The Impact of Rapid Exome Sequencing on Medical Management of Critically Ill Children

Abstract

Objectives:

To evaluate the clinical utility of rapid exome sequencing (rES) in critically ill children with likely genetic disease using a standardized process at a single institution. To provide evidence that rES with should become standard of care for this patient population.

Study design:

We implemented a process to provide clinical-grade rES to eligible children at a single institution. Eligibility included: a) recommendation of rES by a consulting geneticist, b) monogenic disorder suspected, c) rapid diagnosis predicted to affect inpatient management, d) pre-test counseling provided by an appropriate provider, and e) unanimous approval by a committee of 4 geneticists. Trio exome sequencing was sent to a reference laboratory that provided verbal report within 7–10 days. Clinical outcomes related to rES were prospectively collected. Input from geneticists, genetic counselors, pathologists, neonatologists and critical care pediatricians was collected to identify changes in management related to rES.

Results:

54 patients were eligible for rES over a 34 month study period. 46 of these underwent rES, 24 of which (52%) had at least one change in management related to rES. In 20 (43%) patients a molecular diagnosis was achieved, demonstrating that non-diagnostic exomes could change medical management in some cases. 84% were under 1 month old at rES request and mean turnaround time was 9 days.

Conclusions:

rES testing has a significant impact on the management of critically ill children with suspected monogenic disease and should be considered standard of care for tertiary institutions who can provide coordinated genetics expertise.

Exome sequencing is the simultaneous sequencing of all ~20,000 genes in the human genome, and is increasingly a first line diagnostic test for children with multiple congenital anomalies, complex neurodevelopmental phenotypes, and other likely monogenic disorders.^1,2 Numerous studies have demonstrated that ES provides a definitive molecular diagnosis in 30–50% of children with these phenotypes.^3,4,5,6 However, ES is not yet in broad use in pediatric and neonatal intensive care units, despite the fact that these patients are enriched for genetic disease.^4–15 Barriers to the widespread adoption of ES in the ICU setting include the impression that the turnaround time is too long to be useful in the critical care setting; complex test logistics, which requires pretest genetic counseling and may require obtaining samples from both parents; high test costs with poor reimbursement, and just emerging data on how ES impacts clinical management of these children.^16–20

We describe our experience developing the Rapid Inpatient Genomic Testing (RIGhT) study- a clinical program for rapid exome sequencing (rES) within neonatal, pediatric and cardiac ICUs at a single institution. This program was developed as a part of routine clinical care and was not subsidized by research or other funds. Rather than focusing on the diagnostic yield of rES, we evaluated how the results were used by ICU physicians to change medical and surgical management of these critically ill children.

METHODS

This study was performed at Seattle Children’s Hospital. This hospital has a 32 bed NICU that sees approximately 500 patients per year; a 38 bed PICU that sees approximately 2000 admissions per year; and a 20 bed CICU that sees approximately 600 admissions per year. This study was approved by the Seattle Children’s Hospital IRB, Activity ID: CR00003151, IRB ID: STUDY00000553.

Study design and participants:

The RIGhT study began in October 2016. Data is reported through July 2019. In order for a patient to be enrolled in the study, the intensivist first consulted medical or biochemical genetics. All geneticists and intensivists were made aware of the study in October 2016. Recommendations for rES ultimately were made by the consulting geneticist, although the intensive care team and consulting geneticist engaged in collaborative decision making. Initial inclusion criteria were: 1) consultation with a geneticist, 2) suspected monogenic disorder, 3) likelihood of rapid diagnosis altering management, 4) age less than 6 months and critically ill in the ICU, 5) availability of both biological parents for trio sequencing and 6) previous negative chromosomal microarray (CMA). The send out laboratory (GeneDx) performing the sequencing was only able to offer rapid testing for trio sets. If one or both parents were unavailable, testing with an approximately 4 week turnaround time was made available outside of this study. In January 2017 the inclusion criteria were broadened to include children of all ages in an ICU and the prerequisite for CMA was eliminated. The revisions were made based on observations that requiring CMA delayed diagnosis in some patients, the reference laboratory performing exome sequencing could detect CNVs of at least 3 exons, and monogenic disease presents in children older than 6 months.²¹

RIGhT Committee review process:

Referring board-certified geneticists identified the indication for testing, generated a phenotype-driven list of candidate genes, suspected mode of inheritance, and proposed changes in clinical management based on results. Referral information was captured on a standardized 2 page form (Appendix). The major test indication as well as other phenotypic features were recorded using human phenotype ontology (HPO) terms. A laboratory genetic counselor (GC) (SVC) provided an initial review of whether the cases met the inclusion criteria. If the case was appropriate it was then reviewed by a committee of board certified geneticists (HCM, CL, MPA, JTB, AS) with a variety of expertise including dysmorphology, epilepsy, biochemical genetics, vascular and lymphatic disorders and mosaicism. The committee provided review within one business day and unanimous approval was required to proceed.

Genetic counseling and Informed consent:

Two clinical genetic counselors were appointed to provide in person pre-test counseling and all families provided informed consent for clinical ES. All patients and families were given the option of receiving secondary findings. A clinical administrator (coordinated registration of both parents and sample collection logistics.

Sample collection and sequencing:

Peripheral blood samples from the patient and both biological parents were collected and shipped to the laboratory. In cases in which mitochondrial DNA sequencing was required and the patient had undergone recent red blood cell transfusion, a buccal swab was collected. The laboratory also received a pedigree and the consultant geneticist’s notes.

Trio ES was performed by a send out laboratory (GeneDX) that provides a verbal preliminary result within 10 calendar days of receipt of samples. Sequencing was done using the Agilent Clinical Research Exome kit. Targeted regions were sequenced simultaneously on an Illumina HiSeq with 100bp paired end reads. The bidirectional sequence was assembled and aligned to human reference genome build GRCh37/UCSC hg19 and analyzed for sequence variants using a custom developed analysis tool (Xome Analyzer).²²

Return of results and multi-disciplinary case review:

Preliminary verbal results were returned to the consulting geneticist who was then responsible for informing the critical care team and family. Final results were scanned into the electronic health record and returned by the consulting geneticist and/or GC. Cases were classified as molecularly diagnosed when pathogenic or likely pathogenic variant(s) were detected in a gene that explained the patient’s phenotype.²³ Cases in which a pathogenic or likely pathogenic variant explained part, but not all, of the patient’s phenotype were classified as partial diagnoses. Cases were classified as uncertain when there was any variant which was potentially related to patient’s phenotype but there was insufficient evidence to be certain. Cases were classified as non-diagnostic when no disease-associated variants were identified.

All cases were reviewed at monthly multidisciplinary team meetings that included geneticists, GCs, pathologists and neonatologists. Pediatric critical care physicians reviewed cases as needed. Pertinent dates, molecular results and changes in management were recorded in a database. Any change in medication (initiation or discontinuation), laboratory testing, surgical plan, or imaging plan was categorized as a discrete instance of management change. A single patient could have more than one management change related to rES.

All cases were also retrospectively reviewed to confirm previously documented changes in management as well as evaluate for any additional changes in management by a single person. Two neonatologists also independently reviewed each NICU case. One pediatric intensivist reviewed all pediatric and cardiac ICU cases. Any discrepancies were reviewed by the ICU attending and consulting geneticist of record for the case. Only if both attending physicians agreed that the rES results led to the change in management was it recorded. We did not require genetic diagnosis be achieved for there to be a change in medical management. In fact, multiple ICU physicians independently noted that negative results were also helpful in their decision making. Recommendations for cascade family testing, variant of uncertain significance (VUS) resolution testing and identifying recurrence risk for couples was also tracked and recorded, but these were not classified as management changes of the patient.

RESULTS

During the 33 months study period, 60 cases were referred to the laboratory GC by a consulting geneticist for consideration (Figure 1). Six cases were declined because they did not meet inclusion criteria. Fifty-four cases were reviewed by the committee and of those, 46 (85%) were unanimously approved for rES, nineteen of which included mitochondrial DNA testing. The most common reason for exclusion was non-availability of both parents for trio rES and three patients expired prior to sample send out. Multiple congenital anomalies (MCA) with or without congenital heart defect, respiratory failure and heart failure were the primary test indication in about half of the cases (24/46 or 52%). Other common test indications were hydrops, seizures, arthrogryposis, skeletal dysplasias and metabolic abnormalities (Figure 2; available at www.jpeds.com).

Figure 1.

Case selection flow chart for RIGhT study

Figure 2.

The number of diagnostic and non-diagnostic exomes by test indication. The most common category was multiple congenital anomaly both with and without congenital heart defect. “Other” includes all indications which only appeared once in our data such as liver failure and renal failure.

The median patient age at the time of the consulting geneticist’s request was 25 days with a range of 1 day to over 15 years (Table 1; available at www.jpeds.com). Patients had a median 5 day length of stay in the ICU prior to the consulting geneticist’s request to the committee. Over half (56%) of the participants were in the neonatal ICU, 22% were in the pediatric ICU and 22% were in the cardiac ICU. Nine patients (20%) were on extracorporeal membrane oxygenation (ECMO) mechanical cardiopulmonary support in the ICU.

Table 1.

Patient characteristics and prior testing (n=46)

Median age at request (min, mean, max)	25 days (1d, 297d, 15yr)
Median days in ICU prior to genetics consult (min, mean, max)	5 (0, 21, 241)
Type of ICU	Neonatal 26 (56%) Pediatric 10 (22%) Cardiac 10 (22%)
On ECMO	9 (20%)
rES as initial test	21 (46%)
rES as second tier test	25 (54%) - Prior CMA: 20 - Prior single gene or panel test: 7

rES was the first genetic test for 21 patients (46%). Of the 25 patients (54%) that had previous genetic testing, 20 had a prior non-diagnostic SNP array and 7 had prior molecular testing such as a single gene or panel test. Twenty eight patients (61%) had prior or concurrent biochemical testing. Twenty eight cases (61%) had pathology results (biopsy or autopsy) in addition to the rES.

The median turn-around-time from rES request to verbal result was 9 calendar days (range 5 days to 26 days). The median TAT from sample send out to verbal results was 6 calendar days (range 5–10 days). Therefore, the process of committee review, pre-test counseling and sample collection from the patient and parents took about 3 calendar days. Committee review took a maximum of 1 business day. Most of this 3 day period between rES request and sample send out was taken up by pretest counseling and obtaining parental samples, which could be difficult if the parents were not present at the time of consultation. Even with rapid TATs, five patients expired prior to return of verbal results. In two of those patients, a molecular diagnosis was achieved post-mortem.

A molecular diagnosis was made in nearly half of patients (20/46 or 43%) (Table 2). In addition, there were 11 uncertain diagnoses and 4 partial diagnoses (Table 2). Of the confirmed diagnoses, 13 were single nucleotide variants (SNVs), 5 were copy number variants, 1 was a case of maternal uniparental heterodisomy (UPD) and 1 was a compound heterozygote for an autosomal recessive (AR) condition with an SNV and an exonic deletion. Nine diagnoses were de novo dominant, four were inherited autosomal dominant, five were AR, and one was semi-dominant. None were X-linked or mitochondrial. Diagnostic yield by primary test indication is shown in Figure 2.

Table 2.

Cases with a molecular diagnosis by rES.

ID/Sex/Age†	Phenotype (HPO terms)	Disease (OMIM# if available)	Gene/Chromosome	Variant(s)	Inheritance	rES as initial test Y/N
048/ F/ 2	Respiratory failure HP:0002878	LADD syndrome with acinar dysplasia (OMIM#149830)	FGF10	c.524delA, p.M176CfsX5	Autosomal dominant-paternally inherited	Y
028/ F/ 126	Seizures HP:0001250 Abnormal movements HP:0100022	Early infantile epileptic encephalopathy 13 (OMIM# 614558)	SCN8A	c.2549G>A, p.R850Q	Autosomal dominant- de novo	Y
020/ M/ 4	Respiratory failure HP:0002878	1p36 deletion syndrome (OMIM #607872)	1p36.23	(7258622_9097054) x1	Autosomal dominant- de novo	Y
058/ M/ 419	Mitral valve stenosis HP:0001718 GDD HP:0001263	17q21 deletion	17q21	(46314762_48787388)X1	Autosomal dominant- de novo	Y
007/ F/ 36	Hypoplastic left heart HP:0004383 Dysmorphic features HP:0000271	Koolen de Vries syndrome (OMIM #610443)	KANSL1	c.1579_1582delATTG p.1527VfsX50	Autosomal dominant- de novo	N, prior CMA
008/ F/ 4	Complex congenital heart defect HP:0001627 Brain malformation HP:0012443 Dysmorphic features HP:0000271	Chromosome 2q deletion	2q14.2q23.1	(120926106_149857498)x1	Autosomal dominant- de novo	Y
012/ M/ 1	Nonimmune Hydrops Fetalis HP:0001790	Generalized arterial calcification of infancy 1 (OMIM #208000)	ENPP1	c.2662C>T, p. R888W and c.913C>A, p.P305T	Autosomal recessive	Y
016/ M/ 71	Skeletal dysplasia HP:0002652	Spondyloepiphyseal dysplasia congenital (OMIM #183900)	COL2A1	c.1403G>A, p.G468D	Autosomal dominant- de novo	N, prior CMA
022/ F/ 1	Nonimmune Hydrops Fetalis HP:0001790	Noonan syndrome (OMIM #610733) and *Biotinidase deficiency (OMIM #253260)	SOS1 and BTD	c.806T>C, p.M269T and c.1330G>C, p.D444H c.1368A>C, p.Q465H	Autosomal dominant-maternally inherited and Autosomal recessive	N, prior prenatal CMA
027/ F/ 5	Hyperammonemia HP:0008281	Carbonic anhydrase 5a deficiency (OMIM #615751)	CA5A	c.721G>A, p.E241K homozygous	Autosomal recessive	Y
029/ F/ 83	Respiratory insufficiency HP:0002093 Pierre- Robin sequence HP:0000201	LADD syndrome (OMIM #149730)	FGF10	c.577C>T, p.R193X	Autosomal dominant- de novo	N, prior CMA
030/ M/ 1	Respiratory failure HP:0002878 Dysmorphic features HP:0000271	17q23 deletion	17q23	(59290909_61353248)x1	Autosomal dominant- de novo	Y
031/ M/ 2	Arthrogryposis HP:0002804	Congenital myasthenia syndrome (OMIM #616314)	CHRNB1	Exon 8 deletion And c.1218–9_1218–7delCTC	Autosomal recessive	Y(though prior affected fetus with negative CMA)
037/ F/ 16yrs	Renal failure HP:0001919	Juvenile nephronophthisis (OMIM #256100)	2q13 (inclusive of NPHP1)	(110862477_110964737)x0	Autosomal recessive	N, prior single gene testing
047/ F/ 1	Complex congenital heart defect HP:0001627 Heterotaxy HP:0030853 Congenital hydrocephalus HP:0000238	20p11 deletion	20p11	(21,680,345_24,383,453)x1	Autosomal dominant- de novo	N, CMA sent concurrently
054/ F/ 59	Seizures HP:0001250 Jejunal atresia HP:0005235	Chromosome 15 maternal uniparental disomy	Chromosome 15	upd(15)mat	UPD	N, prior CMA
063/ F/ 23	Nonimmune Hydrops Fetalis HP:0001790	Lymphatic malformation (OMIM #617300)	EPHB4	c.2288G>A, p.R763Q	Autosomal dominant-maternally inherited	N, prior CMA
064/ M/ 146	Cardiomyopathy HP:0001638	SCN5A-related dilated cardiomyopathy (OMIM #601154)	SCN5A	c.5129C>T, p. S1710L and c.680T>C, p.L227P	Autosomal semidominant- maternally and paternally inherited	Y, prior CMA
068/ M/ 5	Cardiomyopathy HP:0001638	MYH7-related cardiomyopathy (OMIM #613426)	MYH7	c.2292C>A, p.F764L	Autosomal dominant-paternally inherited	Y
078/ M/ 241	Congenital diaphragmatic hernia HP:0000776 Dysmorphic features HP:0000271 Congenital hypothyroidism HP:0000851	Cutis laxa (OMIM #613177) and *congenital hypothyroidism (OMIM #274700)	LTBP4 And TG	c.14T>G, p. V5G Homozygous And c.3217+5G>A and c.3999C>G, p.I1333M	Autosomal recessive And Autosomal recessive	N, prior CMA
Partial diagnosis
069/ M/ 13yrs	Cardiomyopathy HP:0001638 and intellectual disability HP:0001249	8p12 deletion	8p12	(29317218_32518884)x1	Autosomal dominant-paternally inherited	Y
009/ M/ 163	Liver failure HP:0001399 Hemolytic anemia HP:0001878 Cardiomyopathy HP:0001638	Hereditary spherocytosis (OMIM #182900)	ANK1	c.353C>A, p.A118E	Autosomal dominant-paternally inherited	N, prior HLH panel
074/ M/ 39	Respiratory failure HP:0002878 Hypoalbuminemia HP:0003073	Congenital analbuminemia (OMIM #616000)	ALB	c.412C>T, p.R138X and c.714–2A>G, IVS6–2A>G	Autosomal recessive	Y
035/ F/ 18	Arrhythmia HP:0011675 Cardiomyopathy HP:0001638 Brain malformation HP:0012443 Dysmorphic features HP:0000271	4H leukodystrophy (OMIM #614381)	POLR3B	c.1939G>A, p.E637K and c.237delG, p.M794CfsX16	Autosomal recessive	N, prior CMA

^†age at rES request in days unless otherwise noted

^*Denotes dual diagnosis

Seventy percent opted to receive for secondary findings and in 4 cases a secondary result was found. These included pathogenic or likely pathogenic variants in RET, PMP22 and BRCA2 (x2). In these 4 cases, the primary result was either negative or uncertain.

Of all patients who underwent rES, 52% had a change in clinical management including, in some cases, those with a negative result (Table 3). There were 41 total changes in management in 24 patients- 19 patients with a diagnostic test and 5 with a non-diagnostic test (Figure 3). There were nine changes in testing, nine changes in medication, five changes in imaging and eight changes in surgical planning. In five families, the results led to a change in goals of care such as limiting life-sustaining support or not escalating support. In five cases, the ICU physicians pursued a more aggressive course of care, such as ECMO support (case 032) or invasive diagnostic testing (case 079), because of negative rES results (Table 3).

Table 3.

Patients who experienced changes in management

	ID	Diagnosis or Phenotype	Medication	Testing	Imaging	Surgical Planning	Goals of Care
Neonates ≤28 days	022	Noonan syndrome and biotinidase deficiency	Biotin supplementation	1.Biotinidase activity 2. Coagulation studies	Renal US	-	-
	047	20p11 deletion	1.Levothyroxine 2.Hydrocortisone	Monitored LFTs and bilirubin levels	MRI brain	Proceeded with cardiac surgery	-
	012	Generalized arterial calcification of infancy	Bisphosphonates	Additional planned diagnostic testing cancelled	-	Avoided need for arterial biopsy	-
	031	Congenital myasthenia syndrome	Acetylcholinesterase inhibitors	Electromyography	-	-	-
	008	2q-	-	-	-	Planned surgical intervention for free air in the abdomen was cancelled due to poor neurologic prognosis	Limited life sustaining care
	020	1p36 deletion syndrome	-	1.TSH 2.Audiology evaluation	Renal US	-	-
	068	MYH7-related cardiomyopathy	-	-	-	Proceeded with heart transplant	-
	027	CAVA deficiency	Resumed a non-protein restricted diet and discharged with a no-protein sick day plan.	-	-	-	-
	035	Possible 4H leukodystrophy	-	-	-	Family declined heart transplant	Limited life sustaining care
	063	EPHB4-related lymphatic dysplasia	Sirolimus	-	-	-	-
	032	Cardiomyopathy	-	-	-	-	Escalated care- started on ECMO while awaiting transplant
	046	Respiratory failure	-	-	-	-	Escalated care-maintained continuous renal replacement therapy
Infants 1mo-1yr	074	Congenital analbuminemia	Increased albumin and weaned diuretics	-	-	-	-
	007	KANSL1-related disorder	-	-	Hip US	Family opted to proceed with G-tube placement given diagnosis associated with feeding difficulties	-
	016	Spondyloepiphys eal dysplasia congenita	-	-	Cranial US to evaluate for hydroceph alus	-	-
	054	upd(15)mat	-	Endocrine labs to evaluate for adrenal insufficiency	-	-	-
	029	LADD syndrome	-	-	-	Parents opted for tracheostomy and G-tube placement	-
	028	SCN8A-related early infantile epileptic encephalopathy	AEDs transitioned to sodium channel blocker	-	-	Given poor prognosis for recovery and high risk for sudden death from epilepsy (SUDEP), family opted for tracheostomy	Goal changed from cure to going home with home ventilator
	078	LTBP4-related cutis laxa and congenital hypothyroidism	-	-	-	-	Family agree to no further escalations of care
	044	Seizures					Escalated care-tracheostomy
	043	Post-natal hydrops					Escalated care-continued diuretic therapy for anasarca
	079	Severe ichthyosis					Escalated care-invasive testing including bone marrow and renal biopsies
Children >1yr	058	17q21 deletion	-	-	-	-	Limited life sustaining care
	037	Juvenile nephronophthisis	-	Ophthalmologic evaluation	-	-	-

Figure 3.

In 19 of 26 patients with a diagnostic rES, there was at least one change of clinical management, though most had more than one change. There were 36 changes in 5 categories displayed A(blue sections of pie on the right, % given as a percentage of total changes). Patients with non-diagnostic exomes also had changes of management. Of 22 patients with a non-diagnostic exome, 5 had escalations of care after return of results (orange section of pie on the right, % given as percentage of total changes).

Family cascade genetic testing or other medical evaluation was recommended for six families. In most cases this was an echocardiogram for parents and family members of a child with cardiomyopathy. Family cascade testing or other medical evaluation was not classified as a change in management.

DISCUSSION

We studied trio rES in 46 children in ICUs at a single tertiary institution. We demonstrated an overall diagnostic rate of 43%. An additional 30% had an uncertain result or partial diagnosis. Two patients had dual diagnoses (Case 022: Noonan syndrome and biotinidase deficiency; Case 078: cutis laxa and congenital hypothyroidism). Twenty four participants (52%) experienced a change in medical or surgical management as a direct consequence of the exome sequencing result (Figure 3 and Table 3). This includes 5 patients (032, 046, 044, 043, and 079) in which a non-diagnostic rES result led to in a change in management (Figure 3 and Table 3).

Previous studies that have demonstrated clinical utility of rapid exome or rapid genome sequencing.^4,6–13 However, our study is unique in that all families were provided with pre-test counseling and were given the option to receive secondary findings. Our study did not perform rES in-house, but sent samples out to a laboratory (GeneDX), making it applicable to a larger number of pediatric institutions. Our study was done as a part of routine clinical care and was not subsidized by research or other funds and our study did not consider determining recurrence risk for family planning and family cascade testing/screening as a change in managemen. Our study tracked variant of uncertain significance (VUS) resolution testing. Our results represent an approach to ICU based rapid genomic diagnostics that can be more broadly representative of clinical practice.

Given the high cost of rES and potential for errors in ordering this test, we established a committee of 4 clinical geneticists who had to review each request.¹⁶ Although the inclusion of a review committee added an extra day to the process, the review process was standardized, and testing performed if clinically indicated. Consensus criteria for determining which neonates benefit from rapid testing have only recently emerged, and at the present most health plans in the United States still do not cover this type of testing. We anticipate that results from our study and others will lead to more standardized criteria and health plan coverage changes, obviating the need for review committees, and assisting with coverage for testing.

The median TAT from rES request to verbal result was 9 calendar days. Although this is slower than what has been reported with ultra-rapid genome sequencing,²⁴ it is comparable with several previous reports.^7–13 Previous studies of rapid sequencing have often focused on the speed of the test itself- that is, the DNA sequencing and variant interpretation.^24,25 It is important to note that our median 9 day TAT includes steps (pretest counseling, coordination of parental samples, shipping to the send out laboratory) upstream of DNA extraction. Nonetheless, there are several ways our process could be sped up further. As previously mentioned, removing the requirement of committee review could shorten the process by 1 day. However, the biggest delaying factor was parental coordination, including sample collection, which has been demonstrated in previous studies.^{13, 26} For example, the mothers of several infants were not available at the time of genetics consultation because they remained at the birthing hospital. It is possible that proband-only analysis, which does not require parental samples, could improve TAT without significantly sacrificing diagnostic yield.²⁷ TAT will also continue to improve with the application of more advanced automated variant interpretation algorithms.²⁵

Another source of delay to diagnosis was our requirement of non-diagnostic CMA as an inclusion criterion. Partway through our study, the reference laboratory included CNV detection as a part of rES, and this inclusion criterion was removed. Subsequently, six patients were identified to have a CNV by rES. The smallest detected CNV was a biallelic 102kb deletion of NPHP1 causing juvenile nephronophthisis in case 037 (Table 2). This supports the use of rES as a first line test to detect both SNVs and CNVs to reduce diagnostic delay. This is consistent with previous studies that have suggested higher diagnostic utility for exome sequencing compared with CMA, particularly in hospitalized children.²⁷

In several cases, the results from rES led to medical therapies that would not have otherwise been initiated (Table 3). A neonate (case 031) with arthrogryposis was found to have CHRNB1-related congenital myasthenia syndrome. After confirming pyridostigmine-responsive myasthenia on electromyography, the patient was started on acetylcholinesterase inhibitors which permitted extubation. A hydropic infant (case 012), with possible generalized arterial calcification of infancy based on imaging was able to be placed on bisphosphonates after this diagnosis was molecularly confirmed. A four month old with seizures and abnormal movements (case 028) was changed to a sodium channel blocking anti-epileptic medication after SCN8A-related epilepsy was identified. Sirolimus was started in a neonate with hydrops (case 063) after EPHB4-related primary lymphatic dysplasia was diagnosed.

Primary respiratory failure in neonates was a common indication for rES and highlights the importance of coordination with pathology, as traditionally infants with severe interstitial lung disease have been diagnosed via invasive lung biopsy.²⁸ In case 030, rES was sent to avoid lung biopsy while on ECMO (Table 2). However, the patient clinically worsened and a lung biopsy provided a diagnosis of acinar dysplasia prior to rES results. Life sustaining support was discontinued, thus case 030 represents an example of a diagnostic result that did not change patient management. Nonetheless, the identification of a de novo 17q23 deletion as the cause of this child’s lung disease, even if reported posthumously, provided closure and assistance in family planning.²⁹ In the future rapid genome sequencing, which has the potential for even faster TATs, will likely replace rES as the primary diagnostic test. Indeed several publications have already shown the clinical utility of rapid genome sequencing, though this test is not yet broadly clinically available.^4–15

In four cases, an inherited dominant diagnosis was made. In one of those cases, a neonate with severe cardiomyopathy, (Table 2, case 068), the father was identified to be mosaic for a pathogenic variant by exome sequencing. He was asymptomatic but an echocardiogram was recommended. In two cases, 022 and 063 (Table 2), the parent was diagnosed for the first time. In case 022, the infant was prenatally identified to have hydrops and found to have Noonan syndrome. The patient’s mother had a history of pulmonic stenosis requiring repair in her 20s but was otherwise well and had not previously been diagnosed with Noonan syndrome. In case 063, a maternal family history of lymphedema was identified but the cause was not known. Lastly, case 048 presented with severe respiratory failure and was found to have LADD (Lacrimo-Auriculo-Dento-Digital) syndrome. Her father and other paternal family members had had lacrimal duct stenosis as children but were otherwise healthy.

A major impact of rES results on patient care was guiding goals of care (Table 3). We found that both diagnostic and non-diagnostic rES could change goals of care in both directions-towards more and less aggressive care. For example, a decision to limit aggressive medical intervention was made for a neonate with MCA (case 008) after rES identified a genetic diagnosis consistent with poor neurologic prognosis (chromosome 2q deletion). In contrast, continued aggressive care was chosen for an infant (case 016) with a severe skeletal dysplasia whose rES results (COL2A1-related spondyloepiphyseal dysplasia) predicted normal neurologic outcome. A non-diagnostic rES result led to the decision to begin ECMO support in an infant with hydrops and cardiomyopathy (case 032), as the result reduced the probability of a syndromic diagnosis.

Previous reports of rapid genomic testing, many of which have taken place in the research setting, have primarily reported only diagnostic or non-diagnostic results.^4,6–15 This is not representative of standard clinical genetic test results, which will include VUSs and other inconclusive results. Additional follow up testing to further classify VUSs is commonly required in standard clinical genetic practice. In this study, we tracked both the number of inconclusive results and the health care burden associated with those. Ten patients (22%) had additional actions due to inconclusive results, including 13 additional laboratory or imaging tests. Two patients were enrolled in research studies to further investigate a VUS. We did not include any of this testing as a change in management, but it is important that future studies examining cost-effectiveness of rES track the burden associated with follow up testing. Although this study does not address the cost-effectiveness of rES in the ICU, previous studies have demonstrated the potential for rapid genetic diagnosis to significantly reduce inpatient costs.^4,17

Because all testing was done as trios, we were able to distinguish between inherited and de novo variants. For 11 trios, the pathogenic variant(s) were inherited, conferring a significant recurrence risk for subsequent pregnancies. We chose not to include changes in reproductive risk counseling as a “change in management” in this study. This information, although extremely valuable to families, does not change inpatient management. Additionally, insurance companies do not generally reimburse genetic testing solely for the purpose of family planning. Nonetheless, information regarding recurrence risk counseling is routinely obtained from diagnostic rES and can provide an added benefit to families of children who are critically ill.

Few previous studies of rapid genomic testing within ICUs have reported secondary findings.^4,6–13 In one previous study, 7 of 267 participants had medically-actionable secondary findings.⁹ In our study, 70% of patients opted to receive secondary findings. Four patients (9%) had secondary findings inherited from a parent who also received the results. In all four patients, the rES was non-diagnostic for the primary test indication which led to challenging post-test counseling discussions.²⁶

Our study provides evidence that trio rES for critically ill children with a suspected monogenic disorder has had a significant impact on medical care within our patient population. We have demonstrated that rES has led to a change in management in over 50% of patients tested. Because this test is able to detect both CNVs and SNVs, rES should be considered a first tier test in critically ill children with suspected genetic disease. We believe this test should become standard of care for tertiary institutions that can provide coordinated genetics expertise.

Abbreviations

AR

autosomal recessive

CMA

chromosomal microarray

CNV

copy number variant

ECMO

extracorporeal membrane oxygenation

ES

exome sequencing

GC

genetic counselor

ICU

intensive care unit

MCA

multiple congenital anomalies

rES

rapid exome sequencing

SNV

single nucleotide variant

UPD

uniparental disomy

VUS

variant of uncertain significance