Hemorrhage-Adjusted Iron Requirements, Hematinics and Hepcidin Define Hereditary Hemorrhagic Telangiectasia as a Model of Hemorrhagic Iron Deficiency

BACKGROUND: Iron deficiency anemia remains a major global health problem. Higher iron demands provide the potential for a targeted preventative approach before anemia develops. The primary study objective was to develop and validate a metric that stratifies recommended dietary iron intake to compensate for patient-specific non-menstrual hemorrhagic losses. The secondary objective was to examine whether iron deficiency can be attributed to under-replacement of epistaxis (nosebleed) hemorrhagic iron losses in hereditary hemorrhagic telangiectasia (HHT). METHODOLOGY/PRINCIPAL FINDINGS: The hemorrhage adjusted iron requirement (HAIR) sums the recommended dietary allowance, and iron required to replace additional quantified hemorrhagic losses, based on the pre-menopausal increment to compensate for menstrual losses (formula provided). In a study population of 50 HHT patients completing concurrent dietary and nosebleed questionnaires, 43/50 (86%) met their recommended dietary allowance, but only 10/50 (20%) met their HAIR. Higher HAIR was a powerful predictor of lower hemoglobin (p = 0.009), lower mean corpuscular hemoglobin content (p<0.001), lower log-transformed serum iron (p = 0.009), and higher log-transformed red cell distribution width (p<0.001). There was no evidence of generalised abnormalities in iron handling Ferritin and ferritin(2) explained 60% of the hepcidin variance (p<0.001), and the mean hepcidinferritin ratio was similar to reported controls. Iron supplement use increased the proportion of individuals meeting their HAIR, and blunted associations between HAIR and hematinic indices. Once adjusted for supplement use however, reciprocal relationships between HAIR and hemoglobin/serum iron persisted. Of 568 individuals using iron tablets, most reported problems completing the course. For patients with hereditary hemorrhagic telangiectasia, persistent anemia was reported three-times more frequently if iron tablets caused diarrhea or needed to be stopped. CONCLUSIONS/SIGNIFICANCE: HAIR values, providing an indication of individuals' iron requirements, may be a useful tool in prevention, assessment and management of iron deficiency. Iron deficiency in HHT can be explained by under-replacement of nosebleed hemorrhagic iron losses

Mitotic Arrest in Teratoma Susceptible Fetal Male Germ Cells

Formation of germ cell derived teratomas occurs in mice of the 129/SvJ strain, but not in C57Bl/6 inbred or CD1 outbred mice. Despite this, there have been few comparative studies aimed at determining the similarities and differences between teratoma susceptible and non-susceptible mouse strains. This study examines the entry of fetal germ cells into the male pathway and mitotic arrest in 129T2/SvJ mice. We find that although the entry of fetal germ cells into mitotic arrest is similar between 129T2/SvJ, C57Bl/6 and CD1 mice, there were significant differences in the size and germ cell content of the testis cords in these strains. In 129T2/SvJ mice germ cell mitotic arrest involves upregulation of p27KIP1, p15INK4B, activation of RB, the expression of male germ cell differentiation markers NANOS2, DNMT3L and MILI and repression of the pluripotency network. The germ-line markers DPPA2 and DPPA4 show reciprocal repression and upregulation, respectively, while FGFR3 is substantially enriched in the nucleus of differentiating male germ cells. Further understanding of fetal male germ cell differentiation promises to provide insight into disorders of the testis and germ cell lineage, such as testis tumour formation and infertility

Defective germline reprogramming rewires the spermatogonial transcriptome.

Defective germline reprogramming in Piwil4 (Miwi2)- and Dnmt3l-deficient mice results in the failure to reestablish transposon silencing, meiotic arrest and progressive loss of spermatogonia. Here we sought to understand the molecular basis for this spermatogonial dysfunction. Through a combination of imaging, conditional genetics and transcriptome analysis, we demonstrate that germ cell elimination in the respective mutants arises as a result of defective de novo genome methylation during reprogramming rather than because of a function for the respective factors within spermatogonia. In both Miwi2-/- and Dnmt3l-/- spermatogonia, the intracisternal-A particle (IAP) family of endogenous retroviruses is derepressed, but, in contrast to meiotic cells, DNA damage is not observed. Instead, we find that unmethylated IAP promoters rewire the spermatogonial transcriptome by driving expression of neighboring genes. Finally, spermatogonial numbers, proliferation and differentiation are altered in Miwi2-/- and Dnmt3l-/- mice. In summary, defective reprogramming deregulates the spermatogonial transcriptome and may underlie spermatogonial dysfunction

Critical Period of Nonpromoter DNA Methylation Acquisition during Prenatal Male Germ Cell Development

The prenatal period of germ cell development is a key time of epigenetic programming in the male, a window of development that has been shown to be influenced by maternal factors such as dietary methyl donor supply. DNA methylation occurring outside of promoter regions differs significantly between sperm and somatic tissues and has recently been linked with the regulation of gene expression during development as well as successful germline development. We examined DNA methylation at nonpromoter, intergenic sequences in purified prenatal and postnatal germ cells isolated from wildtype mice and mice deficient in the DNA methyltransferase cofactor DNMT3L. Erasure of the parental DNA methylation pattern occurred by 13.5 days post coitum (dpc) with the exception of approximately 8% of loci demonstrating incomplete erasure. For most loci, DNA methylation acquisition occurred between embryonic day 13.5 to 16.5 indicating that the key phase of epigenetic pattern establishment for intergenic sequences in male germ cells occurs prior to birth. In DNMT3L-deficient germ cells at 16.5 dpc, average DNA methylation levels were low, about 30% of wildtype levels; however, by postnatal day 6, about half of the DNMT3L deficiency-specific hypomethylated loci had acquired normal methylation levels. Those loci normally methylated earliest in the prenatal period were the least affected in the DNMT3L-deficient mice, suggesting that some loci may be more susceptible than others to perturbations occurring prenatally. These results indicate that the critical period of DNA methylation programming of nonpromoter, intergenic sequences occurs in male germline progenitor cells in the prenatal period, a time when external perturbations of epigenetic patterns could result in diminished fertility

DNMT3L Is a Regulator of X Chromosome Compaction and Post-Meiotic Gene Transcription

Previous studies on the epigenetic regulator DNA methyltransferase 3-Like (DNMT3L), have demonstrated it is an essential regulator of paternal imprinting and early male meiosis. Dnmt3L is also a paternal effect gene, i.e., wild type offspring of heterozygous mutant sires display abnormal phenotypes suggesting the inheritance of aberrant epigenetic marks on the paternal chromosomes. In order to reveal the mechanisms underlying these paternal effects, we have assessed X chromosome meiotic compaction, XY chromosome aneuploidy rates and global transcription in meiotic and haploid germ cells from male mice heterozygous for Dnmt3L. XY bodies from Dnmt3L heterozygous males were significantly longer than those from wild types, and were associated with a three-fold increase in XY bearing sperm. Loss of a Dnmt3L allele resulted in deregulated expression of a large number of both X-linked and autosomal genes within meiotic cells, but more prominently in haploid germ cells. Data demonstrate that similar to embryonic stem cells, DNMT3L is involved in an auto-regulatory loop in germ cells wherein the loss of a Dnmt3L allele resulted in increased transcription from the remaining wild type allele. In contrast, however, within round spermatids, this auto-regulatory loop incorporated the alternative non-coding alternative transcripts. Consistent with the mRNA data, we have localized DNMT3L within spermatids and sperm and shown that the loss of a Dnmt3L allele results in a decreased DNMT3L content within sperm. These data demonstrate previously unrecognised roles for DNMT3L in late meiosis and in the transcriptional regulation of meiotic and post-meiotic germ cells. These data provide a potential mechanism for some cases of human Klinefelter's and Turner's syndromes

Naive pluripotency is associated with global DNA hypomethylation

hypomethylatio