Version May 2024
INTRODUCTION — Achieving a molecular diagnosis provides the most comprehensive understanding of a patient's primary immune dysregulation, allowing for tailored interventions and personalized surveillance strategies. The diagnostic process to uncover a molecular etiology, however, can be complicated by numerous testing strategies and modalities, each with their own nuances and limitations. Nevertheless, this trend toward precision medicine requires us to navigate the genetic testing landscape to ultimately improve health outcomes in our patients.
This topic reviews the fundamentals of genetic testing and provides a clinical approach to molecular diagnosis for individuals with suspected primary immunodeficiency or autoinflammatory syndromes (inborn errors of immunity).
Additional helpful discussions include:
●Primary humoral immunodeficiencies (see "Primary humoral immunodeficiencies: An overview")
●Approach to the child with recurrent infections (see "Approach to the child with recurrent infections")
●Genetics terminology (see "Genetics: Glossary of terms")
●Genome sequencing (see "Next-generation DNA sequencing (NGS): Principles and clinical applications")
●Genomic disorders (see "Genomic disorders: An overview")
●Genetic testing (see "Genetic testing")
●Genetic counseling (see "Genetic counseling: Family history interpretation and risk assessment")
●Disclosure of incidental findings from genetic testing (see "Secondary findings from genetic testing")
BENEFITS OF DETERMINING THE UNDERLYING GENETIC ETIOLOGY
Genotype-specific management in primary immunodeficiency — The importance of securing a molecular diagnosis in patients with primary immunodeficiency cannot be understated. Identifying a genetic diagnosis can drive a decision toward hematopoietic cell transplantation (HCT) if the suspected diagnosis is confirmed or lead to use of other treatment modalities. In other cases, such as infants with idiopathic low lymphocytes and abnormal T cell receptor excision circles (TRECs), observation and monitoring rather than treatment may be warranted. In one study of 280 families with primary immunodeficiency, use of whole exome sequencing identified a probable molecular diagnosis in 40 percent and altered the preliminary diagnosis in 55 percent of cases, resulting in a change in clinical management in 25 percent [1]. As of 2021, over 450 genetic defects of immunity have been identified [2,3].
As an example, replacement of immunoglobulin G (IgG) is indicated for the treatment of primary immunodeficiencies characterized by recurrent infections due to a known immune mechanism [4]. The decision to continue IgG replacement therapy can be justified (eg, to a third-party payor) by confirmation of a primary immunodeficiency through identification of a genetic diagnosis.
Management of primary immunodeficiency patients can be impacted by genotype:
●The lymphoid genetic causes of severe combined immunodeficiency (SCID) and SCID variants must be distinguished from primary athymia due to forkhead box N1 (FOXN1) deficiency or complete DiGeorge anomaly since the therapeutic option of choice for the lymphoid defects is HCT, whereas the preferred treatment for the other two conditions is allogeneic thymus transplantation [5,6]. Poor outcomes are known to occur in these patients in the absence of a correct diagnosis [7]. (See "DiGeorge (22q11.2 deletion) syndrome: Clinical features and diagnosis", section on 'Complete DGS' and "Combined immunodeficiencies: An overview", section on 'Genotypes and immunophenotype severity'.)
●Patients with specific forms of SCID that have associated radiation sensitivity should avoid exposure to ultraviolet light, ionizing radiation, and deoxyribonucleic acid (DNA) crosslinking medications [8]. Genotyping is required to identify patients with these forms of SCID. (See "T-B-NK+ SCID: Management".)
●Individuals with gain-of-function (GOF) defects in transmembrane protein 173 (TMEM173) leading to stimulator of interferon genes (STING) associated vasculopathy with onset in infancy (SAVI) [9-11], an autoinflammatory disorder, or signal transducer and activator of transcription 1 (STAT1) causing chronic mucocutaneous candidiasis [12-16] can be treated with ruxolitinib, a selective Janus-associated kinase 1 and 2 (JAK1/2) inhibitor that blocks interferon signaling. STAT1 GOF disease can mimic immune dysregulation-polyendocrinopathy-enteropathy-X-linked (IPEX) syndrome [17]. However, unlike STAT1 GOF disease, IPEX is typically managed with broad immune suppression rather than targeted biologic therapy [18]. (See "Autoinflammatory diseases mediated by interferon production and signaling (interferonopathies)", section on 'STING-associated vasculopathy with onset in infancy (SAVI)' and "Chronic mucocutaneous candidiasis", section on 'Signal transducer and activator of transcription (STAT1) dysfunction' and "IPEX: Immune dysregulation, polyendocrinopathy, enteropathy, X-linked", section on 'IPEX-like syndromes'.)
●Cytotoxic T lymphocyte associated protein 4 (CTLA4) haploinsufficiency can present with autoimmune enteropathy and can be treated with great impact using abatacept, which blocks the costimulatory pathway involved in T cell activation, rather than less specific immunosuppressive medications [19]. (See "Gastrointestinal manifestations in primary immunodeficiency".)
●Some primary immunodeficiencies with associated mucocutaneous candidiasis (eg, dedicator of cytokinesis 8 [DOCK8] deficiency) respond favorably to HCT, whereas others (eg, signal transducer and activator of transcription 3 [STAT3] deficiency and autoimmune regulator [AIRE] deficiency) do not [20,21]. (See "Chronic mucocutaneous candidiasis", section on 'Differential diagnosis' and "Autosomal dominant hyperimmunoglobulin E syndrome", section on 'Genetics' and "Chronic mucocutaneous candidiasis", section on 'Autoimmune regulator deficiency'.)
●In individuals with Mendelian susceptibility to mycobacterial diseases, interferon (IFN) gamma supplementation is not indicated for complete IFN-gamma receptor 1 (IFN-gamma-R1) or IFN-gamma receptor 2 (IFN-gamma-R2) deficiencies but can be beneficial for patients carrying other genotypes, such as complete interleukin 12 receptor gamma 1 (IL-12R-gamma-1) and complete IL-12p40 deficiencies [22]. (See "Mendelian susceptibility to mycobacterial diseases: An overview", section on 'General approach to treatment' and "Mendelian susceptibility to mycobacterial diseases: Specific defects".)
Genotype-specific management in autoinflammatory syndromes — Identification of a molecular defect in patients with autoinflammatory syndromes is essential for optimal management of these conditions as well:
●Colchicine is the first-line treatment for familial Mediterranean fever, a periodic fever syndrome [23].
●In contrast, use of colchicine is not advised in patients with periodic fevers and hyperimmunoglobulin D (IgD) syndrome, due to mevalonate kinase deficiency [24-26]. Instead, IL-1 blockade, etanercept, or IL-6 inhibition are favored [25-31]. (See "Management of familial Mediterranean fever" and "Hyperimmunoglobulin D syndrome: Management".)
●IL-1 blockade with agents such as anakinra, canakinumab, or rilonacept is the recommended first-line treatment in patients with cryopyrin-associated periodic syndromes (CAPS) due to pathogenic variants in NLR family pyrin domain containing 3 (NLRP3) [27,32]. IL-1 blockade is also an option in other autoinflammatory conditions characterized by excess IL-1 signaling, such as pyogenic sterile arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome. (See "Cryopyrin-associated periodic syndromes and related disorders" and "Autoinflammatory diseases mediated by inflammasomes and related IL-1 family cytokines (inflammasomopathies)", section on 'PAPA syndrome'.)
●For individuals with tumor necrosis factor (TNF) receptor-associated periodic syndrome due to defects in TNF receptor superfamily member 1A (TNFRSF1A) (see "Tumor necrosis factor receptor-1 associated periodic syndrome (TRAPS)"), on the other hand, either IL-1 blockade or etanercept is recommended for treatment [33-42], but infliximab or adalimumab are contraindicated [27,34-37].
●GOF variants in NLR family CARD domain containing 4 (NLRC4) result in a clinical presentation that resembles hemophagocytic lymphohistiocytosis (HLH) [43]. However, affected individuals are managed with anakinra rather than the usual HLH-94 or HLH-2004 chemotherapy protocols.
●Lysinuric protein intolerance caused by pathogenic variants in solute carrier family 7 member 7 (SLC7A7) can mimic systemic lupus erythematosus or HLH. The primary management for this condition consists of dietary protein restriction rather than immune suppression [44].
Genotype-specific counseling — Establishing a molecular diagnosis also allows medical care providers to provide affected families and individuals with critical information regarding the natural history of the disease, recurrence risk, and involvement of extra-hematopoietic systems. (See "Genetic counseling: Family history interpretation and risk assessment".)
Data concerning various primary immunodeficiencies collected in registries and cohort studies are helping to understand the natural history of each condition by genotype [45,46]. As an example, disease severity varies by genotype for autosomal recessive chronic granulomatous disease, with p47phox deficiency carrying the best prognosis (milder phenotype and better long-term survival) [47].
Identification of a genetic diagnosis also enables informed counseling of parents regarding recurrence risk for future offspring, as dictated by fundamental Mendelian principles of inheritance, or using empiric risk estimates. Incomplete penetrance or variable expressivity is well documented for some single allelic primary immunodeficiencies, and discussion of this phenomenon with family members becomes difficult without a genetic diagnosis. One example is coatomer protein complex subunit alpha (COPA) syndrome, in which expressivity of the trait is diminished in males compared with females [48].
The presence of a genetic diagnosis can enlighten families regarding expectations related to extra-hematopoietic involvement. As an example, HCT can rescue the profound immune deficiency but does not ameliorate neurologic and developmental problems in patients who have SCID due to defects in DNA repair (eg, DNA ligase 4 [LIG4] and nonhomologous end joining factor 1 [NHEJ1] deficiencies). Similarly, HCT does not reverse type 1 diabetes in forkhead box P3 (FOXP3) deficiency.
CHOOSING WHOM TO TEST — Genetic testing has the highest diagnostic yield in situations where the clinical phenotype is probably caused by one or more known genetic conditions. The diagnostic yield will be higher, for example, if a family member shares a similar disease phenotype or if the pattern of inheritance is evident from the pedigree. In contrast, genetic testing is least helpful in situations where an underlying genetic cause is either unlikely (ie, secondary immunodeficiency suspected) or the disease gene for a known constellation of clinical features has not been identified.
Testing should be initiated first in the affected individual (the proband), with subsequent targeted testing in family members (cascade testing). This approach minimizes the total number of tests and chances for secondary findings in unaffected individuals. If testing is initiated in an unaffected individual, the sensitivity and specificity of testing are reduced, as it becomes difficult to determine if the individual truly has no pathogenic variant or whether a pathogenic variant is present but undetectable by the testing modality.
Assessing the phenotype
Classic primary immunodeficiency phenotypes — Primary immunodeficiencies are usually recognized by the presence of susceptibility to infections. As an example, recurrent infections with polysaccharide-encapsulated organisms, particularly of the respiratory tract, suggest a humoral or primary complement deficiency, whereas opportunistic infections suggest a T cell deficiency. Susceptibility to bacterial and fungal infections, often accompanied by poor wound healing, indicate an underlying defect in phagocytic number or function. (See "Primary humoral immunodeficiencies: An overview" and "Inherited disorders of the complement system" and "Severe combined immunodeficiency (SCID): An overview" and "Combined immunodeficiencies: An overview" and "Primary disorders of phagocyte number and/or function: An overview".)
Other clinical signs may be present that warrant evaluation for the presence of primary immunodeficiency:
●Multiple, unusual, or severe allergies (table 1)
●Severe eczema (table 2)
●Ectodermal dysplasia (table 3)
●Abnormal hair (table 4)
●Early- or very-early-onset inflammatory bowel disease or enteropathy (table 5)
●Early-onset or severe autoimmunity (table 6)
●Early-onset endocrinopathies (table 7)
●Lymphoproliferative disease (table 8)
●Hemophagocytic lymphohistiocytosis (HLH) (table 9)
●Neurologic abnormalities (table 10)
●Skeletal dysplasia (table 11)
Classic autoinflammatory phenotypes — Primary autoinflammatory diseases are typically characterized by recurrent fevers, rashes, articular signs or symptoms, and inflammation of other tissues in the absence of infection or malignancy. Most of these conditions present in childhood. (See "The autoinflammatory diseases: An overview".)
Source of DNA — The vast majority of primary immunodeficiencies and classic autoinflammatory syndromes result from germline variants that are either inherited from a parent or that occurred in the sperm or egg prior to conception. Thus, the variants are present in all tissue types and can be detected in any nucleated cell. Some autoinflammatory syndromes, particularly those caused by activating variants in genes involved in the innate immune system, are somatic mosaic variants, with the most striking example being NLRP3 in patients with neonatal-onset multisystem inflammatory disorder (NOMID)/chronic infantile neurologic cutaneous and articular (CINCA) syndrome [49-52].
Testing for germline variants — Although genetic testing for primary immunodeficiencies and classic autoinflammatory syndromes can be performed using DNA derived from virtually any tissue, most laboratories prefer either blood specimens, cheek (buccal) swabs, or saliva samples. Sequencing requires DNA extraction from any nucleated cell, so nearly any tissue is acceptable. However, cytogenetic analysis (karyotyping) requires that dividing cells are collected, usually from peripheral blood or bone marrow.
Since red blood cells (RBCs) are nonnucleated and do not contain DNA, sequencing of whole blood is primarily dependent upon DNA extracted from the nuclei of white blood cells (WBCs). Thus, in patients with severe leukopenia (often due to either primary immunodeficiency or chemotherapy), whole blood may not contain sufficient DNA, and an alternative tissue is recommended for sample collection.
For individuals who have undergone allogeneic hematopoietic cell transplant (HCT), the peripheral blood DNA often represents the donor rather than the individual's germline. Thus, alternative tissue such as tissue fibroblasts or buccal swabs (containing mostly buccal epithelial cells) are used when performing germline genetic testing after an individual has undergone HCT. Saliva samples can contain a high amount of DNA derived from WBCs (as high as 74 percent) and therefore should not be used in patients who are post-HCT [53].
Testing for somatic variants or mosaicism — Somatic variants are DNA mutations that arose in somatic tissues and are therefore generally restricted to that tissue type or location. Thus, genetic testing must be performed on the "affected" tissue type (bone marrow, WBCs, etc). This type of testing is most commonly performed in autosomal dominant autoinflammatory phenotypes where a gain-of-function (GOF) mechanism is suspected. In one study, nearly 25 percent of patients with primary immunodeficiencies and autoinflammatory disease had evidence for disease-contributing somatic variants or mosaicism [54].
CHOOSING WHICH GENETIC TEST IS BEST
Types of genetic testing — The different types of genetic tests available are reviewed briefly here (table 12) and discussed in greater detail separately. (See "Genetic testing".)
Single-gene analysis — Single-gene analysis involves sequencing and/or deletion/duplication evaluation of one gene for deleterious changes. The sequencing of small regions is most often performed by polymerase chain reaction (PCR) to amplify each of the coding regions of the gene (exons) followed by sequencing. However, alternative methods are used depending upon the particular gene and the architecture of the loci. Sequencing can detect missense variants, nonsense variants, frameshift variants, and splice-site variants, while additional strategies (often referred to as del/dup testing) can be used to evaluate for exonic or whole-gene deletions or duplications.
The strength of single-gene testing is that it is typically a cost-effective approach to molecular diagnosis for an individual when sufficient phenotypic specificity is present. Such may be the case for classic presentations of syndromic immunodeficiencies (such as cartilage-hair hypoplasia; immunodeficiency, centromeric instability, and facial anomalies [ICF] syndrome; Griscelli syndrome; Schimke immunoosseous dysplasia; Roifman syndrome; etc) where extra-immune phenotypic findings make the particular syndrome likely. (See "Syndromic immunodeficiencies".)
The weakness of single-gene testing is that it is usually difficult to select a single gene as the probable cause of the immune perturbation using the phenotypic information alone. Without a high pretest probability in single-gene testing, the time and expense of additional tiered single-gene testing (one gene after another) make this approach clinically impractical.
Gene panels — Panels are typically composed of a set of genes that have clinically overlapping phenotypes or that serve as the cause of a particular disease or group of related conditions. Panels are most helpful when the phenotype is suspicious for an underlying genetic cause (ie, periodic fever syndromes), but numerous genes are linked to the phenotype (termed genetic heterogeneity). (See "Next-generation DNA sequencing (NGS): Principles and clinical applications", section on 'Whole genome, exome, or gene panel'.)
The primary strength of panel testing is that it provides a comprehensive analysis of genes that are known to cause a particular disease while minimizing the chance of secondary findings unrelated to the reason for genetic testing. Panels can also facilitate detection of mosaicism (see 'Testing for somatic variants or mosaicism' above). With the declining prices of genetic testing, many new panels have costs similar to fees for single-gene sequencing, making panels a more popular choice.
One weakness of panel genetic testing is that a panel can become outdated, no longer reflecting a complete gene list for the disease. Patients can also exhibit an atypical presentation of a partially overlapping phenotype (eg, common variable immunodeficiency [CVID] presenting as inflammatory bowel disease [IBD]), in which case a narrow panel may not reveal the molecular etiology.
Panels can contain dozens or even hundreds of genes commonly grouped in broad disease categories such as severe combined immunodeficiency (SCID), syndromic immunodeficiency, CVID, hypogammaglobulinemia, susceptibility to mycobacterial disease, hyperimmunoglobulin M (IgM), or hyperimmunoglobulin E (IgE). Autoinflammatory panels are similarly grouped and include periodic fever syndromes, IBD, autoimmunity syndromes, autoinflammatory syndromes, autoimmune lymphoproliferative disorders, or familial cold autoinflammatory syndrome.
Genome/exome sequencing — Genome sequencing (coding and noncoding DNA, approximately 3.5 gigabases) or exome sequencing (all of the coding exons, approximately 1.5 percent of the genome or approximately 50 megabases) uses an advanced sequencing technique known as next-generation sequencing (NGS), where millions of DNA fragments are sequenced in parallel, exponentially reducing the cost and time per nucleotide.
Numerous advantages of exome and genome sequencing include the ability to detect clinically significant variants in genes that were not initially considered by the clinician and the ability to effectively evaluate for a genetic etiology for multiple diseases states (eg, immunodeficiency and autism) with a single test. As an additional advantage of exome sequencing, many commercial labs will update or reanalyze previously nondiagnostic exomes as new disease-gene associations emerge. Thus, it is possible for a person with an initially nondiagnostic exome to receive a molecular diagnosis years later without additional testing. Aggregated data from cohorts of families with suspected primary immunodeficiencies suggest incremental diagnostic yield with use of exome sequencing plus copy number variant (CNV) analyses over initial gene panel testing if sequencing is performed in a genetically heterogenous population [55].
Primary disadvantages of exome or genome sequencing include the higher initial cost combined with the increased likelihood of finding variants of uncertain clinical significance (VUS). These VUS findings may not only cause psychologic stress for the family, but they might also prompt inappropriate additional medical interventions and unnecessary surveillance. Clinicians ordering exome or genome sequencing should have proper training in interpreting and discussing these results with the family. Evidence-based frameworks have been developed to guide variant interpretation [56,57]. Clinical laboratories generally agree on the interpretation of variants as pathogenic or benign, but laboratory-to-laboratory variation exists for the intermediate categories (likely pathogenic, unknown clinical significance, and likely benign). Providing the laboratory with detailed phenotypic information can aid the interpretation of the clinical significance of the variants detected. Close collaboration among clinicians, bioinformatics, and genetics professionals improves the diagnostic yield from genetic testing and advances the overall interpretation of variant causality or contribution to the immunophenotype [58]. (See "Genetic counseling: Family history interpretation and risk assessment".)
Exome and genome sequencing also carry increased likelihood for identifying medically actionable variations in genes that are unrelated to the primary indication (also called secondary findings) (see "Secondary findings from genetic testing"). The possibility of secondary findings is usually discussed in pretest consultation of clinical genetic testing, and patients can either accept or decline to receive such results.
Exome and genome sequencing also have reduced sequencing fidelity relative to gene panels (see "Next-generation DNA sequencing (NGS): Principles and clinical applications", section on 'Accuracy'). As a result, conventional sequencing should be performed to validate concerning findings. In addition, exome and genome sequencing are not as reliable for detecting deletions and insertions compared with other methods, such as chromosomal microarrays.
Chromosomal microarrays — Microarray analysis allows for the detection of CNVs, which are submicroscopic genomic differences in the number of copies of one or more sections of DNA due to DNA gains or losses (eg, duplications or deletions). The copy number of these segments varies between different persons. Use of computational CNV prediction pipelines and an exome-tiling chromosomal microarray in addition to whole exome sequencing can improve identification of intragenic CNVs [59]. As an example, small, pathogenic CNVs that would have been missed with conventional microarrays were found in 7 of 110 patients in one study [1]. (See "Genomic disorders: An overview", section on 'Copy number variations'.)
Some of the syndromic immunodeficiencies are caused by microdeletions or microduplications, which require a chromosomal microarray for molecular diagnosis. Examples include those with primarily T cell defects (DiGeorge syndrome [microdeletion of 22q11.2] or DiGeorge-like syndrome [terminal deletion with breakpoints at 10p13-p14] [60,61]), as well as those with primarily B cell defects (Wolf-Hirschhorn syndrome [partial deletion of 4p] [62], 18p- syndrome [deletion of the short arm of chromosome 18] [63], 18q- syndrome [deletion of long arm of chromosome 18] [64]). (See "DiGeorge (22q11.2 deletion) syndrome: Epidemiology and pathogenesis" and "Microdeletion syndromes (chromosomes 1 to 11)", section on '4p deletion syndrome (Wolf-Hirschhorn syndrome)' and "Microdeletion syndromes (chromosomes 12 to 22)", section on '18p deletion syndrome'.)
Additionally, several immunodeficiencies may present with deletions on one or both alleles. Examples include deletions in DOCK8 [65], LPS-responsive beige-like anchor protein (LRBA), or CTLA4. Ordering a chromosomal microarray with adequate coverage of these genes is critical for a comprehensive genetics evaluation. (See "Gastrointestinal manifestations in primary immunodeficiency" and "Combined immunodeficiencies: Specific defects", section on 'DOCK8 deficiency'.)
FISH and karyotyping — Fluorescence in situ hybridization (FISH) and karyotyping are less commonly used now that chromosomal microarrays are the preferred first-line test for suspected chromosomal deletions or duplications. However, karyotyping can detect chromosomal translocations, chromosomal inversions, or ring chromosomes. Since these mechanisms, if present, significantly change recurrence risk counseling, karyotyping is the best initial genetic test for suspected aneuploidies. Two common chromosomal aneuploidies that have immune system dysregulation as part of the phenotype include Down syndrome (trisomy 21) and Turner syndrome (45,X or partial X deletions/rings).
Although FISH used to be commonly ordered for DiGeorge syndrome evaluation, it is no longer the best first-line test, since chromosomal microarrays are more sensitive for detecting the deletion, can define the breakpoints, and can simultaneously evaluate for other microdeletion syndromes that can mimic the clinical presentation of DiGeorge syndrome.
RNA sequencing — Also called transcriptome sequencing, ribonucleic acid (RNA) sequencing allows for detection of many potential disease mechanisms missed by other genetic testing modalities, including interrogation of gene expression, detection of novel transcripts, alternatively spliced transcripts or splice isoforms, RNA editing, fusion transcripts, and analysis of allele-specific expression. This powerful tool is primarily restricted to the research setting but is expected to emerge as a clinical test in the coming years. RNA sequencing must be performed with the affected tissue. Thus, the greatest initial clinical utility for this technology may be for disorders of the immune system since white blood cells (WBCs) are one of the most accessible tissues, obtainable by routine phlebotomy. (See "Tools for genetics and genomics: Gene expression profiling".)
Ordering genetic testing — Hundreds of clinical laboratories offer genetic testing. It remains essential to confirm coverage of the gene(s) of interest, including the percent coverage by sequencing and ability to detect deletions or duplications at that loci, when selecting a genetic testing laboratory. Exome coverage for NGS should be at least 90 to 95 percent with a minimum read depth of at least 20x [66]. Several clinical laboratories now offer >99 percent coverage. In addition, bioinformatics resources are critical to variant interpretation and classification, and the best laboratories will have robust and well-curated variant databases combined with easy-to-read, technically appropriate reports. It is reasonable to inquire whether a board-certified medical geneticist from the laboratory is available to discuss the case if questions regarding the test results arise.
Online resources, informed consent, and insurance reimbursement for genetic testing are reviewed in greater detail separately. (See "Genetic testing", section on 'Practical issues' and "Genetic counseling: Family history interpretation and risk assessment", section on 'Informed consent for genetic testing'.)
Online resources for genetic testing availability — Online resources for genetic testing are listed elsewhere. (See "Genetic testing", section on 'Where to test/resources for testing'.)
Informed consent for testing — Informed consent involves far more than merely obtaining a signature on requisition paperwork. It is the cornerstone of patient-centered medicine, and the process ensures that the patient fully understands and agrees to the genetic testing, including the risks, benefits, and limitations of the testing. The importance of informed consent and pretest counseling is summarized elsewhere. (See "Genetic counseling: Family history interpretation and risk assessment", section on 'Informed consent for genetic testing' and "Informed procedural consent".)
One unique aspect of informed consent occurs when ordering a large panel or exome sequencing and includes a discussion of the potential for incidental (secondary) findings that are unrelated to the testing indication but that could have significant medical, psychosocial, or familial consequences (see "Secondary findings from genetic testing"). The discovery of the misattribution of parentage from the ordering of genetic testing is also something that should be discussed during the informed consent process.
Insurance reimbursement
Trends in coverage — In the United States, the trend toward insurance coverage and reimbursement for genetic testing is increasing as the recognition of the role and benefits of genetic testing in routine medical care is growing. Although the cost of DNA sequencing itself has plummeted over the last several years, the costs for interpretation of these complex results have remained expensive. These issues are discussed in greater detail separately. (See "Genetic testing", section on 'Cost and insurance reimbursement'.)
Predetermination letters — Many health care payers require predetermination or prior authorization before some genetic testing is ordered. This practice varies greatly from company to company and from test to test. When required to write a predetermination letter, it is best to specifically state the medical indication for genetic testing and the manner in which the results are expected to change clinical management.
INTERPRETATION OF GENETIC TESTING RESULTS
Potential outcomes of testing — A five-tier classification system [56] is widely used by clinical genetic testing laboratories and recommends reporting predicted variant effects with the modifiers "pathogenic," "likely pathogenic," "uncertain significance," "likely benign," and "benign," based upon criteria using variant evidence (eg, population data, functional data, computational data, segregation data). While single-gene testing is often relatively straightforward, the clinical interpretation of large panels or exome sequencing is complicated by the detection of numerous variants of uncertain clinical significance (VUS), a term describing insufficient or conflicting variant evidence at the time of the report.
A VUS should not be used in clinical decision making. However, efforts to resolve the classification can be undertaken, including the addition of segregation data or the use of additional bioinformatic evidence. While efforts to reclassify the variant are underway, additional monitoring of the patient for the suspected disorder is often pragmatic. When a VUS is reported in a gene related to the primary indication, it is recommended that laboratories suggest periodic inquiry by health care providers to determine if variant classification has changed with new knowledge. The availability of large variant frequency databases has led many VUS to be reclassified as benign. As the use of sequencing tests expands and the identity of variants is increasingly known, the ability for laboratories to make clinically significant classifications is expected to increase. Additional possible outcomes of testing, including classification of variants and secondary findings, are discussed in detail separately. (See "Secondary findings from genetic testing", section on 'Definitions and classification of variants' and "Secondary findings from genetic testing", section on 'Likelihood of detecting a secondary finding' and "Genetic testing", section on 'Acting on the results of testing'.)
Penetrance and expressivity — The ability of a genetic test to predict a disease phenotype (clinical validity) is highly dependent upon the penetrance and expressivity of the variant in question. The impact of penetrance and expressivity on results interpretation is discussed in detail separately. (See "Inheritance patterns of monogenic disorders (Mendelian and non-Mendelian)", section on 'Penetrance and expressivity'.)
Use of variant databases — Interpretation of gene variants hinges upon an understanding of the disease mechanism but also requires bioinformatics expertise to incorporate the allele frequency and population data held in variant databases. Several databases are available to help catalog variants and associated pathogenicity. They are reviewed in detail separately. (See "Next-generation DNA sequencing (NGS): Principles and clinical applications", section on 'Variant databases'.)
Discussing negative results — One common misconception among patients and providers is that a negative genetic test indicates that the patient's disease does not have a genetic cause. It is critical to counsel a family that a negative genetic testing result does not exclude genetic disease, since a variant can be misclassified as benign, missed because it is in a noncoding region, or present in a gene not yet associated with a disease. When discussing negative genetic testing results with a family, it is important to acknowledge that the phenotype may still be caused by an underlying genetic variant and may still have a chance to recur in future pregnancies. Interpretation of negative results is discussed elsewhere. (See "Secondary findings from genetic testing", section on 'Interpretation of 'negative' results'.)
ETHICAL, LEGAL, AND PSYCHOSOCIAL ISSUES — Genetic testing is exceptional compared with other forms of medical or laboratory testing, as the results can not only impact the proband but may also have implications for family members. Often, these family members may not have even been aware the initial testing was ordered. Disclosure of test results to family members, special issues related to testing in children, and addressing misattribution of parentage are discussed in detail separately. (See "Genetic testing", section on 'Ethical, legal, and psychosocial issues' and "Secondary findings from genetic testing", section on 'Children'.)
SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Inborn errors of immunity (previously called primary immunodeficiencies)".)
SUMMARY AND RECOMMENDATIONS
●Benefits of identifying the genetic etiology – Achieving a molecular diagnosis provides the most comprehensive understanding of inborn errors of immunity, allowing for tailored interventions and personalized surveillance strategies. Numerous therapeutic approaches to both immunodeficiency and autoinflammatory disease are dependent upon understanding the molecular etiology. (See 'Introduction' above and 'Benefits of determining the underlying genetic etiology' above.)
●Whom to test – Genetic testing has the highest diagnostic yield in situations where the clinical phenotype is probably caused by one or more known genetic conditions. Testing should be performed in the proband (affected patient) first before pursuing testing in family members. (See 'Choosing whom to test' above.)
●Which test to perform – Many types of genetic testing exist, including single-gene testing, panel testing, exome sequencing, and chromosomal microarrays. Use of exome sequencing plus copy number variant (CNV) analyses may have better diagnostic yield than initial gene panel testing if sequencing is performed in a genetically heterogenous population, whereas exome and genome sequencing is more likely to obtain an optimal diagnostic yield for patients in whom parental consanguinity is known or highly suspected. Ordering a chromosomal microarray remains critical when evaluating for deletion/duplication syndromes (eg, DiGeorge syndrome, Wolf-Hirschhorn syndrome) or when searching for diseases that can be caused by allelic deletions (eg, dedicator of cytokinesis 8 [DOCK8], LPS responsive beige-like anchor protein [LRBA], or cytotoxic T-lymphocyte associated protein 4 [CTLA4] deficiency). (See 'Choosing which genetic test is best' above.)
●Ordering genetic testing – Hundreds of clinical laboratories offer genetic testing. It is important to confirm coverage of the gene(s) of interest, including the percent coverage by sequencing and ability to detect deletions or duplications at that loci, and availability of bioinformatics resources when selecting a genetic testing laboratory. Obtaining informed consent prior to testing is important. (See 'Ordering genetic testing' above.)
●Interpretation of test results – While single-gene testing is often relatively straightforward, the clinical interpretation of large panels or exome sequencing is complicated by the detection of numerous variants of uncertain clinical significance (VUS). Ambiguity arises from uncertainty about whether the variant impacts the function of the gene product and whether and how the gene contributes to human disease. In addition, these variants may reside in genes that are either not related to the patient's phenotype or are not yet implicated in human disease. The uncertainties surrounding VUS makes it difficult to interpret the clinical impact of these variants and complicates the discussion of results. Other challenges include interpretation and discussion of "negative" test results. (See 'Interpretation of genetic testing results' above.)
ACKNOWLEDGMENT — The UpToDate editorial staff acknowledges Bret Bostwick, MD, who contributed to earlier versions of this topic review.
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