Increased methylation levels of the MTHFR gene promoter in Down syndrome.

Impairments of the genes that encode enzymes that are involved in one-carbon metabolism because of the presence of gene polymorphisms can affect the methylation pattern. The altered methylation profiles of the genes involved in cardiogenesis may result in congenital heart defects (CHDs). The aim of this study was to investigate the association between the MTHFR rs1801133, MTHFR rs1801131, MTRR rs1801394, DNMT1 rs2228611, DNMT3A rs1550117, DNMT3B rs1569686, and DNMT3B rs2424913 gene polymorphisms and congenital heart defects in Down syndrome (DS) individuals. The study was conducted on 350 participants, including 134 DS individuals with CHDs (DSCHD+), 124 DS individuals without CHDs (DSCHD-), and 92 individuals with non-syndromic CHD. The genotyping was performed using the PCR-RFLP method. A statistically significant higher frequency of the DNMT3B rs2424913 TT in the DSCHD+ individuals was observed. The DNMT3B rs2424913 TT genotype, as well as the T allele, had significantly higher frequencies in the individuals with DS and atrial septal defects (ASDs) in comparison with the individuals with DS and other CHDs. Furthermore, our results indicate a statistically significant effect of the DNMT3B rs1569686 TT genotype in individuals with non-syndromic CHDs. The results of the study suggest that the DNMT3B rs2424913 TT genotypes may be a possible predisposing factor for CHDs in DS individuals, and especially those with ASDs.

Altered global methylation levels revealed LINE-1 methylation in young mothers of Down syndrome (DS) compared to controls suggesting the possibility of impaired DNA methylation causing abnormal segregation of chromosome 21. Methylene Tetrahydrofolate Reductase (MTHFR) is one of the major enzymes of the folate metabolism pathway. MTHFR gene polymorphism has been associated with maternal risk for DS. Studies have revealed that increased MTHFR promoter methylation results in the reduction of MTHFR protein activity further leading to increased risk of various diseases. The aim of this study is to compare the levels of MTHFR promoter methylation in all three study groups. A total of 120 subjects were recruited for the study and was divided into the following three groups: Group I (mothers of DS without Congenital Heart Defects (CHD), n = 40); Group II (mothers of DS with CHD, n = 40); and Group III (age matched control mothers, n = 40). Genomic DNA was isolated from 2 ml peripheral blood and bisulfite treatment was done to convert all unmethylated cytosines into uracil followed by PCR amplification for MTHFR promoter region and Sanger's sequencing. Results showed that there was a two fold increase in methylated promoter region of MTHFR gene in group II compared to other groups. None of the methylation pattern was observed in the control group. MTHFR promoter methylation affects folate metabolism which is known to play a role in chromosomal breakage, abnormal chromosomal segregation and genomic instability and therefore a developmental defect in the form of congenital cardiac anomaly.

Background: The High-Resolution Melting (HRM) technology has recently been introduced as a rapid and robust analysis tool for the detection of DNA methylation. The methylation status of multiple tumor suppressor genes may serve as biomarkers for early cancer diagnostics, for prediction of prognosis and for prediction of response to treatment. Therefore, it is important that methodologies for detection of DNA methylation continue to evolve. Sensitive Melting Analysis after Real Time - Methylation Specific PCR (SMART-MSP) and Methylation Sensitive - High Resolution Melting (MS-HRM) are two methods for single locus DNA methylation detection based on HRM. Methods: Here, we have assessed the quality of DNA extracted from 30-years-old Formalin Fixed Paraffin Embedded (FFPE) tissue for DNA methylation analysis using SMART-MSP and MS-HRM. The quality assessment was performed on DNA extracted from 54 Non-Small Cell Lung Cancer (NSCLC) samples derived from FFPE tissue, collected over 30 years and grouped into five-years-intervals. For each sample, the methylation level of the CDKN2A (p16) and RARB promoters were estimated using SMART-MSP and MS-HRM assays designed to assess the methylation status of the same CpG positions. This allowed for a direct comparison of the methylation level estimated by the two methods for each sample. Results: The methylation level of the CDKN2A promoter was successfully determined by SMART-MSP and MS-HRM in all of the 54 samples. The same methylation estimates were obtained by the two methods in 46 of samples. The methylation level of the RARB promoter was successfully determined by SMART-MSP in all of samples. When using MS-HRM to assess RARB methylation five samples failed to amplify and 15 samples showed a melting profile characteristic for heterogeneous methylation. Out of the remainder 34 samples, for which the methylation level could be estimated, 27 gave the same result as observed when using SMART-MSP. Conclusion: MS-HRM and SMART-MSP can be successfully used for single locus methylation studies of DNA derived from up to 30-years-old FFPE tissue samples. Furthermore, it can be expected that MS-HRM and SMART-MSP will provide similar methylation estimates when assays are designed to analyze the same CpG positions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4918.

Is respiratory infection mortality in Down's syndrome (DS) individuals due mainly to their congenital heart disease (CHD) or to other factors which subject most mentally retarded persons to risk? Detailed clinical and autopsy records of 137 institutionalized DS patients and 480 non-DS controls over 31 years yielded 42 DS subjects and 13 non-DS controls with congenital heart disease. These were compared to 20 DS patients and 20 controls without CHD. The DS and non-DS patients were matched for age, sex and IQ. DS patients had more CHD; controls had more pulmonary oedema. In neither group was there association between heart disease and death from respiratory infection. Nor did pulmonary oedema contribute importantly to such deaths. Such mortality, however, was associated with young age, short institutionalization and bedridden status. We conclude that respiratory infection death in DS individuals is due not primarily to heart disease but to factors which lead to mortality in the general population of retarded people.

BackgroundThe High Resolution Melting (HRM) technology has recently been introduced as a rapid and robust analysis tool for the detection of DNA methylation. The methylation status of multiple tumor suppressor genes may serve as biomarkers for early cancer diagnostics, for prediction of prognosis and for prediction of response to treatment. Therefore, it is important that methodologies for detection of DNA methylation continue to evolve. Sensitive Melting Analysis after Real Time - Methylation Specific PCR (SMART-MSP) and Methylation Sensitive - High Resolution Melting (MS-HRM) are two methods for single locus DNA methylation detection based on HRM.MethodsHere, we have assessed the quality of DNA extracted from up to 30 years old Formalin Fixed Paraffin Embedded (FFPE) tissue for DNA methylation analysis using SMART-MSP and MS-HRM. The quality assessment was performed on DNA extracted from 54 Non-Small Cell Lung Cancer (NSCLC) samples derived from FFPE tissue, collected over 30 years and grouped into five years intervals. For each sample, the methylation levels of the CDKN2A (p16) and RARB promoters were estimated using SMART-MSP and MS-HRM assays designed to assess the methylation status of the same CpG positions. This allowed for a direct comparison of the methylation levels estimated by the two methods for each sample.ResultsCDKN2A promoter methylation levels were successfully determined by SMART-MSP and MS-HRM in all 54 samples. Identical methylation estimates were obtained by the two methods in 46 of the samples. The methylation levels of the RARB promoter were successfully determined by SMART-MSP in all samples. When using MS-HRM to assess RARB methylation five samples failed to amplify and 15 samples showed a melting profile characteristic for heterogeneous methylation. Twenty-seven of the remaining 34 samples, for which the methylation level could be estimated, gave the same result as observed when using SMART-MSP.ConclusionMS-HRM and SMART-MSP can be successfully used for single locus methylation studies using DNA derived from up to 30 years old FFPE tissue. Furthermore, it can be expected that MS-HRM and SMART-MSP will provide similar methylation estimates when assays are designed to analyze the same CpG positions.

The main objective of this project is to study the possible association between anti-oxidant/redox status and DNA instability in Down syndrome. The activities of 5 antioxidant enzymes were studied in 19 Down syndrome (DS) cases and in age- and sex-matched normal controls. Sister Chromatid Exchanges (SCE) were measured in lymphocyte cultures derived from all DS and control subjects. All DS and control individuals had normal hematological parameters, but the proliferation and mitotic indices were significantly lower in the DS- than in the controls-derived lymphocyte cultures, while the average generation time was higher than that in the controls. The specific activity of superoxide dismutase in the DS individuals was 40% higher than that in the controls, while the specific activity of glutathione S-transferase in the DS group was significantly lower than that in the controls (P ≤ 0.05). Catalase and glutathione peroxides’ activities were not different between the two groups (P > 0.05). SCE rate in the DS derived cultures was significantly higher (P < 0.001) than that of the controls. DS individuals have a higher oxidative stress, higher superoxide dismutase activities and higher rates of SCE in their derived lymphocyte cultures compared to those of the controls. We claim that such differences may have resulted from the over expression of superoxide dismutase gene, leading to imbalanced cellular antioxidant mechanisms and, consequently, resulted in a high concentration of free radicals that destabilized the DNA as expressed by the high rate of SCE.

Chapter 2 - Implications of trisomy 21 on congenital features and health aspects

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.

Abnormal growth in Down syndrome (DS) is reflected by variable reduction in size and simplification in form of many physical traits. This study aimed to compare the thickness of enamel and dentine in deciduous and permanent mandibular incisor teeth between DS and non-DS individuals and to clarify how these tissues contribute to altered tooth size in DS. Sample groups comprised 61 mandibular incisors (29 permanent and 32 deciduous) from DS individuals and 55 mandibular incisors (29 permanent and 26 deciduous) from non-DS individuals. Maximum mesiodistal and labiolingual crown dimensions were measured initially, then the crowns were sectioned midsagittally and photographed using a stereomicroscope. Linear measurements of enamel and dentine thickness were obtained on the labial and lingual surfaces of the crowns, together with enamel and dentine-pulp areas and lengths of the dentino-enamel junction. Reduced permanent crown size in DS was associated with a reduction in both enamel and dentine thickness. After adjustments were made for tooth size, DS permanent incisors had significantly thinner enamel than non-DS permanent teeth. The DS permanent teeth also exhibited significant differences in shape and greater variability in dimensions than the non-DS permanent teeth. Crown dimensions of deciduous incisors were similar in size or larger in DS compared with non-DS deciduous teeth. Enamel and dentine thicknesses of the deciduous teeth were similar in DS and non-DS individuals. The findings indicate that growth retardation in DS reduces both enamel and dentine deposition in the permanent incisors but not in the earlier-forming deciduous predecessors. The results are also consistent with the concept of amplified developmental instability for dental traits in DS.

Congenital heart defects (CHD) are present in most, but not all, cases of Down syndrome (DS). The presence of methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms has been reported as a risk factor for CHD in DS. The aims of the present study were to assess (i) the frequency of MTHFR C677T and A1298C polymorphisms in DS individuals in the Croatian population; (ii) the relationship between the two maternal MTHFR polymorphisms and CHD-affected DS children; and (iii) the transmission frequencies of the variant alleles of the two MTHFR polymorphisms in CHD-affected DS. The study population included 112 DS subjects and 221 controls. CHD were present in 48% of the DS subjects (54/112). The mothers of 107 DS individuals were available for the study; none was a periconceptional folic acid user. Allele transmission was analyzed in 34 complete parent-offspring triads. The frequencies of the allele, individual, and combined genotypes of MTHFR C677T and A1298C in DS subjects were not statistically different compared to the normal healthy Croatian controls. The maternal MTHFR polymorphisms were not found to be a risk factor for DS-related CHD. The allele transmission of the two MTHFR polymorphisms showed no deviations from random segregation. Because the fetus is lost in a great proportion of trisomy 21 pregnancies, both maternal and fetal, not only live-born MTHFR C677T and A1298C, as well as maternal nutrition and lifestyle during pregnancy, should be analyzed to asses the impact on CHD in DS.

Age-dependent deficiency in saliva and salivary antibodies secretion in Down's syndrome

Age and lower folate concentrations are well-known risk factors for cardiovascular disease (CVD), but the potential roles of age and folate deficiency in hyperhomocysteinemia (HHcy), especially in HHcy patients with abnormal methylation levels of key enzyme genes promoter in homocysteinemia (Hcy) pathway, have not been thoroughly evaluated. The purpose of this study was to evaluate the relationship between the promoter methylation levels of six key enzyme genes and age and serum folate level to better understand the pathogenesis of HHcy. In 299 HHcy patients, six key enzyme genes promoter methylation was analyzed by PCR amplification and MethylTargetTM methods. The betaine homocysteine methyltransferase (BHMT), Cystathionine β-synthase (CBS), and Methionine synthasegene (MTR) promoter methylation levels were positively associated with age and a negative correlation was found between CBS promoter methylation level and folate levels. However, these associations were not significant after Bonferroni correction. The stratified analysis showed that the methylation level of CBS gene promoter was positively correlated with age in males, and a positive correlation was also found between BHMT gene promoter methylation level and age in HHcy patients with a history of diabetes or hypertension. Moreover, stratified analysis according to sex revealed that the methylation levels of three CpG regions of BHMT_2, CBS_2, and CBS_3 were positively correlated with age in males after Bonferroni correction. Our data suggested that age and folate deficiency may increase the risk of HHcy by mediating methylation of the promoter regions of key enzyme genes in the one-carbon metabolism pathway.

Altered levels of global DNA methylation and gene silencing through methylation of promoter regions can impact cancer risk, but little is known about their environmental determinants. We examined the association between lifestyle factors and levels of global genomic methylation and IL-6 promoter methylation in white blood cell DNA of 165 cancer-free subjects, 18–78 years old, enrolled in the COMIR (Commuting Mode and Inflammatory Response) study, New York, 2009–2010. Besides self-administrated questionnaires on diet and physical activity, we measured weight and height, white blood cell (WBC) counts, plasma levels of high sensitivity C-reactive protein (hs-CRP), and genomic (LINE-1) and gene-specific methylation (IL-6) by pyrosequencing in peripheral blood WBC. Mean levels of LINE-1 and IL-6 promoter methylation were 78.2% and 57.1%, respectively. In multivariate linear regression models adjusting for age, gender, race/ethnicity, body mass index, diet, physical activity, WBC counts and CRP, only dietary folate intake from fortified foods was positively associated with LINE-1 methylation. Levels of IL-6 promoter methylation were not significantly correlated with age, gender, race/ethnicity, body mass index, physical activity or diet, including overall dietary patterns and individual food groups and nutrients. There were no apparent associations between levels of methylation and inflammation markers such as WBC counts and hs-CRP. Overall, among several lifestyle factors examined in association with DNA methylation, only dietary folate intake from fortification was associated with LINE-1 methylation. The long-term consequence of folate fortification on DNA methylation needs to be further evaluated in longitudinal settings.

Motion perception deficit in Down Syndrome

Down syndrome (DS) is caused by triplication of Human chromosome 21 (Hsa21) and associated with an array of deleterious phenotypes, including mental retardation, heart defects and immunodeficiency. Genome-wide expression patterns of uncultured peripheral blood cells are useful to understanding of DS-associated immune dysfunction. We used a Human Exon microarray to characterize gene expression in uncultured peripheral blood cells derived from DS individuals and age-matched controls from two age groups: neonate (N) and child (C). A total of 174 transcript clusters (gene-level) with eight located on Hsa21 in N group and 383 transcript clusters including 56 on Hsa21 in C group were significantly dysregulated in DS individuals. Microarray data were validated by quantitative polymerase chain reaction. Functional analysis revealed that the dysregulated genes in DS were significantly enriched in two and six KEGG pathways in N and C group, respectively. These pathways included leukocyte trans-endothelial migration, B cell receptor signaling pathway and primary immunodeficiency, etc., which causally implicated dysfunctional immunity in DS. Our results provided a comprehensive picture of gene expression patterns in DS at the two developmental stages and pointed towards candidate genes and molecular pathways potentially associated with the immune dysfunction in DS.