Assessment of aberrant DNA methylation two years after paediatric critical illness: a pre-planned secondary analysis of the international PEPaNIC trial

Figures & data

Figure 1. CONSORT diagram of study participants. Participants included in the epigenetic 2-year follow-up analysis are shown.

Table 1. Demographics and medical characteristics of participants included in the 2-year epigenetic follow-up analysis.

Demographics and medical characteristics of participants	Healthy children N = 392	PICU patients N = 818	p-value	PICU patients Early-PN N = 406	PICU patients Late-PN N = 412	p-value
Demographics
Age at 2-year follow-up (age at swab collection)
– median (IQR) – years	3.8 (2.6–8.2)	3.4 (2.6–7.9)	0.96	3.5 (2.6–8.1)	3.2 (2.6–7.7)	0.60
– mean (SD) – years	6.0 (4.7)	6.0 (4.8)		6.0 (4.6)	6.0 (4.9)
Sex
Male – no (%)	212 (54.1)	475 (58.1)	0.19	238 (58.6)	237 (57.5)	0.75
Female – no (%)	180 (45.9)	343 (41.9)		168 (41.4)	175 (42.5)
Known non-Caucasian race^a – no (%)	32 (8.2)	66 (8.1)	0.95	39 (9.6)	27 (6.6)	0.10
Known non-European origin^a – no (%)	51 (13.0)	144 (17.6)	0.03	82 (20.2)	62 (15.0)	0.05
Medical characteristics
STRONGkids risk level^b			/			0.69
Medium – no (%)	NA	736 (90.0)		367 (90.4)	369 (89.6)
High – no (%)	NA	82 (10.0)		39 (9.6)	43 (10.4)
PeLOD score, first 24 h in PICU^c – mean (SD)	NA	21.0 (11.6)	/	21.0 (11.8)	21.1 (11.4)	0.69
PIM3 score^d – mean (SD)	NA	−3.4 (1.4)	/	−3.4 (1.4)	−3.5 (1.3)	0.48
PIM3 probability of death^e (%) – mean (SD)	NA	6.7 (11.8)	/	6.9 (11.9)	6.4 (11.6)	0.63
Diagnostic category			/			0.71
Surgical
Abdominal – no (%)	NA	63 (7.7)		28 (9.4)	29 (9.2)
Burns – no (%)	NA	2 (0.2)		1 (0.3)	1 (0.3)
Cardiac – no (%)	NA	364 (44.5)		126 (42.4)	138 (43.5)
Neurosurgery-Traumatic brain injury – no (%)	NA	72 (8.8)		28 (9.4)	23 (7.3)
Thoracic – no (%)	NA	41 (5.0)		20 (6.7)	15 (4.7)
Transplantation – no (%)	NA	14 (1.7)		3 (1.0)	7 (2.2)
Orthopaedic surgery-Trauma – no (%)	NA	40 (4.9)		12 (4.0)	7 (2.2)
Other – no (%)	NA	26 (3.2)		8 (2.7)	13 (4.1)
Medical
Cardiac – no (%)	NA	24 (2.9)		8 (2.7)	14 (4.4)
Gastrointestinal-Hepatic – no (%)	NA	5 (0.6)		1 (0.3)	1 (0.3)
Oncologic-Haematologic – no (%)	NA	5 (0.6)		2 (0.7)	3 (1.0)
Neurologic – no (%)	NA	49 (6.0)		16 (5.4)	19 (6.0)
Renal – no (%)	NA	0 (0.0)		0 (0.0)	0 (0.0)
Respiratory – no (%)	NA	78 (9.5)		30 (10.1)	34 (10.7)
Other – no (%)	NA	35 (4.3)		14 (4.7)	13 (4.1)
Malignancy – no (%)	0 (0.0)	39 (4.8)	<0.0001	24 (5.9)	15 (3.6)	0.12
Syndrome^f – no (%)	4 (1.0)	168 (20.5)	<0.0001	81 (20.0)	87 (21.1)	0.67

^a Participants were classified according to race and geographical origin by the investigators. ^b Scores on the Screening Tool for Risk on Nutritional Status and Growth (STRONGkids) range from 0 to 5, with a score of 0 indicating a low risk of malnutrition, a score of 1 to 3 indicating medium risk, and a score of 4 to 5 indicating high risk. ^c Paediatric Logistic Organ Dysfunction (PeLOD) scores range from 0 to 71, with higher scores indicating more severe illness. ^d Paediatric Index of Mortality 3 (PIM3) scores, with higher scores indicating a higher risk of mortality. ^e Paediatric Index of Mortality 3 (PIM3) probability of death. ^f A pre-randomization syndrome or illness a priori defined as affecting or possibly affecting development (see Supplementary Methods S1).

Abbreviations: IQR, interquartile range; NA, not applicable; no, number; PeLOD, paediatric logistic organ dysfunction score; PICU, paediatric intensive care unit; PIM3, paediatric index of mortality 3 score; PN, parenteral nutrition; SD, standard deviation.

Figure 2. Locations of Differentially Methylated Positions and Regions. Bar chart depicting the distribution of DMPs (a), DMRs (b) and CpG sites within DMRs (c) over the gene regions. The blue and red area represent the proportion that is hypomethylated and hypermethylated respectively in the children who had been admitted to the PICU 2 y prior to the follow-up study compared to the healthy control children. For our calculations, we defined the promoter as 0 to 1500 base pairs upstream of the transcription start site and the first exon is reported separately and thus is not included into the gene body. Note: Abbreviations: DMP, differentially methylated position; DMR, differentially methylated region; PICU, Paediatric Intensive Care Unit, UTR: untranslated region

Table 2. Summary of absolute differences in methylation between former PICU patients and healthy children.

Methylation change	Number of CpG sites	Mean	SD	Maximum	Median	IQR
DMP	4047	2.24%	1.42%	8.20%	1.96%	2.09%
DMR^a	4024	1.98%	1.31%	7.41%	1.88%	1.94%
All^b	7404	2.06%	1.36%	8.20%	1.85%	1.98%

^aAll CpG sites within a differentially methylated region were assessed.^bAll CpG sites from DMPs and within DMRs

Abbreviations: DMP, differentially methylated position; DMR, differentially methylated region; SD, standard deviation; IQR, interquartile range.

Table 3. Twenty-five most differentially methylated pathways in former patients 2 y after PICU-admission versus healthy children.

Biological category	KEGG pathway name	Number (percentage^a) of DMGenes	Weighted percentage^b	Classification
Cancer: overview	Pathways in cancer	258 (37.5%)	208.236	differences that may have been present before PICU admission
Cellular community – eukaryotes	Adherens junction	58 (50%)	203.022	physical and neurocognitive development
Drug resistance: antineoplastic	EGFR tyrosine kinase inhibitor resistance	59 (48.8%)	198.821	differences that may have been present before PICU admission
Endocrine system	Oxytocin signalling pathway	91 (43.5%)	196.407	physical and neurocognitive development
Environmental adaptation	Circadian entrainment	59 (44.4%)	180.884	the critical illness phase or the intensive medical care
Signal transduction	Calcium signalling pathway	120 (37.5%)	179.531	physical and neurocognitive development
Cell growth and death	Apoptosis – multiple species	30 (52.6%)	179.012	physical and neurocognitive development
Signal transduction	Phospholipase D signalling pathway	79 (40.9%)	178.855	physical and neurocognitive development
Cancer: specific types	Glioma	48 (46.2%)	178.671	differences that may have been present before PICU admission
Signal transduction	Rap1 signalling pathway	102 (37.6%)	174.075	physical and neurocognitive development
Nervous system	Cholinergic synapse	62 (41.3%)	170.587	physical and neurocognitive development
Sensory system	Inflammatory mediator regulation of TRP channels	54 (42.5%)	169.612	the critical illness phase or the intensive medical care
Signal transduction	Ras signalling pathway	107 (36.3%)	169.488	differences that may have been present before PICU admission
Endocrine system	GnRH signalling pathway	53 (42.4%)	168.34	physical and neurocognitive development
Infectious disease: bacterial	Bacterial invasion of epithelial cells	45 (43.3%)	164.71	differences that may have been present before PICU admission
Endocrine system	Aldosterone synthesis and secretion	54 (41.2%)	164.43	the critical illness phase or the intensive medical care
Signal transduction	cAMP signalling pathway	101 (35.6%)	164.128	physical and neurocognitive development
Infectious disease: bacterial	Yersinia infection	68 (38%)	160.295	differences that may have been present before PICU admission
Transport and catabolism	Endocytosis	106 (34.1%)	158.949	physical and neurocognitive development
Cancer: specific types	Non-small cell lung cancer	40 (43%)	158.662	differences that may have been present before PICU admission
Immune system	Fc gamma R-mediated phagocytosis	50 (40.3%)	157.745	the critical illness phase or the intensive medical care
Nervous system	Glutamatergic synapse	57 (38.8%)	156.773	physical and neurocognitive development
Substance dependence	Morphine addiction	47 (40.5%)	155.996	the critical illness phase or the intensive medical care
Circulatory system	Adrenergic signalling in cardiomyocytes	72 (36.4%)	155.517	the critical illness phase or the intensive medical care
Signal transduction	PI3K-Akt signalling pathway	138 (31.5%)	155.243	physical and neurocognitive development

^aPercentage of genes in the pathway that are differentially methylated. ^b Weighted percentage used to rank pathways and computed as: ln(number of differentially methylated genes in pathway) x (percentage of methylated genes in pathway).

Abbreviations: DMGenes, differentially methylated genes; KEGG, Kyoto Encyclopaedia of Genes and Genomes

Data availability statement

Data sharing is offered under the format of collaborative projects. Proposals can be directed to the corresponding author.