Molecular Genetics Testing

Molecular genetics testing is fundamental in evaluating inherited disorders, somatic or acquired diseases with genetic associations, and pharmacogenetic responses. Genotyping can provide valuable disease diagnosis, prognosis, and progression indicators, guide treatment selection and response, and identify gene-specific therapeutic targets. Human genetic material primarily consists of double-stranded, helical DNA. This molecule has a backbone composed of alternating sugar (deoxyribose) and phosphate groups, with hydrogen bonds linking nitrogenous base pairs. Specifically, adenine (purine) pairs with thymine (pyrimidine), while guanine (purine) pairs with cytosine (pyrimidine), forming the complementary base pairs within the DNA double helix. DNA in human cells is wrapped around histone proteins and packaged into nucleosome units, compacted further to form chromosomes. Somatic cells normally have 23 chromosome pairs, with 1 pair comprised of the sex chromosomes X and Y. Each chromosome has DNA with a terminal stretch of short repeats called “telomeres” and additional repeats in the centromere region. Humans have 2 sets of 23 chromosomes, one derived from the mother’s egg and the other from the father's sperm. Therefore, each egg and sperm is a single or haploid set of 23 chromosomes. Combining the 2 creates a diploid set of human DNA, allowing each individual to possess 2 different sequences, genes, and alleles on each chromosome. Homologous recombination during meiosis generates unique allele combinations in gametes, leading to genetic diversity among offspring in the human population. The complete decoding of the human genome sequence and the development of powerful identification and cloning methods for genes linked to inherited diseases have transformed the practice of molecular genetics and molecular pathology. Advanced molecular analysis methods can now determine presymptomatic individuals' illness risk, detect asymptomatic recessive trait carriers, and prenatally diagnose conditions not yet evident in pregnancy. Molecular genetics techniques are often the only approaches to these puzzles. Thus, genetic tests are powerful tools for diagnosis, genetic consultation, and prevention of heritable diseases. Many genetic tests can analyze gene, chromosome, and protein alterations. A clinician often considers several factors when selecting the appropriate test, including suspected conditions and their possible genetic variations. A broad genetic test is employed when a diagnosis is uncertain, while a targeted test is preferred for suspected specific conditions. Molecular tests look for changes in 1 or more genes. These tests analyze the sequence of DNA building blocks (nucleotides) in an individual's genetic code, a process known as DNA sequencing, which can vary in scope. The targeted single variant test identifies a specific variant in a single gene known to cause a disorder, eg, the gene variant causing β-globin abnormalities that give rise to sickle cell disease. This test assesses the family members of an individual with the known variant to ascertain if they have the familial condition. Single-gene tests examine genetic alterations in 1 gene to confirm or rule out a specific diagnosis, notably when many variants in the gene can cause the suspected condition. Gene panel tests look for variants in multiple genes to pinpoint a diagnosis when a person has symptoms that may fit various conditions or when many gene variants can cause the suspected condition. Whole-exome sequencing or whole-genome sequencing tests analyze the bulk of an individual's DNA to find genetic variations. This approach is useful when a single-gene or panel testing has not provided a diagnosis or when the suspected condition or genetic cause is unclear. This sequencing method is often more cost- and time-effective than performing multiple single gene or panel tests. Chromosomal tests analyze whole chromosomes or long DNA lengths to identify significant alterations, including extra or missing chromosome copies (trisomy or monosomy), large chromosomal segment duplications or deletions, and segment rearrangements (translocations) (see . Trisomy 21 on G-Banded Chromosomal Studies). Chromosomal tests are employed when specific genetic conditions linked to chromosomal changes are suspected. For instance, Williams syndrome results from deleting a chromosome 7 segment. Gene expression tests assess gene activation status in cells, indicating whether genes are active or inactive, with activated genes producing mRNA molecules that serve as templates for protein synthesis. The mRNA produced helps determine which genes are highly active. Too much activity (overexpression) or too little activity (underexpression) of specific genes may suggest particular genetic disorders, including various cancer types. Biochemical tests assess protein or enzyme levels and activity rather than directly analyzing DNA. Abnormalities in these substances may indicate DNA changes underlying a genetic disorder. Heritable mutations are detectable in all nucleated cells and are thus considered germline or constitutional genetic changes. Somatic genetic changes are characteristic of acquired or sporadic diseases like cancer. Both scenarios are investigated using similar molecular biology methods to detect DNA and RNA variations, although the interpretation and utility of the laboratory results often differ significantly. Fluorescent in situ hybridization (FISH), chromosomal microarray analysis (CMA), and cytogenetic analysis (karyotyping) can be used to detect gross mutations like whole- and large-scale gene deletions, duplications, or rearrangements. Conventional karyotyping identifies rearrangements over 5 DNA megabases. FISH has a resolution of 100 kilobases to 1 megabase. Minor alterations, such as single-base substitutions, insertions, and deletions, are detectable with single-strand conformation polymorphism (SSCP) and sequence analysis through next-generation sequencing (NGS). NGS uses genomic DNA (gDNA) or complementary DNA (cDNA) and has 3 modalities: whole genomic DNA, targeted, and exome sequencing. Denaturing high-performance liquid chromatography (DHPLC) can detect small deletions and duplications. Multiplex ligation-dependent probe amplification (MLPA) extends the range of deletions and duplications detected, bridging the gap between FISH or cytogenetic analysis and HPLC. MLPA is particularly useful in identifying complete or single and multiexon deletions or duplications.