jeanflop/post_ocr_correction2 huggingface dataset:
in this family demonstrated the threshold effect in the clinical manifestations of such a-gene disorders since hétérozygotes were clinically and biologically normal. Key words : Hb Taybe, a-globin, hemolytic anémie. Voir Version abrégée (p. 443) Abnormal hemoglobins and thalassemias have been considered historically as two separate genetic entities. In the first case, a structurally abnormal globin subunit is produced, while in the second case the biosynthesis of one of the globin subunits is decreased or abolished. In récent years several abnormal hemoglobins have been recognized as responsible for a phenotype that overlaps both thalassemias and hemolytic anémies due to unstable hemoglobins [1-3]. This is especially well documented for a-chain variants that are expressed within the red blood cell at such a low level that they could not be detected by standard techniques [4, 5]. The clinical features, in the hétérozygotes, are those of thalassemia intermedia: a severe dysérythropoïétique anémie is associated with splenomegaly. In addition, inclusion bodies are observed in the normoblasts of the bone marrow and also in the peripheral red blood cells after splenectomy. Since 1974, this syndrome was recognized as "inclusion body (3-thalassemia" or "dominant thalassemia" [6]. These hypersunstable variants display a structural modification in the part of the hemoglobin molecule involved in the aß Pi contact, mainly in the région encoded by the third exon [7]. A similar situation may also be observed for a few unstable a-chain hemoglobin variants. Nevertheless, due to gène dosage, these abnormal hemoglobins are not expressed clinically to the same extent as their ß-globin counterparts. These unstable a-chain variants may not be detected in the red blood cells of the heterozygous carriers, who are usually asymptomatic. Trace amounts of the abnormal hemoglobin, together with some Hb H, can be observed by electrophoretic methods in patients that are compound hétérozygotes for another a-thalassemic gène. In these patients a dominant Heinz bodies hemolytic hereditary anémie overlaps the a-thalassemic syndrome [8]. In this work we describe Hb Taybe, a new unstable a-chain variant clinically silent in the hétérozygotes and found together with small amounts of Hb H in a splenectomized patient suffering from a
of the hemoglobin molecule involved in the aß Pi contact, mainly in the région encoded by the third exon [7]. A similar situation may also be observed for a few unstable a-chain hemoglobin variants. Nevertheless, due to gène dosage, these abnormal hemoglobins are not expressed clinically to the same extent as their ß-globin counterparts. These unstable a-chain variants may not be detected in the red blood cells of the heterozygous carriers, who are usually asymptomatic. Trace amounts of the abnormal hemoglobin, together with some Hb H, can be observed by electrophoretic methods in patients that are compound hétérozygotes for another a-thalassemic gène. In these patients a dominant Heinz bodies hemolytic hereditary anémie overlaps the a-thalassemic syndrome [8]. In this work we describe Hb Taybe, a new unstable a-chain variant clinically silent in the hétérozygotes and found together with small amounts of Hb H in a splenectomized patient suffering from a hemolytic anémie with mild dysérythropoïétique features. Material and methods Hematological studies were performed by routine procédures. Hemoglobin studies The methods for electrophoretic analysis of hemoglobin included: electrophoresis on cellulose acétate at alkaline pH, citrate agar electrophoresis, isoelectric focusing (IEF) of hemoglobin, electrophoresis of globin chains in urea at pH 6.0 and 9.0 or in the présence of Triton X-100. The techniques used for structural analysis were those previously described [9]. These methods included isolation of the abnormal hemoglobin by flat bed preparative isoelectric focusing, séparation of the globin chains by reversed-phase HPLC, aminoethylation, digestion of the polypeptide chain with trypsin, séparation of the peptides by reversed-phase HPLC and détermination of their amino acid composition by HPLC after precolumn dérivation with phenyl-isothiocyanate (PITC). DNA studies DNA was isolated from leucocytes by phenol-chloroform extraction. • DNA amplification a1 and a2 globin-gene fragments were amplified between primers Pa03 and C2 for a1, Pa03 and C3 for a2, as shown on Figure 7. The spécifique downstream primers C2 and C3 were those described by Dodé et al. [10]. The upstream primer Pa03 (5' ACTCTTCTGGTCCCCACAGA 3') corresponds to nucleotides + 1 to +20 from the cap site and was designed to amplify shorter fragments than
and 9.0 or in the présence of Triton X-100. The techniques used for structural analysis were those previously described [9]. These methods included isolation of the abnormal hemoglobin by flat bed preparative isoelectric focusing, séparation of the globin chains by reversed-phase HPLC, aminoethylation, digestion of the polypeptide chain with trypsin, séparation of the peptides by reversed-phase HPLC and détermination of their amino acid composition by HPLC after precolumn dérivation with phenyl-isothiocyanate (PITC). DNA studies DNA was isolated from leucocytes by phenol-chloroform extraction. • DNA amplification a1 and a2 globin-gene fragments were amplified between primers Pa03 and C2 for a1, Pa03 and C3 for a2, as shown on Figure 7. The spécifique downstream primers C2 and C3 were those described by Dodé et al. [10]. The upstream primer Pa03 (5' ACTCTTCTGGTCCCCACAGA 3') corresponds to nucleotides + 1 to +20 from the cap site and was designed to amplify shorter fragments than those with CI [10]. The reaction mixtures (100 μl) were quite different for ai and a2 amplifications, ai: 1 μg of genomic DNA in 50 mM KCl, 10 mM Tris (pH 8.4), 1.5 mM MgCl2, 10 % dimethylsulfoxide, 0.01 % (w/v) gelatin, 200 μM of each dNTP, 30 pmol of oligonucleotides Pa03 and C2, and 2.5 units of Taq polymerase. a2: 30 pmol of Pa03 and 60 pmol of C2 with 1.0 mM MgCl2 were necessary to obtain the optimal efficiency of amplification. PCR amplifications were performed using a Perkin-Elmer Cetus thermal cycler as follows: a first step of denaturation (5 min at 94 °C), 30 cycles 1 min at 94 °C, 1 min 30 at 57 °C for al, 55 °C for a2 2 min at 72 °C, and a final extension step (7 min at 72 °C). Another cycle of denaturation (10 min at 94 °C) renaturation (30 min at the appropriate annealing temperature) was applied to enhance the formation of heteroduplex molecules. PCR products were analyzed on a 6% polyacrylamide gel in TBE buffer following standard protocols [11]. The specificity of amplification was checked by restriction endonuclease analysis of the PCR products with Apa I [10] according
oligonucleotides Pa03 and C2, and 2.5 units of Taq polymerase. a2: 30 pmol of Pa03 and 60 pmol of C2 with 1.0 mM MgCl2 were necessary to obtain the optimal efficiency of amplification. PCR amplifications were performed using a Perkin-Elmer Cetus thermal cycler as follows: a first step of denaturation (5 min at 94 °C), 30 cycles 1 min at 94 °C, 1 min 30 at 57 °C for al, 55 °C for a2 2 min at 72 °C, and a final extension step (7 min at 72 °C). Another cycle of denaturation (10 min at 94 °C) renaturation (30 min at the appropriate annealing temperature) was applied to enhance the formation of heteroduplex molecules. PCR products were analyzed on a 6% polyacrylamide gel in TBE buffer following standard protocols [11]. The specificity of amplification was checked by restriction endonuclease analysis of the PCR products with Apa I [10] according to the supplier's advice (Boehringer Mannheim, Germany). Direct sequencing of amplified DNA Asymmetric amplifications were carried out in the same conditions as mentioned above, with the same specific PCR primers, 1 pmol C2 or C3, and 50 pmol Cl [10]. Purification of the single strands was obtained with a Centricon 30 (Amicon) procedure elsewhere described [12]. Sequencing reactions were performed using the sequencing kit (US Biochemicals), and Pa03 oligonucleotide served as the sequencing primer. The number of α-globin genes was determined by hybridizing Pst I-digested DNA with α- and β-globin gene probes and BamHI- and Bgl II-digested DNA with α-globin and β-globin gene probes, respectively. These probes were radiolabeled with 32P dCTP by multirandom priming [13]. Case report Hb Taybe was identified in a 22 year-old Israeli Arab woman who has suffered since birth from a hemolytic anemia. At age one year an electrophoretic study of hemoglobin showed 5.4% Hb F and 1.5% Hb A2 and a β-thalassemia trait had been therefore suspected. Nevertheless, this diagnosis could not have explained an anemia varying between 7.5 and 10 g/dl with marked reticulocytosis, enlarged spleen and liver, and persistent jaundice. Erythrocyte enzyme studies had shown an increase in all tested enzyme activities,
30 (Amicon) procedure elsewhere described [12]. Sequencing reactions were performed using the sequencing kit (US Biochemicals), and Pa03 oligonucleotide served as the sequencing primer. The number of α-globin genes was determined by hybridizing Pst I-digested DNA with α- and β-globin gene probes and BamHI- and Bgl II-digested DNA with α-globin and β-globin gene probes, respectively. These probes were radiolabeled with 32P dCTP by multirandom priming [13]. Case report Hb Taybe was identified in a 22 year-old Israeli Arab woman who has suffered since birth from a hemolytic anemia. At age one year an electrophoretic study of hemoglobin showed 5.4% Hb F and 1.5% Hb A2 and a β-thalassemia trait had been therefore suspected. Nevertheless, this diagnosis could not have explained an anemia varying between 7.5 and 10 g/dl with marked reticulocytosis, enlarged spleen and liver, and persistent jaundice. Erythrocyte enzyme studies had shown an increase in all tested enzyme activities, consistent with a young erythrocyte population. The osmotic fragility test and the Ham test had been normal. At age sixteen she underwent splenectomy and cholecystectomy for a symptomatic cholelithiasis. She was reinvestigated one year later: the MCV had increased from 80-90 fl to 110-116 fl. Several immunological abnormalities were observed (high IgG, weakly positive Coombs test with C3 and very weakly with IgG, presence of both antigen I and i). A bone marrow aspiration showed marked increased red cell production with binuclear normoblasts and nuclear bridges. Electron microscopy studies showed increased proliferation of erythrocyte precursors with dyserythropoietic features. Although the morphological changes were compatible with type 1 CDA, there was, in addition to dyserythropoiesis, a marked normal erythropoiesis with many late normoblasts; this raised the possibility that the dyserythropoiesis was a secondary phenomenon. A control Coombs test was found normal. The patient was recently reinvestigated in Paris. Typical hematological data are given in Table I. The blood film showed anisocytosis, target cells, Howell-Jolly bodies, basophilic stippling and Heinz bodies. Table I Hematological data of the propositus RBC (1012/L) Hb (g/dl) 7.3 Hematocrit (%) 24.4 MCV (fl) 117 MCH (pg) 35 MCHC (%) 29.9 Reticulocytes (109/L) 530 The parents of the
(high IgG, weakly positive Coombs test with C3 and very weakly with IgG, presence of both antigen I and i). A bone marrow aspiration showed marked increased red cell production with binuclear normoblasts and nuclear bridges. Electron microscopy studies showed increased proliferation of erythrocyte precursors with dyserythropoietic features. Although the morphological changes were compatible with type 1 CDA, there was, in addition to dyserythropoiesis, a marked normal erythropoiesis with many late normoblasts; this raised the possibility that the dyserythropoiesis was a secondary phenomenon. A control Coombs test was found normal. The patient was recently reinvestigated in Paris. Typical hematological data are given in Table I. The blood film showed anisocytosis, target cells, Howell-Jolly bodies, basophilic stippling and Heinz bodies. Table I Hematological data of the propositus RBC (1012/L) Hb (g/dl) 7.3 Hematocrit (%) 24.4 MCV (fl) 117 MCH (pg) 35 MCHC (%) 29.9 Reticulocytes (109/L) 530 The parents of the propositus are first cousins (Fig. 2). The father is a healthy looking 60 year-old man. He has no history of jaundice or anemia and no cholelithiasis. Physical examination was normal. Laboratory data revealed Hb 13.9 g/dl and MCV 89.2 fl. The RBC morphology was normal with a reticulocyte count of 1.2 %. The mother is 58 year-old with unremarkable physical examination. Laboratory results were as follows: Hb 12.7 g/dl and MCV 84.9. The RBC morphology was normal with a reticulocyte count of 0.9%. By contrast, the sister and a brother of the propositus had a history of anemia since childhood with mild pallor and jaundice. The other brother could not be examined. The propositus' sister had an enlarged spleen (2 cm below the costal margins). Her RBC parameters were as follows: Hb 9.4 g/dl, MCV 84 fl, MCH 24.5 pg and MCHC 29.2 %. Blood smear revealed moderate anisocytosis, tear drop cells, target cells and moderate polychromatophilia. The reticulocyte count was 10 %. Total bilirubin was 2.3 mg %, mostly indirect (1.6 mg %). Physical examination of the brother (18 year-old) was normal except for mild scleral jaundice and enlarged spleen (3 cm below the costal margins). Laboratory data revealed
1.2 %. The mother is 58 year-old with unremarkable physical examination. Laboratory results were as follows: Hb 12.7 g/dl and MCV 84.9. The RBC morphology was normal with a reticulocyte count of 0.9%. By contrast, the sister and a brother of the propositus had a history of anemia since childhood with mild pallor and jaundice. The other brother could not be examined. The propositus' sister had an enlarged spleen (2 cm below the costal margins). Her RBC parameters were as follows: Hb 9.4 g/dl, MCV 84 fl, MCH 24.5 pg and MCHC 29.2 %. Blood smear revealed moderate anisocytosis, tear drop cells, target cells and moderate polychromatophilia. The reticulocyte count was 10 %. Total bilirubin was 2.3 mg %, mostly indirect (1.6 mg %). Physical examination of the brother (18 year-old) was normal except for mild scleral jaundice and enlarged spleen (3 cm below the costal margins). Laboratory data revealed a hemoglobin of 11.7 g/dl and MCV 90 fl. The blood smear showed the same morphological abnormalities as his sister's. Hemoglobin studies Table II describes the electrophoretic pattern of several members of the family. The abnormal hemoglobin was only seen in the propositus who was splenectomized. It could only be detected by IEF. This component amounted to approximately 3-4 % of the total lysate and migrated between Hb A and Hb F, at 1.5 mm from Hb A. Nothing was found in the other experimental conditions. Hb A2 was 2.6 % and Hb F 2.7 %. In addition, trace amounts (ca. 0.5 %) of Hb H were observed. The α/β globin chain biosynthetic ratio was 0.83 after 60 min of incubation (normal range 0.95 ± 0.5). Characterization of the structural abnormality The abnormal Hb was isolated by flat bed preparative IEF. The chains were isolated by RP-HPLC, aminoethylated and submitted to tryptic digestion. The HPLC peptide map showed that peptide αT-5 was more hydrophobic than normal (Fig. 3). Its amino acid composition (not shown) revealed that it contained only one threonine residue instead of two (at position 38 or 39). These data could be explained either by a point mutation
detected by IEF. This component amounted to approximately 3-4 % of the total lysate and migrated between Hb A and Hb F, at 1.5 mm from Hb A. Nothing was found in the other experimental conditions. Hb A2 was 2.6 % and Hb F 2.7 %. In addition, trace amounts (ca. 0.5 %) of Hb H were observed. The α/β globin chain biosynthetic ratio was 0.83 after 60 min of incubation (normal range 0.95 ± 0.5). Characterization of the structural abnormality The abnormal Hb was isolated by flat bed preparative IEF. The chains were isolated by RP-HPLC, aminoethylated and submitted to tryptic digestion. The HPLC peptide map showed that peptide αT-5 was more hydrophobic than normal (Fig. 3). Its amino acid composition (not shown) revealed that it contained only one threonine residue instead of two (at position 38 or 39). These data could be explained either by a point mutation replacing threonine Table II Electrophoretic pattern of several members of the family Hb A2 Hb F HbH Hb % % % Taybe % Proposita (11-1) 2.7 3.0 0.5 4 Mother (I-2) 2.75 0.5 0 Father (I-1) 2.45 0.4 0 II-2 2.45 4.7 0.5 0 II-3 2.35 7.1 0.5 0 Figure 3. HPLC pattern of the tryptic digest of the abnormal α-chain of Hb Taybe. DNA studies Direct sequencing of the α1- and α2-globin genes revealed for the propositus a deletion of 3 bp ACC in the α1 gene (Fig. 4), corresponding to a threonine residue (codon 38 or 39) at the homozygous (α1* / α1*) or heterozygous state (- / α1*), whereas the α2-sequence was normal (nucleotides + 1 to +889). This confirmed the second hypothesis made after protein studies and assigned the abnormality to the α1 gene. The α1α1 genotype was ascertained by Southern blotting as described in methods. The parents were then analyzed, exhibiting the α1 38-39 Thr-deletion on one of their chromosomes. Heterozygosity was first suspected by simply testing the double-stranded amplified products on a non-denaturing 6 % polyacrylamide gel, as shown on Figure 5. Heteroduplex molecules, the formation of which is favored by an additional denaturation-renaturation
3. HPLC pattern of the tryptic digest of the abnormal α-chain of Hb Taybe. DNA studies Direct sequencing of the α1- and α2-globin genes revealed for the propositus a deletion of 3 bp ACC in the α1 gene (Fig. 4), corresponding to a threonine residue (codon 38 or 39) at the homozygous (α1* / α1*) or heterozygous state (- / α1*), whereas the α2-sequence was normal (nucleotides + 1 to +889). This confirmed the second hypothesis made after protein studies and assigned the abnormality to the α1 gene. The α1α1 genotype was ascertained by Southern blotting as described in methods. The parents were then analyzed, exhibiting the α1 38-39 Thr-deletion on one of their chromosomes. Heterozygosity was first suspected by simply testing the double-stranded amplified products on a non-denaturing 6 % polyacrylamide gel, as shown on Figure 5. Heteroduplex molecules, the formation of which is favored by an additional denaturation-renaturation step, have been observed for both parents after amplification of only the α1-globin gene. These additional bands were checked to be α1-specific by restriction mapping of the eluted products with Apa I. They migrated more slowly than the homoduplex molecules owing to the mismatches they contain. The α1 38-39 Thr-deletion was confirmed by direct sequencing, whereas the corresponding α2 sequence was normal. The propositus' sister and brother, who displayed hematological abnormalities, were shown to be, identically, homozygous for the 3 bp deletion. Discussion Some unstable α-chain variants are present at such a low level in the RBC that they are not detected in the heterozygous carriers. Trace amounts of these abnormal hemoglobins, together with some Hb H, can be observed by electrophoresis in patients that are compound heterozygotes for another α-thalassemic gene and/or when they have been splenectomized. These hemoglobins are often referred to as non-deletional α-thalassemias [1-6]. The different hemoglobin variants that may be classified in this group are reported in Table III. Some of them lead 44U C. R. Acad. Sci. Paris, Sciences de la vie/Life sciences, 1994; 317 : 437-44 Hb Taybe: an α-globin defect Figure 4. Direct genomic sequencing of an α1-gene segment of a normal
was confirmed by direct sequencing, whereas the corresponding α2 sequence was normal. The propositus' sister and brother, who displayed hematological abnormalities, were shown to be, identically, homozygous for the 3 bp deletion. Discussion Some unstable α-chain variants are present at such a low level in the RBC that they are not detected in the heterozygous carriers. Trace amounts of these abnormal hemoglobins, together with some Hb H, can be observed by electrophoresis in patients that are compound heterozygotes for another α-thalassemic gene and/or when they have been splenectomized. These hemoglobins are often referred to as non-deletional α-thalassemias [1-6]. The different hemoglobin variants that may be classified in this group are reported in Table III. Some of them lead 44U C. R. Acad. Sci. Paris, Sciences de la vie/Life sciences, 1994; 317 : 437-44 Hb Taybe: an α-globin defect Figure 4. Direct genomic sequencing of an α1-gene segment of a normal subject (lane 1) and the propositus (lane 2), showing a bp deletion corresponding to a threonine residue in the α1-globin gene. Figure 5. Analysis of Pa03-C2 α1-globin gene fragments from normal individuals (lane 1,2), the index case (lane 3) and her parents (lanes 4,5). M: the size marker marker was cut with Hae III. H=homoduplex molecules. h=heteroduplex molecules. Their α1-specificity was checked by Apa I digestion analysis. Hb Quong Sze [α125(H8)Leu→Pro] found in a Chinese patient with a Hb H phenotype and carrying a [--/α1QS] genotype, was the first abnormal hemoglobin of this group described. The sequence of the α2 gene showed that codon 125 was CCG (Pro) instead of CTG (Leu). During biosynthesis studies, the abnormal gene was shown to be transcribed normally but the extremely rapid degradation of the abnormal chain resulted in a functional loss of the mutated α2 gene. Simple heterozygotes for Hb Quong Sze [α1/α2QS] resembled α-thalassemia carriers: there was no evidence of hemolysis nor Heinz bodies. The mutant chain, thus, did not lead to an unstable hemoglobin syndrome but, surprisingly, to an α-thalassemia trait phenotype. This disorder was explained at the molecular level by an alteration of the α1 contact area, resulting in free
cut with Hae III. H=homoduplex molecules. h=heteroduplex molecules. Their α1-specificity was checked by Apa I digestion analysis. Hb Quong Sze [α125(H8)Leu→Pro] found in a Chinese patient with a Hb H phenotype and carrying a [--/α1QS] genotype, was the first abnormal hemoglobin of this group described. The sequence of the α2 gene showed that codon 125 was CCG (Pro) instead of CTG (Leu). During biosynthesis studies, the abnormal gene was shown to be transcribed normally but the extremely rapid degradation of the abnormal chain resulted in a functional loss of the mutated α2 gene. Simple heterozygotes for Hb Quong Sze [α1/α2QS] resembled α-thalassemia carriers: there was no evidence of hemolysis nor Heinz bodies. The mutant chain, thus, did not lead to an unstable hemoglobin syndrome but, surprisingly, to an α-thalassemia trait phenotype. This disorder was explained at the molecular level by an alteration of the α1 contact area, resulting in free uncombined α- and β-globin chains and perhaps in an instability of transiently assembled tetramers [14-16]. In some patients a dominant Heinz bodies hemolytic hereditary anemia overlaps the α-thalassemic syndrome. Hemoglobin Petah Tikva [α110(G17)Leu→Asp], described in Israel among Iraqi Jews, is an unstable variant that yields the phenotype of a mild Hb H disease when present in combination with α-thalassemia trait [8]. In patients having Hb Petah Tikva in association with α-thalassemia the globin chain synthesis ratio was that expected for a mild Hb H disease. Conversely, the carriers of Hb Petah Tikva without associated α-thalassemia were clinically normal. In this case, no Hb H was observed but a substantial excess of total α-chain synthesis relative to β-chain synthesis was found. Such a paradoxical biosynthetic ratio has been C. R. Acad. Sci. Paris, Sciences de la vie/Life sciences, 1994; 317 : 437-44 F. Gaiti et al. Table III Unstable hemoglobin variants responsible for "non deletional α-thalassemias" Variant Structural modification Gene HbH % Référence Hb Evanston α1 (A12) Trp → Arg rightward α-thal2 traces 19 Hb Agrinion α29 (B10) Leu → Pro cc2 traces 25 Hb Taybe α38 or 39 (C4) Thr → al 0.5** (this paper) Hb Adana α59 (G11) Gly