Elza Miyuki Kimura

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.

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.

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.

Determination of beta S haplotypes in patients with sickle-cell anemia in the state of Rio Grande do Norte, Brazil

βS haplotypes were studied in 47 non-related patients with sickle-cell anemia from the state of Rio Grande do Norte, Brazil. Molecular analysis was conducted by PCR/RFLP using restriction endonucleases XmnI, HindIII, HincII and HinfI to analyze six polymorphic sites from the beta cluster. Twenty-seven patients (57.5%) were identified with genotype CAR/CAR, 9 (19.1%) CAR/BEN, 6 (12.8%) CAR/CAM, 1 (2.1%) BEN/BEN, 2 (4.3%) CAR/Atp, 1 (2.1%) BEN/Atp and 1 (2.1%) with genotype Atp/Atp. The greater frequency of Cameroon haplotypes compared to other Brazilian states suggests the existence of a peculiarity of African origin in the state of Rio Grande do Norte.

-thalassemia, HbS, and -globin gene cluster haplotypes in two Afro-Uruguayan sub-populations from northern and southern Uruguay

Hemoglobinopathies are the most common monogenic disorders worldwide; however, they have never been systematically studied from a genetic perspective in Uruguay. In this study, we determined the frequencies of hemoglobin variants in Afro-Uruguayans. A sample of 52 healthy unrelated Afro-Uruguayans from the northern (N = 28) and southern (N = 24) regions of the country was analyzed. Eight individuals (15.4%) were heterozygous for 3,7 thalassemia; seven of them (29.2%) were originally from the southern region, whereas one of them (3.6%) was from the northern region; the differences between both regions were statistically significant (p = 0.016 +/-0.003). The only structural mutation detected was S , which is typical of African populations. Four individuals (10%) were heterozy

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.

Beta‐globin gene cluster haplotypes in Afro‐Uruguayans from two geographical regions (South and North)

The β‐globin gene cluster haplotypes were identified in 52 and 40 chromosomes from two Afro‐Uruguayan populations located in the South and North of the country, respectively. In both regions, the 5′ haplotype 2 (+ − − − −), characteristic of non‐African populations, was the most frequent, reflecting a strong process of admixture in Afro‐Uruguayans (0.355 and 0.262, respectively). The haplotypes 3 (− − − − +) and 4 (− + − − +), characteristics of African sub‐Saharan populations, present inverse frequencies in North and South: whereas in the South haplotype 3 is the second most frequent (0.232), and haplotype 4 presents a low frequency (0.019), in the North haplotype 4 is the third most frequent (0.140), and haplotype 3 only reaches an intermediate frequency (0.088). The pairwise FST and the exact test of differentiation show genetic heterogeneity between both regions. Neis genetic distance show that South and North present affinities with Bantu groups, although the North present the smallest genetic distance with the Mandenka, a Senegalese population. With respect to 3′ haplotypes, haplotype I was the most frequent in both populations, followed by haplotype II, characteristic of sub‐Saharan Africans. The high frequencies of haplotype III‐Asian could indicate admixture with Native American populations. The differences observed between both Uruguayan regions could be explained by microevolutionary events as genetic drift, founder effects, differential admixture, and/or distinct origin of the African slaves introduced in those regions. Am. J. Hum. Biol. 2010.

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.