Role of Next generation sequencing in diagnosis and management of critically ill children with suspected monogenic disorder

doi:10.21203/rs.3.rs-3220389/v1

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Role of Next generation sequencing in diagnosis and management of critically ill children with suspected monogenic disorder

https://doi.org/10.21203/rs.3.rs-3220389/v1

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published 11 Apr, 2024

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Next generation sequencing based diagnosis has emerged as a promising tool for evaluating critically ill neonates and children. However, there is little data on its utility in developing countries. We assessed its diagnostic rate and clinical impact on management of paediatric patients with a suspected genetic disorder requiring critical care. We analyzed 70 children with an illness requiring intensive care and obtained a precise molecular diagnosis in 32 of 70 probands (45.3 %) using diverse sequencing techniques such as clinical exome, whole exome, and whole genome. A significant change in clinical outcome was observed in 13 of 32 (40.6 %) diagnosed probands with a change in medication in 11 subjects and redirection to palliative care in two subjects. Additional benefits included specific dietary management (three cases), avoidance of a major procedure (one case) and better reproductive counselling. Dramatic therapeutic responses were observed in three cases with SCN1A, SCN2A and KCNQ2-related epileptic encephalopathy. A delayed turn-around for sequencing results was perceived as a major limiting factor in the study, as rapid and ultra-rapid sequencing was not available. Achieving a precise molecular diagnosis has great utility in managing critically ill patients with suspected genetic disorders in developing countries.

Biological sciences/Genetics/Medical genetics

Health sciences/Health care/Paediatrics

Next generation sequencing

NGS

critical illness

monogenic

outcome

whole exome sequencing

Next generation sequencing (NGS) technology has emerged as an essential tool for the evaluation of a heterogeneous cohort of individuals with genetic disorders. ^{1 2} It has transformed medical practice by accelerating and simplifying genetic diagnosis by expanding the net of differential diagnosis. ^{3 4} With rapidly decreasing costs, this technology has been increasingly used in clinical practice. There are limited studies assessing the utility of NGS in intensive care units. It is well recognized that these patients are enriched for genetic disease. ⁵ The rapidly evolving clinical situation in the emotionally charged setting of critical care necessitates timely patient identification, skilled assessment, and sensitive handling of complex genetic issues. We describe our experience of using different NGS based techniques in children with critical illness.

The study was conducted at a genetic center in affiliation with the intensive care of a tertiary care hospital in Northern India. The subjects comprised neonates and children (up to 18 years), who had a suspected Mendelian disorder and were admitted in the intensive care unit. The study was approved by the institution ethics committee (EC/07/18/1384).

Study Design: A multidisciplinary working group developed the patient selection criteria with the clinical geneticist as a core coordinator. Standard operating procedures were designed for patient recruitment, informed consent, test performed, report analysis and communication with parents. We enrolled subjects from July 2015 through June 2020 and included patients retrospectively (after discharge from the NICU/PICU), as well as prospectively (during the NICU/PICU admission). All patients had at least one of the following features: one or more congenital malformations, dysmorphic features, abnormalities in growth, neurologic impairment (including encephalopathy, seizures or hypotonia), features suggestive of a metabolic condition, multisystem, unexplained, or atypical presentation. Clinical phenotype was reported in HPO terms. Relevant investigations including metabolic tests, radiographs, echocardiogram, or Magnetic Resonance Imaging (MRI) were performed as part of standard care. If a definitive diagnosis was not established by these investigations, the family was counselled for next-generation sequencing tests. New-borns with suspected chromosomal disorders (e.g. trisomy 18) or specific monogenic conditions (e.g. spinal muscular atrophy) or largely non-genetic (e.g. congenital infections) were excluded.

An expert panel of investigators (SB, RP) determined patient eligibility. A second panel of clinicians (SBM, ICV) participated as required for further assessments and discussions. In case of a disagreement among panel members regarding eligibility, majority approval determined the further course. The overall process of enrollment along with inclusion and exclusion criteria is summarized in esupplement 1.

Data Analysis and Interpretation

Next-generation sequencing was performed as panel/clinical exome/whole exome or genome in solo/trio as feasible and based on recommendations of the geneticist. Clinical exome included analysis of exons of all clinically validated genes listed in OMIM. Variant classification based on the standards of American College of Medical Genetics and Genomics (ACMG) was done with a phenotype driven approach. ⁶ A case was considered diagnosed if a pathogenic or likely pathogenic variant was identified. Variants of Uncertain Significance (VUS) were analysed by clinical correlation, literature review, segregation analysis and raw data reanalysis on a case-to-case basis. Sanger validation, dosage analysis by MLPA / real time PCR, and mRNA studies were done as required to confirm a definitive diagnosis. All novel pathogenic and likely pathogenic variants identified in this study have been deposited in ClinVar.⁷ (Submission Ids: SCV001434393, SCV002097319, SCV002097320, SCV002097321, SCV002097322, SCV002097323, SCV002097324)

Outcome Measures and Statistical Analysis

The primary outcome measure was diagnostic yield. The secondary outcome measure was the clinical impact of the definite molecular genetic diagnosis. This was assessed as impact on change in medication and/or diet (initiation or discontinuation), need/deferral of a major procedure, palliative therapy and genetic counselling. A single patient could have more than one outcome measure change. The number of families opting for prenatal diagnosis during next pregnancy and its outcome was also assessed. Descriptive statistics were used for participant characteristics, referral site, interval between symptom onset and genetic testing, turnaround time of sequencing, time to result, change in management and reproductive counselling. Data collection was complete, and no patients were lost to follow up.

Participant demographics and Indications for testing

Over 5 years, 75 patients were evaluated. Five patients did not meet the study criteria and were excluded (non-genetic aetiology “one”, chromosomal in “two”, specific genetic testing available in two patients). Finally, 70 participants were enrolled for further analysis by NGS.

The study group comprised of seven neonates, 25 infants and 38 children above one year of age. The average age at enrolment was 2.47 years (01 day − 13 years) and the median age was 1.29 yrs. Patients were divided into two groups- group I included 40 patients with a critical illness episode requiring NICU admission and group II had 30 infants/children with a post-neonatal critical illness requiring PICU care. In the NICU cohort, 11 neonates had perinatal asphyxia while seven had hypoglycemia. Other presentations included meconium aspiration syndrome, respiratory distress, cardiac involvement, dyselectrolytemia and sepsis. Seven probands had died before NGS could be done, and the test was performed on stored DNA, whereas 12 patients expired later during the study. The patient characteristics are summarized in Table 1.

Table 1

Demographic data of children enrolled (N = 70)
Criteria	Total 70	Diagnosed 32 (45.7%)	Undiagnosed 38 (54.3%)
Age ( at presentation) Less than one month One mth-1 year More than 1 year	07 25 38	03 (42.9%) 15 (60%) 14 (36.8%)	04 (57.1%) 10 (40%) 24 (63.2%)
Critical illness NICU PICU	40 30	20 (50%) 12 (40%)	20 (50%) 18 (60%)
Gender Male Female	46 24	16 (34.8%) 16 (66.7%)	30 (65.2%) 08 (33.3%)
Consanguineous parents Yes No	8 62	4 (50%) 28 (45.2%)	4 (50%) 34 (54.8%)
Sibling affected Yes No	13 57	5 (38.5%) 27 (47.4%)	8 (61.5%) 30 (52.7%)
Proband alive till completion of study Yes No Test done after death Proband died later	51 19 7 12	21 (41.2%) 11 (57.9%) 6 (85.7%) 5 (41.7%)	30 (58.9%) 8 (42.1%) 1 (14.3%) 7 (58.3%)

Patient Phenotype

The presenting phenotype was neurological in 37 of 70 (52.8%) probands, multi-system in 17 (24.3%), gastrointestinal / hepatic in 5 (7.1%), and haematologic and cardiovascular in 3 (4.3%) each [Fig. 1a]. The remaining presentations included one each of congenital adrenal hypoplasia, diabetes insipidus, pseudo-Bartter syndrome, renal failure and surfactant deficiency. Recurrent seizures were present in 37 probands, neuro-metabolic phenotype in 16 and neuro-regression in six. Global developmental delay (GDD) or intellectual disability (ID) was present in 38 probands at the time of critical care admission. Dysmorphism or malformations were documented in 20 patients.

Interval between critical illness and NGS (Time to testing)

The time gap between critical illness and NGS showed a wide variation ranging from 1 day to 12 years. Nineteen patients were evaluated during critical illness, including 7 in NICU and 12 in PICU. Remaining 51 patients were seen post critical illness. Of the seven neonates who were considered to have a genetic illness, five underwent NGS during the NICU admission, whereas in two neonates, it was done later. NGS was done in 12 infants / children during critical care admission in PICU. In the remaining 53 probands, NGS was done at a later time i.e. post-intensive care illness in NICU/PICU. There was a gap of more than one year between the critical illness and NGS in 25 children. The average turnaround time of standard NGS in the study was 43 days (range 14–56 days) except in one case with neonatal seizures, where the result was provided within two weeks resulting in early seizure control.

Next-generation sequencing results

Clinical exome (CE) comprising od clinically validated OMIM genes was done in 51 patients, whole-exome solo in 13, panel testing in six, and whole-genome in one patient. The test was done predominantly as a “solo” test, except in two patients where “trio” whole-exome sequencing [WES] of the proband, mother and father was done. The diagnostic yield in the present study was 45.7% with a genetic diagnosis established in 32 of 70 probands [Fig. 1b].

A definite genetic diagnosis was established in four out of eight (50%) probands with consanguineous parents. Amongst 13 families, with a previous history of similarly affected sibling, genetic diagnosis was made in five probands (38.5%). The cohort with recurrent seizures had a diagnostic yield of 45.9% and within this cohort, the yield in the sub-group of epileptic encephalopathy was 55%. The cohorts with CNS, GIT, haematologic or multisystem illness did not show any statistical difference in diagnostic yield, though these have a small sample size. Three infants with a predominant CVS phenotype were also included in the study, but no specific diagnosis was obtained by NGS.

The diagnostic yield in the cohorts by phenotype and by type of NGS are presented in Table 2 and Table 3 respectively.

Table 2

Clinical features of the patient cohort and diagnostic yield (N = 70)
Feature	Total 70	Diagnosed 32 [45.7%]	Undiagnosed 38 [54.3%]
Primary phenotype CNS GIT/hepatic Haematologic CVS Multi-system Others (One case each)	37 5 3 3 17 5	16 [43.2%] 1 [20%] 2 [66.7%] 0 9 [53%] 4 [80%]	21[56.8%] 4 [80%] 1 [33.3%] 3 [100%] 8 [47%] 1 [20%]
GDD/Intellectual disability Present Absent	38 32	18 [47.4%] 14 [43.8%]	20 [52.6%] 18[56.2%]
Dysmorphism/ Malformation Present Absent	20 50	7 [35%] 25 [50%]	13 [65%] 25 [50%]
Neuro-regression	6	3 [50%]	3 {50%]
Epileptic Encephalopathy	20	11 [55%]	9 [45%]
Neurometabolic	16	5 [31.2%]	11[68.8%]
Recurrent seizures	37	17 [46%]	20 [54%]

Table 3

Genotypic and phenotypic distribution by type of NGS technique
NGS	OMIM#	Gene	Disorder
NGS	OMIM#	Gene	Phenotype – Neurological (16)	Phenotype-GIT (1) Haematology (2)	Phenotype - Multi-system (9)	Misc (4)
Clinical Exome	219700 610188 266100 603553 237300 163950 221750 612164 617166 304800 202700 616211 311250 300200 607208 252160 241200 613721	CFTR CEP 290 ALDH7A1 PRF1 CPS1 PTPN11 LHX3 STXBP1 FGF12 AVPR2 ELANE WWOX OTC NR0B1 SCN1A MOCS2 KCNJ1 SCN2A	Pyridoxine dependent Epilepsy Hyperammonaemia due to carbamoyl phosphate synthase deficiency EIEE-4 EIEE-47 EIEE-28 Ornithine trans- carbamylase deficiency EIEE-6 Molybdenum cofactor def B EIEE-11	Familial hemophagocytic lymphohistiocytosis-2 Severe congenital neutropenia-1	Joubert syndrome type 5 Noonan syndrome 1 Combined Pituitary Hormone Deficiency type 3 Antenatal bartter syndrome type 2	Cystic Fibrosis Nephrogenic DI CAH with hypogonadotrophic hypogonadism
NGS	OMIM#	Gene	Disorder
NGS	OMIM#	Gene	Phenotype –CNS (16)	Phenotype- GIT(1) Haematology (2)	Phenotype - Multi-system (9)	Misc (4)
Neurology panel	615471	FBXL4	Encephalomyopathic mitochondrial DNA depletion syndrome-13
Epileptic panel	607208	SCN1A	EIEE-6
Clinical exome with del/dup	613720 219700	KCNQ2 CFTR	EIEE7		Cystic Fibrosis
Whole-exome solo	609015 272800 618285 612949 610921 203700 245349	HADHA HEXA CACNA1E SLC25A12 ABCA3 POLG PDHX	Tay-Sachs disease EIEE-69 Lactic acidaemia due to PDX1 deficiency		Trifunctional protein deficiency EIEE39 Mitochondrial DNA depletion syndrome 4A	Surfactant metabolism dysfunction pulmonary, 3
Trio whole-exome	251880 618744	DGUOK UGP2		Mitochondrial DNA depletion syndrome 3	EIEE83
Whole-genome	618580	PIGB	EIEE-80

NGS variant analysis

Ninety-Six variants relevant to the phenotype were detected in the NGS analysis. These included 85 single nucleotide variants (SNV), which comprised of 68 missense (14 homozygous and 40 heterozygous), nine nonsense and eight splice–site variants; along with three in-frame indels, and six out of frame indels and one homozygous exonic deletion.

A “no variant” call was received in 11 of 70 cases, “variant of uncertain significance” in 35, likely pathogenic in 11 and pathogenic in 13 cases, resulting in a preliminary diagnosis in 24 out of 70 cases (34.3%). Clinical correlation, literature review, Sanger validation and segregation analysis assisted in nine variants being reclassified as benign / likely benign, and seven as likely pathogenic variants. A separate consent was sought for study of incidental findings during pretest counselling. ⁸ Four families had consented for the same but no pathogenic variant in genes unrelated to the phenotype was detected.

Fastq files of NGS were re-examined in eight undiagnosed cases and did not result in any change in classification of the variants in seven cases. However, in one case which had already undergone CE in 2016 followed by trio WES in 2018, reanalysis of the data identified a homozygous c.1A > G UGP2 variant, which disrupts the translational start site in the shorter isoform (NM_001001521), reported as pathogenic in Dec 2019. Thus, the final diagnosis was established in 32 of 70 cases (45.7%), which included 24 diagnosed by preliminary NGS analysis, 7 by reclassification of VUS and one after data reanalysis. Of the 32 diagnosed cases, nine were autosomal dominant, 20 autosomal recessive and three X-linked disorders. Seven novel variants have been detected in this study.

Secondary end points

Impact on clinical management

Establishing a definite diagnosis altered the medical management in 11 (34.4%) of 32 patients. These included two cases of SCN1A and one case each of ALDH7A1, AVPR2, CFTR, CPS1, ELANE, KCNQ2, OTC, PTPN11 and SCN2A related genetic disorder [Table 4].

Table 4

Impact of a definitive diagnosis on management
Pt	Phenotype	Gene	Diagnosis	Drugs	Diet/Invasive procedure/ Specialist referral
2 year F	Onset day1, recurrent seizures	ALDH7A1 : c.1411-1412insG, c.187G > T NM_001182.5	Pyridoxine dependent epilepsy	Pyridoxine supplementation
10 mth M	Neonatal onset recurrent vomiting, polyuria	AVPR2 : c.185T > C, hemizygous NM_000054.6	Nephrogenic Diabetes Insipidus	Diuretic, Indomethacin
4 mth F	Onset 1mth, Fever, seizures, resp infection	CFTR : c.1521_1523 delCTT c.1646G > A NM_000492.4	Cystic Fibrosis	Pancreatic enzyme supplementation, azithromycin prophylaxis	Paediatric Gastroenterologist
8 days M	Onset day 3, Lethargy, vomiting, seizures	CPS1 : c.236 + 4A > G, homozygous NM_001875.5	Hyperammonaemia carbamoyl phosphate synthase deficiency	Sodium benzoate, Arginine	Protein restricted diet
2.5 yrs F	Onset 1 year, Recurrent mucosal infections	ELANE : c.182C > T heterozygous NM_001972.4	Severe congenital neutropenia	Inj G-CSF three times a week
1 mth F	Neonatal seizures day 1, encephalopathy	KCNQ2 : c.629G > A heterozygous NM_172107.4	EIEE7	Carbamazepine
5 yrs M	Onset 2 mths, recurrent lethargy, vomiting	OTC : c.386G > A hemizygous NM_00051.6	Ornithine transcarbamylase deficiency	Sodium benzoate	Protein restricted diet
3 mth M	Neonatal, facial dysmorphism, heart disease, juvenile myelomonocytic leukaemia	PTPN11 : c.218C > T, heterozygous NM_002834.5	Noonan syndrome 1	6-mercaptopurine	Avoidance of Haematopoietic stem cell transplant, Endocrinologist
7 mth M	Onset 5 mths, Recurrent seizures	SCN1A : c.765_766 delGT heterozygous NM_001165963.4	EIEE6	Valproate, clobazam, potassium bromide	Ketogenic diet
3 yrs M	Onset 15mths, Febrile seizures, shock	SCN1A : c.1837C > T heterozygous NM_001165963.4	EIEE6	Valproate
5 yrs F	Neonatal seizures day 3	SCN2A : c. 751G > A heterozygous NM_001040142.2	EIEE11	Carbamazepine

Specialized dietary management was advised in three patients; two with urea cycle defect were recommended a protein-restricted diet and one patient with SCN1A related EIEE6 was advised ketogenic diet. Two families opted for palliative care in view of diagnosis of Tay-Sachs disease and congenital surfactant deficiency disease [Fig. 2]. One infant was being considered for haematopoietic stem cell transplant for juvenile myelomonocytic leukaemia, which was withheld after molecular diagnosis of Noonan syndrome 1. Knowledge of the diagnosis provided useful and beneficial information about the natural history and prognosis, allowing accurate counselling about recurrence risk in all probands.

Brief description of some cases with exemplary response to genotype differentiated therapy

One-month old girl with features of epileptic encephalopathy from day 1 on multiple AEDs (phenobarbitone, phenytoin, levetiracetam, topiramate) along with biotin, pyridoxine and riboflavin was diagnosed within 14 days. Extensive metabolic testing with urine GCMS, TMS, serum ammonia, lactate and CSF neurotransmitter analysis was non-diagnostic. A heterozygous missense, previously reported variant in KCNQ2, c.629 G > A, NM_172107.3 (p.Arg210His) causing EIEE-7 was identified. Inclusion of carbamazepine to the management protocol resulted in dramatic control of seizures, which may be considered as an exemplary precision therapy. Parental testing confirmed the “de novo” nature of the variant and other AEDs were gradually tapered. At the 18 months follow up, parents reported that the child was seizure-free from the day of starting of carbamazepine, and has normal motor, language, and cognitive development.

A seven-month boy with recurrent prolonged febrile seizures was diagnosed to have SCN1A related EIEE-6 and subsequent guided therapy with valproate, clobazam, potassium bromide and ketogenic diet resulted in partial control of seizures. In view of the genetic variation, follow up and early intervention for speech was advised.

A five-year girl with seizure onset at day 3 of life, followed by recurrent seizures of tonic-clonic and absence type with mild developmental delay was diagnosed to have SCN2A related EIEE-11. Early-onset seizures in SCN2A are often gain of function mutations which respond well to sodium channel blockers. The child responded to the addition of carbamazepine and is seizure-free at one year follow up.

Reproductive outcomes in families

During the limited study period, ten couples had planned pregnancy and requested for prenatal testing by CVS, which includes two couples requesting for a second time, leading to a total of 12 procedures. Two of these were done for autosomal dominant disorders due to CACNA1E related developmental and epileptic encephalopathy (DEE) 69, and FGF12 related DEE 47 pathogenic variations. Both fetuses were unaffected. The remaining ten tests (in eight families) were done for autosomal recessive disorders. Two fetuses in the same family were consecutively affected, and pregnancy was terminated as per the decision of family. The remaining eight pregnancies were unaffected and were continued to term with birth of normal babies.

A brief summary of diagnosed cases is presented in eSupplement 2

Utility of NGS dataset analysis in critical care setting has been evaluated in recent times and the results appear promising. ^{9 10 11 12 13 14 15 16 17 18 19 20} We examined NGS as a diagnostic tool in 70 critically ill neonates and children suspected to have a monogenic disorder and achieved a diagnostic yield of 45.7%. The impact on management of this definitive diagnosis was observed in 13 of 32 (40.6%) probands with alteration of medication, diet, invasive procedure, and palliative care. The study is unique in that it incorporates a combination of diverse datasets frequently used in clinical practice thus more closely reflecting the actual utility of NGS by the bedside. As most of the studies are from developed nations with access to testing, this is the first study from a developing country context where most genetic testing is not funded and therefore informed, cost effective testing options are essential.

We report a diagnostic rate of 45.7% (32 of 70), slightly higher (50%, 20 of 40) in the NICU cohort as compared to PICU cohort (40%, 12 of 30), though the difference was not statistically significant (p = 0.40). The diagnostic yield of conventional genetic testing without NGS is reported at 26% (34 of 132) in NICU cohort ²¹. The median diagnostic yield is 38.9% on comparing 18 NGS based genomic studies ^{3 9 10 11 12 13 14 15 16 17 18 22 23 24 25 26 27 28} in the intensive care setting. (Table 5)

Table 5

Comparison of the present study and previous studies of NGS in paediatric critical care
S.No	Study population	Type of NGS	Overall diagnostic yield	Time to result (days)	First Author, year
1	Critically ill infants in NICU	rWGS	11/15 (73%)	12	Soden SE ³, 2014
2	Critically ill infants < 4 mths	Rapid GS	20/35 (57%)	23	Willig LK ⁹, 2015
3	Newborns and infants in NICU	Targeted WES	8/20 (40%)	14 to 406	Daoud H ¹⁰, 2016
4	Critically ill infants in ICU	Targeted rWES	7/23 (30%)	12	van Diemen CC ¹¹, 2017
5	Infants < 100 days in intensive care	ES	102/278 (37%), Critical trio 32/63(51%), Trio 13/39 (33%), Solo 57/176 (32%)	13	Meng L ¹², 2017
6	Acutely unwell paediatric patients	rWES	21/40 (52%)	16	Stark Z ¹³, 2018
7	Infants < 4 mths in NICU/PICU	rWGS trio	12/37 (32%)	14	Petrikin JE ¹⁴; 2018
8	Acutely ill inpatient infants	rWGS	18/42 (43%)	2–5	Farnaes L ¹⁵, 2018
9	PICU children,	rWGS trio	10/24 (42%)	9	Mestek-Boukhibar L ¹⁶, 2018
10	PICU children 4mth-18 yrs	rWGS	17/38 (45%)	14	Sanford EF ²², 2019
11	Intensively ill, 1 day-15 yrs in NICU/PICU	WGS trio	40/195 (21%)	21	French CE ¹⁷, 2019
12	Ill newborns in NICU	nGS	0/29 ( 0%), Inconclusive 5/29 (17%)	-	Ceyhan-Birsoy O ¹⁸, 2019
13	Critically ill infants < 4 mths, NICU/PICU CVICU	Total urWGS, rWGS, rWES	48/213 (22%) 11/24 (46%) 18/94 (19%) 19/95 (20%)	4.6–11 4.6 11 11	Kingsmore SF ²³, 2019
14	Exome sequencing in neonates	Diagnostic exome sequencing	25/66 (37.9%)	8	Powis Z ²⁴, 2018
15	Genetic defects in NICU	Medical Exome with CNV analysis	284/2303(12.3%)	-	Yang L ²⁵, 2022
16	Intensive care in < 6 months	Rapid Trio-whole exome sequencing	29/50 (58%)	7	Gubbels CS ²⁶,2020
17	NICU clinical care	Rapid clinical exome	22/80(28%)	13	D'Gama AM ²⁷,2022
18	Critically ill infants	Rapid trio- exome sequencing	6/15 (40%)	16	Wells CF ²⁸,2022
19	Critically ill neonates and children, 1 day-18 yrs, NICU/PICU	NGS (Panel, CE, WES, WGS), solo/trio	32/70 (46%)	43	Present study

In a developing country clinical/ hospital setting with restricted resources and predominant out of pocket expenditure, testing phenotype relevant genes remains the first option. However, with decreasing sequencing costs, trends towards exome sequencing are evolving. ^{1 29} We performed panel testing in six patients, clinical exome sequencing (CES) in 51, WES in 15 and Genome sequencing (GS) in one patient. The diverse datasets analysed closely reflects the changing scenarios in the last decade from evaluating the OMIM morbid genes in CES towards WES/GS. A review of 171 studies by Smith et al.³⁰ reported the use of ES in 93% (159/171), GS in 6% (10/171) and combined ES and GS in 1% (2/171) studies, which is similar to our dataset distribution.

The diagnostic success rates reported by a focused panel approach by Daoud et al. ¹⁰ comprising of 4813 genes in a cohort of 20 NICU babies was 40%. A slightly higher diagnostic success was reported in a study of WGS in children with previous inpatient stay in critical care by Willig et al⁹ with the diagnosis being achieved in 20 out of 35 (57%) cases. We also noticed a similar trend of increasing diagnostic rate with the use of a broader dataset in our study- 20 out of 51 (39.2%) with clinical exome sequencing, 9 of 15 (60%) with ES and the one patient who underwent genome sequencing, achieved a genetic diagnosis.

The incidence of genetic disease among seriously ill infants with disease of unknown aetiology enrolled in the NSIGHT2 trial ²³ was 24% (52 of 213 infants). The diagnostic yield was 46% (11 of 24) in the gravely ill sub-group where ultrarapid WGS (urWGS) reporting in 4.6 days was used, compared to 20% (37 of 189) in the rWES/ rWGS group with TAT of 11 days. Though we were not able to do rapid WES/WGS, we established a definitive molecular diagnosis in 20 of 40 (50%) probands in NICU cohort by standard NGS in 4–6 weeks. The higher diagnostic yield observed in our study could have been due to a small, highly curated cohort where all patients were evaluated by a geneticist prior to NGS testing, leading to increased pre-test probability of a genetic diagnosis. Diagnostic yield has been further augmented by detailed phenotyping (case one), functional testing (case two) and data reanalysis (case three). (eSupplement 2)

In our cohort, seven out of 11 patients who had perinatal asphyxia were confirmed to have a genetic diagnosis. These included patients with CEP290 related Joubert syndrome, STXBP1 related EIEE4, LHX3 related combined pituitary hormone deficiency type 3, CACNA1E related EIEE69, PDHX related pyruvate dehydrogenase E3 binding protein deficiency, UGP2 related EIEE83 and WWOX related EIEE28. Thus, the mere presence of perinatal asphyxia in the history should not be considered as a deterrent to genetic testing. Genetic and metabolic causes of neonatal encephalopathy may be mistaken for hypoxic ischaemic encephalopathy. This may lead to sub-optimal testing and missed opportunities to establish correct diagnosis. An apparent recurrence of perinatal asphyxia was observed in two families; Joubert syndrome type 5 and combined pituitary hormone deficiency, where a previous sibling had died with respiratory distress soon after birth considered to be due to hypoxic ischaemic encephalopathy (HIE). The evaluation to identify genetic and metabolic aetiology in neonatal encephalopathy should be done to distinguish it from HIE as identifying the exact cause of encephalopathy has important implications for treatment as well as recurrence risks. ^{31 32}

Time-to-diagnosis is an essential consideration in critically ill neonates and children. ^{33 34} Soden et al ³ reported a 77 month delay between symptom onset and NGS in ambulant patients with neurodevelopmental delay and a time to diagnosis of 11.5 months by WES, which could be reduced by 10 months by rWGS in high acuity patients. In our study, the time gap between critical illness and ordering NGS varied from one day to 12 years. While 17 (24%) of 70 probands underwent NGS during the critical illness, there was an interval of less than one month in seven (10%), one month to one year in 21 (30%) patients, while 25 (36%) probands underwent testing after a gap of more than one year. Of the 36% (9 of 25) patients with delayed diagnosis of more than one year, precision therapy could have been initiated earlier in one child with EIEE-11 genotype.

The interval between critical illness and NGS could be because of limited appreciation of the benefits of testing in the critical care setting where active management of a sick baby is the prime concern. Additionally, lack of consent by the grieving family who cannot appreciate the utility of testing along with resource and economic limitation also contribute to the delay. We observed that many families come for genetic consultation for recurrence information during the next pregnancy as seen in 20% families in this study. Rapid diagnosis necessitates trio sequencing as previously reported but cost of testing can be challenging in a diagnostic setting as observed in the current study.

The median turnaround time in the present study was 43 days for the standard NGS, which is not optimal for therapeutic utilisation in acute care. We did not have access to rapid or ultrarapid WES/WGS. Turnaround times (TAT) for results appear to have an impact on clinical utility in the critical illness setting. Amongst diagnosed patients, results were considered clinically useful in 25% with standard TAT WES (median 136 days) by Daoud et al¹⁰ and range from 27–95% in rWGS and rWES with TAT of 12–23 days in various studies.^{9 11 12 13 17 23 35} Ultra-rapid WGS with a TAT of 4–5 days reported in NSIGHT1 trial¹⁴ seems to represent an ideal solution; however, the approach may be technically challenging, a shorter TAT adding significantly to costs. However, if trio rapid sequencing (WES/WGS) was done for all patients in this study, an early diagnosis would have been established in 30 of 32 patients who received a definitive diagnosis. The two undiagnosed cases include one family who required mRNA testing to ascertain pathogenicity of the novel splicing variant, and the second proband where the UGP2 gene reported phenotype was not identified at the time of first reporting.³⁶ We observed an impact of the diagnosis on management in 13 of 32 (40.6%) patients in this study. Of those who consented for testing at the time of intensive care admission, a rapid diagnosis by rWES/WGS could have potentially resulted in an earlier and effective control of seizures in five cases by genetic guided precision therapy, along with rapid control of neutropenia in one proband. A recent meta-analysis reported a change in clinical management in 6–27% children and recommended that WGS/WES should be considered a first-line genomic test in suspected genetic disease.³⁷ This information is very relevant while counseling families for rapid genetic testing.

A definitive molecular diagnosis provides valuable information regarding the mode of inheritance and the consequent risk of recurrence in future pregnancies.³⁸ A genetic diagnosis facilitates understanding and acceptance in family members and society, provides closure, reduces guilt and self-blame in parents. ^{39 40} Additionally, it can provide hope for the future and possible enrolment in clinical trials. We have not included the changes in reproductive risk counselling as a change in management of proband, even though the information has been extremely valuable to the families. We faced the peculiar situation three times during the study, where a genetic diagnosis could be established in the proband, but next pregnancy in the mother was too far advanced to offer a prenatal diagnostic test. These families would have benefitted by rapid NGS testing with its shorter TAT.

Limitations

Major hurdles to widespread utilization of NGS in this cohort include long turnaround time, possible requirement of testing of parents and limited diagnostic yield. The present study is limited due to its small sample size. A randomized control group could not be used, which would have allowed the comparison of diagnostic yield using conventional genetic techniques other than NGS, time to diagnosis, cost-effectiveness, health and psychosocial impacts for participating families. Diverse NGS techniques including panel testing, WES as well as WGS have been used in the study, the predominant test utilised being targeted exome focused on OMIM disease-causing genes (Clinical Exome) in 51 of 73 dataset analysis. Clinical Exome (CE) has its limitations, as it does not cover all the known genes (covered in WES) or the non –coding regions (covered in WGS). The turnaround time of nearly 6 weeks of standard NGS is not optimal in critical care setting but was used as we did not have access to rapid sequencing (TAT :7–10 days) or ultra-rapid sequencing (TAT: 2–5 days) at the time of this study.

The usefulness of NGS in critical care illness is being established with increasing awareness and accessibility of the technique in clinical practice. A significant proportion of morbidity and mortality in NICU and PICU is attributed to genetic disorders and an early diagnosis is vital to improve outcomes in this cohort. Our study contributes to establish the role of diverse NGS dataset analysis in critical care illness by achieving a diagnostic rate of 45.7% (32 of 70 probands), with a clinically significant change in outcome in 40.6% (13 of 32 diagnosed probands) by virtue of change in medication, diet, or major change in therapeutic approach. A significant proportion have also been helped by enhanced reproductive options available due to a firm genetic diagnosis. An early diagnosis enabled by rapid or ultra-rapid sequencing will further enhance the clinical outcome in critical care genomics.

Source of support / Funding: None

Data availability statement: The authors confirm that the data supporting the findings of the study are available within the article and its supplementary material. Raw data files will be made available on reasonable request.

Acknowledgements: We would like to thank all the children and families who agreed to be part of this study.

Author Contributions: All the authors have made substantial contributions to the final manuscript including conception and design; acquisition, analysis or interpretation of data. Each author has participated sufficiently in the work and takes public responsibility for appropriate portions of the content. All the authors agree that they would be accountable for all aspects of the work to ensure that questions related to the integrity or accuracy of any part of the work are appropriately investigated and resolved.

SB, ICV and RP made substantial contributions to conception and design.

SB, DG, AS, SBM, ICV and RP contributed to acquisition of data.

SB, SP, SK and RP contributed to analysis and interpretation of data.

SB, SP, ICV and RP contributed in drafting the manuscript and critical revision.

All authors approve the final version to be published.

Funding sources: The study was performed as part of clinical care without research funds.

Ethical approval: The study was approved by the Institution Ethics Committee (EC/07/18/1384).

Competing Interests: The authors report no conflict of interest or competing interests.

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Role of Next generation sequencing in diagnosis and management of critically ill children with suspected monogenic disorder

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Abstract

Figures

Introduction

Materials and Methods

Data Analysis and Interpretation

Outcome Measures and Statistical Analysis

RESULTS

Participant demographics and Indications for testing

Patient Phenotype

Interval between critical illness and NGS (Time to testing)

Next-generation sequencing results

NGS variant analysis

Secondary end points

Impact on clinical management

Brief description of some cases with exemplary response to genotype differentiated therapy

Reproductive outcomes in families

DISCUSSION

Limitations

CONCLUSION

Declarations

References

Additional Declarations

Supplementary Files

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