CAM 204102

Whole Exome and Whole Genome Sequencing for Diagnosis of Patients With Suspected Genetic Disorders

Category:Laboratory   Last Reviewed:July 2018
Department(s):Medical Affairs   Next Review:July 2019
Original Date:October 2013    

Description 
Whole exome sequencing (WES) sequences the portion of the genome that contains protein-coding DNA, while whole genome sequencing (WGS) sequences both coding and noncoding regions of the genome. WES and WGS have been proposed for use in patients presenting with disorders and anomalies that have not been explained by standard clinical workup. Potential candidates for WES and WGS include patients who present with a broad spectrum of suspected genetic conditions.

For individuals who have multiple unexplained congenital anomalies or a neurodevelopmental disorder who receive WES, the evidence includes large case series and within-subject comparisons. Relevant outcomes are test accuracy and validity, functional outcomes, changes in reproductive decision making, and resource utilization. Patients who have multiple congenital anomalies or a developmental disorder with a suspected genetic etiology, but whose specific genetic alteration is unclear or unidentified by standard clinical workup, may be left without a clinical diagnosis of their disorder, despite a lengthy diagnostic workup. For a substantial proportion of these patients, WES may return a likely pathogenic variant. Several large and smaller series have reported diagnostic yields of WES ranging from 25% to 60%, depending on the individual’s age, phenotype, and previous workup. One comparative study found a 44% increase in yield compared with standard testing strategies. Many of the studies have also reported changes in patient management, including medication changes, discontinuation of or additional testing, ending the diagnostic odyssey, and family planning. The evidence is sufficient to determine that the technology results in a meaningful improvement in the net health outcome.

For individuals who have a suspected genetic disorder other than multiple congenital anomalies or a neurodevelopmental disorder who receive WES, the evidence includes small case series and prospective research studies. Relevant outcomes are test accuracy and validity, functional outcomes, changes in reproductive decision-making, and resource utilization. There are increasing reports of use of WES to identify a molecular basis for disorders other than multiple congenital anomalies or neurodevelopmental disorders. The diagnostic yields in these studies range from as low as 3% to 60%. One concern with WES is the possibility of incidental findings. Some studies have reported on the use of a virtual gene panel with restricted analysis of disease-associated genes, and WES data allows reanalysis as new genes are linked to the patient phenotype. Overall, there are a limited number of patients who have been studied for any specific disorder, and clinical use of WES for these disorders is at an early stage. The evidence is insufficient to determine the effects of the technology on health outcomes.

For individuals with a suspected genetic disorder who receive WGS, the evidence includes case series. Relevant outcomes are test accuracy and validity, functional outcomes, changes in reproductive decision making, and resource utilization. WGS has increased coverage and diagnostic yield compared with WES, but the technology is limited by the amount of data generated and greater need for storage and analytic capability. Several authors have proposed that as WGS becomes feasible on a larger scale, it may in the future become the standard first-tier diagnostic test. At present, there is limited data on the clinical use of WGS. The evidence is insufficient to determine the effects of the technology on health outcomes.   

Background 
WHOLE EXOME SEQUENCING AND WHOLE GENOME SEQUENCING
Whole exome sequencing (WES) is targeted next-generation sequencing of the subset of the human genome that contains functionally important sequences of protein-coding DNA, while whole genome sequencing (WGS) uses next-generation sequencing techniques to sequence both coding and noncoding regions of the genome. WES and WGS have been proposed for use in patients presenting with disorders and anomalies not explained by standard clinical workup. Potential candidates for WES and WGS include patients who present with a broad spectrum of suspected genetic conditions.

Given the variety of disorders and management approaches, there are a variety of potential health outcomes from a definitive diagnosis. In general, the outcomes of a molecular genetic diagnosis include (1) impacting the search for a diagnosis, (2) informing follow-up that can benefit a child by reducing morbidity, and (3) affecting reproductive planning for parents and potentially the affected patient.

The standard diagnostic workup for patients with suspected Mendelian disorders may include combinations of radiographic, electrophysiologic, biochemical, biopsy, and targeted genetic evaluations.1 The search for a diagnosis may thus become a time-consuming and expensive process.  

WES and WGS Technology
WES or WGS using next-generation sequencing technology can facilitate obtaining a genetic diagnosis in patients efficiently. WES is limited to most of the protein-coding sequence of an individual (≈85%), is composed of about 20,000 genes and 180,000 exons (protein-coding segments of a gene), and constitutes approximately 1% of the genome. It is believed that the exome contains about 85% of heritable disease-causing mutations. WES has the advantage of speed and efficiency relative to Sanger sequencing of multiple genes. WES shares some limitations with Sanger sequencing. For example, it will not identify the following: intronic sequences or gene regulatory regions; chromosomal changes; large deletions; duplications; or rearrangements within genes, nucleotide repeats, or epigenetic changes. WGS uses techniques similar to WES, but includes noncoding regions. WGS has greater ability to detect large deletions or duplications in protein-coding regions compared with WES, but requires greater data analytics. Technical aspects of WES and WGS are evolving, including the development of databases such as the National Institutes of Health’s ClinVar database (http://www.ncbi.nlm.nih.gov/clinvar/) to catalog variants, uneven sequencing coverage, gaps in exon capture before sequencing, and difficulties with narrowing the large initial number of variants to manageable numbers without losing likely candidate mutations. The variability contributed by the different platforms and procedures used by different clinical laboratories offering exome sequencing as a clinical service is unknown.

In 2013, the American College of Medical Genetics and Genomics, Association for Molecular Pathology, and College of American Pathologists convened a workgroup to develop standard terminology for describing sequence variants. Guidelines developed by this workgroup, published in 2015, describe criteria for classifying pathogenic and benign sequence variants based on 5 categories of data: pathogenic, likely pathogenic, uncertain significance, likely benign, and benign.2 

WES and WGS Testing Services
Several laboratories offer WES and WGS as a clinical service. Illumina offers 3 TruGenome tests: the TruGenome Undiagnosed Disease Test (indicated to find the underlying genetic cause of an undiagnosed rare genetic disease of single-gene etiology), the TruGenome™Predisposition Screen (indicated for healthy patients interested in learning about their carrier status and genetic predisposition toward adult-onset conditions), and the TruGenome™Technical Sequence Data (WGS for labs and physicians who will make their own clinical interpretations). Ambry Genetics offers 2 WGS tests, the ExomeNext and ExomeNext-Rapid, which sequence both the nuclear and the mitochondrial genomes. GeneDx offers WES with its XomeDx™ test. Medical centers may also offer WES and WGS as a clinical service.

Examples of laboratories offering WES as a clinical service and their indications for testing are summarized in Table 1.

Table 1: Examples of Laboratories Offering Whole Exome Sequencing as a Clinical Service

Laboratory Laboratory Indications for Testing
Ambry Genetics, (Aliso Viejo, CA)    

 "The patient's clinical presentation is unclear/atypical disease and there are multiple genetic conditions in the defferential diagnosis."

GeneDx, (Gaithersburg, MD) "a patient with a diagnosis that suggests the involvement of one or more of many different genes, which would, if even available and sequenced individually, be prohibitively expensive'
Baylor Collge of Medicine, (Houston TX)  "used when a patient's medical history and physical exam findings strongly suggest that there is an underlying genetic etiology. In some cases, the patient may have had an extensive evaluation consisting of multiple genetic test, without identifying an etiology."
University of California Los Angeles Health System "This test is intended for use in conjunction with the clinical presentation and other markers of disease progession for the management of patients with rare genetic disorders."
EdgeBio, (Gaithersburg, MD) Recommended "In situations where there has been a diagnostic failure with no discernible path. In situations where there are currently no available tests to determine the status of a potential genetic disease. In situations with atypical findings indicative of multiple disease(s)."
Children's Mercy Hospitals and Clinics, (Kansas City, MO) Provided as a service to families with children who have had an extensive negative work-up for a genetic disease; also used to identify novel disease genes.
Emory Genetics Laboratory, (Atlanta, GA) "Indicated when there is a suspicion of a genetic etiology contributing to the proband's manifestations."

Note that this evidence review does not address the use of WES and WGS for preimplantation genetic diagnosis or screening, prenatal (fetal) testing, or for testing of cancer cells.

Regulatory Status  
Clinical laboratories may develop and validate tests in-house and market them as a laboratory service; laboratory-developed tests must meet the general regulatory standards of the Clinical Laboratory Improvement Amendments. Whole exome or genome sequencing tests as a clinical service are available under the auspices of the Clinical Laboratory Improvement Amendments. Laboratories that offer laboratory-developed tests must be licensed by the Clinical Laboratory Improvement Amendments for high-complexity testing. To date, the U.S. Food and Drug Administration has chosen not to require any regulatory review of this test.  

Policy 

  1. Whole exome sequencing is considered MEDICALLY NECESSARY  for the evaluation unexplained congenital or neurodevelopmental disorder in children when all the following criteria are met:
    • The patient has been evaluated by a board-certified clinician with expertise in clinical genetics and counseled about the potential risks of genetic testing
    • WES results will directly impact patient management and clinical outcome for the individual being tested
    • A genetic etiology is the most likely explanation for the phenotype
    • No other causative circumstances (e.g. environmental exposures, injury, infection) can explain the symptoms
    • Clinical presentation does not fit a well-described syndrome for which single-gene or targeted panel testing (e.g., comparative genomic hybridization/chromosomal microarray analysis) is available
    • The differential diagnosis list and/or phenotype warrant testing of multiple genes and ONE of the following:
      •  WES is more practical than the separate single gene tests or panels that would be recommended based on the differential diagnosis
      • WES results may preclude the need for multiple and/or invasive procedures, follow-up, or screening that would be recommended in the absence of testing
  2. If whole exome sequencing has been previously performed, further genetic tests involving only exome analyses is INVESTIGATIONAL
  3. Whole exome sequencing is INVESTIGATIONAL for all other indications, including but not limited to, tumor sequencing.
  4. Whole genome sequencing is considered INVESTIGATIONAL for all indications.

Policy Guidelines
Beginning in 2015, there will be specific codes for this testing:

81415 Exome (e.g., unexplained constitutional or heritable disorder or syndrome); sequence analysis

81416 sequence analysis, each comparator exome (e.g., parents, siblings) (List separately in addition to code for primary procedure)

81417 re-evaluation of previously obtained exome sequence (e.g., updated knowledge or unrelated condition/syndrome)

81425 Genome (e.g., unexplained constitutional or heritable disorder or syndrome); sequence analysis

81426 sequence analysis, each comparator genome (e.g., parents, siblings) (List separately in addition to code for primary procedure)

81427 re-evaluation of previously obtained genome sequence (e.g., updated knowledge or unrelated condition/syndrome)

Prior to 2015, there are no specific CPT codes for whole exome sequencing. It would likely be reported with the unlisted molecular pathology code 81479.

Benefit Application
Blue Card®/National Account Issues
No applicable information.

Rationale
Validation of the clinical use of any genetic test focuses on 3 main principles: (1) analytic validity, which refers to the technical accuracy of the test in detecting a variant that is present or in excluding a variant that is absent; (2) clinical validity, which refers to the diagnostic performance of the test (sensitivity, specificity, positive and negative predictive values) in detecting clinical disease; and (3) clinical utility (i.e., how the results of the diagnostic test will be used to change management of the patient and whether these changes in management lead to clinically important improvements in health outcomes). 

WHOLE EXOME SEQUENCING IN PATIENTS WITH MULTIPLE CONGENITAL ANOMALIES OR A NEURODEVELOPMENTAL DISORDER
Clinical Context and Test Purpose
The purpose of whole exome sequencing (WES) in patients who have multiple unexplained congenital anomalies or a neurodevelopmental disorder is to establish a molecular diagnosis. The criteria under which diagnostic testing for a genetic or heritable disorder may be considered clinically useful are as follows:

  • A definitive diagnosis cannot be made based on history, physical examination, pedigree analysis, and/or standard diagnostic studies or tests;
  • The clinical utility of a diagnosis has been established (e.g., by demonstrating that a definitive diagnosis will lead to changes in clinical management of the condition, changes in surveillance, or changes in reproductive decision making, and these changes will lead to improved health outcomes); and
  • Establishing the diagnosis by genetic testing will end the clinical workup for other disorders.

The question addressed in this evidence review is: Does WES improve health outcomes when used for the diagnosis of patients with multiple unexplained congenital anomalies or a neurodevelopmental disorder?

The following PICOTS were used to select literature to inform this review.

Patients
The relevant population of interest is patients presenting with multiple unexplained congenital anomalies or a neurodevelopmental disorder that is suspected to have a genetic basis but are not explained by standard clinical workup.

Intervention
The relevant intervention of interest is WES.

Comparators
The relevant comparator of interest is standard clinical workup without WES.

Outcomes
The general outcomes of interest are the accuracy of next-generation sequencing (NGS) compared with Sanger sequencing, the sensitivity and specificity and positive and negative predictive value for the clinical condition, and improvement in health outcomes. Health outcomes include a reduction in morbidity due to appropriate treatment and surveillance, the end of the diagnostic odyssey, and effects on reproductive planning for parents and potentially the affected patient.

False-positive test results can lead to misdiagnosis and inappropriate clinical management. False-negative test results can lead to a lack of a genetic diagnosis and continuation of the diagnostic odyssey. 

Timing
These tests are performed when standard clinical workup has failed to arrive at a diagnosis.

Setting
These tests are offered commercially through various manufacturers.

Analytic Validity
There are relatively few data specific to the analytic validity of WES. NGS techniques used for WES are expected to have high accuracy for mutation detection. However, NGS platforms differ regarding the depth of sequence coverage, methods for base calling and read alignment, and other factors. These factors contribute to potential variability across the platforms and procedures used by different clinical laboratories offering exome sequencing as a clinical service. The American College of Medical Genetics and Genomics has clinical laboratory standards for NGS, including WES.4 The guidelines outline the documentation of test performance measures that should be evaluated for NGS platforms, and note that typical definitions of analytic sensitivity and specificity do not apply for NGS.

Depending on the platform and variant call method used, WES may not accurately detect large insertions and deletions, large copy number variants, and structural chromosome rearrangements due to the short sequence read lengths.4 WES may be less sensitive for the detection of copy number variants than high-resolution microarray testing.5 NGS also has poorer coverage for A/T-rich, G/C-rich, and pseudogene regions, as well as homopolymer stretches.6,7

Clinical Validity
A number of studies have reported on the use of WES in clinical practice (see Table 2). Typically, the populations included in these studies have suspected rare genetic disorders, although the specific populations vary.

Series have been reported with as many as 2,000 patients. The largest reason for referral to a tertiary care center was an unexplained neurodevelopmental disorder. Many patients had been through standard clinical workup and testing without identification of a genetic variant to explain their condition. Diagnostic yield in these studies, defined as the proportion of tested patients with clinically relevant genomic abnormalities, ranged from 25% to as many as 48%. Because there is no reference standard for the diagnosis of patients who have exhausted alternative testing strategies, clinical confirmation may be the only method for determining false-positive and false-negative rates. No reports were identified of incorrect diagnoses, and how often they might occur is unclear.

When used as a first-line test in infants with multiple congenital abnormalities and dysmorphic features, diagnostic yield may be as high as 58%. Testing parent-child trios has been reported to increase diagnostic yield, to identify an inherited variant from an unaffected parent and be considered benign, or to identify a de novo variant not present in an unaffected parent. First-line trio testing for children with complex neurologic disorders was shown to increase the diagnostic yield (29%, plus a possible diagnostic finding in 27%) compared with a standard clinical pathway (7%) performed in parallel in the same patients.8

Clinical Utility
Cohort studies following children from presentation to outcomes have not been reported. There are considerable challenges conducting studies of sufficient size given the underlying genetic heterogeneity, and including follow-up adequate to observe final health outcomes. Studies addressing clinical utility have reported mainly diagnostic yield and management changes. Thus, it is difficult to quantify lower or upper bounds for any potential improvement in the net health outcome owing in part to the heterogeneity of disorders, rarity, and outcome importance that may differ according to identified pathogenic variants. Actionable items following testing in the reviewed studies (see Table 2) included family planning, change in management, change or avoidance of additional testing, surveillance for associated morbidities, prognosis, and ending the diagnostic odyssey.

The evidence reviewed here reflects the accompanying uncertainty, but supports a perspective that identifying a pathogenic variant can (1) impact the search for a diagnosis, (2) inform follow-up that can benefit a child by reducing morbidity and rarely potential mortality, and (3) affect reproductive planning for parents and later potentially the affected child. When recurrence risk can be estimated for an identified variant (eg, by including parent testing), future reproductive decisions can be affected. Early use of WES can reduce the time to diagnosis and reduce the financial and psychological burdens associated with prolonged investigation. 

Table 2. Diagnostic Yields of WES for Congenital Anomalies or a Neurodevelopmental Disorder

(Year)

Patient Population

                  N                     

Design

Yield, n (%)

Additional Information

Yang et al. (2013)9 

Suspected genetic disorder (80% neurologic)  

250 (1% fetus; 50% <5 y;38% 5-18 y; 11% adults) 

Consecutive patients at single center

62 (25) 

Identification of atypical phenotypes of known genetic diseases and blended phenotypes  

Yang et al. (2014)10 

Suspected genetic disorder (88% neurologic or  developmental)

2,000 (45% <5 y; 42% 5-18 y; 12% adults)

Consecutive patients at single center

504 (25)

Identification of novel variants. End of the diagnostic odyssey and change in management

Lee et al. (2014)11

Suspected rare Mendelian disorders (57% of children had developmental delay; 26% of adults had ataxia)

814 (49% <5 y; 15% 5-18 y; 36% adults)

Consecutive patients at single center

213 (26)

Trio (31% yield) vs proband only (22% yield)

Iglesias et al. (2014)12

Birth defects (24%); developmental delay (25%); seizures (32%)

115 (79% children)

Single-center tertiary clinic

37 (32)

Discontinuation of planned testing, changed medical management, and family planning

Soden et al. (2014)13

Children with unexplained neurodevelopmental disorders

119 (100 families)

 

Single-center databasea

53 (45)

 

Change in clinical care or impression in 49% of families

Srivastava et al. (2014)14

Children with unexplained neurodevelopmental disorders

78

Pediatric neurogenetics clinic

32 (41)

Changed medical management, prognostication, and family planning

Farwell et al. (2015)1

Unexplained neurologic disorders (65% pediatric)

500

WES laboratory

152 (30)

Trio (37.5% yield) vs proband only (20.6% yield); 31 (7.5% de novo)

Nolan and Carlson (2016)16

Children with unexplained neurodevelopmental disorders

50

Pediatric neurology clinic

41 (48)

Changed medication, systemic investigation, and family planning

Allen et al. (2016)17

Patients with unexplained early-onset epileptic encephalopathy

50 (95% <1 y)

Single center

11 (22)

2 VUS for follow-up, 11 variants identified as de novo

Stark et al. (2016)18

Infants (≤2 y) with suspected monogenic disorders with multiple congenital abnormalities and dysmorphic features

80

Prospective comparative study at a tertiary center

46 (58)

First-line WES increased yield by 44%, changed clinical management and family planning

Vissers et al. (2017)8

Children with complex neurologic disorders of suspected genetic origin

150

Prospective comparative study at a tertiary center

  • 44 (29) conclusive
  • 41 (27) possible

First-line WES had 29% yield vs 7% yield for standard diagnostic workupb

VUS: variants of uncertain significance; WES: whole exome sequencing.
a Included both WES and whole genome sequencing.
b Standard diagnostic workup included an average of 23.3 physician-patient contacts, imaging studies, muscle biopsies or lumbar punctures, other laboratory tests, and an average of 5.4 sequential gene by gene tests.

Section Summary: Whole Exome Sequencing in Patients With Multiple Congenital Anomalies or a Neurodevelopmental Disorder
The evidence on WES in patients who have multiple congenital anomalies or a developmental disorder with a suspected genetic etiology includes case series. These series have reported diagnostic yields of WES ranging from 22% to 58%, depending on the individual’s age, phenotype, and previous workup. Comparative studies have reported an increase in diagnostic yield compared with standard testing strategies. Thus, for individuals who have a suspected genetic etiology but for whom the specific genetic alteration is unclear or unidentified by standard clinical workup, WES may return a likely pathogenic variant. A genetic diagnosis for these patients is reported to change management, including medication changes, discontinuation of or additional testing, ending the diagnostic odyssey, and family planning.

WES IN PATIENTS WITH A SUSPECTED GENETIC DISORDER OTHER THAN MULTIPLE CONGENITAL ANOMALIES OR A NEURODEVELOPMENTAL DISORDER

Clinical Context and Test Purpose
Most of the literature on WES is on neurodevelopmental disorders in children; however, other potential indications for WES have been reported (see Table 3). These include limb-girdle muscular dystrophy, inherited retinal disease, and other disorders including mitochondrial, endocrine, and immunologic disorders. The yield for unexplained limb-girdle muscular dystrophy and retinal disease is high, but a limited number of patients have been studied to date. 

The purpose of WES in patients who have a suspected genetic disorder other than multiple unexplained congenital anomalies or a neurodevelopmental disorder is to establish a molecular diagnosis. The criteria under which diagnostic testing for a genetic or heritable disorder may be considered clinically useful are as above.

The question addressed in this evidence review is: Does WES improve health outcomes when used for the diagnosis of a suspected genetic condition?

The following PICOTS were used to select literature to inform this review.

Patients
The relevant population of interest is patients presenting with a disorder other than multiple unexplained congenital anomalies or a neurodevelopmental disorder that is suspected to have a genetic basis but is not explained by standard clinical workup.

Intervention
The relevant intervention of interest is WES.

Comparators
The relevant comparator of interest is standard clinical workup without WES.

Outcomes
The general outcomes of interest are the accuracy of NGS compared with Sanger sequencing, the sensitivity and specificity and positive and negative predictive value for the clinical condition, and clinical health outcomes. Health outcomes include a reduction in morbidity due to appropriate treatment and surveillance, the end of the diagnostic odyssey, and effects on reproductive planning for parents and potentially the affected patient.

Timing
The test is performed when standard clinical workup has failed to arrive at a diagnosis.

Setting
These tests are offered commercially through various manufacturers.

Analytic Validity
As described above for use of WES in patients with multiple congenital anomalies or a neurodevelopmental disorder.

Clinical Validity
Studies have assessed WES for a broad spectrum of disorders. The diagnostic yield in patient populations restricted to specific phenotypes ranges from 3% for colorectal cancer to 60% for unexplained limb-girdle muscular dystrophy. Some studies used a virtual gene panel that is restricted to genes that are associated with the phenotype, while others have examined the whole exome, either initially or sequentially. An advantage of WES over individual gene or gene panel testing is that the stored data allows reanalysis as new genes are linked to the patient phenotype. WES has also been reported to be beneficial in patients with atypical presentations.

Table 3. Diagnostic Yields of WES for Conditions Other Than Multiple Congenital Anomalies or a Neurodevelopmental Disorder

Study (Year)

Patient Population

N

Design

Yield, n (%)

Additional Actions

Neveling et al. (2013)19  

Unexplained disorders: blindness, deafness, movement disorders, mitochondrial disorders, hereditary cancer 

 186

Outpatient genetic clinic; post hoc comparison with Sanger sequencing  

3%-52% 

WES increased yield vs Sanger sequencing. Highest yield for blindness and deafness