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The human Fc gamma RIIA gene produces multiple transcripts, including those with (Fc gamma RIIa1) and without (Fc gamma RIIa2) the single exon encoding the transmembrane domain (TM). Previously, a fluorescence-based RT-PCR assay showed lineage-specific differences in Fc gamma RIIA transcript ratios (Fc gamma RIIa2/Fc gamma RIIa1). The mechanism of this lineage-specific expression was investigated in this study. Differential transcript stability does not play a major role, because transcript ratios remained constant in cells with both low (K562) and high (Dami) ratios following actinomycin D treatment. Transient expression studies in K562 and Dami cells using a minigene construct containing a 5.0 kb genomic fragment including the TM exon and adjacent intron and exon sequences showed recapitulation of endogenous transcript ratios. The TM exon was efficiently spliced in by the constitutive splicing machinery in HeLa cells, an Fc gamma RIIA-negative cell line. Lineage-specific TM exon skipping was markedly diminished by two independent minigene mutations: a point mutation of the first nucleotide of the TM exon, and a five basepair intronic deletion near a putative branchpoint. These data demonstrate that cis-acting sequences in or near the TM exon 3' splice acceptor site contribute to lineage-specific differences in Fc gamma RIIA transcript ratios.

Microelectronic DNA chip devices represent an emerging technology for genotyping. We developed methods for detection of single-nucleotide polymorphisms (SNPs) in clinically relevant genes.Primer pairs, with one containing a 5'-biotin group, were used to PCR-amplify the region encompassing the SNP to be interrogated. After denaturation, the biotinylated strand was electronically targeted to discrete sites on streptavidin-coated gel pads surfaces by use of a Nanogen Molecular Workstation. Allele-specific dye-labeled oligonucleotide reporters were used for detection of wild-type and variant sequences. Methods were developed for SNPs in genes, including factor VII, beta-globin, and the RET protooncogene. We genotyped 331 samples for five DNA variations in the factor VII gene,600 samples from patients with beta-thalassemia, and 15 samples for mutations within the RET protooncogene. All samples were previously typed by various methods, including DNA sequence analysis, allele-specific PCR, and/or restriction enzyme digestion of PCR products.Analysis of amplified DNA required 4-6 h. After mismatched DNA was removed, signal-to-noise ratios were5. More than 940 samples were typed with the microelectronic array platform, and results were totally concordant with results obtained previously by other genotyping methods.The described protocols detect SNPs of clinical interest with results comparable to those of other genotyping methods.

The pathophysiology and clinical severity of beta-thalassemia are related to the degree of alpha/non-alpha-chain imbalance. A triplicated alpha-globin gene locus can exacerbate effects of excess alpha-chains caused by a defective beta-globin gene, although this is not observed in all cases. Extensive studies on this condition are lacking. We report a group of 17 patients who are heterozygous for both the alpha alpha alpha(anti-3.7) allele and a mutation in the beta-globin gene cluster. Their clinical phenotypes varied: six had mild anemia with microcytosis and hypochromia, while 11 had more severe anemia with splenomegaly requiring splenectomy (three cases) and blood transfusions (four cases). Different phenotypes were also evident in the presence of the same beta-thalassemia mutation: in one family, two individuals had the same alpha- and beta-globin genotypes but presented with different hematologic phenotypes. In addition, the complex interaction involving a triplicated alpha-globin gene, beta39- and delta+27-thalassemia mutations is studied in a family with two siblings presenting with hemolytic anemia, normal Hb A2 and increased Hb F. Analysis of this series of patients suggests that additional genetic determinants play a role in modulating phenotypic expression in individuals with identical alpha- and beta-globin genotypes. Interaction with a triplicated alpha-gene can play a role in the clinical presentation of patients with defective beta-globin gene expression and should be considered in the diagnosis of atypical cases.

Human erythroblasts are a logical target for studies of expression of transferred globin genes because high-level expression is a prerequisite for gene therapy of hemoglobinopathies. Early erythroid progenitors (erythroid burst-forming units, BFU-E) are readily available from human peripheral blood and can be cultured to produce erythroblasts. However, conditions for efficient transfer into these normal progenitors have not been previously described. Here we demonstrate efficient transfer of the neomycin resistance gene into human peripheral blood BFU-E using the retrovirus vector, N2. We show that liquid culture of mononuclear cells from peripheral blood for 18-24 h prior to retroviral infection leads to increased transfer efficiency of N2 as determined by G418 resistance, and we are able to detect viral DNA by polymerase chain reaction (PCR) analysis. In addition, a second retrovirus, beta(gamma)-SVX, prepared with a human beta-globin gene containing a gamma-globin second exon to facilitate transcript detection and the 3'-enhancer sequence, was also used to determine whether similar results could be obtained when more than one gene is transferred. Using the beta(gamma)-SVX virus, increased transfer efficiency into BFU-E was similarly found after liquid culture for up to 4 days. Expression of the transferred globin gene was also detected by PCR analysis of cDNA made from erythroblast RNA. The human peripheral blood BFU-E system described should allow determination of sequences required for high-level expression of transferred globin and other erythroid genes.