Philippe Lacan

Hemoglobin debrousse (β96[FG3]Leu → Pro): A new unstable hemoglobin with twofold increased oxygen affinity

Hemoglobin Debrousse (β96[FG3]Leu → Pro) is a new unstable variant, with high oxygen affinity responsible, in the steady state, for an apparently well‐compensated chronic hemolytic anemia. The functional properties of this variant are due to the replacement of a leucine residue which is involved in the hydrophobic environment of the proximal side of the heme. This electrophoretically neutral hemoglobin was found as a de novo case in a 6‐year‐old girl suffering from severe anemia with hemolysis and transient aplastic crisis, following infection by parvovirus B19.

Detection of a Thalassemic α-Chain Variant (Hemoglobin Groene Hart) by Reversed-Phase Liquid Chromatography

BACKGROUND Hemoglobin (Hb) Groene Hart [alpha119 (H2)Pro-->Ser (alpha1)], also known as Hb Bernalda, is a nondeletional alpha-thalassemic Hb variant that is frequent in southern Italy and North Africa. This variant is not supposed to be produced in the erythrocytes of carriers. The alpha-thalassemic behavior of this variant has been explained as an impaired interaction between the alpha-globin chain and the alpha-Hb-stabilizing protein. METHODS To separate globin chains, we developed a modified reversed-phase liquid chromatography (RPLC) procedure that uses acetonitrile-water solvents containing up to 3 mL/L trifluoroacetic acid. After RPLC, we characterized the isolated globin chains by electrospray ionization (ESI) mass spectrometry (MS) and analyzed their tryptic peptides with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and nano-LC-ESI-MS/MS. RESULTS RPLC detected an abnormal peak with a retention time substantially greater than that of the wild-type alpha(A)-globin chain. We identified this variant as Hb Groene Hart and found it in the hemolysates of 11 unrelated patients (1 homozygote, 9 heterozygotes, and 1 heterozygote associated with the -alpha(3.7) deletion). These patients possessed abnormal hematologic features suggesting an alpha-thalassemia phenotype. Molecular modeling suggested that the increase in hydrophobicity was due to opening of the GH interhelical segment following replacement of amino acid residue 119 with a nonhelix breaker residue. CONCLUSIONS This method allows the detection of Hb variants at low concentrations, and adjusting the composition of the organic solvents enables the method to identify Hb variants with large changes in hydrophobicity.

Rapid and reliable β-globin gene cluster haplotyping of sickle cell disease patients by FRET Light Cycler and HRM assays

BACKGROUND β-Globin haplotypes are important to predict the clinical development of patients suffering from sickle cell disease (SCD). Five main haplotypes (Benin, Bantu, Senegal, Cameroon and Arabic-Indian) are defined for β(S) chromosomes and their determination usually requires the genotyping by restriction fragment length polymorphism (RFLP) of six to eight single nucleotide polymorphisms (SNPs). However, RFLP is time-consuming and can lead to a misdiagnosis in case of a supplementary SNP on the restriction sequence. We propose a rapid β-globin haplotyping method using fluorescence resonance transfer (FRET) and high resolution melting (HRM) assays. METHODS We have settled a fluorescence resonance energy transfer (FRET) assay for HincII ε, XmnI, HindIII (G)γ, HindIII (A)γ, HincII δ and a high resolution melting (HRM) assay for HincII ψβ. These six SNPs are sufficient in most cases to determine the β(S) haplotype. RESULTS Our methodology allowed us to successfully determine the β-globin haplotypes of 139 patients suffering from sickle cell disease. For some β(S) / β(0)-patients, a supplementary SNP has been identified on the HindIII (G)γ restriction sequence leading to a false-negative RFLP result. CONCLUSION Combination of FRET and HRM assays is a rapid and reliable method for the β-globin gene cluster haplotyping.

THROMBOTIC EVENTS IN COMPOUND HETEROZYGOTES FOR A HIGH AFFINITY HEMOGLOBIN VARIANT: Hb MILLEDGEVILLE [α44(CE2)Pro→Leu (α2)] AND FACTOR V LEIDEN

During routine studies of a patient who presented with venous thrombosis and erythrocytosis, a high affinity hemoglobin (Hb) variant, Hb Milledgeville [a44(CE2)Pro!Leu (a2)], was found. The patient was a 57-year-old French Caucasian man and originated from southeast France. He had had deep venous thrombosis, complicated by pulmonary embolism, four years ago. Medical treatment included heparin followed by an oral anticoagulant for four months. After this last treatment, the patient took daily low doses of aspirin. He was referred to us for biological reevaluation. The hematological parameters are shown in Table 1. Neither spleen nor liver enlargement were found at physical examination, and echographic investigations were negative. Bone marrow biopsy ruled out the occurrence of myeloproliferative disorders. The serum erythropoietin level was normal: 11.2 IU=L (normal range: 3.7–16 IU=L). Routine Hb analyses were performed by standard procedures. Cation exchange high performance liquid chromatography (HPLC) using the VARIANT System with the b-Thalassemia Short Program (Bio-Rad Laboratories, Hercules, CA, USA) revealed the presence of an abnormal Hb which eluted as a shoulder just before Hb A, accounting for 23% of total Hb. Isoelectrofocusing

A novel telomeric (~285 kb) α-thalassemia deletion leading to a phenotypically unusual HbH disease

Many large deletions removing the entire α-globin gene cluster on the short arm of the human chromosome 16 (16p13.3) have been described.[1][1]–[3][2] At the heterozygous state, the resulting phenotype consists in α-thalassemia (α-thal) for relatively short deletions (100 to 356 kb) while an

A Novel Deletion/Insertion Caused by a Replication Error in the β-Globin Gene Locus Control Region

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.

Two New α Chain Variants: Hb Die [α93(FG5)Val→Ala (α1)] and Hb Beziers [α99(G6)Lys→Asn (α1)]

The first new a chain variant here reported, Hb Die [a93(FG5)Val!Ala (a1)], was found during investigations of an anemia prior to surgery for correcting spina bifida. The patient was a 7-year-old girl, living near the town of Die in Southeast France; her parents were of French Caucasian origin. The hematological findings were as follows: RBC 5.8 10/L, hemoglobin (Hb) 11.9 g/dL, MCV 65.5 fL and MCH 20.5 pg; the serum iron level was 6.6 mmol/L (normal values 9–30 mmol/L). Iron treatment associated with erythropoietin was prescribed. The family refused all transfusion therapy. Unfortunately, no hematological data were available after this treatment and no family studies were possible. The second new a chain variant, Hb Beziers [a99(G6)Lys!Asn (a1)], was found during the determination of Hb A1c by high performance liquid chromatography (HPLC) in a diabetic patient. The new Hb variant was detected in a 72-year-old woman, who was also of French Caucasian origin and lived in the city of Beziers in the south of France. The hematological data were as follows: RBC 4.77 10/L, Hb 13.6 g/dL, MCV 86 fL and MCH 28.5 pg; physical examination was normal, without hepatomegaly or splenomegaly.

Characterization of three new deletions in the β-globin gene cluster during a screening survey in two French urban areas.

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.

Hb AUBAGNE [β64(E8)Gly→Ala]: A NEW UNSTABLE β CHAIN VARIANT FOUND IN A FRENCH FAMILY

Only one hemoglobin (Hb) variant has been reported at the internal position 64(E8)Gly of the b-globin chain: Hb J-Calabria, also known as Hb J-Bari and Hb J-Cosenza (!Asp). We here report a new Hb variant at the same position, namely, Hb Aubagne (!Ala), that was found in a young woman suffering from an anemia. The mutation is close to the distal histidine b63(E7) and leads to moderate instability. Routine Hb analyses were performed by isoelectrofocusing (IEF) and high performance liquid chromatography (HPLC) of Hbs and globin chains. Molecular characterization of the mutation was achieved by polymerase chain reaction (PCR), followed by direct DNA sequencing of the PCR products. PCR amplification of exons 1 and 2 of the b-globin gene was carried out using the following primers: 50-TGTACTGATGGTATGGGGC-30 (primer 1) located 225 nucleotides upstream to the Cap site, and 50-AAACGATCCTGAGACTTCC-30 (primer 2) located in the second intron. PCR amplification of exon 3 was performed using: 50-TATCATGCCTCTTTGCACTC-30 (upstream primer 3)

Protein characterization by LC–MS/MS may be required for the DNA identification of a fusion hemoglobin: The example of Hb P-Nilotic

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.