Osteoporosis in beta-thalassemia: Clinical and genetic aspects.

Osteoporosis and osteopenia are frequent complications of thalassemia major (TM) and intermedia (TI). Osteoporosis was found in 23/25 patients with TI and in 115/239 patients with TM. In TM, no association was found with specific polymorphisms in candidate genes (vitamin D receptor, estrogen receptor, calcitonin receptor, and collagen type 1 alpha 1). Osteoporosis in female patients with TM was strongly associated with primary amenorrhea (P < .0001), while in male patients with TM, hypogonadism was not significantly related to bone mineral density (BMD) (P = .0001). Low BMD was also associated with cardiomiopathy (P = .01), diabetes mellitus (P = .0001), chronic hepatitis (P = .0029), and increased ALT (P = .01).

Posttranslational deamidation of proteins: the case of hemoglobin J Sardegna [alpha50(CD8)His-->Asn-->Asp].

Hemoglobin J Sardegna [alpha50(CD8)His-->Asn -->Asp] is a human Hb variant in which a posttranslational deamidation process takes place, transforming an Asn to an Asp residue. This variant, particularly widespread in northern Sardinia, has for the first time been characterized at the DNA level (codon 50 C-->A) on the selectively amplified alpha2-globin gene. We determined the protein and DNA sequences and performed cellulose acetate electrophoresis, isoelectric focusing, globin chain separation, stability tests with isopropanol and heat precipitation, and oxygen affinity analyses on whole blood to fully characterize the variant. A comprehensive review of the deamidation processes involving Asn and Gln residues in mutant proteins is reported, together with a discussion of the molecular mechanisms of such deamidations. Finally, examples of other proteins of clinical importance in which Asn or Gln residues have been implicated by DNA analysis alone are presented. These findings point out the importance of the complete characterization of variant proteins by use of both DNA and protein analyses.

Alpha-thalassemia carrier identification by DNA analysis in the screening for thalassemia.

Differentiation between heterozygous alpha-thalassemia and several phenotypically resembling alleles at the beta-globin gene cluster such as coinherited delta- and beta-thalassemia or gammadelta beta-thalassemia is a critical step in genetic counseling. In this paper we report our experience in the identification of the alpha-thalassemia carrier state using polymerase chain reaction (PCR)-based methods, and the feasibility and simplification of screening for thalassemia using this approach. Alpha-globin genotype was determined by PCR-based method in 526 adult subjects with reduced mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH), normal hemoglobin A2 and F, and normal serum iron. To verify the reliability of the protocol used, in 68 of these subjects we performed globin chain synthesis analysis and in 101 we determined alpha-globin genotype by Southern blot analysis. Five hundred twenty-one (99%) of 526 subjects examined were identified as carriers of one or two alpha-thalassemia alleles. The identification of the alpha-thalassemia carrier state may be fast and accurate by PCR-based method, avoiding other cumbersome and expensive methods such as globin chain synthesis and Southern blot analysis.

Heterozygous beta-thalassemia with thalassemia intermedia phenotype.

In this study we investigated the molecular bases of the beta-thalassemia intermedia phenotype in six patients belonging to two unrelated families of Sardinian descent. Sequence analysis of the beta globin gene from these patients detected, as the sole abnormality, the heterozygosity for the codon 39 nonsense mutation. The A gamma and Ggamma promoters as well as the HS2 and HS3 core sequences of the beta globin LCR from these patients, did not show any non-polymorphic nucleotide variation from the consensus sequence. One of the parents was heterozygous for codon 39 nonsense mutation but showed the beta-thalassemia carrier phenotype; the other was hematologically normal and had an entirely normal beta globin gene sequence. In both families, other members showed the typical hematological phenotype, clinically silent, of heterozygous beta thalassemia. To explain the thalassemia intermedia phenotype, we postulated the presence of an unknown molecular defect interacting with the beta globin gene mutation. Haplotype analysis excluded that this postulated defect lies in the beta globin gene cluster.

Evaluation of an automatic HPLC analyser for thalassemia and haemoglobin variants screening.

In this paper the authors report the evolution of a new automatic HPLC analyser for screening haemoglobinopathies. HbA(2) and F determinations are accurate and reproducible. The analysis time is short (6.5 min) and there is a good separation between the HbA(2) values of beta-thalassemia carriers from normals and alpha-thalassemia carriers, with no overlap between these groups. In addition, the system is also able to detect and quantitate most of the haemoglobin variants, particularly those (HbS, HbC, HbE and Hb Lepore) able to interact with beta-thalassemia and could make haemoglobin electrophoresis unnecessary in all samples. The ease of operation and the limited technical work make this system especially suitable for laboratories with a high workload and allow the cost of screening to be reduced.

Genotype of subjects with borderline hemoglobin A2 levels: implication for beta-thalassemia carrier screening.

In this study, we have defined by molecular analysis, the alpha, beta, and delta globin genotype in a group of individuals with normal or thal-like red cell indices but borderline hemoglobin (Hb)A2 levels, who were identified in a program for beta-thal carrier screening. In 37 of 125 individuals with borderline HbA2 levels, we detected a molecular defect in the beta, in both the delta and the beta, or in the alpha globin gene. Specifically seven of these subjects were carriers of the -101 C T mutation, ten of the IVSI nt6 T C mutation, 16 were double heterozygotes for delta and beta thal, and two had the triple alpha globin gene and two the single alpha globin gene deletion. From these results, we may conclude that subjects with borderline HbA2, particularly when they marry a typical beta-thal carrier, should be extensively investigated in order not to miss heterozygous beta-thalassemia.

Normal individuals with high Hb A2 levels.

Increased haemoglobin (Hb) A2 levels associated with reduced mean corpuscular volume (MCV) and Hb content per cell (MCH) are the most typical features of heterozygous beta thalassaemia. However, double heterozygotes for alpha and beta thalassaemia may have normal MCV and MCH but Hb A2 always in the carrier range. In this report we describe two Sardinian families who have increased Hb A2 levels, normal red blood cell indices and normal globin chain synthesis and in whom DNA sequence analysis of beta and delta globin genes did not reveal any abnormality. Our findings demonstrate the existence of a genetic trait not resulting from a defect of the beta globin gene cluster, transmitted in a dominant manner and manifested as isolated increase of Hb A2.

Human alpha-globin gene expression is silenced by terminal truncation of chromosome 16p beginning immediately 3' of the zeta-globin gene.

The high level expression of the human alpha-globin genes in erythroid tissue appears to require a set of DNaseI hypersensitive sites located upstream of the human alpha-globin gene cluster. These sequences, termed the locus control region (LCR), include two erythroid specific and a number of less restricted DNaseI hypersensitive sites. In this report we describe an individual with alpha-thalassemia associated with a truncation of the short arm of chromosome 16 that removes the LCR region and inactivates the adjacent intact alpha-globin genes. This genetic study supports the critical role of the LCR in the transcriptional activation of the human alpha-globin gene cluster and substantiates the importance of LCR deletions in the etiology of alpha-thalassemia.

HbH disease in Sardinia: molecular, hematological and clinical aspects.

In this study we have defined the molecular basis and correlated the clinical phenotype with the alpha-globin genotype in a large series of patients of Sardinian descent with HbH disease. The most prevalent molecular defect was the deletion of 3 alpha-globin structural genes most commonly the (--/-alpha 3.7) genotype (83.6%) and rarely the (--/-alpha 4.2) genotype (1.4%), followed in decreasing order of incidence by the combination of deletion alpha zero-thalassemia and initiation codon mutation of the alpha 2-gene (--/alpha NcoI alpha = 9.8%), deletion alpha zero-thalassemia and pentanucleotide deletion of IVS-I of the alpha 2-globin gene, (--/alpha HphI alpha = 3.3%) deletion alpha zero-thalassemia and initiation codon mutation of the alpha 1-gene (--/alpha alpha NcoI = 1.3%), a homozygous state for initiation codon mutation of the alpha 2-gene (alpha Nco alpha/alpha NcoI alpha = 0.7%). Patients with the (--/alpha thal alpha) genotypes showed severer clinical and hematological features as compared to those with the (--/-alpha) or those with the (--/alpha alpha thal) genotypes. The single patient with the (alpha Nco alpha/alpha Nco alpha) genotype had a clinical phenotype intermediate between HbH disease and the alpha-thalassemia carrier status. This heterogeneity depends on the fact that the alpha 2-globin gene produces 2-3 times alpha-globin chains than the alpha 1-gene and the single remaining alpha 1-like globin gene in the -alpha 3.7 chromosome has a compensatory increase in the alpha-globin chain output. alpha-Globin gene mapping of HbH disease patients may be useful for predicting the clinical outcome and to improve genetic counseling.

Erythropoiesis following bone marrow transplantation from donors heterozygous for beta-thalassaemia.

This study shows a marked and protracted activation of HbF synthesis in homozygous beta.-thalassaemia patients transplanted from HLA identical siblings heterozygous for beta-thalassaemia, as compared to patients transplanted from normal donors. HbF synthesis in recipients was much higher in relation to the corresponding bone marrow donor values either normal or heterozygous for beta thalassaemia. gamma-chain synthesis and G gamma/A gamma ratio were also studied in peripheral blood BFU-E from recipients and their donors. BFU-E from donors heterozygous for beta-thalassaemia showed higher gamma chain synthesis as compared to normal donors. Peripheral blood BFU-E gamma/beta + gamma ratios and G gamma percentage were higher in recipients than in their corresponding donors both normal or heterozygotes. The marked and protracted reactivation of HbF synthesis in recipients of heterozygous beta-thalassaemia bone marrow most likely results from an increased erythropoietic stress on erythroid progenitors. In order to obtain adequate Hb levels heterozygous beta-thalassaemia bone marrow should produce more red blood cells to compensate for the low MCH. The magnitude of activation of HbF synthesis was very variable. This variability may result from inherited differences in the capacity of reactivation of HbF synthesis of red cell progenitors from heterozygous beta-thalassaemia under stressed erythropoiesis.

Interaction of heterozygous beta zero-thalassemia with single functional alpha-globin gene.

In this study, we analyzed the phenotypic manifestations resulting from the interaction of heterozygous beta zero-thalassemia(beta zero-39 nonsense mutation) with the functional loss of three alpha-globin structural genes in six subjects, of whom four had the [-alpha/--]alpha-globin genotype and two the [--/alpha Th alpha] alpha-globin genotype. The beta-thalassemia defect was in all cases the nonsense mutation at codon 39. The nondeletion alpha-thalassemia alpha th was the initiation codon mutation (AUG----GUG) of the alpha-2 gene. In all these subjects hypochromia and microcytosis were more marked than in beta zero-thalassemia heterozygotes with a full complement of four alpha-globin genes. All but one had moderate anemia. The alpha:beta globin chain synthesis ratios were consistently decreased. No cases had Hb H on electrophoresis. Subjects with [--/alpha Th alpha] alpha-globin genotype had more severe thalassemia-like manifestations than those with [--/-alpha] alpha-globin genotype.

Delineation of the molecular basis of delta- and normal HbA2 beta-thalassemia.

In this study, we used cloning and sequence analysis to define the molecular defect in two delta-thalassemia genes, one associated with reduced output of delta-globin chains (delta +thal) from a Sardinian and the other with a complete suppression of delta-chain production from the affected locus (delta zerp thal) from a Southern Italian. Sequence analysis of the delta +thal gene showed a G----T substitution at the first nucleotide of codon 27 (delta +27) which produces an amino acid change (Ala----Ser) and presumably activates a cryptic splice site located at this position. Therefore, only a fraction of the transcript is processed from this site, as indicated by the clinical phenotype of delta +thal. DNA sequencing of the delta zero thal gene revealed a T----C substitution at position 1 of IVS-1, which abolishes the splicing at this site and thus leads to complete deficiency of normal mRNA explaining the clinical phenotype of delta zero thal. Oligonucleotide analysis was used to confirm the coinheritance of the delta +27 mutation in a group of Sardinians with thalassemia like phenotype and normal HbA2 level who, on the basis of genetic criteria, were supposed to be double heterozygous for delta-thalassemia and beta-thalassemia. The definition of delta-thalassemia defects in each high-risk area facilitates identification of double heterozygotes for delta- and beta-thalassemia by DNA analysis and may thus improve genetic counseling.

Molecular pathology of haemoglobin H disease in Sardinians.

We investigated the molecular basis for haemoglobin H disease in 50 Sardinian patients by restriction endonuclease analysis. We found that the majority (78% of the cases) are due to gene deletion (- -/- alpha). Among those with a combination of deletion and nondeletion defects (- -/alpha alpha th), the most prevalent nondeletion lesion (70% of the nondeletion defects) was the initiation codon mutation of the alpha 2 gene (alpha Nco alpha), previously discovered in this population. Of the remaining patients with the (- -/alpha alpha th) genotype, two showed the IVS-1 splice junction lesion and one a mutation in the alpha 1 gene, removing the Nco I site within the 5' part of the alpha 1 gene, which may arise from a process of gene conversion from the initiation codon mutant of the alpha 2 gene. A single patient had the homozygous state for the initiation codon mutant of the alpha 2 gene. Study of genotype-phenotype correlations indicates that the (alpha Nco alpha) haplotype is associated with a more severe defect in the alpha-globin chain output than that resulting from the (-alpha) haplotype. We may conclude that restriction endonuclease analysis is a powerful method for the definition of the molecular heterogeneity of haemoglobin H disease.

Genetic counseling and genetic heterogeneity in the thalassemias.

In this study, we have compared the hemoglobin A2 levels (Hb A2) of alpha-thalassemia carriers (-alpha/-alpha and -alpha/alpha alpha genotypes) with those of double heterozygotes for delta+ and beta thalassemia genes, who were identified by family studies and polymorphic restriction site analysis within the beta-globin gene cluster. We found that double heterozygotes for the delta+ and beta thalassemia have significantly (p less than 0.001) higher Hb A2 levels as compared with carriers of alpha-thalassemia. This finding has practical implications in the genetic counseling of subjects with a thalassemia-like phenotype associated with normal or borderline Hb A2 levels.

Hemoglobin inclusions in heterozygous alpha-thalassemia according to their alpha-globin genotype.

In this study we have correlated the presence/absence of rare red blood cells with HbH inclusions with the alpha-globin genotype in a group of Sardinian alpha-thalassemia carriers, whose genotype have been defined by alpha-globin gene mapping. We found that the majority of the carriers investigated, including those with the deletion of a single or two alpha-globin genes and those with non-deletion lesions, have rare blood cells with inclusions, with no significant difference in the frequency of positive finding related to the alpha-globin genotype.

Phenotype-genotype correlation in haemoglobin H disease in childhood.

In this study we used restriction endonuclease mapping to characterise the molecular defect responsible for haemoglobin H disease in 14 Sardinian children. The resulting genotypes were then correlated with the respective clinical and haematological phenotypes. We found that patients with the combination of non-deletion alpha(+)-thalassaemia [(alpha alpha)th] and deletion alpha(0)-thalassaemia (-Med) have a more severe phenotype than that resulting from the interaction of deletion alpha(0)-thalassaemia (-Med) and alpha(+)-thalassaemia (-alpha) determinants. Clinically, presentation was earlier and with moderate anaemia or haemolytic crisis, enlargement of the liver and spleen, and thalassaemic bone changes. Haematologically, the anaemia was more severe and there were higher bilirubin levels, reticulocyte counts, Hb H levels, and percentage of red blood cells with inclusion bodies. These results suggest that in those Hb H disease patients with the non-deletion [(alpha alpha)th] determinant, two alpha globin genes produce fewer alpha globin chains than a single alpha globin locus.