Molecular Diagnosis & Gene Therapy

1. Molecular Diagnosis & Gene Therapy Asmarinah Department of Medical Biology Faculty of Medicine, University of Indonesia

2. Molecular diagnosis = nucleid acid-based diagnosis of human disordes is detection of the various pathogenic mutation in DNA and/or RNA sample(change in gene expression) = laboratory medicine + knowledge/technology of molecular genetics  benefiting from the discoveries in the field of molecular biology

3. Discoveries in the field molecular biology which influenced the development of molecular diagnostic

5. Need to know: DNA – RNA - Protein Replication (product: DNA) Transcription (product: mRNA) Translation (product: protein)

6. continued

7. Molecular Dignostic Testing ◘ facilitate: - the detection and characterization of disease - the monitoring the drug response ◘ assist in identification: - genetic modifier: introduced gene which produce a novel protein that can convert the trait of interest - disease susceptibilty

8. The primary function of molecular diagnostics:  detection of mutations and single nucleotide polymophism (SNPs) that are associated with phenotypes ▪ Pauling et al., 1949  discovered single amino acid change in β-globin chain sickle cell anemia •  introduced “molecular disease” • term • set the foundation of molecular diagnosis

9. Sickle-cell disease 1300 1100

10. Methods: • PCR • PCR-RFLP • PCR-ARMS • PCR-ASO. • PCR-DNA sequencing • real time PCR for quantitation • Microarray Mutation detection: • Specimens: • peripheral blood/cord blood lymphocytes • villi choriales • amniotic fluid • semen, hair

11. The discovery of PCR method, (Saiki et al., 1985; Mullis & Faloona, 1987)  greatly facilitated in principle revolutionized molecular diagnosis  foundation for the design and development of mutation detection, based on amplified DNA

12. The most powerfull feature of PCR:  getting the large amount of copies of the target sequence, generated by its exponential amplification

13. PCR-based techniques that can be applied to detect known point mutation or SNPs in DNA: • PCR-ARMS (Amplification Refractory Mutation System) •  based on the principle that a mismatch between the 3’ nucleotide of a PCR primer and the template reduce or prevent primer extention by Taq polimerase • 2. PCR-ASO (Allele-Specific Oligonucleotide) •  based on hybridization of PCR product to allele-specific oligonucleotide probes

14. Schematic of PCR-ARMS assay for detection of single base mutation (underlined)

15. Example: Detection of DHCR7 gene mutation that associated with Smith-Lemli-Opitz syndrome

16. PCR-ASO (Allele-Specific Oligonucleotide) ASO probes is designed to be complementary and specific for various alleles Example 

17. PCR-DNA sequencing  golden standard and definitive experimental procedure for mutation detection

18. DNA sequencing result

19. 174 Mutation in exon 6 of VDAC3 gene in sperm with low motility Sequence of PCR product from sperm with normal motility Posisition 174 : AAG (Lysine) Sequence of PCR product from sperm withlowmotility Posisition 174 : GAG (glutamic acid) (Asmarinah et al, 2005)

20. Real-Time PCR (RT-PCR) • Quantification of the PCR product in real-time, during the exponential phase of the PCR reaction One of methods that widely used: RT-PCR using fluorescent DNA intercalating dyes (such as SYBR Green 1, ethidium bromide)

21. The principle: • The dye incorporates into groove of dsDNA • During the PCR reaction, the amount of double stranded target will increased, paralleled by an increased in SYBR Green I incorporation (fluorescent emission)

22. Quantification of PCR product by RT-PCR method Amplification plot Rn = fluoresence detected at a certain point of reaction – initial fluoresence Ct = threshold cycle By plotting the Ct value of an unknown sample on the standard curve  the amount of target sequence in the sample can be determined (automatically by soft program in RT-PCR maschine)

23. Present-day mutation detection techniques  for high througput mutation analysis : DNA Microarray • The principle: • Oligonucleotides of known sequence are immobilized onto appropriate surface  DNA chip • Hybridization of the target to the microarray • Detection of hybridization, using fluorescent dyes • Quantification by high-resolution fluorescent scanning and will be analyzed by computer software Simultaneous detection of a great nummber of DNA alteration (genome-wide screening)

26. Gene Therapy is a technique for correcting defective genes responsible for disease development. Several approaches for correcting faulty genes: * A normal gene may be inserted into a nonspecific location within the genome to replace a nonfunctional gene. This approach is most common.

28. * An abnormal gene could be swapped for a normal gene through homologous recombination. * The abnormal gene could be repaired through selective reverse mutation, which returns the gene to its normal function. * The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered.

29. Methods in gene therapy: -Using virus as delivering agents: * Retroviruses, that can create double- stranded DNA copies of their RNA genomes * Adenoviruses, viruses with double-stranded DNA genomes that cause respiratory, intestinal, and eye infections in humans. * Adeno-associated viruses, small, single-stranded DNA viruses that can insert their genetic material at a specific site on chromosome 19. * Herpes simplex viruses, double-stranded DNA viruses that infect a particular cell type, neurons. Herpes simplex virus type 1 is a common human pathogen that causes cold sores.

30. Non-viral methods • - Sythetic oligonucleotides • to inactivate the genes involved in the disease process, with: • antisense specific to the target gene to disrupt the • transcription of the faulty gene. • siRNA (small/short interfering or silencing RNA) • to signal the cell to cleave specific unique • sequences in the mRNA transcript of the faulty • gene, disrupting translation of the faulty mRNA, • and therefore expression of the gene

31. Liposome, • an artificial lipid sphere with an aqueous core, • which carries the therapeutic DNA and capable of passing • the DNA through the target cell's membrane.

32. Obstacle factors for gene theraphy becoming an effective treatment for genetic diseases: * Short-lived nature of gene therapy * Immune response * Problems with viral vectors * Multigene disorders

33. References: • Alberts et al., 2002. Molecular Biology of the Cell. 4th ed. • Karp, 2005. Cell and Molecular Biology. 4th ed. • Patrinos & Ansorge, 2006. Molecular Diagnostics. • http://www.ornl.gov/sci/techresources/Human_Genome/medicine/genetherapy.shtml (16 April 2009 jam 15.00 WIB)