Abstract

Mutation screening of the breast and ovarian cancer–predisposition genes BRCA1 and BRCA2 is becoming an increasingly important part of clinical practice. Classification of rare nontruncating sequence variants in these genes is problematic, because it is not known whether these subtle changes alter function sufficiently to predispose cells to cancer development. Using data from the Myriad Genetic Laboratories database of nearly 70,000 full-sequence tests, we assessed the clinical significance of 1,433 sequence variants of unknown significance (VUSs) in the BRCA genes. Three independent measures were employed in the assessment: co-occurrence in trans of a VUS with known deleterious mutations; detailed analysis, by logistic regression, of personal and family history of cancer in VUS-carrying probands; and, in a subset of probands, an analysis of cosegregation with disease in pedigrees. For each of these factors, a likelihood ratio was computed under the hypothesis that the VUSs were equivalent to an “average” deleterious mutation, compared with neutral, with respect to risk. The likelihood ratios derived from each component were combined to provide an overall assessment for each VUS. A total of 133 VUSs had odds of at least 100:1 in favor of neutrality with respect to risk, whereas 43 had odds of at least 20:1 in favor of being deleterious. VUSs with evidence in favor of causality were those that were predicted to affect splicing, fell at positions that are highly conserved among BRCA orthologs, and were more likely to be located in specific domains of the proteins. In addition to their utility for improved genetics counseling of patients and their families, the global assessment reported here will be invaluable for validation of functional assays, structural models, and in silico analyses. Mutation screening of the breast and ovarian cancer–predisposition genes BRCA1 and BRCA2 is becoming an increasingly important part of clinical practice. Classification of rare nontruncating sequence variants in these genes is problematic, because it is not known whether these subtle changes alter function sufficiently to predispose cells to cancer development. Using data from the Myriad Genetic Laboratories database of nearly 70,000 full-sequence tests, we assessed the clinical significance of 1,433 sequence variants of unknown significance (VUSs) in the BRCA genes. Three independent measures were employed in the assessment: co-occurrence in trans of a VUS with known deleterious mutations; detailed analysis, by logistic regression, of personal and family history of cancer in VUS-carrying probands; and, in a subset of probands, an analysis of cosegregation with disease in pedigrees. For each of these factors, a likelihood ratio was computed under the hypothesis that the VUSs were equivalent to an “average” deleterious mutation, compared with neutral, with respect to risk. The likelihood ratios derived from each component were combined to provide an overall assessment for each VUS. A total of 133 VUSs had odds of at least 100:1 in favor of neutrality with respect to risk, whereas 43 had odds of at least 20:1 in favor of being deleterious. VUSs with evidence in favor of causality were those that were predicted to affect splicing, fell at positions that are highly conserved among BRCA orthologs, and were more likely to be located in specific domains of the proteins. In addition to their utility for improved genetics counseling of patients and their families, the global assessment reported here will be invaluable for validation of functional assays, structural models, and in silico analyses. Sequence-based testing for BRCA1 (MIM 113705) and BRCA2(MIM 600185) mutations is now widely available through both commercial laboratories and research studies. Whereas thousands of BRCA1 and BRCA2 truncating mutations have been associated with increased risk of cancer in carriers, the contribution of other BRCA1 and BRCA2 sequence variants to cancer risk remains largely undefined. These “variants of unknown significance” (VUSs) are mainly missense mutations but also include a number of intronic variants and inframe deletions and insertions (IFDIs). The open-access online Breast Cancer Information Core (BIC) Database, which functions as a repository of sequence alterations in BRCA1 and BRCA2, contains >1,500 distinct sequence variants that are currently reported as having unknown clinical significance. To date, classification of the BRCA1 and BRCA2 VUSs as cancer predisposing or neutral has proven problematic, because it is not known whether these subtle changes alter the function of the proteins sufficiently to predispose to cancer. Determination of their disease relevance on the basis of population or family studies has also proved difficult, because most of the alleles encoding these mutations are very rare and, in some cases, population specific. Although identified in a minority (5%–10%) of individuals tested clinically, the number of such tests performed annually worldwide means that a large number of women (and their families) are affected by the VUS issue. In addition, there is evidence to suggest that minority ethnic populations are disproportionately affected. VUSs in the BRCA1 and BRCA2 genes pose significant problems, because patients and physicians do not know whether the VUSs predispose to cancer or are neutral with respect to cancer risk. As a result, carriers of VUSs and their family members cannot take advantage of the risk assessment, prevention, and therapeutic measures that are available to carriers of known deleterious truncating mutations.1Burke W Daly M Garber J Botkin J Kahn MJ Lynch P McTiernan A Offit K Perlman J Petersen G et al.Recommendations for follow-up care of individuals with an inherited predisposition to cancer: II. BRCA1 and BRCA2—Cancer Genetics Studies Consortium.JAMA. 1997; 277: 997-1003Crossref PubMed Google Scholar In addition, carriers of VUSs are sometimes counseled to undergo prophylactic oophorectomy or mastectomy because of the presence of the VUS, but in the absence of any knowledge of the cancer relevance of the VUS. For these reasons, the determination of the clinical relevance of VUSs in BRCA1 and BRCA2 has become an important clinical issue. Although the focus of this article is on BRCA1 and BRCA2, similar concerns and issues are found in virtually all complex, genetically heterogeneous diseases for which genetic testing is performed—for example, colon cancer associated with mutations in mismatch repair genes.2Lucci-Cordisco E Boccuto L Neri G Genuardi M The use of microsatellite instability, immunohistochemistry and other variables in determining the clinical significance of MLH1 and MSH2 unclassified variants in Lynch syndrome.Cancer Biomark. 2006; 2: 11-27PubMed Google Scholar To address the problem of defining which VUSs are deleterious/disease causing and which are neutral, various types of evidence and classification schemes have been proposed.3Goldgar DE Easton DF Deffenbaugh AM Monteiro AN Tavtigian SV Couch FJ Breast Cancer Information Core (BIC) Steering Committee Integrated evaluation of DNA sequence variants of unknown clinical significance: application to BRCA1 and BRCA2.Am J Hum Genet. 2004; 75: 535-544Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 4Billack B Monteiro AN Methods to classify BRCA1 variants of uncertain clinical significance: the more the merrier.Cancer Biol Ther. 2004; 3: 458-459Crossref PubMed Scopus (12) Google Scholar, 5Monteiro AN Couch FJ Cancer risk assessment at the atomic level.Cancer Res. 2006; 66: 1897-1899Crossref PubMed Scopus (14) Google Scholar These include formal assessment of the cosegregation of the variant with disease in pedigrees,6Thompson D Easton DF Goldgar DE A full-likelihood method for the evaluation of causality of sequence variants from family data.Am J Hum Genet. 2003; 73: 652-655Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 7Petersen GM Parmigiani G Thomas D Missense mutations in disease genes: a Bayesian approach to evaluate causality.Am J Hum Genet. 1998; 62: 1516-1524Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 8Zhou XI Iversen Jr, ES Parmigiani G Classification of missense mutations of disease genes.J Am Stat Assn. 2005; 100: 51-60Crossref PubMed Scopus (10) Google Scholar co-occurrence of the variant in question in trans with a known deleterious mutation,9Judkins T Hendrickson BC Deffenbaugh AM Eliason K Leclair B Norton MJ Ward BE Pruss D Scholl T Application of embryonic lethal or other obvious phenotypes to characterize the clinical significance of genetic variants found in trans with known deleterious mutations.Cancer Res. 2005; 65: 10096-10103Crossref PubMed Scopus (50) Google Scholar and a number of in silico approaches that evaluate phylogenetic conservation and severity of the amino acid substitution.10Abkevich V Zharkikh A Deffenbaugh AM Frank D Chen Y Shattuck D Skolnick MH Gutin A Tavtigian SV Analysis of missense variation in human BRCA1 in the context of interspecific sequence variation.J Med Genet. 2004; 41: 492-507Crossref PubMed Scopus (128) Google Scholar, 11Tavtigian SV Samollow PB de Silva D Thomas A An analysis of unclassified missense substitutions in human BRCA1.Fam Cancer. 2006; 5: 77-88Crossref PubMed Scopus (25) Google Scholar, 12Tavtigian SV Deffenbaugh AM Yin L Judkins T Scholl T Samollow PB de Silva D Zharkikh A Thomas A Comprehensive statistical study of 452 BRCA1 missense substitutions with classification of eight recurrent substitutions as neutral.J Med Genet. 2006; 43: 295-305Crossref PubMed Scopus (476) Google Scholar In studies of other genes, evolutionary sequence-conservation analysis involving protein multiple-sequence alignments have been used to show that missense variants (MVs) at highly conserved/invariant residues are more often deleterious, whereas highly variable changes are more likely neutral.13Miller MP Kumar S Understanding human disease mutations through the use of interspecific genetic variation.Hum Mol Genet. 2001; 10: 2319-2328Crossref PubMed Scopus (238) Google Scholar, 14Sunyaev S Ramensky V Koch I Lathe III, W Kondrashov AS Bork P Prediction of deleterious human alleles.Hum Mol Genet. 2001; 10: 591-597Crossref PubMed Scopus (891) Google Scholar For BRCA1, Abkevich et al.10Abkevich V Zharkikh A Deffenbaugh AM Frank D Chen Y Shattuck D Skolnick MH Gutin A Tavtigian SV Analysis of missense variation in human BRCA1 in the context of interspecific sequence variation.J Med Genet. 2004; 41: 492-507Crossref PubMed Scopus (128) Google Scholar developed a predictive algorithm that combines a measure of cross-species conservation, including nonmammalian BRCA1 sequences, with a measure of the degree of chemical change in amino acids,15Grantham R Amino acid difference formula to help explain protein evolution.Science. 1974; 185: 862-864Crossref PubMed Scopus (1539) Google Scholar to identify 50 putative deleterious BRCA1 missense mutations. A number of VUSs have been examined, in terms of their effect on protein, with use of functional assays. Efforts in this area have focused on BRCA1 mutations in the two C-terminal BRCT domains,16Chapman MS Verma IM Transcriptional activation by BRCA1.Nature. 1996; 382: 678-679Crossref PubMed Scopus (423) Google Scholar, 17Monteiro AN August A Hanafusa H Evidence for a transcriptional activation function of BRCA1 C-terminal region.Proc Natl Acad Sci USA. 1996; 93: 13595-13599Crossref PubMed Scopus (414) Google Scholar which assess the transcriptional activation activity of the BRCT domains with use of mammalian and yeast-based models,18Ostrow KL McGuire V Whittemore AS DiCioccio RA The effects of BRCA1 missense variants V1804D and M1628T on transcriptional activity.Cancer Genet Cytogenet. 2004; 153: 177-180Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 19Phelan CM Dapic V Tice B Favis R Kwan E Barany F Manoukian S Radice P van der Luijt RB van Nesselrooij BP et al.Classification of BRCA1 missense variants of unknown clinical significance.J Med Genet. 2005; 42: 138-146Crossref PubMed Scopus (73) Google Scholar, 20Carvalho MA Marsillac SM Karchin R Manoukian S Grist S Swaby RF Urmenyi TP Rondinelli E Silva R Gayol L et al.Determination of cancer risk associated with germ line BRCA1 missense variants by functional analysis.Cancer Res. 2007; 67: 1494-1501Crossref PubMed Scopus (96) Google Scholar and on the E3 ligase activity associated with the N-terminal BARD1-binding domain of BRCA1.21Morris JR Pangon L Boutell C Katagiri T Keep NH Solomon E Genetic analysis of BRCA1 ubiquitin ligase activity and its relationship to breast cancer susceptibility.Hum Mol Genet. 2006; 15: 599-606Crossref PubMed Scopus (80) Google Scholar Examination of BRCA2 function has focused on assays of homology-directed repair and centrosome amplification in response to ectopic expression of full-length, wild-type VUS containing BRCA2 protein.22Wu K Hinson SR Ohashi A Farrugia DJ Wendt P Tavtigian SV Deffenbaugh A Goldgar D Couch FJ Functional evaluation and cancer risk assessment of BRCA2 unclassified variants.Cancer Res. 2005; 65: 417-426PubMed Google Scholar Recently, crystal structures of the BRCA1 BRCT and BRCA2 DNA-binding domains have been used to predict that a number of VUSs predispose to cancer,23Mirkovic N Marti-Renom MA Weber BL Sali A Monteiro AN Structure-based assessment of missense mutations in human BRCA1: implications for breast and ovarian cancer predisposition.Cancer Res. 2004; 64: 3790-3797Crossref PubMed Scopus (88) Google Scholar, 24Karchin R Monteiro AN Tavtigian SV Carvalho MA Sali A Functional impact of missense variants in BRCA1 predicted by supervised learning.PLoS Comput Biol. 2007; 3: e26Crossref PubMed Scopus (46) Google Scholar, 25Yang H Jeffrey PD Miller J Kinnucan E Sun Y Thoma NH Zheng N Chen PL Lee WH Pavletich NP BRCA2 function in DNA binding and recombination from a BRCA2-DSS1-ssDNA structure.Science. 2002; 297: 1837-1848Crossref PubMed Scopus (521) Google Scholar but genetic evidence suggests that some of these predictions are incorrect. Although none of the approaches described above have successfully been used to classify the clinical relevance of BRCA1 or BRCA2 VUSs, a number have shown promise in this regard. The development of methods based on functional assays, crystal structure, phylogenetic and sequence analyses, and other approaches has been hampered by the absence of a large number of VUSs that have been proven to be either deleterious or neutral on the basis of direct genetic evidence that can be used in validation of these various assays. In this article, we examine the genetic evidence for or against disease causality for a large number of variants, with use of a large database of tested individuals collected by Myriad Genetic Laboratories. In addition to analysis of co-occurrence of VUSs with established deleterious mutations and the analysis of VUS cosegregation in individual pedigrees, we apply a novel analysis of personal and family cancer history, using data on >70,000 individuals, to assess the clinical significance of 1,433 distinct sequence variants in BRCA1 and BRCA2. The data analyzed for this article come from the large database of full-sequence testing for mutations in the BRCA1 and BRCA2 genes, performed at Myriad Genetic Laboratories. Since our primary focus was on the analysis of personal and family history, we began with all 70,071 individuals receiving full-sequence tests as of December 2005. To avoid potential confusion over which part of the personal/family history was due to which variant, this data set was reduced by eliminating all individuals who had more than one reported sequence variant (including both VUS and pathogenic mutations), which resulted in a set of 61,270 individuals with at most a single BRCA1 or BRCA2 sequence variant. We then divided the family history data set into several ethnic groups, since we postulated that probands of different ethnic backgrounds are expected to present with different distributions of mutations and different distributions of personal and family histories of cancer. After examining the data set with use of some preliminary logistic regressions to examine heterogeneity as a function of reported ethnicity, we constructed three separate groups that had similar family-history and mutation-prevalence profiles. These groups were: (a) western European, central/eastern European, other/none specified, Native American, and Asian; (b) Ashkenazi Jewish; and (c) Latin American/Caribbean and African American. In the first group, 72% were of European ancestry, 24% did not specify an ethnicity, and 4% were reported as of Asian, Middle Eastern, or Native American origin. Individual probands who reported mixed ancestry between these broad groups (e.g., Western European and Caribbean) were not included in the final data set, leaving 60,529 individual probands for analyses of family history. Table 1 shows the distribution by ethnicity and testing results of these individuals. A total of 1,433 distinct unclassified variants in 4,623 tested probands were identified through sequencing and are the subject of this article.Table 1Results of Full BRCA Sequence Analysis of 60,529 Tested ProbandsNo. (%) of Subjects with GenotypeEthnic GroupWild TypeNeutralVUSBRCA1 DeletionBRCA2 DeletionEuropeanaSee the text for the description of ethnicity for this group.41,899 (78.6)1,993 (3.7)3,972 (7.5)3,102 (5.8)2,334 (4.4)Ashkenazi Jewish3,622 (80.6)381 (8.5)258 (5.7)139 (3.1)91 (2.0)African American/Caribbean 1,667 (60.9)398 (14.5)393 (14.3)176 (6.4)104 (3.8) Total47,188 (78.0)2,772 (4.6)4,623 (7.6)3,417 (5.6)2,529 (4.2)a See the text for the description of ethnicity for this group. Open table in a new tab As before,3Goldgar DE Easton DF Deffenbaugh AM Monteiro AN Tavtigian SV Couch FJ Breast Cancer Information Core (BIC) Steering Committee Integrated evaluation of DNA sequence variants of unknown clinical significance: application to BRCA1 and BRCA2.Am J Hum Genet. 2004; 75: 535-544Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar we assumed that all variants can be classified as deleterious mutations (M) or neutral variants (V). Our aim was to calculate a likelihood ratio (LR) of the formP(Data|M)P(Data|V)for each variant, given the available data. These ratios (or Bayes factors) can be combined with prior probabilities to obtain an absolute probability that each variant is deleterious. In this work, we first focused on the data sources that are most straightforwardly quantifiable from pedigree data: co-occurrence with known deleterious mutations, family history, and cosegregation. These three sources of data are independent, and the LR can therefore be derived by multiplying the LRs from the three components. We then used an admixture model to examine the influence of the type of mutation. To include information on the co-occurrence of a VUS in trans phase with a deleterious mutation as evidence against the variant being disease causing, we followed the likelihood-ratio calculation described in our previous work.3Goldgar DE Easton DF Deffenbaugh AM Monteiro AN Tavtigian SV Couch FJ Breast Cancer Information Core (BIC) Steering Committee Integrated evaluation of DNA sequence variants of unknown clinical significance: application to BRCA1 and BRCA2.Am J Hum Genet. 2004; 75: 535-544Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar We assumed that, for BRCA1, compound heterozygotes for two deleterious mutations are vanishingly rare (occur in the testing population with a frequency of 1 in 10,000), given both biological and genetic evidence that homozygotes or compound heterozygotes for deleterious BRCA1 mutations are embryonically lethal,26Hohenstein P Kielman MF Breukel C Bennett LM Wiseman R Krimpenfort P Cornelisse C van Ommen GJ Devilee P Fodde R A targeted mouse BRCA1 mutation removing the last BRCT repeat results in apoptosis and embryonic lethality at the headfold stage.Oncogene. 2001; 20: 2544-2550Crossref PubMed Scopus (47) Google Scholar, 27Gowen LC Johnson BL Latour AM Sulik KK Koller BH Brca1 deficiency results in early embryonic lethality characterized by neuroepithelial abnormalities.Nat Genet. 1996; 12: 191-194Crossref PubMed Scopus (373) Google Scholar whereas, for BRCA2, we assumed that the probability is 1 in 1,000, the increased frequency reflecting the fact that viable compound heterozygotes have been reported as Fanconi anemia, type D1.28Howlett NG Taniguchi T Olson S Cox B Waisfisz Q Die-Smulders C Persky N Grompe M Joenje H Pals G et al.Biallelic inactivation of BRCA2 in Fanconi anemia.Science. 2002; 297: 606-609Crossref PubMed Scopus (925) Google Scholar, 29Alter BP Rosenberg PS Brody LC Clinical and molecular features associated with biallelic mutations in FANCD1/BRCA2.J Med Genet. 2007; 44: 1-9Crossref PubMed Scopus (169) Google Scholar For these analyses, we used an updated data set based on ∼100,000 tests with observed frequencies of BRCA1 and BRCA2 deleterious mutations of 6.6% and 5.0%, respectively. The phase of the VUS with an observed deleterious mutation was determined through examination of haplotypes of common polymorphisms known in other examples of each variant or when the VUS and the deleterious mutation were in the same PCR fragment, as determined by direct examination of the sequence traces. In some cases, phase could not be determined, and we assumed that the first deleterious mutation was in cis phase and that subsequent different deleterious mutations were in trans phase. The LR for deleterious versus neutral is expressed as(p2)k(1-p2)n-kp1k(1-p1)n-k ,where n is the total number of times the variant was observed, k is the number of those that were in trans with a known deleterious mutation, p1 is the overall frequency of pathogenic mutations in the data set, and p2 is the probability of being a compound heterozygote for two pathogenic mutations (e.g., p1=0.066 and p2=1/10,000). Of the 1,433 VUSs studied here, 476 were observed at least five times, and 248 had at least 10 occurrences. Of the 1,433 variants, 44 were observed to occur with at least one deleterious mutation. We developed likelihood models on the basis of family history,3Goldgar DE Easton DF Deffenbaugh AM Monteiro AN Tavtigian SV Couch FJ Breast Cancer Information Core (BIC) Steering Committee Integrated evaluation of DNA sequence variants of unknown clinical significance: application to BRCA1 and BRCA2.Am J Hum Genet. 2004; 75: 535-544Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar which, given the availability of the large Myriad Genetic Laboratories data set, is an extremely powerful tool for classification of VUSs. The method is based on the difference in personal and family-history profiles between individuals found to carry a true deleterious mutation and those with a wild-type BRCA sequence. The rationale is that mutation prevalence is known to be strongly dependent on certain key factors (e.g., disease status of the proband, age at diagnosis, and number and age of relatives with breast cancer [BRCA] or ovarian cancer), so these characteristics should also predict the probability of a new disease-causing missense mutation, whereas that of a neutral mutation should be independent of family history. We suppose that we have a set of probands drawn from a population with a certain distribution of family history. Suppose first that family history is classified into n types. Let pj be the probability that an individual with a mutation has family history type j, whereas qj is the corresponding probability for neutral variants. For a VUS that is deleterious, the predicted distribution of family history should be the same as that for known pathogenic mutations. The LR for a VUS with family history in category j drawn from the same population is thereforeP(FHj|M,{FH})P(FHj|V,{FH})=pjqj ,where {FH} is the set of all family histories in the study population. In theory, the probabilities pj and qj could be calculated from the proportions of individuals with each category of family history who are found to carry a deleterious mutation. In practice, however, there are large numbers of possible constellations of family history, and the numbers of mutations in some categories would be very small. Instead, we estimated these probabilities by fitting a logistic-regression model. Thus, we fitted a model in which the outcome was the occurrence of a deleterious mutation versus a normal sequence, and summaries of personal and family history were included as covariates. This model then provides predicted probabilities rk that individual k is a mutation carrier, given his or her FH, isrk=P(M|{FH}k) . Let r0 be the corresponding probability under the null hypothesis that the mutation is unrelated to family history or, equivalently, the prior probability of a deleterious mutation in the population. This is estimated by the overall proportion of individuals who have a deleterious mutation (rather than a normal sequence). For example, with use of the data in table 1, for the European group for BRCA1,r0=3,1023,102+41,899=0.069 .Thenpk=P(FHk|M,{FH})=P(M|{FH}k)P({FH}k|{FH})P(M|{FH})=rkP(FHk|{FH})r0andqk=P({FH}k|V,{FH})=P(V|FHk)P([FHk|{FH})P(V|{FH})=(1-rk)P({FH}k|{FH})1-r0. Therefore, the required LR isrk(1-r0)(1-rk)r0 ;that is, the odds ratio (OR) of the predicted probability that the individual with the given family history is a mutation carrier against the corresponding probability under the null hypothesis. Where there are multiple individuals with the same VUS, the LRs can be multiplied. We note that this formulation implicitly assumes that all pathogenic mutations in the same gene confer identical risks—that is, that there is no allelic heterogeneity. To implement this analysis, the logistic-regression model was fitted separately for each ethnic group and separately for BRCA1 and BRCA2. To construct a predictive model to be applied to the VUS probands, we used a logistic-regression model within each ethnic group, comparing the personal and family histories of individuals with known deleterious sequences versus those with normal sequences. The specific factors used in the prediction were categorized asPersonal history of tested individualBRCA1. None,2. Ductal carcinoma in situ (DCIS) only, orDiagnosis of BRCA at age3. ⩾60,4. 50–59,5. 40–49,6. 30–39, or7. 100:1 in favor of being deleterious, 216 had odds of >10:1, and no known variants were excluded at this same threshold. Although we recognize that these summaries may overstate the model’s out-of-sample predictive ability, since the deleterious variants were used to construct the logistic regressions, they do provide reassurance that, in general, the method is performing as we expected. Further confirmation of this is obtained by looking at the similar calculation for 44 variants already classified as neutral and not used in either the logistic regression or in the subsequent analysis of VUSs. Of these 44 variants, 33 had odds against being deleterious of >100:1, and the other 11 all had odds against being deleterious of a lesser magnitude. Elsewhere, we developed a general-pedigree likelihood method for evaluating cosegregation of variants with disease in families that can be used to assess disease causality for unclassified sequence variants.6Thompson D