Vaimse arengu mahajäämuse geneetilised põhjused: X-liiteline vaimse arengu mahajäämus

Üheks kõige sagedasemaks raske puude põhjuseks lastel ja noortel on vaimse arengu mahajäämus (VAM). Vaatamata sellele, et välja on töötatud palju erinevaid uurimis meetodeid, jääb enamik neist patsientidest praegugi veel täpse diagnoosita. Viimastel aastatel on uuringute tähelepanu keskpunkti tõusnud X-kromosoom, kuna on leitud, et võrreldes autosoomidega esineb X-kromosoomis märgatavalt rohkem geene, mis muteerununa põhjustavad VAMi. Artiklis on kirjeldatud teadaolevalt sagedasemaid X-liitelise vaimse arengu mahajäämuse sündroome ning toodud välja autismi seni leitud seosed X-liitelise VAMiga. Eesti Arst 2007; 86 (4): 239–24

Detection of tmRNA molecules on microarrays at low temperatures using helper oligonucleotides

<p>Abstract</p> <p>Background</p> <p>The hybridization of synthetic <it>Streptococcus pneumoniae </it>tmRNA on a detection microarray is slow at 34°C resulting in low signal intensities.</p> <p>Results</p> <p>We demonstrate that adding specific DNA helper oligonucleotides (chaperones) to the hybridization buffer increases the signal strength at a given temperature and thus makes the specific detection of <it>Streptococcus pneumoniae </it>tmRNA more sensitive. No loss of specificity was observed at low temperatures compared to hybridization at 46°C. The effect of the chaperones can be explained by disruption of the strong secondary and tertiary structure of the target RNA by the selective hybridization of helper molecules. The amplification of the hybridization signal strength by chaperones is not necessarily local; we observed increased signal intensities in both local and distant regions of the target molecule.</p> <p>Conclusions</p> <p>The sensitivity of the detection of tmRNA at low temperature can be increased by chaperone oligonucleotides. Due to the complexity of RNA secondary and tertiary structures the effect of any individual chaperone is currently not predictable.</p

Utilising FGF2, IGF2 and FSH in serum-free protocol for long-term in vitro cultivation of primary human granulosa cells

Human granulosa cells acquired as leftover from IVF treatment can be used to study infertility problems and area valuable tool in the research of follicle maturation and ovulation. There is a need for more defined and long-term culture protocols for studying the response of granulosa cells upon treatment with selected hormones/chemicals. In the current study, we tested the effect of adding FGF2, IGF2 and FSH into defined basal medium inorder tofind culture conditions that would support proliferation of cumulus and mural granulosa cells alongwith the expression of common granulosa cell type markers such asFSHR,AMHR2,LHRandCYP19A1. We foundthat FGF2, IGF2 together with FSH helped to retain granulosa cell marker expression while supporting cellsurvival at least for two weeks of culture. The defined serum-free culture conditions for long-term culturing willbe valuable in providing new standards in the research of human granulosa cells

A dual colour FISH method for routine validation of sexed Bos taurus semen

Background: Usage of sexed semen that allows to choose the gender of the calves, is commonly practiced in livestock industry as a profitable breeding alternative, especially in dairy farming. The flow cytometric cell sorting is the only commercially available method for bovine sperm sexing. For validation of the sexing procedure several methods have been developed including sperm fluorescence in situ hybridisation techniques. Latter usually include the use of pre-labelled nucleotides for probe synthesis which is relatively expensive approach compared to combined application of aminoallyl-dUTP and chemical binding of fluorescent dyes. Here a sex determining dual colour bovine sperm fluorescence in situ hybridisation method is presented which is considered more cost-effective technique than the previously reported approaches. Results: The reliability of sex chromosome identifying probes, designed in silico, was proven on bovine metaphase plate chromosomes and through comparison with a commercially available standard method. In the dual colour FISH experiments of unsexed and sexed bovine sperm samples the hybridisation efficiency was at least 98%, whereas the determined sex ratios were not statistically different from the expected. Very few cells carried both of the sex chromosome-specific signals (less than 0.2%). Conclusions: A protocol for a dual colour bovine sperm FISH method is provided which is cost-effective, simple and fast for sex determination of spermatozoa in bull semen samples.Peer reviewe

Target prediction and validation of microRNAs expressed from FSHR and aromatase genes in human ovarian granulosa cells

MicroRNAs (miRNAs) are known post-transcriptional regulators of various biological processes including ovarian follicle development. We have previously identified miRNAs from human preovulatory ovarian granulosa cells that are expressed from the intronic regions of two key genes in normal follicular development: FSH receptor (FSHR) and CYP19A1, the latter encoding the aromatase enzyme. The present study aims to identify the target genes regulated by these miRNAs: hsa-miR-548ba and hsa-miR-7973, respectively. The miRNAs of interest were transfected into KGN cell line and the gene expression changes were analyzed by Affymetrix microarray. Potential miRNA-regulated genes were further filtered by bioinformatic target prediction algorithms and validated for direct miRNA:mRNA binding by luciferase reporter assay. LIFR, PTEN, NEO1 and SP110 were confirmed as targets for hsa-miR-548ba. Hsa-miR-7973 target genes ADAM19, PXDN and FMNL3 also passed all verification steps. Additionally, the expression pattern of the miRNAs was studied in human primary cumulus granulosa cell culture in relation to the expression of their host genes and FSH stimulation. Based on our findings we propose the involvement of hsa-miR-548ba in the regulation of follicle growth and activation via LIFR and PTEN. Hsa-miR-7973 may be implicated in the modulation of extracellular matrix and cell-cell interactions by regulating the expression of its identified targets.Peer reviewe

Genome-wide histone modification profiling of inner cell mass and trophectoderm of bovine blastocysts by RAT-ChIP

Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) has revolutionized our understanding of chromatin-related biological processes. The method, however, requires thousands of cells and has therefore limited applications in situations where cell numbers are limited. Here we describe a novel method called Restriction Assisted Tagmentation Chromatin Immunoprecipitation (RAT-ChIP) that enables global histone modification profiling from as few as 100 cells. The method is simple, cost-effective and takes a single day to complete. We demonstrate the sensitivity of the method by deriving the first genome-wide maps of histone H3K4me3 and H3K27me3 modifications of inner cell mass and trophectoderm of bovine blastocyst stage embryos.Peer reviewe

A dual colour FISH method for routine validation of sexed Bos taurus semen

Abstract Background Usage of sexed semen that allows to choose the gender of the calves, is commonly practiced in livestock industry as a profitable breeding alternative, especially in dairy farming. The flow cytometric cell sorting is the only commercially available method for bovine sperm sexing. For validation of the sexing procedure several methods have been developed including sperm fluorescence in situ hybridisation techniques. Latter usually include the use of pre-labelled nucleotides for probe synthesis which is relatively expensive approach compared to combined application of aminoallyl-dUTP and chemical binding of fluorescent dyes. Here a sex determining dual colour bovine sperm fluorescence in situ hybridisation method is presented which is considered more cost-effective technique than the previously reported approaches. Results The reliability of sex chromosome identifying probes, designed in silico, was proven on bovine metaphase plate chromosomes and through comparison with a commercially available standard method. In the dual colour FISH experiments of unsexed and sexed bovine sperm samples the hybridisation efficiency was at least 98%, whereas the determined sex ratios were not statistically different from the expected. Very few cells carried both of the sex chromosome-specific signals (less than 0.2%). Conclusions A protocol for a dual colour bovine sperm FISH method is provided which is cost-effective, simple and fast for sex determination of spermatozoa in bull semen samples

A speculative outlook on embryonic aneuploidy : Can molecular pathways be involved?

The journey of embryonic development starts at oocyte fertilization, which triggers a complex cascade of events and cellular pathways that guide early embryogenesis. Recent technological advances have greatly expanded our knowledge of cleavage-stage embryo development, which is characterized by an increased rate of whole-chromosome losses and gains, mixoploidy, and atypical cleavage morphokinetics. Embryonic aneuploidy significantly contributes to implantation failure, spontaneous miscarriage, stillbirth or congenital birth defects in both natural and assisted human reproduction. Essentially, early embryo development is strongly determined by maternal factors. Owing to considerable limitations associated with human oocyte and embryo research, the use of animal models is inevitable. However, cellular and molecular mechanisms driving the error-prone early stages of development are still poorly described. In this review, we describe known events that lead to aneuploidy in mammalian oocytes and preimplantation embryos. As the processes of oocyte and embryo development are rigorously regulated by multiple signal-transduction pathways, we explore the putative role of signaling pathways in genomic integrity maintenance. Based on the existing evidence from human and animal data, we investigate whether critical early developmental pathways, like Wnt, Hippo and MAPK, together with distinct DNA damage response and DNA repair pathways can be associated with embryo genomic instability, a question that has, so far, remained largely unexplored.Peer reviewe

Polymorphisms in wolframin (WFS1) gene are possibly related to increased risk for mood disorders

Wolfram syndrome gene (WFS1) has been suggested to have a role in the susceptibility for mood disorders. A 26-fold increased risk for psychiatric disorders in WFS1 mutation carriers has been sug- gested. In this study we tested the hypothesis that the WFS1 gene is related to the risk for mood disorders. We analysed 28 single-nucleotide polymorphisms (SNPs) of the WFS1 gene in 224 unrelated patients with major depressive disorder and bipolar disorder and in 160 healthy control subjects. Patients were further stratified according to their comorbidity with anxiety disorders. We applied arrayed primer extension (APEX)-based genotyping technology followed by association and haplotype analysis. Five SNPs in the WFS1 gene were associated with major depressive disorder, and three SNPs with bipolar disorder. Haplotype analysis revealed a common GTA haplotype, formed by SNPs 684C/ G, 1185C/T and 1832G/A, conferring risk for affective disorders. Specifically, for major depression the GTA haplotype has an OR of 1.59 (p=0.01) and for bipolar disorder an OR of 1.89 (p=0.03). These results support the hypothesis that the WFS1 gene is involved in the genetic predisposition for mood disorders

Karyotype of the blastocoel fluid demonstrates low concordance with both trophectoderm and inner cell mass

Objective To compare the genomic profiles of blastocoel fluid (BF), inner cell mass (ICM), and trophectoderm (TE) cells derived from the same blastocyst. Design Prospective study. Setting Academic and in vitro fertilization units. Patient(s) Sixteen donated cryopreserved embryos at blastocyst stage. Intervention(s) BF, TE, and ICM cells were retrieved from each blastocyst for chromosome analysis by means of next-generation sequencing (NGS). Main Outcome Measure(s) Aneuploidy screening and assessment of mosaicism in BF, TE and ICM samples with subsequent comparison of genomic profiles between the three blastocyst compartments. Result(s) Out of 16 blastocysts, 10 BF samples and 14 TE and ICM samples provided reliable NGS data for comprehensive chromosome analysis. Only 40.0% of BF-DNA karyotypes were fully concordant with TE or ICM, compared with 85.7% concordance between TE and ICM. In addition, BF-DNA was burdened with mosaic aneuploidies and the total number of affected chromosomes in BF was significantly higher compared with the TE and ICM. Conclusion(s) BF-DNA can be successfully amplified and subjected to NGS, but owing to increased discordance with ICM and TE, BF does not adequately represent the status of the rest of the embryo. To overcome biologic and technical challenges associated with BF sampling and processing, blastocentesis would require improvement in both laboratory protocols and aneuploidy calling algorithms. Therefore, TE biopsy remains the most effective way to predict embryonic karyotype, and the use of BF as a single source of DNA for preimplantation genetic screening is not yet advised