Genetic Testing Overview and Applications

Genetic Testing Overview

• Genetic testing assesses inherited genetic variations from parents (germline variation).
• It has broad applications in healthcare, including predicting and managing disease risk.
• The practice has the potential to facilitate accurate and timely diagnosis and treatment for infectious diseases.

Application - Molecular Diagnosis

• Molecular diagnosis involves detecting pathogenic mutations in DNA and/or RNA samples.
• This process facilitates disease detection, diagnosis, sub-classification, prognosis, and monitoring treatment responses.

Molecular Testing

• Direct DNA analysis is possible only when the gene sequence is known.
• Molecular testing offers superior methodology, particularly when a biochemical test is not available.
• DNA testing can be done with any tissue and requires minor amounts of sample.

Cytogenetics

• Cytogenetics studies chromosome structure, behavior in mitosis and meiosis, and factors causing chromosomal changes.
• It is relevant to diagnosing disease status caused by abnormal chromosome number or structure.
• Karyotyping and banding are used for chromosomal analysis.
• Karyotyping involves collecting all chromosome structures.
• Chromosome banding is method of analysis that categorizes individual chromosomes.

Chromosome Analysis - Methods and Procedures

• Cell sources include blood cells, skin fibroblasts, amniotic cells, and chorionic villi.
• Increasing the mitotic index (using colcemid) allows for better observation of cells in mitosis (cell division).
• The process involves cell culture, fixation, spreading of chromosomes onto microscope slides and staining.
• Staining helps differentiate chromosomes for analysis.
• Banding patterns on each chromosome are unique, facilitating analysis of chromosome structure.

Karyotype Analysis - Procedure

• The procedure involves cell counting and analysis of the banding patterns of each chromosome in selected cells.
• Total chromosome counts (minimum 10-15 cells; with suspicion of mosaicism, up to 30 or more) are determined.
• Chromosome analysis involves analyzing 3-5 metaphase spreads for detailed analysis and high-quality banding.
• Banding patterns are represented as an idiogram.

Chromosome Banding

• Chromosome banding is based on the presence of heterochromatin (darkly stained) and euchromatin (lightly stained).
• Various banding techniques include G-banding, C-banding, Q-banding, R-banding, and T-banding.
• Giemsa (G)-banding is a cytogenetic method to visualize condensed chromosomes.

Molecular Cytogenetics

• Molecular cytogenetics locates specific DNA sequences on chromosomes.
• It involves higher resolution analysis of chromosomes, including target DNA sequencing, probes (often 15-50 nucleotides long, chemically synthesized) and more.
• FISH (Fluorescent in situ Hybridization), chromosome painting, CGH (comparative genomic hybridization), molecular karyotyping and Multiplex FISH(M-FISH), Spectral Karyotyping, Array CGH are all methods.

In Situ Hybridization (FISH)

• FISH uses DNA or RNA probes to identify chromosomal locations of genes or cellular locations of mRNA.
• Visualization of DNA or RNA occurs in situ (within the cell).
• Resolution in conventional FISH on metaphase chromosomes typically results in resolution of several megabases.
• Prometaphase chromosomes offer better resolution (1 Mb).

Biochemical Testing

• Biochemical genetic diseases, such as inborn errors of metabolism, disrupt metabolic pathways.
• Diagnostics tests directly measure protein activity (enzymes), metabolite level, and protein quantity/size using tissue samples (blood, urine, amniotic fluid, cerebrospinal fluid).
• Technologies include immunohistochemistry, gas/liquid chromatography/mass spectrometry (LC/GC/MS), and bioassays.

Immunohistochemistry (IHC)

• Immunohistochemistry exploits antibody-antigen interactions to detect and localize specific antigens in tissues.
• Tissue samples are typically frozen, or formalin-fixed paraffin-embedded (FFPE).
• Steps: pretreatment & antigen retrieval, primary antibody addition, secondary antibody application, detection reagent application, visualization.

Molecular Testing Methods

• PCR-based methods amplify target DNA sequences using cycles of denaturation, annealing, and elongation.
• Amplified product analysis can involve digestion with restriction enzymes followed by gel electrophoresis.

Types of Genetic Tests

• Genetic testing ranges from single gene examination to whole genome analysis, selecting tests based on disease type and other factors.
• Types include targeted single variant, single gene, gene panel, genomic sequencing, chromosomal, gene expression, and protein expression.
• Specific examples include newborn screening, diagnostic testing, carrier testing, prenatal testing, preimplantation testing, forensic testing, and research testing.

Single-Nucleotide Polymorphisms (SNPs)

• SNPs are single-base-pair differences in DNA sequence between individuals.
• They are inherited as alleles and widespread throughout genomes.
• SNPs can be found approximately every 1000 base pairs in a comparative analysis.

Advanced Genetic Testing

• Array comparative genomic hybridization (aCGH) assesses single nucleotide polymorphisms (SNPs) and copy number variations.
• SNP microarrays (array) also analyze SNPs and copy number variations.
• Other methods like whole exome sequencing (WES), and whole genome sequencing (WGS) offer detailed analysis.

Microarray Technology

• Microarrays are high-resolution genome-wide screenings used for copy number variants.
• Two different oligonucleotides are used for detecting alleles (with fluorescent dye).
• Results can indicate deletion or duplication of DNA segments.

Next-Generation Sequencing (NGS)

• NGS is a massively parallel sequencing technology, determining the order of nucleotides in entire genomes or targeted DNA/RNA regions.
• The principle involves fragmenting DNA/RNA, adding adapters, sequencing the libraries, and reassembling to form the genetic sequence.

Whole Exome Sequencing

• Whole exome sequencing focuses on exons (coding regions) from the entire genome.
• Exons comprise roughly 1% of the genome.
• This sequencing identifies variations in exon sequences, assisting in disease diagnosis or assessment of disease risk.

Whole Genome Sequencing (WGS)

• WGS resolves the entirety of a patient's genome's detailed nucleotide sequence.
• It identifies variations in the genome and may provide information about regulatory functions within intronic regions.

Ethical, Legal, and Social Issues

• Informed consent and counseling regarding implications & choices are crucial.
• Maintaining patient confidentiality and addressing potential stigma associated with genetic test results is important.

Explore the essentials of genetic testing, including its role in healthcare and the various applications like molecular diagnosis. This quiz covers concepts from germline variations to cytogenetics, enhancing your understanding of disease risk management and treatment strategies.