Haemoglobinopathies with high oxygen affinity. Experience of Erythropathology Cooperative Spanish Group.

Haemoglobinopathies are the world's most frequently found monogenic disorders. In the cases with high oxygen affinity, the decrease in the liberation of the oxygen determines a secondary erythrocytosis. In this work, we present 17 unrelated families of Caucasian race and of Spanish origin, with ten variants of haemoglobin or haemoglobinopathies with high oxygen affinity which were diagnosed in our laboratory. Of the ten haemoglobinopathies, in four (the Hb San Diego, the Hb Johnstown, the Hb Malmö and the Hb Columbia-Missouri), the change of amino acid affects zones of the contact alpha(1)beta(2); in two variants (the Hb Strasbourg and the Hb Syracuse), it affects the unions with 2,3-DPG in the central cavity; in the other two (the Hb Badalona and the Hb La Coruña), the cavity of contact with the group haem is affected; in one (Hb Bethesda), it affects the zone of contact alpha(1)beta(1;) and in one (Hb Olympia), the position 20 of the chain in the helix B in the surface of the protein is affected. In all cases, the change of amino acid, though of different form, facilitates that the quaternary structure of the haemoglobin becomes stable in its relaxed configuration so the transfer of oxygen and the P(50) value are decreased. All cases were sent to our laboratory because of shown erythrocytosis. In the majority of them, the diagnosis was done during an analysis of routine or for being relatives of the first ones.

Oscillations in serum ferritin associated with antiviral therapy in chronic hepatitis C.

BACKGROUND: Hyperferritinemia is often found in patients with chronic hepatitis C (CHC) and is predictive of poorer response to antiviral therapy. OBJECTIVE: To investigate changes in ferritinemia during and after antiviral therapy. PATIENTS AND METHODS: serum ferritin levels were measured in 262 CHC patients (163 males, mean age 48.5 years +/- 10.1) before and during antiviral therapy, and six months post-treatment in all 154 patients with undetectable serum HCV-RNA after therapy completion. RESULTS: Baseline serum ferritin was higher in patients with primary therapeutic failure than in those reaching sustained viral response (330 +/- 291 ng/mL vs. 211 +/- 192 ng/mL, p = 0.002). Serum ferritin transiently increased during therapy from baseline (257 +/- 242 ng/mL vs. 875 +/- 630 ng/mL, p < 0.001). Six months after finishing therapy, serum ferritin decreased under baseline values both in sustained responders (117 +/- 102 ng/mL vs. 211+/- 192 ng/mL, p < 0.001) and, to a lesser extent, in relapsers (217 +/- 174 ng/mL vs. 257 +/- 221 ng/mL, p = 0.047). CONCLUSIONS: Baseline serum ferritin may predict response to antiviral treatment in chronic hepatitis C. Combined antiviral therapy induces a marked increase in serum ferritin that falls below baseline values after sustained viral response, suggesting that the cause of hyperferritinemia in many patients is HCV infection itself rather than iron overload.

Capillary electrophoresis and mutational images of hemoglobin sendagi [Β42 (CD1) PHE → VAL; HBB: C.127T→G].

We report two cases of hemoglobin Sendagi in a Romanian family residing in Spain: a four-year-old boy and his mother, who had been previously diagnosed with another type of congenital hemolytic anemia and had undergone splenectomy in her country during childhood. The unstable hemoglobin variant, hemoglobin Sendagi, is characterized by decreased oxygen affinity caused by replacement of one of the critical amino acid residues, phenylalanine beta 42 (CD1) of the beta-chain, with valine in the heme pocket, resulting in methemoglobin formation. As a result of migratory movements in Europe, new disease-causing hemoglobin variants are emerging in our country. Here, capillary electrophoresis enabled the identification of the variant and a molecular study was used to establish an accurate diagnosis.

[The H63D mutation in the HFE gene is related to the risk of hepatocellular carcinoma]. / La mutacion H63D del gen HFE se asocia con un riesgo aumentado de carcinoma hepatocelular .

AIM: To disclose whether mutations in the HFE gene inducing liver iron overload are related to the risk of hepatocellular carcinoma (HCC) in otherwise predisposed patients. PATIENTS AND METHODS: One hundred and ninety-six patients (161 males) diagnosed with HCC and 181 healthy controls were included in the study. All subjects were white Spaniards.C282Y and H63D mutations in the HFE gene were identified in leucocyte genomic DNA using a polymerase chain reaction (PCR) and specific restriction enzymes. RESULTS (CASES/CONTROLS): 1. Genotype distribution: a) C282Y mutation: homozygotes 1/0, heterozygotes 12/23, wild type 183/158 (p = 0.07, non significant); b) H63D mutation: homozygotes 9/5, heterozygotes 85/52, wild type 102/124 (0dds ratio 2.00, 95% C.I. 1.29-3.12, p = 0.002. Four cases and 6 controls were carriers of heterozygous mixed genotypes. 2. Allele frequencies: a) C282Y mutation: wild type allele 378/339, mutated allele 14/23 (p = 0.11, non significant); b) H63D mutation: wild type allele 289/300, mutated allele 103/62 (0dds ratio 1.72, 95% C.I. 1.19-2.50, p = 0.004). Age at diagnosis, gender and etiology of the underlying liver disease do not influence these findings. CONCLUSION: The C282Y mutation in the HFE gene is not related to the risk of HCC in non-hemochromatosis patients. The H63D mutation is associated with a higher risk of HCC in cirrhotic patients irrespective of their underlying liver disease.

Association of t(9;11)-MLL AF9 and trisomy 8 in an AML-M5 preceded by pancytopenia.

The implication of MLL gene rearrangements in the prognosis of acute myeloblastic leukemia is an issue of considerable current interest. We report a case of a young man who initially presented with a pancytopenia and went on to develop a highly-aggressive acute myeloblastic leukemia. At this time, the karyotypic study revealed trisomy 8, a t(9;11) was demonstrated by fluorescence in situ hybridization (FISH) and the MLL/AF4 rearrangement by reverse transcriptase-polymerase chain reaction (RT-PCR).

Properties of hypotonized, crosslinked and crosslinked- permeabilized rat erythrocytes as potential carrier systems.

The osmotic fragility curves of isotonic rat RBCs, studied at pH 8 to avoid the Hb insolubility, are similar to those in humans at pH 7.4. Hypotonized rat RBCs, either directly or dialysed (0-24 h), are more hemolysis-resistant than isotonic rat RBCs. The discocyte-stomatocyte-spherocyte transformation can be observed with scanning electron microscopy. Protein crosslinking with dimethyl suberimidate can stabilize RBCs. The crosslinking level (60%), the cellular yield (80%), the mechanical and hemolytic resistance and the protective effect of enzyme activities, were studied in crosslinked or crosslinked- permeabilized RBCs after digitonin treatment. The normal discocytic shape of RBCs under scanning electron microscopy becomes stomatocytic in crosslinked and crosslinked- permeabilized RBCs with an erosioned surface.

[Association of alpha and beta thalassemia with alpha gene triplication in one family]. / Asociación de talasemia alpha y beta con triplicación de genes alpha en una misma familia.

UNLABELLED: We describe the haematological data and molecular results of a native family from Cádiz in that one is produced the a within heterozygous beta 0 thalassaemia (IVS-1, nt 1-G-->A), heterozygous alpha+ thalassaemia (-alpha 3.7) and alpha gene triplication (alpha alpha alpha 3.7). PATIENTS AND METHODS) We are studied 7 members to a family composed by father (I1), mother (I2) and five children (II1, II2, II3, II4, II5). The molecular biology study of the alpha gene was realized by Southern blot method using the restriction enzymes Bam HI, Bgl II and Eco RI and hybridized with alpha probe of the plasmid PRB 1 (fragment of 1.5 Kb digested with the enzyme Pst I). The genes were studied by the technique of the polymerase chain reaction (PCR), modified according to designated method "Amplification Refractory Mutation System" (ARMS). RESULTS: The father (I1) presents an interaction of therozygous beta 0 thalassaemia with heterozygous alpha + thalassaemia (beta 0/beta 1;alpha alpha/-alpha 3). The mother (I2) shows an alpha gene triplication (beta A/beta A: alpha alpha alpha 3.7/alpha alpha). Finally the children are expressed 5 possibilities: II4 he is normal (beta A/beta A; alpha alpha/alpha alpha), II2 he has alpha gene triplication (beta A/beta A; alpha alpha/alpha alpha alpha 3.7), II3 he has heterozygous beta 0 thalassaemia (beta 0/beta A; alpha alpha/alpha alpha), II5 he has interaction between heterozygous beta 0 thalassaemia and heterozygous alpha gene triplication (beta 0/beta A; alpha alpha alpha 3.7/alpha alpha) and II1 presents an interaction between a heterozygous beta 0 thalassaemia and together with the lost of one alpha gene in one chromosome he also presents a alpha gene triplication in other one (beta o/beta A; alpha alpha/alpha alpha). The hematological data of II5 corresponds to a intermediate thalassemia with not transfusion dependent feature an opposite to II1 that presents a heterozygous thalassemic trait features with 4 alpha genes. DISCUSSION: The phenotypical expression of the different interactions of these mutations in this family, points out, the relevant role that the unbalance globins chains plays in the pathogenesis and development of the clinical manifestations of the patients with the thalassaemia syndromes.

[The association of beta zero-thalassemia and Hb D Punjab in a family of Indian origin. The second case reported in Spain]. / Asociación de beta zero-talasemia y Hb D-Punjab en una familia de origen hindú. Segundo caso descrito en España.

The present report described the hematologic and molecular study of the second case of Hb D-Punjab associated with a beta zero-thalassemia found in Spain and the first case in which the mutations have been identified at molecular level. A family from India is studied, which is constituted by mother (I2) and 3 children (II1, II2 and II3). The molecular characterization of the hemoglobinopathy was made by electrophoretic and chromatographic techniques and confirmed by sequencing of the beta-globin gene. The mutation causer of the beta-thalassemia was studied by PCR-ARMS. The mother (I2) and one of her child (II2) are carriers of the gene for beta zero-thalassemia owing to the frameshift CD 8/9 mutation (+ G). Other of them (II1) is heterozygous for Hb D-Punjab without beta-thalassemia association. The third child (II3) knows a double heterozygote state for Hb D-Punjab/beta zero-thalassemia (hemoglobin D-thalassemia). In spite of the patient with hemoglobin D-thalassemia has 94.5% of Hb D, without Hb A, the hematologic picture belongs to thalassemia trait with moderate haemolytic anemia, intense microcytosis and hypochromia and numerous target cells. This hematologic picture discloses the mildness of the Hb D-Punjab, but the reliable responsible for the phenotype is the disbalance in the synthesis of globin chains, because of frameshift CD 8/9 mutation (+ G) beta zero-thalassemia mutation.

[HFE gene mutations, hepatic iron content, and histological severity in hepatitis C virus-induced chronic hepatitis].

OBJECTIVE: To study whether any relationship exists between the C282Y and H63D mutations of the HFE gene, iron liver content, and the severity of histological damage in patients with hepatitis C virus (HCV)-induced chronic hepatitis. MATERIAL AND METHODS: In 72 patients diagnosed with HCV-chronic infection, naïve for antiviral therapy, and undergoing liver biopsy, the Knodell index was established, a morphometric evaluation of hepatic hemosiderin deposits was performed by using a semiautomatic method of image analysis, and mutations of the HFE gene were identified through a polymerase chain reaction on leukocyte genomic DNA by using specific restriction enzymes. The control group for the distribution of HFE genetic variants was composed of 181 healthy individuals with the same ethnic and geographical (white Spaniards) origin. RESULTS: (Cases/controls): 1. Genotype distribution: a) mutation C282Y: no homozygotes, 6/23 heterozygotes, 66/158 without the mutation (not significant, n.s.); b) mutation H63D: 2/5 homozygotes, 26/52 heterozygotes, 44/124 without the mutation (n.s.). compound heterozygotes 2/6. 2. Allele frequencies: a) mutation C282Y: 0.042/0.064 (n.s.); b) mutation H63D: 0.208/0.171 (n.s.). Four C282Y heterozygous patients had stainable liver iron (p=0.015 vs patients without mutations). Sixty-six patients were not carriers of the C282Y mutation; among them, 26.9% of 26 carriers and 15% of 40 non-carriers of the H63D mutation had liver stainable iron (n.s.). Knodell index score, gender, age at diagnosis, mode of transmission, and serum and liver iron values were not related to the HFE genotype. CONCLUSIONS: our results suggest that the C282Y mutation, but not the H63D mutation, of the HFE gene is frequently associated with stainable iron in the liver in HCV-related chronic hepatitis. The HFE genotype is not related to the histological severity of the disease.

[A serological survey of rubella among pregnant women in Abidjan (author's transl)]. / Pévalence sérologique de la rubéole chez la femme enceinte à Abidjan.

A serological survey about the prevalence of the rubella hemagglutination inhibiting antibodies among the pregnant women in Abidjan during the year 1975 has shown an average rate of seroconversion of 84 p. 100. This rate depends on age and increases from 70 to 80 p. 100 between 15 and 35 years old. A regression line is fitted, and the goodness of fit is tested. These rates are in accordance with what is presently observed in America, in Europe and in Australia.

[Anthropometric nutritional indices and food intake of a child population of South Ivory Coast (author's transl)]. / Indices anthropomètriques nutritionnels et ration alimentaire d'une population infantile du sud de la Côte-d'Ivoire.

The present study was carried out in order to determine new biochemical or immunological parameters which would be used to make an early diagnosis of protein-caloric malnutrition. For the first phase of the research, based on antropometrics (circumference of the arm, head, weight) and on the quantitative evaluation of daily nutritional food intake, three groups of children between the ages of 1 and 3 were selected according to the following criteria: -- Properly nourished children, -- Malnourished children without physical signs of malnutrition, -- Malnourished children with malnutrition signs. The description of these three groups using the forementioned indicators is reported. This work performed on 652 infants shows the following results: -- Whatever the age, mid-arm circumference and weight are significantly reduced in confirmed cases of malnutrition, but are not useful in detecting malnutrition at an early stage. -- Whatever the age, head circumference does not change with the nutritional status, but it is reduced when the malnutrition has set in. -- Whatever the age, the caloric ration (25% deficit) and the proteic ration (35% deficit) of a malnourished child without signs of malnutrition and those of a well nourished child are different. But those ration does not change with an increasing degree of malnutrition.

Alpha-thalassemia-1 (--CAL mutation) in a Spanish family.

We have detected a second family (five members affected) with a large (32 kb) deletion involving the alpha 1, alpha 2, psi alpha 1, psi alpha 2, psi zeta 1, and zeta-globin genes. This mutation has been previously described in Calabria, Italy, in a child and his mother and has been named alpha-thalassemia--)AL.

Characterization of a new hemoglobin variant: Hb Badalona (beta31[B13]Leu-->Val).

Hemoglobin (Hb) Badalona was identified in a 35-year-old Spanish female and two other family members. All affected subjects presented erythrocytosis and increased oxygen affinity (P(50): 21 mmHg). Hemoglobinopathy was not detected with electrophoretic methods. It was, however, separated and quantified by cation exchange and reverse-phase high-performance liquid chromatography. Hb Badalona accounted for 35% of the total Hb. No significant clinical symptoms were found to be related to this hemoglobinopathy. This is the first case of a Leu-->Val replacement at position beta31(B13) reported in the world literature.

The thalassemia syndromes: molecular characterization in the Spanish population.

This work compiles the results of our research on alpha- and beta-thalassemias, and includes a literature review of the molecular genetics of alpha- and beta-thalassemias in Spain. We studied 1,564 subjects with thalassemia (294 with beta-thalassemia and 1,264 with alpha-thalassemia) by molecular biology techniques. In relation to beta-thalassemia, a total of 15 different mutations were characterized in a study of 308 chromosomes belonging to 294 unrelated subjects. Eleven were homozygotes (22 alleles), three compound heterozygotes (6 alleles), and the remaining 280 were heterozygotes (280 alleles). A total of 86.6% of the alleles identified can be grouped into five different mutations [IVS-I-1 (G-->A), IVS-I-6 (T-->C), IVS-I-110 (G-->A), codon 39 (C-->T), codons 8/9 (+G)]. In 14 subjects (4.5%), all heterozygotes, it was not possible to identify the alteration responsible for the beta-thalassemia. For alpha-thalassemia, 911 subjects showed heterozygous alpha(+)-thalassemia (872 with -3.7 kb; 14 with -4.2 kb; two with the deletion of 3.5 kb of DNA, and 23 with nondeletional alpha-thalassemia). Two hundred and thirty-three subjects had homozygous alpha(+)-thalassemia (223 for -alpha(-3.7)/-alpha(-3.7)); one for -alpha(-4.2)/-alpha(-4.2); six for -alpha(-3.7)/-alpha(-4.2); one for -alpha(-3.5)/-alpha(-3.7); one for alphaalpha(Nco)/alphaalpha(Nco); one for alpha(HPh)/alpha(Hph)). One hundred patients presented with heterozygous alpha(0)-thalassemia (18 of whom were progenitors of patients with Hb H disease). The alpha(0) determinant was found in 20 patients with Hb H disease associated with -alpha(-3.7). From the DNA analysis were identified the - -(MED), - -(SEA), - -(SPAN) deletions and the - -(MA) mutations; in three cases, a break that affects the distal portion of the short arm of chromosome 16; one of these was associated with the ATR-16 (alpha-thal with mental retardation) syndrome. Triplication of the alpha genes (alphaalphaalpha(-3.7)/alphaalpha) was found in 25 subjects, 16 of whom were associated with a heterozygous beta-thalassemia. Only one patient was homozygous for the triplication of alpha genes (alphaalphaalpha(-3.7)/alphaalphaalpha(-3.7)) that was associated with a heterozygous beta-thalassemia. In the Mediterranean region preventive programs for thalassemia, based on the detection of heterozygote carriers and genetic advice, are not sufficient to reduce the incidence of newborns with major thalassemia. Prenatal diagnosis of thalassemias has given a new dimension to the prevention of these, but in order to implement this, a knowledge of the mutations and the incidence of these, is essential. This study, therefore, aims to give a general picture of the molecular genetics of thalassemia and its geographical distribution in our area.

[Molecular heterogeneity of beta thalassemia]. / Heterogeneidad molecular de la beta talasemia.

PURPOSE: To check out the incidence of beta-thalassaemia in different regions of Spain and to demonstrate its heterogeneity. PATIENTS AND METHODS: The study was performed on 60 peripheral blood samples from patients diagnosed of beta-thalassaemia by conventional methods, namely, microcytosis, HbA2 over 3.5%, increased HbF. DNA was isolated with phenol-chloroform and amplified by ARMS technique, and in one case its sequence was established according to Sanger's method. RESULTS: The mutations found in the 67 alleles studied showed similar incidence, although NT1 (IVS 1) mutation was increased and NT110 (IVS 1) mutation was decreased when compared with the findings in other Mediterranean populations. CONCLUSION: Due to historical reasons, the racial characteristics of Spanish people are the product of different ethnic groups, so hereditary disorders are here usually heterogeneous. This study appears to confirm such fact with respect to beta-thalassaemia.

Hb Johnstown [beta 109 (G11) Val-->Leu]: second case described and associated for the first time with beta(0)-thalassemia in two Spanish families.

Hb Johnstown, a high oxygen affinity hemoglobin, was identified in four members from two unrelated Spanish families with erythrocytosis and left-shifted hemoglobin-oxygen dissociation curve. This hemoglobin variant, electrophoretically silent, was analyzed by reverse-phase high-performance liquid chromatography, and the mutation was characterized at the DNA level by beta gene sequencing. In one of these families, two members are affected with Hb Johnstown in association with beta(0)-thalassemia. In these cases the erythrocytosis and low values for P(50) due to Hb Johnstown remain in spite of the beta-thalassemia.

Cleavage of the ALL1 gene in acute lymphoid leukemia before treatment disappears in relapse.

BACKGROUND AND OBJECTIVE: ALL1 gene rearrangements are frequently found in secondary acute leukemias (ALs). A site-specific cleavage of the ALL1 gene in a consensus sequence for topoisomerase II recognition has been considered to be the initial step leading to ALL1 rearrangement and subsequent therapy-related AL. The aim of the present study was to evaluate this cleavage in our patients, to analyze whether it is a laboratory-produced artefact and to check whether it persists or causes a real ALL1 gene rearrangement at relapse. DESIGN AND METHODS: We studied ALL1 rearrangement in 74 cases of AL before treatment by Southern blot avoiding room temperature exposure or delay in processing the samples which could produce ALL1 cleavage. DNA was available for two cases with ALL1 cleavage; it was analyzed by three different Southern blots in one and two in the other. One case with ALL1 cleavage was also studied in relapse. RESULTS: The presence of the cleavage of the ALL1 DNA was found in 3 of 74 (4%) patients. Two of these three patients had the ALL1 cleavage in three and two different analyses. One case was positive for ALL1 cleavage at diagnosis, but negative for both ALL1 cleavage and ALL1 rearrangement at relapse. INTERPRETATION AND CONCLUSIONS: The fact that a constant pattern was obtained from the same patients in different DNA preparations, supports the notion that ALL1 cleavage is not a laboratory artefact. The absence of the cleavage in a sample from a relapsed patient suggests that the subclone with the ALL1 cleavage, in this case, did not play a clear role in the pathogenesis of disease recurrence.

High incidence of the CD8/9 (+G) beta 0-thalassemia mutation in Spain.

BACKGROUND AND OBJECTIVE: In Spain, as in other Mediterranean regions the most common beta-thalassemia mutations are due to point mutations in gene regions that are critical for production of mRNA, such as [IVS-I-nt1 (G-->A), IVS-I-nt6 (T-->C), IVS-I-nt110 (G-->A)] which interrupt normal RNA processing or nonsense mutations [CD39 (C-->T)] which interrupt the translation of mRNA. The frameshift mutation CD8/9 (+G) is a very common allele in Asian Indians but is rare in the Mediterranean regions in which isolated alleles with this mutation have been found in Israel, Greece, Portugal and Turkey. DESIGN AND METHODS: We performed a molecular analysis of 175 chromosomes corresponding to 233 beta-thalassemia patients (221 heterozygous, 10 homozygous and 2 compound heterozygous) who belong to 169 Spanish families. The study of beta-thalassemia was made by PCR-ARMS, the alpha genes by Southern blot, the phenotype of Hb Lepore by enzymatic amplification and the presence of -158 gamma G C-->T mutation by PCR and digestion with the restriction enzyme XmnL. RESULTS: Twenty of these 233 patients showed the beta-thalassemia mutation CD8/9 (+G) (17 were heterozygous, 2 homozygous and in one patient the mutation was associated with a structural variant Hb Lepore Boston). INTERPRETATION AND CONCLUSIONS: These data reveal the heterogeneity of beta-thalassemia in Spain and the relatively high frequency (8.6%) of the frameshift mutation CD8/9 (+G). It is surprising that homozygotes for beta zero-thalassemia due to this mutation with very high Hb F values (around 90%) present a phenotype of intermediate thalassemia.

A new polymorphim (G->A) in the psizeta1 globin gene.

BACKGROUND AND OBJECTIVES: a-globin cluster polymorphisms are obtained with specific restriction enzymes (Xba I, Eco RI, Sac I, Apa I, Bgl II, etc) that can also have implications for genetic analysis. DESIGN AND AND METHODS: We studied three unrelated patients; one from Argentina, one from Spain and one from Australia but of Polish origin. Genomic DNA was digested with several different restriction enzymes and probes, amplified and sequenced with an ABI Prism 310 sequencer. RESULTS: In the three patients an abnormal 26 kb band appeared when they were studied with restriction enzyme Bgl II and z probe. A fragment of 944 bp was amplified with primers that cover from -280 to +714 bp of the recognition sequence of Bgl II enzyme (AGATCT) localized 5' from pseudogene z1. After digestion of this PCR product with Bgl II, two fragments of 714 and 280 bp were produced in normal controls, whereas in patient #1 the PCR fragment was undigested and in patients 2 and 3 both undigested and digested fragments were observed. Sequencing of the PCR fragment showed that in all three patients it was the same polymorphism (G->A) at nucleotide 153171 of the 16 p sequence found in the Bgl II recognition site that changed to AAATCT. INTERPRETATION AND CONCLUSIONS: We describe a new polymorphism in the yz1 first exon Bgl II restriction site (G->A). The polymorphism is associated in cis with haplotype -a3.7. The fragment obtained by PCR enabled us to corroborate the presence of the polymorphism quickly without having to use complicated sequencing techniques.