Translational Molecular Genetics Harold Frucht, M.D. Division of Digestive and Liver Diseases...
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Transcript of Translational Molecular Genetics Harold Frucht, M.D. Division of Digestive and Liver Diseases...
Translational Molecular Genetics
Harold Frucht, M.D.Division of Digestive and Liver Diseases
Columbia University
April 14, 2005
Sporadic Cases (~70%)
Hamartomatous Polyposis Syndromes
(<1%)
Familial Adenomatous
Polyposis (<1%)
Hereditary Non-Polyposis Colorectal
Cancer (3-5%)
Cases with Familial Risk (~25%)
Mechanisms of Cancer Gene Action
M (mitosis)M (mitosis)
GG11
(cell growth)(cell growth)
S (synthesis)S (synthesis)
GG00 (resting) (resting)
Oncogenes:Oncogenes: promote cell growthpromote cell growth
Suppressor genes:Suppressor genes:inhibit cell cycle; promote apoptosis inhibit cell cycle; promote apoptosis
GG22
Mismatch repairMismatch repairgenes:genes: correct correctreplication errorsreplication errors
Modifier genes:Modifier genes:influence cell functioninfluence cell function
Calvert & Frucht, Ann Int Med, 2002;137:603-613
Somatic Mutation (Sporadic Disease)
2 normal copiesof the genein every cell
One copymutatedin cell(1st hit is acquired)
Second copymutatedin cell(2nd hit is also acquired)
Calvert & Frucht, Ann Int Med, 2002;137:603-613
Germline Mutation (Inherited Disease)
One copymutatedin every cell(1st hit is inherited)
Second copymutatedin cell(2nd hit is acquired)
Calvert & Frucht, Ann Int Med, 2002;137:603-613
CIMP = CpG Island Methylator Phenotype
• epigenetic phenomenom
• hypermethylation of the promoter region of the hMLH1 gene
• responsible for MSI in 15% of sporadic colon cancers
APC GeneI1307K Mutation
Germline Mutation of Codon 1307
T-A Transversion(Leucine - Isoleucine Substitution)Causes hypermutability in adjacent sequences resulting in somatic alterations which predispose to colon cancer
Incidence in Ashkenzai Jews 6.1 %Lifetime risk of colon cancer in people with mutation 18-30 %
28%
10%
6%
0%
0% 10% 20% 30%
% with mutation
Non-Jewishpopulation
All Ashkenazi Jews
UnselectedAshkenazi Jews w/
CRC
Ashkenazi Jews w/CRC and familyhistory of CRC
Calvert & Frucht, Ann Int Med, 2002;137:603-613
Inherited Syndromes Predisposing to Colon Cancer
GeneLifetime
Risk of CRC
Familial Adenomatous Polyposis apc ~100%
Hereditary Non-Polyposis Colon Cancer MMR >80%
Peutz-Jeghers Syndrome STK11 2-13%
Juvenile Polyposis SMAD4 ~<50%
Cowden Syndrome PTEN small
Clinical Criteria for Hereditary Non-Polyposis Colorectal Cancer
Amsterdam criteria At least three relatives with colon cancer and all of the following:•One should be the first-degree relative of the other two•Two successive generations should be affected•At least one colon cancer should be diagnosed before the age of 50•FAP should be excluded
Modified Amsterdam criteria
As for the Amsterdam criteria except that the cnacers need to be an HNPCC-associated cancer (colon, endometrium, small bowel, ureter, renal pelvis) instead of specifically colon cancer.
Bethesda criteria Families meeting the Amsterdam criteriaIndividuals with 2 HNPCC-associated cancers, including synchronous or
metachronous cancersIndividuals with colon cancer and a first-degree relative with an HNPCC-
associated cancer and/or colonic adenoma; 1 cancer diagnosed at age < 45 years and the adenoma diagnosed at age < 40 years
Individuals with colon or endometrial cancer diagnosed at < 45 yearsIndividuals with right-sided colon cancer having an undifferentiated pattern
(solid/cribiform) or signet cell histopathology diagnosed at <45 yearsIndividuals with adenomas diagnosed at < 40 years
Calvert & Frucht, Ann Int Med, 2002;137:603-613
Autosomal Dominant Inheritance
• Each child has 50% chance of inheriting the mutation
• No “skipped generations”
• Equally transmitted by men and women
Normal
Affected
HEREDITARY COLON CANCER - Germline Mutation
SPORADIC COLON CANCER - Somatic Mutation
FAMILIAL COLON CANCER - Germline Mutation Causing Hypermutability and Subsequent Somatic Mutation
Germline Mutations - Inherited Disease
APC - Familial Polyposis ColiMMR - HNPCC (Lynch Syndrome)MYH- Familial Polyposis Coli
Somatic Mutations - Sporadic Disease
Oncogenes: myc, ras, srcTumor Suppressors: p53, DCC, APC, MCCMismatch Repair Genes: MSH2, MSH3, MSH6,
MLH1, PMS1, PMS2
“Genetic Polymorphisms” - Familial Disease
APC - Familial Colon Cancer
APC Gene
Germline Mutation - Familial Polyposis Coli
Somatic Mutation - Sporadic Colon Cancer
I1307K Germline Mutation - Familial Colon Cancer
Failure to Diagnose Hereditary Colorectal Cancer and Its Medicolegal Implications
A Hereditary Nonpolyposis Colorectal Cancer Case
Henry T. Lynch, M.D.,* Jane Paulson, J.D.,† Matthew Severin, J.D., Ph.D.,* Jane Lynch, B.S.N.,* Patrick Lynch, J.D., M.D.‡
From the *Department of Preventive Medicine, Creighton University School of Medicine, Omaha, Nebraska, †Paulson & Baisch, Portland, Oregon, and ‡Department of GI Oncology/Digestive Diseases, MD Anderson Cancer Center, Houston, Texas
Diseases of the Colon & Rectum 1999: Jan 42(1); 31-35
The Use and Interpretation of Commercial APC Gene Testing for Familial Adenomatous Polyposis
Francis M. Giardiello, M.D., Jill D. Brensinger, M.S., Gloria M. Petersen, Ph.D., Michael C. Luce, Ph.D., Linda M. Hylind, B.S., R.N., Judith A. Bacon, B.S., Susan V. Booker, B.A., Rodger D. Parker, Ph.D., and Stanley R. Hamilton, M.D.
From the Departments of Medicine (F.M.G., J.D.B., L.M.H., J.A.B., S.V.B.) and Pathology (S.R.H.) and the Oncology Center (F.M.G., G.M.P., S.R.H.), John Hopkins University School of Medicine, Baltimore; the Departments of Epidemiology (G.M.P.) and Health Policy and Management (R.D.P.), John Hopkins University School of Hygiene and Public Health, Baltimore; and the Department of Molecular Biology, LabCorp, Research Triangle Park, N.C. (M.C.L.). Address reprint requests to Dr. Giardiello at Blalock 935, Johns Hopkins Hospital, 600 N. Wofe St., Baltimore, MD 21287-4461.
NEJM 1997; 336:823-27
Background The use of commercially available tests for genes linked to familial cancer has aroused concern about the impact of these tests on patients. Familial adenomatous polyposis is an autosomal dominant disease caused by a germ-line mutation of the adenomatous polyposis coli (APC ) gene that causes colorectal cancer if prophylactic colectomy is not performed. We evaluated the clinical use of commercial APC gene testing.
Methods We assessed indications for APC gene testing, whether informed consent was obtained and genetic counseling was offered
before testing, and the interpretation of the results through telephone interviews with physicians and genetic counselors in a nationwide sample of 177 patients from 125 families who underwent testing during 1995.
Results Of the 177 patients tested, 83.0 percent had clinical features of familial adenomatous polyposis or were at risk for the disease — both valid indications for being tested. The appropriate strategy for presymptomatic testing was used in 79.4 percent (50 of 63 patients). Only 18.6 percent (33 of 177) received genetic counseling before the test, and only 16.9 percent (28 of 166) provided written informed consent. In 31.6 percent of the cases the physicians misinterpreted the test results. Among the patients with unconventional indications
for testing, the rate of positive results was only 2.3 percent (1 of 44).
Conclusions Patients who underwent genetic tests for familial adenomatous polyposis often received inadequate counseling and would have been given incorrectly interpreted results. Physicians should be prepared to offer genetic counseling if they order genetic tests.
Microsatellite Instability (MSI)
• 10% - 15% of sporadic tumors have MSI
• 95% of HNPCC tumors have MSI
• Routine MSI assays soon available
Normal MSI tumor
Electrophoresis gel
Genetic Testing Lab Methods
DNADNA
mRNAmRNA
ProteinProtein
GelGel
NormalNormal MutatedMutatedLinkage Analysis:Linkage Analysis: Probability of Inheritance. Probability of Inheritance.MSI Assays:MSI Assays: Highly predictive for MMR mutation. Highly predictive for MMR mutation.Gene Sequencing:Gene Sequencing: Approaches 100%. Approaches 100%.DGGE:DGGE: Highly sensitive (>90%).SSCP:SSCP: Detects 60%-95% of mutations.
Protein Truncation:Protein Truncation: Point of mutation dependent. Point of mutation dependent. Immunohistochemistry:Immunohistochemistry: Antibody dependent. Antibody dependent.
Calvert & Frucht, Ann Int Med, 2002;137:603-613
History Suggestive of Inherited Colon Cancer
Probable HNPCCProbable FAP
APC genetic test of anaffected individual
Genetic test of anaffected individual
negative positive positive negative
annual endoscopy for all family
members
APC gene testing of family members
HNPCC genetic testing of family
members
continued high risk colon cancer screening of the
individual and all family members
no adenomas
adenomasannual endoscopy
prophylactic colectomy
continued survellance for rectal adenomas and extra-colonic tumors.
consider chemoprevention.
positive
annual endoscopy
no adenomas
negative
Colon Cancer screening as
recommended for the general population
positivenegative
negativepositivefor colon
cancer
colectomy
Calvert & Frucht, Ann Int Med, 2002:137;603-613
FAMILY JW-3911-17-94
Legend: = male; = female; = deceased; = probandSolid figures = cancer; BR = breast cancer; CO = colon cancer; LU = lung cancer;EN = endometrial cancer; number refers to age diagnosis
60heart
disease
8832 52
70’sBR, 70
70’sBR, 70
48
heartdisease
85 60’s
LU, 60
9072
CO, 72
84
uBR, 54
50EN, 42
53EN, 45
2927
CO, 26ascending,Dukes, C
25
Calvert & Frucht, Ann Int Med, 2002:137;603-613
FAMILY JW-379-7-94
Legend: = male; = female; = deceased; = probandSolid figures = cancer; CO = colon cancer; EN = Endometrial cancer; BL = Bladder cancer; BO = Bone cancer; RE = Rectal cancer; number refers to age diagnosis
70 77CO, 59EN, 76
75 90
68RE, 68
73 65CO, 39CO, 64
73BO, 73
4746
CO, 37
27 21
67CO, 39BL, 66
12
65EN, 65
60’s, 70’s
49
Calvert & Frucht, Ann Int Med, 2002:137;603-613
Incidence of Pancreatic Cancer by Number of Affected First Degree Relatives
Klein AP, et al., Cancer Research 2004; 64: 2634-2638
Number of FDRs with Pancreatic Cancer
Incidence (per 100,000) in the U.S. Population
General U.S. (reference) 9
1 41
2 58
3 or more 288
• 10% of patients with pancreatic cancer have a familial aggregation or an inherited predisposition
Clinical Features of Inherited Cancer Syndromes
Feature FAP HNPCC
Age of onset Early Early
No. of adenomas > 100 < 10
Adenoma distribution Total Mainly right side
Cancer distribution Random Mainly right side
Other cancers Periampullary Endometrial, other
Lynch HT et al, Clinical Risk Factors for Colorectal Cancer
Cancer Family Syndromes
Colon (63 %)Endometrium (8/28 %)
Gastric (6 %)Biliopancreatic (4 %)Genitourinary (2 %)
Ovary (1/3 %)Breast (2/6 %)Sarcomas (2 %)Skin (2 %)Small Bowel (1 %)Lung (1 %)Other (2 %)
GENETIC TESTING FOR FAP
1. Linkage Analysis
2. In vitro truncated protein testing (transcription - translation method)
3. Mutation Testing
GENETIC TESTING FOR HNPCC
1. Linkage Analysis
2. Truncated Protein Testing
3. Mutation Testing
4. Microsatellite Instability Testing
Clincial Cancer Screening Recommendations*
Colon Cancer Screening Recommendations
RISK SCREENINGMODALITY
AGE AT WHICHTO BEGIN
FREQUENCY
Average FOBTSigmoidoscopyFOBT and SigmoidoscopyDCBEColonoscopy
5050505050
annuallyevery 5 yearsevery 5 yearsevery 5-10 yearsevery 10 years
First-degree relative with colon cancer or adenomatous polyp at age 60 years
Same as for average risk individuals
40 same as for average risk individuals
Two or more first-degree relatives with colon cancer or adenomatous polyp at age < 60 years
Colonoscopy preferred 40 or 10 years younger than the earlier diagnosis
every 3-5 years
FAP Sigmoidoscopy 10-12 years annually
HNPCC Colonoscopy 20-25 years or 10 years younger than the earliest colon
cancer diagnosis
every 1-2 years
Extracolonic Cancer Screening Recommendations
FAP:Duodenal cancer EGD 20-25 years every 1-3 years
HNPCC:Endometrial and
ovarian cancer
Gastric cancer
Pelvic examTrans vaginal ultrasound
EGD
25-35 years
30-35 years
every 1-2 years
every 1-2 yeas
*DCBE = double contrast barium enema; EGD = esophagogastroduodenoscopy; FOBT = fecal occult blood test.