E.M. Kimura

High prevalence of alpha-thalassemia among individuals with microcytosis and hypochromia without anemia

In order to determine the contribution of alpha-thalassemia to microcytosis and hypochromia, 339 adult outpatients seen at Unicamp University Hospital (with the exception of the Clinical Hematology outpatient clinics), who showed normal hemoglobin (Hb) levels and reduced mean corpuscular volume and mean corpuscular hemoglobin, were analyzed. Ninety-eight were Blacks (28.9%) and 241 were Caucasians (71.1%). In all cases, Hb A2 and F levels were either normal or low. The most common deletional and nondeletional forms of alpha-thalassemia [-alpha3.7, -alpha4.2, --MED, -(alpha)20.5, alphaHphIalpha, alphaNcoIalpha, alphaalphaNcoI and alphaTSAUDI] were investigated by PCR and restriction enzyme analyses. A total of 169 individuals (49.9%) presented alpha-thalassemia: 145 (42.8%) were heterozygous for the -alpha3.7 deletion (-alpha3.7/alphaalpha) and 18 (5.3%) homozygous (-alpha3.7/-alpha3.7), 5 (1.5%) were heterozygous for the nondeletional form alphaHphIalpha (alphaHphIalpha/alphaalpha), and 1 (0.3%) was a --MED carrier (--MED/alphaalpha). Among the Blacks, 56 (57.1%) showed the -alpha3.7/alphaalpha genotype, whereas 12 (12.2%) were -alpha3.7/-alpha3.7 and 1 (1.0%) was an alphaHphIalpha carrier; among the Caucasians, 89 (36.9%) were -alpha3.7/alphaalpha, 6 (2.5%) had the -alpha3.7/-alpha3.7 genotype, 4 (1.7%) presented the nondeletional form (alphaHphIalpha/alphaalpha), and 1 (0.4%) was a --MED carrier. These results demonstrate that alpha-thalassemia, mainly through the -alpha3.7 deletion, is an important cause of microcytosis and hypochromia in individuals without anemia. These data are of clinical relevance since these hematological alterations are often interpreted as indicators of iron deficiency.

Thalassemia intermedia as a result of heterozygosis for ß0-thalassemia and aaa anti-3.7/ aa genotype in a Brazilian patient

We report a case in which the interaction of heterozygosis for both the 0-IVS-II-1 (G->A) mutation and the alpha alpha alpha anti-3,7 allele was the probable cause for the clinical occurrence of thalassemia intermedia. The propositus, a 6-year-old Caucasian Brazilian boy of Portuguese descent, showed a moderately severe chronic anemia in spite of having the -thalassemia trait. Investigation of the alpha-globin gene status revealed heterozygosis for alpha-gene triplication (alpha alpha alpha / alpha alpha). The patients father, also presenting mild microcytic and hypochromic anemia, had the same alpha and genotypes as his son, while the mother, not related to the father and hematologically normal, was also a carrier of the alpha alpha alpha anti-3,7 allele. The present case emphasizes the need for considering the possibility of alpha-gene triplication in -thalassemia heterozygotes who display an unexpected severe phenotype. The -thalassemia mutation found here is being described for the first time in Brazil.

Hemoglobin Hammersmith [β42 (CD1) Phe→ Ser] in a Brazilian girl with congenital Heinz body hemolytic anemia

To the Editor: Akiyama et al. reported in Pediatric Blood & Cancer (article online in advance of print) a new case of Hb Hammersmith (b42 Phe! Ser) in a Japanese female [1]. This is the 12th described case. Patients of different ethnical origins presented with severe hemolytic anemia secondary to the high instability of this hemoglobin variant, in which the amino acid change affects the heme contact [2]. Mutation is always spontaneous (de novo), have a dominant pattern of inheritance and affects females. This leads to the suggestion that an intra-uterine selection against males may be occurring [1,3]. In support to this hypothesis, we would like to report here the 13th case of Hb Hammersmith, also detected in a female child. The patient is a 3-year-old Brazilian female whose parents are clinically and hematologically normal. The propositus, of Italian descent and coming from the Southeastern region of the country, came to our hospital with severe Heinz body hemolytic anemia, hepatosplenomegaly, jaundice, pallor, and red blood cell transfusion-dependence. Peripheral blood smear showed polychromasia, basophilic stippling, anisocytosis, poikilocytosis, and cell fragmentation. Hemoglobin electrophoresis and cation-exchange high performance liquid chromatography did not show any abnormalities, with the exception of high levels of Hb F [4]. Unstable hemoglobin was demonstrated by red blood cell incubation with brilliant cresyl blue and methyl violet, as well as by n-butanol, isopropanol, and heat instability tests. Beta-globin gene sequencing [5] revealed the heterozygotic base substitution corresponding to the Hb Hammersmith (TTT!TCT at the 42nd codon) (Fig. 1). No mutation was found in the patient’s parents. Hematological and biochemical data of this Brazilian family are demonstrated in Table I. Biological paternity was confirmed [6].

Hemoglobin H disease resulting from the association of the – α3.7 rightward deletion and the (αα)ΜΜ deletion in a Brazilian patient

Abstract:  A patient with Hb H disease resulting from the association of the – α3.7 rightward deletion with the rare (αα)ΜΜ deletion, which removes the entire α‐major regulatory element (MRE), is reported. This is the first description of an α‐thalassemic mutation resulting from deletion of the locus‐controlling sequences in the South‐American population.

Identification of Hb Zürich [α2β263(E7)His→Arg] by DNA analysis in a brazilian family

Hb Zurich, in which the β63(E7) histidine is replaced by arginine, was the first unstable hemoglobin (Hb) variant to be analyzed structurally (1-3). The structural alteration in Hb Zurich leads to particularly interesting functional and clinical consequences. The substitution of arginine for the distal histidine at helix E7 causes a marked change in the space where ligand binding occurs (4). The positively charged arginine attaches to the propionate of the heme, leaving the heme pocket wide open, and allowing ready access to the heme iron. This explains the propensity for individuals with this variant to have hemolytic episodes following ingestion of sulfa derivatives (1,5).

Characterization of alpha thalassemic genotypes by multiplex ligation-dependent probe amplification in the Brazilian population

Alpha-thalassemia is the most common inherited disorder of hemoglobin synthesis. Genomic deletions involving the alpha-globin gene cluster on chromosome 16p13.3 are the most frequent molecular causes of the disease. Although common deletions can be detected by a single multiplex gap-PCR, the rare and novel deletions depend on more laborious techniques for their identification. The multiplex ligation-dependent probe amplification (MLPA) technique has recently been used for this purpose and was successfully used in the present study to detect the molecular alterations responsible for the alpha-thalassemic phenotypes in 8 unrelated individuals (3 males and 5 females; age, 4 months to 30 years) in whom the molecular basis of the disease could not be determined by conventional methods. A total of 44 probe pairs were used for MLPA, covering approximately 800 kb from the telomere to the MSLN gene in the 16p13.3 region. Eight deletions were detected. Four of these varied in size from 240 to 720 kb and affected a large region including the entire alpha-globin gene cluster and its upstream regulatory element (alpha-MRE), while the other four varied in size from 0.4 to 100 kb and were limited to a region containing this element. This study is the first in Brazil to use the MLPA method to determine the molecular basis of alpha-thalassemia. The variety of rearrangements identified highlights the need to investigate all cases presenting microcytosis and hypochromia, but without iron deficiency or elevated hemoglobin A₂ levels and suggests that these rearrangements may be more frequent in our population than previously estimated.

β0‐Thalassemia resulting from a novel mutation: β66/u→stop codon

To the Editor: We describe here a novel point mutation which affects the 6th codon of the b-globin gene (GAGpTAG), creating a stop codon instead of a glutamic acid codon. This nonsense mutation was found in a 11-year-old White Brazilian girl with Hb C-b thalassemia. The patients mother was heterozygous for Hb C and the father was heterozygous for the b-thalassemia mutation. This is the ®rst nonsense mutation described in codon 6 of the b-globin gene. b-Thalassemia (b-thal) occurs at high frequencies in individuals of Mediterranean, Middle Eastern, East Indian, African and Southeast Asian descent (1). The b-thal syndromes were brought into Brazil mainly by Italian immigration during the end of the 19th century and the beginning of the 20th century (2). They are usually due to point mutations in the b-globin gene, and 160 different mutations have been described (3). Four of them are responsible for the majority of the b-thal cases in the Brazilian population (b 39, b IVS-I-nt 110, b IVS-I-nt 6 and b IVS-I-nt 1) (4). In this paper we describe a novel point mutation found in a 11-year-old White Brazilian girl with haemoglobin C-b-thal (C-b-thal). Haematological data were determined electronically (Cell Dyn 3.500, Abbott). Hb electrophoresis was performed with standard techniques (5). Hb F was determined by alkali denaturation (6). The b-globin gene was ampli®ed using the polymerase chain reaction (PCR) with primers described elsewhere (7), and direct-sequenced using Thermo Sequenase Cycle Sequencing (Amersham). The results are shown in Table 1. Familial analysis revealed that the mother was heterozygous for Hb C and the father, probably an Italian descendent, was heterozygous for b-thal. In this case, the b-thal resulted from a GpT substitution at the ®rst nucleotide position of the 6th codon (Fig. 1), and created a stop codon in replacement of the glutamic acid codon (GAGpTAG). Premature termination of the globin chain synthesis due to interruption of translation can be caused by either nonsense or frameshift mutations. They usually originate an unstable product which is quickly degraded in the cell (8, 9). Only 14 (8.8%) of the 160 b-thal mutations are nonsense mutations. Only 5 molecular alterations in codon 6 are known (Hb S, Hb C, Hb G-Makassar, Hb Machida and a frameshift ±A from GAG) (3). This is the ®rst thalassemic nonsense mutation described in this codon. Fig. 1. Nucleotide sequence of the PCR-ampli®ed b-globin gene of the studied patient: Lane A, normal control: codon 6 ± GAG (Glu); Lane B, patient: codon 6; there are two mutations GpT, TAG (stop codon) and GpA, AAG (Lys).

Structural alterations of the gamma-globin genes in a Brazilian population.

The g-globin genes are duplicated (g and g, depending on whether there is a glycine or alanine residue at position 136 of the g-globin chain) and are located in the b-globin gene cluster on the short arm of chromosome 11 (11p15.5). They are responsible, together with the a genes, for the production of Hb F (a2g2) during the fetal period of human development. Most of the Hb F structural variants are caused by single amino acid replacements in the g-globin molecule, resulting from single base substitutions in the DNA sequences of the correspondent genes (1). Ninety-nine mutations have so far been described, 45 in the g gene and 54 in the g gene, most of them causing abnormal Hb F with no clinical or hematological manifestations in the carriers (2). As the g genes become silent 6 months after birth, mutations in these genes are less investigated than those in the aor b-globin genes. However, studies on Hb F variants have contributed to a better understanding of the relationship between the structure and function of proteins, and besides, mutations in the g-globin genes may also be used as population genetic markers (1,3).

Haptoglobin study in myasthenia gravis

OBJECTIVE A cross-sectional study of haptoglobin (Hp) in myasthenia gravis (MG) was designed, with the objective to identify its values and correlate them with different disease status. METHOD 46 patients were enrolled in the study, all having disease severity established according to the quantitative myasthenia gravis strength scores (QMGSS). Based on the functional scale determined by Myasthenia Gravis Foundation of America (MGFA) recommendations, patients were classified as having: complete stable remission (CSR; n=10); minimal manifestations-0 (MM0; n=6), minimal manifestations-1 (MM1; n=4); pharmacological remission (PR; n=6). Two other groups participated: thymomatous patients (T; n=10) and patients without imunosuppression or thymectomy, until the assessment for Hp (WIT; n=10). Hp dosage was done by immunonephelometry, blindly to clinical data. Students t-test, Anova test and linear regression were employed for statistical analyses. RESULTS Statistically significant differences occurred between CSR+MM0 x WIT groups (86.62 x 157.57, p<0.001) and PR+MM1 x WIT groups (73.93 x 157.57, p<0.001). Linear regression showed correlation between Hp levels and QMGSS (r=0.759, p<0.001). CONCLUSION Our results suggest that Hp may be useful in clinical practice as a disease severity marker in MG.

A novel α0‐thalassemia deletion in a Brazilian child with Hb H disease [‐‐(Braz)]

Sir, Alpha-thalassemias are among the most common genetic diseases in the world and are characterized by impaired synthesis of the a-globin chains of hemoglobin. Deletions involving the a cluster on chromosome 16p13.3 are the main cause of this condition [1], and although the most common deletions can be detected by PCR, rare or new deletions require more elaborate techniques. Multiplex ligation-dependent probe amplification (MLPA) has been used for this purpose [2–4] and enabled a new, extensive a deletion in trans with the a deletion to be detected in a Brazilian patient with Hb H disease (--/-a). A 3-month-old Brazilian girl of mixed ethnic origin (European and African) was referred to us for investigation of microcytic and hypochromic anemia. Her parents and two sisters were also analyzed. Although they did not present with any clinical complaints, they had hematological changes suggestive of thalassemia. Hematological analysis of the proband and her 2-year-old sister revealed Hb H. The hematological and molecular data for the family studied here are shown in Table 1. A Sysmex hematology analyzer (Sysmex XE-2100, Sysmex, Kobe, Japan) was used for cell counts and hematological indices, and electrophoresis in cellulose acetate in neutral and alkaline pHs and cation-exchange high-performance liquid chromatography (HPLC) (VariantTM, Bio-Rad Laboratories, Hercules, CA, USA) were used in the hemoglobin analysis. Genomic DNA from each member of the family was extracted from peripheral blood leukocytes using a commercial kit (QIAamp DNA Blood Mini Kit, Qiagen, Hilden, Germany). Screening for the seven most common a-thalassemic deletions [-a, -a, -(a), --, --, --, --] was performed by multiplex-PCR [5], and the -a deletion was confirmed by a gap-PCR [6]; a