RESUMO
BACKGROUND: As a result of population growth in African-Caribbean regions of overseas France, and now immigration essentially from North and sub-Saharan Africa to mainland France, neonatal screening for sickle cell disease (SCD) has been performed in France since 1985 in Guadalupe and dependencies, as a universal test. After several pilot studies, screening was gradually extended to mainland France in 1996. Since 2000, the test has been performed at national level for all newborns defined as being "at risk" for SCD based on ethnic origin. METHODS: A dry blood sample is obtained by heel stick and analysed by isoelectric focusing as a first-line method, followed by either high-performance liquid chromatography or acid agar electrophoresis for confirmation, whenever a variant haemoglobin is observed on isoelectric focusing. RESULTS: In 2007, 28.45% of all newborns in mainland France were screened for SCD. Since 1996, a total of 3,890 newborns have been found to have SCD, and they have been followed up by reference paediatricians. CONCLUSION: Although screening for SCD at birth in France is not universal, it appears that missed babies are relatively infrequent. Despite obvious sociological problems inherent to the at-risk population, the follow-up of SCD babies is rather successful. Due to the birth prevalence of SCD in France, especially in comparison with other common genetic diseases, screening all newborns regardless of ethnic origin is an issue that is being addressed.
Assuntos
Anemia Falciforme/diagnóstico , Triagem Neonatal/métodos , Anemia Falciforme/epidemiologia , Coleta de Amostras Sanguíneas/métodos , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Ágar , França/epidemiologia , Hemoglobinopatias/diagnóstico , Hemoglobinopatias/epidemiologia , Humanos , Recém-Nascido , Focalização IsoelétricaRESUMO
Hemoglobin (Hb) Esch, is an alpha1 variant, expressed at less than 5%, resulting from the duplication of the 12 nucleotides corresponding to CD65 through 68. The effect of this insertion is the repetition of the sequence Ala-Leu-Thr-Asn, which corresponds to the last turn of helix E. In this variant the presence of a one-turn elongated helix E causes instability and increased ligand affinity. Hb Esch was characterized by DNA sequencing and confirmed by electrospray mass spectrometry. Functional studies were performed by flash photolysis measurements on a fraction isolated by flatbed isoelectric focusing, which was enriched in the abnormal hemoglobin. Similar to other alpha chain variants due to short insertion (or deletion), Hb Esch probably results from a slipped mispairing mechanism. The stability of such modified proteins depends upon the region which is added or deleted and usually is more stable when involving a flexible loop or complete helix turn(s) near by.
Assuntos
Hemoglobinas Anormais/genética , Hemoglobinas/genética , Mutagênese Insercional , Fragmentos de Peptídeos/genética , Adulto , Sequência de Aminoácidos , Duplicação Gênica , Variação Genética , Hemoglobinas Anormais/biossíntese , Humanos , Masculino , Portugal , Talassemia alfa/diagnóstico , Talassemia alfa/genéticaRESUMO
Hemoglobinopathies have become a significant national health problem in France. The biologists have a pivotal role in the genetic diagnoses. Although sickle cell disease (SCD) is the most frequent abnormality found: not less than 200 new cases are observed each year at birth, many other globin gene variations are found in the various ethnic groups. Since 1995 a neonatal sickle cell screening program has been established for at risk newborns. This programme is supported by the "Association française de dépistage et prévention des handicaps de l'enfant" (AFDPHE). The characterization of hemoglobin genetic variations requires a comprehensive set of laboratory techniques for which we specify here main clinical and technical recommendations.
Assuntos
Hemoglobinas/análise , Análise Química do Sangue/métodos , Análise Química do Sangue/normas , Coleta de Amostras Sanguíneas , Hemoglobinopatias/sangue , HumanosRESUMO
This study identified and characterized a novel delta beta fusion gene in which the delta-globin gene promoter is linked to intact beta-globin coding sequences in a Senegalese family. It results from a 7.4-kb deletion that removes the delta-globin coding sequences, the delta beta intergenic region as well as the beta-globin gene promoter and causes delta(0)beta(+) thalassemia with hemoglobin A expressed at the 11% to 15% range. The phenotype of this naturally occurring delta beta hybrid gene not only clarifies, in an in vivo context, the respective strength of delta- and beta-globin gene promoters, but also emphasizes the importance of beta-globin intragenic sequences in the expression of beta-globin chains. (Blood. 2001;98:1261-1263)
Assuntos
Hemoglobina A/genética , Hemoglobinas Anormais/genética , Deleção de Sequência/genética , Adolescente , Adulto , Criança , Pré-Escolar , Saúde da Família , Feminino , Globinas/genética , Humanos , Masculino , Pessoa de Meia-Idade , Núcleo Familiar , Linhagem , Regiões Promotoras Genéticas/genética , Senegal , Talassemia beta/genéticaRESUMO
UNLABELLED: We report a five-year experience of targeted neonatal screening for sickle cell disease in the northern part of the Paris area as well as the follow-up procedure of screened patients. POPULATION: This geographic area in France is characterized by a high frequency of populations at risk for sickle cell disease. RESULTS: Among 115,480 tested newborns, 250 patients were diagnosed (frequency: 1/462). The quality of the screening was attested by the high frequency (5.34%) of newborn carriers for a hemoglobin abnormality (n = 6168). We developed an optimized strategy which avoids the majority of pitfalls (false positive and false negative responses), except for S/HPFH. More than 95% of sickle cell disease was followed, the majority by medical sickle cell disease experts from hospitals. Only two deaths were recorded during this time period. CONCLUSION: We demonstrate the efficiency of targeting the proposed methodological strategy and the follow-up of affected newborns, a major argument demonstrating the importance of newborn screening.
Assuntos
Anemia Falciforme/diagnóstico , Programas de Rastreamento , Anemia Falciforme/epidemiologia , Reações Falso-Positivas , Feminino , França/epidemiologia , Humanos , Incidência , Recém-Nascido , Masculino , População UrbanaRESUMO
BACKGROUND: The French national programme for the neonatal screening of sickle cell disease (SCD) was set up in 1995. This screening is targeted at newborn infants at risk. Over 5 years, 115,480 newborn infants were tested from 80 maternity departments from the northern part of the Paris area. 250 Patients with SCD were identified--that is, one in 462 newborn infants tested. Carriers for a haemoglobin (Hb) variant are frequent (5.34%). Some uncommon Hb variants were also identified, which gave rise to pitfalls to the testing when associated with HbS: HbKorle-Bu, HbHope, HbBougardirey-Mali, and HbLadésirade (4% of SS-like profiles). OBJECTIVE: An effective screening strategy was developed to avoid these false positive and false negative responses. METHODS: Isoelectric focusing (IEF), the method of primary screening, is rapid and inexpensive. Cation exchange high performance liquid chromatography (CE-HPLC), which is automated, fast, and quantitative was selected as a secondary method. RESULTS: IEF diagnosed normal profiles in 89% of the tested samples from newborn infants. CE-HPLC identified most of the common Hb variants by their retention time and the measure of HbA/HbS ratio, important for the differential diagnosis between an asymptomatic HbS carrier and an HbS/beta+thal compound heterozygote. Furthermore, the high sensitivity of the CE-HPLC detected as little as 0.5% of a Hb variant. This avoided false negatives in samples from premature or transfused newborn infants. All samples with SS-like profiles were confirmed with a second CE-HPLC with another programme. A combination of these three methods confirmed the status of 99.7% of the samples from the tested newborn infants. Some cases required a reverse phase-HPLC method (for gamma-globin or alpha-globin chain variants). Finally, some exceptional samples required confirmation by testing DNA extracted with Güthrie paper for a precise diagnosis. CONCLUSIONS: This effective strategy combining several methods dramatically reduces the risk of errors. Many families are thus spared unnecessary worrying recalls. The only unavoidable cause of false positives remains the HbS/hereditary fetal Hb (HPFH).
Assuntos
Anemia Falciforme/diagnóstico , Triagem Neonatal/métodos , Anemia Falciforme/sangue , Anemia Falciforme/genética , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Erros de Diagnóstico , Triagem de Portadores Genéticos , Testes Genéticos/métodos , Variação Genética , Hemoglobina Falciforme/genética , Hemoglobina Falciforme/isolamento & purificação , Humanos , Recém-Nascido , Sondas de Oligonucleotídeos/genética , Paris , Sensibilidade e EspecificidadeRESUMO
A subset of 299 patients with homozygous sickle cell anaemia, enrolled in the cohort of the French Study Group on sickle cell disease (SCD), was investigated in this study. The majority of patients were children (mean age 10.1 +/- 5.8 yr) of first generation immigrants from Western and Central Africa, the others originated from the French West Indies (20.2%). We report the frequency of the main clinical events (mean follow-up 4.2 +/- 2.2 yr). The prevalence of meningitis-septicaemia and osteomyelitis was, respectively, 11.4% and 12% acute chest syndrome was observed in 134 patients (44.8%). Twenty patients (6.7%) developed stroke with peak prevalence at 10-15 yr of age. One hundred and seventy-two patients (58%) suffered from one or more painful sickle cell crises, while the others (42.5%) never suffered from pain. The overall frequency of acute anaemic episodes was 50.5%, (acute aplastic anaemia 46%; acute splenic sequestration 26%). A group of 27 patients were asymptomatic (follow-up > 3 yr). Epistatic mechanisms influencing SCD were studied. Coinherited alpha-thalassemia strongly reduced the risk of stroke (p <0.001) and increased that of painful crises (p < 0.02). There was a low prevalence of Senegal and Bantu (CAR) betas-chromosomes in patients with meningitis (p <0.04) and osteomyelitis (p < 0.03). Prevalence of Senegal betas-chromosomes was lower in the asymptomatic group of 27 patients (p < 0.02). The patients come from a population of unmixed immigrants in whom the beta-globin gene haplotype strongly reflects the geographic origin and identifies subgroups with a homogenous genetic background. Thus the observed effects might result more from differences in as yet unidentified determinants in the genetic background than from the direct linkage with differences in the beta-globin gene locus.
Assuntos
Anemia Falciforme/complicações , Doença Aguda , Adolescente , Anemia Falciforme/genética , Criança , Pré-Escolar , Estudos de Coortes , Feminino , França/epidemiologia , Genótipo , Humanos , Masculino , Meningite/complicações , Meningite/epidemiologia , Osteomielite/complicações , Osteomielite/epidemiologia , Fenótipo , Fatores de Risco , Sepse/complicações , Sepse/epidemiologia , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/epidemiologia , Talassemia alfa/complicações , Talassemia alfa/genéticaRESUMO
Two new fetal hemoglobin variants affecting the Ggamma chain are reported. Hb F-Clamart was found during investigation of a French newborn who presented with a mild microcytemia. The second variant was found during neonatal screening for hemoglobinopathies of 30,000 babies from a population-at-risk living in the Paris region. It was named Hb F-Ouled Rabah because its structural modification and ethnic distribution is similar to that of Hb D-Ouled Rabah [beta19(B1)Asn-->Lys]. Hb F-Ouled Rabah is clinically silent and occurs at a frequency of ca. 0.1% in newborns originating from Maghreb. Structural characterization of both variants was done by protein chemistry methods, including amino acids analysis and mass spectrometry.
Assuntos
Hemoglobina Fetal/química , Hemoglobina Fetal/genética , Globinas/química , Globinas/genética , Hemoglobinas Anormais/química , Hemoglobinas Anormais/genética , África do Norte/etnologia , Argélia/etnologia , Substituição de Aminoácidos , Cromatografia Líquida de Alta Pressão , Saúde da Família , França/epidemiologia , França/etnologia , Variação Genética , Heterozigoto , Humanos , Recém-Nascido , Peso Molecular , Marrocos/etnologia , Triagem Neonatal , Tunísia/etnologia , Talassemia alfa/genéticaAssuntos
Anemia Falciforme/diagnóstico , Triagem Neonatal , Feminino , França , Humanos , Recém-Nascido , Masculino , População UrbanaRESUMO
The beta thalassemia alleles in 53 thalassemic Indo-Mauritian patients and their families consisting of 23 homozygous beta-thalassemia, 9 HbE/beta-thalassemia, 18 HbS/beta-thalassemia, 1 HbD/beta-thalassemia, 1 deltabeta/beta-thalassemia and 1 HbH/beta-thalassemia from the island of Mauritius were studied. Characterization by polymerase chain reaction-based reverse dot blot hybridization technique revealed that the IVS1-5 (G-->C) mutation accounted for 74% of the beta thalassemic alleles, while six other mutations occurred at much lower frequencies: HbE codon 26 (G-->A); 10.4%, codon 8/9 (+G); 3.5%, codon 30 (AGG-->ACG) also called IVSI (-1).G-->C; 3.5%, codon 15 (G-->A); 3.5%, codon 41/42 (-CTTT); 2.4% and -28 (A-->G); 2.4%. Association of these mutations to specific beta globin gene sequence framework and haplotype allowed to trace their ancestral link. These data are useful in future molecular screening of the population in view of implementing a thalassemia prevention and control program in Mauritius.
Assuntos
Ligação Genética , Globinas/genética , Haplótipos , Mutação , Talassemia beta/genética , Alelos , Humanos , Maurício , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de RestriçãoRESUMO
In humans, studies of the erythroid cell lineage are hampered by difficulties in obtaining sufficient numbers of erythroid progenitors. In fact, these progenitors in bone marrow or peripheral blood are scarce and no specific antibodies are available. We describe a new method which allows proliferation in liquid culture of large numbers of pure normal human erythroid progenitors. CD34+ cells were cultured for 7 d in serum-free conditions with the cytokine mixture interleukin (IL)-3/IL-6/stem cell factor (SCF). This resulted in cell expansion and the appearance of a high proportion of CD36+ cells which were purified on day 7. Methylcellulose clones from these cells were composed of 96.6% late BFU-E and 3.4% CFU-GM. These CD36+ cells could be recultured with the same cytokine mixture plus or minus erythropoietin (Epo) for a further 2-7 d. In both conditions further amplification of CD36+ cells was observed, but Epo induced a more dramatic cell expansion. Glycophorin-positive mature cells appeared only in the presence of Epo, and terminal red cell differentiation was observed after 7 d of secondary culture. Cells obtained from adult CD34+ progenitors mostly contained adult haemoglobin, whereas cord blood-derived cells contained equal proportions of adult and fetal haemoglobin. Activation of STAT5 and tyrosine phosphorylation of the Epo receptor and JAK2 were observed after Epo stimulation of these cells. This new method represents a straightforward alternative to the procedures previously described for the purification of normal erythroid progenitors and is useful in the study of erythropoietic regulation.
Assuntos
Células Precursoras Eritroides/citologia , Antígenos CD34/metabolismo , Antígenos CD36/metabolismo , Diferenciação Celular , Divisão Celular , Separação Celular/métodos , Células Cultivadas , Citocinas/fisiologia , Eritropoetina/fisiologia , Citometria de Fluxo/métodos , Hemoglobinas/análise , HumanosRESUMO
Hereditary persistence of fetal hemoglobin (HPFH) is a group of genetically heterogeneous conditions characterized by the continued expression of fetal hemoglobin in adulthood. These constitute natural models for understanding the mechanism(s) of the hemoglobin switch. Many large deletions in the beta-globin gene cluster and point mutations in one of the fetal globin gene promoters have been described before. In this study we describe a novel C-->A transversion (-114) in the distal CCAAT box of the Ggamma-globin gene promoter associated with the Ggammabeta+-HPFH phenotype in an Algerian family. Individuals heterozygous for this mutation exhibit moderate raise in Hb F levels (0.6-3.5%). Much higher Hb F levels (3.8-11.2%) are observed when a beta(o)-thalassemia allele is present in trans to the hereditary persistence of fetal hemoglobin allele. This novel Algerian HPFH mutation further stresses the importance of the distal CCAAT box in the postnatal regulation of gamma-globin gene expression.
Assuntos
Hemoglobina Fetal/genética , Globinas/genética , Mutação Puntual , Adolescente , Adulto , Idoso , Argélia , Criança , Feminino , Hemoglobina Fetal/metabolismo , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Família Multigênica , LinhagemAssuntos
Cromatografia Líquida de Alta Pressão/instrumentação , Hemoglobinas Glicadas/análise , Automação , Cromatografia Líquida de Alta Pressão/métodos , Diabetes Mellitus/metabolismo , Eritrócitos/metabolismo , Estudos de Avaliação como Assunto , Hemoglobina C/análise , Humanos , Análise de Regressão , Reprodutibilidade dos TestesRESUMO
The two classical methods used to determine the level of Hb F in a hemolysate are herein described. Measurement of this hemoglobin fraction by resistance to alkali denaturation was the first technique introduced; it is today largely replaced by ion exchange high performance liquid chromatography. The limits, pitfalls, and advantages of the two methods are discussed.
Assuntos
Hemoglobina Fetal/análise , Hemoglobinometria/métodos , Álcalis/química , Cromatografia Líquida de Alta Pressão , Hemoglobina Fetal/imunologia , HumanosRESUMO
The Ggamma:Agamma ratio is around 70:30 at the time of birth and usually 40:60 in the trace amounts of Hb F found in the adult. Changes in this ratio are observed in several hemoglobin disorders providing insights on the genetics and molecular pathophysiology of these diseases. Several techniques have been proposed to measure the Ggamma:Agamma ratio. We here describe perfusion chromatography which is now in routine use in one of our laboratories. The method involves a high velocity flow of the mobile phase through a porous reversed phase chromatographic stationary bed and allows us to determine this parameter one order of magnitude faster than with conventional high performance liquid chromatography.
Assuntos
Cromatografia/métodos , Hemoglobina Fetal/química , Cromatografia Líquida de Alta Pressão , Hemoglobina Fetal/análise , Hemoglobina Fetal/imunologia , Globinas/análise , Humanos , Recém-Nascido , PerfusãoRESUMO
A new method to quantify red blood cells (RBCs) containing fetal hemoglobin (HbF), or F cells, by flow cytometry was developed. The use of formaldehyde as fixative agent and sodium dodecyl sulfate (SDS) to permeabilize fixed RBCs resulted in a low staining background, a negligible Hb leakage, and minimal cell clumping. HbF was detected by immunofluorescence with a murine monoclonal antibody directed to the gamma chain of human Hb. The accuracy of the F-cell count was evaluated on mixed-field populations of adult and cord RBCs. The results of flow cytometry were highly correlated with HbF dosage in the hemolysate by high-pressure liquid chromatography (HPLC) (R > 0.99). The sensitivity of the method allowed the study of HbF distribution at the single-cell level in normal controls and patients with sickle-cell disease (SCD). All patients exhibited a heterocellular distribution of HbF, with highly variable percentages of F cells and non-F cells. As an additional and valuable biological parameter in SCD, the mean HbF content per F cell could be deduced from the measurement of HbF level by HPLC and F-cell count by flow cytometry. Results were unchanged after storing the cells for several weeks in an optimized resuspending solution. Since it does not need uncommon reagents or devices and allows the simultaneous detection of membrane antigens by two-color flow cytometry, this method is convenient for routine laboratories as well as for research purposes.
Assuntos
Eritrócitos/química , Hemoglobina Fetal/análise , Citometria de Fluxo/métodos , Adulto , Eritrócitos/imunologia , Sangue Fetal/citologia , Doença da Hemoglobina SC/sangue , HumanosAssuntos
Mutação , Talassemia beta/genética , Alelos , Barein , Mapeamento Cromossômico , Hemoglobinas/genética , Humanos , Oriente Médio , Mutação Puntual , Deleção de SequênciaRESUMO
We have studied the cellular and molecular responses to long-term hydroxyurea (HU) treatment in 29 severely affected young patients with sickle cell disease (mean age, 10.9 +/- 4.1 years). Patients received HU at 20 mg/kg/d on 4 consecutive days per week initially, with a monthly escalated dose avoiding marrow-toxicity (mean steady-state dose, 34.2 +/- 4.6 mg/kg/d) for 12 to 36 months (mean duration, 22 months). The studied parameters were hemoglobin F (HbF), F reticulocytes (F retics), F cells, the amount of HbF per F cell (F/F cell), polymer tendency at 40% and 70% oxygen saturation, and hemolysis. Initial HbF (Fi) was dispersed (from 0.85% to 13.9%). HbF increased in all patients but 1. HbF at maximal response (Fmax) reached a sustained level varying from a 1.5-fold to a 16-fold Fi after a variable delay (6 to 24 months). Fmax was not related to HU dosage, but triangle upF (Fmax - Fi) was strongly correlated to triangle upMCV (MCVmax - MCVi). HbF increase resulted from the increase of both F cells and F/F cell. In this rather short series, Fi and Fmax were not significantly associated with age, gender, or beta-globin haplotype. Neither Fmax nor triangle upF was related to bone marrow reserve, as measured by baseline reticulocyte or neutrophil counts. However, Fmax was highly dependent on Fi. When patients are individualized into three groups according to Fmax (group 1, Fmax >20% [12 patients]; group 2, 10% < Fmax < 20% [11 patients]; group 3, Fmax <10% [5 patients]), Fi is significantly different between groups, being the highest in group 1. In addition, the best responders (group 1) were significantly different from patients in the two other groups with higher levels of total hemoglobin, decreased bilirubin, and decreased polymer tendency.