Methylation profiles of global LINE-1 DNA and the GSTP1 promoter region in children exposed to lead (Pb)

Figures & data

Figure 1. Flowchart of profile for the study participants.

Figure 2. Map of Kabwe showing the 13 health centres (yellow blocks) that were included in the study. The dashed oval-shaped line showed the Pb-polluted area (as exposed area) and outside is Pb-unpolluted area (as control area).

Table 1. Primer sequences used for the analysis.

	Primer sequence
LINE-1	F: 5'-TTTTGAGTTAGGTGTGGGATATA-3' R: 5'-Biot-AAAATCAAAAAATTCCCTTTC-3' S: 5'-AGTTAGGTGTGGGATATAGT-3'
GSTP1	F: 5'-AGTTGCGCGGCGATTTC-3' (sense), R: 5'-GCCCCAATACTAAATCACGACG-3' P: FAM-5'-CGGTCGACGTTCGGGGTGTAGCG-3'-TAMRA
ACTB	F: 5'-TGGTGATGGAGGAGGTTTAGTAAGT-3' R: 5'-AACCAATAAAACCTACTCCTCCCTTAA-3' P: FAM-5'-ACCACCACCCAACACACAATAACAAACACA-3'-TAMRA

F: forward primer; R: reverse primer; S: pyrosequencing primer; P: probe

Table 2. Characteristics of study subjects and blood Pb levels (µg/dL) for exposed (n = 123) and control (n = 63) children enrolled for this study, July-August 2017.

Variable		N	Mean ± SD	Median	Range
Age (Years)	ALL	186	5.30 ± 2.45	5	1–10
	F	92	5.42 ± 2.46	5.5	1–10
	M	94	5.18 ± 2.45	5	1–10
Height (Cm)	F	92	108 ± 18.5	111	70–144
	M	94	106 ± 18.0	106	68–144
Weight (Kg)	F	92	19.0 ± 7.03	17.5	8–41
	M	94	18.0 ± 5.65	17	8–30
BMI (Kg/m^2)	ALL	186	15.6 ± 1.73	15.5	10.6–21.8
	F	92	15.6 ± 1.86	15.6	10.6–21.8
	M	94	15.7 ± 1.59	15.5	11.1–19.8
BLLs (µg/dL)	ALL	186	24.1 ± 18.0	22.4	1.62–74.1
	Exposed area	123	32.4 ± 15.9*	30.7	3.24–74.1
	Control area	63	6.23 ± 4.25	4.5	1.62–19.5
	F	92	26.4 ± 18.3*	24.8	1.73–74.1
	M	94	21.6 ± 17.4	15.2	1.62–66.9
	≤ 2 years	30	33.5 ± 19.3**	31.6	3.25–63.9
	> 2–4 years	52	21.8 ± 17.6	15.1	1.73–65.9
	> 4–7 years	56	23.5 ± 17.5	23.3	1.62–66.8
	> 7 years	48	21.0 ± 16.5	18.9	2.10–74.1

N: number; BLLs: blood Pb levels; SD: standard deviation

* t-test, ** Kruskal-Wallis’s test: p< 0.05

Table 3. Mixed-effects regression analysis of LINE-1 methylation level with blood Pb level and characteristics of the study subjects.

Variables	Estimate	Std Error	Lower 95%	Upper 95%	t Ratio	p-value
BLL (µg/dL)	–0.046	0.017	–0.078	–0.013	–2.760	0.006
Area (exposed)	0.288	0.308	–0.320	0.895	0.930	0.352
Age	–0.305	0.251	–0.799	0.189	–1.220	0.225
Gender [F]	–0.116	0.224	–0.559	0.327	–0.520	0.606
Height (cm)	–0.045	0.099	–0.239	0.149	–0.460	0.648
Weight (kg)	0.198	0.271	–0.336	0.733	0.730	0.465
BMI	–0.514	0.375	–1.253	0.226	–1.370	0.172

Figure 3. LINE-1 methylation level (%) in whole blood (a) by Pb exposure quartiles and (b) relationship with blood Pb level quartiles. *p < 0.05; Wilcoxon rank-sum test between LINE-1 methylation levels and blood Pb quartile levels.

Figure 4. GSTP1 promoter’s methylation levels in whole blood and adjusted odd ratios (a) per area [control area: >3 km far away from the mine site; exposed area: within 3 km of the mine area and windward direction] and (b) per gender. PMR: percent methylation reference; solid bars indicate mean (m), and median (m) within a group of children; #indicates samples with PMR values ≤ 0.01. [a]: adjusted for age, gender, and BMI: [b]: adjusted for area and BMI.