Genetic Testing PDF

Genetic Testing Health Biotechnology Prof. Dr. Attya Bhatti Atta ur Rahman School of Sciences National University of Sciences and Technology  What is Genetic testing? Genetic testing is a tool to assess genetic variations inherited from each parent (germline variation) an...

Genetic Testing Health Biotechnology Prof. Dr. Attya Bhatti Atta ur Rahman School of Sciences National University of Sciences and Technology  What is Genetic testing? Genetic testing is a tool to assess genetic variations inherited from each parent (germline variation) and has multiple potential applications in the healthcare field including its use for predicting and managing the risk of developing several disease Image source: GenepoweRx conditions.  Application- Molecular Diagnosis Molecular diagnosis of human disorders is referred to as the detection of the various pathogenic mutations in DNA and /or RNA samples in order to facilitate – detection, – diagnosis, – sub-classification, – prognosis, and – monitoring response to therapy. 3 Molecular Testing 4  Overview Three main categories: Cytogenetic Testing -Examination of chromosomes and their abnormalities Biochemical Testing - Analyzing the protein instead of the gene Molecular Testing - Direct DNA analysis is possible only when the gene sequence of interest is known 5  Cytogenetics Is the study of the structure and properties of chromosomes, chromosomal behaviour during mitosis and meiosis, chromosomal influence on the phenotype and the factors that cause chromosomal changes. Related to disease status caused by abnormal chromosome number and/or structure.  Methods for chromosomal analysis: Karyotyping and banding The collection of all the chromosomes is referred to as a Karyotype. The method used to analyze the chromosome constitution of an individual, known as chromosome banding. Chromosomes are displayed as a karyogram.  Obtaining and preparing cells for chromosome analysis ❑ Cell source: – Blood cells – Skin fibroblasts – Amniotic cells / chorionic villi ❑ Increasing the mitotic index - proportion of cells in mitosis using colcemid ❑ Synchronizing cells to analyze prometaphase chromosomes  Cytogenetic Testing Chromosomes of a dividing human cell are analyzed Cells from other blood (white blood cells -T lymphocytes) or other tissues such as bone marrow, amniotic fluid, and other biopsies can also be cultured Chromosomes are fixed, spread on microscope slides and then stained Staining allows each of the chromosomes to be individually identified The distinct bands of each chromosome enables analysis of chromosome structure 9  Key procedure In the case of peripheral (venous) blood ❑ A sample is added to a small volume of nutrient medium containing phytoheamagglutinin, which stimulates T lymphocytes to divide. ❑ The cells are cultured under sterile conditions at 37C for about 3 days, during which they divide, and colchicine is then added to each culture. ❑ This drug has the extremely useful property of preventing formation of the spindle, thereby arresting cell division during metaphase, the time when the chromosomes are maximally condensed and therefore most visible. ❑ Hypotonic saline is then added, which causes the red blood cells to lyze and results in spreading of the chromosomes, which are then fixed , mounted on a slide and stained ready for analysis PREPARATION OF CHROMOSOMES Karyotype Analysis Following Steps are involved;  Counting the number of cells, sometimes referred as metaphase spread  Analysis of the banding pattern of each individual chromosome in selected cells.  Total chr. count is determined in 10-15 cells, but if mosaicism is suspected then 30 or more cell count will be undertaken.  Detailed analysis of the banding pattern of the individual chromosomes is carried out in approx. 3-5 metaphase spread, which shows high quality banding.  The banding pattern of each chromosome is specific and shown in the form of Idiogram. MITOTIC CHROMOSOMAL SPREAD  Chromosome Banding  Chromosome banding is developed based on the presence of heterochromatin and euchromatin.  Heterochromatin is darkly stained whereas euchromatin is lightly stained during chromosome staining.  G-banding  C-banding Types of chromosome banding  Q-banding  R-banding  T-banding  Karyogram Giemsa (G)-banding is a cytogenetic method to visualize condensed chromosomes and to attain a visible karyotype using Giemsa stain. 15 Image source: https://www.hematologics.com/services/cytogenetics/  Molecular cytogenetics - locates specific DNA sequences on chromosomes Analysis of the gross structural organization of chromosomes Higher resolution analyses Target DNA Sequence Probe – probes are often 15-50 nucleotides long and are chemically synthesized. Molecular Methods for chromosomal analysis Molecular Cytogenetics ❑ Fluorescent in situ Hybridization (FISH) ❑ Chromosome painting ❑ Comparative Genomic Hybridization (CGH) ❑ Molecular karyotyping and Multiplex FISH(M- FISH) ❑ Spectral Karyotyping ❑ Array CGH In situ hybridization In situ hybridization, DNA probes can be used to determine the chromosomal location of a gene or the cellular location of an mRNA in a process called in situ hybridization. The name is derived from the fact that DNA or RNA is visualized while it is in the cell (in situ). The maximum resolution of conventional FISH on metaphase chromosomes is several megabases. Prometaphase chromosomes can permit 1 Mb resolution. Principles of FISH  Fluorescent IN SITU Hybridization (FISH) A technology in which labeled nucleic acid sequence/ probes are used for the visualization of specific DNA or RNA sequences on mitotic chromosome preparations or in interphase cells. Fluorescently labeled DNA probes to detect or confirm gene or chromosome abnormalities that are generally beyond the resolution of routine Cytogenetics. The sample DNA (metaphase chromosomes or interphase nuclei) is first denatured, a process that separates the complimentary strands within the DNA double helix structure. The fluorescently labeled probe of interest is then added to the denatured sample mixture and hybridizes with the sample DNA at the target site as it reanneals (or reforms itself) back into a double helix. The probe signal can then be seen through a fluorescent microscope and the sample DNA scored for the presence or absence of the signal. Concept: A simple procedure for mapping genes and other DNA sequences is to hybridize a suitable labeled DNA probe against chromosomal DNA that has been denatured in situ.  First step: prepare short sequences of single- stranded DNA that match a portion of the gene (probe) Second step: label probes by attaching fluorescent dye Next: probe to bind to the complementary strand of DNA Image source: NIH Fluorescence In Situ Hybridization Fact Sheet 21  Fluorescent in Situ Hybridization (FISH) Process which vividly paints chromosomes or portions of chromosomes with fluorescent molecules to identify chromosomal abnormalities (e.g., insertions, deletions, translocations and amplifications) help a researcher or clinician identify where a particular gene falls within an individual's chromosomes Image source: https://clgenetics.com/our-services/fluorescence-in-situ-hybridization// 22  Biochemical genetic diseases such as inborn errors of metabolism are present at birth and disrupt a key metabolic pathway. Diagnostic tests can be developed to directly measure protein activity (enzymes), level of metabolites (indirect Biochemical measurement of protein activity), and the size or quantity of protein (structural proteins) Testing Tissue sample in which the protein is present (blood, urine, amniotic fluid, or cerebrospinal fluid) Technologies enable both qualitative detection and quantitative determination: immunohistochemistry, gas/liquid chromatography/mass spectrometry (LC/GC/MS), and bioassays Biochemical Testing Methods 24  Immunohistochemistry Immunohistochemistry (IHC) is a powerful technique that exploits the specific binding between an antibody and antigen to detect and localize specific antigens in cells and tissue, most commonly detected and examined with the light microscope It requires the availability of tissue samples and can be performed on frozen or formalin-fixed paraffin-embedded (FFPE) tissue Step 1: pretreatment and antigen retrieval (AR) (heat/enzymatic digestion) Step 2: addition of primary antibody Step 3: application of a secondary antibody that binds the primary antibody Step 4: addition of a detection reagent to localize the primary antibody Step 5: Visualization 25 Image source: THE SCIENTIST 26  Molecular Testing Direct DNA testing may be the most effective methodology, particularly if the function of the protein is not known and a biochemical test cannot be developed! A DNA test can be performed on any tissue sample and require very small amounts of sample. Several different molecular technologies can be used to perform testing including polymerase chain reaction-based assays (PCR), and hybridization and direct sequencing 27 28 29  PCR Based Method PCR is a commonly used procedure used to amplify targeted segments of DNA through repeated cycles of denaturation (heat-induced separation of double-stranded DNA), annealing (binding of specific primers of the target segment to parent DNA strand), and elongation (extension of the primer sequences to form new copy of target sequence) The amplified product can then be further tested, such as by digestion with a restriction enzyme and gel electrophoresis to detect the presence of a mutation/polymorphism. 30 Genetic testing methods that range from detecting or examining a single gene to the whole genome and can be selected based on disease type and other factors. Image source: https://www.jupiterfamilypractice.com/should-i-get-genetic-testing/ 31  Single-Nucleotide Polymorphisms Single-nucleotide polymorphisms (SNPs, pronounced “snips”), Single-base-pair differences in DNA sequence between individual members of a species. Arising through mutation, SNPs are inherited as allelic variants. Single-nucleotide polymorphisms are numerous and are present throughout genomes. In a comparison of the same chromosome from two different people, a SNP can be found approximately every 1000 bp. 32 33 34  Advanced Genetic Testing Four major genome-wide assays are used to assess single nucleotide polymorphism (SNP) base variants and copy number variations (CNVs): Array comparative genomic hybridization (aCGH) SNP microarray (array) Whole exome sequencing (WES) Whole genome sequencing (WGS) 35  Microarray A high-resolution genome-wide screen for copy number variants! Single nucleotide polymorphism microarrays : SNP arrays involve hybridization of only the patient’s DNA-utilize two different oligonucleotides, one matching each of two variant alleles. Red: more reference DNA than patient DNA, therefore a deletion is present. Green: more patient DNA than reference DNA, therefore a duplication is present. What does yellow represent ? Yellow: equal patient and reference DNA present! 36 Image source: https://www.genomicseducation.hee.nhs.uk/genotes/knowledge-hub/microarray-array-cgh/ 37  Hybridization Array CGH: Allows the detection of very small chromosomal imbalances that cannot be identified through karyotyping (identifies losses or gains of DNA across the whole genome. ) Very small changes in the amount of genetic information are referred to as microduplications (gain of genetic information) or microdeletions (loss of genetic information) Array CGH compares an individual’s DNA with a control sample of DNA (typically from an individual not possessing the disorder of interest) and identifies differences between the two sets of DNA Allows physicians and researchers to relate an individual’s physical and mental characteristics (phenotype) to detailed profile of their genetic makeup (genotype) Principle: one strand of DNA will bind to (or “hybridize”) with another strand having complimentary nucleotide sequence Array CGH is a technique that analyses the entire genome for copy number aberrations (CNA) by comparing patient DNA to a reference DNA 38  Next Generation Sequencing Next-generation sequencing (NGS) is a massively parallel DNA/RNA sequencing technology that offers ultra-high throughput, scalability, and speed The technology is used to determine the order of nucleotides in entire genomes or targeted regions of DNA or RNA Allows for and variant/mutation detection Principle: fragmenting DNA/RNA into multiple pieces, adding adapters, sequencing the libraries, and reassembling them to form a genomic sequence. In principle, the concept is similar to capillary electrophoresis Video: 39  Whole Exome Sequencing Whole genome vs. whole exome sequencing vs. targeted gene sequencing What is exome? DNA contains both coding regions (portions of the DNA that code for proteins, termed exons) and non-coding regions (termed introns) The entirety of human DNA contains roughly 180,000 exons that comprise roughly 1% of the entire genome Since exons code for proteins, they contain most of the errors that occur in DNA sequences that underlie genetic disorders- Variations in an individual’s DNA sequence relative to standards can then be identified (base or copy variants) to help assess the risk of disease or be used along with a patient’s clinical assessment to either confirm or help establish a diagnosis 40  Whole Genome Sequencing WGS is a method for resolving the detailed nucleotide sequence of the entire genome. Ultimate potential to resolve almost the entirety of structural variation in a patient’s genome, including intronic as well as exonic segments of DNA Why study introns when they do not code directly for proteins? Introns may affect the extraction of information from DNA in exonic regions and may have other regulatory functions on genetic networks hence examining sequence variations in these regions has the potential to reveal additional contributors to disease! 41 Image source: https://microbenotes.com/next-generation-sequencing-ngs/ 42  NGS Applications Rapidly sequence whole genomes Deeply sequence target regions Utilize RNA sequencing (RNA-Seq) to discover novel RNA variants and splice sites, or quantify mRNAs for gene expression analysis Analyze epigenetic factors such as genome-wide DNA methylation and DNA-protein interactions Sequence cancer samples to study rare somatic variants, tumor subclones, and more…… Study the human microbiome Identify novel pathogens 43 Molecular Testing Challenging For some genetic diseases, many different mutations can occur in the same gene and result in the same disease, making molecular testing challenging Problem Solving For example, more than 800 mutations in theof cystic Majority CF casesfibrosis are caused by transmembrane conductance regulatorapproximately (CFTR) can 30 mutations, this group of cause cystic mutations is first tested before more fibrosis (CF) comprehensive testing, such as sequencing, is Is sequencing practical here ? performed! It would impractical to sequence the entire CFTR gene to identify the causative mutation since the gene is quite large! 44  Finding Finding genetic disease in unborn child Finding out if people carry genes for a disease and might Finding REASONS pass it on to their children. FOR Screening Screening embryos for disease. GENETIC TESTING Testing Testing for genetic disease in adult before they cause symptoms. Making a diagnosis in a person who has disease Making symptoms Figuring out the type or dose of a medicine that is best for Figuring a certain person.  Types of genetic tests Newborn screening: Newborn screening is used just after birth to identify genetic disorders that can be treated early in life. Test infants for phenylketonuria (a genetic disorder that causes intellectual disability if left untreated) and congenital hypothyroidism (a disorder of the thyroid gland). Diagnostic testing: Diagnostic testing is used to identify or rule out a specific genetic or chromosomal condition. Diagnostic testing can be performed before birth or at any time during a person's life, but is not available for all genes or all genetic conditions.  Types of genetic tests Carrier testing: Carrier testing is used to identify people who carry one copy of a gene mutation that, when present in two copies, causes a genetic disorder. This type of testing is offered to individuals who have a family history of a genetic disorder. If both parents are tested, the test can provide information about a couple's risk of having a child with a genetic condition. Prenatal testing Prenatal testing is used to detect changes in a fetus's genes or chromosomes before birth. This type of testing is offered during pregnancy if there is an increased risk that the baby will have a genetic or chromosomal disorder.  Types of genetic tests Preimplantation testing: Preimplantation testing, also called preimplantation genetic diagnosis (PGD), It is used to detect genetic changes in embryos that were created using assisted reproductive techniques such as in-vitro fertilization. To perform preimplantation testing, a small number of cells are taken from these embryos and tested for certain genetic changes. Only embryos without these changes are implanted in the uterus to initiate a pregnancy. Forensic testing: forensic testing uses DNA sequences to identify an individual for legal purpose. Research testing: research testing involves finding unknown genes, learning how genes work and advancing our understanding of genetic conditions. CONT… Susceptibility / Predictive testing: Susceptibility testing helps determine the likelihood of developing a disease or complication if a specific genetic alteration is present. Susceptibility testing is not a guarantee that disease will develop, but it is a valuable tool in risk assessment and in preventive management (e.g., about 3% of BRCA1 mutation carriers will develop breast cancer by the age of 30, but by age 70, about 85% of women with a BRCA1 mutation will have developed breast cancer  Results of genetic tests mean The results of genetic tests are not always straightforward, which often makes them challenging to interpret and explain. Therefore, it is important for patients and their families to ask questions about the potential meaning of genetic test results both before and after the test is performed. When interpreting test results, healthcare professionals consider a person’s medical history, family history, and the type of genetic test that was done. A positive test result means that the laboratory found a change in a particular gene, chromosome, or protein of interest. Depending on the purpose of the test, this result may confirm a diagnosis, indicate that a person is a carrier of a particular genetic mutation, identify an increased risk of developing a disease (such as cancer) in the future, or suggest a need for further testing Cont… A negative test result means that the laboratory did not find a change in the gene, chromosome, or protein under consideration. This result can indicate that a person is not affected by a particular disorder, is not a carrier of a specific genetic mutation, or does not have an increased risk of developing a certain disease. In some cases, a test result might not give any useful information. This type of result is called uninformative, indeterminate, inconclusive, or ambiguous. Uninformative test results sometimes occur because everyone has common, natural variations in their DNA, called polymorphisms, that do not affect health.  ETHICAL LEGAL AND SOCIAL ISSUES IN GENETIC TESTING Consent: Patient need to sufficiently informed about the implication of genetic screening before they can provide informed consent. The voluntary nature of the screening process must be emphasized. Counseling: To reduce potential psychological distress, counseling should be available to provide information about genetic risk and explain choices regarding genetic testing and further management. The risk of stigma: Misunderstanding of the genetic risk of developing disease can increase stigmatization. This may be around life expectancy, lifestyle choices, or decision about having children.  Cont… Confidentiality: Like other medical information, result from genetic testing are considered confidential, under normal practice, the doctor patient relationship protect against disclosure of genetic information. Disclosure to family members: Doctors face a dilemma when reporting the results of genetic screening. Standard medical practice is based on the principles that doctor’s should focus on their patient and that medical information should remain confidential.