Methylation Status of Imprinted Genes and Repetitive Elements in Sperm DNA from Infertile Males

Stochastic, environmentally and/or genetically induced disturbances in the genome-wide epigenetic reprogramming processes during male germ-cell development may contribute to male infertility. To test this hypothesis, we have studied the methylation levels of 2 paternally (H19 and GTL2) and 5 maternally methylated (LIT1, MEST, NESPAS, PEG3, and SNRPN) imprinted genes, as well as of ALU and LINE1 repetitive elements in 141 sperm samples, which were used for assisted reproductive technologies (ART), including 106 couples with strictly male-factor or combined male and female infertility and 28 couples with strictly female-factor infertility. Aberrant methylation imprints showed a significant association with abnormal semen parameters, but did not seem to influence ART outcome. Repeat methylation also differed significantly between sperm samples from infertile and presumably fertile males. However, in contrast to imprinted genes, ALU methylation had a significant impact on pregnancy and live-birth rate in couples with male-factor or combined infertility. ALU methylation was significantly high-er in sperm samples leading to pregnancy and live-birth than in those that did not. Sperm samples leading to abortions showed significantly lower ALU methylation levels than those leading to the birth of a baby. Copyright (C) 2011 S. Karger AG, Base

Positive selection at codon 38 of the human KCNE1 (= minK) gene and sporadic absence of 38Ser-coding mRNAs in Gly38Ser heterozygotes

<p>Abstract</p> <p>Background</p> <p>KCNE1 represents the regulatory beta-subunit of the slowly activating delayed rectifier potassium channel (IKs). Variants of KCNE1 have repeatedly been linked to the long-QT syndrome (LQTS), a disorder which predisposes to deafness, ventricular tachyarrhythmia, syncope, and sudden cardiac death.</p> <p>Results</p> <p>We here analyze the evolution of the common Gly38Ser variant (rs1805127), using genomic DNAs, complementary DNAs, and HEK293-expressed variants of altogether 19 mammalian species. The between species comparison reveals that the human-specific Gly38Ser polymorphism evolved under strong positive Darwinian selection, probably in adaptation to specific challenges in the fine-tuning of IKs channels. The involved amino acid exchanges (Asp > Gly, Gly > Ser) are moderately radical and do not induce apparent changes in posttranslational modification. According to population genetic analyses (HapMap phase II) a heterozygote advantage accounts for the maintenance of the Gly38Ser polymorphism in humans. On the other hand, the expression of the 38Ser allele seems to be disadvantageous under certain conditions, as suggested by the sporadic deficiency of 38Ser-coding mRNAs in heterozygote Central Europeans and the depletion of homozygotes 38Ser in the Yoruban sample.</p> <p>Conclusion</p> <p>We speculate that individual differences in genomic imprinting or genomic recoding might have contributed to conflicting results of recent association studies between Gly38Ser polymorphism and QT phenotype. The findings thus highlight the relevance of mRNA data in future association studies of genotypes and clinical disorders. To the best of our knowledge, they moreover provide first time evidence for a unique pattern; i.e. coincidence of positive Darwinian selection and polymorphism with a sporadically suppressed expression of one allele.</p

Targeted next-generation sequencing of cancer genes in poorly differentiated thyroid cancer

Poorly differentiated thyroid carcinoma (PDTC) is a rare malignancy with higher mortality than well-differentiated thyroid carcinoma. The histological diagnosis can be difficult as well as the therapy. Improved diagnosis and new targeted therapies require knowledge of DNA sequence changes in cancer-relevant genes. The TruSeq Amplicon Cancer Panel was used to screen cancer genomes from 25 PDTC patients for somatic single-nucleotide variants in 48 genes known to represent mutational hotspots. A total of 4490 variants were found in 23 tissue samples of PDTC. Ninety-eight percent (4392) of these variants did not meet the inclusion criteria, while 98 potentially pathogenic or pathogenic variants remained after filtering. These variants were distributed over 33 genes and were all present in a heterozygous state. Five tissue samples harboured not a single variant. Predominantly, variants in P53 (43% of tissue samples) were identified, while less frequently, variants in APC, ERBB4, FLT3, KIT, SMAD4 and BRAF (each in 17% of tissue samples) as well as ATM, EGFR and FBXW7 (each in 13% of tissue samples) were observed. This study identified new potential genetic targets for further research in PDTC. Of particular interest are four observed ERBB4 (alias HER4) variants, which have not been connected to this type of thyroid carcinoma so far. In addition, APC and SMAD4 mutations have not been reported in this subtype of cancer either. In contrast to other reports, we did not find CTNNB1 variants

Stage-Specific Germ-Cell Marker Genes Are Expressed in All Mouse Pluripotent Cell Types and Emerge Early during Induced Pluripotency

Embryonic stem cells (ESCs) generated from the in-vitro culture of blastocyst stage embryos are known as equivalent to blastocyst inner cell mass (ICM) in-vivo. Though several reports have shown the expression of germ cell/pre-meiotic (GC/PrM) markers in ESCs, their functional relevance for the pluripotency and germ line commitment are largely unknown. In the present study, we used mouse as a model system and systematically analyzed the RNA and protein expression of GC/PrM markers in ESCs and found them to be comparable to the expression of cultured pluripotent cells originated from the germ line. Further, siRNA knockdown experiments have demonstrated the parallel maintenance and independence of pluripotent and GC/PrM networks in ESCs. Through chromatin immunoprecipitation experiments, we observed that pluripotent cells exhibit active chromatin states at GC marker genes and a bivalent chromatin structure at PrM marker genes. Moreover, gene expression analysis during the time course of iPS cells generation revealed that the expression of GC markers precedes pluripotency markers. Collectively, through our observations we hypothesize that the chromatin state and the expression of GC/PrM markers might indicate molecular parallels between in-vivo germ cell specification and pluripotent stem cell generation

Анализ пожарной безопасности магазина торговой сети ПАО "Магнит"

В статье проанализирована система пожарной безопасности магазина торговой сети "Магнит" с целью выявления уязвимых мест, профилактики пожаров и минимизации ущерба от потенциальных возгораний.The article analyzes the fire safety system of the Magnit retail chain store in order to identify vulnerabilities, prevent fires and minimize damage from potential fires

Abnormal hypermethylation at imprinting control regions in patients with S-adenosylhomocysteine hydrolase (AHCY) deficiency

S-adenosylhomocysteine hydrolase (AHCY) deficiency is a rare autosomal recessive disorder in methionine metabolism caused by mutations in the AHCY gene. Main characteristics are psychomotor delay including delayed myelination and myopathy (hypotonia, absent tendon reflexes etc.) from birth, mostly associated with hypermethioninaemia, elevated serum creatine kinase levels and increased genome wide DNA methylation. The prime function of AHCY is to hydrolyse and efficiently remove S-adenosylhomocysteine, the by-product of transmethylation reactions and one of the most potent methyltransferase inhibitors. In this study, we set out to more specifically characterize DNA methylation changes in blood samples from patients with AHCY deficiency. Global DNA methylation was increased in two of three analysed patients. In addition, we analysed the DNA methylation levels at differentially methylated regions (DMRs) of six imprinted genes (MEST, SNRPN, LIT1, H19, GTL2 and PEG3) as well as Alu and LINE1 repetitive elements in seven patients. Three patients showed a hypermethylation in up to five imprinted gene DMRs. Abnormal methylation in Alu and LINE1 repetitive elements was not observed. We conclude that DNA hypermethylation seems to be a frequent but not a constant feature associated with AHCY deficiency that affects different genomic regions to different degrees. Thus AHCY deficiency may represent an ideal model disease for studying the molecular origins and biological consequences of DNA hypermethylation due to impaired cellular methylation status

Spatial, temporal and interindividual epigenetic variation of functionally important DNA methylation patterns

DNA methylation is an epigenetic modification that plays an important role in gene regulation. It can be influenced by stochastic events, environmental factors and developmental programs. However, little is known about the natural variation of gene-specific methylation patterns. In this study, we performed quantitative methylation analyses of six differentially methylated imprinted genes (H19, MEG3, LIT1, NESP55, PEG3 and SNRPN), one hypermethylated pluripotency gene (OCT4) and one hypomethylated tumor suppressor gene (APC) in chorionic villus, fetal and adult cortex, and adult blood samples. Both average methylation level and range of methylation variation depended on the gene locus, tissue type and/or developmental stage. We found considerable variability of functionally important methylation patterns among unrelated healthy individuals and a trend toward more similar methylation levels in monozygotic twins than in dizygotic twins. Imprinted genes showed relatively little methylation changes associated with aging in individuals who are >25 years. The relative differences in methylation among neighboring CpGs in the generally hypomethylated APC promoter may not only reflect stochastic fluctuations but also depend on the tissue type. Our results are consistent with the view that most methylation variation may arise after fertilization, leading to epigenetic mosaicism

Early-life adversity selectively impairs α2-GABAA receptor expression in the mouse nucleus accumbens and influences the behavioral effects of cocaine

Haplotypes of the Gabra2 gene encoding the α2 subunit of the GABAA receptor (GABAAR) are associated with drug abuse, suggesting that α2-GABAARs may play an important role in the circuitry underlying drug misuse. The genetic association of Gabra2 haplotypes with cocaine addiction appears to be evident primarily in individuals who had experienced childhood trauma. Given this association of childhood trauma, cocaine abuse and the Gabra2 haplotypes, we have explored in a mouse model of early life adversity (ELA) whether such events influence the behavioral effects of cocaine and if, as suggested by the human studies, α2-GABAARs in the nucleus accumbens (NAc) are involved in these perturbed behaviors. In adult mice prior ELA caused a selective decrease of accumbal α2-subunit mRNA, resulting in a selective decrease in the number and size of the α2-subunit (but not the α1-subunit) immunoreactive clusters in NAc core medium spiny neurons (MSNs). Functionally, in adult MSNs ELA decreased the amplitude and frequency of GABAAR-mediated miniature inhibitory postsynaptic currents (mIPSCs), a profile similar to that of α2 "knock-out" (α2-/-) mice. Behaviorally, adult male ELA and α2-/- mice exhibited an enhanced locomotor response to acute cocaine and blunted sensitization upon repeated cocaine administration, when compared to their appropriate controls. Collectively, these findings reveal a neurobiological mechanism which may relate to the clinical observation that early trauma increases the risk for substance abuse disorder (SAD) in individuals harbouring haplotypic variations in the Gabra2 gene.</p

Monozygotic twins discordant for constitutive BRCA1 promoter methylation, childhood cancer and secondary cancer

We describe monozygotic twins discordant for childhood leukemia and secondary thyroid carcinoma. We used bisulfite pyrosequencing to compare the constitutive promoter methylation of BRCA1 and several other tumor suppressor genes in primary fibroblasts. The affected twin displayed an increased BRCA1 methylation (12%), compared with her sister (3%). Subsequent bisulfite plasmid sequencing demonstrated that 13% (6 of 47) BRCA1 alleles were fully methylated in the affected twin, whereas her sister displayed only single CpG errors without functional implications. This between-twin methylation difference was also found in irradiated fibroblasts and untreated saliva cells. The BRCA1 epimutation may have originated by an early somatic event in the affected twin: approximately 25% of her body cells derived from different embryonic cell lineages carry one epigenetically inactivated BRCA1 allele. This epimutation was associated with reduced basal protein levels and a higher induction of BRCA1 after DNA damage. In addition, we performed a genome-wide microarray analysis of both sisters and found several copy number variations, i.e., heterozygous deletion and reduced expression of the RSPO3 gene in the affected twin. This monozygotic twin pair represents an impressive example of epigenetic somatic mosaicism, suggesting a role for constitutive epimutations, maybe along with de novo genetic alterations in recurrent tumor development

The FOXP2-Driven Network in Developmental Disorders and Neurodegeneration

The transcription repressor FOXP2 is a crucial player in nervous system evolution and development of humans and songbirds. In order to provide an additional insight into its functional role we compared target gene expression levels between human neuroblastoma cells (SH-SY5Y) stably overexpressing FOXP2 cDNA of either humans or the common chimpanzee, Rhesus monkey, and marmoset, respectively. RNA-seq led to identification of 27 genes with differential regulation under the control of human FOXP2, which were previously reported to have FOXP2-driven and/or songbird song-related expression regulation. RT-qPCR and Western blotting indicated differential regulation of additional 13 new target genes in response to overexpression of human FOXP2. These genes may be directly regulated by FOXP2 considering numerous matches of established FOXP2-binding motifs as well as publicly available FOXP2-ChIP-seq reads within their putative promoters. Ontology analysis of the new and reproduced targets, along with their interactors in a network, revealed an enrichment of terms relating to cellular signaling and communication, metabolism and catabolism, cellular migration and differentiation, and expression regulation. Notably, terms including the words “neuron” or “axonogenesis” were also enriched. Complementary literature screening uncovered many connections to human developmental (autism spectrum disease, schizophrenia, Down syndrome, agenesis of corpus callosum, trismus-pseudocamptodactyly, ankyloglossia, facial dysmorphology) and neurodegenerative diseases and disorders (Alzheimer’s, Parkinson’s, and Huntington’s diseases, Lewy body dementia, amyotrophic lateral sclerosis). Links to deafness and dyslexia were detected, too. Such relations existed for single proteins (e.g., DCDC2, NURR1, PHOX2B, MYH8, and MYH13) and groups of proteins which conjointly function in mRNA processing, ribosomal recruitment, cell–cell adhesion (e.g., CDH4), cytoskeleton organization, neuro-inflammation, and processing of amyloid precursor protein. Conspicuously, many links pointed to an involvement of the FOXP2-driven network in JAK/STAT signaling and the regulation of the ezrin–radixin–moesin complex. Altogether, the applied phylogenetic perspective substantiated FOXP2’s importance for nervous system development, maintenance, and functioning. However, the study also disclosed new regulatory pathways that might prove to be useful for understanding the molecular background of the aforementioned developmental disorders and neurodegenerative diseases