MOLECULAR DIAGNOSTICS PATERNITY TESTING Molecular diagnostics - collection of techniques used to analyse biological markers in the genome and proteome by applying molecular biology to medical testing - techniques are used to: - diagnose and monitor disease - detect risk - decide which therapy will work best for individual patient - by analysing the specifics of the patients and their disease, molecular diagnostics offers the prospect of personalised medicine Molecular diagnostics - these tests are useful in a range of medical specialisms, including: oncology - tumor markers neurology - antigens and antibodies associated with neurodegenerative diseases pulmonary medicine – cystic fibrosis metabolic diseases - phenylketonuria immunology – markers associated with autoimmune diseases infectious disease – chlamydia, influenza virus and tuberculosis prenatal – dynamics of markers in physiological pregnancy and their changes in pathological pregnancy Molecular diagnostics Marker = laboratory demonstrable signs, which disease manifests itself, or from which it is derived Requirements: - associated with disease - determinable in blood, urine or tissue - screening - designed to refine the diagnosis, monitoring of therapy humoral – located in body fluids cellular – receptors on cells, presence of specific proteins in cell membrane or inside cells genetics – in DNA or RNA Molecular diagnostics - strategy Direct DNA/RNA diagnostics - determine whether DNA carries a mutation or not - detection of mutation in genes associated with disease Indirect DNA/RNA diagnostics - use of binding markers in family studies - reveals allele-related illness in the family - use of polymorphic sites of the human genom - today is not used (only for paternity testing) Diagnostics of proteins and their description - whether the protein linked with disease is present or not - changes in conformation of physiologic proteins - presence of receptors Molecular diagnostics - methods Direct DNA/RNA diagnostics - detection of causal mutations in responsible gene always confirm clinical diagnosis (ADVANTAGE) we must know: - gene to be analyzed - standard (wild type) gene sequence 2 procedures: - detection of well known mutations (scoring) - search of unknown mutations (scanning) PCR SSCP on gel (single-strand conformation polymorphism) - changes in DNA primary structure change also secondary structure and it changes the mobility of DNA on the gel DGGE (denaturing gradient gel electrophoresis) - changes in structure influence the speed of denaturing Molecular diagnostics - methods Indirect DNA/RNA diagnostics - paternity testing Diagnostics of proteins and their description We must know: - protein to be analyzed - physiological levels in organism - its structure ELISA (enzyme-linked immunosorbent assay) - detection of antigen (protein), specific antibodies Western blot - electrophoretic separation, detection of specific proteins on membrane 2D electrophoresis Crystallography, NMR, MS-MALDI – determining of structure Molecular genetics markers of selected diseases Oncology Retinoblastoma - 3 % of tumors in children up to 15 years - malignant tumor that rapidly develops from the immature cells of a retina - most common primary malignant intraocular cancer in children, and it is almost exclusively found in young children - AD heredity or sporadically Causes: - mutation in Rb1 gene - „brake“ of cell cycle - tumor suppressor gene Molecular genetics markers of selected diseases Oncology Retinoblastoma Diagnostics: red reflex - checking for a normal reddish-orange reflection from the eye's retina with an ophthalmoscope or retinoscope from approximately 30 cm / 1 foot, usually done in a dark room RT PCR - is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase chain reaction (PCR) Molecular genetics markers of selected diseases Oncology Breast carcinoma - 16 % of women tumors, but 1 % of patients are men - malignant tumor of mammary gland - AD heridity or sporadically symptoms: - area of thickened tissue in the breast, or a lump in the breast - pain in the breast that does not change with the monthly cycle - pitting or redness of the skin of the breast - rash around or on one of the nipples - sunken or inverted nipple - change in the size or shape of the breast Molecular genetics markers of selected diseases Oncology Breast carcinoma Causes: - mutation in BRCA1, BRCA2 gene - receptor HER2/neu - estrogen receptor Diagnostics: self-monitoring mammograms FISH, PCR Molecular genetics markers of selected diseases Neurology Sclerosis multiplex - serious neurodegenerative disease - affects especially girls from 17 to 30 years - is a demyelinating disease in which the insulating covers of nerve cells in the brain and spinal cord are damaged - this damage disrupts the ability of parts of the nervous system to communicate, resulting in a range of signs and symptoms, including physical, mental, and sometimes psychiatric problems Molecular genetics markers of selected diseases Neurology Sclerosis multiplex - is not considered a hereditary disease; however, a number of genetic variations have been shown to increase the risk - some of these genes appear to have higher levels of expression in microglial cells Causes: - changes in human leukocyte antigen (HLA) system - group of genes on chromosome 6 that serves as the major histocompatibility complex (MHC) - mutation in genes IL2RA a IL7RA (receptors for interleukins 2 a 7) Molecular genetics markers of selected diseases Neurology Sclerosis multiplex Diagnostics: - levels of specific antibodies in cerebro-spinal fluid - cerebrospinal fluid is tested for oligoclonal bands of IgG on electrophoresis - MR Molecular genetics markers of selected diseases Neurology Duchenn muscular dystrophy - serious neurodegenerative disease, first signs between 3-6 years, changes in posture, walking on tiptoe symptoms: - muscle weakness, frequent falls, fatigue, difficulty with motor skills (running, hopping, jumping) - enlarged calf muscles (due to increased fat content) - in 13 years invalidity (wheelchair) - expectancy 20 years lighter form: Becker muscular dystrophy cross section of the calf muscle shows extensive replacement of muscle fibers by fat cells Molecular genetics markers of selected diseases Neurology Duchenn muscular dystrophy Cause: - body can´t product muscle protein dystrophin (in correct form) (necessary for proper functioning of muscles) - gene for dystrophin has 79 exons, 70 % frameshift, 30 % mutation de novo - Xp21 Diagnostics: - FISH - MLPA (multiplex ligation-dependent probe amplification) Molecular genetics markers of selected diseases Neurology Duchenn muscular dystrophy Treatment: - exon skipping (antisense oligonucleotides) - structural analogs of DNA - allow faulty parts of the dystrophin gene to be skipped when it is transcribed to RNA for protein production, permitting a shorter but more functional version of the protein to be produced - stem cells replacement Molecular genetics markers of selected diseases Neurology Huntington disease - serious neurodegenerative disease, AD heridity - affects between 40 and 50 years - problems with mobility, leads to dementia Causes: - expansion of trinucleotides repeats - gene for huntingtin, protein accumulates in brain and physiological enzymes are not able to degrade it Diagnostics: triplet primed PCR Molecular genetics markers of selected diseases Neurology Creutzfeldt-Jakob disease - neurodegenerative disease, spongiform encephalopathy - familial, sporadic or iatrogenic - long incubation period, affected is phenotype Causes: - prion (infectious protein particle, PrPSc) changes the structure of standard prion protein (PrPC) to pathological isoform (PrPSc), which accumulates in brain and physiological enzymes can´t degrade it Diagnostics: Western blot ELISA immunohistology – direct detection of PrPSc in brain tissue neurohistopathology Molecular genetics markers of selected diseases Pulmonary diseases Cystic fibrosis - serious incurable disease, „salty children“ - occurrence 1:3000, survival up to 30 years - each 25th human is carrier - multiorgan disease, lung fibrosis leads to death Causes: - mutation in CFTR gene (27 exons) - greatest impact on phenotype: 10, 11, 12, 20, 21, 22 - defect in Na+ ion transfer through membrane of epithelial cells Diagnostics: PCR Molecular genetics markers of selected diseases Prenatal diagnostics Invasive: (= invasion into amnion) AMC, cordocentesis, fetoscopy Non-invasive: ultrasound, biochemical screening biochemical screening – determination of markers level in maternal serum Molecular genetics markers of selected diseases Prenatal diagnostics - performed in 1. and/or 2. trimester First trimester Second trimester PAPP-A protein AFP α-1 fetoprotein βhCG uE3 – non-conjugated estriol SP1 – trophoblast specific β1-glykoprotein -sensitive to +21, +18 - sensitive to cleft palate, intrauterine growth retardation Paternity testing Paternity testing - genomic sequences of each human are unique - DNA typing must be performed efficiently and reproducibly - testing areas: highly polymorphic loci – greater probability that two people don´t have the same locus Codis loci - 13 basic loci of DNA used to create genetic profiles - in paternity testing we performed 16 (greater certainty) - STR markers on different chromosomes genetic profile – combination of several highly polymorphic loci Method: multiple PCR https://www.sciencedirect.com/science/article/pii/S1687157X12000194 Paternity testing paternity index – in each population allele occurs with another frequency - the higher the frequency, the lower the index paternity confirmation - by multiplying paternity indices for each locus get total loci paternity index, which will be converted into the probability (99.9% accuracy) paternity exclusion - a child has a combination of markers, that the father does not have (paternity can be 100% confidently excluded) Thank you for your attention!