An alpha-globin gene initiation codon mutation in a black family with HbH disease

The molecular basis of hemoglobin H disease in a Black family of Canadian origin was investigated. Affected individuals had a combination of deletion and nondeletion alpha-thalassemia mutations on different chromosomes. Cloning and sequencing of the DNA of one member with the nondeletion form revealed a new thalassemia mutation, an A---- G substitution, in the initiation codon of the remaining alpha-globin gene of a rightward (-alpha 3.7) deletion chromosome. This mutation abolished an Ncol restriction site and therefore is detectable in genomic DNA by Southern blot analysis.     

Activation of phenotypic expression of human globin genes from nonerythroid cells by chromosome-dependent transfer to tetraploid mouse erythroleukemia cells

Chromosome-dependent gene transfer mediated by cell fusion was used to show that it is possible to activate phenotypic expression of human alpha globin genes derived from nonerythroid cells. Hybrid cells containing the human alpha globin structural genes were derived by fusion of populations of adult human peripheral blood mononuclear cells (devoid of identifiable erythroid cells) with adenine phosphoribosyl-transferase-deficient mouse erythroleukemia cells that contained close to a tetraploid complement of mouse chromosomes. The hybrid cells retained a near tetraploid complement of mouse chromosomes but had lost 80% of the chromosomes of the human parent cell. All of these hybrid cells and their subclones, which contained human chromosome 16, exhibited synthesis of human alpha globin chains. Human alpha globin mRNA was also demonstrated to be present in one of these hybrid cells by RNA.cDNA molecular hybridization analysis. We conclude that the mechanism responsible for restricting expression of the human globin genes in nonerthroid cells is not irreversible, at least for those globin structural genes that are actively transcribed in erythroid cells during adult life. Moreover, some genetic factor or process in the tetraploid mouse erythroleukemia cell is, under the conditions of our experiments, capable of reactivating phenotypic expression (production of globin chains) of human globin genes derived from nonerythroid hematopoietic cells after chromosome-dependent gene transfer.     

Beta zero-thalassemia in association with a gamma-globin gene quadruplication

We have studied the hematology, hemoglobin composition, and globin gene arrangements in one young Turkish boy with a beta zero-thalassemia homozygosity and in 11 of his relatives. Evidence is presented that the chromosome with the beta zero-thalassemia determinant carries a gamma- globin gene quadruplication, perhaps in a -G gamma-G gamma-G gamma-A gamma-gene arrangement. The eight gamma-globin genes in this patient produced G gamma and A gamma chains in a 95 to 5 ratio, and nearly 99% of the patient's hemoglobin was of the fetal type. The clinical condition resembled that of a thalassemia intermedia. HbF levels in eight beta-thalassemia heterozygotes varied between 0.5 and 4.2% and the percentages of G gamma in this HbF averaged at 87% or 95%; this level is to some extent related to the haplotype of the normal chromosome. All subjects carried four alpha-globin genes; a new BglII polymorphism was observed within the psi alpha-globin gene.     

Deletion of the alpha-globin gene cluster as a cause of acquired alpha-thalassemia in myelodysplastic syndrome

Rarely, myelodysplastic syndrome (MDS) is complicated by an acquired form of alpha-thalassemia (alpha-thalassemia in myelodysplastic syndrome [ATMDS]) characterized by hypochromic, microcytic, anisopoikilocytic red blood cells with hemoglobin H (HbH) inclusions. Acquired mutations in ATRX, a chromatin remodeling gene, have recently been found in 12 patients with typical features of ATMDS, though they have not been detected in MDS patients with similar red blood cell findings but little HbH. The alpha-globin genes themselves have appeared normal in all ATMDS patients studied to date. Here we characterize the molecular defect in a unique MDS patient with rare HbH inclusions in which an abnormal clone lost a greater than 1.9-Mb segment of the telomeric region of the short arm of one allele of chromosome 16, including both alpha-globin genes. Red blood cell changes associated with this acquired somatic genotype (--/alpha alpha) are surprisingly severe, demonstrating that a minor globin chain imbalance may be unexpectedly deleterious during the abnormal erythropoiesis that occurs in the context of MDS.     

Mouse alpha-globin genes and alpha-globin-like pseudogenes are not syntenic.

A genetic polymorphism for a Bgl I endonuclease site near the alpha-globin-like pseudogene alpha-4 of C57BL/6 and C3H/HeN mice was used to show that alpha-4 was not affected by three independent mutations in which the adult globin genes alpha-1 and alpha-2 were deleted. These results indicated that alpha-4 might not be located adjacent to the adult alpha-globin genes on chromosome 11. Restriction endonuclease analysis of DNA of a primary clone of a Chinese hamster--mouse somatic cell hybrid that had lost mouse chromosomes 11 and 18 showed that this clone lacked the adult murine globin genes alpha-1 and alpha-2 but it did contain the alpha-globin-like pseudogenes alpha-3 and alpha-4. These results indicated that the adult alpha-globin genes and alpha-globin-like pseudogenes are not located on the same chromosome. Similar analyses of several other Chinese hamster--mouse somatic cell hybrids that had segregated other mouse chromosomes indicated that the alpha-globin-like pseudogenes alpha-3 and alpha-4 are located on mouse chromosomes 15 and 17, respectively. These data explain why alpha-3 and alpha-4 were not affected by the three independently induced deletion-type mutations that cause alpha-thalassemia in the mouse.     

Hemoglobin synthesis in somatic cell hybrids: globin gene expression in hybrids between mouse erythroleukemia and human marrow cells or fibroblasts.

Somatic cell hybrids were generated by fusion of mouse erythroleukemia cells either to mouse L cells (B82), human fibroblasts (W1-18 VA2), or human marrow fractions enriched in erythroblasts. The hybrid cells were examined for globin gene expression by benzidine staining to detect cytoplasmic hemoglobin, and by molecular hybridization of cellular RNA to globin complementary DNA (cDNA) to detect globin messenger RNA (MRNA). The fibroblast (human or mouse) times erythroleukemia cell hybrids grown in monolayer retained most of the chromosomes of each parent. Neither cytoplasmic hemoglobin nor globin mRNA was detected in dimethylsulfoxide-treated fibroblast times erythroleukemia hybrid cells, indicating extinction of hemoglobin synthesis prior to the formation of cytoplasmic mRNA. The human marrow times mouse erythroleukemia hybrid cells grown in suspension culture contained only a few human chromosomes and exhibited low levels of hemoglobin synthesis which were amplified by 2% dimethylsulfoxide. Mouse (but not human) globin mRNA was demonstrated in these hybrid cells. The results suggest that somatic cell hybrids may be useful in searching for genetic factors which regulate activity of the globin genes.     

Hemoglobin synthesis in somatic cell hybrids: coexpression of mouse with human or chinese hamster globin genes in interspecific somatic cell hybrids of mouse erythroleukemia cells.

Somatic cell hybrids were derived by fusion of mouse erythroleukemia cells with fractionated human marrow enriched in erythroblasts, or with chinese hamster fetal liver erythroid cells. Such interspecific hybrid cells, when isolated in suspension culture, had retained nearly all the mouse chromosomes and had lost most of the human or chinese hamster chromosomes. However, two such hybrids (one human, the other hamster) studied 4-6 weeks after fusion, were found to contain several non-mouse chromosomes. RNA extracted from the human marrow x erythroleukemia hybrid annealed equally to both human and mouse globin complementary DNA, indicating that coexpression of the globin genes of each species had occurred in the hybrid cells. Mouse and human mRNA were found to accumulate only after incubation of the cells in 2% dimethylsulfoxide. The chinese hamster x erythroleukemia hybrid appeared to contain a double complement of mouse chromosomes in addition to several chinese hamster chromosomes. After 7 days of incubation in 2% dimethylsulfoxide, [3H]leucine was incorporated into chinese hamster beta-globin and the mouse globin chains. Thus, globin genes from differentiated cells, when introduced into spontaneously proliferating erythroleukemia cells, may be expressed after exposure of the resulting hybrid cells to an agent capable of inducing hemoglobin synthesis in the erythroleukemia cell.     

The molecular basis of AE-Bart's disease

AE-Bart's disease is a thalassemia intermedia resulting from the interaction between alpha-thalassemia and heterozygous Hb E. In this study we analyzed the alpha-globin genes of 25 patients designated as AE-Bart's disease by starch gel electrophoresis. Twenty-one cases had Hb Constant Spring in addition to Hbs E + A + Bart's, and the remaining four cases had only Hbs E + A + Bart's. DNA mapping revealed the alpha-globin genotype of alpha-thalassemia-1/alpha-thalassemia-2 in four patients who had Hbs E + A + Bart's, whereas the alpha genotype of the remainder is alpha-thalassemia-1/nondeletion alpha-thalassemia. The nondeletion alpha-thalassemia is Hb Constant Spring as indicated by starch gel electrophoresis. Hematologic data and hemoglobin analysis showed that Constant Spring-AE-Bart's disease is a more severe clinical syndrome than AE-Bart's disease.     

Chromosomal localization of human β globin gene on human chromosome 11 in somatic cell hybrids

We have successfully used a DNA.cDNA molecular hybridization assay to directly determine the presence or absence of human beta globin gene sequences in 20 human-mouse somatic cell hybrids, each of which contained a different subset of human chromosomes. The assay is specific for the individual human globin genes and will detect the presence of a globin gene if the relevant chromosome is present in only 10% of the cells of a hybrid population. The content of human chromosomes in each hybrid clone was characterized by Giemsa 11 staining, Giemsa trypsin-Hoechst 33258 staining, and by the use of 22 independent isozyme markers for 17 different human chromosomes. All human chromosomes were present in one or more cell lines devoid of the human beta globin gene except for 6, 8, 9, 11, and 13. Among these latter chromosomes, only chromosome 11 was present in the six hybrid clones that contained the human beta globin gene. In fact, chromosome 11 was the only human chromosome that was present in all of the six hybrid clones found to be positive for the human beta globin gene. Two sister clones, 157-BNPT-1 and 157-BNPT-4, had similar subsets of human chromosomes except that 11 was present only in 157-BNPT-4. 157-BNPT-4 contained the human beta globin gene while 157-BNPT-1 did not. DNA from three hybrid lines was also annealed to purified human gamma globin cDNA; two lines positive for human beta globin gene sequences also contained human gamma globin gene sequences while one line was negative for both beta and gamma gene sequences. On the basis of these results, the human beta and gamma globin genes have been assigned to human chromosome 11.     

Genetics of type II glycogenosis: Assignment of the human gene for acid α-glucosidase to chromosome 17

We have studied somatic cell hybrids between thymidine kinase (EC 2.7.1.75) deficient mouse cells and human diploid fibroblasts for the expression of human acid alpha-glucosidase (EC 3.2.1.20). A deficiency in this enzyme is associated with the type II glycogenosis or Pompe disease. All 30 somatic cell hybrids selected in hypoxanthine/aminopterin/thymidine medium expressed human acid alpha-glucosidase and galactokinase (EC 2.7.1.6) and retained human chromosome 17; counterselection of the same hybrids in medium containing 5-bromodeoxyuridine resulted in the growth of hybrids that concordantly lost the expression of human acid alpha-glucosidase and galactokinase as well as human chromosome 17. Hybrids between thymidine kinase-deficient mouse cells and fibroblasts from a patient with Pompe disease that contained human chromosome 17 were found not to express human acid alpha-glucosidase. Because we have already shown that hybrids between mouse peritoneal macrophages and GM54VA simian virus 40-transformed human cells selectively retain human chromosome 17 and lose all other human chromosomes, we tested 13 independent mouse macrophage x GM54VA hybrid clones, including two that retained human chromosome 17 and no other human chromosomes, for the expression of human acid alpha-glucosidase and galactokinase. All 13 hybrid clones were found to express these human enzymes. Thus, we conclude that the gene coding for human acid alpha-glucosidase is located on human chromosome 17.     

Regional assignment of genes for human α-globin and phosphoglycollate phosphatase to the short arm of chromosome 16

The human α-globin and phosphoglycollate phosphatase (EC 3.1.3.18) genes have been regionally localized to the short arm of human chromosome 16 (HC16). This was accomplished by fusing mouse fibroblasts (A9) to human fibroblasts that contain a reciprocal translocation between the long arms of chromosomes 16 and 11. The murine A9 cells are deficient in adenine phosphoribosyltransferase (APRT), an enzyme present on the long arm of HC16 (HC16q). Hybrid cells were grown in selection culture medium that required the cells to retain human APRT. Therefore, the hybrids exhibited stable retention of the entire HC16 or the rearranged chromosome containing HC16q. We isolated five independent primary and secondary hybrid cell lines which retained either HC16 or HC16q at a high frequency. The presence of human α-globin genes in the various clones was established directly by DNA extraction and hybridization to a cDNA probe for human α-globin genes. Autoradiographs showed that hybrid cells containing the long arm, but not the short arm, of HC16 showed only the background mouse bands. Hybrid cells that retained the entire HC16 demonstrated the band(s) containing the human α-globin genes. Hybrid cells that contained HC16 with its α-globin genes were then placed in culture medium that contained diaminopurine, which is lethal for cells containing APRT. These counter-selected hybrid cells had lost HC16 and also lost the human α-globin genes as determined by blot hybridization. The presence of α-globin gene sequences in the hybrid clones was concordant with HC16 only and not with any other human chromosome. These results confirm the assignment of α-globin genes to HC16 and localize the genes to the short arm. We also assign the locus for phosphoglycollate to the short arm of HC16.     

The expression of human α-like globin genes in transgenic mice mediated by bacterial artificial chromosome

After screening a bacterial artificial chromosome of human genomic DNA library with human HS-40, zeta-, alpha-, and theta-globin probes, a 110-kb clone bearing the whole human alpha-globin gene cluster was obtained and rare restriction endonuclease mapping was performed. The bacterial artificial chromosome DNA was isolated, and transgenic mice were generated. Three founders were detected from 35 newborn mice. The copy numbers were 1, 2, and 2, and the expression of human alpha-globin genes in various tissues at different developmental stages in the transgenic mice was assayed. The human alpha-globin mRNA can be detected in bone marrow, kidney, liver, brain, but not in muscle, testis, or thymus. The human zeta-globin genes were switched off, and the alpha-globin genes were switched at day 11.5 in mouse embryo, indicating that developmental stage-specific expression of the alpha-like globin genes was properly regulated. The human alpha-globin mRNA ranged between 17-68% of the endogenous mouse alpha-globin, suggesting that the expression of human alpha-globin genes is integration site-dependent in transgenic mice. The ratio of human alpha(2)- and alpha(1)-globin gene expression in adult transgenic mouse is about 2.5:1 similar to the expression in human.     

HS-48 alone has no enhancement role on the expression of human alpha-globin gene cluster

To investigate the in vivo function of the newly defined DNase I hypersensitive site HS-48 on the whole human alpha-globin gene cluster, the region containing all the other known 5 hypersensitive sites HS-4 to HS-40 was deleted from a 117 kb bacterial artificial chromosome clone bearing the whole human alpha-globin gene cluster. Transgenic mice were generated from this construct. The RNase protection assays showed that with HS-48 left and all the other 5 hypersensitive sites deleted, the expression of human alpha-like globin genes was completely silenced in embryonic, fetal and adult stages in all tissues. This finding indicates that HS-48 alone has no enhancer activity on the expression of human alpha-like globin genes, and that the region of HS-4 to HS-40 already contains all the upstream cis-elements needed for regulating human alpha-like globin genes.     

Only adult hemoglobin is produced in fetal nonerythroid x MEL cell hybrids

In order to test whether the ontogenetic origin of human parental cells influences the expression of globin genes, we did fusions of mouse erythroleukemia (MEL) cells with fetal or adult nonerythroid cells and we assessed globin expression in hybrids. For selection of hybrid clones we used a monoclonal antibody that recognizes a human chromosome 11-linked cell surface marker. Globin expression was assessed with fluorescent antibody labeling, globin isoelectric focusing, and S1 nuclease mapping. Chromosome 11-containing hybrids from human nonerythroid cells (adult or fetal lymphoid cells; fetal fibroblasts) produced human adult (beta) but not fetal (gamma) globin chains. These results suggest that the MEL x nonerythroid cell hybrids express the adult human globin independent of whether the human cells derive from the fetal or from the adult stage of human development. Our findings, together with previous observations in MEL x fetal erythroid or MEL x HEL hybrids, show that the globin program of the parental human cell is the main determinant of the expression of fetal human globin in these hybrids.