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Polycythemia vera (PV) and essential thrombocytosis (ET) are clonal chronic myeloproliferative disorders originating from a multipotent stem cell. Bone marrow examinations reveal chromosomal abnormalities in 15-43% of PV patients and 5% of ET patients, but no specific recurring abnormality has been found to date. We aimed to find cytogenetic aberrations in PV and ET by comparative genomic hybridization (CGH), a relatively new molecular cytogenetic technique. In this study, CGH analysis was performed on peripheral blood leukocytes of 12 PV patients and 8 ET patients. One patient (8.3%) with PV had an abnormal karyotype with a deletion in 7q11.2 and one patient with ET (12.5%) had a gain in 18p. Peripheral blood analysis by CGH revealed a low frequency of cytogenetic abnormalities in PV and ET patients. However, using CGH we were able to detect two cytogenetic aberrations that were not reported previously in these disorders.

We describe a man with pericentric inversion 9 and constitutive heterochromatin, and a high disomy rate in his sperm cells (with all probes analyzed). The disomy rate was estimated with the following probes: 8, 9, 18, X, and Y, and was significantly higher than that in control sperm cells, while chromosome 9 showed the highest disomy frequency. The probes of X and Y together showed the same disomy frequency as X and Y alone, which indicates the same nondisjunction rate in the first meiotic division. We suggest that the interchromosomal effect found in this man differed from other findings in sperm cells of men carrying an inversion in terms of the difference in the length of the heterochromatin between the two chromosomes 9. Also, it is well known that the effect of inversion 9 with increased heterochromatin is highly variable and may even vary in members of the same family.

It has recently been reported that in human sperm cells, the centromeres are clustered in a chromocenter in the interior region of the nucleus. The aim of the present study was to determine the intra-chromocenter organization of the five centromeres of the acrocentric chromosomes responsible for the biosynthesis of rRNA. The acrocentric centromeres were labeled by fluorescence in situ hybridization (FISH) after mild decondensation of the sperm nuclei to preserve the tail structure. The tail was used as a topographical marker for the orientation of the nucleus. The following results were obtained: (a) the association among the five centromeres was higher than expected from random distribution; (b) all the centromeres observed were randomly located within the chromocenter, occupying about 87% of the total area of the internal nucleus; (c) a major subpopulation of centromeres was located in a preferred area occupying 8.3% of the total nuclear area, with a peak 0.6 microm on the lateral axis and 1.0 microm on the apical side of the longitudinal axis; and (d) The dispersion of the centromeres was not influenced by the degree of the nuclear decondensation. We conclude that in human sperm nuclei, the acrocentric centromeres are organized within a nonlocalized structural element in the chromocenter. The chromocenter can range from an expanded size of 87% of the whole nucleus to a preferred size of 8.3% independent of the degree of nuclear decondensation. These findings have important implications for nuclear function (rRNA) that is not directly related to sperm cell function or early embryo development.

Background Fetal cells exfoliate in the uterine cavity during early pregnancy and are a potential source of material for NIPD. Aims This study was designed to test the hypothesis that fetal cells obtained from the uterine cervix during the first trimester of pregnancy could be utilized for prenatal diagnosis of chromosomal aneuploidy. Study design Fetal cells retrieved from the distal endocervical canal during the first trimester of pregnancy were hybridized with chromosome 21 specific FISH probes and analyzed with an automated fluorescence microscope. Subjects and outcome measures Cells with 3 copies of chromosome 21 were detected in 5 out of 5 trisomy 21 pregnancies. Results The number of trisomic cells detected ranged from 1 to 27 with a median value of 5. Conclusions FISH-based scanning can identify trisomy 21 pregnancies by analysis of routine cervical brushings. The approach offers the potential for non-invasive prenatal diagnosis as early as 5 weeks gestation.

Objective. The aim of this study was to report the clinical features, management, and outcome of two cases of complete hydatidiform mole with a coexisting viable fetus and to review the literature. Patients. In this article, we report on the well-documented follow-up of 2 cases of twin pregnancies with complete hydatidiform mole and a viable fetus, both of which ended with the delivery of a normal infant at 41 and 26 weeks of gestation. It is of interest that both pregnancies were achieved following induction of ovulation with hMG/hCG. Since 1977, the year in which complete and partial moles were characterized as distinct pathologic entities, 15 cases (including our 2) have been reported. Results. Persistent GTT developed in eight patients (53.3%) and four patients (27.7%) developed metastatic disease. Seventy-five percent patients with persistent GTT were treated with single-agent chemotherapy. The median gestational age of the patients with subsequent persistent GTT was 34.5 weeks compared to 38 weeks in the patients without persistent GTT. Conclusion. Complete hydatidiform mole and coexistent fetus is a rare occurrence and is associated with an increased risk of persistent gestational trophoblastic tumor. Based on currently available information, it seems that in the presence of a stable pregnancy, normal karyotype, and a normal sonogram it is reasonable to allow the pregnancy to continue.

Recently, a working-model of a stepwise malignant transformation in the molecular pathogenesis of multiple myeloma (MM) was proposed, involving the tumor suppressor gene TP53 and retinoblastoma gene (RB1) as prominent components of cell cycle control. To further define the role of TP53 and RB1 in disease progression, we retrospectively analyzed by fluorescence in situ hybridization (FISH) cytological material from 16 patients who underwent sequential bone marrow biopsies during the course of their disease. For TP53, no deletions were detected at presentation or during follow-up. It is possible that the patients reported here represent a subset with relatively long survival, and therefore did not demonstrate the TP53 deletions that had been reported in patients with a very poor prognosis. For RB1, monoallelic deletion was demonstrated in nine patients. In each case, the deletion appeared already in the first biopsy analyzed. The presence of a deletion did not affect the rate of tumor progression or the length of follow-up, and thus prognosis. Monoallelic deletions of RB1 appear to be a frequent and early event in the pathogenesis of MM, without obvious relevance for disease progression.

In this study we evaluated the replication pattern and cell-cycle dynamics of cells from patients considered to have a premalignant condition (monoclonal gammopathy, or MGUS) and patients with multiple myeloma (MM), as well as healthy controls. We applied the fluorescence in situ hybridization (FISH) technique with the TP53, RB-1 and 21q22 loci on the patient's cells. Asynchrony was determined by the presence of one single and one set of double dots in the same cell. The rate of asynchronic replication was significantly higher in the cells from MM patients, with intermediate value in the cells from MGUS, while the lowest rate was in cells from controls. We suggest that these results may reflect the changes in gene replication and cell-cycle progression that occur in premalignant and malignant cells.

BACKGROUND Loss of replication synchrony during the S-phase of the cell cycle has been shown to be associated with aneuploidy in malignant cells. METHODS The replication pattern of cervical cells obtained from normal patients and from patients with preinvasive and invasive carcinoma of the cervix was evaluated. The fluorescence in situ hybridization technique was applied to Papanicolaou smear slides using p53 and 21q22 loci. Asynchrony was determined by the presence of one single and one set of double dots in the same cell. RESULTS The rate of asynchrony in low grade squamous intraepithelial lesions was not significantly different from that of normal controls. However, the rate of asynchrony was significantly higher in the high grade squamous intraepithelial lesions and even higher in the invasive carcinoma. CONCLUSIONS The authors believe these results reflect impairment of the replication control of homologous loci and that this phenomenon may be correlated to the phenotype of the tumor. Cancer 1998;83:1966-1971. © 1998 American Cancer Society.

We have used FISH to determine the level of synchronisation in replication timing of four pairs of alleles, unrelated to chromosome 21 (p53, HER2, RB1, and c-myc), in foetal (amniotic fluid) cell samples of Down syndrome and in normal foetuses. All samples derived from the Down syndrome subjects showed large temporal differences in replication timing, in contrast to the high level of synchrony shown in all samples of normal individuals. Thus, as judged by four independent loci which are not associated with chromosome 21, the additional chromosome in the Down syndrome genome induces changes in the replication pattern of an allelic pair: from a synchronous pattern characteristic to concomitantly expressed alleles to an unsynchronised one shown by alleles displaying an allele-specific mode of expression.

The clinical significance of mosaicism trisomy 20 detected prenatally following amniocentesis remains uncertain, due to the rarity of liveborn cases with inconsistent clinical findings, the short postnatal follow-up, and failure in evaluating other fetal tissues for the presence of the trisomy. We report on a 15 month-old 46,XX chromosome constitution in white blood cells, while skin fibroblasts demonstrated trisomy 20 mosaicism (54%) by fluorescence in situ hybridization (FISH) analysis. Clinical examination of the baby showed only minor phenotypic signs (bilateral epicanthal folds, delayed closure of fontanel with no other gross anomalies), but demonstrated a considerable developmental delay in gross and fine motor skills along with hypotonicity. This is the second oldest described liveborn with trisomy 20 mosaicism confirmed in skin fibroblasts. This cytogenetic aberration along with her developmental delay suggests that the two findings are related and that aberration affects various fetal tissues and is not confined to extra-embryonic tissue as suggested previously. Yet, an undiagnosed condition may be the cause of the child's developmental delay. Based on this case and following a review of the literature we suggest that when mosaic trisomy 20 is identified in amniocytes, further evaluation is required. Cord blood should be analyzed preferably by FISH. During counseling the parents should be advised of an additional risk, such as developmental delay, even when fetal cord karyotype and detailed ultrasonic scan are normal.

This study was undertaken in an attempt to determine the significance of elevated maternal serum human chorionic gonadotropin (MShCG), in the presence of an otherwise normal screen with respect to fetal malformations, chromosomal aberrations, and pregnancy outcome. Targeted ultrasound findings and perinatal outcome of 298 women in whom serum hCG was > or = 2.5 MOM and who were screen-negative for Down syndrome (the study group) were compared with a control group of 229 women in whom serum hCG as well as the other parameters were within the normal range. Genetic amniocentesis was performed in 125 women from the study group. Ultrasonically detected malformations were significantly more frequent among the study group (12 vs. 1, P = 0.01). Pregnancy complications were similar in the two groups, with the exception of pre-eclampsia-toxaemia, which was significantly more frequent in the study group (5 vs. 0, P = 0.02). There was one case of an abnormal karyotype (47,XXY). Although genetic amniocentesis does not appear warranted, isolated elevated MShCG levels during the second trimester screening was associated with an increased risk of fetal anomalies detected by ultrasound and of toxaemia of pregnancy.

Chromosomal aberrations can be detected in 50% of patients with chronic lymphocytic leukemia (CLL). A role for tumor suppressor genes in the genesis of lymphoid tumors has been reported. In B-CLL, p53 gene mutations were found in 10-15% of the patients. We used fluorescence in situ hybridization (FISH) to detect p53 deletion in B-CLL. We also correlated the cytogenetic findings with the clinical course. In situ hybridization to interphase nuclei showed monallelic p53 deletion in 6 of 23 patients (26%). The percentage of cells with one p53 signal ranged from 12 to 100. A statistically significant correlation between p53 deletion and progression of CLL was demonstrated. We conclude that FISH is a sensitive and reliable method to detect deletion of specific genes (i.e., p53) in CLL. The finding of p53 deletion is associated with disease progression.

Cellular senescence limits proliferation of potentially detrimental cells, preventing tumorigenesis and restricting tissue damage. However, the function of senescence in nonpathological conditions is unknown. We found that the human placental syncytiotrophoblast exhibited the phenotype and expressed molecular markers of cellular senescence. During embryonic development, ERVWE1-mediated cell fusion results in formation of the syncytiotrophoblast, which serves as the maternal/fetal interface at the placenta. Expression of ERVWE1 caused cell fusion in normal and cancer cells, leading to formation of hyperploid syncytia exhibiting features of cellular senescence. Infection by the measles virus, which leads to cell fusion, also induced cellular senescence in normal and cancer cells. The fused cells activated the main molecular pathways of senescence, the p53- and p16–pRb-dependent pathways; the senescence-associated secretory phenotype; and immune surveillance-related proteins. Thus, fusion-induced senescence might be needed for proper syncytiotrophoblast function during embryonic development, and reuse of this senescence program later in life protects against pathological expression of endogenous fusogens and fusogenic viral infections.

Essential thrombocytosis (ET) is a clonal, chronic myeloproliferative disorder (MPD) originating from a multipotent stem cell. To date no specific cytogenetic marker has been found in ET. It was recently reported that chromosomal aberrations have been detected by fluorescence in situ hybridization (FISH) in patients with normal karyotypes or nonanalyzable metaphases. Therefore, we evaluated whether trisomies 8 and 9, which are commonly found in MPDs, can be detected in ET by FISH and compared the results with chromosome analysis. Peripheral blood mononuclear cells of patients with essential thrombocytosis were studied by classical chromosome banding and by FISH. We used biotin labeled α satellite of chromsome 8 and biotin labeled β satellite of chromosome 9 as probes for the FISH studies. FISH detected 5 patients with trisomy 8 and 5 with trisomy 9 of the 18 patients evaluated. No trisomy was found by cytogenetic studies. The trisomies were detected by FISH in only a minority of the cells. No correlation was found between the presence of a trisomy and clinical characteristics. FISH is a sensitive method for the detection of trisomes 8 and 9 in patients with ET. The common finding of these chromosomal aberrations in MPD suggests that genes associated with myeloid proliferation are located on these chromosomes. Standardization of interphase cytogenetics is needed before this technique can be accepted for routine use in the clinic.

Cytogenetic and fluorescent in situ hybridization (FISH) studies were performed on several formalin-fixed tissues obtained from four fetuses diagnosed at amniocentesis as 45,XO-Turner syndrome. Three of the four were phenotypically normal and one had malformations. The three phenotypically normal cases were found to have an additional normal cell line, which may explain their ability to survive, at least to the time of pregnancy termination well into the second trimester. The abnormal 45,XO fetus was not found to be mosaic in all of the tissues examined. In 45,XO cases in which no malformation is detected, the possibility of mosaicism should be raised and thus the counselling should be modified accordingly.

Interleukin-11 (IL-11) is a novel cytokine that has been shown to stimulate human hematopoietic progenitors including the CD34+ CD33- DR- early progenitors. IL-11 has little effect on its own but it synergizes with other hematopoietic growth factors. We investigated the recovery of human myeloid progenitors incubated with IL-11 alone or in combination with other cytokines, including stem cell factor (SCF), interleukin-3 (IL-3) and granulocyte macrophage colony-stimulating factor (GM-CSF) following their in vitro treatment with ARA-C (10(-9) M) or Eilatin (10(-7) M). IL-11 in combination with IL-3 and GM-CSF markedly increased CFU-C colony growth pre- and post-ARA-C or Eilatin incubation from CML and normal individual bone marrow (BM) cells. Similarly, IL-11 alone or in combination with other cytokines increased cell recovery following 7-day suspension culture. A decrease in BCR/ABL fusion product was observed (by FISH analysis) after incubation of BM cells from CML patients in liquid culture for 7 days with 10(-9) M ARA-C or 10(-7) M Eilatin in the presence of IL-11 alone or in combination with other cytokines. These results indicate that following cytoreductive therapy IL-11 may enhance to a greater extent the growth of normal myeloid progenitors than the malignant clone and may, therefore, be of clinical importance for CML patients treated with chemotherapeutic agents.

Objective Primary sclerosing cholangitis (PSC) is a chronic cholestatic disorder that involves inflammatory and fibrotic changes in the bile ducts. Up to 80% of patients have concomitant inflammatory bowel disease (IBD) with colitis. PSC patients are predisposed to develop hepatobiliary, colonic and other extrahepatic malignancies, probably related to inflammatory processes that might promote carcinogenesis. Telomerase is an enzyme complex that lengthens telomeres and has enhanced expression in numerous malignancies. In this study, we evaluated the TERC gene copy number, the proportion of cells in senescence and the amount of fragmentation in the senescent state. Methods Fluorescence in situ hybridization (FISH) for the TERC gene was applied to lymphocytes retrieved from PSC (N = 19), colitis (N = 20) and healthy control patients (N = 20) to determine the TERC copy number. On the same FISH slides, cells stained with DAPI were also analyzed for senescence-associated heterochromatin foci (SAHF) status, including the number of cells with fragments and the number of SAHF fragments in each cell. Results A higher TERC gene copy number was observed in cells from PSC patients compared to colitis and control group patients. It was also higher in the colitis than in the control group. Significantly more cells in the senescent state and more fragmentation in each cell were observed in the PSC group compared to colitis and control groups. Conclusion The TERC gene copy number and the number of cells in the senescent state were increased in PSC patients compared to the colitis and control groups. These findings are probably related to the genetic instability parameters that reflect the higher tendency of this patient group to develop malignancies.

The putative oncogenes BCL-1, BCL-2, and BCL-3 are commonly rearranged by translocations to the immunoglobulin genes in B-cell malignancies. However, Southern blotting rarely detected their involvement in chronic lymphocytic leukemia (CLL). This discrepancy could stem from some unique features of the oncogenesis of CLL or be due to shortcomings of Southern blotting. We have therefore evaluated the role of fluorescence in situ hybridization (FISH) in the detection of these oncogenes in CLL. Twenty consecutive CLL patients were studied by FISH for the detection of BCL-1, BCL-2, or BCL-3 rearrangement and for the presence of trisomy 12. Selected patients were also evaluated by classical cytogenetic techniques and by Southern blot analysis. Juxtaposition of JH and BCL-1 was demonstrated in 10 (50%), BCL-2 in three (15%), and BCL-3 in four (20%) of the patients. Trisomy 12 was detected by FISH in 11 (55%) patients. The coexistence of trisomy 12 and translocation of the BCL-1 oncogene was common. Three of the patients had chromosomal aberrations compatible with those detected by FISH. In contrast, in none of the five patients selected by their positive FISH findings was a rearrangement demonstrated by Southern blotting. We conclude that FISH is a sensitive method for the detection of oncogene involvement in CLL. Mainly BCL-1, but also BCL-2 and BCL-3, are commonly translocated to the immunoglobulin heavy chain locus on chromosome 14. These translocations are often associated with trisomy 12. These findings indicate that the BCL oncogenes are commonly involved in CLL and lend support to the multi-hit theory of cancer development.

The malignant plasma cells of multiple myeloma (MM) have a low proliferative activity and therefore cytogenetic studies of the disease have been severely limited. We evaluated the role of fluorescence in situ hybridization (FISH) in the detection of numerical chromosomal abnormalities in early stages of myeloma and the applicability of the method to stored archival slides. Old air-dried bone marrow smears from 15 myeloma patients obtained at presentation were probed with α satellite DNA sequences to chromosomes 3 and 7. Numerical chromosome aberrations were found in eight (53%) of the patients, including six (of 12) with trisomy 7, and two (of eight) with trisomy 3. This study demonstrates that FISH is a sensitive method for the detection of numerical aberrations in myeloma and for the study of old slides for retrospective analysis.

Five cases of supernumerary marker chromosomes were identified in prenatal diagnosis as derived from chromosomes 18, X, and Y. One unexpected finding was in a case where the PCR was positive for the SRY gene while fluorescence in situ hybridization was positive for two X centromeres. In another case with an X derived supernumerary marker the newborn was phenotypically normal. Two women with fetal mar(18) and mar(Xp) decided to terminate the pregnancy. The fifth pregnancy had a karyotype of 46,XX,-15,+der(15)t(Y:15)(q11,23;p13). A phenotypically normal girl was born at term.

Background. Malignant thymoma is composed of neoplastic epithelial cells and small lymphocytes. Rarely, patients also may have peripheral T-lymphocytosis. These lymphocytes have been considered nonneoplastic because of their microscopic appearance and immunophenotype, as well as gene rearrangement studies. Methods. A 42-year-old man developed lymphocytosis 3 years after the completion of intensive combined chemoradiotherapy protocol for lymphocytic thymoma. These peripheral blood lymphocytes were evaluated phenotypically and genotypically. Results. Immunophenotyping established that the cells were CD3 positive, CD4 negative, CD8 negative, T-cell receptor (TCR)-alpha/beta negative, and TCR-gamma/delta positive. Gene rearrangement studies with TCR-delta probe confirmed the monoclonality of these cells. Chromosome analysis showed deletion of chromosome Y. The clinical course was progressive and had the features of malignant lymphoma. Conclusions. To the authors' knowledge, this is the first report of a patient with thymoma in whom monoclonal proliferation of T-gamma/delta peripheral blood lymphocytes was confirmed immunophenotypically and genotypically. These monoclonal TCR-gamma/delta lymphocytes may belong to the malignant clone of the thymoma; however, the possibility that they represent an evolution of a second lymphatic malignancy cannot be excluded.

Individuals with trisomy 21 have an increased risk of developing leukemia and premature dementia. They also have a higher rate of telomere loss. The aim of the study was to compare telomere length and the hTERC gene copy number, which encodes the telomerase RNA subunit, in amniocytes of trisomy 21 conceptions and normal pregnancies. A quantitative fluorescence-in-situ protocol (Q-FISH) was used to compare telomere length in amniocytes cultured from 11 trisomy 21 conceptions and from 14 normal pregnancies. Quantification was conducted using novel computer software. Fluorescence in situ hybridization (FISH) was used to assess the percentage of cells with additional copies of hTERC. We found that the immunofluorescence intensity, which represents telomere length, was significantly lower in amniocytes from trisomy 21 conceptions compared to the control group. The trisomy 21 group had a higher number of cells with additional copies of hTERC. This observation could be one of the cytogenetic parameters that represent a state of genetic instability and might play a role in the pathomechanism of typical features of Down syndrome, such as dementia and malignancy.

Introduction Intrauterine growth restriction (IUGR) is a significant cause of both short- and long-term morbidity and mortality. IUGR secondary to placental dysfunction is correlated with telomere shortening. Telomerase is an enzyme complex that elongates telomeres. One of its components is encoded by the telomerase RNA component gene (TERC), which serves as the RNA template for the addition of telomeric repeats. We hypothesized decreased TERC gene copy number in IUGR placentas as part of the mechanism of telomere shortening in placental dysfunction. Methods We estimated the gene copy number of the TERC gene at 3q26 by applying FISH to trophoblasts of placental biopsies from five pregnancies with IUGR caused by placental insufficiency and compared them to placentas from five gestational-age matched, uncomplicated pregnancies. Results Significantly lower TERC gene copy number was observed in IUGR trophoblasts on the same chromosome and on other chromosomes, compared to the control samples (p Conclusions The TERC gene copy number is decreased in IUGR trophoblasts. These results support the observations of telomere shortening and decreased telomerase activity in IUGR placentas. We suggest that these findings might play a role in the pathophysiology of IUGR, perhaps by promoting senescence in trophoblasts of IUGR placentas.

Focal amplification of specific regions of the genome creates high copy number and expression of oncogenes in tumors. By applying fluorescence in situ hybridization (FISH) to leukocytes of hepatitis C (HCV) patients and non-Hodgkin lymphoma (NHL) patients, we estimated gene dosage of the TERC gene at 3q26.3. Higher TERC copy numbers were found in NHL at diagnosis compared to HCV patient groups. Higher TERC copy numbers were also observed in NHL patient at diagnosis and relapse compared to patients in remission. We believe that the TERC gene amplification is involved in the process of genetic instability leading to tumor genesis such as in NHL.

Hepatitis C virus (HCV) infection is found in 80% to 90% of patients with essential mixed cryoglobulinemia (EMC) type II, which is associated with monoclonal IgMk produced by monoclonal B cells. It was investigated whether bcl-2 rearrangement is associated with the clonal B-cell proliferation of EMC induced by hepatitis C. The study groups were composed of 15 patients with HCV and EMC, 12 patients with HCV without EMC, and 7 patients with chronic liver disease (CLD) unrelated to HCV. Fluorescence in situ hybridization with probes was applied to JH and to bcl-2 to study whether JH/bcl-2 translocation was present in these patients. Thirteen of 15 (86%) of patients with HCV-related EMC had the JH/bcl-2 translocation, a significantly higher rate than in HCV patients without EMC (16%; P

Although the comprehensive evaluation of the fetal heart includes echocardiography by an experienced pediatric cardiologist, economic constraints sometimes dictate the need to select patients.To analyze the usefulness of fetal echocardiography in the detection of congenital heart disease according to the referral indication.This retrospective survey relates to all 3965 FE studies performed in our center from January 2000 to December 2004. The diagnosed cardiac anomalies were classified as significant and non-significant malformations. All FE studies were done by a single operator (A.L.) at Meir Medical Center, a referral center for a population of about 400,000. The 3965 FE studies were performed for the following indications: abnormal obstetric ultrasound scans, maternal and family history of cardiac malformations, medication use during the pregnancy, and maternal request. The relative risk of detecting CHD was calculated according to the various referral indications.Overall, 228 (5.8%) cases of CHD were found. The most common indication for referral was suspicion of CHD during a four-chamber view scan in a basic system survey or during a level II ultrasound survey. No correlation was found between maternal age and gestational age at the time of scanning and the likelihood of finding CHD.Our data suggest that a suspicious level II ultrasound orthe presence of polyhydramnios is an important indication for FE in the detection of significant CHD.

Objective Telomeres shorten and aggregate with cellular senescence and oxidative stress. Telomerase and its catalytic component human telomerase reverse-transcriptase regulate telomere length. The pathogenesis of preeclampsia and intrauterine growth restriction involves hypoxic stress. We aimed to assess telomere length in trophoblasts from pregnancies with those complications. Study Design Placental specimens from 4 groups of patients were studied: severe preeclampsia, intrauterine growth restriction, preeclampsia combined with intrauterine growth restriction, and uncomplicated (control). Telomere length and human telomerase reverse-transcriptase expression were assessed by using quantitative fluorescence-in-situ protocol and immunohistochemistry. Results Telomere length was significantly lower in preeclampsia, intrauterine growth restriction, and preeclampsia plus intrauterine growth restriction placentas. More aggregates were found in preeclampsia, but not in intrauterine growth restriction placentas. Human telomerase reverse-transcriptase was significantly higher in the controls compared with the other groups. Conclusion Telomeres are shorter in placentas from preeclampsia and intrauterine growth restriction pregnancies. Increased telomere aggregate formation in preeclampsia but not in intrauterine growth restriction pregnancies, implies different placental stress-related mechanisms in preeclampsia with or without intrauterine growth restriction.

Broken chromosomes can acquire new telomeres by "telomere capture" (TC), and it has become possible to investigate the terminus in cytogenetically visible telomere rearrangements. The TC phenomenon was observed in malignant conditions. We evaluated the TC rate in hepatitis C virus (HCV) patients compared to non-Hodgkin's lymphoma patients, as well as relative to a control group. For this purpose, we used two Cytocell probes, 15qter and 13qter. Higher TC rates were found in the three study groups relative to the control group. Our results showed that HCV patients have some of the components that can initiate the cascade of events leading to malignancies.

We report the results of screening for Down's syndrome (DS) in older women using published rate schedules based on maternal serum alpha-fetoprotein (MSAFP) and age. Five hundred and seventeen patients aged 35 years and older, who were referred for a mid-trimester genetic amniocentesis, were first tested for MSAFP and then underwent an amniocentesis. Individual risks for DS, combining MSAFP and age, were derived using three different published rate schedules. Theoretical selection for amniocentesis was made using the cut-off level of the average collective risk for a 35-year-old woman (1:380 at live birth or 1:270 at amniocentesis). Six affected pregnancies (five with DS and one with trisomy 18), which were diagnosed prenatally, were all found to be at a higher risk than the specified cut-off. These cases would have been diagnosed in any event, using any of the published rate schedules. According to these rate schedules, between 39 and 45 per cent of the patients would be in the lower risk group and therefore would have been counselled not to undergo amniocentesis. Further studies should be conducted in order to reach conclusive screening policies for DS in older women.

Telomeres are nucleoprotein structures located at the termini of chromosomes that protect the chromosomes from fusion and degradation. Hepatocyte cell-cycle turnover may be a primary mechanism of telomere shortening in hepatitis C virus (HCV) infection, inducing fibrosis and cellular senescence. HCV infection has been recognized as potential cause of B-cell lymphoma and hepatocellular carcinoma. The present study sought to assess relative telomere length in leukocytes from patients with chronic HCV infection, patients after eradication of HCV infection (in remission), and healthy controls. A novel method of manual evaluation was applied. Leukocytes derived from 22 patients with chronic HCV infection and age- and sex-matched patients in remission and healthy control subjects were subjected to a fluorescence-in-situ protocol (DAKO) to determine telomere fluorescence intensity and number. The relative, manual, analysis of telomere length was validated against findings on applied spectral imaging (ASI) in a random sample of study and control subjects. Leukocytes from patients with chronic HCV infection had shorter telomeres than leukocytes from patients in remission and healthy controls. On statistical analysis, more cells with low signal intensity on telomere FISH had shorter telomeres whereas more cells with high signal intensity had longer telomeres. The findings were corroborated by the ASI telomere software. Telomere shortening in leukocytes from patients with active HCV infection is probably due to the lower overall telomere level rather than higher cell cycle turnover. Manual evaluation is an accurate and valid method of assessing relative telomere length between patients with chronic HCV infection and healthy subjects.

In vitro fertilization cycles yield a low percentage of pregnancies. Eighty-five to ninety percent of the transferred embryos do not implant, and the abortion rate approaches 30%. Aneuploidy is assumed to be responsible for a major portion of this pregnancy wastage. The purpose of this study was to determine if there was any correlation between morphology and chromosomal content of unfertilized oocytes and rejected embryos. To assess the chromosomal content of oocytes and embryos, we used the method described by Tarkowski in 1966. Sixty oocytes fron 28 women, aged between 27 and 41 years, were analyzed. Sixty-seven percent were aneuploid; of these, 23.35% were hyperhaploid, 23.35% were hypohaploid, 8.35% were hyperdiploid, 3.35% were diploid, and 8.35% showed premature chromosome condensation. Of 20 preimplantation embryos analyzed, 80% were aneuploid, 10% were diploid, 5% were haploid, and 5% showed structural anomaly. Correlation was found between maternal age and aneuploidy in oocytes and between morphology and genetic balance in preimplantation embryos.

Down syndrome (DS) is a multifactorial disorder with a high predisposition to leukemia and other malignancies. A change in the replication pattern from synchronous in normal genes to asynchronous in DS amniocytes has previously been reported. The objective of this study was to evaluate additional molecular cytogenetic factors which could re-emphasize the high correlation between DS cells and genetic instability. We found a higher rate of random aneuploidy in chromosomes 9 and 18 and a higher rate of asynchronous replication in the subtelomeric region or DS leukocytes than in cells from normal newborns. In addition, the telomere capture phenomenon was observed in the DS leukocytes but not in normal controls. The molecular cytogenetic factors observed in the DS individuals are known to correlate with genomic instability and with predisposition to cancer.

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia of adults in Western countries. The most frequent recurring chromosomal aberrations identified in B-CLL patients are trisomy 12 and deletions of 13q, 17p, and 11q. Cases with deletions of 11q and 17p have a poor prognosis, whereas cases with deletions in 13q have a favorable prognosis. It was previously shown that CLL patients with trisomy 12 and del(13)(q14) have a higher rate of asynchronous replication of normal structural genes when compared to those with normal karyotypes. We studied the replication pattern of the structural locus 21q22 and the imprinted gene SNRPN and its telomere (15qter) and the random aneuploidy of chromosomes 9 and 18 in CLL patients with trisomy 12 and deletions of 11q and 17p, and compared the results to those of CLL patients without these aberrations and to healthy controls. Random aneuploidy rate was higher in the group of patients with trisomy 12 as compared to all other groups. The replication pattern with higher asynchronous pattern was found in both aberration groups compared to the CLL patients without the aberrations and to the control group with involvement of 21q22 and 15qter, whereas the highest synchronous group was found in the 2 aberrations CLL patient groups compared to the other groups with the imprinted locus SNRPN. The existence and significance of chromosomal aberrations in CLL have a deleterious effect on the processes of cell cycle and gene replication and may have biological and prognostic implications.

In this study, we applied the fluorescent in situ hybridization (FISH) technique and compared the common numerical abnormalities with chromosomes 13, 16, 18, 21, X, and Y in spontaneous to artificial abortion. This would cover about 75% of the common aneuploidy in spontaneous abortion.Placentas were taken from 59 patients with a first trimester spontaneous abortion and 61 patients who underwent an elective first trimester pregnancy termination. The range of growth was from 5 to 12 gestational weeks. Placentas were processed according to direct chorionic villi preparation. Direct dual color FISH was performed according to Vysis protocol with the probes for the following chromosomes: 13, 16, 18, 21, X, and Y.The aneuploidy rate in spontaneous abortion was 55.9% and in artificial abortion 8.2%. There was a significant difference between the two groups in the aneuploidy rate (P = 6 x 10(-9)).FISH is a rapid, efficient, and relatively inexpensive tool in detecting aneuploidy in placentas from cases of spontaneous abortions. Our rate of detected aneuploidy is compatible with other reports in which conventional cytogenetics was utilized.

Telomeric regions of the human genome are of particular interest, because rearrangements of these regions are difficult to identify by conventional chromosome banding technology. With the advent of molecular cytogenetic techniques such as fluorescence in situ hybridization (FISH), it has been possible to investigate the terminus in cytogenetically visible terminal deletions and telomere rearrangements. We investigated telomere capture and aneuploidy rates in chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML) patients, as well as in healthy control subsets. Using a FISH technique, we estimated the random aneuploidy and telomere capture of the 21q22, SNRPN, and 15qter loci. Higher aneuploidy rates were found in the leukocytes of CLL and CML patients, compared with the control group, for the 21q22 and SNRPN loci. There was no difference in the aneuploidy rate between the CML and CLL groups. Telomere capture was found in the two groups (CLL and CML), but not in the control group. We propose that the telomere capture phenomenon is much more common than has been reported in the literature; however, its prognostic significance is yet to be established.

Objective Because the effect of granulocyte colony-stimulating factor (G-CSF), which is widely used for allogeneic stem cell transplantation, on DNA function and stability has not yet been unequivocally elucidated, the aim of this study was to determine whether G-CSF leads to epigenetic and/or genetic modifications. Materials and methods Molecular cytogenetic techniques based on fluorescence in situ hybridization technology were used. Results Lymphocytes of G-CSF mobilized donors displayed epigenetic (altered replication timing of alleles) and genetic (aneuploidy) alterations similar to those observed in lymphocytes of cancer patients. Specifically, in the donors' lymphocytes, biallelically expressed genes (TP53 and AML1) and a repetitive noncoding DNA sequence associated with chromosome segregation (CEN17) showed loss of synchrony in allelic replication timing (allele-specific replication). Each displayed a highly asynchronous pattern of allelic replication similar to that characterizing monoallelic expressed genes. This non–locus-specific epigenetic phenomenon, which also affects DNA sequences associated with chromosome segregation, was accompanied by aneuploidy. Although the loss of replication synchrony in the lymphocytes of G-CSF mobilized donors was a transient epigenetic modification, aneuploidy remained unchanged. The G-CSF effect also was observed after G-CSF administration in vitro. 5-Azacytidine, a DNA methylation blocking agent, inhibited G-CSF in vitro induction of allele-specific replication. Conclusion G-CSF, probably via changes in DNA methylation capacity, leads to cancer-characteristic DNA modifications in lymphocytes of normal mobilized donors.

To determine whether low maternal serum concentrations of human chorionic gonadotropin (hCG) were associated with poor pregnancy outcome.Between 1999 and 2000, 20,880 women underwent triple test screening in our hospital. The levels of hCG were detected by fluorescent immunoassay. Low hCG levels (or = 0.20 MoM in our center) were considered to be a marker for increased risk for trisomy 18 or adverse pregnancy outcome. Each patient completed a questionnaire regarding fetal karyotype, complications of pregnancy and pregnancy outcome.Low maternal serum concentrations of hCG were detected in 119 pregnancies (0.57%). Of these, 19 (16%) were found to be missed abortions. The distribution of the remaining 100 cases was as follows: 72% had an isolated low hCG level, 24% had a low hCG level and a combination of hCG + alpha-fetoprotein0.80 MoM, and 4% had a low hCG level and a combination of hCG + unconjugated estriol0.80 MoM. No trisomy 18 or other chromosomal abnormalities were detected in our patient population. However, there were perinatal complications.Based on our screened population, a combination of multiple analytes seemed to be a better marker than an isolated finding of low maternal serum concentrations of hCG with regards to abnormal fetal karyotype, specifically trisomy 18, although it did determine a high-risk group in terms of complications of pregnancy.

In this study we describe two patients with congenital absence of alpha-fetoprotein (AFP). The pathological examination results, including an immunohistochemical stain, which define qualitatively the levels of AFP detected by the biochemical studies and the comparative genomic hybridization (CGH) are enclosed. A description of the suggested functions of AFP and the means of its production are set forth. An explanation is suggested for the lack of symptoms in a newborn with undetectable levels of AFP and the mechanism by which this condition might occur.

Multiple myeloma represents a malignant proliferation of plasma cells derived from a single clone. It is well known that alkylating agents are capable of inducing myelodysplastic syndromes (MDS) and acute myelocytic leukemias (AML). This risk of both diseases in patients with multiple myeloma has been estimated to be 10-20% after 10 years. We aimed to evaluate the time course and the type of genetic abnormalities in melphalan-treated patients in the chronic stage of the disease. We applied fluorescence in situ hybridization methods with probes to 5q31 and 7q31 to mononuclear peripheral blood leukocytes of 18 melphalan-treated patients and compared the results to those of 8 untreated myeloma patients. We found three patients (17%) with a 5q31 deletion in their peripheral white blood cells, but no 7q31 deletion. These findings suggest that 5q- occurs before the overt development of MDS/AML and raise important concerns regarding long-term treatment of myeloma patients with alkylating agents. Also, the performance of cytogenetic evaluation should be considered before autologous transplantation. The clinical and biological implications of these findings should be evaluated in larger clinical and laboratory studies.

Benign cystic teratoma, also known as dermoid cyst, is the most common of all ovarian neoplasms accounting for 10%–20% of all ovarian cysts. Most have a normal 46,XX karyotype. Less than 2% can undergo malignant transformation. By using the comparative genomic hybridization technique on nine benign ovarian cysts we were able to show two cases with chromosomal aberrations not seen before in dermoid cysts but known to be involved in malignant ovarian tumors. We speculate that these are the tumors carrying the potential for malignant transformation.

Xeroderma pigmentosum (XP) is a rare autosomal recessive syndrome. Laboratory investigations have failed to detect any consistent anomaly in cells from XP heterozygotic subjects, although examples of behavior intermediate between normal and XP cells have been reported. To estimate random aneuploidy we applied fluorescence in situ hybridization (FISH) with alpha-satellite probes for chromosomes 8 and 9 and replication pattern for TP53 (p53), ERBB2 (HER-2/neu), and MYCN (N-MYC) loci and for the imprinted SNRPN locus. A significantly higher rate of aneuploidy rate was observed in XP patients and carriers than in controls. The asynchrony pattern was significantly higher in XP carriers and patients with all three coding loci analyzed and significantly lower in XP patients and carriers with the imprinted locus SNRPN than in the control group. Molecular cytogenetic parameters such as random aneuploidy and replication pattern, which are known to reflect chromosomal instability, may be part of the tumorigenesis process. In XP patients and carriers, this genetic instability may represent a potential for developing malignancies.

We hypothesize that coordination between the two DNA parental sets in somatic cells is essential for the stability of the diploid genome, and that its disruption is associated with the many alterations observed in the various cancerous phenotypes. As coordination between two allelic counterparts is well exemplified by synchrony in replication timing, we examined, in blood cells of patients suffering from various hematologic malignancies, replication patterns of five loci. These loci were three cancer-implicated genes (TP53, AML1, and RB1) and two nontranscribed sequences engaged in chromosome segregation. All five loci normally display synchrony in allelic replication timing. In addition, in order to exemplify an asynchronous mode of allelic replication, we followed the replication of allelic counterparts of an imprinted gene (SNRPN), which is distinguished by its asynchronous mode of allelic replication (allele-specific replication). Allelic replication patterns were studied by fluorescence in situ hybridization (FISH), which has been shown to distinguish between nonreplicated and replicated regions of the genome in interphase cells, based on the structure of the specific hybridization signals that are being detected. Using the FISH replication assay we observed, for all loci which normally exhibit synchrony in allelic replication, loss of synchrony when present in blood cells of patients with hematologic malignancies. The loss of synchrony in allelic replication in patients' cells was accompanied by aneuploidy (chromosome losses and gains), the hallmark of cancer. We were able to reinstate the normal pattern of replication in the patients' cells by introducing an inhibitor of DNA methylation. It thus appears loss of allelic coordination is an epigenetic alteration characterizing cancer, which is easily identified by simple cytogenetic means and has a potential use in both cancer investigation and detection.

In this study, we used spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) as complementary techniques for the analysis of two therapy-related secondary myelodysplastic syndrome (t-MDS) cases with complex karyotypes, previously analyzed by G-banding. Different types of SKY's cytogenetic contributions include confirmation of G-banding results, identification of partially characterized rearrangements, identification of marker chromosomes unidentified by G-banding, and detection of cryptic reciprocal translocations. In particular, the ability of SKY to clarify a number of markers led to the comprehension of clonal evolution. The common aberration found in these two t-MDS cases was the fragility of chromosome 5 and monosomy of chromosome 18. We clearly present that the use of SKY combined with conventional G-banding analysis and FISH has assisted in the identification of important chromosomal events that may play a key role in the development of t-MDS.

While most allelic pairs of DNA replicate synchronously during the S phase of the cell cycle, some genes normally replicate asynchronously, i.e., genes on the X chromosome and imprinted genes. The replication control mechanism is unknown but was shown to be impaired in malignancies and chromosomal trisomies where replication pattern becomes asynchronous.To determine the level of asynchronization in replication timing of cells from patients with microdeleted genomes.We applied monocolor fluorescent in situ hybridization with different probes on leukocytes from microdeleted genomes.All samples derived from the microdeleted genomes showed significantly higher levels of an asynchronized pattern compared to normal individuals.Even a "small" genetic imbalance (microdeletion) can interfere with gene replication and cell cycle progression, as previously shown in full trisomies.

We assessed possible inter- and intraindividual variations in the frequency of disomy in sperm cells from three men with abnormal sperm analysis parameters. Mono- and dual-color fluorescence in situ hybridization (FISH) was applied to sperm cells from different samples of the men. Four men with a normal sperm profile were used as controls. The samples were taken separately over a period of 6 months. FISH probes used for the disomy rate analysis were clones from the satellite region of chromosomes 8, 18, X, and Y. The study group showed a significantly higher disomy rate compared with the control group, whereas there was no significant difference in the disomy rate between three different samples from the same individuals. These results suggest that the sampling time has no importance in assessing the rate of nondisjunction in sperm cells.

Comparative genomic hybridization (CGH) was applied to samples taken from various sites of placentas originating from complicated pregnancies: 24 with intrauterine growth restriction (IUGR), one with multiple fetal malformation, one with toxemia, one with hydrocephalus and two with undetectable maternal serum alpha-fetoprotein (MSAFP). One of the most common aberrations in the IUGR cases was the addition of a whole or part of the X chromosome. Other aberrations such as additional Y chromosome or of 13(q22) or loss of chromosome 17 also appeared in different cases. In one IUGR case trisomy 8 (in one site) and 47,XXY (in all sites) were detected. In the two cases with undetectable MSAFP monosomy 16 was found. Some of the results were also confirmed by the FISH technique. In all the control cases (six normal and five with aneuploidy) CGH concurred with the known karyotype. Our results demonstrate the usefulness of the CGH technique in the genetic evaluation of fresh and paraffin embedded placentas in problematic pregnancies even when morphology is normal. However, it is very important to take multiple samples from different sites of the placenta.

Objective A study was conducted to evaluate the sensitivity of combining a second trimester triple test and targeted ultrasound in order to detect Down syndrome in women undergoing amniocentesis over 35 years of age. Methods Women over 35 years of age underwent a triple test and an ultrasound examination for chromosomal markers immediately prior to genetic amniocentesis. Results One thousand and six women were examined. Four hundred and thirty seven were triple test-positive and in 195 cases ultrasonographic abnormalities were observed. Thirteen had Down syndrome and eight had other chromosomal abnormalities. All women with Down syndrome babies were triple test-positive and seven also had ultrasonographic markers. Three of eight women who had babies with chromosomal aberrations other then Down syndrome were also triple test-positive. Conclusions The use of the triple test as a screening tool in our population would reduce the number of amniocenteses by 60%, while no cases of Down syndrome would be missed. Ultrasonographic markers have added little to this population. Three non-Down syndrome chromosomal abnormalities and two Down syndrome mosaic cases would be missed by this approach. Copyright © 2002 John Wiley & Sons, Ltd.