Molecular Genetics Testing

Excerpt

Genetic molecular testing has become a fundamental method for evaluating a growing number of 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 interrogate targets for gene-specific therapies. The majority of genetic material is DNA, composed of two strands of a sugar-phosphate backbone bound together by hydrogen bonds between two purines and two pyrimidines attached to the sugar molecule, deoxyribose, in a double helix.

DNA in human cells is wrapped around histone proteins and packaged into nucleosome units, which are compacted further to form chromosomes. There are 23 pairs of chromosomes, two of which are the sex chromosomes X and Y. Each chromosome is a single length of DNA with a stretch of short repeats at the ends called telomeres and additional repeats in the centromere region. In humans, there are two sets of 23 chromosomes, a mixture of DNA from the mother's egg and the father's sperm. Therefore, each egg and sperm is a single or haploid set of 23 chromosomes. The combination of the two creates a diploid set of human DNA, allowing each individual to possess two different sequences, genes, and alleles on each chromosome, one from each parent. Each child has a unique combination of alleles because of homologous recombination between homologous chromosomes during meiosis in the development of gametes (egg and sperm cells). This creates genetic diversity within the human population.

The completion of the full human genome sequence, the identification, and cloning of numerous genes associated with inherited and acquired conditions and diseases, plus the advent of powerful methods for molecular analysis of these genes in clinical specimens, have revolutionized the practice of molecular genetics and molecular pathology. With the aid of these techniques, it is now possible to identify illness risk in individuals who are not yet showing symptoms, identify asymptomatic carriers of recessive traits, and make prenatal diagnoses for diseases that might not develop in pregnancy. Molecular genetic, nucleic acid-based techniques are often the only approaches available for these applications. As such, they offer a powerful tool for diagnosis, genetic consultation, and prevention of heritable diseases.

Many types of genetic tests are available to analyze changes in genes, chromosomes, or proteins. A healthcare provider will consider several factors when selecting the appropriate test, including what condition or conditions are suspected and the genetic variations typically associated with those conditions. A test that looks at many genes or chromosomes may be used if a diagnosis is unclear. However, a more focused test may be done if a specific condition is suspected.

Molecular tests look for changes in one or more genes. These types of tests determine the order of DNA, building blocks (nucleotides) in an individual's genetic code, and a process called DNA sequencing. These tests can vary in scope. The targeted single variant is a single variant test that looks for a specific variant in one gene. The selected variant is known to cause a disorder (for example, the specific variant in the HBB gene that causes sickle cell disease). This type of test is often used to test family members of someone known to have a particular variant, to determine whether they have a familial condition. Single-gene tests look for any genetic changes in one gene. These tests are typically used to confirm (or rule out) a specific diagnosis, particularly when many variants in the gene can cause the suspected condition. Gene panel tests look for variants in more than one gene. This type of test is often used to pinpoint a diagnosis when a person has symptoms that may fit a wide array of conditions or when variants in many genes can cause the suspected condition.

Whole exome sequencing/whole genome sequencing tests analyze the bulk of an individual's DNA to find genetic variations. This test is typically used 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 lengths of DNA to identify large-scale changes. Changes that can be found include an extra or missing copy of a chromosome (trisomy or monosomy, respectively), a large piece of a chromosome that is added (duplicated) or missing (deleted), or rearrangements (translocations) of segments of chromosomes. Certain genetic conditions are associated with specific chromosomal changes, and a chromosomal test can be used when one of these conditions is suspected. (For example, Williams syndrome is caused by a deletion of a section of chromosome 7).

Gene expression tests look at which genes are turned on or off (expressed) in different types of cells. When a gene is turned on (active), the cell produces a molecule called mRNA from the instructions in the genes, and the mRNA molecule is used as a blueprint to make proteins. Gene expression tests study the mRNA in cells to determine which genes are active. Too much activity (overexpression) or too little activity (underexpression) of certain genes can be suggestive of particular genetic disorders, such as many types of cancer. Biochemical tests do not directly analyze DNA, but they study the amount or activity level of proteins or enzymes that are produced from genes. Abnormalities in these substances can indicate that there are changes in the DNA that underlie a genetic disorder.

Mutations associated with heritable disorders are detectable in all nucleated cells and thus are considered germline or constitutional genetic changes. Somatic genetic changes are characteristic of acquired or sporadic diseases, such as cancer. The molecular biology methods applied to investigate these two scenarios are very similar and focus on detecting DNA and RNA variations. However, the interpretation and utility of the laboratory results may be quite distinct.

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 is limited to detecting rearrangements involving more than 5 Mb of DNA. The resolution of the FISH technique, using fluorescent probes, is about 100kb-1Mb in size. The minor changes as single-base substitutions, insertions, and deletions can be detected with single-strand conformation polymorphism (SSCP) and sequence analysis through next-generation sequencing (NGS), a procedure that can use genomic DNA or complementary DNA (cDNA) with three modalities that include whole genomic DNA, targeted or exome sequencing; the denaturing high-performance liquid chromatography (DHPLC) is valid for detection of small deletions and duplications too; the extension of the deletions and duplications detected by multiplex ligation-dependent probe amplification (MLPA)is in some way in between of those identified by FISH or cytogenetic analysis and HPLC, i.e., MLPA is useful for detection of complete or single and multiexon deletions or duplications.