Elucidating how and to what extent CpG islands (CGIs) are methylated in germ cells is essential to understand genomic imprinting and epigenetic reprogramming1-3. Here, we present the first integrated epigenomic analysis of mammalian oocytes, identifying over a thousand CGIs methylated in mature oocytes. We show that these CGIs depend on DNMT3A and DNMT3L4-5, but are not distinct at the sequence level, including in CpG periodicity6. They are preferentially located within active transcription units and are relatively depleted in H3K4me3, supporting a general transcription-dependent mechanism of methylation. Very few methylated CGIs are fully protected from post-fertilisation reprogramming but, surprisingly, the majority exhibits incomplete demethylation in E3.5 blastocysts. Our study shows that CGI methylation in gametes is not entirely related to genomic imprinting, but is a strong factor in determining methylation status in preimplantation embryos, suggesting a need to reassess mechanisms of post-fertilization demethylation.
Idiopathic infantile arterial calcification (IIAC; OMIM 208000) is characterized by calcification of the internal elastic lamina of muscular arteries and stenosis due to myointimal proliferation. We analyzed affected individuals from 11 unrelated kindreds and found that IIAC was associated with mutations that inactivated ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). This cell surface enzyme generates inorganic pyrophosphate (PP(i)), a solute that regulates cell differentiation and serves as an essential physiologic inhibitor of calcification.
Genomic imprinting requires the differential marking by DNA methylation of genes in male and female gametes. In the female germline, acquisition of methylation imprint marks depends upon the de novo methyltransferase Dnmt3a and its cofactor Dnmt3L, but the reasons why specific sequences are targets for Dnmt3a and Dnmt3L are still poorly understood. Here, we investigate the role of transcription in establishing maternal germline methylation marks. We show that at the Gnas locus, truncating transcripts from the furthest upstream Nesp promoter disrupts oocyte-derived methylation of the differentially methylated regions (DMRs). Transcription through DMRs in oocytes is not restricted to this locus but occurs across the prospective DMRs at many other maternally marked imprinted domains, suggesting a common requirement for transcription events. The transcripts implicated here in gametic methylation are protein-coding, in contrast to the noncoding antisense transcripts involved in the monoallelic silencing of imprinted genes in somatic tissues, although they often initiate from alternative promoters in oocytes. We propose that transcription is a third essential component of the de novo methylation system, which includes optimal CpG spacing and histone modifications, and may be required to create or maintain open chromatin domains to allow the methylation complex access to its preferred targets.[Keywords: Genomic imprinting; DNA methylation; oocytes; transcription] Supplemental material is available at http://www.genesdev.org.
, MD; the GACI Study GroupBackground-Generalized arterial calcification of infancy has been reported to be frequently lethal, and the efficiency of any therapy, including bisphosphonates, is unknown. A phosphate-poor diet markedly increases survival of NPP1 null mice, a model of generalized arterial calcification of infancy. Methods and Results-We performed a multicenter genetic study and retrospective observational analysis of 55 subjects affected by generalized arterial calcification of infancy to identify prognostic factors. Nineteen (34%) patients survived the critical period of infancy. In all 8 surviving patients tested, hypophosphatemia due to reduced renal tubular phosphate reabsorption developed during childhood. Eleven of 17 (65%) patients treated with bisphosphonates survived. Of 26 patients who survived their first day of life and were not treated with bisphosphonates only 8 (31%) patients survived beyond infancy. Forty different homozygous or compound heterozygous mutations, including 16 novel mutations in ENPP1, were found in 41 (75%) of the 55 patients. Twenty-nine (71%) of these 41 patients died in infancy (median, 30 days). Seven of the 14 (50%) patients without ENPP1 mutations died in infancy (median, 9 days). When present on both alleles, the mutation p.P305T was associated with death in infancy in all 5 cases; otherwise, no clear genotype-phenotype correlation was seen. Conclusion-ENPP1 coding region mutations are associated with generalized arterial calcification of infancy in Ϸ75% of subjects. Except for the p.P305T mutation, which was universally lethal when present on both alleles, the identified ENPP1 mutations per se have no discernable effect on survival. However, survival seems to be associated with hypophosphatemia linked with hyperphosphaturia and also with bisphosphonate treatment. ENPP1 encodes a type II transmembrane glycoprotein ectoenzyme that forms homodimers of identical disulfidebonded subunits. 7 NPP1 has an extracellular catalytic domain as well as somatomedin B-like and substrate-binding or substrate-specifying nuclease-like domains. 8 NPP1 regulates soft tissue calcification and bone and joint cartilage mineralization by generating PP i , which not only serves as an essential physiological inhibitor of hydroxyapatite crystal growth 9 but also is a suppressor of chondrogenesis. 10 In artery smooth muscle cells, deficiencies of NPP1 (or of extracellular PP i without NPP1 deficiency in ank/ank mice homozygous for functional inactivation of the PP i transporter ANK) promote chondrogenic transdifferentiation in vivo and also in vitro under circumstances where excess of an inorganic phosphate (P i ) source is provided. 10,11 Although the pathophysiologic role of NPP1-mediated PP i generation in GACI has come to light within recent years, the factors accounting for the variation of the GACI phenotype including the presence or absence of intracerebral artery calcification and periarticular calcification, early death in utero and long-term survival are not known. 12 PP i and ...
The hypomyelinating leukodystrophies X-linked Pelizaeus-Merzbacher disease (PMD) and Pelizaeus-Merzbacher-like disease (PMLD) are characterized by nystagmus, progressive spasticity, and ataxia. In a consanguineous family with PMLD, we performed a genomewide linkage scan using the GeneChip Mapping EA 10K Array (Affymetrix) and detected a single gene locus on chromosome 1q41-q42. This region harbors the GJA12 gene, which encodes gap junction protein alpha 12 (or connexin 46.6). Gap junction proteins assemble into intercellular channels through which signaling ions and small molecules are exchanged. GJA12 is highly expressed in oligodendrocytes, and, therefore, it serves as an excellent candidate for hypomyelination in PMLD. In three of six families with PMLD, we detected five different GJA12 mutations, including missense, nonsense, and frameshift mutations. We thereby confirm previous assumptions that PMLD is genetically heterogeneous. Although the murine Gja12 ortholog is not expressed in sciatic nerve, we did detect GJA12 transcripts in human sciatic and sural nerve tissue by reverse-transcriptase polymerase chain reaction. These results are in accordance with the electrophysiological finding of reduced motor and sensory nerve conduction velocities in patients with PMLD, which argues for a demyelinating neuropathy. In this study, we demonstrate that GJA12 plays a key role in central myelination and is involved in peripheral myelination in humans.
Linkage analyses have implicated chromosome 7p21.3 as a susceptibility region for inflammatory bowel disease (IBD). Recently, the mouse phenotype with diarrhea and goblet cell dysfunction caused by anterior gradient protein 2 dysfunction was reported (European patent WO2004056858). The genes encoding for the human homologues AGR2 and AGR3 are localized on chromosome 7p21.3. The gene structures were verified and mutation detection was performed in 47 IBD patients. A total of 30 single nucleotide polymorphisms (SNPs) were tested for association to ulcerative colitis (UC, N ¼ 317) and Crohn's disease (CD, N ¼ 631) in a German cohort and verified in a UK cohort of 384 CD and 311 UC patients. An association signal was identified in the 5 0 region of the AGR2 gene (most significant SNP hcv1702494, nominal P TDT ¼ 0.011, P case/control ¼ 0.0007, OR ¼ 1.34, combined cohort). The risk haplotype carried an odds ratio of 1.43 in the German population (P ¼ 0.002). AGR2 was downregulated in UC patients as compared to normal controls (Po0.001) and a trend toward lower expression was seen in carriers of the risk alleles. Luciferase assays of the AGR2 promoter showed regulation by the goblet cell-specific transcription factors FOXA1 and FOXA2. In summary, AGR2 represents an interesting new avenue into the etiopathophysiology of IBD and the maintenance of epithelial integrity.
Generalized arterial calcification of infancy (GACI), is characterized by calcification of the internal elastic lamina of large and medium-sized arteries and stenosis due to myointimal proliferation. Although survival to adulthood has been reported, most patients die within the first six months of life. Recently, we found mutations of ENPP1 coding for ecto-nucleotide pyrophosphatase/phosphodiesterase 1 to be associated with GACI in 8 of 11 families. In this study, we analyzed ENPP1 in affected individuals of another 12 unrelated families. We identified 11 novel homozygous or compound heterozygous mutations in 10 of the 12 new families. The mutations (1 nonsense, 7 missense, 1 single amino acid deletion, and 2 frame shift mutations) were scattered over the whole coding region with a slightly more condensed distribution within the catalytic and nuclease-like domain as compared to the first survey. In this study, three mutations were found repeatedly in apparently unrelated patients, 7 x c.913C>A (p.Pro305Thr) and c.2662C [corrected]>T (p.Arg888Trp) as well as c.2320C>T (p.Arg774Cys) each twice. However, haplotype analysis suggested a founder effect of British extraction for mutation c.913C>A (p.Pro305Thr). The fact that the two other mutations c.2662C [corrected]>T (p.Arg888Trp) and c.2320C>T (p.Arg774Cys) occurred twice within a single allele also suggests a single founder. This study confirms the role of ENPP1 mutations as the main cause of GACI and adds considerably to the mutational spectrum of ENPP1.
Genomic imprinting is the epigenetic marking of gene subsets resulting in monoallelic or predominant expression of one of the two parental alleles according to their parental origin. We describe the systematic experimental verification of a prioritized 16 candidate imprinted gene set predicted by sequence-based bioinformatic analyses. We used Quantification of Allele-Specific Expression by Pyrosequencing (QUASEP) and discovered maternal-specific imprinted expression of the Kcnk9 gene as well as strain-dependent preferential expression of the Rarres1 gene in E11.5 (C57BL/6 x Cast/Ei)F1 and informative (C57BL/6 x Cast/Ei) x C57BL/6 backcross mouse embryos. For the remaining 14 candidate imprinted genes, we observed biallelic expression. In adult mouse tissues, we found that Kcnk9 expression was restricted to the brain and also was maternal-specific. QUASEP analysis of informative human fetal brain samples further demonstrated maternal-specific imprinted expression of the human KCNK9 orthologue. The CpG islands associated with the mouse and human Kcnk9/KCNK9 genes were not differentially methylated, but strongly hypomethylated. Thus, we speculate that mouse Kcnk9 imprinting may be regulated by the maternal germline differentially methylated region in Peg13, an imprinted non-coding RNA gene in close proximity to Kcnk9 on distal mouse chromosome 15. Our data have major implications for the proposed role of Kcnk9 in neurodevelopment, apoptosis and tumourigenesis, as well as for the efficiency of sequence-based bioinformatic predictions of novel imprinted genes.
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