Hemoglobinopathies are hereditary disorders of the hemoglobin molecule with a high prevalence worldwide. Brazil has a prevalence of 0.1 to 0.3% of newborns with sickle cell anemia and 20.0 to 25.0% of heterozygous alpha2 thalassemia among African Brazilians. In the present study, we investigated the presence of variant hemoglobins and alpha2(3.7 Kb) and alpha2(4.2 Kb) thalassemia in newborns from Salvador, Bahia, Brazil. Samples of umbilical cord blood from a total of 590 newborns were analyzed, of which 57 (9.8%) were FAS; 36 (6.5%) FAC; one (0.2%) SF; and five (0.9%) FSC. One hundred fourteen (22.2%) newborns had alpha2(3.7 Kb) thalassemia, of whom 101 (19.7%) were heterozygous and 13 (2.5%) homozygous, showing statistical significance for hematological data between newborns with normal alpha genes and alpha2(3.7 Kb) thalassemia carriers. The alpha2(4.2 Kb) thalassemia was not found. Frequencies found in the present study confirm that hemoglobinopathies are a public health problem in Brazil, emphasizing the need for neonatal screening and genetic counseling programs.
Hemoglobinopathies; Sickle Cell Anemia; Thalassemia; Newborn Infant
Hemoglobinopatias são alterações hereditárias na molécula de hemoglobina com prevalência mundial elevada. O Brasil apresenta prevalência de 0,1 a 0,3% para recém-nascidos com anemia falciforme e freqüência de 20,0 a 25,0% para a ocorrência de heterozigotos da talassemia alfa2 entre indivíduos afro-descendentes. O presente estudo investigou a presença de hemoglobinas variantes e talassemia alfa2(3.7Kb) e alfa2(4.2Kb) em recém-nascidos de Salvador, Bahia, Brasil. Analisamos o sangue do cordão umbilical de 590 recém-nascidos, sendo 57 (9,8%) com padrão FAS; 36 (6,5%) FAC; um (0,2%) SF e cinco (0,9%) FSC. Cento e catorze (22,2%) apresentaram talassemia alfa2(3.7Kb), dos quais 101 (19,7%) foram heterozigotos e 13 (2,5%) homozigotos, mostrando significância estatística para os dados hematológicos entre recém-nascidos com genes alfa normais e portadores de talassemia alfa2(3.7Kb). A talassemia alfa2(4.2Kb) não foi encontrada. As freqüências descritas neste trabalho confirmam que as hemoglobinopatias são um problema de Saúde Pública no Brasil, enfatizando a importância dos programas de triagem neonatal e aconselhamento genético.
Hemoglobinopatias; Anemia Falciforme; Talassemia; Recém-Nascido
Hemoglobinopathies in newborns from Salvador, Bahia, Northeast Brazil
Hemoglobinopatias em recém-nascidos de Salvador, Bahia, Nordeste do Brasil
Elisângela Vitória AdornoI; Fábio David CoutoI; José Pereira de Moura NetoI; Joelma Figueiredo MenezesII; Marco RêgoIII; Mitermayer Galvão dos ReisI; Marilda Souza GonçalvesI, II
ICentro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Brasil
IIFaculdade de Farmácia, Universidade Federal da Bahia, Salvador, Brasil
IIIFaculdade Medicina, Universidade Federal da Bahia, Salvador, Brasil
Hemoglobinopathies are hereditary disorders of the hemoglobin molecule with a high prevalence worldwide. Brazil has a prevalence of 0.1 to 0.3% of newborns with sickle cell anemia and 20.0 to 25.0% of heterozygous a2 thalassemia among African Brazilians. In the present study, we investigated the presence of variant hemoglobins and a23.7 Kb and a24.2 Kb thalassemia in newborns from Salvador, Bahia, Brazil. Samples of umbilical cord blood from a total of 590 newborns were analyzed, of which 57 (9.8%) were FAS; 36 (6.5%) FAC; one (0.2%) SF; and five (0.9%) FSC. One hundred fourteen (22.2%) newborns had a23.7 Kb thalassemia, of whom 101 (19.7%) were heterozygous and 13 (2.5%) homozygous, showing statistical significance for hematological data between newborns with normal a genes and a23.7 Kb thalassemia carriers. The a24.2 Kb thalassemia was not found. Frequencies found in the present study confirm that hemoglobinopathies are a public health problem in Brazil, emphasizing the need for neonatal screening and genetic counseling programs.
Hemoglobinopathies; Sickle Cell Anemia; Thalassemia; Newborn Infant
Hemoglobinopatias são alterações hereditárias na molécula de hemoglobina com prevalência mundial elevada. O Brasil apresenta prevalência de 0,1 a 0,3% para recém-nascidos com anemia falciforme e freqüência de 20,0 a 25,0% para a ocorrência de heterozigotos da talassemia a2 entre indivíduos afro-descendentes. O presente estudo investigou a presença de hemoglobinas variantes e talassemia a23.7Kb e a24.2Kb em recém-nascidos de Salvador, Bahia, Brasil. Analisamos o sangue do cordão umbilical de 590 recém-nascidos, sendo 57 (9,8%) com padrão FAS; 36 (6,5%) FAC; um (0,2%) SF e cinco (0,9%) FSC. Cento e catorze (22,2%) apresentaram talassemia a23.7Kb, dos quais 101 (19,7%) foram heterozigotos e 13 (2,5%) homozigotos, mostrando significância estatística para os dados hematológicos entre recém-nascidos com genes a normais e portadores de talassemia a23.7Kb. A talassemia a24.2Kb não foi encontrada. As freqüências descritas neste trabalho confirmam que as hemoglobinopatias são um problema de Saúde Pública no Brasil, enfatizando a importância dos programas de triagem neonatal e aconselhamento genético.
Hemoglobinopatias; Anemia Falciforme; Talassemia; Recém-Nascido
Hemoglobinopathies are genetic globin gene disorders, characterized by the presence of variant hemoglobin and a decrease or absence of globin chain synthesis, known as thalassemia 1,2.
Hemoglobin S is the most common variant hemoglobin, and results from a single amino acid substitution of valine for glutamic acid at the sixth position of the b-globin chain; sickle cell anemia carriers are characterized by homozygosity of S hemoglobin. The hemoglobin S gene has a high frequency among Africans and African descendents, as well as in India, Greece, and the United States 3. Primary studies in Brazil revealed a high prevalence of hemoglobin disorders. The sickle cell trait (AS) was reported in 6.6% of blacks in the State of São Paulo, in Southeast Brazil 4. When the study was extended to the general population, frequencies of 2.7% of AS and 0.09% of sickle cell disease (0.07 % HbSS and 0.02% HbSC) were observed. These frequencies varied widely according to the degree of racial admixture in the different regions of the country. In the South of Brazil, a frequency of 1.2% of HbS gene was shown among newborns 5. On the other hand, in the Northeast, frequencies of 5.1% of sickle cell trait (FAS) and 0.2% with sickle cell disease (FSC) were reported among newborns in the State of Pernambuco 6 and the State of Bahia, the frequency of AS genotype varies from 7.4% to 15.7%, according to the population group studied 7.
Hemoglobin C (HbC) is a variant hemoglobin in which lysine replaces glutamic acid at the sixth amino acid position of the b-globin chain 2. HbC has a prevalence of 3% among African-Americans and about 1-3% among Puerto Ricans 8,9. In Brazil, HbC is the second most common variant hemoglobin and has been found around 2.2% to 5.2% when the heterozygous genotype (AC) has been considered in Bahia 7.
Thalassemia syndromes are found worldwide, especially a- and b-thalassemia 10,11. In Southeast Brazil, a frequency of 1.3% of b-thalassemia trait and 0.1% of b-thalassemia major was reported for the general population 12, while a2-thalassemia by a 3.7 kb DNA deletion (a23.7Kb-thalessemia) varied from 20.0% to 25.0% in black populations 13, and Borges et al. 14 found 49.9% of a-thalassemia in adult outpatients seen at the University of Campinas Hospital with microcytosis and hypochromia without anemia. In the Northeast, a23.7Kb-thalessemia was investigated in 106 pregnant women with AC and AA hemoglobin pattern, showing a 21.7% heterozygous and 0.9% homozygous rate for this alteration 15.
The Consensus Conference Panel convened by the National Institutes of Health (United States) in 1987 recommended newborn screening for hemoglobinopathies in order to decrease morbidity and mortality associated with sickle cell disease. Streptococcus pneumoniae is a common cause of death in sickle cell patients and the early diagnosis of sickle cell disease could alert clinicians to potential clinical complications of the disease and allow prompt clinical care and prophylactic therapy with vaccines and antibiotics 16,17.
In Brazil, not all States have consolidated neonatal screening programs for hemoglobinopathies, despite a ruling mandating them by the Ministry of Health in June 2001 (Portaria GM/MS 822 Diário Oficial da União 2001; 7 jun). The sickle cell disease screening program in Minas Gerais State was considered a model for South America by Serjeant 18. This program found 3.2% of screened babies with sickle cell trait, 1.3% heterozygous for HbC, and 0.08% with sickle cell disease. Other States have developed excellent newborn screening programs, such as Bahia, Pernambuco, Maranhão, the Federal District (Brasília), São Paulo, Rio de Janeiro, Espírito Santo, Santa Catarina, Paraná, Mato Grosso do Sul, and Rio Grande do Sul (Technical Advisory Group on Neonatal Screening, Ministry of Health, Brazil). In California (United States), a newborn hemoglobinopathy screening program evaluated two million newborns and found 492 with sickle cell disease, while 290 had hemoglobinopathy SS, 143 SC, and 47 Sb+ thalassemia 19. The screening programs' implementation is an important step for increasing survival, reducing hospitalization, and minimizing expenses associated with sickle cell disease 20,21. Based on the high frequency of hemoglobinopathies in Salvador, Bahia, Brazil, we investigated the presence of variant hemoglobin and a23.7 Kb and a24.2 Kb thalassemias among newborns babies in order to identify potential associations between hemoglobin abnormalities and hematological characteristics.
Material and methods
From February to June 2000, a cross-sectional epidemiological study analyzed 590 neonates delivered by vaginal birth at the Tsylla Balbino Maternity Clinic located in Salvador, Bahia, Brazil. Information about newborns and gestational age was obtained from mothers by a questionnaire and patient records.
Newborns with gestational age less than thirty-eight weeks were considered premature 22. Racial composition was determined by observation of facial characteristics and color of mammillae and scrotum 23.
Umbilical cord blood samples were collected by midwives under supervision of maternity clinic physician staff. After clamping, blood was drawn into a Vacutainer tube (Becton-Dickinson Corkeysville, Maryland, United States) containing EDTA and transported to the Pathology and Molecular Biology Laboratory, Gonçalo Moniz Research Center, Oswaldo Cruz Foundation (FIOCRUZ). Hematological analysis was performed by automated cell counter (Coulter T-890 Coulter Corporation, Florida, United States) and DNA was isolated from leukocytes (GFXTM Genomic Blood DNA Purification KIT Amersham Pharmacia Biotech, United States).
Hemoglobin profile was analyzed by High Performance Liquid Chromatography HPLC Variant Hemoglobin Testing System (Bio-Rad Laboratories California, United States). HPLC analyses were interpreted by comparing peak retention times with those obtained for the AFCS control, utilizing the sickle cell kit for hemoglobin screening in newborns.
The -a23.7Kb and -a24.2 deletions were identified by polymerase chain reaction (PCR), using specific primers and reaction conditions as previously described 24.
The statistical analyses were conducted in the Epi Info software, version 6.04. Statistical significance was established at P > 0.05.
The study was approved by the Institutional Review Board/Ethics Committee of the Gonçalo Moniz Research Center, FIOCRUZ, after the newborn's parent or guardian had signed the informed consent form.
The Tsylla Balbino Maternity Clinic is the largest public maternity ward in Salvador. During the study period, 2,958 children were born and 590 newborns (19.9% of total birth cohort) were analyzed, after statistical sample calculation. Of those analyzed, 63 (10.8%) had premature delivery and 523 (89.2%) had normal gestational age; 296 (51.3%) were female and 281 (48.7%) male. Racial composition was determined for 578 babies: 98 (17.0%) were classified as white, 309 (53.4%) as mulatto, and 171 (29.6%) as black. Mean weight was 3.152 kg (± 0.530).
Hematological data and hemoglobin profiles
Hemoglobin profiles were determined in 581 of the 590 newborns: 480 (82.6%) had the normal profile FA, and 101 (17.4%) presented variant hemoglobins, of which 57 (9.8%) were heterozygous for HbS (FAS), and 38 (6.5%) were heterozygous for HbC (FAC). One (0.2%) baby was homozygous for HbS (FS) and five (0.9%) were double heterozygous for HbS and HbC (FSC).
There was no statistical difference in the gestational ages of newborns with respect to normal and variant hemoglobin patterns. The racial distribution and hemoglobin types of 502 newborns are shown in Table 1. Of the 47 newborns found to have the FAS pattern, 24 (51.1%) were mulatto, 15 (31.9%) were black, and eight (17.0%) were white.
Newborns with normal and variant hemoglobin had statistically similar hematological characteristics, except for the FA and FAC groups, which were statistically different for PVC, RBC, and MCHC values, with p-values of 0.031, 0.003, and 0.0009, respectively (Table 2).
a2 thalassemia analyses
Of the 590 newborns, 514 were analyzed for a2 thalassemia: 400 (77.8%) had normal a-genes (aa/aa) and 101 (19.7%) were heterozygous (a/aa) and 13 (2.5%) homozygous (-a/a-) for the a23.7Kb deletion. There was no statistical difference between presence of a23.7Kb thalassemia and premature delivery.
Among the 114 newborns identified as a23.7Kb thalassemia carriers, 21 (18.4%) were whites, 57 (50.0%) were mulattos, and 36 (31.6%) blacks. Table 3 shows the statistical analysis of hematological parameters of newborns with normal a-genes and a23.7Kb thalassemia carriers.
Analysis of hemoglobin profiles and presence of a23.7Kb thalassemia was performed on 451 of 590 newborns. Of the 377 newborns with FA pattern, 295 (78.2%) had normal a-genes, 72 (19.1%) were heterozygous, and ten (2.7%) homozygous.
Of the 43 newborns with FAS profile, 32 (74.4%) had normal a-genes, nine (20.9%) were heterozygous, and two (4.7%) were homozygous; of the 27 newborns with pattern FAC, 22 (81.5%) had normal a-genes and five (18.5%) were heterozygous; among the four newborns with FSC pattern, three (75.0%) had normal a-genes and one (25.0%) was heterozygous for a23.7Kb thalassemia. The newborn with FS pattern had normal a-genes.
Table 4 shows the hematological data from newborns with normal and variant hemoglobin genotypes, as well as for a23.7Kb thalassemia carriers.
There was no statistical difference in the hematological characteristics of newborns with FA and FSC hemoglobin profile and thalassemia a23.7Kb carriers.
The frequencies of variant hemoglobin found in this study (Hb FAS 9.8%; Hb FAC 6.5%; FS 0.2%, and FSC 0.9%) are the highest described in Brazil. The high frequencies of variant hemoglobin are probably due to the high rate of racial admixture in the Bahian population, with a strong African gene component introduced by the African slave trade in Brazil. Furthermore, the Tsylla Balbino Maternity ward serves the majority of low-income women in Salvador, who are almost exclusively blacks or mulattos 23. Among the newborns analyzed, only six (5 FSC and 1 FS) required special care, representing the symptomatic group that can develop severe clinical conditions and need early treatment. The 47 newborns identified as FAS displayed a racial distribution of 17.0% whites, 53.4% mulattos and 29.6% blacks. These variations of racial distribution of hemoglobin S among the newborns groups highlight the need for universal neonatal hemoglobinopathy screening in the Brazilian population. The comparison of hematological data among newborns with HbC and those with normal hemoglobin showed statistical significance for PVC, RBC, and MCHC due to increased blood viscosity, demonstrating the influence of HbC on hematological parameters 8,9.
The high frequencies of a23.7Kb thalassemia (heterozygous 19.7%; homozygous 2.5%) are similar to those found in previous studies conducted in Brazilian populations 11. The presence of a23.7Kb thalassemia was not associated with premature delivery. However, van der Dijs et al. 25 speculated that the presence of a2 thalassemia could decrease the capacity to extract oxygen from maternal circulation and could thus be a contributing factor to early delivery of newborn a2 thalassemia carriers. Newborns with FAS and FAC pattern, such as the babies heterozygous for a23.7Kb thalassemia, did not present symptoms of severe anemia. However, they still need genetic counseling 26.
We did not identify any newborns with a24.2Kb thalassemia in our study, suggesting that this type of thalassemia is rare or absent in Bahia. The frequency of a23.7Kb thalassemia among FAS newborns (20.9% of which were heterozygous and 4.7% of which were homozygous) could be beneficial for these S hemoglobin carriers and for the sickle cell anemia patients in Bahia. The a2 thalassemia is considered a beneficial factor, since this condition is associated with less severe clinical manifestations among sickle cell disease carriers 8,9. It decreases MCHC and the rate of hemolysis, resulting in higher Hb, PCV, and RBC values. Therefore, some complications such as leg ulcers, renal pathology, and strokes become less frequent. However, the frequency of other complications such as osteonecrosis and retinopathy may be increased 27.
The comparison of hematological data among newborns with a-normal genes and those with a23.7Kb thalassemia showed statistical differences in all hematological parameters analyzed, except for MCHC. The hematological analysis of newborns with FA and FAS hemoglobin groups and a23.7Kb thalassemia carriers showed statistically significant differences in RBC and MCH; the comparison of the FA and FAC groups and a23.7Kb thalassemia carriers showed significant differences in MCH and MCHC. The hematological differences found between newborns with normal a-genes and a23.7Kb thalassemia carriers could be intensified by the presence of variant hemoglobins. Furthermore, differentiation within the Bahian pediatric population needs to be developed in order to determine whether the presence of a23.7Kb thalassemia could be responsible for intensifying the hematological pattern of anemia, especially in child with iron deficiency, avoiding erroneous therapy.
In Bahia, the presence of hemoglobinopathies is a public health problem, and the early diagnosis of variant hemoglobin carriers provides an opportunity for counseling and early clinical follow-up of the child, both of which contribute to reduce child morbidity and mortality rates.
E. V. Adorno participated in the blood sample collection in the hospital, hemoglobin analysis, DNA extraction, molecular analysis, and drafting of the manuscript. F. D. Couto, J. F. Menezes and J. P. Moura Neto participated in the blood sample collection in the hospital, hemoglobin analysis, and DNA extraction. M. Rego participated in the statistical analysis. M. G. Reis participated in the interpretation of the results and drafting and revision of the manuscript. M. S. Gonçalves supervised the laboratory analysis, interpretation of the results, and drafting and revision of the manuscript.
We wish to thank all the parents for allowing their newborns to participate in the study, Dr. Edeltrudes do Espírito do Santo (head of the Tsylla Balbino Maternity Clinic), the clinical staff who helped with sample collection, and Craig Milroy and Fabíola Nascimento da Conceição, who reviewed the English version of this article.
Submitted on 28/May/2003
Final version resubmitted on 18/Feb/2004
Approved on 01/Mar/2004
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