Whole Exome Sequencing

review is: Does the use of WES improve health outcomes when used for the diagnosis of children with multiple unexplained congenital anomalies or a neurodevelopmental disorder of unknown etiology following standard workup?


Summary of comments and response
With few exceptions, the comments provided did not suggest any substantive changes to the key questions or scope of the review. The comments are summarized in Table 2. For the purpose of organizing the framework of review, we have considered incidental findings under safety, as it is not directly related to the reason for ordering WES. We acknowledge that incidental findings can provide benefit as well as harms, and will discuss this balance in the discussion of the review.
Comment 5: Suggest clarifications in Key Questions 3a, 3b, and 3d We do not want to over specify and risk leaving out relevant evidence.
Comment 6: Suggest addressing costs associated with false negatives in non-WES/non-WGS pathways All costs associated with all pathways will be considered if the data is presented.
Comment 7: For discussing VUS, suggest use of the term "uncertain" instead of "unknown".
We have made this change.
Comment 8: Suggest adding over diagnosis and discrimination to harms We have added employment or insurance discrimination to the list of harms. We will consider overdiagnosis in the analysis of the contextual question on re-analysis of WES WES may be helpful in these scenarios. however, these reasons are not included as indications of a genetic disorder in the citations we identified.

Amy Yuen, MD, PhD
List of relevant articles Thank you for this information.
Our search identified all the suggested articles; we will evaluate them against the final study selection criteria for inclusion.

Jessie Conta, MS, LCGC Laboratory Genetic Counselor, Supervisor
Change "How many patients receive reports on ACMGdefined medically actionable incidental findings after WES testing?" to "For patients who opt-in to receive ACMGdefined medically actionable incidental findings, how many have such findings identified by WES testing?" Thank you for your comments. We did not make this change because our preliminary evidence scan suggests that studies that report this outcome generally do not specify whether the patients opted to receive them, and we do not want to exclude relevant evidence.
Clarify in background that patients can opt-in/opt-out of receiving ACMG-defined medically actionable incidental findings.
We added a sentence to this affect at the end of paragraph 3 in the background.
Add as key question: What is the impact (positive or negative) of pre-test counseling and consent by a genetics expert prior to WES testing?
This question is outside of the scope of this review.
Consider including whole genome sequencing.
See response to first comment above.
 Improvement in prognostic certainty  Family/reproductive planning  Initiation of palliative and/or hospice care In addition, we would note that clinical utility is not limited to patients with positive diagnostic results. A negative test result may also result in clinical utility, suggesting the lack of a known genetic basis for the disease.

Comment 3: Suggest that Key Question 2 (Health Outcomes) should define health outcomes of interest
For a number of reasons, including the heterogeneity of the patient population with "suspected genetic diseases," there are not as well-defined measures of "health outcomes" as in many other disease states.
Health outcomes are often measured in life years or quality-adjusted life years (QALYs); however, numerous publications have highlighted how challenging these are to measure in rare genetic diseases. Other health outcomes of interest may include: length of stay; hospitalizations; and various survey tools to assess individual benefits and burden of healthcare utilization.

Comment 4: In Key Question 3 and the analytical framework, suggest that incidental findings should not be considered solely as a harm.
While secondary or incidental findings are unrelated to the primary reason for testing, the American College of Medical Genetics and Genomics (ACMG) recommends that all laboratories conducting clinical sequencing offer to report pathogenic and likely pathogenic variants for a short list of carefully chosen genes and conditions. Patients (and parents) should receive pre-test counseling and be given the opportunity to "opt out" if they do not wish to the lab to carry out the analysis. There is no additional charge associated with these analyses. The ACMG recommendations set a standard for laboratory practices, by limiting to incidental findings that meet a high threshold of clinical utility ("unequivocally pathogenic mutations in genes where pathogenic variants lead to disease with very high probability and where evidence strongly supports the benefits of early intervention"). Certainly, there is potential for harm based on unexpected and unwanted results. However, we would still suggest carefully framing questions around incidental findings to weigh the benefits and harms.
In addition, some laboratories do report true incidental findings. These are defined by the following:  Pathogenic or likely pathogenic variants that occur within a possibly diagnostic pattern of inheritance (e.g. de novo, homozygous, compound heterozygous, hemizygous)  Patient's reported phenotype made available to the lab does not include features of the inferred genetic disorder  Results are expected to be actionable before age 18, such as change in management or surveillance  Specifically excludes late onset conditions for which there is no established effective therapy An example of an incidental finding might be the detection of a known pathogenic variant in G6PD, enabling avoidance of triggering agents in the future. In contrast to the ACMG recommended gene list for secondary findings, true incidental findings are encountered by the laboratory in their normal analysis workflows. Reporting of such variants gives the providers the opportunity to evaluate whether the variants are clinically impactful and is likely to be largely beneficial to the patients. Pre-test counseling can alert patients and families that incidental findings are occasionally observed.

Comment 5: Suggest clarifications in Key Questions 3a, 3b, and 3d
In Key Question 3a, we suggest that harms caused by erroneous results should distinguish between the laboratory results and the final diagnostic interpretation by the doctor.
In Key Question 3b, we suggest clarifying the question, as we believe it could have multiple interpretations. Currently the question focuses two issues: uncertain results and negative results. Uncertain results could include the finding of a variant of uncertain significance within an appropriate inheritance pattern (e.g. de novo, homozygous, compound heterozygous, hemizygygous). Uncertain results could also include findings where there is not a clear clinical correlation in the patient either due to age (not yet manifesting all symptoms) or where additional biomarker correlations are advised (e.g. in a neurometabolic disorder). The potential harms in these results could be the need for additional testing or surveillance and the psychologic distress of the uncertainty. The benefits, however, may outweigh these harms if the molecular diagnosis is confirmed as they will then enable all the needed change in management associated with the diagnosis.
Negative results, in contrast, are not inherently harmful since they do only reduce the pre-test probability of specific diagnoses and do not "rule out" strongly suspected clinical diagnoses. Exome and genome testing largely have the equal or superior analytical sensitivity compared to standard genetic tests, so they are not expected to leave many patients with false negative diagnoses compared to standard tests. The current exception is the detection of low-level mosaicism for small variants which generally requires high depth NGS. Low level mosaicism for small variants is not detected by Sanger sequencing tests and some other clinical platforms. It usually requires strong clinical suspicion so that targeted testing can be used. Low-level mosaicism for CNVs is detected by some array platforms and WGS but not WES.
In Key Question 3d, we suggest that "harm to family relationships" could be explained further by adding additional specificity. First, there may be some benefits within a family that can be recognized. These include: improvements in empowerment; control; effective resource planning; improved family decision quality; improved family functioning, communication, and medical planning; individual health behavior changes (e.g., diet); improved overall satisfaction with care; and improved coping with burdens of disease.
Some of the potential harms could include: increased perceived uncertainty, risk, social vulnerability, and stigma; perceived or experienced insurance/employment discrimination; and increased anxiety or worry, depression, stress/distress. There is a specific potential for harm in the unwanted disclosure of misattributed parentage, and providers should identify this risk in pre-test counseling. The American Society of Human Genetics in its 2015 Points to Consider recommends avoiding disclosure of misattributed parentage, most easily at the stage of lab reporting, while recognizing dissenting views in the literature [Botkin et al PMID: 26140447].

Comment 6: Suggest addressing costs associated with false negatives in non-WES/non-WGS pathways
In Key Question 4c, we suggest that there is also a need to account for the costs of false negatives in non-WES pathways (i.e., the cases in which a genetic diagnosis is achievable but fails due to lack of use of WES or WGS).

Comment 7: Suggest clarifications in Contextual Question 2.
For discussing VUS, suggest use of the term "uncertain" instead of "unknown" (Richards et al 2015 PMID: 25741868).
In addition, there are at least 2 categories for test outcome when VUS are reported that should be considered:  Likely Positive (i.e., has strong phenotype overlap and may be confirmed by further investigation (measuring an informative surrogate);  Inconclusive (i.e., no further analysis can adjudicate whether the VUS is causal in the patient). Table 1 In the bullet highlighting "misdiagnosis," suggest that "overdiagnosis" should also be included.

Comment 8: Comments on Outcomes in
In the bullet indicating psychosocial harms, suggest addition of discrimination (e.g., insurance or employment-related).

Comment 9: Introduction
In the first paragraph discussion of clinical signs of a genetic disease, we suggest adding some additional indicators:  Non-specific phenotypes (e.g. infantile hypotonia) that do not correspond to a specific disorder  Clinical phenotypes and diagnoses with known extensive locus heterogeneity  Known genetic disorders but targeted testing has been negative  Atypical clinical course (e.g., unexpected severity, duration, response to therapy, unusual adverse events, etc)  Rare and specific clinical or laboratory abnormalities or common laboratory values far outside the normal range  Atypical or complex combinations of clinical abnormalities Thank you again for the opportunity to comment. If you have any questions or if we could be of assistance in clarifying any of the comments above, please do not hesitate to reach out to us. Who are children with multiple unexplained congenital anomalies or a neurodevelopmental disorder of unknown etiology following standard workup

Interventions of interest are:
Whole exome sequencing with trio testing when possible

Comparators of interest are:
Standard clinical workup without whole exome sequencing

Relevant outcomes include:
Test validity Functional outcomes Changes in reproductive decision making Resource utilization Individuals: Who are children with a suspected genetic disorder other than multiple congenital anomalies or a neurodevelopmental disorder of unknown etiology following standard workup

Interventions of interest are:
Whole exome sequencing with trio testing when possible

Comparators of interest are:
Standard clinical workup without whole exome sequencing

Relevant outcomes include:
Test validity Functional outcomes Changes in reproductive decision making Resource utilization

Individuals:
Who are critically ill infants with a suspected genetic disorder of unknown etiology following standard workup

Interventions of interest are:
Rapid whole genome sequencing with trio testing when possible

Comparators of interest are:
Standard clinical workup without whole exome or whole genome sequencing

Relevant outcomes include:
Test Who are children with a suspected genetic disorder other than multiple unexplained congenital anomalies or a neurodevelopmental disorder of unknown etiology following standard workup

Interventions of interest are:
Whole genome sequencing with trio testing when possible

Comparators of interest are:
Standard clinical workup without whole exome sequencing

Relevant outcomes include:
Test validity Functional outcomes Changes in reproductive decision making Resource utilization

Overview by Evidence Review Indications
Indication 1: Individuals who are children with multiple unexplained congenital anomalies or a neurodevelopmental disorder of unknown etiology following standard workup who are evaluated with whole exome sequencing with trio testing when possible.
The evidence is sufficient to determine that the technology results in a meaningful improvement in the net health outcome. Indication 2: Individuals who are children with a suspected genetic disorder other than multiple congenital anomalies or a neurodevelopmental disorder of unknown etiology following standard workup who are evaluated with whole exome sequencing with trio testing when possible.
The evidence is insufficient to determine the effects of the technology on health outcomes. Indication 3: Individuals who are critically ill infants with a suspected genetic disorder of unknown etiology following standard workup who are evaluated with rapid whole genome sequencing with trio testing when possible.
The evidence is sufficient to determine that the technology results in a meaningful improvement in the net health outcome. Indication 4: Individuals who are children who are not critically ill with multiple unexplained congenital anomalies or a neurodevelopmental disorder of unknown etiology following standard workup and who are evaluated with whole genome sequencing with trio testing when possible.
The evidence is sufficient to determine that the technology results in a meaningful improvement in the net health outcome. Indication 5: Individuals who are children with a suspected genetic disorder other than multiple unexplained congenital anomalies or a neurodevelopmental disorder of unknown etiology following standard workup and who are evaluated with whole genome sequencing with trio testing when possible.
The evidence is insufficient to determine the effects of the technology on health outcomes. https://app.evidencestreet.com 3/35

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 variants. 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 a greater ability to detect large deletions or duplications in protein-coding regions compared with WES but requires greater data analytics. 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

RATIONALE
The evidence review was created in September 2013 and has been updated regularly with searches of the MEDLINE database. The most recent literature update was performed through August 6, 2018.
This review was informed in part by a TEC Special Report (2013) on exome sequencing for patients with suspected genetic disorders. 3, Evidence reviews assess whether a medical test is clinically useful. A useful test provides information to make a clinical management decision that improves the net health outcome. That is, the balance of benefits and harms is better when the test is used to manage the condition than when another test or no test is used to manage the condition.
The first step in assessing a medical test is to formulate the clinical context and purpose of the test. The test must be technically reliable, clinically valid, and clinically useful for that purpose. Evidence reviews assess the evidence on whether a test is clinically valid and clinically useful. Technical reliability is outside the scope of these reviews, and credible information on technical reliability is available from other sources.

Whole exome sequencing for children with multiple congenital anomalies or a neurodevelopmental disorder of unknown etiology following standard workup Clinical Context and Test Purpose
The purpose of whole exome sequencing (WES) in children who have multiple unexplained congenital anomalies or a neurodevelopmental disorder of unknown etiology following standard workup 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 (eg, 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 the use of WES improve health outcomes when used for the diagnosis of children with multiple unexplained congenital anomalies or a neurodevelopmental disorder of unknown etiology following standard workup?
The following PICO were used to select literature to inform this review.

Patients
The relevant population of interest is children presenting with multiple unexplained congenital anomalies or a neurodevelopmental disorder that are suspected to have a genetic basis but are not explained by standard clinical workup.
Intervention https://app.evidencestreet.com 5/35 The relevant intervention of interest is WES with trio testing when possible.

Comparators
The following practice is currently being used to diagnose multiple unexplained congenital anomalies or a neurodevelopmental disorder: standard clinical workup without WES.
A standard clinical workup for an individual with a suspected genetic condition varies by patient phenotype but generally involves a thorough history, physical exam (including dysmorphology and neurodevelopmental assessment, if applicable), routine laboratory testing, and imaging. If the results suggest a specific genetic syndrome, then established diagnostic methods relevant for that syndrome would be used.

Outcomes
There is no reference standard for the diagnosis of patients who have exhausted alternative testing strategies, therefore diagnostic yield will be the clinical validity outcome of interest.
The health outcomes of interest are 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.

Study Selection Criteria
For the evaluation of clinical validity of WES, studies that met the following eligibility criteria were considered: · Reported on the diagnostic yield or performance characteristics such as sensitivity and specificity of WES; · Patient/sample clinical characteristics were described; children with congenital abnormalities or neurodevelopmental disorders were included; · Patient/sample selection criteria were described; · Included at least 20 patients.

Technically Reliable
Assessment of technical reliability focuses on specific tests and operators and requires review of unpublished and often proprietary information. Review of specific tests, operators, and unpublished data are outside the scope of this evidence review and alternative sources exist. This evidence review focuses on the clinical validity and clinical utility.

Clinically Valid
A test must detect the presence or absence of a condition, the risk of developing a condition in the future, or treatment response (beneficial or adverse).
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 had suspected rare genetic disorders, although the specific populations vary.
Series have been reported with as many as 2000 patients. The most common reason for referral to a tertiary care center was an unexplained neurodevelopmental disorder. Many patients had been through standard clinical https://app.evidencestreet.com 6/35 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 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. 4,

Clinically Useful
A test is clinically useful if the use of the results informs management decisions that improve the net health outcome of care. The net health outcome can be improved if patients receive correct therapy, or more effective therapy, or avoid unnecessary therapy, or avoid unnecessary testing.

Direct Evidence
Direct evidence of clinical utility is provided by studies that have compared health outcomes for patients managed with and without the test. Because these are intervention studies, the preferred evidence would be from randomized controlled trials (RCTs).
No RCTs assessing the use of WES to diagnose multiple unexplained congenital anomalies or a neurodevelopmental disorder were identified.

Chain of Evidence
Indirect evidence on clinical utility rests on clinical validity. If the evidence is insufficient to demonstrate test performance, no inferences can be made about 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.

Section Summary: Whole Exome Sequencing for Children with Multiple Congenital Anomalies or a Neurodevelopmental Disorder of Unknown Etiology Following Standard Workup
The evidence on WES in children who have multiple congenital anomalies or a developmental disorder with a suspected genetic etiology of unknown etiology following standard workup 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 for children with a Suspected Genetic Disorder Other than multiple congenital anomalies or a neurodevelopmental disorder of unknown etiology following standard workup 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 purpose of WES in patients who have a suspected genetic disorder other than multiple unexplained congenital anomalies or a neurodevelopmental disorder of unknown etiology following standard workup is to establish a molecular diagnosis. The criteria under which diagnostic testing for a genetic or heritable disorder may be considered clinically useful are stated above.
The question addressed in this evidence review is: Does WES improve health outcomes when used for the diagnosis of a suspected genetic condition other than multiple congenital anomalies or a neurodevelopmental disorder of unknown etiology following standard workup?
The following PICO were used to select literature to inform this review.

Patients
The relevant population of interest is children 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. Specific tests were described in the preceding section on WES.
Comparators https://app.evidencestreet.com 10/35 The following practice is currently being used to diagnose a suspected genetic disorder other than multiple unexplained congenital anomalies or a neurodevelopmental disorder: standard clinical workup without WES.
Standard clinical workup was described in a preceding section.

Outcomes
There is no reference standard for the diagnosis of patients who have exhausted alternative testing strategies, therefore diagnostic yield will be the clinical validity outcome of interest.
The health outcomes of interest are 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.

Study Selection Criteria
For the evaluation of clinical validity of WES, studies that met the following eligibility criteria were considered: · Reported on the diagnostic yield or performance characteristics such as sensitivity and specificity of WES; · Patient/sample clinical characteristics were described; · Patient/sample selection criteria were described; · Included at least 20 patients.

Technically Reliable
Assessment of technical reliability focuses on specific tests and operators and requires review of unpublished and often proprietary information. Review of specific tests, operators, and unpublished data are outside the scope of this evidence review and alternative sources exist. This evidence review focuses on the clinical validity and clinical utility.

Clinically Valid
A test must detect the presence or absence of a condition, the risk of developing a condition in the future, or treatment response (beneficial or adverse).
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 (see Table 3). Some studies used a virtual gene panel that is restricted to genes 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. The purpose of the gaps tables (see Tables 4 and 5) is to display notable gaps identified in each study. This information is synthesized as a summary of the body of evidence and provides the conclusions on the sufficiency of the evidence supporting the position statement. Reclassification of diagnostic or risk categories not reported; 5. Adverse events of the test not described (excluding minor discomforts and inconvenience of venipuncture or noninvasive tests). e Follow-Up key: 1. Follow-up duration not sufficient with respect to natural history of disease (true positives, true negatives, false positives, false negatives cannot be determined).

Clinically Useful
A test is clinically useful if the use of the results informs management decisions that improve the net health outcome of care. The net health outcome can be improved if patients receive correct therapy, or more effective therapy, or avoid unnecessary therapy, or avoid unnecessary testing.

Direct Evidence
Direct evidence of clinical utility is provided by studies that have compared health outcomes for patients managed with and without the test. Because these are intervention studies, the preferred evidence would be from RCTS.
No RCTs assessing the use of WES to diagnose a suspected genetic disorder other than multiple unexplained congenital anomalies or a neurodevelopmental disorder were identified.

Chain of Evidence
Indirect evidence on clinical utility rests on clinical validity. If the evidence is insufficient to demonstrate test performance, no inferences can be made about clinical utility.
A genetic diagnosis for an unexplained disorder can alter management in several ways: such a diagnosis may lead to including genetic counseling and ending the diagnostic odyssey and may affect reproductive decision making.
Because the clinical validity of WES for this indication has not been established, a chain of evidence cannot be constructed.

Section Summary: WES for a Suspected Genetic Disorder Other Than Multiple Congenital Anomalies or a Neurodevelopmental Disorder
There is an increasing number of reports assessing use of WES identify a molecular basis for disorders other than multiple congenital anomalies or neurodevelopmental disorders. The diagnostic yields in these studies ranged from 3% for colorectal cancer to 60% for trio (parents and child) analysis of limb-girdle muscular dystrophy. Some studies have reported on the use of a virtual gene panel with restricted analysis of diseaseassociated genes, and the authors noted that WES data allows reanalysis as new genes are linked to the patient phenotype. Overall, a limited number of patients have been studied for any specific disorder, and study of WES in these disorders is at an early stage with uncertainty about changes in patient management.

Whole Genome Sequencing
The purpose of whole genome sequencing (WGS) in patients with a suspected genetic disorder of unknown etiology following standard workup is to establish a molecular diagnosis from either the coding or noncoding regions of the genome. The criteria under which diagnostic testing for a genetic or heritable disorder may be considered clinically useful are stated above.
The question addressed in this evidence review is: Does WGS improve health outcomes when used for the diagnosis of patients with a suspected genetic disorder of unknown etiology following standard workup without whole exome or whole genome sequencing?
The following PICO were used to select literature to inform this review.

Patients
The relevant populations of interest are: · Critically ill infants presenting with any of a variety of disorders and anomalies suspected to have a genetic basis but not explained by standard workup. For examples, patients may have a phenotype that does not correspond with a specific disorder for which a genetic test targeting a specific gene is available. Specifically for critically ill infants, the population would also include patients for whom specific diagnostic tests available for that phenotype are not accessible within a reasonable timeframe. Petrikin (2018) identified the critically ill infants that are appropriate for rapid testing as meeting the following inclusion criteria: multiple congenital anomalies; abnormal laboratory test suggests a genetic disease or complex metabolic phenotype; abnormal response to standard therapy for a major underlying condition; significant hypotonia; or persistent seizures. Exclusion criteria included: an infection with normal response to therapy; isolated prematurity; isolated unconjugated hyperbilirubinemia; Hypoxic Ischemic Encephalopathy; confirmed genetic diagnosis explains illness; Isolated Transient Neonatal Tachypnea; or nonviable neonates.
· Children who are not critically ill with multiple unexplained congenital anomalies or a neurodevelopmental disorder of unknown etiology following standard workup · Children with a suspected genetic disorder other than multiple unexplained congenital anomalies or a neurodevelopmental disorder of unknown etiology following standard workup

Interventions
The relevant interventions being considered include: · rapid WGS with trio testing when possible · WGS with trio testing when possible Several laboratories offer WGS as a clinical service. Medical centers may also offer rapid WGS or standard WGS as a clinical service.
The median time for standard WGS is several weeks. The median time-to-result for rapid WGS is approximately 5 days or less.
Note that this evidence review does not address the use of WGS for preimplantation genetic diagnosis or screening, prenatal (fetal) testing, or for testing of cancer cells.

Comparators
The following practice is currently being used to diagnose a suspected genetic disorder: standard clinical workup without WES or WGS.
Standard clinical workup was described in a preceding section.

Outcomes
Outcomes of interest are as described above for use of WES in patients with multiple congenital anomalies or a neurodevelopmental disorder. For critically ill infants, rapid diagnosis is important therefore, in addition to the outcomes described in the previous section, time to diagnosis and time to discharge are also outcomes of interest.

Study Selection Criteria
For the evaluation of clinical validity of WGS, studies that met the following eligibility criteria were considered: · Reported on the diagnostic yield or performance characteristics such as sensitivity and specificity of rapid WGS or WGS; · Patient/sample clinical characteristics were described; · Patient/sample selection criteria were described; · Included at least 20 patients.

Technically Reliable
Assessment of technical reliability focuses on specific tests and operators and requires review of unpublished and often proprietary information. Review of specific tests, operators, and unpublished data are outside the scope of this evidence review and alternative sources exist. This evidence review focuses on the clinical validity and clinical utility.

Clinically Valid
A test must detect the presence or absence of a condition, the risk of developing a condition in the future, or treatment response (beneficial or adverse).
Studies have shown that WGS can detect more pathogenic variants than WES, due to an improvement in detecting copy number variants, insertions and deletions, intronic single nucleotide variants, and exonic single nucleotide variants in regions with poor coverage on WES. A majority of studies described methods for interpretation of WGS indicating that only pathogenic or likely pathogenic variants were included in the diagnostic yield and that variants of uncertain significance were not reported (see Tables 6,7 and 8). In some studies, the genes examined were those previously associated with the phenotype, while other studies were research-based and conducted more exploratory analysis. 32 It has been noted that genomes sequenced with WGS are available for future review when new variants associated with clinical diseases are discovered.
The use of WGS and rapid WGS has been studied in critically ill children in several observational studies, both prospective and retrospective, and one RCT. Studies are described in Table 6. The RCT is discussed in more detail in the following 'Clinically useful' section. One study included only infants with cardiac defects and had a diagnostic yield of 6% with WGS. The remaining studies included phenotypically diverse but critically ill infants and had yields of between 30% and 60%. Nine of 20 WGS diagnoses were diseases that were not part of the differential at time of enrollment The use of WGS has been studied in children who are not critically ill with multiple unexplained congenital anomalies or a neurodevelopmental disorder of unknown etiology following standard workup in several observational studies, both prospective and retrospective. Studies are described in Table 7. The diagnostic yield of WGS has been between 20% and 40%. Additional indirect evidence is available from studies reporting diagnostic yield of WES in a similar population as summarized above, and it is reasonable to expect that WGS is likely to result in similar or better diagnostic yield for pathogenic or likely pathogenic variants as compared with WES. The use of WGS has been studied in children with a suspected genetic disorder other than multiple unexplained congenital anomalies or a neurodevelopmental disorder in several observational studies, both prospective and retrospective. Studies are described in Table 8. The diagnostic yield of WGS has been between 9% and 55%. However, these studies include mixed indications with heterogenous populations and include little information about associated changes in management following genetic diagnosis. Not compared to other tests in use for same purpose. d Outcomes key: 1. Study does not directly assess a key health outcome; 2. Evidence chain or decision model not explicated; 3. Key clinical validity outcomes not reported (sensitivity, specificity and predictive values); 4. Reclassification of diagnostic or risk categories not reported; 5. Adverse events of the test not described (excluding minor discomforts and inconvenience of venipuncture or noninvasive tests). e Follow-Up key: 1. Follow-up duration not sufficient with respect to natural history of disease (true positives, true negatives, false positives, false negatives cannot be determined).

Clinically Useful
A test is clinically useful if the use of the results informs management decisions that improve the net health outcome of care. The net health outcome can be improved if patients receive correct therapy, or more effective therapy, or avoid unnecessary therapy, or avoid unnecessary testing.

Direct Evidence
Direct evidence of clinical utility is provided by studies that have compared health outcomes for patients managed with and without the test. Because these are intervention studies, the preferred evidence would be from RCTs. , blinded, and pragmatic trial comparing trio rWGS with standard genetic tests to standard genetic tests alone with a primary outcome of proportion of NICU/PICU infants receiving a genetic diagnosis within 28 days. Parents of patients and clinicians were unblinded after 10 days and compassionate cross-over to rWGS occurred in 5 control patients. The study was designed to enroll 500 patients in each group but was terminated early due to loss of equipoise on the part of study clinicians who began to regard standard tests alone as inferior to standard tests plus trio rWGS. Intention-to-treat analyses were reported, i.e., crossovers were included in the group to which they were randomized. The trial required confirmatory testing of WGS results which lengthened the time to rWGS diagnosis by 7-10 days. Study characteristics are shown in Table 11 and results are shown in Table 12.
Tables 13 and 14 display notable gaps identified in each study.

Chain of Evidence
Indirect evidence on clinical utility rests on clinical validity. If the evidence is insufficient to demonstrate test performance, no inferences can be made about clinical utility.
Clinical validity is established based on the meaningful diagnostic yield associated with WGS when a genetic etiology is uncertain after standard workup. Studies on rapid WGS and WGS report changes in management that would improve health outcomes. The effect of WGS results on health outcomes are the same as those with WES, including avoidance of invasive procedures, medication changes to reduce morbidity, discontinuation of or additional testing and initiation of palliative care or reproductive planning. A chain of evidence linking meaningful improvements in diagnostic yield and changes in management expected to improve health outcomes supports the clinical value of WGS for both critically ill infants with a suspected genetic disorder and for children who are not critically ill with multiple unexplained congential anomalies or a neurodevelopmental disorder when there is an unknown etiology following standard workup.

Section Summary: Whole Genome Sequencing
For critically ill infants, disease may progress rapidly and genetic diagnoses must be made quickly. Rapid WGS has increased coverage compared to WES. One RCT comparing rapid trio WGS (rWGS) with standard genetic tests to diagnose suspected genetic disorders in critically ill infants funded by NIH has been conducted. The study was terminated early due to loss of equipoise on the part of study clinicians who began to regard standard tests alone as inferior to standard tests plus trio rWGS. The rate of genetic diagnosis within 28 days of enrollment was higher for rWGS versus standard tests (31% vs 3%; p=0.003) and the time to diagnosis was shorter (13 days versus 107 days; p=0.002). The age at hospital discharge and mortality rates were similar in the https://app.evidencestreet.com 28/35 two groups. An ongoing RCT (n=1000) is comparing rWGS to rWES with completion expected in December 2018. Several retrospective and prospective observational studies with sample sizes ranging from about 23 to 65 and in total including more than 200 infants reporting on diagnostic yield for rWGS included phenotypically diverse but critically ill infants and had yields of between 30% and 60% and reports of changes in management such as avoidance of invasive procedures, medication changes, discontinuation of or additional testing and initiation of palliative care.
WGS has been studied in non-critically ill children with congenital abnormalities and development delays of unknown etiology following standard workup. The diagnostic yield for WGS has been reported between 20% and 40%. Additional indirect evidence is available from studies reporting diagnostic yield and change in management results of WES in a similar population, and it is reasonable to expect that WGS is likely to result in similar or better diagnostic yield for pathogenic or likely pathogenic variants and similar changes in management as compared with WES.
WGS has also been studied in children with a suspected genetic disorder other than multiple unexplained congenital anomalies or a neurodevelopmental disorder of unknown etiology following standard workup. The diagnostic yield of WGS has been between 9% and 55%. However, these studies include mixed indications with heterogenous populations and include little information about associated changes in management following genetic diagnosis.

Summary of Evidence
For individuals who are children with multiple unexplained congenital anomalies or a neurodevelopmental disorder of unknown etiology following standard workup who receive WES with trio testing when possible, the evidence includes large case series and within-subject comparisons. Relevant outcomes are test 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 are children with a suspected genetic disorder other than multiple congenital anomalies or a neurodevelopmental disorder of unknown etiology following standard workup who receive WES with trio testing when possible, the evidence includes small case series and prospective research studies. Relevant outcomes are test validity, functional outcomes, changes in reproductive decision making, and resource utilization. There is an increasing number of reports evaluating the 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%. 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, a limited number of patients have been studied for any specific disorder, and clinical use of WES for these disorders is at an early stage with uncertainty about changes in patient management. The evidence is insufficient to determine the effects of the technology on health outcomes.
For individuals who are critically ill infants with a suspected genetic disorder of unknown etiology following standard workup who receive rapid WGS (rWGS) with trio testing when possible, the evidence includes an RCT and case series. Relevant outcomes are test validity, functional outcomes, changes in reproductive decision making, and resource utilization. One RCT comparing rapid trio WGS (rWGS) with standard genetic tests to diagnose suspected genetic disorders in critically ill infants was terminated early due to loss of equipoise. The rate of genetic diagnosis within 28 days of enrollment was higher for rWGS versus standard tests (31% vs 3%; p=0.003). Changes in management due to test results were reported in 41% vs 21% (p=0.11) of rWGS vs control patients; however, 73% of control subjects received broad genetic tests (eg, NGS panel testing, WES, or WGS) as part of standard testing. Several retrospective and prospective studies including more than 200 infants in total have reported on diagnostic yield for rWGS including phenotypically diverse but critically ill infants and had yields of between 30% and 60% for pathogenic or likely pathogenic variants. Studies have also reported associated changes in patient management for patients receiving a diagnosis from rWGS, including avoidance of invasive procedures, medication changes to reduce morbidity, discontinuation of or additional testing and initiation of palliative care or reproductive planning. A chain of evidence linking meaningful improvements in diagnostic yield and changes in management expected to improve health outcomes supports the clinical value of rWGS. The evidence is sufficient to determine that the technology results in a meaningful improvement in the net health outcome.
For individuals who are children who are not critically ill with multiple unexplained congenital anomalies or a neurodevelopmental disorder of unknown etiology following standard workup who receive WGS with trio testing when possible, the evidence includes case series. Relevant outcomes are test validity, functional outcomes, changes in reproductive decision making, and resource utilization. In studies of children with congenital abnormalities and development delays of unknown etiology following standard clinical workup, the yield of WGS has been between 20% and 40%. Additional indirect evidence is available from studies reporting diagnostic yield and change in management results of WES in a similar population, and it is reasonable to expect that WGS is likely to result in similar or better diagnostic yield for pathogenic or likely pathogenic variants and similar changes in management as compared with WES. The evidence is sufficient to determine that the technology results in a meaningful improvement in the net health outcome.
For individuals who are children with a suspected genetic disorder other than multiple unexplained congenital anomalies or a neurodevelopmental disorder of unknown etiology following standard workup who receive who receive WGS with trio testing when possible, the evidence includes case series. Relevant outcomes are test validity, functional outcomes, changes in reproductive decision making, and resource utilization. WGS has also been studied in other genetic conditions with yield ranging from 9% to 55%. Overall, a limited number of patients have been studied for any specific disorder, and clinical use of WGS as well as information regarding meaningful changes in management for these disorders is at an early stage. The evidence is insufficient to determine the effects of the technology on health outcomes.

American College of Medical Genetics and Genomics
The American College of Medical Genetics and Genomics (ACMG) has recommended that diagnostic testing with whole exome sequencing (WES) and whole genome sequencing (WGS) should be considered in the clinical diagnostic assessment of a phenotypically affected individual when 48, : "a. The phenotype or family history data strongly implicate a genetic etiology, but the phenotype does not correspond with a specific disorder for which a genetic test targeting a specific gene is available on a clinical basis.
b. A patient presents with a defined genetic disorder that demonstrates a high degree of genetic heterogeneity, making WES or WGS analysis of multiple genes simultaneously a more practical approach. c. A patient presents with a likely genetic disorder but specific genetic tests available for that phenotype have failed to arrive at a diagnosis. d. A fetus with a likely genetic disorder in which specific genetic tests, including targeted sequencing tests, available for that phenotype have failed to arrive at a diagnosis. " ACMG has recommended that for screening purposes: WGS/WES may be considered in preconception carrier screening, using a strategy to focus on genetic variants known to be associated with significant phenotypes in homozygous or hemizygous progeny.
ACMG has also recommended that WGS and WES not be used at this time as an approach to prenatal screening or as a first-tier approach for newborn screening.
ACMG guidelines (2014) on the clinical evaluation and etiologic diagnosis of hearing loss stated that for individuals with findings suggestive of a syndromic genetic etiology for hearing loss, "pretest genetic counseling should be provided, and, with patient's informed consent, genetic testing, if available, should be ordered to confirm the diagnosis-this testing may include single-gene tests, hearing loss sequencing panels, WES, WGS, chromosome analysis, or microarray-based copy number analysis, depending on clinical findings." 49, ACMG (2016) updated its recommendations on reporting incidental findings in WGS and WES testing. 50 ACMG determined that reporting some incidental findings would likely have medical benefit for the patients and families of patients undergoing clinical sequencing, recommending that, when a report is issued for clinically indicated exome and genome sequencing, a minimum list of conditions, genes, and variants should be routinely evaluated and reported to the ordering clinician. The 2016 update added 4 genes and removed of 1 gene resulting in an updated secondary findings minimum list including 59 medically actionable genes recommended for return in clinical genomic sequencing.

American Academy of Neurology et al
The American Academy of Neurology and American Association of Neuromuscular and Electrodiagnostic Medicine (2014) issued evidence-based guidelines on the diagnosis and treatment of limb-girdle and distal dystrophies, which made the following recommendations (see Table 15). 51, Table 15. Guidelines on LGMD Recommendation LOE Diagnosis · For patients with suspected muscular dystrophy, clinicians should use a clinical approach to guide genetic diagnosis based on the clinical phenotype, including the pattern of muscle involvement, inheritance pattern, age at onset, and associated manifestations (e.g., early contractures, cardiac or respiratory involvement). B · In patients with suspected muscular dystrophy in whom initial clinically directed genetic testing does not provide a diagnosis, clinicians may obtain genetic consultation or perform parallel sequencing of targeted exomes, whole-exome sequencing, whole-genome screening, or next-generation sequencing to identify the genetic abnormality. C Management of cardiac complications · Clinicians should refer newly diagnosed patients with (1) limb-girdle muscular dystrophy (LGMD)1A, LGMD1B, LGMD1D, LGMD1E, LGMD2C-K, LGMD2M-P, … or (2) muscular dystrophy without a specific genetic diagnosis for cardiology evaluation, including electrocardiogram (ECG) and structural evaluation (echocardiography or cardiac magnetic resonance imaging [MRI]), even if they are asymptomatic from a cardiac standpoint, to guide appropriate management.

B
Recommendation LOE · If ECG or structural cardiac evaluation (e.g., echocardiography) has abnormal results, or if the patient has episodes of syncope, near-syncope, or palpitations, clinicians should order rhythm evaluation (e.g., Holter monitor or event monitor) to guide appropriate management. B · Clinicians should refer muscular dystrophy patients with palpitations, symptomatic or asymptomatic tachycardia or arrhythmias, or signs and symptoms of cardiac failure for cardiology evaluation. B · It is not obligatory for clinicians to refer patients with LGMD2A, LGMD2B, and LGMD2L for cardiac evaluation unless they develop overt cardiac signs or symptoms. B Management of pulmonary complications · Clinicians should order pulmonary function testing (spirometry and maximal inspiratory/expiratory force in the upright and, if normal, supine positions) or refer for pulmonary evaluation (to identify and treat respiratory insufficiency) in muscular dystrophy patients at the time of diagnosis, or if they develop pulmonary symptoms later in their course. B · In patients with a known high risk of respiratory failure (e.g., those with LGMD2I …), clinicians should obtain periodic pulmonary function testing (spirometry and maximal inspiratory/expiratory force in the upright position and, if normal, in the supine position) or evaluation by a pulmonologist to identify and treat respiratory insufficiency. B · It is not obligatory for clinicians to refer patients with LGMD2B and LGMD2L for pulmonary evaluation unless they are symptomatic. C · Clinicians should refer muscular dystrophy patients with excessive daytime somnolence, nonrestorative sleep (e.g., frequent nocturnal arousals, morning headaches, excessive daytime fatigue), or respiratory insufficiency based on pulmonary function tests for pulmonary or sleep medicine consultation for consideration of noninvasive ventilation to improve quality of life. B LOE: level of evidence; LGMD: limb-girdle muscular dystrophy.

U.S. Preventive Services Task Force Recommendations
Not applicable.

Medicare National Coverage
There is no national coverage determination. In the absence of a national coverage determination, coverage decisions are left to the discretion of local Medicare carriers.

Ongoing and Unpublished Clinical Trials
Some currently unpublished trials that might influence this review are listed in Table 16.