Sex chromosome markers: characterization using fluorescence in situ hybridization and review of the literature.

Fluorescence in situ hybridization (FISH) using biotin labeled X- and Y-chromosome DNA probes was utilized in the analysis of 23 sex chromosome-derived markers. Specimens were obtained through prenatal diagnosis, because of a presumptive diagnosis of Ullrich-Turner syndrome, mental retardation, and minor anomalies or ambiguous genitalia; three were spontaneous abortuses. Twelve markers were derived from the X chromosome and eleven from the Y chromosome; this demonstrates successfully the value and necessity of FISH utilizing DNA probes in the identification of sex chromosome markers. Both fresh and older slides, some of which had been previously G-banded, were used in these determinations. We have also reviewed the literature on sex chromosome markers identified using FISH.

Mosaic trisomy 16 ascertained through amniocentesis: evaluation of 11 new cases.

Trisomy 16, once thought to result uniformly in early pregnancy loss, has been detected in chorionic villus samples (CVS) from on-going pregnancies and was initially ascribed to a second, nonviable pregnancy. Prenatally detected trisomy 16 in CVS and its resolution to disomy has led to the reexamination of the viability of trisomy 16. This study evaluates 11 cases of mosaic trisomy 16 detected through second trimester amniocentesis. In 9 of the 11 cases, amniocenteses were performed in women under the age of 35 because of abnormal levels of maternal serum alpha-fetoprotein (MSAFP) or maternal serum human chorionic gonadotropin (MShCG). The other two amniocenteses were performed for advanced maternal age. Five of the 11 pregnancies resulted in liveborn infants, and six pregnancies were electively terminated. The liveborn infants all had some combination of intrauterine growth retardation (IUGR), congenital heart defects (CHD), or minor anomalies. Two of them died neonatally because of complications of severe congenital heart defects. The three surviving children have variable growth retardation, developmental delay, congenital anomalies, and/or minor anomalies. In the terminated pregnancies, the four fetuses evaluated by ultrasound or autopsy demonstrated various congenital anomalies and/or IUGR. Cytogenetic and fluorescent in situ hybridization studies identified true mosaicism in 5 of 10 cases examined, although the abnormal cell line was never seen in more than 1% of cultured lymphocytes. Placental mosaicism was seen in all placentas examined and was associated with IUGR in four of seven cases. Maternal uniparental disomy was identified in three cases. Mosaic trisomy 16 detected through amniocentesis is not a benign finding but associated with a high risk of abnormal outcome, most commonly IUGR, CHD, developmental delay, and minor anomalies. The various outcomes may reflect the diversity of mechanisms involved in the resolution of this abnormality. As 80% of these patients were ascertained because of the presence of abnormal levels of MSAFP or MShCG, the increased use of maternal serum screening should bring more such cases to clinical attention.

Pneumomediastinum in pregnancy: two case reports and a review of the literature, pathophysiology, and management.

Pneumomediastinum, free air trapped in the mediastinal connective tissue, is a rare complication of pregnancy, occurring most frequently in the second stage of labor. Symptoms are often not noted until after delivery. Occurrence before and in the first stage of labor, as seen in the two cases reported here, is more uncommon. One case history is the first report of the coexistence of pneumomediastinum and pneumothorax in pregnancy. The prognosis for spontaneous pneumomediastinum in pregnancy is favorable. Pathophysiologic mechanisms, diagnosis, and management are discussed, and a review of the literature is presented.

Prospects for human gene therapy.

Retroviral vectors containing marker genes and the sequence for human proteins have been used to transduce cultured lymphocytes, which have then been reinfused into patients. Circulating hematopoietic progenitor cells from human fetal cord blood obtained at the time of term and premature deliveries as early as 19 weeks of gestation have been shown to express such transduced genes in vitro. Cord blood cells from fetal sheep sampled and transduced ex vivo and transfused back in utero expressed marker genes up to two years after birth. Although the efficiency of gene transfer into cells and their long-term expression need to be improved, the potential exists for treating some genetic diseases after prenatal diagnosis either in utero or shortly after birth.

Prospects for gene therapy.

Experiments in animal models and human cells in vitro suggest that gene transfer using retroviral vectors may be useful to treat genetic diseases and to gain information that may improve treatment of other common diseases such as cancer. The approach to treatment of genetic diseases by inserting genes into bone marrow cells and experimental models, and a novel application of gene transfer technology to cancer research are discussed herein.

p-Isothiocyanatophenyl 6-phospho-alpha-D-mannopyranoside coupled to albumin. A model compound recognized by the fibroblast lysosomal enzyme uptake system. 1. Chemical synthesis and characterization.

We have developed a simple synthesis for a conjugate of albumin and isothio-nitrophenyl alpha-D-mannopyranoside to study the requirements of the fibroblast lysosomal enzyme recognition system. p-aminophenyl 6-phospho-alpha-D-mannopyranoside was prepared in two ways: (1) phosphorylation of isothio-nitrophenyl alpha-D-mannopyranoside and subsequent reduction of the nitro group by catalytic hydrogenation and (2) direct phosphorylation of p-aminophenyl alpha-D-mannopyranoside. Mannosides were phosphorylated in a reaction with phosphoryl chloride, pyridine, and water at 0 degrees C for 1 h, by a procedure selective for primary hydroxyl groups. Purified p-a minophenyl 6-phospho-alpha-D-mannopyranoside was characterized by chromatographic, enzymatic, and 13C nuclear magnetic resonance spectroscopic methods. Isothio-Isothiocyanatophenyl 6-phospho-alpha-D-mannopyranoside and the p-isothiocyanatophenyl glycosides of alpha-mannose, alpha-glucose, alpha- and beta-galactose, and alpha-L-fucose were formed by reaction of the respective p-aminophenyl glycosides with thiophosgene. Incubation of the p-isothiocyanatophenyl glycosides with bovine serum albumin at pH 8.5, 25 degrees C, for 18 h generally resulted in the coupling, primarily through lysine residues, of up to 20-30 mol of glycoside per mol of protein. Biological properties of the conjugates in the fibroblast lysosomal enzyme recognition system are described in the accompanying paper.

Biosynthesis of yeast mannan. Properties of a mannosylphosphate transferase in Saccharomyces cerevisiae.

A homogenate of mechanically broken, freshly grown Saccharomyces cerevisiae X2180 cells catalyzes the transfer of mannosylphosphate units from guanosine diphosphate mannose to reduced alpha1 leads to 2-[3H]mannotetraose to yield reduced mannosylphosphoryl [3H]-mannotetraose. The product is analogous in structure to the phosphorylated mannan side chains, which suggests that the enzymic activity is involved in mannoprotein biosynthesis in the intact cell. The mannosylphosphate transferase activity, localized in a membrane fraction obtained by differential centrifugation at 100,000 x g, was solubilized by Triton X-155 and purified 250-fold by ammonium sulfate precipitation and by ion exchange and gell filtration chromatographies. The enzyme requires MN2+ OR Co2+ ions for activity and is stimulated by various detergents. The mnn2 and mnn3 mannan mutants of S. cerevisiae possess normal levels of mannosylphosphate transferase activity, whereas the mnn4 mutant cells contain very low, if any, activity. This is consistent with a previous conclusion that the mnn4 mutation affects the mannosylphosphate transferase activity, whereas the mnn2 and mnn3 strains possess phosphate-deficient mannans because they are unable to synthesize the appropriate side chain precursors. A new mannan mutant class with the mnn4 chemotype was isolated, but the mutation proved to be recessive and nonallelic with the mnn4 locus. This new locus is designated mnn6.

p-Isothiocyanatophenyl 6-phospho-alpha-D-mannopyranoside coupled to albumin. A model compound recognized by the fibroblast lysosomal enzyme uptake system. 2. Biological properties.

A conjugate of p-aminophenyl 6-phospho-alpha-D-mannopyranoside and bovine serum albumin was shown to interact with the uptake system for lysosomal enzymes in cultured human diploid fibroblasts. Radioiodinated conjugate containing 20 mol of mannose 6-phosphate/mol of albumin was taken up by the cells and degraded to trichloroacetic acid soluble fragments which were released into the medium. Unlabeled conjugate, mannose 6-phosphate, and a lysosomal enzyme, L-iduronidase, inhibited the uptake of the 125I-labeled conjugate (Ki = 2 X 10(-8), 5 X 10(-6), and 1.5 X 10(-9) M, respectively). Conversely, the uptake of L-iduronidase was competitively inhibited by the mannose 6-phosphate conjugate as well as by free mannose 6-phosphate; however, higher concentrations of these compounds were required (Ki = 10(-6) and 5 X 10(-5) M, respectively). These results suggest that although L-iduronidase and the conjugate are bound to the same receptor by mannose 6-phosphate residues, the uptake of the enzyme involves some additional structure that is not shared by the conjugate. Internalization of the radiolabeled mannose 6-phosphate albumin conjugate was observed only in human diploid fibroblast strains. An SV-40 transformed line of human fibroblasts as well as three permanent rodent fibroblast lines (CHO, NRK, and L cells) failed to take up the conjugate, presumably because they were deficient in receptors or in the ability to internalize receptor-conjugate complexes.

Evidence for structural heterogeneity from molecular cytogenetic analysis of dicentric Robertsonian translocations.

Most Robertsonian translocations are dicentric, suggesting that the location of chromosomal breaks leading to their formation occur in the acrocentric short arm. Previous cytogenetic and molecular cytogenetic studies have shown that few Robertsonian translocations retain ribosomal genes or beta-satellite DNA. Breakpoints in satellite III DNA, specifically between two chromosome 14-specific subfamilies, pTRS-47 and pTRS-63, have been indicated for most of the dicentric 14q21q and 13q14q translocations that have been studied. We have analyzed the structure of 36 dicentric translocations, using several repetitive DNA probes that localize to the acrocentric short arm. The majority of the translocations retained satellite III DNA, while others proved variable in structure. Of 10 14q21q translocations analyzed, satellite III DNA was undetected in 1; 6 retained one satellite III DNA subfamily, pTRS-47; and 3 appeared to contain two 14-specific satellite III DNA sub-families, pTRS-47 and pTRS-63. In 10/11 translocations involving chromosome 15, the presence of satellite III DNA was observed. Our results show that various regions of the acrocentric short arm, and, particularly, satellite III DNA sequences, are involved in the formation of Robertsonian translocations.

Retrovirus-mediated transfer and expression of an allogeneic major histocompatibility complex class II DRB cDNA in swine bone marrow cultures.

The possibility of inducing transplantation tolerance by somatic gene transfer is under investigation in our miniature swine model. As a crucial step in this project, we have used a retroviral vector engineered to express both a drug-resistance gene (Neo) and a swine class II DRB cDNA to transduce porcine bone marrow (BM) cells. Analysis of cultured swine fibroblasts exposed to high-titer viral supernatants demonstrated that drug resistance had been conferred and that transferred vector sequences were transcribed appropriately. Similar transduction studies with swine BM demonstrated the transfer of drug resistance to as high as 14% of colony-forming unit-granulocyte-macrophage (CFU-GM). Using polymerase chain reaction (PCR) of cDNA, vector-derived allogeneic DRB transcripts were detected in colonies derived from primitive CFU-Mix and high proliferative potential-colony-forming cell (HPP-CFC), as well as in drug-resistant GM colonies grown from transduced bone marrow (BM) maintained in long-term BM cultures (LTBMCs) for up to 5 weeks. These results indicate that a significant proportion of both colony-forming progenitors and LTBMC-initiating cells were transduced with the DRB-recombinant retroviral vector and that both vector-derived genes were expressed in the differentiated progeny of these cells.

Type 2 Gaucher disease with hydrops fetalis in an Ashkenazi Jewish family resulting from a novel recombinant allele and a rare splice junction mutation in the glucocerebrosidase locus.

Gaucher disease, the deficiency of the lysosomal enzyme glucocerebrosidase (EC 3.2.1.45), is frequently encountered in the Ashkenazi Jewish population. Carrier screening for Gaucher disease by enzyme analysis performed during a routine pregnancy indicated that both Ashkenazi parents were carriers. Screening for four common Gaucher mutations was subsequently performed on fetal and parental DNA. None of the common Ashkenazi mutations were identified. However, when exons 9-11 were amplified and digested with NciI to detect the L444P mutation, it appeared that the mother and the fetus had an unusual allele and that the expected paternal allele was not present. When the fetal amniocytes were found to have less than 2% of the normal glucocerebrosidase activity and a fetal sonogram revealed hydrops fetalis, the pregnancy was terminated. The diagnosis of severe type 2 Gaucher disease was confirmed at autopsy. Ultrastructural studies of epidermis from the fetus revealed the characteristic disruption of lamellar bilayers, diagnostic for type 2 Gaucher disease. In subsequent studies of the fetal DNA, long-template polymerase chain reaction amplification revealed one appropriately sized band (approximately 6.5 kb) and one smaller (approximately 5.2 kb) band. Sequencing of the approximately 5.2-kb fragment identified a novel fusion allele resulting from recombination between the glucocerebrosidase gene and its pseudogene beginning in intron 3. This fusion allele was inherited from the father. The result was confirmed by Southern blot analysis using the enzyme S8tII. Sequencing of the 6.5-kb fragment identified a previously described, although rare, T-to-G splice junction mutation in intron 10 of the maternal allele, which introduced an NciI site. The couple had a subsequent pregnancy which was also found to be affected. This case study identifies a novel recombinant allele and an unusual splice junction mutation, and demonstrates that even in the Ashkenazi population, screening for common mutations may not accurately identify the most severe forms of the disease.

Gene therapy: efforts at developing large animal models for autologous bone marrow transplant and gene transfer with retroviral vectors.

Two new large animal models, non-human primates and fetal sheep, have been developed in an effort to determine the feasibility of using retroviruses for gene therapy. The retroviral vectors N2 and SAX have been used to introduce the genes for neomycin phosphotransferase (neoR, conferring resistance to the antibiotic G418) and human adenosine deaminase (ADA; EC 3.5.4.17), respectively. Varying levels of human ADA activity have been detected in six of the eight SAX-treated monkeys analysed. In the monkey with the greatest activity, human ADA levels approximately 0.5% of endogenous monkey ADA levels were detected. By in situ hybridization, roughly one in 100 bone marrow cells were found to express vector DNA. Sheep have been used for studies of the infectability of fetal blood progenitors in vivo. Blood cells were treated with the N2 vector at the 96th day of gestation, and marrow cells were assayed for the presence of G418-resistant haematopoietic progenitors, starting from one week after birth (62 days after treatment). Up to 33% of colony-forming progenitors were drug resistant initially and, although the proportion of resistant colony-forming units declined, a level of 10% has been found 153 days after transplantation. Human bone marrow has also been treated with the N2 vector, resulting in 1-2% G418-resistant progenitors.

Gene transfer into humans--immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction.

BACKGROUND AND METHODS: Treatment with tumor-infiltrating lymphocytes (TIL) plus interleukin-2 can mediate the regression of metastatic melanoma in approximately half of patients. To optimize this treatment approach and define the in vivo distribution and survival of TIL, we used retroviral-mediated gene transduction to introduce the gene coding for resistance to neomycin into human TIL before their infusion into patients--thus using the new gene as a marker for the infused cells. RESULTS: Five patients received the gene-modified TIL. All the patients tolerated the treatment well, and no side effects due to the gene transduction were noted. The presence and expression of the neomycin-resistance gene were demonstrated in TIL from all the patients with Southern blot analysis and enzymatic assay for the neomycin phosphotransferase coded by the bacterial gene. Cells from four of the five patients grew successfully in high concentrations of G418, a neomycin analogue otherwise toxic to eukaryotic cells. With polymerase-chain-reaction analysis, gene-modified cells were consistently found in the circulation of all five patients for three weeks and for as long as two months in two patients. Cells were recovered from tumor deposits as much as 64 days after cell administration. The procedure was safe according to all criteria, including the absence of infections virus in TIL and in the patients. CONCLUSIONS: These studies demonstrate the feasibility and safety of using retroviral gene transduction for human gene therapy and have implications for the design of TIL with improved antitumor potency, as well as for the possible use of lymphocytes for the gene therapy of other diseases.