Supplementary Table 1: Laboratory Protocols Used in This Study

Supplementary Table 1: Laboratory protocols used in this study

Laboratory Cells / Culture Nonsense RNA extraction Dnase 1 Amount of cDNA cDNA PCR amplification PCR conditions 2 PCR product Sequencing acronym tissue conditions mediated method treatment total RNA synthesis synthesis template analysis (collection decay used in cDNA primer protocol method) inhibition synthesis OUH LCL LCL With and Qiagen RNeasy RNase-Free 3µg Gene Invitrogen 2μl of the cDNA 94oC, 3 min Agarose gel Sequencing of cDNA + RPMI without Minikit 1 DNase specific Super-script mixture 35 cycles of pool using + 10% serum puromycin I1(Qiagen) II 1 Taq (Roche) 94oC, 30s ABI3730xl + 1% Pen/strep 200μg /ml 60oC, 30s for 2 hours 72oC, 45c then 72oC, 5 min

FPGMX Fresh blood 1ml blood With and Qiagen RNeasy RNase-Free 1µg Random Invitrogen 2μl of the cDNA 94oC, 2 min Agilent or Qiaxell Sequencing of cDNA (EDTA tubes + 9 ml RPMI without Minikit 1 DNase hexamers Super-script mixture 3 cycles of automated pool or purified or Heparin + 20% serum puromycin I1(Qiagen) II 1 GoTaq (Promega) 94oC, 30 s electrophoresis bands using tubes for + 1% Pen/strep 200μg /ml xoC, 40 s or agarose gel and ABI3730xl PHA) + PHA (100μg/ for 8 hours 72oC, 1 min gel purification of ml) 3 cycles of aberrant bands 72 hrs 94oC, 30 s (QIAquick) (x-2)oC, 40 s 72oC, 1 min 30 cycles of 94oC, 30 s (x-4)oC, 40 s 72oC, 1 min then 72oC, 7 min IOV LCL LCL None Qiagen RNeasy RNase-Free 1µg Random Invitrogen 3μl of the cDNA 95°C 8 min Agarose gel and gel Sequencing of cDNA + RPMI Minikit 1 DNase hexamers Superscript mixture 38 cycles of purification of purified bands using + 13% serum I1(Qiagen) III; AmpliTaq gold 94°C 40 s aberrant bands ABI3130 + 2mM l- 50°C for 60 (Applied Biosystems) x°C 45 s (QIAquick) and/or glutamine min 72°C 45 s; DHPLC "double- then 72°C 10 strand multiple min fragments" application CBCS Fresh blood N/A N/A TriZOL N/A 0.5-1µg Random Applied 1l of the cDNA 98C 30 s Agarose gel, PCR- Sequencing of cDNA (EDTA or hexamers Biosystems mixture 35 cycles of Blunt-TOPO cloning purified bands using Citrate Multiscribe 1 Phusion (Finnzymes) 98C 15 s ABI3730 tubes) 56C 20 s 72C 30 s then 72C 5 min HVH Fresh blood N/A N/A TriZOL and RNA RNeasy Kit 1µg Random Applied 2μl of the cDNA 94°C 5 min, Agarose gel Sequencing of cDNA (EDTA cleanup with and RNase- hexamers Biosystems mixture 35 cycles of pool using tubes) Qiagen RNeasy Free DNase and oligo dT High EcoTaq 94°C 30 s ABI3130xl Mini Kit I1(Qiagen) Capacity (Ecogen) x°C 30 s RNA-to- 72°C 30 s cDNA 72°C 7 min Master Mix 1 AAS Fresh blood N/A N/A TriZOL N/A 1-5µg Random Invitrogen 2μl of the cDNA 95oC, 2 min Agarose gel Sequencing of cDNA (EDTA hexamers Superscript mixture 38 cycles of pool using ABI310xl, tubes) III1 AmpliTaq 95oC, 30 s allele-specific (Applied Biosystems) 56oC, 30 s sequencing 72oC, 60 s then 72oC 2 min ICO Fresh blood 500 μL PBL Puromycin TriZOL N/A 0.5-1µg Random Invitrogen 2μl of the cDNA 96oC 5 min Agarose gel Sequencing of cDNA (EDTA + 7mL RPMI 250g/ml hexamers Superscript mixture 35 cycles of pool using ABI3130 tubes) + 10% serum for 4-6 hours II 1 MegaMix (Microzone) 96oC 30s-60 s + 1% Pen/strep xoC + PHA (10 g per 30s-60 s ml RPMI 72oC 60-120s medium) then 72oC, 10 72-120 hrs min

1 According to manufacturers' protocols

2 x=melting temperature depending on the pair of primers used in the reaction

LCL: lymphoblastoid cell lines

Pen/strep: penicillin-streptomycin

PHA: phytohaemaglutinin Supplementary Table 2: Methodology and primer sequences used to assay variants

GENE VARIANT (HGVS) Exon Method Group cDNA primers Forward PCR primers (5’-3’) Location of Reverse PCR primers (5’-3’) Location Expected Sequencing primers, if different /Intron Forward of Reverse band size from PCR primers (5’-3’) Location Primer Primer (bp) of Variant

BRCA1 c.301+6T>C I6 RT-PCR, IOV random sequencing, CAAGGAACCTGTCTCCACAAAGTG exon 3 AAGTCTTTTGGCACGGTTTCTG exon 7 319 DHPLC hexamers

random CAAGGAACCTGTCTCCACAAAGTG exon 3 CGTCTTTTGAGGTTGTATCCGCTG exon 8 436 hexamers

random GAAAGGGCCTTCACAGTGTC exon 5 TCTTTTGGCACGGTTTCTGT exon 7 244 hexamers BRCA1 c.441+2T>A I7 RT-PCR, FPGMX sequencing CAGAAGAAAGGGCCTTCACA exon 5 TTTGGCATTATCAACTGGCTTATC exon 11 2670 CAGCTTGACACAGGTTTGGA random (forward exon 6) hexamers GATCAGCATTCAGATCTACC CAGCTTGACACAGGTTTGGA exon 6** GATCAGCATTCAGATCTACC exon 11** 757 (reverse, exon 11)

BRCA1 c.548-8delT I8 RT-PCR, OUH TGCAAGTTTG sequencing TAACCTTGGAACTGTGAGAAC exon 8 CAGAATCCAAACTGATTTCATC exon 10 192 AAACAGAAC

155 TGCAAGTTTG AAAACTTTTATTGATTTATTTTTTGG intron 8 CAGAATCCAAACTGATTTCATC exon 10 if intron AAACAGAAC retention 217 TGCAAGTTTG AAATATTTCTAGTTGAATATCTG intron 8 CAGAATCCAAACTGATTTCATC exon 10 if intron AAACAGAAC retention

RT-PCR, random CTGTTGCTCCTCCACATCAA BRCA1 c.4097-15T>C I11 FPGMX ACCGTTGCTACCGAGTGTCT exon 11 CCCTGCTCACACTTTCTTCC exon 16 1168 sequencing hexamers (reverse, exon 15) BRCA1 c.4184_4185 E12 I12 RT-PCR, FPGMX CAAAAGCGTCCAGAAAGGAG exon 11 CCCTGCTCACACTTTCTTCC exon 16 1370 ACCGTTGCTACCGAGTGTCT +2del sequencing random (forward, exon 11) hexamers CTGTTGCTCCTCCACATCAA ACCGTTGCTACCGAGTGTCT exon 11** CTGTTGCTCCTCCACATCAA exon 15** 879 (reverse, exon 15)

RT-PCR, random BRCA1 c.4357+1G>A I13 FPGMX GAAAATAATCAAGAAGAGCAAAGCA exon 11 CCCTGCTCACACTTTCTTCC exon 16 847 sequencing hexamers BRCA1 c.4358-4delA I13 RT-PCR, OUH GGCAAGTAAG sequencing CTGCGAAATCCAGAACAAAG exon 13 TGTTGCTCCTCCACATCAAC exon 15 290 ATGTTTC

285 GGCAAGTAAG GTAGATTTGTTTTCTCATTCCA intron 13 TGTTGCTCCTCCACATCAAC exon 15 if intron ATGTTTC retention BRCA1 c.4987-2A>G I16 RT-PCR, FPGMX random GGCCTTTCTGCTGACAAGTT exon 14 ATTCTCTTGCTCGCTTTGGA exon 20 853 AAGACAGAGCCCCAGAGTCA sequencing hexamers (forward exon 16) GACCTTGGTGGTTTCTTCCA AAGACAGAGCCCCAGAGTCA exon 16** GACCTTGGTGGTTTCTTCCA exon 20** 504 (reverse, exon 20)

RT-PCR, random BRCA1 c.5074G>C E17 IOV CCCCAGAAGAATTTATGCTCGTG exon 16/17 TTCTCTTGCTCGCTTTGGACC exon 20 290 sequencing hexamers

RT-PCR, random BRCA1 c.5075-107A>G I17 ICO GGGTCAACAAAAGAATGTCCA exon 16 TCTTCCATTGACCACATCTCC exon 20 300 sequencing hexamers BRCA1 c.5333A>G E22 RT-PCR, random sequencing ICO GTTTGCCAGAAAACACCACA exon 17 CACAGGTGCCTCACACATCT exon 24 496 hexamers

random FPGMX TGAAGTCAGAGGAGATGTGG exon 20 TGGTAGAGTGCTACACTGTC exon 24 338 hexamers random RT-PCR, hexamers and BRCA2 c.3G>A E2 HVH AGCGTGAGGGGACAGATTT exon 1 AGTCAGCCCTTGCTCTTTGA exon 3 380 sequencing anchored- oligo (dT)18 RT-PCR, random TCTGGAGCGGACTTATTTACCAAA BRCA2 c.316+5G>A I3 AAS AAGAGAGGCCAACATTTTTTGAAA exon 2 GGTGTCTGACGACCCTTCACA exon 6/7 514 sequencing hexamers TCTGGAGCGGACTTATTTACCAAG BRCA2 c.425+33A>G I4 RT-PCR, random sequencing ICO TCAGCTGGCTTCAACTCCAATAA exon 3 GAACTAAGGGTGGGTGGTGTAGC exon 7 402 hexamers random hexamers and HVH GGATCCAAAGAGAGGCCAAC exon 2 TGAAACAAACTCCCACATACCA exon 6 488 anchored- oligo (dT)18 GATCACGAATTCTCCAGCAGCTGAA GATCACGGATCCAAATATGTTTTGGTG pSPL3 vector-specific primers: intron 4 exon 7 BRCA2 c.426-22G>T I4 minigene* CBCS N/A ATTTGTGAGTAC TCTGACGACC 648 5’-TCTGAGTCACCTGGACAACC-3’ 5’-ATCTCAGTGGTATTTGTGAGC-3’ GATCACGAATTCTCCAGCAGCTGAA GATCACGGATCCAAATATGTTTTGGTG pSPL3 vector-specific primers: intron 4 exon 7 BRCA2 c.516+18T>C I6 minigene* CBCS N/A ATTTGTGAGTAC TCTGACGACC 648 5’-TCTGAGTCACCTGGACAACC-3’ 5’-ATCTCAGTGGTATTTGTGAGC-3’ random RT-PCR, hexamers and BRCA2 c.632-16A>C I7 HVH CCGCTGTACCAATCTCCTGT exon 3 TTCCAATGTGGTCTTTGCAG exon 10 589 sequencing anchored- oligo (dT)18 RT-PCR, random BRCA2 c.992A>T E10 ICO TCAAAGAGAAGCTGCAAGTCA exon 9 TTCCTCAGAATTGTCCCAAAA exon 11 1236 sequencing hexamers

RT-PCR, random BRCA2 c.1096T>G E10 FPGMX CAAATCAAAGAGAAGCTGCAA exon 9 ACCTTTGAGCTTGTCTGACA exon 11 1634 sequencing hexamers CAGCCACCACCACACAGA RT-PCR, random (forward, exon 10) BRCA2 c.1788T>C E10 FPGMX CAAATCAAAGAGAAGCTGCAA exon 9 ACCTTTGAGCTTGTCTGACA exon 11 1634 sequencing hexamers TCTGGTTTTCAGGCACTTCA (reverse, exon 11) CCAATTTCAAATCACAGTTTTGGAGGT RT-PCR, random (forward, exon 11) BRCA2 c.3156A>C E11 FPGMX CAGTGGCTTCTTCATTTCAGG exon 10 GGAGTGCTTTTTGAAGCCTTT exon 12 9282 sequencing hexamers GGAGTGCTTTTTGAAGCCTTT (reverse, exon 12) random RT-PCR, hexamers and BRCA2 c.7397C>T E14 HVH AGGCTTCAAAAAGCACTCCA exon 12 TCCACCATCAGCCAACTGTA exon 16 842 sequencing anchored- oligo (dT)18 random RT-PCR, hexamers and BRCA2 c.7435+6G>A I14 HVH AGGCTTCAAAAAGCACTCCA exon 12 TCCACCATCAGCCAACTGTA exon 16 842 sequencing anchored- oligo (dT)18 CTAACAGTACTCGGCCTGCTC RT-PCR, random (forward, exon 19) BRCA2 c.8421G>T E19 FPGMX ATGGAAAGGGATGACACAGC exon 18 CTGATGATGGACGCCAAATA exon 23 956 sequencing hexamers CAGATTCCATGGCCTTCCTA (reverse, exon 22) BRCA2 c.8754+3G>C E21 RT_PCR, random CBCS CGGCCTGCTCGCTGGTAT exon 19 GCCTTCCTAATTTCCAACTGGATCTG exon 22 503 sequencing hexamers

pSPL3 vector-specific primers: GATCACGAATTCTTCCTGGAAAACTT GATCACCTCGAGTTAGGGTAGAGGAT minigene* CBCS N/A intron 20 intron 21 5’-TCTGAGTCACCTGGACAACC-3’ ATAGCA TATCAAGTACA 5’-ATCTCAGTGGTATTTGTGAGC-3’

* Minigene primers listed in the body of the table are those used for the cloning of the specific exons into the minigene vector. The following vector-specific primers were used for the final PCR: Forward primer: 5- GTGAACTGCACTGTGACAAGCTGC-3; Reverse primer: 5-CACCTGAGGAGTGAATTGGTCG-3 **Primers used for a nested PCR Supplementary Table 3: Bioinformatic prediction of cryptic splice site usage for variants associated with splicing aberrations in vitro*

Score for variant sequence

SSF MaxEnt NNSPLICE GeneSplicer Predicted location of Location of cryptic splice Gene Variant Site alternative splice site site(s) utilized in vitro [0-100] [0-12] [0-1] [0-15]

≥70 ≥0 ≥0.4 ≥0

Exon 6 - c.226 71.44 4.65 — —

BRCA1 c.301+6T>C D Exon 6 - c.292 Exon 6 - c.292 74.93 5.63 0.65 —

Intron 6 - c.301+106 73.84 1.23 — —

Exon 7 - c.379 72.36 3.77 0.54 — D Exon 7 - c.379 (donor); BRCA1 c.441+2T>A Intron 7 - c.441+91 79.7 — — — Exon 8 - c.445 (acceptor) A Exon 8 - c.445** — 4.78 0.63 2.9

Intron 11 - c.4097-1 78.16 5.73 0.57 —

Exon 12 - c.4112 — 0.35 — —

Exon 12 - c.4114 — 0.22 — —

Intron 12 - c.4185+9 — 1.63 — —

BRCA1 c.4184_4185+2del D Intron 12 - c.4185+11 None - exon 12 skipping — 1.3 — —

Intron 12 - c.4185+29 — 0.29 — —

Intron 12 - c.4185+31 — 1.3 — —

Intron 12 - c.4185+57 — 3.71 — 0.49#

Intron 12 - c.4185+78 70.04 1.69 — —

BRCA1 c.4357+1G>A D Intron 13 - c.4357+71 None - exon 13 skipping 73.58 5.42 0.89 —

Intron 16 - c.4987-88 70.68 2.73 — —

Intron 16 - c.4987-39 — 0.42 — —

BRCA1 c.4987-2A>G A Intron 16 - c.4987-37 None - exon 17 skipping 72.04 — — —

Exon 17 - c.5048 73.68 — — —

Intron 17 - c.5074+1 75.96 4.36 — 3.95

BRCA1 c.5074G>C D Intron 17 - c.5074+55 Intron 17 - c.5074+153 78.13 — — — Intron 17 - c.5074+60 79.17 — — — Intron 17 - c.5074+153** — 0.56 — 1.62

Intron 3 - c.316+17 — 1.91 — —

BRCA2 c.316+5G>A D Intron 3 - c.316+51 None - exon 3 skipping 74.47 — — —

Intron 3 - c.316+100 71.17 — — —

Exon 21 - c.8682 — 2.69 — —

Intron 21 - c.8754+8 73.27 5.73 0.57 # — BRCA2 c.8754+3G>C D Intron 21 - c.8754+46 Intron 21 - c.8754+46 90 8.68 1 6.64

Intron 21 - c.8754+87 — 4.82 — —

* Predictions for cryptic sites consistent with the observed in vitro results are noted in bold. – No predicted site. D = Donor site A = Acceptor site

** Acceptor cryptic splice site location falls outside the region analyzed, input sequence increased on the basis on results from RT-PCR analysis to generate scores shown in Table.

# Score for alternative splice site was not generated for wildtype sequence input. Supplementary Table 4: Posterior Probability of Variant Pathogenicity as estimated using Multifactorial Likelihood Analysis

Likelihood ratios derived from analysis of a Myriad dataset** Likelihood ratios derived from analysis of ENIGMA datasets***

Classification Frequency Total # Instances based on LLR LLR based of LR based ENIGMA Posterior Multifactorial Observatio of co- Posterior Combined Variant Intron-Exon based on co- mutations on co- co- LR based on Odds for Probability Likelihood LLR based on Total LLR Total LR from ns of the occurrence Odds of a Interpretation (HGVS Location Prior on occurrence in relevant occurrence segregatio co- Causality of a Classification Gene Cosegregatio from Myriad Myriad variant in reported variant of Multifactorial nomenclat (missense A- Probability* report of with a gene, in with a n segregation (all LRs variant according to n data information information the by being Analysis, ure) GVGD score) Family deleterious reporting deleterious pedigrees data combined) being the IARC 5 combined ENIGMA deleterious Frequency Data History mutation laboratory mutation available deleterious Class system^ datasets sites and Splicing (p1) Results

Class 1 Not Class 1 Not Pathogenic / Pathogenic / BRCA1 c.301+6T>C Intron 6 0.0021221 0.0007456 0.0007450 Low Clinical Low Clinical 0.26 ND ND ND ND 1 1 0 0.058 1.061 1 0.002 6 2 7 Significance Significance Class 5 Intron 7 Pathogenic c.441+2T> within splice BRCA1 (observed A consensus 1.0475142 25.140342 0.9617449 Class 4 Likely splicing site 0.96 ND ND ND ND 1 1 0 0.045 1.048 0 1 9 86 5 Pathogenic aberration) c.548- 1.0338607 0.3632483 0.2664579 Class 3 Class 3 BRCA1 Intron 8 8delT 0.26 ND ND ND ND 1 1 0 0.033 1.034 0 1 0 5 4 Uncertain Uncertain c.4097- 3.6034491 1.2660767 0.5587086 Class 3 Class 3 BRCA1 Intron 11 15T>C 0.26 ND ND ND ND 1 1 0 0.045 1.048 1 3.44 4 3 8 Uncertain Uncertain Exon12- Intron 12 Class 5 c.4184_418 spanning Pathogenic BRCA1 5+2del splice (observed consensus 2.1893048 52.543316 0.9813235 Class 4 Likely splicing site 0.96 ND ND ND ND 1 1 0 0.045 1.048 1 2.09 6 57 3 Pathogenic aberration) Class 5 Intron 13 Pathogenic c.4357+1G within splice (multifactorial, BRCA1 >A consensus observed site 4786300.9 5013718.5 120329246 0.9999999 Class 5 splicing 0.96 6.09 0.6 ND 6.68 23 1 0 0.045 1.048 0 1 9 .23 9 Pathogenic aberration) c.4358- 1.0338607 0.3632483 0.2664579 Class 3 Class 3 BRCA1 Intron 13 4delA 0.26 ND ND ND ND 1 1 0 0.033 1.034 0 1 0 5 4 Uncertain Uncertain Class 5 Intron 16 Pathogenic c.4987- within splice BRCA1 (observed 2A>G consensus 1.0475142 25.140342 0.9617449 Class 4 Likely splicing site 0.96 ND ND ND ND 1 1 0 0.045 1.048 0 1 9 86 5 Pathogenic aberration) Class 5 c.5074G>C Pathogenic Exon 17 BRCA1 p.Asp1692 (observed (C65) His 1.0641430 1.1291401 4.8137028 0.8279925 Class 3 splicing 0.81 -0.045 0.073 ND 0.027 18 1 0 0.058 1.061 0 1 8 8 9 Uncertain aberration) c.5075- 1.0627528 0.3733996 0.2718798 Class 3 Class 3 BRCA1 Intron 17 107A>G 0.26 ND ND ND ND 1 2 0× 0.030 1.063 0 1 8 6 3 Uncertain Uncertain c.5333A>G Class 2 Likely BRCA1 p.Asp1778 Exon 22 (C0) 0.3090295 0.030; 0.3337152 0.0033708 0.0033595 Not Class 2 Likely Gly 0.01 -0.64 0.13 ND -0.51 43 2 0 0.045 1.080 0 1 9 6 4 Pathogenic Not Pathogenic Exon 2, c.3G>A BRCA2 disrupts start p.Met1Ile 2.1445923 51.470217 0.9809415 Class 4 Likely Class 4 Likely codon 0.96 ND ND ND ND 1 1 0 0.068 1.072 1 2.00 9 39 7 Pathogenic Pathogenic c.316+5G> 1.7378008 5.6436016 1.9828870 0.6647543 Class 3 Class 4 Likely BRCA2 Intron 3 A 0.26 0.18 0.06 ND 0.24 29 1 0 0.028 1.028 1 3.16 7 7 2 Uncertain Pathogenic 1, in cis (BRCA2 c.425+33A BRCA2 Intron 4 c.9018C>A Class 2 Likely >G p.Tyr3006X 0.041; 0.0155544 0.0054650 0.0054353 Not Class 2 Likely 0.26 ND ND ND ND 1 4 ) 0.026 1.140 2 0.01 8 9 8 Pathogenic Not Pathogenic c.426- 1.0238076 0.3597162 0.2645524 Class 3 Class 3 BRCA2 Intron 4 22G>T 0.26 ND ND ND ND 1 1 0 0.024 1.024 0 1 5 0 1 Uncertain Uncertain c.516+18T> 1.0238076 0.3597162 0.2645524 Class 3 Class 3 BRCA2 Intron 6 C 0.26 ND ND ND ND 1 1 0 0.024 1.024 0 1 5 0 1 Uncertain Uncertain c.632- 0.2511886 0.2693486 0.0946360 0.0864543 Class 3 Class 3 BRCA2 Intron 7 16A>C 0.26 -0.65 0.05 ND -0.6 43 1 0 0.068 1.072 0 1 3 0 1 Uncertain Uncertain

c.992A>T Class 2 Likely BRCA2 Exon 10 (C0) p.Lys331Ile 0.8222426 0.8561451 0.0086479 0.0085737 Not Class 2 Likely *Prior Probability based on location of variant relative to splice junction (intronic variants, Easton et al 2007) or physicochemical characteristics of encoded missense substitution (exonic variants, Tavtigian et al 2008).

** Log likelihood ratio (LLR) estimates derived from analysis of 70,000 BRCA1 and BRCA2 tests as reported in Easton et al 2007.

***Mutation detection rate for relevant laboratory reporting the variant; co-occurrence likelihood ratios were calculated separately where two laboratories detected the same variant.

^Classifications as described in Plon et al (Plon, et al., 2008)

× co-occurrence with mutation in BRCA2