Bayes Analysis Provides Evidence of Pathogenicity for the BRCA1 c.135-1G>T (IVS3-1) and BRCA2 c.7977-1G>C (IVS17-1) Variants Displaying In Vitro Splicing Results of Equivocal Clinical Significance

Abstract

Although in vitro splicing assays can provide useful information about the clinical interpretation of sequence variants in high-risk cancer genes such as BRCA1 and BRCA2, results can sometimes be difficult to interpret. The BRCA1 c.135-1G>T (IVS3-1G>T) variant has been shown to give rise to an in-frame deletion of exon 5 (BRCA1 c.135_212del) that is predicted to encode 26 amino acids. BRCA2 c.7977-1G>C (IVS17-1G>C) was shown to increase the expression of two naturally occurring transcripts that contain frameshifts (BRCA2, c.7977_8311del (exon 18 deletion); BRCA2, c.7806_8331del (exon 17&18 deletion)). In this study we conducted multifactorial likelihood analysis to evaluate the clinical significance of these two variants, including assessing variant segregation in families by Bayes analysis, and breast tumor pathology features suggestive of positive mutation status. Multifactorial analysis provided strong evidence for causality for both of these variants. The Bayes scores from a single family with BRCA1 c.135-1G>T was 9528:1, and incorporation of pathology features gave an overall likelihood of causality of 28108:1. The Bayes scores from five informative families with BRCA2 c.7977-1G>C was 47401:1, and the combined Bayes-pathology odds of causality was 29389:1. Multifactorial likelihood analysis indicates that the BRCA1 c.135-1G>T and BRCA2 c.7977-1G>C variants are disease-associated mutations which should be managed clinically in the same fashion as classical truncating mutations.

INTRODUCTION

A previous study by members of our group assessed possible splicing aberrations associated with seven different splice site region genetic alterations in BRCA1 (MIM# 113705) and BRCA2 (MIM# 600185), using in vitro splicing assays conducted on lymphoblastoid cell lines (Tesoriero, et al., 2005). Five of the variants were found to express transcripts that would be predicted to produce a truncated protein, and were thus considered to be pathogenic. However, the clinical interpretation of two variants was unresolved. BRCA1 c.135-1G>T (BIC nomenclature IVS3-1G>T, reported as BRCA1 IVS4-1G>T) was shown to give rise to an in-frame deletion of exon 5, predicted to result in loss of 26 amino acids in the encoded protein (BRCA1 p.Phe46_arg71del). The BRCA2 c.7977-1G>C (reported as BIC nomenclature IVS17-1G>C) was shown to increase the expression of two naturally occurring transcripts that contain frameshifts (BRCA2 c.7977_8311del (exon 18 deletion); BRCA2 c.7806_8311del (exon 17&18 deletion)). It was suggested by the authors that further investigation was required to assess the clinical significance of these variants. Indeed, from interpretation of the suggestions put forward in a recent review on assessment of splicing aberrations for classification of sequence variants, these variants would be considered Class 4 likely pathogenic variants on the basis of the previously reported non quantitative in vitro splicing results, and thus further study using statistical approaches is recommended to confirm their pathogenicity (Spurdle, et al., 2008a). In this study, we used a multifactorial likelihood approach (Chenevix-Trench, et al., 2006; Goldgar, et al., 2004; Spurdle, et al., 2008b) to assess causality of these two variants.

MATERIALS AND METHODS

Pedigrees with variants under study were ascertained by the Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab) (Mann, et al., 2006). Participants provided written informed consent, and relevant ethics committees approved the study. Genotyping information obtained from the central kConFab database had been generated by previous kConFab research studies and/or core genotyping of BRCA1/2 sequence variants in kConFab probands and their family members. The multifactorial likelihood model described by Goldgar et al (Goldgar, et al., 2004) was applied. Methods and example calculations are shown in (Spurdle, et al., 2008b). In summary, Bayes analysis was used to estimate likelihood of causality based on variant segregation in families, as described previously (Thompson, et al., 2003). The Bayes factor was included directly as a likelihood ratio (LR) score for the pedigree analysis component of the multifactorial likelihood analysis. A combination of pathology slide review (SL and LdaS for BRCA1, RB for BRCA2), and pathology report abstraction was used to assess the tumours of variant carriers for histopathological features of BRCA1 or BRCA2 mutation carriers. Derivation of probabilities and multifactorial likelihood scoring for pathology features was as described previously (Chenevix-Trench, et al., 2006; Spurdle, et al., 2008b). BRCA2 pathology LRs were derived from tubule formation scores only (and not ER and Grade), since tubule formation is considered to be a more robust pathological measure than overall grade for scores derived from multiple sources. Scores were defined as: 1, present in 75% + of tumor; 2, present in 10–75% of tumor; 3, present in <10% tumor. The segregation Bayes LR (Thompson, et al., 2003) and the pathology LR (Chenevix-Trench, et al., 2006; Spurdle, et al., 2008b) were multiplied to calculate an overall multifactorial likelihood ratio, assuming statistical independence of the sources of information.

HGVS nomenclature is based on the following genomic sequences (GenBank): BRCA1 NM_007294.3; BRCA2 NM_000059.1. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence, according to journal guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1.

RESULTS AND DISCUSSION

Results from multifactorial likelihood analysis are shown in Table 1.

Table 1.

Results from Multifactorial Likelihood Analysis

BIC Nomenclature	HGVS Nomenclature	Bayes Factor- By family	Overall Bayes Factor	ER Status	Grade	Tubule Formation Score	LR Pathology**	Overall LR Pathology	Overall Odds For causality
BRCA1 IVS3-1G>T*	BRCA1 c.135-1G>T (p.Phe46_Arg71del)	9528	9528	Negative	3		2.95	2.95	28108:1
				N/A	2		N/A
				N/A	3		N/A
				N/A	3		N/A
				N/A	3		N/A
				N/A	3		N/A
BRCA2 IVS17-1G>C	BRCA2 c.7977-1G>C	20.23	47401	Positive	1	N/A	N/A	0.62	29389:1
		4.59		Negative	3	N/A	N/A
				N/A	2	N/A	N/A
		0.94		N/A	1	N/A	N/A
		21.46		N/A	3	3	1.2
				N/A	2 or 3#	1	0.5
				N/A	2	N/A	N/A
				Negative	3	3	1.2
		25.32		N/A	3	3	1.2
				Positive	3	3	1.2
				N/A	3	N/A	N/A
				Positive	3	2	0.5
				Positive	2	3	1.2

^*Previously reported in Tesoriero et al (2005) as BRCA1 IVS4-1G>T

^**LR for pathology was derived from ER and grade for BRCA1, and tubule formation only for BRCA2 (see Methods).

^#A previous and a subsequent tumor from this individual were both reported to be grade 3, but material from these diagnoses was unavailable for review.

ER=estrogen receptor; LR=likelihood ratio; N/A = not available.

Note that calculations using figures presented in the table will result in slight differences in overall Bayes Factor scores, and odds for causality, due to rounding error. HGVS nomenclature is based on the following genomic sequences (GenBank): BRCA1 NM_007294.3; BRCA2 NM_000059.1. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence, according to journal guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1.

The Bayes scores from the single family with BRCA1 c.135-1G>T was 9528:1, providing strong evidence of causality for this variant. ER and grade information was available from a single tumour displaying a basal-like morphology, and the combined scores gave an overall likelihood of causality of 28108:1. While likelihood scores could not be derived from the tumors of five additional carriers due to missing information on ER status, four of them displayed features commonly associated with BRCA1 mutation status, namely basal-like morphology (high grade, confluent necrosis, marked nucleur pleiomorphism, prominent lymphocytic infiltrate, pushing borders; three tumors), or high-grade ductal morphology (one tumor).

The Bayes scores from five informative families with BRCA2 c.7977-1G>C was 47401:1, overwhelmingly in evidence of causality. As shown in Table 1, there were 13 tumors available from BRCA2 variant carriers, and only partial information was available for pathology features previously reported to be weakly predictive of BRCA2 mutation status: tubule formation (Chenevix-Trench, et al., 2006); and ER status and grade combined (Bane, et al., 2007)). Slide review revealed the low tubule formation that is weakly predictive of BRCA2 mutation status for 5 of 7 tumours examined, equating to a likelihood ratio of 0.62. Similarly, although the majority of tumours examined were ER-positive (4/6), or high grade (8/13), only 2 of 6 tumours had the combined ER-positive plus high grade features reported to be weakly predictive of BRCA2 mutation status (Bane, et al., 2007), and only one of these was scored to have low tubule formation. These discrepant findings highlight the need for more specific and sensitive markers of BRCA2 mutation status for use in multifactorial likelihood analysis. Nevertheless, the overall odds for causality provided convincing evidence for pathogenicity for this variant, with a combined Bayes-pathology odds of 29389:1.

In this analysis we did not incorporate the prior probability of pathogenicity based on in silico analysis of sequence conservation and the domain(s) disrupted by the splicing aberrations (Tavtigian, et al., 2008), as it was not clear what the appropriate prior probability would be. However, given the strong evidence from co-segregation and pathology combined, the posterior probability for these variants would be greater than 99%, and would thus be considered class 5 pathogenic variants (Plon, et al., 2008).

In summary, multifactorial likelihood analysis indicates that the BRCA1 c.135-1G>T and BRCA2 c.7977-1G>C variants are disease-associated mutations which should be managed clinically in the same fashion as classical truncating mutations (Plon, et al., 2008).