Clinically Applicable Models to Characterize BRCA1 and BRCA2 Variants of Uncertain Significance

Andrew D Spearman; Kevin Sweet; Xiao-Ping Zhou; Jane McLennan; Fergus J Couch; Amanda Ewart Toland

doi:10.1200/JCO.2008.17.8228

. 2008 Sep 29;26(33):5393–5400. doi: 10.1200/JCO.2008.17.8228

Clinically Applicable Models to Characterize BRCA1 and BRCA2 Variants of Uncertain Significance

Andrew D Spearman ¹, Kevin Sweet ¹, Xiao-Ping Zhou ¹, Jane McLennan ¹, Fergus J Couch ¹, Amanda Ewart Toland ¹

PMCID: PMC2651073 PMID: 18824701

Abstract

Purpose

Twenty percent of individuals with a strong family and/or personal history of breast and ovarian cancer carry a deleterious mutation in BRCA1 or BRCA2. Identification of mutations in these genes is extremely beneficial for patients pursuing risk reduction strategies. Approximately 7% of individuals who have genetic testing of BRCA1 and BRCA2 carry a variant of uncertain significance (VUS), making clinical management less certain. The majority of identified VUS occur only in one to two individuals; these variants are not able to be classified using current classification models with segregation analysis components.

Methods

To develop a clinically applicable method that can predict the pathogenicity of VUS that does not require familial information or segregation analysis, we identified characteristics of breast or ovarian tumors that distinguished sporadic tumors from tumors with BRCA1 or BRCA2 mutations. Study participants included individuals with known deleterious mutations in BRCA1 or BRCA2 and individuals with classified or unclassified BRCA variants.

Results

We applied the models to 57 tumors with 43 different deleterious BRCA mutations and 57 tumors with 54 unique classified and unclassified BRCA variants. Of the 33 previously unclassified VUS studied, we found evidence of neutrality for 21.

Conclusion

Our models showed 98% sensitivity and 76% specificity for predicting classified DNA changes. We classified 64% of unknown variants as neutral. Classification of VUS as neutral will have immediate benefit for those individuals and their family members. These models are adaptable for the clinic and will be useful for individuals with limited available family history.

INTRODUCTION

Mutations in BRCA1 and BRCA2 confer a greatly increased lifetime risk for breast and ovarian cancer. The number of individuals who have had genetic testing of BRCA1 and BRCA2 in the United States now exceeds 70,000.¹ Individuals who carry a mutation in BRCA1 or BRCA2 can increase the likelihood of detecting cancers at an earlier stage by more stringent surveillance and can decrease their chance of developing cancer through the use of prophylactic surgery and/or chemopreventive agents. Of the high-risk individuals who have had mutation analyses through Myriad Genetic Laboratories (Salt Lake City, UT), approximately 7% have a variant of uncertain significance (VUS) in BRCA1 or BRCA2.²^-⁴ VUS are typically missense or potential splice site changes of uncertain biologic or clinical relevance. They are more frequent in the minority ethnic populations.³ More than 1,500 VUS have been identified; most have only been reported in one to two individuals.

Through prediction studies, some BRCA VUS have been classified as neutral polymorphisms, and a small number of BRCA VUS have been classified as likely deleterious. Many of the predicted deleterious VUS are located in highly conserved functional domains or affect transcript start or splice sites. Still, the majority of variants have yet to be classified.⁴^-¹¹ Several of the algorithms used to characterize VUS use familial segregation analysis or study variants in multiple unrelated individuals to provide evidence for or against causality. One difficulty with this approach is that the vast majority of reported VUS have been observed in only one or two individuals, which makes them ineligible for segregation analysis. Also, population-based studies in BRCA carriers have shown that more than 50% of BRCA-positive individuals may not have a close family member with a BRCA-related cancer who would be available for segregation analysis.¹² Finally, some individuals with BRCA VUS have BRCA-related cancers segregating on both sides of the family. Thus, it is important to develop additional means to classify VUS that do not rely on segregation and can be applied in various clinical settings.

To address this need, we developed four BRCA VUS classification models that use clinical information typically available from pathology and mutation testing reports combined with protein prediction model data, cancer type, and tumor loss of heterozygosity (LOH) data. We applied these models to 57 tumors with deleterious BRCA mutations and 57 tumors with classified and/or unclassified variants.

METHODS

Human Samples

Studies were approved by local institutional review boards. All study participants signed informed consent for this research. Paired normal and tumor tissues were ascertained through one of the following three sources: The Ohio State University Comprehensive Cancer Center Clinical Cancer Genetics Program, the Mayo Clinic Familial Cancer Program, and the University of California, San Francisco Familial Risk Shared Resource. Eligibility criteria included a diagnosis of breast or ovarian cancer, available normal and tumor DNA, available pathology records, and a BRCA testing report showing a BRCA1 and/or BRCA2 deleterious mutation, VUS, or a reclassified variant. Hematoxylin and eosin slides from tumors were reviewed by a pathologist, and areas comprising greater then 70% tumor cells were microdissected from sections or were cored. Normal DNA was isolated from blood or histologically normal breast tissue. Lymphoblastoid cell lines were established on a subset of individuals.

Genomic DNA was isolated from fixed archival tissue by removal of paraffin using xylene and ethanol washes. Protein was removed by proteinase K treatment for 48 hours at 55°C in nucleic acid lysis buffer. Samples were phenol/chloroform extracted and ethanol precipitated.

Allele-Specific LOH

We used restriction fragment length polymorphism studies and sequence analysis to conduct allele-specific LOH analysis. When a mutation or variant altered a restriction enzyme site, polymerase chain reaction (PCR) products were digested with the appropriate enzyme, separated on an agarose gel, and stained with ethidium bromide. Bands were visualized on an Alphaimager (Alpha Innotech, San Leandro, CA). For mutations resulting in deletions of more than 20 base pairs, PCR products were run directly on a 1.5% agarose gel. LOH was assessed visually for restriction fragment length polymorphism analysis. When the results were visually ambiguous, ratios of cut to uncut band of normal and tumor DNA were taken using band intensity. Samples were scored as loss if the ratios were greater than 1.5 or less than 0.67 according to criteria used in other studies.¹³^,¹⁴ To avoid undercalling LOH, we scored samples as uncertain if ratios fell between 0.67 and 0.76 or 1.35 and 1.50. For changes not resulting in an altered restriction site, PCR products were treated with ExoSap-It (USB, Cleveland, OH) and sequenced. Sequence traces in the forward and reverse direction were compared between control DNA and normal DNA and tumor DNA containing the sequence change using DNAstar 3.0 (DNASTAR, Inc, Madison, WI). Loss was determined visually by two reviewers and consisted of at least 30% difference between the two alleles compared with normal carrier ratios.

Evolutionary Conservation

We used align grantham variation deviation analysis (A-GVGD) to identify missense changes of potential functional significance (http://agvgd.iarc.fr/alignments.php).⁶^,¹⁵ A-GVGD uses a combination of the physical properties of amino acids and protein sequence alignments from multiple species to predict if missense substitutions are more likely to be neutral or affect protein function. Because deleterious missense changes have not been reported outside of conserved BRCA domains,⁴ we applied A-GVGD only to missense changes occurring in the following three conserved domains: BRCA1 ring domain (amino acids 1 to 102), BRCA1 breast cancer C-terminal domain (amino acids 1641 to 1863), or BRCA2 DNA binding domain (amino acids 2401 to 3200). A-GVGD analysis assigns a probability class to each variant studied ranging from class (C) 0 variants, which are expected to be neutral, to C65 variants, which are expected to affect protein function. Using data from 1,400 VUS,⁴ prior probabilities of a change being deleterious for the A-GVGD classes of variants mapping to conserved regions were as follows: C65 = 0.81; C35, C45, and C55 = 0.66; C15 and C25 = 0.29; and C0 = 0.01.¹⁶ From these likelihood probabilities, we calculated odds of being deleterious for the A-GVGD classes falling into conserved domains as follows: C0 = 0.01; C15 and C25 = 0.41; C35, C45, and C55 = 1.5; and C65 = 4.26. All other missense changes were given a probability of being deleterious of 0.01.⁴

Splicing Studies

To determine whether any VUS affected normal splicing patterns and, therefore, could be functional changes, we used ESEfinder and SpliceSiteFinder to measure the theoretical effect on splicing.¹⁷^,¹⁸ These programs are used to identify exonic splice enhancer motifs (ESEfinder) or putative splice sites (SpliceSiteFinder). We isolated RNA from lymphoblastoid cell lines from individuals carrying putative splice site mutations for use in splicing studies. We performed reverse transcriptase PCR using primer sets from the two flanking exons as well as primer sets between the flanking exon and the potentially aberrantly spliced exon using cDNA from patients and wild-type controls. We noted aberrant splicing in cases where bands of sizes other than those seen in 11 wild-type cDNA controls were present. To confirm abnormal splicing, we sequenced any extra bands.

Observation With a Deleterious Mutation

Because homozygous BRCA1 mutations are expected to be lethal and homozygous BRCA2 mutations are expected to result in lethality or phenotypes such as Fanconi anemia, the presence of a deleterious mutation in trans with a variant suggests its neutrality.⁶ We used data from mutation reports and the literature to determine whether variants in our study had been observed in trans with a deleterious mutation. For our models, we used the published estimates of the probabilities of two deleterious mutations occurring in trans in BRCA1 and BRCA2 of P = .0001 and P = .001, respectively.⁵

Model Phenotypes

To identify tumor characteristics that would be useful in discriminating deleterious variants from nondeleterious variants, we searched the literature for studies comparing characteristics of breast and ovarian tumors from BRCA1 and BRCA2 mutation-positive and mutation-negative individuals. We identified a number of characteristics available in most pathology reports that could be used in a prediction model (Table 1). ⁵^,¹⁹^-⁵⁶ When a considerable range of frequency estimates was observed for a characteristic, we calculated an average frequency based on raw numbers from all of the studies. Not surprisingly, the features predictive of mutation status varied between both breast and ovarian cancer and between BRCA1 and BRCA2. As a result, we created likelihood odds for four predictive models, one for each gene and tumor type, that differ only between gene/cancer site and model-specific likelihood odds (Appendix Table A1, online only; see also Appendix, online only).

Table 1.

Frequency of Histopathologic Characteristics of BRCA1, BRCA2, and Sporadic Tumors

Characteristic	Sporadic Frequency (%)		BRCA1 Frequency (%)		BRCA2 Frequency (%)
Characteristic	Range Reported in Literature	Frequency Used for Weighting	Range Reported in Literature	Frequency Used for Weighting	Range Reported in Literature	Frequency Used for Weighting
Breast
BRCA1 loss	20-40	30	68-100	80	NA
BRCA2 loss	22-50	30	NA		38-100	70
Medullary	1-3	1.5	11-13	12	ND
Triple negative	10-25	16	80	80	ND
ER negative	13-37	26	63-90	83	ND
PR positive	58-68	65	16-21	20	ND
HER-2 positive	12-35	20	0-3	3	0-3	3
HER-2 negative	70-88	80	97-100	97	97-100	97
ER positive/grade 1	27-42	30	0-2.4	2	5-17	7
ER positive/grade 2	11-28	27	10-17	10	22-45	41
ER positive/grade 3	9-17	13	12-28	13	28-30	28
ER negative/grade 1	3-14	5	0-1	1	1-4	2
ER negative/grade 2	12-13	13	9-13	13	2-17	5
ER negative/grade 3	12-16	16	62-91	65	9-18	16
Grade 1	18-56	22	0-9	2.5	11-22	17
Grade 2	23-49	42	16-26	23	29-49	43
Grade 3	21-49	36	66-100	71	38-64	47
Ovarian
BRCA1 loss	40-66	40	72-100	90	NA
BRCA2 loss	30-42	30	NA		80-100	80
Serous	29-47	34	40-100	50	39-100	60
Mucinous	11-36	17	0-3	2	0-2	1
Endometrioid	5-33	26	NA		0-29	18
Grade 1	10-19	13	1-3	2	0-3	3
Grade 2	21-34	31	22-27	26	13-14	14
Grade 3	50-55	54	72-75	72	81-87	87
Stage I	24		3-17	17	0-6	6
Stage II	17		10-16	10	0-6	6
Stage III	40		69-70	69	82-100	82
Stage IV	19		3-11	6	0-7	5

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Abbreviations: NA, not applicable comparison; ND, not calculated because the literature suggests no differences from sporadic rates; ER, estrogen receptor; PR, progesterone receptor; HER-2, human epidermal growth factor receptor 2.

Statistical Methods

To derive odds of pathogenicity for each variant, we used a modified multifactorial approach that combines the odds of causation of independent variables.⁴^,⁵^,⁷^,⁹ Odds for each independent variable were determined from frequencies reported in the literature or published odds, with the exception of truncating mutations and splice site alterations for which we calculated odds of being deleterious of 1,000:1 if splicing defects or truncating mutations were identified and odds of 0.01 if splicing defects were not observed. The product of the likelihoods of independent variables was obtained to provide an overall odds of pathogenicity for each sequence alteration (additional details are in Appendix). On the basis of previous studies, we used a cutoff of 1,000:1 in favor of being deleterious and a cutoff of 100:1 in favor of being neutral for final classification status.⁴^,⁵ When two independent tumors with the same sequence alteration were identified, independent factors for both were combined and considered in the final data interpretation. We calculated odds both with and without LOH data because LOH data may not be typically available clinically and the frequency of LOH in neutral BRCA variants has not been well documented. If a prediction of variant status was only significant in the combined odds using LOH data, we scored the variant as suspected neutral or suspected deleterious accordingly.

RESULTS

Identification of Study Criteria for the Models

We collected tumor and normal tissue and histopathologic and clinical data from the medical records and mutation reports of 102 individuals with BRCA mutations or characterized or uncharacterized variants (Table 2). The study group consisted of 57 tumors with 43 unique deleterious mutations and 57 tumors with 54 unique variants (Tables 2, 3, and 4; Appendix Tables A2 and A3, online only).

Table 2.

Study Population

Factor	No. of Mutations			No. of VUS
Factor	BRCA1	BRCA2	Total	BRCA1	BRCA2	Total
Breast tumors	33	17	50	21	29	50
Ovarian tumors	3	4	7	1	6	7
Unique change	25	18	43	19	35	54
Previously classified	NA	NA	NA	9	12	21
Previously unclassified	NA	NA	NA	10	23	33
Patients^*	33	17	50	21	34	55
Patients with two or more changes	2	0		2	4
Bilateral cancer	3	0	3	1	1	2

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Abbreviations: VUS, variant of uncertain significance; NA, not applicable.

Note that the No. of patients on study (n = 102) does not equal the total No. of tumors studied (n = 114) or the total No. of patients in this table (n = 105). Some individuals have bilateral cancers; other study participants have more than one change identified and are counted in more than one category.

Table 3.

Examples of Characteristics for Predictive Breast Models

Sequence Change	LOH	Conserved Domain	A-GVGD Class	Splice Defect	In Trans	Age at Diagnosis (years)	Grade	ER Status	PR Status	HER-2 Status	Histology
BRCA1
1294del40	WT	NA	NA	ND	NA	40	3	−	−	+	IDC
IVS13+1 G>A	None	NA	NA	Yes	No	41	2	−	−	−	IDC
C61G	WT	Yes	C65	ND	No	29	3	−	−	−	IDC
Y856H	None	No	C0	ND	Yes	45	3	+	+	+	DCIS
BRCA2
2041delA	None	NA	NA	ND	NA	34	3	+	+	−	IDC
IVS8-12delTA	None	NA	NA	No	No	44	1	+	+	−	IDC
N517S	None	No	C0	ND	No	45	1	+	+	ND	IDC
L2106P	None	No	C0	ND	No	57	3	+	+	−	IDC

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NOTE. Examples of phenotypes from eight individuals with different types of BRCA1 and BRCA2 sequence changes are shown. The full list of tumors and sequence alterations studied is provided in Appendix Table A2.

Abbreviations: LOH, loss of heterozygosity; A-GVGD, align grantham variation, grantham deviation; ER, estrogen receptor; PR, progesterone receptor; HER-2, human epidermal growth factor receptor 2; WT, wild-type allele lost; NA, not applicable; ND, no data; −, negative; +, positive; IDC, invasive ductal carcinoma; DCIS, ductal carcinoma in situ.

Table 4.

Examples of Characteristics for Predictive Ovarian Models

Gene	Mutation Type	Change	Conserved Domain	LOH	A-GVGD Class	In Trans	Stage	Grade	Age at Diagnosis (years)	Histology
BRCA1	Truncating	1135insA	NA	WT	NA	No	ND	3C	59	Papillary serous
BRCA1	Missense	P334L	No	WT	C0	Yes	ND	ND	39	Unspecified
BRCA2	Truncating	7297delCT	NA	None	NA	NA	ND	3C	22	Serous
BRCA2	Missense	M784V	No	V	C0	No	IIIC	3	45	Unspecified
BRCA2	Missense	V1643A	No	WT	C0	No	IIIC	ND	50	Serous

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NOTE. Examples of phenotypes from different BRCA1 and BRCA2 sequence alterations seen in ovarian tumors are shown. The full list of phenotypes from all sequence alterations studied is provided in Appendix Table A3.

Abbreviations: LOH, loss of heterozygosity; A-GVGD, align grantham variation, grantham deviation; NA, not applicable; WT, wild-type allele lost; ND, no data; V, variant or mutant allele lost.

LOH

One characteristic of BRCA1 and BRCA2 mutation tumors that has been used previously to assess pathogenicity of BRCA VUS is tumor LOH.⁵^,¹¹^,²⁵ LOH occurs more frequently in BRCA mutation–positive tumors than sporadic tumors and is thought to occur predominantly on the nonmutated chromosome (Table 1).⁵^,¹¹^,²⁵^,²⁶^,²⁸ LOH in tumors with a neutral BRCA1 or BRCA2 variant is predicted to be random for loss of variant or wild-type allele; however, LOH in this group has not been well studied. To determine whether our observed rates of LOH were comparable to published rates, we successfully conducted allele-specific LOH studies on 56 of 57 tumors from carriers of known deleterious mutations. We detected LOH of the wild-type BRCA1 allele in 85% of breast tumors (28 of 33 tumors) and 100% of ovarian tumors (three of three tumors; Tables 3 and 4; Appendix Tables A2 and A3). We observed LOH of the wild-type BRCA2 allele in 63% of breast tumors (10 of 16 tumors) and 25% of ovarian tumors (one of four tumors). We also observed loss of the mutant allele in one BRCA1 breast tumor and in one BRCA2 breast tumor (Appendix Table A2). The BRCA1 frequencies of LOH are similar to published rates. However, the BRCA2 frequencies of loss were slightly lower than expected and lower than the frequencies used in other studies.⁵ We were also surprised by the high observed frequency of LOH of the mutant allele in breast tumors (one of 33 tumors for BRCA1 and one of 16 tumors for BRCA2). On the basis of our data and previous published reports, we used a frequency of LOH to generate likelihood ratios of the wild-type allele of 79% and 69% for BRCA1- for BRCA2-positive breast tumors, respectively, and 89% and 79% for BRCA1- and BRCA2-positive ovarian tumors, respectively, in our prediction models (Appendix Table A1). For deleterious mutations, we assumed that 1% of the time there would be loss of the mutant allele, as has been suggested in the literature.⁵^,¹¹^,²⁵^,²⁶^,²⁸

We successfully assessed allele-specific LOH in 55 of 57 tumors with BRCA VUS (Tables 2 and 3; Appendix Tables A2 and A3). Twenty-nine percent of tumors (six of 21 tumors) with BRCA1 VUS showed LOH of the wild-type allele, 14% of tumors (three of 21 tumors) showed LOH of the variant allele, and 57% of tumors (12 of 21 tumors) showed no imbalance. For BRCA2, 26% of breast tumors (nine of 34 tumors) showed wild-type allele loss, 23% of tumors (eight of 34 tumors) showed variant allele loss, and 50% of tumors (17 of 34 tumors) showed no imbalance. Variants BRCA1 IVS2-14 C>T, S127N, and V1804D and BRCA2 A1170V, E2856A, H1966Y, I2285V, M784V, N1878K, and Y42C were lost in tumors providing evidence for neutrality.

Sequence-Based Methods to Determine Pathogenicity

On the basis of a study of more than 1,433 BRCA VUS, pathogenic VUS are most likely to occur in regions that are highly conserved across species, occur in specific BRCA protein domains, and/or affect splicing.⁴ Five BRCA1 and nine BRCA2 missense changes fell in or near highly conserved domains. For these variants, we used A-AVGD, a statistical algorithm validated and widely used for the study of BRCA VUS, to assign classes of C0 (neutral) to C65 (likely deleterious).⁵^,⁶^,⁹^,¹⁵^,¹⁶ A-AVGD classified 10 of these variants as C0 or neutral (Tables 2 and 3; Appendix Tables A2 and A3). Three VUS and one suspected mutation were assigned a higher class and given an increased likelihood of being deleterious (Tables 5 and 6; Appendix Tables A4 and A5, online only).

Table 5.

Examples of Breast Tumor Odds

Sequence Change	Odds												Odds	Odds, No LOH	Interpretation
Sequence Change	LOH	A-GVGD/Mutation	Splice	In Trans	Age at Diagnosis	Triple Negative	ER Status	PR Status	HER-2	Grade/ER	Grade	HP	Odds	Odds, No LOH	Interpretation
BRCA1
1294del40	5.26	1,000	1	1	3.4	1	1	2.29	0.15	4.1	1	1	2.5 × 10⁴	4,788	D
IVS13+1 G>A	0.285	1	1,000	1	3.4	5	1	1	1	1	0.55	1	2,665	9,350	D^*
C61G	5.26	4.26	1	1	15.3	5	1	1	1	1	1.97	1	3,377	642	DS^*
Y856H	0.285	0.01	1	0.0001	3.4	1	0.23	0.31	0.15	1	1.97	1	2 × 10⁻⁸	7 × 10⁻⁸	N
BRCA2
2041delA	0.428	1,000	1	1	4.97	1	1	1	1.2	2.2	1	1	5,616	1.3 × 10⁴	D
IVS8-12delTA	0.428	1	0.01	1	2.89	1	1	1	1.2	0.23	1	1	0.003	0.008	N
N517S	0.428	0.01	1	1	2.89	1	1	1	1	0.23	1	1	0.003	0.007	N
L2106P	0.428	0.01	1	1	2.07	1	1	1	1.2	2.2	1	1	0.023	0.054	U

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NOTE. Examples of odds generated from phenotypic data of Table 3 are shown. The final odds are calculated by combining odds for each of the independent variables. The full list of odds for each sequence variation and tumor studied is provided in Appendix Table A4.

Abbreviations: LOH, loss of heterozygosity; A-GVGD, align grantham variation, grantham deviation; ER, estrogen receptor; PR, progesterone receptor; HER-2, human epidermal growth factor receptor 2; HP, histopathology; D, deleterious; DS, suspected deleterious; N, neutral; U, uncertain.

Reclassified as deleterious by other studies.

Table 6.

Examples of Ovarian Tumor Odds

Gene	Change	Odds							Combined Odds	Odds, No LOH	Interpretation
Gene	Change	LOH	A-GVGD/ Mutation	In Trans	Stage	Grade	Histology	Age	Combined Odds	Odds, No LOH	Interpretation
BRCA1	1135insA^*	4.45	1,000	1	1	1.33	1.47	11.8	102,662.3	23,070.2	D
BRCA1	P334L	4.45	0.01	0.0001	1	1	1	7.06	0.0000314	0.0000071	N^†
BRCA2	7297delCT	0.428	1,000	1	1	1.61	1.76	0.52	630.65	1,473.47	D
BRCA2	M784V	0.067	0.01	1	2.05	1.61	1	4.05	0.009	0.1337	SN
BRCA2	V1643A	4.6	0.01	1	2.05	1	1.76	7.92	1.3145	0.2858	U

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NOTE. Examples of odds generated from phenotypes listed in Table 4 are shown. The final odds are calculated by combining odds for each of the independent variables. The full list of odds from all sequence alterations and ovarian tumors studied is provided in Appendix Table A5.

Abbreviations: LOH, loss of heterozygosity; A-GVGD, align grantham variation, grantham deviation; D, deleterious; N, neutral; SN, suspected neutral; U, uncertain.

Change was also seen in a different individual with breast cancer.

^†

Reclassified as neutral by other studies.

Splicing Studies

Using ESEfinder and SpliceSiteFinder, none of the missense variants were predicted to affect splicing (Appendix Tables A4 and A5). To study splicing defects of intronic variants, we isolated RNA from lymphoblastoid cell lines for four individuals with unclassified intronic VUS and from one individual with an intronic variant classified as deleterious. cDNA from 11 individuals not carrying the variants and genomic DNA from the carriers were used as controls. None of the four VUS studied (BRCA1 IVS2-14 C>T and IVS20-14 C>G and BRCA2 IVS23+9 C>T and IVS8-12delTA) showed splicing defects in contrast to the intervening sequence variant previously classified as deleterious (BRCA1 IVS5-11 G>T; data not shown).

Prediction of Deleterious Status

To classify BRCA VUS, we developed four weighted models that incorporated allele-specific LOH, tumor histopathology, evolutionary prediction and splicing, and observation with a deleterious mutation (Tables 5 and 6; Appendix Tables A4 and A5). Because LOH may not be as readily available as some of the other parameters used in our models and the frequencies of LOH in tumors with neutral BRCA variants have not been well documented, we calculated combined odds with and without LOH status (Tables 5 and 6; Appendix Tables A4 and A5). The combined sensitivity of our four models was 98% for known deleterious mutations (42 of 43 deleterious mutations were predicted as deleterious or suspected deleterious), and the specificity was 76% for previously classified neutral changes (16 of 21 neutral VUS were predicted neutral or suspected neutral).⁴^-⁶^,⁸^,⁹^,⁵⁷^-⁶² The known sequence alterations not classified as deleterious or neutral were all classified as uncertain; none were incorrectly classified. Of previously unclassified variants, 64% (21 of 33 variants) were classified as neutral by our models.

DISCUSSION

We developed four models for the prediction of BRCA VUS that do not require family history information or segregation analysis. To our knowledge, we describe the first models for the characterization of BRCA VUS that use ovarian tumor histopathology to characterize VUS. Using these models, we characterized 21 BRCA VUS as neutral. Of known deleterious mutations, none were incorrectly classified as neutral, and of predicted neutral variants, none were incorrectly classified as deleterious. For known or predicted deleterious mutations, our models showed 98% sensitivity. No VUS in this study were predicted to be deleterious.

In our study, one known deleterious truncating BRCA1 mutation was not classified as deleterious or suspected deleterious. The tumor sample was from a 75-year-old woman. It did not show LOH and had pathologic characteristics (estrogen receptor [ER] positive and progesterone receptor [PR] positive) more typical of a non-BRCA1, sporadic tumor observed in women of this age. It is possible that this tumor is a phenocopy and not a result of the patient's inherited BRCA1 mutation.

A few VUS, such as BRCA2 IVS13+5 G>C and BRCA1 L1764P, showed odds of greater than 20:1 in favor of being deleterious and warrant additional studies for characterization. L1764P has shown suggestive odds for being deleterious in other studies.⁴^,⁵⁸ A number of VUS were close to a cutoff of 100:1 in favor of a neutral classification. These are likely to be neutral; however, further studies are needed to clarify their status.

These models can be readily adapted for use in the clinical setting or for an online clinical prediction tool because all of the weighting factors, except for LOH, are available from mutation and pathology reports, online programs, and published resources. Thus, these models offer a significant advantage over other models currently used to predict VUS status. Recently, a study by the kConFab group proposed a similar model using tumor histopathology as one feature to classify BRCA VUS.⁶³ The main differences between our models and the kConFab model are that the kConFab model included segregation analysis, a different protein prediction method, and immunohistochemical staining of basal cytokeratins. Their model also did not evaluate VUS found in ovarian tumors. One advantage of our model over other models is that it does not require segregation analysis or additional laboratory analysis. For example, basal cytokeratin staining is not yet commonly used in many pathology laboratories for breast cancer diagnosis purposes. However, ER, PR, and human epidermal growth factor receptor 2 staining are part of routine practice for breast cancer pathology. In addition, a recent article suggests that, although basal cytokeratins can help to predict BRCA1 mutation–positive tumors, cytokeratin staining itself was not as good a predictor as age of onset and ER, PR, and human epidermal growth factor receptor 2 status.⁴² Seventy-two percent of the 20 VUS in the kConFab study, including some previously characterized variants, and 64% of the 33 unclassified VUS in our study were classified. Together, these studies demonstrate that inclusion of histopathology can be powerful for the characterization of BRCA VUS.

Individuals and their at-risk family members who carry a BRCA VUS often delay making decisions about their cancer risk management or make management decisions based on uncertain information. These individuals are also not able to take full advantage of prevention and therapeutic strategies typically offered to deleterious mutation carriers. Classification of 21 previously unclassified VUS using the models described here has the potential to help the individuals who carry these variants. Our models should also be useful for the classification of additional BRCA VUS in individuals from whom limited family history is available.

AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

The author(s) indicated no potential conflicts of interest.

AUTHOR CONTRIBUTIONS

Conception and design: Andrew D. Spearman, Fergus J. Couch, Amanda Ewart Toland

Financial support: Amanda Ewart Toland

Administrative support: Kevin Sweet

Provision of study materials or patients: Kevin Sweet, Xiao-Ping Zhou, Jane McLennan, Fergus J. Couch

Collection and assembly of data: Andrew D. Spearman, Amanda Ewart Toland

Data analysis and interpretation: Andrew D. Spearman, Amanda Ewart Toland

Manuscript writing: Andrew D. Spearman, Kevin Sweet, Amanda Ewart Toland

Final approval of manuscript: Andrew D. Spearman, Kevin Sweet, Xiao-Ping Zhou, Jane McLennan, Fergus J. Couch, Amanda Ewart Toland

Acknowledgments

We thank Drs. S. Barsky, R. Jimenez, M. Abdel-Rahman, and B.-M. Ljung for reviewing pathology. J. Ziegler, B. Crawford (University of California, San Francisco [UCSF] Familial Risk Shared Resource), C. Reeder (The Ohio State University [OSU] Comprehensive Cancer Center [CCC] Tissue Procurement Shared Resource), K. Chew (UCSF Tissue Shared Resource), K. Gault, M. Means, A. D’Souza, I. Comeras (OSU Clinical Cancer Genetics), and S. Nagy, (OSU Human Cancer Genetics Sample Bank) were instrumental in sample ascertainment and preparation. Finally, we thank the OSU CCC Nucleic Acids Shared Resource for sequencing support.

Appendix

Variants of Uncertain Significance Characterization Models

We created four models to characterize BRCA1 and BRCA2 variants of uncertain significance, one each for BRCA1 breast cancer, BRCA1 ovarian cancer, BRCA2 breast cancer, and BRCA2 ovarian cancer. The models differ only in the input variables and the weighted odds associated with each variable. The input characteristics and their weighted odds are listed for each model in Appendix Table A1. The overall likelihood odds of causality for each variant were calculated by obtaining the product of the combined independent variables. When nonindependent variables were present (eg, estrogen receptor status and estrogen receptor status/grade), the variable with the highest odds for being deleterious was used in the calculation. When data from multiple tumors with the same variant were combined, the odds for align grantham variation, grantham deviation/mutation, splice site, age at diagnosis, and in trans with mutation were used only once in the combined odds.

We developed models with and without loss of heterozygosity (LOH) data because LOH data may not readily be available clinically, and we wished to determine whether the models would perform well without these data. A sequence change was considered to be deleterious if the odds without LOH data were 1,000:1 or higher. A sequence change was considered to be suspected deleterious if the odds were 1,000:1 or higher for the odds including the LOH data but the odds not including the LOH data were lower than 1,000:1. A sequence change was considered to be neutral if the odds of deleterious without LOH data were 0.01:1 or lower. A sequence change was considered to be suspected neutral if the odds of deleterious with LOH data were 0.01 or lower but the odds of deleterious without LOH data were higher. Sequence changes falling between odds of deleterious of 1,000:1 and 0.01:1 were scored as uncertain. The concordance in calls between the odds with and without the LOH data was 79% for deleterious mutations (34 of 43 mutations) and 90% for previously predicted neutral variants (19 of 21 variants). These data suggest that these models will perform well with or without the LOH data.

Sensitivity and Specificity of Models

The sensitivity of our four models in predicting known deleterious mutations was calculated using the combined odds without LOH data. Forty-two (98%) of 43 known deleterious mutations were scored as deleterious or suspected deleterious using our models. If we used only mutations scored as deleterious on the odds no LOH, 88% of the mutations (38 of 43 mutations) were scored as deleterious. Our two ovarian models were 100% sensitive for predicting ovarian tumors with deleterious mutations (three of three BRCA1 mutations and four of four BRCA2 mutations) using the odds without LOH. The specificity of our four models was 76% for previously predicted neutral variants (16 of 21 variants).

Table A1.

Odds Used for VUS Classification

Characteristic	Odds
Characteristic	BRCA1	BRCA2
Breast
No LOH	0.285	0.428
Loss of wild type	5.26	4.6
Loss of variant	0.067	0.067
Medullary	8.0	NA
Triple negative	5.0	NA
ER positive	0.23	NA
ER negative	3.2	NA
PR positive	0.31	NA
PR negative	2.29	NA
HER-2 positive	0.15	0.15
HER-2 negative	1.2	1.2
ER positive/grade 1	0.067	0.23
ER positive/grade 2	0.37	1.5
ER positive/grade 3	NA	2.2
ER negative/grade 1	0.2	0.4
ER negative/grade 2	NA	0.38
ER negative/grade 3	4.1	NA
Grade 1	0.11	0.77
Grade 2	0.55	NA
Grade 3	1.97	1.3
Diagnosis at > 60 years^*	1.25	1.55
Diagnosis at 50-59 years^*	1.67	2.07
Diagnosis at 40-49 years^*	3.40	2.89
Diagnosis at 30-39 years^*	9.65	4.97
Diagnosis at < 30 years^*	15.3	4.71
Unconserved domain	0.01	0.01
Conserved C0	0.01	0.01
Conserved C15, C25	0.41	0.41
Conserved C35, C45, C55	1.5	1.5
Conserved C65	4.26	4.26
Splice defect	1,000	1,000
Not affecting splicing	0.01	0.01
Truncating mutation	1,000	1,000
In trans with mutation	0.0001	0.001
Ovarian
No LOH	0.167	0.428
Loss of wild type	4.45	4.6
Loss of variant	0.05	0.067
Serous	1.47	1.76
Mucinous	0.11	0.06
Endometrioid	NA	0.69
Grade 1	0.15	0.23
Grade 2	NA	0.45
Grade 3	1.33	1.61
Stage I	0.71	0.25
Stage II	0.59	0.35
Stage III	1.73	2.05
Stage IV	0.32	0.26
Diagnosis at > 60 years^*	4.6	4.52
Diagnosis at 50-59 years^*	11.8	7.92
Diagnosis at 40-49 years^*	18.0	4.05
Diagnosis at < 40 years^*	7.06	0.52
Unconserved domain	0.01	0.01
Conserved C0	0.01	0.01
Conserved C15, C25	0.41	0.41
Conserved C35, C45, C55	1.5	1.5
Conserved C65	4.26	4.26
Splice defect	1,000	1,000
Not affecting splicing	0.01	0.01
Truncating mutation	1,000	1,000
In trans with mutation	0.0001	0.001

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Abbreviations: VUS, variant of uncertain significance; LOH, loss of heterozygosity; NA, not applicable because no significant differences were identified; ER, estrogen receptor; PR, progesterone receptor; HER-2, human epidermal growth factor receptor 2; C, align-grantham variation, grantham deviation class.

Odds for age of diagnosis as previously reported.

Table A2.

BRCA1 and BRCA2 Breast Tumor Characteristics

Sequence Change	Sample	Race	Conserved Domain	A-GVGD Class	LOH	Splice Defect	In Trans	Age at Diagnosis (years)	Grade	ER Status	PR Status	HER-2 Status	Histology
BRCA1 truncating
1135insA	1463	White	NA	NA	WT	ND	NA	38	3	−	−	−	IDC
1240delC	0409	White	NA	NA	WT	ND	NA	38	2	−	−	−	DCIS
1294del40	4439	White	NA	NA	WT	ND	NA	ND	4	−	−	ND	IDC
1294del40	127345	White	NA	NA	WT	ND	NA	40	3	−	−	+	IDC
1294del40	78081	White	NA	NA	WT	ND	NA	51	1	−	−	ND	IDC
1294del40	169184	White	NA	NA	WT	ND	NA	32	ND	−	−	−	ND
1547del10	21538	White	NA	NA	WT	ND	NA	33	3	−	−	+	IDC
1623del5	71759	White	NA	NA	WT	ND	NA	39	2	−	−	+	IDC
1675delA	10950	White	NA	NA	WT	ND	NA	36	3	−	−	ND	IDC
1793delA	12313	Hispanic	NA	NA	WT	ND	NA	37	3	−	−	−	IDC
187delAG	45251a^*	Ash J	NA	NA	WT	ND	NA	35	3	−	−	+	IDC
187delAG	45251b^*	Ash J	NA	NA	WT	ND	NA	36	3	−	−	−	IDC
187delAG	5042	Ash J	NA	NA	WT	ND	NA	35	3	−	−	−	IDC
2530delAG	458	White	NA	NA	WT	ND	NA	30	2	−	−	−	IDC
2553delC	4724	Hispanic	NA	NA	WT	ND	NA	27	3	−	ND	ND	IDC
2576delC	61933	White	NA	NA	WT	ND	NA	58	3	−	−	−	IDC
2576delC	175158	White	NA	NA	WT	ND	NA	47	ND	+	+	−	IDC
2800delAA	24042a^*	White	NA	NA	WT	ND	NA	39	3	ND	ND	ND	IDC
2800delAA	24042b^*	White	NA	NA	None	ND	NA	47	2	−	−	−	IDC
3118delA	11984	Hispanic	NA	NA	V	ND	NA	32	3	−	−	−	IDC
3600del11	34406	White	NA	NA	WT	ND	NA	37	2	−	−	−	Unspecified
5382insC	20758	Ash J	NA	NA	WT	ND	NA	61	2	−	−	ND	IDC
E143X	34642	White	NA	NA	WT	ND	NA	32	3	−	−	−	IDC
Q563X	61051	White	NA	NA	WT	ND	NA	47	3	+	−	−	IDC
S868X	176811	White	NA	NA	None	ND	NA	74	3	+	+	−	ILC
Q1408X	34642	White	NA	NA	None	ND	NA	29	ND	−	−	−	Unspecified
BRCA1 splice defect
IVS4-1 G>T	2364a^*	White	NA	NA	WT	Yes^†	No	48	3	−	−	ND	DCIS, LCIS
IVS4-1 G>T	2364b^*	White	NA	NA	WT	Yes^†	No	49	1	+	+	−	DCIS, LCIS
IVS13+1 G>A	98453	White	NA	NA	None	Yes^‡	No	41	2	−	−	−	IDC
IVS15+1 G>A	2-112-140	White	NA	NA	WT	Yes^‡	No	ND	4	−	−	ND	IDC
IVS5-11 T>G	45949	White	NA	NA	WT	Yes	No	59	2	−	−	−	Medullary
BRCA1 intronic
IVS12+1 0G>C	78081	White	NA	NA	None	ND	No	51	1	−	−	ND	IDC
IVS20-14 C>G	168788	White	NA	NA	None	No	No	34	3	+	+	−	IDC
IVS2-14 C>T	64703	White	NA	NA	V	No	No	44	1	+	+	−	IDC
IVS2-6 T>C	15007	White	NA	NA	None	ND	No	38	ND	+	+	−	DCIS
BRCA1 missense
C61G	14068	White	Yes	C65	WT	ND	No	32	3	−	−	+	IDC
C61G	19882	White	Yes	C65	WT	ND	No	29	3	−	−	−	IDC
E597K	98286	White	No	C0	None	ND	No	45	1	+	+	ND	IDC
E736A	15102	Asian	No	C0	None	ND	No	44	ND	+	+	−	DCIS
I1275V	4061	White	No	C0	WT	ND	No	37	3	−	−	−	IDC
K1109N	0477a^*	White	No	C0	None	ND	No	35	ND	ND	ND	ND	DCIS
K1109N	0477b^*	White	No	C0	None	ND	No	49	2	+	+	−	IDC
L1764P	4744	White	Yes	C35	WT	ND	No	32	3	−	−	+	IDC
P1637L	175158	White	Close	C0	WT	ND	No	47	ND	+	+	−	IDC
P1776H	154045	White	No	C0	WT	ND	No	48	3	+	+	−	ND
P1776H	133886	White	No	C0	WT	ND	No	55	ND	ND	ND	ND	Unspecified
P334H	34402	White	No	C0	None	ND	No	38	2	+	+	−	IDC
S127N	4060	Af Am/N Am	No	C0	V	ND	No	38	ND	ND	ND	ND	IDC
V1247I	11092	Asian	No	C0	None	ND	No	40	3	+	+	−	IDC
V1804D	16719	White	Yes	C0	V	ND	No	58	2	+	−	+	IDC
V1804D	73481	White	Yes	C0	ND	ND	No	59	1	+	+	ND	IDC
V772A	78081	White	No	C0	WT	ND	Yes	47	1	−	−	ND	IDC
T1310K	176831	White	No	C0	None	ND	No	45	ND	ND	ND	ND	DCIS
Y856H	1995	Asian	No	C0	None	ND	Yes	45	3	+	+	+	DCIS
Y856H	4294	Asian	No	C0	None	ND	Yes	47	ND	ND	ND	ND	DCIS
BRCA2 truncating
2041delA	46662	White	NA	NA	None	ND	NA	34	3	+	+	−	IDC
3036del4	4208	White	NA	NA	U	ND	NA	ND	2	+	+	−	IDC
3036del4	4310	Asian	NA	NA	WT	ND	NA	36	3	+	+	+	IDC
3036del4	542	White	NA	NA	WT	ND	NA	43	ND	+	+	ND	IDC
3972del4	5-077	White	NA	NA	WT	ND	NA	ND	ND	+	+	ND	IDC
4206ins4	26949	White	NA	NA	None	ND	NA	37	1	+	+	−	LCIS
4361del4	2609	White	NA	NA	WT	ND	NA	ND	ND	−	+	−	IDC
5270delTG	169989	White	NA	NA	None	ND	NA	46	2	+	−	ND	IDC
6174delT	4317	Ash J	NA	NA	WT	ND	NA	46	3	−	−	−	IDC
6503delTT	35962	White	NA	NA	WT	ND	NA	46	2	+	+	+	IDC
7990del3ins2	15494	White	NA	NA	WT	ND	NA	41	3	+	−	−	IDC
8765delAG	5702	White	NA	NA	ND	ND	NA	50	ND	−	−	ND	IDC
8803delC	80659	White	NA	NA	None	ND	NA	36	ND	+	+	ND	Unspecified
9481insA	94467	White	NA	NA	WT	ND	NA	64	2	+	+	+	IDC
Q321X	3-744	White	NA	NA	V	ND	NA	ND	4	+	+	−	IDC
S1882X	3553	Asian	NA	NA	WT	ND	NA	37	3	+	+	−	IDC
Y1894X	11008	White	NA	NA	WT	ND	NA	26	ND	+	+	−	IDC
BRCA2 intronic
IVS13+5 G>C	16004	White	NA	NA	WT	ND	No	46	2	+	+	−	IDC
IVS23+9 C>T	12673	White	NA	NA	WT	No	No	40	2	+	+	+	IDC
IVS8-12delTA	64703	White	NA	NA	None	No	No	44	1	+	+	−	IDC
BRCA2 missense
A1170V	143226	White	No	C0	ND	ND	No	52	3	−	+	+	IDC
A2351G	2664	Asian	No	C0	None	ND	No	61	3	−	−	−	IDC
D1352Y	11193	White	No	C0	WT	ND	No	47	2	+	+	−	IDC
E2856A	5047	White	Yes	C0	V	ND	No	45	2	+	+	−	IDC
H1966Y	46345	White	No	C0	V	ND	No	44	3	−	−	−	IDC
I2285V	4112	White	No	C25	V	ND	No	ND	2	+	+	+	IDC
K1434I	34415	White	No	C15	None	ND	No	58	ND	+	−	ND	DCIS
L2106P	161491	White	No	C0	None	ND	No	57	3	+	+	−	IDC
L929S	14388	Af Am	No	C0	None	ND	Yes	37	2	+	+	−	IDC
M2676T	565	White	Yes	C0	None	ND	No	49	ND	+	ND	ND	IDC
N1878K	14573a^*	White	No	C0	V	ND	No	63	1	+	−	−	IDC
N1878K	14573b^*	White	No	C0	V	ND	No	67	2	+	+	−	IDC
N517S	11859	White	No	C0	None	ND	No	45	1	+	+	ND	IDC
N588D	0772	White	No	C0	WT	ND	No	56	2	+	+	+	IDC
N987I	14388	Af Am	No	C0	None	ND	Yes	37	2	+	+	−	IDC
P1819S	4146	White	No	C0	None	ND	No	57	1	+	+	−	IDC
P655R	11631	Ash J	No	C0	None	ND	No	38	3	+	+	−	IDC
R2034C	2098	White	No	C0	None	ND	Yes	75	3	−	−	+	IDC
R2418G	26664	White	Yes	C0	None	ND	No	61	2	+	+	−	IDC
R2502H	98245	White	Yes	C0	None	ND	No	58	3	+	+	−	IDC
R2973C	2382	White	Yes	C25	WT	ND	No	52	2	+	+	−	IDC
S1424C	155088	White	No	C0	None	ND	No	55	ND	ND	ND	ND	ND
S2483N	25006	White	Yes	C0	None	ND	No	48	1	+	+	−	IDC
S384F	6996	White	No	C0	WT	ND	Yes	49	2	+	+	+	IDC
T2681R	25705	White	Yes	C0	WT	ND	No	34	3	−	−	−	IDC
T3211K	59457	White	Yes	C0	None	ND	No	52	3	−	−	−	ND
Y42C	4620	White	No	C0	V	ND	No	40	ND	−	−	−	IDC

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Abbreviations: A-GVGD, align grantham variation deviation analysis; LOH, loss of heterozygosity; ER, estrogen receptor; PR, progesterone receptor; HER-2, human epidermal growth factor receptor 2; NA, not applicable; WT, loss of wild-type allele; ND, no data; −, negative; IDC, invasive ductal carcinoma; DCIS, ductal carcinoma in situ; +, positive; Ash J, Ashkenazi Jewish; V, loss of variant allele; ILC, invasive lobular carcinoma; LCIS, lobular carcinoma in situ; Af Am, African American; N Am, Native American; U, uncertain loss.

Individual has bilateral breast cancer, and both tumors were included.

^†

Change shown to affect splicing by others.

^‡

Change predicted to affect splicing by SpliceSiteFinder.

Table A3.

Ovarian Tumor Characteristics

Sequence Change	Sample	Race	Conserved Domain	LOH	A-GVGD Class	In Trans	Stage	Grade	Age at Diagnosis (years)	Histology
BRCA1 truncating
1135insA	24127	White	NA	WT	NA	No	ND	3C	59	Papillary serous
262delT	0690	White	NA	WT	NA	No	ND	3C	63	Endometrioid
3600del11	10945	White	NA	WT	NA	No	ND	3	41	Serous
BRCA1 missense
P334L	6167	White	No	WT	C0	Yes	ND	ND	39	Unspecified
BRCA2 truncating
6307insA	4945	Af Am	NA	None	NA	NA	ND	ND	ND	Unspecified
7297delCT	0947	White	NA	None	NA	NA	ND	3C	22	Serous
7990del3ins2	540	White	NA	None	NA	NA	ND	ND	46	Unspecified
9481insA	23722	White	NA	WT	NA	NA	ND	3C	59	Papillary serous
BRCA2 missense
A1170V	97594	White	No	V	C0	No	ND	ND	59	ND
D1420Y	0690	White	No	None	C15	Yes	ND	3C	63	Endometrioid
L2721H	6167	White	Yes	WT	C25	No	ND	ND	39	Unspecified
M784V	11073	Asian	No	V	C0	No	IIIC	3	45	Unspecified
S1172L	5701	White	No	None	C15	No	IIIC	ND	58	Serous
S326R	5701	White	No	None	C0	Yes	IIIC	ND	58	Serous
V1643A	76,049	White	No	WT	C0	No	IIIC	ND	50	Serous

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Abbreviations: LOH, loss of heterozygosity; A-GVGD, align grantham variation, grantham deviation; NA, not applicable; WT, loss of wild-type allele; ND, no data; Af Am, African American; V, loss of variant or mutant allele.

Table A4.

Prediction of Deleterious Status for BRCA1 and BRCA2 Breast Tumors

Sequence Change	Sample	Odds												Odds	Odds, No LOH	Interpretation
Sequence Change	Sample	LOH	A-GVGD/Mutation	Splice	In Trans	Age at Diagnosis	Triple Negative	ER Status	PR Status	HER-2 Status	Grade/ER	Grade	HP	Odds	Odds, No LOH	Interpretation
BRCA1 truncating
1135insA^*	1463	5.26	1,000	1	1	9.65	5	1	1	1	1	1.97	1	4.9 × 10⁵	9.5 × 10⁴	D
1240delC	0409	5.26	1,000	1	1	9.65	5	1	1	1	1	0.55	1	1.4 × 10⁵	2.6 × 10⁴	D
1294del40	4439	5.26	1,000	1	1	1	1	1	2.29	1	4.1	1	1	4.9 × 10⁴	9,389	D
1294del40	127345	5.26	1,000	1	1	3.4	1	1	2.29	0.15	4.1	1	1	2.5 × 10⁴	4,788	D
1294del40	78081^†	5.26	1,000	1	1	1.67	1	1	2.29	1	0.2	1	1	4,023	765	DS
1294del40	169184	5.26	1,000	1	1	9.65	5	1	1	1	1	1	1	2.5 × 10⁵	4.8 × 10⁴	D
1294del40	combo	765.5	1,000	1	1	9.65	5	1	11.53	0.15	3.362	1	1	2.1 × 10⁸	2.8 × 10⁵	D
1547del10	21538	5.26	1,000	1	1	9.65	1	1	2.29	0.15	4.1	1	1	7.1 × 10⁴	1.4 × 10⁴	D
1623del5	71759	5.26	1,000	1	1	9.65	1	3.2	2.29	0.15	1	1	1	3.1 × 10⁴	5,834	D
1675delA	10950	5.26	1,000	1	1	9.65	1	1	2.29	1	4.1	1	1	4.8 × 10⁵	9 × 10⁴	D
1793delA	12,313	5.26	1,000	1	1	9.65	5	1	1	1	1	1.97	1	4.9 × 10⁵	9.5 × 10⁴	D
187delAG	45251a^‡	5.26	1,000	1	1	9.65	1	1	2.29	0.15	4.1	1	1	7.1 × 10⁴	1.4 × 10⁴	D
187delAG	45251b^‡	5.26	1,000	1	1	9.65	5	1	1	1	1	1.97	1	4.9 × 10⁵	9.5 × 10⁴	D
187delAG	5042	5.26	1,000	1	1	9.65	5	1	1	1	1	1.97	1	4.9 × 10⁵	9.5 × 10⁴	D
187delAG	combo	145.53	1,000	1	1	9.65	25	1	2.29	0.15	4.1	3.88	1	1.9 × 10⁸	1.3 × 10⁶	D
2530delAG	458	5.26	1,000	1	1	9.65	5	1	1	1	1	0.55	1	1.4 × 10⁵	2.7 × 10⁴	D
2553delC	4724	5.26	1,000	1	1	15.3	1	1	1	1	4.1	1	1	3.3 × 10⁵	6.3 × 10⁴	D
2576delC	61933	5.26	1,000	1	1	1.67	5	1	1	1	1	1.97	1	8.7 × 10⁴	1.6 × 10⁴	D
2576delC	175158	5.26	1,000	1	1	3.4	1	0.23	0.31	1.2	1	1	1	1,530	291	DS
2576delC	combo	27.66	1,000	1	1	3.4	5	0.23	0.31	1.2	1	1.97	1	7.9 × 10⁴	2,865	D
2800delAA	24042a^‡	5.26	1,000	1	1	9.65	1	1	1	1	1	1.97	1	9.9 × 10⁴	1.9 × 10⁴	D
2800delAA	24042b^‡	0.285	1,000	1	1	3.4	5	1	1	1	1	0.55	1	2,665	9,350	D
2800delAA	combo	1.499	1,000	1	1	9.65	5	1	1	1	1	1.08	1	7.8 × 10⁴	5.2 × 10⁴	D
3118delA	11984	0.0667	1,000	1	1	9.65	5	1	1	1	1	1.97	1	6,340	9.5 × 10⁴	D
3600del11^*	34406	5.26	1,000	1	1	9.65	5	1	1	1	1	0.55	1	1.4 × 10⁵	2.7 × 10⁴	D
5382insC	20758	5.26	1,000	1	1	1.25	1	1	2.29	1	4.1	1	1	6.2 × 10⁴	1.2 × 10⁴	D
E143X	34642	5.26	1,000	1	1	9.65	5	1	1	1	1	1.97	1	4.9 × 10⁵	9.5 × 10⁴	D
Q563X	61051	5.26	1,000	1	1	3.4	1	0.23	2.29	1.2	1	1.97	1	2.2 × 10⁴	4,233	D
S868X	176811	0.285	1,000	1	1	1.25	1	0.23	0.31	1.2	1	1.97	1	60	210	U
Q1408X	34642	0.285	1,000	1	1	15.3	5	1	1	1	1	1	1	4.9 × 10⁴	1.8 × 10⁵	D
BRCA1 splicing
IVS4-1 G>T	2364^‡a^‡	5.26	1	1,000	1	3.4	1	1	2.29	1	4.1	1	1	1.7 × 10⁵	3.2 × 10⁴	D
IVS4-1 G>T	2364^‡b^‡	5.26	1	1,000	1	3.4	1	0.23	0.31	1.2	1	0.11	1	168	32	U
IVS4-1 G>T	combo	27.66	1	1,000	1	3.4	1	0.23	0.71	1.2	4.1	0.11	1	8,314	300	DS^§
IVS13+1 G>A	98453	0.285	1	1,000	1	3.4	5	1	1	1	1	0.55	1	2,665	9,350	D^§
IVS15+1 G>A	2-112-140	5.26	1	1,000	1	1	1	1	2.29	1	4.1	1	1	4.9 × 10⁴	9,389	D^§
IVS5-11 T>G	45949	5.26	1	1,000	1	1.67	5	1	1	1	1	0.55	4.3	1 × 10⁵	1.9 × 10⁴	D^§
BRCA1 intronic
IVS12+10 G>C	78081^*	0.285	1	1	1	1.67	1	1	2.29	1	0.2	1	1	0.218	0.765	U
IVS20-14 C>G	168788	0.285	1	0.01	1	9.65	1	0.23	0.31	1.2	1	1.97	1	0.0045	0.0162	NS
IVS2-14 C>T	64703	0.0667	1	0.01	1	3.4	1	1	0.31	1.2	1	0.11	1	0.0009	0.001	N
IVS2-6 T>C	15007	0.285	1	1	1	9.65	1	0.23	0.31	1.2	1	1	1	0.235	0.826	U
BRCA1 missense
C61G	14068	5.26	4.26	1	1	9.65	1	1	2.29	0.15	4.1	1	1	304	58	U
C61G	19882	5.26	4.26	1	1	15.3	5	1	1	1	1	1.97	1	3,377	642	DS
C61G	combo	27.66	4.26	1	1	15.3	5	1	2.29	0.15	4.1	1.97	1	2.5 × 10⁴	904	DS
E597K	98286	0.285	0.01	1	1	3.4	1	0.23	0.31	1	1	0.11	1	7.6 × 10⁻⁵	0.00027	N^§
E736A	15102	0.285	0.01	1	1	3.4	1	0.23	0.31	1.2	1	1	1	0.00008	0.0029	N
I1275V	4061	5.26	0.01	1	1	9.65	5	1	1	1	1	1.97	1	4.99	0.95	U^§
K1109N	0477^‡a^‡	0.285	0.01	1	1	9.65	1	1	1	1	1	1	1	0.028	0.097	U
K1109N	0477^‡b^‡	1	0.01	1	1	3.4	1	1	2.29	1.2	0.37	1	1	0.092	0.035	U
K1109N	combo	0.285	0.01	1	1	9.65	1	1	2.29	1.2	0.37	1	1	0.074	0.098	U^§
L1764P	4744	5.26	1.5	1	1	9.65	1	1	2.29	0.15	4.1	1	1	107	20.4	U^§
P1637L	175158	5.26	0.01	1	1	3.4	1	0.23	0.31	1.2	1	1	1	0.015	0.0029	N
P1776H	154045	5.26	0.01	1	1	3.4	1	0.23	0.31	1.2	1	1.97	1	0.030	0.057	U
P1776H	133886	5.26	0.01	1	1	1.67	1	1	1	1	1	1	1	0.088	0.017	U
P1776H	combo	145.5	0.01	1	1	3.4	1	0.23	0.31	1.2	1	1.97	1	0.159	0.006	N
P334H	34402	0.285	0.01	1	1	9.65	1	1	0.31	1.2	0.37	1	1	0.003	0.01	N
S127N	4060	0.0667	0.01	1	1	9.65	1	1	1	1	1	1	1	0.0064	0.097	NS
T1310K	176831	0.285	0.01	1	1	3.4	1	1	1	1	1	1	1	0.0097	0.034	NS
V1247I	11092	0.285	0.01	1	1	3.4	1	0.23	0.31	1.2	1	1.97	1	0.0016	0.0057	N^§
V1804D	16719	0.0667	0.01	1	1	1.67	1	1	2.29	0.15	0.37	1	1	0.00014	0.014	NS
V1804D	73481	1	0.01	1	1	1.67	1	1	0.31	1	0.067	1	1	0.00034	0.00034	N
V1804D	combo	0.0667	0.01	1	1	1.67	1	1	0.71	0.15	0.025	1	1	2.9 × 10⁻⁶	4.4 × 10⁻⁵	N^§
V772A	78081^‡^†	5.26	0.01	1	0.0001	3.4	1	1	2.29	1	0.2	1	1	8.2 × 10⁻⁶	1.6 × 10⁻⁶	N^§
Y856H	1995	0.285	0.01	1	0.0001	3.4	1	0.23	0.31	0.15	1	1.97	1	2 × 10⁻⁸	7 × 10⁻⁸	N
Y856H	4294	0.285	0.01	1	0.0001	3.4	1	1	1	1	1	1	1	9 × 10⁻⁷	3 × 10⁻⁶	N
Y856H	combo	0.08	0.01	1	0.0001	1	1	0.23	0.31	0.15	1	1.97	1	5.7 × 10⁻⁹	7 × 10⁻⁸	N^§
BRCA2 truncating
2041delA	46662	0.428	1,000	1	1	4.97	1	1	1	1.2	2.2	1	1	5,616	1.3 × 10⁴	D
3036del4	4208	1	1,000	1	1	1	1	1	1	1.2	1.5	1	1	1,800	1,800	D
3036del4	4310	4.6	1,000	1	1	4.97	1	1	1	0.15	2.2	1	1	7,544	1,640	D
3036del4	542	4.6	1,000	1	1	2.89	1	1	1	1	1	1	1	1.3 × 10⁴	2,890	D
3036del4	combo	21.16	1,000	1	1	2.89	1	1	1	0.18	3.3	1	1	3.6 × 10⁴	1,717	D
3972del4	5-077	4.6	1,000	1	1	1	1	1	1	1	1	1	1	4,600	1,000	D
4206ins4	26949	0.428	1,000	1	1	4.97	1	1	1	1.2	0.23	1	1	587	1,371	D
4361del4	2609	4.6	1,000	1	1	1	1	1	1	1.2	1	1	1	5,520	1,200	D
5270delTG	169989	0.428	1,000	1	1	2.89	1	1	1	1	1.5	1	1	1,855	4,335	D
6174delT	4317	4.6	1,000	1	1	2.89	1	1	1	1.2	1	1.3	1	2.1 × 10⁴	4,508	D
6503delTT	35962	4.6	1,000	1	1	2.89	1	1	1	0.15	1.5	1	1	2,991	650	DS
7990del3ins2^*	15494	4.6	1,000	1	1	2.89	1	1	1	1.2	2.2	1	1	3.5 × 10⁴	7,630	D
8765delAG	5702	0.428	1,000	1	1	2.07	1	1	1	1	1	1	1	886	2,070	D
8803delC	80659	0.428	1,000	1	1	4.97	1	1	1	1	1	1	1	2,127	4,970	D
9481insA	94467	4.6	1,000	1	1	1.55	1	1	1	0.15	1.5	1	1	1,604	349	DS
Q321X	3-744	0.0667	1,000	1	1	1	1	1	1	1.2	2.2	1	1	176	2,640	D
S1882X	3553	4.6	1,000	1	1	4.97	1	1	1	1.2	2.2	1	1	6 × 10⁴	1.3 × 10⁴	D
Y1894X	11008	4.6	1,000	1	1	4.97	1	1	1	1.2	1	1	1	2.6 × 10⁴	5,652	D
BRCA2 intronic
IVS13+5 G>C	16004	4.6	1	1	1	2.89	1	1	1	1.2	1.5	1	1	23.93	5.2	U
IVS23+9 C>T	12673	4.6	1	0.01	1	2.89	1	1	1	0.15	1.5	1	1	0.03	0.0065	N
IVS8-12delTA	64703	0.428	1	0.01	1	2.89	1	1	1	1.2	0.23	1	1	0.003	0.008	N
BRCA2 missense
A1170V^*	143226	1	0.01	1	1	2.07	1	1	1	0.15	1	1.3	1	0.027	0.027	N^‖
A2351G	2664	0.428	0.01	1	1	1.55	1	1	1	1.2	1	1.3	1	0.010	0.024	NS
D1352Y	11193	4.6	0.01	1	1	2.89	1	1	1	1.2	1.5	1	1	0.239	0.05	U
E2856A	5047	0.0667	0.01	1	1	2.89	1	1	1	1.2	1.5	1	1	0.0034	0.52	NS^§
H1966Y	46345	0.0667	0.01	1	1	2.89	1	1	1	1.2	1	1.3	1	0.003	0.045	NS
I2285V	4112	0.0667	0.01	1	1	1	1	1	1	0.15	1.5	1	1	0.001	0.002	N^§
K1434I	34415	0.428	0.01	1	1	2.07	1	1	1	1	1	1	1	0.009	0.027	NS
L2106P	161491	0.428	0.01	1	1	2.07	1	1	1	1.2	2.2	1	1	0.023	0.054	U
L929S	14388^¶	0.428	0.01	1	0.001	4.97	1	1	1	1.2	1.5	1	1	3.8 × 10⁻⁵	8.9 × 10⁻⁵	N^§
M2676T	565	0.428	0.01	1	1	1	1	1	1	1	1	1	1	0.012	0.029	U
N1878K	14573^‡a^‡	0.0667	0.01	1	1	1.55	1	1	1	1.2	0.23	1	1	0.0003	0.004	N
N1878K	14573^‡b^‡	0.0667	0.01	1	1	1.55	1	1	1	1.2	1.5	1	1	0.002	0.028	NS
N1878K	combo	0.0045	0.01	1	1	1.55	1	1	1	1.44	0.345	1	1	3.4 × 10⁻⁵	0.008	N
N517S	11859	0.428	0.01	1	1	2.89	1	1	1	1	0.23	1	1	0.003	0.007	N
N588D	0772	4.6	0.01	1	1	2.07	1	1	1	0.15	1.5	1	1	0.02	0.005	N
N987I	14388^¶	0.428	0.01	1	0.001	4.97	1	1	1	1.2	1.5	1	1	3.8 × 10⁻⁵	8.9 × 10⁻⁵	N^§
P1819S	4146	0.428	0.01	1	1	2.07	1	1	1	1.2	0.23	1	1	0.002	0.006	N^§
P655R	11631	0.428	0.01	1	1	4.97	1	1	1	1.2	2.2	1	1	0.056	0.131	U
R2034C	2098	0.428	0.01	1	0.001	1.55	1	1	1	0.15	1	1.3	1	0.00001	2 × 10⁻⁶	N^§
R2418G	26664	0.428	0.01	1	1	1.55	1	1	1	1.2	1.5	1	1	0.011	0.028	U
R2502H	98245	0.428	0.01	1	1	2.07	1	1	1	1.2	2.2	1	1	0.023	0.054	U
R2973C	2382	4.6	0.41	1	1	2.07	1	1	1	1.2	1.5	1	1	7.03	1.53	U^§
S1424C	155088	0.428	0.01	1	1	2.07	1	1	1	1	1	1	1	0.009	0.008	N
S2483N	25006	0.428	0.01	1	1	2.89	1	1	1	1.2	0.23	1	1	0.003	0.008	N
S384F	6996	4.6	0.01	1	0.001	2.89	1	1	1	0.15	1.5	1	1	2.9 × 10⁻⁵	6.5 × 10⁻⁶	N^§
T2681R	25705	4.6	0.01	1	1	4.97	1	1	1	1.2	1	1	1	0.274	0.059	U
T3211K	59457	0.428	0.01	1	1	2.07	1	1	1	1	1	1	1	0.009	0.021	NS
Y42C	4620	0.0667	0.01	1	1	2.89	1	1	1	1.2	1	1	1	0.002	0.035	NS^§

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Abbreviations: LOH, loss of heterozygosity; A-GVGD, align grantham variation, grantham deviation; ER, estrogen receptor; PR, progesterone receptor; HER-2, human epidermal growth factor receptor 2; HP, histopathology; D, deleterious; DS, suspected deleterious; Combo, odds combined for all samples with variant; N, neutral; U, uncertain; NS, suspected neutral.

Mutation also seen in a different individual with ovarian cancer.

^†

Individual has both BRCA1mutation and two BRCA1 variants.

^‡

Individual has bilateral breast cancer; both tumors were tested.

^§

Reclassified as neutral or deleterious by other studies.⁴,⁵,⁶,⁸,⁹,⁵⁷-⁶²

^‖

Variant characterized as neutral when data are combined with individual 97594 with ovarian cancer.

^¶

Individual has both BRCA2L929S and N987I variants.

Table A5.

Prediction of Deleterious Status in BRCA1 and BRCA2 Ovarian Tumors

Sequence Change	Sample	LOH	A-GVGD/Mutation	Splice	In Trans	Stage	Grade	HP	Age at Diagnosis	Combined Odds	Odds, No LOH	Interpretation
BRCA1 truncating
1135insA^*	24127	4.45	1,000	1	1	1	1.33	1.47	11.8	102,662.3	23,070.2	Deleterious
262delT	0690^†	4.45	1,000	1	1	1	1.33	1	4.6	27,225.1	6,118	Deleterious
3600del11^*	10945	4.45	1,000	1	1	1	1.33	1.47	18	156,603.51	35,192	Deleterious
BRCA1 missense
P334L	6167^‡	4.45	0.01	1	0.0001	1	1	1	7.06	0.0000314	0.0000071	Neutral^§
BRCA2 truncating
6307insA	4945	0.428	1,000	1	1	1	1	1	1	428	1,000	Deleterious
7297delCT	0947	0.428	1,000	1	1	1	1.61	1.76	0.52	630.65	1,473.47	Deleterious
7990del3ins2^*	540	0.428	1,000	1	1	1	1	1	4.05	1,733.4	4,050	Deleterious
9481insA	23722	4.6	1,000	1	1	1	1.61	1.76	7.92	103,233.72	22,442.11	Deleterious
BRCA2 missense
A1170V^*	97594	0.067	0.01	1	1	1	1	1	7.92	0.00528	0.0792	Neutral^‖
D1420Y	0690^†	0.428	0.01	1	0.001	1	1.61	0.69	4.52	0.0000215	0.00005	Neutral^§
L2721H	6167^‡	4.6	0.41	1	1	1	1	1	0.52	0.9807	0.2132	Uncertain
M784V	11073	0.067	0.01	1	1	2.05	1.61	1	4.05	0.009	0.1337	Neutral suspected
S1172L	5701^¶	0.428	0.01	1	1	2.05	1	1.76	7.92	0.1223	0.2857	Uncertain^§
S326R	5701^¶	0.428	0.01	1	0.001	2.05	1	1.76	7.92	0.000122	0.00028	Neutral^§
V1643A	76049	4.6	0.01	1	1	2.05	1	1.76	7.92	1.3145	0.2858	Uncertain

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Abbreviations: LOH, loss of heterozygosity; A-GVGD, align grantham variation, grantham deviation; HP, histopathology.

Change was also seen in a different individual with breast cancer.

^†

Individual carries both BRCA1 262delT and BRCA2 variant D1420Y.

^‡

Individual carries both BRCA1 P334L and BRCA2 L2721H.

^§

Reclassified as neutral by other studies.⁴,⁶⁰,⁶²

^‖

Variant characterized as neutral when data are combined with data from individual 143226 with breast cancer.

^¶

Individual carries both BRCA2S1172L and S326R.

published online ahead of print at www.jco.org on September 29, 2008

Supported by the National Institutes of Health, National Cancer Institute, Bay Area Breast Spore Career Development Award No. P50 CA582017 (A.E.T.), Mayers Summer fellowships (A.D.S.), and internal funds from the Ohio State University Comprehensive Cancer Center (A.E.T.).

Authors’ disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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31.Honrado E, Benitez J, Palacios J: Histopathology of BRCA1- and BRCA2-associated breast cancer. Crit Rev Oncol Hematol 59:27-39, 2006 [DOI] [PubMed] [Google Scholar]
32.Bane AL, Beck JC, Bleiweiss I, et al: BRCA2 mutation-associated breast cancers exhibit a distinguishing phenotype based on morphology and molecular profiles from tissue microarrays. Am J Surg Pathol 31:121-128, 2007 [DOI] [PubMed] [Google Scholar]
33.Haffty BG, Yang Q, Reiss M, et al: Locoregional relapse and distant metastasis in conservatively managed triple negative early-stage breast cancer. J Clin Oncol 24:5652-5657, 2006 [DOI] [PubMed] [Google Scholar]
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35.Rakha EA, El-Sayed ME, Green AR, et al: Prognostic markers in triple-negative breast cancer. Cancer 109:25-32, 2007 [DOI] [PubMed] [Google Scholar]
36.Tischkowitz M, Brunet J-S, Begin LR, et al: Use of immunohistochemical markers can refine prognosis in triple negative breast cancer. BMC Cancer 7:134, 2007 [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Bauer K, Brown M, Cress RD, et al: Descriptive analysis of estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and HER2-negative invasive breast cancer, the so-called triple negative phenotype. Cancer 109:1721-1728, 2007 [DOI] [PubMed] [Google Scholar]
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39.Foulkes WD, Metcalfe K, Sun P, et al: Estrogen receptor status in BRCA1- and BRCA2-related breast cancer: The influence of age, grade and histological type. Clin Cancer Res 10:2029-2034, 2004 [DOI] [PubMed] [Google Scholar]
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44.Breast Cancer Linkage Consortium: Pathology of familial breast cancer: Differences between breast cancers of BRCA1 or BRCA2 mutations and sporadic cases. Lancet 349:1505-1510, 1997 [PubMed] [Google Scholar]
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50.Neuhausen SL, Marshall CJ: Loss of heterozygosity in familial tumors from three BRCA1-linked kindreds. Cancer Res 54:6069-6072, 1994 [PubMed] [Google Scholar]
51.Gras E, Cortes J, Diez O, et al: Loss of heterozygosity on chromosome 13q12-q14, BRCA-2 mutations and lack of BRCA-2 promoter hypermethylation in sporadic epithelial ovarian tumors. Cancer 92:787-795, 2001 [DOI] [PubMed] [Google Scholar]
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54.Piek JM, Torrenga B, Hermsen B, et al: Histopathological characteristics of BRCA1- and BRCA2-associated intraperitoneal cancer: A clinic-based study. Fam Cancer 2:73-78, 2003 [DOI] [PubMed] [Google Scholar]
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57.Tesoriero AA, Wong EM, Jenkins MA, et al: Molecular characterization and cancer risk associated with BRCA1 and BRCA2 splice site variants identified in multiple-case breast cancer families. Hum Mutat 26:495, 2005 [DOI] [PubMed] [Google Scholar]
58.Carvalho MA, Marsillac SM, Karchin R, et al: Determination of cancer risk association with germ line BRCA1 missense variants by functional analysis. Cancer Res 67:1494-1501, 2007 [DOI] [PMC free article] [PubMed] [Google Scholar]
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62.National Human Genome Research Institute: Breast Cancer Information Core. http://research.nhgri.nih.gov/projects/bic/
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[r57] 57.Tesoriero AA, Wong EM, Jenkins MA, et al: Molecular characterization and cancer risk associated with BRCA1 and BRCA2 splice site variants identified in multiple-case breast cancer families. Hum Mutat 26:495, 2005 [DOI] [PubMed] [Google Scholar]

[r58] 58.Carvalho MA, Marsillac SM, Karchin R, et al: Determination of cancer risk association with germ line BRCA1 missense variants by functional analysis. Cancer Res 67:1494-1501, 2007 [DOI] [PMC free article] [PubMed] [Google Scholar]

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[r62] 62.National Human Genome Research Institute: Breast Cancer Information Core. http://research.nhgri.nih.gov/projects/bic/

[r63] 63.Spurdle AB, Lakhani SR, Healey S, et al: Clinical classification of BRCA1 and BRCA2 DNA sequence variants: The value of cytokeratin profiles and evolutionary analysis—A report from the kConFab Investigators. J Clin Oncol 26:1657-1663, 2008 [DOI] [PubMed] [Google Scholar]

PERMALINK

Clinically Applicable Models to Characterize BRCA1 and BRCA2 Variants of Uncertain Significance

Andrew D Spearman

Kevin Sweet

Xiao-Ping Zhou

Jane McLennan

Fergus J Couch

Amanda Ewart Toland

Abstract

Purpose

Methods

Results

Conclusion

INTRODUCTION

METHODS

Human Samples

Allele-Specific LOH

Evolutionary Conservation

Splicing Studies

Observation With a Deleterious Mutation

Model Phenotypes

Table 1.

Statistical Methods

RESULTS

Identification of Study Criteria for the Models

Table 2.

Table 3.

Table 4.

LOH

Sequence-Based Methods to Determine Pathogenicity

Table 5.

Table 6.

Splicing Studies

Prediction of Deleterious Status

DISCUSSION

AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

AUTHOR CONTRIBUTIONS

Acknowledgments

Appendix

Variants of Uncertain Significance Characterization Models

Sensitivity and Specificity of Models

Table A1.

Table A2.

Table A3.

Table A4.

Table A5.

REFERENCES

ACTIONS

PERMALINK

RESOURCES

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