Table 1.
POLE and POLD1 exonuclease domain germline variants identified in the study (population MAF <1%).
| Genetic variant (motif/DNA binding cleft) (Suppl. Fig. S1 and Fig. 2)a | dbSNP rs # population MAFNFE (%)b | Evolutionary conservation (PhyloP/PhastCons)c In silico prediction (REVEL)d |
Proband’s phenotype and cosegregation data (Suppl. Figs. S2 and S3) | Mutator phenotype in yeast (Fig. 2)e | Tumor sequencing analysis (mutational burden and signature contribution) (Fig. 3 and Suppl. Tables S6 and S7) | ACMG/AMP variant classificationf42 (InterVar/gInterVar-adjusted) |
|---|---|---|---|---|---|---|
| POLE | ||||||
| c.861T>A; p.Asp287Glu (Flanking Exo I/Yes) | rs139075637 (0.1710)h |
Moderately conserved (1.189/1) Benign (0.286) |
Fam A: BC 40 Noncarrier: Mother (OC 53) Fam B: BC 59; CRC 59 Cancer-affected carriers vs. total cancer-affected individuals:i 7/13 (2/6 in one same family) |
Yes (+++) |
Fam A–II.3 (BC): 185.7 Mut/Mb; 16.3% MMR-d sig |
Likely benign (PM1, PM2, BS2 and BP4)/Likely benign (PM1, BP4, PS3_moderate, BS1, BS4_supporting) |
| c.881T>G; p.Met294Arg (Flanking Exo I/Yes) | n.a. |
Conserved (9.279/1) Damaging (0.816) |
Fam C: OC 36; EC 36 Carriers: mother (CRC 42; EC 55), uncle (CRC 67) Fam D: CRC 37 Carriers: uncle (CRC 57, 59, 69) Total meiosis: 3 across 1 family |
Yes (++) |
Fam C–II.2 (OC): 249 Mut/Mb; 15.3% MMR-d + POLE sig (SBS14) Fam C–II.2 (EC): 300 Mut/Mb; 95.4% MMR-d sigs Fam C–I.4 (CRC): 11 Mut/Mb; 23.1% POLE sig (SBS10) |
Uncertain significance (PM1, PM2)/Pathogenic (PM1, PM2, PP1, PP3, PS3_supporting, PP4_strong) |
| c.919A>G; p.Ile307Val (outside Exo/No) | n.a. |
Conserved (7.979/1) Benign (0.337) |
Fam E: Melanoma (×4) 81 Carriers: sister (BC 83), sister (melanoma 55) |
Yes |
Fam E–II.1 (Melanoma): 32 Mut/Mb; 21.6% MMR-d sigs |
Uncertain significance (PM1, PM2)/(PM2) |
| c.1007A>G; p.Asn336Ser (outside Exo/No) |
rs5744760 (0.0197)j |
Conserved (7.979/1) Damaging (0.425) |
Fam F: CRC 38 Fam G: Melanoma 48 |
No effect40 | n.a. | Benign (PM1, BS1, BS2 and BP6)/Likely benign (BS1, BS3_supporting, PP3) |
| c.1138G>T; p.Gly380Cys (outside Exo/No) | rs199746481 (0.0051) |
Conserved (7.867/0.878) Damaging (0.531) |
Fam H: BC (×2) 61 Fam I: 10–20 Polyps 72 |
Yes (++) | Fam H–II.4 (BC): 0.7 Mut/Mb |
Uncertain significance (PM1, PM2, PP3)/(PP3, PS3_supporting) |
| c.1274A>G; p.Lys425Arg (Exo IV/Yes) | rs757186755 (0.0025) |
Conserved (7.979/1) Damaging (0.457) |
Fam J: BC 46; OC 48, EC 48 Total reported carriers:k 8 |
n.p. | n.a. | Uncertain significance (PM1 and PM2)/(PM1, PP3) |
| c.1277C>T; p.Ala426Val (Exo IV/Yes) | rs374920539 (0.0034) |
Conserved (9.953/1) Damaging (0.392) |
Fam K: Renal cancer 49; CRC 44; adenomas. Carriers: paternal uncle (CRC 73), father (CRC 60) Total meiosis: 3 across 1 family |
Yes (++) |
Fam K–II.1 (CRC): 41 Mut/Mb; 16.3% MMR-d sigs |
Uncertain significance (PM1 and PM2)/(PM1, PP3, PS3_supporting, PP1) |
| POLD1 | ||||||
| c.921T>G; p.Ile307Met (outside Exo/No) | n.a. |
Nonconserved (−2.516/0.00) Benign (0.268) |
Fam L: OC 40 | Not conserved in S. pombe | n.a. |
Uncertain significance (PM1, PM2 and BP4)/(PM2 and BP4) |
| c.973A>G; p.Ile325Val (Exo I/Yes) | rs558345043 (0.0017) |
Moderately conserved (2.440/0.984) Benign (0.041) |
Fam M: BC 33 | Not conserved in S. pombe | n.a. |
Uncertain significance (PM1, PM2, and BS2)/(PM1, BP4) |
| c.1054C>T; p.Arg352Cys (outside Exo/No) | rs762330164 (0.0032) |
Conserved (4.348/1) Benign (0.299) |
Fam N: 5 adenomatous polyps |
n.p. | TCGA (CRC): 35 Mut/Mb, 71% MMR-d sigs. COSMIC (Lymph): 24.7 Mut/Mb; non-POLE/D1, non-MMR-d sigs |
Uncertain significance (PM1 and PM2)/Likely benign (BP4, BP5) |
| c.1562G>A; p.Arg521Gln (outside Exo/Yes) | rs143076166 (0.0238)l |
Conserved (7.044/1) Benign (0.278) |
Fam O: CRC (×2) 34 | n.p. | Fam-6 (CRC)7,43: 39 Mut/Mb; 14.5% MMR-d sigs (14.51%) |
Uncertain significance (PM1 and PM2)/Likely benign (PM1, BS2_supporting and BP4) |
| c.1573C>T; p.Arg525Trp (outside Exo/Yes) | rs201804732 (0.0010) |
Moderately conserved (3.030/1) Benign (0.267) |
Fam P: BC 35, 49, 53; CRC 69 |
Not conserved in S. pombe | TCGA (GC): 13.7 Mut/Mb; non-POLE/D1, non-MMR-d sigs (other DNA repair defects) | Uncertain significance (PM1,PM2 and BS2)/(PM1, PM2, BP4) |
ACMG/AMP American College of Medical Genetics and Genomics/Association for Molecular Pathology, CRC colorectal cancer, EC endometrial cancer, GC gastric cancer, gnomAD Genome Aggregation Database, Lymph lymphoid neoplasm, MAF minor allele frequency, MMR-d mismatch repair deficiency, n.a. not available; NFE non-Finnish Europeans, n.p. not performed, OC ovarian cancer, SBS single base signature, sig(s) signature(s).
aRefSeq GRCh37: POLE (NM_006231.2; NP_006222.2) and POLD1 (NM_001256849.1; NP_001243778.1).
bPopulation MAF obtained from gnomAD non-Finnish, noncancer Europeans (gnomAD v.2.1.1).
cPhyloP/PhastCons values were obtained from alignments of 100 vertebrate sequences. The higher the score, the more conserved the site is (PhyloP range score: −20 to +10; PhastCons: 0 to 1).
dIn silico prediction according to REVEL ≥0.35.
eYeast variant rate increase (+, p = 0.01; ++, p = 0.01–0.001; +++, p < 0.001 indicate the differences with the wildtype control, calculated using the Mann–Whitney nonparametric test).
fAccording to the standard guidelines for the clinical interpretation of genetic variants (ACMG/AMP recommendations) using the automatic InterVar software.
gACMG/AMP–based manual curation (new evidence considered is underlined). Details are shown in Supplementary Methods and Supplementary Table S3: PM2, absent or <1/100,000 in general population (gnomAD noncancer data set); PP1, cosegregation with disease in multiple affected family members (at least three meiosis across one family); PP3, in silico prediction indicates that the variant could be damaging (REVEL ≥ 0.35); PP4, the patient’s phenotype is highly specific for a disease with a single genetic etiology (moderate: when hyper/ultramutation and POLE/D1-associated mutational signatures 14 or 20 were detected in the tumor; strong: when hyper/ultramutation and the POLE/D1-associated mutational signature 10 were detected in the tumor); PS3, functional studies are supportive of a damaging effect on the gene (supporting: when a mutator phenotype in S. pombe was observed with p = 0.01–0.001; moderate: when a mutator phenotype in S. pombe was observed with p < 0.001). BS1, allele frequency is greater than expected for disorder (considering MAF > 0.1% in any gnomAD v2.1.1 noncancer subpopulation); BS2_supporting, observed in ≥10 healthy adult individuals (above 60 years of age) for a dominant (heterozygous) disorder (not applied when BS1 was considered); BS3, functional studies showed no damaging effect on protein function (supporting: when a mutator phenotype in S. pombe was not observed); BS4_supporting, lack of segregation in more than three cancer-affected members (same or related phenotype) of a family; BP4, in silico prediction of pathogenicity estimated by REVEL suggests no impact (REVEL < 0.30). BP5, variant found in a case with an alternate molecular basis for disease (for tumors with somatic POLE/D1 variants: when an MMR-proficient tumor was not hyper/ultramutated or when an DNA repair–proficient tumor was hypermutated but mutational signatures 10, 14, or 20 were not identified. For tumors from carrier families: same criteria as defined in the previous sentence for tumors with somatic variants but applied to two different tumors to avoid a false classification due to the presence of a phenocopy).
hMAF in European non-Finnish, noncancer population (gnomAD): 202/118,154 individuals (60 above 60 years of age).
iCharacteristics of POLE c.861T>A (p.Asp287Glu) carrier families are shown in Supplementary Table S4.
jMAF in African, noncancer population (gnomAD): 623/23,530 individuals (2.6%); 10 homozygous carriers.
kPOLE c.1274A>G (p.Lys425Arg) carriers from our study and previously reported.3,10,35,36
lMAF in European non-Finnish, noncancer population (gnomAD): 28/117,648 individuals (10 above 60 years of age).
Families carrying the corresponding POLE/POLD1 variant, tumors studied for the analysis of mutational burden and signatures, and ACMG/AMP variant classification results, are highlighted in bold. Cosegregation data (carriers, non-carriers, meiosis) are shown in italics.