Molecular Medicine in Clinical Practice

1. Molecular Medicine in Clinical Practice Dr. Osama . I . Nassif , FRCPC Associate Professor and Consultant Pathologist Department of Pathology, Faculty of Medicine King Abdullaziz University Hospital

2. Introduction • Sources of DNA in clinical practice: • Any nucleated cell in the body • Blood • Tumor sample (tissue or aspirate) • Body discharge • Hair root, semen, or body fluid • Chorionic villi and amnionic fluid • Mouth wash

3. Introduction • DNA isolation • RNA isolation

4. Introduction • Molecular Bio-techniques • Blotting • Southern • Northern • Western • Hybridization • PCR, RT-PCR • DNA sequencing • cDNA cloning • Recombinant protein

5. Introduction • Molecular Bio-techniques has many applications in several fields of clinical practice including: • Medical genetics • Fetal and neonatal medicine • Medical microbiology • Infectious diseases • Medical oncology • Hematology • Anatomical pathology and tumor diagnosis • Therapeutics • Forensic pathology

6. Applications of Molecular Bio-techniques in Medical Genetics • Analysis and characterization of genes abnormalities leading to disease. • Understanding genetic diseases pathogenesis • Detection of gene mutation (mutational analysis) • Study of genetic diseases pattern of inheritance • Diagnosis and screening of genetic diseases • Prenatal diagnosis • Identification of diseases carrier to help in genetic and pre-marriage counseling.

7. Medical Genetics • Four major categories of genetic disorders: • (1) disorders related to mutant genes of large effect. most of these follow the classic Mendelian patterns of inheritance, they are also referred to as Mendelian disorders. • (2) diseases with multifactorial (polygenic) inheritance. These are influenced by both genetic and environmental factors • (3) chromosomal disorders, includes diseases that result from genomic or chromosomal mutations • (4) single-gene disorders with nonclassic patterns of inheritance.

8. Mutational Analysis • It means the identification of changes in DNA which produce disease or dysfunction. • Several methods can be used to detect gene mutation including PCR, southern blotting, Pulsed-Field Gel Electrophoresis (PFGE):, FISH, cytogenetic, DNA sequencing. • Factors that determine the type of methods to be used include: • Nature and size of mutation • Mutation knowledge • The frequency of mutation in the population of interest (hot spot mutation) • Size of the gene of interest • Nature of the available sample for testing

9. Mutational Analysis • Detecting DNA deletion: • Very small deletions can be detected by PCR (e.g. cystic fibrosis) • Larger deletion (e.g. αthalassaemia) can be detected by Southern blotting • The largest deletion (e.g. contiguous gene syndrome) can be detected by PFGE or FISH

10. Mutational Analysis • Detecting point mutation: • These occur more frequently than deletion • They are more difficult to identify because they are small, and heterogeneous. • PCR is the most useful technique in detecting these mutation if they are known in family of interest.

11. Mutational Analysis • DNA sequencing • Chromosomal analysis • Karyotyping • FISH

12. Applications of Molecular Bio-techniques in Medical Genetics

13. Diagnosis of Genetic Diseases • Two general methods are used: • Cytogenetic analysis and • Molecular analysis.

14. Diagnosis of Genetic Diseases Prenatal chromosome analysis: • This should be offered to all patients who are at risk of cytogenetically abnormal progeny. • It can be performed on cells obtained by amniocentesis, on chorionic villus biopsy, or on umbilical cord blood. • indications are the following: • Advanced maternal age (>34 years) because of greater risk of trisomies • A parent who is a carrier of a balanced reciprocal translocation, robertsonian translocation, • A previous child with a chromosomal abnormality • A parent who is a carrier of an X-linked genetic disorder (to determine fetal sex)

15. Diagnosis of Genetic Diseases Postnatal chromosome analysis: • This is performed on peripheral blood lymphocytes. • Indications are as follows: • Multiple congenital anomalies. • Unexplained mental retardation or developmental delay. • Suspected aneuploidy (e.g., features of Down syndrome). • Suspected unbalanced autosome (e.g., Prader-Willi syndrome). • Suspected sex chromosomal abnormality (e.g., Turner syndrome). • Suspected fragile X syndrome. • Infertility (to rule out sex chromosomal abnormality). • Multiple spontaneous abortions.

16. Diagnosis of Genetic Diseases • Many genetic diseases are caused by subtle changes in individual genes that cannot be detected by karyotyping. • Traditionally the diagnosis of single-gene disorders has depended on the identification of abnormal gene products (e.g., mutant hemoglobin or enzymes) or their clinical effects, such as anemia or mental retardation (e.g., phenylketonuria). • Now it is possible to identify mutations at the level of DNA and offer gene diagnosis for several mendelian disorders. • Examples of inherited diseases that can be detected by PCR

17. Diagnosis of Genetic Diseases The advantages of molecular diagnosis of genetic disorders: • It is remarkably sensitive. • The amount of DNA required for diagnosis by molecular hybridization techniques can be readily obtained from 100,000 cells. • The use of PCR allows several million-fold amplification of DNA or RNA, making it possible to use as few as 100 cells or 1 cell for analysis. • Tiny amounts of whole blood or even dried blood can supply sufficient DNA for PCR amplification. • DNA-based tests are not dependent on a gene product that may be produced only in certain specialized cells (e.g., brain) or expression of a gene that may occur late in life. • Virtually all cells of the body of an affected individual contain the same DNA, each postzygotic cell carries the mutant gene. • These two features have profound implications for the prenatal diagnosis of genetic diseases because a sufficient number of cells can be obtained from a few millilitres of amniotic fluid or from a biopsy of chorionic villus that can be performed as early as the first trimester.

18. Diagnosis of Genetic Diseases • There are two approaches to the diagnosis of single-gene diseases by DNA based technology: • Direct detection of mutations and • Indirect detection based on linkage of the disease gene with a harmless "marker gene."

19. Diagnosis of Genetic Diseases • Direct Gene Diagnosis: “diagnostic biopsy of the human genome” • Direct gene diagnosis is possible only if the mutant gene and its normal counterpart have been identified and cloned and their nucleotide sequences are known. • One technique relies on: • some mutations alter or destroy certain restriction sites on DNA • e.g.: detecting the mutation of gene encoding factor V. This protein is involved in the coagulation pathway, and a mutation affecting the factor V gene is the most common cause of inherited predisposition to thrombosis.

20. Direct gene diagnosis: detection of coagulation factor V mutation by PCR. Base substitution in an exon destroys one of the two Mnl1 restriction sites. The mutant allele therefore gives rise to two, rather than three, fragments by PCR analysis.

21. Diagnosis of Genetic Diseases • Allele-specific oligonucleotide hybridization "dot blot" test: • e.g.: α1 antitrypsin deficiency • Direct gene diagnosis by using PCR and an allele-specific oligonucleotide probe. • Base change converts a normal α1 antitrypsin (allele M) to a mutant (Z) allele.

22. Two synthetic oligonucleotide probes, one corresponding in sequence to the normal allele (M probe) and the other corresponding to the mutant allele (Z probe), are lined up against normal and mutant genes • The PCR products from normal individuals, those heterozygous for the Z allele or homozygous for the Z allele, are applied to filter papers in duplicate, and each spot is hybridized with radiolabeled M or Z probe. A dark spot indicates that the probe is bound to the DNA.

23. Diagnosis of Genetic Diseases • Mutations that affect the length of DNA (e.g., deletions or expansions) can be detected by PCR analysis. • e.g.: the fragile X syndrome (associated with trinucleotide repeats)

24. With PCR, the differences in the size of CGG repeat between normal and premutation gives rise to products of different sizes and mobility. With a full mutation, the region between the primers is too large to be amplified by conventional PCR. In Southern blot analysis the DNA is cut by enzymes that flank the CGG repeat region, and is then probed with a complementary DNA that binds to the affected part of the gene. A single small band is seen in normal males, a higher-molecular-weight band in males with premutation, and a very large (usually diffuse) band in those with the full mutation.

25. Diagnosis of Genetic Diseases • Indirect DNA Diagnosis: Linkage Analysis • large number of genetic diseases, including some that are relatively common, information about the gene sequence is lacking. • Therefore, alternative strategies are to track the mutant gene on the basis of its linkage to detectable genetic markers.

26. Diagnosis of Genetic Diseases • Principle: • to determine whether a given fetus or family member has inherited the same relevant chromosomal region(s) as a previously affected family member. • the success of such a strategy depends on the ability to distinguish the chromosome that carries the mutation from its normal homologous counterpart. • This is accomplished by finding naturally occurring variations or polymorphisms in DNA sequences.

27. Diagnosis of Genetic Diseases • Restriction Fragment Length Polymorphisms (RFLPs). • Background: • examination of DNA from any two persons reveals variations in the DNA sequences. • Most of these variations occur in noncoding regions of the DNA and are hence phenotypically silent. • these single base pair changes may abolish or create recognition sites for restriction enzymes, thereby altering the length of DNA fragments produced after digestion with certain restriction enzymes. • Using appropriate DNA probes that hybridize with sequences in the vicinity of the polymorphic sites, it is possible to detect the DNA fragments of different lengths by Southern blot analysis. • RFLP refers to variation in fragment length between individuals that results from DNA sequence polymorphisms.

28. RFLP: This technique is to distinguish family members who have inherited both normal chromosomes from those who are heterozygous or homozygous for the mutant gene.

29. RFLP analysis for the presence of the sickle-cell locus. Genomic DNA is isolated and digested with the restriction enzyme MstII. One MstII site is lost at the sickle-cell locus. The DNA is then Southern blotted and analyzed with a b-globin-specific probe corresponding to sequences at the 5'-end of the gene.

30. Diagnosis of Genetic Diseases • Length polymorphisms: • Background: • Human DNA contains short repetitive sequences of noncoding DNA. • the number of repeats affecting such sequences varies greatly between different individuals, the resulting length polymorphisms are quite useful for linkage analysis. • These polymorphisms are often subdivided on the basis of their length into: • Microsatellite repeats (usually less than 1 kb and are characterized by a repeat size of 2 to 6 base pairs). • Minisatellite repeats (these are larger 1 to 3 kb and the repeat is usually 15 to 70 base pairs) • These stretches of DNA can be used quite effectively to distinguish different chromosomes

31. allele C is linked to a mutation responsible for autosomal dominant polycystic kidney disease (PKD). Application of this to detect progeny carrying the disease gene is illustrated in one hypothetical pedigree

32. Diagnosis of Genetic Diseases • Limitations of linkage studies: • For diagnosis, several relevant family members must be available for testing. • Key family members must be heterozygous for the polymorphism • Normal exchange of chromosomal material between homologous chromosomes (recombination) during gametogenesis may lead to "separation" of the mutant gene from the polymorphism pattern with which it had been previously coinherited. This may lead to an erroneous genetic prediction in a subsequent pregnancy.

33. Diagnosis of Genetic Diseases • Molecular diagnosis by linkage analysis has been useful in the antenatal or presymptomatic diagnosis of disorders such as Huntington disease, cystic fibrosis, and adult polycystic kidney disease. • In general, when a disease gene is identified and cloned, direct gene diagnosis becomes the method of choice. • If the disease is caused by several different mutations in a given gene direct gene diagnosis is not possible, and linkage analysis remains the preferred method.

34. Applications of Molecular Bio-techniques in Medical Oncology

35. Molecular Biology for Medical Oncology • Diagnosis • Cancer screening and early detection • Evaluation of cancer risk • Treatment • Follow up and detection of residual tumor • Prognosis • Research and cancer pathogenesis

36. Molecular Diagnosis of Cancer • Molecular techniques can be used for: • Cancer diagnosis • Ancillary tools for cancer diagnosis • Subclassification of tumors

37. Molecular Diagnosis of Cancer • The gold standard test for cancer diagnosis of almost all tumors is tissue diagnosis. • PCR and/or Southern blot can be used in diagnosing B and T cell lymphomas. • PCR-based detection of T-cell receptor or immunoglobulin genes rearrangement allow distinction between monoclonal (neoplastic) and polyclonal (reactive) proliferations.

38. Molecular Diagnosis of Lymphoma Gene Rearrangement

39. Molecular Diagnosis of Lymphoma Gene Rearrangement

40. Molecular Diagnosis of Lymphoma • The normal circulating lymphocytes are polyclonal. • Because of the multiplicity of the gene rearrangement involved, the changes will not be detected at DNA level for polyclonal population. • The presence of a monoclonal population will usually mean there is a hematological or immunological disorder involving these cells. • Gene rearrangement indicates a clonal population • DNA mapping patterns are able to detect monoclonal population in B or T lymphocytes because the same gene rearrangement is now present in large number of cells

41. Molecular Diagnosis of Lymphoma • TCR-beta gene rearrangements of the DNAs extracted from cells. • The BamHI-, EcoRI-, and HindIII-digested DNA were hybridized to a probe specific for the joint region of TCR-beta gene. • Lanes P denote DNAs from this patient and Lanes N from lymphocytes of normal control. • Arrows denoted rearranged bands and bar, germline bands.

42. Molecular Techniques as Ancillary Tools for Cancer Diagnosis • RT-PCR, FISH, or cytogentics can be used to detect certain translocation or gene amplification that specific for some cancer. • These findings can be used as ancillary tool to help in soft tissue and hematological diagnosis.

43. Molecular Techniques as Ancillary Tools for Cancer Diagnosis

44. Ancillary Tools for Cancer Diagnosis

45. Subclassification of Tumors • Acute myelobalstic leukemia can be classified based on Cytogenetic findings. • Molecular techniques can help in subclassifications of non-Hodgkin's lymphomas, and pediatric sarcoma.

46. Molecular Biology for Medical Oncology • Cancer screening and early detection • Evaluation of cancer risk • Table of familial cancer

47. Follow up and detection of residual tumor • Detection of BCR-ABL by PCR gives a measure of minimal residual leukemia in patients treated for CML.

48. Evaluation of Prognosis and Response to Treatment • FISH or PCR can be used to detect amplification of HER2-nue in breast cancer patient. • PCR or cytogenetics can be used to detect amplification of C-myc in neuroblastoma patient.

49. Molecular Biology and Cancer Therapeutics

50. Anticancer “Smartbombs” Tyrosine kinase inhibitors Gleevec Iressa Monoclonal antibodies - CML - GIST Antiangiogenesis Herceptin anti-VEGF thalidomide Cetuximab Rituximab Retinoids COX-2 inhibitors Celecoxib All-trans retinoic acid - Breast Cancer