Out of 308 BRCA1-positive patients, 173 had breast cancer (56.2%), 115 had ovarian/peritoneal cancer (37.3%), and 14 patients had cancer of an unknown origin (4.5%) which were requested from other hospitals for BRCA1/BRCA2 genes tests. Two patients had dual primary cancers (breast/ovary and breast/peritoneal cancer). Out of 222 BRCA2-positive patients, 168 had breast cancer (75.7%), 39 had ovarian/peritoneal cancer (17.6%), and eight patients had cancer of an unknown origin (3.6%). Breast cancer was the most common, followed by ovarian and fallopian tube cancer. Especially, the proportion of breast cancer in BRCA2-positive patients (76.6%) was higher than that of breast cancer in BRCA1-positive patients (56.2%) (p<0.0001). In the same context, gynecologic cancer, ovarian, fallopian tubal, and peritoneal cancer were more common in BRCA1-positive patients than in BRCA2-positive patients (37.3% vs. 17.6%, respectively; p<0.0001). Family histories were dominant contributing factors for breast cancer, especially in the BRCA2-positive group. The mean ages were 46 (range 21-77) years old for BRCA1-positive patients and 48 (range 27-78) years old for BRCA2-positive patients (Table 2).
Variant Type Classification.
BRCA1 and BRCA2 genes were analyzed for all coding exons in all patients. We classified the detected pathogenic and likely pathogenic variants as possessing nonsense, frameshift, missense, synonymous, intronic, or amino acid deletion mutations. In the BRCA1 gene, there were 84 total variants, and frameshift variants were most common (39/84, 46.4%), followed by nonsense variations (30/84, 35.7%). More than 80% of variants in the BRCA1 gene conferred a loss of function. There were 81 total variants in the BRCA2, and 54 (66.7%) were frameshift variants.
We defined recurrent variants as variants that were found in more than five unrelated cases. There were 14 recurrent variants of the BRCA1 gene. Among them, seven were frameshift, four were nonsense, two were intronic, and the remaining was a missense variant. In the BRCA2 gene, there were seven recurrent variants. Among them, four were frameshift variants, and the other three were nonsense variants (Figure 1).
Detected Recurrent Variants of BRCA1/2 and Classified Groups.
There were 84 PV/LPVs of the BRCA1 gene, which were found in 308 patients. Among them, 14 variants were detected in more than five unrelated patients. Of all detected BRCA1 PV/LPVs, 64.3% (198/308) were recurrent variants. Considering the 14 recurrent variants, seven of which were frameshift type variants, four were nonsense, two were intronic, and one was a missense variant. The two most frequently detected variants were c.3627dup 20-22 and c.390C>A 23,24, which were both found in 29 people. The variants analyzed in this study are described in Table 1. Moreover, we classified variants into groups from A to C according to selection criteria based on their population-specific prevalence. Group A, which included pan-ethnic variants, included eight variants, c.3627dup 20-22, c.390C>A 23,24, c.2433del 25,26, c.302-2A>C 27,28, c.5030_5033del 20,28, c.5080G>T 27,29, c.5445G>A 20,30, and c.5467+1G>A 22,31. There were three Asian-prevalent variants in group B, c.5470_5477del 32,33, c.5496_5506delinsA 22, and c.3442del 34. In group C, there were three Korean-prevalent variants, c.1831del 35, c.5339T>C 26,36-38, and c.922_924delinsT 22,39. The grouped variants are listed in Table 3.
There were 80 PV/ LPVs of the BRCA2 gene, which were found in 222 patients. Among them, seven variants were detected in more than five unrelated patients. Four variants were frameshift type, and the remaining three variants were nonsense type. The most frequently detected variant was c.7480C>T, found in 45 people (Table 2). We divided these recurrent variants into groups A to C, as described above. Two BRCA2 gene variants were stratified into group A, c.7480C>T 22,40 and c.3744_3747del 41-43. There were two variants, c.10150C>T 44-46 and c.5576_5579del 47-49, in group B and three variants, c.1399A>T39,50,51, c.3018del 52, and c.6724_6725del 53,54 in group C (Table 3).
Haplotype analysis of Patients with a BRCA1 gene variant.
We defined the linkage block as that which harbored the detected variant. Even if there was a significant block that did not harbor the variant, we did not count the block as a haplotype block. If the result of the analysis revealed that there was no absolute block, we assumed the block size was shorter than the distance between the two SNPs. There were 14 variants of BRCA1 detected in more than five people. We compared them against a sample of the normal Korean population (n=397) through haplotype and SNP chip analysis.
In group A, the BRCA1 variant c.390C>A had an 8.9kb haplotype block, c.3627dup had a 0.8kb haplotype block, and c.5467+1G>A had a 1.3 kb haplotype block. The c.5030_5033del and c.5080G>T variants had the same-sized haplotype block, 4.0 kb. These two variants are closely located, so near SNPs were linked in both variants. In BRCA1 c.2433del, which was detected in eight patients, we tried to find the linkage block. However, the start of the block was defined, but the end of the block was not found due to lopsided SNPs. The c.302-2A>C and c.5445G>A variants had no rest specimen to be analyzed.
In group B, the BRCA1 c.5496_5506delinsA variant had a 1.3kb sized linkage block, and c.3442del had a 0.4 kb linkage block. There were only two patients with c.5470_5477del, so we could not analyze the haplotype block this variant due to small number.
With respect to group C, the linkage block of c. 1831del, which was found in six patients, was 23.0 kb long. The variant c.922_924delinsT showed a 2.9 kb-sized block. For the c.5339T>C variant, the linkage block was very long, 74.5kbs. This variant was detected in 20 unrelated patients, and we could analyze eight samples. The most common block size among the eight analyzed was 74.5 kb. The size of the linkage block that covered more than 50% of patients (the major block) was 398.8 kb, which exceeded the size of the BRCA1 gene. This variant was detected and reported mostly in the Korean population. The representative variants of each group are summarized in Figure 2.
Haplotype analysis of Patients with BRCA2 gene variants.
There were seven recurrent variants in the BRCA2 gene. In group A, there were two variants, c.7480C>T and c.3744_3747del, which were found in all ethnicities. The BRCA2 c.7480C>T variant was most detected in this study but also detected in others 22. Forty-five patients with this variant were analyzed with 24 SNPs and compared to a sample of the normal Korean population (n=397). However, there were no meaningful blocks or linkages due to the longer length between any two SNPs. Therefore, we could guess that the block size was less than 6.0 kb based on the SNPs around the variant. After analyzing 14 patients using the Korea Biobank Array, we concluded that the common block of this variant was 4.7 kb long. The result of haplotyping for the c.3744_3747del variant showed a common block size of 12.3 kb in 17 patients. Especially, the SNP positioned at 32929387 (rs169547) was highly located by T in patients (84.6%), and the odds ratio (OR) was extremely high, 7,945 (95% confidence interval [CI], 415.14 to 152053.25; p value, 0.0001).
Regarding Group B, including variants primarily found in Asia, the c.5576_5579del variant was analyzed in 12 patients and showed a most common block sized 4.0 kb. It could be expanded to 123.1 kb to cover more than 50% of patients. This variant has actually been reported in all ethnicities 47-49,55 but is usually detected in the Japanese population and people with Japanese ancestry. In this context, this variant was suggested to be a Japanese founder variant 55. The BRCA2 variant c.10150C>T could neither be analyzed by haplotyping nor by SNP chip analysis because of its terminal location and lack of specimens.
In group C, the c.1399A>T variant, which has been mostly reported in the Korean population, was detected in 26 unrelated patients in this study. Compared to a sample of the normal Korean population (n = 1,099), there was a highly suspicious haplotype, sized over 21.0 kb, harboring c.1399A>T. The OR for this linkage block was 273.85 (95% CI, 37.4721 to 2001.3852; p value <0.0001). After Korea Biobank Array analysis, the common block of this variant could be defined in 35.5 kb, and the major block was 144.5 kb long. It appears as though the original block was the longest, and recombinations were generated in other shorter blocks. The BRCA2 c.3018del variant, which was found in six patients, had a 3.5 kb-sized block, and the c.6724_6725del variant, found in seven patients, had a long block sized 21.0 kb.
Characteristics of BRCA1 and BRCA2 Korean founder variants.
The sites of disease in patients with c.5339T>C in the BRCA1 gene and that of other BRCA1-positive patients were similar, breast cancer (p=0.7213) and gynecologic cancer (p=0.0986) (Table 4). However, there were three patients (25.0%) with c.5339T>C in the BRCA1 gene and bilateral breast cancer, and this risk could be higher than other BRCA1-positive breast cancer patients (1.9%) (p<0.0001). Moreover, the hormone receptor status of breast cancer in those with c.5339T>C was different compared to other BRCA1-positive patients. BRCA1 germline variants are well-known to be associated with negative hormone receptor status. Of those with the BRCA1 Korean founder variant, ten patients showed positive, and four patients showed negative hormone status.
Twenty-two patients with c.1399A>T in the BRCA2 gene had breast cancer, and two had gynecologic cancer. Seven patients had both breast and gynecologic cancer, but no patients with both cancers in founder variant of BRCA2 gene. It was not statistically significant. The hormone receptor status in those with c.1399A>T was not different compared to other BRCA2-positve patients. Patients with founder variants in the BRCA2 gene had more family history of gynecologic cancer (p=0.0055).