Prevalence of G6PD deficiency and molecular characterization of G202A, A376G and C563T polymorphisms in newborns in Southeastern Brazil / Prevalência da deficiência de G6PD e caracterização molecular dos polimorfismos G202A, A376G e C563T em neonatos no Sudeste do Brasil

ABSTRACT Objective To evaluate the prevalence of G6PD deficiency and characterize G202A, A376G and C563T polymorphisms in neonates using molecular assays. Methods A total of one thousand samples were tested through quantitative analysis of enzyme activity, detecting 25 G6PD-deficient individuals. Patients identified as deficient were submitted to molecular analysis quantitative real-time polymerase chain reaction - (qPCR) to investigate the presence of variants associated with the deficiency. Results The total prevalence of G6PD deficient was 2.5%. Of the 25 samples identified as deficient, 21 were submitted to qPCR assay to analyze the presence of G202A, A376G and C563T variants. All samples showed the G202A/A376G genotype, characterizing G6PD A- phenotype. Conclusion The prevalence of G6PD deficiency in the present study was similar to that observed in other study populations in Brazil. Molecular analysis identified in all patients the presence of the genetic polymorphism G202A/A376G, more common in the Brazilian population with G6PD deficiency, which is directly estimated by enzyme activity level.

RESUMO Objetivo Avaliar a prevalência da deficiência de G6PD e caracterizar, por ensaios moleculares, os polimorfismos G202A, A376G e C563T em recém-nascidos. Métodos Foram testadas mil amostras por meio da análise quantitativa da atividade enzimática, detectando 25 portadores de deficiência de G6PD. Os pacientes identificados como deficientes foram submetidos à análise molecular reação em cadeia da polimerase em tempo real (qPCR) para pesquisa da presença das variantes associadas à deficiência. Resultados A prevalência total de deficientes de G6PD foi de 2,5%. Das 25 amostras identificadas como deficientes, 21 foram submetidas ao qPCR, para análise da presença das variantes G202A, A376G e C563T. Todas as amostras apresentaram o genótipo G202A/A376G, caracterizando fenótipo G6PD A-. Conclusão A prevalência da deficiência da G6PD no presente estudo foi semelhante à verificada em outras populações de estudo no Brasil. A análise molecular identificou em todos os pacientes a presença do polimorfismo genético G202A/A376G, mais comum na população brasileira portadora da deficiência de G6PD, que é diretamente estimada pelo nível de atividade enzimática.

Polimorfismo genético da glicose-6-fosfato desidrogenase na população da região de Araraquara, Estado de São Paulo / Genetic polymorphism of glucose-6-phosphate dehydrogenase in the population of Araraquara, State of São Paulo

The most important role played by the enzyme Glucose-6-Phosphate Dehydrogenase (G6PD) in erythrocyte metabolism is in generating energy and reducing power used to protect the cell against oxidative attack. G6PD deficiency is the erythroenzymopathy that most frequently causes hemolytic anemia, and more than 130 molecular variants have already been identified. The aim of this study was to analyze the genetic mutations in the G6PD-deficient adult males in the population of the region of Araraquara, São Paulo State. Out of 5087 male blood donors, 89 were deficient for G6PD, as confirmed by assaying the enzyme activity and electrophoresis on cellulose acetate. Thus, a frequency of 1.75% of G6PD-deficient patients was found, this value being similar to other investigations in São Paulo state. Molecular analysis was performed by amplification of genomic DNA with specific primers and digestion with restriction enzymes. In 96.6% of the patients, the G6PD A¯ variant was observed, with mutations at residues 376(A-G) and 202(G-A). Mean G6PD specific activity among the patients was 1.31 IU.g Hb-1.min-1 at 37ºC, that is 10.8% of the normal activity of the G6PD B enzyme. The variant forms G6PD A¯680(G-T) and 968(T-C) were not found. In 3.4% of the deficient individuals, the G6PD Mediterranean variant was found, with a mutation at 563(C-T). In these cases,mean enzymatic activity was 0.25 IU.g Hb-1.min-1 at 37ºC, or 2.1% of the enzymatic activity of G6PD B. Theuse of traditional techniques, allied to the identification of the different molecular variants, is important for the understanding of the structural and functional properties and hemolytic behavior of the red blood cells of the patient...

A importância da enzima Glicose-6-fosfato desidrogenase (G6PD) no metabolismo eritrocitário está na obtenção de energia calórica e redutora para a proteção celular contra agressões oxidativas. A deficiência de G6PD é a eritroenzimopatia que causa mais frequentemente anemia hemolítica, com mais de 130 variantes moleculares identificadas. O objetivo deste estudo foi realizar a análise molecular da deficiência de G6PD em uma população masculina adulta da região de Araraquara, SP, para a identificação das mutações genéticas. Nos 5087 doadores de sangue do sexo masculino pesquisados, foram encontrados 89 deficientes de G6PD, confirmados pela determinação da atividade enzimática e eletroforese em acetato de celulose, com frequência de 1,75%, valores semelhantes aos encontrados por outros pesquisadores no Estado de São Paulo. A análise molecular realizada pela amplificação do DNA genômico com iniciadores específicos e digestão com enzimas de restrição, demonstrou que 96,6% dos deficientes apresentaram a variante G6PD A¯, com as mutações 376(A-G) e 202(G-A) e atividade enzimática média de 1,31 UI.g de Hb-1.min-1 a 37ºC, correspondendo a 10,8% da atividade enzimática da enzima normal G6PD B. Não foram encontradas as formas variantes G6PD A¯ 680(G-T) e 968(T-C). Em 3,4% dos indivíduos deficientes, foi encontrada a variante G6PD Mediterrânea, mutação 563(C-T) e atividade enzimática média de 0,25 UI.g de Hb-1.min-1 a 37ºC, correspondendo a 2,1% da atividade enzimática da G6PD B. A utilização das técnicas tradicionais, aliadas à identificação da variante molecular, são importantes na compreensão das propriedades estruturais, funcionais e comportamento hemolítico dos glóbulos vermelhos do paciente...

G6PD deficiency in Latin America: systematic review on prevalence and variants

Plasmodium vivax radical cure requires the use of primaquine (PQ), a drug that induces haemolysis in glucose-6-phosphate dehydrogenase deficient (G6PDd) individuals, which further hampers malaria control efforts. The aim of this work was to study the G6PDd prevalence and variants in Latin America (LA) and the Caribbean region. A systematic search of the published literature was undertaken in August 2013. Bibliographies of manuscripts were also searched and additional references were identified. Low prevalence rates of G6PDd were documented in Argentina, Bolivia, Mexico, Peru and Uruguay, but studies from Curaçao, Ecuador, Jamaica, Saint Lucia, Suriname and Trinidad, as well as some surveys carried out in areas of Brazil, Colombia and Cuba, have shown a high prevalence (> 10%) of G6PDd. The G6PD A-202A mutation was the variant most broadly distributed across LA and was identified in 81.1% of the deficient individuals surveyed. G6PDd is a frequent phenomenon in LA, although certain Amerindian populations may not be affected, suggesting that PQ could be safely used in these specific populations. Population-wide use of PQ as part of malaria elimination strategies in LA cannot be supported unless a rapid, accurate and field-deployable G6PDd diagnostic test is made available.

Prevalence of thalassaemia, iron-deficiency anaemia and glucose-6-phosphate dehydrogenase deficiency among Arab migrating nomad children, southern Islamic Republic of Iran

This study investigated the prevalence of iron-deficiency anaemia, glucose-6-phosphate dehydrogenase [G6PD] deficiency and beta-thalassaemia trait among Arab migrating nomad children in southern Islamic Republic of Iran. Blood samples were analysed from 134 schoolchildren aged < 18 years [51 males, 83 females]. Low serum ferritin [< 12 ng/dL] was present in 17.9% of children [21.7% in females and 11.8% in males]. Low haemoglobin [Hb] correlated significantly with a low serum ferritin. Only 1 child had G6PD deficiency. A total of 9.7% of children had HbA2 >/= 3.5 g/ dL, indicating beta-thalassaemia trait [10.8% in females and 7.8% in males]. Mean serum iron, serum ferritin and total iron binding capacity were similar in males and females. Serum ferritin index was as accurate as Hb index in the diagnosis of iron-deficiency anaemia. A high prevalence of beta-thalassaemia trait was the major potential risk factor in this population

Thalassaemia and glucose-6-phosphate dehydrogenase deficiency in sickle-cell disorder patients in Taiz, Yemen

A pilot study was conducted to determine the prevalence and haematological characteristics of the interaction between thalassaemia or/and glucose-6-phosphate dehydrogenase [G6PD] deficiency in patients with sickle-cell disorder [SCD] in Taiz city, Yemen, where the prevalence of sickle-cell trait [HbAS] is 8.2%. Blood samples were collected from 31 SCD patients. Complete blood count and haemoglobin electrophoresis, G6PD activity and serum ferritin were determined. Thalassaemia was found in 6 patients [19.4%] and G6PD deficiency [6 mild and 1 severe] was detected in 7 patients [22.6%] The frequency of thalassaemia and/or G6PD deficiency with SCD was high and this may have an effect on the seventy of the clinical course of SCD in Taiz. The study should be repeated with DNA analysis to define the nature of the globin gene defect and to clarify its role in the severity of SCD

Preliminary report on neonatal screening for congenital hypothyroidism, congenital adrenal hyperplasia and glucose-6-phosphate dehydrogenase deficiency: a chandigarh experience.

Objective To establish newborn screening in Indian scenario that could lay a framework for future such initiatives. Three disorders namely, congenital hypothyroidism (CH), congenital adrenal hyperplasia (CAH) and glucose-6- phosphate dehydrogenase deficiency (G-6-PDD) were selected for a preliminary study for newborn screening. Methods Heel-prick blood samples were collected from liveborn neonates at 24–48 h of birth as a part of a screening program after prior written consent from the parents. Blood levels of glucose-6-phosphate-dehydrogenase enzyme (G-6- PD), thyroid-stimulating hormone (TSH) and 17-α-OH progesterone (17-OHP) were measured using DELFIA time resolved fluoroimmunoassay. Results Six thousand eight hundred and thirteen (6,813) neonates (86.3%), out of a total of 7,893 live births in our institute during the period May’2007 through July’2009, were screened for CAH, CH and G6PD deficiency. Major reason for missing samples was early discharge of the neonates and admission to the neonatal intensive care unit. G-6-PD deficiency was confirmed in 61 cases, congenital hypothyroidism (CH) in 2 cases and congenital adrenal hyperplasia (CAH) in 1 neonate, accounting for an incidence of 1/112 for G-6-PDD, 1/ 3400 for CH and 1/6813 for CAH. Conclusions Preliminary data on prevalence of various genetic disorders viz. G-6-PDD, CH and CAH in the population of this region revealed that G-6-PDD is most prevalent disorder followed by CH and CAH. More efforts need to be undertaken to create awareness and emphasis on significance of preventive testing to make screening a successful program in India.

Thalassemia, iron and G6PD deficiency in Lor migrating nomad children, Southern Iran

Ferropenia and consequent iron deficiency anemia [IDA], beta-thalassemia, and glucose 6-phosphate dehydrogenase [G6PD] deficiency are three main common hematologic problems in Iran. This study was conducted on the prevalence of these problems in Lor migrating nomads ethnic group in southern Iran. From June to October 2006, the blood samples of 79 Lor migrating nomadic children including 53 [67.1%] male and 26 [32.9%] female were checked for iron indices and G6PD deficiency. The family history of favism, thalassemiaand, signs and symptoms in relation to anemia of participants were evaluated. RBC count, different types of Hb, Hct, MCV, MCH, MCHC, RDW, SI, TIBC and SF were determined immediately after blood sampling. Fourteen [17.7%] children had SF<12 ng/mL while the prevalence of this low serum ferritin was higher in females than males [19.2% vs. 17%]. The low hemoglobin [Hb] level had statistical correlation with the low serum ferritin level. Among all participants, the prevalence of G6PD deficiency was 10.1%, and all of them were male children. The prevalence of beta-thalassemia was 2.5% and all were male. The prevalence of IDA was 17.7%. Although IDA figure is less than those reported in other developing countries [25-35%]; but it shows that Lor tribes in southern Iran are still behind the health status of developed countries [5-8%]. Even the prevalence of beta-thalassemia is not very high, but regarding the devastating potential risk of Cooley's anemia; a careful performance of Iranian thalassemia program is recommended. It seems that G6PD deficiency is prevalent in Lor nomads, so establishment of educational programs and investigation on their dietary habits seem to be a good way to prevent the favism occurrence

Study of glucose-6-phosphate dehydrogenese [G6PD] gene mutations in deficient patients of Fars and Esfahan provinces

Glucose-6-phosphate dehydrogenase [G6PD] deficiency is the most common human enzyme defect, being present in more than 400 million people worldwide. The aim of this study was the molecular analysis of common G6PD mutations, including Mediterranean, Chatham, Cosenza and A-[G202A/A367G] in the patient with favism in Fars and Esfahan provinces. In this basic study, 96 non-relative patients with G6PD deficiency [34 from Fars and 62 from Esfahan province] were studied. Genomic DNA was analyzed by PCR-RFLP and product electrophoresis method for known mutations such as Mediterranean [C-T] nt, Chatham, Cosenza and A202 [G-A]/367 [A-G] mutation. Of 96 samples, 79 [82.3%] and 8 [8.3%] had G6PD Mediterranean and G6PD Chatham, respectively. None of the samples had Cosenza and A-[G202A/A367G] mutation. This study showed that G6PD Mediterranean is the most prevalent mutation in Iran

Genetic disorders and malaria in Indo-China region.

High prevalence of malaria in Southeast Asia including Thailand is believed to be a major public health problem to the population in this area since time immemorial. Adaptation of the population in this area following the principle of natural selection coupled with genetic disorders can be expected. Some good examples for natural selection of malaria are the co-existence of high prevalence of thalassaemia as well as glucose-6-phosphate dehydrogenase deficiency. In this report, general aspects of some important genetic disorders and malaria in Indo-China area (Thailand, Laos, Cambodia, Myanmar, Vietnam, Yunnan and Manipur) are summarized and discussed.

Screening extended families for a genetic X-linked disorder, glucose 6 phosphate dehydrogenase deficiency

To test an alternative approach to population based program for identifying Glucose 6 Phosphate Dehydrogenase [G6PD] deficient Individuals in Pakistan where consanguineous marriage is common. This study was conducted at Armed Forces Institute of Pathology Rawalpindi and Pathology Department Government Lady Reading Hospital Peshawar. Five large families from Northern Pakistan, 03 with an index case of G6PD deficiency, 02 without such history [control] were screened for G6PD deficiency. All apparently healthy members of the families, both male and female of all ages, of the last three generations were included in the study. A commercial qualitative screening kit from Sigma Chemical Co. Ltd England was used for screening the individuals for G6PD deficiency. In the control families, no individual with G6PD deficiency was found among 101 individuals tested out of 159 living members. In the 03 families with an index case of G6PD deficiency 155 were tested out of 229 family member and 52 [33.5%] were found G6PD deficient. While in population screening out of 800 apparently healthy adult male subjects screened for G6PD deficiency, 47 [5.9%] were found glucose 6 phosphate dehydrogenase deficient. Testing extended families is feasible and highly cost effective way of screening for X- linked genetic disorder like Glucose 6 Phosphate Dehydrogenase deficiency in communities in which consanguineous marriage is common

Prevalence of Glucose-6-phosphate Dehydrogenase Deficiency and Haemoglobin S in High and Moderate Malaria Transmission Areas of Muheza; North-Eastern Tanzania

Glucose-6-phosphate dehydrogenase (G6PD) deficiency and haemoglobin S (HbS) are very common genetic disorders in sub Saharan Africa; where malaria is endemic. These genetic disorders have been associated with protection against malaria and are therefore under strong selection pressure by the disease. In November-December 2003; we conducted a cross-sectional survey to determine the prevalence of G6PD deficiency and HbS in the population and relate these to malaria infection and haemoglobin levels in lowland and highland areas of differing malaria transmission patterns of Muheza; Tanzania. Blood samples from 1959 individuals aged 6 months to 45 years were collected. A total of 415 (21) and 1181 (60) samples were analysed for G6PD deficiency and HbS; respectively. Malarial parasite prevalence was 17.2(114/1959) in the highlands and 39.6(49/1959) in the lowlands. Lowlands had higher prevalence of G6PD deficiency and HbS than highlands (G6PD deficiency = 11.32(24/212) versus 4.43(9/203); P = 0.01; and HbS = 16.04(98/611) versus 6.32(36/570); P = 0.0001). Logistic regression model showed an association between G6PD deficiency and altitude [lowlands] (Odds ratio [OR] 3.4; 95CI=1.49; 7.90; P=0.004). In the lowlands; G6PD deficient individuals had lower mean haemoglobin (10.9g/dl) than normal ones (12.8g/dl); P = 0.01. These findings show that high malaria transmission in the lowlands might have selected for G6PD deficiency and HbS

Regional experience with newborn screening for sickle cell disease, other hemoglobinopathies and G6PD deficiency

Newborn screening for sickle cell disease, other hemoglobinopathies and G6PDdeficiency is one of the most important means of decreasing mortality and morbidity in high prevalence areas. Nine years experience in newborn screening in Qatif Central Hospital are summarized. Patients and All newborns in Qatif Central Hospital had cord blood screening for sickle cell disease, other hemoglobinopathies and G6PD deficiency using alkaline and electrophoresis, agar gel electrophoresis for sickle cell disease and fluoresecent screening technique for G6PD deficiency. Families of infants with minor hemoglobinopathies and G6PD deficiency were informed about the results in the well baby clinic. From December 1992 to December 2001, 24 012 newborn were screened. 21 858 [91.03%] were Saudi and 2154 [8.97%] were non-Saudi. In the Saudi hemoglobin electrophoresis patterns, AF [normal] was found in 49.52%, hemoglobin FS [sickle cell disease] + FS Bart's [sickle cell disease with alpha thalassemia] in 2.57%, hemoglobin AFS [sickle cell trait] + AFS Bart's [sickle cell trait with alpha thalassemia] in 21.14%, and alpha thalassemia [based on elevated Bart's hemoglobin 2%] in 35.68%. G6PD deficiency was found in 37.02% and 21.27% in males and females, respectively. Of 563 Saudi newborn with a presumptive diagnosis of sickle cell disease, 48 [8.5%] did not come to the hematology clinic or were not contactable. The diagnosis of sickle cell anemia or sickle thalassemia was confirmed in 513 patients, and 2 cases were found to have sickle cell trait on repeat testing. Many parents found it hard to accept the initial diagnosis and the resulting impact on their relationship with one another. Prevention and early identification of sickle cell disease, other major hemoglobinopathies and G6PD deficiency remains the cornerstone of management of these diseases. The main barriers to successful neonatal screening for hemoglobinopathies are the level of the education and deficiency in manpower. We recommend including newborn screening for hemoglobinopathies and G6PD deficiency in the national hypothyroidism screening program in the eastern province and the establishment of a special center for hemoglobinopathies with a high standard of medical care in Qatif

community-based study of common hereditary blood disorders in Oman

We assessed the prevalence of three common hereditary blood disorders [sickle-cell and beta-thalassaemia traits and glucose 6-phosphate dehydrogenase deficiency] among the Omani population. We interviewed a representative sample of 6103 Omani households and blood samples from 6342 children aged 0-5 years were collected. About 27% of Omani males had inherited glucose-6-phosphate dehydrogenase deficiency [compared with 11% of females] while countrywide prevalence rates for the sickle-cell and beta-thalassaemia traits were estimated to be 5.8% and 2.2% respectively and showed no significant gender differences. There was a significant association between all three disorders and region of the country

Investigaçäo genético-epidemiológica e molecular da deficiência de G-6-PD em uma comunidade brasileira / G-6-PD deficiency in a Brazilian community: an investigation involving epidemiological genetics and molecular techniques

Teve por objetivo estudar a deficiência de G-6-PD em uma comunidade do interior do Estado de Säo Paulo (Bragança Paulista). Durante 36 meses foram selecionados 4.621 doadores de sangue do sexo masculino, detectando-se 80 deficientes em G-6-PD. A análise molecular foi realizada em 70 deficientes näo consangüíneios mediante a amplificaçäo de DNA por PCR seguida de digestäo por enzimas de restriçäo e análise de polimorfismo de conformaçäo em hélice simples (SSCP). Em 98,6 por cento dos casos, foi identificada a mutaçäo G-6-PD A- (202 G->A), por digestäo do exon 4 com Nla III. Verificou-se a presença de mutaçäo mais rara no exon 9, por SSCP. Näo foi constatado caso da variante Mediterrânea. Tais resultados mostraram que a variante A- (202->A), quase que exclusiva, foi introduzida na comunidade näo apenas por descedentes de africanos, como também pelos imigrantes italianos, espanhóis e portugueses. A contribuiçäo italiana em termos da variante Mediterrânea de G-6-PD foi menor do que a sua participaçäo em termos de talassemia beta, provavelmente devido à origem no Norte da Itália.

Neonatal screening for sickle cell disease, glucose-6-phosphate dehydrogenase deficiency and alpha-thalassemia in qatif and al hasa

Screening programs to determine the frequency of sickle cell, glucose-6-phosphate dehydrogenase deficiency and alpha-thalassemia gene are available in Saudi Arabia, although not used frequently. Greater use of these programs will decrease the morbidity and mortality of Saudi children affected by these disorders. Neonatal hemoglobin electrophoresis and glucose-6-dehydrogcnase Fluorescent spot tests were performed on newborn babies delivered between December 1992 and December 1993 at the Qatif Central Hospital and at the King Fahad Hospital in Al Hasa. Cord blood samples were collected from babies born in these two hospitals. Babies born in other hospitals had blood collected in their first visit to Qatif primary care centers at the time of vaccination. All specimens were sent to Dammam Central Laboratory. The diagnosis of sickle cell and alpha-halassemia was based on cellulose acetate electrophoresis and confirmed by agar gel electrophoresis, and glucose-6-phosphate dehydrogenase was confirmed by fluorescent spot test. Results: A total of 12,220 infants, including 11,313 Saudis [92.6%], were screened over a 12-month period. The common phcnotypes detected in these infants included AF, AF Bart's, SFA, SFA Bart's, FS and FS Bart's. In the Saudi infants, homozygous sickle cell disease was detected in 2.35% and 1.08% in Qatif and Al Hasa, respectively. The frequencies of sickle cell gene were 0.1545% and 0.1109% in Qatif and Al Hasa. alpha-thalassemia gene based on an elevated level of Hb Bart's were 28% and 16.3% in Qatif and Al Hasa. The screening for G6PD deficiency revealed a high prevalence of 30.6% and 14.7% in Qatif and Al Hasa. In the non-Saudi infants, the frequencies were low. The outcome of this study indicates that the Saudi populations in Qatif and Al Hasa are at risk for hemoglobinopathies and G6PD. Neonatal screening programs are essential and cost effective and should be maintained as a routine practice

Medical genetics in Thailand.

The development of medical genetics as a discipline in Thailand is relatively recent. It originated through interest in hematological disorders which occur in high frequency, namely the thalassemias and hemoglobinopathies, together with glucose-6-phosphate dehydrogenase deficiency. Although a complete registry of genetic diseases in Thailand has not yet been established, considerable data has accumulated through special interest groups.

Prevalence of hemoglobin E, alpha-thalassemia and glucose-6-phosphate dehydrogenase deficiency in 1,000 cord bloods studied in Bangkok.

Thalassemia hemoglobinopathies and glucose-6-phosphate dehydrogenase (G-6-PD) deficiency are prevalent in Thailand. We studied the prevalence of these disorders from 1,000 cord bloods collected during 14 months period, using EDTA as anticoagulant. Red blood cell G-6-PD quantitative assay was performed in all male subjects. Nine hundred and eighty five specimens were available for hemoglobin (Hb) typing by starch gel electrophoresis. Further evaluation by cellulose acetate electrophoresis and follow up were made in the cases who had Hb E and/or high level of Hb Bart's. It was found that out of 505 males, 61 cases (12.08%) had G-6-PD deficiency. Among 985 cases studied for Hb typing, 61.92% revealed normal Hb type AF while Hb E was present in 18.68% and Hb Bart's designated alpha-thalassemias were present in 25.18% respectively. Of these 985 cases, 18.78% had low Hb Bart's level ie detectable to 8.2% consistent with alpha-thal2, Hb Constant Spring (CS) or alpha-thal1 trait. Ten cases (1.02%) had high levels of Hb Bart's ranging from 16.1-35% without or with Hb CS and E, and further follow-up revealed homozygous Hb CS, Hb A-E-Bart's, Hb H and Hb H with Hb CS disease. The other 53 cases (5.38%) had low level of Hb Bart's with Hb E consistent with alpha-thalassemia trait with Hb E trait. There were 127 cases (12.89%) who had only Hb E trait and 3 cases (0.3%) who had Hb F and E without Hb A initially.