Double heterozygosity for the codon beta 39 C-to-T nonsense mutation and a triplicate alpha-globin gene locus can cause "dominantly" inherited beta-thalassemia intermedia.

BACKGROUND: A beta-thalassemia intermedia phenotype can be caused by multiple genotypes. METHODS: We studied a family where the mother was hematologically normal and both father and daughter had beta-thalassemia intermedia. RESULTS: Both affected individuals were heterozygous for a codon 39 CAG-to-TAG mutation. They also were heterozygous for a triplicate alpha-globin gene locus (alphaalphaalpha(anti 3.7)). CONCLUSIONS: This compound heterozygous condition of a beta39 C-to-T mutation and triplicate alpha-globin gene increases alpha:beta-globin chain imbalance and accounts for the presence of beta-thalassemia intermedia. The proband received both an abnormal beta-globin gene and a triplicate alpha-globin locus from her father. Although the phenotype seems to be dominantly inherited, because of independent segregation of the alpha- and beta-globin genes, it is more accurately an example of polygenic inheritance.     

Jaundice and alpha gene triplication in beta-thalassemia: association or causation?

There are few studies investigating alpha globin gene triplications in beta-thalassemia in Asian Indians and its effect on phenotype, which was the primary aim of this study. Gap-PCR was performed in order to detect common alpha thalassemia determinants (-alpha(3.7), -alpha(4.2) and alpha alpha alpha(anti 3.7) triplication). Alpha-triplication was detected in 15.4% (10/65) of patients with thalassemia intermedia, 8.8% (4/45) of those with thalassemia minor and in 2.7% (2/74) of healthy controls. The severity of jaundice was higher in thalassemia intermedia cases with alpha-triplication and two of the alpha-triplication cases had a marked increase in serum bilirubin following intercurrent illness. Thus, alpha globin gene triplication is important genetic determinant underlying thalassemia intermedia in North Indians. Patients with alpha-triplication may develop prominent jaundice with marked increase in serum bilirubin following antecedent aggravating factors.     

Triple alpha genes in association with sickle cell and beta-thalassaemia gene in the Saudi population

This paper describes the case of a 6-year-old Saudi male who had sickle cell heterozygosity, beta +-thalassaemia and possessed three alpha-genes of the haplotype alpha alpha alpha anti-3.7/as diagnosed by restriction endonuclease studies using Hpa I, Bam HI, Bgl II, Hind III and Xba I. Since the iron level was found to be normal, it is proposed that the coexistence of beta-thalassaemia with triple alpha-genes in Hb S heterozygotes may be the cause of the anemia. A possible mechanism for severe anaemia is presented.     

Prevalence of -alpha(3.7) and alpha alpha alpha(anti3.7) alleles in sickle cell trait and beta-thalassemia patients in Mexico

The aim of this study was to determine the frequency of alpha-globin gene mutations in three groups of Mexican unrelated individuals. The first two groups were normal and sickle cell trait individuals from the Costa Chica region, a place with a 12.8% frequency of HbS carriers, and the third group comprised of Mexican mestizo patients with beta-thalassemia. We searched for -alpha(3.7) and -alpha(4.2) alpha(+)-thalassemia deletion alleles, as well as the alpha alpha alpha(anti3.7) triplication through long-gap PCR. The alleles -alpha(3.7) and alpha alpha alpha(anti3.7) were found in the heterozygote state only; 19% of the normal subjects had the -alpha(3.7) allele, and 2% showed the alpha alpha alpha(anti3.7) allele. In individuals with the sickle cell trait, 17% had the -alpha(3.7) deletion, and the alpha alpha alpha(anti3.7) triplication was observed in 3% of these individuals. We revealed that 16% of the subjects with beta-thalassemia showed the -alpha(3.7) deletion and 28% the alpha alpha alpha(anti3.7) triplication. The -alpha(4.2) deletion was not detected in any individual. The frequency of the -alpha(3.7) allele was roughly the same in the three groups studied; this can be explained by the fact that the three groups have common genes from Africa and the Mediterranean, where a high prevalence of alpha(+)-thalassemia has been observed. To our knowledge, the frequency of alpha alpha alpha(anti3.7) triplication observed in the Mexican beta-thalassemia patients is the highest reported. As the -alpha(3.7) and alpha alpha alpha(anti3.7) alleles are very common in our selected populations, we believe that there is a need to investigate systematically the alpha-globin gene mutations in all hemoglobinopathies in the Mexican population.     

Haplotypes and levels of fetal hemoglobin and G gamma to A gamma ratios in Mediterranean patients with thalassemia minor and major

This study concerned the gamma chain composition of Hb F and the haplotypes of 44 patients with beta-thalassemia major or intermedia and many of their relatives. Seventeen patients came from Northern (Turkish) Cyprus, 12 from the Istanbul area, and 15 from Macedonia and Bulgaria. Analysis of the A gamma T-G gamma-A gamma I ratio was made by HPLC, while haplotyping involved seven restriction sites. Specific haplotypes were present in certain populations; haplotype I [1] is the dominant type among North Cypriot thalassemia patients. Numerous types were seen in the patients from the Balkan countries. A direct relationship between the A gamma to G gamma ratios and the haplotypes, which exists among black beta-thalassemia heterozygotes [3], was also observed among these Mediterranean patients, although such analyses were considerably complicated by extensive blood transfusion therapy. Haplotypes without the Hinc II restriction site within the psi beta gene were associated with lower G gamma values than those that had this polymorphic site. The A gamma T chain was observed in a small number of beta-thalassemia homozygotes and heterozygotes. Three thalassemia chromosomes with slightly different haplotypes and one normal chromosome with a related haplotype were associated with the gamma 75 Ile----Thr substitution. A few patients with a thalassemia intermedia were heterozygotes for beta-thalassemia with either haplotypes V or VII [1] while the "nonthalassemic" chromosome had a haplotype I, which is the most common "beta-thalassemic" haplotype among the Mediterranean population(s). Detailed analyses of this chromosome have not been completed.     

Biochemical and molecular aspects of beta-thalassemia types in northern Sardinia

Forty-three patients with beta-thalassemia from Northern Sardinia (31 severe and polytransfused, six follow-up babies, five adults with mild thalassemia who were not transfusion dependent, and a young transfused patient was also affected by a disease of intermediate severity) were studied in order to establish the fetal hemoglobin composition, restriction fragment length polymorphism haplotypes at the beta-globin gene cluster, and the type(s) of mutation. Haplotype II was prevalent, [56/86 chromosomes (65%)], haplotype I was also fairly common, [22/86 chromosomes (25%)], while other types were relatively rare. The nonsense mutation at codon 39 was nearly exclusive, [76/80 chromosomes (95%)]. Other beta-thalassemia mutations occurred on chromosomes with haplotypes III, IX, X, and perhaps V, and a new type related to II. The mutated A gamma T gene was associated with type II, X, and the new type. Type IX was linked to a beta(0) gene and to an Xmn I site 5' to the G gamma gene, to a high G gamma globin level, and to a disease of mild severity. Type III was associated with a beta(+)-thalassemic gene. The (0)39 mutation linked to type II was associated with thalassemia intermedia in three patients.     

Molecular pathogenesis and clinical variability of beta-thalassemia syndromes among Indians

Sixty-four homozygous beta-thalassemia patients comprising 40 patients with beta-thalassemia major and 24 patients with beta-thalassemia intermedia were investigated for the nature of their beta-thalassemia mutations, associated alpha-thalassemia, and XmnI polymorphism in the gamma gene which are known to affect the clinical course of the disease. This study was undertaken to look for the contribution of these associated factors in reducing the clinical severity of homozygous beta-thalassemia from a severe disease to a beta-thalassemia intermedia phenotype. Clinical severity of these patients was assessed by the degree of transfusion dependency and the age at which the patient presented with symptoms. Globin chain synthetic ratio was taken as the biochemical pointer of severity of the disease. Eleven different beta-thalassemia mutations were encountered among 128 beta-thalassemia chromosomes. It was observed that the nature of the beta-thalassemia mutations was not very different between the beta-thalassemia major and beta-thalassemia intermedia groups in our patients, but co-inheritance of one or more alpha-globin gene deletions (-alpha(3.7)) and the presence of the XmnI polymorphism were associated with lesser severity of the disease in Indians.     

A novel basis for delta beta-thalassemia in a Chinese family

We have studied a Chinese family in which beta-thalassemia and delta beta-thalassemia were found in simple and compound heterozygous states. The delta beta-thalassemia heterozygote (the mother) had 22.3% hemoglobin F, of which 40% was G gamma and 60% A gamma; globin chain studies showed an alpha/beta + gamma ratio of 1.36. The compound heterozygote for delta beta-thalassemia and beta-thalassemia (the child) had the clinical picture of thalassemia intermedia and an alpha/beta + gamma ratio of 4.44. Gene mapping studies were performed using DNA from the affected child. Seventy kilobases of DNA in the beta- globin gene cluster starting upstream from the epsilon-globin gene and ending downstream from the beta-globin gene were mapped, and no detectable deletions or rearrangements were detected. In addition, heterozygosity was detected at multiple polymorphic restriction sites in and 3' to the beta-globin gene, which excludes the possibility of a deletion of the entire beta-globin gene cluster. This is the first example of a nondeletion delta beta-thalassemia associated with increased expression of both G gamma and A gamma genes.     

Do alpha deletions influence hydroxyurea response in thalassemia intermedia?

Thalassemia intermedia patients show variable phenotypes. Hydroxyurea (HU) may benefit some of the thalassemia intermedia cases (1), however, the parameters influencing the response to HU have not been reported. In this study, the molecular parameters, alpha-globin and beta-globin genotype and the Xmn I polymorphism, were correlated with the HU response. Twenty patients with thalassemia intermedia were given HU (10-20 mg/kg) and responses were evaluated over a one year period. Twelve patients (60%) showed a good response to therapy with a significant increase in Hb and HbF levels and with elimination of the transfusion requirement in four patients. Four out of the twelve (33%) patients were positive for -alpha(3.7) deletions whereas none of the 8 non-responders were positive for alpha deletions. One each of the responders and non-responders were positive for alpha alpha alpha(anti-3.7) triplication. Three (25%) responsive and one non-responsive patients were homozygous for the IVS1-1 (G-->T) mutation. Three of the responsive patients with alpha deletions were also homozygous positive for Xmn I polymorphism. Thus, in addition to acting in synergy with the XmnI polymorphism, alpha deletions may be an independent factor predicting good response to HU in thalassemia intermedia, although this needs to be confirmation in larger studies.     

Globin chain synthesis in beta-thalassemia with normal hemoglobins A2 and F.

Biosynthetic studies were performed in a patient with beta-thalassemia intermedia heterozygous for both beta-thalassemia with normal hemoglobins A2 and F and beta-thalassemia with increased Hb A2, in his both parents, one sister and one brother. In propositus the alpha/beta ratio was 1.68. In his mother with normal Hb A2, this value was 1.21. In contrast, in his father who had increased Hb A2, the alpha/beta ratio was 1.07, possibly due to combination of alpha- and beta-thalassemia. In his sister who had increased Hb A2, alpha/beta ratio was 1.57. In his brother with normal Hb A2 (2.5%) ratio was 0.6 indicating the presence of an alpha-thalassemia gene. Similar beta-thalassemic syndromes found in other countries are discussed.     

Molecular basis of beta-thalassemia intermedia in a southern Italian region (Puglia).

We investigated the molecular bases for a mild phenotype by alpha-, beta- and gamma-globin gene analyses in 22 patients with transfusion-independent thalassemia intermedia (15) or a late-presenting form of thalassemia major (7) originating from Puglia, a region of southern Italy. Twenty-two patients with thalassemia major served as controls. The beta+ IVS-I nt 6 of the beta-globin gene and the C----T substitution at position -158 5' of the G gamma-globin gene were detected more frequently in patients with thalassemia intermedia or late-presenting thalassemia major considered together as compared to those affected by typical transfusion-dependent thalassemia major. Three of 15 patients with thalassemia intermedia had the triple alpha-globin gene arrangement in the heterozygous (2) or homozygous state (1) in association with heterozygous beta zero-thalassemia. From these results, we may conclude that the inheritance of a mild beta-thalassemia allele such as the beta+ IVS-I nt 6 mutation, in the homozygous or heterozygous state, the coinheritance with homozygous beta zero-thalassemia of the -158 (C----T) G gamma gene promoter mutation and the presence of heterozygous beta-thalassemia/triple alpha-globin gene arrangement are the most common reasons accounting for the development of attenuated forms of beta-thalassemia in Puglia.     

Hb H (beta4) disease in Cukurova, Southern Turkey

In this study, 32 patients with Hb H (beta(4)) disease have been identified. Three different alpha-thalassemia-1 (thal) determinants; nine with the -17.4 kb (MED I) type, 12 with the -20.5 kb type and 10 with the -26.5 kb (MED II) type were characterized. Of the 32 patients, 19 had the 3.7 kb deletion and one had the 4.2 kb deletion in trans to alpha-thal-1 determinants. Only one patient, homozygous for the polyadenylation signal (poly A) site (PA 1) mutation, was identified to be associated with Hb H disease. The other patient had the poly A (PA 1) mutation in trans to the MED I (-17.4 kb) determinant. The 5 nt (nucleotide) deletion was present in three patients, two of them in the same family; this mutation was found in association with the MED II (26.5 kb deletion). The other patient had the -5 nt mutation in trans to the MED I (-17.4 kb) determinant. An unstable hemoglobin (Hb) variant [Hb Adana, codon 59 (CA)] was present in association with the alpha-thal-1 deletion (20.5 kb) in two adults and caused a severe type of Hb H disease. Five patients with Hb H disease had the genotype - -(MED II)/alpha(PA 2)alpha one had a Hb S heterozygosity (- -(MED II)/alpha(PA 2)alpha + Hb AS). A patient with Hb H disease (- -(MED I)/-alpha(3.7)) also had Hb S trait.     

Different types of beta-thalassemia intermedia. A genetic study in 20 patients.

20 patients with beta-thalassemia intermedia classified according to the results of genetic studies are presented. (1) 9 patients with beta-thalassemia intermedia homozygous for beta-thalassemia with increased Hb-A2 are reported. (2) 8 patients with beta-thalassemia intermedia, 3 homozygous for beta-thalassemia with normal levels of Hbs.A2 and F, 5 heterozygous for both this and beta-thalassemia with increased Hb-A2 are presented. (3) 2 families with beta-thalassemia intermedia heterozygous for both beta-thalassemia with increased HbA2 and 'silent" beta-thalassemia are reported. Two different varieties are presented.     

The effect of iron overload in the hearts of patients with beta-thalassemia

Background and hypothesis: An important complication of beta-thalassemia is iron deposition in cardiac tissues resulting in fibrosis and dysfunction. Our aim was the investigation of the possible clinical effect of iron loading in the heart of patients with beta-thalassemia prior to the appearance of symptoms of depressed systolic function. Methods: Thirty-five patients with beta-thalassemia, of whom 24 had the major type (Group 1) and 11 had the intermedia type (Group 2) were studied. Eleven age- and gender-matched controls were also studied (Group 3). All patients were evaluated echocardiographically and were shown to have normal left ventricular systolic function and dimensions. Serum ferritin, atrial natriuretic peptide (ANP), left atrial diameter (LAD), peak early mitral inflow velocity (E), peak late mitral inflow velocity (A), E/A ratio, deceleration time of the mitral inflow E wave (DT), and isovolumic relaxation time (IVRT) were measured. Results: Univariate analysis showed that both groups of patients had similarly increased LAD and ANP plasma levels. Group 1 had a higher E/A ratio (2.27 ± 0.88) SS than Group 2 (1.69 ± 0.47, p = 0.05) and Group 3(1.50 ± 0.38, p = 0.01). Serum ferritin was significantly higher in Group 1 (3.526 ± 0.352) than in Group 2 (2.808 ± 0.288, p < 10^–5) and Group 3 (2.139 ± 0.124, p<10^–5). Multivariate analysis showed that ANP is a factor that is affected by the LAD and E/A ratio and that serum ferritin levels affect the LAD and E/A ratio. Conclusions: Although LAD and ANP levels are increased in patients with beta-thalassemia, the increased serum ferritin levels of patients seem to affect left atrial size and E/A ratio. ANP secretion is consecutively affected by these factors.     

Genetic interactions in thalassemia intermedia: Analysis of β-Mutations, α-Genotype, γ-Promoters,and β-LCR hypersensitive sites 2 and 4 in Italian patients

In order to verity the genetic factors influencing the clinical expression of β-thalassemla we have studied 292 Kalian patients, 165 with thalassemia intermedia and 127 with thalassemia major. The β-globin gene mutations were defined in all cases. The number of α-globin genes and the integrity of specific control regions of the β-globin cluster—γ promoters and β-Locus Control Region (β-LCR)—were studied in selected cases. Homozygosity for mild mutations (group I) accounts for 24% of the intermedia patients and it is not represented among major patients. Forty-four percent of intermedia patients had combinations of mild/severe (group II) mutations and 32% had homozygosity or double heterozygosity for severe mutations (group III). Seventy-six percent of patients with thalassemia major were classified in group III end 24% in group II. Deletion type —α^3.7 thalassemia, assessed in a part of the cases, was found in 5% of thalassemia major and 19.5% of Intermedia patients in groups II and III. Structural analysis of γ promoters and β-LCR HS2 and HS4 regions, carried out in order to look for alterations associated with Hb F increase, did not reveal new mutations. Only rare polymorphic changes were observed at the HS2 and HS4 level. The ? 158 γ C T change was found with an increased incidence in intermedia patients in groups II and III. A subset of 10 β-thalassemia heterozygotes with mild intermedia phenotype resulted from coinheritance of a triplicated α-locus. We have been unable to find a molecular basis for the benign clinical course in approximately 20% of patients with thalassemia intermedia. Other genetic or acquired factors must be hypothesized which ameliorate the clinical condition.     

Clinical heterogeneity of silent-gene B-thalassemia among Indians

Summary This paper describes six Indian families in which silent — gene beta-thalassemia was encountered by us in the past 2 years. This stresses the importance of globin-chain synthesis in the diagnosis of beta-thalassemia. Of these six cases, five were diagnosed retrospectively when they had a homozygous beta-thalassemia offspring. The beta/alpha synthetic ratios ranged from 0.45 to 0.60. Four belong to group-I silent betathalassemia and two cases to group II. The severity of thalassemia major in the offspring of these silent carriers cannot be predicted.     

Haplotypes and α globin gene analyses in sickle cell anaemia patients from Kenya

Over 60 patients from the Luo and Luhya tribes of Western Kenya, aged 1-23 years, with severe sickle cell anaemia were evaluated through haematological and gene mapping analyses. Nearly all (56 of 58 tested) were homozygous for haplotype 20 (Antonarakis et al, 1984) which is also frequently present in SS patients of the Central African Republic. All patients had a severe haemolytic anaemia with low Hb F levels and low levels of G gamma chains. An alpha-thalassaemia-2 heterozygosity (-alpha/alpha alpha; -3.7 kb deletion) was present in 26 of 53 patients tested; one patient was a homozygote [f(-alpha) = 0.255]. The alpha-thal-2 was type I in all but one subject with this deficiency; the one exception had an alpha-thal-2 heterozygosity, type II. Heterozygosity for the alpha-thal-2 did not affect the clinical condition nor the haematology; Hb F levels were somewhat lower in SS patients with -alpha/alpha alpha than in those with alpha alpha/alpha alpha. A high frequency was observed for the absence of an Xba I restriction site 5' to the zeta globin gene; the frequency of this anomaly [f(Xba I-)] was estimated at 0.39 for the chromosome with two alpha globin genes and at 0.74 for that with the alpha-thal-2 deletion. An Apa I restriction site polymorphism was observed in the IVS-II of the alpha 2 globin gene; 13 alpha 2 genes of 53 normal (alpha alpha/) chromosomes had this restriction site which was absent in the hybrid alpha globin gene of the -alpha/chromosome.     

Plasma chitotriosidase activity in patients with beta-thalassemia

Variable increases in chitotriosidase levels have been reported in Italian patients with beta-thalassemia major and intermedia. We measured plasma chitotriosidase levels in Israeli patients with beta-thalassemia to ascertain its use as a universal marker of disease and/or response to therapy. Chitotriosidase levels in 39 adults (16-53 years; 30 with beta-thalassemia major, 9 with intermedia), and in 14 children (0.7-15 years; 12 with beta-thalassemia major, 2 with intermedia) were compared with other measures of disease, such as ferritin, hemoglobin, liver function tests, and genotype. Plasma chitotriosidase levels were normal (0.37 +/- 0.04 mU/mL) in all children. Twelve adults (31%) had elevated levels (>1.33 mU/mL): 11 patients (37%) with thalassemia major and 1 patient (11%) with thalassemia intermedia. A significant correlation was only found between plasma chitotriosidase levels and ferritin levels, and with mean number of transfusions per year. The patient with the highest chitotriosidase (1,440 nmol/mL/hr) had the highest ferritin (5,175 microg/L), required the most transfusions per year (40), and had abnormal liver tests. Normal chitotriosidase levels in the pediatric cohort and increased levels in only some adults may reflect status of iron overload in macrophages; thus there may be a role for monitoring chitotriosidase in patients with beta-thalassemia. Our results confirm results of the Italian cohort; however, in the latter, a more universal correlation was noted and chitotriosidase levels were much higher.     

Hydroxyurea in thalassemia intermedia—a promising therapy

Pharmacological agents such as hydroxyurea (HU) have been known to cause induction of fetal hemoglobin and possibly may alleviate the symptoms in thalassemia intermedia patients. Thirty-seven patients with beta-thalassemia intermedia were enrolled to assess response to HU therapy. Major response was defined as transfusion independence or hemoglobin rise of more than 20 g/l and minor response as rise in hemoglobin of 10-20 g/l or reduction in transfusion frequency by 50%. The median age was 10 years (range: 4-50 years) and median follow-up was 12 months (range: 4-36 months). Twenty-six patients (70.2%) showed response to HU therapy. Seventeen patients (45.9%) were major responders, and nine patients (24.3%) showed minor response. There was no correlation of response with beta-thalassemia mutation or XmnI polymorphism; however, the presence of alpha(3.7) deletion was associated with major response in three patients. Mean fetal hemoglobin (HbF) levels rose on HU therapy. Older age, low baseline F cell percent, and low baseline HbF levels (below 10%) were predictors of poor response. Response was evident within 1 month of starting HU therapy in the majority of responders. Thus, a short trial of HU therapy can predict durable response.     

A 65 bp duplication/insertion in exon II of the beta globin gene causing beta0-thalassemia

We describe a patient originating from Ghana who had combined heterozygous alpha (4.2)thalassemia, alpha alpha alpha anti3.7 triplication, the common delta globin variant HbA2' and a new 65 bp duplication/insertion in exon II of the b globin gene causing beta (0)-thalassemia.