DMPK hypermethylation in sperm cells of myotonic dystrophy type 1 patients

International audienceMyotonic dystrophy type 1 (DM1) is an autosomal dominant muscular dystrophy that results from a CTG expansion (50–4000 copies) in the 3′ UTR of the DMPK gene. The disease is classified into four or five somewhat overlapping forms, which incompletely correlate with expansion size in somatic cells of patients. With rare exception, it is affected mothers who transmit the congenital (CDM1) and most severe form of the disease. Why CDM1 is hardly ever transmitted by fathers remains unknown. One model to explain the almost exclusive transmission of CDM1 by affected mothers suggests a selection against hypermethylated large expansions in the germline of male patients. By assessing DNA methylation upstream to the CTG expansion in motile sperm cells of four DM1 patients, together with availability of human embryonic stem cell (hESCs) lines with paternally inherited hypermethylated expansions, we exclude the possibility that DMPK hypermethylation leads to selection against viable sperm cells (as indicated by motility) in DM1 patients

FMR1 Epigenetic Silencing Commonly Occurs in Undifferentiated Fragile X-Affected Embryonic Stem Cells

Fragile X syndrome (FXS) is the most common heritable form of cognitive impairment. It results from epigenetic silencing of the X-linked FMR1 gene by a CGG expansion in its 5′-untranslated region. Taking advantage of a large set of FXS-affected human embryonic stem cell (HESC) lines and isogenic subclones derived from them, we show that FMR1 hypermethylation commonly occurs in the undifferentiated state (six of nine lines, ranging from 24% to 65%). In addition, we demonstrate that hypermethylation is tightly linked with FMR1 transcriptional inactivation in undifferentiated cells, coincides with loss of H3K4me2 and gain of H3K9me3, and is unrelated to CTCF binding. Taken together, these results demonstrate that FMR1 epigenetic gene silencing takes place in FXS HESCs and clearly highlights the importance of examining multiple cell lines when investigating FXS and most likely other epigenetically regulated diseases

Uncovering the Role of Hypermethylation by CTG Expansion in Myotonic Dystrophy Type 1 Using Mutant Human Embryonic Stem Cells

CTG repeat expansion in DMPK, the cause of myotonic dystrophy type 1 (DM1), frequently results in hypermethylation and reduced SIX5 expression. The contribution of hypermethylation to disease pathogenesis and the precise mechanism by which SIX5 expression is reduced are unknown. Using 14 different DM1-affected human embryonic stem cell (hESC) lines, we characterized a differentially methylated region (DMR) near the CTGs. This DMR undergoes hypermethylation as a function of expansion size in a way that is specific to undifferentiated cells and is associated with reduced SIX5 expression. Using functional assays, we provide evidence for regulatory activity of the DMR, which is lost by hypermethylation and may contribute to DM1 pathogenesis by causing SIX5 haplo-insufficiency. This study highlights the power of hESCs in disease modeling and describes a DMR that functions both as an exon coding sequence and as a regulatory element whose activity is epigenetically hampered by a heritable mutation

Establishment of Homozygote Mutant Human Embryonic Stem Cells by Parthenogenesis

<div><p>We report on the derivation of a diploid 46(XX) human embryonic stem cell (HESC) line that is homozygous for the common deletion associated with Spinal muscular atrophy type 1 (SMA) from a pathenogenetic embryo. By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19), monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome. Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation. As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve.</p></div

Methylation levels at <i>H19</i>, <i>SNRPN</i> and <i>MEST</i> imprinted loci.

<p>Bisulfite single colony sequencing was performed on 3 different imprinted regions (<i>H19</i>, SNRPN and <i>MEST</i>) to determine methylation levels in normal (WT) and SZ-SMA5 HESC lines. Each line represents a single DNA molecule. Full circles correspond to methylated CpGs while empty circles represent unmethylated CpGs.</p

Whole genome view of Cytoscan SNP Array data.

<p>Genome wide SNP array results obtained from (A) reference DNA from a male with a normal karyotype (46; XY); and (B) SZ-SMA5 HESC line DNA. The X axis represents chromosomes 1–22, X and Y. (I) The Y axis represents the copy number, determined by the log2 ratio (grey dots) on the left side of the graph, and it's smoothed ratio (red line) on the right. The expected copy number is 2 for autosomal chromosomes (log2 of 0 and smooth signal of 2). The log2 ratio and the smooth signal are determined from both the nonpolymorphic copy number probes and the polymorphic SNP probes. (II) The Y-axis corresponds to homozygote calls (AA or BB) and heterozygote calls (AB). Allele peaks of 1, 0, and -1 indicate a copy number of two, while allele peaks of 0.5 and -0.5 indicate a copy number of one. Allele peaks are calculated from SNP probes. The distinction between XY (reference DNA) and XX (SZ-SMA5) cells is clearly illustrated by the difference in X chromosome copy number. In addition, the overall 0.49% inherent heterozygote call error rate in SZ-SMA5 is below even the expected array genotyping error of ~1% (as determined by dividing the number of heterozygous calls by the total number of SNP probes on the array). Therefore, these data indicate that SZ-SMA5 features a completely homozygous diploid genome. (C) Fraction of SNP heterozygote calls in WT male reference and SZ-SMA5 DNA. Chromosomes are indicated in the X axis and the Y axis indicates the fraction of heterozygous SNP calls per total SNP calls on each chromosome.</p

Characterization of SZ-SMA5.

<p>(A) Expression of undifferentiated cell specific markers by immunostaining for <i>OCT4</i> (red nuclear staining, merged onto Hoechst (blue)), for the cell surface marker <i>Tra 1–60</i> (red, merged onto Hoechst (blue)); and (B) for alkaline phosphatase activity (AP). (C) Expression of undifferentiated cell specific markers: <i>OCT4</i>, <i>REX1</i>, <i>NANOG</i> and <i>SOX2</i> in SZ-SMA5 and a wild-type (WT) HESC control by RT-PCR. <i>GAPDH</i> expression served as a loading control. (D) Teratoma sections stained by H&E from SZ-SMA5 demonstrating multi-cellular structures derived from the three different embryonic germ layers. (E) A representative normal 46(XX) karyotype in SZ-SMA5 as identified by Giemsa staining of 20 different spreads of metaphase chromosomes.</p