Outpatient Primary Care Genetic Testing Primer: What to Order and Testing Considerations

Abstract

The field of genetics has evolved rapidly over the last few decades, from testing methods to genetic diagnoses, bringing new genetic testing guidelines and considerations for health care providers. Overall geneticists are limited in number and availability, particularly in non-academic settings, and many patients first present to a primary care provider. Here, we aim to review various modalities of genetic testing, their indications, limitations, and other pretest considerations for the primary care provider. In addition, we comment on the limitations of direct-to-consumer (DTC) genetic testing, which has seen a rise in popularity among the general population.

Introduction

Human DNA is packaged in 46 chromosomes, each with many bands and subregions. Within those areas are hundreds of genes that each have a specific nucleotide sequence. Available genetic testing targets various levels of genetic changes, from chromosome duplications or deletions to single nucleotide changes, with testing techniques and methods changing significantly over the last few decades. Concurrently, identified genetic conditions, now more than 5,000 rare disorders,¹ have expanded rapidly and identification of novel pathogenic changes continues at a fast pace. This has led to increased medical indications for genetic testing and further considerations towards the approach of broad genetic testing. This is true for both medical genetic testing and DTC genetic testing, including 23andMe, among others.

Types of Genetic Testing

Karyotype

Karyotyping was initially introduced in the 1950s, involving the pairing of the 22 autosomal chromosomes and the pair of sex chromosomes, allowing for diagnosis of chromosome number changes, such as monosomy X or trisomy 21.² In the decades that followed, various chromosome banding techniques were introduced, including the widely used Giemsa, or G-banding technique (Figure 1). This allowed for diagnosis of smaller chromosomal changes in the range of 5–10 million base pairs, or mega-bases, such as Cri-du-Chat Syndrome (5p- or a deletion of the short arm of the 5^th chromosome), as well as other larger chromosomal rearrangements like translocations or inversions.

Figure 1.

G-banded karyotype of a normal male karyotype (A) compared to that of a patient with trisomy 21 (B) with white arrow and a patient with Cri-du-Chat Syndrome or deletion of 5p (C) with red arrow.

Fluorescence in situ Hybridization (FISH)

Developed in the 1980s, FISH used fluorescently labeled DNA probes to bind to specific nucleotide sequences of interest (Figure 2). This led to the ability to diagnose chromosome microdeletion and microduplication syndromes like 22q11.2 deletion syndrome (formerly known as DiGeorge syndrome). This technique is also utilized widely in cancer genetics, helping identify common chromosome changes in various cancer lines, often with prognostic or clinical management implications. Due to the fast turnaround time compared to other genetic studies, FISH testing remains useful for screening of common trisomies (trisomy 13, 18, 21), sex chromosome differences (XO Turner syndrome and XXY Klinefelter syndrome), and specific microdeletion or duplication syndromes. FISH testing is limited by the targeted nature of the test, as providers must select which probes or related condition the patient should be tested for.

Figure 2.

(A and B) FISH aneuscreen for X/Y/18. X chromosome in green, Y chromosome in red, chromosome 18 in aqua. A-Normal male individual with one X chromosome, one Y chromosome, and two copies of chromosome 18, notated as nuc ish(DXZ1x1, DYZ3x1, D18Z1x2). (B)Male with Trisomy 18 with one X chromosome, one Y chromosome, and three copies of chromosome 18, notated as nuc ish(DXZ1x1, DYZ3x1, D18Z1x3. (C and D) FISH for 22q11.2 microdeletion also known as DiGeorge syndrome. Red marker for the 22q11.2 area. Green marker for the 22q13.3 area that is used as a control. (C) Normal individual without a 22q11.2 deletion with 2 red and 2 green markers. (D) Individual with 22q11.2 deletion with 1 red marker and 2 green markers.

Chromosomal Microarray (CMA)

Later development in the 1990s and 2000s led to array-based comparative genomic hybridization and single nucleotide polymorphism (SNPs) arrays, involving thousands of probes on a slide that could be hybridized to patient DNA. The different ratios between patient DNA and each probe DNA could be visualized to detect copy number variants (CNVs) in the whole genome at a resolution of 25–250 thousand base pairs or kilo-bases.¹ This improved diagnostic yield of testing, with smaller detection resolution than banded karyotype and wider coverage than targeted FISH.³ However, this type of testing is unable to identity balanced chromosomal rearrangements, for which karyotype is required. CMA is also known as molecular karyotyping or whole genome microarray.

DNA Sequencing

In 2003, the Human Genome Project completed the first rough DNA sequence of the human genome, identifying over 20,000 protein-coding genes and 2.1 million SNPs.⁴ This then led to further development of sequencing technology, massively parallel sequencing, and improvement in efficiency and cost of DNA sequencing. Research on single gene disorders and identification of new disease genes grew exponentially. There are now many types of gene panels available, as well as whole exome and whole genome sequencing (WES/WGS).

Single and multigene panels are available through a variety of laboratories for targeted, disease-focused sequencing. Gene panels are often grouped by similar clinical phenotype. Broader testing includes WES, which analyzes all exons or known protein encoding regions. Comparatively, WGS analyzes total genetic material encompassing protein encoding and non-protein encoding regions. This broadens the diagnostic potential to include CNVs, upstream and downstream regulatory elements and other intronic regions, or non-protein encoding regions. However, many of these non-protein encoding regions are not well characterized and their pathogenicity remain unknown. WGS is starting to become more widely available, though was previously largely in the research realm. DNA sequencing is limited in identifying triplet repeat expansion disorders, disorders of methylation, or uniparental disomy, which includes conditions like Fragile X syndrome, Beckwith-Wiedemann syndrome, and Prader Willi syndrome, respectively. Separate molecular tests can identify these types of syndromes.

Common Indications for Genetic Testing

Genetic testing is recommended for a wide range of indications, with the most common being global developmental delay (GDD), intellectual disability (ID), autism spectrum disorders (ASD), and multiple congenital anomalies (MCA). Global developmental delay is defined as significant developmental delay in 2+ developmental domains (e.g. speech and language, social, gross motor) that can be a predictor for future intellectual disability.⁵ Intellectual disability is defined as significant limitations in intellectual functioning and adaptive behavior per the American Association on Intellectual and Developmental Disability. Autism spectrum disorders are defined by the DSM V criteria and require formal evaluation for diagnosis. Congenital anomalies have varying definitions, typically involving structural or functional anomalies present since birth that impact the patient and often require medical intervention.⁶

Current Recommendations for Genetic Testing

Genetic testing recommendations (Table 1) continue to evolve as evidence accumulates implicating genetic etiologies. Most recently in July 2021, the American College of Medical Genetics and Genomics (ACMG) published guidelines recommending WES/WGS as first line or second line genetic testing for those with GDD, ID, and MCA, citing a diagnostic yield of 38%, compared to 21% for prior standard genetic testing,^6,7 as well as the potential for a more timely diagnosis. It is acknowledged that this may currently be cost limiting, but declines in test costs are anticipated with improved technology, health plan coverage, and cost savings compared to the step by step tiered genetic testing approach that would have been previously pursued. This recommendation did not include those with isolated ASD.

Table 1.

Summary of genetic testing recommendations and indications. Metabolic screening is not universally recommended but is recommended by some. ^*WES/WGS is recommended as first line testing or second line testing after CMA.

WES-Whole Exome Sequencing. WGS-Whole Genome Sequencing. GDD-Global developmental delay. ID-Intellectual disability. ASD-Autism spectrum disorders. MCA-Multiple congenital anomalies. FISH-fluorescence in situ hybridization. CMA-Chromosome Microarray

Indications	Recommended 1st line testing
GDD/ID	WES/WGS* and fragile X if male
MCA	WES/WGS*
ASD	CMA and fragile X if male
Suspicion for particular underlying genetic disorder	Targeted testing
Noninvasive prenatal testing	FISH
Concern for balanced chromosomal translocations, inversions, etc.	Karyotype

Prior to this, in 2010, the American Society of Human Genetics (ASHG) recommended CMA as the first line diagnostic genetic test for those with GDD, ID, ASD, and MCA, citing a diagnostic yield of 15–21%.^8–10 In 2013 the ACMG published guidelines endorsing the same.¹¹ Similar guidelines were released by the American Academy of Pediatrics (AAP) and the American Academy of Child and Adolescent Psychiatry, in the years following.^5,12,13 For those with GDD, ID, or ASD, fragile X screening is also indicated, particularly in males.^10,14 Other types of screening, including metabolic testing and MECP2 (for Rett phenotypes) are generally felt to be second line tests and are not routinely recommended unless there are suggestive physical exam findings or clinical symptoms.¹⁰ There has been debate regarding targeted metabolic testing based on concerning signs or symptoms versus broad metabolic screening in this population.^5,10

Prior to the above guidelines, G-banded karyotype was former first line genetic test. Yield of karyotype testing after normal CMA results is low, 0.78–1.3%, and is not routinely indicated as second line testing.³ Uniparental disomy screening has been suggested in those with GDD or ID, however, did not have high clinical diagnostic utility.¹⁵

For those patients with clinical presentations that are highly suggestive of a particular genetic syndrome, targeted testing is the first line recommendation (e.g. particular syndromes, familial conditions).

Indications for Referral

Indications for geneticist involvement commonly include ID, GDD, ASD, and MCA, as well as high risk abnormal newborn screens, patients with dysmorphic features, and familial genetic conditions. The timing of referrals to genetics can be dependent on comfort of the primary care provider (PCP), specialist availability, and insurance testing coverage. Given the significant advances in genetics, many PCPs report not feeling comfortable with the current guidelines for indicated genetic testing, the ordering process, or the management of results.^16,17 Overall, ordering of genetic testing appears to be underutilized in the primary care setting.¹⁸ Of patients with a diagnosis of ASD, ID, or GDD, only 32% of families reported a history of having undergone genetic testing.¹⁹ It is also worthwhile to note that many health plans have requirements for genetic testing, which can include the specialty of the ordering provider or prior authorization and the genetics team may be most logistically able to navigate this. Unfortunately, many PCPs report not having access to genetic services, with over 53% of PCPs reporting limited access.²⁰ This may improve with the recent broader adaptation of virtual medical care.

Genetic Testing Considerations

Pre-Test Counseling

Prior to any genetic testing, genetic testing implications, and potential results should be discussed. This includes potential variants of uncertain significance (VUS), carrier status, secondary findings, paternity, consanguinity, and the Genetic Information Nondiscrimination Act (GINA). These considerations are reviewed below. Patients can proceed with, decline, or decline certain aspects of testing, once these issues are addressed. In addition, the diagnostic yield of genetic testing can be limited and expectations of diagnostic results should be reviewed with families prior to testing.

Variants of Uncertain Significance

Genetic variants are classified into 5 main categories: pathogenic, likely pathogenic, benign, likely benign, and VUS. Pathogenic and likely pathogenic variants are associated with disease and meet ACMG criteria for pathogenicity. Benign and likely benign are not associated with disease. Variants are classified as a VUS due to limited information and are often eventually found to be benign or likely benign as more evidence emerges with time. Over time VUS findings may be re-characterized based on the literature, animal models, or family cohorts. In the meantime, these results are unsatisfactory and can be frustrating for patients and families.

Parentage

Genetic testing has become standard of care for a variety of indications and often testing is more useful if a patient sample is analyzed with parental samples. This can be done from the onset or later on to help classify the pathogenicity of variants identified in the patient. It can also shed light on recurrence risk and future family planning. However, it can also reveal non-paternity or consanguinity, and families should be counseled prior to testing regarding these potential results and implications. Patient-only testing can be performed medically to avoid non-paternity concerns, however DTC genetic testing typically contains an ancestry component that would potentially reveal parentage based on matched relatives within the ancestry platform. DTC testing that has inadvertently exposed unexpected parentage has been reported numerous times in mainstream media, including cases related to in vitro fertilization with embryo mix ups or alternate sperm use.

Pediatric Carrier Screening

Pediatric screening for carrier status or adult onset conditions without implications in childhood is not currently recommended per AAP, ACMG, and ASHG guidelines.^21,22 These conditions do not have implications during childhood and medical monitoring for these adult onset conditions begin in adulthood. The recommendation aims to support future decisional autonomy regarding testing for particular conditions or carrier status and avoid the harm of stigma, discrimination, or emotional burden of early testing. This recommendation extends to DTC services, particularly with concerns for validity of testing and the need for adequate, as well as developmentally appropriate, pre- and post-test counseling.

With increasing utilization of WES/WGS, carrier testing, and predisposition/risk screening, related ethical concerns are increasing. WES/WGS testing by definition is comprehensive and will sequence genes for adult onset disorders (e.g. high risk genes for Alzheimer’s disease), cancer predisposition genes (e.g. BRCA1/2 associated with breast and ovarian cancers), carrier status for various conditions (e.g. Cystic Fibrosis) and other medically actionable findings that may not be related to the initial reason for obtaining testing. To address this, the ACMG publishes guidance for genes to be reported in secondary findings on WES/WGS. The latest release from 2021 includes 73 genes. This list will be updated annually by the ACMG Secondary Findings Maintenance Working Group, who take into consideration the “actionability, severity, penetrance, impact and burden of available treatment modalities or screening recommendations.”²³ During pre-test counseling for WES/WGS, secondary findings are explained and offered for families to opt in or out of.^24,25

Genetic Information Nondiscrimination Act

Pre-test counseling also involves discussing the federal Genetic Information Nondiscrimination Act (GINA), which prevents employers and health insurance companies from discrimination based on genetic information.²⁶ This does not apply to disability insurance, life insurance, or long-term care insurance. These types of insurances can utilize medical information, including genetic information, for underwriting, pricing, and other aspects of insurance products. GINA does not extend to certain federal programs and groups, including the United States military and medical care through the Department of Veterans Affairs, or the Indian Health Service.

Direct-to-Consumer Genetic Testing

Direct-to-Consumer (DTC) genetic testing is marketed directly to customers, often via television, the internet, or telemarketing. Interested individuals can purchase these products online or in stores, send DNA samples to the company, and receive their genetic testing results directly. This process does not require the involvement of a health care provider or genetic counselor. The testing performed varies between companies, but can include medically pertinent testing like BRCA1/2, other cancer predisposition genes, carrier screening, and known risk genes for conditions like Alzheimer’s disease. It can also include other genetic traits (e.g. eye color) and ancestry information. Common DTC genetic testing providers include 23andMe, EasyDNA, Helix, among others.

Direct-to-Consumer testing has increased in the last five years²⁷ and patients are presenting results primarily to their PCPs for additional guidance and decision-making.²⁸ Because the majority of these DTC genetic testing companies do not require complete pre- and post-test genetic counseling, many individuals pursue testing without full understanding of potential implications,⁷ leaving the bulk of this counseling to be done after receiving results, if at all.

Most DTC testing is based on SNP-chip genotyping, not DNA sequencing.²⁹ This process involves using thousands of probes on a slide that hybridized to patient DNA. The different ratios between patient DNA and each probe DNA can be visualized to detect genetic variants. This type of testing is well suited for common genetic variations, but can be very inaccurate for rare pathogenic variants. On analysis, there has been a 40%–96% false positive rate.^30,31 These companies often do not confirm these rare variants with Sanger sequencing or other internal methods of validation,³¹ which is the standard for medical genetic testing. This is particularly relevant as some DTC companies have been granted FDA clearance to report BRCA1/2 variants, associated with increased breast cancer risk, and other medically actionable genes.³² The rare variants identified may or may not appear in the direct report to the customer, but will be available in the raw genetic data. Many individuals request this raw genetic data, approximately 60–70%^28,33 by some reports, to have it analyzed through a third party. In addition to the high false positive rate associated with the raw data itself, third party analyses can vary widely in their classification of a particular variant as pathogenic, benign, uncertain, etc., based on their own definitions. The ACMG and the Association for Molecular Pathology have strict variant classification standards. The ACMG has defined criteria for variant classification and recommends result interpretation by a board-certified clinical molecular geneticist or molecular genetic pathologist.³⁴ There have been reports of raw data analyses that have characterized variants as pathogenic that would have been benign variants or VUS per ACMG criteria.³⁵

When clinical decisions are made based on presumed pathogenic variants, whether from false positive data or inaccurate classification, unnecessary and at times harmful medical interventions are pursued. There are a number of case reports regarding patients who were thought to have a pathogenic variant by DTC testing and underwent multiple ED visits, subspecialty workup, extensive imaging, and even surgery. Subsequent confirmatory sequencing by a certified clinical laboratory of the “pathogenic variant” did not confirm DTC testing findings.³³ In addition to the unnecessary medical interventions, patients and their families undergo emotional, social, and potentially financial stressors related to the false test results from DTC testing.^33,36

In general, reputable genetic testing laboratories have certification requirements, including certification through clinical laboratory improvement amendments (CLIA) and the College of American Pathologists (CAP), and must be in good standing of these rigorous requirements. They will confirm variants with internal validation or a secondary testing method and report the coverage of sequencing for particular genes. While DTC genetic testing can be informative and is directly accessible to the consumer, it has many limitations, and patients with medical concerns related to potential genetic causes should undergo genetic testing through appropriate channels.

Conclusion

Many patients initially present to their PCP with genetics related concerns and unfortunately genetic specialists can be limited in number or availability, making continued PCP education and familiarity with genetic testing modalities, indications for testing or referral, considerations for pre-test counseling, and limitations of DTC testing very important. Common referrals for genetic testing include DD/ID, MCA, ASD, patients with dysmorphic features, and familial genetic conditions. The first line testing recommendation for DD/ID and MCA is WES/WGS, providing a diagnostic rate of 38% in certain indications, in contrast to the prior diagnostic rates of 10–20%. Other modalities of genetic testing continue to be important in various contexts.

Due to implications of genetic testing outside of the patient’s current medical concerns, pretest counseling is essential for patients or the parents of patients who are minors. Unfortunately pre- and post-test counseling is often limited in DTC testing, which also has other limitations including high false positive rates for rare genetic variants and genetic variant interpretation concerns. With advances in medicine, genetics has become a substantial part of patient care and PCP familiarity with genetic testing options and indications allow providers to deliver appropriate, up-to-date healthcare.