A prognostic method for the litter size in Berkshire pigs based on DNA methylation of IGFBP4 gene

2018 ◽  
Vol 98 (4) ◽  
pp. 845-851
Author(s):  
Seulgi Kwon ◽  
Sang Mi An ◽  
Go Eun Yu ◽  
Jung Hye Hwang ◽  
Da Hye Park ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Litter Size ◽  
Dna Sequences ◽  
Epigenetic Modification ◽  
Methylation Status ◽  
Follicular Development ◽  
Restriction Enzymes ◽  
Important Trait ◽  
Specific Restriction ◽  
Polymerase Chain

Litter size is an important trait in the pig industry. Therefore, a lot of effort has been put into improving this trait. DNA methylation is an essential epigenetic modification present in unique DNA sequences. Alterations in methylation can affect transcription and phenotypic variation. The purpose of this study was to investigate the effect of DNA methylation on litter size. Methylation-specific restriction enzymes are simple and useful tools for detecting DNA methylation status. We used a pair of methylation-sensitive isoschizomers, which have the same recognition site, HpaII and MspI. Insulin-like growth factor binding protein 4 (IGFBP4) is a key regulator of ovarian follicular development and fetal growth in eutherian mammals. In this study, we discovered that IGFBP4 was hyper-methylated in the uterus tissue of a larger litter size group using bisulfite sequencing, and validated the positive relationship between the methylation status of IGFBP4 and the total number born of pigs using the porcine methylation-specific restriction enzyme polymerase chain reaction (PMP) assay. We suggest that the IGFPB4 gene can be used as a prognostic biomarker for hyperprolific sows and that the PMP assay is a useful tool for methylation status screening.

scholarly journals Correlation of methylation status in MTHFR promoter region with recurrent pregnancy loss

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Mai Mahmoud Shaker ◽  
Taghreed Abdelmoniem shalabi ◽  
Khalda said Amr
Keyword(s):  
Dna Methylation ◽  
Dna Sequences ◽  
Promoter Region ◽  
Pregnancy Loss ◽  
Recurrent Pregnancy Loss ◽  
Methylation Status ◽  
Control Group ◽  
Case Group ◽  
Methyl Radical ◽  
Fertile Women

Abstract Background DNA methylation is an epigenetic process for modifying transcription factors in various genes. Methylenetetrahydrofolate reductase (MTHFR) stimulates synthesis of methyl radical in the homocysteine cycle and delivers methyl groups needed in DNA methylation. Furthermore, numerous studies have linked gene polymorphisms of this enzyme with a larger risk of recurrent pregnancy loss (RPL), yet scarce information is available concerning the association between epigenetic deviations in this gene and RPL. Hypermethylation at precise DNA sequences can function as biomarkers for a diversity of diseases. We aimed by this study to evaluate the methylation status of the promoter region of MTHFR gene in women with RPL compared to healthy fertile women. It is a case–control study. Hundred RPL patients and hundred healthy fertile women with no history of RPL as controls were recruited. MTHFR C677T was assessed by polymerase chain reaction-restriction fragment length polymorphism (RFLP). Quantitative evaluation of DNA methylation was performed by high-resolution melt analysis by real-time PCR. Results The median of percentage of MTHFR promoter methylation in RPL cases was 6.45 [0.74–100] vs. controls was 4.50 [0.60–91.7], P value < 0.001. In the case group, 57 hypermethylated and 43 normo-methylated among RPL patients vs. 40 hypermethylated and 60 normo-methylated among controls, P< 0.005. Frequency of T allele in C677T MTHFR gene among RPL patients was 29% vs. 23% among the control group; C allele vs. T allele: odds ratio (OR) = 1.367 (95% confidence interval (CI) 0.725–2.581). Conclusion Findings suggested a significant association between hypermethylation of the MTHFR promoter region in RPL patients compared to healthy fertile women.

scholarly journals Exploring Differentially Methylated Genes in Vulvar Squamous Cell Carcinoma

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3580
Author(s):  
Shatavisha Dasgupta ◽  
Patricia C. Ewing-Graham ◽  
Sigrid M. A. Swagemakers ◽  
Thierry P. P. van den Bosch ◽  
Peggy N. Atmodimedjo ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Dna Sequences ◽  
Cpg Island ◽  
Epigenetic Modification ◽  
Vulvar Squamous Cell Carcinoma ◽  
Differentially Methylated Genes ◽  
Methylation Profiling

DNA methylation is the most widely studied mechanism of epigenetic modification, which can influence gene expression without alterations in DNA sequences. Aberrations in DNA methylation are known to play a role in carcinogenesis, and methylation profiling has enabled the identification of biomarkers of potential clinical interest for several cancers. For vulvar squamous cell carcinoma (VSCC), however, methylation profiling remains an under-studied area. We sought to identify differentially methylated genes (DMGs) in VSCC, by performing Infinium MethylationEPIC BeadChip (Illumina) array sequencing, on a set of primary VSCC (n = 18), and normal vulvar tissue from women with no history of vulvar (pre)malignancies (n = 6). Using a false-discovery rate of 0.05, beta-difference (Δβ) of ± 0.5, and CpG-island probes as cut-offs, 199 DMGs (195 hyper-methylated, 4 hypo-methylated) were identified for VSCC. Most of the hyper-methylated genes were found to be involved in transcription regulator activity, indicating that disruption of this process plays a vital role in VSCC development. The majority of VSCCs harbored amplifications of chromosomes 3, 8, and 9. We identified a set of DMGs in this exploratory, hypothesis-generating study, which we hope will facilitate epigenetic profiling of VSCCs. Prognostic relevance of these DMGs deserves further exploration in larger cohorts of VSCC and its precursor lesions.

scholarly journals DNA Methylation in Solid Tumors: Functions and Methods of Detection

2021 ◽  
Vol 22 (8) ◽  
pp. 4247
Author(s):  
Andrea Martisova ◽  
Jitka Holcakova ◽  
Nasim Izadi ◽  
Ravery Sebuyoya ◽  
Roman Hrstka ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Solid Tumors ◽  
Epigenetic Modification ◽  
Single Gene ◽  
Restriction Enzymes ◽  
Methylation Analysis ◽  
Cpg Dinucleotides ◽  
Sequencing Technologies ◽  
Genome Level ◽  
Dna Methylation Analysis

DNA methylation, i.e., addition of methyl group to 5′-carbon of cytosine residues in CpG dinucleotides, is an important epigenetic modification regulating gene expression, and thus implied in many cellular processes. Deregulation of DNA methylation is strongly associated with onset of various diseases, including cancer. Here, we review how DNA methylation affects carcinogenesis process and give examples of solid tumors where aberrant DNA methylation is often present. We explain principles of methods developed for DNA methylation analysis at both single gene and whole genome level, based on (i) sodium bisulfite conversion, (ii) methylation-sensitive restriction enzymes, and (iii) interactions of 5-methylcytosine (5mC) with methyl-binding proteins or antibodies against 5mC. In addition to standard methods, we describe recent advances in next generation sequencing technologies applied to DNA methylation analysis, as well as in development of biosensors that represent their cheaper and faster alternatives. Most importantly, we highlight not only advantages, but also disadvantages and challenges of each method.

scholarly journals Detection of LINE-1 hypomethylation in cfDNA of Esophageal Adenocarcinoma Patients

2020 ◽  
Vol 21 (4) ◽  
pp. 1547 ◽  
Author(s):  
Elisa Boldrin ◽  
Matteo Curtarello ◽  
Marco Dallan ◽  
Rita Alfieri ◽  
Stefano Realdon ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Esophageal Adenocarcinoma ◽  
Methodological Approach ◽  
Methylation Status ◽  
Dna Amplification ◽  
Restriction Enzymes ◽  
Poor Quality ◽  
Bisulfite Conversion ◽  
Dna Hypomethylation ◽  
Methylation Patterns

DNA methylation plays an important role in cancer development. Cancer cells exhibit two types of DNA methylation alteration: site-specific hypermethylation at promoter of oncosuppressor genes and global DNA hypomethylation. This study evaluated the methylation patterns of long interspersed nuclear element (LINE-1) sequences which, due to their relative abundance in the genome, are considered a good surrogate indicator of global DNA methylation. LINE-1 methylation status was investigated in the cell-free DNA (cfDNA) of 21 patients, 19 with esophageal adenocarcinoma (EADC) and 2 with Barrett’s esophagus (BE). The two BE patients and one EADC patient were also analyzed longitudinally. Methylation status was analyzed using restriction enzymes and DNA amplification. This methodology was chosen to avoid bisulfite conversion, which we considered inadequate for cfDNA analysis. Indeed, cfDNA is characterized by poor quality and low concentration, and bisulfite conversion might worsen these conditions. Results showed that hypomethylated LINE-1 sequences are present in EADC cfDNA. Furthermore, longitudinal studies in BE suggested a correlation between methylation status of LINE-1 sequences in cfDNA and progression to EADC. In conclusion, our study indicated the feasibility of our methodological approach to detect hypomethylation events in cfDNA from EADC patients, and suggests LINE-1 methylation analysis as a new possible molecular assay to integrate into patient monitoring.

scholarly journals DNA Methylation Validation Methods: a Coherent Review with Practical Comparison

2019 ◽  
Vol 21 (1) ◽  
Author(s):  
Šárka Šestáková ◽  
Cyril Šálek ◽  
Hana Remešová
Keyword(s):  
Dna Methylation ◽  
Accurate Method ◽  
Primer Design ◽  
Clinical Diagnostics ◽  
Bisulfite Conversion ◽  
Design And Optimization ◽  
Validation Methods ◽  
Specific Restriction ◽  
Polymerase Chain ◽  
Pcr Conditions

Abstract Here, we present a practical overview of four commonly used validation methods for DNA methylation assessment: methylation specific restriction endonucleases (MSRE) analysis, pyrosequencing, methylation specific high-resolution DNA melting (MS-HRM) and quantitative methylation specific polymerase chain reaction (qMSP). Using these methods, we measured DNA methylation levels of three loci in human genome among which one was highly methylated, one intermediately methylated and one unmethylated. We compared the methods in terms of primer design demands, methods’ feasibility, accuracy, time and money consumption, and usability for clinical diagnostics. Pyrosequencing and MS-HRM proved to be the most convenient methods. Using pyrosequencing, it is possible to analyze every CpG in a chosen region. The price of the instrument may represent the main limitation of this methodology. MS-HRM is a simple PCR-based method. The measurement was quick, cheap and very accurate. MSRE analysis is based on a methylation specific digestion of DNA. It does not require a bisulfite conversion of DNA as the other methods. MSRE analysis was very easy to perform, however, it was not suitable for intermediately methylated regions and it was also quite expensive. qMSP is a qPCR-based method that uses primers designed specifically for methylated and unmethylated alleles of a chosen region. This was the least accurate method and also the primer design and optimization of PCR conditions were highly demanding.

scholarly journals Discrimination of Diploid Crucifer Species Using PCR-RFLP of Chloroplast DNA

HortScience ◽  
2004 ◽  
Vol 39 (7) ◽  
pp. 1575-1577 ◽  
Author(s):  
Claudia Cunha ◽  
Muhammet Tonguç ◽  
Phillip D. Griffiths
Keyword(s):  
Chloroplast Dna ◽  
Dna Sequences ◽  
Raphanus Sativus ◽  
Fragment Length Polymorphism ◽  
Diploid Species ◽  
Restriction Enzymes ◽  
Chain Reaction ◽  
Species Identity ◽  
Pcr Rflp ◽  
Polymerase Chain

Chloroplast DNA (cpDNA) was used to identify polymorphisms between crucifer species using the polymerase chain reaction-random fragment-length polymorphism (PCR-RFLP) technique. Ten primer pairs based on cpDNA gene sequences were used to amplify cpDNA fragments in Brassica oleracea L., B. rapa L., B. nigra (L.) Koch, B. napus L., B. carinata Braun, B. juncea (L.) Czern, and Raphanus sativus L. accessions. Amplified DNA sequences were then digested using 11 restriction enzymes to identify polymorphisms between the 7 species. Of the 110 combinations, 38 generated polymorphisms that discriminated one or more of the species. Genotyping of these polymorphisms in 10 accessions of each of the diploid species (B. oleracea, B. nigra, B. rapa and R. sativus) did not reveal segregating polymorphisms among accessions within species, indicating that they can be used to help determine species identity. Ten accessions of each of the amphidiploids B. napus, B. carinata and B. juncea were genotyped to infer their maternal ancestry. The diploid source of cpDNA in B. carinata was B. nigra in all accessions tested and B. rapa for nine of ten B. juncea accessions tested. Two B. napus accessions amplified polymorphisms shared with B. rapa, and eight accessions produced unique polymorphisms from neither B. rapa, B. oleracea or B. nigra. The polymorphisms identified in this study can be used to help confirm identity of the diploid crucifer species for taxonomic and conservation studies.

scholarly journals The expression and mutation of <i>BMPR1B</i> and its association with litter size in small-tail Han sheep (<i>Ovis aries</i>)

2021 ◽  
Vol 64 (1) ◽  
pp. 211-221
Author(s):  
Yu-Liang Wen ◽  
Xiao-Fei Guo ◽  
Lin Ma ◽  
Xiao-Sheng Zhang ◽  
Jin-Long Zhang ◽  
...  
Keyword(s):  
Litter Size ◽  
Quantitative Polymerase Chain Reaction ◽  
Follicular Development ◽  
Follicular Phase ◽  
Ovis Aries ◽  
Negative Effects ◽  
Single Nucleotide ◽  
Ovarian Granulosa Cell ◽  
Polymerase Chain ◽  
Genotype And Allele Frequencies

Abstract. Previous studies have shown that BMPR1B promotes follicular development and ovarian granulosa cell proliferation, thereby affecting ovulation in mammals. In this study, the expression and polymorphism of the BMPR1B gene associated with litter size in small-tail Han (STH) sheep were determined. The expression of BMPR1B was detected in 14 tissues of STH sheep during the follicular phase as well as in the hypothalamic–pituitary–gonadal (HPG) axis of monotocous and polytocous STH sheep during the follicular and luteal phases using quantitative polymerase chain reaction (qPCR). Sequenom MassARRAY® single nucleotide polymorphism (SNP) technology was also used to detect the polymorphism of SNPs in seven sheep breeds. Here, BMPR1B was highly expressed in hypothalamus, ovary, uterus, and oviduct tissue during the follicular phase, and BMPR1B was expressed significantly more in the hypothalamus of polytocous ewes than in monotocous ewes during both the follicular and luteal phases (P<0.05). For genotyping, we found that genotype and allele frequencies of three loci of the BMPR1B gene were extremely significantly different (P<0.01) between the monotocous and polytocous groups. Association analysis results showed that the g.29380965A>G locus had significant negative effects on the litter size of STH sheep, and the combination of g.29380965A>G and FecB (Fec – fecundity and B – Booroola; A746G) at the BMPR1B gene showed that the litter size of AG–GG, AA–GG, and GG–GG genotypes was significantly higher compared with other genotypes (P<0.05). This is the first study to find a new molecular marker affecting litter size and to systematically analyze the expression of BMPR1B in different fecundity and physiological periods of STH sheep.

scholarly journals Hypomethylation in the promoter region of <i>ZPBP</i> as a potential litter size indicator in Berkshire pigs

2019 ◽  
Vol 62 (1) ◽  
pp. 69-76
Author(s):  
Sang Mi An ◽  
Seulgi Kwon ◽  
Jung Hye Hwang ◽  
Go Eun Yu ◽  
Deok Gyeong Kang ◽  
...  
Keyword(s):  
Litter Size ◽  
Promoter Region ◽  
Methylation Status ◽  
Uterine Tissue ◽  
Genome Wide ◽  
Size Groups ◽  
Increase Productivity ◽  
Specific Restriction ◽  
Berkshire Pigs ◽  
Epigenetic Biomarker

Abstract. In pigs, litter size is typically defined as the total number of piglets born (TNB) or the number of piglets born alive (NBA). Increasing pig litter size is of great economic interest as a means to increase productivity. The capacity of the uterus is a critical component of litter size and may play a central role in prolificacy. In this study, we investigated litter-size-related epigenetic markers in uterine tissue from Berkshire pigs with smaller litter size groups (SLGs) and larger litter size groups (LLGs) using genome-wide bisulfite sequencing (GWBS). A total of 3269 differentially methylated regions (DMRs) were identified: 1566 were hypermethylated and 1703 hypomethylated in LLG compared to SLG. The zona pellucida binding protein (ZPBP) gene was significantly hypomethylated in the LLG promoter region, and its expression was significantly upregulated in uterine tissue. Thus, the methylation status of ZPBP gene was identified as a potential indicator of litter size. Furthermore, we verified its negative correlation with litter size traits (TNB and NBA) in whole blood samples from 172 Berkshire sows as a blood-based biomarker by a porcine methylation-specific restriction enzyme polymerase chain reaction (PMP) assay. The results suggest that the methylation status of the ZPBP gene can serve as a valuable epigenetic biomarker for hyperprolific sows.

scholarly journals Association of Breastfeeding Duration with Susceptibility to Allergy, Influenza, and Methylation Status of TLR1 Gene

Medicina ◽  
2019 ◽  
Vol 55 (9) ◽  
pp. 535
Author(s):  
Ma’mon M. Hatmal ◽  
Nada N. Issa ◽  
Walhan Alshaer ◽  
Hamzeh J. Al-Ameer ◽  
Omar Abuyaman ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Promoter Region ◽  
Methylation Status ◽  
Breastfeeding Duration ◽  
Toll Like Receptor ◽  
Chain Reaction ◽  
Blood Samples ◽  
Three Samples ◽  
Gene Level ◽  
Polymerase Chain

Background and Objectives: This study aimed to investigate the possible association between exclusive breastfeeding duration during early infancy and susceptibility to allergy and influenza in adulthood. Furthermore, we also investigated the association of breastfeeding duration with DNA methylation at two sites in the promoter of the toll-like receptor-1 (TLR1) gene, as well as the association between DNA methylation of the toll-like receptor-1 (TLR1) gene and susceptibility to different diseases. Materials and Methods: Blood samples were collected from 100 adults and classified into two groups according to breastfeeding duration (<6 months and ≥6 months) during infancy. Subjects were asked to complete a questionnaire on their susceptibilities to different diseases and sign a consent form separately. Fifty-three samples underwent DNA extraction, and the DNA samples were divided into two aliquots, one of which was treated with bisulfite reagent. The promoter region of the TLR1 gene was then amplified by polymerase chain reaction (PCR) and sequenced. Results: We found a significant association between increased breastfeeding duration and a reduction in susceptibility to influenza and allergy, as well asa significant reduction in DNA methylation within the promoter of the TLR1 gene. No association was found between DNA methylation and susceptibility to different diseases. Conclusions: The findings demonstrate the significance of increased breastfeeding duration for improved health outcomes at the gene level.

scholarly journals Faulty homocysteine recycling in diabetic retinopathy

2020 ◽  
Vol 7 (1) ◽  
Author(s):  
Renu A. Kowluru ◽  
Ghulam Mohammad ◽  
Nikhil Sahajpal
Keyword(s):  
Dna Methylation ◽  
Diabetic Retinopathy ◽  
Methylenetetrahydrofolate Reductase ◽  
Epigenetic Modification ◽  
Mitochondrial Dynamics ◽  
Methylation Status ◽  
Mitochondrial Damage ◽  
Diabetic Patients ◽  
Retinal Microvasculature ◽  
Increased Risk

Abstract Background Although hyperglycemia is the main instigator in the development of diabetic retinopathy, elevated circulating levels of a non-protein amino acid, homocysteine, are also associated with an increased risk of retinopathy. Homocysteine is recycled back to methionine by methylenetetrahydrofolate reductase (MTHFR) and/or transsulfurated by cystathionine β-synthase (CBS) to form cysteine. CBS and other transsulfuration enzyme cystathionine-γ-lyase (CSE), through desulfuration, generates H2S. Methionine cycle also regulates DNA methylation, an epigenetic modification associated with the gene suppression. The aim of this study was to investigate homocysteine and its metabolism in diabetic retinopathy. Methods Homocysteine and H2S levels were analyzed in the retina, and CBS, CSE and MTHFR in the retinal microvasculature from human donors with established diabetic retinopathy. Mitochondrial damage was evaluated in retinal microvessels by quantifying enzymes responsible for maintaining mitochondrial dynamics (fission-fusion-mitophagy). DNA methylation status of CBS and MTHFR promoters was examined using methylated DNA immunoprecipitation technique. The direct effect of homocysteine on mitochondrial damage was confirmed in human retinal endothelial cells (HRECs) incubated with 100 μM L-homocysteine. Results Compared to age-matched nondiabetic control human donors, retina from donors with established diabetic retinopathy had ~ 3-fold higher homocysteine levels and ~ 50% lower H2S levels. The enzymes important for both transsulfuration and remethylation of homocysteine including CBS, CSE and MTHFR, were 40–60% lower in the retinal microvasculature from diabetic retinopathy donors. While the mitochondrial fission protein, dynamin related protein 1, and mitophagy markers optineurin and microtubule-associated protein 1A/1B-light chain 3 (LC3), were upregulated, the fusion protein mitofusin 2 was downregulated. In the same retinal microvessel preparations from donors with diabetic retinopathy, DNA at the promoters of CBS and MTHFR were hypermethylated. Incubation of HRECs with homocysteine increased reactive oxygen species and decreased transcripts of mtDNA-encoded CYTB. Conclusions Compromised transsulfuration and remethylation processes play an important role in the poor removal of retinal homocysteine in diabetic patients. Thus, regulation of their homocysteine levels should ameliorate retinal mitochondrial damage, and by regulating DNA methylation status of the enzymes responsible for homocysteine transsulfuration and remethylation, should prevent excess accumulation of homocysteine.

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