Alveolar soft part sarcoma--reciprocal translocation between chromosome 17q25 and Xp11. Report of a case with metastases at presentation and review of the literature.

The molecular pathogenesis of alveolar soft part sarcoma, a rare tumor with uncertain histogenesis, was elucidated recently and was shown to be due to a translocation between chromosome 17q25 and Xp11 resulting in a fusion product between TFE3 (a transcription factor gene) at chromosome Xp11 and a novel gene designated as ASPL at chromosome 17q25. This results in the transcriptional dysregulation in the pathogenesis of this neoplasm. Of the 12 cases reported so far, the translocation was due to non-reciprocal translocation in 11 cases with only one case demonstrating a reciprocal translocation with respective fusion products. We report yet another case with reciprocal translocation between chromosomes 17q25 and Xp11 with TFE3/ASPL fusion product who presented with metastatic disease. A standard cytogenetic analysis of primary tumor cells with G-banding revealed an abnormal karyotype: 46, X, t(X;17)(p11;q25)[15]/46,XX[5]. PCR analysis of the frozen tumor tissue revealed a type 1 fusion product as described in the literature. We demonstrate a rare cytogenetic abnormality in ASPS, namely reciprocal translocation between chromosomes 17q25 and Xp11 with demonstration of molecular fusion product between TFE3 and ASPL in a patient who initially presented with pulmonary metastases.

Age dependent aneuploidy and telomere length of the human vascular endothelium.

RATIONALE: Aneuploidy and telomere length are two major parameters that have been associated with cellular senescence in vitro. In order to explore the role of aneuploidy and telomere length in aging of the human vasculature, we studied these two parameters in direct preparations of endothelial cells of the human abdominal aorta. METHODS: Using fluorescent in situ hybridization on 'touch prep' slides, we evaluated aneuploidy of two autosomes (chromosomes 6 and 16) and sex chromosomes in non cultured endothelial cells of the abdominal aorta as a function of the donor's age. RESULTS: We found that the frequency of aneuploidy of vascular endothelial cells significantly increased with age. This was expressed by age-dependent tetrasomy (r(s)=0.56, P=0.006 for chromosome 6; and r(s)=0.54, P=0.008 for chromosome 16), and age dependent loss of the Y chromosome (r(s)=0.85, P=0.0003). In addition, we found that telomere length was inversely correlated with age (r=-0.38, P=0.008). DATA INTERPRETATION: These findings suggest that indicators of cellular senescence, earlier observed in vitro, are also expressed in the human vascular endothelium in vivo. Aneuploidy and telomere attrition might thus play a role in the aging of the human vasculature.

Sex chromosome mosaicism in gonads of a fetus with cystic hygroma and deletion of the short arm of Y chromosome including loss of SRY.

The SRY gene on the short arm of the Y chromosome is necessary for male development. Without SRY, patients with 46,XY karyotype develop as females, fail to achieve normal puberty and have dysgenic gonads and a high incidence of gonadoblastoma. Here we report a female fetus, aborted at 17 weeks of pregnancy, with a non-mosaic 46,X,del(Y)(p11.2).ish del(Y)(SRY-) karyotype diagnosed by classical cytogenetics and fluorescence in situ hybridization (FISH). Ovarian tissue was full of oocytes and mitotic figures. FISH studies of ovarian tissues with X and Y centromere probes revealed extensive sex chromosome mosaicism, manifested by loss of the Y chromosome and polysomy of the X chromosome. We propose that X chromosome polysomy is a post-zygotic event that arises to facilitate gonadal differentiation in the absence of all factors necessary for normal gonadal development.

Loss of chromosome 13 in cultured human vascular endothelial cells.

In the vascular endothelium of human beings, telomere length is negatively related while the frequency of aneuploidy is positively related to donor age. Both in culture and in vivo the frequency of aneuploidy increases as telomere length is shortened. In this study we explored the relation between telomere length and aneuploidy in cultured human umbilical vein endothelial cells (HUVEC) by: (a) karyotype analysis and fluorescent in situ hybridization (FISH), (b) measurement of the terminal restriction fragments (TRF), and (c) assessment of replicative senescence by the expression of beta-galactosidase. Of 8 HUVEC strains, 7 cell strains lost chromosome 13, as shown by metaphase analysis and FISH of interphase cells. Five strains gained chromosome 11. In addition, five HUVEC strains became hypotetraploid shortly after the loss of chromosome 13. The loss of chromosome 13 was observed as early as PD 20, when mean TRF length was greater than 9 kb and the percentage of cells positive for beta-galactosidase was relatively low. The almost uniform loss of chromosome 13 suggests that this unique type of aneuploidy of HUVEC is the result of a progressive expression of clones with survival advantage.

Telomere attrition of the human abdominal aorta: relationships with age and atherosclerosis.

Little is known about the turnover rate (i.e. the rate of replication and death) of cells in the intima and media of human arteries as a function of age and atherosclerosis. One indicator of the replicative history of cells is telomere length. In this work we explored the rate of telomere attrition as a function of age and atherosclerosis in cells of the human abdominal aorta. Telomere length, measured by the terminal restriction fragment using Southern analysis, was determined in the intima and media of the distal (infrarenal) versus proximal (suprarenal) segments of the abdominal aorta. Telomere length was then correlated with age and atherosclerotic grade. The rate of age-dependent telomere attrition was higher in both the intima and media of the distal versus proximal abdominal aorta. In addition, telomere length was negatively correlated with atherosclerotic grade. However, after adjustment for age, this relationship was not statistically significant. The high rate of age-dependent telomere attrition in the distal abdominal aorta probably reflects enhanced cellular turnover rate due to local factors such as an increase in shear wall stress in this vascular segment.

Telomeres and essential hypertension.

The dynamics of telomere attrition in human beings might shape the course of age-dependent, complex genetic traits. One of these traits is essential hypertension. Age-dependent telomere attrition could lead to critically shortened telomeres and aneuploidy (ie, the loss or gain of chromosomes) with a resultant mosaicism that will be variably expressed in different cells and tissues. The chromosomal instability and loss of heterozygosity resulting from this process would promote an age-dependent expression of variant genes that harbor susceptibility for essential hypertension or genes that accelerate the process of aging.

Telomeres, hidden mosaicism, loss of heterozygosity, and complex genetic traits.

Telomeres appear to function as an endogenous timing mechanism in human beings. Telomere attrition not only provides a satisfactory explanation for some aspects of aging, it might also resolve enigmatic features of complex genetic traits that are age-dependent. If, with the passage of time, telomere attrition in human beings leads to genomic instability and particularly the loss of chromosomes, then the age dependency of phenotypic expressions of complex genetic traits might result from the temporal loss of heterozygosity and the consequent expression of disease-causing genes. In this way, telomere attrition might play a role not only in aging, but also in the diverse expression of complex genetic traits, such as essential hypertension, non-insulin-dependent diabetes mellitus, atherosclerosis, and cancer.

Synchrony in telomere length of the human fetus.

Telomere length, measured by terminal restriction fragments, was examined in tissues from human fetuses of gestational ages estimated as 15-19 weeks. The length of telomeres was similar in most fetal tissues. However, there were significant variations in telomere length among fetuses, with no apparent relationship between gestational age and telomere length. We conclude that synchrony in telomere length exists among tissues of the human fetus. This synchrony is apparently lost during extrauterine life.

Shortened telomere length in white blood cells of patients with IDDM.

IDDM is a polygenic and autoimmune disorder in which subsets of white blood cells (WBCs) are engaged in the destruction of beta-cells of the pancreas. The mechanisms that account for the abnormal behavior of these cells in IDDM are not fully understood. By measuring the mean length of telomeres of WBCs from patients with IDDM, we tested the concept that telomeres might play a role in IDDM. We examined the lengths of the terminal restriction fragments (TRFs) of DNA of WBCs from 234 white men comprising 54 patients with IDDM, 74 patients with NIDDM, and 106 control subjects. When adjusted for age, the TRF length from WBCs of patients with IDDM was significantly shorter than that of nondiabetic control subjects (mean +/- SE: 8.6 +/- 0.1 vs. 9.2 +/- 0.1, P = 0.002). No significant difference was observed between the TRF length from WBCs of patients with NIDDM versus nondiabetic subjects. Neither the duration nor the complications of IDDM (i.e., nephropathy and hypertension) had an effect on the TRF length of WBCs from patients with IDDM. The shortened TRF length of WBCs of patients with IDDM likely reflects a marked reduction in the TRF length of subsets of WBCs that play a role in the pathogenesis of IDDM.

Interstitial insertion of Y-specific DNA sequences including SRY into chromosome 4 in a 45,X male child.

A 45,X chromosome complement was found in the lymphocytes and skin fibroblast cultures of a male infant with minor facial anomalies and gastrointestinal abnormalities. Fluorescence in situ hybridization (FISH) studies with DNA probes specific for the entire Y chromosome (painting) and SRY identified insertion of a short piece of Y chromosome DNA, including the SRY region, into a der(4) chromosome at 4p15. FISH studies with DNA probes specific for Wolf-Hirschhorn syndrome (WHS) and telomere of 4p indicated that these 2 regions were intact and that the insertion of Y DNA had occurred proximal to the WHS region. High-resolution chromosome analysis performed after FISH studies showed an altered banding pattern of 4p at the region of insertion. The typical Giemsa dark band of 4p15 was consistently replaced by a gray band; this probably indicates deletion of the distal part of 4p15. The consequences of the double-chromosome anomaly in this patient were discussed in relation to his phenotype.

Familial transmission of a deletion of chromosome 21 derived from a translocation between chromosome 21 and an inverted chromosome 22.

Chromosome analysis of a newborn boy with Down syndrome resulted in the identification of a family with an unusual derivative chromosome 22. The child has 46 chromosomes, including two chromosomes 21, one normal chromosome 22, and a derivative chromosome 22. Giemsa banding and fluorescent in situ hybridization (FISH) studies show that the derivative chromosome is chromosome 22 with evidence of both paracentric and pericentric inversions, joined to the long arm of chromosome 21 from 21q21.2 to qter. The rearrangement results in partial trisomy 21 extending from 21q21.2 to 21q terminus in the patient. The child's mother, brother, maternal aunt, and maternal grandmother are all carriers of the derivative chromosome. All have 45 chromosomes, with one normal chromosome 21, one normal chromosome 22, and the derivative chromosome 22. The rearrangement results in the absence of the short arm, the centromere, and the proximal long arm of chromosome 21 (del 21pter-21q21.2) in carriers. Carriers of the derivative chromosome in this family have normal physical appearance, mild learning disabilities and poor social adjustment.

Reflections on telomeres, growth, aging, and essential hypertension.

Here we review the "telomere hypothesis of cellular aging." We propose that this hypothesis is relevant to our understanding of the roles of genetics as well as growth and development in the etiology of essential hypertension and its cardiovascular complications. Elements of this hypothesis and the speculations that we make can be directly tested using tissues (cells) obtained from human beings.

Uniparental disomy explains the occurrence of the Angelman or Prader-Willi syndrome in patients with an additional small inv dup(15) chromosome.

A patient with Angelman syndrome and a 46,XY/47,XY,+inv dup(15)(pter-->q11: q11-->pter) karyotype and a patient with Prader-Willi syndrome and a 46,XY/47,XY,+inv dup(15)(pter-->q12: q12-->pter) karyotype were investigated with molecular markers along chromosome 15. Paternal uniparental isodisomy was found for all informative markers in the first case which indicates that this, rather than the presence of the extra chromosome, is the cause of the Angelman syndrome phenotype. Similarly, the PWS patient showed maternal uniparental distomy with absence of PWS region material on the inv dup(15) chromosome. If (1) marker chromosomes are an occasional by product of 'rescuing' a trisomic fertilisation, or (2) if duplication of the normal homologue in a zygote which has inherited a marker in place of the normal corresponding chromosome 'rescues' an aneuploid fertilisation, or (3) if the presence or formation of a marker chromosome increases the probability of non-disjunction, then uniparental disomy might be found occasionally in other subjects with de novo marker chromosomes.

Partial trisomy 11q in a female infant with Robin sequence and congenital heart disease.

We describe the clinical and cytogenetic findings in a female infant with partial trisomy 11q, Robin sequence, cardiac anomalies, and other minor malformations. We compare the phenotypic similarities of our case to a series by Pihko et al. (1981), who reported on 20 cases with partial trisomy 11q with similar associated craniofacial and cardiac defects. We conclude that genetic etiologies for patients diagnosed with the Robin sequence may be more common than previously believed and that initial karyotyping should be performed to aid both diagnosis and clinical management. In addition, the pattern of Robin sequence and cardiac defects may be specifically suggestive of partial trisomy 11q.

High-level expression of enzymatically active human Cu/Zn superoxide dismutase in Escherichia coli.

Expression of human Cu/Zn superoxide dismutase (SOD) with activity comparable to the human erythrocyte enzyme was achieved in Escherichia coli by using a vector containing a thermoinducible lambda PL promoter and a beta-lactamase-derived ribosomal binding site. The recombinant human SOD was found in the cytosol of disrupted bacteria and represented greater than 10% of the total bacterial protein. The enzyme was purified to homogeneity by salt precipitation, gel filtration chromatography, and ion exchange chromatography. The active enzyme was obtained in high yield only when 1 mol of copper and 1 mol of zinc were incorporated into each mol of subunit during bacterial growth or by reconstitution of the apoenzyme. Human Cu/Zn SOD produced in bacteria has an apparent subunit molecular mass of 19 kDa on NaDodSO4/polyacrylamide gels. The native enzyme behaves as a dimer of 32 kDa as determined by gel filtration. Sequence analysis of the NH2 terminus revealed that the first 14 amino acids corresponded to authentic human SOD except that the NH2-terminal alanine was not acetylated. Thus, the bacterial processing system readily removes the NH2-terminal methionine residue from recombinant human SOD.

Inhibition of lactogenic activities of ovine prolactin and human growth hormone (hGH) by a novel form of a modified recombinant hGH.

A recombinant analog of human GH (hGH) lacking 13 amino acids at the amino-terminus (Met14hGH) inhibited the hGH- or ovine PRL (oPRL)-stimulated proliferation of Nb2 lymphoma cells and bovine PRL-stimulated fat synthesis and alpha-lactalbumin secretion in explants from bovine lactating mammary gland. The inhibition was competitive in nature, and in Nb2 cells could be abolished by an excess of hGH or oPRL. Inhibition of oPRL-stimulated proliferation of Nb2 cells by Met14hGH could also be specifically abolished by anti-hGH monoclonal antibodies. Met14hGH had no growth-stimulating activity in Nb2 cells and was not cytotoxic. It also did not affect glucose uptake by the mammary gland explants. Met14hGH competed with [125I]hGH for binding to intact Nb2 cells, IM-9 lymphocytes, solubilized microsomal fraction from lactating bovine mammary gland, and microsomal fraction from the liver of female virgin rats, but its affinity for those receptors was 2 orders of magnitude lower than the affinity of hGH. Since Met14hGH used in most experiments contained about 25% impurities and degradation products, a small amount of it was further purified by immunoaffinity chromatography. Two purified fractions, one consisting of a single 20K protein and the other accompanied by a small amount of 25K protein, were obtained. Both fractions exhibited increased inhibition of hGH- or oPRL-stimulated proliferation of Nb2 cells, thus indicating that the inhibitory activity results from the intact Met14hGH molecule. To the best of our knowledge, this is the first report describing the inhibition of lactogenic hormone activities by a modified hGH.

Preferential transcription and nuclear transport of globin gene sequences, as control steps leading to final differentiation of murine erythroleukemic cells.

Murine erythroleukemic (MEL) cells undergo a specific program of differentation in vitro, which is mainly characterized by accumulation of globin mRNA. These cells serve as a model system to study in detail the expression of a specific gene product at the transcriptional and post-transcriptional level. In this report we describe experiments in which the transcription rate of globin and non-globin genes, as well as their cytoplasmic appearance, was measured during the differentiation process. Two independent steps for regulating the abundance of globin mRNA were observed. On the transcriptional level we have observed that, in contrast to the transcription of globin genes, the transcription rate of non-globin genes is dramatically reduced throughout the period of induction. When the rate of cytoplasmic appearance was measured newly synthesized globin RNA molecules were found to be preferentially transported into the cytoplasm. It was shown that the reduction in cytoplasmic appearance of nonglobin genes is not a result of a shut-off in their transcriptional activity. In cells treated with 12-O-tetradecanoylphorbol 13-acetate the transcription rate remains constant while a continuous reduction in the cytoplasmic appearance is observed. These two independent phenomena which affect the non-globin genes, i.e. the suppression of their transcription and reduced cytoplasmic appearance, lead to the reduction in the relative amounts of the stable poly(A)-rich mRNA population and to the accumulation of globin sequences in the cytoplasm of the differentiated erythroid cells. These observations are in agreement with our previous model, which claimed that disappearance of the stable poly(A)-rich mRNA population is an obligatory process leading to the final differentiation of MEL cells.

Evaluation of immunological methods for detection of bovine growth hormone (BGH) produced in E. coli.

The use of several immunological methods for studies on synthesis of bovine growth hormone (BGH) by E. coli is described here. The ELISA procedure was shown to be the least sensitive and unfit for assaying BGH in E. coli extracts. The solid-phase radioimmunoassay (RIA) proved to be highly sensitive, but since E. coli extract itself (not containing BGH) interfered with the immunological reaction, its use for measuring BGH was practically limited. The best adequate procedure proved to be radioimmunoassay in solution, which was not adversely affected by the E. coli extract and was sufficiently sensitive to detect nanogram quantities of BGH. The size of the BGH produced by normal bacterial cells was investigated by protein fractionation, transfer to nitrocellulose paper and detection by anti-BGH serum. This method was also served for semi-quantitative determination of BGH in the bacterial extract.

Screening for highly active plasmid promoters via fusion to beta-galactosidase gene.

A plasmid containing promoter-deleted inactive beta-galactosidase gene [1] was used to select promoters of the pEP 121 plasmid [2]. Colonies of cells harboring reactivated beta-galactosidase gene were identified by their red color on McConkey plates. The quantitative amounts of beta-galactosidase produced in each clone were estimated by assaying enzyme activity and by measuring the specific beta-galactosidase protein following fractionation of total cells' proteins on polyacrylamide gel. A wide range of enzyme activities was observed. The most active promoter isolated was shown to promote beta-galactosidase production more efficiently, compared with the original beta-galactosidase promoter, amounting to 20% of all cell proteins. Such highly active promoters may be utilized in the future, to promote expression of cloned genes in bacteria.

Expression of a cloned bovine growth hormone gene in Escherichia coli minicells.

The synthesis of polypeptides in Escherichia coli minicells, directed by a pBR322 plasmid and its derivative-carrying bovine growth hormone cDNA insert, was studied. Two polypeptides coded by the ampicillin-resistance (Apr) gene (32 000 and 28 000 daltons) and a tetracycline-resistance (Tcr) polypeptide (36 000 daltons) were identified by insertion inactivation. Two additional polypeptides of 37 000 and 34 000 daltons of as yet unknown function were detected in all extracts regardless of the presence of the Apr or Tcr genes in the plasmid. The pBR322-BGH recombinant plasmid coded for several novel polypeptides, among them one of 46 000 daltons, presumably a fused product of the BGH and beta-lactamase genes. This protein, however, was not secreted into the periplasmic space of the cells as was the beta-lactamase.