Table 1. Sample re-analysis pilot study.
| Sample | Cancer history (proband and 1st and 2nd degree relatives) | Clinical criteria | Previous analysis | I2HCP analysis | Coding non-synonymous infrequent detected variants | Functional prediction |
|---|---|---|---|---|---|---|
| Sample_R1 | Proband: endometrial c (59y). Mother: breast c (41y), colon c (42y). Maternal aunt: endometrial c (45y). Maternal uncle: Gastric (57) | HNPCC, Amsterdam Criteria | Conserved MLH1/MSH2 and non-evaluable MSH6 expression in proband’s tumor. MSH6 genetic analysis negative | 1) Lynch genes set | MSH2:NM_000251:exon3:c.G437T:p.G146V | Neutral |
| MSH6:NM_000179:exon9:c.3952_3960del:p.1318_1320del | Deleterious | |||||
| 2) Whole I2HCP | EPCAM:NM_002354:exon3:c.G267C:p.Q89H | Deleterious | ||||
| APC:NM_001127510:exon11:c.G1139A:p.R380Q | Deleterious | |||||
| PMS2:NM_000535:exon11:c.C1169T:p.A390V | Neutral | |||||
| FANCC:NM_000136:exon2:c.C77T:p.S26F | Deleterious | |||||
| SDHD:NM_003002:exon1:c.G34A:p.G12S | Neutral (ClinVar:PAT) | |||||
| FANCM:NM_020937:exon20:c.C4799T:p.T1600I | Deleterious | |||||
| BRCA1:NM_007294:exon10:c.G3119A:p.S1040N | Neutral | |||||
| BBRCA1:NM_007294:exon10:c.C2521T:p.R841W | Deleterious | |||||
| MN1:NM_002430:exon1:c.G56A:p.G19D | Deleterious | |||||
| Sample_R2 | Proband: Malignant fibrous histiocytoma (an undifferentiated pleomorphic sarcoma), a glioblastoma multiforme and 3 neurofibromas | NF1 | None | 1) NF1 genes set | No candidate variant detected | |
| 2) Whole I2HCP | Whole CDKN2A gene deletion. Validated by MLPA | Deleterious OMIM: 155755 | ||||
| ATM:NM_000051:exon50:c.C7375G:p.R2459G | Neutral | |||||
| Sample_R3 (V11) | Proband: breast c (46y), skin c (49y), uterine c (60y), skin c (60y), thyroidectomy due to multinodular hyperplasia (45y). Father: gastric c (55y). Sister: ovarian c (Brenner t., 39y). Sister’s son: breast c (34y, BRCA2 VUS c.5729A>T carrier). | HBOC | BRCA1/2 genetic analysis negative | 1) HBOC genes set | No candidate variant detected | |
| 2) Whole I2HCP | FANCL:NM_001114636:exon14:c.1111_1114dupATTA:p.Thr372Asnfs* | Deleterious | ||||
| BARD1:NM_000465:exon11:c.C2191T:p.R731C | Deleterious | |||||
| CHEK2:NM_007194:exon14:c.C1525T:p.P509S | Neutral | |||||
| PMS2:NM_000535:c.1866G>A:p.M622I | Insight: Class 1 | |||||
| Sample_R4 (V30) | Proband: breast c (46y). Mother: breast c (77y).Maternal aunt: ovarian c (58y). Maternal uncle: CCR (67y). Paternal aunt-1: ovarian c (56y). Paternal aunt-2: ovarian c ? Paternal uncle: CCR ? | HBOC | BRCA1/2 genetic analysis negative | 1) HBOC genes set | PALB2:NM_024675:exon9:c.G2993A:p.G998E | Deleterious |
| PALB2:NM_024675:exon5:c.G2014C:p.E672Q | Neutral | |||||
| 2) Whole I2HCP | SUFU:NM_016169:exon7:c.G856A:p.E286K | Neutral | ||||
| Sample_R5 | Proband: 2 breast c (62, 69y), endometrial c (77y). Son: prostate c (64y). Mother: breast c (75y). Father: colon c (52y). Sister: colon c (49y). Brother: prostate c (74y). | HNPCC, Amsterdam Criteria | MSH2 and MSH6 LoE in proband’s endometrial tumor. MSH2 & MLH1 genetic analysis negative | 1) Lynch genes set | No candidate variant detected | |
| 2) Whole I2HCP | PPM1D:NM_003620:exon6:c.1736_1737insA:p.T579fs | Deleterious | ||||
| XPC:NM_004628:exon2:c.C142T:p.L48F | Neutral | |||||
| WRN:NM_000553:exon9:c.G1149T:p.L383F | Neutral | |||||
| WRN:NM_000553:exon25:c.G2983A:p.A995T | Neutral | |||||
| MLH3:NM_014381:exon2:c.G1870C:p.E624Q | Deleterious | |||||
| PALB2:NM_024675:exon5:c.C2135T:p.A712V | Neutral | |||||
| Sample_R6 | Proband: colon c (43y). Mother: colon c (73y). Maternal uncle-1: colon c (22y). Maternal uncle-2: colon c (41y). Maternal aunt: colon c (73y) | HNPCC, Amsterdam Criteria | Microsatellite stability in proband’s tumor. MSH2 & MLH1 genetic analysis negative | 1) 3 Lynch genes set | No candidate variant detected | |
| 2) Whole I2HCP | BMPR1A:NM_004329:exon4:c.A223G:p.T75A | Deleterious | ||||
| FH:NM_000143:exon7:c.C926T:p.P309L | Deleterious | |||||
| RAD50:NM_005732:exon16:c.C2548T:p.R850C | Deleterious | |||||
| ATM:NM_000051:exon37:c.A5558T:p.D1853V | Deleterious | |||||
| SLX4:NM_032444:exon15:c.A5501G:p.N1834S | Neutral | |||||
| Sample_R7 (V21) | Proband: colon c (39y). Mother: gastric c (61y). Maternal grandfather: skin c (60y). | HNPCC, Bethesda criteria | Microsatellite instability and MLH1 LoE in proband’s tumor. MLH1 genetic analysis negative | 1) Lynch genes set | APC:NM_000038:exon16:c.T3920A:p.I1307K | Neutral |
| 2) Whole I2HCP | ERCC6:NM_000124:exon10:c.C1996T:p.R666C | Deleterious | ||||
| ERCC6:NM_000124:exon18:c.A3122C:p.Q1041P | Neutral | |||||
| DDB2:NM_000107:exon9:c.G1228A:p.A410T | Neutral | |||||
| ATM:NM_000051:exon41:c.A6084T:p.Q2028H | Neutral | |||||
| FANCM:NM_020937:exon21:c.A5627G:p.N1876S | Neutral | |||||
| EXO1:NM_003686:exon10:c.G1378C:p.V460L | Neutral | |||||
| Sample_R8 | Proband: Biliary tract c (43y). Father: colon c (52y). Brother: colon c (42y). Sister: breast c (55y) | HNPCC, Amsterdam Criteria | MSI-H and MLH1 LoE in proband’s tumor. MLH1 genetic analysis negative | 1) Lynch genes set | No candidate variant detected | |
| 2) Whole I2HCP | ERCC4:NM_005236:c.2624A>G:p.E875G | Deleterious | ||||
| Sample_R9 | Proband: colon c (59y), lung c (67y). Father: colon c (68y). Sister: colon c (45y). Sister’s son: leukaemia (25y). Brother: colon c 63(y) | HNPCC, Amsterdam Criteria | MLH1 LoE in proband’s tumor. Absence of MLH1 somatic methylation. MLH1 genetic analysis negative | 1) Lynch genes set | PMS2:NM_000535:c.1866G>A:p.M622I | Insight: Class 1 |
| 2) Whole I2HCP | PRSS1:NM_002769:exon5:c.G617C:p.C206S | Deleterious | ||||
| PALB2:NM_024675:exon9:c.G2993A:p.G998E | Deleterious | |||||
| Sample_R10 | Proband: colon c (31y). Father: 3 colon c (67, 67 and 68y). Paternal aunt: endometrial c (50y) | HNPCC, Amsterdam Criteria | MLH1 LoE in proband’s tumor. Absence of MLH1 somatic methylation. MSH2 & MLH1 genetic analysis negative | 1) Lynch genes set | No candidate variant detected | |
| 2) Whole I2HCP | EXO1:NM_130398:c.1918C>G:p.P640A | Neutral | ||||
| PTPN11:NM_002834:exon14:c.C1658T:p.T553M | Neutral | |||||
| FANCA:NM_000135:exon27:c.C2574G:p.S858R | Neutral | |||||
| FLCN:NM_144997:exon9:c.G979A:p.A327T | Neutral | |||||
| MN1:NM_002430:exon1:c.G3550C:p.E1184Q | Deleterious | |||||
| Sample_R11 | Proband: colon c (58y). Mother: colon c (70y). Brother: colon c (47y) | HNPCC, Amsterdam Criteria | MSH6 LoE in proband’s tumor. MSH2 & MSH6 genetic analysis negative | 1) Lynch genes set | No candidate variant detected | |
| 2) Whole I2HCP | BARD1:NM_000465:exon7:c.G1670C:p.C557S | Neutral | ||||
| MRE11A:NM_005590:exon13:c.C1475A:p.A492D | Deleterious | |||||
| SLX4:NM_032444:exon15:c.A5501G:p.N1834S | Neutral | |||||
| BRCA1:NM_007294:exon10:c.G3119A:p.S1040N | Deleterious | |||||
| Sample_R12 | Proband: ovary c (35y). Mother: endometrial c (79y). Maternal uncle 1: colon c (63y). Maternal uncle 2: prostate c (79y) | HNPCC, Amsterdam Criteria | MSH2/MSH6 LoE in proband’s tumor. MLH1, MSH2 & MSH6 genetic analysis negative | 1) Lynch genes set | No candidate variant detected | |
| 2) Whole I2HCP | EXO1:NM_130398:c.2276G>A:p.G759E | Neutral | ||||
| HNF1A:NM_000545:exon4:c.C827G:p.A276G | Deleterious | |||||
| BRCA2:NM_000059:exon11:c.G4258T:p.D1420Y | Neutral | |||||
| PALB2:NM_024675:exon7:c.C2590T:p.P864S | Neutral | |||||
| PALB2:NM_024675:exon4:c.G232A:p.V78I | Neutral | |||||
| PRKAR1A:NM_002734:exon3:c.G221A:p.R74H | Deleterious | |||||
| Sample_R13 | Proband: colon c (37y). Father: colon c (57y). Grandfather: colon c (60y) | HNPCC, Amsterdam Criteria | MSH6 LoE in proband’s tumor. MLH1, MSH2 & MSH6 genetic analysis negative | 1) Lynch genes set | POLE:NM_006231:exon42:c.A5797G:p.I1933V | Neutral |
| 2) Whole I2HCP | FANCD2:NM_001018115:exon28:c.G2702T:p.G901V | Neutral | ||||
| ATM:NM_000051:exon22:c.C3161G:p.P1054R | Deleterious | |||||
| PALB2:NM_024675:exon7:c.C2590T:p.P864S | Neutral | |||||
| Sample_R14 (V39) | Proband: Multiples schwannomas | Schwannomatosis | NF2 and SMARCB1 genetic analysis negative | 1) NF2 genes set | No candidate variant detected | |
| 2) Whole I2HCP | BARD1:NM_000465:exon11:c.A2146G:p.T716A | Neutral | ||||
| ERCC6:NM_000124:exon16:c.G2924A:p.R975Q | Deleterious | |||||
| SUFU:NM_016169:exon8:c.G1018T:p.A340S | Neutral | |||||
| ATM:NM_000051:exon17:c.T2572C:p.F858L | Neutral | |||||
| ATM:NM_000051:exon22:c.C3161G:p.P1054R | Deleterious | |||||
| FANCA:NM_000135:exon42:c.C4249G:p.H1417D | Deleterious | |||||
| FANCA:NM_000135:exon27:c.T2507A:p.F836Y | Neutral |
Results of the re-analysis of 14 samples, including 4 samples from the validation set (V11, V30, V21 and V39). The I2HCP analysis encompasses the rare exonic non-synonymous variants detected in: 1) the genes included in the predefined gene group on the basis of clinical manifestations (Supplementary Table S1) and 2) the variants found in all genes of the panel. 1000G frequencies <0.01% have been used to filter rare variants. Variants highlighted in bold could be responsible for the developed phenotype.
Further analyses are needed to evaluate the impact of these variants. Functional prediction is based on data from Polyphen2 HDIV and HVAR, SIFT, PROVEAN and MutationTaster and a variant is marked as deleterious if three or more predictors classified it as damaging.