Supplementary Information
Case / Sex / Primary carcinoma / Age, y / Criteria / Tumour features / Relevant family history / Additional comments / Ref1 / F / Colon / 25 / BG / MSI, MLH1-, LOH / None / Extensive monoallelic methylation / 1
H166 / F / Colon (Asc)
Endometrium / 38
44 / BG / MSI, MLH1-, LOH
MSI, MLH1-, ROH / None / Japanese heritage. Extensive methylation / 2
H403 / M / Colon (Trans) / 28 / BG / MSI / No FDRs / Japanese heritage. Extensive methylation / 2
H450 / F / Colon (Asc) / 23 / BG / NA / No FDRs / Japanese heritage. Extensive methylation / 2
H628 / M / Colon (Desc)
Colon (Asc) / 17
29 / BG / NA
MSI, MLH1- / No FDRs / Japanese heritage. Extensive methylation / 2
VT / F / Caecum
Endometrium
Melanoma
Breast (infiltrating duct) / 46
53
57
63 / BG / MSI, MLH1-, LOH, BRAFV600E
MSI, MLH1-, ROH
NA
MSI, MLH1-, LOH / Mother, colon 64y / Caucasian heritage. Monoallelic methylation with some mosaicism / 3
TT / M / Caecum
Colon (Desc)
Duodenum (3x sync)
Ampulla of Vater / 43
44
51
59 / BG / MSI, MLH1-, LOH
ROH
MSI, MLH1-, LOH
MSI, MLH1-, LOH
All were BRAF WT / Mother, Endometrium 55y / Mediterranean heritage. Monoallelic methylation with some mosaicism. No methylation in spermatozoa. / 3; 4
ST / M / Colon / 39 / BG / MSI, MLH1-, LOH, BRAF WT, KRAS+ / None / Caucasian heritage. Extensive methylation arising de novo on maternal allele. Complete allelic inactivation. / 5
A / F / Endometrium
Colon (Asc)
Rectum,
Skin sarcoma / 45
59
60
68 / BG / MSI, MLH1-
MSI, MLH1-, L724W, KRAS+
MSI, MLH1-, S108R
MSI, MLH1-
All BRAF WT / No FH. Non-Mendelian transmission of epimutation to one of three sons (unaffected at age 48y) who had inherited the affected maternal allele. / Caucasian heritage. Extensive monoallelic methylation in proband and methylation of maternal allele in son. Complete allelic inactivation in both. No methylation, plus allelic reactivation in spermatozoa of son. / 6; 7
B / F / Colon
Rectum / 41
45 / BG / MSI, MLH1-, BRAF WT
MSI, MLH1-, BRAF WT / None / Caucasian heritage. De novo methylation of maternal allele. Complete allelic inactivation. / 6
2 / M / Epidermoid (lip)
Colon / 34
35 / BG / NA
MSI, MLH1-, LOH / None / Spanish heritage. De novo epimutation. / 8
1 / M / Colon (Asc)
Colon (Sig) & rectum / 33
47, 47 / BG / MSI, MLH1-, BRAF WT / Brother, gastric 51y / Dutch heritage. Extensive methylation / 9
2 / F / Colon (Sig)
Colon (Trans) / 58
59 / BG / MSI, MLH1-, BRAF WT
MSI, MLH1-, BRAF WT / Son, hyperplastic polyp 34y / Dutch heritage. Extensive methylation / 9
3 / M / Rectum / 41 / BG / MSI, MLH1-, BRAF WT / Mother, colon 59y; Brother, polyps 44y / Dutch heritage. Some mosaicism / 9
4 / M / Colon (Trans) / 39 / BG / MSI, MLH1-, BRAF WT / Carrier Mother, CRC at 65y.
Carrier Sister, unaffected at 44y. / Dutch heritage. Dominant inheritance of mosaic epimutation from mother. Linked to a 280-375 kb duplication encompassing MLH1 and neighbouring genes. / 9; 10
5 / F / Rectum
Colon (Sig) / 40
41 / BG / MSI, MLH1- / Mother, cervical 33y, colon 64y (MSS, MLH1+) / Dutch heritage. Extensive monoallelic methylation arising de novo on maternal allele / 9
6 / M / Colon (Asc)
Rectum / 40
44 / BG / MSI, MLH1- / Father, polyps 50y / Dutch heritage. Extensive monoallelic methylation. / 9
7 / M / Colon (Trans) / 33 / BG / MSI, MLH1- / None / Dutch heritage. Extensive monoallelic methylation. / 9
8 / M / Colon (Asc)
Skin
Cyst, sebaceous gland / 35
42, 48
49 / BG / MSI, MLH1- / No FDR / Dutch heritage. Mosaic. Partial allelic silencing demonstrated. / 9
9 / F / Colon (Asc) / 37 / BG / MSI, MLH1- / Father, renal 41y / Dutch heritage. De novo on maternal allele, some mosaicism / 9
10 / F / Colon (left flexure) / 30 / BG / MSI, MLH1- / None / Dutch heritage. Extensive monoallelic methylation. / 9
11 / M / Colon (Asc & Trans) / 46, 46 / BG / MSI, MLH1- / None / Dutch heritage. Some mosaicism / 9
12 / F / Colon (Asc) / 35 / BG / MSI, MLH1-, BRAF WT / None / Dutch heritage. Some mosaicism / 9
(H10) / M / Colorectal / 41 / BG / MSI, MLH1- / None / Chinese heritage. Unconfirmed extensive methylation by MSP / 11
(H29) / F / Colorectal / 37 / Am I / MSI, MLH1- / Positive FH, unspecified / Chinese heritage. Unconfirmed extensive methylation by MSP / 11
(H32) / M / Colorectal / 51 / Am I / MSI, MLH1+ / Positive FH, unspecified / Chinese heritage. Unconfirmed partial methylation by MSP / 11
(H42) / M / Colorectal / 65 / Am I / MSI, NA / Positive FH, unspecified / Chinese heritage. Unconfirmed partial methylation by MSP / 11
(H46)† / M / Colorectal / 48 / Am I / MSI, MLH1+ / Positive FH, unspecified / Chinese heritage. Unconfirmed partial methylation by MSP / 11
N2 / F / Colon
Endometrium / 35
45 / BG / MSI, MLH1-, ROH / Finnish heritage. Extensive hemiallelic methylation. Complete allelic inactivation. / 12
F36 / F / Colon
Endometrium / 22, 45
40 / BG / MSI, MLH1- / Father, multiple CRC at 41y, 49y, 69y and skin cancer at 56y. / Finnish heritage. Linked to a 6.4kb deletion from 68bp upstream of ATG start site to intron 2. Complete allelic inactivation. / 12
4 / M / Colorectal / 37 / BG / MSI, MLH1- / None / Dutch heritage. De novo. / 13
5 / F / Colorectal (x3 sync)
Endometrium
Colorectal
Colorectal
Ureter / 48
52
58
60
62 / BG / All MSI. N/A for IHC. / Positive, unspecified. / Dutch heritage. / 13
ID60 / F / Colorectal
Endometrium / 33
52 / BG / MSI, MLH1 methylated / Sister, endometrium 37y / Dutch heritage. / 14
ID36 / F / Colorectal
Pancreas / 60
62 / None / MSI, MLH1 methylated, CIMP+, BRAFV600E. Pancreas, MSS. / None / Dutch heritage. / 14
YT / M / Colorectal / 18 / BG / MSI, MLH1-, LOH, BRAF WT / None / Sri Lankan heritage. Arose de novo on maternal allele. / 7
BF / F / Colorectal / 20 / BG / MSI, MLH1-, LOH, BRAF WT, KRAS+ / None / African American/ Eastern Indian / Caucasian heritage. Arose de novo on paternal allele. / 7
84-1 / F / Rectum / 35 / BG / N/A / None / Indian heritage. Mosaic (8% methylation) monoallelic methylation. / 15
291-1 / M / Colorectal / 39 / BG / MLH1- / None / African heritage. Dense monoallelic methylation. / 15
Family 16 / F / Colon / 41 / Am I / MSI, MLH1-, LOH / Brother, colon 46y, MSI, MLH1-.
Mother, endometrium 42y, rectum 58y, caecum 64y. / Caucasian heritage from Western Australia. Dominantly inherited epimutation transmitted across 3 generations, linked to c.-27C>A and c.85G>T variants. / 16
A / M / Colorectal / 35 / BG / MSI, PMS2- (MLH1 NI) / None / Dense hemillelic methylation.
Family B / F / Colorectal
Colorectal
Adenoma and HPs / 55,
56
59, 62 / Am I / MSI, (MLH1 NI), BRAF WT
MSS
MSI, MLH1-, BRAF WT / Carrier son, CRC 40y, MSI, MLH1-. Carrier daughter, adenoma (MSS, BRAF V600E), HP, 44y. Non-carrier daughter adenoma (MSS, MLH1+), 36y. / French heritage. Mosaic epimutation identified in mother and 2 of 5 children (both affected). Epimutation segregated with a particular genetic allele, but no causative genetic alteration identified to date. / 17
836 / Colon / 31 / MSI, MLH1-, LOH / “High-level methylation detected.” / 18
2286 / Colon / 41 / MSI, MLH1-, LOH / High-level methylation. Allelic loss of expression. / 18
(2138) / Colon / 39 / MSI / “High-level methylation detected.” / 18
(2255) / Colon / 47 / MSI / “High-level methylation detected.” / 18
(61) / Colon / 41 / N/A / “High-level methylation detected.” / 18
(1492) / Colon / 36 / N/A / “High-level methylation detected.” / 18
(2007) / Colon / 39 / N/A / “High-level methylation detected.” / 18
2235 / Colon / 37 / MSI, MLH1-, LOH / “Medium-level methylation detected.” / 18
1 / M / Colon (sig)
Colon (hep flex) / 32
34 / BG / Both MSI, MLH1-, BRAF WT / None
Daughter inherited affected paternal allele but had no epimutation due to erasure. / Dense de novo monoallelic soma-wide methylation, complete allelic expression loss. Absence of methylation in spermatozoa, indicating complete germline erasure. / 19
34 / F / Colon (sig)
Colon (hep flex)
Endometrium / 29
44
49 / BG / N/A, LOH
MLH1-, BRAF WT
MSI, MLH1-, BRAF WT / Mother, breast 77y. / 19
G46 / F / Gastric / 60 / None / N/A / None / Chinese heritage. Mosaic methylation 10%. / 20
CCFR-A / M / Colon (rectosigmoid) / 48 / BG / Either MSI/MLH1- unspecified / None / Filipino heritage. Monoallelic methylation 36%. / 21
CCFR-B / F / Colon
Colon (Trans) / 55
56 / BG / Either MSI/MLH1- unspecified / None / Caucasian Australian. Monoallelic methylation, 17%. / 21
CCFR-C / M / Colon (rectosigmoid) / 41 / BG / MSI, MLH1- / None / Japanese heritage. Monoallelic methylation, 45%. / 21
CCFR-D / M / Caecum / 43 / BG / MSI, MLH1- / None / Causacian. Mosaic, 11-15% methylation. / 21
CCFR-E / M / Kidney
Colon (Asc) / 42
47 / BG / Either MSI/MLH1- unspecified / None / Caucasian. Monoallelic methylation 46%. / 21
CCFR-H / F / Colon (Asc) / 43 / Am I / Either MSI/MLH1- unspecified / Extensive FH on maternal side, including mother CRC 43y, maternal uncle CRC 40y. / Caucasian Australian. Mosaic methylation 32% linked to the c.-27C>A and c.85G>T variants. Unrelated to WA Family 16. / 21
CCFR-N / M / Colon (Desc)
Caecum / 50
52, 54 / Am I / MSI, MLH1- / Carrier son CRC 22y. But CRC also presented in non-carrier relatives. / Caucasian. Monoallelic mosaic 4% methylation linked to c.27G>A variant transmitted to affected son. / 21
CCFR-P / F / Colon (Asc) / 69 / None / Either MSI/MLH1- unspecified / None / Caucasian. Monoallelic low-level 2% methylation. / 21
(CCFR-O) / F / Colon (Sig) / 45 / BG / Either MSI/MLH1- unspecified / None / Caucasian. Low-level methylation 2%. / 21
(CCFR-Q) / M / Colon (Asc)
Skin / 39
44 / BG / MSI, MLH1- / No FDR / Caucasian. Low-level methylation 3%. / 21
Supplementary Table 1. Reported index cases with a confirmed constitutional MLH1 epimutation.
Clinical, tumour and molecular features are summarized for cases with a constitutional MLH1 epimutation in the chronological order in which they were identified. Cases in parentheses are considered herein as probable carriers of an MLH1 epimutation, but are currently unconfirmed based on the information published. Green font is used to highlight one familial case where non-Mendelian intergenerational transmission of the epimutation was proven. Blue font highlights familial cases where the epimutation has been linked to a genetic defect on the epimutated allele and/or autosomal dominant inheritance of the epimutation has been demonstrated by linkage to or segregation with, a particular genetic allele between generations. † Personal communication with the authors. M, male; F, female; y, years; Asc, ascending; Desc, descending; Sig, sigmoid; Trans, transverse; Hep flex, hepatic flexure; Sync, synchronous; Am, Amsterdam criteria; BG, Bethesda guidelines; Mod, modified; Rev, Revised; C; criterion within specified clinical guidelines, MSI, microsatellite unstable; MSS, microsatellite stable; IHC, immunohistochemistry; LOH, loss of heterozygosity; ROH, retention of heterozygosity; WT, wild-type; KRAS+, positive for KRAS hot-spot mutation within codon 12 or 13; CIMP+, positive for CpG island methylator phenotype; HP, hyperplastic polyp; FH, family history; FDRs, first degree relatives; MSP, methylation-specific PCR; NA, not available for study. Analyses of tumours for MLH1 expression and the nature of the second hit were not assessed in some cases due to the lack of available specimen. The mean age of onset of the first colorectal cancer in the confirmed cases (excluding those in parentheses) is 39.2 ±11.1 years and for all cases listed is 40.1 ±10.7 years.
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