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# Clinical utility of 24-h rapid trio-exome sequencing for critically ill infants

## Abstract

Genetic diseases are a leading cause of death in infants in the intensive care setting; therefore, rapid and accurate genetic diagnosis is desired. To validate 24-h trio-exome sequencing (TES), samples from probands and their parents were processed by the AmpliSeq /Ion S5XL platform in a hospital clinical laboratory. Infants from the intensive care unit (ICU) suspected of having a genetic disease were enrolled. Regular and 24-h TES using the Agilent SureSelect capture kit/Illumina platform were performed on all samples in parallel. Of 33 enrolled infants, 23 received positive results with rapid TES, and an additional two diagnoses were achieved with regular TES. Among the 23 diagnosed patients, 10 experienced changes in medical management, such as hematopoietic stem cell transplant. Ten diagnosed cases were discharged prior to receiving the regular TES results; six received timely symptom control, and four withdrew medical support. Rapid TES enabled faster time to diagnosis, which resulted in an overall decrease in length of hospital stay. The 24-h TES can serve as a rapid response tool for patients with suspected monogenic disorders and can guide clinical decision-making in urgent cases.

## Introduction

Genetic disease diagnosis in infants can be extremely challenging to diagnose because the symptoms and characteristics may not appear early in life and may progress rapidly1,2,3. It is critical to diagnose these children as soon as possible to enable timely interventions, thus reducing mortality and unessential intensive care, as well as minimizing the anxiety of patient families. Patients without a diagnosis often embark on a diagnostic odyssey, including multiple specialist consultations, invasive investigations, or further laboratory tests, imposing both economic and physical burdens on patients and their families, sometimes even after the patient is deceased4,5.

Currently, comprehensive clinical panel testing or exome sequencing requires weeks to obtain genetic test results6,7. It is difficult to break the 1-week turn-around-time (TAT) barrier because conventional high throughput sequencing platforms run cases by batch, reducing flexibility for individual cases. In principle, it has been shown that genome sequencing as a first-tier genetic test can provide a higher diagnostic yield than conventional genetic testing in a clinically heterogeneous cohort8. Furthermore, rapid genome sequencing can decrease infant morbidity and hospitalization costs in acutely ill inpatient infants4,9,10. However, current rapid trio-genome sequencing is cost-prohibitive for most patients in developing countries. Moreover, data analysis of genomes has not fully matured due to its high complexity. Therefore, because rapid trio-exome sequencing can, by comparison, produce results in a short amount of time and is less expensive, it is the ideal testing strategy for critically ill infants in the pediatrics/neonate intensive care unit (PICU/NICU).

Here we present a rapid 24-h trio-exome sequencing (TES) pipeline that permits early genetic diagnosis with a TAT of approximately 24 h, which is comparable to the record of rapid genome sequencing9,11 but with only a small fraction of its cost. Among a group of critically ill infants who were highly likely to have diseases of genetic etiology, we successfully identified molecular diagnoses in 23 of 33 cases. Furthermore, treatment plans were modified in 10/23 of the diagnosed patients, and the overall hospitalization time decreased in four NICU/PICU patients. Overall, this study showed that clinical trio-exome can be achieved with a 24 h TAT and at a reasonable cost and may enhance the clinical utility of genomic sequencing for critically ill patients.

## Results

### Clinical characteristics

Twenty-two males and 11 females, ranging in age from 2 to 210 days old, with a mean of 55.97 days and a median of 51 days were enrolled in this study. Ten of the participants were neonates from the NICU, and 23 were from the PICU. There were 14 (43%) patients with metabolic-related diseases, 8 (24%) of whom had neuromuscular disorders, 8 (24%) of whom had immunodeficiency diseases, and 3 (9%) of whom had dysmorphic conditions with multiple congenital anomalies. Patients stayed in the ICU from 1 to 72 days, with an average of 21.8 days and a median of 12 days; only 10 (30%) stayed less than one week (Table 1; Supplementary Table 13).

### Timeline of the rapid TES

The median TAT for rapid TES was 24 h (22–27 h); it was 10 days (9–12 days) for regular TES (Table 1). The workflow and time requirements for rapid TES were as follows (Fig. 1b): blood samples were sent to the laboratory via the hospital’s internal delivery system, and genomic DNA extraction and DNA quality determination required 1 h. The library preparation and quantification required 7 h. Library enrichment and chip loading took another 7 h. Then, 2.5 h were allotted for sequencing and approximately, 5.5 h for alignment and variant calling in S5 XL servers. The server capacity was expanded to increase the speed of the base alignment and variant calling. VCF files were then loaded on the Fudan Pipeline version 2.0 for automatic analysis and evaluated by senior reviewers. An oral provisional report with candidate pathogenic variants was supplied to the clinician within one hour. Sanger sequencing for patients and parents was performed for positive diagnostic variants. Then, written clinical reports of positive rapid TES cases were issued immediately. For regular TES tests, clinical reports were issued for both positive and negative cases.

### Genetic test results

Rapid TES resulted in the study, 23 of 33 cases with diagnostic findings, including 15 males and eight females, seven (30.4%) were neonates from the NICU, and 16 (69.9%) were from the PICU. Of those receiving a diagnosis, 11 (78.5%) were patients with metabolic-related diseases, four (50%) had neuromuscular disorders, five (62.5%) had immunodeficiency diseases, and three (100%) patients had dysmorphic conditions with multiple congenital anomalies. Detailed information for these patients is provided in Supplementary Table 1. After obtaining the parallel regular TES test results, two additional diagnoses were made in the ten negative cases. The minimal false-negative ratio was 6.06% (2/33). Regular TES identified a homozygous frameshift pathogenic variant of the MTHFR gene (NM_005957: c.1267dupG, p.E423Gfs*6) in case 6, which caused homocystinuria due to MTHFR deficiency. The rapid TES data provided only three reads in the patient and two reads in the parents at this position. In contrast, in case 19, a hemizygous frameshift pathogenic variant of the MTM1 gene (NM_000252: c.1446_1447delTG, p.C482*) was identified, causing X-linked myotubular myopathy. The read depths of the pathogenic variant in the MTM1 gene in rapid TES were lower than 20× in the proband and his parents (11, 8, and 15, respectively, counted in IGV) (Supplementary Table 2).

### Impact of the genetic diagnosis on clinical procedures

The influence of the genetic diagnosis was subdivided into four different outcomes: specific treatment initiation, medication modification, palliative therapy, and deceased. The influence on changed management in ten patients is summarized in Table 2. Five diagnosed patients (case 5: IL10RA; case 16: IL7R; case 25: TCIRG1; case 32: IFNGR1; case 33: CD40LG) were recommended to undergo hematopoietic stem cell transplant (HSCT). Four patients reached clinical remission, and one patient (case 32) died of multiple organ dysfunction syndromes (MODS) due to the failure of the transplant.

Three infants who suffered seizures were found to have de novo pathogenic variants in SCN2A (case 17), KCNQ2 (case 9) and STXBP1 (case 8), respectively. These pathogenic variants cause early infantile epileptic encephalopathy. Antiepileptic medicines were adjusted in case 17 with sodium valproate oral solution (Debakin) plus levetiracetam (Caplan), in case 9 with sodium valproate syrup and in case 8 with 10% levetiracetam and topiramate capsules (Topiramate).

Case 4 was genetically diagnosed with SERAC1 gene mutation, which caused 3-methylglutaconic aciduria with deafness, encephalopathy, and Leigh-like syndrome. The initial symptom was only abnormal coagulation, with no characteristics leading to any clinical diagnosis. After the rapid genetic test, physicians reassessed and confirmed the diagnosis. The patient was then treated with a vitamin cocktail to control the metabolic disorder, and then was arranged to receive a follow-up visit in clinic. Case 13 was diagnosed with glycogen storage disease II, and the patient was discharged on day 8 with suggested treatment of enzyme replacement. With this diagnosis, muscle biopsy, electromyography (EMG), laboratory biochemical tests and other tests were avoided. In addition, case 1 was admitted to the PICU because of infantile muscular hypotonia and seizures and was then discharged after diagnosis of a GALC pathogenic variant with follow-up on an out-patient basis (Supplementary Table 1).

The average length of ICU stay in diagnosed patients from rapid TES was 19.4 days, and the median was 8 days (Table 1). Ten cases were discharged before the regular TES results were received, six of whom (cases 1, 8, 9, 13, 17, and 20) received timely symptom control, and four of whom underwent medical support withdrawal (case 3, 10, 15, and 18) (Supplementary Fig. 1, Table 3). These four patients were withdrawn from the ICU within three days after receiving the genetic diagnoses from rapid TES. Compared with the 10 days median TAT of the regular TES test, there were cumulatively at least 31 hospitalization days were saved (8, 7, 7, and 7days, respectively) in these four cases.

### Deceased and undiagnosed cases

Of the diagnosed cases, 11 patients died (Fig. 1c, E, F). In the five patients who died in the ICU, case 11 had lethal neonatal CPT II deficiency; case 12 carried a FOXC2 mutation, with severe clinical symptoms, and died within 72 h; case 14 had a CYBB mutation and died due to BCG vaccine-induced infection before the return of the TES results; case 28, a boy obtained in vitro fertilization, had lethal type III congenital disorder of glycosylation with COG6 mutation, whose parents insisted to keep him in ICU until death at 72 days; and case 32 with compound heterozygous mutation of IFNGR1 gene, who failed HSCT and died after two months. Six patients were discharged from the hospital and later died (Table 1, Fig. 1c, E). The families of case 3, 10, 15, and 18 withdrew medical support within 72 h after clinical confirmation of the diagnoses of severe metabolic diseases. Case 7 was discharged and died on day 38, case 31 was discharged on day 21 and died shortly after.

Of the eight undiagnosed individuals, two of them died in the ICU on day 1 (case 22) and day 7 (case 23). The families of case 21 and 29 withdrew treatment, and the patients were discharged from the hospital on day 26 and 43 for economic reasons (Supplementary Table 3).

### Patient care and genetic counseling

Special services, such as multidisciplinary team care, palliative care, special diets or medications, imaging studies, surgical procedures, and essential genetic counseling, were provided to the diagnosed families. Two families (case 1 and 18) plan to have another child. Prenatal testing was advised. Eight probands harboring de novo pathogenic variants were also advised to receive counseling before the next pregnancy.

### Phenotype analysis

To evaluate whether special clinical presentations were more likely to be associated with a molecular diagnosis in the ICU, the HPO terms of the diagnosed patients were analyzed. There were 14/24 with neuromuscular problems, 9/17 with metabolic abnormalities, and 8/16 with immune system abnormalities among the diagnosed cases (Supplementary Table 4).

## Discussion

Genetic diseases are the leading causes of death in infants requiring intensive care12,13,14. Here we show the feasibility of rapid clinical exome sequencing to provide genetic diagnoses for a wide range of clinical presentations. Using clinical exome sequencing, some published paper described the genetic diagnoses’ impact on clinical decision making in pediatric patients, and their notable effect on medical management among a group of critically ill infants who were suspected to have genetic disorders15,16. It was more efficient than chromosomal microarray detection17,18. A systematic literature review (January 2011–August 2017) and meta-analysis in 37 studies, comprising 20,068 children with suspected genetic diseases, showed that the diagnostic rate and clinical utility of WGS/WES were greater than CMA18.

The time window for which many life-saving treatments can be most effective for some diseases is very limited. Therefore, developing a rapid diagnostic method for such diseases is an urgent need. The current clinical trio-exome cost is around $1500 with TAT of 2–3 weeks, and the cost of trio-genome sequencing is far higher (around$4500) with similar TAT. Miller et.al.11 and Clark et al.9 reported a 26-h, and then a 19-h rapid genome sequencing genetic diagnosis system with highly specialized equipment, institute improved analytic tools, and extremely dedicated professionals, which unfortunately cannot be easily replicated in most places. We try to provide a convenient and reproducible rapid trio-WES sequencing for patients stayed in ICU using a personal genome sequencer (Ion Torrent S5 XL) in a laboratory located in the hospital.

### Reporting summary

Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.

## Data availability

The pathogenic variants have been submitted to ClinVar. The accession numbers were listed in Supplementary Table 9. This study is compliant with the ‘Guidance of the Ministry of Science and Technology (MOST) for the Review and Approval of Human Genetic Resources’, which requires formal approval for the export of human genetic material or data from China.

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## Acknowledgements

We are very grateful to the patients’ families. We also thank the clinicians for taking care of the patients and our genetics laboratory teams who contributed to this study. Great thanks are extended to Dr. Fan Xia from Baylor College of Medicine, who provided suggestions for the rapid TES method and guidance on the manuscript. The sequencing reagents were funded by the Ministry of science and technology national key research and development program (2018YFC0116903, Prof. Zhou), Science and technology commission of Shanghai (18411962000, B.W.), Shen Kang Hospital Development Center Clinical Science and technology innovation project of Shanghai (SHDC12017110, W.Z.), and the sequencing platform was supported by the project of Shanghai Key Laboratory of Birth Defects (13DZ2260600). The corresponding author had full access to all the data in this study and the final responsibility for the decision to submit the manuscript for publication.

## Author information

Authors

### Contributions

W.H.Z. conceptualized and designed the study, coordinated the study overall, and revised the manuscript; H.J.W. co-designed the study, co-drafted the initial manuscript, and revised the manuscript; Y.Y.Q. performed the experiments, collected and interpreted the data, co-drafted the initial manuscript, and revised the manuscript; B.B.W. developed the laboratory, interpreted the data, and critically reviewed the manuscript; L.Y. enrolled the patients and was involved in the clinical disposition with the clinicians; G.P.L. and G.Q.C. treated the patients in the ICU and discussed the genetic results with multidisciplinary teams; Q.Q. and Y.L.L. developed the algorithm and data analysis pipeline, and they revised the manuscript; P.Z. performed the laboratory work and analyzed and interpreted the data. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.

### Corresponding authors

Correspondence to Bingbing Wu or Wenhao Zhou.

## Ethics declarations

### Competing interests

The authors declare no competing interests.

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Wang, H., Qian, Y., Lu, Y. et al. Clinical utility of 24-h rapid trio-exome sequencing for critically ill infants. npj Genom. Med. 5, 20 (2020). https://doi.org/10.1038/s41525-020-0129-0

• Accepted:

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• DOI: https://doi.org/10.1038/s41525-020-0129-0

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