Denise M. Oliveira

The deletion of SOX8 is not associated with ATR-16 in an HbH family from Brazil

The most common cause of a-thalassaemia are deletions in the a-globin gene cluster, taking away one or more a-globin genes. However, an increasing number of rare deletions have been described that cause monosomy of the telomeric region of chromosome 16 in individuals with a-thalassaemia only, or a-thalassaemia and a variety of developmental abnormalities and mild mental retardation, known as the ATR-16 syndrome (Daniels et al, 2001) (Horsley et al, 2001). ATR-16 is defined as a contiguous gene syndrome resulting from haploinsufficiency of the a-globin gene cluster and genes involved in mental retardation (MR). To date, only few cases have been described which result from pure monosomy for a deletion of 16p. The region on chromosome 16p for which haploinsufficiency leads to the dysmorphic features and MR typical for ATR-16, has been narrowed down to a region 0Æ9–1Æ5 Mb from the telomere (Harteveld et al, 2007), encompassing SOX8 at 0Æ97 Mb, which is generally assumed to be a candidate gene for ATR-16 (Meyer et al, 1997; Pfeifer et al, 2000; Takada & Koopman, 2003). Here we describe a family of Brazilian origin investigated by multiplex ligation dependent probe amplification (MLPA) because of a suspected large deletion based on the presence of HbH and Inclusion Bodies while only the common )a (Rightward) deletion was observed. The family consisted of three generations. The father (FI.1) was a carrier of the )a deletion and the mother (FI.2) presented with microcytic hypochromic anaemia, showing none of the seven most common a-thalassaemia deletions detected by gap-polymerase chain reaction (PCR) (Tan et al, 2001). They had six children, three were heterozygous for the )a allele (FII.3, 5, 6), one (FII.2) presented with the same microcytic hypochromic anaemia observed in the mother without any abnormalities on gap-PCR, while the two index patients (FII.1 and FII.4) with HbH disease had only the )a deletion detected by gap-PCR (Tan et al, 2001). Finally the son of propositus FII.1 (FIII.1 in Table I) had inherited the thalassaemia trait from the paternal grandmother. The MLPA analysis revealed an extensive deletion, 0Æ97– 1Æ05 Mb in size, removing the most telomeric MLPA-probe, located between position 45 799–45 856 [UCSC Genome Browser (May 2004) chromosome 16p13Æ3] up to probe 42 (at position 976 591–976 640) (Fig 1). The deletion breakpoint is located between position 976 640 and 1 053 683. The MLPA-probes 41 and 42 at the 3¢ end of the breakpoint, located in exon1 and exon3 of SOX8 respectively, were both deleted, indicating the complete absence of expression of this three-exon gene on the deletion allele. The carriers in this family had the largest a-thalassaemia deletion, without having the features of ATR-16. The proband (FII.1) is working as a security agent in a private company; his sister (FII.4), with

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].

Three New α-Globin Variants: Hb Itapira [α30(B11)Glu→Val (α1)], Hb Bom Jesus Da Lapa [α30(B11)Glu→Ala (α1)] and Hb Boa Esperança [α16(A14)Lys→Thr (α2)]

Three novel α-globin variants were found during a screening program for hemoglobinopathies in blood donors at the UNICAMP Hematology and Hemotherapy Center, Campinas, State of São Paulo, Southeastern Brazil. They were named for the town of origin of the carrier as Hb Itapira [α30(B11)Glu→Val], Hb Bom Jesus da Lapa [α30(B11)Glu→Ala] and Hb Boa Esperança [α16(A14)Lys→Thr]. Hb Itapira, like Hb Bom Jesus da Lapa, shows an electrophoretic mobility similar to that of Hb S [β6(A3)Glu→Val, GAG→GTG] at alkaline pH; it is associated with a triplicate α-globin allele (αααanti 3.7) and corresponds to only 5.5% of the total hemoglobin (Hb). Hb Boa Esperança, found in two different individuals, moves faster than Hb A and exhibits an abnormal functional performance.

Prevalence of α-thalassemia 3.7 kb deletion in the adult population of Rio Grande do Norte, Brazil

α-Thalassemia, arising from a defect in α-globin chain synthesis, is often caused by deletions involving one or both of the α-genes on the same allele. With the aim of investigating the prevalence of α-thalassemia 3.7 kb deletion in the adult population of Rio Grande do Norte, 713 unrelated individuals, between 18 and 59 years-of-age, were analyzed. Red blood cell indices were electronically determined, and A2 and F hemoglobins evaluated by HPLC. PCR was applied to the molecular investigation of α-thalassemia 3.7 kb deletion. Eighty (11.2%) of the 713 individuals investigated presented α-thalassemia, of which 79 (11.1%) were heterozygous (-α3.7/αα) deletions and 1 (0.1%) homozygous (-α3.7/-α3.7). Ethnically, heterozygous deletions were higher (24.8%) in Afro-Brazilians. Comparison of hematological parameters between individuals with normal genotype and those with heterozygous α+-thalassemia showed a statistically significant difference in the number of erythrocytes (p < 0.001), MCV (p < 0.001), MCH (p < 0.001) and Hb A2 (p = 0.007). This study is one of the first dedicated to investigating α-thalassemia 3.7 kb deletion in the population of the State Rio Grande do Norte state. Results obtained demonstrate the importance of investigating this condition in order to elucidate the causes of microcytosis and hypochromia.

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.

Hb H disease resulting from the association of an αº-thalassemia allele [-(α)20.5] with an unstable α-globin variant [Hb Icaria]: first report on the occurrence in Brazil

Hb H Disease is caused by the loss or inactivation of three of the four functional α-globin genes. Patients present chronic hemolytic anemia and splenomegaly. In some cases, occasional blood transfusions are required. Deletions are the main cause of this type of thalassemia ( α-thalassemia). We describe here an unusual case of Hb H disease caused by the combination of a common α0 deletion [-( α) 20.5 ] with a rare point mutation (c.427T > A), thus resulting in an elongated and unstable α-globin variant, Hb Icaria, (X142K), with 31 additional amino-acid residues. Very high levels of Hb H and Hb Barts were detected in the patients red blood cells (14.7 and 19.0%, respectively). This is the first description of this infrequent association in the Brazilian population.

Hb S-São Paulo: A new sickling hemoglobin with stable polymers and decreased oxygen affinity

Hb S-São Paulo (SP) [HBB:c.20A>T p.Glu6Val; c.196A>G p.Lys65Glu] is a new double-mutant hemoglobin that was found in heterozygosis in an 18-month-old Brazilian male with moderate anemia. It behaves like Hb S in acid electrophoresis, isoelectric focusing and solubility testing but shows different behavior in alkaline electrophoresis, cation-exchange HPLC and RP-HPLC. The variant is slightly unstable, showed reduced oxygen affinity and also appeared to form polymers more stable than the Hb S. Molecular dynamics simulation suggests that the polymerization is favored by interfacial electrostatic interactions. This provides a plausible explanation for some of the reported experimental observations.

Haptoglobin Genotypes in Sickle-Cell Disease

We compared the frequencies of the haptoglobin (Hp) genotypes of 775 Brazilian patients with sickle-cell disease divided into the following age groups: 3 months-5 years, 6-10 years, 11-15 years, 16-20 years, and over 20 years. The last group (>20 years) was also compared with a healthy control group and was further divided into subgroups including only subjects aged 21-30 years (V.a and Control.a) and over 30 years (V.b and Control.b). There was no significant difference in the frequencies of the Hp genotypes between the different patient groups or between the patients and controls. However, the Hp2-2 genotype was always less frequent than the Hp1-1 genotype in the patient groups, whereas the opposite was observed in healthy controls. The frequency of Hp2-2 was 25.0% in patients in the 21-30 years age group and fell to 19.5% in those over 30 years. In the controls, the corresponding frequency was around 28%. Although our results do not allow us to conclude that Hp genotypes on their own confer greater or lesser selective advantage on sickle-cell disease patients in the population studied, this polymorphism may, when combined with other genetic and environmental factors, contribute to the clinical diversity observed in this disease.

Identificação e caracterização de variantes novas e raras da hemoglobina humana

Hemoglobin structural abnormalities are among the most commonly found human genetic diseases. The Laboratory of Hemoglobinopathies in the Clinical Pathology Department of the Medical Sciences School of the State University in Campinas - Unicamp, Sao Paulo, Southeastern Brazil, carried out, in its 27 years of activity, about 130,000 diagnoses. As expected, hemoglobins S, C and D were the most frequently observed variants, but an expressive number of other abnormal, novel and rare hemoglobins, was also detected. These findings are summarized in the present article.

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.