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BRCA1/2 VUS RECLASSIFICATION GUIDE

What is the purpose of this guide?

This guide was developed to provide the clinical community with a listing of specific BRCA1/2 variants, formerly classified as V ariants of U ncertain S ignificance (VUS), which have been subjected to additional analyses as part of research studies, resulting in potential changes in classification. Given that each laboratory testing company uses its own threshold for rescoring DNA variants, the information provided in this guide may be, but is not always, included as part of the test report you receive. You may wish to use information in this guide to help guide clinical recommendations for your patients.

How were BRCA VUS classified?

In 2008, an international VUS working group of researchers and clinicians meeting at the International Agency for Research on Cancer (IARC) of the World Health Organization developed a quantitative model, called the Multifactorial Likelihood Model, to provide a score that represents the clinical relevance of DNA VUS in the BRCA1 and BRCA2 genes. This model was applied to BRCA1/2 variants for which sufficient data was available to generate a score that indicates clinical relevance of the BRCA variant. In addition, several functional studies that also provide qualitative data have been validated and published, and where available, this information is provided. While these two types of data cannot currently be combined into one score, they can be used to assess consistency in the classification of VUS.

Who developed this guide?

This guide was developed by Dr. Noralane M. Lindor and Dr. Fergus J. Couch in collaboration with Dr. Susan T. Vadaparampil as part of an NIH SPORE grant P50 CA116201 awarded to the Mayo Clinic. This guide was developed in response to a 2013 survey of cancer genetic counselors preferences and needs regarding VUS results.

What information is contained in this guide?

The first part of this guide gives a brief overview of the current approaches to reclassify VUS. The next section provides clinical management recommendations based on the IARC multifactorial likelihood model. Finally, the tables at the end of this guide list specific BRCA test results that were initially reported as a VUS, but have been assigned a score that reflects the likelihood of that variant being non-pathogenic or pathogenic based on the integration of multiple lines of data.

How is a VUS classified using the IARC multifactorial likelihood model?

The Quantitative Approach

Multiple types of variant-specific data can be combined together as joint probabilities in an equation to produce a Bayesian “posterior probability” score. To be useable in this model, each type of evidence has an empiric likelihood of being found in BRCA mutation carriers versus being found in non-BRCA mutation carriers. Depending on availability of information (a limiting factor for many variants still), the multifactorial likelihood model may use different pieces of information including:

(1) Cosegregation - the way a VUS tracks with disease within families;(2) Sequence - the way the DNA sequence is conserved across different organisms

and the degree to which the amino acid change is predicted to affect protein charge or conformation;

(3) Pathology - the way a VUS correlates with characteristic pathology features of the cancers (e.g., triple negative markers);

(4) Co-occurrence - whether this VUS is seen in conjunction with other recognized BRCA mutations, as biallelic BRCA1 mutations are thought to be lethal and biallelic BRCA2 mutations cause Fanconi anemia; and,

(5) History of cancer – the family history and personal history of cancer defined by age of diagnosis and type of cancer and how this compares to histories of families with known pathogenic BRCA1/2 mutations.

Can functional studies be used to reclassify VUS?

Multiple laboratories are engaged in efforts to determine if specific VUS undermine normal BRCA gene functions. Because the BRCA genes have so many different functions, various functional assays have been developed to test multiple specific subcellular functions. A given pathogenic mutation may appear to wipe out some normal functions on some assays, but may appear quite normal if the assay conducted does not address a function knocked out by that particular variant. Thus, caution is needed when interpreting reports that indicate “normal function” while interpreting those of “loss of a normal function” as more convincing evidence of a pathogenic variant. It is essential that any functional assay be validated by testing its performance in known neutral and known pathogenic variants, and that the assay used is appropriate to the variant’s location within the gene. At present, functional data derived from studying VUS can be used to support the direction that other types of evidence suggest, or to call that impression into question if they disagree. Some argue that a variant that causes loss of function on a validated assay that reflects more global cellular function is sufficient to call a variant pathogenic. Currently functional studies are not integrated into the multifactorial likelihood model, as empiric likelihood ratios are required and are not available for most assays.

Because of the limitations of both the quantitative method and the functional studies, many VUS may remain as VUS for a long time to come.

What is the best way to use this guide in clinical practice?

In 2008, a group of clinical experts working with IARC lead by Dr. Sharon Plon from Baylor College of Medicine recommended when a posterior probability score reaches a certain threshold of certainty it may be reasonable to use the score to guide clinical decision-making. Further, they proposed genetic test results could be divided into 5 categories to guide health care decisions as shown in Table 1 [Plon SE et al. Hum Mutat 29, 1282-91 (2008)].

A Word of Caution

It is important to add words of caution regarding the use of this model and these tables. In arriving at the numbers for each VUS, a number of educated assumptions had to be made and because of this, there is the possibility that a VUS can be misclassified. To protect against the possibility of over-interpreting one line of evidence, the group that developed these new classifications [Plon et al, 2008] insisted that more than one line of information was required before a VUS could be placed into Class 1, 2, 4, or 5 categories, but errors in reclassification are still possible. Supplemental functional assays have been used to evaluate some of the same variants. If there is discordance with the integrated likelihood score, this is noted in the tables.

Table 1: IARC Class and Clinical ImplicationsIARCClass Definition Posterior

Probability Clinical Testing Surveillance Recommendations

5Definitely

pathogenic >0.99Test at-risk relatives for the variant

Full high-risk surveillance

4Likely

pathogenic 0.95-0.99Test at-risk relatives for the variant

Full high-risk surveillance

3 Uncertain 0.05-0.949

Do not use as predictive testing in

at-riskrelatives

Counsel based on family history and other risk

factors

2Likely not

pathogenic 0.001-0.049


at-riskrelatives


factors

1Not

pathogenic <0.001


at-riskrelatives


factors

Proposed classification for DNA sequence variants and correlation of clinical recommendation with probability that any given alteration is deleterious (i.e., increases chance for disease related to malfunction of a gene). Adapted from Plon et al., 2008

Clarification regarding use of the following tables

The following section presents three tables addressing BRCA1, BRCA2, and BRCA1 and BRCA2 candidate splicing variants. VUS are class 3, so what is shown on these tables are former Class 3 variants that have now moved to Class 1, 2, or Class 4, 5. In some cases, an IARC score has not yet been generated, but functional data is available. These variants also are included in the table with an n/a in the IARC column.

How to Search the Tables

Different nomenclatures have been used across laboratories and over time, so these tables include alternative terminologies for each variant. The variants are organized numerically. Be sure the codon and exons in your report match the variant you are looking for. The IARC class, based only on the multifactorial likelihood ratio model, is shown for each variant and the clinical management for that class is shown in Table 1. If validated functional studies exist, results of those are shown in the final column and where these are discordant with the score from the multifactorial model, this is noted.

Classification of BRCA1

Technical note: GenBank Reference BRCA1 U14680.1 Nucleotide numbering in “HGVS: DNA level” reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence, according to journal guidelines

(www.hgvs.org/mutnomen). The initiation codon is codon 1. Nucleotide numbering in “BIC-DNA level” refers to the original nomenclature for BRCA1 and BRCA2 before adoption of HGVS standards where BRCA1 +119 and BRCA2 +228 correspond to the A of the ATG translation initiation

codons in the reference sequence.

EXON Codon HGVS:protein level HGVS: DNA level

BIC: DNAlevel

Reference IARCClass Functional Studies

2 M18T p.Met18Thr c.53T>C 172T>C 16, 19 4

Homology directed recombination and single- strand annealing pathway

assays both abnormal supports pathogenicity

2 L22S p.Leu22Ser c.65T>C 184T>C 16 53 T37K p.Thr37K c.110C>A 229C>A 16 53 C39R p.Cys39Arg c.115T>C 234T>C 16 53 C44S p.Cys44Ser c.130T>A 249T>A 16 53 C44Y p.Cys44Tyr c.131G>A 250G>A 16 53 K45Q p.Lys45Gln c.133A>C 252A>C 16 1

5 C61G p.Cys61Gly c.181T>G 300T>G 16, 19 5



5 D67Y p.Asp67Tyr c.199G>T 318G>T 16, 19 1


assays both normal supports non pathogenicity

7 Y105C p.Tyr105Cys c.314A>G 433A>G 16 17 I124V p.Ile124Val c.370A>G 489A>G 16 1

7 N132K p.Asn132Lys c.396C>A 515C>A 16, 19 1



7 P142H p.Pro142His c.425C>A 544C>A 16 17 E143K p.Glu143Lys c.427G>A 546G>A 16 18 Q155E p.Gln155Glu c.463C>G 582C>G 16 1

8 Y179C p.Tyr179Cys c.536A>G 655A>G 16, 19 1



9 S186Y p.Ser186Tyr c.557C>A 676C>A 16, 19 1



9 V191I p.Val191Ile c.571G>A 690G>A 16 111 L246V p.Leu246Val c.736T>G 855T>G 16 111 A280G p.Ala280Gly c.839C>G 958C>G 16 111 M297I p.Met297Ile c.891G>A 1010G>A 16 111 P334H p.Pro334His c.1001C>A 1120C>A 16 211 P334L p.Pro334Leu c.1001C>T 1120C>T 16 111 Q356R p.Gln34Arg c.1067A>G 1186A>G 16 111 D369del p.Asp369del c.1105_1107del 1225del3 16 111 D369N p.Asp369Asn c.1105G>A 1224G>A 16 111 D420Y p.Asp420Tyr c.1258G>T 1377G>T 16 111 N473S p.Asn473Ser c.1418A>G 1537A>G 16 1






BIC: DNAlevel


11 F486L p.Phe486Leu c.1456T>C 1575T>C 16 111 R496H p.Arg496His c.1487G>A 1606G>A 16 111 R496C p.Arg496Cys c.1486C>T 1605C>T 16 111 R504H p.Arg504His c.1511G>A 1630G>A 16 111 N550H p.Asn550His c.1648A>C 1767A>C 16 111 E597K p.Glu597Lys c.1789G>A 1908G>A 16 111 A622V p.Ala622Val c.1865C>T 1984C>T 16 111 D642H p.Asp642His c.1924G>C 2043G>C 16 111 K654Q p.Lys654Gln c.1960A>C 2079A>C 16 211 L668F p.Leu668Phe c.2002C>T 2121C>T 16 111 D693N p.Asp693Asn c.2077G>A 2196G>A 16 111 N723D p.Asn723Asp c.2167A>G 2286A>G 16 111 E736A p.Glu736Ala c.2207A>C 2326A>C 16 211 V772A p.Val772Ala c.2315T>C 2434T>C 16 111 Q804H p.Gln804His c.2412G>C 2531G>C 16 111 N810Y p.Asn810Tyr c.2428A>T 2547A>T 16 111 K820E p.Lys820Gln c.2458A>G 2577A>G 16 111 T826K p.Thr826Lys c.2477C>A 2596C>A 16 111 R841W p.Arg841Trp c.2521C>T 2640C>T 16 111 E842G p.Glu842Gly c.2525A>G 2644A>G 16 111 Y856H p.Tyr856His c.2566T>C 2685T>C 16 111 K862E p.Lys862Glu c.2584A>G 2703A>G 16 111 R866C p.Arg866Cys c.2596C>T 2715C>T 16 111 P871L p.Pro871Leu c.2612C>T 2731C>T 16 111 G890V p.Gly890Val c.2669G>T 2788G>T 16 111 V920A p.Val920Ala c.2759T>C 2878T>C 16 211 I925L p.Ile925Leu c.2773A>C 2892A>C 16 111 M1008I p.Met1008Ile c.3024G>A 3143G>A 16 111 M1008V p.Met1008Val c.3022A>G 3141A>G 16 111 R1028H p.Arg1028His c.3083G>A 3202G>A 16 111 E1038G p.Gln1038Gly c.3113A>G 3232A>G 16 111 S1040N p.Ser1040Asn c.3119G>A 3238G>A 16 111 I1044V p.Ile1044Val c.3130A>G 3249A>G 16 111 P1099L p.Pro1099Leu c.3296C>T 3415C>T 16 111 S1101N p.Ser1101Asn c.3302G>A 3421G>A 16 111 K1109N p.Lys1109Asn c.3327A>C 3446A>C 16 111 S1140G p.Ser1140Gly c.3418A>G 3537A>G 16 111 D1155H p.Asp1155His c.3463G>C 3582G>C 16 111 K1183R p.Lys1183Arg c.3548A>G 3667A>G 16 111 Q1200H p.Gln1200His c.3600G>C 3719G>C 16 211 R1203Q p.Arg1203Gln c.3608G>A 3727G>A 16 111 E1214K p.Glu1214Lys c.3640G>A 3759G>A 16 111 P1238L p.Pro1238Leu c.3713C>T 3832C>T 16 111 V1247I p.Val1247Ile c.3739G>A 3858G>A 16 111 E1250K p.Glu1250Lys c.3748G>A 3867G>A 16 1






BIC: DNAlevel


11 S1266T p.Ser1266Thr c.3797G>C 3916G>C 16 111 I1275V p.Ile1275Val c.3823A>G 3942A>G 16 111 R1347G p.Arg1347Gly c.4039A>G 4158A>G 16 111 T1349M p.Thr1349Met c.4046C>T 4165C>T 16 111 M1361L p.Met1361Leu c.4081A>C 4200A>C 16 113 H1402Y p.His1402Tyr c.4204C>T 4323C>T 16 113 E1419Q p.Glu1419Gln c.4255G>C 4374G>C 16 113 R1443G p.Arg1443Gly c.4327C>G 4446C>G 16 114 N1468H p.Asn1468His c.4402A>C 4521A>C 16 114 aR1495M p.Arg1495Met c.4484G>T 4603G>T 16 515 S1512I p.Ser1512Ile c.4535G>T 4654G>T 16 115 V1534M p.Val1534Met c.4600G>A 4719G>A 16 115 D1546N p.Asp1546Asn c.4636G>A 4755G>A 16 115 D1546Y p.Asp1546Tyr c.4636G>T 4755G>T 16 116 L1564P p.Leu1564Pro c.4691T>C 4810T>C 16 116 S1613G p.Ser1613Gly c.4837A>G 4956A>G 16 116 P1614S p.Pro1614Ser c.4840C>T 4959C>T 16 116 M1628T p.Met1628Thr c.4883T>C 5002T>C 16 116 P1637L p.Pro1637Leu c.4910C>T 5029C>T 16 1

16 M1652I p.Met1652Ile c.4956G>A 5075G>A 11, 16 1No functional effect on

multiple assays supports non pathogenicity

16 M1652T p.Met1652Thr c.4955T>C 5074T>C 11, 16 1No functional effect on


16 F1662S p.Phe1662Ser c.4985T>C 5104T>C 11, 16 1No functional effect on


17 E1682K p.Glu1682Lys c.5044G>A 5163G>A 11, 16 1No functional effect on


17 T1685A p.Thr1685Ala c.5053A>G 5172A>G 11, 16 5 Strong functional impact demonstrated

17 T1685I p.Thr1685Ile c.5054C>T 5173C>T 16 517 V1688del p.Val1688del c.5181_5183delGTT 5181del3 16 5

17 M1689R p.Met1689Arg c.5066T>G 5185T>G 11, 16 4 Strong functional impact demonstrated

17 D1692H p.Asp1692His c.5074G>C 14 3Functional study suggests cryptic splice leading to

frameshift so may be Class 5

18 R1699Q p.Arg1699Gln c.5096G>A 5215G>A 11, 16 5 Strong functional impact demonstrated

18 R1699W p.Arg1699Trp c.5095C>T 5214C>T 11, 16 5 Strong functional impact demonstrated

18 G1706A p.Gly1706Ala c.5117G>C 5236G>C 11, 16 1No functional effect on


18 G1706E p.Gly1706Glu c.5117G>A 5236G>A 16 5

18 A1708E p.Ala1708Glu c.5123C>A 5242C>A 11, 16 5 Strong functional impact demonstrated

18 S1715R p.Ser1715Arg c.5143A>C 5262A>C 11, 16 5 Strong functional impact demonstrated






BIC: DNAlevel


19 T1720A p.Thr1720Ala c.5158A>G 5277A>G 11, 16 1No functional effect on


20 G1738R p.Gly1738Arg c.5212G>A 5331G>A 11, 16 5 Strong functional impact demonstrated

20 R1751Q p.Arg1751Gln c.5252G>A 5371G>A 16 1

21 L1764P p.Leu1764Pro c.5291T>C 5410T>C 11, 16 5 Strong functional impact demonstrated

21 I1766S p.Ile1766Ser c.5297T>G 5416T>G 11, 16 5 Strong functional impact demonstrated

21 M1775K p.Met1775 Lys c.5324T>A 5443T>A 11, 16 5 Strong functional impact

demonstrated

21 M1775R p.Met1775Arg c.5324T>G 5443T>G 16, 19 5



21 P1776H p.Pro1776His c.5327C>A 5446C>A 16 2

22 C1787S p.Cys1787Ser c.5359T>A 5478T>A 11, 16 5

WARNING!! Functional data discordant with IARC class. No

functional effect on multiple assays. Note: 2004 family data showed this variant in cis with G1788D, so C1787S might not be driver of the original scoring

22 G1788V p.Gly1788Val c.5363G>T 5482G>T 11, 16 5 Strong functional impact demonstrated

23 V1804D p.Val1804Asp c.5411T>A 5530T>A 11, 16 1No functional effect on


24 V1838E p.Val1838Glu c.5513T>A 5632T>A 11, 16 5 Strong functional impact demonstrated

24 I1858L p.Ile1858Leu c.5572A>C 5691A>C 16 124 P1859R p.Pro1859Arg c.5576C>G 5695C>G 16 1

18 Y1703S p.Tyr1703Ser c.5108A>C 5227A>C 13 n/a

Not in LOVD or BIC. Transcription activation assay

abnormal so suspected pathogenic based only on that

19 W1718L p.Trp1703Leu c.5153G>T 5272G>T 13 n/a



21 G1770V p.Gly1770Val c.5309G>T 5428G>T 13 n/a







EXON CODONHGVS:

protein level

HGVS: DNA level

BIC: DNAlevel


2 R18H p.Arg18His c.53G>A 281G>A 1 1

3 Y42C p.Tyr42Cys c.125A>G 353A>G 1 1

DNA change shown to have no effect on BRCA2 DNA repair

function via Homologous Recombination supports non

pathogenicity3 N56T p.Asn56Thr c.167A>C 395A>C 1 13 A75P p.Ala75Pro c.223G>C 451G>C 1 16 P168T p.Pro168Thr c.502C>A 730C>A 1 1

10 N319T p.Asn319Thr c.956A>C 1184A>C 1 1



pathogenicity10 S326R p.Ser326Arg c.978C>A 1206C>A 1 1

10 N372H p.Asn372His c.1114C>A 1342C>A 1 1



pathogenicity10 P375S p.Pro375Ser c.1123C>T 1351C>T 1 110 S384F p.Ser384Phe c.1151C>T 1379C>T 1 1

10 E462G p.Glu462Gly c.1385A>G 1613A>G

1 1



pathogenicity10 A487E p.Ala487Glu c.1440C>A 1688C>A 2 2 Not in IARC LOVD

10 K513R p.Lys513Arg c.1538A>G 1766A>G

1 110 N517S p.Asn517Ser c.1550A>G 1778A>

G1 2

10 C554W p.Cys554Trp c.1662T>G 1890T>G 1 110 T582P p.Thr582Pro c.1744A>C 1972A>C 1 110 N588D p.Asn588Asp c.1762A>G 1990A>

G1 2

10 G602R p.Gly602Arg c.1804G>A 2032G>A

1 110 K607T p.Lys607Thr c.1820A>C 2048A>C 1 210 T630I p.Thr630Ile c.1889C>T 2117C>T 1 111 P655R p.Pro655Arg c.1964C>G 2192C>G 1 111 D806H p.Asp806His c.2416G>C 2644G>C 1 111 V894I p.Val894Ile c.2680G>A 2908G>

A1 1

11 L929S p.Leu929Ser c.2786T>C 3014T>C 1 111 D935N p.Asp935Asn c.2803G>A 3031G>

A6 1 Not in IARC LOVD

11 N987I p.Asn987Ile c.2960A>T 3188A>T 1 111 L1019V p.Leu1019Val c.3055C>G 3283C>G 1 111 N1102Y p.Asn1102Tyr c.3304A>T 3532A>T 1 111 S1172L p.Ser1172Leu c.3515C>T 3743C>T 1 111 R1190W p.Arg1190Trp c.3568C>T 3796C>T 1 111 G1194D p.Gly1194Asp c.3581G>A 3809G>

A1 1

11 N1228D p.Asn1228Asp c.3682A>G 3910A>G

1 111 C1265S p.Cys1265Ser c.3793T>A 4021T>A 1 111 D1280V p.Asp1280Val c.3839A>T 4067A>T 1 1





EXON CODONHGVS:

protein level

HGVS: DNAlevel

BIC: DNAlevel Reference IARC

Class Functional Studies11 V1306I p.Val1306Ile c.3916G>A 4144G>A 1 111 I1349T p.Ile1349Thr c.4046T>C 4274T>C 1 111 D1352Y p.Asp1352Tyr c.4054G>T 4282G>T 1 211 T1354M p.Thr1354Met c.4061C>T 4289C>T 1 111 C1365Y p.Cys1365Tyr c.4094G>A 4322G>A 1 111 Q1396R p.Gln1396Arg c.4187A>G 4415A>G 1 111 D1420Y p.Asp1420Tyr c.4258G>T 4486G>T 1 111 K1434I p.Lys1434Ile c.4301A>T 4529A>T 1 211 F1524V p.Phe1524Val c.4570T>G 4798T>G 1 111 G1529R p.Gly1529Arg c.4585G>A 4813G>A 1 111 K1690N p.Lys1690Asn c.5070A>C 5298A>C 1 111 S1733F p.Ser1733Phe c.5198C>T 5426C>T 1 111 S1760A p.Ser1760Ala c.5278T>G 5506T>G 1 211 G1771D p.Gly1771Asp c.5312G>A 5540G>A 1 111 P1819S p.Pro1819Ser c.5455C>T 5683C>T 1 111 L1904V p.Leu1904Val c.5710C>G 5938C>G 1 111 H1918Y p.His1918Tyr c.5752C>T 5980C>T 1 111 I1929V p.Ile1929Val c.5785A>G 6013A>G 1 111 R2034C p.Arg2034Cys c.6100C>T 6328C>T 1 111 N2048I p.Asn2048Ile c.6143A>T 6371A>T 1 111 H2074N p.His2074Asn c.6220C>A 6448C>A 1 111 R2108H p.Arg2108His c.6323G>A 6551G>A 1 111 N2113S p.Asn2113Ser c.6338A>G 6566A>G 1 111 T2250A p.Thr2250Ala c.6748A>G 6976A>G 1 111 G2274V p.Gly2274Val c.6821G>T 7049G>T 1 212 I2285V p.Ile2285Val c.6853A>G 7081A>G 1 112 D2312V p.Asp2312Val c.6935A>T 7163A>T 4 1 Not in IARC LOVD

13 R2318Q p.Arg2318Gln c.6953G>A 7181G>A 1 2

R2336H p.Arg2336His c.7007G>A c.7235G> A 10, 15 5Predicted pathogenic based on embryonic stem cell functional

assay14 A2351G p.Ala2351Gly c.7052C>G 7280C>G 1 214 G2353R p.Gly2353Arg c.7057G>C 7285G>C 2 1 Not in IARC LOVD

14 Q2384K p.Gln2384Lys c.7150C>A 7378C>A 1 114 L2396F p.Leu2396Phe c.7188C>A 7416G>T 4 1 Not in IARC LOVD

14 K2411T p.Lys2411Thr c.7232A>C 7460A>C 4 1 Not in IARC LOVD

14 R2418G p.Arg2418Gly c.7252A>G 7480A>G 1 214 N2436I p.Asn2436Ile c.7307A>T 7535A>T 1 114 K2446E p.Lys2446Glu c.7336A>G 7564A>G 5 2 Not in IARC LOVD

14 K2472T p.Lys2472Thr c.7415A>C 7643A>C 1 1

15 T2515I p.Thr2515Ile c.7544C>T 7772C>T 1 1

DNA change shown to have no effect on DNA repair function

via Homologous Recombination; supports non

pathogenicity





EXON CODONHGVS:

protein level

HGVS: DNA level


Class Functional Studies

L2510P 7, 15 3

Posterior probability model still lists as Class 3 but

CLINVAR lists as deleterious and functional study [3]) does

show disruption of BRCA2 repair function via

homologous recombination; mouse embryonic stem cell assay predicts pathogenic as

16 P2589H p.Pro2589His c.7766C>A 7994C>A 2 2 Not in IARC LOVD

17 G2609D p.Gly2609Asp c.7826G>A 8054 G>A 3 4

Not in IARC LOVD; DNA change shown to disrupt BRCA2 DNA

repair function via Homologous Recombination, supports

pathogenicity

H2623R 7 3

Not yet reclassified in IARC LOVD but CLINVAR lists as deleterious and functional

study [3] does show disruption of BRCA2 repair function via homologous recombination

17 W2626C p.Trp2626Cys c.7878G>C 8106G>C 1,15 5

Listed in ClinVar as unknown (Class 3); DNA change shown to disrupt BRCA2 DNA repair

function via Homologous Recombination, supports

pathogenicity; mouse embryonic stem cell assay

supports pathogenic as well

17 I2627F p.Ile2627Phe c.7879A>T 8107A>T 1 5 Listed in ClinVar as unknown (Class 3)

17 L2647P p.Leu2647Pro c.7940T>C 8168T>C 1 4

DNA change shown to disrupt DNA repair function via

Homologous Recombination, supports pathogenicity

17 L2653P p.Leu2653Pro c.7958T>C 8186T>C 1 5



17 aR2659K p.Arg2659Lys c.7976G>A 8204G>A 1 5

18 aE2663V p.Glu2663Val c.7988A>T 8216A>T 1 5

18 D2665G p.Asp2665Gly c.7994A>G 8222A>G 1 2



pathogenicity18 M2676T p.Met2676Thr c.8027T>C 8255T>C 2 2 Not in IARC LOVD

18 L2688P p.Leu2688Pro c.8063T>C 8291T>C 3 4



pathogenicity18 A2717S p.Ala2717Ser c.8149G>T 8377G>T 1 1

18 T2722R p.Thr2722Arg c.8165C>G 8393C>G 1 5







EXON CODONHGVS:

protein levelHGVS: DNAlevel


Class Functional Studies

18 D2723H p.Asp2723His c.8167G>C 8395G>C 1 5



18 aD2723G p.Asp2723Gly c.8168A>G 8396A>G 1 5



18 K2729N p.Lys2729Asn c.8187G>T 8415G>T 1 1



pathogenicity.

18 G2748D p.Gly2748Asp c.8243G>A 8471G>A 1 5



18 A2770T p.Ala2770Thr c.8308G>A 8536G>A 1 2

19 R2787H p.Arg2787His c.8360G>A 8588G>A 1 2



pathogenicity20 R2842H p.Arg2842His c.8525G>A 8753G>A 1 1

20 E2856A p.Glu2856Ala c.8567A>C 8795A>C 1 1



pathogenicity20 Q2858R p.Gln2858Arg c.8573A>G 8801A>G 1 221 R2888C p.Arg2888Cys c.8662C>T 8890C>T 1 1

21 V2908G p.Val2908Gly c.8723T>G 8951T>G 1 2



pathogenicity22 E2947K p.Glu2947Lys c.8839G>A 9067G>A 5 2 Not in IARC LOVD

22 D2965H p.Asp2965His c.8893G>C 9121G>C 2 2 Not in IARC LOVD

22 V2969M p.Val2969Met c.8905G>A 9133G>A 4 1 Not in IARC LOVD

22 R2973C p.Arg2973Cys c.8917C>T 9145C>T 1 1



pathogenicity

E3002K 7 3

Posterior probability model still lists as Class 3 but

CLINVAR lists as deleterious and functional study (Guidugli L 2012) does show disruption of BRCA2 repair function via homologous recombination

24 R3052W p.Arg3052Trp c.9154C>T 9382C>T 1 5







EXON CODONHGVS:

protein levelHGVS: DNA

levelBIC: DNA

level Reference IARCClass Functional Studies

24 P3063S p.Pro3063Ser c.9187C>T 9415C>T 1 2



pathogenicity

24 V3079I p.Val3079Ile c.9235G>A 9463G>A 1 1Initially listed in ClinVar as

deleterious but now corrected to indicate non-pathogenic

25 Y3092C p.Tyr3092Cys c.9275A>G 9503A>G 1 2



pathogenicity

25 D3095E p.Asp3095Glu c.9285C>G 9513C>G 1 4



25 Y3098H p.Tyr3098His c.9292T>C 9520T>C 1 1



pathogenicity

17 N3124I p.Asn3124Ile c.9371A>T 9599A>T 3 4



pathogenicity26 D3170G p.Asp3170Gly c.9509A>G 9737A>G 1 126 C3198R p.Cys3198Arg c.9592T>C 9820T>C 1 1

27 K3326X p.Lys3326X c.9976A>T 10204A>T 1 1



pathogenicity27 T3349A p.Thr3349Ala c.10045A>G 10273A>G 1 1

Classification of BRCA1 and 2 candidate splicing variants




Gene Exon Codon HGVS: DNA levelBIC: DNA

levelReference IARC

ClassFunctional

studies

BRCA1 IVS2+1G>A c.80+1G>A 21 n/aMini-gene splicing

assay predicts pathogenic

BRCA1 3 IVS2-11delT c.81-11delT 200-11delT 4 1

BRCA1 3 IVS2-13C>G c.81-13C>G 200-13C>G 4 1

BRCA1 c.132C>T 21 n/aMini-gene splicing


BRCA1 5 IVS5+3A>G c.212+3A>G 331+3A>G 4 5

BRCA1 10 IVS10+1delG c.670+1delG 21 n/aMini-gene splicing


BRCA1 6 IVS6+7G>A c.301+7G>A 420+7G>A 4 1

BRCA1 7 IVS6-1G>C c.302-1G>C 421-1G>C 4 5

BRCA1 7 IVS6-3C>G c.302-3C>T 421-3C>G 4 5

BRCA1 7 IVS7+2T>A c.441+2T>A 14 4

Functional study shows cryptic splice

site leads to frameshift so may

be class 5

BRCA1 9 IVS8-17G>T c.548-17G>T 667-17G>T 4 1

BRCA1 9 IVS9+4A>G c.593+4A>G 712+4A>G 17 2

BRCA1 12 IVS12+1G>A c.4185+1G>A 21 n/aMini-gene splicing


BRCA1 12 IVS11-1G>A c.4097-1G>A 4216-1G>A 4 5

BRCA1 12 IVS11-11T>C c.4097-11T>C 4216-11T>C 4 1

BRCA1 12 c.4184_4185+2del 14 4



be class 5

BRCA1 12 IVS12+10G>C c.4185+10G>C 4304+10G>C 4 1

BRCA1 13 IVS13+1G>A c.4357+1G>A 4476+1G>A 4, 14 5


site leads to frameshift supports

pathogenicity

BRCA1 15 IVS15+1G>A c.4675+1G>A 4794+1G>A 4 5

BRCA1 16 IVS15-7C>T c.4676-7C>T 4795-7C>T 17 2

BRCA1 17 IVS17-2A>G c.4987-2A>G 14 4



be class 5

BRCA1 17 IVS16-20A>G c.4987-20A>G 5106-20A>G 4 1

BRCA1 17 IVS17+1G>A c.5074+1G>A 5193+1G>A 4 5

BRCA1 17 IVS17-1G>C c.5075-1G>C 21 n/aMini-gene splicing


Classification of BRCA1 and 2 candidate splicing variants




Gene Exon Codon HGVS: DNA levelBIC: DNA

level ReferenceIARCClass

Functional studies

BRCA1 18 IVS17-9A>T c.5075-9A>T 5194-9A>T 4 2

BRCA1 18 IVS18+1G>T c5152+1G>T 5271+1G>T 6 5

BRCA1 19 IVS18-13A>G c.5153-13A>G 5272-13A>G 4 1

BRCA1 19 IVS18-1G>C c.5153-1G>C 5272-1G>C 4 5

BRCA1 19 IVS18-6C>A c.5153-6C>A 5272-6C>A 4 1

BRCA1 20 IVS19-12G>A c.5194-12G>A 5313-12G>A 17 5

BRCA1 20 IVS20+1G>A c.5277+1G>A 5396+1G>A 4 5

BRCA1 22 IVS21-8C>T c.5333-8C>T 5452-8C>T 4 2

BRCA1 23 IVS23+5G>C c.5467+5G>C 5586G>C 17 2

BRCA2 6 IVS5-2A>G c.476-2A>G 704-2A>G 4 5

BRCA2 6 IVS6+1G>T c.516+1G>T 744+1G>T 17 4

BRCA2 7 IVS7+2T>G c.631+2T>G 864+2T>G 15 n/a

Internal deletion of 105 amino acids and

seen in Fanconi anemia

BRCA2 9 IVS8-12delTA c.791delTA 910-12delTA 18 2

BRCA2 12 IVS11-20T>A c.6842-20T>A 7070-20T>A 4 1

BRCA2 13 IVS13+1G>C c.7007+1G>C 7235+1G>C 17 4

BRCA2 15 IVS14-14T>G c.7436-14T>G 7664G>C 17 2

BRCA2 16 IVS15-1G>A c.7618-1G>A 7846-1G>A 17 5

BRCA2 19 IVS19+1G>A c.8487+1G>A 8715+1G>A 4 5

BRCA2 21 IVS20-16C>G c.8633-16C>G 8861-16C>G 4 2

BRCA2 21 IVS21+3G>C c.8754+3G>C 14 3



be class 4

BRCA2 21 IVS21+4A>G c.8754+4A>G 8982+4A>G 4 4

BRCA2 22 IVS22+1G>T c.8953+1G>T 9181+1G>T 17 5

BRCA2 23 P3039P c.9117G>A 9345G>A 4 5

BRCA2 25 IVS24-1G>C c.9257-1G>C 9485-1G>C 4 5

BRCA2 25 IVS25+9A>C c.9501+9A>C 9729+9A>C 4 1

BRCA2 27 IVS26-20C>T c.9649-20C>T 9877-20C>T 4 1

Online resources that may be useful when you are faced with an unclassified variantNote: all these databases are dependent on ongoing curation and may be incomplete or out of date.

E vidence-based Network for the Interpretation of Germline Mutant Alleles – ENIGMAhttp://enigmaconsortium.org/

The Breast Cancer Information Core (BIC)An international collaborative effort to have an open access on-line breast cancer mutation

database hosted by National Human Genome Research Institute. Requires first time registration, but is free.http://research.nhgri.nih.gov/bic/ <http://research.nhgri.nih.gov/bic/>

CLINVAR (Clinically Relevant Sequence Variations)Supported by National Center for Biotechnology Information at the National Institutes for Health. ClinVar archives submitted information from individuals as well as public resources, but neither curates content nor modifies it independent of an explicit submission. It duly catalogues discrepancies in interpretations.

o For BRCA1: https://www.ncbi.nlm.nih.gov/clinvar?term=BRCA1 o For BRCA2: <https://www.ncbi.nlm.nih.gov/clinvar/?term=BRCA2>

Leiden Open Variome Database is open to all. Dr. Sean Tavtigian has set up a special page for BRCA Class 3 variants that have been reclassified using the Multifactorial Likelihood Model.

o For BRCA1: http://brca.iarc.fr/LOVD/variants.php?select_db=BRCA1&action=view_unique

o For BRCA2: http://brca.iarc.fr/LOVD/variants.php?select_db=BRCA2&action=view_unique

PubMed/Google ScholarCan type your variant directly into either search engine, also be sure to search using different variations of nomenclature.http://www/ncbi.nlm.nih.gov/PubMed http://scholar.google.com/

Exome Variant ServerNHBLI Exome Sequencing Project lists variants found in individuals without cancer. http://evs.gs.washinton.edu/EVS/

BRCA1 CircosThe BIC also hosts a BRCA1 functional study database, curated by Dr. Alvaro Monteiro http://lgdfm3.ncifcrf.gov/bic/BIC%20Circos%20Database%20Table/recordlist.php

This work was supported by the Breast Cancer Spore CA 116201Dr. Fergus J. Couch, Ph.D. - Mayo Clinic, Rochester MNDr. Noralane M. Lindor, M.D. - Mayo Clinic, Scottsdale, AZDr. Susan T. Vadaparampil, Ph.D. - Moffitt Cancer Center, Tampa, FL

References used in the Tables:1) For detail on this variant:<http://brca.iarc.fr/LOVD/variants.php?select_db=BRCA2&action=view_unique>2) Lindor NM, et al., A review of a multifactorial probability-based model for classification of BRCA1 and BRCA2 variants of uncertain significance (VUS). Hum Mutat. <http://www.ncbi.nlm.nih.gov/pubmed/21990134> 2012 Jan;33(1):8-21.3) Guidugli L et al.,. A classification model for BRCA2 DNA binding domain missense variants based on homology-directed repair activity. Cancer Research. 73(1):265-75, 2013 Jan 1.4) Easton DF et al., A systematic genetic assessment of 1,433 sequence variants of unknown clinical significance in the BRCA1 and BRCA2 breast cancer-predisposition genes. Am J Hum Genet.<http://www.ncbi.nlm.nih.gov/pubmed/17924331> 2007 Nov;81(5):873-83.5) Walker LC et al., Detection of splicing aberrations caused by BRCA1 and BRCA2 sequence variants encoding missense substitutions: implications for prediction of pathogenicity. Hum Mutat.<http://www.ncbi.nlm.nih.gov/pubmed/20513136>2010 Jun;31(6):E1484-505.6) Spurdle AB 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.<http://www.ncbi.nlm.nih.gov/pubmed/18375895> 2008 Apr 1;26(10):1657-63.7) For detail on this variant: https://www.ncbi.nlm.nih.gov/clinvar/?term=BRCA2<https://www.ncbi.nlm.nih.gov/clinvar/?term=BRCA2>8) For detail on this variant: https://www.ncbi.nlm.nih.gov/clinvar?term=BRCA1<https://www.ncbi.nlm.nih.gov/clinvar?term=BRCA1>9) Wu K et al., Functional evaluation and cancer risk assessment of BRCA2 unclassified variants. Cancer Res.<http://www.ncbi.nlm.nih.gov/pubmed/15695382> 2005 Jan 15;65(2):417-26.10) Farrugia DJ et al., Functional assays for classification of BRCA2 variants of uncertain significance. Cancer Res. <http://www.ncbi.nlm.nih.gov/pubmed/18451181> 2008 May 1;68(9):3523-31. doi: 10.1158/0008-5472.CAN- 07-1587.11) Lee MS et al., Comprehensive analysis of missense variations in the BRCT domain of BRCA1 by structural and functional assays. Cancer Research. 70(12):4880-90, 2010 Jun 15.12) Millot GA et al., ENIGMA Consortium Functional Assay Working Group. A guide for functional analysis of BRCA1 variants of uncertain significance. [Review]> Human Mutation. 33(11):1526-37, 2012 Nov.13) Quiles F et al., Functional and structural analysis of C-terminal BRCA1 missense variants. PLoS ONE [Electronic Resource]. 8(4):e61302, 2013.14) Thomassen M et al., Characterization of BRCA1 and BRCA2 splicing variants: a collaborative report by ENIGMA consortium members. Breast Cancer Research & Treatment. 132(3):1009-23, 2012 Apr.15) Biswas K et al., A comprehensive functional characterization of BRCA2 variants associated with Fanconi anemia using mouse ES cell-based assay. Blood. <http://www.ncbi.nlm.nih.gov/pubmed/21719596> 2011 Sep 1;118(9):2430-42.16) For details on this variant:<http://brca.iarc.fr/LOVD/variants.php?select_db=BRCA1&action=view_unique>17) Whiley PJ et al., . Splicing and multifactorial analysis of intronic BRCA1 and BRCA2 sequence variants identifies clinically significant splicing aberrations up to 12 nucleotides from the intron/exon boundary. Hum Mutat.<http://www.ncbi.nlm.nih.gov/pubmed/21394826> 2011 Jun;32(6):678-87.18) Spearman AD et al., Clinically applicable models to characterize BRCA1 and BRCA2 variants of uncertain significance. <http://www.ncbi.nlm.nih.gov/pubmed/18824701> J Clin Oncol. 2008 Nov 20;26(33):5393-400. Erratum in: J Clin Oncol. 2009 May 10;27(14):241519) Towler WI, et al.,. Analysis of BRCA1 Variants in Double-Strand Break Repair by Homologous Recombination and Single-Strand Annealing (pages 439–445) Human Mutation Volume 34, Issue 3, pages 439– 445, March 201320) Biswas K et al., Functional evaluation of BRCA2 variants mapping to the PALB2-binding and C-terminal DNA-binding domains using a mouse ES cell-based assay. Hum Mol Genet.<http://www.ncbi.nlm.nih.gov/pubmed/22678057?dopt=Abstract> 2012 Sep 15;21(18):3993-4006.21) Steffensen AY et al., Functional characterization of the BRCA1 gene variants by mini-gene splicing assay. European J Hum Genet (2014) 1-7. 22.22) Plon SE et al., Sequence variant classification and reporting: recommendations for improving the interpretation of cancer susceptibility genetic test results. Hum Mutat. 2008 Nov;29(11):1282-91.

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