Kentaro Yamasaki

Identification of Pregnancy-Associated MicroRNAs in Maternal Plasma

BACKGROUND Several placental microRNAs (miRNAs) have been identified as pregnancy-associated molecules with the potential for use in estimating the condition of the placenta. Our understanding of these novel molecules is still limited, however. The aim of this study was to isolate and characterize pregnancy-associated miRNAs in maternal plasma. METHODS By microarray-based screening of 723 human miRNAs, we selected miRNAs that exhibited signal intensities >100 times higher in placental tissues than in the corresponding whole blood samples. Subsequent quantitative real-time reverse-transcription PCR revealed miRNAs produced predominantly in the placenta that showed significantly decreased concentrations in maternal plasma after delivery. These miRNAs were identified as pregnancy-associated miRNAs. RESULTS We selected 82 miRNAs produced predominantly in the placenta and identified 24 as pregnancy-associated miRNAs. The genes encoding these miRNAs included 16 that are clustered on 19q13.42 and 5 clustered on 14q32. As the pregnancy progressed into the third trimester, the plasma concentrations of cell-free chromosome 19-derived miRNAs (has-miR-515-3p, has-miR-517a, has-miR-517c, has-miR-518b, and has-miR-526b) increased significantly (P = 0.0284, 0.0069, 0.0125, 0.0284, and 0.0093, respectively, Wilcoxon signed rank test), whereas that of cell-free has-miR-323-3p on chromosome 14q32.31 showed no change (P = 0.2026). CONCLUSIONS In addition to the known pregnancy-associated miRNAs, we identified new pregnancy-associated miRNAs with our microarray-based approach. Most of the genes encoding these miRNAs were clustered on 19q13.42 or 14q32, which are critical regions for placental and embryonic development. These new pregnancy-associated miRNAs may be useful molecular markers for monitoring pregnancy-associated diseases.

Characterization of placenta-specific microRNAs in fetal growth restriction pregnancy.

The aim of this study was to characterize placenta‐specific microRNAs in fetal growth restriction (FGR) pregnancy.

Increased level of cell-free placental mRNA in a subgroup of placenta previa that needs hysterectomy.

The purpose of this study was to investigate whether cell‐free placental mRNA levels have the potential to predict a placenta previa resulting in hysterectomy.

Clinical application of fetal sex determination using cell-free fetal DNA in pregnant carriers of X-linked genetic disorders

As the first step in prenatal diagnosis of X-linked genetic disorders, chorionic villus sampling (CVS) for fetal sex determination is generally performed at 11–13 weeks of gestation. However, as the procedure-related miscarriage rate of CVS is 0.5–1.0%, non-invasive methods such as PCR of cell-free fetal DNA (cff-DNA) in maternal plasma are preferable. Here, we determined fetal sex at 9–12 weeks of gestation using PCR of cff-DNA in three pregnant carriers of Duchenne muscular dystrophy. The fetal sex was accurately determined in all three cases, as confirmed by ultrasound and amniocentesis at 16 weeks (for the two female fetuses) and CVS at 12 weeks (for the one male fetus). This procedure could avoid unnecessary CVS in female fetuses.

Clinical outcome of infants with confined placental mosaicism and intrauterine growth restriction of unknown cause.

The purpose of this study was to know a role of confined placental mosaicism (CPM) in perinatal outcome and postnatal growth and development of infants with intrauterine growth restriction (IUGR). We selected 50 infants with IUGR (<−2.0 SD) from 3,257 deliveries in a regional medical center during the past 10‐year period, and carried out cytogenetic and molecular analyses in their placenta and cord blood. Of the 50 infants, 8 had CPM (CPM group) and were composed of five single (CPM2, 7, 13, 22, and 22), one double (CPM7/13), and one quadruple trisomy (CPM2/7/15/20), and one partial monosomy [del(2)(p16)]. The origin of an extra chromosome of trisomy was maternal in six cases of CPM, paternal in one, and undetermined in one. Uniparental disomy in disomic cell lines was ruled out in all these mosaics. We also compared clinical parameters for perinatal outcome between CPM group and infants without evidence of CPM (non‐CPM group), such as maternal and gestational age, birth weight, Apgar score, cord blood pH, gender, and uterine artery patterns by Doppler ultrasonography, as well as weight, height, and developmental quotient (DQ) by Denver Developmental Screening Test at age 12 months. Phenotypic abnormalities were noted in two infants with CPM and three infants of non‐CPM group: One with CPM22 had ASD and hypospadias, one with CPM7/13 had Russell–Silver syndrome (RSS), and one without CPM had polydactyly, and two without CPM had RSS. All but one infant with CPM are alive at age 12 months. Among the clinical parameters, the detection rate of a notch waveform pattern of the uterine artery was significantly higher in the CPM group (P < 0.05). However, no significant difference was noted in perinatal outcome of pregnancy and in DQ at age 12 months between the two groups. Interestingly, short stature (<−2 SD) at age 12 months was more frequently seen in CPM group (7/8 infants with CPM vs. 8/15 infants without CPM), although no statistically significant difference was obtained. The information obtained will be useful for perinatal care and genetic counseling for infants with IUGR and CPM.

Atp10a, the mouse ortholog of the human imprinted ATP10A gene, escapes genomic imprinting

The mouse Atp10a gene is located at the border of an imprinted domain distal to the p-locus on mouse chromosome 7. The localization of Atp10a neighboring the maternally expressed gene Ube3a in the imprinted domain and an unusual inheritance pattern of the obesity phenotype with a p-locus deletion have suggested that Atp10a might be imprinted and associated with body fat. Recently, its human ortholog, ATP10A, was identified as the second imprinted gene with maternal expression in the human chromosome 15q11-q13 imprinted domain. To elucidate the imprinting status of Atp10a, we performed expression analysis in various tissues from reciprocal crosses between C57BL/6 and PWK (divergent strains of Mus musculus) mice. The results revealed that Atp10a was biallelically expressed in all tissues examined. Furthermore, there was no differential methylation in the CpG island and no antisense transcripts of the gene. These findings suggest that the mouse Atp10a gene escapes genomic imprinting.

Copy number variation of the antimicrobial-gene, defensin beta 4, is associated with susceptibility to cervical cancer

The aim of this study was to investigate association between copy number variation of the defensin beta 4 gene (DEFB4) and susceptibility to cervical cancer in a population at high risk of persistent oncogenic human papillomavirus (HPV) infection. The study subjects comprised 204 women with cervical cancer, a population having a high risk of persistent oncogenic HPV infection (cervical cancer group), and 200 healthy women from the general population (control group). Copy number variation of DEFB4 in each test sample was determined by relative quantitation using the comparative CT (ΔΔCT) method. Differences between the two groups were evaluated. The median DEFB4 copy number in the cervical cancer group was four and in the control group was five (P=2.77e–4, t-test). The odds ratio of cervical cancer in individuals with four DEFB4 copies or less was higher (odds ratio 2.02; 95% confidence interval odds ratio 1.36–3.02), compared with that in individuals with five or more copies (odds ratio 0.49; 95% confidence interval odds ratio 0.33–0.74). We found copy number variation of DEFB4 was a host genetic factor conferring susceptibility to cervical cancer. A lower DEFB4 copy number was associated with susceptibility to cervical cancer.

The gene TSGA14, adjacent to the imprinted gene MEST, escapes genomic imprinting.

We identified the gene TSGA14, encoding the testis-specific protein A14 and located 50 kb proximal to the imprinted gene MEST in a head-to-head orientation. TSGA14 has at least two transcripts: a long-type (l-type) transcript, and a short-type (s-type) transcript. Since the COPG2IT1 gene in the vicinity of MEST has been reported to be imprinted, we presumed that TSGA14 might also be imprinted. We thus analyzed the imprinting status of TSGA14 l-type and s-type transcripts in various fetal tissues. TSGA14 l-type transcript, which consists of 11 exons and encodes a l-type isoform with 373 amino acids, is biallelically expressed in the fetal tissues including the testis. TSGA14 s-type transcript, which consists of three exons and encodes a s-type isoform with 54 amino acids, also showed biallelic expression in the fetal brain and liver. No allele-specific methylation in the TSGA14 CpG island was detected. The fact that COPG2 and TSGA14, both neighbors of MEST, escape genomic imprinting suggests that the 7q32 imprinted region may be small and not similar to other imprinted domains, such as those at 15q11-13 and 11p15.5.

The possibility of microarray-based analysis using cell-free placental mRNA in maternal plasma

The purpose of this study is to investigate a possibility of overall assessment of cell‐free (CF) placental mRNAs in maternal plasma.

Human papillomavirus DNA in plasma of patients with HPV16 DNA-positive uterine cervical cancer.

OBJECTIVES The squamous cell carcinoma antigen is considered the most accurate serologic tumor marker for uterine cervical carcinoma. However, serum squamous cell carcinoma antigen levels were found to correlate significantly with clinical severity of atopic dermatitis and chronic renal failure. The present study was conducted in patients with human papillomavirus 16 DNA-positive uterine cervical cancer to determine the plasma level of human papillomavirus 16 DNA and the diagnostic values of plasma human papillomavirus DNA in these patients. METHODS Forty-three human papillomavirus 16-positive patients with cervical intraepithelial neoplasia or uterine cervical squamous cell carcinoma were recruited in this study. The diagnosis was cervical cancer in 20 patients, high-grade squamous intraepithelial lesions in 21, low-grade squamous intraepithelial lesions in 1 and negative for intraepithelial lesion or malignancy in 3 patients. Before any treatment, blood samples were collected from all patients. For analysis of human papillomavirus DNA in plasma of patients with cervical cancer, quantitative polymerase chain reaction fluorescent assay for human papillomavirus 16 was performed using human papillomavirus 16 primers and SYBR Green dye using the LightCycler 480 SW1.5 apparatus. RESULTS Plasma human papillomavirus 16 DNA was detected in only 30.0% of the patients with human papillomavirus 16-positive cervical cancer and in none of normal controls. The copy number of plasma human papillomavirus 16 DNA was higher in patients with invasive cancer than in those with cervical intraepithelial neoplasia (CIN3), micro-invasive cancer and in normal individuals. CONCLUSIONS These results indicated that the plasma human papillomavirus DNA level could be potentially used as a marker of low-invasive cervical cancer tumors in patients with normal squamous cell carcinoma antigen levels before treatment.