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NRAS gene study

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alaa alahmadi

on 16 November 2015

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Transcript of NRAS gene study


Discussion
Presentation outlines...
• Introduction

• Objectives

• Methodology

• Results

• Discussion

• Conclusion & Recommendation

• References
Supervisor Co-Supervisor


Lab Supervisor

DNA Sequencing
PCR
Software
Analysis
DNA Extraction
Sample
Collection
Methodology
[Using Big Dye Termination]
[Polymerase Chain
Reaction]
NanoDrop 2000c Spectrophotometer
QIAamp DNA FFPE kit
Polymerase Chain Reaction
Gel Electrophoresis preparations
and Gel documentation using UV illuminator
(*)Life technologies,BigDye Kit.
3130 Genetic Analyzer
Results
In hotspot (61), a substitution mutations were found in CAA.
A-base G-base
Following figures are some of the mutated ‘hot spot’:
Objectives
Predisposing Genetic Factors for Thyroid Cancer
Introduction
Thyroid Gland Cells
Diagnosis of Thyroid Cancer
Treatment of Thyroid Cancer
Pathway and Genes involved in TC
Procedure of DNA Extraction*

Agarose gel image
1
2
3
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
Data Analysis
References

Alaa Gh. Alahmadi

By
Dr. Etimad A. Huwait
Proof. Mamdooh A. Gari

Dr. Hans-Juergen Schulten

Thyroid gland
Thyroid gland
is a butterfly-shaped gland that is locates in the front of the neck below the larynx


It consists of two lobes lying on either side of the trachea and connected by a narrow band of tissue called the isthmus.
Follicular cells
Use iodine from the blood to make and secretion thyroid hormones, thyroxine (T4) and triiodothyronine (T3) which are responsible for regulation of body metabolism.
follicular cell
Parafollicular cells
Make calcitonin hormone that lowers the concentration of calcium in the blood when it rises above the normal value.
( C cells )
Overview
Predisposing Genetic Factors
for Thyroid Cancer

Molecular Biology of Thyroid Cancer
Thyroid Gland Disorders
Physical examination
Blood tests
Imaging tests
Biopsy

Surgery
Radioactive iodine therapy
Thyroid hormone therapy
External beam radiotherapy
Chemotherapy
Prevalence of Thyroid Cancer

Ten Most Common Cancers among Saudi adult (2009)
Iodine deficiency is the commonest cause of thyroid disorders, resulting in a number of thyroid diseases that can be diagnosed based on individual features and symptoms.

Disorders in thyroid can affect body weight, energy and cholesterol level, heart and memory. They affect thyroid functions in two ways either
hypothyroidism
or
hyperthyroidism
.
The worldwide incidence levels for thyroid cancer vary from country to another, that is may be refer to age, sex, ethnicity and geographic region.

In 2004, thyroid cancer affected 3% of women (one of the top ten cancers in women) and 1% of men.
Thyroid Cancer in Saudi Arabia...
Papillary Thyroid Carcinoma(PTC)
Microscopically, there are two variant of PTC :
Follicular variant of papillary thyroid carcinoma (FVPTC) which is the most common variant of PTC. It characterizes by larger tumor size.
Papillary thyroid microcarcinoma (PTMC) which is defined as subset of PTC in which the tumor cells are equal or less than 1 cm in diameter and it is making up to 24% of all thyroidectomies.
Thyroid Cancer Staging
Stage 1 :
cancer is inside the thyroid and less than 2cm in diameter.

Stage 2
: cancer becomes large within thyroid or spreads to tissues adjunct to the thyroid gland.

Stage 3
:

cancer invades lymph nodes.

Stage 4
: in which distant metastases occur.
Uncontrollable

Controllable

Exposure to ionizing radiation and to chemical carcinogenic substrates
low iodine intake
Obesity
Age and gender
Family history
Geographic and ethnic variations
The mitogen-activated protein kinase (MAPK)
pathway is one of the most important molecular signaling pathways; it is involved in cell growth, proliferation, differentiation and cell survival.

Unphysiological activation has been demonstrated to be essential for incidence of thyroid cancer.
RAS Genes Family


KRAS, HRAS and NRAS are members of human RAS gene family. These genes encode similar proteins known as G-proteins (p21) with molecular weight of 21 kilo Daltons (KD).
All RAS genes have similar structure and identical sequence in region r
esponsible for GTP/GDP binding and GTPase activity.

They differ significantly only in
the C-terminal 25 amino acids
and their
location on chromosomes
.

Mutational analysis
of the NRAS gene in different histological types of benign and malignant thyroid lesions
Find the correlation of genetic alteration with clinical pathology of the disease.
381 patients who underwent thyroidectomy or lobotectomy for proliferative thyroid lesions at KAUH and KFSH&RC were sent to Center of Excellence in Genome Medicine & Research Unit and considered to be utilized in this study after collecting demographic data, family history of thyroid disease and clinicopathological data.
Formalin-fixed paraffin-embedded (FFPE) and fresh tissues sections were used in this study.
The primer sequences were designed to amplify the mutational hotspot regions in NRAS gene in exon 2 (codon 12 & 13) and exon3 (codon 61) using touchdown PCR protocol.
Primers sequence for PCR
Statistical
analysis
Associations of age and gender of patients and tumor stage with mutational status including NRAS codon 61 mutations in FVPTC were evaluated using SPSS statistics program.
Detection of NRAS codon 12, 13 and 61 mutations
Classification of
Thyroid Cancer
It originates from follicular cells.

It accounts 75–85% of all TC cases.

Cancer cells grow slowly and are usually noninvasive.
Follicular Thyroid Carcinoma (FTC)
It originates from parafollicular cells.

It accounts for 5-10% of TC.

It grows slowly and can invade nearby tissues or spread to distance organ.
Medullary Thyroid Carcinoma
(MTC)
It originates from follicular cells.

It accounts 10-20% of all TC.

Cancer cells tend to grow slowly.
Anaplastic Thyroid Carcinoma (ATC)
It originates from follicular cells.

It is an aggressive and rare form with frequency less than 2% of TC.

Cancer cells tend to grow and spread very quickly.
Papillary Thyroid Carcinoma ( PTC )
Thyroid Neoplastic
diseases
Follicular Adenoma
Encapsulated benign neoplasm that originates from follicular cells.

The most common clinical features of FA are a painless in thyroid but if it is increase in size, it causes hemorrhage and necrosis and becomes painful.
Thyroid Carcinoma
It is the most common endocrine cancer.

It shows higher incidence in female than male with a male: female ratio about 1:3 in the majority of countries.
1 One primary tumor was not staged
almost all micro PTCs cases were classified as stage I tumors except two cases in stage III and one case in stage IV tumor. The majority of cases were stage I tumors in PTC, FVPTC, and FTC. All eight non-WDTC cases were stage IV tumors
NRAS mutational survey in 381 cases in proliferative thyroid lesions
C-base A-base
Glutamine Arginine
Glutamine lysine
Correlation of genetic alteration with clinical pathology of the disease
Fisher`s exact test was applied to the study the correlation between NRAS codon 61 mutations screening cases in FVPTC and age, gender and tumor stage.


On the other hand, we found a trend significance between age and NRAS codon 61 mutations (P = 0.066) in patients younger than 45 years.
RAS and BRAF mutational survey in 381 cases in proliferative thyroid lesions
RAS mutations
have varying frequency patterns in the different types of cancers, but mutations in
codon 61 of NRAS
were shown by many studies to be present in
higher frequency than other RAS mutations in follicular thyroid cancer
(Prior et al., 2012; Fukahori et al., 2012; Nikiforov, 2011; Schulten et al., 2013 )



In contrast to BRAF mutations
which are found in malignant thyroid lesions only, RAS mutations are found in both malignant and benign thyroid lesions at different frequencies and they are commonly linked to follicular histology. This findings correspond with the results of Rivera et al. study in 2010 that indicated the association between the mutations of NRAS codon 61 and the follicular variant of PTC (Zhu et al., 2003, Di Cristofaro et al., 2006, Rivera et al., 2010).

No
hashimoto`s thyroiditis
was found to have RAS mutations in codon 12, 13& 61 or BRAF exon 15 in our case series. These findings correspond with results of other studies indicating that we can clearly distinguish HT from PTC by the absence of any RAS mutation (Sadow et al., 2010; Kang et al., 2007).


For nodular goiter
, prevalence of RAS mutations differed among different studies, i.e. 21% HRAS codon 12 (Namba et al., 1990), 20% KRAS codon 13 (1/5) (Horie et al., 1995). Our study identified a 9% prevalence of RAS mutations in HRAS codon 13, KRAS 12, and NRAS 61.

FA
molecular genetics and role in the histopathogenesis of TC is not completely understood. However, there is a hypothesis which states that FAs possess atypical nuclear characteristics of TC that have a follicular histology and become malignant through acquisition of NRAS codon 61 mutations (Vasko et al., 2003).
In FVPTC patients,
RAS and BRAF mutations combine are significantly associated with lower in comparison to higher tumor stage (p = 0.033) .

A trend association of RAS and BRAF mutations was revealed with younger age (p = 0.056) but no association was revealed with gender (p = 1). These findings indicate that mutational screening in patients with a FVPTC may gain clinical relevance.
FVPTC with a RAS mutation are frequently encapsulated and more related to FA than those with a BRAF mutation which are commonly infiltrative and resemble more PTCs (Gupta et al., 2012; Rivera et al., 2010).



Our findings showing that FVPTCs with either a RAS or a BRAF mutation are commonly associated with lower stages may be in part attributed to the fact that our patients are on average younger compared to other studies and other cancer genes than RAS and BRAF contribute more in older than in younger patients to thyroid cancer progression (Gupta et al., 2012; Rivera et al., 2010).



When comparing our data with others we have to keep in mind that staging of differentiated thyroid tumors is related to age (45 years threshold). Furthermore, our data are based on a relatively small number of FVPTC samples and more studies are needed to confirm our findings.
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• Kang, D.Y., Kim, K. H., Kim, J. M., Kim, S. H., Kim, J. Y., Baik, H. W. and Kim, Y. S. (2007) High Prevalence of Ret, Ras, and Erk Expression in Hashimoto's Thyroiditis and in Papillary Thyroid Carcinoma in the Korean Population, Thyroid, vol. 11: 1031-1037.
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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 research, vol. 12: 4179-4183.
• Schulten, H.J., Salama, S., Al-Ahmadi, A., Al-Mansouri, Z., Mirza, Z., Al-Ghamdi, Kh., Al-Hamour, O., Huwait, E., Gari, M., Al-Qahtani, M., Al-Maghrabi, J.(2013) Comprehensive Survey of HRAS, KRAS, and NRAS Mutations in Proliferative Thyroid Lesions from An Ethnically Diverse Population, Anticancer research, vol. 33: 4779-4784.


First and foremost, I must acknowledge my limitless thanks to Allah for giving me the strength and patience to complete my research.

I would like to express my sincere gratitude to my supervisors Dr. Etimad A. Huwait and Prof. Mamdouh A. Gari for their continuous support, patience, motivation, enthusiasm, and immense knowledge. Their guidance helped me in all the time of research and writing of this thesis.

I am also extremely indebted to Dr. Hans-Juergen Schulten for his continuous guidance in the practical work and supporting me whenever I needed, under his guidance I successfully overcame many difficulties and learned a lot.

I wish to express my deep sense of gratitude and indebtedness to my family for their love, care, support and prayers.

I also would like to express my wholehearted thanks to all my beloved friends for their kindness and moral support along the way of my thesis.

Last but not least, deepest thanks go to King AbdulAziz City for Science and Technology for supporting and funding this research.

acknowledgments
Samples Collection
Parafollicular cell
Autoimmune disorders
1) Hashimoto's thyroiditis
antibody attack and destroys thyroid gland cells resulting hypothyroidism.
antibodies produce against thyroid stimulating hormone receptor (TSHR) resulting hyperthyroidism.
2) Graves' disease
Goiter and hyperplastic
an enlargement in thyroid gland. It may be associated with normal thyroid hormone level, hypothyroidism or with hyperthyroidism.
Neuroblastoma RAS viral (v-ras) oncogene homolog (NRAS)
It is one of RAS family members, it named for its initial identification in human neuroblastoma cells in 1983.

It produce protein involves in cell growth, proliferation, differentiation and cell survival.
It is located on the short (p) arm of chromosome1 at position 13.2 and span 12.43Kb in length of DNA.

It has seven exons, four of seven exons are coding exons.
Gel Electrophoresis
Online NCBI’s BLAST database
Sequence result for NRAS exon 2
No significant association was observed between NRAS codon 61 mutational status and lower stage vs. higher stage (I+II vs. III+IV) (P = 0.17). Also, no significant association was observed between NRAS codon 61 mutational status and gender(P= 0.57).
THANK
YOU
Risk factors of thyroid cancer
All three oncogenic mutations are in amino acid regions that are identical between the three isoforms and assumed to generate equivalent effects on protein activity.


Point mutation in one of these RAS genes is found in ~ 30% of all tumors cases.


In cancer, point mutations occur in the RAS gene resulting in either an
increased affinity for GTP
(mutations in codons 12 and 13, exon 2) or
inactivation of the autocatalytic GTPase function
(mutations in codon 61, exon 3).

The image shows codon 12 & 13 in patient's sample, there are no mutation in these codons.
NRAS codon 61& tumor stage
NRAS codon 61& gender
Conclusion, Recommendations and Publication
Conclusion
Moderate frequency of RAS mutations, particularly NRAS codon 61 mutations in follicular variants of thyroid lesions, in consequence supporting the hypothesis that benign thyroid tumors with follicular histology like FA that harbor a RAS mutation may represent precursor lesions for malignant follicular variant of PTC.

Our data are in line with reports identifying NRAS codon 61 mutations in a minority of TC with follicular histology.

FVPTC harboring either a RAS or BRAF mutations are in general of lower stage needs further investigation to sustain these findings.

Recommendations
We recommend to increase numbers of FVPTC cases in further studies to examine the relation between RAS mutation and tumor stage in more detail.

Also, we recommend that the pathologist can distinguish between NRAS positive and NRAS negative mutations in FA and FVPTCs patients to strengthen the hypothesis that NRAS mutations may be predictive in FA to turn malignant.

We recommend furthermore to perform microarray expression analyses in FA and FVPTC with NRAS mutations to study and compare gene expression profiles in these tumors.

Publication
• Schulten, H., Salama, S., Al-Ahmadi, A., Al-Mansouri, Z., Mirza, Z., Al-Ghamdi, Kh., Al-Hamour, O., Huwait, E., Gari, M., Al-Qahtani, M., Al-Maghrabi, J.(2013) Comprehensive Survey of HRAS, KRAS, and NRAS Mutations in Proliferative Thyroid Lesions from An Ethnically Diverse Population, 33: 4779-4784.

• Alotibi, R., Al-Ahmadi, A., Ata, M., Karim, S., Huwait, E., Gari, M., Al-Qahtani, M., Schulten, H., Maghrabi, J. (2014) High-density microarray expression profiling in conventional papillary thyroid carcinomas with versus without a BRAF mutation, 15(Suppl 2):P21.

•Al-Ahmadi, A., Alotibi, R., Al-Quaiti, M., Ashgan, F., Narasimhan, K., Huwait, E., Gari, M., Qahtani, M., Al-Maghrabi, J., Schulten, H. (2014) Frequent microdeletions in conventional papillary thyroid carcinoma detected by high-density oligonucleotide microarrays, 15(Suppl 2):P62.
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