Validation of DNA test for hip dysplasia failed in Danish Labrador Retrievers.

Canine hip dysplasia is characterized by poor hip joint conformation and laxity. The disease is a complex trait influenced by both genetics and environment. Diagnosis and quantification of hip dysplasia are performed by radiographic examination of the hip joint and the diagnosis is used for making breeding decisions in many breeds. A prognostic genetic test (the Dysgen test) based on seven associated SNPs has been developed in a study based on Spanish Labrador Retrievers. In our study this test has been evaluated in 39 Danish Labrador Retrievers with known radiographic hip score: 14 with hip dysplasia (grade D or E) and 25 without hip dysplasia (grade A or B). There was no significant correlation between the Dysgen test results and the radiographic hip status (P = 0.3203) in these dogs, indicating that Dysgen test results obtained for Danish Labrador Retrievers have no prognostic value.

Efficient retrovirus-mediated transfer of the multidrug resistance 1 gene into autologous human long-term repopulating hematopoietic stem cells.

Pre-clinical studies indicate that efficient retrovirus-mediated gene transfer into hematopoietic stem cells and progenitor cells can be achieved by co-localizing retroviral particles and target cells on specific adhesion domains of fibronectin. In this pilot study, we used this technique to transfer the human multidrug resistance 1 gene into stem and progenitor cells of patients with germ cell tumors undergoing autologous transplantation. There was efficient gene transfer into stem and progenitor cells in the presence of recombinant fibronectin fragment CH-296. The infusion of these cells was associated with no harmful effects and led to prompt hematopoietic recovery. There was in vivo vector expression, but it may have been limited by the high rate of aberrant splicing of the multidrug resistance 1 gene in the vector. Gene marking has persisted more than a year at levels higher than previously reported in humans.

Globin composition and synthesis of hemoglobins in developing fetal mice erythroid cells.

Fetal mouse erythropoiesis proceeds initially in yolk-sac blood islands (8 to 12 days) and, subsequently, in liver (12 to at least 16 days). Yolksac cells synthesize three hemoglobins, Hb E(I), Hb E(II) and Hb E(III). Hb E(I) has x- and y-globin chains; Hb E(II) has alpha and y; HB E(III), alpha and z. No detectable beta-globin is formed in these cells. Liver erythroid cells form only adult hemoglobin, composed of alpha- and beta-chains.

Disorders of human hemoglobin.

Studies of the human hemoglobin system have provided new insights into the regulation of expression of a group of linked human genes, the gamma-delta-beta-globin gene complex in man. In particular, the thalassemia syndromes and related disorders of man are inherited anemias that provide mutations for the study of the regulation of globin gene expression. New methods, including restriction enzyme analysis and cloning of cellular DNA, have made it feasible to define more precisely the structure and organization of the globin genes in cellular DNA. Deletions of specific globin gene fragments have already been found in certain of these disorders and have been applied in prenatal diagnosis.

Hemoglobin synthesis in beta-thalassemia: the properties of the free alpha-chains.

The decrease in hemoglobin A (HbA, alpha(2)beta(2)) synthesis in the erythroid cells of patients with beta-thalassemia is due to a selective defect in beta-chain synthesis. Since alpha-chains continue to be formed at a normal rate in these cells, this results in a marked relative excess of alpha-chain synthesis over beta- and gamma-chain synthesis. The alpha-chains uncombined with beta- or beta-like-chains (delta, gamma) will be referred to as free alpha-chains. The experiments presented in this paper show that these free alpha-chains are capable of combining with beta-chains to form HbA and are, therefore, structurally normal. Alternatively, in the absence of added beta-chains, alpha-chains aggregates of various sizes are formed. Peripheral blood from patients with beta-thalassemia was incubated with radioactive amino acids and hemolysates were prepared. Column chromatography demonstrates that a majority of the free alpha-chains are not present in HbA. They are strongly bound to carboxymethylcellulose resin at pHs from 7.0 to 10.0, and do not elute with HbA. However, when chemically prepared hemoglobin H (Hbbeta(4)) is added to the fresh hemolysates, the free alpha-chains are readily recovered in the HbA peak. This indicates that the free alpha-chains are able to combine normally with beta-chains to form HbA. Freshly labeled hemolysates were also subjected to Sephadex G-100 chromatography. The free alpha-chains eluted as a broad peak migrating between myoglobin and hemoglobin, consistent with their forming alpha-chain aggregates of various mol wt between 16,000 and 64,000. It is suggested that the chromatographic behavior of the free alpha-chains reported herein simply reflects the chemical properties of normal alpha-chains in the absence of adequate numbers of beta- or gamma-chains. The tendency of these free alpha-chains to aggregate may lead to their intracellular precipitation and the subsequent destruction of the cells containing them.

Localization of the site of recombination in formation of the Lepore Boston globin gene.

The site of crossover between the delta- and beta-globin gene sequences resulting in Lepore Boston globin gene formation has been localized at the DNA Level. Probes specific for detecting the intervening sequences (IVS) of the delta- and beta-globin genes were used to map the origin of cellular DNA fragments of a patient homozygous for hemoglobin Lepore Boston. Restriction endonuclease analysis and hybridization of this DNA to IVS specific probes show that most, if not all, of the large intervening sequences (IVS 2) in Lepore DNA are derived from the beta-globin gene IVS 2. In addition, the crossover occurs in a region of DNA in which the delta- and beta-genes have almost complete nucleotide homology, and might be expected to pair most tightly during meiosis.

Heterogeneity of DNA fragments associated with the sickle-globin gene.

We have examined the genetic polymorphism previously reported to be associated with the sickle-cell (beta s) gene. The polymorphism involves an alteration of the DNA sequence 3' to the beta-globin gene as detected with the restriction endonuclease, Hpa I. In normal individuals, the beta-globin gene is contained within a DNA fragment of 7.6 kilobases (kb), whereas 87% of individuals with sickle-cell anemia have been reported to have the beta s-gene associated with a 13.0-kb Hpa I fragment. We have studied this polymorphism in 31 New York Black individuals homozygous for sickle-cell anemia to ascertain its genetic and biochemical significance and to evaluate its potential use in the prenatal diagnosis of sickle-cell disease. Our results show only a 58% association of the beta s-gene and the 13.0-kb Hpa I fragment, as well as the presence of additional variants involving the Hpa I site. In addition, the 13.0-kb fragment is also found associated with the beta c- and beta A-genes. Thus, the Hpa I polymorphism probably represents a change in DNA not specifically associated with the beta s-gene, and appears to antedate the beta s-and beta c-mutations.

Quantitation of human gamma globin genes and gamma globin mRNA with purified gamma globin complementary DNA.

Complementary DNA (cDNA) specific for gamma-globin nucleotide sequences has been prepared by hybridizing total cDNA made from cord blood messenger RNA (mRNA) as template to an excess of normal adult human globin mRNA and recovering the single-stranded cDNA from hydroxylapatite. The specificity of the gamma cDNA for gamma mRNA sequences is strongly supported by the hybridization of this cDNA at low Cot values (Co, concentration of RNA and t, time in seconds) to RNA samples containing large amounts of functional gamma globin mRNA and the lack of hybridization to RNA samples containing little, if any, gamma-globin mRNA. The absence of cross-hybridization of gamma cDNA with alpha, beta, and delta mRNAs is demonstrated by the complete hybridization of the gamma cDNA to mRNA samples completely lacking either alpha or beta and delta mRNA. An estimate of the number of gamma-globin genes in human cellular DNA was obtained by hybridization of purified gamma cDNA to DNA from spleen and white blood cells of normal and beta-thalassemia subjects and measurement of the percent of gamma cDNA hybridized at saturation. The results indicate that there are between one and two gamma-globin genes per total haploid gene DNA equivalent obtained from both normal and beta-thalassemia subjects. These values are consistent with genetic evidence for the presence of multiple gamma gene loci in human cells. The finding that the number of gamma-globin genes in beta-thalassemia DNA is similar to that in nonthalassemia DNA indicates that a deletion of gamma-globin genes cannot account for either the inadequate gamma-globin synthesis or indirectly for the decreased or absent beta-globin synthesis in beta-thalassemia cells.

Preferential binding of beta s globin chains associated with stroma in sickle cell disorders.

Sickle cell anemia (SS) is associated with abnormalities of the red cell membrane and decreased red cell deformability. The present study assesses globin chain binding to stroma in SS, sickle cell trait (AS), and nonsickling (AA) cells. The results indicate that there is preferential binding of newly synthesized beta(S) globin to red cell stroma in SS cells and preferential binding of beta(S) to stroma compared to beta(A) in AS cells. These studies show that beta(S) globin binding to stroma accompanies the membrane abnormalities in SS and AS patients.

Balanced globin chain synthesis in hereditary persistence of fetal hemoglobin.

In two black families with the hereditary persistence of fetal hemoglobin (HPFH) gene there are eight A-F heterozygotes and two double heterozygotes for sickle cell trait and HPFH. These patients are clinically asymptomatic and have homogeneous acid elution smears. Measurement of globin chain synthesis in peripheral blood demonstrates balanced production of a alpha and non-alpha (beta plus gamma) chains. In these patients, the balance is achieved by increased gamma globin production and increased activity of the remaining beta globin allele. In two patients, one A-F and the other S-F there is also balanced globin synthesis in the bone marrow. In a double heterozygote for HPFH and beta-thalassemia, anemia (Hb: 11.5 g/100 ml) is associated with a moderate degree of globin chain imbalance. There is a correlation between balanced globin chain synthesis and the absence of anemia in patients with HPFH.

A new polymorphism in the human beta-globin gene useful in antenatal diagnosis.

A new polymorphism in the beta-globin is described, using the restriction enzyme Asu I. A radioactive probe specifically representing the large intervening sequence (IVS 2) of the beta-globin gene has been used to detect this polymorphism. Normally, a 0.8-kilobase fragment containing beta-IVS 2 is generated by Asu I; however, a 1.0-kilobase fragment is seen in association with 18% of beta A-genes, and 38% of beta-thalassemia genes in an Israeli population studied. By contrast, the Asu I polymorphism has rarely been seen in blacks examined to date. An additional Asu I change is seen the the delta-globin gene with a delta-IVS probe. The beta-Asu I polymorphism is shown to be useful in the antenatal diagnosis of beta-thalassemia.

Translation and stability of human globin mRNA in Xenopus oocytes.

Human globin messenger RNA (mRNA) prepared from erythroid cells of patients with sickle cell anemia has been translated in Xenopus laevis oocytes. Addition of hemin to the injected mRNA causes total globin synthesis to increase and the ratio of alpha- to betas-globin synthesis (alpha/betas ratio) to approach unity. To determine the effect of the length of the poly-(A) segment on human globin mRNA stability, 10 S globin mRNA was fractionated into poly-(A)-poor fractions by oligo (dT)-cellulose column chromatography. When oocytes are injected with each of these fractions, translation of the poly-(A)-rich globin mRNA is sustained for a longer period than that of the poly-(A)-poor mRNA. Regardless of the mRNA fraction injected, the alpha/betas ratio of the synthesized globin decreases as the injected oocytes are incubated for longer periods. The results indicate that in frog oocytes poly-(A)-rich mRNA has greater translational stability than poly-(A)-poor mRNA, AND beta-mRNA has greater stability than alpha-mRNA with comparable poly-(A) content.

Diminished synthesis of an alpha chain mutant, hemoglobin I (alpha-16 lys leads to glu).

In patients heterozygous for abnormal hemoglobins there is usually less than 50% of the mutant hemoglobin present in peripheral blood. The synthetic rates of alpha-chain mutants compared to alpha(A) have not been reported to date. In this study the production of alpha(A)- and alpha(I)-chains has been measured in peripheral blood and bone marrow of two patients with approximately 30% hemoglobin I, an alpha-chain abnormality (alpha(16 lys --> glu)). The results suggest that the decreased amount of alpha(I) compared to alpha(A) is due solely to diminished biosynthesis of the alpha(I)-chains. The relative rates of synthesis of alpha(I)- and alpha(A)-chains are similar in both nucleated red cells and reticulocytes indicating that no change occurs during erythroid cell maturation which preferentially affects either alpha(I) or alpha(A) production.

Defects in DNA and globin messenger RNA in homozygotes for hemoglobin Lepore.

Globin messenger RNA (mRNA) isolated from three patients homozygous for hemoglobin Lepore is shown to have a marked reduction of the amount of beta-like globin mRNA (Lepore-globin mRNA sequences) compared with alpha-globin mRNA by molecular hybridization. The relative amounts of alpha- and Lepore mRNA are similar to the amounts of alpha- and Lepore globin synthesized in intact cells and by isolated mRNA in a cell-free system. It is also demonstrated that Lepore-globin mRNA can completely hybridize to full-length or nearly full-length beta-globin specific complementary DNA and protect it from nuclease digestion, indicating close homology between the delta-mRNA sequences present in Lepore mRNA and the beta-complementary-DNA probe. We have also quantitated the numbers of beta-like globin gene sequences in genomic Lepore DNA by molecular hybridization and demonstrated a reduction in their number consistent with the Lepore gene being a delta beta-gene fusion product.

Developmental-stage-specific expression and regulation of an amphotropic retroviral receptor in hematopoietic cells.

Expression of the transmembrane receptor protein Ram-1 may be critical to optimizing retroviral gene transfer. Ram-1 acts as both a sodium-dependent phosphate transporter and a receptor for amphotropic retroviruses. We previously reported detectable Ram-1 in murine hematopoietic fetal liver cells (FLC) despite resistance of these cells to amphotropic retroviral transduction (infection). We document here that Ram-1 expression is completely absent in murine yolk sac cells from days 9.5 through 13.5 of ontogeny and first appears at low levels in midgestational FLC between days 13.5 and 14.5. In addition, Ram-1 expression is detected only in more differentiated populations within FLC, day 14.5, and not in those highly enriched for stem cells, indicating developmental regulation of Ram-1 during murine hematopoiesis. Others have reported the in vitro use of phosphate-free medium as a stimulus to increase levels of Ram-1 mRNA in nonhematopoietic cells. We now demonstrate that Ram-1 poly(A)+ mRNA increases significantly following culture of FLC in phosphate-free medium. Further, transduction of FLC in phosphate-free medium with an amphotropic retrovirus containing the multiple drug resistance gene leads to gene transfer not observed previously. These data demonstrate that (i) the normal resistance of FLC to amphotropic transduction is most likely due to an insufficient number of Ram-1 molecules for efficient retroviral recognition and binding, and (ii) Ram-1 can be upregulated by increasing the need for phosphate transport across the cell membrane.

Erythroleukemia (K562) cells contain a functional beta-globin gene.

K562 cells are human erythroid cells that synthesize embryonic and fetal globins but not adult beta-globin. A cloned beta-globin gene was isolated from K562 cells and transfected into HeLa cells. The RNA transcripts produced were comparable in both amount and size to those obtained with a normal beta-globin gene.

An ikaros-containing chromatin-remodeling complex in adult-type erythroid cells.

We have previously described a SWI/SNF-related protein complex (PYR complex) that is restricted to definitive (adult-type) hematopoietic cells and that specifically binds DNA sequences containing long stretches of pyrimidines. Deletion of an intergenic DNA-binding site for this complex from a human beta-globin locus construct results in delayed human gamma- to beta-globin switching in transgenic mice, suggesting that the PYR complex acts to facilitate the switch. We now show that PYR complex DNA-binding activity also copurifies with subunits of a second type of chromatin-remodeling complex, nucleosome-remodeling deacetylase (NuRD), that has been shown to have both nucleosome-remodeling and histone deacetylase activities. Gel supershift assays using antibodies to the ATPase-helicase subunit of the NuRD complex, Mi-2 (CHD4), confirm that Mi-2 is a component of the PYR complex. In addition, we show that the hematopoietic cell-restricted zinc finger protein Ikaros copurifies with PYR complex DNA-binding activity and that antibodies to Ikaros also supershift the complex. We also show that NuRD and SWI/SNF components coimmunopurify with each other as well as with Ikaros. Competition gel shift experiments using partially purified PYR complex and recombinant Ikaros protein indicate that Ikaros functions as a DNA-binding subunit of the PYR complex. Our results suggest that Ikaros targets two types of chromatin-remodeling factors-activators (SWI/SNF) and repressors (NuRD)-in a single complex (PYR complex) to the beta-globin locus in adult erythroid cells. At the time of the switch from fetal to adult globin production, the PYR complex is assembled and may function to repress gamma-globin gene expression and facilitate gamma- to beta-globin switching.

Restriction endonuclease sensitivity of DNA containing globin genes in different murine erythroleukemia cell lines.

The arrangement of the globin structural genes has been examined in murine erythroleukemia cells, strain DS19, and several related inducer-resistant variant cell lines. One fragment larger than 20 kilobases and six globin gene-containing fragments between 10 and 1.9 kilobases in size are detected in EcoRI-cleaved purified DNA prepared from strain DS19. By comparison, when isolated nuclei are digested with EcoRI, only two globin gene-containing fragments are detected, one greater than 20 kilobases and the other 1.9 kilobases. Of seven cell lines derived from DS19 and resistant to inducers, six had similar patterns to DS19 of globin gene-containing EcoRI-generated DNA fragments from nuclei and from purified DNA. One cell line, DR10, a DMSo-resistant cell line, lacks the 1.9 kilobase fragment after digestion of either nuclei or purified DNA. The 1.9 kilobase fragment hybridizes with alpha-globin cDNA but not with the beta-globin cDNA, suggesting either rearrangement or deletion of an alpha-globin gene-like fragment in DR10 DNA.

In vivo human brachial artery elastic mechanics: effects of smooth muscle relaxation.

BACKGROUND: The effects of smooth muscle relaxation on arterial wall mechanics are controversial. We used a new, in vivo, noninvasive technique to measure brachial artery wall mechanics under baseline conditions and following smooth muscle relaxation with nitroglycerin (NTG). METHODS AND RESULTS: Eight healthy, normal subjects (6 male, 2 female; age 30+/-3.1 years) participated in the study. The nondominant brachial artery was imaged through a water-filled blood pressure cuff using an external ultrasound wall-tracking system at baseline and following 0.4 mg sublingual NTG. Simultaneous radial artery pressure waveforms were recorded by tonometry. Transmural pressure (TP) was reduced by increasing water pressure in the cuff. Brachial artery area, unstressed area, compliance, stress, strain, incremental elastic modulus (Einc), and pulse wave velocity (PWV) were measured over a TP range from 0 to 100 mm Hg. Baseline area versus TP curves generated 30 minutes apart were not significantly different. NTG significantly shifted area versus TP (P<0.0001) and compliance versus TP (P<0.001) curves upward, whereas the Einc versus TP (P<0.05) and PWV versus TP (P<0. 01) curves were shifted downward. NTG also significantly shifted stress versus strain (P<0.01) and Einc versus strain (P<0.01) curves to the right. CONCLUSIONS: We conclude that brachial artery elastic mechanics can be reproducibly measured over a wide range of TP and smooth muscle tone using a new noninvasive ultrasound technique. Smooth muscle relaxation with NTG increases isobaric compliance and decreases isobaric Einc and PWV in the human brachial artery.

Phase I trial of retroviral-mediated transfer of the human MDR1 gene as marrow chemoprotection in patients undergoing high-dose chemotherapy and autologous stem-cell transplantation.

PURPOSE: Normal bone marrow cells have little or no expression of the MDR p-glycoprotein product and, therefore, are particularly susceptible to killing by MDR-sensitive drugs, such as vinca alkaloids, anthracyclines, podophyllins, and paclitaxel and its congeners. Here we report the results of a phase I clinical trial that tested the safety and efficacy of transfer of the human multiple drug resistance (MDR1, MDR) gene into hematopoietic stem cells and progenitors in bone marrow as a means of providing resistance of these cells to the toxic effects of cancer chemotherapy. PATIENTS AND METHODS: Up to one third of the harvested cells of patients who were undergoing autologous bone marrow transplantation as part of a high-dose chemotherapy treatment for advanced cancer were transduced with an MDR cDNA-containing retrovirus; these transduced cells were reinfused together with unmanipulated cells after chemotherapy. RESULTS: High-level MDR transduction of erythroid burst-forming unit (BFU-E) and colony-forming unit-granulocyte macrophage (CFU-GM) derived from transduced CD34+ cells was shown posttransduction and prereinfusion. However, only two of the five patients showed evidence of MDR transduction of their marrow at a low level at 10 weeks and 3 weeks, respectively, posttransplantation. The cytokine-stimulated transduced cells may be out-competed in repopulation by unmanipulated normal cells that are reinfused concomitantly. The MDR retroviral supernatant that was used was shown to be free of replication-competent retrovirus (RCR) before use, and all tests of patients' samples posttransplantation were negative for RCR. In addition, no adverse events with respect to marrow engraftment or other problems related to marrow transplantation were encountered. CONCLUSION: These results indicate the feasibility and safety of bone marrow gene therapy with a potentially therapeutic gene, the MDR gene.