Dr. Fahd Al-Mulla©2007

• Understand that diseases (in most cases) are phenotypic representation of proteins quality and quantity disorders, either at DNA, transcriptional, translational or post translational levels

• Understand the molecular and cellular basis of common diseases:1. Genetic disorders (Down’s, heamoglobinopathies, Cancer)2. Metabolic disorders (see clinical chemistry lectures)3. Ion channels disorders (Cystic Fibrosis, Arrythmias)4. Trafficking disorders (Familial Hypercholesterolemia)5. Signaling disorders (Diabetes Mellitus)6. Structural proteins defects (Osteogenesis Imperfecta)7. Microbial virulence and Immune response.

• Define and describe the disciplines of molecular biology, molecular pathology and molecular genetics

• Comprehend that understanding the molecular and cellular basis of diseases leads to more comprehensive diagnosis, prognosis, tailored therapies and potential cures. Examples, STI571 uses in CML, Genetherapy.

What is Disease

• 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 goodat 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?

Molecular BasisOf Diseases

EnvironmentAnd genes

Changes an Enzymee.g. Phenylalanine hydroxylaseSplice site mutation leading to reduced amountCausing phenylketonuria

Changes an Enzyme inhibitore.g. 1-AntitrypsinMissense mutation that impair secretion from liver To serum causing Emphysema and Liver disease

Changes a receptore.g. Low density lipoprotein receptorDeletion or point mutation that reduce synthesis, Or transport to the cell surface or binding to low density lipoproteinCausing Familial hypercholesterolemia

Change a transport or carrier protein1.e.g. HaemoglobinMutations in splice sites (commonest) leading to Reduced -globin.causing -Thalassemia in -Thalassemia the -globin gene is usually deleted2.e.g. Cystic fibrosis transmembrane conductanceRegulator. Deletions or point mutation causingCystic fibrosis.

Changes in Hemostasise.g. Factor VIII deletions, insertions, nonsenseMutation reduce synthesis or abnormal factor VIIICausing Hemophilia A.

Changes in structuralProteins1.e.g. collagen, DeletionsOr point mutation thatProduce reduced amountOf normal collagen orNormal amounts of mutantCollagen. Causing Osteogenesis imperfecta2.e.g cell membrane FibrillinMissense mutations causingMarfan syndromeOr deletion of dystrophin geneCausing Duchene muscular Dystrophy

Growth regulatione.g.Rb causingRetinoblastoma etc

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

• 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.

• A pathologist is unable to define the molecules and how they interact to produce the disease represented by what is observed microscopically. This is the job of a molecular pathologist.

• 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

• Who is a molecular geneticist? Clinical geneticist?

• Diagnosis:

– 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.

• Prognosis:– Associates specific molecules or a set of molecules with the probable outcome of

a disease.

• Treatment– Enables new treatment modalities for specific diseases. The concept of

custom/tailored therapy

Relevance of Molecular Pathology

Example of Diagnostic powers of Molecular Pathology

Sickle cell anaemia

Sickle cell anaemia

Power of Molecular Pathology

• What is the importance of knowing the mutation for the patient and the offspring?

• PGD

Other examples of uses of Molecular Pathology in diagnosis

• Bladder Cancer– FISH

NORMAL

Bladder Ca

Prognosis and Molecular Pathology

1 year 1 year

metastases5-years

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

WHY DO SOE PEOPLE WITH THE SAME CANCER TYPE AND STAGE HAVE DIFFERENT PROGOSIS?

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,"

Example of Prognostic powers of Molecular PathologyPlease read: Alizadeh Nature, al-mulla Mod pathol 2006 (I made copies for you)

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).

The DLBCL expression matrix, with rows and columns ordered according to a hierarchical clustering applied separately to the rows and columns.

Gene chip

Locations of the breakpoints in the ABL and BCR genes and structure of the chimeric mRNAs derived from the various breaks.

9q34

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 isM-bcr giving p210 BCR-ABL chimera). In others remaining ALL etcthe breakpoint differ giving chimeras of p190 to p230 respectively

22

Example of application of Molecular Pathology in “Tailored therapy”

9/22 translocation

9

22

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.

Other examples of tailored therapy

• HER-2/Neu amplification and treatment of breast cancer by Herceptin

• Pharmacogenomics

Molecular Methods helps in DIAGNOSIS, predicting PROGNOSIS and tailoring THERAPY to your patients needs.

They have there limitations and you need to understand their probabilistic nature

•Expense•Expertise

We are part of a multidisplinary team “Remember that taking a GoodHistory allows you to get a diagnosis in 80-90% of cases without the Need of using Fancy technology”

ANY QUESTIONS?