N. S. Smetanina

Variability in the fetal hemoglobin level of the normal adult.

We analyzed blood samples from more than 200 normal adults, and quantified their Hb F by cation‐exchange high‐performance liquid chromatography. In several subjects with slightly elevated Hb F (0.4–4.3%), we determined the Gγ levels in the Hb F and DNA sequence variations in the locus control region II and in the Gγ and Aγ promoters. About 25% of the ∼200 normal teenaged high school students had elevated Hb F; detailed analyses of some 20 students, selected at random, identified most as females with a homozygosity for the C→T variation at position ‐158 (Gγ). One 11‐year‐old boy was heterozygous for the A→G change at position ‐161 (Gγ); he and two of his relatives had ∼4% Hb F, high Gγ values, and a high level of (mainly) Gγ‐mRNA. Nearly 40 normal adults from Macedonia and from Georgia (mostly Caucasians) were tentatively identified as Swiss HPFH heterozygotes because slightly elevated Hb F levels were observed at least once. Many of these persons were heterozygous or homozygous for the C→T mutation at ‐158 (Gγ), and a few carried a γ‐globin gene triplication. The C→T change appears to be an important factor predisposing the adult to increased Hb F production. Evidence suggests a gene dose effect in (mildly) anemic adults; however, other factors besides the C→T change at ‐158 (Gγ), including factors not linked to the β‐globin region, may cause an increase in γ‐chain synthesis.

Hb Lepore-Baltimore (δ68Leu-β84Thr) and Hb Lepore-Washington-Boston (δ87Gln-βIVS-II-8) in Central Portugal and Spanish Alta Extremadura

Abstract Hb Lepore is one of the most common abnormal haemoglobins in Caucasians in Central Portugal and in the Spanish Alta Extremadura (0.28% in a survey of school children). A group of 19 Portuguese and 14 Spanish Hb Lepore carriers (all unrelated) was characterised at the molecular level by the polymerase chain reaction, sequencing and restriction enzyme analysis. The Portuguese and one Spanish carrier were heterozygous for Hb Lepore-Baltimore, whereas all other Spanish subjects were Hb Lepore-Washington-Boston carriers. Sequencing of the Hb Lepore-Baltimore gene further established the crossover at δ68-β84, a region two codons (CDs) shorter than that previously described and easily confirmed by digestion with MaeI and BanI. Data from haplotype analysis suggest that this crossover occurred as an independent event on the lberian Peninsula. The haematological data were similar in both groups except for the levels of Hb F and the Gγ chain, which were significantly higher in the Hb Lepore-Baltimore heterozygotes. Quantification of the globin chains and the mRNA transcripts showed that the δβ gene is transcribed at a higher level than the δ gene with levels of translation giving rise to 10%–15% of Hb Lepore. The different levels of Hb F observed in the two groups are the results of the higher transcription rate of the γ genes in Hb Lepore-Baltimore heterozygotes and an apparently less efficient translation of Gγ genes in Hb Lepore-Washington-Boston heterozygotes.

The dominant β-thalassaemia in a Spanish family is due to a frameshift that introduces an extra CGG codon (=arginine) at the 5' end of the second exon

We have discovered a Spanish family with a dominant type of β‐thalassaemia. Carriers are characterized by mild anaemia, hypochromia, microcytosis, elevated Hb A2 and Hb F levels, reticulocytosis, and splenomegaly. The molecular basis of this condition is the introduction of a CGG triplet between codons 30 and 31 of the β gene; this was determined by sequencing of amplified DNA and confirmed by dot‐blot analysis. The abnormal mRNA (βTh‐mRNA) is stable and present in quantities similar to that of normal βA‐mRNA. cDNA fragments derived from βTh‐ and βA‐mRNAs can be separated on a denaturing polyacrylamide gel electrophoresis because the βTh fragment is three nucleotides (nts) longer than the βA fragment. The βTh‐mRNA translates into a β chain that is 147 amino acid residues long and carries an extra arginine residue between residues 30 and 31. This βX chain has not been detected. It may be unstable and does not bind to the α chain. It probably is continuously digested by proteolytic enzymes in red cell precursors in the bone marrow. The abnormal chain probably binds haem that is excreted after proteolysis causing a darkening of the urine.

The alpha / beta and alpha 2 / alpha 1-globin mRNA ratios in different forms of alpha-thalassemia.

The present study provides information about the alpha / beta and alpha 2 / alpha 1-mRNA ratios in reticulocytes of normal adults and individuals with different alpha-globin gene deficiencies; it found its origin in analytical data of blood samples from a Laotian couple and their newborn baby. The father carried the 4.2 kb deletion on one chromosome and a TAA --> CAA mutation at the terminating codon of the alpha 2 gene (Hb Constant Spring or CS) on the other chromosome. The mother had the 3.7 kb deletion on one chromosome and a TA A --> TAT mutation at the terminating codon of the alpha 2-globin gene (Hb Paksé) of the second chromosome. The baby was a compound heterozygote for the two termination codon mutations. The mRNA data for this family were compared to those for persons with several well-defined alpha-globin gene deficiencies. The results confirm the importance of the alpha 2 alpha 1-mRNA for the synthesis of alpha chains in alpha-thalassemia-2 homozygotes (-alpha/-alpha) and in patients with Hb H disease due to the deletion of three alpha-globin genes (-alpha/--). Furthermore, the MRNA production of the alpha 1-globin gene on the chromosome with the alpha CS mutation (alpha CS alpha) is only one-half of that by the alpha 2 alpha 1-globin gene of a chromosome with a 3.7 or 4.2 kb deletion, explaining the greater severity of, and higher Hb H level in Hb H patients with the alpha CS alpha condition (alpha CS alpha/--) as compared to those with the three gene deletion (-alpha/--). The methodology could be useful as a preliminary screening for the presence of point mutations leading to the functional loss of a single alpha-globin gene, provided common deletional alleles have been excluded.

Hb Costa Rica or α2β277(EF1)His→Arg: The first example of a somatic cell mutation in a globin gene

Abstract We have identified a minor hemoglobin component (∼5%) in the blood of a healthy Costa Rican female, but not in her mother and two brothers (father not studied), that has an His→Arg replacement at position β77 (Hb Costa Rica). No other amino acid replacements were observed and no β- or γ-chain-like peptides were present. Hb Costa Rica has a normal stability. Sequence analyses of numerous polymerase chain reaction (PCR)-amplified segments of DNA that contain exon 2 of the β gene failed to identify a CAC→CGC (His→Arg) mutation. The same was the case when cDNA was sequenced, indicating that a β-Costa Rica-mRNA could not be detected with this procedure. Gene mapping of genomic DNA with BglII, BamHI, and HindIII gave normal fragments only and with the same intensity as observed for the fragments of a normal control. The quantities of the β chain variants Hb J-Iran and Hb Fukuyama with related mutations at β77 vary between 30% and 45% in heterozygotes, whereas that of Hb F-Kennestone with the same His→Arg mutation but in the Gγ-globin gene, is a high 40%–45% (as percentage of total Gγ) in a heterozygous newborn. These different observations exclude a heterozygosity of the A→G mutation at codon β77, as well as a deletion comparable to that of Hbs Lepore or Kenya, or a β-globin gene duplication, and point to a nontraditional inheritance of Hb Costa Rica. Allele-specific amplification of cDNA with appropriate primers identified the presence of a low level of mutated mRNA in the reticulocytes of the patient, which was confirmed by dotblot analysis of the same material with 32P-labeled probes. Comparable amplification products were not observed in genomic DNA. The A→G mutation apparently occurred in a somatic cell at a relatively early stage in the development of the hematopoietic cell system, and Hb Costa Rica accumulated through rapid cell divisions in patchy areas in the bone marrow (somatic mosaicism). An unequal distribution of Hb Costa Rica over the red cells supports this possibility.

Identification of Several α-Globin Gene Variations in a Small Laotian Family

The present study concerns the identification of four α-globin gene deficiencies, one α1-globin gene mutation, and one β-globin gene mutation in a Laotian couple and their newborn baby. The parents we

Comparison of the Relative Quantities of γ-mRNAs and Fetal Hemoglobin in SS Patients with Different Haplotypes

We have studied the relative levels of γ-mRNA [%γ/(γ + β)], ^Gγ- and ^Aγ-mRNAs [%^Gγ/(^Gγ + ^Aγ)], hemoglobin (Hb) F, and the ^Gγ and ^Aγ chains in some 50 patients with sickle cell anemia (SS) and with different haplotypes. As expected, the Hb F levels varied greatly and were high in patients with the Saudi Arabian-Indian haplotype. Similarly, the ^Gγ values varied greatly (from 19.5 to 76.5%) and depended on the haplotypes. A rare haplotype, named Mor, was found in 3 SS patients, 1 of whom was a homozygote Mor/Mor; this haplotype is associated with the lowest ^Gγ value (19.5% in the homozygote) and with a C→T mutation at position –202 of the ^Aγ promoter. The levels of γ-mRNA roughly parallel those of Hb F, but older patients have increased levels of mRNA, which appears not to be efficiently translated into Hb F. Similar observation have been reported for other hemoglobinopathies such as δβ-thalassemia heterozygotes and Hb Lepore heterozygotes. The relative quantity of ^Gγ-mRNA was closely related to that of the ^Gγ chain in the 15 patients who were studied; the ^Gγ- to ^Aγ-mRNA ratio did not change with age.

Alpha-, beta-, and gamma-mRNA levels in beta-thalassemia; transcriptional and translational differences in heterozygotes, homozygotes, and compound heterozygotes.

We have determined the relative levels of alpha-, beta-, and gamma- (G gamma- and A gamma-) mRNAs in the reticulocytes of patients with mild beta-thalassemia intermedia due to combinations of promoter mutations and a classical type of beta-thalassemia, as well as in their relatives. The expected differences in the alpha/beta-mRNA ratio confirmed the mild suppression of beta-mRNA synthesis, particularly in heterozygotes for the -101 (C-->T) promoter mutation and the large increase in the relative gamma-mRNA level in compound heterozygotes. A significant discrepancy between Hb F and gamma-mRNA levels, observed in previously published studies, was confirmed indicating a less efficient gamma-mRNA translation. When the two different gamma-mRNA (G gamma- and A gamma-) levels were determined it was observed that in beta-thalassemia heterozygotes the extra gamma-mRNA was primarily of the G gamma type suggesting a more efficient translation of the A gamma-mRNA. This difference disappeared in homozygotes and compound heterozygotes: both mRNAs (G gamma- and A gamma-) translate with an equal efficiency.

mRNA analysis in reticulocytes of subjects with Hb D, Hb Porto Alegre, Hb E, and different types of unstable hemoglobin variants

Using a reverse transcription‐polymerase chain reaction (RT‐PCR) technique we determined the α2/α1, α/β, and γ/β mRNA ratios in reticulocytes of 11 patients with seven different unstable β chain variants, of 4 patients with two unstable α chain variants, in hemoglobin (Hb) D, Hb Porto Alegre, and Hb E heterozygotes, and in 8 patients with Hb X‐β°‐thalassemia (thal) (three D‐β°‐thal, one Porto Alegre‐β°‐thal, one Lulu Island‐β°‐thal, and three E‐β°‐thal). In addition, we determined the βx/βA mRNA ratios (X = unstable) in some Hb D heterozygotes and in 6 subjects with an unstable β chain variant. Normal α/β and βx/βA mRNA ratios were found in all heterozygotes tested, indicating that the respective mutations did not alter the stability of the mRNAs. The α/β mRNA ratio in four Hb E heterozygotes averaged 4.21 (normal, 4.47, and that in 2 patients with Hb E‐β°‐thal and four α‐globin genes (αα/αα) averaged a high 22.4. The γ mRNA level in the Hb E heterozygotes was <1% but varied greatly in patients with Hb E‐β°‐thal; the α/(γ + β) mRNA ratios in the 2 patients were 15.5 and 16.7, respectively. The large differences in α/β and α/(γ + β) mRNA ratios in reticulocytes of subjects with AE and with E‐β°‐thal may be due to differences in the levels of normally‐spliced βE and abnormally‐spliced βE mRNAs. Only the latter is unstable and is preferentially produced in bone marrow and reticulocytes of Hb E‐β°‐thal patients, where it is rapidly degraded.

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

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.