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

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

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

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

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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

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

Sickle-cell disease

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

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

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

PCR-based techniques that can be applied to detect known point mutation or SNPs in DNA: 1.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

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

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

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

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

DNA sequencing result

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

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)

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)

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)

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)

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. Gene Therapy

* 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. * An abnormal gene could be swapped for a normal gene through homologous recombination.

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.

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

-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.

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

References: Alberts et al., Molecular Biology of the Cell. 4 th ed. Karp, Cell and Molecular Biology. 4 th ed. Patrinos & Ansorge, Molecular Diagnostics. medicine/genetherapy.shtml (16 April 2009 jam WIB) medicine/genetherapy.shtml (16