sars-cov-2 polymorphisms and multisystem inflammatory … · 2020. 9. 7. · prepublication release...

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©2020 American Academy of Pediatrics

SARS-CoV-2 Polymorphisms and Multisystem Inflammatory Syndrome in Children (MIS-C)

Juanita Pang, BSc, Florencia A.T. Boshier, PhD, Nele Alders, MD, Garth Dixon, PhD, Judith Breuer, MD

DOI: 10.1542/peds.2020-019844

Journal: Pediatrics

Article Type: Research Brief

Citation: Pang J, Boshier FAT, Alders N, Dixon G, Breuer J. SARS-CoV-2 polymorphisms and multisystem inflammatory syndrome in children (MIS-C). Pediatrics. 2020; doi: 10.1542/peds.2020-019844

This is a prepublication version of an article that has undergone peer review and been accepted for publication but is not the final version of record. This paper may be cited using the DOI and date of access. This paper may contain information that has errors in facts, figures, and statements, and will be corrected in the final published version. The journal is providing an early version of this article to expedite access to this information. The American Academy of Pediatrics, the editors, and authors are not responsible for inaccurate information and data described in this version.

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SARS-CoV-2 Polymorphisms and Multisystem Inflammatory Syndrome in Children (MIS-C)

Juanita Panga*, BSc, Florencia A.T. Boshiera*, PhD, Nele Aldersb, MD, Garth Dixonc, PhD, Judith Breuera, MD

* These authors contributed equally to this work.

Affiliations: aDepartment of Infection, Immunity and Inflammation, UCL Great Ormond Street Institute of Child Health, University College London, London, United Kingdom. bDepartment of Infectious Disease, Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom. cDepartment of Microbiology, Virology and Infection Control, Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom.

Address correspondence to: Judith Breuer, Research Department of Infection UCL, Infection and Immunity, The Cruciform Building, Gower street , WC1E 6BT, +44-203 108 2030

Conflict of Interest Disclosures (includes financial disclosures): The authors have no example conflicts of interest to disclose.

Funding/Support: No funding was secured for this study.

Role of Funder/Sponsor: The funders had no role in the design and conduct of the study.

Contributors’ Statement Page

Juanita Pang and Florencia A.T. Boshier: completed the data analysis, literature search, drafted the initial manuscript, reviewed and revised the manuscript, approved the final manuscript as submitted and agree to be accountable for all aspects of the work.

Nele Alders and Garth Dixon: collected the data, reviewed the manuscript, approved the final manuscript as submitted and agree to be accountable for all aspects of the work.

Judith Breuer: conceived the study, completed the data interpretation and literature search, approved the final manuscript as submitted and agrees to be accountable for all aspects of the work.




Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes Coronavirus

Disease 2019 (COVID-19), was first identified in Wuhan, China in December 2019. It rapidly

spread across the world and on 11th March 2020, the WHO declared COVID-19 as a global

pandemic1.

In adults, SARS-CoV-2 can manifest as severe interstitial pneumonia and hyperinflammation

with approximately 3-5% of infections requiring admission to critical care2,3. In contrast, severe

illness and death due to SARS-CoV-2 infection in children is rare4. However recently, a small

number of cases of shock and multisystem inflammation have been reported in children who

have either been tested positive for SARS-CoV-2 (by PCR or serology) or had epidemiological

links to it. This new syndrome, which has overlapping features of Kawasaki disease, is called the

Multisystem Inflammatory Syndrome in Children (MIS-C)5.

The exact pathogenesis of MIS-C is as yet unknown. However, it has been suggested that part of

the SARS-CoV-2 viral spike (S) protein may resemble a superantigen which could drive the

development of MIS-C and trigger cytokine storms in adults6. Specifically, polymorphic residues

in S including A831V and D839Y/N/E which are predicted to enhance binding affinity to the

TCR have been observed in lineages circulating in Europe and North America, where most MIS-

C cases have been described. In addition, the 614G Spike protein polymorphism may be

associated with increased transmission and altered SARS-CoV-biology7. Phylogenetic

comparisons of SARS-CoV-2 viral sequences from MIS-C patients and non MIS-C cases is

necessary to identify the significance of viral polymorphisms in the etiology of MIS-C.




To this end, we compare SARS-CoV-2 viral sequences found in 5 children diagnosed with MIS-

C to sequences from 8 non-MIS-C children and 130 community cases in North London.

Methods

Sample collection and sequencing

We sequenced SARS-CoV-2 from children hospitalized for COVID-19 in London between late-

March and mid-May 2020. Of 61 hospitalized children with COVID-19, confirmed by

polymerase chain reaction (PCR) for SARS-CoV-2 RNA in nasopharyngeal aspirates, and/or by

the presence of SARS-CoV-2 antibodies (Meso Scale Discovery (MSD) Chemiluminescent

binding assay), 36 were diagnosed with MIS-C based on the case definition of Royal College of

Pediatrics and Child Health8,9. Those classified as not having MIS-C were asymptomatically

infected or had predominantly respiratory symptoms with no evidence of multi-organ

involvement, widespread inflammation, shock or Kawasaki-like symptoms. Eleven of the 36

MIS-C patients were positive for SARS-CoV-2 viral RNA, the remainder being positive for

antibodies to SARS-CoV-2 (Meso Scale Discovery (MSD) Chemiluminescent binding assay).

All 25 non-MIS-C patients were positive for SARS-CoV-2 viral RNA. Full length SARS-CoV-2

genome sequences were obtained from 5 children classified as MIS-C and 8 non-MIS-C children

(Table 1) using SureSelectXT target enrichment and Illumina sequencing. Reads generated were

quality checked and mapped to the SARS-COV-2 reference genome (NC_045512) using BWA

v0.7.17. Sequences are available on GISAID (Accession ID: EPI_ISL_479777 to

EPI_ISL_479789). Demographics of the 5 MIS-C and 8 non-MIS-C children are summarized in

Table 1.




Phylogenetics

We reconstructed a maximum likelihood phylogenetic tree of all sequences using RAxML v8.0.0

using model GTRGAMMA and 1000 bootstrap replicates10. In addition, single nucleotide

polymorphisms (SNPs) were identified by comparison with SARS-CoV-2 reference genome

(NC_045512) and classified as synonymous/ non-synonymous.

Statistical Analysis

Categorical data is presented as proportions. Comparison of categorical data between groups

(MIS-C, non MIS-C and community cases) was made by Chi-square (chi.test) in R11.

Results

The maximum likelihood phylogeny of 13 pediatric SARS-CoV-2 cases and 130 SARS-CoV-2

sequences generated from community cases across North London is shown in Figure 1A. We

found no clustering of viral sequences from MIS-C patients (red) or non MIS-C patients (blue)

in relation to other local sequences (black).

We observed no SNPs unique to the MIS-C or to the other childhood cases and no difference in

the distribution of SNPs between MIS-C, non MIS-C and community cases as depicted in Figure

1 B. The proportion of non-synonymous SNPs did not differ in the MIS-C, non MIS-C and

community cases (Chi-squared test, df = 2, p = 0.64).

All childhood cases were D839 and A831 as were all of the locally circulating samples. The

majority of PIMS-ST (3/5), non PIMS-ST (6/8) and community cases (118/130) were 614G

positive.




Discussion

Understanding the immunopathological etiology of MIS-C is useful in effective management and

treatment of the disease. This report compares viral sequences from children diagnosed with

MIS-C to viral sequences from children without MIS-C as well as the wider London community.

Overall, the data suggest that the viruses causing MIS-C in our patients are representative of

locally circulating SARS-CoV-2. We found no evidence for an association of MIS-C with the

presence of new or unusual sequence polymorphisms. This suggests that alternative factors, such

as host-genetics, may trigger MIS-C. Further studies are required to address this.

Study Approval

This study was approved by Great Ormond Street Hospital (Clinical Audit Number #2857) and

PHE Research Ethics and Governance Group (REGG) (R&D NR0195).

Acknowledgments

This work was supported by COG-UK, The James Black Charitable Foundation and the UCLH

and GOSH NIHR biomedical research centers. JP is supported by the Rosetrees Foundation and

FTB by a Wellcome Trust Collaborative Award to JB. We thank Richard Goldstein, Kathryn

Harris, Julianne Brown, Jack Lee, Rachel Williams, Helena Tutill and Sunando Roy for their

contribution to this work. We should also like to acknowledge the contribution of the UCL

Pathogen Genomics Unit, UCL Genomics, and the Great Ormond Street Hospital Departments of

Infectious Disease, and Microbiology, Virology and Infection Control.

We declare no competing interests.




References

1 World Health Organization. WHO Timeline - COVID-19. https://www.who.int/news-room/detail/27-04-2020-who-timeline---covid-19 (accessed May 30, 2020).

2 CDC. Severe Outcomes Among Patients with Coronavirus Disease 2019 (COVID-19) — United States, February 12–March 16, 2020. MMWR. Morbidity and Mortality Weekly Report. 2020. https://www.cdc.gov/mmwr/volumes/69/wr/mm6912e2.htm (accessed June 1, 2020).

3 WHO. Coronavirus disease (COVID-19) situation report – 162. 2020. https://www.who.int/docs/default-source/coronaviruse/20200630-covid-19-sitrep-162.pdf?sfvrsn=e00a5466_2 (accessed July 5, 2020).

4 Ludvigsson JF. Systematic review of COVID-19 in children shows milder cases and a better prognosis than adults. Acta Paediatrica 2020; 109: 1088–95.

5 European Centre for Disease Prevention and Control. Rapid risk assessment: Paediatric inflammatory multisystem syndrome and SARS -CoV-2 infection in children. 2020; published online May 15. https://www.ecdc.europa.eu/en/publications-data/paediatric-inflammatory-multisystem-syndrome-and-sars-cov-2-rapid-risk-assessment.

6 Cheng MH, Zhang S, Porritt RA, Arditi M, Bahar I. An insertion unique to SARS-CoV-2 exhibits superantigenic character strengthened by recent mutations. Immunology, 2020 DOI:10.1101/2020.05.21.109272.

7 Zhang L, Jackson CB, Mou H, et al. The D614G mutation in the SARS-CoV-2 spike protein reduces S1 shedding and increases infectivity. Microbiology, 2020 DOI:10.1101/2020.06.12.148726.

8 RCPCH. Guidance: Paediatric multisystem inflammatory syndrome temporally associated with COVID-19. 2020. https://www.rcpch.ac.uk/sites/default/files/2020-05/COVID-19-Paediatric-multisystem-%20inflammatory%20syndrome-20200501.pdf.

9 RCPCH. COVID-19 - clinical management of children admitted to hospital with suspected COVID-19. https://www.rcpch.ac.uk/sites/default/files/generated-pdf/document/COVID-19---clinical-management-of-children-admitted-to-hospital-with-suspected-COVID-19.pdf.

10 Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 2014; 30: 1312–3.

11 R Core Team. R: A language and environment for statistical computing. Vienna, Austria.: R Foundation for Statistical Computing, 2019 https://www.R-project.org/ (accessed Sept 1, 2019).




Patient 1 2 3 4 5 6 7 8 9 10 11 12 13

Age 12y1m 5y8m 14y8m 15y0m 8y9m 5y11m 8y11m 1y8m 2y11m 10m 10y3m 1y4m 1m

Days in hospital 10 15 18 82 8 44 14 19 284 268 22 87 23 Days in ICU 3 0 10 16 2 - 11 19 - - - - 22

Sex M F M F M F F M M M M F M

Ethnicity Black African White Mixed Asian Asian Asian Asian Black African Other Other Black African Other White

Co-morbidities Seizure disorder

Seizure disorder,

right upper lobectomy

Previously well

Previously well

Previously well

Metabolic disease

Bronchiectasis, lobectomy

Hepatoblastoma, chronic lung

disease, BiPAP

Metastatic neuroblastoma

Ex-prem 23weeks, short

gut; chronic lung disease

T- cell lymphoma

Primitive neuroectodermal

tumour

Ex- prem 32weeks + 6 days, cleft

palate Clinical presentation

Borderline MIS-C MIS-C MIS-C MIS-C MIS-C Asymptomatic Respiratory

deterioration Respiratory deterioration

Upper respiratory tract infection

Upper respiratory tract infection

Enterobacter line infection Asymptomatic Respiratory

deterioration Fever Y Y Y Y Y N Y Y Y Y Y N N Fatigue Y Y Y Y Y N Y N Y N N N N Cough N Y Y Y Y N Y Y Y Y N N Y URT symptoms N N Y Y Y N N Y Y Y N N Y Dyspnoea Y Y Y Y Y N Y N Y N N N Y Abdominal pain N N Y N N N Y Y N N Y N N Vomiting N Y Y Y Y N Y Y N Y Y N N Diarrhoea N N Y Y Y N Y Y N N N N N Headache N N N N Y N Y N N N Y N N Seizure Y Y N N N N N N N N N N N Meningitis N N N N N N N N N N N N N Anosmia N N Y N N N N N N N N N N Rash N N N Y Y N N N N N N N N Oedema N N Y Y N N N N N N N N N Shock requiring resuscitation Mild Y Y Y Y N N N N N N N N

Mechanical ventilation Y N Y Y Y N Y N N N N N Y

Highest neutrophil count, x 10^9/L

3,98 11,58 15,46 30,34 13,91 0.94 5,67 0.13 2.3 3,52 2,74 2.39 8,49

Lowest lymphocyte count, x 10^9/L

0,57 3,98 1,26 0,35 0,47 1.47 0,19 0.66 0.43 3,58 0,19 0.51 1,18

Highest CRP, mg/L 158 323 449 290 311 <5 284 253 49 <5 28 <5 63

Highest ferritin, ug/L - - 1452 63626 990 - 366 2496 754 9,5 786 - 789

Highest D-dimers, ug/L - - 6742 14798 7085 212 1737 2332 - 415 265 - 1160

Highest fibrinogen, g/L 3,4 7.49 12.8 5 6,6 2.4 6,5 5,7 - 2,6 5,7 3,3 3,9

Lowest albumin, g/L 34 34 29 <20 27 40 25 29 29 27 34 36 20

Highest LDH, U/L - - 1905 5764 1018 - 1022 685 602 - 423 868 -

Highest ALT, U/L 108 6 121 482 101 104 41 44 85 89 25 147 59


5’ UTR ORF1ab S ORF3a

E

M

ORF6

ORF7

ORF8

N

3’ UTR

ORF10

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No PIMS-TS

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uenc

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noMIS-C No MIS-C

MIS-C

No MIS-C

Figure 1: Characteristics of SARS-CoV-2 sequences from children with and without MIS-C(A) Phylogenetic tree of sequences analysed in this study. Tips coloured in red = from MIS-C children, blue = from non-MIS-C children, black = other sequences from London with no association to MIS-C. (B) Frequency of occurrence of single nucleotide polymorphisms. Top: 130 London Samples in yellow. Bottom: 13 Paediatric samples from GOSH, red = 5 MIS-C samples, blue = 8 non MIS-C samples. The x-axis is annotated with a map of the reading frames in the viral genome.




originally published online September 9, 2020; Pediatrics Juanita Pang, Florencia A.T. Boshier, Nele Alders, Garth Dixon and Judith Breuer

(MIS-C)SARS-CoV-2 Polymorphisms and Multisystem Inflammatory Syndrome in Children

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