Gene expression profiling during erythroid differentiation of K562 cells.

We studied the temporal changes in gene expression in K562 cells at intervals from 2 to 48 h following induction using differential display polymerase chain reaction and gene expression arrays. More than 110 cDNA fragments representing 86 unique mRNAs were either up- or downregulated during erythroid differentiation. Sixty-one of the differentially expressed cDNA fragments had more than 95% homology to known GenBank sequences; 21 represented cDNA sequences with only dbEST or high-throughput gene-screening database matches. Four fragments had no database matches. Using gene expression arrays, 73 differentially expressed genes were observed. Unique expressed sequence tags (ESTs) were used to "clone" two novel genes from available databases and their tissue expression was examined. Erythroid maturation in induced K562 cells is associated with differential expression of many genes. Some differentially expressed clones were transcription factors and 25 expressed fragments with open reading frames were found whose function remains unknown.

Induction of globin synthesis in K562 cells is associated with differential expression of transcription factor genes.

Globin gene switching may be mediated by proteins expressed during different stages of development. Their identification may clarify the mechanisms of the conversion from fetal to adult globin production and lead to new approaches to reversing or retarding the gamma- to beta-globin gene switch. To explore this hypothesis, K562 erythroleukemia cells were induced to differentiate with 1.25, 2.5, and 5 mM sodium butyrate and gene expression was studied after 24, 48, and 72 h. Erythroid differentiation was verified by benzidine staining and by measuring the activity of a transduced A gamma-globin gene promoter linked to a luciferase reporter gene. Using differential display polymerase chain reaction (PCR), total mRNA extracted from induced cells at each time point of induction was reverse transcribed in the presence of A, G, and C anchored primers and 16 arbitrary primers, calculated to amplify approximately 50% of expressed genes. Amplified mRNAs from induced and uninduced cells were separated in polyacrylamide gels and compared. More than 110 cDNA fragments which appeared to represent either up- or downregulated mRNA species in induced K562 cells were identified. Sixty-four of these fragments had more than 95% homology to known GenBank sequences. Seventeen fragments with characteristics of transcription factors were cloned. These include differentiation-related gene-1 (drg-1), PAX 3/forkhead transcription factor, HZF2 which is a Kruppel-related zinc finger protein, three helix-loop-helix proteins (heir-1, Id3, and GOS8), alpha-NAC transcriptional coactivator, LIM domain protein, and trophoblast hypoxia regulating factor. Differential expression of all 17 fragments over 72 h was confirmed by reverse Northern dot blot analysis, semiquantitative PCR using nested primers, and Northern analysis. Erythroid maturation in induced K562 cells is associated with differential expression of numerous genes. Some encode transcription factors that could effect the initiation of HbF synthesis. Almost half of the differentially expressed clones contained cDNAs of unidentified open reading frames and these are the object of continued study.

Double heterozygosity for the codon beta 39 C-to-T nonsense mutation and a triplicate alpha-globin gene locus can cause "dominantly" inherited beta-thalassemia intermedia.

BACKGROUND: A beta-thalassemia intermedia phenotype can be caused by multiple genotypes. METHODS: We studied a family where the mother was hematologically normal and both father and daughter had beta-thalassemia intermedia. RESULTS: Both affected individuals were heterozygous for a codon 39 CAG-to-TAG mutation. They also were heterozygous for a triplicate alpha-globin gene locus (alphaalphaalpha(anti 3.7)). CONCLUSIONS: This compound heterozygous condition of a beta39 C-to-T mutation and triplicate alpha-globin gene increases alpha:beta-globin chain imbalance and accounts for the presence of beta-thalassemia intermedia. The proband received both an abnormal beta-globin gene and a triplicate alpha-globin locus from her father. Although the phenotype seems to be dominantly inherited, because of independent segregation of the alpha- and beta-globin genes, it is more accurately an example of polygenic inheritance.

Genetic studies suggest a multicentric origin for Hb G-Coushatta [beta22(B4)Glu-->Ala].

Hb G-Coushatta [beta22(B4)Glu-->Ala] is found in geographically separated ethnic groups. Commonest along the Silk Road region of China but also present in the North American Coushatta, we sought to determine whether this variant had a unicentric or multicentric origin. We examined the haplotype of the beta-globin gene cluster in two Chinese families and in five Louisiana Coushatta heterozygous for this mutation. Chinese and Louisiana Coushatta had different haplotypes associated with the identical Hb G mutation. These haplotypes were defined by the presence of a HindIII restriction site in the Agamma-globin gene and AvaII restriction site in the beta-globin gene in Chinese subjects and their absence in the Louisiana Coushatta. We found a CAC at codon beta2 (beta-globin gene framework 1 or 2) linked to the Hb G-Coushatta gene in Chinese, and a CAT (framework 3) in Louisiana Coushatta, indicating different beta-globin gene frameworks. Both the Hb G-Coushatta mutation (GAA-->GCA) and the codon 2 CAC-->CAT polymorphism are normal delta-globin gene sequences, suggesting the possibility of gene conversion. We conclude that Hb G-Coushatta had at least two independent origins. This could be due to separate mutations at codon beta22 in Chinese and Louisiana Coushatta, a mutation at this codon and a beta-->delta conversion, or two beta-->delta gene conversion events.

Beta-globin gene haplotype in Hb SC disease.

We asked the question, is the haplotype found with the sickle hemoglobin gene associated with different hematological characteristics in patients who were combined heterozygotes for sickle hemoglobin and hemoglobin C (Hb SC disease)? In 73 adults with Hb SC disease, a Benin haplotype chromosome was present in 56%, and Bantu (or Central African Republic; CAR), Senegal, and atypical haplotype chromosomes were found in 25%, 6%, and 12%, respectively. No significant difference were found in hematological characteristics or fetal hemoglobin levels of patients with Benin/C, CAR/C, Senegal/C, and atypical/C haplotypes. There were 71% C I, 18% C II, and 11% other beta(c) haplotypes. Fetal hemoglobin levels are lower in Hb SC disease than in sickle-cell anemia. Perhaps because haplotype has no discernible effect on fetal hemoglobin level in Hb SC disease, it does not modulate its hematological features.

Two missense mutations in the beta-globin gene can cause severe beta thalassemia. Hemoglobin Medicine Lake (beta 32[B14]leucine-->glutamine; 98 [FG5] valine-->methionine).

We studied the molecular basis of transfusion-dependent hemolytic anemia in an infant who rapidly developed the phenotype of beta thalassemia major. DNA sequence of one beta-globin gene of the proband revealed two mutations, one for the moderately unstable hemoglobin (Hb) Köln and another for a novel codon 32 cytosine-thymidine-guanine-->cytosine-adenine-guanine transversion encoding a leucine-->glutamine mutation. A hydrophilic glutamine residue at beta 32 has an uncharged polar side chain that could potentially distort the B helix and provoke further molecular instability. This new hemoglobin was called Hb Medicine Lake. Biosynthesis studies showed a deficit of beta-globin synthesis with early loss of beta-globin chains. An abnormal unstable hemoglobin, globin chain, or tryptic globin peptide was not present, demonstrating the extreme lability of this novel globin. Hb Medicine Lake mRNA was present, but an aberrantly spliced message was not. Absence of an abnormal beta-globin gene in the mother makes it likely that a de novo mutation occurred in the proband. The molecular pathogenesis of Hb Medicine Lake illustrates a mechanism whereby the phenotype of a genetic disorder, like the mild hemolytic anemia associated with a hemoglobinopathy, can be modulated by a coincident mutation in the same gene.

Beta-thalassemia in southwestern Iran.

Seventeen unrelated beta-thalassemia patients or carriers from Southwestern Iran were examined for the beta-globin gene mutations by polymerase chain reaction amplification of the beta-globin gene and direct genomic sequencing, or by the method of allele-specific oligonucleotide hybridization. Their clinical and hematological characteristics were also recorded. Of 26 potential thalassemia-causing chromosomes examined, 10 different mutations were found. The IVS-II-1 (G-->A) mutation was the most frequent (31%) followed by the IVS-I-6 (T-->C) mutation (15%). Eight mutations were initially described in Mediterranean populations and two were of Kurdish origin. Four of these mutations, both initially described in the Mediterranean region, are reported here for the first time in Iranians. The unexpectedly high number of different mutations that account for beta-thalassemia in this region of Iran suggest migration of chromosomes from distant places and genetic admixture.

G gamma A gamma (beta+) hereditary persistence of fetal hemoglobin: the G gamma -158 C-->T mutation in cis to the -175 T-->C mutation of the A gamma-globin gene results in increased G gamma-globin synthesis.

Hereditary persistence of fetal hemoglobin (HPFH) can be generally classified into deletional and nondeletional forms. The family described in the present study has characteristics of both types of HPFH. The proband is a healthy 30-year-old black woman. Analysis of her hemoglobin revealed 40.4% HbS, 40.9% HbF (G gamma/A gamma ratio 0.53), 16.8% HbA, and 1.9% HbA2. All of her hematologic indices were normal, and the distribution of HbF in her red cells was pancellular. Family studies demonstrated that the proband has one chromosome 11 bearing the beta s-globin gene and the other bearing a G gamma A gamma (beta+) HPFH determinant in cis to the beta A-globin gene. Gene mapping studies of the region between the G gamma- and beta-globin genes were normal. However, when the A gamma and G gamma promoters were amplified by polymerase chain reaction (PCR) and sequenced, the A gamma promoter was found to have the T-->C mutation at -175, and the G gamma promoter region was found to have the C-->T mutation at -158. The -158 C-->T mutation has been associated with elevated G gamma levels and high HbF in hemolysis, although its role in causing these effects is unclear. The present study suggests that this mutation can also enhance G gamma-globin expression in cis to the -175 T-->C mutation in the absence of hemolysis. We suggest that the alteration of the A gamma gene octamer binding site by the -175 mutation, as well as the loss of a putative G gamma "silencer" caused by the -158 mutation may account for this phenotype. We propose calling these linked mutations the G gamma A gamma(beta+) HPFH.

Beta-thalassemia intermedia with exceptionally high hemoglobin A2: relationship to mutations in the beta-gene promoter.

Small deletions of the 5' portion of the beta-globin gene that remove the promoters but stop 3' to the delta-globin gene are recognized as the sole cause of beta-thalassemia with exceptionally high hemoglobin A2 (HbA2) levels. Two patients with beta-thalassemia intermedia and exceptionally high levels of HbA2 (10.4 and 12.0%) were examined. One patient was a combined heterozygote for the -88 C----T and a novel -87 C----A mutation, while the other was homozygous for the -29 A----G beta(+)-thalassemia mutation. The remainder of the beta genes were normal. There was no evidence for deletions involving the 5' portion of the beta gene or the region between the beta and delta genes. Gene mapping studies excluded the possibility of a beta delta-anti-Lepore hemoglobin gene with beta promoters and delta coding sequences. There were no mutations in the promoters of the G gamma or A gamma-globin genes that have been associated with the hereditary persistence of HbF phenotype. The delta-globin gene promoters were normal from codon 17 to position -145 relative to the mRNA capping site. There appears to be considerable heterogeneity of HbA2 and HbF levels in patients who are homozygous or mixed heterozygotes for mutations in the TATA box and other promoter elements of the beta-globin gene. The capacity for proteolysis within the erythrocyte may vary among individuals. The authors hypothesize that in the exceptionally high HbA2 beta-thalassemia intermedia phenotype, proteolysis of superfluous alpha-globin chains is less efficient than in patients with customary levels of HbA2.(ABSTRACT TRUNCATED AT 250 WORDS)

Slowly exchanging calcium binding sites unique to cardiac/slow muscle troponin C.

Evidence is presented for the existence of slowly exchanging Ca2(+)-binding sites in troponin C (CTNC) of cardiac and slow twitch skeletal muscles. These sites were revealed in the course of experiments aimed at measuring the Ca2(+)-binding properties of TNC in the myofilament lattice. 45Ca bound to chemically skinned muscle fibers or myofibrils of cardiac and soleus muscles was eluted by EGTA in a two-exponential timecourse with a slow phase of a rate constant of about 2 x 10(-4)/s. The slow phase was not found in skinned fiber or myofibrils of psoas, a fast skeletal muscle. However, skinned psoas fibers in which the native TNC was replaced by CTNC exhibited the slow 45Ca elution characteristic of soleus and cardiac preparations, indicating that the slowly-exchanging sites are located in CTNC. These sites are tentatively identified as the Ca2(+)-Mg2+ sites of CTNC on the basis that the slow phase was observed under conditions known to restrict Ca2+ binding to the Ca2(+)-Mg2+ sites.