The goal of molecular diagnostic testing is to provide definitive diagnoses for suspected or unknown genetic conditions. A precise diagnosis is important for determining what caused a particular birth defect, for making an accurate cancer diagnosis, for assessing predisposition to adult disorders, or for providing potential therapeutic targets for present and future treatments. There are dozens of molecular-genetic techniques that are currently utilized, all with the common purpose of determining pathologic variations in the primary nucleotide sequence in the affected subject. Clinical molecular testing can be divided into those tests that look at the sequence of a specific single gene, or a more shotgun approach where panels of genes or whole exomes/genomes are sequenced in order to identify the cause of the presenting condition. However, whole exome/genome sequencing provides the individual not only with a diagnosis that explains current symptoms, but also incidental medically actionable health information that may have significant implications for the patient and his/her family. Incidental actionable medical information includes detecting carrier status for a known mendelian disorder, finding susceptibility genes for various cancers, such as BRCA1/BRCA2, or discovering genetic changes that affect response to certain drugs.

Most of the testing in molecular genetics has focused on region(s) of DNA that encode proteins (see Chapter 1, Figure 1-1).¹ These coding regions are called exons. The terms “mutation” and “variant” are used here interchangeably and may be pathogenic, benign or of unknown significance. We can interpret nucleotide variations easier if we find pathogenic mutations (pathogenic variants) that alter protein structure. On the other hand, many mutations can also occur outside of the coding regions of the gene, and these may involve regulatory components of the genome, such as promoters and enhancers, that regulate gene expression, or affect messenger RNA stability (mutations in the 5′ or 3′ untranslated gene regions, known as UTRs). Mutations can partially affect gene function by rendering a protein less efficient to perform its functions (androgen receptor mutations that cause incomplete androgen insensitivity syndrome), by completely abolishing the function of the protein (CYP21A2 mutations that cause the classic form of congenital adrenal hyperplasia), by interfering with the function of the native protein (dominant negative mutations of fibrillin that cause Marfan syndrome), or by making the protein more active (fibroblast growth factor receptor 3 gene gain of function mutations that cause achondroplasia). Mutations can result from changes in a single nucleotide (point mutation) or multiple nucleotides (deletions, duplications, insertions). Point mutations within the coding region of the gene (exons) may have multiple consequences. It may cause no change in the amino acid (neutral or synonymous mutation and likely benign), may change amino acid (missense or nonsynonymous mutation which may or may not be pathogenic), or may introduce a stop codon (nonsense mutation which usually tend to be pathogenic). Point mutations outside of the exonic regions can affect splicing of exons, which leads to variant RNA transcripts that may encode proteins with no function. Insertions, deletions, and duplications involve a change in the number of nucleotide residues, and will change the amino acid composition of the protein, and therefore its function, or truncate the protein due to premature insertion of a stop codon. When reading a molecular genetic report that identifies a nucleotide variation that differs from the “reference genome” (a nonaffected control), it is important to determine whether such variation is pathogenic, or not. Many nucleotide variations cause nonsynonymous amino acid changes that are benign (not causing protein dysfunction). The population frequency of a particular variant can help distinguish benign from pathogenic nucleotide variation. A nucleotide variation that is rare, <0.5% in the population, is more likely to be pathogenic in an individual with a rare disorder, than variant that is present in more than 20% of the population. Other clues to whether a particular variant is pathogenic includes whether the changed amino acid is highly conserved among different species, whether the changed amino acid change is predicted to disrupt protein function, or whether the particular gene that harbors the variant makes physiologic sense from previous animal research showing the observed phenotype.

Genetic testing can be used in different settings. It can either be applied to an individual with clear phenotype who seeks answers about the genetic etiology of their disease, or it can be used to screen populations for a highly prevalent condition. Cystic fibrosis has a relatively high carrier frequency (1 in 29) in the Caucasian population, making it appropriate for a population based screening program. Among Ashkenazi (Eastern European) Jews, the carrier frequency approaches 1 in 4 for at least one of 19 different diseases common in this ethnic group. Whole exome/genome sequencing blurs the distinction between diagnostic and screening testing, as the information obtained from such testing provides both diagnostic and screening information. For any genetic testing, pre- and posttest genetic counseling by a genetic counselor or genetic physician is of utmost importance. Genetic counseling should include information on the testing procedure, the possible results, the potential uncertainties in testing, and how the results may impact the patient and their family.

Polymerase Chain Reaction

Polymerase chain reaction (PCR) and PCR-based applications represent the most common methodologies used in genetic testing. This technology is based on the discovery that DNA polymerase from Thermus aquaticus, also known as Taq polymerase, resists high temperatures. The discovery of Taq polymerase led to the development of PCR (Figure 16-1). Each PCR cycle involves denaturing DNA double strands into single strands at high temperature (~95°C), annealing short region-specific oligonucleotides that bracket the region of interest, followed by extension synthesis of targeted regions using Taq DNA polymerase. The PCR amplification is exponential, and creates several billion copies of uniquely targeted DNA fragments. PCR can amplify almost any genomic region with small starting amounts of DNA. It can be used to amplify all the exons within a specific gene for sequencing. Quantitative PCR is a modification of the PCR technique that quantitates the amount of DNA or RNA in the sample, and can be used to detect deletions or duplications, and determine RNA expression levels, as well as viral and bacterial loads.