Polymerase chain reaction

PCR is a quick and efficient way to amplify genetic material from any source, and very small samples of genetic material can be used to assist in the diagnosis of a broad spectrum of diseases. A sample of DNA from nearly any site can be used for analysis, although the sample type is generally determined by the disease or disorder that is being investigated. DNA is denatured with heat, a process that creates inverse copies of single-stranded DNA. With the single-stranded DNA serving as a template, a polymerase enzyme then synthesizes identical copies of double-stranded DNA. The process is repeated through many cycles, creating more than 1 billion copies of the DNA under study.14 PCR is useful in diagnosing infectious diseases, including bacterial, fungal, viral, and parasitic infections; it is particularly helpful in the case of infectious agents that are difficult to isolate with normal culture techniques.15 Additionally, in some situations, the time required to obtain traditional culture results can delay proper treatment, as in cases of sepsis or meningitis. Emerging PCR-based technology has made it possible to detect pathogens more rapidly in such circumstances, reducing the time to results from days to hours, with better patient outcomes.16 The pathogens that are routinely identified through PCR testing are listed in Table 2.17-19 Laboratories commonly use PCR as the first step in isolating genetic material, which can then undergo further testing with other methodologies.14 This is often done in the diagnosis of genetic abnormalities or malignancies caused by certain known mutations. Providers should be aware that false-positive and false-negative results are a concern with PCR testing because of the ease of sample contamination, and that sensitivity and specificity vary greatly depending on the disease for which PCR is being used.15 

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Fluorescence in situ hybridization

In FISH, fluorescence-tagged probes are used to detect specific DNA sequences, which can then be visualized under the microscope. This process makes it possible to identify where a particular gene or gene portion is located within the chromosomes of a cell.20 FISH can detect structural rearrangements such as translocations, inversions, and insertions, and the loss of chromosomal regions, including microdeletions.21 The specimen types usually submitted to the laboratory are peripheral blood or bone marrow, but solid tissue can also be used in the analysis of tumor cytogenetics, so FISH has a unique role in confirming cancer types without the need for actively dividing cells.22 FISH testing is most useful in evaluating malignancies, especially hematologic malignancies, but is also used to determine HER2/neu overexpression in breast cancers.22 Accurately and efficiently identifying genetic alterations that lead to malignancy can facilitate early detection, diagnosis, prognosis, and treatment. Although FISH is a very useful test when used in the correct setting, it is not as effective for investigative purposes as some other genetic testing techniques. Generally, it is used to verify the presence of a suspected chromosomal aberration, or as a supplement in the prognosis and treatment of certain malignancies, rather than to search for a genetic abnormality.21,22 Reproductive specialists may use FISH (or PCR) with embryos of couples who have a known inherited disorder, as a screening tool before in vitro fertilization. Post-implant karyotype is still recommended, however, because the results of FISH may be inaccurate.23 

Whole-exome sequencing 

The exome is the portion of DNA that codes for proteins, and it is the location of about 85% of the mutations that lead to disease-related traits.24 The starting material for WES is double-stranded nuclear DNA. This can be obtained from whole blood, a buccal swab, or a hair follicle.25 WES is useful in identifying variants in the exome associated with conditions such as, but not limited to, Mendelian disorders (e.g., Huntington disease, familial Mediterranean fever, type I Charcot-Marie-Tooth disease, sickle cell anemia, cystic fibrosis, alpha-1-antitrypsin deficiency) and in evaluating severe intellectual disability or developmental delay.26,27 Once variants are found, they can be connected to clinical phenotypes that have already been identified, allowing a diagnosis. The disadvantage is that not every clinical phenotype has been identified, leaving the possibility of a patient with unanswered questions and no diagnosis.28 

WES is useful when all other diagnostic avenues have been exhausted; it may then help identify a clinically apparent abnormality. (Table 3) lists examples of genetic diseases for which WES is appropriate, or for which an alternate test is preferable.10,22,23,26,27,29-36) In addition, WES is useful in identifying diseases and disorders that are not yet clinically apparent or that have not yet reached full clinical expression, thus preventing a misdiagnosis.26,27 Because incidental findings are possible, patients must be prepared for unexpected results, either an unexpected disease state or a genetic variant about which little is currently known. Counseling is needed before and after testing to help patients understand the range and types of results that may be obtained, as well as to determine which results warrant action, which results call for increased screening or surveillance, and which results are of little concern. 

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Considerations for patient education

Genetic testing can be a fearful, daunting, overwhelming, exciting, or joyful experience. In many cases, the emotions of people who pursue genetic testing can be described with more than just one of these words, or others. Therefore, the patients undergoing most avenues of genetic testing require some counseling and education. Although some genetic tests, such as PCR and karyotype, can yield straightforward results, others often do not provide results that are easily interpreted. Genetic counselors have the expertise to offer an informed interpretation, but PCPs are the educators who must explain to patients why genetic testing and counseling should be considered. Patient education is needed even before an appointment with a genetic counselor is arranged. It is important to educate patients regarding all the possibilities, such as false positives, false negatives, incidental findings, unexpected results, and variants of unknown significance. It is also important to inform them that the symptoms of a mutation-caused disease can vary significantly between different people. Furthermore, explaining the value of discovering a genetic disease is an essential part of patient education. If a specific disease is identified, the element of uncertainty is removed, and interventions may be available that will mitigate symptoms. Helping patients think about the potential benefits and challenges of identifying a disease or disease risk should be part of the role of every PCP, before genetic counseling or testing is undertaken. Although a genetic counselor will provide invaluable information to the patient and family, the PCP will coordinate the patient’s ongoing care, so that an interdisciplinary team approach to genetic testing is desirable for optimal outcomes. Test results may enhance the team’s performance by indicating which subspecialty collaboration would be most helpful. 


Providing patients with information about genetic testing will become more commonplace as our understanding of the field of genetics continues to grow. Patients may seek information from other resources, such as the Internet or a friend, before talking to their PCP. Thus, the PCP must be knowledgeable about genetic testing because many patients will present with a rudimentary understanding of the subject. Acquiring an understanding of genetic testing will also allow PCPs to make the best possible use of this resource in patient care, whether it be in counseling, making a diagnosis, developing a prognosis, providing targeted screening, elaborating on a consult with a genetic counselor, or making an appropriate referral. 

Gina R. Brown, MPAS, PA-C, is an associate professor at Wichita State University and a PA in family practice in Wichita, KS; Seth Metzler, MPA, PA-C, is a PA in family practice in Salina, KS; Trisha Desjardins, MPA, PA-C, is a PA in orthopedic trauma in Bangor, ME; and Brittany Seiler, MPA, PA-C, is a PA in family practice in Wichita, KS.


The authors would like to thank Claire Chappell, PA-C, Brandi Jensen, PA-C, and Melissa Short, PA-C, for providing the initial foundational direction for this article.


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