Double Heterozygosity for Germline Mutations in Chinese Breast Cancer Patients

Simple Summary About 5–10% of breast cancers are related to heredity. Very often, affected individuals will carry only a single mutation defect in any of the hereditary breast and ovarian cancer syndrome (HBCO)-related genes. Breast cancer patients who are double heterozygous (DH) for different HBCO-related genes are rare. In this study, we provide real-world data for the Chinese population based on our high-risk referral patients from the Hong Kong Hereditary Breast Cancer Family Registry. DH germline mutations were identified in nine patients (0.25%) and associated with a higher prevalence of bilateral breast cancers in the Chinese population compared to other populations. A more stringent surveillance program and possibly a more aggressive treatment plan for all DH families could be tailored to specific family needs in the local area. Abstract Double pathogenic mutations occurring in an individual are considered a rare event. The introduction of a multiple-gene panel at Hong Kong Hereditary Breast Cancer Family Registry has allowed the identification of pathogenic variants in multiple genes, providing more information on clinical management and surveillance to the proband and their family members. Breast cancer patients who are double heterozygous (DH) for different hereditary breast and ovarian cancer syndrome (HBCO)-related genes were identified from a cohort of 3649 Chinese patients. Nine patients (0.25%) were observed to have germline DH mutations in ATM, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, MSH6, PALB2, and TP53. Three probands were diagnosed with unilateral breast cancer, two patients were diagnosed with bilateral breast cancer, and four patients had multiple primary cancers. The median age for breast cancer diagnosis was an early age of 36 years. Chinese DH carriers did not show worse phenotypes or have a significantly downhill clinical presentation. However, seven out of nine (77.8%) of our DH carriers harbored a BRCA1 mutation, and four of them (44.4%) developed bilateral breast cancer, suggesting Chinese DH individuals may have a higher chance of having bilateral breast cancer than other populations (p = 0.0237).


Introduction
Hereditary breast and ovarian cancer syndrome is an inherited cancer-predisposition syndrome, predominantly caused by mutations in the BRCA genes.Women carrying BRCA1 and BRCA2 pathogenic variants have a significant lifetime risk of breast and ovarian cancers, with rates of up to 85% and 65%, respectively [1].Men with these mutations have a 1% greater lifetime risk of breast cancer than male non-carriers [2], in addition to a risk of developing prostate carcinomas.
DH mutations are uncommon, particularly in other non-Ashkenazi and Slavic individuals.A recent study on Brazilians identified 1.2% DH cases from among a cohort of 1156 early-onset breast cancer patients and no significant differences in age of BC onset and risk for bilateral BC in DH carriers of simple heterozygote (SH) mutations [15].However, in a Caucasian population, the proportion of DH BRCA females identified was 0.1%, and they might develop breast cancer at an earlier age and have more severe disease than SH BRCA mutation carriers [16].Only a few DH cases have been reported in the Asian population [17][18][19], and the corresponding cancer risk is still unknown.DH of BRCA mutations was identified in 93 females from 32,295 BRCA1/2 mutation carriers listed in the CIMBA database [20].
To characterize the nature of DH and its clinical phenotypes in the Chinese populations, we systematically evaluated the frequency and clinical characteristics of DH and compared them with those of carriers of SH mutation.This was conducted in order to create a more suitable surveillance program and treatment plan for DH families.

Participants and Selection Criteria
Germline mutation screening was performed on 3649 high-risk Chinese breast cancer patients recruited through the Hong Kong Hereditary Breast Cancer Family Registry from March 2007 to August 2022.Individuals were included in this study if they met any of the following criteria: (1) they had been diagnosed with breast cancer at any age and had at least one first-or second-degree relative with breast and/or ovarian cancer, regardless of age; (2) they had been diagnosed with breast cancer at or before 45 years of age; (3) they had bilateral breast cancer; (4) they had triple-negative breast cancer; or (5) they were male with breast cancer.All participants recruited gave their consent for the study, and the research was conducted in accordance with the Declaration of Helsinki.

DNA Extraction and Sequencing
Genomic DNA was pooled and sequenced with a 30-gene panel (Color Genomics Laboratory, Burlingame, CA, USA) or a 93-gene DHS-001Z human breast cancer panel (Qiagen, Hilden, Germany) using MiSeq or NextSeq (Illumina, San Diego, CA, USA) instruments after extraction from peripheral blood using the QIAamp DNA Blood Mini Kit or the QIAsymphony DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.The minimum sequencing depth and median coverage were typically 50-fold with 200-300X.All detected pathogenic variants were further confirmed and validated via conventional Sanger bi-directional DNA sequencing.

Variant Interpretation and Annotation
Variant-calling bioinformatics was performed, and paired sequencing reads were mapped to human reference genome sequence GRCh37/hg19, as previously described [21,22].Variants with a minor allele frequency of at least 1%, as reported by the 1000 Genomes Project [23], were excluded from manual variant curation.The variants were described according to the recommendations of the Human Genome Variation Society (HGVS) nomenclature (http://www.HGVS.org/varnomen,(accessed on 4 July 2022)) and further cross-checked with the Mutalyzer Name Checker (http://mutalyzer.nl).

Statistical Analysis
Fisher's exact test was used to analyze the relationship between clinicopathological variables and mutation status.The threshold of statistical significance was set at a p-value of <0.05 for all analyses.Data analyses were conducted using the statistical software program R (version 3.4.2) [24].

Results
In this cohort of 3649 high-risk Chinese breast cancer patients, pathogenic or likely pathogenic (P/LP) DH germline mutations were identified in 9 (0.25%) Chinese probands with breast cancer.These mutations involved ATM, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, MSH6, PALB2, and TP53 genes (Table 1).Seven out of nine (77.8%) DH carriers carried at least one P/LP BRCA1 mutation variant.The mean and median ages of first breast cancer diagnosis were 39.8 and 36, respectively, ranging from 30 to 69 years.Out of the nine DH carriers, a total of four (44.4%) had bilateral breast cancer, and four out of these nine subjects (44.4%) had other cancers in addition to the breast cancer.Three patients had breast and ovarian cancers, and two of them had an additional third or fourth primary cancers of the larynx and liver.Among the DH carriers, 44.4% had a family history of breast or ovarian cancer, while only 22.2% had a family history of prostate cancer.The majority of the breast cancers were invasive ductal carcinoma (IDC) (92.3%), and 54.5% of them were high-grade.Around 55% of the cases were ER/PR+, while only 27% were HER2+.Overall, 40% of cases were triple-negative breast cancers.Among seven families, 19 family members were available for the genetic test, and only one family had more than one DH carrier.This was the sister of a proband in family 003 who had personally diagnosed breast cancer at 39.In the same generation, three out of four siblings, who did not come for genetic test, had their breast cancers diagnosed at a young age (<45).The pedigrees and detailed surveillance managements of these families are detailed in Figure 1 and the Supplementary Materials.There were no consanguineous families in our cohort.In this cohort of 3649 high-risk Chinese breast cancer patients, 511 probands were found to have P/LP germline simple heterozygote (SH) mutations from our HBOC-related gene panel.The age of diagnosis, personal history of multiple cancers, bilateral breast cancer, family history of cancers, and tumor characteristics were compared between DH mutation carriers and SH mutation carriers.In this small collection of DH individuals, the ages of first cancer diagnosis (p = 0.213) (Figure 2) and all other clinicopathologic characteristics were similar between DH and DM carriers (Table 2).DH carriers did not show worse phenotypes or more significantly downhill clinical presentations than SH mutation carriers.Patients with DH mutations had a greater family history of ovarian cancers than SH mutation carriers (p = 0.031), but there was no significant difference between DH and SH mutation carriers on those with a family history of breast and prostate cancer.Symbols are used as follows: male-square; female-circle; marriage-square and circle linked by a horizontal line; cancer affected individuals-refer to legends; proband-pointed with an arrow; mutation status-"+" tested mutation carrier and "-" tested non-carrier; deceased-circle or square with a line running through it; Roman numerals symbolize number of siblings of same gender.* only including 1st and 2nd degree.
In this cohort of 3649 high-risk Chinese breast cancer patients, 511 probands were found to have P/LP germline simple heterozygote (SH) mutations from our HBOC-related gene panel.The age of diagnosis, personal history of multiple cancers, bilateral breast cancer, family history of cancers, and tumor characteristics were compared between DH mutation carriers and SH mutation carriers.In this small collection of DH individuals, the ages of first cancer diagnosis (p = 0.213) (Figure 2) and all other clinicopathologic characteristics were similar between DH and DM carriers (Table 2).DH carriers did not show worse phenotypes or more significantly downhill clinical presentations than SH mutation carriers.Patients with DH mutations had a greater family history of ovarian cancers than SH mutation carriers (p = 0.031), but there was no significant difference between DH and SH mutation carriers on those with a family history of breast and prostate cancer.
A previous study on a Jewish population reported no significant differences in breast cancer occurrence between DH and SH carriers: 46.7% of DH women had a personal history of breast carcinoma, while 40.2% of carriers of a single mutation had such a personal history (p = 0.801) [26].Another study compared the age at breast cancer diagnosis with DH and SH in the BRCA1 and BRCA2 genes.The ages were similar regarding to DH versus SH in the BRCA1 gene (p = 0.231), but the age of diagnosis was, on average, 4.5 years earlier for DH individuals than SH patients in the BRCA2 gene (p < 0.001) [20].This study also demonstrated that patients with two inherited pathogenic mutations were more likely to develop ovarian cancer than those carrying a BRCA2 pathogenic variant alone [20].In our small cohort, our patients with DH and SH mutations showed that the DH mutation carriers also had stronger family histories of ovarian cancer than SH mutation carriers (p = 0.031); however, the relationship between having ovarian cancer and DH may not be that simple, as all three of our DH carriers with both breast and ovarian cancers carried the BRCA1 mutation instead of the BRCA2 mutation.In our registry, patients with both breast and ovarian cancers were more likely to carry a BRCA1 mutation (24.7%) than a BRCA2 mutation (10.8%) [27].The higher incidence of developing a personal and family history of ovarian cancer may not be solely related to DH, as there were modifiers such as a Chinese population trend or bias within the cohort in our registry.
A study of eight DH individuals suggested that DH individuals develop breast cancer at an earlier age and have more severe disease than those with SH in the BRCA gene [28].Two studies on DH in the CHEK2 gene or the ATM gene with BRCA mutations demonstrated that the presence of second gene defects did not further increase the risk of disease in those patients who already carried SH in the BRCA genes [16,29].On the other hand, another report on 17 DH breast cancer patients stemming from Russia, Belarus, and Poland showed no association with an earlier age at diagnosis for the patients with DH.These Eastern European families did not have a stronger family history than the SH families [12].A single BRCA1/PALB2 DH case showed no associations with the severity of the phenotype compared to SH carriers [30].In our cohort, there was no significant difference in these clinicopathological parameters between DH and SH carriers (Table 2).
Aggregated data showed bilateral breast cancers were not particularly prevalent in non-Asian DH carriers.Among a total of 7/81 (8.6%) non-Asian DH patients with bilateral breast cancers reported in the literatures, including Australian, Caucasian, French Canadian, Hispanic, Italian, Scottish, and Slavic individuals, the frequency of bilateral breast cancers was only 10.8% (7/65, see Supplementary Table S1).However, bilateral breast cancers were observed in 44.4% (4/9) of patients in our cohort, suggesting Chinese with DH may have a higher chance of having bilateral breast cancer than other populations (p = 0.0237).For our locality, we suggest conducting more frequent surveillance imaging or taking aggressive prophylactic measures for patients and family members who are DH.
In conclusion, the clinical oncological features of DH individuals might be ethnically specific.Carriers of DH mutations were not noticeably different from SH carriers with respect to an earlier onset and a worsening phenotype.For the Chinese population in our cohort of high-risk referral patients of the Hong Kong Hereditary Breast Cancer Family Registry, DH was associated with a higher prevalence of bilateral breast cancers.Chinese individuals with DH may have a higher chance of having bilateral breast cancer than other populations.Although the validity of this observation might need to be confirmed using a larger cohort, and though it might be premature at this stage to recommend a general management strategy for all DH families, tight surveillance and possibly more-aggressive treatment plans could be tailored to specific family needs in our locality.

Conclusions
We conducted a review of double heterozygous (DH) carriers among a high-risk breast cancer cohort in Hong Kong and evaluated whether DH carriers exhibited a worse phenotype or a significantly deteriorating clinical presentation compared to single heterozygous (SH)-mutation carriers.However, we found that DH carriers did not show a worse phenotype or a significantly deteriorating clinical presentation when compared to SH mutation carriers.Based on the review of DH cases available in the literature, it appears that Chinese individuals with DH mutations may have a higher chance of developing bilateral breast cancer compared to other populations.However, whether it is worth recommending a more stringent surveillance program and a more aggressive treatment plan for DH families may require confirmation using a larger cohort.
Author Contributions: All authors contributed to this study's conception and design.A.K. designed and coordinated prospective data collection for the Hong Kong Hereditary Breast Cancer Family Registry.C.Y.S.H. retrieved data, interpreted the results, and drafted the manuscript for this particular study.A.K., C.-H.A., and E.S.K.M. reviewed the manuscript.All authors have read and agreed to the published version of the manuscript.

Figure 1 .
Figure1.The pedigrees of individuals from family 1 to family 9 who carried double mutations.Symbols are used as follows: male-square; female-circle; marriage-square and circle linked by a horizontal line; cancer affected individuals-refer to legends; proband-pointed with an arrow; mutation status-"+" tested mutation carrier and "-" tested non-carrier; deceased-circle or square with a line running through it; Roman numerals symbolize number of siblings of same gender.* only including 1st and 2nd degree.

Figure 2 .
Figure 2. Boxplot showing the age of onset of cancer among patients carrying double heterozygous (DH) mutations and simple mutations (SM).* represented the extreme values in the data set.

Figure 2 .
Figure 2. Boxplot showing the age of onset of cancer among patients carrying double heterozygous (DH) mutations and simple mutations (SM).* represented the extreme values in the data set.

Table 1 .
Characteristics of DH carriers.

Table 1 .
Cont. affected individuals-refer to legends; proband-pointed with an arrow; mutation status-"+" tested mutation carrier and "-" tested non-carrier; deceased-circle or square with a line running through it; Roman numerals symbolize number of siblings of same gender.* only including 1st and 2nd degree.
* only including 1st and 2nd degree.Cancers 2024, 16, x FOR PEER REVIEW 5 of 12 Figure 1.The pedigrees of individuals from family 1 to family 9 who carried double mutations.Symbols are used as follows: male-square; female-circle; marriage-square and circle linked by a horizontal line; cancer

Table 2 .
Characteristics of double heterozygous (DH) mutation and simple heterozygous mutation (SH) carriers.

Table 2 .
Characteristics of double heterozygous (DH) mutation and simple heterozygous mutation (SH) carriers.