Alterations in DNA methylation associate with fatty liver and metabolic abnormalities in a multi-ethnic cohort of pre-teenage children

Figures & data

Table 1. Child characteristics by top tertile liver fat content (PDFF).

Child Characteristics	All with PDFF (n = 79)	Top Tertile PDFF ≥ 1.7%	PDFF < 1.7% (n = 50)	p-value
		(n = 29)
Age (years) (median and IQR)	9.2 (8.1–9.8)	9.6 (8.4–10.6)	8.8 (7.9–9.5)	0.02
Male Sex, n (%)	35 (44.3)	14 (40)	21 (60)	0.59
Race				0.81
White	24 (30.4)	8 (33.3)	16 (66.7)
Black	51 (64.5)	19 (37.3)	32 (62.7)
Other	4 (5.1)	2 (50)	2 (50)
Non-Hispanic Ethnicity	72 (91.1)	26 (36.1)	46 (63.9)	0.72
Maternal pre-pregnancy BMI (kg/m^2) (n = 77)	31.6 (23.7–44.7)	31.9 (27.4–35.9)	31.1 (23.3–46.7)	0.56
Child Body Mass Index (BMI) (kg/m[^2]) (n = 78)	19.3 (15.6–23.9)	22.7 (20.3–29.6)	17.4 (15.4–20.9)	<0.0001
BMI %tile (n = 78)	86.5 (41.7–96)	95 (85–99)	64 (30.5–92)	0.0004
BMI %tile Category (n = 78)
Obese (> 95%), n (%)	29 (37.2)	18 (62.1)	11 (22.5)	.002
Overweight (> 85th to < 95th), n (%)	11 (14.1)	4 (13.8)	7 (14.3)
Normal (> 5th to <85th), n (%)	34 (43.6)	6 (20.7)	28 (57.1)
Underweight (<5th), n (%)	4 (5.1)	1 (3.4)	3 (6.1)
Waist Circumference (inches) (n = 76)	24 (22–29.9)	29.8 (24.5–32.5)	23 (21–25.)	<0.0001
Waist to Hip Ratio (n = 75)	1.0 (0.9–1)	1.0 (0.90–1.0)	1.0 (0.9–1.0)	0.18
Systolic Blood Pressure (mmHg) %tile (n = 78)	66 (46–88.3)	74.5 (46.5–91.8)	65 (43.5–88.3)	0.35
Diastolic Blood Pressure (mmHg)%tile (n = 78)	53 (28.3–74)	51.5 (30.8–73)	54.5 (24.5–74.3)	0.84
Hypertension, n (%) (n = 78)	15 (19.2)	7 (25)	8 (16)	0.63
Elevated blood pressure, n (%)	7 (9)	2 (7.1)	5 (10)
Laboratory Data
Glycosylated Haemoglobin (HbA1c%) (n = 77)	5.4 ± 0.4	5.5 ± 0.3	5.4 ± 0.4	0.15
Total Bilirubin (mg/dL) (n = 76)	0.6 (0.4–0.8)	0.6 (0.4–0.7)	0.7 (0.5–0.8)	0.22
AST (U/L) (n = 77)	28 (24–30)	26.5 (25.5–29)	28 (23–32)	0.63
ALT (U/L) (n = 77)	17 (15–20)	18 (15–21.5)	16 (14–19)	0.01
Triglycerides (mg/dL) (n = 78)	45 (37–63)	51 (40–67.5)	43.5 (35–54)	0.08
Total Cholesterol (mg/dL) (n = 78)	157 (132–169)	154.5 (122.7–166)	157 (133.7–169.3)	0.33
HDL (mg/dL) (n = 78)	56 (45–66)	51 (44–63.5)	58.5 (48–67)	0.12
LDL (mg/dL) (n = 78)	88.5 (75–96)	85 (72.5–97)	90.5 (81–96)	0.54
TG/HDL ratio (n = 78)	0.85 (0.58–1.17)	0.97 (0.7–1.45)	0.78 (0.52–1.1)	0.07

AST, aspartate aminotransferase; ALT, alanine aminotransferase; BMI, body mass index; HDL, high density lipoprotein; HTN, hypertension; LDL, low density lipoprotein; IQR, interquartile range; OW, overweight; PDFF, proton density fat fraction; SD, standard deviation; TG, triglycerides.

Table 2. Imaging characteristics of children by top tertile liver fat content (PDFF).

Characteristics (median ± IQR)	All with PDFF (n = 79)	Top Tertile PDFF ≥ 1.7% (n = 29)	PDFF < 1.7% (n = 50)	p-value
Liver Stiffness Measurement (kPa) (n = 74)	4.3 (3.7–4.9)	4.4 (3.7–5.3)	4.2 (3. – 4.6)	0.33
MRE (kPa) (n = 78)	2.5 (2.2–2.6)	2.3 (2.2–2.5)	2.5 (2.3–2.7)	0.047
Subcutaneous to Visceral Fat Ratio (n = 78)	0.84 (0.77–0.88)	0.87 (0.81–0.92)	0.82 (0.74–0.88)	0.03
Visceral to Subcutaneous Fat Ratio (n = 78)	0.16 (0.12–0.23)	0.13 (0.09–0.2)	0.18 (0.12–0.26)	0.03
MR Subcutaneous Fat Area (cm2) (n = 75)	51.4 (24.9–156.6)	157.5 (83.1–199.5)	30.8 (21.4–61.2)	<0.001
MR Visceral Fat Area (cm2) (n = 75)	11 (6.3–22.3)	20.2 (9.9–35.8)	8.2 (4.9–13.7)	0.001

IQR, interquartile range; OW, overweight; PDFF, proton density fat fraction; MRE, magnetic resonance elastography; MR, magnetic resonance

Table 3. Significant DMR associated with top tertile liver fat content (by MRI-PDFF).

Gene	# of CpG	Chr.	max. β	Area	FDR P-value	Gene Ontology/Annotation
MROH6	13	Chr 8	−0.007	PROMOTER	3.3 x 10^−[^8]	IL18, body fat percentage/Variant may regulate inflammasome
CDH24	4	Chr 14	−0.0031	PROMOTER	1.2 x 10^−[^7]	Cell morphogenesis, cell-cell junction assembly/Binds catenins, alterations implicated in several cancers
WFIKKN2	20	Chr 17	0.005	PROMOTER	1.4 x 10^−[^7]	Skeletal system development, TGFβ-R signalling pathway/Loss of function variant associated with glucose, haemoglobin A1c and HOMA-IR in humans
FGFBP1	9	Chr 4	−0.0067	GENE_BODY	2.4 x 10^−[^7]	Signal transduction, cell-cell signalling/Increased gene expression associated with familial hypertension
TMEM232	14	Chr 5	−0.013	PROMOTER	8.9 x 10^−[^6]	Membrane component/Variant associated with atopic dermatitis
KCNG2	6	Chr 18	0.008	GENE_BODY	1.1 x 10^−[^5]	Ion transport, regulation of insulin secretion/Regulation of neurotransmitters, heart rate, transport
AKAP8L	6	Chr 19	−0.007	PROMOTER	1.1 x 10^−[^5]	mRNA processing, mitotic chromosome condensation/Regulation of H3K4 histone methyltransferase complexes, apoptosis, DNA replication
WIZ	6	Chr 19	−0.007	DOWNSTREAM	1.1 x 10^−[^5]	Regulation of transcription, cell cycle/Aberrant methylation associated with atherosclerosis risk subtype
SLC6A20	4	Chr 3	−0.005	INTRON	4.3 x 10^−[^5]	Amino acid transport, glycine transport/Variants associated with increased risk for diabetes, kidney transporters
SCO2	6	Chr22	−0.005	PROMOTER	4.7 x 10^−[^5]	In utero embryonic development, copper transport/Cytochrome c oxidase assembly, mitochondrial copper delivery pathways

Analysis adjusted for mother’s pre-pregnancy body mass index, mother’s age at delivery, child sex, maternal smoking and cell proportions. DMR, differentially methylation region; Chr, chromosome; FDR, false discovery rate; TSS, transcription start site.

Figure 1. Ingenuity pathway analysis. Top 30 Canonical pathways of comparison analysis of genes overlap with most significant DMRs in children with top-tertile ALT, PDFF and NAFLD.

Figure 2. IPA Results for upstream regulators.

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