Human globin gene analysis for a patient with beta-o/delta beta-thalassemia.

Abstract

Complementary DNA (cDNA) was prepared with RNA-dependent DNA polymerase from human globin messenger RNA (mRNA). Annealing and translation experiments with total mRNA from circulating cells from a patient with heterozygous betaP/heterozygous beta-delta0 thalassemia (30/,6_0-thalassemia) The common genetic disease 13-thalassemia ((3-thal) is characterized by a reduced production of hemoglobin A (HbA, a2132), which results from the reduced synthesis of ,B-globin chains relative to a-globin chains, thus causing an imbalance in globin chain production and hence abnormal erythropoiesis (1). The (3-thalassemias show heterogeneity, and there are now several well-defined conditions that form this group.In some cases there is a partial deficiency of ,3-chain production (,3+-thal), in others a total deficiency of (3-chain production (,B0-thal), and finally there is a condition in which both the 6 chains of HbA2 (a262) and the 13-chains of HbA are not produced (6500-thal). It has been demonstrated previously that in many of these forms of (3-thalassemia there is a reduction in the amount of ,B-globin messenger RNA (mRNA). This is seen by the lack of mRNA translation activity in vivo and in homologous cell-free systems (2, 3), in heterologous cell-free systems (4-6), and by hybridization with complementary DNA (cDNA) prepared from globin mRNA with viral RNAdependent DNA polymerase (7-9). There is evidence, however, that in at least one variant of (-thalassemia, that originating in the Ferrara region of Italy, inactive globin Abbreviations: Dot, product of Do (initial concentration of cDNA in mol of nucleotide X liter-') and time (sec); Cot, product of CO (initial concentration of total DNA in mol of nucleotide X liter-') and time (sec). mRNA for ,B-globin chains is present (10,11). In all other types studied there appears to be a direct correspondence between the amount of (3-mRNA present and the amount of ,B-globin chain synthesized, both in homozygotes and heterozygotes.The (3, 6, and y-globin genes are thought to be adjacent to one another in a small region of a single chromosome (1,12 Recently it has been shown by cDNA/DNA hybridization techniques that in at least some cases the severe form of athalassemia is the result of a gene deletion involving all or a considerable part of the a-globin genes (13,14). In the present study we have applied similar techniques to see if the (3-globin genes are present in an individual heterozygous for both 30°-and 6300-thalassemia ((0/6(3O-thalassemia

Transcriptional and Post‐transcriptional Defects in β0‐Thalassaemia

“…Both the first and last defects have been proposed for certain rare forms of Po-thalassaemia, a gene deletion in Gopo-thalassaemia [2,3] and a translational defect in Po-thalassaemia of the Ferrara type [4,5]. Many Po-thalassaemic patients, however, appear to have no 8-globin mRNA in circulating reticulocytes, although the gene is present [6,7]. This could be due to a transcriptional or a posttranscriptional defect.…”

“…The two patients selected were from a series of more than 15 Po-thalassaemia patients with no detectable mRNAp in circulating red blood cells, as measured by hybridization to cDNA [6,7]. Both were of Southern Italian origin, of parents with the high HbA2 type of thalassaemia trait.…”

“…cDNAap is about 55 % cDNA, and 45 % cDNAp; cDNAHbH is approcimately 80% cDNAp as judged by hybridization to ~o/GoPo-thalassaemia RNA as previously described [6]. For cDNA-DNA hybridization experiments a full-size cDNAp of high purity (90-95 %) was prepared by methods previously described [2,6].…”

Demonstration of Two alpha-Globin Genes per Human Haploid Genome for Normals and Hb J Mexico

“…The fractions were incubated at 70 "C for 30 min in 1 ml of 1 M perchloric acid and, after the addition of 10 ml of Triton-based scintillant, the radioactivity in each slice was measured is 15.08 x, which must be corrected for the background resistance of the probe to S 1 nuclease digestion (1.96 %> and for the fact that the probe only hybridises to 85 % with mRNA,, giving a corrected plateau value of 15.44%. The t112 of the reaction is 4.3 h, giving a Do tllz after normalisation for salt concentration of 71.55 x mol s I-' [13,19], and hence a kinetic complexity of 233 700 daltons.…”

“…The complexity of the cDNA can be determined from the rate of hybridisation using the equations of Bishop and Freeman [19] as previously applied by us to human globin gene analysis [13,15].…”