Genetic modifiers of fetal hemoglobin affect the course of sickle cell disease in patients treated with hydroxyurea.

The course of sickle cell disease (SCD) is modified by polymorphisms boosting fetal hemoglobin (HbF) synthesis. However, it has remained an open question how these polymorphisms affect patients who are treated with the HbF-inducing drug hydroxyurea/ hydroxycarbamide. The German SCD registry offers the opportunity to answer this question, because >90% of patients are treated according to national guidelines recommending the use of hydroxyurea in all patients above 2 years of age. We analyzed the modifying effect of HbF-related genetic polymorphisms in 417 patients with homozygous SCD >2 years old who received hydroxyurea. HbF levels were correlated with higher total hemoglobin levels, lower rates of hemolysis, a lower frequency of painful crises and of red blood cell transfusions. The minor alleles of the polymorphisms in the γ-globin promoter (rs7482144), BCL11A (rs1427407) and HMIP (rs66650371) were strongly associated with increased HbF levels. However, these associations did not translate into lower frequencies of vaso-occlusive events which did not differ between patients either carrying or not carrying the HMIP and BCL11A polymorphisms. Patients on hydroxyurea carrying the γ-globin promoter polymorphism demonstrated substantially higher hemoglobin levels (P<10-4) but also higher frequencies of painful crises and hospitalizations (P<0.01) when compared to patients without this polymorphism. Taken together, these data indicate that the γ-globin, HMIP and BCL11A polymorphisms correlate with increased HbF in SCD patients on hydroxyurea. While HbF is negatively correlated with the frequency of painful crises and hospitalizations, this was not observed for the presence of known HbF-boosting alleles.

Glucose-6-phosphate dehydrogenase activity measured by spectrophotometry and associated genetic variants from the Oromiya zone, Ethiopia.

BACKGROUND: The study aimed to gain first data on the prevalence of G6PD enzyme deficiency measured by spectrophotometry and associated genetic variants in Jimma and surroundings, Ethiopia. The area is a Plasmodium vivax endemic region, but 8-aminoquinolines such as primaquine are not recommended as G6PD testing is not available. METHODS: Healthy volunteers were recruited at Jimma University, Ethiopia. Enzyme activity was tested by spectrophotometry at the University of Ulm, Germany. A G6PD RDT (Binax NOW® G6PD, Alere, USA) was additionally performed. The G6PD gene was analysed for polymorphisms in a sub-population. Tests for haemoglobinopathies and the presence of malaria parasites were conducted. RESULTS: No severe or moderate (cut-off 60%) G6PD deficiency was found in 206 volunteers. Median male activity was 6.1 U/g Hb. Eleven participants (5.4%) showed activities between 70 and 80%. No haemoglobinopathy was detected. None of the subjects showed asymptomatic parasitaemia. One G6PD-A+ variant (A376G) and one new non-synonymous mutation (G445A) were found. CONCLUSIONS: As the prevalence of G6PD deficiency seems low in this area, the use of 8-aminoquinolines should be encouraged. However, reliable G6PD testing methods have to be implemented and safe cut-off levels need to be defined.

A case of anti-Rd causing fetal anemia.

BACKGROUND: Rd (SC4) is a low-frequency antigen of the Scianna blood group system. Only very few reports on anti-Rd in pregnancy exist. Mild to moderate hemolytic disease of the newborn caused by anti-Rd has been reported. This report may add further information on the clinical significance of anti-Rd for the fetus. CASE REPORT: In a case of severe fetal anemia (hemoglobin concentration, 3.0 g/dL) repeated intrauterine transfusions were required. The strongly positive direct antiglobulin test (DAT) of the fetal red blood cells led to the diagnosis of hemolytic disease. The routine antibody screen was negative, extended testing revealed a maternal anti-Rd with a titer of 256. Both the newborn and her father were confirmed to carry the SC*01.04 allele. CONCLUSION: Anti-Rd can cause fetal anemia. Low-frequency antigens including Rd are normally not present on screening cells. Antibodies directed against low-frequency antigens will usually not be detected by routine antibody screening in pregnancy. Thus, in cases of fetal anemia the DAT should always be included in the diagnostic workup.

Severe glucose-6-phosphate dehydrogenase deficiency leads to susceptibility to infection and absent NETosis.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymatic disorder of red blood cells in human subjects, causing hemolytic anemia linked to impaired nicotinamide adenine dinucleotide phosphate (NADPH) production and imbalanced redox homeostasis in erythrocytes. Because G6PD is expressed by a variety of hematologic and nonhematologic cells, a broader clinical phenotype could be postulated in G6PD-deficient patients. We describe 3 brothers with severe G6PD deficiency and susceptibility to bacterial infection. OBJECTIVE: We sought to study the molecular pathophysiology leading to susceptibility to infection in 3 siblings with severe G6PD deficiency. METHODS: Blood samples of 3 patients with severe G6PD deficiency were analyzed for G6PD enzyme activity, cellular oxidized nicotinamide adenine dinucleotide phosphate/NADPH levels, phagocytic reactive oxygen species production, neutrophil extracellular trap (NET) formation, and neutrophil elastase translocation. RESULTS: In these 3 brothers strongly reduced NADPH oxidase function was found in granulocytes, leading to impaired NET formation. Defective NET formation has thus far been only observed in patients with the NADPH oxidase deficiency chronic granulomatous disease, who require antibiotic and antimycotic prophylaxis to prevent life-threatening bacterial and fungal infections. CONCLUSION: Because severe G6PD deficiency can be a phenocopy of chronic granulomatous disease with regard to the cellular and clinical phenotype, careful evaluation of neutrophil function seems mandatory in these patients to decide on appropriate anti-infective preventive measures. Determining the level of G6PD enzyme activity should be followed by analysis of reactive oxygen species production and NET formation to decide on required antibiotic and antimycotic prophylaxis.

Impact of genotype on endocrinal complications in ß-thalassemia patients.

In ß-thalassemia, certain mutations cause a complete absence of ß-globin chain synthesis, termed ß0-thalassemia, while others may allow certain ß-globin production and are termed ß+- or ß++-thalassemia. The homozygous state results in severe anemia, which requires regular blood transfusion. By contrast, frequent blood transfusion can in turn lead to iron overload, which may result in several endocrinal complications. The present study aimed to investigate the impact of genotype on the development of endocrine complications in ß-thalassemia patients. A cross-sectional study was conducted on 100 thalassemia patients >10 years. A data abstraction form was designed to capture the appropriate information from the individual medical records, including full clinical, laboratory, transfusion and chelation data. The genotype of the patients was identified by the DNA sequencing technique. Growth retardation and hypogonadism were the most prominent endocrinal complications (70 and 67%, respectively) followed by hypothyroidism, diabetes mellitus and hypoparathyrodism (8, 8 and 7%, respectively). The most common mutations identified were IVS-1-110, IVS-1-1 and IVS-1-6 (63, 47 and 41%, respectively). Patients with the ß0ß0 genotype had a significantly higher prevalence of growth retardation, hypogonadism, hypothyroidism and hypoparathyrodism compared to those with the ß0ß+ and ß+ß+ genotypes (P<0.001, P<0.001, P<0.001 and P=0.037, respectively). Patients with the homozygous IVS-11-745 mutation had a significantly higher prevalence of diabetes (P=0.001). The underlying genetic defect in thalassemia patients is a contributing factor for the development of endocrinal complications, as patients with the more severe defects have a greater rate of iron loading through higher red cell consumption.

A new (G)γ-globin variant causing low oxygen affinity: Hb F-Brugine/Feldkirch [(G)γ105(G7)Leu→His; HBG2: c.317T>A].

In two unrelated families, several newborns developed cyanosis within the first days of life. For all of them, consecutive arterial blood gas analyses showed a right shift of the saturation curve, suggesting the presence of a hemoglobin (Hb) variant. A new (G)γ-globin variant was detected, namely (G)γ105(G7)Leu → His; HBG2: c.317T > A, that we named Hb F-Brugine/Feldkirch after the place of origin of the two families. This T to A conversion results in a leucine to histidine amino acid change at codon 105 of the (G)γ-globin gene and caused a Hb variant with lowered oxygen affinity. The γ to ß switch proceeded normally.

A novel type of congenital hypochromic anemia associated with a nonsense mutation in the STEAP3/TSAP6 gene.

STEAP3/TSAP6 encodes a ferrireductase that is involved in the acquisition of iron by developing erythroblasts and steap3/tsap6 null-mice display severe microcytic anemia. We report a family in which 3 siblings born to healthy parents display transfusion-dependent hypochromic anemia. A nonsense STEAP3/TSAP6 was identified in the siblings at the heterozygous state. This mutation was inherited from their father while no mutation was found in their mother. A large variability of expression was found between normal alleles in a control population, confirming a previous report that STEAP3/TSAPS6 is an expressed quantitative trait locus (e-QTL). Determination of the relative allele expression showed that the "normal" allele was expressed at a significantly higher level in the father than in the affected siblings relative to the shared mutated allele. The blood level of STEAP3/TSAP6 mRNA was severely reduced in the siblings, while both parents were in the lower range of normal controls. The STEAP3/TSAP6 protein was also reduced in lymphocytic cell lines from the patients. Collectively, our data support the hypothesis that STEAP3/TSAP6 deficiency leads to severe anemia in the affected siblings and results from the combination of a mutated allele inherited from their father and a weakly expressed allele inherited from their mother.

Hemoglobinopathies: clinical manifestations, diagnosis, and treatment.

BACKGROUND: Hemoglobinopathies are among the most common inherited diseases around the world. They have become much more common recently in northern and central Europe, including Germany, due to immigration. METHOD: Selective review of the literature with consideration of national guidelines. RESULTS: The hemoglobinopathies encompass all genetic diseases of hemoglobin. They fall into two main groups: thalassemia syndromes and structural hemoglobin variants (abnormal hemoglobins). α- and ß-thalassemia are the main types of thalassemia; the main structural hemoglobin variants are HbS, HbE and HbC. There are many subtypes and combined types in each group. The highly variable clinical manifestations of the hemoglobinopathies range from mild hypochromic anemia to moderate hematological disease to severe, lifelong, transfusion-dependent anemia with multiorgan involvement. Stem-cell transplantation is the preferred treatment for the severe forms of thalassemia. Supportive, rather than curative, treatment consists of periodic blood transfusions for life, combined with iron chelation. Drugs to treat the symptoms of sickle-cell disease include analgesics, antibiotics, ACE inhibitors and hydroxyurea. Blood transfusions should be given only when strictly indicated. More than 90% of patients currently survive into adulthood. Optimally treated patients have a projected life span of 50 to 60 years. CONCLUSION: Hemoglobinopathies are a public health issue in today's multiethnic German population. Adequate care of the affected patients requires a wide variety of diagnostic and therapeutic measures.

Dihydrofolate reductase deficiency due to a homozygous DHFR mutation causes megaloblastic anemia and cerebral folate deficiency leading to severe neurologic disease.

The importance of intracellular folate metabolism is illustrated by the severity of symptoms and complications caused by inborn disorders of folate metabolism or by folate deficiency. We examined three children of healthy, distantly related parents presenting with megaloblastic anemia and cerebral folate deficiency causing neurologic disease with atypical childhood absence epilepsy. Genome-wide homozygosity mapping revealed a candidate region on chromosome 5 including the dihydrofolate reductase (DHFR) locus. DHFR sequencing revealed a homozygous DHFR mutation, c.458A>T (p.Asp153Val), in all siblings. The patients' folate profile in red blood cells (RBC), plasma, and cerebrospinal fluid (CSF), analyzed by liquid chromatography tandem mass spectrometry, was compatible with DHFR deficiency. DHFR activity and fluorescein-labeled methotrexate (FMTX) binding were severely reduced in EBV-immortalized lymphoblastoid cells of all patients. Heterozygous cells displayed intermediate DHFR activity and FMTX binding. RT-PCR of DHFR mRNA revealed no differences between wild-type and DHFR mutation-carrying cells, whereas protein expression was reduced in cells with the DHFR mutation. Treatment with folinic acid resulted in the resolution of hematological abnormalities, normalization of CSF folate levels, and improvement of neurological symptoms. In conclusion, the homozygous DHFR mutation p.Asp153Val causes DHFR deficiency and leads to a complex hematological and neurological disease that can be successfully treated with folinic acid. DHFR is necessary for maintaining sufficient CSF and RBC folate levels, even in the presence of adequate nutritional folate supply and normal plasma folate.

Frequency of congenital dyserythropoietic anemias in Europe.

Congenital dyserythropoietic anemias (CDAs) are rare hereditary disorders characterized by ineffective erythropoiesis and striking abnormalities of erythroblast morphology. The mutated genes are known for the most frequent types, CDA I and II, but data about their frequency do not exist. The objective of this retrospective study was to estimate the frequency of CDA I and II, based on all cases reported in the last 42 yr in publications and identified registries or surveys. Reports were collected of 124 and 377 confirmed cases of CDA I and CDA II cases, respectively. The cumulated incidence of both types combined varied widely between European regions, with minimal values of 0.08 cases/million in Scandinavia and 2.60 cases/million in Italy. CDA II is more frequent than CDA I, with an overall ratio of approximately 3.2, but the ratio also varied between different regions. The most likely explanations for the differences are both differences in the availability of advanced diagnostic procedures and different levels of the awareness for the diagnosis of the CDAs. The estimations reported here are most probably below the true incidence rates, because of failure to make the correct diagnosis and to underreporting. Limited data do not suggest differing levels of risk in identified ethnic groups.

Hemoglobinopathies: a longitudinal study over four decades.

BACKGROUND: Hemoglobinopathies are among the most common hereditary diseases worldwide, with high prevalence in the Mediterranean basin, Africa, and Asia. Although they are rare in the indigenous central European population, they have become much more common in Germany recently through the immigration of millions of people from endemic regions. METHODS: In a long-term study (1971-2007), 100,621 hemoglobin analyses were performed and retrospectively evaluated. Basic clinical and hematological information were provided by the participating physicians. The hemoglobin defects were characterized with hematological and biochemical methods, as well as by DNA analysis in selected cases (from the mid-1980's onward). 73% of the analyses were performed in patients with an immigration background, 27% in patients of German ethnic origin. RESULTS: 34,228 persons, or 34% of those studied, were found to have a hemoglobinopathy. Most cases involved thalassemia syndromes (25,798 cases, 25.6%); the second most common type was a structural abnormality of hemoglobin (8,430 cases, 8.4%). This study provides the first broad overview of the occurrence, spectrum, and geographical distribution of hemoglobinopathies in Germany. CONCLUSIONS: These data show that hemoglobinopathies are a relevant health problem in the population of Germany today. This is not an epidemiological study, and thus it is unknown to what extent these data are representative. Because hemoglobin defects are of widely diverse genetic and clinical types, specialized laboratory analysis is needed to diagnose them correctly and provide a basis for proper therapeutic decisions.

The high oxygen-affinity Hemoglobin Johnstown [(beta 109(G11) Val-->Leu] in a German kindred with an elevated erythrocyte hemoglobin content: potential interaction with HFE mutations.

A 23-year-old man with an elevated blood hemoglobin was found to have hemoglobin (Hb) Johnstown, a high oxygen-affinity hemoglobin and an elevated red cell hemoglobin content. The hemoglobin mutation was present in all family members who consented to molecular investigation. No elevation of the erythropoietin level was found in the carriers. A high ferritin level was observed in one family member. When carriers of the hemoglobin mutation were analyzed for mutations in the HFE-genes (C282Y, H63D and S65C), only the individual with the high ferritin level was a compound heterozygous for the H63D/S65C genotype. This genotype normally does not confer the hemochromatosis phenotype but may contribute to iron overload when present in an individual with increased hemoglobin synthesis. The original report of this hemoglobin variant was in the United States. Additional descriptions followed from Spain and Argentina. The family in this report is the first described in Central Europe carrying the beta109 (Val-->Leu) mutation.

A new type of transfusion-dependent congenital dyserythropoietic anemia.

Cases of congenital dyserythropoietic anemia (CDA) that do not conform to any of the three classical types often present diagnostic difficulties and are at risk of developing secondary hemochromatosis. Here, we report a case of a six year old boy with transfusion dependency and gross abnormalities of the erythroblasts.

Codon 104(-G), a dominant beta0-thalassemia-like phenotype in a German Caucasian family is associated with mild chronic hemolytic anemia but influenced in severity by co-inherited genetic factors.

Codon 104(-G), a heterozygous frameshift mutation in exon 2 of HBB, resulted in a dominantly inherited beta0-phenotype with mild anemia in a German kindred, and thalassemia intermedia in the index patient. A co-inherited a gene triplication, long-term transfusion therapy, and ineffective erythropoiesis were confounding factors.

Molecular genetic analyses in familial and sporadic congenital primary erythrocytosis.

Dominant mutations in the erythropoietin receptor (EPOR) gene account for only about 15% of cases of primary congenital erythrocytosis. To search for molecular alterations in patients with this disorder. Sixteen patients with Epo <10 mU/mL were studied, 3 were related. Analyses included EPOR and JAK2 gene sequencing, quantitative PRV-1 RT-PCR, and erythroid colony assays. A novel sporadic EPOR 1453G->A (Trp439Stop) mutation was detected. All familial cases, varied in phenotype, presented the EPOR 1414C->G (Tyr426Stop) mutation. JAK2 mutations are not involved in the pathogenesis of primary congenital erythrocytosis.

Congenital dyserythropoietic anemia type I (CDA I): molecular genetics, clinical appearance, and prognosis based on long-term observation.

Congenital dyserythropoietic anemia type I (CDA I) is a rare autosomal recessive disorder with ineffective erythropoiesis and iron overloading. More than 100 cases have been described, but with the exception of a report on a large Bedouin tribe, these reports include only small numbers of cases, and no data on the lifetime evolution of the disease are available. Since 1967, we have been able to follow 21 cases from 19 families for up to 37 years. Twenty-one patients with a confirmed diagnosis of CDA I exhibited chronic macrocytic anemia of variable severity, requiring regular red cell transfusions only in 2 individuals. Four developed gallstones before the age of 30 years. Fifteen of 16 cases alive at the time of analysis showed mutations of at least one allele from exons 6 to 28 within CDAN1. Iron overloading is to be expected in all patients. In 9 patients, iron depletion was started between the ages of 7 and 36 years. Splenectomy, which was performed in 7 patients, did not result in improvement of hemoglobin values. Five patients were treated with interferon alpha-2a, and all responded with a rise in hemoglobin concentration of between 25 and 35 g/L (2.5 and 3.5 g/dL) starting within 4 weeks.

Mutations in the von Hippel-Lindau (VHL) tumor suppressor gene and VHL-haplotype analysis in patients with presumable congenital erythrocytosis.

BACKGROUND AND OBJECTIVES: Congenital erythrocytoses or polycythemias are rare and heterogeneous. A homozygous mutation (C598T->Arg200Trp) in the von Hippel-Lindau (VHL) gene was originally identified as the cause of the endemic Chuvash polycythemia. Subsequently this and other mutations in the VHL gene were also detected in several patients of different ethnic origin. Haplotype analyses of the VHL gene suggested a common origin for the Chuvash-type mutation. DESIGN AND METHODS: Thirty-four patients with presumable congenital erythrocytosis due to an unknown underlying disorder were examined for VHL gene mutations and VHL region haplotypes. RESULTS: Four patients were homozygous and one patient heterozygous for the Chuvash-type mutation. One additional patient presented a previously not described heterozygous mutation G311->T VHL in exon 1. The haplotype analyses were in agreement with recently published data for three of the four patients with homozygous mutations as well as for the patient with a heterozygous Chuvash-type mutation. One patient of Turkish origin with homozygous Chuvash-type mutation had a haplotype not previously found in individuals with Chuvash-type mutation. INTERPRETATION AND CONCLUSIONS: These results confirm that mutations in the VHL gene are responsible for a substantial proportion of patients with congenital erythrocytoses. Erythrocytoses due to a C598->T mutation of the VHL gene are not geographically restricted. The majority of patients with Chuvash polycythemia share a common VHL gene haplotype. The different haplotype in one of the patients with Chuvash-type mutation indicates that this mutation was not spread only from a single founder but developed independently in other individuals.

Limited influence of haemoglobin variants on Plasmodium falciparum msp1 and msp2 alleles in symptomatic malaria.

Haemoglobin (Hb) S, HbC, and alpha(+)-thalassaemia confer protection from malaria. Accordingly, these traits may influence the multiplicity of infection (MOI) of Plasmodium falciparum and the presence of distinct parasite genotypes. In 840 febrile children in northern Ghana, we typed the P. falciparum merozoite surface protein genes (msp1, msp2) and examined effects of the Hb variants on MOI and parasite diversity. HbAC, HbAS, heterozygous, and homozygous alpha(+)-thalassaemia occurred in 21, 5, 29 and 4% of the children, respectively. Plasmodium falciparum was detected in 95%. The haemoglobinopathies did not influence MOI, nor did the Hb type bias the distribution of the msp allelic families. However, IC type parasites were most common among patients with homozygous alpha(+)-thalassaemia (93%), less frequent in heterozygotes (89%), and least frequent in alpha-globin normal children (84%, P(chi2 trend) = 0.03). The opposite was seen for Mad20 type parasites (34%, 47%, 53%, P(chi2 trend) = 0.02). Only a few of the 72 individual msp alleles were selected by the haemoglobinopathies. HbC and alpha(+)-thalassaemia are frequent in northern Ghana. In symptomatic children, the effect of Hb variants on parasite multiplicity and diversity appears to be limited. This may reflect an actual lack of influence or indicate abrogation in symptomatic malaria.

Familial polycythemia vera with Budd-Chiari syndrome in childhood.

Polycythemia vera is a myeloproliferative disorder that, in most cases, occurs sporadically with a median age at presentation of 60 years. Familial cases are very rare and usually manifest in elderly family members. The Budd-Chiari syndrome, characterized by the obstruction and occlusion of the suprahepatic veins, is a rare typical complication in polycythemia vera patients. To date, only two children or adolescents with polycythemia vera and Budd-Chiari syndrome have been described. Here, we report an 11-year-old girl with Budd-Chiari syndrome as the initial symptom of familial polycythemia vera, which was also found in the girl's grandmother. Details of the diagnostic procedures used and the clinical course are reported. The patient underwent orthotopic liver transplantation and is being treated with hydroxyurea. The available literature on familial polycythemia vera and polycythemia vera in childhood with and without Budd-Chiari syndrome is reviewed.