also showed that patients with BRCA mutations had a younger age of onset, more advanced stage, and higher risk of axillary lymph node metastasis than those without mutations. The mutation prevalence of BRCA is distinct in different countries. In this study, BRCA-related families accounted for 25.9% of the 27 familial BC families. In a German study including 21,401 families with familial breast or ovarian cancers, the percentage of BRCA-related families was 24.0%44. According to another study57, which analyzed comparative families with ≥ 2 cases of breast and/or ovarian cancer among first- and second-degree relatives, the percentage of BRCA-related families was 46.2% in 78 Caucasian families, 68.9% in 29 Ashkenazi Jewish families, and 27.9% in 43 African families. The prevalence of BRCA1/2 mutations in this study was comparably higher than those reported in other Chinese familial BC cohorts. According to previous studies6,22,56, the prevalence of BRCA1/2 mutations was 12.7%–19.1% in Chinese familial BC patients, which is distinct from the prevalence of BRCA1/2 mutations in this study. The disparity might be caused by differences in the study populations and the methods of statistical analyses. In our study, we focused on the prevalence of BRCA1/2 mutations in each hereditary BC family, including both familial BC patients and their direct relatives. The prevalence of BRCA1/2 mutation was significantly higher compared to those studies including familial BC patients only. In addition, different geographical areas, ethnic groups, genetic testing methods, as well as limited sample sizes might have contributed to the disparity in the prevalence of BRCA1/2 mutations. We plan to enroll more hereditary BC families in the future to validate our findings.

We found that the mutation frequency of the BRCA2 gene was 1.09 times that of the BRCA1 gene in these BC families. In other reports based on a Chinese population, Zhang et al.46 reported in 409 Chinese familial BC patients that the BRCA2 mutation frequency was 1.7 times (6.6%/3.9%) higher than the BRCA1 mutation frequency. However, this result was inconsistent with other reported findings, which indicated that the mutation frequency of the BRCA1 gene was considerably higher than that of the BRCA2 gene in European and American populations57,58. Another important difference is the penetrance of BRCA. It is well-documented that Western women who carry a pathogenic BRCA1 or BRCA2 mutation may have a 57%–65% or 45%–49% risk of developing BC by the age of 70 years59,60. Women of Ashkenazi Jewish and Icelandic descent who carry a BRCA1/2 mutation have a BC risk as high as 70% by the age of 70 years3,61,62. However, the breast cancer risk for BRCA1/2 mutation carriers is only 35%–49% in women from Australia, the UK, and the Republic of Korea3,63,64. A study based on a Chinese population65 reported that the estimated cumulative risks of BC by the age of 70 years were 37.9% for BRCA1 mutation carriers and 36.5% for BRCA2 mutation carriers. The differences might be predominantly derived from the disparities in ethnic groups, which should be thoroughly investigated in a large-scale random case-control trial, especially in the Chinese population.

Of the 116 subjects in this study, 48.6% were found to exclusively carry mutations in non-BRCA genes. Testing for non-BRCA genes increased the detection of hereditary BC families by 22.2%. Similar results were also found in other reports. According to the Lin et al.66 study, the mutation prevalence of BRCA1/2 in Han Chinese patients with early onset or with a significant family history was 15.0%, and there was a 7.5% mutation of non-BRCA genes in women who tested negative for BRCA1/2 mutations. In another study of German BC patients, extended testing beyond BRCA1/2 also identified a deleterious mutation in an additional 6% of patients67. Our previous findings also showed that the percentage of possible disease-causing mutation carriers among BC patients with a family history increased from 21.3% to 27.7% when the sequenced genes were increased from 6 to 2022. Therefore, broader panel testing including more genes would significantly increase the detection percentage of mutation carriers and enhance the screening efficiency for hereditary BC.

We also found that non-BRCA-mutated BC was more likely to be accompanied by benign breast diseases. Benign breast disease is an important risk factor for the development of BC. It has been reported that women with severe atypical epithelial hyperplasia of the breasts were twice as likely to develop BC as women without such diseases68. The non-BRCA gene mutation has a weaker pathogenic effect on carcinogenesis than the BRCA gene69,70. It is reasonable that BC gradually develops from benign breast disease upon stimulation from non-BRCA gene mutations. This could also explain the trend to some degree that the average age of onset of BC patients with non-BRCA mutations was older than that of BRCA-mutated BC patients (55.84 ± 10.50 vs. 45.50 ± 9.24). These findings indicated that genetic variants of those non-BRCA genes also played an important role in the development of hereditary BC. These genetic variants should be further evaluated to predict the hereditary BC predisposition of high risk individuals.

In this study, 80.8% (21/26) of the mutated genes were DDR genes. Among these, 71.4% of DDR genes were involved in the HR pathway. In addition to BRCA1/2 genes, the mutated HR genes also included ATM, BAP1, BARD1, BRIP1, BLM, CHEK2, FANCD2, FANCI, MRE11A, NBN, PALB2, RAD50, and RAD51C. The interaction between BRCA1-BARD1, the BRCA2-PALB2 complex, and the recombinant enzyme RAD51 is an important aspect in the HR process71. The most important function of HR genes is to repair DNA double strand breaks (DSBs), which are the most serious type of DNA damage72,73. Breast cells with homologous recombination repair defects may not be able to initiate HR to repair DSBs. Abnormal repair may lead to chromosome loss, transposition, and other changes. Over time, under the influence of multiple carcinogenic factors, the accumulation of errors leads to the development of BC74. The mutated MMR genes included MLH1, MSH2, MSH6, EPCAM, and PMS2, accounting for 23.8% of the DDR genes. MMR genes prevent mutational events through correction of mismatched bases during DNA replication. Genetic defects in the DNA MMR system result in DNA replication errors, including base substitutions and insertion-deletion loops, known as microsatellite instability75. Germline mutations in MMR genes can give rise to Lynch syndrome (LS), an autosomal-dominant cancer predisposition syndrome that increases the risk for several forms of malignancy, including colorectal (lifetime cancer risk, 70%–80%), endometrial (50%–60%), stomach cancer (13%–19%), and ovarian cancer (9%–14%). BC incidence has been found to be increased in patients with Lynch syndrome76. MMR genes belong to low penetrance genes associated with BC. Studies have suggested that there might be a functional overlap between the MMR and FA-BRCA pathways77,78. Furthermore, 19.2% of the mutated genes were driver genes, including APC, CDH1, RET, STK11, and TP53. Driver gene mutations promote cancer progression and have major impacts on patient clinical outcomes. Further research on these genes in BC tissue may be warranted. Other studies have shown that the mutation clonality of driver genes was prognostic and predictive for BC patients79,80.

Conclusions

In conclusion, this study primarily compared germline mutation profiling among 27 Chinese familial/hereditary BC families to comprehensively evaluate the genetic variants and clinical significance of 43 BC predisposition genes with different penetration rates in carcinogenesis. We found that in addition to BRCA1/2, genetic variants in non-BRCA genes, especially DDR genes, played significant roles in the development of Chinese familial/hereditary BC, which implied the indispensable significance of more extensive multiple-gene panel testing in genetic screening of hereditary BC families. People with non-BRCA gene mutations are more likely to suffer from BC accompanied by benign breast diseases because non-BRCA gene mutations have a weaker pathogenic effect on carcinogenesis than BRCA genes. Therefore, more intensive mammary screening of non-BRCA mutation-bearing individuals in hereditary BC families is recommended to increase the efficacy of early diagnosis and early treatment of BC. However, this study was limited by a small sample size from a single center. A larger multicenter study in a Chinese population should be conducted to validate the findings of this study.

Supporting Information

Grant support

This work was supported by the National Natural Science Foundation of China (Grant Nos. 82072588, 82002601, 81872143, and 81702280); the National Science and Technology Support Program of China (Grant Nos. 2015BAI12B15 and 2018ZX09201015); the National Key Research and Development Program of China; the Net Construction of Human Genetic Resource Bio-bank in North China (2016YFC1201703), the Projects of Science and Technology of Tianjin (Grant Nos. 13ZCZCSY20300 and 18JCQNJC82700), and the Key Project of Tianjin Health and Family Planning Commission (Grant No. 16KG126).

Conflict of interest statement

No potential conflicts of interest are disclosed.

Author contributions

Conceived and designed the analysis: Jinpu Yu and Juntian Liu.

Collected the data: Li Dong, Hailian Zhang, Huan Zhang, Yingnan Ye, Yanan Cheng, Lei Han, Lijuan Li, Lijuan Wei, and Shixia Li.

Contributed data or analysis tools: Lei Han and Yandong Cao.

Performed the analysis: Li Dong, Hailian Zhang, and Huan Zhang.

Wrote the paper: Li Dong, Hailian Zhang, and Huan Zhang.

Other contribution: Xishan Hao gave guidance and help on the design of this study.

Acknowledgments

The authors thank Campian Jian Li from Washington University School of Medicine for providing constructive suggestions to this manuscript.

Footnotes

  • ↵*Li Dong and Hailian Zhang contributed equally to this work.

  • Received January 5, 2021.
  • Accepted April 7, 2021.
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