Supplementary Online Content
Hansford S, Kaurah P, Li-Chang H, et al. Hereditary diffuse gastric cancer syndrome: CDH1 mutations and beyond. JAMA Oncol. Published online February 12, 2015. doi:10.1001/jamaoncol.2014.168.
eMethods 1. CDH1 Gene Screening eMethods 2. Penetrance Analysis eMethods 3. Multiplexed Sequencing Analysis Using Illumina’s TruSeq Custom Amplicon Assay eMethods 4. Polymerase Chain Reaction Protocol and Primer Sequences eMethods 5. Secondary Analysis of Panel Data eMethods 6. Immunohistochemistry Protocol and Description of Antibodies Used eReferences. Supplemental Methods References eTable 1. CDH1 Mutations Described to Date eTable 2. Families in Penetrance Analysis eTable 3. Genes Selected for Custom Panel Sequencing Based on Association With Upper Gastrointestinal Syndromes eTable 4. Novel and Rare Missense Mutations Detected in HDGC Families Using Custom UGI Gene Panel and Predicted Impact From in Silico Methods eTable 5. Summary of Families With CDH1 Mutations in This Study eTable 6. Cumulative Risk % (95% CI) and Relative Risk of Gastric and Breast Cancer in CDH1 Germline Mutation Carriers eFigure 1. Description of Current Techniques Used in Discovering Pathogenic, Germline Susceptibility Variants eFigure 2. HDGC Mutational Profile eFigure 3. Photomicrographs of the Tumor of the Proband in Family 25 With the CTNNA1 Germline Mutation eFigure 4. Schematic View of Genes With Pathogenic and Likely Pathogenic Mutations Identified Using Panel-Based Sequencing eReferences This supplementary material has been provided by the authors to give readers additional information about their work.
© 2015 American Medical Association. All rights reserved.
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eMethods 1. CDH1 gene screening
Genomic DNA was extracted from peripheral blood, saliva or paraffin embedded sections using
the Gentra Puregene DNA purification Kit (Qiagen), Oragene DNA kit (DNA Genotek) or DNA
FFPE tissue kit (Qiagen), respectively, according to the manufacturers’ protocols. Screening for
mutations was performed by direct sequencing of all exons and multiplex ligation-dependant
probe amplification with previously described primers and reaction conditions (1,2). In short, 16
individual PCR reactions were performed on each sample for a full mutational screen of all 16
exons and splice junctions of the CDH1 gene. Amplified PCR products were bi-directionally
sequenced on the Applied Biosystems 3130xl Genetic Analyzer (Life Technologies) using the Big
Dye v3.1 Terminator Cycle Sequencing Kit (Life Technologies). Samples with no significant point
mutations or minor insertions/deletions were tested for copy number variations using multiplex
ligation dependent probe amplification (MRC-Holland).
eMethods 2. Penetrance analysis
We used the pedigree information to estimate the penetrance of CDH1 using the MENDEL
program (3). Ascertainment of families was on the basis of multiple affected members with an
index case that tested positive for a truncating CDH1 mutation. Thus the conditional likelihood of
the pedigree was maximized given the phenotype of the family and genotype of the index case at
ascertainment, where the phenotype of the pedigree is the phenotype of all individuals in the
family (vital status, current age/age at death, cancer status and age at diagnosis) and their
relationships:
L(Pedigree|Ascertained Pedigree) = L(pedigree) / L(Ascertained pedigree)
We parameterized the model in terms of log relative risk for GC and BC in mutation carriers
compared with population risks. This assumes that cancer incidences in mutation carriers is the
same for all families, whatever their ethnic origin. Non-carriers of the deleterious mutation in
each family were assumed to develop the disease according to the population incidence rates.
The GC relative risks (RR) were estimated separately for men and women and allowed to vary
with age using 2 age groups: 10–39 and 40-79. The RR of breast cancer was estimated for
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women only and assumed to be constant with age. Other models with either 3 or 4 age groups for
GC risk and 2 or 3 age groups for BC risk were also tested. The model selected was the most
parsimonious model that fits the data well. The CDH1 mutant allele was assumed to be rare in
the general population with a frequency of 0.001.
The cumulative risk or penetrance was calculated from the cumulative incidence λ(t): n (t) iktk exp(k ) k1
Where ik is the incidence in the kth age band of length tk and βk is log relative risk in the kth age
band.
Cumulative risk of penetrance and 95% confidence intervals were calculated from the cumulative
incidence A(t), where:
n Ʌ(t)= ∑iktkexp(βk) k=1
n n 2 2 1/2 var Ʌ(t)= ∑i kt kvar(βk)exp(2βk) + 2 ∑ikijtkTkTj[ var(βk) var (βj)] exp(βk) (βj)corr((βk, βj) k=1 k=1
th th Where ik = incidence in the k age-band of length Tk and βk = ln (RR) in k age band.
The cumulative risk F(t) is given by:
F(t) = 1 - exp(-Ʌ(t)) With 95% CI = 1- exp(-Ʌ(t)) ± 1.96 √var Ʌ(t)
eMethods 3. Multiplexed sequencing analysis using TruSeq Custom Amplicon assay.
Genomic DNA Quantification. DNA samples were quantified to ensure minimum input
requirements of 250ng were met for sequencing (Qubit dsDNA broad range kit). All samples
were of sufficient quality and quantity and were included in the downstream multiplexed
sequencing assay. Multiplexed sequencing analysis was performed using the TruSeq custom
amplicon (TSCA) assay (Illumina, San Diego, CA) on 115 germline DNA samples divided into 7
runs, to sequence simultaneously with additional samples of interest from concurrent projects.
An average of 42 samples were multiplexed per run. Custom-pooled oligonucleotides were
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hybridized to individual samples to generate template libraries and sample-specific indices were
then added to each template library by PCR. A bead-based normalization technique eliminated
the need for downstream quantification methods. Samples were then pooled prior to loading onto
the MiSeq Platform for simultaneous amplification, cluster generation and bi-directional
sequencing of 251 cycles per amplicon. Seven germline DNA samples with known pathogenic
CDH1 mutations were included in the assay to determine the reliability of the data analysis
software.
eMethods 4. Polymerase-chain reaction protocol and primer sequences.
PCR was performed with variant-specific primers designed using Integrated DNA Technologies
online software (CTNNA1 (N71*) F5’-TTGTAAGGTTTACTGGGTCTTCA-3’ and R5’-
GGTTAACTAAACCCATGCATCAA-3’; CTNNA1 (R129X) F5’-
TGAAAACTCTTAAACTAAATTTGTGC-3’ and R5’-AAAACATCTCTGGTCCATTGAGA-3’;
BRCA2 (N1287*) F5’-GTGGGTTTGCAATTTATAAAGCAG-3’ and R5’-
TAACTTACCAGAAGCTTGTTTCC-3’; SDHB (S163P) F5’-GCTGAGGTGATGATGGAATCT-3’
and R5’-ACCACACTCCTGGCAATCATCT-3’; STK11 (F354L) F5’-GAGGAGCTGGGTCGGAAA-
3’ and R5’-TGGCCGAGTCAGCAGAG-3’; PALB2 (V398*) F5’-
GAAAGTGAGATTCTAAGTCAACCTAAG-3’ and R5’-TTCTTGACATCCAAATGACTCTG-3’;
MSR1 (R293X) F5’-AGTACCTTGACAGATGACTAACC-3’ and R5’-
CCCTACACATGTACCTGGATG-3’; ATM E1267* F5’-
TGTAAAACGACGGCCAGTGCTACTGAACAAGGTCCCATTT-3’ and R5’-
CAGGAAACAGCTATGACCCAGTCCTCTTGAATCTGATTAGC-3; ATM R521* F5’-
GAGGTCAAACCTAGAAAGCTCA-3’ and R5’-GTGTGTGTCTGTGTGTGTTTATC-3’; ATM
Y2791* F5’ - GCTGAATGATCATCAAATGCTCT- 3’ and R5’ - ATGGCTTATTAAAGCTGACAGC-
3’). High Fidelity Taq Polymerase (Life Technologies, Carlsbad, CA) was used during PCR
according to manufacturer’s instructions. Post-sequencing cleanup was performed using ExoSAP
(Affymetrix, Santa Clara, CA) prior to tagging with M13 primer sequences and Bigdye
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fluorescence (Life Technologies) and sequencing on a 3130xl Genetic Analyzer (Applied
Biosystems, Carlsbad, CA).
eMethods 5. Secondary Analysis of panel data
All sequences in the raw data files from 25 cases were first trimmed by 225 base pairs to account
for sequence masking done by the MiSeq software in the event that amplicon regions were
shorter than 250 base pairs. Samples were then aligned (Burrows-Wheeler Alignment tool-0.5.9)1
to a custom genome then repositioned for variant calling on the full set of loci comprised by the
amplicon coordinates. Reads with greater than 5 mismatches were filtered out, and those
remaining were called for SNVs using SNVmix2 (4,5) after passing thresholds for base quality
(>10) and mapping quality (>20). Each SNV position was tested using binomial exact test, with
the background distribution defined by the reference allele frequency from the entire amplicon
coordinate list. The resulting p-value was corrected for the number of total tests using the
Benjamini Hochberg FDR4 procedure and any resulting q-value <0.00001 were considered
TRUE mutations. Indels were called using SAMtools5 pileup, separating lines containing short
indels and filtered on a minimum SNP quality score (>300), number of variant reads (>3) and
variant “allele” frequency (≥0.1). Coding indels were annotated using information in Ensembl
release 67.
eMethods 6. Immunohistochemistry protocol and description of antibodies used.
Formalin-fixed paraffin-embedded tissue blocks containing whole sections of tumor were obtained
from a surgical resection specimen corresponding to the proband of families 16 and 25, found to
carry truncating CTNNA1 mutations. Sections (4μm in thickness) were deparafinized, rehydrated,
and stained using the semi-automated Ventana Discovery® XT System (Ventana Medical
Systems, Tucson, AZ, USA). Antigen retrieval was performed using Cell Conditioning Solution 1
(Ventana). The primary antibodies used were EP1793Y (Rabbit monoclonal for alpha-catenin;
1:200 dilution; Abcam, Cambridge, UK), JC/70A (Mouse monoclonal for CD31; 1:100 dilution;
Dako, Carpinteria, CA, USA), CAM5.2 (Mouse monoclonal for cytokeratin; 1:5 dilution; BD
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Biosciences, Franklin Lakes, NJ, USA) and NCH-38 (Mouse monoclonal for E-cadherin; 1:25
dilution; Dako). Pre-diluted UltraMap® (Ventana) anti-Rabbit and anti-Mouse horseradish
peroxidase secondary antibodies were used, and signal detection was performed using the
UltraMap® DAB Detection Kit (Ventana). Two pathologists (DS and HL) assessed the sections
for the presence or absence of staining of E-cadherin and alpha-catenin within tumor cells. The
cytokeratin and CD31 stains were used to delineate epithelial cells and endothelial cells,
respectively. Histologic images were obtained using the Olympus DP21® digital camera
(Olympus, Shinkuju, Japan).
SUPPLEMENTAL METHODS REFERENCES
1. Kaurah P, MacMillan A, Boyd N et. al. Founder and recurrent CDH1 mutations in families with hereditary diffuse gastric cancer. JAMA. 2007 ;297(21):2360-72. Epub 2007 Jun 3.
2. Oliveira C, Senz J, Kaurah P, et. al. Germline CDH1 deletions in hereditary diffuse gastric cancer families. Hum Mol Genet. 2009;18(9):1545-55
3. Lange K, Weeks D, Boehnke M. Programs for Pedigree Analysis: MENDEL, FISHER, and dGENE. Genet Epidemiol. 1988;5(6):471-2.
4. Li H. and Durbin R. Fast and accurate short read alignment with Burrows-Wheeler Transform. Bioinformatics. 2009;25:1754-60.
5. Shah S, Morin RD, Khattra J, et al. Mutational evolution in a lobular breast tumour profiled at single nucleotide resolution. Nature. 2009;461, 809-813.
6. Goya R, Sun MG, Morin RD, Leung G, Ha G, Wiegand KC, Senz J, Crisan A, Marra MA, Hirst M, Huntsman D, Murphy KP, Aparicio S, Shah SP. SNVMix: predicting single nucleotide variants from next-generation sequencing of tumors. Bioinformatics. 2010 Mar 15;26(6):730-6
7. Li H, Handsaker B, Wysoker A, et al. The Sequence alignment/map (SAM) format and SAMtools. Bioinformatics. 2009;25, 2078-9.
Downloaded From: https://jamanetwork.com/ on 10/02/2021 eTable 1. CDH1 Mutations Described-To-Date CDH1 Mutation Location Mutation Type Ethnicity or (Country) Reference
5'UTR(-117)G>A Promoter 5'UTR-Unclassified Variant (Canada) 1
5'UTR(-71)C > G Promoter 5'UTR-Unclassified Variant (Canada) 1,2
-63C>A Promoter 5'UTR-Unclassified Variant Italian 3
chr16:del 67328695-67328 Current study 844 (150bp) 5'UTR and Exon 1 Large Deletion Hispanic (reported in 4)
chr16:del 67324886- Current study 67330557 (5671bp) Exon 1 and 2 Large Deletion Lithuanian (reported in 4)
chr16:del 67193822- Current study 67387415 (193 593bp) Exon 1 and 2 Large Deletion German-Hungarian; Caucasian (reported in 4) 1-?_163+?del Exons 1 and 2 Large Deletion Eastern European Current study 2T>C (M1T) Exon 1 Missense-Pathogenic Unknown 5
3G>A (start site) (M1I) Exon 1 Missense-Pathogenic Scottish-Irish Current study
3G>C (start site) (M1I) Exon1 Missense-Pathogenic Scottish-Irish 6
Missense-Unclassified 8C>G (P3R) Exon 1 Variant Unknown 7 41delT Exon 1 Deletion (Canada) 1 44_46delTGC Exon 1 Deletion Chinese 8 45_46insT Exon 1 Insertion European 9 46_48insTGC Exon 1 Insertion Unknown 5
48+1G>A Intron 1 Splice Site (United Kingdom); French-Canadian 10, Current study Intron 1 Splice Site Caucasian; Irish 11,12
Downloaded From: https://jamanetwork.com/ on 10/02/2021 49-2A>G
49-2A>C Intron 1 Splice Site Unknown 13 53delC Exon 2 Deletion Caucasian 14 55_74del20 Exon 2 Deletion Unknown 15 59G>A Exon 2 Nonsense British-Irish 11 personal communication- 67C>T Exon 2 Nonsense Unknown Caldas 70G>T Exon 2 Nonsense European 16
Missense-Unclassified 79C>T (P27S) Exon 2 Variant Italian Current study
Missense-Unclassified 88C>A (P30T) Exon 2 Variant Unknown 7
Missense-Unclassified 185G>T (G62V) Exon 3 Variant Japanese 17,18 187C>T Exon 3 Nonsense Caucasian; Unknown 6,15,19 190C>T Exon 3 Nonsense Maori 16
283C>T Exon 3 Nonsense Unknown; (France); (France) 20-23
Missense-Unclassified 286A>G (I96V) Exon 3 Variant Hispanic Current study 353C>G (T118R) Exon 3 Missense-Pathogenic Caucasian 13, 24 164-?_387+?del Exon 3 Large Deletion Japanese 25 164-?_387+?del Exon 3 Large Deletion (France) 23 377delC Exon 3 Deletion German; Unknown 21,26a 382delC Exon 3 Deletion Italian; Lebanese 27, Current study
447_453delCAGAAGA Exon 4 Deletion English Current study 469delG Exon 4 Deletion France 23
Downloaded From: https://jamanetwork.com/ on 10/02/2021 489C>A Exon 4 Nonsense Unknown 15
Missense-Unclassified 515C>G (P172R) Exon 4 Variant Finnish 2 517insA Exon 4 Insertion ??? 28 531+2T>A Intron 4 Splice Site (France) 29 532-18C>T Intron 4 Potential Splice-Site (England); (Portugal) 30
Missense-Unclassified 554A>T (E185V) Exon 5 Variant Unknown 31 586G>T Exon 5 Nonsense European 16
Missense-Unclassified 604G>A (V202I) Exon 5 Variant Chinese 8 18,24; personal communication 641T>C (L214P) Exon 5 Missense-Pathogenic European Caldas
Missense-Unclassified 670C>T (R224C) Exon 5 Variant Italian 3 687+1G>A Intron 5 Splice Site European 27 687+1G>T Intron 5 Splice Site Algerian Current study
Missense-Unclassified 695C>G (S232C) Exon 6 Variant Unknown 31
13,21,32; Current 715G>A Exon 6 Missense-Splice Site Filipino; Caucasian; Korean; Hispanic study
Missense-Unclassified 731A>G (D244G) Exon 6 Variant Korean 33 753insG Exon 6 Insertion Unknown 5
Missense-Unclassified 808T>G (S270A) Exon 6 Variant Unknown 34 811_812delins12 Exon 6 Deletion-Insertion Hindustan 15
Missense-Unclassified 820G>A (G274S) Exon 6 Variant Italian 35
Downloaded From: https://jamanetwork.com/ on 10/02/2021 832G>A Exon 6 Splice Site Pakistani; Unknown 9, 36 832+1G>T Exon 6 Splice Site France; Swedish-german 23; Current study 833-2A>G Intron 6 Splice Site Caucasian 37; Current study
Missense-Unclassified 27, 18, 24,38; 892G>A (A298T) Exon 7 Variant European Current study 940A>T Exon 7 Nonsense Mexican-European Current study 1003C>T Exon 7 Nonsense Swedish; Caucasian; Korean 6,32,39,40 1008G>T Exon 7 Missense-Splice Site Maori 41 1008+1G>A Exon 7 Splice Site (France) 23
chr 16: del 68845689- 68869444 (275kb) Exon 7-16 Large Deletion Japanese 25 personal communication - 1009-2delAG Exon 8 Splice Site Caucasian Caldas 1009-2A>G Exon 8 Splice Site Caucasian Current study 1005delA Exon 8 Deletion Russian 42 1018A>G (T340A) Exon 8 Missense-Pathogenic European; Chinese 9,18,24,30,43,44 1023T>G Exon 8 Nonsense Unknown 5 1063delT Exon 8 Deletion Caucasian 6 1064_1065insT Exon 8 Insertion Caucasian 27 1107delC Exon 8 Deletion Hispanic 13 1118C>T (P373L) Exon 8 Missense-Pathogenic Italian 45 1134del8,ins5 Exon 8 Splice Site Caucasian; Unknown 15,27,46 13,21,23,29; Current study; Unknown;Swedish- personal Norwegian/Jordanian; Caucasian (x2 communication- 1137G>A Exon 8 Splice Site families); German; Arabic Caldas 1137G>T Exon 8 Splice Site Native American Current study 1137+1G>A Intron 8 Splice Site African-American; Unknown 16; Current study
Downloaded From: https://jamanetwork.com/ on 10/02/2021 Current study 1189A>T Exon 9 Nonsense Caucasian (reported in 47) 25,27; Current 1212delC Exon 9 Deletion English-Japanese; Japanese study
Missense-Unclassified 1223C>T (A408V) Exon 9 Variant Unknown 7 1226T>C (W409R) Exon 9 Missense-Pathogenic European 23
Missense-Unclassified 1243A>C (I415L) Exon 9 Variant Japanese 48 1285C>T (P429S) Exon 9 Missense-Pathogenic Caucasian 6,18
Missense-Unclassified 1297G>A (D433N) Exon 9 Variant Unknown 7
1302_1303insA, 1306_1307delTT Exon 9 Insertion-Deletion Austrian 49 1397_1398delTC Exon 10 Deletion English/Caucasian 13b,21 1404delC Exon 10 Deletion Unknown 15
Missense-Unclassified 1460T>C (V487A) Exon 10 Variant Korean 33 10c;50; personal communication - 1466_1467insC Exon 10 Insertion Caucasian; (United Kingdom) Caldas 1470_1483del14 Exon 10 Deletion (France) 23 1472_1473insA Exon 10 Insertion European 9 1476_1477delAG Exon 10 Deletion Caucasian; Unknown 15,23
1488_1494delCGAGGAC Exon 10 Deletion Unknown 16 1507C>T Exon 10 Nonsense Chinese 51
1565+1G>A Intron 10 Splice Site Scottish-Dutch-Swedish-English 52; Current study 1565+1G>C Intron 10 Splice Site Caucasian Current study
Downloaded From: https://jamanetwork.com/ on 10/02/2021 1565+1G>T Intron 10 Splice Site Caucasian; Arabic 14; Current study
15,23,40; Current 1565+2dupT Intron 10 Insertion/Splice Site Caucasian; (French); Unknown study 1566-?_1711+?del Exon 11 Large Deletion French Caucasian (France) 23 1566-?_1711+?del Exon 11 Large Deletion Japanese 53 1582delG Exon 11 Deletion French Caucasian 22 1588_1589insC Exon 11 Insertion European 16 1595G>A Exon 11 Nonsense (France) 23 1610delC Exon 11 Deletion Spanish 54 1619_1620insG Exon 11 Insertion German 55 1679C>G Exon 11 Missense-Splice Site (France);Caucasian 23; Current study 1682_1683insA Exon 11 Insertion Irish 21 1710delT Exon 11 Deletion African-American 14 1711_1712insG Exon 11 Insertion European 19 1711+5G>A Exon 11 Splice Site European 27 personal communication 1734delC Exon 11 Deletion Carlos Caldas 1748T>G (L583R) Exon 12 Missense-Pathogenic Creole 15
Missense-Unclassified 1774G>A (A592T) Exon 12 Variant (Sweden) 18,55,56 1779_1780insC Exon 12 Insertion Caucasian 27
6,14,19,57; Current 1792C>T Exon 12 Nonsense Caucasian; Irish-English; Unknown study
Missense-Unclassified 1795A>T (T599S) Exon 12 Variant Korean 16,32
Missense-Unclassified 1813A>G (R605G) Exon 12 Variant Unknown 7
Missense-Unclassified 1849G>A (A617T) Exon 12 Variant Caucasian 18,30,58
Downloaded From: https://jamanetwork.com/ on 10/02/2021 Missense-Unclassified 1876T>A (F626V) Exon 12 Variant Unknown 59 1895_1896delAC Exon 12 Deletion Northern European 60 1901C>T Exon 12 Missense-Splice Site English; Unknown; Maori 13,21,30,46 1913G>A Exon 12 Nonsense Spanish 21/61 1942G>T Exon 13 Nonsense (United Kingdom) 10 2058_2059delTG Exon 13 Deletion Brazilian Current study 2064_2065delTG Exon 13 Deletion (USA) 6,27,40 2095C>T Exon 13 Nonsense Maori; Chinese 13,41 Current study; personal communication - 2100delT Exon 13 Deletion English-Scottish Caldas 2161C>G Exon 13 Splice Site Hispanic 6 2164+2T>A Exon 13 Splice Site (France) 23 2164+5G>A Exon 13 Splice Site Unknown 21 2195G>A Exon 14 Missense-Splice Site Caucasian; Turkish 15,21,27 2245C>T (R749W) Exon 14 Missense-Pathogenic Columbian 21,24,27,31 personal Missense-Unclassified communication - 2248G>A Exon 14 Variant Caldas 2265T>A Exon 14 Nonsense Unknown Current study 2269G>A (E757K) Exon 14 Missense-Pathogenic Portuguese 4,24, 31 2275G>T Exon 14 Nonsense Iranian 62 2276delG Exon 14 Deletion Unknown 6
chr16:del 67416845- 67424923 (8078bp) Exon 14-16 Large Deletion European 4 2287G>T Exon 14 Nonsense Unknown 63 2295+5G>A Intron 14 Splice Site European 14 2310delC Exon 15 Deletion Scottish/English; Unknown 27; Current study
Downloaded From: https://jamanetwork.com/ on 10/02/2021 Missense-Unclassified 2329G>A (D777N) Exon 15 Variant 39 2343A>T (E781D) Exon 15 Missense-Pathogenic English; (France) 21,23 2381_2382insC Exon 15 Insertion Maori 41 2386delC Exon 15 Deletion (France) 23 2396C>G (P799R) Exon 15 Missense-Pathogenic German 18,24,55 2398delC Exon 15 Deletion Irish/English/Scottish 10,21,23,40d 2399delG Exon 15 Deletion (France) 64 personal communication- 2400delG Exon 15 Deletion (United Kingdom) Caldas
Missense-Unclassified 2413G>A (D805N) Exon 15 Variant Jewish Current study 2430delT Exon 15 Deletion English Current study 2440-6C>G Intron 15 Splice Site 13 2494G>A (V832M) Exon 16 Missense-Pathogenic Japanese 7,18,65
chr16:del 67424298- 67425126 (828 bp) Exon 16 Large Deletion Central European 4 aDenoted as 372delC bDenoted as 1391_1392delTC cDenoted as 1465insC
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Family Proband (Ca No. of GC Cases; DGC No. of BC Cases; LBC CDH1 Other type, age @ dx) Casesb (Age Range at Casesb (Age Range at Mutation Cancers in Diagnosis, y) Diagnosis, y) Family
F1 DGC (40) 1;0 (24) 0 Del 50-UTR- Pancreatic exon 1 (150 bp)
F2 DGC (29) 2;2 (19;45) 1;1 (65) (45_46 insT)
F3 Obligate carrier 9;2 (45-65) 0 IVS 01-2A>G - (80)
F4 Unaffected (54) 5;1 (36-58) 0 49-2A>G Lung, Skin
F5 DGC (69) 5;0 (34-57) 0 49-2A>G
F6 DGC (37) 2(37;50) 0 59 G>A Colon
F7 DGC (45) 3;0 (50-63) 1;0 (35) 67 C>T Rectal; Ovarian F8 DGC (38) 3;2 (39-46) 1;0 (52) 70 G>T
F9 LBC (39) 2;0 (24, 52) 2;0 (38, 39) 187C>T Colorectal 67; Pancreatic 74 F10 DGC (33) 5;2 (48-85) 3;0 (43-64) 187C>T Colon x3; En x2, PR, Pa
Downloaded From: https://jamanetwork.com/ on 10/02/2021 F11 DGC (39) 6;1 (43-?) 1;0 (50) 283 C>T -
F12 DGC (37) 4; 2 (15-58) 1;0 (49) 372delC
F13 DGC (32) 5;1 (45-65) 2;0 (55, 55) 382 delC Lung 75
F14 DGC (30) 1;0 (75) 0 382delC -
F15 DGC (61) 5;1 (39-88) 1;0 (45) 447-453 del Colorectal CAGAAGA (57; 58) Leukemia 48 Prostate (57;68) Sarcoma 17 F16 DGC (40) 3;0 (36-50) 1;0 (50) IVS5(+1)G>A H&N NOS 66 F17 LBC (53) 3;1 (44-48) 3;0 (60-71) IVS5(+1)G>A Melanoma x2 Myeloma (70) Ovarian Bladder F18 DGC (47) 3;3 (31-55) 0 586 G>T
F19 DGC (49) 3;1 (20-43) 1;0 641T>C Skin, rectal; larynx
F20 DGC (39) 11;1 (19-75) 3;0 (50-70) 688 -2 A>G Ca NOS (?) (IVS6-2 A>G)
F21 DGC (40) 14;8 (23-70) 0 832G>A
Downloaded From: https://jamanetwork.com/ on 10/02/2021 F22 DGC (57) 8;2 (25-67) 2;1 (45;54) C1003T Melanoma (27); lymphoma (53); Liver (18) NOS (50;63) F23 DGC (31) 4;1(48-58) 0 C1003T -
F24 LBC (51) 2;2 (21;43) 0 R335X (1005) Lymphoma
F25 DGC (42) 4;4 (14-32) 0 1008 G>T
F26 DGC (32) 9;3 (33-74) 0 1008 G>T Colorectal
F27 DGC (45) 13;1 (16-60) 0 1008 G>T
F28 unaffected 5;2 (39-52) 1;0(35) IVS7 -2 A>G -
F29 DGC (58) 2;1 (34;50) 1064 ins(T)
F30 DGC (27) 0 0 1063 delT
F31 DGC (42) 2;1 (37,48) 0 1137 G>A Lymphoma (60s?) F32 DGC (42) 7;2 (26-63) 2;0 (21;48) 1137 G>A Non- Hodgkins Lymphoma (67)
Downloaded From: https://jamanetwork.com/ on 10/02/2021 F33 DGC (38) 3;0 (35-46) 1;0 (59) 1137 G>T -
F34 DGC (44) 5;2 (21-72) 1;0 (75) 1137 G>A Lung
F35 DGC (36) 3;0 (32-42) 0 intron 8 1137 - +1
F36 LBC (47) 0 1;0 (32) 1189 A>T Family was identified due to several cases of GC on paternal side, mutation was found on maternal side F37 DGC (61) 4;1 (32-41) 3;0 (47-59) 1212 delC Colorectal 59 F38 LBC (51) 5;0 (31-60) 3:0 (51-70)
F39 DGC (49) 7;2 (31-56) 0 c.1302_1303in sA, 1306_1307del TT
F40 DGC (37) 2;0 (31;62) 0 1397 delTC Ca NOS x3
F41 DGC (32) 2; 1 (34;40) 0 1472_1473 insA)
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F42 DGC (32) 1;0 (40) 0 1476delAG
F43 Unaffected 4;2 (42-75) 0 1466- 1467insC
F44 DGC (55) 2;0 (58;76) 2;1 (67;74) 1565+1 G>A Leukemia (IVS10+1 (50) G>C)
F45 Unaffected 3;1 (?-70) 0 1565+1 G>T
F46 DGC (40) 9;4 (38-82) 2;1 (42;53) 1588insC Leukemia, lung; pancreatic; Ca NOS x7 F47 DGC (52) 2;0 (41;49) 0 1682insA Colorectal (72;81) F48 DGC (29) 2;0 (19;45) 1;0 (65) 1710delT
F49 DGC (43) 9;7 (27-67) 0 1711insG
F50 DGC (32) 3;1 (20-42) 0 1792 C>T
F51 DGC (59) 3;1 (41-55) 3;0 (33-67) 1792 C>T )
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F52 DGC (37) 1;1 (11) 0 1792 C>T
F53 Unaffected 5;1 (31-54) 0 1895 delAG (exon 12)
F54 LBC (65:75) 2;1 (34;45) 0 C1901T in exon 12
F55 DGC (24) 1;0 (46) 1;1 (57) Lymphoma 44 2016 delTG Ovarian 59 Exon 13 Colorectal 89 F56 DGC (46) 0 1:0 (79) Ovarian 2058_59 del (35) TG in exon 13 Colorectal (55) F57 DGC (37) 1;0 (73) 2;0 (65;66) 2061 delTG exon 13
F58 DGC (20) 2;0 (29;?) 0 Melanoma 2100delT x2 (?) exon 13 Colorectal (38;75) Esophageal (51;82)
Downloaded From: https://jamanetwork.com/ on 10/02/2021 F59 DGC (24) 4;2 (28-43) 0 C2095T exon13
F60 DGC (44) 2;0 (38;44) 0 IVS13 +5 G/A
F61 DGC (47) 3;0 (36-49) 1;0(48) 2195 G>A Sarcoma 15 (R732Q) in Hodgkin’s exon 14 15 Leukemia 3
F62 DGC (66); LBC 5;0 (32-66) 4;0 (40-75) 2195G>A (66) (R732Q) exon 14
F63 unaffected 2;1 (33;38) 0 2265T>A nonsense in exon 14
F64 DGC (50) 5;0 (35-62) 2;1 (74;75) 2276 delG Colorectal (exon 14) (65) Esophageal (55) Hodgkin’s (75)
Downloaded From: https://jamanetwork.com/ on 10/02/2021 F65 DGC (51) 1;1 (52) 4;2 (52-?) 2287C>T Pancreatic 62 Thyroid 51 Colon 60s NOS 77
F66 DGC (42) 2;1 (34;40) 1;1 (52) 2310 delC
F67 obligate carrier 8;1 (42-79) 1;0 (75) 2311 delC
F68 LBC (54) 22;11 (24-?) 17;4 (35-?) 2398 delC
F69 DGC (39) 2;1 (27;48) 2398 delC
F70 DGC (63) 3;1 (35-75) 0 2398 delC Colorectal (68) Ca NOS (25) F71 DGC (38 & 43) 2;0 (35;55) 0 chr16:deletion Colorectal 67 193 822-67 (76) 387 415 (193 593bp)
F72 DGC (35) 1;1 (33) 0 Del exon 14– 16 (8078 bp)
F73 Unaffected 4;3 (29-52) 0 Del exon 16
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F74 LBC (61) 3;1 (28-40) 0 chr16:deletion 67 193 822-67 387 415 (193 593bp)
F75 DGC (38;43) 1;0 (35) 0 chr16:deletion Colorectal; 67 193 822-67 Skin 387 415 (193 593bp)
Downloaded From: https://jamanetwork.com/ on 10/02/2021 eTable3. Genes selected for custom panel based on association with uppergastrointestinal syndrome
Syndrome Gene Mutation Status Penetrance Carney-Stratakis syndrome SDHB Heterozygous High SDHC Heterozygous High SDHD Heterozygous High Colorectal carcinoma & Polyposis MUTYH Homozygous High Esophageal adenocarcinoma/Barrett's esophagous AKAP12 Heterozygous High CTHRC1 Heterozygous Intermediate FOXF1 Heterozygous Intermediate MSR1 Heterozygous Intermediate Familial adenomatous polyposis APC Heterozygous High Gastric Cancer ARID1A Heterozygous High BCL2L10 Heterozygous High BRCA1 Heterozygous High BRCA2 Heterozygous High CASP10 Heterozygous High CDH1 Heterozygous High CTNNA1 Heterozygous High FAT4 Heterozygous Unknown FHIT Heterozygous High HSPA5 Heterozygous High IDH1 Heterozygous Unknown IDH2 Heterozygous Unknown PSCA Heterozygous High PTEN Heterozygous High Cowden syndrome PTEN Heterozygous High SDHB Heterozygous High SDHD Heterozygous High Gastrointestinal-type polyposis MSH3 Heterozygous High Hereditary mixed polyposis syndrome GREM1 Heterozygous High SCG5 Heterozygous High TGFR2 Heterozygous High Juvenile polyposis, Pancreatic cancer BMPR1A Heterozygous High SMAD4 Heterozygous High
Downloaded From: https://jamanetwork.com/ on 10/02/2021 Lynch syndrome EPCAM Heterozygous High MLH1 Heterozygous High MSH2 Heterozygous High MSH3 Heterozygous High MSH6 Heterozygous High PMS1 Heterozygous High PMS2 Heterozygous High Pancreatic cancer ATM Heterozygous Intermediate CDKN2A Heterozygous High CFTR Heterozygous High CHEK2 Heterozygous Intermediate PALB2 Heterozygous High PRSS1 Heterozygous High SPINK1 Heterozygous High TP53 Heterozygous High Peutz-Jeghers syndrome STK11 Heterozygous High *Collaborating projects AKR7A3 Heterozygous Unknown GAB2 Heterozygous Unknown ITIH2 Heterozygous Unknown MAP3K6 Heterozygous Unknown MCCC1 Heterozygous Unknown PRR5 Heterozygous Unknown PXN Heterozygous Unknown SCARF2 Heterozygous Unknown SLC22A4 Heterozygous Unknown
*genes included in study based on collaboarative projects and possible involvement in UGI cancer (unpublished data) Bolded genes included based on direct suggested involvement in gastric cancers
Downloaded From: https://jamanetwork.com/ on 10/02/2021 eTable 4: Novel and rare missense mutations detected in HDGC families using custom UGI gene panel and predicted impact using in silico methods Gene Mutation Coverage ID (Chromosome) Coordinate dbSNP ID Frequency Ref. Var. Consequence Depth PolyPhen PROVEAN SIFT Score P20 AKAP12 (chr.6) 151673820 - - GA G1432R 2388 Probably Damaging Neutral Damaging P37 AKAP12 (chr.6) 151674257 - - GC Q1577H 2233 Probably Damaging Neutral Damaging P70 AKAP12 (chr.6) 151670490 - - G A E322K 1679 Probably Damaging Deleterious Damaging P19 AKR7A3 (chr.1) 19615041 rs145878095 0% C T E55K 2397 Probably Damaging Deleterious Damaging P3 ARID1A (chr.1) 27089742 rs138418893 0% G A A900T 246 Probably Damaging Neutral Damaging P68 ATM (chr.11) 108141988 - - T C S978P 734 Probably Damaging Deleterious Damaging P38 BMPR1A (chr.10) 88679061 - - TC L334S 4012 Probably Damaging Deleterious Damaging P86 BMPR1A (chr.10) 88676988 - - T C V258A 1194 Probably Damaging Deleterious Damaging P101 BMPR1A (chr.10) 88676994 - - T C V260A 1146 Probably Damaging Deleterious Damaging P56 BRCA2 (chr.13) 32915190 rs41293501 0% C A A2233D 915 Probably Damaging Damaging Damaging P68 BRCA2 (chr.13) 32945102 - - A G K2833E 20 Probably Damaging Neutral Damaging P34 CASP10 (chr2) 202074096 - - C G S366C 2663 Probably Damaging Deleterious Tolerated P8 CFTR (chr.7) 117149143 rs115545701 0% CT R74W 211 Probably Damaging Neutral Tolerated P59 CFTR (chr.7) 117251657 - - TA H1054Q 2692 Probably Damaging Deleterious Damaging P89 CFTR (chr.7) 117250625 rs149279509 0% A G Y1014C 100 Probably Damaging Deleterious Damaging P94 CFTR (chr.7) 117250575 rs1800111 0% GC L997F 220 Probably Damaging Tolerated Tolerated P100 CFTR (chr.7) 117176630 rs191456345 0% AG R258G 133 Probably Damaging Deleterious Damaging P24 EPCAM (chr.2) 47601029 rs146480420 0% GC Q89H 15 Probably Damaging Neutral Damaging P43 ITIH2 (chr.10) 7759650 rs138977046 0% G A V177M 2008 Probably Damaging Deleterious Damaging P3 MLH1 (chr.3) 37035088 - - AC N17T 185 Probably Damaging Deleterious Damaging P24 MSH2 (chr.2) 47641548 - - CA N311K 425 Probably Damaging Deleterious Damaging P24 MSH2 (chr.2) 47698172 rs149796110 0% TC I577T 250 Probably Damaging Deleterious Damaging P112 PMS1 (chr.2) 190660586 rs61756360 0% CT T75I 166 Probably Damaging Deleterious Damaging P84 PRSS3 (chr.9) 33796794 - - A G Y79C 184 Probably Damaging Deleterious Damaging P36 PTEN (chr.10) 89624228 - - T G M1R 1552 Probably Damaging Deleterious Damaging P55 PTEN (chr.10) 89624228 - - T G M1R 1372 Probably Damaging Deleterious Damaging P33 SLC22A4 (chr.5) 131676347 - - GA G512R 248 Probably Damaging Deleterious Tolerated
Downloaded From: https://jamanetwork.com/ on 10/02/2021 eTable 5. Summary of Families with CDH1 Mutations in This Study Family Criteriaa Proband No. of GC No. of BC Ethnicity CDH1 Mutation (Age at Cases; DGC Cases; LBC Location Mutationc Type Reference Diagnosis, Casesb (Age Casesb (Age y) Range at Range at Diagnosis, y) Diagnosis, y) F1 1 DGC (38 & 2;2 (30,35) 0 Hungarian Exons 1 chr16:del 67 193 Large Oliveria 43) / and 2 822 - 67 387 415 deletion 2009e German (193 593bp) F2 4 LBC (61) 3;2 (28-40) 0 Caucasian Exons 1 chr16:del 67 193 Large Oliveria and 2 822 - 67 387 415 deletion 2009e (193 593bp) F3 1 DGC (34) 1;0 (?) 0 Lithuanian Exons 1 chr16:del 67 324 Large Oliveria and 2 886 - 67 330 557 deletion 2009e (5671bp) F4 1 DGC (40) 2;2 (24, 39) 0 Mexican Exon 1 chr16:del 67 328 Large Oliveria 695 - 67 328 844 deletion 2009e (150bp) F5 1 DGC (48) 1 (45) 0 Eastern Exons 1 1-?_163+?del Large Novel European and 2 Deletion F6 1 DGC & LCIS 2;1 (32-54) 2;1 (32-50) Scottish/ Exon 1 3G>A (M1I) Missense- Novel (32); DGC Irish Pathogenicd (49) F7 1 DGC (74) 5;2 (52-72) 0 French- Intron 1 48 +1G>A Splice Site Petridis Canadian 2013 F8 3 Unaffected – 1 (30) 0 Italian Exon 2 79C>T (P27S) Missense- Novel obligate Unclassified carrier Variant F9 3 DGC (29) 0 0 Hispanic Exon 3 286A>G (I96V) Missense- Novel Unclassified Variant F10 1 DGC (30) 1;0 (75) 0 Lebanese Exon 3 382delC Deletion Brooks- Wilson 2004 F11 1 DGC (42) 5;1 (39-88) 1;1 (45) Irish/ Exon 4 447_453del Deletion Novel English CAGAAGA F12 3 DGC (30) 1;1 (30) 0 Algerian Intron 5 687+1G>T Splice Site Novel F13 3 DGC (31) Not known Exon 6 715G>A (G239R) Missense- Kaurah Splice Site 2007; More 2007
Downloaded From: https://jamanetwork.com/ on 10/02/2021 F14 1 DGC (46) 3;1 (46-64) 1;0 (70) Swedish/ Exon 6 832+1G>T Splice Site Benusiglio German 2012 F15 1 DGC (41) 12;2 (29-75) 0 Not known Intron 6 833-2A>G Splice Site Novel F16 3 DGC (37) 0 0 Not known Exon 7 892G>A (A298T) Missense- Brooks- Unclassified Wilson Variant 2004 F17 1 DGC (39) 4;1 (39-49) 3;0 (?-64) Mexican/ Exon 7 940A>T Nonsense Novel European F18 4 Unaffected 5;2 (39-52) 1;0(35) Caucasian Exon 8 1009-2A>G Splice Site Novel F19 4 LBC (46) 1; 1 (69) 2;2 (29-46) German Exon 8 1137G>A Splice Site Kaurah 2007; More 2007; Frebourg 2006 F20 1 Unaffected 7;2 (26-63) 1;0 (?) Arabic Exon 8 1137G>A Splice Site Kaurah 2007; More 2007; Frebourg 2006 F21 1 Unaffected 4;3 (35-40) 1;0 (59) Native Exon 8 1137G>T Splice Site Novel American F22 1 DGC (36) 4;1 (32-42) 0 Not known Intron 8 1137+1G>A Splice Site Guilford 1999 F23 4 LBC (47) 0 2;1 (32-47) Not known Exon 9 1189A>T Nonsense Hebbard 2009e F24 4 LBC (51) 4;0 (31-68) 5;1 (51-87) Japanese Exon 9 1212delC Deletion Brooks- wilson 2003; Yamada 2011 F25 4 LBC (53) 1;0 (40) 6;1 (30-60) Northern Intron 10 1565+1G>A Splice site Schrader European 2008e F26 4 DGC (55) 2;1 (55,76) 2;1 (67,74) Caucasian Intron 10 1565+1G>C Splice Site Novel F27 1 Unaffected 2;1 (30-57) 0 Arabic Intron 10 1565+1G>T Splice Site Humar 2002 F28 1 DGC (39) 3;1 (39-58) 0 Not known Intron 10 1565+2dupT Splice Site Rogers 2008 F29 1 DGC (47) 3;1 (47-64) 1;0 (61) Caucasian Exon 11 1679C>G Missense- Benusiglio
Downloaded From: https://jamanetwork.com/ on 10/02/2021 (T560R) Splice Site 2012 F30 4 Unaffected 6;4 (40-61) 1;1 (58) Not known Exon 12 1792C>T Nonsense Gayther 1998; Suriano 2005 F31 4 DGC (59) 5;1 (41-59) 3;0 (36-67) Irish/ Exon 12 1792C>T Nonsense Gayther English 1998; Suriano 2005 F32 1 DGC (31) 2;0 (23;?) 0 Hispanic Exon 7 1914G>A Nonsense Van (W638X) Domesela ar 2007
F33 1 DGC (46) 2;1 (46-71) 0 Brazilian Exon 13 2058_2059delTG Deletion Novel F34 1 DGC (20) 2;2 (29-?) 0 English/ Exon 13 2100delT Deletion Novel Scottish F35 1 Unaffected 2;1 (33-38) 0 Not known Exon 14 2265T>A Nonsense Novel F36 4 DGC (42) 3;2 (32-70) 1;1 (52) Not known Exon 15 2310delC Deletion Brooks- Wilson 2004 F37 1 Unaffected 4;0 (33-65) 0 Jewish Exon 15 2413G>A Missense- Novel (D805N) Unclassified Variant F37 1 DGC (45) 3;1 (45-66) 0 English Exon 15 2430delT Deletion Novel Abbreviations: GC, gastric cancer; DGC, diffuse gastric cancer; LCIS, lobular carcinoma in situ; BC, breast cancer; LBC, lobular breast cancer. aCriteria 1-4 as per 2010 International Gastric Cancer Linkage Consortium guidelines (Fitzgerald et al.) and described in Table 1. bRefers to number of confirmed cases. cNumbering is based on CDH1 cDNA sequence (GenBank NM_004360.3) with A in start ATG (methionine) corresponding to position 1. Amino acid changes for missense mutations denoted in parentheses. dNucleotide change disrupts the start codon. eNovel mutations identified in families ascertained and tested during the time period of this study that were published separately by collaborators. Note: The family IDs in eTable 5 do not correspond with family IDs in eTable 2.
Downloaded From: https://jamanetwork.com/ on 10/02/2021 eTable 6: Cumulative Risk % (95% Confidence Interval) and Relative Risk of Gastric and Breast Cancer in CDH1 germline mutation carriers.
Gastric Breast Age (yrs) Male RR Female RR Female RR 10-29 4 (0-13) 1937 2 (0-9) 1068 0(0-1) 30-39 10 (3-26) 526 17 (9-32) 2813 3(1-6) 40-49 25 (12-50) 309 25 (13-47) 392 11 (9-14) 50-59 32 (14-64) 42 33 (16-63) 149 21(16-26) 60-69 48(24-80) 35 41 (19-74) 47 32 (26-39) 70-79+ 70(40-94) 35 56 (27-90) 47 42 (35-51) Breast Cancer 10-49 7.7 50+ 7.4
Downloaded From: https://jamanetwork.com/ on 10/02/2021 Whole Genome Single Gene Screen Multiplex Custom- Sequencing Panel Sequencing (i.e. HDGC families)
Single Sample Single Sample Up to 96 Samples
$7,000 ($4,700) $2500 ($400) $4,000 ($300)
Illumina HiSeq Platform Sanger Sequencing Illumina MiSeq Platform or MLPA
4-6 weeks 2-4 weeks <1 week
> 150Gb <1Gb 10-15Gb Repeat with new gene of interest of gene new Repeatwith (dependent on size of panel)
MiSeq Reporter Software On-machine data analysis Bioinformatics Pipeline >40% Families 60% families with Filtering and detection of genomic variants with pathogenic unknown genetic variant susceptibility Selection of candidate variants • Protein truncating variants • Pathogenic SNPs Carrier testing for Selection of candidate variants unaffected family • functional relevance members No candidate variants Validate • relation of gene to disease Submit for WGS Sanger Sequencing
PTG for mutation carriers Segregation & LOH analysis
eFig 1: Description of current techniques used in discovering pathogenic, germline susceptibility variants
Downloaded From: https://jamanetwork.com/ on 10/02/2021 CDH1 Mutations 41.9%
Unknown CDH1 promoter Genetic Risk methylation 54% <1% BRCA2 (1)
CDH1 deletions SDHB (1) 3.8% PRSS1 (1)
PALB2 (1)
CTNNA1 (2) Unknown STK11 (3) Genetic Risk 48% ATM (3)
MSR1 (4)
eFig 2. HDGC mutational profile, including novel and rare potentially pathogenic mutation in genes: BRCA2, SDHB, PRSS1, PALB2, CTNNA1, STK11, ATM, MSR1. Number of families found to carry such variants is indicated in parentheses.
Downloaded From: https://jamanetwork.com/ on 10/02/2021 eFig 3: Photomicrographs of the tumor of the proband in Family 25 with the CTNNA1 germline mutation. A) The tumor consisted of diffuse adenocarcinoma (asterix) proliferating underneath the benign gastric mucosa (arrow; H&E, 40x). B) The neoplastic cells were arranged individually and in small clusters within pools of mucin and stroma (H&E, 200x). The cells had foamy cytoplasm and mild nuclear atypia (inset, H&E, 400x). C) E-cadherin expression was preserved in both the tumor (asterix) and in the benign epithelium (arrow, E-cadherin immunohistochemistry, 400x). D) E-cadherin expression in the tumor was cytoplasmic and membranous (E-cadherin, 200x and 400x[inset]). E) Alpha-catenin was expressed in the benign epithelium (arrow), but was lost in the tumor (asterix, alpha-catenin, 200x). F) The endothelium within the tumor expressed alpha-catenin (arrowhead, alpha-catenin, 200x), but no staining was present in the tumor cells themselves (inset, 400x), suggesting loss of heterozygosity at the CTNNA1 locus.
Downloaded From: https://jamanetwork.com/ on 10/02/2021 Downloaded From: https://jamanetwork.com/ on 10/02/2021 C G B A BRCA2 eFig E 1 Legend: 1 4: , (C) 1 Sema c 1 Interact with Interact with Interact with Interact with RAD51 RAD51 SDHB 7 TAN 33 1 BRCA2 BRCA2 reductase reductase Fumarate Fumarate PALB2 PALB2
view of genes with pathogenic and likely pathogenic muta ons iden fied using panel-based sequencing. (A)
, (D) 21 NTR 167 22 39 β-catenin NLS STK11 200 143 147 VH1 VH1 319 Iron-sulfur Iron-sulfur , (E) binding
Novel Novel Truncating Mutation Dimerization repeat
611 HEAT Interact with Interact with ATM 1002 domain domain MRG15 MRG15
BRC Repeats Repeats BRC , (F) PRSS1, (G)
RAD51
Leucine ferredoxin ferredoxin Alpha-helical Alpha-helical 224 Zipper
765 NLS 871 WD40/YVTN-repeat domain domain WD40/YVTN-repeat α-ac nin BRCA2 & RAD51 binding binding & RAD51 BRCA2 377
PALB2 2097 FAT FAT 275 2085 VH2 VH2 280 , (H)
2482 2390 H NLS MSR1 Adhesion Adhesion domain domain
D 2525
586
2715 PI3-K 2610 OB1 1
1185 Rare Missense; pathogenic pathogenic Missense; Rare 1186 2952 OB2 697 49 NLS 55 F- actin binding domain domain binding
FATC Protein OB3
63 VH3 and H 3056
1 3200 Kinase Vinculin RAD51
NLS NLS 276 3350 848 triple-helix F Collagen
1 309 3390 3418 Phosphoryla on/
peptidase domain domain peptidase Farnesyla on Cysteine Ser428 345 Domain Domain
SRCR SRCR Cys430 /serine /serine 436
CTNNA1 450 451 247 , (B) References
1. Bacani JT, Soares M, Zwingerman R, et.al. CDH1/E-cadherin germline mutations in early- onset gastric cancer. J Med Genet. 2006;43(11):867-72. Epub 2006 Jun 26. 2. Avizienyte E, Launonen V, Salovaara R et.al. E-cadherin is not frequently mutated in hereditary gastric cancer. J Med Genet. 2001 Jan;38(1):49-52. 3. Corso G, Pedrazzani C, Pinheiro H, et.al. E-cadherin genetic screening and clinico- pathologic characteristics of early onset gastric cancer. Eur J Cancer. 2011;47(4):631-9. doi: 10.1016/j.ejca.2010.10.011. Epub 2010 Nov 22. 4. Oliveira C, Senz J, Kaurah P, et.al. Germline CDH1 deletions in hereditary diffuse gastric cancer families. Hum Mol Genet. 2009 May 1;18(9):1545-55. doi: 10.1093/hmg/ddp046. Epub 2009 Jan 24. 5. Guilford P, Humar B, Blair V. Hereditary diffuse gastric cancer: translation of CDH1 germline mutations into clinical practice. Gastric Cancer. 2010;13(1):1-10. 6. Suriano G, Yew S, Ferreira P, et.al. Characterization of a recurrent germ line mutation of the E-cadherin gene: implications for genetic testing and clinical management. Clin Cancer Res. 2005;11(15):5401-9. 7. Schrader KA, Masciari S, Boyd N, et.al. Germline mutations in CDH1 are infrequent in women with early-onset or familial lobular breast cancers. J Med Genet. 2011 Jan;48(1):64-8. doi: 10.1136/jmg.2010.079814. Epub 2010 Oct 4. 8. Chen QH, Deng W, Li XW, et.al. Novel CDH1 germline mutations identified in Chinese gastric cancer patients. World J Gastroenterol. 2013 Feb 14;19(6):909-16. 9. Oliveira C, Bordin MC, Grehan N, et.al. Screening E-cadherin in gastric cancer families reveals germline mutations only in hereditary diffuse gastric cancer kindred. Hum Mutat. 2002;19(5):510-7. 10. Petridis C, Shinomiya I, Kohut K, et.al. Germline CDH1 mutations in bilateral lobular carcinoma in situ. Br J Cancer. 2013 Dec 24. [Epub ahead of print] 11. Richards FM, McKee SA, Rajpar MH et.al. Germline E-cadherin gene (CDH1) mutations predispose to familial gastric cancer and colorectal cancer. Hum Mol Genet. 1999; 8(4):607-10. 12. Moran CJ, Joyce M, McAnena OJ. CDH1 associated gastric cancer: a report of a family and review of the literature. Eur J Surg Oncol. 2005;31(3):259-64. 13. More H, Humar B, Weber W, et.al. Identification of seven novel germline mutations in the human E-cadherin (CDH1) gene. Hum Mutat. 2007;28(2):203. 14. Humar B, Toro T, Graziano F et.al. Novel germline CDH1 mutations in hereditary diffuse gastric cancer families. Hum Mutat. 2002;19(5):518-25. 15. Kluijt I, Siemerink EJ, Ausems MG, Dutch Working Group on Hereditary Gastric Cancer. et.al. CDH1-related hereditary diffuse gastric cancer syndrome: clinical variations and implications for counseling. Int J Cancer. 2012;131(2):367-76. doi: 10.1002/ijc.26398. Epub 2011 Oct 23. 16. Guilford PJ, Hopkins JB, Grady WM, et.al. E-cadherin germline mutations define an inherited cancer syndrome dominated by diffuse gastric cancer. Hum Mutat. 1999; 14(3):249-55. 17. Shinmura K, Kohno T, Takahashi M, et.al. Familial gastric cancer: clinicopathological characteristics, RER phenotype and germline p53 and E-cadherin mutations. Carcinogenesis. 1999 Jun;20(6):1127-31. 18. Suriano G, Seixas S, Rocha J, et.al. A model to infer the pathogenic significance of CDH1 germline missense variants. J Mol Med (Berl). 2006;84(12):1023-31. Epub 2006 Aug 16. 19. Gayther SA, Gorringe KL, Ramus SJ, et.al. Identification of germ-line E-cadherin mutations in gastric cancer families of European origin. Cancer Res. 1998;58(18):4086-9. 20. Dussaulx-Garin L, Blayau M, Pagenault M, et.al. A new mutation of E-cadherin gene in familial gastric linitis plastica cancer with extra-digestive dissemination. Eur J Gastroenterol Hepatol. 2001;13(6):711-5. 21. Kaurah P, MacMillan A, Boyd N et. al. Founder and recurrent CDH1 mutations in families with hereditary diffuse gastric cancer. JAMA. 2007 ;297(21):2360-72. Epub 2007 Jun 3.
Downloaded From: https://jamanetwork.com/ on 10/02/2021 22. Xie ZM, Li LS, Laquet C,et. al. Germline mutations of the E-cadherin gene in families with inherited invasive lobular breast carcinoma but no diffuse gastric cancer. Cancer. 2011 15;117(14):3112-7. Epub 2011 Jan 26. 23. Benusiglio PR, Malka D, Rouleau E, et. al. CDH1 germline mutations and the hereditary diffuse gastric and lobular breast cancer syndrome: a multicentre study. J Med Genet. 2013 Jul;50(7):486-9. Epub 2013 May 25. 24. Mateus AR, Simões-Correia J, Figueiredo J, et.al. E-cadherin mutations and cell motility: a genotype-phenotype correlation. Exp Cell Res. 2009;315(8):1393-402. doi: 10.1016/j.yexcr.2009.02.020. Epub 2009 Mar 4. 25. Yamada H, Shinmura K, Ito H, et.al. Germline alterations in the CDH1 gene in familial gastric cancer in the Japanese population. Cancer Sci. 2011;102(10):1782-8. doi: 10.1111/j.1349-7006.2011.02038.x. Epub 2011 Aug 19. 26. Keller G, Vogelsang H, Becker I, et.al. Diffuse type gastric and lobular breast carcinoma in a familial gastric cancer patient with an E-cadherin germline mutation. Am J Pathol. 1999 Aug;155(2):337-42. 27. Brooks-Wilson AR, Kaurah P, Suriano G et. al. Germline E-cadherin mutations in hereditary diffuse gastric cancer: assessment of 42 new families and review of genetic screening criteria. J Med Genet. 2004 ;41(7):508-17. 28. Masciari S, Larsson N, Senz J et. al. Germline E-cadherin mutations in familial lobular breast cancer. J Med Genet. 2007;44(11):726-31. Epub 2007 Jul 27. 29. Frebourg T, Oliveira C, Hochain P, et. al. Cleft lip/palate and CDH1/E-cadherin mutations in families with hereditary diffuse gastric cancer. J Med Genet. 2006; 43(2):138-42. Epub 2005 Apr 14. 30. Suriano G, Oliveira C, Ferreira P, et.al. Identification of CDH1 germline missense mutations associated with functional inactivation of the E-cadherin protein in young gastric cancer probands. Hum Mol Genet. 2003;12(5):575-82. 31. Simões-Correia J, Figueiredo J, Lopes R,et.al. E-cadherin destabilization accounts for the pathogenicity of missense mutations in hereditary diffuse gastric cancer. PLoS One. 2012;7(3):e33783. doi: 10.1371/journal.pone.0033783. Epub 2012 Mar 21. 32. Kim S, Chung JW, Jeong TD,et.al. Searching for E-cadherin gene mutations in early onset diffuse gastric cancer and hereditary diffuse gastric cancer in Korean patients. Fam Cancer. 2013;12(3):503-7. doi: 10.1007/s10689-012-9595-6. 33. Yoon KA, Ku JL, Yang HK, et.al. Germline mutations of E-cadherin gene in Korean familial gastric cancer patients. J Hum Genet. 1999;44(3):177-80. 34. Ikonen T, Matikainen M, Mononen N, et.al. Association of E-cadherin germ-line alterations with prostate cancer. Clin Cancer Res. 2001;7(11):3465-71. 35. Garziera M, De Re V, Geremia S, et.al. A novel CDH1 germline missense mutation in a sporadic gastric cancer patient in north-east of Italy. Clin Exp Med. 2013;13(2):149-57. doi: 10.1007/s10238-012-0184-7. Epub 2012 Apr 29. 36. Pharoah PD, Guilford P, Caldas C; International Gastric Cancer Linkage Consortium. Incidence of gastric cancer and breast cancer in CDH1 (E-cadherin) mutation carriers from hereditary diffuse gastric cancer families. Gastroenterology. 2001;121(6):1348-53. 37. Matsukuma KE, Mullins FM, Dietz L, et.al. Hereditary diffuse gastric cancer due to a previously undescribed CDH1 splice site mutation. Hum Pathol. 2010;41(8):1200-3. 38. Johnston JJ, Rubinstein WS, Facio FM, et. al. Secondary variants in individuals undergoing exome sequencing: screening of 572 individuals identifies high-penetrance mutations in cancer-susceptibility genes. Am J Hum Genet. 2012 Jul 13;91(1):97-108. doi: .1016/j.ajhg.2012.05.021. Epub 2012 Jun 14. 39. Jonsson BA, Bergh A, Stattin P, et.al. Germline mutations in E-cadherin do not explain association of hereditary prostate cancer, gastric cancer and breast cancer. Int J Cancer. 2002 Apr 20;98(6):838-43. 40. Rogers WM, Dobo E, Norton JA, et.al. Risk-reducing total gastrectomy for germline mutations in E-cadherin (CDH1): pathologic findings with clinical implications.Am J Surg Pathol. 2008;32(6):799-809. 41. Guilford P, Hopkins J, Harraway J, et. al. E-cadherin germline mutations in familial gastric cancer. Nature. 1998;392(6674):402-5.
Downloaded From: https://jamanetwork.com/ on 10/02/2021 42. Tsukanov AC, Shelygin IuA, Kashnikov VN, et.al. [Molecular genetics study of hereditary predisposition to diffuse gastric cancer in Russian patients]. Vopr Onkol. 2013;59(5):580-4. 43. Kim HC, Wheeler JM, Kim JC, et.al. The E-cadherin gene (CDH1) variants T340A and L599V in gastric and colorectal cancer patients in Korea. Gut. 2000;47(2):262-7. 44. Zhang Y, Liu X, Fan Y, et.al. Germline mutations and polymorphic variants in MMR, E- cadherin and MYH genes associated with familial gastric cancer in Jiangsu of China. Int J Cancer. 2006;119(11):2592-6. 45. Roviello F, Corso G, Pedrazzani C,et.al. Hereditary diffuse gastric cancer and E-cadherin: description of the first germline mutation in an Italian family. Eur J Surg Oncol. 2007;33(4):448-51. Epub 2006 Nov 28. 46. Oliveira C, Ferreira P, Nabais S, et.al. E-Cadherin (CDH1) and p53 rather than SMAD4 and Caspase-10 germline mutations contribute to genetic predisposition in Portuguese gastric cancer patients. Eur J Cancer. 2004;40(12):1897-903. 47. Hebbard PC, Macmillan A, Huntsman D, et al. Prophylactic total gastrectomy (PTG) for hereditary diffuse gastric cancer (HDGC): the Newfoundland experience with 23 patients. Ann Surg Oncol. 2009;16:1890–1895 48. Wang Y, Song JP, Ikeda M, et.al. Ile-Leu substitution (I415L) in germline E-cadherin gene (CDH1) in Japanese familial gastric cancer. Jpn J Clin Oncol. 2003;33(1):17-20. 49. Mayrbaeurl B, Keller G, Schauer W, et.al. Germline mutation of the E-cadherin gene in three sibling cases with advanced gastric cancer: clinical consequences for the other family members. Eur J Gastroenterol Hepatol. 2010;22(3):306-10 50. Barber ME, Save V, Carneiro F, et. al. Histopathological and molecular analysis of gastrectomy specimens from hereditary diffuse gastric cancer patients has implications for endoscopic surveillance of individuals at risk. J Pathol. 2008;216(3):286-94 51. Jiang Y, Wan YL, Wang ZJ, et.al. [Germline E-cadherin gene mutation screening in familial gastric cancer kindreds]. Zhonghua Wai Ke Za Zhi 2004 7;42(15):914-7. Chinese. 52. Schrader KA, Masciari S, Boyd N,et.al. Hereditary diffuse gastric cancer: association with lobular breast cancer. Fam Cancer. 2008;7(1):73-82 53. Sugimoto S, Yamada H, Takahashi M, et.al. Early-onset diffuse gastric cancer associated with a de novo large genomic deletion of CDH1 gene. Gastric Cancer. 2013Jun 29. [Epub ahead of print] 54. Rodriguez-Sanjuan JC, Fontalba A, Mayorga M, et.al. A novel mutation in the E-cadherin gene in the first family with hereditary diffuse gastric cancer reported in Spain. Eur J Surg Oncol. 2006 Dec;32(10):1110-3. Epub 2006 Jul 25. 55. Keller G, Vogelsang H, Becker I, et.al. Germline mutations of the E-cadherin(CDH1) and TP53 genes, rather than of RUNX3 and HPP1, contribute to genetic predisposition in German gastric cancer patients. J Med Genet. 2004;41(6):e89. 56. Salahshor S, Hou H, Diep CB, et.al. A germline E-cadherin mutation in a family with gastric and colon cancer. Int J Mol Med. 2001;8(4):439-43. 57. Shah MA, Salo-Mullen E, Stadler Z, et.al. De novo CDH1 mutation in a family presenting with early-onset diffuse gastric cancer. Clin Genet. 2012;82(3):283-7. doi: 10.1111/j.1399- 0004.2011.01744.x. Epub 2011 Aug 2. 58. Ascaño JJ, Frierson H Jr, Moskaluk CA, et.al. Inactivation of the E-cadherin gene in sporadic diffuse-type gastric cancer. Mod Pathol. 2001;14(10):942-9. 59. Sarrió D, Moreno-Bueno G, Hardisson D, et.al. Epigenetic and genetic alterations of APC and CDH1 genes in lobular breast cancer: relationships with abnormal E-cadherin and catenin expression and microsatellite instability. Int J Cancer. 2003;106(2):208-15. 60. Shenoy S, Palmer C, Dunsworth T et.al. Hereditary diffuse gastric cancer: genetics, prophylactic total gastrectomy, and follow up. Gastrointestinal Cancer: Targets and Therapy 2011:1 15–19 61. Van Domselaar F, Correa D, Vaccaro C, et.al. [Hereditary diffuse gastric cancer (HDGC): presentation of a family with a new mutation of the CDH1 gene]. Acta Gastroenterol Latinoam. 2007;37(3):158-63. Spanish. 62. Ghaffari SR, Rafati M, Sabokbar T, et.al. A novel truncating mutation in the E-cadherin gene in the first Iranian family with hereditary diffuse gastric cancer. Eur J Surg Oncol. 2010;36(6):559-62. doi: 10.1016/j.ejso.2010.04.001. Epub 2010 May 14.
Downloaded From: https://jamanetwork.com/ on 10/02/2021 63. Charlton A, Blair V, Shaw D, et.al. Hereditary diffuse gastric cancer: predominance of multiple foci of signet ring cell carcinoma in distal stomach and transitional zone. Gut. 2004;53(6):814-20. 64. Caron O, Schielke A, Svrcek M,et.al. Usefulness of prophylactic gastrectomy in a novel large hereditary diffuse gastric cancer (HDGC) family. Am J Gastroenterol. 2008 Aug;103(8):2160-1 65. Yabuta T, Shinmura K, Tani M,et.al. E-cadherin gene variants in gastric cancer families whose probands are diagnosed with diffuse gastric cancer. Int J Cancer. 2002 Oct 10;101(5):434-41.
Downloaded From: https://jamanetwork.com/ on 10/02/2021