Characterization and clinical implications of marker chromosomes identified at prenatal diagnosis.

Eighteen fetuses with marker chromosomes were detected at diagnostic amniocentesis in our laboratory among 15 781 amniocentesis samples. Using combined approaches, conventional cytogenetics including special stain techniques and fluorescence in situ hybridization (FISH), we successfully characterized 15 of them, which assisted subsequent genetic counselling. Six marker chromosomes were of sex chromosome origin, each of which substituted a missing sex chromosome, and 12 were supernumerary marker chromosomes (SMCs). Nine of the SMCs were proven to be of autosomal origin. Of those autosomal SMCs, five originated from chromosome 15, two from chromosome 18, one from chromosome 12 and one from chromosome 1. Among 16 marker chromosomes with adequate follow-up information, 50% were benign including four sex chromosome markers and four autosomal markers. Two thirds of de novo marker chromosomes were associated with abnormal outcomes, while all inherited ones were benign regardless of their parental origin. Our study demonstrated that molecular characterization of prenatal marker chromosomes is of great significance in facilitating phenotype-genotype correlation.

Prenatal diagnosis of Smith-Lemli-Opitz syndrome in a pregnancy with low maternal serum oestriol and a sex-reversed fetus.

A cytogenetically normal male fetus was subsequently found to have female external genitalia, a cardiac malformation and mid-trimester intra-uterine growth retardation by ultrasound examination. The maternal serum oestriol level was low. The combination of low oestriol and sonographic findings suggested Smith Lemli Opitz syndrome (SLO), which was confirmed by a markedly increased amniotic fluid level of 7-dehydrocholesterol. We review the differential diagnosis of apparent sex reversal in a fetus and low maternal serum oestriol level. To further examine the specificity of low maternal oestriol level as a marker for SLO a follow-up study of 12141 pregnancies screened for Down syndrome using three biochemical markers: alpha-fetoprotein, beta-human chorionic gonadotrophin and oestriol was performed. 26 pregnancies had an oestriol level that was 0.25 MoM or less. SLO was not diagnosed clinically in any of the liveborn children ascertained through a low maternal oestriol level. Nine of the pregnancies ended in spontaneous miscarriage. Although the frequency of SLO in pregnancies with low maternal oestriol levels or sex-reversed fetuses is unknown, the diagnosis of SLO should, nevertheless, be considered in both clinical settings.

Constitutional inversion of chromosome 7 and hematologic cancers.

Nonrandom aberrations of chromosome 7 have been described in various hematopoietic disorders. We describe here two unrelated families with the same constitutional inversion of chromosome 7 [inv(7)(q11.2q22)]. The probands in both families had acute leukemia and cytogenetic analysis revealed that the inversion was the sole cytogenetic abnormality in the bone marrow at diagnosis. There is a history of hematologic diseases in one of these families that included a son who is a carrier of this constitutional inversion. The distal inversion breakpoint lies within the common region of chromosome loss identified in some myeloid diseases. These observations raise the possibility that this inherited chromosome rearrangement could result in a mutation of a tumor suppressor gene and possibly represent a predisposing event for the development of leukemia in these individuals.

Interphase FISH and morphologic analysis of AML.

Interphase cytogenetic analysis of peripheral white blood cells from a patient with acute myelogenous leukemia (AML) who was previously diagnosed with trisomy 8 was performed for the purpose of documenting clonal response to intensive chemotherapy. DNA in situ hybridization was performed using a probe specific for chromosome 8 alpha-satellite DNA sequences. Cells were stained with Wright stain prior to interphase analysis and photographed to allow correlation of cell morphology with abnormal karyotype. Prior to chemotherapy, the patient's leukocyte differential contained 8% blasts; interphase analysis revealed 23% trisomy 8 cells, including mature granulocytes. Forty days after the start of chemotherapy, at which point the patient had attained clinical remission, interphase analysis revealed only 2% cells with three signals, which was not statistically significant when compared with our own series of controls. The use of interphase FISH analysis in this case provides additional evidence that some leukemic blasts may be capable of limited differentiation in vivo and also suggests a differential sensitivity to chemotherapy between cytogenetically normal and abnormal hematopoietic precursor cells.

Mosaic 5p tetrasomy.

We report on a mildly abnormal 5-year-old girl with seizures, psychomotor retardation, and areas of hyperpigmentation who had a supernumerary marker chromosome in fibroblasts which was identified as an i(5p). To our knowledge, this is the first reported case of tetrasomy 5p. She shares in common some, but not all, manifestations of the dup (5p) syndrome. Cytogenetic analysis of relatives showed that the phenotypically apparently normal mother, maternal grandmother, and a brother of the proband also had a marker chromosome in their lymphocytes which was unrelated to the i(5p).

Relationship between mitotic delay and the minimum dose rate of X irradiation required to stop cell proliferation.

When cells are subjected to irradiation, their progression through the cell cycle can be arrested. If the arrested cells are subjected to additional damage, their period of arrest is prolonged. Under continuous low-dose-rate irradiation, the cumulative nature of arrest time leads to a geometric increase in the arrest time as a function of the dose rate. Above a certain cell-line-specific dose rate, the arrest duration becomes infinite and cell proliferation ceases. We find that the lowest dose rate (critical dose rate) required to stop cell proliferation during continuous irradiation is the reciprocal of the mitotic delay per gray of high-dose-rate irradiation. The calculated critical dose rates for X rays agree with those measured by Mitchell et al. (Radiat. Res. 79, 537-551, 1979), provided that the critical dose rate is identified with the minimum dose rate which allows no more than one population doubling. This specific identification of the critical dose rate is explained on the basis of cell cycle kinetics. A kinetic mechanism for cell cycle arrest and recovery leads to the critical dose rate as the reciprocal sensitivity for G2 arrest.

Longitudinal cytogenetic study of metaphase and interphase cells in childhood monosomy 7 syndrome.

A 15-year-old male with myelodysplastic syndrome (MDS) characterized by monosomy 7 was cytogenetically evaluated by metaphase karyotyping and fluorescence in situ hybridization (FISH) of interphase cells at six different points during the course of his disease. At diagnosis, there was complete agreement between metaphase and interphase findings. Interphase analysis alone provided important cytogenetic information on the first specimens received following intensive combination chemotherapy and bone marrow transplantation where metaphase analyses were uninformative. The detection of a minor post-treatment monosomy 7 population by interphase but not metaphase studies may have identified minimal residual disease prior to recurrence of MDS. From this longitudinal study, it is concluded that metaphase and interphase cytogenetic analyses form complementary approaches and that use of both provides greater analytical power when appropriate chromosome markers are available.

Epidermal growth factor receptor expression in a retinoic acid-treated human melanoma cell line.

Treatment of a human cell line (HXG-2), established from a metastatic melanoma, with retinoic acid (RA) induced morphologic differentiation and eliminated its cloning capacity in soft agar. With the v-erb B oncogene as a probe, slot blot hybridization of genomic DNA from parental HXG-2 cells did not show epidermal growth factor (EGF) receptor gene amplification as compared with normal diploid fibroblasts. Analysis of RNA as well as EGF receptor determinations from HXG-2 and RA-treated HXG-2 cells showed essentially no differences, indicating that RA treatment does not modulate EGF receptor gene expression. Although enhanced EGF receptor expression is found in some advanced-stage melanomas, RA-induced changes in the transformation phenotype of cell line HXG-2 probably do not result from modulation of the EGF-mediated pathway.

Enhanced G2 chromatid radiosensitivity in dyskeratosis congenita fibroblasts.

Dyskeratosis congenita (DC) is an inherited disorder characterized by reticular pigmentation of the skin, dystrophic nails, mucosal leukoplakia, and a predisposition to cancer in early adult life. In the majority of cases, DC is an X-linked recessive trait. However, one or more autosomal form(s) of DC may exist. Although excessive spontaneous chromatid breakage has been reported in DC, it is not a consistent cytological marker for this disorder. We examined the frequency and specificity of X-irradiation-induced G2 chromatid breakage in fibroblasts from three unrelated DC patients (two males and one female). Metaphase cells from DC patients had significantly more chromatid breaks (16-18-fold and 17-26-fold at 50 and 100 rad X-irradiation, respectively) and chromatid gaps (10-12-fold and 6-7-fold at 50 and 100 rad, respectively) than those from two different controls. Analysis of banded chromosomes revealed a nonrandom distribution of chromatid aberrations in DC but not in controls, a distribution corresponding to some of the known breakpoints for cancer-specific rearrangements, constitutive fragile sites, and/or loci for cellular proto-oncogenes. The significance of this finding for cancer predisposition in DC patients is uncertain, but the increased susceptibility of X-irradiation-induced chromatid breakage may serve as a cellular marker of diagnostic value.

Spontaneous and 4-nitroquinoline 1-oxide-induced G2 chromosome aberrations in lymphoblasts from familial melanoma patients.

Lymphoblastoid cell lines established from three unrelated kindreds with familial melanoma (FM) were cytogenetically analyzed for spontaneous and 4-nitroquinoline-1-oxide (4NQO)-induced chromosome aberrations. There were no significant differences between control, FM, or xeroderma pigmentosum (XP) cell lines for spontaneous aberrations. As a group, FM patients, as well as XP patients, had significantly higher 4NQO-induced aberrations than controls when metaphase cells were analyzed 2.5 hours after treatment during the G2 phase of the cell cycle. When selected cell lines were analyzed 18 hours after 4NQO treatment, the frequency of chromosome aberrations in FM cells returned to spontaneous levels, but XP cells retained significantly elevated aberration frequencies. The wide variability for chromosome aberrations within the control. FM relative, and FM patient groups during G2, however, indicated that analysis of total breakage rates alone would not be predictive of susceptibility to FM. Heterogeneity for carcinogen-induced chromosome breakage between some cancer-prone individuals and the possible significance of site-specific chromosome aberrations are discussed.

Bleomycin hypersensitivity in dyskeratosis congenita fibroblasts, lymphocytes, and transformed lymphoblasts.

We studied the responses of several dyskeratosis congenita (DC) cell lines to the DNA strand-cleaving and base-damaging agent bleomycin. Fibroblasts, peripheral blood lymphocytes, and transformed lymphoblasts of six DC patients and an obligate DC heterozygote showed more chromatid breaks than did respective controls exposed to various concentrations of bleomycin during the G2 phase of the cell cycle (P less than 0.0001). Unsynchronized DC fibroblasts in culture also showed decreased survival, compared to normals, following bleomycin treatment. DC lymphocytes treated with bleomycin for the final 24 h of culture showed more chromatid- and chromosome-type damage than did normals (P less than 0.0001) or G0-treated DC lymphocytes. Spontaneous chromosome breakage was normal in all six DC cell lines. The ability to distinguish affected and heterozygous DC cells without spontaneous chromosome instability from normals on the basis of their bleomycin hypersensitivity provides a marker for future studies of the pathogenesis of this disorder.

In situ genetic complementation analysis of cells with generalized peroxisomal dysfunction.

Most patients with Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease and hyperpipecolic acidemia are characterized by a deficiency of peroxisomes. We have developed a simple cytological method for the in situ detection of genetic complementation among and between these patients who are clinically and biochemically defined as having generalized peroxisomal dysfunction. This technique should facilitate both complementation studies in these disorders and investigations into the biogenesis of peroxisomes.

Incidental detection of premature centromere separation in amniocytes associated with a mild form of Roberts syndrome.

Premature centromere separation (PCS) was detected in amniocytes after an amniocentesis was done because of markedly elevated maternal serum alpha-fetoprotein values in a healthy primiparous young woman. PCS has been associated with the Roberts-SC phocomelia syndrome (RS). By 23 weeks' gestation, ultrasonic evaluations did not reveal abnormal fetal development. The pregnancy continued and a male infant was born with mild manifestations of RS. PCS was confirmed in cord blood lymphocytes. This case illustrates that PCS, when detected in amniotic fluid cell cultures, requires a thorough evaluation.

Effect of differentiating agents on nucleolar organizer region activity in human melanoma cells.

A human cell line established from a metastatic melanoma had both multiple numerical and structural chromosome aberrations including one to two copies of a submetacentric marker chromosome with an insertion of an active nucleolar organizer region (NOR). Treatment of this cell line with retinoic acid (RA) induced morphologic differentiation and reduced the cellular saturation density concomitant with a significant decrease in Ag-NOR activity. RA-treated cells grown in the absence of this differentiating agent, however, displayed a return to normal Ag-NOR activity, indicating the effect of this chemical on ribosomal genes is reversible.

The in vitro growth, heterotransplantation, and immunohistochemical characterization of the blastemal component of Wilms' tumor.

Wilms' tumor has been proposed to originate from a developmental abnormality of the metanephric blastema. This undifferentiated component of Wilms' tumors has previously eluded efforts for in vitro growth. Blastema from a "classical" Wilms' tumor was transplanted into nude mice and passaged through 12 generations of heterotransplantation. Tumors from heterotransplants were grown for 12 serial passages in a serum-free growth medium supplemented with hormones and conditioned media from human kidney proximal tubule cells. The blastema initially grew on a collagen-fetal calf serum matrix as multicellular spheroids, and the cells proliferating from the rim of the spheroids had a flattened shape. Pulse-labeling with bromodeoxyuridine (BrdU) identified the proliferating cell population as blastemal in origin. Except for a loss of extracellular matrix, ultrastructural studies demonstrated morphologic similarities in the cultured cells, compared with the primary tumor and heterotransplants. Lectin histochemical stains for the peanut lectin (PNA) and immunohistochemical stains for cytokeratin (CYTO), vimentin (VIM), and epithelial membrane antigen (EMA) were performed on the original tumor, successive heterotransplants, and cells grown in vitro. The PNA stained the surface of the blastemal cells after sialidase digestion in the original tumor, heterotransplants, and cultured cells. The blastema of the original tumors was negative for CYTO and EMA but reactive for vimentin. This lack of differentiation was maintained in heterotransplants through 12 passages. However, blastemal cells demonstrated coexpression of CYTO and VIM intermediate filaments when grown in a serum-free medium on a matrix material. These studies demonstrate that the blastemal component of Wilms' tumor can be successfully grown in culture, passaged in nude mouse heterotransplants, and shown to undergo early stages of blastemal differentiation in vitro by growth in serum-free medium. This in vitro system provides a model for testing the factors that influence the growth and differentiation of the blastemal component of Wilms' tumors.

Isochromosome 11q in a case of Ewing's sarcoma.

A low passage Ewing's sarcoma cell line established from a metastatic lesion was cytogenetically analyzed. The modal karyotype was 44,X, -8,i(11q), -15, +12. Other cells had random chromosome aneuploidy superimposed on this karyotype. No cell had a structural rearrangement involving 11q24 or 22q12 as described in other patients with Ewing's sarcoma, but all cells had an isochromosome 11q.

The in vitro growth, heterotransplantation, and differentiation of a human rhabdomyosarcoma cell line.

A human rhabdomyosarcoma (RMS) cell line was established from a case of childhood small cell sarcoma, which in vivo showed no evidence of differentiation, but which demonstrated myogenic differentiation in tissue culture. In a serum-free culture medium, the tumor cells demonstrated continuous growth without ultrastructural or biochemical evidence of differentiation. Heterotransplanted RMS cells gave rise to tumors in nude mice which also showed no myogenic differentiation. However, RMS cells grown in the presence of either retinoic acid (5 microns), phorbol ester (1 nM), prostaglandin E1 (10 ng/ml), or 2% fetal calf serum gave rise to myotubes with a biochemical shift in the creatine kinase isoenzyme pattern from the embryonic to the mature skeletal muscle form. The karyotype of the RMS cells revealed a translocation of Chromosomes 2 and 13, which may represent a nonrandom aberration unique to this morphologic subtype. In addition, the RMS cells gave evidence for gene amplification in the form of double minute chromosomes. This human RMS cell line provides a valuable in vitro system for study of myogenesis and factors which induce differentiation.

Combined phenotypic and genotypic analysis of ringed sideroblasts in acquired idiopathic sideroblastic anemia.

Bone marrow from an 81-year-old male with acquired idiopathic sideroblastic anemia was found to be mosaic for 45,X/46,XY cell lines. Analysis of ringed sideroblasts for the presence or absence of a quinicrine fluorescent Y body indicated that all sideroblastic cells had lost the Y chromosome. The demonstration that the ringed sideroblasts were cytogenetically abnormal in this patient provides evidence that the cytogenetic changes often found in patients with sideroblastic anemia may not be due to randomly acquired chromosome aberrations accompanying tissue aging unrelated to the disease process.

Asymmetric skeletal anomalies in siblings.

We describe two siblings with asymmetric limb reduction malformations. Such anomalies are usually considered to result from sporadic events, but the recurrence in siblings without any identifiable teratogenic insult suggests a genetic etiology. This finding becomes important when parents are counseled about future pregnancies. The use of prenatal diagnostic techniques during subsequent pregnancies should be considered.

BS I-B4 isolectin as a probe for an investigation of membrane alterations and transformation phenotypes of mouse L cells.

BS I-B4, an alpha-D-galactopyranosyl-binding isolectin from Bandeiraea simplicifolia seeds, was found to interact differently with transformed mouse L cells and non-transformed mouse 3T3 cells. The lectin induces detachment of 3T3 cells but increases adhesiveness and clustering of L cells. However, the induced cell aggregation does not lead to cell fusion. A variant clone of L cells, resistant to BS I-B4, which had lost the capacity for agglutination in the presence of the lectin, was isolated. Fluorescence binding studies of this variant suggest a lesion involving alpha and beta-D-galactopyranosyl units on its cell-surface structures. Although the variant cells form colonies in a methylcellulose medium, they do not produce tumours, as do the parental cells, when transplanted in athymic nude mice. The results demonstrate that alterations in cell membrane glyco-conjugates play an important role in tumourigenesis of animal cells, but anchorage-independent growth in vitro, as one of the transformation phenotypes, cannot be correlated absolutely with tumourigenicity in vivo.