Kiyonori Miura

Age Related Changes in 5‐methylcytosine Content in Human Peripheral Leukocytes and Placentas: an HPLC‐based Study

The goal of the present study was to investigate inter‐individual and age‐dependent variation of global DNA methylation in human tissues. In this work, we examined 5‐methyldeoxycytidine (metC) content by HPLC in human peripheral blood leukocytes obtained from 76 healthy individuals of ages varying from 4 to 94 years (yr), and 39 human placentas from various gestational stages. The HPLC analysis revealed a significant variation of metC across individuals and is consistent with the previous findings of age‐dependent decrease of global methylation levels in human tissues. The age‐dependent decrease of metC was relatively small, but statistically highly significant (p= 0.0002) in the aged group (65.9 ± 8.9 [mean age ± SD] yr; n = 22) in comparison to the young adult group (19.3 ± 1.4 yr; n = 21). Males showed a subtle but statistically significant higher mean metC content than females. In contrast to the peripheral blood samples, DNA extracted from placentas exhibited gestational stage‐dependent increase of methylation levels that appeared to inversely correlate with the expression levels of human endogenous retroviruses. These data may be helpful in further studies of DNA methylation, such as inheritance of epigenetic patterns, environment‐induced changes, and involvement of epigenetic changes in disease.

Characterization of the human nasal embryonic LHRH factor gene, NELF , and a mutation screening among 65 patients with idiopathic hypogonadotropic hypogonadism (IHH)

AbstractAs the mouse nasal embryonic LHRH factor gene (Nelf) encodes a guidance molecule for the migration of the olfactory axon and gonadotropin-releasing hormone neurons, its human homolog, NELF, is a candidate gene for Kallmann syndrome, a disease of idiopathic hypogonadotropic hypogonadism (IHH) with anosmia or hyposmia. We report here characterization of NELF and results of mutation analysis in 65 IHH patients. Assembling EST clones, RACE, and sequencing showed that NELF mapped to 9q34.3 is composed of 16 exons and 15 introns with a 1,590-bp ORF encoding 530 amino acids. RT-PCR on a fetal brain cDNA library revealed five alternatively spliced variants. Among them, NELF-v1 has 93-94% identity at the amino acid level to mouse/rat Nelf, and four other transcripts are also highly conserved among the three species. A 3.0-kb transcript is expressed most highly in the adult and fetal brain, testis, and kidney, indicating that NELF plays a role in the function of these tissues. Mutation screening detected in a patient with IHH one novel heterozygous missense mutation (1438A>G, T480A) at the donor-splice site in exon 15 of NELF. As this mutation was not found in 100 normal control individuals, T480A may be associated with IHH. Four other novel SNPs (102C>T and 1029C>T within the coding region, and two IVS14+47C>T and IVS15+41G>A) were also identified in NELF.

Detection of cell free placental DNA in maternal plasma: direct evidence from three cases of confined placental mosaicism

Fetal cells are consistently found in the maternal circulation, and polymerase chain reaction based studies have led to the identification of cell free fetal DNA (fetal DNA) in maternal blood. Approximately 1.2 nucleated fetal cells/ml of whole blood from women carrying a male fetus were detectable,1 and relative enrichment of fetal DNA was detected in the maternal plasma and serum.2 The amount of fetal DNA in the maternal blood increases with progression of pregnancy, and 3.4–6.2% of the total maternal plasma DNA during pregnancy was of fetal origin.3 Therefore, cell free fetal DNA in pregnant women’s plasma is useful for non-invasive prenatal diagnosis, especially for detection of fetal sex,3,4 RhD blood type,5–7 and gene mutations of paternal origin.8–10 Previous studies indicated that pregnant women with pre-eclampsia,11 placenta previa12 and fetal chromosome abnormalities13 tend to have elevated levels of fetal DNA in their plasma. Since functional or structural abnormalities of the placenta and destruction of the trophoblast may be associated with these diseases,14 it is suggested that cell free fetal DNA is of placental origin. This implies that quantitative analysis of fetal DNA may be valuable to screen for placental dysfunction. Ng et al15 recently reported that placental mRNA is present in the maternal circulation, and suggested that the same might occur for placental DNA,16 However, no direct evidence has been given for placenta derived cell free fetal DNA in the maternal blood, although its clinical use is growing.17nnConfined placental mosaicism, which is defined by the presence of abnormal karyotypes only in the placenta while the fetus itself is usually diploid,18 may occur through a loss of the extra chromosome in a trisomic zygote during an early mitotic cell division in only the …

Do monochorionic dizygotic twins increase after pregnancy by assisted reproductive technology

AbstractAlthough monochorionic (MC) dizygotic twins (DZT) are extremely rare in natural pregnancy, six pairs of such twins have successively been reported in a recent short period. All six cases of MC DZT were the products of pregnancy by assisted reproductive technology (ART). In this overview, we summarize these six cases and discuss possible mechanisms of this twinning and clinical implications of confined blood cell chimerism (CBC). The placental MC membrane was diagnosed ultrasonographically in all cases and pathologically in four. The presence of CBC was confirmed in four cases by haplotyping at polymorphic marker loci in peripheral blood leukocytes, karyotyping of lymphocytes and skin fibroblasts, and/or ABO blood group typing. As CBC is attributable to placental vessel anastomosis between DZT, it may become a risk factor for twin-twin transfusion syndrome (TTTS), mortality, and for other complications in twins. MC DZT may produce psychological trauma, especially in a girl/woman when she grows up and is known to be chimeric for a male karyotype and vice versa, although genital organs are generally normal—unlike freemartin in cattle. In addition, CBC in twins may mislead physicians when genotyping for a disease-susceptibility test is performed in medical practice in the near future. Blood group chimera may also cause confusion if a blood transfusion is necessary. Therefore, sufficient informed consent prior to ART and genetic counseling before/after birth are absolutely necessary for improved quality of life. It is most likely that all six cases are the consequence of fusion between two outer cell masses from two zygotes. The ART used in the six MC DZT included in vitro fertilization-embryonic transfer (IVF-ET) into the uterus, FSH-induced superovulation followed by intrauterine insemination, and/or intracytoplasmic sperm injection (ICSI). The use of an ovulation-inducing agent and implantation of several fertilized eggs at close sites are probably the events common among these cases. Assisted hatching, simultaneous ET, the use of eggs that have developed to the blastcyst stage, and cell culture procedures that lead to changes of the nature of cell surface, all may increase the chance of a cell fusion. This “chance hypothesis” can simply explain why MC DZT are very rare in natural pregnancy. Large-scale research on the prevalence of ART-associated MC DZT and long-term follow-up of the twins are essential.

Microarray comparative genomic hybridization (CGH)-based prenatal diagnosis for chromosome abnormalities using cell-free fetal DNA in amniotic fluid

AbstractCell-free fetal DNA (cffDNA) in the supernatant of amniotic fluid, which is usually discarded, can be used as a sample for prenatal diagnosis. For rapid prenatal diagnosis of frequent chromosome abnormalities, for example trisomies 13, 18, and 21, and monosomy X, using cffDNA, we have developed a targeted microarray-based comparative genomic hybridization (CGH) panel on which BAC clones from chromosomes 13, 18, 21, X, and Y were spotted. Microarray-CGH analysis was performed for a total of 13 fetuses with congenital anomalies using cffDNA from their uncultured amniotic fluid. Microarray CGH with cffDNA led to successful molecular karyotyping for 12 of 13 fetuses within 5 days. Karyotypes of the 12 fetuses (one case of trisomy 13, two of trisomy 18, two of trisomy 21, one of monosomy X, and six of normal karyotype) were later confirmed by conventional chromosome analysis using cultured amniocytes. The one fetus whose molecular-karyotype was indicated as normal by microarray CGH actually had a balanced translocation, 45,XY,der(14;21)(q10;q10). The results indicated that microarray CGH with cffDNA is a useful rapid prenatal diagnostic method at late gestation for chromosome abnormalities with copy-number changes, especially when combined with conventional karyotyping of cultured amniocytes.

Complete hydatidiform mole and normal live birth following intracytoplasmic sperm injection

AbstractA twin pregnancy with complete hydatidiform mole (HM) and preterm birth of a normal female infant after intracytoplasmic sperm injection (ICSI) conception was experienced. ICSI due to severe oligozoospermia was performed on three ova, and three embryos with confirmed two proneclei (2PN) were subsequently transferred to the uterus. At 7 weeks of gestation, molar pregnancy as well as a viable fetus was recognized. At 33 weeks, the pregnancy was terminated due to preterm labor. Dichorionic pregnancy consisting of a normal fetus and placenta in one chorionic membrane and complete HM in the other was recognized. Cytomolecular analysis indicated that the complete HM genome was derived from duplication of a single sperm, and a normal neonate was from biparental genomes. It should be noted that ICSI can avoid incomplete HM (mostly triploid) due to multi-sperm fertilization but might not be able to avoid complete HM (paternal diploid) although such a risk is very low. This is the second report of this condition and is accompanied by the first well-described molecular analysis.

Congenital diaphragmatic hernia: an evaluation of the prognostic value of the lung-to-head ratio

PurposeA retrospective analysis of the prognostic significance of the lung-to-head ratio (LHR) on the outcome of fetuses with left-sided congenital diaphragmatic hernia (CDH).MethodsA total of 12 fetuses with isolated left CDH without any chromosomal abnormalities were included in this study. Twelve LHR measurements could retrospectively be calculated from the last available ultrasonographic recordings before birth. The relationship between the LHR and fetal outcome and gestational age was investigated. The cutoff levels as previously published were applied to determine their predictive value in this population. The association between other prenatal predictive variables and the fetal outcome was also determined. Survival was defined as being discharged from the hospital.ResultsThe overall survival rate was 75%, and a statistically significant difference was observed between the mean LHR of the survivors and the mean LHR of the nonsurvivors (1.81 vs. 0.43), whereas the mean gestational age of these two groups did not differ. The LHR was not gestational age dependent with regard to its ability to predict the fetal outcome. The cutoff levels LHR <1, 1-1.4, and >1.4 showed good applicability for predicting fetal outcome within the present study population, with 100% survival if LHR >1.4 and 75% mortality if LHR <1. The intrathoracic position of the stomach, mediastinal shift, and polyhydramnios as individual variables and early diagnosis (<25 weeks gestation) all turned out to be poor sonographic predictors of fetal outcome.ConclusionThe LHR proved to be a good predictor of fetal outcome, independent of gestational age at the time of measurement. To further substantiate our observations, a prospective multicenter study is warranted.

A Monozygotic Conjoined Twin Pregnancy Discordant for Laterality of Cleft Lip

Conjoined twins discordant for phenotype of cleft lip are reported. One had right-sided cleft lip, while the co-twin had left-sided cleft lip. Both twins had a normal 46,XY karyotype. Genotyping at 50 polymorphic loci revealed that the twins were identical (monozygotic). Our analysis suggests that the laterality of cleft lip is more affected by the process of twinning than by genetic factors.