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BRAF Study

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reem alotibi

on 23 December 2014

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Transcript of BRAF Study

Presentation outlines
• Introduction
• Objectives
• Methodology
• Results
• Discussion
• Conclusion
• Future Plans
• References
Supervised by:

Co-supervised by:

Lab supervisor:
Reem Mansour Alotibi
DNA Sequencing
DNA Extraction
Patient Selection & Sample
Material and Methods
[Using Big Dye Termination]
[Polymerase Chain
A total of 115 patients who underwent thyroidectomy or lobotectomy for proliferative thyroid lesions between 2008-2012 were included in this study. Of the 115 patients, 70% were female and 30% were male. The age of patients ranged between 15 and 87 years, with an average age of 51 years.
Patient Samples
NanoDrop 2000c Spectrophotometer
Extraction mini kit
To amplify millions of copies of the wanted gene fragment by using specific oligonucleotides.

Repetitive cycles of denaturation, annealing and extension.

PCR and Gel Electrophoresis
Eppendorf Thermalcycler
Gel Electrophoresis preparations
and Gel documentation using UV illuminator
Online NCBI’s BLAST database
(*)Life technologies,BigDye Kit.
(*) Life technologies, 3130 Genetic Analyzer
Out of 115 patient, 72 patients were found positive for a mutation in exon 15 of BRAF.
In codon 600, most of the BRAF mutations were common point mutation in this codon leading to substitution of valine by glutamic acid.
In codon 601, One PTC point mutation in this codon resulted in substitution of lysine by glutamic acid.
Significant findings of our study include rare BRAF mutations in PTC that in cases of T599insT and K601E seem to confer a certain risk for progressive TC.

The expression study focusing on affected genes that are differentially expressed between BRAFwt and BRAFmut conventional PTCs.

Our microarray expression study provides a detailed overview of 237 differentially expressed genes between BRAFwt and BRAFmut PTCs resulting in identification of molecular biomarkers which are of further interest for basic molecular genetics and translational studies in PTCs.
Future Plans
Mutational analysis of BRAF that is involved in deregulating of the MAPK pathway in PTC
Microarray Gene expression analysis in PTC
The main reason for the distribution of young age of patients with BRAF mutation in our study could be related to the region structure consisting of younger aged population.

Our study identified one mutation (K601E) in PTC. This mutation has been related to PTC histology of TC (Trovisco
et al.,

Beside the V600E mutation, BRAF mutations are usually linked to non-aggressive tumors of TC (Nikiforov and Nikiforova, 2011); still other mutations as T599insT and K601E were reported to associate with advanced cases of TC (Gauchotte
et al.,
Molecular genetic analysis of susceptibility genes potentially impact in thyroid diseases in Saudi Arabia
Thyroid Cancer
v-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF)
of thyroid cancer
Differentiated Thyroid Cancer
Undifferentiated Thyroid Cancer

Fine needle aspiration cytology (FNAC).
Ultrasound Imaging.
Computerized Tomography Scan (CT).
Blood hormone test.
Diagnosis of Thyroid Cancer
Symptoms of Thyroid Cancer
Thyroid cancer is the fastest increasing cancer in both men and women. It is the most common malignant tumor of the endocrine system located within the thyroid gland.

Thyroid cancer occurs in all age groups, about 2 of every 3 people diagnosed with thyroid cancer are between ages 20 and 55.
Etiology of Thyroid cancer

BRAF is a serine-threonine kinase that belongs to the family of RAF proteins, which are intracellular effectors of the MAPK signaling cascade.

BRAF is located on the long (q) arm of chromosome 7 at band 34.
Consist of 18 exons
Sequence length : 766 AA.
BRAF mutations are the most common alterations found in thyroid cancer, with a prevalence that varies from 23 to 62% among different studies and populations under investigation.

In 2002, it was shown to be (mutated) in human cancers (Davies, et al., 2002).

BRAF-activating mutations are restricted to the kinase domain located in exons 11 and 15.

Dr. Etimad A. Huwait

Prof. Mamdouh A. Gari

Dr. Hans-Juergen Schulten
lumps or nodules in front of the neck.
enlarged lymph nodes in the neck.
problems with swallowing.
hoarseness or voice changes.
pain or discomfort in the neck.
chronic cough.
(*) SmartTec molecular website: FFPE extraction universal kit.
Procedure of DNA Extraction*

Gel image
* Genentech Biotechnology
First Purification
Remove any contamination from the DNA and have PURE DNA.
Cycle Sequencing
Make number of different sized fragments from the DNA, using big dye terminator.
Second Purification
Same as first purification.
DNA Extraction
Software Analysis
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Barnier, J. V., Papin, C., Eychène, A., Lecoq, O. and Calothy, G. (1995) The Mouse B-Raf Gene Encodes Multiple Protein Isoforms with Tissue-Specific Expression, Journal of Biological Chemistry, vol. 40: 23381-23389.
Bollag, G., Tsai, J., Zhang, J., Zhang, C., Ibrahim, P., Nolop, K. and Hirth, P. (2012) Vemurafenib: The First Drug Approved for Braf-Mutant Cancer, Nature Reviews Drug Discovery, vol. 11: 873-886.
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Schulten, H.-J., Al-maghrabi, J., Al-ghamdi, K., Salama, S., Al-muhayawi, S., Chaudhary, A., Hamour, O., Abuzenadah, A., Gari, M. and Al-qahtani, M. (2011) Mutational Screening of Ret, Hras, Kras, Nras, Braf, Akt1, and Ctnnb1 in Medullary Thyroid Carcinoma, Anticancer Res, vol. 12: 4179-4183.
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Wellbrock, C., Karasarides, M. and Marais, R. (2004) The Raf Proteins Take Centre Stage, Nature Reviews Molecular Cell Biology, vol. 11: 875-885.
Xing, M. (2007) Braf Mutation in Papillary Thyroid Cancer: Pathogenic Role, Molecular Bases, and Clinical Implications, Endocrine reviews, vol. 7: 742-762.
Zebisch, A. and Troppmair, J. (2006) Back to the Roots: The Remarkable Raf Oncogene Story, Cellular and Molecular Life Sciences CMLS, vol. 11: 1314-1330.
Thank You!
Molecular genetic analysis of susceptibility genes potentially impact in thyroid diseases in Saudi Arabia

Papillary Thyroid Cancer (PTC)

Follicular Thyroid Cancer (FTC)
begins in the follicular cells of the thyroid.
PTC is the most common type of thyroid cancer.
It accounts for about 80% .

begins in the follicular cells of the thyroid.
It accounts for about 10-15% .
Anaplastic Thyroid Cancer (ATC)
begins in the follicular cells of the thyroid.
ATC is extremely rare. It accounts for only 1–2% of all thyroid cancers.
Medullary thyroid cancer (MTC)
begins in the C cells of the thyroid.
MTC is associated with genetic inheritance.
MTC is not very common, occurring in less than 5 % of patients who are diagnosed with thyroid cancer.
A wide variation in incidence of thyroid cancer according to age, sex, ethnicity and geographic region was observed.

Thyroid cancer ranked second among female population and fourteenth among male population

More than 7 of 10 people diagnosed with thyroid cancer are female.
Pathophysiology of Thyroid Cancer
There are several recurring mechanisms of tumorigenesis that have been identified in the various types of thyroid cancer.
Family history of thyroid disease

Genetics Factors

Age and Gender

Environmental and life-style

Diet: Low in iodine levels

Exposure to radiation

Risk Factors for Thyroid cancer
BRAF has three conserved regions (CR1, CR2 and CR3)*.
MAPK Pathway
Mitogen-activated protein kinases (MAPK) pathway refers to a cascade of protein kinases that are highly conserved in evolution and play central roles in signal transduction in all eukaryotic cells.

MAPK pathway plays a fundamental role in cellular proliferation, differentiation, apoptosis, and survival.
MAPK signaling*
First technique employed:
Mutational analysis in proliferative thyroid diseases with Sanger sequencing on genomic DNA
Affymetrix GeneChip arrays are the most popular high density oligonucleotide expression arrays and are used by thousands of researchers worldwide.
Base-Pairing, hybridization between nitrogen bases.
Thousands of spotted samples known as probes (with known identity and length of 25 base pairs) are immobilized on a solid support (a microscope glass slides or silicon chips or nylon membrane).
The spots can be DNA, cDNA, or oligonucleotides. These are used to determine complementary binding of the unknown sequences thus allowing parallel analysis for gene expression and gene discovery.

Day 1 protocol
Day 2 protocol
Day 3 & 4 protocol
Material and Methods
Second employed technique:
Gene expression analysis using oligonucleotide microarrays
Patient Selection & Sample
Patient Samples
For free tissue samples and Formalin-fixed paraffin-embedded (FFPE)
25 patients and 7 normal samples were utilized in this protocol.

RNA extraction from fresh tissues sections derived from:
* The Pathology Departments of the King Abdulaziz University, Jeddah.
* King Faisal Specialist Hospital & Research Center, Jeddah.

RNA concentration can be processed is 100ng/µl.
Day 2 protocol
* MENA= Middle East and North Africa
1* mean age of all 72 patients with BRAF mutations : 40.1 years
Following figures from electropheropgram are some of the mutated ‘hot spots’:
In codon 599, a rare 3-base pair insertion at this codon found in PTC resulting in duplication of threonine (heterozygous).
PCA scatter plot wherein each dot represents a sample with a group specific color
Distance between dots is a dimensional measure for the similarity of the respective expression profiles of the samples. Ellipsoids are a measure to visualize distance of relationships between samples of a group. Red, normal thyroid; blue, BRAFwt PTCs, green, BRAFmut PTCs.
Hierarchial cluster analysis of 237 genes which were differentially expressed (FDR p-value ≤ 0.05 and fold change > 2.0) between BRAFwt and BRAFmut PTCs.
Gene expression of TN samples is included in analysis. Color scheme, red for comparably higher and blue for comparably lower expression. Green branches, TN samples; purple branches, BRAFwt PTCs; red branches, BRAFmut PTCs.
237 significant differentially expressed genes were scanned.
CR1contains the cysteine-rich domain (CRD) and most of the Ras binding domain (RBD).
CR2 is rich in serine and threonine residues, and its an inhibitory phosphorylation site.
CR3 has the kinase domain.
One of the mutations (T599insT) identified in this study, in the aggressive PTC case was reported few years ago to be an activation mutation pilocytic astrocytomas (Jones
et al.,

T599insT has a similar effect as V600E which is elevated kinase activity (Eisenhardt
et al.
, 2011).

T599insT results in disruption of the inhibitory conformation of the activation loop.

Of notice, a considerable number of sporadic mutations in BRAF exon 15 affects or co-affects codon 599 in TC (Chiosea et al., 2009). It was reported that these mutations also elevate the activity of the BRAF (Eisenhardt et al., 2011).
In comparison to other microarray studies using different PTC histological subtypes, the number of genes that are common between our and other studies might be low.

An expression array study was done on the basis of enhanced stringent threshold criteria, more than 80 up- and downregulated genes in the BRAFmut group in comparison to PTCs with other gene aberrations and the 40 most up- and downregulated genes have an overlap of 40 % to our list of 237 differentially expressed genes (Giordano et al., 2005).
The high frequency of BRAF mutations in PTC suggests that inhibition of BRAF activity by newly developed RAF kinase inhibitors may offer a new strategy in the treatment of these tumors.
Since all patients in this study came from the western region (Jeddah), other regions of KSA especially the Eastern region must be investigated to provide us information about the BRAF mutation status in TC there.
comparing the results with our findings and to take BRAF mutational screening into the repertoire of clinical tools for thyroid carcinoma treatment.
We also recommend pathway analysis of differentially expressed genes to investigate they role in TC regard to BRAFmut status.
Demographic and clinicopathological features of BRAFwt and BRAFmut PTCs
First and foremost, I must acknowledge my limitless thanks to Allah, the Ever-Thankful, for His help and bless.

I am grateful to some people, who worked hard with me from the beginning till the completion of the present research particularly my supervisors Dr. Etimad H. Huwait, Prof. Mamdouh A. Gari and Dr. Hans-Juergen Schulten they have been always generous during all phases of the research.

I would like to take this opportunity to say warm thanks to all my beloved friends, who have been so supportive along the way of doing my thesis.

I also would like to express my thanks to my family for their generous support they provided me throughout my entire life and particularly through the process of pursuing the master degree.

A special thanks goes to King AbdulAziz City for Science and Technology for supporting and funding this research.
3130 Genetic Analyzer*
Taken from Cancer Incidence and Survival Report Saudi Arabia, 2007
Taken from "Genetics Home Reference"
(*) Atlas of Genetics and Cytogenetics in Oncology and Haematology.
Taken from "Solve the thyroid puzzle"
Taken from "Fermoy Medical Hall"
1* fold change in BRAFwt PTCs compared to BRAFmut PTCs
Schulten, H.-J., Salama, S., Al-Mansouri, Z., Alotibi, R., Al-Ghamdi, K., Al-Hamour, O. A., Sayadi, H., Al-Aradati, H., Al-Johari, A. & Huwait, E. (2012)
Braf Mutations in Thyroid Tumors from an Ethnically Diverse Group
, Hered Cancer Clin Pract, vol. 10.

Alotibi, R., Al-Ahmadi, A., Ata, M., Karim, S., Huwait, E., Gari, M., Al-Qahtani, M., Schulten, H.-J. & Al-Maghrabi, J. (2014)
High-Density Microarray Expression Profiling in Conventional Papillary Thyroid Carcinomas with Versus without a Braf Mutation
, BMC Genomics, vol. Suppl 2: P21.

Al-Ahmadi, A., Alotibi, R., Al-Quaiti, M., Ashgan, F., Narasimhan, K., Huwait, E., Gari, M., Al-Qahtani, M., Al-Maghrabi, J. & Schulten, H.-J. (2014)
Frequent Microdeletions in Conventional Papillary Thyroid Carcinoma Detected by High-Density Oligonucleotide Microarrays
, BMC Genomics, vol. Suppl 2: P62.
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