The relative levels of βA and βS mRNAs in Hb S heterozygotes and in patients with Hb S‐β+ ‐thalassaemia or Hb S‐β+ ‐HPFH combinations

Dimovski, Aleksandar; Efremov, Dimitar G.; Gu, Lihao; Huisman, T. H. J.

doi:10.1111/j.1365-2141.1994.tb04921.x

Cited by 20 publications

(6 citation statements)

References 14 publications

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“…mRNA was reverse transcribed to cDNA with a Gene Amp RNA kit (Perkin Elmer Cetus, Norwalk, Conn., USA) and amplified [7,8], The fragment obtained was 352 bp long [7] and contained codon 6: GAG in PA and GTG in ps. The PA and Ps fragments were characterized after enzymatic digestion with Bsu I, an Mst II isoshizomer (New England Biolabs, Beverly, Mass., USA).…”

Section: Resultsmentioning

confidence: 99%

“…The two oligo nucleotide primers were: 5' (direct) positions + 11 through + 29 located in the 5' untranslated region 5'-CTGACACAACTGTGTT-CAC-3'; 3' (reverse) positions + 493 through + 475 located in exon 2 5'-TGAAGTTCTCAGGATCCAC-3'. The PCR was done as recom mended in the original publication [7],…”

Section: Hb Analysismentioning

confidence: 99%

“…Total RNA was isolated from the reticulocytes of the freshly col lected blood sample as described by Chomczynski and Sacchi [6]. Reverse transcription followed by PCR was used for the amplifica tion of specific PA-and ps-globin cDNA fragments as described pre viously by Dimovski et al [7] and Efremov et al [8]. The two oligo nucleotide primers were: 5' (direct) positions + 11 through + 29 located in the 5' untranslated region 5'-CTGACACAACTGTGTT-CAC-3'; 3' (reverse) positions + 493 through + 475 located in exon 2 5'-TGAAGTTCTCAGGATCCAC-3'.…”

Section: Hb Analysismentioning

confidence: 99%

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Sickle Cell Anemia Identified in a Multiple-Transfused Patient through Analysis of mRNA with an RT-PCR-Based Technique

Smetanina

Gu²,

Leonova³

et al. 1995

Acta Haematol

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Section: Resultsmentioning

confidence: 99%

Section: Hb Analysismentioning

confidence: 99%

Section: Hb Analysismentioning

confidence: 99%

See 1 more Smart Citation

Sickle Cell Anemia Identified in a Multiple-Transfused Patient through Analysis of mRNA with an RT-PCR-Based Technique

Smetanina

Gu²,

Leonova³

et al. 1995

Acta Haematol

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“…This was recently demonstrated with an RT-PCR procedure show ing equal quantities of pA-and (3s-mRN As in Hb S hetero zygotes with or without an additional a-thal [11], In con trast, other base changes will affect the rate of transcrip tion, prematurely terminate translation, or create a non functional mRNA producing phenotypes of mild to se vere P-thal. Studies by Lim et al [38] and Baserga and Benz [39] The slight increase in a/p-mRNA ratio observed in the 3 subjects with deletional p-thal mutants and four a-globin genes (average 5.22) is difficult to explain.…”

Section: Discussionmentioning

confidence: 99%

Globin mRNA in β-Thalassemia Heterozygotes with Different β-Thalassemia Alleles and in Heterozygotes for Hereditary Persistence of Fetal Hemoglobin

1996

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Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine the α2/α1-, α/β-, and γ/β-mRNA ratios in subjects with β -thalassemia (β-thal), hereditary persistence of fetal hemoglobin (HPFH), and normal adults. The α- and β -globin gene mutations were characterized with gene mapping, PCR, and DNA sequencing. The average α2/α1-mRNA ratio was the same in normal adults and β-thal heterozygotes with four α-globin genes (2.61-2.63) or with an α-thal-2 trait (1.48-1.55). The average α/β -mRNA ratios were 4.47 and 3.84 in normal adults with four α-globin genes and with α-thal-2 trait (-α/αα), respectively. There was an increase of ∼ 50% in ιs β -thal heterozygotes with transcriptional mutants [-88 (C→T) and -29 (A→G)] with lower alues (∼ 25%) in those with α-thal-2 trait (-α/αα). High α/β ratios were also observed for heterozygotes for nonsense or frameshift mutants located in exon 1 or exon 2. Increases of -150-165% were seen in subjects with RNA processing defects; an exception was the IVS-I-110 (G→ A) mutation with a normal value in the heterozygote. The increases were also less pronounced in heterozygotes for the codon (CD) 121 (G→T) mutation and the CDs 134-137 insertion/deletion. Normal α/(γ+β) values were seen in 3 heterozygotes each with a different deletion involving part of the β -globin gene. The presence of the silent β -thal allele, -101 (C→T), in trans to a CD 8 (-AA) allele has a minor effect on the α/β-mRNA ratio. The α/β -mRNA ratio in HPFH heterozygotes was ∼ 145% of normal, but with a γ-mRNA level of 35.4-44.7% the calculated α/(γ+β) ratio became as in normal adults. The RT-PCR methodology appears useful in expression studies in β -thal (and HPFH) and values of mRNA appear to correspond to the type of prevailing mutation(S) and concomitant α-thal.

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“…To determine the ratio of mRNA ␣2/␣1, we used RNase protection assay as previously published (9). To quantify the relative amounts of normal and mutant mRNA, we used RT-PCR based on published methods (10). At first, 0.5 g of RNA was reverse transcribed using 0.5 g of oligo dT primer (GIBCO) in 1X PCR buffer (GIBCO), 0.2 mM dNTP, 1.5 mM MgC1 2 , 10 mM DTT, 40 U of RNA guard (Amersham Pharmacia Biotech), with 400 U of M-MLV reverse transcriptase (GIBCO) by incubation at 42°C for 60 min followed by inactivation of reverse transcriptase at 94°C for 5 min.…”

mentioning

confidence: 99%

The Biologic Implications of a Rare Hemoglobin Mutant That Decreases Oxygen Affinity

2001

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Blood from seven newborns, a 13-y-old, and seven adult family members with a suspected hemoglobinopathy because of unexplained cyanosis was obtained for analysis to determine Hb oxygen affinity and to characterize and quantify the Hb variants. Their oxygen saturation was 76 to 84%. The P 50 was 30.3 Ϯ 2.9 for the newborns and 32.5 Ϯ 2.6 mm Hg for their related adults. In the same order, the plasma erythropoietin was 7.4 Ϯ 2.9 and 15.9 Ϯ 3.7 mU/mL, whereas 2,3-diphosphoglycerate was 16.1. Ϯ 2.9 and 15.9 Ϯ 3.7 mol/g Hb. In four of the newborns with increased P 50 , the mother had a normal P 50 (27 mm Hg), which indicated a greater maternal oxygen affinity than the fetus with no adverse effects on the fetus. Genetic analysis of ␣-globin genes demonstrated a heterozygous mutation on the ␣2 gene [␣94(G1)Asp3 His] for each of the newborns and their related adults. The same mutation was found on the ␣1 gene in an adolescent and her father. The mRNA measurements showed that the ␣2-to ␣1-globin mRNA mean ratio was 2.5, ␣2 mutant globin mRNA/total ␣2-globin mRNA was 45.0%, whereas the ␣1 mutant globin mRNA/total ␣1-globin mRNA was 37.8%. The level of ␣2 mutant globin/total ␣-globin was 27.3 Ϯ 1%, and ␣1 mutant globin/total ␣-globin was 23.8 Ϯ 1%. The percentage of synthesized ␣2 and ␣1 mutant globins was 27.5 Ϯ 2 and 26.1 Ϯ 1, respectively. The ratio of the ␣2/␣1 mutant globins was 1.1, which corresponded to a ratio at the mRNA level of ␣2/␣1 of 2.5 Ϯ 0.5, which suggested that there is a less efficient translation of the ␣2 mRNA than ␣1 mRNA. The reversal of the physiologic fetomaternal oxygen affinity had no effects on fetal development. Abbreviations ODC, Hb-oxygen dissociation curve DPG, 2,3-diphosphoglycerate P 50 , PO 2 required to achieve a saturation of 50% at pH 7.4 and 37°CA rare mutant Hb with a low oxygen affinity designated as Sunshine Seth [␣94(G 1 ) Asp3 His] (1) was detected in a series of newborn infants as well as in a 13-y-old and her father. The infants, because of persistent cyanosis, were transferred soon after birth to a neonatal intensive care unit to investigate the cause of their Hb desaturation. The cyanotic infants and their parents provided an opportunity to evaluate the physiologic implications of low oxygen Hb affinity during the perinatal period as well as carry out genetic analysis to further the knowledge of the of human ␣-globin gene expression.The two human ␣-globin genes ␣1 and ␣2 are coexpressed in normal erythroid cells and encode identical ␣-globin protein products. The ␣2 gene encodes a 2-3-fold higher level of mRNA than the ␣1 gene (2). Because of the identical ␣-globin produced, the relative levels of expression are difficult to determine. Despite some data that suggest posttranscriptional/ translational modifications balancing the amount of protein expressed from these mRNA (3), it is still controversial how the difference in ␣2 and ␣1 mRNA levels are reflected at the protein level (4). Because of a rare occasion in which an identical mutation in both the ␣1 and ␣2 genes existed ...

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The relative levels of β^A and β^S mRNAs in Hb S heterozygotes and in patients with Hb S‐β⁺ ‐thalassaemia or Hb S‐β⁺ ‐HPFH combinations

Cited by 20 publications

References 14 publications

Sickle Cell Anemia Identified in a Multiple-Transfused Patient through Analysis of mRNA with an RT-PCR-Based Technique

Sickle Cell Anemia Identified in a Multiple-Transfused Patient through Analysis of mRNA with an RT-PCR-Based Technique

Globin mRNA in β-Thalassemia Heterozygotes with Different β-Thalassemia Alleles and in Heterozygotes for Hereditary Persistence of Fetal Hemoglobin

The Biologic Implications of a Rare Hemoglobin Mutant That Decreases Oxygen Affinity

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