C. L. Harteveld

Evaluating five dedicated automatic devices for haemoglobinopathy diagnostics in multi-ethnic populations.

We have tested five haemoglobin (Hb) separation apparatuses, dedicated to haemoglobinopathy diagnostics. These are the four high performance liquid chromatography devices: VARIANT II™, HA 8160, G7, Ultra2 and the Capillary Electrophoresis apparatus from Sebia. In the first place, we focussed on the capacity of all apparatuses to detect the most common structural variants relevant for public health, these being HbS, HbC, HbE, HbD‐Punjab and HbO‐Arab. We then compared how the high HbA2β‐thalassaemia carriers were identified. All apparatuses were able to identify carriers of these traits with the expected sensitivity and specificity. With the primary goal of a high degree of conformity in basic diagnostics of haemoglobinopathies, we present the interpretation and the significance of the results on all apparatuses, and we comment on the unavoidable problems and solutions.

A novel polyadenylation signal mutation in the α2‐globin gene causing α thalassaemia

Summary. In a family of Indian origin we have identified a deletion of two bases at the polyadenylation signal sequence of the α2‐globin gene (AATAAA AATA). Three individuals heterozygous for this mutation display an αo‐thalassaemialike phenotype. Single‐stranded conformation analysis and automatic sequencing showed no additional mutations in either α1‐ or α2‐globin genes. A previously described polyadenylation sequence mutation (AATAAA AATAAG), αTSaudiα, causes HbH disease in homozygotes. In this study the patients heterozygous for the AATA(‐AA) mutation show a similar phenotype observed in the αTSaudiα heterozygotes. This confirms the observation that the inefficient transcriptional termination due to mutations of the polyadenylation sequence of the α2‐gene might interfere with the α1‐gene expression.

Molecular spectrum of beta-thalassemia in the Iranian Province of Hormozgan.

Prevention of β-thalassemia implies knowledge of the molecular spectrum occurring in the population at risk. This knowledge is necessary, especially when a prevention protocol is applied to a multiethnic population. For this purpose, we have recently analyzed a large population of Iranian patients living in the Province of Hormozgan in Iran, and a small group of Iranian patients living in The Netherlands. We have found a different mutation spectrum in both populations as compared to the data obtained by other authors for the Iranian regions of Tehran, Fars, Sistan Balouchestan, Bushehr, and Khouzestan. The IVS-I-5 (G → C) is the most frequent mutant in the province of Hormozgan (69%), followed by the IVS-II-1 (G → A) (9.6%), while the IVS-I-1 (G → A) was the most frequent defect found in the Iranian population sample in The Netherlands. The IVS-II-745 (C →G) mutation in cis with the 5′UTR (untranslated region) +20 (C → T) transition was observed in two unrelated, transfusion-dependent homozygotes, living in the Hormozgan Province where, in contrast with populations living in other provinces of Iran, no IVS-I-110 (G → A) or IVS-I-1 (G → A) mutations were found. We report the molecular spectra of our population samples and compare them with the mutation spectra observed in the Iranian populations by other authors. We discuss the severe phenotype of the patients homozygous for the IVS-II-745 (C → G) mutation, linked in cis to the 5′UTR +20 (C→ T) transition. Molecular analysis using commercial kits is briefly compared with denaturing gradient gel electrophoresis, emphasizing the value of a rapid method of detection for molecular defects in areas where many mutations occur.

Phenotype variability of the dominant β-thalassemia induced in four Dutch families by the rare cd121 (G→T) mutation

Abstractu2002Eight patients who were carriers of β-thalassemia induced by the cd121 (G→T) mutation are described in four nonrelated Dutch families. This mutant, which is considered rare and inherited in a dominant manner, is expressed in a different way among each of the four families and even among carriers of the same family. The symptoms vary from an hemolytic anemia of intermediate gravity with hepatosplenomegaly, inclusion bodies and erythroblastosis, to a mild anemia with minor hematological abnormalities. We report the analytical procedures used for the detection of the mutant, the hematological and clinical data of the four families and discuss the variable physiopathology of this molecular defect. We also compare the variation in fetal hemoglobin expression in relation to the haplotypes of the β-gene cluster and to the different hematological conditions. The presence of this rare mutant in four nonrelated Dutch families could derive from a single mutation or from multiple events. The existence of the four mutations in three different haplotypes suggests the occurrence of at least two independent events. The presence of five abnormal hemoglobins and the β-thalassemia defect on different haplotypes at cd121 also suggests a relatively increased rate of mutations at this particular site.

The molecular spectrum of beta-thalassemia and abnormal hemoglobins in the allochthonous and autochthonous dutch population.

The prevalence at birth of hemoglobin defects in the autochthonous North-European population is low. However, the long immigration and colonial history of the Netherlands has resulted in a group of about 1–2 million ‘autochthonous‘ inhabitants, with Asian, South-European or African ancestors, in whom a moderate birth prevalence of globin gene mutations can be expected. Furthermore, at least 10% of the Dutch population consists of recent immigrants from different countries with high birth prevalence of hemoglobinopathies. Because of the endogamous partner choice, which is prevalent in this population, the risk for homozygous progeny remains elevated. At least 100,000 carriers of hemoglobinopathies of recent allochthonous origin are present in the Netherlands, and the number of homozygous children is rising. Prevention by prenatal diagnosis requires a suitable protocol and knowledge about the molecular defects present in the country. Therefore we have analyzed a large number of patients and carriers, both at the hematological and at the DNA level. Our survey revealed 47 different β-thalassemia determinants, characterized on 223 independent chromosomes from individuals of different ethnic origins. As expected, the most prevalent mutations were largely represented. The cd39 (C→T) mutation was found in 70% of the immigrants from Morocco, Sardinia and other Central-West-Mediterranean regions while the IVS-I-110 (G→A) was prevalent in the East-Mediterranean populations. The IVS-I-5 (G→C) mutation was found in 45% of the patients of Indonesian origin. We also registered 308 independent chromosomes with common structural defects (HbS, HbC, HbE, Hb Lepore, Hb Constant Spring and HbD Punjab) and 33 chromosomes with 19 different, less frequent, rare or very rare mutants. Seven structural mutants were described for the first time and published separately. Furthermore, 139 independent chromosomes with deletional and nondeletional α-thalassemia defects were characterized.

α-Thalassaemia as a result of a novel splice donor site mutation of the α1-globin gene

We describe the characterization of an α+‐thalassaemia determinant as a result of a transition of G→A of the donor splice consensus site sequence of the first intron of the α1‐globin gene (α1IVS I‐1). The mutation was found in combination with the South‐East Asian α0‐thalassaemia deletion in an haemoglobin (Hb)H patient and her sister, both of Thai origin. Sequencing of the abnormally spliced mRNA product revealed the presence of a cryptic splice site in exon 1 of the α1‐globin gene. No normally spliced α1mRNA was detected. The abnormally spliced mRNA product from the α1‐gene carrying the mutation does not lead to functional protein and causes a mild HbH‐disease phenotype when in combination with the deletion type α0‐thalassaemia.

An IVS1–116 (A→G) acceptor splice site mutation in the α2 globin gene causing α+ thalassaemia in two Dutch families

We report the characterization of an α+‐thalassaemia determinant due to a transition Au2003→u2003G of the acceptor splice consensus site sequence (IVS1–116) of the first intron of the α2‐globin gene. The mutation, found in two apparently unrelated Dutch Caucasian families, was detected by DGGE analysis followed by direct sequencing. Haplotype analysis suggests a common origin of the mutation in both families. The disruption of the acceptor splice site consensus sequence interferes with the correct splicing and leads to the retention of the first intron in the abnormally spliced mRNA. The α+‐thalassaemia phenotype observed in the carriers is caused by the absence of functional mRNA which cannot be replaced by the abnormally spliced mRNA. The low amounts of abnormal mRNA found in reticulocytes is, most probably, due to the post‐transcriptional instability which follows the presence of a termination codon in the retained intronic sequence. This situation is often associated with a decreased mRNA stability as observed for several nonsense mutations of the β‐globin gene.

Atypical HbH disease in a Surinamese patient resulting from a combination of the −SEA and −α3.7 deletions with HbC heterozygosity

The first case of haemoglobin H (HbH) disease in combination with haemoglobin C (HbC) is reported in a man of Surinamese origin. Only haemoglobin A (HbA) and HbC were detected by electrophoresis. The amount of HbC was much less than expected in HbC heterozygotes. The synthesis ratio (βAu2003+βC/α) indicated an α‐thalassaemia defect with two non‐functional α genes, which did not correlate with the degree of haemolysis and anaemia displayed by the patient. The DNA analysis of the α‐genes clusters revealed a defect combination −SEA/−α3.7. The haematological data and the physiopathology of this atypical case are compared with the typical HbH disease found in a first cousin of the propositus. Data on the globin chains expression and on the formation of βA and βC homotetramers in HbH/HbC disease are presented.

Hb Utrecht [α2 129(H12)Leu → Pro], a new unstable α2‐chain variant associated with a mild α‐thalassaemic phenotype

We describe a new α2‐globin gene point mutation found in six individuals of a three‐generation Dutch family. The mutant, which is associated with a mild α‐thalassaemic phenotype, is not detectable at the protein level. The α2 cd129 (CTGu2003→u2003CCG) transition was found by molecular analysis using denaturing gradient gel electrophoresis (DGGE) and single‐strand conformation analysis (SSCA) followed by direct sequencing of the α2‐globin gene. Southern analysis revealed a triplication of the ζ‐gene in cis with the mutant α‐globin gene.

Paediatric allogeneic bone marrow transplantation for homozygous β -thalassaemia, the Dutch experience

Summary:We reviewed the results of the Dutch paediatric bone marrow transplant (BMT) program for children receiving HLA-identical BMT for β-thalassaemia major over an 18-year period. In all, 19 patients underwent a total of 21 transplants in our treatment centre between July 1984 and February 2002. Eight females (age 0.3–12 years; median 5 years) and 11 males (age 0.8–18 years; median 6 years) were included. Information, prospectively collected, included molecular defects, donor genotype, β/α-globin expression rates, serum ferritin levels, hepato-splenomegaly, chelation history, virology screening, liver pathology together with post-transplant outcome inclusive of leucocyte chimerism. In total, 11 patients received standard busulphan/cyclophosphamide (Bu/Cy) conditioning, with or without ATG. Stable engraftment was seen in 5/11 with late rejection occurring in six patients. Of these, two children underwent a second successful SCT. For this group, overall event-free survival (EFS) and disease-free survival (DFS) were 90 (10/11) and 64% (7/11), respectively. The probability of rejection was 55%. Subsequent addition of melphalan to the conditioning regimen resulted in long-term stable engraftment in all patients with an EFS/DFS for this group of 90% (9/10). Treatment-related mortality, irrespective of conditioning, was low at 5% (1/19 patients). Veno-occlusive disease (VOD) occurred in 19% (4/21 transplants) and acute GvHD in 19% (4/21 transplants). Post-BMT β/α synthetic ratio measurement monitored donor erythroid engraftment and predicted rejection with a return to transfusion dependency. Maintained full donor chimerism is indicative of stable engraftment both for leucocyte and erythroid lineages, whereas mixed donor chimerism is not. Our results emphasise the importance of the conditioning regimen and post-transplant chimerism surveillance predictive of rejection or long-term stable engraftment.