DNA methylation profiling in Trisomy 21 females with and without breast cancer.

Background: Down Syndrome (DS) is the most common chromosome anomaly in humans and occurs due to an extra copy of chromosome 21. The malignancy profile in DS is unique, since DS patients have a low risk of developing solid tumors such as breast cancer however they are at higher risk of developing acute myeloid leukemia and acute lymphoblastic leukemia. Methods: In this study, we investigated DNA methylation signatures and epigenetic aging in DS individuals with and without breast cancer. We analyzed DNA methylation patterns in Trisomy 21 (T21) individuals without breast cancer (T21-BCF) and DS individuals with breast cancer (T21-BC), using the Infinium Methylation EPIC BeadChip array. Results: Our results revealed several differentially methylated sites and regions in the T21-BC patients that were associated with changes in gene expression. The differentially methylated CpG sites were enriched for processes related to serine-type peptidase activity, epithelial cell development, GTPase activity, bicellular tight junction, Ras protein signal transduction, etc. On the other hand, the epigenetic age acceleration analysis showed no difference between T21-BC and T21-BCF patients. Conclusions: This is the first study to investigate DNA methylation changes in Down syndrome women with and without breast cancer and it could help shed light on factors that protect against breast cancer in DS.

Obesity in Qatar: A Case-Control Study on the Identification of Associated Risk Factors.

Obesity is an emerging public health problem in the Western world as well as in the Gulf region. Qatar, a tiny wealthy county, is among the top-ranked obese countries with a high obesity rate among its population. Compared to Qatar's severity of this health crisis, only a limited number of studies focused on the systematic identification of potential risk factors using multimodal datasets. This study aims to develop machine learning (ML) models to distinguish healthy from obese individuals and reveal potential risk factors associated with obesity in Qatar. We designed a case-control study focused on 500 Qatari subjects, comprising 250 obese and 250 healthy individuals- the later forming the control group. We obtained the most extensive collection of clinical measurements for the Qatari population from the Qatar Biobank (QBB) repertoire, including (i) Physio-clinical Biomarkers, (ii) Spirometry, (iii) VICORDER, (iv) DXA scan composition, and (v) DXA scan densitometry readings. We developed several machine learning (ML) models to distinguish healthy from obese individuals and applied multiple feature selection techniques to identify potential risk factors associated with obesity. The proposed ML model achieved over 90% accuracy, thereby outperforming the existing state of the art models. The outcome from the ablation study on multimodal clinical datasets revealed physio-clinical measurements as the most influential risk factors in distinguishing healthy versus obese subjects. Furthermore, multiple feature ranking techniques confirmed known obesity risk factors (c-peptide, insulin, albumin, uric acid) and identified potential risk factors linked to obesity-related comorbidities such as diabetes (e.g., HbA1c, glucose), liver function (e.g., alkaline phosphatase, gamma-glutamyl transferase), lipid profile (e.g., triglyceride, low density lipoprotein cholesterol, high density lipoprotein cholesterol), etc. Most of the DXA measurements (e.g., bone area, bone mineral composition, bone mineral density, etc.) were significantly (p-value < 0.05) higher in the obese group. Overall, the net effect of hypothesized protective factors of obesity on bone mass seems to have surpassed the hypothesized harmful factors. All the identified factors warrant further investigation in a clinical setup to understand their role in obesity.

Memory CD4+ T cells lacking expression of CCR7 promote pro-inflammatory cytokine production in patients with diffuse cutaneous systemic sclerosis.

Systemic sclerosis (SSc) is a predominantly T-cell-mediated autoimmune disorder with a characteristic sequence of Th1 and Th2 inflammation resulting in fibrosis. The contribution of differentiated memory T-cell subpopulations and methylation of CpG regions of Th1- or Th2-specific transcription factor genes on the inflammatory cytokine signature in SSc is not well understood. The study aimed to investigate phenotypic differentiation, the cytokine signature, sensitivity of memory T cells to in vitro suppression by autologous regulatory T cells (Tregs), and methylation of Th1- and Th2-specific transcription factor genes in patients with limited (lcSSc) and diffuse cutaneous SSc (dcSSc) compared to healthy donors (HD). Phenotype/intracellular cytokine production and methylation of Th1- and Th2-specific transcription factor genes were determined by flow cytometry and epigenetic analysis, respectively, and compared between patients with lcSSc, dcSSc and HD. Discrimination of CD4+ T cells that lack CCR7 expression revealed that CCR7- CD4+ memory T cells and effectors are producers of intracellular TNFα, IL-13 and IL-4, particularly in dcSSc. A proportional increase in CCR7- memory T cells was demonstrated by SSc-derived CD4+ T-cells after insufficient suppression by Tregs. A higher level of methylation of GATA3 or STAT4 (Th2- and Th1-specific transcription factor genes, respectively) was observed in dcSSc. An abundance of specific CD4+ memory T-cell subpopulations strongly contributes to the production of pro-inflammatory cytokines in dcSSc. Our results suggest that therapeutic concepts should focus more intensively on the memory phenotype to control T cell-mediated inflammation in SSc patients.

Allele-specific methylation of imprinted genes in fetal cord blood is influenced by cis-acting genetic variants and parental factors.

AIM: To examine the effects of genetic variation, parental age and BMI on parental allele-specific methylation of imprinted genes in fetal cord blood samples. METHODOLOGY: We have developed SNP genotyping and deep bisulphite sequencing assays for six imprinted genes to determine parental allele-specific methylation patterns in diploid somatic tissues. RESULTS: Multivariate linear regression analyses revealed a negative correlation of paternal age with paternal MEG3 allele methylation in fetal cord blood. Methylation of the maternal PEG3 allele showed a positive correlation with maternal age. Paternal BMI was positively correlated with paternal MEST allele methylation. In addition to parental origin, allele-specific methylation of most imprinted genes was largely dependent on the underlying SNP haplotype. CONCLUSION: Our study supports the idea that parental factors can have an impact, although of small effect size, on the epigenome of the next generation, providing an additional layer of complexity to phenotypic diversity.

Limiting Dilution Bisulfite Pyrosequencing®: A Method for Methylation Analysis of Individual DNA Molecules in a Single or a Few Cells.

Bisulfite-based methods for DNA methylation analysis of small amounts of DNA from a limited number of cells are technologically challenging. Degradation of genomic DNA by bisulfite treatment, contamination with foreign DNA, and biases in the amplification of individual DNA molecules can generate results, which are not representative of the starting sample. Limiting dilution (LD) bisulfite Pyrosequencing(®) (BSP) is a relatively simple technique to circumvent these problems. The bisulfite-treated DNA of a single or a few cells is diluted to an extent, that only a single DNA target molecule is present in the reaction. Then each individual DNA molecule in the starting sample is separately amplified and analyzed by Pyrosequencing. This allows the detection of rare alleles that are easily masked when pools of DNA target molecules are analyzed. Amplicons containing a heterozygous single nucleotide polymorphism (SNP) allow one to delineate the parental origin of the recovered molecules in addition to their methylation status. The number of cells (DNA target molecules) in the starting sample determines the dilution level and the number of reactions that have to be performed. LD-BSP allows methylation analysis of small cell pools (i.e., 5-10 microdissected cells) and even individual cells. The primers and PCR conditions described here have been successfully employed to analyze the methylation status of up to eight target genes in individual 2-16 cell embryos, germinal vesicle (GV) oocytes, and haploid sperms.

Comparative methylation profiles and telomerase biology of mouse multipotent adult germline stem cells and embryonic stem cells.

Recently, several groups described the isolation of mouse spermatogonial stem cells (SSCs) and their potential to develop to embryonic stem cell (ESC)-like cells, so-called multipotent germline stem cells (mGSCs). We were the first to derive such mGSCs from SSCs isolated from adult mouse testis and, therefore, called these mGSCs multipotent adult germline stem cells (maGSCs). Here, we comparatively analyzed gene-specific and global DNA methylation profiles as well as the telomerase biology of several maGSC and male ESC lines. We show that undifferentiated maGSCs are very similar to undifferentiated male ESCs with regard to global DNA methylation, methylation of pluripotency marker gene loci, telomerase activity and telomere length. Imprinted gene methylation levels were generally lower in undifferentiated maGSCs than in undifferentiated male ESCs, but, compared with undifferentiated mGSCs derived by other groups, more similar to those of male ESCs. Differentiation of maGSCs increased the methylation of three of the four analyzed imprinted genes to almost somatic methylation patterns, but dramatically decreased global DNA methylation. Our findings further substantiate the pluripotency of maGSCs and their potential for regenerative medicine.