Norio Miharu

Meiotic segregation analysis of a 14;21 Robertsonian translocation carrier by fluorescence in situ hybridization

Abstract. Meiotic segregation of chromosomes 14 and 21 in sperm from a 14;21 Robertsonian translocation carrier was analyzed with dual-color FISH using two locus-specific DNA probes (Tel 14q and LSI 21). The frequency of normal or chromosomally balanced sperm, resulting from alternate segregation, was 88.42%. The frequency of unbalanced sperm, resulting from adjacent segregation, was 11.25%. These observed frequencies deviated significantly from the theoretical frequencies (33.33% and 66.67%, respectively) based on random chromosome segregation, with sperm resulting from alternate segregation being preferentially produced in the translocation carrier. With respect to the chromosomally unbalanced sperm, the frequency of 21q disomic sperm was 2.45%, which is in agreement with the frequencies of unbalanced fetuses or offspring at the time of amniocentesis or at term (0–4.3%) reported by others. Although the frequency of 14 or 21 nullisomic sperm should be theoretically equal to that of 14q or 21q disomic sperm in both the carrier and controls, the frequency of nullisomic sperm was significantly higher than that of disomic sperm in the carrier (P=0.0009 for chromosome 14, P<0.0001 for chromosome 21) but not in the controls (P=0.091 for chromosome 14, P=0.74 for chromosome 21). This evidence suggests the occurrence of maturation arrest during spermatogenesis of the carrier.

Quantitation of fetal DNA in maternal serum in normal and aneuploid pregnancies

Abstract. We investigated whether the amount of circulating cell-free fetal DNA in maternal serum is influenced by fetal karyotype, using real-time quantitative polymerase chain reaction assay. Serum samples were obtained from pregnant women at gestational ages ranging from 15 to 17 weeks, prior to their undergoing amniocentesis. In total, we examined 70 samples consisting of 55 cases of pregnancy with 46,XY, 5 cases with 47,XY,+21, 3 cases with 47,XY,+18, a single case with 46,XY,dup(1) and 2 cases with twins of 46,XY, and 4 cases with 46,XX which were used as negative controls. We measured the concentration of the SRY sequence as a molecular marker for fetal DNA. The SRY sequence was detectable and measurable when the fetuses were male except for one case with 47,XY,+18. This case showed fetal growth retardation and bradycardia. No amplification signals of the SRY sequence were detected when the fetuses were female. The mean concentration of fetal DNA in maternal serum was 31.5 copies/ml in the pregnancy with 46,XY, 23.5 copies/ml in the pregnancies with 47,XY,+21 and 21.5 copies/ml in the pregnancies with 46,XY,+18. There were no significant differences in the concentration of fetal DNA between pregnancies with fetuses of normal karyotype and those with fetuses of abnormal karyotype.

Cell-free Fetal DNA in Maternal Circulation after Amniocentesis

After amniocentesis, 5–20% of patients have evidence of fetal-maternal hemorrhage as indicated by increases in maternal serum α-fetoprotein (1)(2)(3)(4)(5) or by the Betke–Kleihauer test (6)(7)(8). The Betke–Kleihauer test can differentiate fetal from maternal erythrocytes by the relative resistance of hemoglobin F-containing cells to acid elution, and it is the most popular method of diagnosing and assessing the severity of fetal-maternal hemorrhage (9). The reliability of this test has been questioned, however, because numerous sources of error are associated with it (10). These sources of error possibly contribute to the wide variation in the reported incidence of fetal-maternal hemorrhage; a more accurate method of assessing fetal-maternal hemorrhage is therefore required in the clinical setting of rhesus D-negative pregnant women. The discovery of cell-free fetal DNA in maternal serum and plasma has opened a new avenue for noninvasive prenatal diagnosis and has provided a useful marker of complicated pregnancies (11)(12)(13)(14)(15)(16). The analysis of fetal DNA in maternal serum or plasma has afforded diagnoses of fetal rhesus D status (12) and single-gene disorders (13), as well as the determination of fetal gender (14)(15). Cell-free DNA may be liberated directly from the fetal-placental interface into the maternal circulation (16), or it may be transferred into the maternal …

Detection of chromosomal abnormalities in uterine leiomyoma using conventional cytogenetic method and interphase fluorescence in situ hybridization

Seventy-nine uterine leiomyomas were examined using a conventional cytogenetic method and fluorescence in situ hybridization (FISH) for detection of chromosomal abnormalities of chromosome 12. Nine (17.6%) of 51 tumor samples examined showed chromosomal abnormalities by conventional cytogenetic analysis. Rearrangements of chromosome 12 were detected in two tumors. Other tumors showed abnormalities affecting chromosomes 1, 4, 6, 7, 10, 13, 14, and 22. For FISH, the whole-chromosome painting probe and the D12Z3 probe specific for the centromeric region were used to detect structural and numerical abnormalities of chromosome 12. Of forty-one tumor samples, six showed structural aberrations and four showed numerical aberrations of chromosome 12 by FISH analysis. Of the tumors with structural aberrations identified by FISH, two had normal karyotypes, two showed structural rearrangements of chromosome 12 cytogenetically, and two could not be analyzed because of an insufficient number of metaphases. There were no correlations between the cytogenetic data and clinical parameters. The results indicate that chromosomal abnormalities are important in the biology of at least some types of uterine leiomyomas, and that FISH is a useful complement to conventional cytogenetic analysis in the study of solid tumors.

Chromosomal FISH analysis of unfertilized human oocytes and polar bodies

AbstractThe incidence of chromosomal aneuploidy was studied in 208 unfertilized metaphase II human oocytes from an in vitro fertilization program by fluorescence in situ hybridization using probes for chromosomes 18, 21, and X. Chromosome spreads were prepared by a gradual fixation-air-drying method. We obtained analyzable results from 183 oocytes and 93 polar bodies; 167 oocytes (91%) were normal, 11 (6%) were diploid, and 5 (3%) were aneuploid. Of the five aneuploid oocytes, four involved chromosome 21, and one involved the X chromosome. In this study, oocyte aneuploidy caused by both nondisjunction of bivalent chromosomes and predivision of univalent chromosomes was observed. The aneuploidy rate (9.8%) in the oocytes from women aged ≧35 years was significantly higher than that (0.7%) in those aged 23 to 34 years (P= 0.0017).

Functional disomy for Xq22-q23 in a girl with complex rearrangements of chromosomes 3 and X.

A 5‐year‐old girl with developmental and growth retardation is reported with complex chromosome rearrangements consisting of a partial Xq deletion and an abnormal chromosome 3 with multiple breakpoints. GTG‐banding, and multiplex and conventional FISH studies showed that a 6.6‐Mb Xq22‐q23 segment was inserted into 3q, in addition to three intrachromosomal insertions in chromosome 3. Her karyotype was thus interpreted as 46,X,der(X)(Xpter→Xq22::Xq23→Xqter),der(3)(3pter→3p26::3p12→3q25.3::3p12→3p26::Xq22→Xq23::3q25.3→3qter). Replication R‐banding study showed that the der(X) was inactivated in all blood lymphocytes analyzed. Methylation‐specific PCR at the androgen receptor gene (HUMARA) locus at Xq11‐q12 showed a skewed inactivation pattern with the active/inactive X chromosome ratio of 92/8. These data indicated the presence, in the majority of cells, of a functioning Xq22‐q23 segment in both the normal X and the der(3) chromosomes. Her growth retardation, developmental delay, and other minor anomalies were most likely caused by dosage effects of the genes in the functionally disomic Xq22‐q23 region. Despite the presence of two active copies of the proteolipid protein 1 gene (PLP1), she did not show the symptoms of Pelizaeus‐Merzbacher disease, a subset of which has been known to be caused by the duplication of PLP1.

Fetal diagnosis of galactosialidosis (protective protein/cathepsin A deficiency)

The fetal diagnosis of galactosialidosis is performed by measuring carboxypeptidase (cathepsin A) activity in cultured villous cells and by immunofluorescence analysis with an antibody against an oligopeptide corresponding to the N-terminal domain of the human mature protective protein. Neither carboxypeptidase activity nor immunofluorescence was detected in cultured villous cells derived from an at-risk fetus or in cultured fibroblasts derived from the sister with galactosialidosis. Neuraminidase and beta-galactosidase activities were also confirmed to be deficient or low. A direct assay system for protective protein/cathepsin A is useful for the accurate prenatal diagnosis of galactosialidosis.

Current status of non‐invasive prenatal testing in Japan

The purpose of this study was to report the 3‐year experience of a nationwide demonstration project to introduce non‐invasive prenatal testing (NIPT) of maternal plasma for aneuploidy, and review the current status of NIPT in Japan.

Detection of aneuploidy in human spermatozoa using fluorescence in situ hybridization (FISH)

SummaryFluorescence in situ hybridization (FISH) with chromosome specific alpha-satellite DNA probes was used to estimate the rates of aneuploidy of chromosomes 1, 17, 18, X and Y in human ejaculated sperm. Sperm samples were collected from six donors, and biotinylated DNA probes, D1Z5, D17Z1, D18Z1, DXZ1 and DYZ3 were hybridized to interphase sperm which had been pretreated with dithiothreitol to expand their nuclei. A minimum of 3,000 sperm per donor were analyzed. The hybridization efficiency was 99.68% for all the five probes. The frequencies of aneuploidy for chromosomes 1, 17 and 18 were 0.65%, 0.66% and 0.61% respectively. For XX-and YY-sperm the frequencies were 0.28% and 0.27% respectively. To estimate the diploidy and disomy rates, a mixture of D17Z1 and D18Z1 were used as probes, and the frequency of diploid sperm was calculated to be 0.27%. After subtraction of the diploidy rate, the disomy rates for chromosomes 1, 17, and 18 were estimated to be 0.38%, 0.39% and 0.33%, respectively. The proportion of X- and Y-bearing sperm were 49.9% and 49.66%, consistent with an expected 1 : 1 ratio.

Detection of chromosomal abnormalities of chromosome 12 in uterine leiomyoma using fluorescence in situ hybridization

SummaryFifty uterine leiomyomas were examined using conventional cytogenetic method and fluorescence in situ hybridization (FISH) for detection of chromosomal, abnormalities of chromosome 12. Of the 50 tumors, nine were examined using FISH on the non-cultured samples. Two (4.0%) of 50 tumor samples examined showed chromosomal abnormalities of chromosome 12 by the conventional cytogenetic analysis. For FISH, the whole-chromosome painting probe and D12Z3 probe specific for the centromeric region were used. Of the 50 cultured samples, 10 showed structural aberrations and four showed numerical aberrations of chromosome 12 by FISH analysis. Of the nine non-cultured samples, four showed structural abnormalities of chromosome 12, all of which also showed structural abnormalities of chromosome 12 on the cultured samples. These results indicate that chromosomal abnormalities of chromosome 12 are important in the biology of at least some types of uterine leiomyoma, and that FISH is a useful complement to the conventional cytogenetic analysis in the study of solid tumors.