The future is here: Integrating genetics into the pediatric pulmonary clinic

Abstract

Recognition of underlying genetic etiologies of disease is increasing at an exponential rate, likely due to greater access to and lower cost of genetic testing. Monogenic causes of disease, or conditions resulting from a mutation or mutations in a single gene, are now well recognized in every subspecialty, including pediatric pulmonary medicine; thus, it is important to consider genetic conditions when evaluating children with respiratory disease. In the pediatric pulmonary clinic, genetic testing should be considered when multiple family members present with similar or related clinical features and when individuals have unusual clinical presentations, such as early-onset disease or complex, syndromic features. This review provides a practical guide for genetic diagnosis in the pediatric pulmonary setting, including a review of genetic concepts, considerations for test selection and results in interpretation, as well as an overview of genetic differential diagnoses for common pediatric pulmonary phenotypes. Genetic conditions that commonly present to the pediatric pulmonary clinic are reviewed in a companion article by Yonker et al.

1 |. INTRODUCTION

Recognition of underlying genetic etiologies of disease is increasing at an exponential rate, likely due to greater access to and lower cost of genetic testing. Monogenic causes of disease, or conditions resulting from a mutation or mutations in a single gene, are now well recognized in every subspecialty, including pediatric pulmonary medicine; thus, it is important to consider genetic conditions when evaluating children with respiratory disease.

In the pediatric pulmonary clinic, genetic testing should be considered when multiple family members present with similar or related clinical features and when individuals have unusual clinical presentations, such as early-onset disease or complex, syndromic features. This review provides a practical guide for genetic diagnosis in the pediatric pulmonary setting, including a review of genetic concepts, considerations for test selection and results in interpretation, as well as an overview of genetic differential diagnoses for common pediatric pulmonary phenotypes. Genetic conditions that commonly present to the pediatric pulmonary clinic are reviewed in a companion article by Yonker et al.

2 |. GENETIC CONCEPTS OVERVIEW

Some fundamental concepts to consider when evaluating a patient and their family for a genetic condition are the inheritance pattern, disease penetrance, and expressivity of a disorder. Knowing the inheritance pattern of a genetic condition can aid in pinpointing a suspected diagnosis based on a patient’s family history. Classical inheritance patterns include autosomal dominant, autosomal recessive, X-linked, and mitochondrial. In addition, understanding the penetrance of a condition, or the proportion of individuals who carry a pathogenic genetic variant that will become symptomatic is important when evaluating a family. Reduced penetrance is common, particularly in autosomal dominant conditions, and can make determining an inheritance pattern more difficult.¹

Further complicating the genetic evaluation of a patient is the fact that genetic conditions often display variable expressivity, meaning that not all individuals with a specific genetic condition will have the same clinical features. The presentation and severity of some conditions can vary widely, even amongst family members. Another genetic phenomenon that is observed more rarely is the concept of genetic anticipation, which describes conditions that have the potential to increase in severity with each subsequent generation.¹ While a thorough family history can be a useful tool, many genetic conditions, particularly those with recessive inheritance, often occur in individuals with no family history of the disease. In addition, some autosomal dominant conditions, such as alveolar capillary dysplasia and congenital central hypoventilation syndrome, are typically caused by de novo mutations, or those that occur in germ cells or early in embryogenesis. Therefore, these conditions also present in the absence of family history.

The concepts of variable expressivity and genetic anticipation are exemplified in Figure 1A which represents a family impacted by a short telomere syndrome with autosomal dominant inheritance. Short telomere syndromes describe a group of conditions that are caused by the presence of excessively short telomere length for age. Dyskeratosis congenita is the severe, syndromic phenotype, while milder presentations include isolated features of pulmonary fibrosis, aplastic anemia, or liver cirrhosis.² The affected individuals in the family in Figure 1A present with diverse clinical features, which demonstrates the variable expressivity of the condition. In addition, anticipation can be observed, characterized by younger ages of onset and a more severe presentation among later generations. In contrast, the family in Figure 1B shows the autosomal recessive inheritance of primary ciliary dyskinesia, which is characterized by affected individuals in only one generation.

FIGURE 1.

Pedigrees demonstrating autosomal dominant and autosomal recessive inheritance. Arrow: indicates proband; Circles: indicate female family members; Squares: indicate male family members; Diagonal line: indicates individual is deceased

Another consideration is the fact that many pulmonary genetic conditions have clinical features that overlap with common multifactorial disorders, or conditions that manifest due to a combination of environmental exposures and a predisposing genetic background. For example, findings such as bronchiectasis and interstitial lung disease are often multifactorial in origin. However, for these presentations, it is important to rule out monogenic conditions, such as cystic fibrosis and primary ciliary dyskinesia, as natural history, treatment, and reproductive risk for these conditions are well established.

3 |. TEST SELECTION

Genetic testing can be a powerful and cost-effective clinical tool. When ordering a genetic test, there are many things to consider to ensure that the appropriate test is being selected. As of 2020, over 65 000 clinical genetic tests offered by more than 560 laboratories, were listed in the Genetic Testing Registry.³ With so many options to choose from, it is important to select a test from a reputable lab with gene content that is relevant to the patient’s clinical presentation. Some qualifications to consider are that laboratories should adhere to government regulations for preforming clinical testing and have the appropriate certification to do so, have testing options designed by experts in genetics, and have transparent methodologies and limitations available to providers.

Some clinical findings are essentially pathognomonic for specific genetic conditions, such as the presence of multiple arteriovenous malformations (AVMs) and its association with hereditary hemorrhagic telangiectasia (HHT).⁴ However, many other phenotypes, including respiratory distress, interstitial lung disease, and pneumothorax have a wider differential diagnosis. Therefore, for a patient with AVMs, it may be easy to select an HHT gene panel that includes all of the known relevant genes, but for a condition such as neonatal respiratory distress, which can have various genetic and nongenetic causes, it may be necessary to gather additional phenotypic information to guide test selection. When initiating genetic testing, it is generally recommended to start by testing for the most common genetic conditions with the highest clinical suspicion and then expanding to more comprehensive genetic tests, such as whole-exome or genome sequencing, if necessary. Table 1 provides genetic differential diagnoses for various pulmonary phenotypes that can have multiple underlying genetic etiologies.

TABLE 1.

Monogenic genetic differential diagnosis for pediatric pulmonary phenotypes

Diffuse lung disease	Pneumothorax	Immunodeficiency
• Alveolar capillary dysplasia5	• Autosomal recessive polycystic kidney disease25,26	• 22q11.2 Microdeletion syndrome38
• Ataxia-telangiectasia6		• Ataxia-telangiectasia39
• Brain-lung-thyroid syndrome7,8	• Birt-Hogg-Dubé syndrome27,28	• Bloom syndrome40
• Cystic fibrosis9	• Cutis Laxa29	• Chediak-Higashi syndrome41
• Dyskeratosis congenita/short telomere syndromes2,10	• Cystic fibrosis30	• Hermansky-Pudlak syndrome42
	• DICER1-related disorders31	• Hypohidrotic ectodermal dysplasia43
• FLNA-associated periventricular heterotopia11	• FLNA-associated periventricular heterotopia11	• Nijmegen breakage syndrome44
• Gaucher disease12		• Primary immunodeficiencies*,21
• Hermansky-Pudlak syndrome13,14	• Homocystinuria32	• Trisomy 2145
• Interstitial lung and liver disease15	• Loeys-Dietz syndrome33
• Marfan syndrome16	• Marfan syndrome34
• Myhre syndrome17	• Primary ciliary dyskinesia35
• Niemann-Pick disease18,19	• Tuberous sclerosis36
• Primary ciliary dyskinesia20	• Vascular Ehlers Danlos syndrome37
• Primary immunodeficiencies*,21
• Protein alveolar proteinosis22
• Surfactant deficiencies23
• Williams syndrome24
Respiratory distress of the newborn	Bronchiectasis	Pulmonary arterial hypertension
• Alveolar capillary dysplasia5	• Ataxia-telangiectasia6	• Alveolar capillary dysplasia5
• Autosomal recessive polycystic kidney disease25,26	• Cystic fibrosis9	• FLNA-associated periventricular heterotopia11
	• Primary ciliary dyskinesia20	• Gaucher disease52,53
• Brain-lung-thyroid syndrome46,47	• Primary immunodeficiencies*,21	• Hereditary hemorrhagic telangiectasia54,55
• Inborn errors of metabolism48	• Pseudohypoaldosteronism51	• BMPR2-associated pulmonary arterial hypertension56
• Neuromuscular disorders48,49	• Marfan syndrome16
• Primary ciliary dyskinesia20,50		• Pulmonary veno-occlusive disease57
• Surfactant deficiency23		• Sickle cell anemia58

Note: References are disease reviews or pulmonary publications. Several of these conditions are discussed in detail in the companion article to this review (Yonker et al).

^*Primary immunodeficiencies (PID) encompass over 180 monogenic disorders that cause an intrinsic defect in the immune system. Picard et al⁵⁹ provide a more detailed discussion of primary immunodeficiencies.

Today, the majority of clinical genetic tests, are performed via next-generation sequencing. When a specific genetic disorder is suspected, targeted gene panel testing or single gene sequencing is generally recommended as an initial testing strategy. Whole exome sequencing, which examines all of the coding (exonic) regions, or whole-genome sequencing, which interrogates the entire genome, can be also used as a first-tier testing strategy in cases with a large differential diagnosis. Analysis of a large amount of data generated from whole exome and whole genome sequencing requires provider input of clinical information, so detailed phenotyping is essential for these studies.

Another type of testing used commonly in the pediatric setting, particularly when syndromic features are present or in the context of neurodevelopmental disorders, is chromosomal microarray analysis. Microarray analysis looks for deleted or duplicated chromosomal segments, known as copy number variants, which are a common cause of genetic syndromes and other inherited conditions. Microarrays can be used independently of next-generation sequencing assays or as a complement to gene panel or exome testing. In particular, when a single likely pathogenic or pathogenic variant in a gene with autosomal recessive inheritance is detected by sequencing analysis, it may be necessary to reflex to a microarray or other assay for detecting copy number variants to determine if there is a deletion or duplication on the opposite allele. As sequencing technologies improve, some assays can now detect copy number variants or other complex types of the variant. When a copy number variant is detected via next-generation sequencing, microarray analysis can also be used to clarify the breakpoints of a deletion or duplication.

Some additional assays are designed to more accurately detect certain types of mutations and knowing the type of genetic variant that causes disease in a specific gene, or the mutation spectrum is essential to selecting the right test. Until sequencing technologies advance to detect all types of genetic variation, additional tests such as methylation studies, karyotype, and polymerase chain reaction (PCR), remain necessary diagnostic tools for certain genetic conditions. Types of genetic tests that are commonly used in the pediatric pulmonary clinic are outlined in Figure 2.

FIGURE 2.

Outline of commonly used genetic testing options

A practical example of the importance of test methodology that is relevant to the pulmonary clinic is congenital central hypoventilation syndrome (CCHS), which is caused by mutations in the PHOX2B gene. While Sanger sequencing and next-generation sequencing of the PHOX2B gene are clinically available, CCHS usually results from an expansion of a polyalanine triplet repeat region in the gene. These expansions can be missed by traditional sequencing methods and targeted fragment length analysis or long-range PCR is often necessary to characterize this region.⁶⁰ Therefore, a negative result from a panel or whole-exome test is not a true negative unless the testing laboratory employs specific methods to analyze the repetitive region.

Another layer beyond gene content and methodology is the need to consider the indication for testing. For example, due to the high carrier rate of cystic fibrosis in the general population, the CFTR gene is included on many carrier testing panels that are offered as part of prenatal or preconception counseling. Although these panels are sequencing the CFTR gene, this is not an appropriate test to select for a patient with suspected cystic fibrosis as the analysis of the data and what types of genetic variants are reported differs in the carrier versus diagnostic setting.

4 |. RESULTS INTERPRETATION

Genetic variants are alterations in the genetic code that differ from a reference sequence and can refer to a variety of changes, such as a substitution of a single nucleotide base pair or large deletions of a chromosomal segment. Every individual has thousands of genetic variants, the majority of which have no known clinical impact. The goal of clinical genetic testing is to identify genetic variants that may have an impact on health and to provide molecular diagnosis for individuals with specific clinical features.

When interpreting a genetic testing report, it is essential to understand genetic variant nomenclature, which can be complex, particularly for genes with significant historical data. For example, the most common pathogenic variant in the CFTR gene is traditionally referred to as ΔF508; however, based on current naming guidelines⁶¹ this variant would be reported as c.1521_1523delCTT (p.Phe508del). Being able to interpret that ΔF508 is equivalent to p.Phe508del is critical to caring for these patients, since carrying this variant has implications for prognosis and treatment.⁶² Gene names can also change over time such as with the NKX2.1 gene, which causes a brain-lung-thyroid syndrome that was previously known as thyroid transcription factor-1 (TTF1).

Another key aspect of results interpretation is the classification of genetic variants. Laboratories generally classify variants based on internal data, case data, and functional evidence published in the scientific literature, and information available in public databases. For the majority of laboratories, this data is used to classify variants into one of five categories: pathogenic, likely pathogenic, uncertain significance, benign, and likely benign. It is essential for providers who are ordering genetic testing to have some knowledge of how genetic variants are classified and interpreted by testing laboratories so that patients can be accurately counseled as to the meaning of their results. Variants of uncertain significance, or those without sufficient evidence to determine if they are benign or pathogenic, are commonly reported, particularly on panel testing, and over or under the interpretation of these findings by clinicians can lead to misdiagnosis and inappropriate care.^63,64

While there are standards and guidelines for variant interpretation, genetic information is dynamic.⁶⁴ A seemingly pathogenic mutation can eventually be reclassified as a benign variant or vice versa. More modest changes in classification, such as a variant of uncertain significance being upgraded to a likely pathogenic variant, are very common. Therefore, genetic testing results require periodic follow up to determine if the interpretation of the findings has changed.

For some assays, such as whole-exome sequencing and whole-genome sequencing, it may be necessary to request a reanalysis of a patient’s sequencing data as the knowledge about the association of genes and genetic variants to disease expands over time. This is of particular relevance in the fairly young field of pediatric pulmonary genetics that, excluding well-characterized conditions such as cystic fibrosis, is likely to show a significant increase in understanding of gene-disease relationships in the coming years.

Depending on the assay, genetic testing results can be positive, meaning a genetic variant or genetic variants were identified that are consistent with a molecular diagnosis; uncertain, meaning that a genetic variant or variants were identified but there is not sufficient data to determine their clinical significance; or negative, meaning no potentially clinically significant variants were identified. When results are uncertain, it can sometimes be helpful to test additional family members or to perform additional clinical studies to better correlate the findings.

When ordering exome or genome sequencing, the inclusion of samples from additional family members, particularly the patient’s biological parents, can aid in the analysis. The inclusion of parental samples allows the laboratory to determine if two variants identified in the same gene occur on opposite chromosomes or on the same chromosome (in cis or in trans). In genes associated with autosomal recessive inheritance, it is necessary for pathogenic changes to be present on opposite chromosomes (in trans) to cause disease, usually as a result of one variant being inherited from each parent. In addition, the inclusion of parental samples allows for the identification of de novo variants, which are those that occurred as new changes in the patient and were not inherited from either parent. Samples from affected family members can help to track the segregation of variants within a family. Testing of additional family members can also be helpful after other types of genetic testing, such as gene panel tests or microarray analysis, to clarify uncertain findings or confirm that variant inheritance is consistent with the mechanism of disease.

Negative test results can be useful by ruling out genetic conditions that may be associated with a severe prognosis or extrapulmonary involvement. However, negative genetic testing results do not necessarily rule out that an underlying genetic etiology exists in a patient, even in the context of whole-exome or whole-genome sequencing. All genetic testing has limitations that may lead to false-negative results and patients should be informed of this residual risk. These limitations are due to both the technological and analytical limitations of the testing assay, as well as gene-disease associations that have yet to be discovered. Even for well-established genetic conditions, there are cases where individuals meeting clinical diagnostic criteria do not have identifiable mutations. For example, the detection rate for individuals meeting the clinical diagnostic criteria for hereditary hemorrhagic telangiectasia (HHT) is roughly 85%.⁶⁵ Therefore, when these individuals have negative genetic testing, it does not mean that they do not have HHT and associated screening may still be necessary for patients and family members. The residual risk after a negative test result varies based on the clinical indication and the type of testing. Detection rates of genetic testing can also vary by ethnicity. In genetics as well as many other areas of medicine, individuals of European descent are often the most well-studied population. Therefore, negative results in other ethnicities may be less informative than negative results in individuals of European ancestry.

While the results of genetic testing can be complex, communicating these results is of particular importance as these findings often have implications for other family members.

5 |. PRACTICAL CONSIDERATIONS

The implications for a family are a substantial consideration when ordering genetic tests. Although the cost has dropped significantly in recent years, genetic analyses are still relatively expensive and have widely variable coverage by insurance companies. In addition, genetic results can have implications for family members beyond the index case, including the need for clinical screening of at-risk relatives or carrier testing to determine their reproductive risk. Because genetic information has the potential to impact other family members, particularly when a positive finding is uncovered, it is important to communicate genetic results in a way that is understandable to patients so that they can effectively pass this information along. In general, patients need to understand which family members may be at risk, what the implications are, and how those family members can access appropriate medical care and/or genetic counseling. One effective way to aid patients in communicating with family members is to provide patients with a letter explaining the genetic results and how at-risk family members can obtain a further evaluation.

Incidental findings including, but not limited to, the detection of genetic variants unrelated to the testing indication, such as those that predispose to certain types of cancer, and the uncovering of unexpected familial relationships, such as nonpaternity or unknown consanguinity, are an additional complication. It is important that patients are properly educated regarding these possibilities, that informed consent is obtained, and that ordering providers are prepared to discuss these findings should they arise.

Another practical consideration concerns payment for genetic tests, which often cost hundreds to thousands of dollars. Insurance coverage of genetic testing has expanded greatly in recent years, although, just as for other expensive medical procedures or prescriptions, what is covered and how much is covered varies widely. Fortunately, many genetic testing companies have resources to aid with billing, can interface directly with insurance companies, and offer out of pocket maximums for patients.

Because genetic information is highly sensitive health data that can sometimes be predictive of an individual’s health risks even before symptoms appear, discrimination based on genetic test results is another concern for patients. In 2008 the United States passed the Genetic Information Nondiscrimination Act (GINA) which protects Americans from discrimination by their employers or health insurance companies based on their genetic information. It does not protect against discrimination for life or long-term disability insurance. Patients should be educated about what is and is not protected during the consenting process for genetic testing.

Because selecting the appropriate test, obtaining consent, and discussing the results of genetic testing can be complex, involving a medical geneticist or genetic counselor is recommended whenever possible. It can also be helpful to contact genetic testing laboratories, many of which employ genetic counselors that can aid in test selection and results in interpretation.

6 |. CLINICAL UTILITY

Although genetic testing can be complex and has limitations, making a genetic diagnosis can have clinical utility for many reasons. It can help with clinical management by clarifying the likely disease progression and prognosis as well as guiding screening measures and treatment options. For example, if a patient presenting with pneumothorax is found to have an underlying diagnosis of Marfan syndrome, they will need regular cardiac imaging to screen for aortic root dilation and referral to an ophthalmologist to assess for ocular complications.^66,67 Genetic diagnosis can also alleviate uncertainty for families, identify other family members who are at risk to be affected and provide information about the recurrence risk.

In addition to these benefits, genetic testing will have increased utility as the field of precision medicine advances. For many new targeted treatments and gene therapies to be employed, it will be necessary to identify specific underlying molecular defects or disease-causing genetic variants. At this point, the majority of genetic conditions do not have targeted therapies, however, such therapies have already begun to have a large impact on patients with cystic fibrosis. Individuals with specific CFTR mutations are eligible for treatment with CFTR modulator drugs, such as ivacaftor, lumacaftor, tezacaftor, and the recently approved elexacaftor/ivacaftor/tezacaftor combination therapy, which works to improve or restore function of the defective protein, thereby offering a significant therapeutic advantage over the traditional standard of care of symptom management.⁶²

Additional examples of genetically-guided therapies include those developed for neuromuscular disorders, some of which progress to respiratory failure and require significant pulmonary care. Duchenne muscular dystrophy patients frequently have mutations that result in premature termination of translation, which is often amenable to treatment with exon skipping drugs. These drugs work by skipping an additional exon in the patient’s messenger RNA, which ultimately restores the reading frame of the gene that was disrupted by the original mutation, allowing for some production of the dystrophin protein and thus improving muscle function.⁶⁸

Spinal muscular atrophy (SMA), which is caused by biallelic loss of function of the SMN1 gene is also now amenable to genetic-based therapies. Nusinersen is an antisense oligonucleotide that works by increasing the output of functional protein produced from the SMN2 gene, which is a homolog of SMN1 that normally shows reduced protein production.^69,70 Recently, a gene therapy, onasemnogene abeparvovec-xioi, that works by introducing a functional copy of the SMN1 gene via a viral vector, was approved by the FDA to treat children under 2 years of age with SMA.^69,70 As drug development progresses and more genetic variants are functionally characterized, more targeted treatments will become available to patients, thus increasing the need for accurate genetic diagnosis.

7 |. CONCLUSION

Although genetic testing results can be complex and at times unclear, it will be of increasing importance for providers across all specialties to incorporate genetics into clinical practice as a growing number of patients are diagnosed with genetic conditions. Table 2 provides a list of publicly available resources that have useful information about genetic conditions, genes and variants, and genetic testing. The continued incorporation of genetics into the pediatric pulmonary clinic will help to provide a more accurate diagnosis, prognosis, management, and treatment for these patients.

TABLE 2.

Publicly available genetics resources

Resource	General description	Website
Gene-disease information resources
GeneReviews	• In depth information about genetic conditions, including clinical features, diagnostic criteria, management recommendations, differential diagnosis, and additional resources.	https://www.ncbi.nlm.nih.gov/books/NBK1116/
	• Written by disease area experts and updated periodically.
Genetics Home Reference	• Brief summaries of gene function, gene-disease associations, clinical features, associated genes, disease inheritance, and disease prevalence.	https://ghr.nlm.nih.gov/
	• Uses mostly patient friendly language and has general genetics resources for patients.
Online Mendelian Inheritance in Man (OMIM)	• Gene-disease associations, inheritance patterns, and phenotype descriptions.	https://www.ncbi.nlm.nih.gov/omim
Clinical Genome Resource (ClinGen)	• Expert curation of gene-disease associations and clinical actionability.	https://www.clinicalgenome.org/
Variant interpretation resources
Human Genome Variation Society (HGVS)	• Guidelines and recommendations for nomenclature of genetic variants.	https://varnomen.hgvs.org/
Genome Aggregation Database (GnomAD)	• Population database with frequency of genetic variants in various subpopulations.	https://gnomad.broadinstitute.org/
	• Also shows gene level information, such as gene constraint and tissue specific transcript expression.
ClinVar	• Database of genetic variants with classifications and interpretations based on assessment by various submitters, including clinical labs and researchers.	http://www.clinvar.com/
UCSC Genome Browser	• Transcript and conservation information with the ability to view data from many sources for a specific gene or region.	https://genome.ucsc.edu/
Genotype-Tissue Expression (GTEx)	• Shows tissue specific expression of genes and their different isoforms.	https://gtexportal.org/home/
Genetic testing resources
American College of Medical Genetics (ACMG)	• Guidelines for diagnosis and management of genetic conditions and use of genetic testing.	https://www.acmg.net/
Genetic Testing Registry (GTR)	• International database of clinically available genetic tests.	https://www.ncbi.nlm.nih.gov/gtr/
Laboratory Websites	• Genetic testing menus with information about methodology and limitations.
Genetic counseling resources
National Society of Genetic Counselors (NSGC)	• Search for genetic counselors providing either in person or telecounseling services.	https://www.findageneticcounselor.com/
	• Patient resources about genetic counseling and genetic testing.