Transcript Document

Dr. Fahd Al-Mulla
©2012
http://www.al-mulla.org
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Understand that diseases (in most cases) are phenotypic
representation of proteins quality and quantity disorders,
either at DNA, transcriptional, translational or post
translational levels.
Define and describe the disciplines of molecular biology,
molecular pathology and molecular genetics.
Understand the molecular and cellular basis of common
diseases.
Comprehend that understanding the molecular and cellular
basis of diseases leads to more comprehensive
Theranostics.
What is Disease
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dis·ease (dĭ-zēz')
n.
A pathological condition of a part, organ, or system of an organism resulting
from various causes, such as infection, genetic defect, or environmental stress,
and characterized by an identifiable group of signs or symptoms.
A condition or tendency, as of society, regarded as abnormal and harmful.
Obsolete. Lack of ease; trouble.
Part of your job is to build good experience in associating symptoms
and signs with disease names. This is an art and you have to be good
at it.
Once you become proficient, you will think:
•What caused the disease? Endogenous reaction/defect or exogenous
factor(s)
•How do I diagnose it
•What is the molecular basis for the disease?
•Why do diseases behave differently in different people?
Changes an Enzyme
e.g. Phenylalanine hydroxylase
Splice site mutation leading to reduced amount
Causing phenylketonuria
Growth regulation
e.g.Rb causing
Retinoblastoma etc
Changes in structural
Proteins
1.e.g. collagen, Deletions
or point mutation that
Produce reduced amount
of normal collagen or
Normal amounts of mutant
Collagen. Causing
Osteogenesis imperfecta
2.e.g cell membrane Fibrillin
Missense mutations causing
Marfan syndrome
Or deletion of dystrophin gene
Causing Duchene muscular
Dystrophy
Changes an Enzyme inhibitor
e.g. 1-Antitrypsin
Missense mutation that impair secretion from liver
To serum causing Emphysema and Liver disease
Changes a receptor
e.g. Low density lipoprotein receptor
Deletion or point mutation that reduce synthesis,
Or transport to the cell surface or binding to low
density lipoprotein
Causing Familial hypercholesterolemia
Change a transport or carrier protein
1.e.g. Haemoglobin
Mutations in splice sites (commonest) leading to
Reduced -globin.causing -Thalassemia in
-Thalassemia the -globin gene is usually deleted
2.e.g. Cystic fibrosis transmembrane conductance
Regulator. Deletions or point mutation causing
Cystic fibrosis.
Changes in Hemostasis
e.g. Factor VIII deletions, insertions, nonsense
Mutation reduce synthesis or abnormal factor VIII
Causing Hemophilia A.
What is Molecular Pathology?
• Pathology: is the study of diseases.
• Molecular biology: the study of molecules in biological systems that
are responsible for normal biological traits or behaviors i.e.: DNA
replication, transcription and translation in normal cells.
• Molecular pathology: an evolving field that examines and identifies
the molecules involved in specific diseases.
• Integrates knowledge and techniques applied in molecular biology to
pathology.
Molecular Pathology: Rationale
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Classical pathologists examine tissue sections stained with Haematoxilin
and Eosin (H&E) and other stains, and is able to know the issues
origin, organization and what disease it represents. However, this is an
art involving human skill, not science.
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A pathologist is unable to define the molecules and how they interact
to cause the disease. This is the job of a molecular pathologist.
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The molecular pathologist utilizes techniques from molecular biology to
study differences between normal and diseased tissue at the molecular
level, so that the specific molecules associated with the disease maybe
identified.
We work as members of multidisciplinary team
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Who is a molecular geneticist? Clinical geneticist?
Molecular Pathology
Theranostics
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Diagnosis:
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Looking at the disease from the small molecules point of view
Elucidates the causes of the disease (viruses, hereditary, disruptions of the normal control
processes, such as the cell-cycle, apoptosis etc…)
Provides a more comprehensive understanding of a disease, it’s natural history, and
progression.
Provides an understanding of the overall complexity of the disease.
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Prognosis:
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Associates specific molecules or a set of molecules with the probable outcome of a disease.
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Treatment
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Enables new treatment modalities for specific diseases. The concept of custom/tailored
therapy or personalised medicine.
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Predictive Medicine; Human Genome project
Other examples of uses of Molecular
Pathology in diagnosis
• Bladder Cancer: Chromosomal instability
– FISH
NORMAL
Bladder Ca
Other examples of uses of Molecular
Pathology in diagnosis
•Find the mutations in unknown syndromes
•Exome/mitochondrial Sequencing now is affordable $2500
Sci Transl Med 25 January 2012:
Vol. 4, Issue 118, p. 118ra10
Sci. Transl. Med.
Research Article
Molecular Diagnosis of Infantile Mitochondrial Disease
with Targeted Next-Generation Sequencing
Sarah E. Calvo
Power of Molecular Pathology
• What is the importance of knowing the
mutation for the patient and the offspring?
PGD
Twitter Follow
@GeneTLC
Prognosis and Molecular Pathology
WHY DO SOME PEOPLE WITH THE SAME CANCER TYPE
AND STAGE HAVE DIFFERENT PROGOSIS?
1 year
1 year
5-years
metastases
Al-Mulla F, Hagan S, Behbehani AI, Bitar MS, George SS, Going JJ,
Garcia JJ, Scott L, Fyfe N, Murray GI, Kolch W.
Raf kinase inhibitor protein expression in a survival analysis of
colorectal cancer patients.
J Clin Oncol. 2006 Dec 20;24(36):5672-9.
al-mulla Mod pathol 2006
Example of Prognostic powers of Molecular Pathology
Please read: Alizadeh Nature, al-mulla Mod pathol 2006 (I made copies for you)
If we have learned something from the 1990 is that cancer is a genetic disease requiring a
Large number of genetic alterations for progression. Studying single genes or a single translocation is a
futile process. In the years to come we are using more powerful comparative techniques such as gene
chips and proteomics, which as I will show you unveil our blindfold.
By arraying nearly 18,000 genes on a glass chip about
twice the size of a postage stamp and recording the
expression patterns of those genes, researchers have
obtained detailed molecular portraits of a form of
lymphoma.
The gene expression profiling experiments revealed that
diffuse large-cell B-cell lymphoma (DLBCL) is actually at
least two distinct forms of cancer.
"This work shows that the molecular portrait of a
tumor that we get from DNA microarray analysis
can actually be interpreted as a much clearer,
more detailed picture of the tumor's biology and
that the new things we can see in this picture
really make a difference for the patient,"
Gene chip
The DLBCL expression matrix, with rows
and columns ordered according to a
hierarchical clustering applied separately to
the rows and columns.
Survival curves of the two groups defined by the low
or high expression of genes in the first cluster from
tree harvesting. Group 1 has low gene expression, and
group 2 has high gene expression. The survival in the
groups is significantly different (p = 2.4 × 10-5).
Immunohistochemistry Algorithm
Clin Cancer Res 2009;15(17) September 1, 2009
GCB: Germinal
Center B-cell like
(Good prognosis)
ABC: Activated
B-cell like
(Poor prognosis)
Example of application of Molecular Pathology in
“Tailored therapy”
Personalized Medicine
• All patients with a specific disorder are treated the
same. However, People are different and diseases
respond differently!
• Treat specific disorder depending on disrupted
pathway.
• BRAF mutation in Melanoma
• Imatinib/Gleevec in CML and GIST (c-KIT)
• HER-2/Neu amplification and treatment of breast
cancer by Herceptin
• CetuxiMab benefit in KRAS wild type GGT
• Pharmacogenomics (Warfarin and CYP2C9+VKORC1 Variants)
Sorafenib and
Dacarbazine
CetuxiMab benefit in wild type
KRAS oncogene GGT
Karapetis et al., N Engl J Med 2008; 359:1757-1765
MAPK pathway
EGFR
Locations of the breakpoints in the ABL and BCR
genes and structure of the chimeric mRNAs derived
from the various breaks.
9q34
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Never find exon 1 in of ABL EVEN IF RETAINED IN THE GENOMIC FUSION
A2 is always fused. In the majority of CML and 1/3 of ALL (ph+) the break point is
M-bcr giving p210 BCR-ABL chimera). In others remaining ALL etc
the breakpoint differ giving chimeras of p190 to p230 respectively
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9/22 translocation
The drug competes with ATP for its specific binding site in the kinase domain. Thus, whereas the physiologic binding of
ATP to its pocket allows Bcr-Abl to phosphorylate selected tyrosine residues on its substrates (left diagram), a synthetic
ATP mimic such as STI571 fits this pocket equally well but does not provide the essential phosphate group to be
transferred to the substrate (right diagram). The downstream chain of reactions is then halted because, with its tyrosines
in the unphosphorylated form, this protein does not assume the necessary conformation to ensure association with its
effector.
• Gleevec STI-571 is currently in phase II-III and is showing unprecedented success in PH+ ALL and CML. Although it
will not be useful for PH- leukemias. This is a good example for tailored therapy put forward by understanding the
molecular basis of diseases.
Molecular Methods helps in DIAGNOSIS, predicting PROGNOSIS
and tailoring THERAPY to your patients needs.
They have their limitations and you need to understand their probabilistic
nature
•Expense
•Expertise
We are part of a multidisplinary team “Remember that taking a Good
History allows you to get a diagnosis in 80-90% of cases without the
Need of using Fancy technology”
ANY QUESTIONS?