A new highly unstable alpha chain variant causing alpha(+)-thalassemia: Hb Zurich Albisrieden [alpha59(E8)Gly-->Arg (alpha2)

A new alpha-globin mutation causing persistent mild hypochromic microcytosis and erythrocytosis is described. Hb Zurich Albisrieden [alpha59(E8)Gly-->Arg (alpha2)] is not detected at the protein level and leads to alpha(+)-thalassemia (thal).     

A moderately severe alpha-thalassemia condition resulting from a combination of the alpha2 polyadenylation signal (AATAAA-->AATA- -) mutation and a 3.7 Kb alpha gene deletion in an Australian family

We have recently studied a family with a rare combination of two abnormal alpha-globin genes. The combination of a two-base (AA) deletion in the alpha2 polyadenylation signal (poly A) (AATAAA-->AATA- -) and a 3.7 kb alpha gene deletion, found in two children, resulted in a moderately severe thalassemic condition. Both parents and three siblings were tested and the hematological condition and molecular findings are presented. The father was born in India with Portuguese and British ancestry; the mother is of Dutch ancestry. All three siblings were born in Australia.     

Hb Bronte or alpha93(FG5)Val-->Gly: a new unstable variant of the alpha2-globin gene,associated with a mild alpha(+)-thalassemia phenotype

We report a new unstable variant identified in three carriers of a family from East Sicily; it was named Hb Bronte after the place from which the family originated. DNA sequencing from nucleotides -181 to +894 (alpha1) and to +884 (alpha2) revealed a GTG-->GGG substitution at codon 93 of the alpha2-globin gene. The MCV and MCH values were at the lower end of the normal range in the carriers. On cation exchange high performance liquid chromatography (HPLC), the Hb A2 level was apparently increased to around 6%, and a small abnormal peak (0.3-0.4%) was detected after Hb A2. Two abnormal bands were detected by cellulose acetate electrophoresis: a major band (about 3-4%) migrated between Hb A and Hb F; a minor band (<1%) migrated between Hb A2 and carbonic anhydrase. Normal values of Hb A2 were detected by DEAE microchromatography. On reversed phase HPLC the variant chain was not detected, and most likely it was eluted with the alpha chain peak. The isopropanol stability test was very slightly positive in the carriers. Hemolytic symptoms were absent with the exception of indirect bilirubin, which was at high borderline in 2/3 carriers. In biosynthesis in vitro, the specific activity of the alpha chains was much higher than that of the beta-globin chains, and the alpha/beta biosynthetic ratio in the mother and proband was of the beta-thalassemia (thal) type (2.24 and 2.54, respectively). Time course experiments showed that the increase of the 3H-specific activity of the peak containing normal and variant alpha chains was not linear and was much higher than that of beta chains; moreover, the alpha/beta biosynthetic ratio varied during the 2 hours incubation.     

Hb Aghia Sophia [alpha62(E11)Val-->0 (alpha1)], an "in-frame" deletion causing alpha-thalassemia

In this report we describe a case of Hb H disease due to the interaction of the --(MED 1) deletion with a new alpha(+)-thalassemia determinant. The molecular analysis of the proband's genomic DNA was carried out by polymerase chain reaction amplification and sequencing of both alpha genes of the alpha(+)-thalassemia chromosome and revealed a deletion of codon 62 of the alpha1 gene. This DNA triplet codes for a valine residue at the E11 alpha helix, which is located in the interior of the heme pocket. Substitutions of valine E11 with other amino acid residues in the alpha as well as beta polypeptide chains lead, in the heterozygous carrier, either to Hb M disease or to congenital non-spherocytic hemolytic anemia. We assume that the deletion of valine at alpha62(E11) disrupts the conformation of the alpha chain to such an extent that the mutated subunit is rapidly removed by proteolysis. The final result is an alpha-thalassemia phenotype rather than an unstable hemoglobin syndrome. This conclusion is supported by the apparent absence of an abnormal alpha chain in the peripheral blood of the patient.     

Observation of a rare hemoglobin variant [Hb Lulu island, beta107(G9)Gly-->Asp, GGC-->GAC] co-inherited with a beta+-thalassemia mutation [IVS-I-110 (G-->A)] or in the heterozygous state in a Greek-Albanian family

We report clinical, hematological, biochemical, functional and molecular studies carried out on two first cousins from a Greek-Albanian family who have clinical and hematological findings consistent with the diagnosis of thalassemia intermedia. DNA studies determined that they had co-inherited a common Mediterranean beta-thalassemia (thal) mutation, IVS-I-110 (G-->A), in trans to a beta-globin gene mutation at codon 107 (GGC-->GAC), predicted to give rise to a rare unstable beta chain variant Hb Lulu Island or beta107(G9)Gly-->Asp.     

A novel alpha-thalassemia nonsense mutation in codon 23 of the alpha2-globin gene (GAG-->TAG) in a Tunisian family

Herein we describe a novel alpha-thalassemia (thal) point mutation in the alpha2-globin gene, found in a 3-year-old Tunisian girl who had Hb Bart's (gamma4) at birth, later on presenting with moderate anemia, microcytosis and hypochromia. She had a normal Hb A2 level and no abnormal hemoglobin (Hb) fraction. After excluding most of the common Mediterranean mutations, the alpha2-globin gene was sequenced and found to have a point mutation in the heterozygous state that creates a premature stop signal for translation (GAG-->TAG or Glu-->Term) at codon 23. The same mutation was also found in the mother in the heterozygous state, while the father had a normal sequence. The presence of the mutation was also confirmed by nucleotide sequencing of the opposite strand. Since the mutation creates a restriction site for the BfaI enzyme, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based assay was established for screening purposes.     

Hb Cardarelli [beta86(F2)Ala-->Pro]: a new unstable and hyperaffine variant in association with beta(+)-thalassemia

Hb Cardarelli [beta86(F2)Ala-->Pro] is a new unstable and high oxygen affinity variant found in several members of a family from Naples, Southern Italy. A detailed structural and functional characterization of the variant was performed on two subjects, at both the protein and DNA level. The first patient exhibited 43% of the variant hemoglobin (Hb) without major hematological problems. The proband showed 82% of the abnormal Hb in association with beta(+)-thalassemia (thal) that caused relevant erythrocytosis requiring frequent phlebotomies. Structural investigation of the Hb variant by mass spectrometric methodologies identified the amino acid replacement as Ala-->Pro at beta86. The corresponding DNA mutation GCC-->CCC at codon 86 of the beta-globin gene was assessed by both DNA sequencing and amplification refractory mutation system (ARMS) techniques. Functional studies carried out on whole blood and diluted hemolysates from both patients demonstrated increased oxygen affinity, decreased Bohr effect, reduced heme-heme interaction and nearly halved 2,3-diphosphoglycerate (2,3-DPG) and chloride effects.     

Thalassemia intermedia due to co-inheritance of beta0/beta(+)-thalassemia and (- -SEA) alpha-thalassemia/Hb Westmead [alpha122(H5)His > Gln (alpha2)] in a Chinese family

Two brothers from a Chinese family with beta-thalassemia intermedia who harbor both alpha- and beta-globin gene defects are described. They are both compound heterozygous for codons 41/42 (-CTTT) beta0-thalassemia and nt - 28 (A > G) beta(+)-thalassemia mutations together with concurrent (- -SEA) alpha-thalassemia (SEA) deletion. One sibling also harbors Hb Westmead, giving an unusual genotype of beta0/beta(+)-thalassemia and (- -SEA) alpha-thalassemia/Hb Westmead. With respect to the age at presentation and transfusion requirement, this subject shows a milder clinical phenotype than his brother, most probably explainable by the presence of Hb Westmead in addition to the SEA deletion, which causes a further amelioration of the alpha-chain excess and hence a less severe disease. For areas with high prevalence of both alpha- and beta-thalassemia mutations, their interactions should always be considered in genotype phenotype correlation. Moreover, routine laboratory diagnostic strategy for non-deletional alpha-globin gene mutations in the Chinese may need to include Hb Westmead, as it is a common alpha-globin gene mutation in our population apart from Hb Constant Spring and Hb Quong Sze.     

A rare thalassemic syndrome caused by interaction of Hb Adana [alpha59(E8)Gly-->Asp] with an alpha+-thalassemia deletion: clinical aspects in two cases

Hb Adana is a highly unstable and rare alpha-globin hemoglobin (Hb) variant, to date described in only three families, in interaction with other alpha-thalassemia (alpha-thal) deletions. We describe the clinical and hematological findings in two cases from independent families of Albanian origin, who have an interaction of the codon 59 (Gly-->Asp) alpha2-globin gene variant in trans to a 3.7 kb alpha(+)-thal deletion (alpha(codon 59)alpha/-alpha). We report their presenting symptoms and laboratory findings as well as complications and differences in their clinical management. Both cases can be characterized as thalassemia intermedia and illustrate the problems associated with selecting the most appropriate options for patient management, especially in cases with rare underlying genotypes.     

Hb Sallanches [alpha104(G11)Cys-->Tyr, TGC-->TAC (alpha2)]: an unstable hemoglobin variant found in an Indian child

We report the fourth observation of Hb Sallanches [alpha104(G11)Cys-->Tyr, TGC-->TAC (alpha2)], an unstable alpha chain variant of intermediate severity in the homozygous state. Heterozygosity occasionally produces mild hypochromia and microcytosis in some patients. A balanced beta/alpha ratio, found in previously reported cases, points to unstable alphabeta dimers formed as a result of the Cys-->Tyr substitution at the alpha1beta1 contact site in this hemoglobin (Hb) variant. Our patient, and the previous two of the three cases reported in patients of Pakistani origin, points to a common population stock, separated by the mass population migration which occurred during the partition of Pakistan and India in 1947.     

Hb A2-Pasteur-Tunis [delta59(E3)Lys-->Asn, AAG-->AAC]: a new delta chain variant detected by DNA sequencing in a Tunisian carrier of the codon 39 (C-->T) beta0-Thalassemia mutation

We describe a new delta-globin variant, Hb A2-Pasteur-Tunis [delta59(E3)Lys-->Asn, AAG-->AAC]. This hemoglobin (Hb) displayed an electrophoretic mobility faster than normal Hb A2 and was expressed at 2.2 %. The molecular defect was characterized by DNA sequencing and confirmed by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-designed protocol. Hb A2-Pasteur-Tunis was found in a carrier of a codon 39 (C-->T) beta0-thalassemia (thal), presenting with a normal Hb A2 level. Phenotype and genotype investigations revealed that the total Hb A2 level of the patient was that expected for a minor beta-thal (4.8%).     

First description in Tunisia of a point mutation at codon 119 (CCT-->TCT) in the alpha1-globin gene: Hb Groene Hart in association with the -alpha3.7 deletion

Herein we describe the case of a Tunisian girl who presented with 3% Hb Bart's (gamma4) at birth. At the age of 3 years, she showed microcytosis and hypochromia in the absence of iron deficiency. The first step of molecular analysis was to test for the common Mediterranean mutations and the classical -alpha3.7 deletion was found in the heterozygous state. Since this finding could not explain the level of Hb Bart's at birth, or the hypochromia and microcytosis, all the alpha-globin genes were sequenced. This revealed a rare point mutation at codon 119 (CCT-->TCT) in the alpha1-globin gene, identified for the first time in Tunisia, and which has previously been described as an unstable hemoglobin (Hb) variant named Hb Groene Hart [alpha119(H2)Pro-->Ser (alpha1)]. Here the -alpha3.7/alpha(alpha)119(CCT-->TCT) genotype is responsible for the alpha-thalassemia (thal) trait phenotype.     

Haemoglobin J-Biskra: a new mildly unstable α1 gene variant with a deletion of eight residues (α50–57, α51–58 or α52–59) including the distal histidine

Single point mutation, which accounts for 92% of the 700 known variants, is the most frequent genetic defect responsible for abnormal haemoglobins. Small deletions (or insertions) involving from one to five residues are also observed, but in only approximately 5% of cases. The remaining variants produce fusion or extended haemoglobins. A deletion of eight residues, which included the distal histidine and its neighbours (α50–57, α51–58 or α52–59), was found in Hb J-Biskra. This new α-chain variant was mildly unstable in vitro only and there was no haematological or biochemical evidence of haemolysis in the affected family members. 24 nucleotides were missing in a region of the α1 gene showing an identical sequence of eight nucleotides at both ends. Several starting points could therefore lead to the same nucleotide and aminoacid remaining sequence. This deletion is the largest up to now reported in a haemoglobin molecule which is expressed at an almost normal level in the red blood cell. Comparison of the DNA sequences near to the deleted (or inserted) regions in the various haemoglobins carrying this type of abnormality almost always revealed the presence of a sequence that was hypothesized to slow down progression of the replication fork, and of repeats that may lead to possible secondary structures favouring slipped mispairing.     

Rare thalassemic syndrome caused by interaction of Hb Questembert (alpha1 codon 131, TCT>CCT, Ser>Pro) with an alpha-thalassemia-2 deletion: implications for diagnosis and management

Abnormal globin chain biosynthesis may result in deficient quantity (thalassemia) or structural variation (abnormal hemoglobins) and traditionally, they represent two phenotypically distinct groups of disorders. However, the phenotypic expression of unstable hemoglobin variants often combine features of thalassemia together with variable peripheral hemolysis. To achieve definitive diagnosis in a child presenting with hemolytic anemia along with features associated with thalassemia intermedia, we evaluated clinical, hematological, biochemical, globin biosynthetic and molecular data. Definitive diagnosis was achieved by DNA analysis which characterized the proband to be a compound heterozygote for a common alpha-thalassemia-2 deletion (3.7 kb) and Hb Questembert (alpha131[H14] Ser>Pro) caused by a C>T mutation in codon 131 of the alpha1 globin gene in trans. The phenotype of thalassemia intermedia with marked dyserythropoiesis, found in patients inheriting alpha-thalassemia mutations along with unstable alpha-globin variants (i.e., alpha-thalassemic hemoglobinopathies), represents a distinct type of thalassemic syndrome. The proband in this study additionally had variable peripheral hemolysis, presumably related to characteristics of the unstable Hb Questembert. There is minimal experience for the management of such atypical cases and this case illustrates that it is probably insufficient to monitor clinical status in patients with such hemoglobinopathies based only on the levels of hemoglobin.     

Hb adana or α259(E8)Gly→Aspβ2, A severely unstable α1-globin variant,observed in combination with the -(α)20.5 KB α-thal-1 deletion in two Turkish patients

We have identified a severely unstable hemoglobin variant through sequencing of amplified DNA involving the α₁-globin gene; the mutation is located in codon 59 (CC G CA G) andresults in a Gly—Asp replacement. This amino acid substitution concerns a glycine residue at an internal position in the E helix, which is in close contact with a glycine residue of the B helix; introduction of the larger and charged aspartic acid residue greatly affects the stability of the molecule. This variant was present in association with a common α-thalassemia-1 deletion [-(α)20.5 kb] in two adults and caused a severe type of Hb H disease with anemia, low levels of Hb A₂, increased → chain, and Hb Bart's. In vitro chain synthesis in reticulocytes showed a high specific activity of the variant α chain. Only a minute quantity of Hb H was present but instead about 10% of Hb Bart's was observed. The increased synthesis of γ chains was likely due to specific characteristics of a chromosome with haplotype #3, which was present in both patients. The same family was studied 18 years ago [1]; the improved methodology presently available has led to a corrected diagnosis for these patients. © 1993 Wiley-Liss, Inc.     

Two new unstable haemoglobins leading to chronic haemolytic anaemia: Hb Caruaru [β122 (GH5) Phe→Ser], a probable case of germ line mutation,and Hb Olinda [β22 (B4) ‐ 25 (B7)], a deletion of a 12 base‐pair sequence

We describe here two new unstable β‐globin variants, Hb Caruaru and Hb Olinda, found in northeastern Brazil, both associated with chronic haemolytic anaemia. Haemoglobin Caruaru is caused by a single base substitution at codon 122 (TT C→TC C), possibly originating from the germ line cells of the patient’s grandmother. Haemoglobin Olinda is also a de novo mutation, caused by a 12 bp deletion leading to the removal of the 22nd to the 25th residues of the normal β‐globin chain.