Detection of mutations in the beta-globin gene by colorimetric staining of DNA microarrays visualized by a flatbed scanner.
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Chromatography, High Pressure Liquid↗
Biomedical subjects
Henrik Birgens
Publications and source records attributed to Henrik Birgens.
Detection of mutations using microarrays of poly(C)10-poly(T)10 modified DNA probes immobilized on agarose films.
Allele-specific hybridization to a DNA microarray can be a useful method for genotyping patient DNA. In this article, we demonstrate that 13- to 17-base oligonucleotides tagged with a poly(T)10-poly(C)10 tail (TC tag), but otherwise unmodified, can be crosslinked by UV light irradiation to an agarose film grafted onto unmodified glass. Microarrays of TC-tagged probes immobilized on the agarose film can be used to diagnose mutations in the human beta-globin gene, which encodes the beta-chains in hemoglobin. Although the probes differed widely regarding melting point temperature ( approximately 20 degrees C), a single stringency wash still gave sufficiently high discrimination signals between perfect match and mismatch probes to allow robust mutation detection. In all, 270 genotypings were performed on patient materials, and no genotype was incorrectly classified. Quality control experiments conducted using a target DNA specific for the TC tag of the immobilized probes showed that the spotting and hybridization procedure had a variance of 20%, indicating that signal differences as low as twofold could be detected between perfect match and mismatch. Together, our results show that the use of microarrays of TC-tagged probes that have been immobilized on agarose films grafted onto glass is a robust and inexpensive genotyping method.
Alleles↗
Inherited hemoglobin disorders in Guinea-Bissau, West Africa: a population study.
The determination of the prevalence of inherited hemoglobin (Hb) disorders in endemic areas is important in order to develop programs for their control and management. The aim of this study is to determine the prevalence of inherited Hb diseases in Guinea-Bissau which is situated on the west coast of Africa, between Senegal and Guinea. One thousand and fifty-seven blood samples were collected and analyzed with high performance liquid chromatography (HPLC) for detection of beta-thalassemia (thal) and Hb variants, and by gap polymerase chain reaction (gap-PCR) for the detection of deletions in the alpha-globin genes. We found 4.7% children were heterozygous for Hb S [beta6(A3)Glu-->Val, GAG -->GTG], 0.2% were homozygous for Hb S, and 0.3% were heterozygous for Hb C [beta6(A3)Glu-->Lys, GAG -->AAG]. One child had heterozygous beta+-thal, 13.8% were heterozygous for the -alpha3.7 deletion, 1.5% were homozygous for the -alpha3.7 deletion, and 1.5% were heterozygous for the -alpha4.2 deletion. We recommend national screening programs to focus primarily on sickle cell disease, since beta-thal is rare, and the observed alpha-thal deletions are of minor genetic importance.
Anemia, Sickle Cell↗
Rituximab in chronic cold agglutinin disease: a prospective study of 20 patients.
Chronic cold agglutinin disease (CAD) is an acquired autoimmune hemolytic anemia. Previous therapeutic modalities, including alkylating cytostatics, interferon and prednisolone, have been disappointing. However, several case reports and small-scaled studies have demonstrated promising results after treatment with rituximab. We performed a phase II multicentre trial to investigate the effect of rituximab in CAD, including 20 patients studied from October 2002 until April 2003. Thirteen patients had idiopathic CAD and seven patients had CAD associated with a malignant B-cell lymphoproliferative disease. Rituximab was given in doses of 375 mg/m(2) at days 1, 8, 15 and 22. Sixteen patients were followed up for at least 48 weeks. Four patients were excluded after 8, 16, 23 and 28 weeks for reasons unrelated to CAD. Nine patients (45%) responded to the treatment, one with complete response (CR), and eight with partial response. Eight patients relapsed, one patient was still in remission at the end of follow-up. There were no serious rituximab-related side-effects. Our study confirms previous findings of a favourable effect of rituximab in patients with CAD. However, few patients will obtain CR and, in most patients, the effect will be transient.
Aged↗
Beta-thalassemia due to a novel nonsense mutation at codon 37 (TGG-->TAG) found in an Afghanistani family.
We have identified and characterized a novel beta-thalassemic mutation in an Afghanistanifamily. The molecular pathology consists of a single base substitution (TGG-->TAG) at codon 37 of the beta-globin gene, giving rise to a stop codon (TAG). Premature stop of translation results in a truncated protein and usually the phenotype of beta-thalassemia (thal) major in homozygous individuals. However, this was not the case in our proband, who was homozygous for the codon 37 mutation. He presented with the phenotype of thalassemia intermedia with a hemoglobin (Hb) level of 8.1 g/dL and no previous history of blood transfusions. High performance liquid chromatography (HPLC) analysis showed exclusively Hb F except for a Hb A2 level within normal limits. Subsequent analysis demonstrated homozygosity for the XmnI Ggamma polymorphism and heterozygosity for a deletional alpha-thal (alphaalpha/-alpha(-3.7)). These findings might, at least partly, explain the beta-thal intermedia phenotype observed in the proband.
Adult↗
Hemochromatosis mutations in the general population: iron overload progression rate.
The progression rate of iron overload in hereditary hemochromatosis in individuals in the general population is unknown. We therefore examined in the general population iron overload progression rate in C282Y homozygotes. Using a cohort study of the Danish general population, The Copenhagen City Heart Study, we genotyped 9174 individuals. The 23 C282Y homozygotes identified were matched to 2 subjects each of 5 other HFE genotypes with respect to sex, age, and alcohol consumption. As a function of biologic age, transferrin saturation increased from 50% to 70% from 25 to 85 years of age and from 70% to 80% from 35 to 80 years of age in female and male C282Y homozygotes, respectively. Equivalently, ferritin levels increased from 100 to 500 microg/L and decreased from 800 to 400 microg/L in female and male C282Y homozygotes. As a function of 25 years follow-up irrespective of age, transferrin saturation and ferritin levels increased slightly in male and female C282Y homozygotes. None of the C282Y homozygotes developed clinically overt hemochromatosis. In conclusion, individuals in the general population with C282Y homozygosity at most demonstrate modest increases in transferrin saturation and ferritin levels, and clinically overt hemochromatosis is rare. Therefore, C282Y homozygotes identified during population screening, and not because of clinically overt hemochromatosis, at most need to be screened for manifestations of hemochromatosis every 10 to 20 years.
Adult↗
Spinal cord compression due to extramedullary hematopoiesis in polycythemia vera.
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Adult↗
Imatinib mesylate in idiopathic and postpolycythemic myelofibrosis.
Imatinib mesylate targets the adenosine triphosphate (ATP)-binding sites of the protein tyrosine kinase domains associated with Bcr-abl, the platelet-derived growth factor (PDGF) and c-kit. In idiopathic myelofibrosis (IMF) PDGF is considered to be one of the growth factors responsible for the development of bone marrow fibrosis. Recently, it has been shown that imatinib has antifibrogenic effect on bone marrow fibrosis in chronic myelogenous leukemia. Treatment with imatinib alone in IMF has been associated with significant side effects. In this study, the safety and efficacy of imatinib therapy in IMF, either administered as a single agent or in combination with hydroxyurea (HU) and/or alpha-interferon (IFN-alpha) are evaluated. Eleven patients (median age, 63 years; range, 33-82 years) with IMF (n = 8) or postpolycythemic myelofibrosis (PPMF) (n = 3) were studied All patients had been treated with HU (n = 9) and/or IFN (n = 7) before study entry. In all but one patient, treatment with these agents was discontinued when imatinib therapy was instituted. One patient continued IFN when treatment with imatinib was started. Imatinib was given at a dose of 400 mg/day. Nine patients were in an advanced disease phase. The patients have been followed for a median period of 2 months (range, 0.5-12 months). Treatment with imatinib has been stopped in six patients (55%), because of overt side effects (n = 4), recurrence of transitory dizziness and visual defects owing to a rising platelet count (n = 1), or the occurrence of an acute subdural hemorrhage that was evacuated without neurological deficits (n = 1). In nine patients imatinib treatment was followed by a rise in leukocyte and platelet counts that required combination with HU or IFN. The combined treatment modalities were followed by a rapid decrease in cell counts and were well tolerated apart from IFN side effects. A beneficial effect of imatinib was documented in three patients. It is concluded that leukocytosis and thrombocytosis are seen in most patients with myelofibrosis during treatment with imatinib. Combination therapy with HU or IFN seems safe and well tolerated and followed by a decrease in disease activity. A subgroup of patients in an early disease phase might benefit from imatinib therapy alone.
Adult↗
[Prevalence of hemochromatosis-associated mutations in the hemochromatosis gene in the Danish population].
INTRODUCTION: Hereditary haemochromatosis is an autosomal recessive condition characterised by systemic iron overload. In Northern Europe, 85-90% of patients with haemochromatosis are homozygous for the C282Y mutation in the HFE gene. In the present study, we determined the prevalence of the haemochromatosis-associated mutations, C282Y and H63D, in the Danish general population. MATERIAL AND METHODS: We genotyped 9174 individuals from a random sample of the Danish general population (The Copenhagen City Heart Study), stratified by gender and age in 10-year age groups, for the presence of C282Y and H63D. RESULTS: In The Copenhagen City Heart Study, 0.25% (95% confidence interval: 0.16-0.38%) were homozygous for C282Y, 1.4% (1.2-1.7%) were compound heterozygous for C282Y/H63D, and 9.2% (8.6-9.9%) were heterozygous for C282Y alone. Accordingly, the allele frequencies of C282Y and H63D in the Danish population were 5.6% (5.3-5.9%) and 12.7% (12.2-13.2%). All in all, more than 10% of the Danish population are either homozygous or heterozygous for C282Y, whereas about 24% are either homozygous or heterozygous for H63D. CONCLUSION: A prevalence of 0.25% homozygotes and > 10% heterozygotes for C282Y makes hereditary haemochromatosis the potentially most common inherited disorder in Denmark. However, in new studies from USA of the association between genotype and disease in unselected populations the penetrance is very low. On this background, population screening for the presence of these mutations is not advisable.
Adult↗