β141 Leu is not deleted in the unstable haemoglobin Atlanta-Coventry but is replaced by a novel amino acid of mass 129 daltons

Reinvestigation of the structure of the beta-chain of Hb Atlanta-Coventry (beta 75 Leu----Pro, beta 141 Leu deleted) confirmed the presence of two abnormalities; however, analysis of the aberrant beta Co14 tryptic peptide by liquid secondary ion mass spectrometry indicated that the beta 141 Leu (mass 113 daltons) was not deleted but replaced by a novel amino acid of mass 129 daltons. The new amino acid in peptide beta Co14 was uncharged at pH 6.5, more hydrophillic than leucine and susceptible to cleavage by both chymotrypsin and carboxypeptidase A. We propose that the new residue is likely to be hydroxyleucine and that it results from post-translational oxidation of beta 141 Leu as a consequence of perturbation of the haem environment caused by the beta 75 Leu----Pro mutation in the E helix (E19). This proposal is entirely consistent with recent DNA analysis which showed that beta At-Co was not the product of a third beta-globin gene and that neither of the two beta-globin genes, beta A nor beta Atlanta, contained a deletion of the beta 141 Leu codon. We have subsequently found this modified amino acid at position beta 141 in two other unstable haemoglobins, both of which involve mutations on the haem side of the E helix.     

Haemoglobin Perth: β32 (B14) Leu→Pro, An Unstable Haemoglobin Causing Haemolysis

S ummary . An unstable haemoglobin, Hb-Perth, β32 (B14) Leu→Pro, is described. The patient had a haemolytic anaemia with a T _½⁵¹Cr RBC survival of 8 days, Hb 10–12 g/100 ml and had post splenectomy Heinz body formation.     

A new unstable haemoglobin variant: Hb Shanghai [β131 (H9)Gl→Pro] found in China

A 34-year-old woman patient was found to have a chronic hereditary haemolytic anaemia. No abnormal haemoglobin band was detected by conventional electrophoresis, but a slow beta chain could be separated on urea-carboxymethyl cellulose chromatography. Investigations of the patient's haemoglobin revealed an unstable component. Analyses of chemical structure, including isolation and TPCK trypsin digestion of the abnormal globin chain. HPL chromatography, amino acid composition as well as sequence determination of the abnormal peptide, indicated that a glutamine was replaced by a proline at position beta 131 (H9). Biosynthesis studies demonstrated a normal rate of synthesis but relatively fast degradation of the mutant beta chain. The new variant is named as Hb Shanghai according to the place where it was discovered.     

A single nucleotide deletion in codon 123 of the β-globin gene causes an inclusion body β-thalassaemia trait: a novel elongated globin chain βMakabe

The beta-globin gene from a Japanese individual with an inclusion body beta-thalassaemia trait has been characterized by gene cloning and DNA sequencing. An adenine deletion was detected at the first position of codon 123 (ACCCC) of one allele whereas the other allele had a normal sequence. Heterozygosity for this mutation in the patient was confirmed by Southern blots of the genomic DNA digested with HphI, the recognition site of which is eliminated by this deletion. This one base deletion results in the shift of a reading frame in such a manner that the normal termination codon is out of phase. This frameshift mutation results in the synthesis of an elongated beta-globin chain with 10 extra amino acid residues and with an altered C-terminus. Analysis of labelled globin chains using CM-cellulose column chromatography failed to demonstrate any abnormal protein, thereby suggesting that the beta-globin chain variant is highly unstable and probably degrades rapidly after synthesis. This event will lead to an accumulation of free alpha-chains precipitating in the red blood cells and an inclusion body beta-thalassaemia phenotype would ensue.     

Hereditary persistence of fetal haemoglobin (HPFH) in conjunction with a chromosomal translocation involving the haemoglobin β locus

An HPFH syndrome was found in a woman and her daughter who also carry a 'balanced' cyclic translocation of chromosome segments involving four chromosomes, with one break point located in the region of the Hb beta locus. This HPFH is characterized by 5% and 8% Hb F in peripheral blood, uneven distribution of Hb F in the red cells, and a G gamma/G gamma + A gamma ratio of 0.4. The mapping of the non alpha gene cluster shows no detectable deletion in the entire gamma-delta-beta-globin gene region.     

Haemoglobin Siriraj, β-7 (βA4) Glu→ Lys, in a Chinese Subject in Taiwan

Abstract. Haemoglobin Siriraj, β-7 Glu→Lys, was reported in members of a Thai family by Tuchinda, Beale and Lehmann [Brit. med. J. 1 : 1583–1585, 1965]. The same variant now is reported in a normal Chinese subject originating from the Chinese province of Honan. Relative amounts of haemoglobins A_o and Siriraj were 67 to 33. Incidence of Siriraj in Chinese subjects is low; only one subject was found among 160,000 Chinese tested.     

Possible factors influencing the haemoglobin and fetal haemoglobin levels in patients with β-thalassaemia due to a homozygosity for the IVS-I-6 (T→C) mutation

Summary. We have collected haematological, haemoglobin (Hb) and DNA sequence data for 29 patients with a homozygosity for the IVS-I-6 (TC) mutation with the intention of identifying factors contributing to the observed variability in the severity of the disease. None of the patients had received blood transfusion therapy for at least 6 months prior to the study. Hb levels varied from 5·0 to 9·9 g/dl. Patients with high Hb F (more than 1·5 g/dl or <20%) had high total Hb levels (7·5–9·7 g/dl) but some with low Hb F also had high total Hb levels; two had a concomitant α-thalassaemia-2 (α-thal-2) heterozygosity. An inverse correlation between the Hb F and Hb A₂ levels was observed. The majority of the patients were homozygous for haplotype VI (49/58 chromosomes) but haplotypes IV (2/58) and VII (7/58) were also present. The only haplotype IV homozygote had high Hb F levels with high ^Gγ values and the CT mutation at position – 158 in the ^Gγ promoter, while both high and low Hb F levels were observed among patients with haplotypes VI and VII. Analysis of sequence variations in regulatory regions included the 5 hypersensitive sites (HS) 4, 3 and 2 of the locus control region (LCR), the ^Gγ and ^Aγ 5 flanking regions, the second intervening sequence (IVS-II), and the 5 β-globin gene region in two patients with high Hb F (one homozygote each for haplotypes VI and IV), and in two patients with low Hb F levels (one homozygote each for haplotypes VI and VII). Haplotype specific differences were observed in the LCR 5 HS-2 and in the ^Gγ and ^Aγ flanking and IVS-II regions; however, no differences were present between the low and high Hb F-producing haplotype VI chromosomes, suggesting a major role for factors which are not linked to the β-globin gene cluster in mediating γ-globin gene expression in patients with this type of β-thal.     

β2-glycoprotein 1 (β2GP1) enhances cardiolipin binding activity but is not the antigen for antiphospholipid antibodies

Some investigators have reported that a serum protein, beta 2-glycoprotein 1 (beta 2GP1), either alone or in combination with negatively charged phospholipid, may be the antigen for anticardiolipin (aCL) antibodies. To examine these reports further, ELISA tests, inhibition experiments, Ouchterlony and Western blot techniques were used to examine anticardiolipin binding to beta 2GP1. Sera from patients with the antiphospholipid syndrome (APS) and syphilis were studied, as well as whole IgG immunoglobulin and affinity purified (a.p.) IgG aCL antibodies. Results showed no binding of aCL antibodies to beta 2GP1 in the absence of cardiolipin. beta 2GP1 caused enhanced binding of aCL antibodies to cardiolipin, but this enhancement was not observed in inhibition experiments. Binding to cardiolipin occurred in the absence of beta 2GP1. Enhancement of cardiolipin binding activity by beta 2GP1 was observed for APS, but not for syphilis. We conclude that beta 2GP1 is not the antigen for aCL antibodies, nor is it likely that the antibody recognizes shared beta 2GP1-cardiolipin epitopes. Instead, this protein may make cardiolipin more available for aCL binding on solid surfaces by some yet undefined mechanism. This effect may not extend to aqueous suspensions.     

Location and PCR-based detection of three polymorphisms of the human erythrocyte β-spectrin gene (SPTB)

Summary We describe a patient with both haemophagocytic syndrome and acute myocarditis probably associated with parvovirus B19 infection. The patient had a marked neutrophilia instead of neutropenia more usually observed in virus-associated haemophagocytic syndrome (VAHS). Endogenous serum concentrations of macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), and tumour necrosis factor-α (TNF-α) were higher than normal, suggesting that these cytokines may be involved in the genesis of the observed syndrome.     

Dominant β-thalassaemia trait in a Portuguese family is caused by a deletion of (G)TGGCTGGTGT(G) and an insertion of (G)GCAG(G) in codons 134, 135, 136 and 137 of the β-globin gene

We have studied a Portuguese family with a dominant beta-thalassaemia trait that was present in one member of each of three generations. It was characterized by a moderate anaemia, microcytosis and hypochromia, anisopoikilocytosis, Heinz body formation in peripheral red cells, splenomegaly, and a blood transfusion requirement during pregnancy. Sequence analyses of amplified DNA detected a deletion of (G) TG.GCT.GGT.GT(G) at codons 134-137 (Val.Ala.Gly.Val) and the insertion of (G)GC.AG(G) (Gly.Arg) at the same location. Thus, the resulting beta chain has an abnormal structure only at codons 134-137 and is two residues shorter than the normal 146 residues. This chain could not be detected in circulating red cells and must be degraded rapidly by proteolysis because the Heinz bodies consisted mainly of alpha chains.     

Composition of the intra-erythroblastic precipitates in thalassaemia and congenital dyserythropoietic anaemia (CDA): identification of a new type of CDA with intra-erythroblastic precipitates not reacting with monoclonal antibodies to α- and β-globin chains

Ultrathin sections of bone marrow cells from two patients with homozygous β-thalassaemia, two patients with haemoglobin H (HbH) disease, a patient with congenital dyserythropoietic anaemia (CDA) type III and two patients with severe congenital dyserythropoietic anaemia of an unusual type were reacted with mouse monoclonal antibodies against various globin chains and the reaction visualized using a gold-labelled goat antibody against mouse IgG. The multiple rounded intra-erythroblastic inclusions found in homozygous β-thalassaemia reacted with the monoclonal antibody against α-globin chains but not β-globin chains, thus confirming that they consisted of precipitated α-globin chains. The branching intra-erythroblastic inclusions found in HbH disease and CDA type III reacted with the monoclonal antibody against β-globin chains but not α-globin chains, indicating that they consisted of precipitated β-globin chains. The two patients with severe CDA had been transfusion-dependent since infancy, had a normal α:β globin chain synthesis ratio or parents with normal red cell indices, displayed prominent dysplastic changes in their erythroblasts, and had intra-erythroblastic inclusions resembling those seen in homozygous β-thalassaemia. However, unlike those in β-thalassaemia, the inclusions in these two patients did not react with the monoclonal antibody against either α- or β-globin chains. The inclusions reacted with antibody against ζ-globin chains, but detailed studies in one of the patients indicated that the antigen involved was not ζ-globin. These patients have features not reported in the condition known as dominantly inherited inclusion body β-thalassaemia and appear to suffer from a novel type of CDA in which the intra-erythroblastic inclusions may consist of some non-globin protein or structurally-abnormal α-globin chains.     

A Leu262Pro mutation in the integrin beta(3) subunit results in an alpha(IIb)-beta(3) complex that binds fibrin but not fibrinogen

Platelet retraction of a fibrin clot is mediated by the platelet fibrinogen receptor, alpha(IIb)beta(3). In certain forms of the inherited platelet disorder, Glanzmann thrombasthenia (GT), mutant alpha(IIb)beta(3) may interact normally with fibrin yet fail to support fibrinogen-dependent aggregation. We describe a patient (LD) with such a form of GT. Platelets from LD supported normal clot retraction but failed to bind fibrinogen. Platelet analysis using flow cytometry and immunoblotting showed reduced but clearly detectable alpha(IIb)beta(3), findings consistent with type II GT. Genotyping of LD revealed 2 novel beta(3) mutations: a deletion of nucleotides 867 to 868, resulting in a premature stop codon at amino acid residue 267, and a T883C missense mutation, resulting in a leucine (Leu) 262-to-proline (Pro) substitution. Leu262 is highly conserved among beta integrin subunits and lies within an intrachain loop implicated in subunit association. Leu262Probeta(3) cotransfected with wild-type alpha(IIb) into COS-7 cells showed delayed intracellular maturation and reduced surface expression of easily dissociable complexes. In human embryonic kidney 293 cells, Leu262Probeta(3) formed a complex with endogenous a(v) and retracted fibrin clots similarly to wild-type beta(3). The same cells, however, were unable to bind immobilized fibrinogen. The molecular requirements for alpha(IIb)beta(3) to interact with fibrin compared with fibrinogen, therefore, appear to differ. The region surrounding beta(3) Leu262 may maintain beta(3) in a fibrinogen-binding, competent form, but it appears not to be required for receptor interactions with fibrin.     

A 5' splice region G → C mutation in exon 3 of the human β-spectrin gene leads to decreased levels of β-spectrin mRNA and is responsible for dominant hereditary spherocytosis (spectrin Guemene-Penfao)

We studied a family with autosomal dominant hereditary spherocytosis (HS) associated with a mild spectrin deficiency. Linkage analysis using two microsatellite markers (D14S63 and D14S271) very close to the β-spectrin gene (SPTB) showed that HS co-segregated with alleles of these microsatellite markers and the linkage between the marker and HS was statistically significant. The presence of a β-spectrin protein polymorphism (β-spectrin Vay; A1880V) in trans of the HS allele was not itself deleterious, but allowed the detection of decreased membrane expression of the spherocytic β-spectrin allele in two HS-affected subjects. Direct sequencing of the coding exons of the β-spectrin gene in one affected subject showed the presence of a G → C transversion at the terminal nucleotide of exon 3, which did not change the leucine codon 100 (CTG → CTC). The presence of the mutation was confirmed by restriction enzyme digestion at the DNA level in all affected SH members of the family. The G → C mutation severely reduced the utilization of the 5' splice site and resulted in aberrant mRNA splicing with intron 3 retention.     

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 (β^Th-mRNA) is stable and present in quantities similar to that of normal β^A-mRNA. cDNA fragments derived from β^Th- and β^A-mRNAs can be separated on a denaturing polyacrylamide gel electrophoresis because the β^Th fragment is three nucleotides (nts) longer than the β^A fragment. The β^Th-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 β^X 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.     

A novel TC deletion resulting in Pro(260)-->Stop in the human LCAT gene is associated with a dominant effect on HDL-cholesterol

Human lecithin:cholesterol acyltransferase (LCAT) plays a key role in the biogenesis of circulating high-density lipoprotein-cholesterol (HDL-C) and reverse cholesterol efflux. We investigated the molecular defect in the LCAT gene in a family with low levels of HDL-C. The proband, a 53-year-old woman from Oklahoma City, had a HDL-C level of 0.21 mmol/l. The LCAT activity in the proband was 5 nmol/ml/h and cholesterol esterification rate was 54.2 nmol/ml/h, consistent with LCAT deficiency. Analysis of polymerase chain reaction (PCR) amplified subgenomic fragments of LCAT DNA on polyacrylamide gels revealed heteroduplex bands in the proband and three other affected individuals in exon 6. DNA sequence analyses of the proband's LCAT gene identified a 2 base pair deletion (TC) (base pairs 4544-4545, corresponding to amino acid 255) in the heteroduplex allele, thereby converting Pro(260) to a premature stop codon and a predicted truncated protein of 260 amino acids. This is approximately 60% of the length of the normal translated protein. The heterozygous individuals also revealed significant reduction in apolipoprotein A-1 levels compared with the unaffected family members (n=4). The marked reduction in HDL-C in the proband and sibling suggests a dominant effect of this mutation on HDL-C levels. Furthermore, because the deletion results in a heterozygous allele that can be detected by a simple PCR reaction and polyacrylamide gel-size fractionation, it may be possible to rapidly screen susceptible individuals for the presence of this mutation.     

Lack of sequence variations in the C4b-BP β-chain in patients with type III protein S deficiency bearing the Ser 460 to Pro mutation: description of two new intragenic isomorphisms in the C4b-BP β-chain gene (C4BPB)

Type III protein S (PS) deficiency, characterized by low levels of free PS and normal total PS levels, is often associated with the Ser 460 to Pro substitution. However, some patients bearing this mutation have normal PS levels, suggesting that another gene defect may account for this phenotype. We postulated that this defect was located in the C4b-BP β-chain gene (C4BPB) and searched for a mutation in the coding regions of this gene in 35 propositi with type III PS deficiency and bearing the Ser 460 to Pro mutation. No mutations explaining the phenotype of type III PS deficiency were identified. We did, however, find two frequent nucleotide changes, one being located in the donor splice site of intron d and the second in the codon corresponding to Asn 137. We used these two polymorphisms to establish C4BPB gene haplotype in five informative type III PS-deficient families and exclude a role of the C4BPB gene in this phenotype of three of them. Finally, increased C4b-BP β-chain levels were not responsible for the phenotype of type III PS deficiency as the C4BPB haplotype did not correlate with C4b-BP β-chain levels.     

A novel missense mutation (1060G --> C) in the phosphoglycerate kinase gene in a Japanese boy with chronic haemolytic anaemia,developmental delay and rhabdomyolysis

We report the case of a 3-year-old Japanese boy with phosphoglycerate kinase 1 (PGK1) deficiency (Online Mendelian Inheritance in Man entry 311800). The patient had anaemia and jaundice at birth, necessitating exchange transfusions for 2 d. After one red blood cell transfusion at age 2 months, his Hb level was 8-9 g/dl, his reticulocyte counts were 300-500 x 109/l, and his total bilirubin level was 25.65-42.75 micro mol/l. The patient suffered two episodes of respiratory infection-associated haemolytic crisis and rhabdomyolysis during early infancy. At age 3.0 years, his developmental milestones (developmental quotients measured using the Tsumori-Inage methods) score was 49% (normal 74-131%), and his height was below average by -2.0 standard deviations. The diagnosis of PGK1 deficiency was made based on his remarkably low (< 10% of normal) erythrocyte PGK enzyme activity level and the identification of a novel missense (1060G-->C) PGK1 gene mutation. This mutation results in the Ala-353Pro amino acid substitution, which has been designated PGK Kyoto. The patient developed the full clinical symptoms of PGK1 deficiency including haemolytic anaemia, myopathy, central nervous system disorder and growth retardation, which is unusual.     

Occurrence of haemoglobin Norfolk (alpha2 57 (E6) Gly leads to Asp beta2) at the level of 33% in an Italian family from Calabria.

An abnormal, fast-moving haemoglobin was observed in 5 healthy subjects of a family from Calabria (southern Italy). In all these carriers the abnormal haemoglobin, which structural studies identified as Hb Norfolk (alpha2 57 (E6) Gly leads to Asp beta2) [4], occurs at a level averaging 33% of the total haemoglobin. Biosynthetic studies showed no evidence for unbalance of the globin chain synthetic ratio. In order to account for the observed percentages of Hb Norfolk, current concepts about the alpha-globin chain genetic system are reviewed, and different genic arrangements which would be in agreement with the experimental findings are discussed.