Early-life tobacco exposure is causally implicated in aberrant RAG-mediated recombination in childhood acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children, yet few environmental risk factors have been identified. We previously found an association between early-life tobacco smoke exposure and frequency of somatic deletions of 8 leukemia driver genes among childhood ALL patients in the California Childhood Leukemia Study. To expand analysis genome-wide and examine potential mechanisms, we conducted tumor whole-genome sequencing in 35 ALL patients, including 18 with high prenatal tobacco exposure and 17 with low exposure as determined by established epigenetic biomarkers. High tobacco exposure patients had significantly more structural variants (P < .001) and deletions (P = .001) genome-wide than low exposure patients. Investigation of off-target RAG recombination revealed that 41% of deletions in the high tobacco exposure patients were putatively RAG-mediated (full RAG motif identified at one or both breakpoints) compared with only 21% in the low exposure group (P = .001). In a multilevel model, deletions in high tobacco exposure patients were 2.44-fold (95% CI:1.13-5.38) more likely to be putatively RAG-mediated than deletions in low exposure patients. No point mutational signatures were associated with prenatal tobacco exposure. Our findings suggest that early-life tobacco smoke exposure may promote leukemogenesis by driving development of somatic deletions in pre-leukemic lymphocytes via off-target RAG recombination.

Genetic variation near GRB10 associated with bone growth and osteosarcoma risk in canine and human populations.

BACKGROUND: Canine and human osteosarcoma are similar in clinical presentation and tumor genomics. Giant breed dogs experience elevated osteosarcoma incidence, and taller stature remains a consistent risk factor for human osteosarcoma. Whether evolutionarily conserved genes contribute to both human and canine osteosarcoma predisposition merits evaluation. METHODS: A multi-center sample of childhood osteosarcoma patients and controls underwent genome-wide genotyping and imputation. Ancestry-adjusted SNP associations were calculated within each dataset using logistic regression, then meta-analyzed across the three datasets, totaling 1091 patients and 3026 controls. Ten regions previously associated with canine osteosarcoma risk were mapped to the human genome, spanning ∼6 Mb. We prioritized association testing of 5985 human SNPs mapping to candidate osteosarcoma risk regions detected in Irish wolfhounds, the largest dog breed studied. Secondary analyses explored 6289 additional human SNPs mapping to candidate osteosarcoma risk regions identified in Rottweilers and greyhounds. RESULTS: Fourteen SNPs were associated with human osteosarcoma risk after adjustment for multiple comparisons, all within a 42 kb region of human Chromosome 7p12.1. The lead variant was rs17454681 (OR=1.25, 95 %CI: 1.12-1.39; P=4.1×10-5), and independent risk variants were not observed in conditional analyses. While the associated region spanned 2.1 Mb and contained eight genes in Irish wolfhounds, associations were localized to a 50-fold smaller region of the human genome and strongly implicate GRB10 (growth factor receptor-bound protein 10) in canine and human osteosarcoma predisposition. PheWAS analysis in UK Biobank data identified noteworthy associations of the rs17454681 risk allele with varied measures of height and pubertal timing. CONCLUSIONS: Our comparative oncology analysis identified a novel human osteosarcoma risk allele near GRB10, a growth inhibitor that suppresses activated receptor tyrosine kinases including IGF1R, PDGFRB, and EGFR. Epidemiologists may benefit from leveraging cross-species comparisons to identify haplotypes in highly susceptible but genetically homogenous populations of domesticated animals, then fine-mapping these associations in diverse human populations.

Inferring causal cell types of human diseases and risk variants from candidate regulatory elements.

The heritability of human diseases is extremely enriched in candidate regulatory elements (cRE) from disease-relevant cell types. Critical next steps are to infer which and how many cell types are truly causal for a disease (after accounting for co-regulation across cell types), and to understand how individual variants impact disease risk through single or multiple causal cell types. Here, we propose CT-FM and CT-FM-SNP, two methods that leverage cell-type-specific cREs to fine-map causal cell types for a trait and for its candidate causal variants, respectively. We applied CT-FM to 63 GWAS summary statistics (average N = 417K) using nearly one thousand cRE annotations, primarily coming from ENCODE4. CT-FM inferred 81 causal cell types with corresponding SNP-annotations explaining a high fraction of trait SNP-heritability (~2/3 of the SNP-heritability explained by existing cREs), identified 16 traits with multiple causal cell types, highlighted cell-disease relationships consistent with known biology, and uncovered previously unexplored cellular mechanisms in psychiatric and immune-related diseases. Finally, we applied CT-FM-SNP to 39 UK Biobank traits and predicted high confidence causal cell types for 2,798 candidate causal non-coding SNPs. Our results suggest that most SNPs impact a phenotype through a single cell type, and that pleiotropic SNPs target different cell types depending on the phenotype context. Altogether, CT-FM and CT-FM-SNP shed light on how genetic variants act collectively and individually at the cellular level to impact disease risk.

A noncoding regulatory variant in IKZF1 increases acute lymphoblastic leukemia risk in Hispanic/Latino children.

Hispanic/Latino children have the highest risk of acute lymphoblastic leukemia (ALL) in the US compared to other racial/ethnic groups, yet the basis of this remains incompletely understood. Through genetic fine-mapping analyses, we identified a new independent childhood ALL risk signal near IKZF1 in self-reported Hispanic/Latino individuals, but not in non-Hispanic White individuals, with an effect size of ∼1.44 (95% confidence interval = 1.33-1.55) and a risk allele frequency of ∼18% in Hispanic/Latino populations and <0.5% in European populations. This risk allele was positively associated with Indigenous American ancestry, showed evidence of selection in human history, and was associated with reduced IKZF1 expression. We identified a putative causal variant in a downstream enhancer that is most active in pro-B cells and interacts with the IKZF1 promoter. This variant disrupts IKZF1 autoregulation at this enhancer and results in reduced enhancer activity in B cell progenitors. Our study reveals a genetic basis for the increased ALL risk in Hispanic/Latino children.

In Utero Origins of Acute Leukemia in Children.

Acute leukemias, mainly consisting of acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), comprise a major diagnostic group among hematologic cancers. Due to the early age at onset of ALL, particularly, it has long been suspected that acute leukemias of childhood may have an in utero origin. This supposition has motivated many investigations seeking direct proof of prenatal leukemogenesis, in particular, twin and "backtracking studies". The suspected in utero origin has also focused on gestation as a critical window of risk, resulting in a rich literature on prenatal risk factors for pediatric acute leukemias. In this narrative review, we recount the circumstantial and direct evidence for an in utero origin of childhood acute leukemias.

Evaluation of the genetic basis of familial-associated early-onset hematologic cancers in an ancestral/ethnically diverse population.

Genetic predisposition to hematologic malignancies has historically been addressed utilizing patients recruited from clinical trials and pedigrees constructed at major treatment centers. Such efforts leave unexplored the genetic basis of variations in risk by race/ethnic group shown in population-based surveillance data where cancer registration, compulsory by law, delivers universal enrollment. To address this, we performed exome sequencing on DNA isolated from newborn bloodspots derived from sibling pairs with early-onset cancers across California in which at least one of the siblings developed a hematologic cancer, using unbiased recruitment from the full state population. We identified pathogenic/likely pathogenic (P/ LP) variants among 1,172 selected cancer genes that were private or present at low allele frequencies in reference populations. Within 64 subjects from 32 families, we found 9 LP variants shared between siblings, and an additional 7 such variants in singleton children (not shared with their sibling). In 8 of the shared cases, the ancestral origin of the local haplotype that carries P/LP variants matched the dominant global ancestry of study participant families. This was the case for Latino sibling pairs on FLG and CBLB, non-Latino White sibling pairs in TP53 and NOD2, and a shared GATA2 variant for a non-Latino Black sibling pair. A new inherited mutation in HABP2 was identified in a sibling pair, one with diffuse large B-cell lymphoma and the other with neuroblastoma. Overall, the profile of P/LP germline variants across ancestral/ethnic groups suggests that rare alleles contributing to hematologic diseases originate within their race/ethnic origin parental populations, demonstrating the value of this discovery process in diverse, population-based registries.

Novel insight into the etiology of ischemic stroke gained by integrative multiome-wide association study.

Stroke, characterized by sudden neurological deficits, is the second leading cause of death worldwide. Although genome-wide association studies (GWAS) have successfully identified many genomic regions associated with ischemic stroke (IS), the genes underlying risk and their regulatory mechanisms remain elusive. Here, we integrate a large-scale GWAS (N = 1 296 908) for IS together with molecular QTLs data, including mRNA, splicing, enhancer RNA (eRNA), and protein expression data from up to 50 tissues (total N = 11 588). We identify 136 genes/eRNA/proteins associated with IS risk across 60 independent genomic regions and find IS risk is most enriched for eQTLs in arterial and brain-related tissues. Focusing on IS-relevant tissues, we prioritize 9 genes/proteins using probabilistic fine-mapping TWAS analyses. In addition, we discover that blood cell traits, particularly reticulocyte cells, have shared genetic contributions with IS using TWAS-based pheWAS and genetic correlation analysis. Lastly, we integrate our findings with a large-scale pharmacological database and identify a secondary bile acid, deoxycholic acid, as a potential therapeutic component. Our work highlights IS risk genes/splicing-sites/enhancer activity/proteins with their phenotypic consequences using relevant tissues as well as identify potential therapeutic candidates for IS.

Epigenomic signature of major congenital heart defects in newborns with Down syndrome.

BACKGROUND: Congenital heart defects (CHDs) affect approximately half of individuals with Down syndrome (DS), but the molecular reasons for incomplete penetrance are unknown. Previous studies have largely focused on identifying genetic risk factors associated with CHDs in individuals with DS, but comprehensive studies of the contribution of epigenetic marks are lacking. We aimed to identify and characterize DNA methylation differences from newborn dried blood spots (NDBS) of DS individuals with major CHDs compared to DS individuals without CHDs. METHODS: We used the Illumina EPIC array and whole-genome bisulfite sequencing (WGBS) to quantitate DNA methylation for 86 NDBS samples from the California Biobank Program: (1) 45 DS-CHD (27 female, 18 male) and (2) 41 DS non-CHD (27 female, 14 male). We analyzed global CpG methylation and identified differentially methylated regions (DMRs) in DS-CHD versus DS non-CHD comparisons (both sex-combined and sex-stratified) corrected for sex, age of blood collection, and cell-type proportions. CHD DMRs were analyzed for enrichment in CpG and genic contexts, chromatin states, and histone modifications by genomic coordinates and for gene ontology enrichment by gene mapping. DMRs were also tested in a replication dataset and compared to methylation levels in DS versus typical development (TD) WGBS NDBS samples. RESULTS: We found global CpG hypomethylation in DS-CHD males compared to DS non-CHD males, which was attributable to elevated levels of nucleated red blood cells and not seen in females. At a regional level, we identified 58, 341, and 3938 CHD-associated DMRs in the Sex Combined, Females Only, and Males Only groups, respectively, and used machine learning algorithms to select 19 Males Only loci that could distinguish CHD from non-CHD. DMRs in all comparisons were enriched for gene exons, CpG islands, and bivalent chromatin and mapped to genes enriched for terms related to cardiac and immune functions. Lastly, a greater percentage of CHD-associated DMRs than background regions were differentially methylated in DS versus TD samples. CONCLUSIONS: A sex-specific signature of DNA methylation was detected in NDBS of DS-CHD compared to DS non-CHD individuals. This supports the hypothesis that epigenetics can reflect the variability of phenotypes in DS, particularly CHDs.

Evaluating genomic polygenic risk scores for childhood acute lymphoblastic leukemia in Latinos.

The utility of polygenic risk score (PRS) models has not been comprehensively evaluated for childhood acute lymphoblastic leukemia (ALL), the most common type of cancer in children. Previous PRS models for ALL were based on significant loci observed in genome-wide association studies (GWASs), even though genomic PRS models have been shown to improve prediction performance for a number of complex diseases. In the United States, Latino (LAT) children have the highest risk of ALL, but the transferability of PRS models to LAT children has not been studied. In this study, we constructed and evaluated genomic PRS models based on either non-Latino White (NLW) GWAS or a multi-ancestry GWAS. We found that the best PRS models performed similarly between held-out NLW and LAT samples (PseudoR2 = 0.086 ± 0.023 in NLW vs. 0.060 ± 0.020 in LAT), and can be improved for LAT if we performed GWAS in LAT-only (PseudoR2 = 0.116 ± 0.026) or multi-ancestry samples (PseudoR2 = 0.131 ± 0.025). However, the best genomic models currently do not have better prediction accuracy than a conventional model using all known ALL-associated loci in the literature (PseudoR2 = 0.166 ± 0.025), which includes loci from GWAS populations that we could not access to train genomic PRS models. Our results suggest that larger and more inclusive GWASs may be needed for genomic PRS to be useful for ALL. Moreover, the comparable performance between populations may suggest a more oligogenic architecture for ALL, where some large effect loci may be shared between populations. Future PRS models that move away from the infinite causal loci assumption may further improve PRS for ALL.

Gene-Environment Analyses Reveal Novel Genetic Candidates with Prenatal Tobacco Exposure in Relation to Risk for Childhood Acute Lymphoblastic Leukemia.

BACKGROUND: Associations between maternal tobacco exposure during pregnancy and childhood acute lymphoblastic leukemia (ALL) have yielded mixed results. This may be due to biases in self-reported smoking or other differences in individual-level risk factors. We utilized a biological marker of maternal tobacco exposure to evaluate the association between maternal tobacco exposure during pregnancy, genetics, and subsequent childhood ALL risk in two large population-based studies of childhood ALL in California. METHODS: Maternal exposure to tobacco smoke was assessed with a validated methylation marker (cg05575921) of the aryl hydrocarbon receptor repressor (AHRR) gene in newborn dried blood spots. We adjusted for sex, birthweight, gestational age, mode of delivery, year of birth, AHRR quantitative trait locus (mQTL) rs77111113, and a polygenetic risk score for childhood ALL. We additionally adjusted for principal components in a gene-environment interaction testing method that incorporates gene-only and environment-only effects along with interactions. RESULTS: AHRR hypomethylation overall was not associated with childhood ALL. In gene-environment interaction testing, several genetic variants displayed significant interaction with AHRR hypomethylation and childhood ALL. CONCLUSIONS: Our results suggest that novel candidates in PTPRK and DPP6 may play a role in tobacco-related leukemogenesis. Further research is necessary to better understand the effects of tobacco and these variants on childhood ALL risk. IMPACT: Despite the lack of an overall "main effect," tobacco exposure during pregnancy affects childhood ALL risk depending on specific genetic variants.

Evaluating Genomic Polygenic Risk Scores for Childhood Acute Lymphoblastic Leukemia in Latinos.

The utility of polygenic risk score (PRS) models has not been comprehensively evaluated for childhood acute lymphoblastic leukemia (ALL), the most common type of cancer in children. Previous PRS models for ALL were based on significant loci observed in genome-wide association studies (GWAS), even though genomic PRS models have been shown to improve prediction performance for a number of complex diseases. In the United States, Latino (LAT) children have the highest risk of ALL, but the transferability of PRS models to LAT children has not been studied. In this study we constructed and evaluated genomic PRS models based on either non-Latino white (NLW) GWAS or a multi-ancestry GWAS. We found that the best PRS models performed similarly between held-out NLW and LAT samples (PseudoR 2 = 0.086 ± 0.023 in NLW vs. 0.060 ± 0.020 in LAT), and can be improved for LAT if we performed GWAS in LAT-only (PseudoR 2 = 0.116 ± 0.026) or multi-ancestry samples (PseudoR 2 = 0.131 ± 0.025). However, the best genomic models currently do not have better prediction accuracy than a conventional model using all known ALL-associated loci in the literature (PseudoR 2 = 0.166 ± 0.025), which includes loci from GWAS populations that we could not access to train genomic PRS models. Our results suggest that larger and more inclusive GWAS may be needed for genomic PRS to be useful for ALL. Moreover, the comparable performance between populations may suggest a more oligo-genic architecture for ALL, where some large effect loci may be shared between populations. Future PRS models that move away from the infinite causal loci assumption may further improve PRS for ALL.

Epigenomic signature of major congenital heart defects in newborns with Down syndrome.

Background: Congenital heart defects (CHDs) affect approximately half of individuals with Down syndrome (DS) but the molecular reasons for incomplete penetrance are unknown. Previous studies have largely focused on identifying genetic risk factors associated with CHDs in individuals with DS, but comprehensive studies of the contribution of epigenetic marks are lacking. We aimed to identify and characterize DNA methylation differences from newborn dried blood spots (NDBS) of DS individuals with major CHDs compared to DS individuals without CHDs. Methods: We used the Illumina EPIC array and whole-genome bisulfite sequencing (WGBS) to quantitate DNA methylation for 86 NDBS samples from the California Biobank Program: 1) 45 DS-CHD (27 female, 18 male) and 2) 41 DS non-CHD (27 female, 14 male). We analyzed global CpG methylation and identified differentially methylated regions (DMRs) in DS-CHD vs DS non-CHD comparisons (both sex-combined and sex-stratified) corrected for sex, age of blood collection, and cell type proportions. CHD DMRs were analyzed for enrichment in CpG and genic contexts, chromatin states, and histone modifications by genomic coordinates and for gene ontology enrichment by gene mapping. DMRs were also tested in a replication dataset and compared to methylation levels in DS vs typical development (TD) WGBS NDBS samples. Results: We found global CpG hypomethylation in DS-CHD males compared to DS non-CHD males, which was attributable to elevated levels of nucleated red blood cells and not seen in females. At a regional level, we identified 58, 341, and 3,938 CHD-associated DMRs in the Sex Combined, Females Only, and Males Only groups, respectively, and used machine learning algorithms to select 19 Males Only loci that could distinguish CHD from non-CHD. DMRs in all comparisons were enriched for gene exons, CpG islands, and bivalent chromatin and mapped to genes enriched for terms related to cardiac and immune functions. Lastly, a greater percentage of CHD-associated DMRs than background regions were differentially methylated in DS vs TD samples. Conclusions: A sex-specific signature of DNA methylation was detected in NDBS of DS-CHD compared to DS non-CHD individuals. This supports the hypothesis that epigenetics can reflect the variability of phenotypes in DS, particularly CHDs.

Novel insight into the etiology of ischemic stroke gained by integrative transcriptome-wide association study.

Stroke, characterized by sudden neurological deficits, is the second leading cause of death worldwide. Although genome-wide association studies (GWAS) have successfully identified many genomic regions associated with ischemic stroke (IS), the genes underlying risk and their regulatory mechanisms remain elusive. Here, we integrate a large-scale GWAS (N=1,296,908) for IS together with mRNA, splicing, enhancer RNA (eRNA) and protein expression data (N=11,588) from 50 tissues. We identify 136 genes/eRNA/proteins associated with IS risk across 54 independent genomic regions and find IS risk is most enriched for eQTLs in arterial and brain-related tissues. Focusing on IS-relevant tissues, we prioritize 9 genes/proteins using probabilistic fine-mapping TWAS analyses. In addition, we discover that blood cell traits, particularly reticulocyte cells, have shared genetic contributions with IS using TWAS-based pheWAS and genetic correlation analysis. Lastly, we integrate our findings with a large-scale pharmacological database and identify a secondary bile acid, deoxycholic acid, as a potential therapeutic component. Our work highlights IS risk genes/splicing-sites/enhancer activity/proteins with their phenotypic consequences using relevant tissues as well as identify potential therapeutic candidates for IS.

Multi-ancestry genome-wide association study of 4069 children with glioma identifies 9p21.3 risk locus.

BACKGROUND: Although recent sequencing studies have revealed that 10% of childhood gliomas are caused by rare germline mutations, the role of common variants is undetermined and no genome-wide significant risk loci for pediatric central nervous system tumors have been identified to date. METHODS: Meta-analysis of 3 population-based genome-wide association studies comprising 4069 children with glioma and 8778 controls of multiple genetic ancestries. Replication was performed in a separate case-control cohort. Quantitative trait loci analyses and a transcriptome-wide association study were conducted to assess possible links with brain tissue expression across 18 628 genes. RESULTS: Common variants in CDKN2B-AS1 at 9p21.3 were significantly associated with astrocytoma, the most common subtype of glioma in children (rs573687, P-value of 6.974e-10, OR 1.273, 95% CI 1.179-1.374). The association was driven by low-grade astrocytoma (P-value of 3.815e-9) and exhibited unidirectional effects across all 6 genetic ancestries. For glioma overall, the association approached genome-wide significance (rs3731239, P-value of 5.411e-8), while no significant association was observed for high-grade tumors. Predicted decreased brain tissue expression of CDKN2B was significantly associated with astrocytoma (P-value of 8.090e-8). CONCLUSIONS: In this population-based genome-wide association study meta-analysis, we identify and replicate 9p21.3 (CDKN2B-AS1) as a risk locus for childhood astrocytoma, thereby establishing the first genome-wide significant evidence of common variant predisposition in pediatric neuro-oncology. We furthermore provide a functional basis for the association by showing a possible link to decreased brain tissue CDKN2B expression and substantiate that genetic susceptibility differs between low- and high-grade astrocytoma.

Associations between early-life and in utero infections and cytomegalovirus-positive acute lymphoblastic leukemia in children.

Childhood infections and cytomegalovirus (CMV) are associated with pediatric acute lymphoblastic leukemia (ALL). CMV dysregulates the host immune system and alters the immune response to subsequent antigenic exposures. We suspect that this immune dysregulation contributes to increased numbers of symptomatic infections in childhood allowing for expansion of pre-leukemic clones. We explored the association between childhood infections, maternal infections during pregnancy and CMV-positive ALL. Using a droplet digital PCR assay, we screened diagnostic ALL bone marrow samples from the California Childhood Leukemia Study (1995-2015) for the presence of CMV DNA identifying CMV-positive and CMV-negative cases. We performed a case-only analysis (n = 524) comparing the number and types of childhood infections and maternal infections during pregnancy between CMV-positive and CMV-negative ALL cases using logistic regression. With increasing numbers of infections in the first 12 months of life, children were more likely to classify to the highest tertile of CMV DNA in the bone marrow at diagnosis (OR: 1.04, 95% CI: 1.01-1.08). Specifically, those reporting cough or flu in the first 12 months were more likely to be CMV-positive at ALL diagnosis (OR: 2.15, 95% CI: 1.06-4.37 and OR: 2.06, 95% CI: 1.17-3.63 respectively). Furthermore, those with a history of maternal infection during pregnancy were more likely to be CMV-positive (OR: 2.12, 95% CI: 1.24-3.62). We hypothesize that children with underlying immune dysregulation develop more symptomatic infections in childhood and ultimately CMV-positive ALL; this underlying immune dysregulation may be due to early immune system alterations via CMV exposure (in utero or early infancy) proposing a potential link between CMV and ALL etiology.

Clonal hematopoiesis and risk of prostate cancer in large samples of European ancestry men.

Little is known regarding the potential relationship between clonal hematopoiesis (CH) of indeterminate potential (CHIP), which is the expansion of hematopoietic stem cells with somatic mutations, and risk of prostate cancer, the fifth leading cause of cancer death of men worldwide. We evaluated the association of age-related CHIP with overall and aggressive prostate cancer risk in two large whole-exome sequencing studies of 75 047 European ancestry men, including 7663 prostate cancer cases, 2770 of which had aggressive disease, and 3266 men carrying CHIP variants. We found that CHIP, defined by over 50 CHIP genes individually and in aggregate, was not significantly associated with overall (aggregate HR = 0.93, 95% CI = 0.76-1.13, P = 0.46) or aggressive (aggregate OR = 1.14, 95% CI = 0.92-1.41, P = 0.22) prostate cancer risk. CHIP was weakly associated with genetic risk of overall prostate cancer, measured using a polygenic risk score (OR = 1.05 per unit increase, 95% CI = 1.01-1.10, P = 0.01). CHIP was not significantly associated with carrying pathogenic/likely pathogenic/deleterious variants in DNA repair genes, which have previously been found to be associated with aggressive prostate cancer. While findings from this study suggest that CHIP is likely not a risk factor for prostate cancer, it will be important to investigate other types of CH in association with prostate cancer risk.

Meta-Analysis of DNA Methylation Datasets Shows Aberrant DNA Methylation of Thyroid Development or Function Genes in Down Syndrome.

Background: In Down syndrome (DS), there is high occurrence of congenital hypothyroidism (CH) and subclinical hypothyroidism (SH) early in life. The etiology of CH and early SH in DS remains unclear. Previous research has shown genome-wide transcriptional and epigenetic alterations in DS. Thus, we hypothesized that CH and early SH could be caused by epigenetically driven transcriptional downregulation of thyroid-related genes, through promoter region hypermethylation. Methods: We extracted whole blood DNA methylation (DNAm) profiles of DS and non-DS individuals from four independent Illumina array-based datasets (252 DS individuals and 519 non-DS individuals). The data were divided into discovery and validation datasets. Epigenome-wide association analysis was performed using a linear regression model, after which we filtered results for thyroid-related genes. Results: In the discovery dataset, we identified significant associations for DS in 18 thyroid-related genes. Twenty-one of 30 significant differentially methylated positions (DMPs) were also significant in the validation dataset. A meta-analysis of the discovery and validation datasets detected 31 DMPs, including 29 promoter-associated cytosine-guanine dinucleotides (CpG) with identical direction of effect across the datasets, and two differentially methylated regions. Twenty-seven DMPs were hypomethylated and promoter associated. The mean methylation difference of hypomethylated thyroid-related DMPs decreased with age. Conclusions: Contrary to our hypothesis of generalized hypermethylation of promoter regions of thyroid-related genes-indicative of epigenetic silencing of promoters and subsequent transcriptional downregulation, causing biochemical thyroid abnormalities in DS-we found an enrichment of hypomethylated DMPs annotated to promoter regions of these genes. This suggests that CH and early SH in DS are not caused by differential methylation of thyroid-related genes. Considering that epigenetic regulation is dynamic, we hypothesize that the observed thyroid-related gene DNAm changes could be a rescue phenomenon in an attempt to ameliorate the thyroid phenotype, through epigenetic upregulation of thyroid-related genes. This hypothesis is supported by the finding of decreasing methylation difference of thyroid-related genes with age. The prevalence of early SH declines with age, so hypothetically, epigenetic upregulation of thyroid-related genes also diminishes. While this study provides interesting insights, the exact origin of CH and early SH in DS remains unknown.