Association between telomere length and Plasmodium falciparum malaria endemicity in sub-Saharan Africans.

Leukocyte telomere length (LTL) varies significantly across human populations, with individuals of African ancestry having longer LTL than non-Africans. However, the genetic and environmental drivers of LTL variation in Africans remain largely unknown. We report here on the relationship between LTL, genetics, and a variety of environmental and climatic factors in ethnically diverse African adults (n = 1,818) originating from Botswana, Tanzania, Ethiopia, and Cameroon. We observe significant variation in LTL among populations, finding that the San hunter-gatherers from Botswana have the longest leukocyte telomeres and that the Fulani pastoralists from Cameroon have the shortest telomeres. Genetic factors explain ∼50% of LTL variation among individuals. Moreover, we observe a significant negative association between Plasmodium falciparum malaria endemicity and LTL while adjusting for age, sex, and genetics. Within Africa, adults from populations indigenous to areas with high malaria exposure have shorter LTL than those in populations indigenous to areas with low malaria exposure. Finally, we explore to what degree the genetic architecture underlying LTL in Africa covaries with malaria exposure.

Clonal landscape and clinical outcomes of telomere biology disorders: somatic rescuing and cancer mutations.

Telomere biology disorders (TBD), caused by pathogenic germline variants in telomere-related genes, present with multi-organ disease and a predisposition to cancer. Clonal hematopoiesis (CH) as a marker of cancer development and survival in TBD is poorly understood. Here, we characterized the clonal landscape of a large cohort of 207 TBD patients with a broad range of age and phenotype. CH occurred predominantly in symptomatic patients and in signature genes typically associated with cancers: PPM1D, POT1, TERT promoter (TERTp), U2AF1S34, and/or TP53. Chromosome 1q gain (Chr1q+) was the commonest karyotypic abnormality. Clinically, multiorgan involvement and CH in TERTp, TP53, and splicing factor genes associated with poorer overall survival. Chr1q+, and splicing factor or TP53 mutations significantly increased the risk of hematologic malignancies, regardless of the clonal burden. Chr1q+ and U2AF1S34 mutated clones were pre-malignant events associated with the secondary acquisition of mutations in genes related to hematologic malignancies. Like known effects of Chr1q+ and TP53-CH, functional studies demonstrated that U2AF1S34 mutations primarily compensated for aberrant upregulation of TP53 and interferon pathways in telomere-dysfunctional hematopoietic stem cells, highlighting the TP53 pathway as a canonical route of malignancy in TBD. In contrast, somatic POT1/PPM1D/TERTp-CH had distinct trajectories unrelated to cancer development. With implications beyond TBD, our data show that telomere dysfunction is a strong selective pressure for CH. In TBD, CH is a poor prognostic marker associated with worse overall survival. The identification of key regulatory pathways that drive clonal transformation in TBD allows the identification of patients at a higher risk of cancer development.

The distribution and accumulation of the shortest telomeres in telomere biology disorders.

Individuals with telomere biology disorders (TBDs) have very short telomeres, high risk of bone marrow failure (BMF), and reduced survival. Using data from TBD patients, a mean leukocyte Southern blot telomere length (TL) of 5 kilobases (kb) was estimated as the 'telomere brink' at which human survival is markedly reduced. However, the shortest telomere, not the mean TL, signals replicative senescence. We used the Telomere Shortest Length Assay (TeSLA) to tally TL of all 46 chromosomes in blood-derived DNA and examined its relationship with TBDs. Patients (n = 18) had much shorter mean TL (TeSmTL) (2.54 ± 0.41 kb vs. 4.48 ± 0.52 kb, p < 0.0001) and more telomeres <3 kb than controls (n = 22) (70.43 ± 8.76% vs. 33.05 ± 6.93%, p < 0.0001). The proportion of ultrashort telomeres (<1.6 kb) was also higher in patients than controls (39.29 ± 10.69% vs. 10.40 ± 4.09%, p < 0.0001). TeS <1.6 kb was associated with severe (n = 11) compared with non-severe (n = 7) BMF (p = 0.027). Patients with multi-organ manifestations (n = 10) had more telomeres <1.6 kb than those with one affected organ system (n = 8) (p = 0.029). Findings suggest that TBD clinical manifestations are associated with a disproportionately higher number of haematopoietic cell telomeres reaching a telomere brink, whose length at the single telomere level is yet to be determined.

Genetics and geography of leukocyte telomere length in sub-Saharan Africans.

Leukocyte telomere length (LTL) might be causal in cardiovascular disease and major cancers. To elucidate the roles of genetics and geography in LTL variability across humans, we compared LTL measured in 1295 sub-Saharan Africans (SSAs) with 559 African-Americans (AAms) and 2464 European-Americans (EAms). LTL differed significantly across SSAs (P = 0.003), with the San from Botswana (with the oldest genomic ancestry) having the longest LTL and populations from Ethiopia having the shortest LTL. SSAs had significantly longer LTL than AAms [P = 6.5(e-16)] whose LTL was significantly longer than EAms [P = 2.5(e-7)]. Genetic variation in SSAs explained 52% of LTL variance versus 27% in AAms and 34% in EAms. Adjustment for genetic variation removed the LTL differences among SSAs. LTL genetic variation among SSAs, with the longest LTL in the San, supports the hypothesis that longer LTL was ancestral in humans. Identifying factors driving LTL variation in Africa may have important ramifications for LTL-associated diseases.

Measurement of Telomere Length for Longitudinal Analysis: Implications of Assay Precision.

Researchers increasingly wish to test hypotheses concerning the impact of environmental or disease exposures on telomere length (TL), and they use longitudinal study designs to do so. In population studies, TL is usually measured with a quantitative polymerase chain reaction (qPCR)-based method. This method has been validated by calculating its correlation with a gold standard method such as Southern blotting (SB) in cross-sectional data sets. However, in a cross-section, the range of true variation in TL is large, and measurement error is introduced only once. In a longitudinal study, the target variation of interest is small, and measurement error is introduced at both baseline and follow-up. In this paper, we present results from a small data set (n = 20) in which leukocyte TL was measured twice 6.6 years apart by means of both qPCR and SB. The cross-sectional correlations between qPCR and SB were high at both baseline (r = 0.90) and follow-up (r = 0.85), yet their correlation for TL change was poor (r = 0.48). Moreover, the qPCR data but not the SB data showed strong signatures of measurement error. Through simulation, we show that the statistical power gain from performing a longitudinal analysis is much greater for SB than for qPCR. We discuss implications for optimal study design and analysis.

Telomeres and COVID-19.

The medical, public health, and scientific communities are grappling with monumental imperatives to contain COVID-19, develop effective vaccines, identify efficacious treatments for the infection and its complications, and find biomarkers that detect patients at risk of severe disease. The focus of this communication is on a potential biomarker, short telomere length (TL), that might serve to identify patients more likely to die from the SARS-CoV-2 infection, regardless of age. The common thread linking these patients is lymphopenia, which largely reflects a decline in the numbers of CD4/CD8 T cells but not B cells. These findings are consistent with data that lymphocyte TL dynamics impose a limit on T-cell proliferation. They suggest that T-cell lymphopoiesis might stall in individuals with short TL who are infected with SARS-CoV-2.

Telomere length tracking in children and their parents: implications for adult onset diseases.

Adults with comparatively short or long leukocyte telomere length (LTL) typically continue to display comparatively short or long LTL throughout life. This LTL tracking stems from the inability of person-to-person variation in age-dependent LTL shortening during adulthood to offset the wide interindividual LTL variation established prior to adult life. However, LTL tracking in children is unstudied. This study aimed to examine LTL shortening rates and tracking in children and their parents. Longitudinal study in children (n = 67) and their parents (n = 99), whose ages at baseline were 11.4 ± 0.3 and 43.4 ± 0.4 yr, respectively. LTL was measured by Southern blotting at baseline and ∼14 yr thereafter. LTL displayed tracking in both children [intraclass correlation coefficient (ICC) = 0.905, P < 0.001] and their parents (ICC = 0.856, P < 0.001). The children's rate of LTL shortening was twice that of their parents (40.7 ± 2.5 bp/yr; 20.3 ± 2.1 bp/yr, respectively; P < 0.0001). LTL tracking applies not only to adulthood but also to the second decade of life. Coupled with previous work showing that the interindividual variation in LTL across newborns is as wide as in their parents, these findings support the thesis that the LTL-adult disease connection is principally determined before the second decade of life, perhaps mainly at birth.-Benetos, A., Verhulst, S., Labat, C., Lai, T.-P., Girerd, N., Toupance, S., Zannad, F., Rossignol, P., Aviv, A. Telomere length tracking in children and their parents: implications for adult onset diseases.

Short Leukocyte Telomere Length Precedes Clinical Expression of Atherosclerosis: The Blood-and-Muscle Model.

RATIONALE: Short telomere length (TL) in leukocytes is associated with atherosclerotic cardiovascular disease (ASCVD). It is unknown whether this relationship stems from having inherently short leukocyte TL (LTL) at birth or a faster LTL attrition thereafter. LTL represents TL in the highly proliferative hematopoietic system, whereas TL in skeletal muscle represents a minimally replicative tissue. OBJECTIVE: We measured LTL and muscle TL (MTL) in the same individuals with a view to obtain comparative metrics for lifelong LTL attrition and learn about the temporal association of LTL with ASCVD. METHODS AND RESULTS: Our Discovery Cohort comprised 259 individuals aged 63±14 years (mean±SD), undergoing surgery with (n=131) or without (n=128) clinical manifestation of ASCVD. In all subjects, MTL adjusted for muscle biopsy site (MTLA) was longer than LTL and the LTL-MTLA gap similarly widened with age in ASCVD patients and controls. Age- and sex-adjusted LTL (P=0.005), but not MTLA (P=0.90), was shorter in patients with ASCVD than controls. The TL gap between leukocytes and muscle (LTL-MTLA) was wider (P=0.0003), and the TL ratio between leukocytes and muscle (LTL/MTLA) was smaller (P=0.0001) in ASCVD than in controls. Findings were replicated in a cohort comprising 143 individuals. CONCLUSIONS: This first study to apply the blood-and-muscle TL model shows more pronounced LTL attrition in ASCVD patients than controls. The difference in LTL attrition was not associated with age during adulthood suggesting that increased attrition in early life is more likely to be a major explanation of the shorter LTL in ASCVD patients. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02176941.

Telomere length dynamics in early life: the blood-and-muscle model.

Telomere length (TL) trajectories in somatic tissues during human growth and development are poorly understood. We examined a blood-and-muscle model during early life, focusing on TL trajectories in leukocytes, representing the highly proliferative hematopoietic system, and skeletal muscle, a minimally proliferative tissue. Leukocyte TL (LTL) and skeletal muscle TL (MTL) were measured in 28 fetuses and 73 children. LTL and MTL were highly variable across individuals (sd: fetal LTL = 0.72 kb, MTL = 0.72 kb; children LTL = 0.81 kb, MTL = 0.82 kb) but were highly correlated within individuals (fetuses, r = 0.76, P < 0.0001; children, r = 0.87, P < 0.0001). LTL was shorter than MTL in fetuses (10.63 vs. 11.01 kb; P = 0.0004) and children (8.46 vs. 9.40 kb; <0.0001). The LTL-MTL gap was smaller in fetuses than children. TL in children was inversely correlated with body mass index (BMI) (LTL: -0.047 ± 0.016 kb/BMI, P < 0.005; MTL: -0.037 ± 0.017 kb/BMI, P = 0.03). We conclude that variations in TL across adults and differences in TL between somatic tissues are largely established in early life. Because TL plays a significant role in aging-related diseases, insight into the factors that fashion TL in somatic tissues during early development should contribute to an understanding of the relationship of TL with these disease and longevity in humans.-Sabharwal, S., Verhulst, S., Guirguis, G., Kark, J. D., Labat, C., Roche, N. E., Martimucci, K., Patel, K., Heller, D. S., Kimura, M., Chuang, D., Chuang, A., Benetos, A., Aviv, A. Telomere length dynamics in early life: the blood-and-muscle model.

Telomere Length and the Cancer-Atherosclerosis Trade-Off.

Modern humans, the longest-living terrestrial mammals, display short telomeres and repressed telomerase activity in somatic tissues compared with most short-living small mammals. The dual trait of short telomeres and repressed telomerase might render humans relatively resistant to cancer compared with short-living small mammals. However, the trade-off for cancer resistance is ostensibly increased age-related degenerative diseases, principally in the form of atherosclerosis. In this communication, we discuss (a) the genetics of human telomere length, a highly heritable complex trait that is influenced by genetic ancestry, sex, and paternal age at conception, (b) how cancer might have played a role in the evolution of telomere biology across mammals, (c) evidence that in modern humans telomere length is a determinant (rather than only a biomarker) of cancer and atherosclerosis, and (d) the potential influence of relatively recent evolutionary forces in fashioning the variation in telomere length across and within populations, and their likely lasting impact on major diseases in humans. Finally, we propose venues for future research on human telomere genetics in the context of its potential role in shaping the modern human lifespan.

Shorter telomere length in Europeans than in Africans due to polygenetic adaptation.

Leukocyte telomere length (LTL), which reflects telomere length in other somatic tissues, is a complex genetic trait. Eleven SNPs have been shown in genome-wide association studies to be associated with LTL at a genome-wide level of significance within cohorts of European ancestry. It has been observed that LTL is longer in African Americans than in Europeans. The underlying reason for this difference is unknown. Here we show that LTL is significantly longer in sub-Saharan Africans than in both Europeans and African Americans. Based on the 11 LTL-associated alleles and genetic data in phase 3 of the 1000 Genomes Project, we show that the shifts in allele frequency within Europe and between Europe and Africa do not fit the pattern expected by neutral genetic drift. Our findings suggest that differences in LTL within Europeans and between Europeans and Africans is influenced by polygenic adaptation and that differences in LTL between Europeans and Africans might explain, in part, ethnic differences in risks for human diseases that have been linked to LTL.

Positive Association Between Vitamin D Serum Levels and Naevus Counts.

Lower vitamin D serum levels are linked to increased melanoma risk and poorer survival. Naevus counts are associated with both melanoma risk and survival and to leucocyte telomere length. Vitamin D is also linked to telomere biology with higher levels of vitamin D in individuals with longer leucocyte telomere length despite adjusting for age. Using the TwinsUK data, we explored the association between naevus count, leucocyte telomere length and vitamin D serum levels. Increasing vitamin D levels were associated with increasing naevus count: serum levels were 73.3 nmol/l in individuals with less than 50 naevi compared to 78.8 nmol/l in individuals with more than 50 naevi (p?=?0.002). In the final regression model, using naevus count as a continuous variable, vitamin D remained associated with higher naevus counts despite adjustment for age, weight, height, season of sampling and twin relatedness (p?=?0.02). Further adjustment for leucocyte telomere length, decreased the magnitude of the association but it remained significant so leucocyte telomere length is not the sole driver of this association. Having large numbers of naevi is associated with higher vitamin D serum levels.

Correlation of Leukocyte Telomere Length Measurement Methods in Patients with Dyskeratosis Congenita and in Their Unaffected Relatives.

Several methods have been employed to measure telomere length (TL) in human studies. It has been difficult to directly compare the results from these studies because of differences in the laboratory techniques and output parameters. We compared TL measurements (TLMs) by the three most commonly used methods, quantitative polymerase chain reaction (qPCR), flow cytometry with fluorescence in situ hybridization (flow FISH) and Southern blot, in a cohort of patients with the telomere biology disorder dyskeratosis congenita (DC) and in their unaffected relatives (controls). We observed a strong correlation between the Southern blot average TL and the flow FISH total lymphocyte TL in both the DC patients and their unaffected relatives (R² of 0.68 and 0.73, respectively). The correlation between the qPCR average TL and that of the Southern blot method was modest (R² of 0.54 in DC patients and of 0.43 in unaffected relatives). Similar results were noted when comparing the qPCR average TL and the flow FISH total lymphocyte TL (R² of 0.49 in DC patients and of 0.42 in unaffected relatives). In conclusion, the strengths of the correlations between the three widely used TL assays (qPCR, flow FISH, and Southern blot) were significantly different. Careful consideration is warranted when selecting the method of TL measurement for research and for clinical studies.

A short leucocyte telomere length is associated with development of insulin resistance.

AIMS/HYPOTHESIS: A number of studies have shown that leucocyte telomere length (LTL) is inversely associated with insulin resistance and type 2 diabetes mellitus. The aim of the present longitudinal cohort study, utilising a twin design, was to assess whether shorter LTL predicts insulin resistance or is a consequence thereof. METHODS: Participants were recruited between 1997 and 2000 through the population-based national Danish Twin Registry to participate in the GEMINAKAR study, a longitudinal evaluation of metabolic disorders and cardiovascular risk factors. Baseline and follow-up measurements of LTL and insulin resistance over an average of 12 years were performed in a subset of the Registry consisting of 338 (184 monozygotic and 154 dizygotic) same-sex twin pairs. RESULTS: Age at baseline examination was 37.4 ± 9.6 (mean ± SD) years. Baseline insulin resistance was not associated with age-dependent changes in LTL (attrition) over the follow-up period, whereas baseline LTL was associated with changes in insulin resistance during this period. The shorter the LTL at baseline, the more pronounced was the increase in insulin resistance over the follow-up period (p < 0.001); this effect was additive to that of BMI. The co-twin with the shorter baseline LTL displayed higher insulin resistance at follow-up than the co-twin with the longer LTL. CONCLUSIONS/INTERPRETATION: These findings suggest that individuals with short LTL are more likely to develop insulin resistance later in life. By contrast, presence of insulin resistance does not accelerate LTL attrition.

Leukocyte Telomere Length and Risks of Incident Coronary Heart Disease and Mortality in a Racially Diverse Population of Postmenopausal Women.

OBJECTIVE: Telomeres are regions at the ends of chromosomes that maintain chromosomal structural integrity and genomic stability. In studies of mainly older, white populations, shorter leukocyte telomere length (LTL) is associated with cardiometabolic risk factors and increased risks of mortality and coronary heart disease (CHD). On average, African Americans (AfAm) have longer LTL than whites, but the LTL-CHD relationship in AfAm is unknown. We investigated the relationship of LTL with CHD and mortality among AfAm. APPROACH AND RESULTS: Using a case-cohort design, 1525 postmenopausal women (667 AfAm and 858 whites) from the Women's Health Initiative had LTL measured in baseline blood samples by Southern blotting. CHD or mortality hazards ratios were estimated using race-stratified and risk factor-adjusted Cox proportional hazards models. There were 367 incident CHD (226 mortality) events in whites, whereas AfAm experienced 269 incident CHD (216 mortality) events during median follow-up of 13 years. Shorter LTL was associated with older age, current smoking, and white race/ethnicity. In whites, each 1 kilobase decrease in LTL was associated with 50% increased hazard of CHD, hazard ratio=1.50 (95% confidence interval, 1.08-2.10), P=0.017. There was no association between CHD and LTL in AfAm. White women with shorter LTL had higher risks of mortality. In contrast, shorter LTL was weakly associated with decreased mortality hazard in AfAm. CONCLUSIONS: As one of the largest prospective studies of LTL associations with incident CHD and mortality in a racially diverse sample, our study suggests differences in LTL associations with CHD and mortality between white and AfAm postmenopausal women.

Increased attrition of leukocyte telomere length in young adults is associated with poorer cognitive function in midlife.

Evidence for an association of leukocyte telomere length (LTL) with cognitive function, predominantly in older adults, is inconsistent. No report has examined the association of LTL dynamics (age-specific LTL and its attrition rate) with cognitive function. We aimed to examine the association of LTL dynamics over 13 years in young adulthood with cognitive function in midlife. 497 individuals who had LTL measured at ages 28-32 and 41-46 years were assessed at ages 48-52 for global cognitive function and its five specific component domains with a NeuroTrax computerized test battery. Multivariable regression and logistic models were applied for cognition treated as a continuous and categorical variable, respectively. We found that LTL attrition (adjusted for sex, baseline LTL and potential confounders including socioeconomic variables) was inversely associated with global cognition (standardized ß = -.119, p = .004) and its component domains: information processing speed (ß = -.102, p = .024), visual-spatial function (ß = -.102, p = .017) and memory (ß = -.093, p = .045), but less so for the attention and executive domains. The multivariable-adjusted odds ratio for low global cognition comparing the upper versus lower thirds of LTL attrition was 2.12 (95 % CI 1.11-4.08, p for trend = .023). There was no association of baseline or follow-up LTL with cognition. No effect modification was evident for sex, smoking or inflammatory markers. In conclusion, faster LTL attrition in young adulthood was associated with poorer global and domain-specific cognitive function in midlife, suggesting that more rapid LTL attrition may be predictive of cognitive aging in healthy young adults.

The heritability of leucocyte telomere length dynamics.

BACKGROUND: Leucocyte telomere length (LTL) is a complex trait associated with ageing and longevity. LTL dynamics are defined by LTL and its age-dependent attrition. Strong, but indirect evidence suggests that LTL at birth and its attrition during childhood largely explains interindividual LTL variation among adults. A number of studies have estimated the heritability of LTL, but none has assessed the heritability of age-dependent LTL attrition. METHODS: We examined the heritability of LTL dynamics based on a longitudinal evaluation (an average follow-up of 12 years) in 355 monozygotic and 297 dizygotic same-sex twins (aged 19-64 years at baseline). RESULTS: Heritability of LTL at baseline was estimated at 64% (95% CI 39% to 83%) with 22% (95% CI 6% to 49%) of shared environmental effects. Heritability of age-dependent LTL attrition rate was estimated at 28% (95% CI 16% to 44%). Individually unique environmental factors, estimated at 72% (95% CI 56% to 84%) affected LTL attrition rate with no indication of shared environmental effects. CONCLUSIONS: This is the first study that estimated heritability of LTL and also its age-dependent attrition. As LTL attrition is much slower in adults than in children and given that having a long or a short LTL is largely determined before adulthood, our findings suggest that heritability and early life environment are the main determinants of LTL throughout the human life course. Thus, insights into factors that influence LTL at birth and its dynamics during childhood are crucial for understanding the role of telomere genetics in human ageing and longevity.

DCAF4, a novel gene associated with leucocyte telomere length.

BACKGROUND: Leucocyte telomere length (LTL), which is fashioned by multiple genes, has been linked to a host of human diseases, including sporadic melanoma. A number of genes associated with LTL have already been identified through genome-wide association studies. The main aim of this study was to establish whether DCAF4 (DDB1 and CUL4-associated factor 4) is associated with LTL. In addition, using ingenuity pathway analysis (IPA), we examined whether LTL-associated genes in the general population might partially explain the inherently longer LTL in patients with sporadic melanoma, the risk for which is increased with ultraviolet radiation (UVR). RESULTS: Genome-wide association (GWA) meta-analysis and de novo genotyping of 20 022 individuals revealed a novel association (p=6.4×10(-10)) between LTL and rs2535913, which lies within DCAF4. Notably, eQTL analysis showed that rs2535913 is associated with decline in DCAF4 expressions in both lymphoblastoid cells and sun-exposed skin (p=4.1×10(-3) and 2×10(-3), respectively). Moreover, IPA revealed that LTL-associated genes, derived from GWA meta-analysis (N=9190), are over-represented among genes engaged in melanoma pathways. Meeting increasingly stringent p value thresholds (p<0.05, <0.01, <0.005, <0.001) in the LTL-GWA meta-analysis, these genes were jointly over-represented for melanoma at p values ranging from 1.97×10(-169) to 3.42×10(-24). CONCLUSIONS: We uncovered a new locus associated with LTL in the general population. We also provided preliminary findings that suggest a link of LTL through genetic mechanisms with UVR and melanoma in the general population.

Estimating telomere length from whole genome sequence data.

Telomeres play a key role in replicative ageing and undergo age-dependent attrition in vivo. Here, we report a novel method, TelSeq, to measure average telomere length from whole genome or exome shotgun sequence data. In 260 leukocyte samples, we show that TelSeq results correlate with Southern blot measurements of the mean length of terminal restriction fragments (mTRFs) and display age-dependent attrition comparably well as mTRFs.

Comparative anatomy of chromosomal domains with imprinted and non-imprinted allele-specific DNA methylation.

Allele-specific DNA methylation (ASM) is well studied in imprinted domains, but this type of epigenetic asymmetry is actually found more commonly at non-imprinted loci, where the ASM is dictated not by parent-of-origin but instead by the local haplotype. We identified loci with strong ASM in human tissues from methylation-sensitive SNP array data. Two index regions (bisulfite PCR amplicons), one between the C3orf27 and RPN1 genes in chromosome band 3q21 and the other near the VTRNA2-1 vault RNA in band 5q31, proved to be new examples of imprinted DMRs (maternal alleles methylated) while a third, between STEAP3 and C2orf76 in chromosome band 2q14, showed non-imprinted haplotype-dependent ASM. Using long-read bisulfite sequencing (bis-seq) in 8 human tissues we found that in all 3 domains the ASM is restricted to single differentially methylated regions (DMRs), each less than 2kb. The ASM in the C3orf27-RPN1 intergenic region was placenta-specific and associated with allele-specific expression of a long non-coding RNA. Strikingly, the discrete DMRs in all 3 regions overlap with binding sites for the insulator protein CTCF, which we found selectively bound to the unmethylated allele of the STEAP3-C2orf76 DMR. Methylation mapping in two additional genes with non-imprinted haplotype-dependent ASM, ELK3 and CYP2A7, showed that the CYP2A7 DMR also overlaps a CTCF site. Thus, two features of imprinted domains, highly localized DMRs and allele-specific insulator occupancy by CTCF, can also be found in chromosomal domains with non-imprinted ASM. Arguing for biological importance, our analysis of published whole genome bis-seq data from hES cells revealed multiple genome-wide association study (GWAS) peaks near CTCF binding sites with ASM.