Clinicopathological and Molecular Profiles of Colorectal Tumours with BRAF mutation Weiqi Li BSc THE UNIVERSITY OF WESTERN AUSTRALIA 2006 This thesis is presented for the degree of Master of Medical Sciences at the University of Western Australia Supervisor: Associate Professor Barry Iacopetta School of Surgery and Pathology, University of Western Australia
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Clinicopathological and Molecular Profiles of
Colorectal Tumours with BRAF mutation
Weiqi Li BSc
THE UNIVERSITY OF WESTERN AUSTRALIA
2006
This thesis is presented for the degree of Master of Medical
Sciences at the University of Western Australia
Supervisor: Associate Professor Barry Iacopetta
School of Surgery and Pathology, University of Western Australia
2
Abstract
Introduction
BRAF is a member of the RAF family that encodes serine/threonine kinases of
the RAS/RAF/MAP kinase pathway. Recently, the BRAF V600E hotspot
mutation has been implicated in about 10% of colorectal cancers (CRC). It
occurs frequently in CRC with microsatellite instability (MSI+) caused by
promoter hypermethylation of the mismatch repair gene hMLH1, but has never
been observed in MSI+ tumours from patients with the familial CRC syndrome
referred to as hereditary nonpolyposis colorectal cancer (HNPCC). This opens
the possibility of using BRAF mutation screening to assist in the detection of
HNPCC individuals at the population level. BRAF mutations are inversely
associated with KRAS mutations and could define a subgroup of CRC with
distinctive phenotypic features.
Aims
The primary aim of this study was to identify the clinicopathological and
molecular features of CRC with BRAF mutation. The secondary aim was to
determine the frequency of BRAF mutation in CRC from younger patients who
were being screened as part of a population-based study into the prevalence of
HNPCC in the state of Western Australia.
Methods
A consecutive and well characterized series of 275 stage I-IV colorectal
tumours was evaluated for BRAF, KRAS and TP53 mutations, as well as MSI. A
large (n=780) series of CRCs from young (<60 years) patients was also
analyzed for BRAF mutation and MSI. All mutations and MSI status were
3 determined using fluorescent-single stranded conformation polymorphism (F-
SSCP) analysis.
Results and Conclusions
BRAF mutations were identified in 8.4% of a consecutive series of CRC. These
were mutually exclusive with KRAS mutations but no clear association with the
presence of TP53 mutation was observed. Mutations in BRAF were 5-10-fold
more frequent in tumours located in the proximal colon and having poor
histological grade, mucinous appearance and the presence of infiltrating
lymphocytes. BRAF mutant tumours were also 10-fold more likely to be MSI+
and frequently methylated. Such morphological features remained after
stratification for MSI and methylator phenotypes, suggesting that BRAF
mutation identifies a CRC subgroup with distinctive phenotypic properties
independently of MSI status.
Amongst 55 MSI+ cases identified in younger (<60 yrs) patients from the
HNPCC screening study, only 5 (9%) harboured a BRAF mutation. These could
therefore be excluded from further follow-up as possible HNPCC individuals.
Similar strong associations between BRAF mutation and proximal tumour site,
poor histological grade and mucinous appearance were found for younger and
older patients. In contrast, BRAF mutations were far more common in MSI+
tumours from older patients (50% vs 9%, P<0.0001). This important observation
suggests that the molecular phenotype of MSI+ tumours varies according to
patient age.
4 Our study has clarified the clinicopathological and molecular features of CRC
with BRAF mutations. It also provides evidence that associations between
BRAF mutation and MSI+ are age-related. Incorporation of BRAF mutation
analysis for young (<60 years) CRC patients could aid in further refinement of
population-based screening programs for HNPCC.
5 PUBLICATIONS ARISING FROM THIS THESIS
1. Li WQ, Kawakami K, Ruszkiewicz A, Bennett G, Moore J, Iacopetta B
(2006) BRAF mutations are associated with distinctive clinical,
pathological and molecular features of colorectal cancer independently of
microsatellite instability status. Molecular Cancer 5:2
2. Iacopetta B, Li WQ, Grieu F, Ruszkiewicz A, Kawakami K (2006) BRAF
mutation and gene methylation frequencies of colorectal tumours with
microsatellite instability increase markedly with patient age. Gut (in
press)
6 ACKNOWLEDGEMENTS
It has been a great pleasure conducting my Master of Medical Science in the
Department of Surgery and Pathology. The past year and a half has been a
wonderful journey filled with joy and warmth brought about by many people.
First of all, I would like to extend my gratitude to my supervisor Barry Iacopetta
for his excellent supervision that is not only filled with scientific knowledge but
also with an abundance of patience, kindness and motivation. His amicable
spirit has created an excellent studying environment for my master degree.
I would also like to thank Fabienne Grieu for her inexhaustible assistance
around the laboratory. Her ever-smiling face made lab work felt so much less
tedious and there is always plenty of coffee and cookies to nourish my tired
brain! Thank you for your wonderful friendship!
Natasha Watson for her assistance in gathering the tissue samples.
Not forgetting Sophia Ang for her friendship, thought-provoking discussions and
lunch companionship!
Everyone else in the department including Shaoying Li, Norman Rong and
Maggie Weedon for assisting me in one way or another.
The team at the Oncology Research Institute, National University of Singapore,
for their advice and friendship, including Dr. Richie Soong, Nur Diyanah Anuar,
Peiyi Chong, Swee Siang Ng, Michelle Goh and Tiling Chang.
7 SPECIAL DEDICATION
In Loving Memory of My late Mother,
Even though you knew you were losing the battle to cancer, your lovely smile
never ceases. That, together with your love, has always been an inspiration to
me. I miss you Mum!
To My Dad and Little Brother,
The past two years have been extremely difficult with mum’s passing. However,
your undying love, support and belief in me have carried me through the tough
times. I am blessed to have the both of you.. Thank you for staying strong for
me! I love you both!
To my Fiancé Ben,
You are the reason for me to smile again! Thank you for all the laughter and
CHAPTER 3: Results BRAF mutations are associated with distinctive clinical, pathological and molecular features of colorectal cancer independently of microsatellite instability status 343.1 Introduction 35
3.2 Results 36
3.3 Discussion 43
3.4 Conclusion 45
10
CHAPTER 4: Results BRAF mutation in tumours from patients aged <60 years 464.1 Introduction 46
4.2 BRAF mutations and clinicopathological features of
tumours in patients aged <60 years 48
4.3 Clinicopathological characteristics of tumours with BRAF
mutations: comparison between young and old
colorectal cancer patients 50
4.4 Discussion 52
CHAPTER 5: General Discussion 5.1 BRAF mutations and phenotypic properties of CRC 55
5.2 BRAF mutations and screening for HNPCC 57
5.3 Limitations of this study 59
5.4 Conclusions 60
5.5 Future work 61
References 63
11 LIST OF TABLES
Table 1.1 Amsterdam I and II criteria for the identification of
HNPCC cases (source: Vasen et al., 1991; Vasen et
al., 1999)
22
Table 1.2 Bethesda guidelines for testing colorectal tumours
for MSI (source: Rodriguez-Bigas et al., 1997; Umar
et al., 2004)
23
Table 2.1 Primer sequences, annealing temperatures and
PCR product sizes
31
Table 2.2 SSCP gel conditions for the mutation analyses of
BAT-26, BRAF, KRAS and TP53
32
Table 3.1 Associations between BRAF mutation and
clinicopathological features of colorectal cancer
39
Table 3.2 Associations between BRAF mutation and
molecular features of colorectal cancer
40
Table 3.3 Clinicopathological and molecular features of BRAF
mutant colorectal cancers stratified according to
microsatellite instability status
41
Table 3.4 Clinicoptahological and molecular features of BRAF
mutant colorectal cancers stratified according to
methylator phenotype status
42
Table 4.1 Associations between BRAF mutation and
clinicopathological features of colorectal cancer in
patients aged <60 years
49
12 Table 4.2 Clinicopathological characteristics and MSI status of
tumours with BRAF mutations in young (<60 yrs)
and old (≥60 yrs) colorectal cancer patients
51
13 LIST OF FIGURES
Figure 1.1 The general structure of (a) MAPK pathway and
(b) ERK pathway (source: Kolch, 2000)
15
Figure 1.2 The BRAF protein and signal transduction
(Source: Pollock & Meltzer, 2002)
16
Figure 2.1 SSCP analysis of BRAF, KRAS, BAT-26 and
TP53 genes
33
Figure 3.1 (A) Representative F-SSCP gel used to detect
BRAF mutations in colorectal cancer. WT, wild
type; M, mutation. (B) DNA sequencing gel result
confirms the presence of a 1799T to A mutation
giving rise to the V600E mutation.
38
14 CHAPTER 1 INTRODUCTION
1.1 Mitogen-activated protein kinase (MAPK) cascade
Cancer is a disease of the genome, triggered by the accumulation of genetic
errors that eventually transform a normal cell into a tumour cell (Pollock &
Meltzer, 2002a). Multiple physiological processes are governed by the mitogen-
activated protein kinase (MAPK) cascade (Figure 1.1a) - a conserved signaling
system that transduces extracellular signals into the nucleus via a cascade of
(Loukola et al., 2001; Peltomaki, 2003). Germline mutations in MLH1 and MSH2
constitute about 90% of all MMR gene mutations (Lynch & Chapelle, 1999;
Domingo et al., 2004b). These genes are normally responsible for correcting
errors in the length of microsatellites (nucleotide repeat regions) produced
during the replication of DNA (Lynch & Chapelle, 2003). The presence of
somatic alterations in the length of microsatellites (referred to as microsatellite
instability, or MSI+) and the absence of MMR protein expression detected by
immunohistochemistry (IHC) are both hallmarks of HNPCC (Wang et al., 2003;
Baudhuin et al., 2005).
20 Colonoscopy screening performed at 3-year intervals is able to reduce CRC-
related mortality by about 65% in HNPCC families (Jarvinen et al., 2000). At
present, the International Collaborative Group on HNPCC (ICG-HNPCC)
recommends that ‘at-risk’ individuals in HNPCC families undergo colonoscopic
surveillance every 1-2 years, beginning at the age of 25 years or 5 years
younger than the youngest affected family member, whichever is earliest
(Vasen et al., 1993). Faecal occult blood testing is offered in alternate years or
to subjects unwillingly to undergo colonoscopy. Screening for endometrial
carcinoma is recommended from 30-35 years of age (Jarvinen et al., 1995).
Extended surgery has been recommended for patients with proven HNPCC
because of the increased risk of metachronous CRC.
Testing for germline mutation of MMR genes is important as it allows exclusion
of healthy family members carrying the wild-type allele from unnecessary
surveillance programs (Wolf et al., 2005). The following guidelines have been
proposed to help identify patients with a high probability of a MMR germline
mutation.
1.4.3 Guidelines for detection of HNPCC
The detection of suspected HNPCC cases is often difficult as the syndrome
lacks well-defined pre-symptomatic characteristics (Lynch et al., 2003; Aaltonen
et al., 1998). HNPCC is usually recognized by the occurrence of cancers over
multiple generations and at an early age of onset (average age of onset <45
years). A strong family history has therefore become the primary diagnostic tool
(Lynch & de la Chapelle, 2003). In order to standardize diagnostic criteria, the
21 ICG-HNPCC developed the original Amsterdam criteria (I) as shown in Table
1.1 below (Umar et al., 2004; Aaltonen et al., 1998). Since then, revision has
been made to include small families (Amsterdam criteria II). These criteria were
pivotal in identifying kindreds that eventually led to association of the HNPCC
syndrome with germline MMR gene mutations (Rodriguez-Bigas et al., 1997).
22 Table 1.1 Amsterdam I and II criteria for the identification of HNPCC cases
(source: Vasen et al., 1991; Vasen et al., 1999)
Amsterdam Criteria I Amsterdam Criteria II
1. Three or more family members
with CRC and all of the following
features:
2. One is a first-degree relative of the
other two
1. At least three relatives must have
a cancer associated with HNPCC
(CRC, endometrial, stomach,
ovary, ureter or renal-pelvis,
brain, small bowel, heptobiliary
tract, or skin 3. At least two successive
generations must be affected 2. One must be a first-degree
relative of the other two 4. At least one of the relatives with
CRC must have received the
diagnosis before the age of 50
years
3. At least two successive
generations must be affected
4. At least one of the relatives with
cancer associated with HNPCC
should have received the
diagnosis before the age of 50
5. FAP should have been excluded
in any relatives with CRC
5. FAP must have been excluded.
6. Tumours should be pathologically
verified whenever possible
In clinical practice, MSI testing is used as a marker for underlying MMR
dysfunction. To identify which patients are appropriate for MSI testing, the
National Cancer Institute (NCI) developed a set of criteria known as the
Bethesda Guidelines (Table 1.2) during the International Workshop on HNPCC
in 1996 and later revised in 2002 (Rodriguez-Bigas et al., 1997).
23 Table 1.2 Bethesda guidelines for testing colorectal tumours for MSI
(source: Rodriguez-Bigas et al., 1997; Umar et al., 2004)
Bethesda Guidelines Revised Bethesda Guidelines
1. Amsterdam I criteria met
2. Individuals with more than one
HNPCC cancer
3. CRC and first-degree relative with
CRC/HNPCC cancer, one cancer
younger than 45 years or one
adenoma younger than 40
4. CRC/endometrial cancer younger
than age 45
5. Right-sided CRC, undifferentiated,
younger than 45
6. Signet ring CRC younger than 45
7. Adenomas younger than 40 years
1. CRC diagnosed in a patient under
the age of 50
2. Presence of synchronous,
metachronous colorectal, or other
HNPCC-associated tumours,
regardless of age
3. CRC with the MSI-H histology
diagnosed in a patient who is less
than 60 years of age
4. CRC diagnosed in one or more
first-degree relatives with an
HNPCC-related tumour*, with one
of the cancers being diagnosed
under age 50 years
5. CRC diagnosed in two or more
first- or second-degree relatives
with HNPCC-related tumours,
regardless of age.
*HNPCC-related tumours include colorectal, endometrial, stomach, ovarian,
pancreas, ureter and renal pelvis, biliary tract, and brain (usually glioblastoma
as seen in Turcot syndrome) tumours, sebaceous gland adenomas and
keratoacanthomas in Muir-Torre syndrome, and carcinoma of the small bowel
(Lin et al., 1998).
24 Despite the availability of improved diagnostic criteria and guidelines for
identification and molecular testing, the detection of HNPCC patients at the
population level remains difficult. The sensitivity of the Amsterdam criteria is
compromised by the amount of time and resources needed to obtain a
comprehensive family history required to assess the possible genetic risks.
These lead to inaccuracies when reporting people at risk of CRC (Mitchell et al.,
2004). It has been estimated that only 10-20% of individuals at high risk for
HNPCC are being referred for further evaluation (Terdiman et al., 2002). The
specificity of MSI testing is limited by the occurrence of MSI+ in 15% of sporadic
CRC cases. These arise due to somatic inactivation of the MMR genes,
particularly methylation-induced transcriptional silencing of MLH1 (Thibodeau et
al., 1993; Umar et al., 2004).
Current recommendations for the detection of HNPCC includes an initial testing
of tumours for the presence of MSI+ combined with IHC for the absence of
MMR protein expression. If loss of gene expression is found, this allows
germline mutation testing to be targeted to the relevant gene (Salovaara et al.,
2000; Domingo et al., 2004a). This complementary MSI/IHC approach may
increase the sensitivity and specificity when diagnosing HNPCC. However, this
molecular-based approach may not be sufficiently efficient, cost effective or
available in routine clinical practice to allow HNPCC screening in the entire
colorectal cancer population (Halvarsson et al., 2004; Domingo et al., 2004b).
1.4.4 BRAF in HNPCC
Recently, investigators have reported a strong association between V600E
BRAF mutation and MMR deficiency (Rajagopalan et al., 2002; Davies et al.,
25 2002; Yuen et al., 2002). This association was seen exclusively in sporadic
MSI+ tumours, but not in MSI+ tumours from HNPCC (Deng et al., 2004; Wang
et al., 2003; Koinuma et al., 2004; Kambara et al., 2004; Nagasaka et al., 2004;
Domingo et al., 2004a; Miyaki et al., 2004; McGivern et al., 2004). This
exclusivity of BRAF mutation for sporadic but not familial MSI+ CRC could
therefore be used as a strategy to help identify HNPCC families.
1.5 AIMS
Hereditary nonpolyposis colorectal cancer is the most common form of familial
bowel cancer (Lynch et al., 2003). It is often left undiagnosed due to the lack of
distinguishing morphological features, thus leaving other family members with
germline mutations to the risk of cancer at an early age (Lynch et al., 2003;
Aaltonen et al., 1998). There is firm evidence that routine colonoscopic
screening, improves the survival rate of individuals with HNPCC syndrome
(Jarvinen et al., 2000), thus justifying the need to detect this genetic condition in
the population.
Currently, suspected HNPCC cases are identified primarily based on family
history as defined by the Amsterdam criteria (Vasen et al., 1991; Vasen et al.,
1999). However, the feasibility and accuracy of following these guidelines have
been proven less than ideal (Mitchell et al., 2004). An alternative approach
which involves complementary IHC-based screening for loss of MMR protein
expression and molecular screening for MSI+ has been proposed (Halvarsson
et al., 2004). Although this approach increases the sensitivity and specificity in
diagnosing HNPCC suspects, its implementation at the population level needs
26 to be evaluated for cost-effectiveness (Halvarsson et al., 2004; Domingo et al.,
2004b).
The recent finding that V600E BRAF mutation is frequently present in sporadic
MSI+ but not HNPCC MSI+ tumours suggests a possible strategy that may
simplify the detection of HNPCC families. The aims of this work are therefore:
1. To evaluate the clinical, pathological and molecular phenotype of
colorectal tumours with BRAF mutations
2. To compare the clinical, pathological and molecular features of colorectal
tumours with BRAF mutation in younger and older patients
3. To determine the frequency of BRAF mutation in the younger patient
population that is likely to be the target of screening for HNPCC
27 Chapter 2 MATERIALS & METHODS
2.1 Case selection
A consecutive series of 275 stage I - IV colorectal tumours investigated in this
study were obtained from the Colorectal Unit of the Royal Adelaide Hospital.
The tumour samples were snap frozen in liquid nitrogen within 20-40 min after
resection and stored at -70ºC prior to DNA extraction.
Another series of 780 stage I - IV paraffin-embedded colorectal tumour samples
were also studied. These were obtained from three major public teaching (Sir
Charles Gairdner, Royal Perth, Fremantle) and two private hospitals (St John of
God at Murdoch and Subiaco) in Western Australia. Cases selected from these
five institutes were diagnosed between 2000 and 2004 and a patient age at
diagnosis of <60 years was the basis for selection.
Clinical data available for both colorectal tumour series included patient age and
sex, while pathology data included nodal involvement, tumour site, histological
grade, mucinous histology and the presence of infiltrating lymphocytes.
2.2 Ethics approval
Ethics approval was been obtained from each of the West Australian public
teaching (Sir Charles Gairdner, Royal Perth, Fremantle) and major private
hospitals (St John of God) for access to archival paraffin-embedded tumour
blocks for the purposes of phenotypic analysis.
28 2.3 DNA extraction from paraffin-embedded tissue sections
Two 25 µm sections of paraffin embedded tissues from each case were placed
in a 1.5 ml Eppendorf tube containing 300 µl of digestion buffer (50 mH Tris
HCL, 1 mM EDTA, 0.5 % Tween 20 at pH 8.5). The tubes were heated at 94°C
for 10 min in a water bath to melt the paraffin before centrifuging for 10 min at
12,000 rpm to separate the tissues from the paraffin. The tubes were allowed to
cool at 4°C for approximately 2 hours until a firm crust of paraffin was formed.
This was removed and the tissue transferred to a new 1.5 ml Eppendorf tube
containing 200 µl of fresh digestion buffer. Twenty µl of Proteinase K from a 20
mg/ml stock dissolved in digestion buffer were then added and the mixture
incubated in a rotating oven at 55°C for 48 hours. The Proteinase K reaction
was then inactivated by heating the tubes at 94°C in a water bath for 10 min.
The samples were centrifuged and the resulting clear solution containing DNA
was transferred into a new 1.5 ml Eppendorf tube and stored at 4°C for use
within several weeks.
2.4 PCR for MSI screening
The MSI status of each tumour was evaluated by fluorescent-single stranded
conformation polymorphism (F-SSCP) analysis of the BAT-26 mononucleotide
repeat (Iacopetta et al., 1998). The PCR reaction was carried out in a 16 µl
reaction mix containing 1x polymerization buffer, 1x Q-Solution, 200 µM of each
dioxynucleotide triphosphate (dNTP), 3 mM MgCl2 (Qiagen, Melbourne), 0.5 µM
of each HEX-labeled BAT-26 primer (Geneworks, Adelaide; primer sequences
listed in Table 2.1) and 0.5U Taq DNA Polymerase (Qiagen, Melbourne).
Reactions were ‘hot started’ by the addition of 1 µl genomic DNA at 94°C prior
to commencement of cycling. PCR amplification was carried out using the
29 following conditions: 35 cycles of 94°C for 30 sec, 46°C for 30 sec, and 70°C for
30 sec; followed by a final extension at 70°C for 10 min.
2.5 PCR for KRAS mutation screening
Mutations in KRAS codons 12 and 13 were detected by F-SSCP analysis
(Wang et al., 2003a). The KRAS gene was amplified in a 14 µl mix containing
1x polymerization buffer, 1x Q-Solution, 200 µM of each dNTP, 3 mM MgCl2
(Qiagen, Melbourne), 0.5 µM of each HEX-labeled KRAS primer (Geneworks,
Adelaide; primer sequences listed in Table 2.1), 0.5U Taq DNA Polymerase
(Qiagen, Melbourne) and 1 µl of DNA. Amplification was performed using the
same cycling conditions as described in Section 2.4 except that the annealing
temperature was 54°C.
2.6 PCR for TP53 mutation screening
Exons 5, 7 and 8 of TP53 tumour suppressor gene were screened for mutations
using F-SSCP as described previously (Soong & Iacopetta., 1997). The reaction
mix for each exon was 14 µl containing 1x polymerization buffer, 1x Q-Solution,
200 µM of each dNTP, 2.5 mM MgCl2 (Qiagen, Melbourne), 0.5 µM of each
HEX-labeled primer (Geneworks, Adelaide; respective primer sequences listed
in Table 2.1), 0.5U Taq DNA Polymerase (Qiagen, Melbourne) and 1 µl of DNA.
Amplification was carried out using the same cycling conditions as described in
Section 2.4 except the extension time for TP53 exon 5 was 45 seconds and the
annealing temperatures were 60°C, 60°C and 56°C, respectively, for TP53
exons 5, 7 and 8.
30 2.7 PCR for BRAF mutation screening
A hotspot V600E mutation site in exon 15 of the human BRAF gene was
identified in previous studies (Davies et al., 2002). This mutation was identified
here by F-SSCP analysis. The BRAF gene was amplified in a 14 µl mix
containing 1x polymerization buffer, 1x Q-Solution, 200 µM of each dNTP, 3 mM
MgCl2 (Qiagen, Melbourne), 0.5 µM of each HEX-labeled BRAF primer
(Geneworks, Adelaide; Davies et al., 2002; primer sequences listed in Table
2.1), 0.5U Taq DNA Polymerase (Qiagen, Melbourne) and 0.8 µl of DNA.
Amplification was carried out following the same cycling conditions as described
in Section 2.4 except that the annealing temperature was 60°C.
2.8 Screening for CpG island methylation
CIMP phenotype of tumours included in this work was determined in a previous
study headed by A/Prof Iacopetta (Kawakami et al., 2003).
31 Table 2.1 Primer sequences, annealing temperatures and PCR product
sizes.
Primer Sequence
Annealing
Temperature (ºC)
PCR product size (bp)
BAT26 F 5’-TTGGATATTGCAGCAGTCAG-3’ 46 136 BAT26 R 5’-GCTCCTTTATAAGCTTCTTCA-3’ BRAF F 5’-TCATAATGCTTGCTCTGATAGGA-3’ 60 224 BRAF R 5’-GGCCAAAAATTTAATCAGTGGA-3’ KRAS F 5’-GACTGAATATAAACTTGTGG-3' 54 107 KRAS R 5’-CTATTGTTGGATCATATTCG-3'
TP53
Exon 5 F 5'-TCTTCCTGCAGTACTCCCCT-3' 60 205 Exon 5 R 5'-AGCTGCTCACCATCGCTATC-3' Exon 7 F 5'-TTGTCTCCTAGGTTGGCTCT-3' 60 136 Exon 7 R 5'-GCTCCTGACCTGGAGTCTTC-3' Exon 8 F 5'-TCCTGAGTAGTGGTAATCTA-3' 56 157 Exon 8 R 5'-GCTTGCTTACCTCGCTTAGT-3'