Three new mutations account for the prevalence of glucose 6 phosphate deshydrogenase (G6PD) deficiency in Tunisia.

A previous study on G6PD deficiency carried out on Tunisian population, led to the finding of seven different mutations with the prevalence of G6PD A- variant. This present study reports 23 new unrelated deficient subjects studied at the molecular level to determine the mutation that causes G6PD deficiency. Using PCR-SSCP of coding regions followed by direct sequencing of abnormal pattern, three new mutations were detected. Two of them are polymorphic intronic mutations. The first is IVS-V 655C-->C/T, found in four female subjects with mild deficiency of class III variant. The second is IVS-VIII 43 G-->A, found in three male subjects with mild deficiency of class III variant. The third mutation is in the exon region so that it changes the primary structure of the molecule. It is cited for the first time and named G6PD Tunisia. This variant affects the exon 7 of the gene at genomic position 15435 G→T. Its cDNA position is 93 G→G/T, it changes arg 246 to leu. This mutation was found in one heterozygote female with deficiency of class II who have had hemolytic anemia due to ingestion of fava beans. Finally, G6PD Med variant, reported before in three cases, was also found in five other cases (four heterozygote females and one male hemizygote). These findings first enlarge the spectre of mutations to be ten variant mutations, characterizing the Tunisian population and also contribute with hemoglobin gene research in our laboratory to trace the whole genetic map of Tunisian population.

Molecular characterization of erythrocyte glucose-6-phosphate dehydrogenase deficiency in Tunisia.

Screening of G6PD deficiency was carried out on 79 unrelated subjects (32 females and 47 males), all coming from out consultation. DNA from deficient subject (11 females and 30 males) was analyzed for the presence of G6PD mutation. Known mutations were studied by the appropriate restriction enzyme digestion of fragment amplified by PCR. Where the mutation could not be identified in this way, the samples were subjected to SSCP analysis and abnormal fragments were sequenced. Through these methods, seven different mutations have been identified. Among deficient females, eight had the African variant A-(tow of them were homozygous) and three had the Mediterranean variant, one of them was homozygous and have had a haemolytic crisis after ingestion of fava beans showing at birth manifestation of neonatal jaundice. Among deficient males, four were hospitalized and transfused after a haemolytic crisis due to ingestion of fava beans. All of them have had manifestation of neonatal jaundice. Of them, one carried the Mediterranean variant and three others had the African variant A-. Among the remaining deficient males, 15 had A-variant, two had the Aurès mutation. SSCP analysis of nine mild deficient males, revealed the presence of the association of 1311 CT/93 TC in two subjects, a newly described silent mutation in the exon 12 associated with the polymorphism in the intron 11 93 TC in one subject and tow single intronic base deletion. The first is IVS V 17 (-C) found in two subjects and the second is IVS VIII 43 (-G) encountered in four subjects.

Short insertion in a hemoglobin chain: Hb Esch, an unstable alpha1 variant with duplication of the sequence Ala65-Leu-Thr-Asn68.

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.

[Good practices for the study of hemoglobin]. / Bonnes pratiques de l'étude de l'hémoglobine.

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.

Abnormal hemoglobins: laboratory methods.

Laboratory methods allowing the detection and characterization of hemoglobin variants are reviewed. Protein chemistry techniques such as isoelectrofocusing, electrophoreses under various experimental conditions, cation exchange and reversed phase high performance liquid chromatography, are the most frequently used for the detection of variants. When associated with a few additional data they may lead to a presumptive diagnosis. DNA studies are also developed in many laboratories. Final identification of a variant may be achieved either by molecular biology techniques or by protein sequence analysis in which mass spectrometry now occupies a key position.

Hb Mont Saint Aignan [beta128(H6)Ala-->Pro]: a new unstable variant leading to chronic microcytic anemia.

Hb Mont Saint-Aignan [beta128(H6)Ala-->Pro] is a mildly unstable variant, associated with hemolytic anemia, marked microcytosis and increased alpha/beta biosynthetic ratio (1.55 versus 1.1 +/- 0.1 in the control). The abnormal chain was isolated by selective precipitation with isopropanol and the structural modification determined by protein chemistry methods (reversed phase high performance liquid chromatography and mass spectrometry). Possible mechanisms underlying the beta(+)-thalassemia-like expression of this variant are discussed.

Sickle cell anemia and beta-globin gene cluster haplotypes in Colombia.

We studied 46 unrelated sickle cell anemia patients from the western region of Colombia which has the largest Black population of the country. Twenty-three children and 23 adults were studied. The distribution of haplotypes in the children was 58% Bantu, 38% Benin, and 4% Senegal, and in the adults it was 59.4% Bantu, 35.1% Benin, and 5.5% Senegal (p = 0.920). All 92 chromosomes had typical African haplotypes, Bantu 55.5%, Benin 34.8%, Senegal, 4.3%, and Cameroon, 5.4%. Our results suggest a lack of differential survival among patients with sickle cell anemia and typical beta-globin gene cluster haplotypes. They also agree closely with historical data that indicate that most African slaves brought to Colombia originated from Angola (Bantu population) and the Sao Thomó Island in the Bight of Benin (Central West Africa).

Two new variants with the same substitution at position beta122: Hb Bushey [beta122(GH5)Phe-->Leu] and Hb Casablanca [beta65(E9)lys-->Met; beta122(GH5)Phe-->Leu].

Hb Bushey, found in a Chinese baby and his father, is a new variant with a point mutation leading to the substitution Phe-->Leu at position beta122. Hb Casablanca, found in a family from Morocco, is a further example of a hemoglobin variant that carries two abnormalities in the same chain; the first is identical to that of Hb Bushey and the second to that of Hb J-Antakya [beta65 (E9)Lys-->Met]. Structural abnormalities of both Hbs were determined by protein chemistry methods including electrospray and tandem mass spectrometry. Their stability and oxygen binding properties were found to be identical to those of Hb A. Various mechanisms that may lead to two point mutations in the same chain are reviewed briefly.

HbF in the adult: could its composition discriminate normal from abnormal foetal globin gene expression?

The relative proportions of the two gamma chain species (G gamma and A gamma) have been reinvestigated in newborns, during the physiological switch from foetal to adult haemoglobin, and in adults with some persistent expression of HbF. In newborns, with about 80% HbF, the G gamma percentage was close to 70% while in adult RBC, with less than 0.5% HbF, the G gamma chain was almost non-detectable and may reflect the completion of the foetal to adult switch. Conversely, in adult patients with HbF above 0.6%, usually accompanying some degree of marrow stimulation, the relative ratio of G gamma varied between 40 and 60%, independently of HbF level. This ratio corresponds to what has been described in the literature as being the adult type of HbF. In all the cases where we found higher levels of G gamma, the results could be explained by the presence of a specific genetic background such as the Senegalese haplotype in sickle cell disease.

Two hemoglobin variants with an alteration of the oxygen-linked chloride binding: Hb Antananarivo [alpha1(NA1)Val-->Gly] and Hb Barbizon [beta144(HC1)Lys-->Met].

We here report two new, clinically silent, hemoglobin variants in which the structural modification disturbs the oxygen-linked chloride binding. Hb Antananarivo [alpha1(NA1)Val-->Gly] was found during a systematic hematological study in a 24-year-old woman, who originates from Madagascar. Hb Barbizon [beta144 (HC1)Lys-Met] was found in several members of a French family. The oxygen binding properties of Hb Barbizon were similar to those of Hb Antananarivo showing, in vitro, a decreased chloride effect as compared to Hb A. In Hb Barbizon, the replacement of lysine beta144 by a methionine residue decreased from 4 to 2 the excess positive charges in the central cavity, thus leading to a reduction of about half of the chloride effect. For Hb Antananarivo, the mechanism is unclear since there is no difference in the number of positive charges in the central cavity but alterations are likely at the alpha1alpha2 interface.

Determination of Hb F levels: the routine methods.

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.

Gamma chain heterogeneity: determination of Hb F composition by perfusion chromatography.

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.

Hemoglobins in Togolese newborns: Hb S, Hb C, Hb Bart's, and alpha-globin gene status.

The gene frequency of the most important hemoglobin (Hb) abnormalities is reported in a population of 171 Togolese newborns. Hb phenotypes, hematological parameters, and the more frequently described alpha-gene deletions were analyzed. Structural abnormalities of beta-globin were observed in 35.7% of the children with a gene frequency of 0.105 for beta(S) and 0.091 for beta(C). The frequency of the different alpha-globin genotypes was alpha alpha/ = 0.71, -alpha/ = 0.28, and alpha alpha alpha/ = 0.01. All of the individuals homozygous for the -alpha genotypes, and most of the heterozygous individuals, carried Hb Bart's. Within the alpha alpha/alpha alpha and the -alpha/alpha alpha groups, several individuals with or without Hb Bart's were found; they did not differ from the others by their red blood cell (RBC) parameters but by their levels of fetal hemoglobin (Hb F). The African alpha2 polymorphism marker, characterized by the replacement of G by TCGGCCC at position 7238 (EMBL HSHBA4, 1993) and of T 7174 by G, was found in 21 newborns. The mean value of Hb F was calculated for each genotype, the mean (G)gamma percentage was 69.4 +/- 4.0%, and the gene frequency of the AgammaT marker was 0.10; this marker was linked to the normal beta-globin cluster.

Hb Nancy and Hb Osler: two distinct genetic variants with identical clinical and hemoglobin phenotype.

Three hemoglobin variants (Hb Nancy, Osler and Fort Gordon), carrying the same Tyr-->Asp substitution at position beta 145 (HC2), have been independently described in 1975 in patients with marked polycythemia. The first one was found in a French caucasian family from Lorraine, and the two others in African Americans. Two unrelated individuals with Hb Osler have been recently reinvestigated at the DNA level and surprisingly, in their beta gene, codon 145 was found to be AAT which encodes for asparagine and not for aspartic acid, the aspartate at the protein level resulting, thus, from a very efficient posttranslational event. We reinvestigated a patient from the family of Hb Nancy and found that codon 145 was GAT, encoding for aspartate. This demonstrates that Hb Nancy is genetically distinct from Hb Osler despite an almost identical phenotype.