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Sickle cell disease is a genetic disorder with a large variability in the pattern and severity of clinical manifestations. Different genetic modulators have been identified but very few epidemiologic data are available on these modifier genes in Senegal. This study aimed to determine their prevalence in a Senegalese sickle cell disease pediatric population. The following genetic parameters were genotyped in 295 sickle cell disease children of the Dakar pediatric hospital: sickle cell disease genotype [βS/βS (HBB: c.20A>T), βS/βC (HBB: c.19G>A), βS/β0-thalassemia (β0-thal)], XmnI polymorphism, the five most common α-thalassemia (α-thal) deletions and the A(-) and Betica glucose-6-phosphate-dehydrogenase (G6PD) deficient variants. Despite very few βS/βC and βS/β0-thal children (1.0% each), a novel frameshift β0-thal mutation was characterized: HBB: c.265_266del; p.Leu89Glufs*2. The -α3.7 (rightward) deletion was the only α-thal deletion identified in this cohort (12.0% allelic frequency). Most of βS/βS patients (61.9%) were homozygous for the XmnI polymorphism and assumed to carry a Senegal/Senegal βS haplotype. The remaining haplotypes were predominantly of the Benin type. While the Betica G6PD variant was quite frequent (13.0%), a low frequency of the A(-) variant was detected (1.0-2.0%). The systematic genotyping of the -α3.7 deletion and of the G6PD Betica variant in sickle cell disease patients from Senegal could be useful to identify patients at risk for several complications, such as cerebral vasculopathy, where it has been demonstrated that a normal α-globin genotype and G6PD deficiency are predisposing factors. These patients should be eligible for a transcranial Doppler examination that is not routinely offered in Senegal.

Because of the increased hemolytic rate, a significant proportion of patients with sickle cell disease (SCD) are prone to develop cholelithiasis. The present study investigated the role of several genetic factors (UGT1A1 promoter (TA)One hundred and fifty-eight children (2-18 yr old) regularly followed at the University Hospital of Lyon (France) were included. A multivariate Cox model was used to test the associations between cholelithiasis and the different parameters analyzed.We confirmed that alpha-thalassemia and low basal reticulocyte (RET) count were independent protective factors for cholelithiasis while 7/7, 8/8 and 7/8 UGT1A1 (TA)Our findings demonstrate that UGT1A1 (TA)

We report two new variants of the δ-globin gene: Hb A(2)-Saint-Etienne [δ14(A11)Leu→Pro] and Hb A(2)-Marseille [δ22(B4)Ala→Lys]. The first variant has a low rate of expression, the second results from a double nucleotide mutation on the same codon.

Le diagnostic du deficit en α-1 antitrypsine (A1AT) repose sur la focalisation isoelectrique des proteines seriques et sur la mesure de la concentration serique. Cependant, la focalisation isoelectrique est techniquement delicate et une concentration fortement abaissee en A1AT sans bande anormale en focalisation isoelectrique ne permet pas de differencier un variant instable d’un variant dit « nul » (c’est-a-dire sans aucune expression phenotypique) a l’etat heterozygote. Dans la presente etude, nous avons compare les resultats du dosage, du phenotype et du genotype de l’A1AT chez 50 patients. Les alleles A1AT normaux (Pi*M1 a Pi*M4) ou deficitaires les plus courants (Pi*S et Pi*Z) ont ete parfaitement identifies au phenotypage. En revanche, le genotypage s’est avere necessaire pour caracteriser : (i) certains alleles A1AT plus rares (S-Munich, X-Christchurch) ; (ii) un allele nul et ; (iii) deux nouveaux alleles A1AT non encore decrits dans la litterature. En conclusion, meme si le genotypage A1AT n’est generalement pas necessaire, il est indispensable pour resoudre les cas complexes et pour obtenir des temoins valides pour la focalisation isoelectrique.

We report here two new beta(0)-thalassemic mutations. In the first case, a deletion of two nucleotides (-CC) at codon 142 was found in a French Caucasian woman. In the second case, an insertion of a single nucleotide (+T) at codon 45 was found in a Turkish girl. In both cases, no dominant thalassemia-like phenotype was observed.

We have identified a deletion of 125 bp (α-α(Δ125)) (NG_000006.1: g.37040_37164del) in the α-globin gene cluster in a Kabyle population. A combination of singlex and multiplex polymerase chain reaction (PCR)-based assays have been used to identify the molecular defect. Sequencing of the abnormal PCR amplification product revealed a novel α1-globin promoter deletion. The endpoints of the deletion were characterized by sequencing the deletion junctions of the mutated allele. The observed deletion was located 378 bp upstream of the α1-globin gene transcription initiation site and leaves the α2 gene intact. In some patients, the α-α(Δ125) deletion was shown to segregate with Hb S (HBB: c.20A>T) and/or Hb C (HBB: c.19G>A) or a β-thalassemic allele. The α-α(Δ125) deletion has no discernible effect on red cell indices when inherited with no other abnormal globin genes. The family study demonstrated that the deletion is heritable. This is the only example of an intergenic α2-α1 non coding DNA deletion, leaving the α2-globin gene and the α1 coding part intact.

We present here seven new hemoglobin (Hb) variants identified during routine Hb analysis. All of them are caused by a missense mutation except Hb Saint Chamond, which results from an in-frame deletion of the asparagine residue at β80. All these variants are clinically silent in the heterozygous state but two of them (Hb Cap d'Agde and Hb Dompierre) may be unstable, whereas Hb Nîmes could present a very slightly elevated oxygen affinity. These data are to be confirmed by appropriate biochemical tests.

A new mutation of the beta-globin gene initiation codon, ATG--AAG (Met--Tyr), is reported in a man originating from the southeast of France. Typical hematological findings of beta-thalassemia (thal) trait were found. We emphasize the importance of characterizing uncommon beta-thal mutations for genetic counseling.

We report a new mutation on the α2-globin gene causing α(+)-thalassemia (α(+)-thal) with a deletion of a single nucleotide (T) at amino acid residue 43 [HBA2:c.130delT or HBA2:c.131delT]. This frameshift deletion gives rise to a premature termination codon at codon 47.

We report a new β-globin chain variant: Hb Meylan [β73(E17)Asp → Phe; HBB: c.220G>T; c.221A>T]. The new variant results from a double nucleotide mutation at the same codon. The possible molecular mechanisms are discussed.

We present two case reports in which an HBD mutation is present with a rare α hemoglobinopathy that substantially complicates the associated phenotype. In the first case, a new δ-globin variant, Hb A2-Pierre-Benite [δ83(EF7)Gly→Arg; HBD: c.250G>C] is associated with Hb Groene Hart [α119(H2)Pro→Ser (α1); HBA1: c.358C>T], an α-thalassemic variant. In the second case, a δ(+)-thalassemic variant, δ4(A1)Thr→Ile; HBD: c.14C>T, is associated with a newly described deletion of the hypersensitive site 40 (HS-40) region on the α-globin gene cluster. In both patients, a δ-globin mutation was suspected because of an abnormally low Hb A2 level, whereas the α hemoglobinopathy was sought to explain the slight microcytosis and hypochromia presented by the probands.

Background Deletions represent about 5% of the mutations in the β-globin gene cluster. We report here the screening for such deletions in the two French urban areas of Paris and Lyon between 2003 and 2010. Methods Semi-quantitative PCR methods were used for the first screening of deletions. Thereafter, a specific gap-PCR, eventually followed by DNA sequencing, was used for precise identification. Results 285 patients bore a deletion or recombination event in the β-globin gene cluster. Hbs Lepore or anti-Lepore were detected in 99 patients. Among the remaining 186 patients, 132 bore a deletion that could be fully identified. The most prevalent deletions were the Ghanaian HPFH-2 (n = 46), the Sicilian (δβ) 0 -thal (n = 22) and the Spanish (δβ) 0 -thal (n = 12). The other characterized deletions were the: HPFH-3, HPFH-1, Filipino, Senegalese, Corfu, Kabilian, − 1.39 kb, Indian − 619 bp and − 468 bp. Interestingly, three new deletions were fully characterized: a − 7719 bp deletion, a − 27,825 bp deletion with a 25 bp insertion and a − 125 bp deletion. Conclusions The present study emphasizes the importance to detect deletions in the β-globin gene cluster, particularly for at risk couples. The new − 27,825 bp deletion illustrates the complexity to understand the transcriptional regulation of fetal to adult hemoglobin switch.

Deletions in the β-globin locus control region (β-LCR) lead to (εγδβ)(0)-thalassemia [(εγδβ)(0)-thal]. In patients suffering from these rare deletions, a normal hemoglobin (Hb), phenotype is found, contrasting with a hematological thalassemic phenotype. Multiplex-ligation probe amplification (MLPA) is an efficient tool to detect β-LCR deletions combined with long-range polymerase chain reaction (PCR) and DNA sequencing to pinpoint deletion breakpoints. We present here a novel 11,155 bp β-LCR deletion found in a French Caucasian patient which removes DNase I hypersensitive site 2 (HS2) to HS4 of the β-LCR. Interestingly, a 197 bp insertion of two inverted sequences issued from the HS2-HS3 inter-region is present and suggests a complex rearrangement during replication. Carriers of this type of thalassemia can be misdiagnosed as an α-thal trait. Consequently, a complete α- and β-globin gene cluster analysis is required to prevent a potentially damaging misdiagnosis in genetic counselling.

DNA analysis is currently the easiest way to identify a hemoglobin variant in most cases. Nevertheless, in case of complex gene rearrangements, mass spectrometry studies may be required to orientate the DNA diagnosis. The present report shows the use of mass spectrometry techniques prior to DNA analysis for the identification of the rare P-Nilotic fusion hemoglobin. Complete protein analysis is performed by liquid chromatography–tandem mass spectrometry on the abnormal globin chain isolated by reversed-phase liquid chromatography.

We present here five new hemoglobin (Hb) variants which have been identified during routine Hb analysis before their genotypic characterization. Four of these result from a classical missense mutation: Hb Canuts [alpha85(F6)Asp--His (alpha1)], Hb Ambroise Pare [alpha117(GH5)Phe--Ile (alpha2)], Hb Beaujolais [beta84(EF8)Thr--Asn] and HbA(2)-North Africa [delta59(E3)Lys--Met]. The last one, Hb Monplaisir [beta147 (Tyr-Lys-Leu-Ala-Phe-Phe-Leu-Leu-Ser-Asn-Phe-Tyr-158-COOH)], results from a frameshift mutation at the stop codon of the beta-globin gene which leads to a modified C-terminal sequence in the beta-globin chain. None of these variants seem to have a particular clinical expression in the heterozygous state. The circumstances of the discovery of these five new Hb variants emphasize the fact that an association of techniques is necessary for a complete screening of Hb variants during routine Hb analysis. Globin chain separation by reversed phase liquid chromatography (RP-LC) appears to be the most relevant method.

Despite the fact that mutations in the human beta-globin gene cluster are essentially point mutations, a significant number of large deletions have also been described. We present here four new large deletions in the beta-globin gene cluster that have been identified on patients displaying an atypical hemoglobin phenotype (high HbF) at routine analysis. The first deletion, which spreads over 2.0 kb, removes the entire beta-globin gene, including its promoter, and is associated with a typical beta-thal minor phenotype. The three other deletions are larger (19.7 to 23.9 kb) and remove both the delta and beta-globin genes. Phenotypically, they look like an HPFH-deletion as they are associated with normal hematological parameters. The precise localization of their 5' and 3' breakpoints gives new insights about the differences between HPFH and (deltabeta)(0)-thalassemia at the molecular level. The importance of detection of these deletions in prenatal diagnosis and newborn screening of hemoglobinopathies is also discussed.

Two new fetal hemoglobin (Hb F) variants affecting the (G)gamma chain are reported: Hb F-Saint-Etienne [G gamma 79(EF3)Asp--His] and Hb F-Lyon [G gamma 97(FG4)His--Arg]. These new Hb variants were found during a neonatal screening for hemoglobinopathies but characterized a few months later by our reference laboratory. The corresponding mutations are located on the external part of the Hb molecule and seem to be clinically silent.

A new hemoglobin (Hb) variant in the heterozygous state, Hb Al-Hammadi Riyadh [codon 75 (GAC--GTC); alpha75(EF4)Asp--Val (alpha2)] corresponding to an A--T transversion on the second exon of the alpha2-globin gene, is described. The variant was characterized by DNA sequencing and mass spectrometry (MS). The variant was found during a routine Hb analysis for anemia in a 16-month-old boy who lived in Riyadh, Kingdom of Saudi Arabia.

A new G(gamma) hemoglobin (Hb) variant, Hb F-Bron [gamma20(B2)Val--Ala] on the first exon of the G(gamma)-globin gene is described. The variant was characterized by DNA sequencing and mass spectrometry (MS). Hematological abnormalities included hypochromia and microcytosis and were probably caused by an interaction with an alpha-thalassemia (thal) (3.7 kb) deletion in the heterozygous state.

Two new hemoglobin (Hb) variants: Hb Brem-sur-Mer [codon 9 (TCT--TAT); beta9(A6)Ser--Tyr] on the first exon of the beta-globin gene and Hb Passy [codon 81 (TCC--CCC); alpha81(F2)Ser--Pro (alpha2)] on the second exon of the alpha2-globin gene, are described. The two variants were characterized by DNA sequencing and mass spectrometry (MS). Hematological abnormalities: microcytosis and hypochromia were found only in the carrier of Hb Passy. In the absence of an association with an alpha-thalassemic deletion or mutation, the mutation 81(F2)Pro could induce a possible alpha-thalassemia (thal).

Among more than 800 hemoglobin (Hb) variants currently described in the HBVar database of the Globin Gene Server (1), variants with elongated chains are very rare. Standard protein techniques such as ion-exchange HPLC and isoelectric focusing (IEF) on polyacrylamide gel can detect many Hb variants (2), but correct identification of single mutated, inserted, or deleted amino acid residues requires more sophisticated techniques, such as electrospray ionization mass spectrometry (ESI-MS) or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (3)(4)(5). The interpretation of DNA sequencing in the presence of inserted nucleotide sequences in the heterozygous state can be difficult and requires direct and reverse sequencing. Protein analysis by MS can be used to check results from DNA sequencing and also can detect posttranslational changes such as acetylation (NH2 terminus), deamidation, methionine oxidation, Hb addition products, or artifacts. ESI-MS and MALDI-TOF MS combined with specific tryptic digestion for peptide mass mapping are rapid and sensitive techniques to confirm inserted amino acid residues. We applied these techniques to the identification of a novel Hb variant with a five-amino acid insertion in the β-globin chain. These techniques allowed us to confirm the DNA sequencing results. Standard Hb analysis was performed by ion-exchange HPLC, IEF on polyacrylamide gel, and reversed-phase (RP)-HPLC of globin chains (6)(7). RP-HPLC was performed with a Vydac C4 analytical column (The Separations Group) with CH3CN–H2O containing 1 mL/L trifluoroacetic acid (TFA) as the mobile phase (7 …

Two new beta-globin chain variants: Hb Tripoli: codon 26, GAG--GCG [beta26(B8)Glu--Ala] and Hb Tizi-Ouzou: codon 29, GGC--AGC [beta29(B11)Gly--Ser] are described on the first exon of the beta-globin gene. The two variants are characterized by DNA sequencing and mass spectrometry (MS). Hematological abnormalities were found in the two carriers. The presence of microcytosis and hypochromia is explained by an additional homozygous 3.7 kb alpha(+) thalassemic deletion for the carrier of Hb Tizi-Ouzou. Hb Tizi-Ouzou showed a slight instability in vitro. The same hematological abnormalities associated with anemia are difficult to explain for Hb Tripoli's carrier in the absence of an alpha-globin genes abnormality and could suggest a possible abnormal splicing.

Subjects heterozygous for point mutations or short deletions at the polyadenylation (poly A) site of the β‐globin gene have mild β+‐thalassemia (thal) [[1]]. Several β+‐thal point mutations have be...

During routine studies of a patient who presented with venous thrombosis and erythrocytosis, a high affinity hemoglobin (Hb) variant, Hb Milledgeville [α44(CE2)Pro→Leu (α2)], was found. The patient...

We propose a new method for the quantitative determination of carbon dioxide released by a biological microgenerator: a suspension of human polymorphonuclear leukocytes (PMNL). This method is based on the reverse isotope dilution analysis by gas chromatography/isotope ratio mass spectrometry of 13CO2 released by PMNL in a controlled isotope abundance atmosphere containing 3% CO2. 13CO2 release is effective after PMNL stimulation in the presence of [13C]glucose, labeled on positions 1, 2 or 6. The validation of this method is carried out by the measurement of the isotope ratio 13CO2/12CO2 using known amounts of [13C]sodium hydrogen carbonate and the comparison with theoretical isotope abundances derived from various CO2 equilibria. Complete release of CO2 is achieved by the acidification of the medium. This method requires only few cells, displays high sensitivity and specificity and can be applied to the analysis of large series of samples using an automatic sample injector. In addition, this method can also be applied to other types of biological microgenerators of carbon dioxide.