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Background The epigenetic regulation of the renin–angiotensin–aldosterone system (RAAS) potentially plays a role in the pathophysiology underlying the high burden of hypertension in sub-Saharan Africans (SSA). Here we report the first epigenome-wide association study (EWAS) of plasma renin and aldosterone concentrations and the aldosterone-to-renin ratio (ARR). Methods Epigenome-wide DNA methylation was measured using the Illumina 450K array on whole blood samples of 68 Ghanaians. Differentially methylated positions (DMPs) were assessed for plasma renin concentration, aldosterone, and ARR using linear regression models adjusted for age, sex, body mass index, diabetes mellitus, hypertension, and technical covariates. Additionally, we extracted methylation loci previously associated with hypertension, kidney function, or that were annotated to RAAS-related genes and associated these with renin and aldosterone concentration. Results We identified one DMP for renin, ten DMPs for aldosterone, and one DMP associated with ARR. Top DMPs were annotated to the PTPRN2, SKIL, and KCNT1 genes, which have been reported in relation to cardiometabolic risk factors, atherosclerosis, and sodium-potassium handling. Moreover, EWAS loci previously associated with hypertension, kidney function, or RAAS-related genes were also associated with renin, aldosterone, and ARR. Conclusion In this first EWAS on RAAS hormones, we identified DMPs associated with renin, aldosterone, and ARR in a SSA population. These findings are a first step in understanding the role of DNA methylation in regulation of the RAAS in general and in a SSA population specifically. Replication and translational studies are needed to establish the role of these DMPs in the hypertension burden in SSA populations.

Summary Background African populations are going through health transitions due to rapid urbanisation and international migration. However, the role of biological ageing in the emerging burden of cardiometabolic diseases among migrant and non-migrant Africans is unknown. We aimed to examine differences in epigenetic-age acceleration (EAA) as measured by four clocks (Horvath, Hannum, PhenoAge, and GrimAge) and their associations with cardiometabolic factors among migrant Ghanaians residing in Europe and non-migrant Ghanaians residing in Ghana using cross‑sectional data. Methods In this population-based cross-sectional RODAM substudy, recruitment of urban participants in Ghana was done in two cities (Kumasi and Obuasi), whereas recruitment in rural areas was done in 15 villages in the Ashanti region. In Europe, participants were recruited from the cities of Amsterdam (Netherlands), Berlin (Germany), and London (UK). The method and location of participant recruitment varied according to country and city. Participants were included in the RODAM study if they were older than 25 years, had completed the RODAM study questionnaire, were physically examined, and had blood samples taken. In the present subsample, data for DNA-methylation (DNAm) had to be available for the participants. We did not specify any exclusion criteria. We used genome-wide DNAm data from Ghanaians to quantify EAA. We assessed the correlation between DNAm-based age measures and chronological age, and then we did linear regressions to investigate the associations between EAA and body-mass index (BMI), fasting blood glucose (FBG), blood pressure, alcohol consumption, smoking status, physical activity, and one-carbon metabolism nutrients among migrant and non-migrant populations. We replicated our findings among rural–urban sibling pairs from the India Migration Study and among indigenous South Africans from the PURE-SA-NW study. Findings Between Feb 2, 2012, and Sept 30, 2014, 736 individuals participated in the RODAM epigenetics substudy, of which 12 (2%) were excluded during DNAm quality control, and a further 12 (2%) were excluded because of genotypic and phenotypic sex discordance. 712 (97%) of 736 participants were included in the analysis; 365 (51%) of these 712 participants were migrants and 347 (49%) were non-migrants. We found that migrant Ghanaians had lower EAA than non-migrant Ghanaians (intrinsic EAA Horvath –0·34 years vs 0·35 years; extrinsic EAA Hannum –0·86 years vs 0·90 years; PhenoAge acceleration –1·68 years vs 1·77 years; and GrimAge acceleration –0·18 years vs 0·19 years). Within migrant Ghanaians, higher FBG was positively associated with EAA measures, with the adjusted regression β for intrinsic EAA being 0·30 (95% CI 0·01 to 0·59) for migrants and 0·12 (−0·04 to 0·28) for non-migrants, for extrinsic EAA being 0·31 (0·05 to 0·56) for migrants and 0·08 (−0·06 to 0·22) for non-migrants, for PhenoAge acceleration being 0·39 (0·07 to 0·71) for migrants and 0·14 (−0·01 to 0·32) for non-migrants, and for GrimAge acceleration being 0·18 (0·01 to 0·34) for migrants and 0·12 (0·03 to 0·21) for non-migrants. Within non-migrant Ghanaians, higher BMI and vitamin-B9 (folate) intake were negatively associated with EAA measures. Our findings on FBG, BMI, and folate were replicated in the independent cohorts. Interpretation Our study shows that migration is negatively associated with EAA among Ghanaians. Moreover, cardiometabolic factors are differentially associated with EAA within migrant and non-migrant subgroups. Our results call for context-based interventions for cardiometabolic diseases among transitioning populations that account for the effects of biological ageing. Funding European Commission under the Framework Programme and European Research Council Consolidation.

Molecular mechanisms at the intersection of inflammation and cardiovascular diseases (CVD) among Africans are still unknown. We performed an epigenome-wide association study to identify loci associated with serum C-reactive protein (marker of inflammation) among Ghanaians and further assessed whether differentially methylated positions (DMPs) were linked to CVD in previous reports, or to estimated CVD risk in the same population. We used the Illumina Infinium® HumanMethylation450 BeadChip to obtain DNAm profiles of blood samples in 589 Ghanaians from the RODAM study (without acute infections, not taking anti-inflammatory medications, CRP levels PC, BTG4 and PADI1 showed trends of associations with estimated CVD risk, we identified a separate DMP in MORC2 that was associated with CRP levels ≤10 mg/L, and we successfully replicated 65 (24%) of previously reported DMPs. All DMPs with gene annotations (13) were biologically linked to inflammation or CVD traits. We have identified epigenetic loci that may play a role in the intersection between inflammation and CVD among Ghanaians. Further studies among other Africans are needed to confirm our findings.

Aim: We assessed epigenome-wide DNA methylation (DNAm) differences between migrant and non-migrant Ghanaians. Materials & methods: We used the Illumina Infinium® HumanMethylation450 BeadChip to profile DNAm of 712 Ghanaians in whole blood. We used linear models to detect differentially methylated positions (DMPs) associated with migration. We performed multiple post hoc analyses to validate our findings. Results: We identified 13 DMPs associated with migration (delta-beta values: 0.2–4.5%). Seven DMPs in CPLX2, EIF4E3, MEF2D, TLX3, ST8SIA1, ANG and CHRM3 were independent of extrinsic genomic influences in public databases. Two DMPs in NLRC5 were associated with duration of stay in Europe among migrants. All DMPs were biologically linked to migration-related factors. Conclusion: Our findings provide the first insights into DNAm differences between migrants and non-migrants.

Background: Recent findings in neuroimaging and epigenetics offer important insights into brain structures and biological pathways of altered gene expression associated with posttraumatic stress disorder (PTSD). However, it is unknown to what extent epigenetic mechanisms are associated with PTSD and its neurobiology in youth. Methods: In this study, we combined a methylome-wide association study and structural neuroimaging measures in a Dutch cohort of youths with PTSD (8–18 years of age). We aimed to replicate findings in a similar independent U.S. cohort. Results: We found significant methylome-wide associations for pediatric PTSD (false discovery rate p < .05) compared with non-PTSD control groups (traumatized and nontraumatized youths). Methylation differences on nine genes were replicated, including genes related to glucocorticoid functioning. In both cohorts, methylation on OLFM3 gene was further associated with anterior hippocampal volume. Conclusions: These findings point to molecular pathways involved in inflammation, stress response, and neuroplasticity as potential contributors to neural abnormalities and provide potentially unique biomarkers and treatment targets for pediatric PTSD.

Sub-Saharan African (SSA) migrants in Europe experience psychosocial stressors, such as perceived discrimination (PD). The effect of such a stressor on health could potentially be mediated via epigenetics. In this study we performed an epigenome-wide association study (EWAS) to assess the association between levels of PD with genome-wide DNA methylation profiles in SSA migrants. The Illumina 450 K DNA-methylation array was used on whole blood samples of 340 Ghanaian adults residing in three European cities from the cross-sectional Research on Obesity and Diabetes among African Migrants (RODAM) study. PD was assessed using sum scores of the Everyday Discrimination Scale (EDS). Differentially methylated positions and regions (DMPs and DMRs) were identified through linear regression analysis. Two hypo-methylated DMPs, namely cg13986138 (CYFIP1) and cg10316525(ANKRD63), were found to be associated with PD. DMR analysis identified 47 regions associated with the PD. To the best of our knowledge, this survey is the first EWAS for PD in first generation SSA migrants. We identified two DMPs associated with PD. Whether these associations underlie a consequence or causal effect within the scope of biological functionality needs additional research.

Recent findings in neuroimaging and epigenetics offer important insights into brain structures and biological pathways of altered gene expression associated with posttraumatic stress disorder (PTSD). However, it is unknown to what extent epigenetic mechanisms are associated with PTSD and its neurobiology in youth. Therefore, we combine genome-wide epigenetic and structural neuroimaging measures in a Dutch cohort of youth with PTSD (ages 8-18 years). We aimed to replicate findings in a similar independent American cohort. We found significant methylome-wide associations for pediatric PTSD (FDR p

Purpose: We describe the clinical implementation of genome-wide DNA methylation analysis in rare disorders across the EpiSign diagnostic laboratory network and the assessment of results and clinical impact in the first subjects tested. Methods: We outline the logistics and data flow between an integrated network of clinical diagnostics laboratories in Europe, the United States, and Canada. We describe the clinical validation of EpiSign using 211 specimens and assess the test performance and diagnostic yield in the first 207 subjects tested involving two patient subgroups: the targeted cohort (subjects with previous ambiguous/inconclusive genetic findings including genetic variants of unknown clinical significance) and the screening cohort (subjects with clinical findings consistent with hereditary neurodevelopmental syndromes and no previous conclusive genetic findings). Results: Among the 207 subjects tested, 57 (27.6%) were positive for a diagnostic episignature including 48/136 (35.3%) in the targeted cohort and 8/71 (11.3%) in the screening cohort, with 4/207 (1.9%) remaining inconclusive after EpiSign analysis. Conclusion: This study describes the implementation of diagnostic clinical genomic DNA methylation testing in patients with rare disorders. It provides strong evidence of clinical utility of EpiSign analysis, including the ability to provide conclusive findings in the majority of subjects tested.

BACKGROUND: Type 2 diabetes (T2D) results from a complex interplay between genetics and the environment. Several epigenome-wide association studies (EWAS) have found DNA methylation loci associated with T2D in European populations. However, data from African populations are lacking. We undertook the first EWAS for T2D among sub-Saharan Africans, aiming at identifying ubiquitous and novel DNA methylation loci associated with T2D. METHODS: The Illumina 450k DNA-methylation array was used on whole blood samples of 713 Ghanaian participants (256 with T2D, 457 controls) from the cross-sectional Research on Obesity and Diabetes among African Migrants (RODAM) study. Differentially methylated positions (DMPs) for T2D and HbA1c were identified through linear regression analysis adjusted for age, sex, estimated cell counts, hybridization batch, array position and body mass index (BMI). We also did a candidate analysis of previously reported EWAS loci for T2D in non-African populations, identified through a systematic literature search. RESULTS: Four DMPs [cg19693031 (TXNIP), cg04816311 (C7orf50), cg00574958 (CPT1A), cg07988171 (TPM4)] were associated with T2D after correction for inflation by possible systematic biases. The most strongly associated DMP—cg19693031, TXNIP (P = 2.6E-19) —showed hypomethylation in T2D cases compared with controls. Two out of the four DMPs [cg19693031 (TXNIP), cg04816311 (C7orf50)] remained associated with T2D after adjustment for BMI, and one locus [cg07988171 (TPM4)] that has not been reported previously. CONCLUSIONS: In this first EWAS for T2D in sub-Saharan Africans, we have identified four DMPs at epigenome-wide level, one of which is novel. These findings provide insight into the epigenetic loci that underlie the burden of T2D in sub-Saharan Africans.

The original version of this article unfortunately contained a mistake. The following correction has therefore been made in the original: The spelling of Michael A. Levy’s name was incorrect. The corrected author list is given above. The original article has been corrected.

Autologous transplantation of spermatogonial stem cells is a promising new avenue to restore fertility in infertile recipients. Expansion of the initial spermatogonial stem cell pool through cell culturing is a necessary step to obtain enough cells for effective repopulation of the testis after transplantation. Since in vitro propagation can lead to (epi-)genetic mutations and possibly malignant transformation of the starting cell population, we set out to investigate genome-wide DNA methylation status in uncultured and cultured primary testicular ITGA6+ sorted cells and compare them with germ cell tumor samples of the seminoma subtype. Seminomas displayed a severely global hypomethylated profile, including loss of genomic imprinting, which we did not detect in cultured primary testicular ITGA6+ cells. Differential methylation analysis revealed altered regulation of gamete formation and meiotic processes in cultured primary testicular ITGA6+ cells but not in seminomas. The pivotal POU5F1 marker was hypomethylated in seminomas but not in uncultured or cultured primary testicular ITGA6+ cells, which is reflected in the POU5F1 mRNA expression levels. Lastly, seminomas displayed a number of characteristic copy number variations that were not detectable in primary testicular ITGA6+ cells, either before or after culture. Together, the data show a distinct DNA methylation patterns in cultured primary testicular ITGA6+ cells that does not resemble the pattern found in seminomas, but also highlight the need for more sensitive methods to fully exclude the presence of malignant cells after culture and to further study the epigenetic events that take place during in vitro culture.

Beckwith-Wiedemann syndrome (BWS) is caused due to the disturbance of imprinted genes at chromosome 11p15. The molecular confirmation of this syndrome is possible in approximately 85% of the cases, whereas in the remaining 15% of the cases, the underlying defect remains unclear. The goal of our research was to identify new epigenetic loci related to BWS. We studied a group of 25 patients clinically diagnosed with BWS but without molecular conformation after DNA diagnostics and performed a whole genome methylation analysis using the HumanMethylation450 Array (Illumina). We found hypermethylation throughout the methylome in two BWS patients. The hypermethylated sites in these patients overlapped and included both non-imprinted and imprinted regions. This finding was not previously described in any BWS-diagnosed patient. Furthermore, one BWS patient exhibited aberrant methylation in four maternally methylated regions—IGF1R, NHP2L1, L3MBTL, and ZDBF2—that overlapped with the differentially methylated regions found in BWS patients with multi-locus imprinting disturbance (MLID). This finding suggests that the BWS phenotype can result from MLID without detectable methylation defects in the primarily disease-associated loci (11p15). Another patient manifested small but significant aberrant methylation in disease-associated loci at 11p near H19, possibly confirming the diagnosis in this patient. Electronic supplementary material The online version of this article (10.1186/s13148-019-0649-6) contains supplementary material, which is available to authorized users.

Aim: Fetal alcohol spectrum disorder (FASD) involves prenatal growth delay, impaired facial and CNS development and causes severe clinical, social-economic burdens. Here, we aim to detect DNA-methylation aberrations associated with FASD and potential FASD diagnostic and prognostic biomarkers. Patients & methods: The FASD diagnosis was established according to golden-standard protocols in a discovery and independent replication cohort. Genome-wide differential methylation association and replication analyses were performed. Results: We identified several loci that were robustly associated with FASD or one of its sub phenotypes. Our findings were evaluated using previously reported genome-wide surveys. Conclusion: We have detected robust FASD associated differentially methylated positions and differentially methylated regions for FASD in general and for FASD subphenotypes, in other words on growth delay, impaired facial and CNS development.

Background: Familial hypercholesterolemia (FH) is a monogenic disorder characterized by elevated low-density lipoprotein cholesterol (LDL-C). A FH causing genetic variant in LDLR, APOB, or PCSK9 is not identified in 12–60% of clinical FH patients (FH mutation-negative patients). We aimed to assess whether altered DNA methylation might be associated with FH in this latter group Methods: In this study we included 78 FH mutation-negative patients and 58 FH mutation-positive patients with a pathogenic LDLR variant. All patients were male, not using lipid lowering therapies and had LDL-C levels >6 mmol/L and triglyceride levels

SETD1B is a component of a histone methyltransferase complex that specifically methylates Lys-4 of histone H3 (H3K4) and is responsible for the epigenetic control of chromatin structure and gene expression. De novo microdeletions encompassing this gene as well as de novo missense mutations were previously linked to syndromic intellectual disability (ID). Here, we identify a specific hypermethylation signature associated with loss of function mutations in theSETD1Bgene which may be used as an epigenetic marker supporting the diagnosis of syndromicSETD1B-related diseases. We demonstrate the clinical utility of this unique epi-signature by reclassifying previously identifiedSETD1BVUS (variant of uncertain significance) in two patients.

Cow&rsquo, s milk allergy (CMA) is an early-onset allergy of which the underlying genetic factors remain largely undiscovered. CMA has been found to co-occur with other allergies and immunological hypersensitivity disorders, suggesting a shared genetic etiology. We aimed to (1) investigate and (2) validate whether CMA children carry a higher genetic susceptibility for other immunological hypersensitivity disorders using polygenic risk score analysis (PRS) and prospective phenotypic data. Twenty-two CMA patients of the Dutch EuroPrevall birth cohort study and 307 reference subjects were genotyped using single nucleotide polymorphism (SNP) array. Differentially genetic susceptibility was estimated using PRS, based on multiple P-value thresholds for SNP inclusion of previously reported genome-wide association studies (GWAS) on asthma, autism spectrum disorder, atopic dermatitis, inflammatory bowel disease and rheumatoid arthritis. These associations were validated with prospective data outcomes during a six-year follow-up in 19 patients. We observed robust and significantly higher PRSs of asthma in CMA children compared to the reference set. Association analyses using the prospective data indicated significant higher PRSs in former CMA patients suffering from asthma and related traits. Our results suggest a shared genetic etiology between CMA and asthma and a considerable predictive sensitivity potential for subsequent onset of asthma which indicates a potential use for early clinical asthma intervention programs.

Background: Epigenome-wide association studies (EWAS) have identified DNA methylation loci involved in adiposity. However, EWAS on adiposity in sub-Saharan Africans are lacking despite the high burden of adiposity among African populations. We undertook an EWAS for anthropometric indices of adiposity among Ghanaians aiming to identify DNA methylation loci that are significantly associated.Methods: The Illumina 450k DNA methylation array was used to profile DNA methylation in whole blood samples of 547 Ghanaians from the Research on Obesity and Diabetes among African Migrants (RODAM) study. Differentially methylated positions (DMPs) and differentially methylation regions (DMRs) were identified for BMI and obesity (BMI ≥ 30 kg/m2), as well as for waist circumference (WC) and abdominal obesity (WC ≥ 102 cm in men, ≥88 cm in women). All analyses were adjusted for age, sex, blood cell distribution estimates, technical covariates, recruitment site and population stratification. We also did a replication study of previously reported EWAS loci for anthropometric indices in other populations.Results: We identified 18 DMPs for BMI and 23 for WC. For obesity and abdominal obesity, we identified three and one DMP, respectively. Fourteen DMPs overlapped between BMI and WC. DMP cg00574958 annotated to gene CPT1A was the only DMP associated with all outcomes analysed, attributing to 6.1 and 5.6% of variance in obesity and abdominal obesity, respectively. DMP cg07839457 (NLRC5) and cg20399616 (BCAT1) were significantly associated with BMI, obesity and with WC and had not been reported by previous EWAS on adiposity.Conclusions: This first EWAS for adiposity in Africans identified three epigenome-wide significant loci (CPT1A, NLRC5 and BCAT1) for both general adiposity and abdominal adiposity. The findings are a first step in understanding the role of DNA methylation in adiposity among sub-Saharan Africans. Studies on other sub-Saharan African populations as well as translational studies are needed to determine the role of these DNA methylation variants in the high burden of adiposity among sub-Saharan Africans.

Posttraumatic stress disorder (PTSD) is a debilitating psychiatric disorder that may develop after a traumatic event. Here we aimed to identify epigenetic and genetic loci associated with PTSD. We included 73 traumatized police officers with extreme phenotypes regarding symptom severity despite similar trauma history: n = 34 had PTSD and n = 39 had minimal PTSD symptoms. Epigenetic and genetic profiles were based on the Illumina HumanMethylation450 BeadChip. We searched for differentially methylated probes (DMPs) and differentially methylated regions (DMRs). For genetic associations we analyzed the CpG-SNPs present on the array. We detected no genome-wide significant DMPs and we did not replicate previously reported DMPs associated with PTSD. However, GSE analysis of the top 100 DMPs showed enrichment of three genes involved in the dopaminergic neurogenesis pathway. Furthermore, we observed a suggestive association of one relatively large DMR between patients and controls, which was located at the PAX8 gene and previously associated with other psychiatric disorders. Finally, we validated five PTSD-associated CpG-SNPs identified with the array using sanger sequencing. We subsequently replicated the association of one common SNP (rs1990322) in the CACNA1C locus with PTSD in an independent cohort of traumatized children. The CACNA1C locus was previously associated with other psychiatric disorders, but not yet with PTSD. Thus, despite the small sample size, inclusion of extreme symptom severity phenotypes in a highly homogenous traumatized cohort enabled detection of epigenetic and genetic loci associated with PTSD. Moreover, here we showed that genetically confounded 450K probes are informative for genetic association analysis.

Item does not contain fulltext Oculopharyngeal muscular dystrophy (OPMD) is caused by trinucleotide repeat expansion mutations in Poly(A) binding protein 1 (PABPN1). PABPN1 is a regulator of mRNA stability and is ubiquitously expressed. Here we investigated how symptoms in OPMD initiate only at midlife and why a subset of skeletal muscles is predominantly affected. Genome-wide RNA expression profiles from Vastus lateralis muscles human carriers of expanded-PABPN1 at pre-symptomatic and symptomatic stages were compared with healthy controls. Major expression changes were found to be associated with age rather than with expression of expanded-PABPN1, instead transcriptomes of OPMD and elderly muscles were significantly similar (P

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by ptosis, dysphagia and proximal limb weakness. Autosomal-dominant OPMD is caused by a short (GCG)(8-13) expansions within the first exon of the poly(A)-binding protein nuclear 1 gene (PABPN1), leading to an expanded polyalanine tract in the mutated protein. Expanded PABPN1 forms insoluble aggregates in the nuclei of skeletal muscle fibres. In order to gain insight into the different physiological processes affected in OPMD muscles, we have used a transgenic mouse model of OPMD (A17.1) and performed transcriptomic studies combined with a detailed phenotypic characterization of this model at three time points. The transcriptomic analysis revealed a massive gene deregulation in the A17.1 mice, among which we identified a significant deregulation of pathways associated with muscle atrophy. Using a mathematical model for progression, we have identified that one-third of the progressive genes were also associated with muscle atrophy. Functional and histological analysis of the skeletal muscle of this mouse model confirmed a severe and progressive muscular atrophy associated with a reduction in muscle strength. Moreover, muscle atrophy in the A17.1 mice was restricted to fast glycolytic fibres, containing a large number of intranuclear inclusions (INIs). The soleus muscle and, in particular, oxidative fibres were spared, even though they contained INIs albeit to a lesser degree. These results demonstrate a fibre-type specificity of muscle atrophy in this OPMD model. This study improves our understanding of the biological pathways modified in OPMD to identify potential biomarkers and new therapeutic targets.

Hypohidrotic ectodermal dysplasia (HED) can be caused by mutations in the X-linked ectodysplasin A (ED1) gene or the autosomal ectodysplasin A-receptor (EDAR) and EDAR-associated death domain (EDARADD) genes. X-linked and autosomal forms are sometimes clinically indistinguishable. For genetic counseling in families, it is therefore important to know the gene involved. In 24 of 42 unrelated patients with features of HED, we found a mutation in ED1. ED1-negative patients were screened for mutations in EDAR and EDARADD. We found mutations in EDAR in 5 of these 18 patients. One mutation, p.Glu354X, is novel. In EDARADD, a novel variant p.Ser93Phe, probably a neutral polymorphism, was also found. Clinically, there was a difference between autosomal dominant and autosomal recessive HED patients. The phenotype in patients with mutations in both EDAR alleles was comparable to males with X-linked HED. Patients with autosomal dominant HED had features comparable to those of female carriers of X-linked HED. The teeth of these patients were quite severely affected. Hypohidrosis and sparse hair were also evident, but less severe. This study confirms Chassaing et al's earlier finding that mutations in EDAR account for approximately 25% of non-ED1-related HED. Mutations leading to a premature stop codon have a recessive effect except when the stop codon is in the last exon. Heterozygous missense mutations in the functional domains of the gene may have a dominant-negative effect with much variation in expression. Patients with homozygous or compound heterozygous mutations in the EDAR gene have a more severe phenotype than those with a heterozygous missense, nonsense or frame-shift mutation.

Background Cow’s milk allergy (CMA) is a common disease in infancy. Early environmental factors are likely to contribute to CMA. It is known that epigenetic gene regulation can be altered by environmental factors. We have set up a proof of concept study, aiming to detect epigenetic associations specific with CMA. Methods We studied children from the Dutch EuroPrevall birth cohort study (N = 20 CMA, N = 23 controls, N = 10 tolerant boys), age and gender matched. CMA was challenge proven. Bisulfite converted DNA (blood) was analyzed using the 450K infinium DNA-methylation array. Four groups (combined, girls, boys and tolerant boys) were analysed between CMA and controls. Statistical analysis and pathway-analysis were performed in “R” using IMA, Minfi and the global-test package. Differentially methylated regions in DHX58, ZNF281, EIF42A and HTRA2 genes were validated by quantitative amplicon sequencing (ROCHE 454®). Results General hypermethylation was found in the CMA group compared to control children, while this effect was absent in the tolerant group. Methylation differences were, among others, found in regions of DHX58, ZNF281, EIF42A and HTRA2 genes. Several of these genes are known to be involved in immunological pathways and associated with other allergies. Conclusion We show that epigenetic associations are involved in CMA. Although, the statistical power of our study is limited and our sample was based on whole blood, we were still able to detect feasible loci and pathways. Therefore our findings might contribute to future diagnostic or therapeutic interventions for specific CMA. Further studies have to confirm the findings of our study. Electronic supplementary material The online version of this article (doi:10.1186/s13601-016-0105-z) contains supplementary material, which is available to authorized users.

Background Cow’s milk allergy (CMA) is the most common allergic disease in infancy. It is not clear, whether infants with CMA have an increased risk of developing other allergic diseases later in life, the so-called “allergic march”. We aimed to detect genetic associations of CMA using reported single nucleotide polymorphisms (SNP) in other allergic diseases and genetic mutations within the filaggrin (FLG) gene. Both to investigate possible causes of CMA, which also suggests an “allergic march”. Methods Thirty children from the Dutch EuroPrevall birth cohort study with CMA in infancy and twenty-three healthy controls were studied. Six candidate SNPs were selected (minor allele frequency 10–50 % combined with a large effect) based on the literature. Thirteen FLG candidate mutations were selected spread over repeats 1, 3, 4, 5, 6, 7, 9 and 10 respectively. Results We found two SNP’s, rs17616434 (P = 0.002) and rs2069772 (P = 0.038), significantly associated with CMA. One is located near the toll like receptor 6 (TLR6) gene, which functionally interacts with toll-like receptor 2, and is associated with an increased risk of other allergic diseases. One is located at the Interleukin 2 (IL2) locus. Twelve FLG amplicons were analyzed, but showed no significant enrichment. Nevertheless, we did observe more FLG mutations in the CMA-group compared to controls. Conclusion We significantly associated two SNPs with CMA, suggesting that variation in the TLR6 and IL2 genes contribute to the expression of CMA. In addition, since TLR6 and IL2 were earlier associated with other later onset allergies, this also favours the “allergic march” hypothesis. We observed more FLG mutations in the CMA-group, albeit we found no statistical significant enrichment of FLG mutations. Further studies are necessary to investigate the role of common variants and FLG or other skin barrier gene mutations in CMA. Electronic supplementary material The online version of this article (doi:10.1186/s13601-016-0096-9) contains supplementary material, which is available to authorized users.

Contains fulltext : 181501.pdf (Publisher’s version ) (Open Access) BACKGROUND: Muscle fibrosis characterizes degenerated muscles in muscular dystrophies and in late onset myopathies. Fibrotic muscles often exhibit thickening of the extracellular matrix (ECM). The molecular regulation of this process is not fully understood. In oculopharyngeal muscular dystrophy (OPMD), an expansion of an alanine tract at the N-terminus of poly(A)-binding protein nuclear 1 (PABPN1) causes muscle symptoms. OPMD patient muscle degeneration initiates after midlife, while at an earlier age carriers of alanine expansion mutant PABPN1 (expPABPN1) are clinically pre-symptomatic. OPMD is characterized by fibrosis in skeletal muscles but the causative molecular mechanisms are not fully understood. METHODS: We studied the molecular processes that are involved in OPMD pathology using cross-species mRNA expression profiles in muscles from patients and model systems. We identified significant dysregulation of the ECM functional group, among which the procollagen C-endopeptidase enhancer 1 gene (PCOLCE) was consistently down-regulated across species. We investigated PCOLCE subcellular localization in OPMD muscle samples and OPMD model systems to investigate any functional relevance of PCOLCE down-regulation in this disease. RESULTS: We found that muscle degeneration in OPMD is associated with PCOLCE down-regulation. In addition to its known presence at the ECM, we also found PCOLCE within the nucleus of muscle cells. PCOLCE sub-cellular localization changes during myoblast cell fusion and is disrupted in cells expressing mutant expPABPN1. Our results show that PCOLCE binds to soluble PABPN1 and co-localizes with aggregated PABPN1 with a preference for the mutant protein. In muscle biopsies from OPMD patients we find that extracellular PCOLCE is depleted with its concomitant enrichment within the nuclear compartment. CONCLUSIONS: PCOLCE regulates collagen processing at the ECM. Depletion of extracellular PCOLCE is associated with the expression of expPABPN1 in OPMD patient muscles. PCOLCE is also localized within the nucleus where it binds to PABPN1, suggesting that PCOLCE shuttles between the ECM and the nucleus. PCOLCE preferentially binds to expPABPN1. Nuclear-localized PCOLCE is enriched in muscle cells expressing expPABPN1. We suggest that nuclear entrapment of PCOLCE and its extracellular depletion represents a novel molecular mechanism in late-onset muscle fibrosis.

The choice for a polyadenylation site determines the length of the 3 0 -untranslated region (3 0 -UTRs) of an mRNA. Inclusion or exclusion of regulatory sequences in the 3 0 -UTR may ultimately affect gene expression levels. Poly(A) binding protein nuclear 1 (PABPN1) is involved in polyadenylation of pre-mRNAs. An alanine repeat expansion in PABPN1 (exp-PABPN1) causes oculopharyngeal muscular dystrophy (OPMD). We hypothesized that previously observed disturbed gene expression patterns in OPMD muscles may have been the result of an effect of PABPN1 on alternative polyadenylation, influencing mRNA stability, localization and translation. A single molecule polyadenylation site sequencing method was developed to explore polyadenylation site usage on a genome-wide level in mice overexpressing exp-PABPN1. We identified 2012 transcripts with altered polyadenylation site usage. In the far majority, more proximal alternative polyadenylation sites were used, resulting in shorter 3 0 -UTRs. 3 0 -UTR shortening was generally associated with increased expression. Similar changes in polyadenylation site usage were observed after knockdown or overexpression of expanded but not wild-type PABPN1 in cultured myogenic cells. Our data indicate that PABPN1 is important for polyadenylation site selection and that reduced availability of functional PABPN1 in OPMD muscles results in use of alternative polyadenylation sites, leading to large-scale deregulation of gene expression.

Gene regulatory networks give important insights into the mechanisms underlying physiology and pathophysiology. The derivation of gene regulatory networks from high-throughput expression data via machine learning strategies is problematic as the reliability of these models is often compromised by limited and highly variable samples, heterogeneity in transcript isoforms, noise, and other artifacts. Here, we develop a novel algorithm, dubbed Dandelion, in which we construct and train intraspecies Bayesian networks that are translated and assessed on independent test sets from other species in a reiterative procedure. The interspecies disease networks are subjected to multi-layers of analysis and evaluation, leading to the identification of the most consistent relationships within the network structure. In this study, we demonstrate the performance of our algorithms on datasets from animal models of oculopharyngeal muscular dystrophy (OPMD) and patient materials. We show that the interspecies network of genes coding for the proteasome provide highly accurate predictions on gene expression levels and disease phenotype. Moreover, the cross-species translation increases the stability and robustness of these networks. Unlike existing modeling approaches, our algorithms do not require assumptions on notoriously difficult one-to-one mapping of protein orthologues or alternative transcripts and can deal with missing data. We show that the identified key components of the OPMD disease network can be confirmed in an unseen and independent disease model. This study presents a state-of-the-art strategy in constructing interspecies disease networks that provide crucial information on regulatory relationships among genes, leading to better understanding of the disease molecular mechanisms., Author Summary The identification of gene regulatory networks can provide vital information on biological processes. Despite numerous advancements in developing machine learning strategies, the stochastic nature of such biological systems complicates the construction of robust and reliable network structures. In recent years, the use of cross-species datasets enabled scientists to better understand the molecular mechanisms that are associated with human disorders. However, it also presents a challenge in dealing with especially difficult mapping of protein orthologues, alternative transcript splicing, noise, or other artifacts. Here, we developed a novel algorithm for constructing interspecies disease networks that provide accurate predictive value over the disease phenotype and gene expression. We show that the disease-association of potential key regulators that play a role in interspecies disease networks can be reproduced and validated in an unseen and independent model system. This study presents a novel strategy for constructing networks that can be translated across species whilst providing a comprehensive view of regulatory relationships associated with the disease.

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease caused by an alanine tract expansion mutation in poly(A) binding protein nuclear 1 (expPABPN1). To model OPMD in a myogenic and physiological context, we generated mouse myoblast cell clones stably expressing either human wild type (WT) or expPABPN1 at low levels. Transgene expression is induced on myotube differentiation and results in formation of insoluble nuclear PABPN1 aggregates that are similar to those observed in patients with OPMD. Quantitative analysis of PABPN1 in myotube cultures revealed that expPABPN1 accumulation and aggregation is greater than that of the WT protein. We found that aggregation of expPABPN1 is more affected than WT PABPN1 by inhibition of proteasome activity. Consistent with this, in myotube cultures expressing expPABPN1, deregulation of the proteasome was identified as the most significantly perturbed pathway. Differences in the accumulation of soluble WT and expPABPN1 were consistent with differences in ubiquitination and rate of protein turnover. This study demonstrates, for the first time to our knowledge, that, in myotubes, the ratio of soluble/insoluble expPABPN1 is significantly lower compared with that of the WT protein. We suggest that this difference can contribute to muscle weakness in OPMD.

Closure of the ductus arteriosus (DA) at birth is essential for the transition from fetal to postnatal life. Before birth the DA bypasses the uninflated lungs by shunting blood from the pulmonary trunk into the systemic circulation. The molecular mechanism underlying DA closure and degeneration has not been fully elucidated, but is associated with apoptosis and cytolytic necrosis in the inner media and intima. We detected features of histology during DA degeneration that are comparable to Hutchinson Gilford Progeria syndrome and ageing. Immunohistochemistry on human fetal and neonatal DA, and aorta showed that lamin A/C was expressed in all layers of the vessel wall. As a novel finding we report that progerin, a splicing variant of lamin A/C was expressed almost selectively in the normal closing neonatal DA, from which we hypothesized that progerin is involved in DA closure. Progerin was detected in 16.2%±7.2 cells of the DA. Progerin-expressing cells were predominantly located in intima and inner media where cytolytic necrosis accompanied by apoptosis will develop. Concomitantly we found loss of α-smooth muscle actin as well as reduced lamin A/C expression compared to the fetal and non-closing DA. In cells of the adjacent aorta, that remains patent, progerin expression was only sporadically detected in 2.5%±1.5 of the cells. Data were substantiated by the detection of mRNA of progerin in the neonatal DA but not in the aorta, by PCR and sequencing analysis. The fetal DA and the non-closing persistent DA did not present with progerin expressing cells. Our analysis revealed that the spatiotemporal expression of lamin A/C and progerin in the neonatal DA was mutually exclusive. We suggest that activation of LMNA alternative splicing is involved in vascular remodeling in the circulatory system during normal neonatal DA closure.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset progressive muscle disorder caused by a poly-alanine expansion mutation in the Poly(A) Binding Protein Nuclear 1 (PABPN1). The molecular mechanisms that regulate disease onset and progression are largely unknown. In order to identify molecular pathways that are consistently associated with OPMD, we performed an integrated high-throughput transcriptome study in affected muscles of OPMD animal models and patients. The ubiquitin-proteasome system (UPS) was found to be the most consistently and significantly OPMD-deregulated pathway across species. We could correlate the association of the UPS OPMD-deregulated genes with stages of disease progression. The expression trend of a subset of these genes is age-associated and therefore, marks the late onset of the disease, and a second group with expression trends relating to disease-progression. We demonstrate a correlation between expression trends and entrapment into PABPN1 insoluble aggregates of OPMD-deregulated E3 ligases. We also show that manipulations of proteasome and immunoproteasome activity specifically affect the accumulation and aggregation of mutant PABPN1. We suggest that the natural decrease in proteasome expression and its activity during muscle aging contributes to the onset of the disease.