Download a genetic and epidemiological study of hereditary non

A GENETIC AND EPIDEMIOLOGICAL
STUDY OF HEREDITARY NON-POLYPOSIS
COLORECTAL CANCER IN COLORECTAL
CARCINOMA PATIENTS PRESENTING TO
A TERTIARY CARE HOSPITAL
Department of SGE
NIMS Hyderabad
23-03-2009
THIS STUDY IS BEING DONE
IN COLLABORATION WITH
CENTER FOR DNA FINGERPRINTING AND DIAGNOSTICS
(CDFD)
Abbreviations
 CRC
colorectal carcinoma
 HNPCC Hereditary Non-polyposis Colorectal Cancer
 APC
 MSI
 MMR
 PCR
adenomatous polyposis coli
micro satellite instability
mismatch repair
polymerase chain reaction
Outline




Background
Aim
Materials
Methods
 DNA isolation
 Genomic
 Tumor
 Microsatellite instability
 Immunohistochemistry for MMR genes
 PCR
 Sanger’s sequencing
What is HNPCC
(Lynch Syndrome)
 Definition is based on demonstration of
mutation in MMR genes
 No clinical definition
 Amsterdam’s criteria helps in clinically
defining HNPCC
Amsterdam II criteria:
What is Amsterdam's criteria
What are Bethesda guidelines
Tests for HNPCC
 Screening tests
 Confirmatory tests
Background
 Colorectal carcinoma in India is biologically a
different disease compared to western
population.





Occurs in young age
Rectal carcinoma constitutes half of the cases
Incidence is less
Majority of young CRC patients are sporadic
Late presentation
 No studies on CRC genetics from India
 No reported cases of HNPCC from India
Background
 90% of CRC in west occur after 50years
 India 50-60% <50years
 WHY?
Background
 In west 10% occur in young
 30% of these are HNPCC
 This stimulated us to study about HNPCC
and colorectal carcinoma in our own
population
Vogelstein’s pathway
Background
 HNPCC is caused by MSI, the hallmark of
HNPCC
 MSI is caused by MMR genes mutation
 MMR genes are also called caretaker genes
 MMR gene mutation causes HNPCC
Background
 Bethesda guidelines are for MSI screening
 It is likely that Bethesda guidelines are not
useful in Indian population.
 This hypothesis will be either proved or
disproved by our study.
Life time risk of cancer in
HNPCC
Primary objective
 To study the incidence of HNPCC in CRC




patients presenting to NIMS.
To study the mutations causing HNPCC
To identify any new mutation
Study the usefulness of MSI & IHC in
diagnosing HNPCC
To screen first degree relatives of HNPCC
patients for MMR mutation
Secondary objective
 To assess age distribution , sex distribution ,
site distribution of CRC
 To assess pathologic characteristics of CRC
 To assess stage of presentation
INCLUSION CRITERIA
1. CRC, diagnosed in patients < 50 years
2. Synchronous or metachronous CRC or other
tumors associated with HNPCC, regardless
of age
3. CRC with of MSI-H or specific histology
diagnosed < 60 years.
4. HNPCC related cancer <50yrs in 1 first
degree relative
5. HNPCC associated tumor at any age in two
first- or second-degree relatives
Exclusion criteria
 All patients with neoadjuvant CT/RT are
excluded from study.
 All patients who are HIV/HBsAg +ve.
 Patients with FAP
Methods
 DNA isolation
 Genomic
 Tumor
 Microsatellite instability
 Immunohistochemistry for MMR genes
 PCR
 Sanger’s sequencing
Case 1
 24yr female with bleeding PR
 Sigmoid carcinomas at two synchronous
locations-T2N0M0
 Left hemi-colectomy T2N0M0 x2
 3 yrs later - multiple carcinomas
 Caecum –T3N0M0
 Duodenum-T2N0M0
 Jejunum –T2N0M0
DNA isolation
 Genomic DNA
 Blood – easy to isolate
 Normal tissue
 Tumor DNA
 H&E stained 5micron slide
 Identify tumor cells
 Scrape the cells
 Isolate DNA
DNA isolation from blood
 3ml blood
 Potassium EDTA bottle
 Store at 40⁰ c
 Should not be frozen
 If frozen - should not be thawed
 Blood should not be stored for more than 7
days because yield will come down
DNA isolation from blood
 Centrifuge blood after adding erythrocyte






lysis buffer –30min
Centrifuge @10000 rpm for 10min
Discard supernatant fluid
WBC pellet
Add proteinkinase , DDS
Incubate at 37⁰ C for 12hours
Purification of DNA
DNA isolation from blood
 Purification is a lengthy procedure
 WBC pellet is digested with proteinkinase
 Intra cellular contents are released
 Repeated purification and centrifugation
 Protein heaviest at bottom
 DNA next to protein
 Need careful extraction
 Once DNA is isolated it can be stored at -20⁰ c
for a long time for many reactions
 Genomic DNA is highly concentrated
 Should be very careful otherwise it will
contaminate other reactions
PCR
 First described in 1985, Nobel Prize for Kary
Mullis in 1993
 The technique was made possible by the
discovery of Taq polymerase, the DNA
polymerase that is used by the bacterium
Thermus aquaticus, discovered in hot springs.
PCR
 The primary materials, or reagents, used in PCR
are:
 DNA nucleotides, the building blocks for the new DNA
 Template DNA, the DNA sequence that you want to
amplify
 Primers, single-stranded DNAs between 20 and 50
nucleotides long (oligonucleotides) that are
complementary to a short region on either side of the
template DNA
 DNA polymerase, a heat stable enzyme that drives, or
catalyzes, the synthesis of new DNA
PCR
Sample tray and micropipettor. Each tray holds 96
samples
PCR
 The result is a dramatic amplification of a the
DNA that exists between the primers.
 The amount of amplification is 2 raised to the
n power;
 n represents the number of cycles that are
performed.
 After 20 cycles, this would give approximately
1 million fold amplification. After 40 cycles
the amplification would be 1 x 1012
STEP-1
 Denaturation at around 94°C :
 During the denaturation, the double strand
melts open to single stranded DNA, all
enzymatic reactions stop (for example the
extension from a previous cycle).
STEP-2
 Annealing at around 54°C :
 Hydrogen bonds are constantly formed and
broken between the single stranded primer
and the single stranded template. If the
primers exactly fit the template, the
hydrogen bonds are so strong that the primer
stays attached
STEP-3
 Extension at around 72°C :
 The bases (complementary to the template)
are coupled to the primer on the 3' side (the
polymerase adds dNTP's from 5' to 3', reading
the template from 3' to 5' side, bases are
added complementary to the template)
THERMOCYCLER
Sitamahalakshmi
AGAROSE GEL ELECTROPHORESIS
A method used to
separate macromolecules
like proteins and nucleic
acids (ie DNA/RNA)
based
on their size and electric
charge
Sequencing
 “Sequencing” means finding the order of
nucleotides on a piece of DNA .
 Nucleotide order determines Amino acid
order, and by extension, protein structure and
function (proteomics)
 An alteration in a DNA sequence can lead to
an altered or non functional protein, and
hence to a harmful effect
Sequencing
 Historically there are two main methods of
DNA sequencing:

Maxam & Gilbert, using chemical
sequencing

Sanger, using dideoxynucleotides.
 Modern sequencing equipment uses the
principles of the Sanger technique.
The Sanger Technique
 Uses dideoxynucleotides (dideoxyadenine,
dideoxyguanine, etc)
 These are molecules that resemble normal
nucleotides but lack the normal -OH group.
 Because they lack the -OH (which allows
nucleotides to join a growing DNA strand),
replication stops.
Normally, this would
be where another phosphate
Is attached, but with no -OH
group, a bond can not form
and replication stops
The Sanger method requires
 Multiple copies of single stranded template DNA
 A suitable primer (a small piece of DNA that can
pair with the template DNA to act as a starting
point for replication)
 DNA polymerase (an enzyme that copies DNA,
adding new nucleotides to the 3’ end of the
template
 A ‘pool’ of normal nucleotides
 A small proportion of dideoxynucleotides labeled
in some way ( radioactively or with fluorescent
dyes)
 The template DNA pieces are replicated,
incorporating normal nucleotides, but
occasionally and at random dideoxy (DD)
nucleotides are taken up.
 This stops replication on that piece of DNA
 The result is a mix of DNA lengths, each ending
with a particular labeled DDnucleotide.
 Because the different lengths ‘travel’ at different
rates during electrophoresis, their order can be
determined.
A Nanodrop readout :DNA purity & concentration
is checked by spectrophotometer
Preparing the wells
 The Sample wells are loaded with DNA to be
sequenced. Great care needs to be taken to
ensure that each sample goes into its
assigned well.
 Reagents are added (water, dye, primers) in
required amounts
 The sample wells are ‘spun’ to ensure that
the DNA and reagents are mixed and at the
bottom of the sample wells.
The samples are run
through a cycle
sequencing process to
get the fluorescent
dyes incorporated by
the DNA.
The DNA and reagents
are alternately heated
and cooled over a
2 1/2 hour period.
Mlh1 amplicons


Exon 1; amplicon 1
5’ UTR:
Tggggctggatggcgtaagctacagctgaaggaagaacgtgagcacgaggcactgaggtg
 Exon 1:
ATTGGCTGAAGGCACTTCCGTTGAGCATCTAGACGTTTCCTTGGCTCTT
CTGGCGCCAAA
ATGTCGTTCGTGGCAGGGGTTATTCGGCGGCTGGACGAGACAGTGGTG
AACCGCATCGCG
GCGGGGGAAGTTATCCAGCGGCCAGCTAATGCTATCAAAGAGATGATT
GAGAACTG
 Intron1:
gtacggagggagtcgagccgggctcacttaagggctacgacttaacgggccgcgtcactc
aatggcgcggacacgcctctttgcccgggcagaggcatgtacagcgcatgcccacaacgg
cggaggccgccgggttccctgacgtgccagtcaggccttctccttttccgcagaccgtgtgt
Microsatellites
 Microsatellites also known as variable
nucleotide tandem repeats (VNTRs), are loci
throughout the genome in which a short
motif, such as a dinucleotide or
mononucleotide sequence, is repeated at
least several times.
 AAAAAAAAAAAAAAAAAAAAAAAAAAAAA
 CACACACACACACACACACACACACACACA
Microsatellites
 Microsatellites refer to long segments of
nontranscribed DNA that are composed of a
repeating mononucleotide (eg,
AAAAAAAAAAAAAAAA) or dinucleotide
sequences (eg, GTGTGTGTGTGTGT).
 These long DNA sequences provide a simple
indicator of genetic mutation rate and risk
because mutations are readily apparent in
these long repeating sequences.
MSI
 MSI is the hallmark of lynch syndrome
occuring in >90% of lynch syndrome.
Aaltonen L.A et.al science 1993;260:812-816
Altonen et.al cancer res-1994;54;1645
MSI
 Only 20% to 25% of patients who have MSI-H
colon cancer have an MMR gene mutation
and Lynch syndrome .
Barnetson RA, Tenesa A, et al. . N Engl J Med 2006;356(26):
2751–63.
MSI
 The finding of microsatellite instability should
lead to genetic testing because it is found in
more than 90% of patients who have Lynch
syndrome,but in only 15% to 20% of patients
who have sporadic colon cancer.
 ICG-HNPCC
Mismatch Repair Genes
 MMR are called caretaker genes because of
their important role in policing the integrity
of the genome and correcting DNA
replication errors.
 MMR genes that undergo a loss of function
contribute to carcinogenesis by accelerating
tumor progression.
Mismatch Repair Genes
 Mutations in MMR genes produce
microsatellite instability.
 Microsatellites are repetitive sequences of
DNA that appear to be randomly distributed
throughout the genome.
Mismatch Repair Genes
 Stability of microsatellite sequences is a good
measure of the general integrity of the genome.
 MMR gene mutations result in errors in S phase
when DNA is newly synthesized and copied.
 Microsatellite instability exists in 10% to 15% of
sporadic tumors and in 95% of tumors in patients
with HNPCC. Even so, only 50% of patients
diagnosed with HNPCC have readily identifiable
MMR mutations.
Mismatch Repair Genes
 Stability of these sequences is a good measure of
the general integrity of the genome.
 MMR gene mutations result in errors in S phase
when DNA is newly synthesized and copied.
 Microsatellite instability exists in 10% to 15% of
sporadic tumors and in 95% of tumors in patients
with HNPCC. Even so, only 50% of patients
diagnosed with HNPCC have readily identifiable
MMR mutations.
Age
</=20
21-30
31-40
41-50
51-60
61-70
71-80
total
Number
2
4
9
10
8
4
3
41
Observations
 Total number of cases till now
 Male
 Female
 <50 yrs
 >50 yrs
 Rectal ca
 Colonic
21
20
25
16
20
21
41
Site
Caecum
Ascending colon
Hepatic flx.
Transverse colon
Splenic flex.
Descending colon
Sigmoid colon
Rectum
Total
Number
4
4
2
+1
1
4+1
3
20
41
Sex distribution
number
male
female
FAMILY HYSTORY
 SIGNIFICANT IN 10 PATIENTS
 1st degree relatives with cancer 8 patients
 Second primary in 2 patients
Patient no 14
Patient no 14
Patient no 6,8
Patient no 2,3,4,5 for 5 exons
Patient no 2,3,4,5 for 5 exons
Our plan of work for next 3 months
 12 patients suspected HNPCC – priority
 Complete investigation
 DNA isolation
 Tumor
 Genomic





MSI
IHC
DNA amplification of specific gene all exons
Sequencing
Final result
Next
 All patients who are included in study –
 MSI
 IHC
 MSI+
 IHC +
 DNA-SEQUENCING
 FINAL RESULT
THANK YOU