Tiêu đề MBDx L6 - Molecular Testing on Genetic Disorders.pdf ✅

MT 3 | L6: MOLECULAR TESTING ON GENETIC DISORDERS LECTURER: MA. LOURDES L. BAJARIAS, RMT, MD MBDx: MAGALLANES, BCA. & OCLADINA, ZK. GENETIC DISORDERS • Residing in the nucleus, especially in eukaryotic cells, are chromosomes – thread like structures, made up of CHON and molecule of DNA. • DNA and RNA are called critical molecules of life because DNA holds genetic information that is important for the maintenance of life. This information can be passed down to the offspring. • Genes – segments of DNA. The basic unit of heredity/inheritance. Because they contain instructions that would determine the person’s physical and biological trait. This can be passed down to offsprings. They code proteins, that are important in determining the human’s trait. • Humans have approximately 20,000 protein coding gene. MUTATIONS • Changes, alterations, deviations in the DNA sequence of an organism MAY RESULT FROM • Errors in DNA replication during cell division • Exposure to mutagens – agents that cause mutations or a viral infection. (e.g., Somatic mutation, Germline mutation) SOMATIC MUTATION somatic cells or body cells. It happens during the embryonic life/human being. It only affects the somatic cells in a specific areas GERMLINE MUTATION happens before fertilization. It can be passed down to generation to generation. It has larger effects as opposed to somatic mutation. GENETIC DISORDERS • occurs when a mutation affects an individual’s genes. TYPES OF GENETIC DISORDERS CHROMOSOMAL ABNORMALITIES Chromosomes are missing or changed. Down syndrome[1] & Turner syndrome[2] SINGLE – GENE DEFECT Mutations affect one gene Sickle cell disease[3] & cystic fibrosis MULTIFACTORIAL/ COMPLEX[4] Mutations in two or more genes Cancer diabetes [1] Has an extra copy of Chromosome 21, hence aka Trisomy 21 [2] There is a defect on one of the X chromosomes. [3] Affected gene is the hemoglobin, beta gene HBB gene. [4] Not only genetic, but also the lifestyle factors. MOLECULAR DIAGNOSTICS • Detection of genomic variants (mutations/alterations) in DNA or RNA samples by laboratory tests • Combines molecular genetics and laboratory medicine. • An increasingly useful, cost-effective, sensitive, and non- invasive tool. • All disease has a genetic component SIGNIFICANCE OF MOLECULAR TESTING SIGNIFICANCE OF MOLECULAR TESTING • Symptomatic[1] – diagnosis, prognosis, subclassification, progression, pharmacogenetic response • Asymptomatic – carriers of heritable disease, probably in those at risk • Prenatal – diagnosis of genetic disease prenatal, likelihood of parents passing on genetic mutation. MOLECULAR TESTING DIFFERS FROM TRADITIONAL MEDICINE TESTING • Results are relevant not just to the patient but the patient’s relatives or family as well. • May be used solely for personal decision and not just for medical care. MOLECULAR GENETIC TESTING METHODS DIRECT TESTING[1] INDIRECT TESTING[2] positive identification of the disease-causing genetic alterations that establish a person’s genetic status relied on linkage analysis (DNA sequences serve as markers to track a gene mutation) Direct Testing looking at the DNA right away. Indirect Testing – using other methods to see if there is a mutation DIRECT TESTING 1. MUTATION ANALYSIS • To identify the presence of a specific mutation or a set of known mutations using a panel of common mutations[1] • Detects large deletions and large trinucleotide repeat expansions. The mutation is compared with a panel of common mutation, to check if the DNA or the segment has a mutation EXAMPLE SPECIFIC MUTATION Sickle Cell Anemia SET OF KNOWN MUTATIONS Cystic Fibrosis carrier detection LARGE DELETIONS Duchenne Muscular Dystrophy Becker Muscular Dystrophy LARGE TIRNUCLEOTIDE REPEAT EXPANSIONS Fragile X Syndrome Myotonic Dystrophy SOUTHERN BLOT • It is a crude method of mutation Analysis. • Discovered by Edwin Southern, hence the name. • Sample from blood, cheek swab, etc. STEPS FOR SOUTHERNBLOT METHOD 1 Purify the DNA sample and digest it into smaller fragments using restrictive enzymes to facilitate further analysis 2 Expose the fragmented DNA to denaturing agents, such as sodium hydroxide, to separate the strands, making them accessible for further processing. 3 Load the denatured DNA fragments onto a diffusion medium, typically an agarose gel, and perform electrophoresis to separate the fragments based on size. Smaller fragments will migrate further through the gel. 4 Transfer the separated DNA fragments from the agarose gel to a nitrocellulose filter for immobilization. This step also involves the removal of excess reagents to prepare for subsequent procedures. 5 Employ tags, such as fluorochromes, radioactive isotopes, or chemical labels, to visualize the movement of the DNA fragments on the nitrocellulose filter. 6 Prepare probes that are complementary to the target mutation within the DNA fragments. Hybridization occurs when the probes attach to the mutation, indicating its presence in the sample. 7 Analyze the hybridized DNA fragments to infer the presence of the mutation, confirming its existence within the sample. STEPS SUMMARIZED [1] DNA Digestion [2] Gel Electrophoresis [3] Blotting [4] Probe labeling [5] Hybridization & Washing [6] Detection 2. MUTATION SCANNING (SCREENING) • TWO-STEP PROCESS: 1. Screening of DNA segment via one of a variety of techniques o Single-strand conformational polymorphism[1] o Denaturing gradient gel electrophoresis o Denaturing gradient high-pressure liquid chromatography 2. Further analyzed by sequence analysis OFTEN USED WHEN • Mutations are distributed throughout a gene. • When most families have different mutations • Sequence analysis would be excessively time consuming.
LESSON 6: MOLECULAR TESTING ON GENETIC DISORDERS 2 MBDx: MAGALLANES, BCA. & OCLADINA, ZK. SINGLE-STRAND CONFORMATIONAL POLYMORPHISM STEPS FOR SSCP 1 Begin by aiming to identify mutations within the DNA sample prior to sequencing 2 Recognize that single-stranded DNA molecules possess distinct conformations or shapes. Abnormalities in the DNA sequence can result in alterations to these conformations. 3 Distinguish between mutant and non-mutant DNA samples based on differences in their conformational structures. 4 Initiate the process with denaturation, separating the DNA strands. Upon cooling, the single-stranded DNA molecules will reassume their native conformations. 5 Employ electrophoresis (non-denaturing gel eletrophoresis) to analyze the DNA samples. Mutant DNA molecules will exhibit distinct conformational differences compared to non- mutant counterparts. 6 Observe and interpret the electrophoretic patterns to identify mutations within the DNA sample based on variations in conformational structures. 7 Utilize the information obtained from SSCP analysis to guide further sequencing efforts, focusing on regions with identified mutations for detailed genetic analysis. 3. SEQUENCE ANALYSIS (SEQUENCING) • Nucleotide sequence for a DNA segment is determined. • Maybe the entire gene or a portion such as select exons. • Considered as the gold standard for molecular genetic testing by many. • Interpretation of the significance of a sequence alteration may not be straightforward. INTERPRETATION OF SEQUENCE ALTERATION RESULTS: TYPES OF SEQUENCE ALTERATIONS THAT MAY BE DETECTED. • Pathogenic sequence alteration reported in literature. • Sequence alteration predicted to be pathogenic but not reported in literature. • Unknown sequence alteration of unpredictable clinical significance • Sequence alteration predicted to be benign but not reported in the literature. • Benign sequence alteration reported in the literature. POSSIBILITIES IF A SEQUENCE ALTERATION IS NOT DETECTED • Patient does not have the mutation in the tested gene (e.g. a sequence alteration exists in another gene at another locus) WATCH DNA SEQUENCING (YOUTUBE VIDEO) • DNA sequencing is the process of working out the order of the building blocks, or bases, in a strand of DNA. 1. Preparation of DNA Fragments: • DNA is cut into smaller pieces and inserted into plasmid DNA, then placed into bacterial cells to produce multiple copies. • Isolation of DNA from bacteria. 2. Preparation for Sequencing Reaction: • Isolated DNA is transferred to a plate. • A mixture of ingredients including free DNA bases (A, C, G, T), DNA polymerase enzyme, DNA primers, and labeled terminator bases are added. 3. Sequencing Reaction: • DNA is heated (96oc) to separate into single strands. • (50oc) Primers bind to plasmid DNA. • (60oc) DNA polymerase binds to the primer DNA and starts making a new DNA strand by adding unlabeled DNA bases. • Terminator bases are added, stopping DNA polymerase from adding more bases. 4. Fragmentation and Separation: • Heating and cooling process repeated to create fragments of DNA with different lengths. • Fragments are separated by length using electrophoresis, arranging them from shortest to longest. 5. Detection and Recording: • Fragments pass through a capillary tube containing a gel. • Laser illuminates terminator bases, detected by a camera. • Each terminator base is labeled with a different color. • Sequencing machine records the color of terminator bases as a series of blocks. 6. Sequence Analysis: • Colored blocks are converted into letters (A, C, G, T) to determine the sequence of DNA fragments. Shortest DNA fragments are read first, and longest fragments are read last. 4. OTHERS (PROTEIN TRUNCATION TESTING & METHYLATION) INDIRECT TESTING • Used when direct DNA analysis is not possible because the gene is not known. • Relies on linkage analysis[1] . Statistical method used to map out gene in the chromosome. • DNA sequences serve as markers to track gene mutations. Since the mutation is unknown, it utilizes markers. In linkage analysis, we look for the linked genes. • Longer process and complicated. Linkage analysis in the family involves studying the inheritance pattern of genetic markers within a family to identify regions of the genome that are associated with a particular trait or disease. By comparing the genetic markers with the trait of interest among family members, researchers can map the location of genes responsible for the trait or disease. This analysis helps in understanding genetic diseases, identifying carrier status, and developing strategies for prevention or treatment. MEDICAL TESTING PARADIGM • Genetic tests provide information that directly influences medical care. 1. DIAGNOSTIC TESTING – confirms diagnosis in a symptomatic person. 2. PREDICTIVE TESTING – to identify genetic alteration in at- risk asymptomatic relatives. o Presymptomatic (100% surely will get the disease/disorder) o Predisposition (< 100% risk of getting the disease/disorder) GENETIC COUNSELING PARADIGM • Genetic tests provide information for personal decision making. 1. PREDICTIVE TESTING - for diseases in which no medical interventions exist (lifestyle decisions, therapy) o PDM: Change lifestyle. 2. CARRIER TESTING - to identify carriers of autosomal recessive gene mutations and X-linked recessive gene mutations (reproductive decision) o PDM: don’t give birth, or birth planning 3. PRENATAL TESTING - is used to evaluate fetus at high risk for genetic disorder (termination) o PDM: Pregnancy termination GENETIC EVALUATION • Gather information on a patient or family with suspected or known genetic disorder. • Diagnose by physical exam and genetic testing. • Perform risk assessment. GENETIC COUNSELING • Educating patients • Conveying genetic risks • Providing for informed decision making • Offering psychosocial support and referral • Referring geographically dispersed at-risk family members to local genetic services. APPLICATIONS 1. DIAGNOSTIC TESTING • Confirm a diagnosis, monitor prognosis of a disease or response to treatment. • Usually Performed on symptomatic patients. • Single gene DNA tests are absolutely disease specific. • One must weigh the test against more traditional methods with regard to cost, convenience, and utility. • Advantageous for early or atypical clinical presentations (e.g., Cystic fibrosis) READ MOLBIO LAB NOTES FOR PCR TESTING. SINCE PCR TESTING IS UNDER DIAGNOSTIC TESTING 2. CARRIER SCREENING • Detection of recessive mutations in healthy individuals for purposes of genetic counseling and family planning • Typically offered to those with: o Family history of a genetic disorder o Ethnic groups like Ashkenazi Jews, with an increased risk of specific genetic condition
LESSON 6: MOLECULAR TESTING ON GENETIC DISORDERS 3 MBDx: MAGALLANES, BCA. & OCLADINA, ZK. CYSTIC FIBROSIS • Inherit one copy from each parents of the CFTR gene. • Father and Mother carriers = child has 25% chance of CF • Mother has C, Father is a carrier = child has 50% chance of CF. 3. PREIMPLANTATION TESTING • Testing the embryo after in vitro fertilization • Offered for: o Determine the sex of the embryo for sex-linked disorders. o To identify single gene defects o Used in chromosomal disorders (usually the reason for early pregnancy miscarriages) Not all sex-linked disorders are testable right away. Testing the sex can help narrow down the mutation being detected. 4. PRENATAL TESTING • Detection of genetic disease in the fetus • Purpose: o Therapy can be instituted promptly at birth. o For psychological reassurance of a couple o Practical option of pregnancy termination o Noninvasive prenatal testing (NIPT) around 10-12 weeks of gestation. Harder but safer 5. NEWBORN SCREENING • Aims to identify relatively prevalent inherited defects in otherwise asymptomatic individuals to know if there are defects that will manifest as the person progress in their development. • The goal is to ascertain affected babies early in life so that the treatment (dietary and pharmaceutical) can be initiated before irreversible damage occurs.) • Most widespread use of genetic testing • Blood collection is done after 24 hours of life but not later than 3 days from complete delivery. GENERAL TESTS: NBS EXPANDED NBS • Congenital Hypothyroidism (CH) • Congenital Adrenal Hyperplasia (CAH) • Galactosemia (GAL) • Phenylketonuria (PKU) • Glucose-6-Phosphate- Dehydrogenase Deficiency (G6PD Deficiency) • Maple Syrup Urine Disease (MSUD) • Cystic Fibrosis • Biotinidase Disease • Organic Acid Disorder • Fatty Acid Oxidation Disorders • Amino Acid Disorders • Urea Cycle Disorder • Hemoglobin Disorders 6. PREDECTIVE AND PRESYMPTOMATIC TESTING • To identify the individuals at risk of getting a disease prior to onset of symptoms • Involves the use of the two genetic testing paradigms (Genetic evaluation & Genetic counseling). • Useful if with family history of a specific disease and an intervention is available to prevent the onset of disease or minimize disease severity. • Late-onset dominant disorders at risk: desire to know their status before its clinical onset to make informed reproductive, employment, and lifestyle decisions. o Ex: Heritable cancer syndromes (breast cancer), Huntington Disease • Huntington’s Disease Symptoms: o Physical: ▪ Involuntary movements ▪ Difficulty swallowing ▪ Loss of coordination o Mental & Emotional Changes: ▪ Depression and mood swings ▪ Difficulty learning & reasoning. ▪ Memory loss 7. FORENSICS • For identity testing, especially for legal purposes o Criminal investigations o Questions of paternity o Identification after catastrophic events RISK AND LIMITATION • Challenges remain even as molecular testing advances. • Risks o Can be physical, emotional, social, financial, ethical, genetic discrimination. • Limitations o If a person will show symptoms (when, how) o Severity of symptoms o How the disorder will progress o Lack of treatment strategies o Informed Consent o More people in the field Transcriber’s note: Transes is from Block C’s Lecture. Please do have the time to cross-check your own notes if there is other missing information, or not quite elaborated topic in this transes. Thank You and God Bless!