De novo deletion within the telomeric region flanking the human alpha globin locus as a cause of alpha thalassaemia

We have identified and characterized a Scottish individual with alpha thalassaemia, resulting from a de novo 48 kilobase (kb) deletion from the telomeric flanking region of the alpha globin cluster which occurred as a result of recombination between two misaligned repetitive elements that normally lie approximately 83 kb and 131 kb from the 16p telomere. The deletion removes two previously described putative regulatory elements (HS-40 and HS-33) but leaves two other elements (HS-10 and HS-8) intact. Analysis of this deletion, together with eight other published deletions of the telomeric region, showed that they all severely downregulated alpha globin expression. Together they defined a 20.4-kb region of the human alpha cluster, which contains all of the positive cis-acting elements required to regulate alpha globin expression. Comparative analysis of this region with the corresponding segment of the mouse alpha globin cluster demonstrated conserved non-coding sequences corresponding to the putative regulatory elements HS-40 and HS-33. Although the role of HS-40 as an enhancer of alpha globin expression is fully established, these observations suggest that the role of HS-33 and other sequences in this region should be more fully investigated in the context of the natural human and mouse alpha globin loci.     

A novel deletion causing (epsilon gamma delta beta) degrees thalassaemia in a Chilean family

We describe a novel deletion causing (epsilongammadeltabeta) degrees thalassaemia segregating in three generations of a Chilean family of Spanish descent. Heterozygotes for the deletion were all affected by neonatal haemolytic anaemia. The deletion of 152,569 bp extends from 77 kb upstream of the epsilon gene to 31 kb downstream of the beta gene, and includes the entire beta-globin gene cluster and two upstream olfactory receptor genes. Comparison of the sequences of the deletion junction with those of the flanking normal DNA suggests that the deletion results from a non-homologous recombination event. The insertion of 16 'orphan' nucleotides in the deletion junction creates a perfect inverted repeat of 12 nucleotides, forming a 12-bp stem with a four-nucleotide loop that could have contributed to the illegitimate recombination. The 3' breakpoint is located within an L1 family repeat that contains a perfect 160-bp palindrome, and is in close proximity to the 3' breakpoints of five other deletions in the beta cluster - Indian (HPFH-3), Italian (HPFH-4) and Vietnamese GgammaAgamma (deltabeta) degrees HPFH, German and Belgian Ggamma (Alphagammadeltabeta) degrees thalassaemia.     

A reverse dot-blot method for rapid detection of non-deletion alpha thalassaemia

A reverse dot blot method based on membrane-bound allele-specific oligonucleotides as hybridization targets for amplified alpha-gene fragments has been developed for the rapid detection of four non-deletion alpha thalassaemia defects found in the Chinese. Since these non-deletion defects account for 22 8% of haemoglobin H disease, a sensitive, specific and rapid screening method should be of value.     

Complex interactions of deltabeta hybrid haemoglobin (Hb Lepore-Hollandia) Hb E (beta(26G-->A)) and alpha+ thalassaemia in a Thai family

Haemoglobin Lepore-Hollandia is an extremely rare condition in which a small deletion gives rise to a deltabeta hybrid, beta-like globin. There are two single reports of patients from South Pacific Islands and Bangladesh. We describe a family from central Thailand, in which this Hb Lepore-Hollandia interacts with a common beta globin variant (beta(E) resulting from the codon 26, G-->A mutation) and alpha(+) thalassaemia (alpha(3.7)). This intriguing interaction caused a troublesome diagnosis, as the two proband brothers were diagnosed as having Hb E/beta thalassaemia. Molecular analysis of genomic DNA performed in this study allowed the definitive diagnosis of this complicated interaction. Such studies are required in the diagnosis of thalassaemia and haemoglobinopathies for particular regions like South-east Asia, where many different genotypes may give rise to haemoglobin disorders.     

Hb H hydrops fetalis syndrome associated with the interaction of two common determinants of alpha thalassaemia (--MED/(alpha)TSaudi(alpha))

To date, more than 35 single or oligonucleotide mutations of the alpha genes that cause alpha thalassaemia have been described. Their interactions give rise to widely variable clinical manifestations, from a mild hypochromic, microcytic anaemia to a lethal intrauterine anaemia associated with hydrops fetalis. Understanding the molecular genetics enables accurate genotyping, genetic counselling and prenatal testing for the most severe forms of alpha thalassaemia. Here we show for the first time that the interaction between two relatively common forms of alpha thalassaemia (--MED/(alpha)TSaudi(alpha)) may be associated with a clinically severe form of alpha thalassaemia, Hb H hydrops fetalis.     

Diagnosis of thalassaemia by non-isotope detection of α/β and ζ/α mRNA ratios

Summary. The α/β and ζ/α messenger RNA (mRNA) ratios in the thalassaemia syndromes were investigated by polymerase chain reaction (PCR) with silver staining of the PCR products. In this study we used the PCR to amplify cDNA copies of circulating erythroid cell mRNA in order to measure the relative amounts of α-, β- and ζ-globin contained within. Quantitation was performed by scanning the silver stain of specific globin cDNA bands. We found that there were significant differences of α/β-mRNA and ζ/α-mRNA in patients with Hb H disease and α-thalassaemia-1 compared to normal subjects. There was a marked increase in the α/β-mRNA ratio but not in the ζ/α-mRNA ratio in patients with β-thalassaemia. In two β-thalassaemia cases abnormal increases of ζ-globin bands were noted and they were confirmed through DNA analysis to be combined with α-thalassaemia-1. This method provides a simple, rapid and non-radioactive approach to detect thalassaemia syndromes, and can help to screen cases of β-thalassaemia with α-thalassaemia-1.     

A novel 7.9 kb deletion causing alpha+-thalassaemia in two independent families of Indian origin

We describe the characterization of a novel 7.9 kb deletion that eliminated one of the duplicated alpha-globin genes, causing an alpha+-thalassaemia phenotype in two independent carriers of Suriname-Indian origin. The molecular characterization of the deletion breakpoint fragment revealed neither involvement of Alu repeat sequences nor the presence of homologous regions prone to recombination, suggesting a non-homologous recombination event. This alpha+-thalassaemia deletion was found to give rise to an atypical haemoglobin H (HbH) disease characterized by a non-transfusion-dependent moderate microcytic hypochromic anaemia in combination with a poly adenylation signal mutation of the alpha-globin gene (alpha2 AATAAA --> AATA-- --).     

A variant of spectrin low-expression allele αLELY carrying a hereditary elliptocytosis mutation in codon 28

Summary. Allele α^LELY is a low-expression allele of the erythroid spectrin α-gene. It carries mutations in exon 40 (α^V/41 polymorphism) and intron 45, respectively, and is associated with partial skipping of exon 46. The latter phenomenon is thought to impair the recruitment of α-chains by β-chains, and would eventually account for the low-expression character. When it occurs in trans to an α-allele responsible for hereditary elliptocytosis (α^HE allele; α^HE/α^LELY diplotype), allele α^LELY enhances the severity of elliptocytosis. Because allele α^LELY is widespread, we anticipated that it would occasionally carry HE determinants. These variants of allele α^LELY will be designated α^HE-LELY alleles. We report two families with the same α^HE-LELY allele. The HE component was the known α28 Arg → His mutation. This α^HE-LELY allele was investigated within the α^HE-LELY/α^LELY diplotype, a diplotye not described before. Except for the neonatal period, the presentation was mild. In a consistent manner, the α^LELY component in cis of the α^HE mutation counteracted the like component in trans .     

Hb Woodville, a rare alpha-globin variant, caused by codon 6 mutation of the alpha1 gene

Since 1995, the national programme for the prevention and control of severe thalassaemia has been implemented in Thailand. This programme is composed of the population screening in pregnant women and couples by osmotic fragility, HbE screening and the confirmation test using haemoglobin analyses by electrophoresis or chromatography. Thereafter, several hitherto unidentified haemoglobins (Hbs) with structural defects are increasingly described and these variants are now easily studied using DNA technology. In this study, the authors describe the haematology and molecular analyses in a 28-yr-old healthy female who was identified as having an exceptionally 'high HbA2' from haemoglobin analysis. Subsequent analyses demonstrated that observed atypical 'HbA2' was, in fact, a rare innocuous alpha-globin variant, called Hb Woodville [alpha 2 6(A4); Asp --> Tyr]. For the first time, this abnormal Hb species is characterised at the molecular level.     

Efficacy of an analysis of lymphocyte subsets in predicting the clinical response to α-interferon therapy in thalassaemia patients with chronic infection by hepatitis C virus: a pilot study

Summary. α-interferon (α-IFN) has been used to treat chronic non-A non-B hepatitis in thalassaemic patients with response rates from 45% to 83%. Unfortunately, treatment with α-IFN is associated with side-effects which have a negative effect on the quality of life of the patient. Therefore it would be useful if we could distinguish in advance those patients who would benefit from such therapy from those who would not. In the present study we found that the modification of lymphocyte subsets 20 h after the administration of the first dose of α-IFN revealed that relative numbers of T helper lymphocytes (CD4⁺) increased in three non-responding patients and decreased in five responding patients, whereas those of T suppressor lymphocytes (CD8⁺), and natural killer cells (CD57⁺. CD16⁺) decreased in non-responding patients and increased in responding patients. Therefore analysis of the lymphocyte subsets CD4, CD8, CD57 and CD16 before and 20 h after the administration of α-IFN can be used to predict the clinical response to treatment with α-IFN.     

The -308 TNFalpha gene polymorphism in severe acute alcoholic hepatitis: identification of a new susceptibility marker

Background:  This study investigated whether genetic polymorphisms in the tumor necrosis factor alpha (TNFα) gene's promoter region play a role in severe acute alcoholic hepatitis (AAH).
Methods:  One-hundred and fifty patients (58 AAH patients, 45 cirrhosis group free-AAH, 47 healthy group) were genotyped for 3 TNFα polymorphisms (−238, −308, −863) using a polymerase chain reaction-restriction fragment length polymorphism technique. Serum TNFα levels were determined.
Results  The TNFα−308 allele A frequency was significantly lower in AAH group (0.09), than cirrhosis (0.28) ( p  < 0.001), and healthy groups ( p  < 0.001). The TNFα−308 A/G, A/A genotypes were significantly lower in AAH group, than cirrhosis ( p  = 0.005), and healthy groups ( p  < 0.001). For AAH group, there were no clinical, biological, and serum TNFα differences between the −308 G/G and A/G, A/A genotype patients, apart higher transaminase in the former group ( p  = 0.02). AAH and cirrhosis groups did not differ for the frequency of TNFα−238, −863 polymorphisms. The specific genotype did not appear to have any influence on the therapeutic response following corticotherapy or posttreatment 6-month survival. In the AAH group, nonsurvivors had higher TNFα levels than survivors (7.9 ± 8.8 vs. 3.3 ± 1.6 pg/ml, p  = 0.01).
Conclusions:  These results attest to the involvement of TNFα-related genetic factors in susceptibility to AAH.     

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.     

The C-->G transition in the alpha 2-globin gene of a normal alpha alpha-chromosome is responsible for the Hb G-Philadelphia variant in Sardinians

Sequencing of alpha-globin genes of 18 Sardinian heterozygotes for the Hb G-Philadelphia [alpha 68(E17)Asn-->Lys] variant, with four active alpha genes and circulating level of the variant of about 27%, showed the AAC-->AAG change at codon 68 of the alpha 2-globin gene (alpha(G)alpha/alpha alpha). Two heterozygotes with level of about 37% were the carriers of the same mutation on the same alpha 2 gene, and of the alpha 2 alpha 1 hybrid gene, because of the 3.7-kb deletion, in trans (alpha(G)alpha/-alpha(3.7)). In Black people, the same C-->G mutation occurs on the hybrid gene (-alpha(G)3.7), whereas in Caucasians the Lys for Asn change is because of the C-->A transversion occurring on the alpha 2 gene of a normal alpha alpha arrangement. The identification of the C-->G mutation on the normal alpha alpha chromosome points to an undescribed genotype for this rather common variant, which is probably because of the high rate of recombination between the duplicated alpha-globin genes.     

Alpha+ -thalassaemia and pregnancy in a malaria endemic region of Papua New Guinea

The effect of maternal alpha+ -thalassaemia on pregnancy was assessed in the north coastal region of Papua New Guinea (PNG), where malaria is hyperendemic and alpha+ -thalassaemia is extremely common. In a prospective study of 987 singleton hospital deliveries, we correlated maternal alpha-globin genotype with markers of reproductive fitness (age in primigravidae, gravidity, pregnancy interval and the number of miscarriages and stillbirths), Plasmodium falciparum(P. falciparum) infection of the mother and placenta, maternal haemoglobin, preterm delivery and birthweight. The frequency of the -alpha genotype in mothers was 0.61. Markers of reproductive fitness were similar in women with and without alpha+ -thalassaemia. Median haemoglobin concentration during pregnancy and after delivery was about 1.0 g/dl lower in homozygous alpha+ -thalassaemia than in women with a normal alpha- globin genotype (P < or = 0.001). The frequency of placental P. falciparum infection and systemic malaria infection after delivery showed no consistent relationship to alpha-globin genotype. The frequency of preterm delivery and low birthweight did not vary significantly according to maternal alpha-globin genotype. Maternal alpha+ -thalassaemia does not affect reproductive fitness or susceptibility to malaria during pregnancy. Although median haemoglobin concentration was significantly lower in mothers homozygous for alpha+ -thalassaemia than those with a normal alpha-globin genotype, this did not result in an adverse outcome of pregnancy.     

The molecular basis of β thalassaemia in Punjabi and Maharashtran Indians includes a multilocus aetiology involving triplicated α-globin loci

Summary We have analysed 201 β-thalassaemia (β-thal) genes from natives of the Punjab (156) and Maharashtra states of India and found the causative mutation in 200 of them. The most common β-globin gene mutations differed significantly between these two groups and between these groups and Indian immigrants in the U.S.A. and the U.K. In the Punjabi Indians the IVS-1, nt 1 (G–T) mutation accounted for nearly one-quarter of β-thal genes, whereas it was 5% or less in the other groups. Likewise, the cap+ 1 mutation was much more prevalent in the Punjabis, whereas the nonsense codon 15 allele had a higher frequency in the Maharashtrans of the Bombay region. The common IVS-1, nt5 allele had a frequency of 60% of β-thal genes in the Maharastrans, 35% in North American immigrants, and only 23% in the Punjabis. Two-thirds of all β-thal genes in Punjab were found in the merchant caste (Khatri-Arora), whereas the menial caste (Shudra) was highly represented among those with β-thal genes in Maharashtra. Two novel β-globin alleles were each found once; a frameshift codon 55 (+ A) in Maharashtrans and a frameshift codons 47–48 (+ ATCT) in Punjabis.
Of three Punjabi patients with β-thal intermedia in whom only a single severe β-globin gene mutation was found, two had six α-globin genes (homozygosity for a triplicated α-globin locus) instead of the normal α-globin gene number of four. Thus, these two individuals had a multilocus aetiology of β-thal and their parents have the unusual recurrence risk of 1 in 8 for conceiving a third with β-thal intermedia. Since 15% of 126 α-globin clusters studied in Punjabis contained either single (10%) or triplicated (5%) α-globin genes, the α-globin gene number is a frequent modifier of the phenotype of β-thal in this ethnic group.