Maria de Fátima Sonati

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription–PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.

Sickle Cell Disease in a Brazilian Population from Sao Paulo: A Study of the βs Haplotypes

In this study we have determined the frequency of βs haplotypes in a Brazilian sickle cell disease population from Sao Paulo, Brazil, by analyzing sequence variations in the immediate 5’ fl

Polymorphism of human haptoglobin and its clinical importance

Haptoglobin (Hp) is a plasma glycoprotein, the main biological function of which is to bind free hemoglobin (Hb) and prevent the loss of iron and subsequent kidney damage following intravascular hemolysis. Haptoglobin is also a positive acute-phase protein with immunomodulatory properties. In humans, the HP locus is polymorphic, with two codominant alleles (HP1 and HP2) that yield three distinct genotypes/phenotypes (Hp1-1, Hp2-1 and Hp2-2). The corresponding proteins have structural and functional differences that may influence the susceptibility and/or outcome in several diseases. This article summarizes the available data on the structure and functions of Hp and the possible effects of Hp polymorphism in a number of important human disorders.

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.

alpha-Globin genes: thalassemic and structural alterations in a Brazilian population

Seven unrelated patients with hemoglobin (Hb) H disease and 27 individuals with alpha-chain structural alterations were studied to identify the alpha-globin gene mutations present in the population of Southeast Brazil. The -alpha3.7, --MED and -(alpha)20.5 deletions were investigated by PCR, whereas non-deletional alpha-thalassemia (alphaHphalpha, alphaNcoIalpha, alphaalphaNcoI, alphaIcalpha and alphaTSaudialpha) was screened with restriction enzymes and by nested PCR. Structural alterations were identified by direct DNA sequencing. Of the seven patients with Hb H disease, all of Italian descent, two had the -(alpha)20.5/-alpha3.7 genotype, one had the --MED/-alpha3.7 genotype, one had the --MED/alphaHphalpha genotype and three showed interaction of the -alpha3.7 deletion with an unusual, unidentified form of non-deletional alpha-thalassemia [-alpha3.7/(alphaalpha)T]. Among the 27 patients with structural alterations, 15 (of Italian descent) had Hb Hasharon (alpha47Asp-->His) associated with the -alpha3.7 deletion, 4 (of Italian descent) were heterozygous for Hb J-Rovigo (alpha53Ala-->Asp), 4 (3 Blacks and 1 Caucasian) were heterozygous for Hb Stanleyville-II (alpha78Asn-->Lys) associated with the alpha+-thalassemia, 1 (Black) was heterozygous for Hb G-Pest (alpha74Asp-->Asn), 1 (Caucasian) was heterozygous for Hb Kurosaki (alpha7Lys-->Glu), 1 (Caucasian) was heterozygous for Hb Westmead (alpha122His-->Gln), and 1 (Caucasian) was the carrier of a novel silent variant (Hb Campinas, alpha26Ala-->Val). Most of the mutations found reflected the Mediterranean and African origins of the population. Hbs G-Pest and Kurosaki, very rare, and Hb Westmead, common in southern China, were initially described in individuals of ethnic origin differing from those of the carriers reported in the present study and are the first cases to be reported in the Brazilian population.

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.

The genetics of blood disorders: hereditary hemoglobinopathies.

OBJECTIVE To summarize recently published data on the pathophysiology, diagnosis and treatment of sickle cell diseases and beta-thalassemias, the most relevant hereditary hemoglobinopathies in the global population. SOURCES Searches were run on the MEDLINE and SCIELO databases, limited to the period from 2003 to May 2008, using the terms hereditary hemoglobinopathies, sickle cell diseases and beta-thalassemia. Two books and two chapters were also included. SUMMARY OF THE FINDINGS More than 2,000 articles were identified; those providing the most important information and broadest views were selected. CONCLUSIONS Morbidity and mortality rates from sickle cell diseases and beta-thalassemia are still very high and represent an important challenge. Increased understanding of pathophysiological aspects has lead to significant improvements in treatment and prevention of these diseases.

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

Characterization of beta-thalassemia mutations in patients from the state of Rio Grande do Norte, Brazil

35 unrelated individuals were studied for characterization as either heterozygous or homozygous for beta-thalassemia. Molecular analysis was done by PCR/RFLP to detect the mutations most commonly associated with beta-thalassemia (β0IVS-I-1, β+IVS-I-6, and β039). In the patients who showed none of these mutations, the beta-globin genes were sequenced. Of the 31 heterozygous patients, 13 (41.9%) had the β+IVS-I-6 mutation, 15 (48.4%) the β0IVS-I-1 mutation, 2 (6.5%) the β+IVS-I-110 mutation and 1 (3.2%) the β+IVS-I-5 mutation. IVS-I-6 was detected in the four homozygotes. The mutation in codon 39, often found in previous studies in Brazil, was not detected in the present case. This is the first study aiming at identifying mutations that determine beta-thalassemia in the state of Rio Grande do Norte.

Haptoglobin polymorphism and diabetic nephropathy in Brazilian diabetic patients

Diabetic nephropathy (DN) has an important impact on morbidity/mortality in diabetic patients. Genetic factors are probably involved in the development of this microvascular complication. Haptoglobin (Hp) is a genetically polymorphic glycoprotein that forms stable complexes with plasma-free hemoglobin (Hb) providing protection against heme-induced oxidative stress and kidney damage. The aim of the present study was to investigate the existence of association between the Hp genotypes and the presence of DN in Brazilian diabetic patients. The Hp genotypes of 265 patients, 95 type 1 diabetes mellitus (DM1) sufferers with at least 10 years of disease and 170 type 2 diabetes mellitus (DM2) sufferers with at least 5 years of disease were determined by allele-specific PCR; both groups included patients with and without DN. Hp allele and genotype frequencies were compared among the patient groups and between the patient groups and a control group of 142 healthy individuals. No association between Hp genotypes and DN could be demonstrated. Additionally, urinary albumin excretion values and the presence or absence of systemic arterial hypertension (SAH) were compared among the patient groups. Again, no significant correlations were found. The Hp polymorphism could not be associated with DN in the population studied here.