DNA methylation at a nutritionally sensitive region of the PAX8 gene is associated with thyroid volume and function in Gambian children

Abstract

PAX8 is a key thyroid transcription factor implicated in thyroid gland differentiation and function, and PAX8 gene methylation is reported to be sensitive to the periconceptional environment. Using a novel recall-by-epigenotype study in Gambian children, we found that PAX8 hypomethylation at age 2 years is associated with a 21% increase in thyroid volume and an increase in free thyroxine (T4) at 5 to 8 years, the latter equivalent to 8.4% of the normal range. Free T4 was associated with a decrease in DXA-derived body fat and bone mineral density. Furthermore, offspring PAX8 methylation was associated with periconceptional maternal nutrition, and methylation variability was influenced by genotype, suggesting that sensitivity to environmental exposures may be under partial genetic control. Together, our results demonstrate a possible link between early environment, PAX8 gene methylation and thyroid gland development and function, with potential implications for early embryonic programming of thyroid-related health and disease.

Fig. 1.. Study overview.

QC, quality control.

Fig. 2.. PAX8 region of interest.

The PAX8 gene extends from chr2:113,215,997 to 113,278,921 (hg38) and contains 12 exons and 11 introns. Exons 8 and 9 of PAX8 are shown, as well as the first two exons of an isoform of the PAX8-AS1 antisense long noncoding RNA. CpGs are shown in the top track. The four CpGs highlighted in red were analyzed in this study (chr2:113,235,186; 113,235,228; 113,235,251; and 113,235,267). The “Known DMRs” (differentially methylated regions) track highlights regions identified in the following studies: putative MEs displaying systemic interindividual variation identified in (i) Silver et al. (25) and (ii) Kessler et al. (23); (iii) DMR associated with gestational famine (27), Gambian SoC (35), and maternal folic acid supplementation (35); and (iv) DMRs associated with Gambian SoC in an additional study (31). The SNP track denotes variants within 2000 bp of the CpGs of interest that were called from the methyl-seq data. The SNP highlighted in red is close to our region of interest and tags an LD block encompassing it (see fig. S11). This is the SNP used for the genotype analyses (rs10193733; chr2:113,235,047 T > C).

Fig. 3.. DNA methylation in the study population at the PAX8 CpGs of interest.

Individuals were sorted by methylation level at CpG chr2:113,235,228 (indicated by dots), which was used to select the high (red) and low (blue) PAX8 methylation groups. Methylation range across the four CpGs of interest is indicated by the boxes.

Fig. 4.. C alleles at rs10193733 are associated with decreased DNA methylation variation at the PAX8 region of interest.

DNA methylation at CpGs is shown for the PAX8 region of interest (delimited by red brackets) and flanking 200-bp region, split by rs10193733 genotype. Numbers in brackets denote the counts of individuals with each genotype at the SNP (total N = 523).

Fig. 5.. Proposed model linking PAX8 methylation and expression, thyroid function and development, and phenotype.

(1) PAX8 methylation is influenced by periconceptional nutritional factors, sex, and genotype. (2) PAX8 methylation is set early in embryonic development. (3) PAX8 methylation sets a trajectory for thyroid gland development: PAX8 is expressed from gestational days 20 to 22 in humans, around the same time that thyroid progenitor cells begin specification in the endoderm (15). PAX8 methylation state (or related epigenetic factors) alters PAX8 expression that influences thyroid gland development. (4) PAX8 methylation is inversely associated with thyroid size and free T4 in mid-childhood. (5) Free T4 is inversely associated with fat and BMD in mid-childhood. (6) PAX8 methylation is inversely associated with PAX8-AS1 expression. (7) There is a divergence between blood and thyroid methylation in adults.