Improvement of medical care in a cohort of newborns with sickle-cell disease in North Paris: impact of national guidelines.

We conducted a retrospective study on newborns with sickle-cell disease (SCD), born 1995-2009, followed in a multicentre hospital-based network. We assessed patient outcomes, medical care and compliance with the national guidelines published in December 2005. Data from 1033 patients (742 SS/Sß°-thalassaemia) with 6776 patient-years of follow-up were analysed (mean age 7·1 ± 3·9 years). SCD-related deaths (n = 13) occurred only in SS-genotype patients at a median age of 23·1 months, mainly due to acute anaemia (n = 5, including 2 acute splenic sequestrations) and infection (n = 3). Treatment non-compliance was associated with a 10-fold higher risk of SCD-related death (P = 0·01). Therapeutic intensification was provided for all stroke patients (n = 12), almost all patients with abnormal transcranial Doppler (TCD) (n = 76) or with >1 acute chest syndrome/lifetime (n = 64) and/or ≥3 severe vaso-occlusive crises/year (n = 100). Only 2/3 of patients with baseline haemoglobin <70 g/l received intensification, mainly for other severity criteria. Overall, hydroxycarbamide was under-prescribed, given to 2/3 of severe vaso-occlusive patients and 1/3 of severely anaemic patients. Nevertheless, introduction of the on-line guidelines was concomitant with an improvement in medical care in the 2006-2009 cohort with a trend towards increased survival at 5 years, from 98·3% to 99·2%, significantly increased TCD coverage (P = 0·004) and earlier initiation of intensification of therapy (P ≤ 0·01).

Impact of glucose-6-phosphate dehydrogenase deficiency on sickle cell anaemia expression in infancy and early childhood: a prospective study.

In patients with sickle cell anaemia (SCA), concomitant glucose-6-phosphate dehydrogenase (G6PD) deficiency is usually described as having no effect and only occasionally as increasing severity. We analysed sequential clinical and biological data for the first 42 months of life in SCA patients diagnosed by neonatal screening, including 27 G6PD-deficient patients, who were matched on sex, age and parents' geographic origin to 81 randomly selected patients with normal G6PD activity. In the G6PD-deficient group, steady-state haemoglobin was lower (-6·2 g/l, 95% confidence interval (CI), [-10·1; -2·3]) and reticulocyte count higher (247 × 10(9) /l, 95%CI, [97; 397]). The acute anaemic event rate was 3 times higher in the G6PD-deficient group (P < 10(-3) ). A higher proportion of G6PD-deficient patients required blood transfusion (20/27 [74%] vs. 37/81 [46%], P < 10(-3) ), for acute anaemic events, and also vaso-occlusive and infectious events. No significant between-group differences were found regarding the rates of vaso-occlusive, infectious, or cerebrovascular events. G6PD deficiency in babies with SCA worsens anaemia and increases blood transfusion requirements in the first years of life. These effects decrease after 2 years of age, presumably as the decline in fetal haemoglobin levels leads to increased sickle cell haemolysis and younger red blood cells with higher G6PD activity.

Advantage of HbA1c assay by HPLC D-10 versus cobas integra 400 in a population carrier for HbS and HbC.

BACKGROUND: Several studies demonstrate significant bias in analytical methods used to measure glycohemoglobin. The clinical importance of that fact is evident when HbA1c overestimation leads to aggressive glucose management, resulting in more frequent hypoglycaemic episodes. Our study was aimed to compare two automated instruments (Integra 400 and D-10) in the evaluation of HbA1c in the Tunisian population. METHODS: Samples of 205 Tunisian diabetic patients were collected. The HbA1c assay was done simultaneously with a first generation immunoturbidimetric assay on an INTEGRA 400 (ROCHE) and using ionic exchange high pressure liquid chromatography (HPLC) on a D-10 system (BIO-RAD). RESULTS: Correlation is determined by linear regression analysis: D-10 = 0.921*(Integra 400) +1.125; coefficient of correlation (r) = 0.946. This r increases to 0.973 when samples of carriers for HbS and HbC (n = 9) are filtered out. For the carrier patients, significant differences in the percentage of HbA1c were observed relating to the methodology used. CONCLUSIONS: Laboratories must be aware of hemoglobin variant interferences on their methods of assessment of glycated hemoglobin. Using ion-exchange HPLC to control glycated hemoglobin seems to be essential to prevent mis-management in diabetic patients and to permit the diagnosis of the presence of HbS in patients.

Unsuspected glucose-6-phosphate dehydrogenase deficiency presenting as symptomatic methemoglobinemia with severe hemolysis after fava bean ingestion in a 6-year-old boy.

We report the occurrence of symptomatic methemoglobinemia in a previously healthy boy, who presented with severe acute hemolysis after fava bean ingestion. The methemoglobinemia revealed a previously unrecognized glucose-6-phosphate dehydrogenase (G6PD) deficiency. We discuss the pathophysiology of severe methemoglobinemia when associated with acute hemolysis, favism, and the common African G6PD A-variant [G6PD, VAL68MET, ASN126ASP]. In conclusion, screening for G6PD deficiency must be considered in symptomatic methemoglobinemia, especially in young boys, when associated with intravascular hemolysis.

[Flowcharts for the diagnosis and the molecular characterization of hemoglobinopathies]. / Arbres décisionnels pour le diagnostic et la caractérisation moléculaire des hémoglobinopathies.

The diagnosis of the main hemoglobinopathies (HbS, HbC, HbE, heterozygous beta-thalassemia) is easy for laboratories using Hb electrophoresis and/or cation-exchange high performance liquid chromatography (CE-HPLC) methods. However, the diagnosis of alpha-thalassemias and of rare Hb variants is much more complex. Six French laboratories, forming together the "Pathologie héréditaire de l'érythrocyte" network, routinely practice the molecular diagnosis of hemoglobinopathies. These laboratories have shared their experiences to propose flowcharts for the phenotypical diagnosis and the molecular characterization of the main hereditary Hb pathologies. These flowcharts are applicable to any single patient with an adult erythropïesis (more than two-years-old), that is to say after the fetal to adult (gamma>beta) Hb commutation, when the HbF level is stabilized.

Genetic variants in the noncoding region of RPS19 gene in Diamond-Blackfan anemia: potential implications for phenotypic heterogeneity.

Mutations in the RPS19 gene have been identified in 25% of individuals affected by Diamond-Blackfan anemia (DBA), a congenital erythroblastopenia characterized by an aregenerative anemia and a variety of malformations. More than 60 mutations in the five coding exons of RPS19 have been described to date. We previously reported a mutation (c.-1 + 26G>T) and an insertion at -631 upstream of ATG (c.-147_-146insGCCA) in the noncoding region. Because DBA phenotype is extremely heterogeneous from silent to severe and because haploinsufficiency seems to play a role in this process, it is likely that genetic variations in the noncoding regions affecting translation of RPS19 can modulate the phenotypic expression of DBA. However, to date, very few studies have addressed this question comprehensively. In this study, we performed detailed sequence analysis of the RPS19 gene in 239 patients with DBA and 110 of their relatives. We found that 6.2% of the patients with DBA carried allelic variations upstream of ATG: 3.3% with c.-1 + 26G>T; 2.5% with c.-147_-146insGCCA; and 0.4% with c.-174G>A. Interestingly, the c.-147_-146insGCCA, which has been found in a black American and French Caribbean control population, was not found in 500 Caucasian control chromosomes we studied. However, it was found in association with the same haplotype distribution of four intronic polymorphisms in our patients with DBA. Although a polymorphism, the frequency of this variant in the patients with DBA and its association with the same haplotype raises the possibility that this polymorphism and the other genetic variations in the noncoding region could play a role in DBA pathogenesis.

Effect of transfusion therapy on cerebral vasculopathy in children with sickle-cell anemia.

This retrospective study assessed the long-term effect of transfusional exchange therapy on MRA/MRI abnormalities in 24 homozygous sickle-cell anemia (HbSS) children presenting with abnormal brain MRA. The median time elapsed from baseline to last available MRA was 29 months. Follow-up MRAs showed improvement, stabilization or worsening of cerebrovascular lesions in 11, 6 and 7 patients respectively. Complete normalization of MRA was observed in 6 patients within a mean time of 1.4 years, but stenosis recurred at the same location in the 4 patients in whom transfusion therapy was discontinued. Baseline severe stenosis/occlusion of large cerebral arteries and occurrence of moyamoya syndrome were significantly associated with an absence of improvement of the cerebral vasculopathy. These data emphasize the heterogeneity of the course of cerebrovasculopathy in SS children receiving chronic transfusion. Further studies are needed to determine whether different therapeutic approaches have to be considered according to these different evolutive patterns in SS children.

Variants of the mannose-binding lectin gene in the Benin population: heterozygosity for the p.G57E allele may confer a selective advantage. 2007.

Human mannose- binding lectin (MBL) plays an important role in innate immunity. MBL deficiency is associated with mutations in the promoter region and in exon 1 of the MBL2 gene. Such deficiency has been correlated with elevated incidence of infections in infancy and in immunocompromised adults. We determined the distribution profile of the MBL2 gene variants in the general population of Benin (West Africa) and in a vulnerable subset of children with sickle cell disease (SCD) (SS homozygotes). Five hundred forty-two healthy individuals (274 newborns, 268 adults) and 128 patients with SCD (35 newborns, 93 children) were screened for the common variant alleles in the MBL2 secretor haplotype region (exon 1 and promoter). The p.G57E variant allele was the most frequent allele compared to p.G54D (27.5% vs. 1.6%, respectively). The p.R52C allele was not found in this population. There was no difference in allele or genotype frequencies between healthy newborns and newborns with SCD. Alleles associated with MBL deficiency were more frequent in adults than in newborns (69.8% vs. 57.3%, respectively; p=0.002). This enrichment was exclusively due to an elevated proportion of heterozygotes for the p.G57E allele (47.0% vs. 35.3%,respectively; p=0.004), supporting a potential selective advantage of this genotype. Our results, compared to those reported in other African countries, support the implication of the MBL2 gene in various major infections in Africa, such as meningitis and tuberculosis in HIV- positive patients.

Molecular basis of alpha-thalassemia in Algeria.

An epidemiological molecular study was carried out to evaluate the spectrum and allelic frequency of alpha-thalassemia (alpha-thal) defects in Algeria. A series of 153 randomly selected blood donors was screened for 10 alpha-thal alleles described in the Mediterranean area. In addition, six unrelated cases with hematological and biochemical data suggestive of Hb H disease were investigated. Our data revealed an allele frequency of 4.6%. The presence of alpha(0)-thal determinants (-alpha(20.5) and --MED I) was observed both in Hb H patients and in the randomly collected samples. Overall, the -alpha(3.7) deletion was the most prevalent allele (2.9%), followed by the alpha(Nco I)alpha (HBA2:c.1A>G) allele (0.6%) and by the alpha(Hph I)alpha (HBA2:c.95 + 2_95 + 6delTGAGG), -alpha(20.5), --(MED I) alleles (0.3% each). The -alpha(4.2) deletion was observed in only one Hb H patient. These results outline the heterogeneity of the alpha-thal alleles in Algeria which reflects the anthropological history of the country. Because of their frequency, alpha-thal alleles are probably frequent modulators of prevalent beta-globin gene-related hemoglobinopathies in Algeria.

Modulation of erythroid adhesion receptor expression by hydroxyurea in children with sickle cell disease.

BACKGROUND: We investigated adhesion receptor levels on red blood cells, reticulocytes and erythroid progenitors from children with sickle cell disease treated or not with hydroxyurea. DESIGN AND METHODS: Four groups of patients were investigated: (i) children receiving hydroxyurea for severe vaso-occlusive events (n=26); (ii) untreated children with a history of vaso-occlusive events (n=20); (iii) children with no history of vaso-occlusive events (n=28); and (iv) healthy African controls (n=27). Expression of adhesion receptors was analyzed by flow cytometry with specific mono-clonal antibodies. RESULTS: Reticulocytes and/or red blood cells from the children with sickle cell disease showed significantly higher expression of CD36, alpha 4beta 1, Lu/BCAM than those from controls, whatever the severity of the disease, as well as less marked increases in expression of ICAM-4, CD47 and CD147. Under hydroxyurea treatment, the expression of CD36, alpha 4beta 1 and ICAM-4 (to a lesser extent) was decreased, but surprisingly the expression of Lu/BCAM (and also CD47 and CD147 to a lesser extent) was significantly increased. Alterations of levels of adhesion receptors could be recapitulated in two-phase liquid cultures of erythroid progenitors from controls and untreated children with a history of vaso-occlusive disease, grown in the absence or presence of hydroxyurea. CONCLUSIONS: Our results suggest that hydroxyurea acts during erythroid development and modulates adhesion receptor expression and function differently, possibly by acting on gene expression and the signaling cascade leading to receptor activation.

Variants of the mannose-binding lectin gene in the Benin population: heterozygosity for the p.G57E allele may confer a selective advantage.

Human mannose-binding lectin (MBL) plays an important role in innate immunity. MBL deficiency is associated with mutations in the promoter region and in exon 1 of the MBL2 gene. Such deficiency has been correlated with elevated incidence of infections in infancy and in immunocompromised adults. We determined the distribution profile of the MBL2 gene variants in the general population of Benin (West Africa) and in a vulnerable subset of children with sickle cell disease (SCD) (SS homozygotes). Five hundred forty-two healthy individuals (274 newborns, 268 adults) and 128 patients with SCD (35 newborns, 93 children) were screened for the common variant alleles in the MBL2 secretor haplotype region (exon 1 and promoter). The p.G57E variant allele was the most frequent allele compared to p.G54D (27.5% vs. 1.6%, respectively). The p.R52C allele was not found in this population. There was no difference in allele or genotype frequencies between healthy newborns and newborns with SCD. Alleles associated with MBL deficiency were more frequent in adults than in newborns (69.8% vs. 57.3%, respectively; p = 0.002). This enrichment was exclusively due to an elevated proportion of heterozygotes for the p.G57E allele (47.0% vs. 35.3%, respectively; p = 0.004), supporting a potential selective advantage of this genotype. Our results, compared to those reported in other African countries, support the implication of the MBL2 gene in various major infections in Africa, such as meningitis and tuberculosis in HIV-positive patients.

The role of the G6PD AEth376G/968C allele in glucose-6-phosphate dehydrogenase deficiency in the seerer population of Senegal.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is common in tropical Sub-Saharan countries. The allele most frequently associated with G6PD deficiency in this a region is G6PD 376G/202A. Here, we show that, the prevalence of G6PD deficiency is 12% in the Sereer ethnic group from Senegal ant that the 376G/968C genotype is predominant; the frequency of the 376G/202A genotype is very low in this ethnic group.

Decreased plasma endothelin-1 levels in children with sickle cell disease treated with hydroxyurea.

Plasma endothelin-1 (ET-1) is elevated in patients with sickle cell disease (SCD). Hydroxyurea (HU) is the only drug with demonstrated clinical efficacy in SCD. Here we show that treatment with HU results in a decreased concentration of circulating ET-1 which is not correlated with the HU-induced increase in HbF level. Blunting of the ET-1 vasoconstrictive stimulus could contribute to the beneficial effects of HU.

Special management of insulin lispro in continuous subcutaneous insulin infusion in young diabetic children: a randomized cross-over study.

OBJECTIVE: To compare the safety, efficacy and management of insulin lispro (LP) with regular human insulin (RH) in young diabetic children treated with continuous subcutaneous insulin infusion (CSII). STUDY DESIGN: 27 very young diabetic children (age 4.6 +/- 2.2 years) treated with CSII participated in an open-label, randomized cross-over multicenter study comparing 2 periods of 16 weeks of CSII with LP or RH. RESULTS: Mean daily basal rate was significantly higher during the LP period (p = 0.04). No differences were seen in changes in HbA1c levels, number of hypoglycemic events, cutaneous infections and catheter occlusions. There was no significant difference between the two treatments for preprandial and postprandial glucose values, although prandial glucose excursions tended to be lower with LP (significant at dinner, p = 0.01). Mean blood glucose levels were significantly higher at 0.00 and 3.00 a.m. during LP therapy (p < 0.05). No episode of ketoacidosis occurred during LP treatment. More parents indicated that LP made their own and the child's daily life easier (p = 0.02) and preferred LP (p = 0.01). CONCLUSIONS: LP in CSII therapy in children is safe, as effective as RH, improved postprandial excursions, met the needs of young children in their daily life well, and gained their parents' satisfaction and preference. However, a shorter duration of LP resulted in hyperglycemia during the first part of the night, which must be compensated for by increasing nocturnal basal rates during this time.

Origin of Hb A2' (Hb B2) [delta16(A13)Gly --> Arg (GGC --> CGC)].

On a field trip to the Dogon country (le Pays Dogon) in central Mali, we detected a high frequency of the Hb A2 abnormality, reaching higher numbers among blacksmiths (up to 12.4%) living in the same villages. In this report, by direct nucleotide sequencing and employing a polymerase chain reaction-restriction fragment length polymorphism approach, we show that the Hb A2 variant observed in the Dogon population is indeed Hb A2', also called Hb B2, and that in all of the cases the abnormal delta-globin gene is linked to a unique haplotype. The same haplotype was found linked to Hb A2' in the Herero population belonging to the South African Bantu-speaking Blacks from Namibia. Although the unique origin of this mutation in Africa is a possibility, a recurrent mutational event cannot be excluded because the linked beta cluster haplotype is one of the two major haplotypes found in all African populations. A study of populations from other regions of Africa is required to clarify this issue.

Foetal haemoglobin in normal healthy adults: relationship with polymorphic sequences cis to the beta globin gene.

We evaluated the effects of polymorphic markers within the beta globin gene cluster on HbF expression in two groups. These groups were randomly selected from a survey of HbF distribution in a large population study of unrelated healthy Algerian adults (n=827). The first group contained individuals with normal HbF levels (0.1-0.5%) and the second group contained individuals with raised HbF levels (0.8-2.3%). Of the various polymorphic markers analysed, only the -309 G gamma A-->G, the -158 G gamma C-->T, the G gamma IVS2 TC (TG)(9) AG (TG)(2) (CG)(2) and the -540 beta (AT)(9) T(5) sequence configurations were significantly associated with increased HbF levels. More than 84% of the subjects with elevated HbF levels carried one or several of these four marker configurations, suggesting that the beta globin gene cluster exerts a significant effect on HbF expression in healthy individuals.

A novel rearrangement of the human fetal globin genes leading to a six gamma-globin gene haplotype.

Haematological as well as gene mapping data are reported for three members of a Portuguese Caucasian family with high G(gamma)-globin levels. A gamma-globin gene sextuplication of the G(gamma)AG(gamma)AG(gamma)AG(gamma)AG(gamma)A(gamma) type was present in the proband and her father. Comparison of gene mapping data with quantitative results of fetal haemoglobin (HbF) analysis provided an explanation for the extremely high G(gamma)-globin levels (> 90%) in the HbF from the two mentioned individuals. This rearrangement, for which a generation mechanism is proposed, is the first gamma-globin gene sextuplication described in the literature.