Peter van Delft

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.

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.

A novel 7·9 kb deletion causing α+‐thalassaemia in two independent families of Indian origin

Summary. We describe the characterization of a novel 7·9u2003kb deletion that eliminated one of the duplicated α‐globin genes, causing an α+‐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 α+‐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 α‐globin gene (α2 AATAAAu2003→u2003AATA‐‐u200a‐‐).

Hb Geldrop St. Anna [β94(FG1)Asp→Tyr]: a New Hemoglobin Variant Observed in a Diabetic Patient

An abnormal hemoglobin (Hb) fraction was observed during a high performance liquid chromatographic (HPLC) Hb A1c control for diabetes mellitus in a 56-year-old north European woman. Family analyses revealed the abnormal fraction in three of her five siblings and in her son. Elevated Hb and packed cell volume (PCV) values and red blood cell (RBC) counts were present in all carriers. No histories of anemia, hemolytic or circulatory episodes were reported. The abnormal Hb fraction estimated at 40%, migrated just below Hb F on alkaline electrophoresis and overlapped the Hb A2 peak on cation exchange HPLC. Direct sequencing of the β-globin genes revealed a new GAC→TAC transversion in heterozygous form at codon 94 of the β-globin gene. Based on the hematological/biochemical data and the decreased P50 value, we conclude that the new variant is a stable Hb associated with a slightly elevated oxygen affinity.

A novel α0-thalassemia deletion in a Greek patient with HbH disease and β-thalassemia trait: A novel α0-thalassemia deletion

Objectives:u2002 To determine the molecular basis in a Greek child suspected of having HbH disease and β‐thalassemia trait.

Hb 'T LANGE LAND [β136(H14)Gly → Arg]: A NEW HEMOGLOBIN VARIANT DESCRIBED IN A DUTCH PATIENT OF CHINESE ORIGIN

Carriers of hemoglobin (Hb) variants that are not associated with dominant phenotypes, are usually diagnosed by chance during laboratory analysis. The detection methods are mainly Hb electrophoresis or Hb chromatography that separate the Hb fraction by their speci®c electric charge or chromatographic behavior. However, many variants behave in a similar way on both separation methods because different abnormal Hbs often have very similar electric charge or hydrophobic-hydrophilic interaction. To date, molecular characterization of Hb variants can be rapidly achieved by DNA sequencing. We describe the hemoglobinopathy analysis performed on a family who was sent to our laboratory by the department of Clinical Chemistry of the t Lange Land Hospital, Zoetermeer, The Netherlands, where a formal diagnosis of Hb S carrier status, done elsewhere, was found to be inconsistent. The proband, a Dutch male of Chinese ancestry, aged 51, his wife and son, were thoroughly studied at the hematological, biochemical, and molecular level. Hematology was performed according to standard procedures (1). Genomic DNA was isolated by selective lysis and high salt extraction (2). Molecular analysis of the b-globin gene was done by direct sequencing of polymerase chain reaction HEMOGLOBIN, 25(3), 331±336 (2001)

Gγ −37 (A→T): A New Nondeletional Hereditary Persistence of Fetal Hemoglobin Determinant Associated with the Rare Codon 91 (+T) δ0-Thalassemia

We recently described a rare frameshift mutation in the δ-globin gene in a Dutch patient, in association with a new mutation of the Gγ-globin gene promoter [Gγ −37 (A→T)] with a moderately elevated Hb F level of 2.3%. The δ mutation at codon 91 (+T) has been described once before in our laboratory in 1989, in a complex Belgian family with Gγ (Aγδβ)0-thalassemia (thal) and moderately elevated Hb F levels, without the Gγ (Aγδβ)0-thal deletion in some individuals. Analysis of the patients from 1989 revealed the presence of the same Gγ-globin gene mutation and moderately elevated Hb F in all patients, who were also carriers of the δ-globin gene frameshift. Further analysis demonstrated that the two mutations were in linkage with the same haplotype in both the Belgian family and the recently found patient, confirming the association of the elevated Hb F expression with the new Gγ-globin gene mutation.

Hb DELFZICHT [α9(A7)Asn→Lys (α1)]: A NEW, CLINICALLY SILENT HEMOGLOBIN VARIANT OBSERVED IN A DUTCH PATIENT

a Gene point mutations inducing a-thalassemia (thal) or abnormal hemoglobins (Hbs) are frequently observed at the Hemoglobinopathies Laboratory (Leiden, The Netherlands). Mutations causing structural changes are usually detectable at the electrophoretic or chromatographic level. Some of these, if not associated with clinical symptoms, remain undetected or are observed by chance in elderly patients being monitored for diabetes mellitus by Hb A1c analysis. A blood sample from a 69-year-old Dutch female, collected in Na2 EDTA, was sent to our laboratory after an abnormal Hb pattern was observed during Hb A1c analysis at the Department of Clinical Chemistry of the Delfzicht Hospital, Delfzijl, The Netherlands. The complete hemoglobinopathy analysis was done as previously described. Electrophoretic analyses of the Hb fractions included starch gel electrophoresis (pH 8.6), isoelectrofocusing (IEF) on polyacrylamide gel, cellulose acetate electrophoresis at alkaline pH, and citrate agar gel electrophoresis. Globin chain electrophoreses were done on polyacrylamide gel in 6 M urea at pH 6.0 and 9.0 and in presence of Triton X-100 (UT). The percentage of the abnormal components was determined by cation exchange high performance liquid chromatography (HPLC) on the VARIANT System (Bio-

Contents Vol. 1, 1998

93 Translating Advances in Human Genetics into Disease Prevention and Health Promotion. First Annual Conference on Genetics and Public Health, Atlanta, Ga., May 13–15, 1998 Khoury, M.J. (Atlanta, Ga.); Puryear, M. (Rockville, Md.); Thomson, E. (Bethesda, Md.); Bryan, J. (Washington, D.C.) 109 Book Review No. 1