BRCA1 Variant Confers Intermediate Cancer Risk
BRCA1 Variant Confers Intermediate Cancer Risk
Background Clinical classification of rare sequence changes identified in the breast cancer susceptibility genes BRCA1 and BRCA2 is essential for appropriate genetic counselling of individuals carrying these variants. We previously showed that variant BRCA1 c.5096G>A p.Arg1699Gln in the BRCA1 transcriptional transactivation domain demonstrated equivocal results from a series of functional assays, and proposed that this variant may confer low to moderate risk of cancer.
Methods Measures of genetic risk (report of family history, segregation) were assessed for 68 BRCA1 c.5096G>A p.Arg1699Gln (R1699Q) families recruited through family cancer clinics, comparing results with 34 families carrying the previously classified pathogenic BRCA1 c.5095C>T p.Arg1699Trp (R1699W) mutation at the same residue, and to 243 breast cancer families with no BRCA1 pathogenic mutation (BRCA-X).
Results Comparison of BRCA1 carrier prediction scores of probands using the BOADICEA risk prediction tool revealed that BRCA1 c.5096G>A p.Arg1699Gln variant carriers had family histories that were less 'BRCA1-like' than BRCA1 c.5095C>T p.Arg1699Trp mutation carriers (p<0.00001), but more 'BRCA1-like' than BRCA-X families (p=0.0004). Further, modified segregation analysis of the subset of 30 families with additional genotyping showed that BRCA1 c.5096G >A p.Arg1699Gln had reduced penetrance compared with the average truncating BRCA1 mutation penetrance (p=0.0002), with estimated cumulative risks to age 70 of breast or ovarian cancer of 24%.
Conclusions Our results provide substantial evidence that the BRCA1 c.5096G>A p.Arg1699Gln (R1699Q) variant, demonstrating ambiguous functional deficiency across multiple assays, is associated with intermediate risk of breast and ovarian cancer, highlighting challenges for risk modelling and clinical management of patients of this and other potential moderate-risk variants.
The clinical classification of rare sequence changes identified in the high-risk breast cancer susceptibility genes BRCA1 and BRCA2 is essential for appropriate genetic counselling of individuals carrying these variants. Classification of BRCA1 and BRCA2 variants was facilitated by the development of a multifactorial likelihood model, which provides a quantitative estimate of pathogenicity by assessing measures of genetic and other features of variant carriers relative to characteristics observed for classical high-risk mutations. Moreover, this quantitative assessment of risk has been linked to clinical management guidelines to provide a basis for standardised variant reporting, variant classification and management of families with such variants. The multifactorial likelihood methodology has been applied in multiple studies, with more than 200 BRCA1 or BRCA2 variants now classified using this approach. However, the multifactorial approach is designed to distinguish high-risk mutations from variants with no or little clinical significance, and it is likely that additional methods are required to detect and validate BRCA1 or BRCA2 rare variants associated with more modest risks than the average penetrance reported for classical mutations in these genes, that is, 65% risk of breast cancer and 39% risk of ovarian cancer to age 70 years for BRCA1 mutations, and 45% risk of breast cancer and 11% risk of ovarian cancer to age 70 years for BRCA2 mutations.
We previously showed that the variant BRCA1 R1699Q (c.5096G>A p.Arg1699Gln) located in the BRCA1 carboxyl terminal (BRCT) regions of the transcriptional transactivation domain (TAD) demonstrated equivocal results from a series of functional assays, when compared with wild-type control and known pathogenic missense mutation BRCA1 A1708E (c.5123C>A p.Ala1708Glu) which was null in all assays. In particular, this variant displayed intermediate transcriptional transactivation activity in human 293T and T47D cell lines and wild-type centrosome amplification function, but behaved as a deleterious mutation when assayed for formation of nuclear foci and trypsin sensitivity. There is also inconsistency in assay results from other functional studies, including discrepancies between yeast and mammalian transcriptional transactivation assay results in a single report, and categorisation of R1699Q as a variant with strong functional effect due to compromised peptide binding activity and specificity, and compromised transcriptional activity in yet another study. Most recently, Chang et al performed an extensive study of the R1699Q substitution using mouse embryonic stem (ES) cell-based functional assays, and demonstrated that this variant affected mouse ES cell survival and differentiation, and was unable to rescue embryonic lethality of Brca1-null mice. However, this study also demonstrated that the variant did not cause significant cell-cycle defects, and had no effect on genomic stability, but it was suggested that abrogated repression of oncomir miR-155 was the underlying mechanism for BRCA1-mediated tumour suppression. The equivocal behaviour of this variant can be explained at a protein level, as demonstrated by protein modelling predictions shown in supplementary figure S1. R1699 is located in the linker connecting the BRCT repeat domain, and participates in a salt bridge between the BRCT repeats. The loss of salt-bridging interactions and steric strain associated with accommodating a tryptophan substitution contributes to conformational instability of the R1699W (c.5095C>T p.Arg1699Trp) pathogenic mutation and, subsequently, disrupts transcriptional transactivation function. By contrast, substitutions with little or no effect on structures, such as R1699Q, may be fully or partially active in these assays. Moreover, R1699 lies in a conserved phosphopeptide-binding groove of the BRCA1 repeat, and plays an important role in phosphopeptide recognition through its interaction. Specifically, our protein modelling results directly comparing R1699Q and R1699W show that the volume of R1699W is likely to cause steric clashes with the phosphopeptide, whereas, the smaller surface and volume presentation of R1699Q will not cause steric clashes, but may modestly alter phosphopeptide recognition (see supplementary figure S1). These modelling predictions explain the experimental results from biophysical assays of BACH1 binding affinity which demonstrated that R1699W leads to a significant 160-fold reduction in affinity compared with wild-type, whereas, the reduction is only 24-fold for R1699Q.
We previously proposed that the R1699Q variant has partial abrogation of BRCA1 functions, and may confer low to moderate risk of cancer that would be better measured using pooled family studies. In a study assessing pathogenicity of 1433 variants based on family history, co-occurrence and cosegregation data from a large dataset derived from clinical testing at Myriad Genetic Laboratories, the combined odds that BRCA1 R1699Q was a pathogenic variant compared with neutral/no clinical significance was 2.5:1, based on a sample of 16 family histories with cosegregation data on only three of these. By contrast, BRCA1 R1699W at the same residue was classified as pathogenic, with odds in favour of pathogenicity of 39 978:1. Bioinformatic analysis shows that the arginine at position 1699 is conserved through tunicate, but the severity of the amino acid substitution is much less marked for glutamine (Grantham deviation 43) compared with tryptophan (Grantham deviation 101). Accordingly, the Align-GVGD algorithm (http://brca.iarc.fr) classifies R1699Q as a C35, while R1699W falls in the most severe C65 category for missense alterations. Based on an analysis of the same Myriad dataset, C35 variants were estimated to have a prior probability of pathogenicity of 0.66, while C65 variants were associated with a prior probability of 0.81.
Mohammadi et al assessed the likelihood of causality by cosegregation analysis of a single family, and reported a likelihood ratio (LR) of 1.4 for R1699Q. In another genetic study of several BRCA1/2 sequence variants, Gomez Garcia et al examined the R1699Q and R1699W variants as part of a model-building exercise that incorporated family history, and estimated the probability of pathogenicity to be 0.87 for R1699Q and >0.99 for R1699W. Although this model classified both variants as pathogenic mutations, the authors noted that R1699Q did not cosegregate completely with disease in one of three of the families in which such data were available.
In summary, a number of different studies to date indicate that the R1699Q variant demonstrates inconsistent or inconclusive results at the functional and genetic level. In an extension of our previous study, we confirmed the intermediate transcriptional transactivation activity of BRCA1 R1699Q in the 293T cell line relative to pathogenic variant R1699W at the same residue, and then initiated large-scale genetic studies to assess if this intermediate function might translate to the lower risk of breast and ovarian cancer in families for R1699Q compared with R1699W.
Abstract and Introduction
Abstract
Background Clinical classification of rare sequence changes identified in the breast cancer susceptibility genes BRCA1 and BRCA2 is essential for appropriate genetic counselling of individuals carrying these variants. We previously showed that variant BRCA1 c.5096G>A p.Arg1699Gln in the BRCA1 transcriptional transactivation domain demonstrated equivocal results from a series of functional assays, and proposed that this variant may confer low to moderate risk of cancer.
Methods Measures of genetic risk (report of family history, segregation) were assessed for 68 BRCA1 c.5096G>A p.Arg1699Gln (R1699Q) families recruited through family cancer clinics, comparing results with 34 families carrying the previously classified pathogenic BRCA1 c.5095C>T p.Arg1699Trp (R1699W) mutation at the same residue, and to 243 breast cancer families with no BRCA1 pathogenic mutation (BRCA-X).
Results Comparison of BRCA1 carrier prediction scores of probands using the BOADICEA risk prediction tool revealed that BRCA1 c.5096G>A p.Arg1699Gln variant carriers had family histories that were less 'BRCA1-like' than BRCA1 c.5095C>T p.Arg1699Trp mutation carriers (p<0.00001), but more 'BRCA1-like' than BRCA-X families (p=0.0004). Further, modified segregation analysis of the subset of 30 families with additional genotyping showed that BRCA1 c.5096G >A p.Arg1699Gln had reduced penetrance compared with the average truncating BRCA1 mutation penetrance (p=0.0002), with estimated cumulative risks to age 70 of breast or ovarian cancer of 24%.
Conclusions Our results provide substantial evidence that the BRCA1 c.5096G>A p.Arg1699Gln (R1699Q) variant, demonstrating ambiguous functional deficiency across multiple assays, is associated with intermediate risk of breast and ovarian cancer, highlighting challenges for risk modelling and clinical management of patients of this and other potential moderate-risk variants.
Introduction
The clinical classification of rare sequence changes identified in the high-risk breast cancer susceptibility genes BRCA1 and BRCA2 is essential for appropriate genetic counselling of individuals carrying these variants. Classification of BRCA1 and BRCA2 variants was facilitated by the development of a multifactorial likelihood model, which provides a quantitative estimate of pathogenicity by assessing measures of genetic and other features of variant carriers relative to characteristics observed for classical high-risk mutations. Moreover, this quantitative assessment of risk has been linked to clinical management guidelines to provide a basis for standardised variant reporting, variant classification and management of families with such variants. The multifactorial likelihood methodology has been applied in multiple studies, with more than 200 BRCA1 or BRCA2 variants now classified using this approach. However, the multifactorial approach is designed to distinguish high-risk mutations from variants with no or little clinical significance, and it is likely that additional methods are required to detect and validate BRCA1 or BRCA2 rare variants associated with more modest risks than the average penetrance reported for classical mutations in these genes, that is, 65% risk of breast cancer and 39% risk of ovarian cancer to age 70 years for BRCA1 mutations, and 45% risk of breast cancer and 11% risk of ovarian cancer to age 70 years for BRCA2 mutations.
We previously showed that the variant BRCA1 R1699Q (c.5096G>A p.Arg1699Gln) located in the BRCA1 carboxyl terminal (BRCT) regions of the transcriptional transactivation domain (TAD) demonstrated equivocal results from a series of functional assays, when compared with wild-type control and known pathogenic missense mutation BRCA1 A1708E (c.5123C>A p.Ala1708Glu) which was null in all assays. In particular, this variant displayed intermediate transcriptional transactivation activity in human 293T and T47D cell lines and wild-type centrosome amplification function, but behaved as a deleterious mutation when assayed for formation of nuclear foci and trypsin sensitivity. There is also inconsistency in assay results from other functional studies, including discrepancies between yeast and mammalian transcriptional transactivation assay results in a single report, and categorisation of R1699Q as a variant with strong functional effect due to compromised peptide binding activity and specificity, and compromised transcriptional activity in yet another study. Most recently, Chang et al performed an extensive study of the R1699Q substitution using mouse embryonic stem (ES) cell-based functional assays, and demonstrated that this variant affected mouse ES cell survival and differentiation, and was unable to rescue embryonic lethality of Brca1-null mice. However, this study also demonstrated that the variant did not cause significant cell-cycle defects, and had no effect on genomic stability, but it was suggested that abrogated repression of oncomir miR-155 was the underlying mechanism for BRCA1-mediated tumour suppression. The equivocal behaviour of this variant can be explained at a protein level, as demonstrated by protein modelling predictions shown in supplementary figure S1. R1699 is located in the linker connecting the BRCT repeat domain, and participates in a salt bridge between the BRCT repeats. The loss of salt-bridging interactions and steric strain associated with accommodating a tryptophan substitution contributes to conformational instability of the R1699W (c.5095C>T p.Arg1699Trp) pathogenic mutation and, subsequently, disrupts transcriptional transactivation function. By contrast, substitutions with little or no effect on structures, such as R1699Q, may be fully or partially active in these assays. Moreover, R1699 lies in a conserved phosphopeptide-binding groove of the BRCA1 repeat, and plays an important role in phosphopeptide recognition through its interaction. Specifically, our protein modelling results directly comparing R1699Q and R1699W show that the volume of R1699W is likely to cause steric clashes with the phosphopeptide, whereas, the smaller surface and volume presentation of R1699Q will not cause steric clashes, but may modestly alter phosphopeptide recognition (see supplementary figure S1). These modelling predictions explain the experimental results from biophysical assays of BACH1 binding affinity which demonstrated that R1699W leads to a significant 160-fold reduction in affinity compared with wild-type, whereas, the reduction is only 24-fold for R1699Q.
We previously proposed that the R1699Q variant has partial abrogation of BRCA1 functions, and may confer low to moderate risk of cancer that would be better measured using pooled family studies. In a study assessing pathogenicity of 1433 variants based on family history, co-occurrence and cosegregation data from a large dataset derived from clinical testing at Myriad Genetic Laboratories, the combined odds that BRCA1 R1699Q was a pathogenic variant compared with neutral/no clinical significance was 2.5:1, based on a sample of 16 family histories with cosegregation data on only three of these. By contrast, BRCA1 R1699W at the same residue was classified as pathogenic, with odds in favour of pathogenicity of 39 978:1. Bioinformatic analysis shows that the arginine at position 1699 is conserved through tunicate, but the severity of the amino acid substitution is much less marked for glutamine (Grantham deviation 43) compared with tryptophan (Grantham deviation 101). Accordingly, the Align-GVGD algorithm (http://brca.iarc.fr) classifies R1699Q as a C35, while R1699W falls in the most severe C65 category for missense alterations. Based on an analysis of the same Myriad dataset, C35 variants were estimated to have a prior probability of pathogenicity of 0.66, while C65 variants were associated with a prior probability of 0.81.
Mohammadi et al assessed the likelihood of causality by cosegregation analysis of a single family, and reported a likelihood ratio (LR) of 1.4 for R1699Q. In another genetic study of several BRCA1/2 sequence variants, Gomez Garcia et al examined the R1699Q and R1699W variants as part of a model-building exercise that incorporated family history, and estimated the probability of pathogenicity to be 0.87 for R1699Q and >0.99 for R1699W. Although this model classified both variants as pathogenic mutations, the authors noted that R1699Q did not cosegregate completely with disease in one of three of the families in which such data were available.
In summary, a number of different studies to date indicate that the R1699Q variant demonstrates inconsistent or inconclusive results at the functional and genetic level. In an extension of our previous study, we confirmed the intermediate transcriptional transactivation activity of BRCA1 R1699Q in the 293T cell line relative to pathogenic variant R1699W at the same residue, and then initiated large-scale genetic studies to assess if this intermediate function might translate to the lower risk of breast and ovarian cancer in families for R1699Q compared with R1699W.
