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1.
J Clin Pathol ; 73(1): 14-16, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31434698

RESUMO

AIMS: Untranslated regions (UTRs) play an important role in post-transcriptional regulation of gene expression, including by modulating messenger RNA (mRNA) transport out of the nucleus, translation efficiency, subcellular localisation and stability. Any mutation in this region could alter the stability of mRNA and thereby affect protein synthesis. We analysed if a mutation located in the α complex protected region of the α1 globin gene could cause non-deletional α-thalassaemia by affecting post-transcriptional stability (mRNA stability). METHODS: A total of 14 patients without anaemia, normal or slight microcytosis and hypochromia (medium concentration haemoglobin [MCH] <27 pg) were studied. Haemoglobin subtypes were screened using capillary zone electrophoresis and ion-exchange high-performance liquid chromatography (VARIANT II ß-Thalassaemia Short Program). The most common α-globin mutations were identified by multiplex PCR (Alpha-Globin StripAssay kit) and the molecular characterisation by automatic sequencing of alpha globin genes. RESULTS: All of them shown a novel transversion mutation in nt 778 (C>A), which is located in the 3' UTR in the α complex protected region [HBA1: c.*+46C>A]. CONCLUSIONS: This mutation is in the αRNAmin binding site, so a single nucleotide substitution in this region can decrease mRNA stability by potentially compromising the binding of α-complex protein to αRNAmin, favouring the decay of α-globin mRNA via erythroid cell-enriched endoribonuclease cleavage. In this case, it is a non-deletional α-thalassaemia. However, in silico and empirical studies predicted that it could be a silent polymorphism. Functional studies should be carried out to confirm whether it is a pathological mutation or a silent polymorphism.


Assuntos
Regiões 3' não Traduzidas , Mutação , Polimorfismo Genético , Estabilidade de RNA , RNA Mensageiro/genética , alfa-Globinas/genética , Talassemia alfa/genética , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Pré-Escolar , Análise Mutacional de DNA/métodos , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Multiplex , Fenótipo , RNA Mensageiro/metabolismo , Fatores de Risco , alfa-Globinas/metabolismo , Talassemia alfa/sangue , Talassemia alfa/diagnóstico
2.
Hemoglobin ; 43(1): 52-55, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31106603

RESUMO

This study reports the case of 2-year-old Northeastern Thai girl with ß-thalassemia (ß-thal) disease who has required regular blood transfusions since she was 8 months old. Hemoglobin (Hb) analysis by high performance liquid chromatography (HPLC) separated Hb A2/E (16.5%), Hb F (22.7%), Hb A (51.8%) and an abnormal peak (Hb X) found at a retention time (RT) of 5.05 min. (C-window) with 2.8%. Multiplex gap-polymerase chain reaction (gap-PCR) revealed heterozygous α-thalassemia-2 (α-thal-2) (-α3.7/αα; NG_000006.1: g.34164_37967 del3804). This patient was suspected of having a ß-globin chain variant and Hb E (HBB: c.79G>A) according to the high Hb F level and disease presentations. Surprisingly, Hb Mahasarakham (the geographic origin of the proband), a novel single nucleotide deletion (-G) at the first nucleotide of codon 121 (HBB: c.364delG), was identified by direct DNA sequencing and secondary confirmation by PCR-restriction fragment length polymorphism (PCR-RFLP). This novel mutation causes a frameshift mutation and added 10 more residues to the ß-globin chain that was elongated to 156 amino acids. Molecular basis of this novel mutation in the heterozygous state is required to confirm the mode of inheritance.


Assuntos
Heterozigoto , Mutação , Domínios Proteicos/genética , Talassemia alfa/diagnóstico , Talassemia alfa/genética , Globinas beta/genética , Alelos , Substituição de Aminoácidos , Sequência de Bases , Pré-Escolar , Análise Mutacional de DNA , Eritrócitos/patologia , Feminino , Hemoglobinas Anormais/genética , Humanos , Globinas beta/química
3.
Hemoglobin ; 43(1): 4-6, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31084368

RESUMO

Interest in α-globin point mutations has increased in the past few years because nondeletional variations can affect protein function and stability, giving rise to hemoglobin (Hb) variants that present a wide spectrum of phenotypes, from asymptomatic forms to hemolytic anemia. We describe a novel α1-globin gene variant, which we have named Hb Milano [α109(G16)Leu→Pro (CTG>CCG); HBA1: c.329T>C]. We performed high performance liquid chromatography (HPLC) to carry out Hb analysis, capillary electrophoresis (CE) for Hb separation and quantitation of Hb subtypes, two tests on stroma-free lysates for evaluating Hb stability, multiplex ligation-dependent probe amplification (MLPA) to detect deletions/duplications within the α gene cluster and Sanger sequencing of the α-globin genes. No abnormal Hb variants were identified by HPLC and CE. Isopropanol and stability tests were negative. The peripheral blood film showed no inclusions such as Hb H or Heinz bodies. Multiplication ligation-dependent probe amplification of the α-globin gene cluster detected a heterozygosity for the -α3.7 (rightward) deletion. Direct sequencing of the α-globin genes identified the Hb Milano variant on the HBA1 gene. No mutations were found on the HBA2 gene. The clinical consequences of the Hb Milano variant differ based on the genotype: according to our study, the hematological parameters range from a marked microcythemia with mild anemia if the variant is coinherited with an α gene deletion, to mild microcytosis when the variant is not associated with α gene deletions.


Assuntos
Substituição de Aminoácidos , Grupo com Ancestrais do Continente Europeu/genética , Genótipo , Hemoglobina A Glicada/genética , Hemoglobinas Anormais/genética , Mutação , alfa-Globinas/genética , Adulto , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Itália , Fenótipo , Talassemia alfa/diagnóstico , Talassemia alfa/genética
4.
Pediatr Blood Cancer ; 66(8): e27807, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31094093

RESUMO

BACKGROUND: The Uganda Sickle Surveillance Study provided evidence for a large sickle burden among HIV-exposed infants in Uganda. To date, however, no large scale screening program has been developed for Central or East Africa. METHODS: A 3-year targeted sickle cell screening project in Uganda was designed by the Ministry of Health to (1) determine sickle cell trait and disease prevalence within high-burden districts, (2) document the prevalence among HIV-exposed and nonexposed children, (3) confirm previously suggested HIV comorbidity, and (4) estimate the co-inheritance of known genetic modifiers of sickle cell disease. RESULTS: A total of 163 334 dried blood spot samples collected between April 2015 and March 2018 were analyzed, including 112 352 samples within the HIV Early Infant Diagnosis program. A high burden with >1% sickle cell disease was found within targeted East Central and Mid-Northern districts, in both HIV-exposed and nonexposed children. Based on crude birth-rate data, 236 905 sickle cell trait births and 16 695 sickle cell disease births will occur annually in Uganda. Compared to sickle cell disease without HIV, the odds ratio of having sickle cell disease plus HIV was 0.50 (95% confidence interval = 0.40-0.64, P < .0001). Alpha-thalassemia trait and G6PD deficiency were common with sickle cell disease, but with different geospatial distribution. CONCLUSIONS: High sickle cell burden and potential HIV comorbidity are confirmed in Uganda. Genetic modifiers are common and likely influence laboratory and clinical phenotypes. These prospective data document that targeted sickle cell screening is feasible and effective in Uganda, and support development of district-level comprehensive care programs.


Assuntos
Anemia Falciforme/diagnóstico , Genes Modificadores , Deficiência de Glucosefosfato Desidrogenase/diagnóstico , Infecções por HIV/diagnóstico , Programas de Rastreamento/métodos , Talassemia alfa/diagnóstico , Anemia Falciforme/complicações , Anemia Falciforme/epidemiologia , Anemia Falciforme/genética , Pré-Escolar , Comorbidade , Feminino , Seguimentos , Deficiência de Glucosefosfato Desidrogenase/complicações , Deficiência de Glucosefosfato Desidrogenase/epidemiologia , Deficiência de Glucosefosfato Desidrogenase/genética , HIV/genética , HIV/isolamento & purificação , Infecções por HIV/complicações , Infecções por HIV/epidemiologia , Infecções por HIV/genética , Humanos , Lactente , Recém-Nascido , Masculino , Prevalência , Prognóstico , Estudos Prospectivos , Talassemia alfa/complicações , Talassemia alfa/epidemiologia , Talassemia alfa/genética
5.
Hemoglobin ; 43(1): 73-75, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31104519

RESUMO

A regulatory single nucleotide polymorphism (rSNP), the Aγ (+25 G>A) (rs368698783) (NG_000007.3: g47783G>A) located in the HBG1 proximal promoter, is a significant predictor of clinical severity by elevating Hb F levels in ß-thalassemia (ß-thal). In this study, the presence of the Aγ (+25 G>A) and Aγ (+25 A>A) genotypes was investigated in four subgroups from a total of 611 subjects, including 88 α-thalassemia (α-thal) carriers (group A), 162 ß-thal carriers of point mutations (group B), 57 carriers of ß-thal deletions (group C) and 152 non thalassemic individuals (group D). The result is that the genotypes G>A and A>A exhibit significantly high levels of Hb F compared with the genotype G>G in both groups B and C, while no significant difference was observed in both groups A and D. We assume that the effect of Aγ (+25 G>A) polymorphism on Hb F production is only under erythropoietic stress characteristic for ß-globin chain deficiency.


Assuntos
Alelos , Eritropoese/genética , Hemoglobina Fetal/genética , Genes Modificadores , Polimorfismo de Nucleotídeo Único , Globinas beta/genética , Talassemia beta/diagnóstico , Talassemia beta/genética , Índices de Eritrócitos , Feminino , Genótipo , Humanos , Fatores de Transcrição Kruppel-Like/genética , Masculino , Análise de Sequência de DNA , Talassemia alfa/diagnóstico , Talassemia alfa/genética
6.
Hemoglobin ; 43(1): 66-68, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30843739

RESUMO

We describe a proband originating from Al-Quneitra Province, Syria. His hematology data was as follows: Hb A 24.7%, Hb F 71.1%, Hb A2 4.2%. Molecular analysis, based on DNA sequencing of the ß-globin gene mutation, showed for the first time a compound heterozygous IVS-I-1 (G>A) (HBB: c.92+1G>A)/IVS-II-705 (T>G) (HBB: c.316-146T>G) on the ß-globin gene. A reverse hybridization technique revealed that the proband was also an α-thalassemia (α-thal) -α3.7 (rightward) deletion carrier. Haplotypes analysis for the seven polymorphic restriction sites showed that the compound heterozygous mutations, IVS-I-1/IVS-II-705, were linked to two haplotypes: I [+ - - - - + +] and VI [- + + - - - +], respectively. Our results showed, for the first time, the presence of rare ß-thalassemia (ß-thal) IVS-II-705 (T>G) mutation associated with IVS-I-1 (G>A). Our findings suggest the presence of these mutations resulted from past migrations.


Assuntos
Alelos , Íntrons , Mutação , alfa-Globinas/genética , Talassemia alfa/diagnóstico , Talassemia alfa/genética , Globinas beta/genética , Talassemia beta/diagnóstico , Talassemia beta/genética , Adolescente , Análise Mutacional de DNA , Índices de Eritrócitos , Família , Haplótipos , Heterozigoto , Humanos , Masculino , Linhagem , Síria , Talassemia alfa/sangue , Talassemia beta/sangue
7.
Int J Lab Hematol ; 41(3): 397-403, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30830998

RESUMO

INTRODUCTION: The standard screening method for alpha thalassaemia is the examination of HbH preparation. It is labour intensive and poorly standardized. The development of a rapid strip immunochromatographic test (ICT) for haemoglobin Barts offers a fast, user friendly and cost-effective alternative screening tool. METHOD: A total of 180 subjects with results of the thalassaemia screen and genetic testing were included. Results of the ICT and HbH preparation were correlated with genetic results to determine the performance characteristics of the tests and the effect of mean sample age on results. RESULTS: Of 180 subjects, 111 carried alpha thalassaemia mutations and 69 participants had normal genetic results. The ICT had a sensitivity of 63.06% for all alpha gene mutations and 100% for both heterozygous alpha0 and HbH disease, with a specificity of 91.30%. Examination of HbH preparation had a sensitivity of 34.23% overall, detecting 89% of heterozygous alpha0 and 100% of HbH disease with a specificity of 98.55%. Sample age did not affect overall results. CONCLUSIONS: The ICT is a sensitive screening method for significant alpha mutations and detects the majority of homozygous alpha+ and nondeletional mutations. It demonstrates greater correlation with genetic testing than HbH preparation and could replace HbH preparation in the screening algorithm for alpha thalassaemia.


Assuntos
Imuno-Histoquímica/métodos , Talassemia alfa/diagnóstico , Alelos , Estudos Transversais , Grupos Étnicos , Genótipo , Humanos , Programas de Rastreamento , Mutação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA , alfa-Globinas/genética , Talassemia alfa/sangue , Talassemia alfa/epidemiologia , Talassemia alfa/genética
8.
Hemoglobin ; 43(1): 69-72, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30810399

RESUMO

The α+-thal deletion of 3.557 kb (NG_000006.1: g.32745_36301del, -αMAL3.5), involving the entire α2-globin gene, was identified in a Chinese family by multiplex ligation-dependent probe amplification (MLPA) followed by gap-polymerase chain reaction (gap-PCR) and sequencing. The proband, a compound heterozygote for this mutant gene and the Southeast Asian (- -SEA; NG_000006.1: g.26264_45564del19301) deletion, had a phenotype of Hb H disease [hemoglobin (Hb) 7.6 g/dL, mean corpuscular volume (MCV) 60.0 fL, Hb H (ß4) 0.7%, Hb Bart's (γ4) 2.4% and Hb A2 1.1%]; one of her sisters with same genotype showed a similar phenotype. Another two family members, who were carriers of this mutant gene, had a hematological phenotype of a silent α-thal. The 5' and 3' breakpoints of this deletion are located at the Y2 and Y1 boxes, respectively, therefore, it probably originated from an unequal crossover between these two homologous boxes. This mutation constitutes an additional heterogeneous defect causing α-thal in the Chinese population and would be valuable for elucidating the arrangement in the human α-globin gene cluster.


Assuntos
Heterozigoto , Mutação , alfa-Globinas/genética , Talassemia alfa/diagnóstico , Talassemia alfa/genética , Adolescente , Adulto , Idoso , Alelos , Criança , Análise Mutacional de DNA , Feminino , Hemoglobina A Glicada/genética , Hemoglobina A2/genética , Humanos , Masculino , Pessoa de Meia-Idade , Família Multigênica , Linhagem , Adulto Jovem , Talassemia alfa/sangue
9.
Lab Med ; 50(3): 306-312, 2019 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-30806653

RESUMO

BACKGROUND: Methods for detecting the complex genetic characteristics of α- and ß-thalassemias are required for preventing and controlling the outbreak of new cases. METHODS: We evaluated the accuracy and practical utility of microarray for simultaneous detection of α- and ß-thalassemias. A total of 102 DNA specimens, which represented 25 different genotypes, were tested in parallel using the microarray and reference methods used in the thalassemia laboratory of the Associated Medical Sciences-Clinical Services Center (AMS-CSC), Chiang Mai, Thailand. RESULTS: A total of 100 (98.0%) DNA specimens were completely concordant between the microarray and reference methods, whereas discrepancies between the different methods were observed in only 2 DNA specimens with homozygous hemoglobin E (HbE). CONCLUSIONS: The microarray appeared to be a fast, easy to perform, and accurate method for simultaneous detection of α- and ß-thalassemias in Thailand and Southeast Asian countries. However, this technique needs to be improved and validated in a larger number of specimens with homozygous HbE before further routine laboratory use.


Assuntos
Técnicas de Genotipagem/métodos , Análise em Microsséries/métodos , Técnicas de Diagnóstico Molecular/métodos , Talassemia alfa/diagnóstico , Talassemia beta/diagnóstico , Genótipo , Humanos , Mutação , Tailândia
10.
Ann Acad Med Singapore ; 48(1): 5-15, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30788489

RESUMO

INTRODUCTION: Haemoglobinopathy testing is performed for carrier screening and evaluation of microcytic anaemia. We evaluated the effectiveness of thalassaemia screening tests at our institution and suggest ways of improving the testing algorithm. MATERIALS AND METHODS: A total of 10,084 non-antenatal and 11,364 antenatal samples with alkaline gel electrophoresis (AGE), capillary electrophoresis (CE), haemoglobin H (HbH) inclusion test, mean corpuscular haemoglobin (MCH) and mean corpuscular volume (MCV) were retrospectively reviewed. A subgroup of 187 samples with genetic testing was correlated with HbH inclusions and MCH/ MCV. The effect of iron deficiency on percentage hemoglobin A2 (HbA2) was studied. RESULTS: HbH inclusion test showed low sensitivity of 21.43% for α-thalassaemia mutations but higher sensitivity of 78.95% for --SEA deletion. By receiver operating characteristic (ROC) analysis, MCH ≤28 pg or MCV ≤80 fl for non-antenatal samples and MCH ≤27 pg or MCV ≤81 fl for antenatal samples had >98% sensitivity for HbH inclusions. Above these thresholds, the probability that HbH inclusions would be absent was <99% (negative predictive value [NPV] >99%). MCH ≥28 pg had 100% sensitivity (95% CI 95.63%-100%) for α-thalassaemia mutations and 97.68% calculated NPV in the antenatal population. Detection of haemoglobin variants by CE correlated highly with AGE (99.89% sensitivity, 100% specificity). Severe iron deficiency reduced HbA2 in hemoglobin (P <0.001) and α-thalassaemia (P = 0.0035), but not in ß-thalassaemia. CONCLUSION: MCH/MCV thresholds have adequate sensitivity for α-thalassaemia in the antenatal population, and genotyping plays an important role as HbH inclusion test shows low sensitivity. CE without AGE, may be used as initial screening for haemoglobin variants. Our study provides contemporary data to guide thalassaemia screening algorithms in Singapore.


Assuntos
Inclusões Eritrocíticas/patologia , Hemoglobina H/análise , Complicações Hematológicas na Gravidez/diagnóstico , Talassemia alfa/diagnóstico , Eletroforese das Proteínas Sanguíneas , Eletroforese Capilar , Índices de Eritrócitos , Feminino , Testes Genéticos , Humanos , Masculino , Programas de Rastreamento , Gravidez , Complicações Hematológicas na Gravidez/sangue , Estudos Retrospectivos , Sensibilidade e Especificidade , Singapura , Talassemia alfa/sangue
11.
Medicine (Baltimore) ; 97(52): e13557, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30593129

RESUMO

This study is a retrospective analysis of the prenatal genetic diagnosis results of fetuses with high risk of major thalassemia to provide information for clinical genetic counseling and to better control the birth of major thalassemia child in Hakka population. Totally, 467 fetuses in at-risk pregnancies were collected from Meizhou people's hospital from January 2014 to December 2017. Genomic DNAs were extracted from peripheral blood of the couples and villus, amniotic fluid or cord blood of the fetuses. DNA-based diagnosis was performed using polymerase chain reaction (PCR) and flow-through hybridization technique. Follow-up visits were done half a year after the fetuses were born. Around 467 fetus at-risk pregnancies were performed prenatal diagnosis. We detected 88 CVS samples, 375 amniocentesis fluid samples and, 4 cord blood samples. The 356 fetuses in α-thalassemia families consisted of 69 (19.38%) with Bart's hydrops syndrome, 20 (5.62%) fetuses with Hb H disease, and 184 (51.68%) fetuses with heterozygote. And the 111 fetuses in ß-thalassemia families consisted of 31 (27.93%) thalassemia major, 51 (45.95%) fetuses with heterozygote. There are 13 fetuses with α+ß-thalassemia, including 2 cases with severe ß-thalassemia. DNA-based testing prenatal diagnosis of thalassemia was found to be highly reliable. Our findings provide key information for clinical genetic counseling of prenatal diagnosis for major thalassemia in Hakka pregnant women. Our work plays an important role in the prevention and control of thalassemia in Hakka population. We will also combine other techniques to further improve our molecular prenatal diagnostic capabilities, including the next-generation sequencing (NGS), Sanger sequencing and MLPA.


Assuntos
Testes Genéticos/métodos , Diagnóstico Pré-Natal/métodos , Talassemia alfa/diagnóstico , Talassemia beta/diagnóstico , Adolescente , Adulto , China/etnologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Gravidez , Estudos Retrospectivos , Análise de Sequência de DNA/métodos , Adulto Jovem , Talassemia alfa/embriologia , Talassemia alfa/genética , Talassemia beta/embriologia , Talassemia beta/genética
12.
Hematology Am Soc Hematol Educ Program ; 2018(1): 353-360, 2018 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-30504332

RESUMO

The α-thalassemia trait, associated with deletions removing both α-globin genes from 1 chromosome (genotype ζ αα/ζ--), is common throughout Southeast Asia. Consequently, many pregnancies in couples of Southeast Asian origin carry a 1 in 4 risk of producing a fetus inheriting no functional α-globin genes (ζ--/ζ--), leading to hemoglobin (Hb) Bart's hydrops fetalis syndrome (BHFS). Expression of the embryonic α-globin genes (ζ-globin) is normally limited to the early stages of primitive erythropoiesis, and so when the ζ-globin genes are silenced, at ∼6 weeks of gestation, there should be no α-like globin chains to pair with the fetal γ-globin chains of Hb, which consequently form nonfunctional tetramers (γ4) known as Hb Bart's. When deletions leave the ζ-globin gene intact, a low level of ζ-globin gene expression continues in definitive erythroid cells, producing small amounts of Hb Portland (ζ2γ2), a functional form of Hb that allows the fetus to survive up to the second or third trimester. Untreated, all affected individuals die at these stages of development. Prevention is therefore of paramount importance. With improvements in early diagnosis, intrauterine transfusion, and advanced perinatal care, there are now a small number of individuals with BHFS who have survived, with variable outcomes. A deeper understanding of the mechanism underlying the switch from ζ- to α-globin expression could enable persistence or reactivation of embryonic globin synthesis in definitive cells, thereby providing new therapeutic options for such patients.


Assuntos
Transfusão de Sangue Intrauterina , Hemoglobinas Anormais , Hidropisia Fetal , Assistência Perinatal/métodos , Talassemia alfa , Hemoglobinas Anormais/genética , Hemoglobinas Anormais/metabolismo , Humanos , Hidropisia Fetal/diagnóstico , Hidropisia Fetal/genética , Hidropisia Fetal/metabolismo , Hidropisia Fetal/terapia , Talassemia alfa/diagnóstico , Talassemia alfa/genética , Talassemia alfa/metabolismo , Talassemia alfa/terapia
13.
Nan Fang Yi Ke Da Xue Xue Bao ; 38(10): 1250-1254, 2018 Sep 30.
Artigo em Chinês | MEDLINE | ID: mdl-30377127

RESUMO

OBJECTIVE: To develop a rapid preimplantation genetic diagnosis method for α-thalassemia SEA deletion based on blastocyst cell whole genome amplification (WGA) combined with short fragment Gap-PCR. METHODS: Using multiple displacement amplification (MDA) WGA technique, we established a double-fluorescent PCR system of the housekeeping genes GAPDH and ß-actin for WGA quality testing, and a genotyping PCR system of mutant and normal short sequences for α-thalassemia SEA deletion. The sensitivity and accuracy of this method for diagnosis of α-thalassemia SEA deletion were evaluated by detecting lymphocyte samples containing different cell numbers from carriers of SEA deletion. The applicability of this method was evaluated by testing of 12 blastocyst biopsy samples. RESULTS: Detection of lymphocyte samples with different cell numbers using the method developed in this study revealed no ADO in 3-cell samples, and the product quantity of WGA became stable for 4-cell samples. Genotyping of the 10 blastocyst biopsy samples with successful WGA showed a genotype of --SEA/αα in 5 samples and αα/αα in the other 5 samples, which were consistent with the verification results. CONCLUSIONS: The method developed in this study is a complete testing process for 4-6 blastocyst biopsy cells to allow rapid, accurate, and cost-effective PGD genotyping of α-thalassemia SEA deletion using short fragment gap-PCR.


Assuntos
Blastocisto , Deleção de Genes , Reação em Cadeia da Polimerase/métodos , Diagnóstico Pré-Implantação/métodos , Deleção de Sequência/genética , Talassemia alfa/genética , Ásia Sudeste , Feminino , Estudo de Associação Genômica Ampla/métodos , Humanos , Gravidez , Sensibilidade e Especificidade , Talassemia alfa/diagnóstico
14.
Blood Adv ; 2(21): 3035-3044, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30425067

RESUMO

In sub-Saharan Africa, inherited causes of anemia are common, but data are limited regarding the geographical prevalence and coinheritance of these conditions and their overall contributions to childhood anemia. To address these questions in Malawi, we performed a secondary analysis of the 2015-2016 Malawi Micronutrient Survey, a nationally and regionally representative survey that estimated the prevalence of micronutrient deficiencies and evaluated both inherited and noninherited determinants of anemia. Children age 6 to 59 months were sampled from 105 clusters within the 2015-2016 Malawi Demographic Health Survey. Hemoglobin, ferritin, retinol binding protein, malaria, and inflammatory biomarkers were measured from venous blood. Molecular studies were performed using dried blood spots to determine the presence of sickle cell disease or trait, α-thalassemia trait, and glucose-6-phosphate dehydrogenase (G6PD) deficiency. Of 1279 eligible children, 1071 were included in the final analysis. Anemia, iron deficiency, and malaria were common, affecting 30.9%, 21.5%, and 27.8% of the participating children, respectively. α-Thalassemia trait was common (>40% of children demonstrating deletion of 1 [33.1%] or 2 [10.0%] α-globin genes) and associated with higher prevalence of anemia (P < .001). Approximately 20% of males had G6PD deficiency, which was associated with a 1.0 g/dL protection in hemoglobin decline during malaria infection (P = .02). These data document that inherited blood disorders are common and likely play an important role in the prevalence of anemia and malaria in Malawian children.


Assuntos
Anemia/diagnóstico , Transtornos Herdados da Coagulação Sanguínea/diagnóstico , Malária/diagnóstico , Anemia/complicações , Anemia/epidemiologia , Anemia/patologia , Anemia Falciforme/complicações , Anemia Falciforme/diagnóstico , Transtornos Herdados da Coagulação Sanguínea/complicações , Transtornos Herdados da Coagulação Sanguínea/epidemiologia , Pré-Escolar , Análise Discriminante , Feminino , Genótipo , Deficiência de Glucosefosfato Desidrogenase/complicações , Deficiência de Glucosefosfato Desidrogenase/diagnóstico , Deficiência de Glucosefosfato Desidrogenase/genética , Humanos , Malária/complicações , Malária/epidemiologia , Malaui/epidemiologia , Masculino , Prevalência , Índice de Gravidade de Doença , Talassemia alfa/complicações , Talassemia alfa/diagnóstico , Talassemia alfa/genética
15.
BMJ Case Rep ; 20182018 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-30366888

RESUMO

A 29-year-old nulliparous woman with a dichorionic diamniotic (DCDA) twin pregnancy was referred to our hospital at 16 weeks' gestation for prenatal diagnosis. She was diagnosed of Haemoglobin H Constant Spring (Hb H CS; --SEA/αCSα) and her husband of alpha thalassemia-1 trait (--SEA/αα). Detailed ultrasound showed that left twin had fetal anaemia and early signs of hydrops while the right one was normal. Both twins were female. Amniocentesis in each sac was performed for prenatal diagnosis of thalassemia after a proper counselling with the couple. DNA analysis confirmed that the left fetus was affected with haemoglobin Bart's hydrops fetalis (--SEA/--SEA) while the right one was alpha thalassemia-1 trait (--SEA/αα). Selective feticide with intracardiac injection of KCl was successfully performed on the hydropic fetus. Identification of the affected fetus is crucial for selective termination. Family counselling about the procedure and complications is also necessary.


Assuntos
Doenças Fetais/diagnóstico , Talassemia alfa/diagnóstico , Adulto , Feminino , Doenças Fetais/genética , Doenças Fetais/terapia , Predisposição Genética para Doença/genética , Hemoglobinas Anormais/genética , Humanos , Hidropisia Fetal/diagnóstico , Hidropisia Fetal/genética , Gravidez , Redução de Gravidez Multifetal/métodos , Diagnóstico Pré-Natal/métodos , Gêmeos Dizigóticos , Talassemia alfa/genética , Talassemia alfa/terapia
16.
Hemoglobin ; 42(3): 171-177, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-30192689

RESUMO

Detection of α-thalassemia-1 (α-thal-1) carriers provides valuable insight for genetic consulting in prevention and control programs for couples who are at risk of conceiving a fetus with severe thalassemia, both Hb Bart's hydrops fetalis and hemolytic Hb H disease. The traditional method is complicated, time-consuming and requires high instrument cost and expertise. Loop-mediated isothermal amplification (LAMP) based on pH-sensitive dye technology, shows all the characteristics required of a real-time analysis with simple operation for potential use in the clinical diagnosis of high incidence α-thal-1 [Southeast Asian (SEA) or - -SEA deletion]. Four primers specific for six distinct regions of the α-globin gene deletion were designed and analyzed by LAMP using the pH-indicator dye, phenol red. The amplification of the - -SEA deletion changed the color of phenol red from pink to orange. The diagnostic ability of detection of the - -SEA deletion by pH-sensitive LAMP was validated using both known and unknown blood samples and compared to the conventional polymerase chain reaction (PCR) method. Color inspection of pH-sensitive LAMP products could clearly identify the - -SEA deletion. There was no cross reaction with a normal α-globin gene, α-thal-1 Thai (- -THAI deletion), α-thal-2 [-α3.7 (rightward) and -α4.2 (leftward) deletion] and ß-thalassemia (ß-thal). Detection of the SEA deletion by pH-sensitive LAMP was consistent as compared to conventional PCR. The pH-sensitive LAMP method developed for this deletion carrier diagnosis has high sensitivity, specificity, simplicity, and requires simple instrumentation that makes it applicable for resource-limited laboratories in rural areas of developing countries.


Assuntos
Triagem de Portadores Genéticos/métodos , Deleção de Sequência , Talassemia alfa/diagnóstico , Humanos , Concentração de Íons de Hidrogênio , Fenolsulfonaftaleína , Diagnóstico Pré-Natal , Sensibilidade e Especificidade , Talassemia alfa/genética
17.
Clin Lab ; 64(7): 1279-1287, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30146840

RESUMO

BACKGROUND: The aim of this study was to use a lectin microarray to detect differential glycan profiling of serous glycoprotein in iron overload thalassemia patients. METHODS: This study enrolled iron overload α/ß-thalassemia patients and no iron overload α/ß-thalassemia patients. Lectin microarray was used to detect the alteration of protein glycosylation. The reliability of the lectin microarray results was verified by the lectin blotting technique. Expression level of hepcidin, erythropoietin, ferritin, and transferrin were measured by western blotting. Data were analyzed using the SPSS 16.0 software. RESULTS: In this study, 19 differentiating lectins were screened from the iron overload α-thalassemia group, and 15 were screened from the iron overload ß-thalassemia group. The agglutinin blotting technique demonstrated that the results of the Aleuria aurantia lectin (AAL), Lens culinaris agglutinin (LCA), and Wheat germ agglutinin (WGA) agglutinin affinity for serum glycoproteins were consistent with the results of the lectin microarray. In iron overload thalassemia groups, expression levels of erythropoietin and ferritin were increased, but hepcidin and transferrin were significantly reduced. CONCLUSIONS: The differentially expressed glycoprotein database of iron overload thalassemia was successfully created, and the specific glycan patterns of serous glycoprotein might be efficient biomarkers for diagnosis or progression of iron overload thalassemia.


Assuntos
Glicoproteínas/sangue , Sobrecarga de Ferro/sangue , Talassemia alfa/sangue , Talassemia beta/sangue , Adolescente , Adulto , Bases de Dados de Proteínas , Feminino , Ferritinas/sangue , Ferritinas/metabolismo , Glicoproteínas/metabolismo , Glicosilação , Hepcidinas/sangue , Hepcidinas/metabolismo , Humanos , Sobrecarga de Ferro/diagnóstico , Lectinas/metabolismo , Masculino , Pessoa de Meia-Idade , Análise Serial de Proteínas/métodos , Reprodutibilidade dos Testes , Transferrina/metabolismo , Adulto Jovem , Talassemia alfa/diagnóstico , Talassemia beta/diagnóstico
18.
J Immunoassay Immunochem ; 39(3): 323-336, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29985765

RESUMO

Hemoglobin F (HbF) in blood lysate can be accurately measured by various methods, including immunoassay. In this study, we have produced polyclonal antibody (pAb) against HbF and established a modified sandwich-type ELISA for HbF quantification in blood lysates. The modified sandwich ELISA utilized anti-γ-globin monoclonal antibody clones Thal N/B as the capture antibody (Ab) coated on solid-phase, fluorescein isothiocyanate (FITC)-labeled pAb as the detecting Ab, and HPR-labeled anti-FITC Ab as the signal-generating Ab. By using an optimized blood lysate dilution, the HbF could be measured with no interference from hemoglobin Bart's (Hb Bart's) and hemoglobin Portland (Hb Portland 1) presented in α-thalassemia carriers. HbF levels measured by the modified sandwich ELISA were comparable to those quantified by the standard cation-exchange high performance liquid chromatography. We suggested that this modified sandwich ELISA was able to accurately measure HbF levels even in α-thalassemia carriers containing Hb Bart's and Hb Portland 1 and be an alternative method for HbF measurement.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Hemoglobina Fetal/análise , Hemoglobinas Anormais/análise , Talassemia alfa/sangue , Talassemia alfa/diagnóstico , Animais , Humanos , Coelhos
19.
Arch Gynecol Obstet ; 298(2): 307-311, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29948167

RESUMO

PURPOSE: The aim of the present study was to report experiences with invasive prenatal diagnosis of α-thalassemia for the prevention of Hb Bart's hydrops fetalis syndrome in the Guangxi Zhuang Autonomous Region, China. METHODS: Pregnant women and their partners who tested positive for α0-thalassemia or were diagnosed with HbH diseases were counseled and suggested to undergo a prenatal diagnostic procedure for α-thalassemia. Fetal material was obtained by chorionic villus sampling (CVS) between 9 and 13 weeks of gestation, by amniocentesis between 16 and 24 weeks of gestation and by cordocentesis after 24 weeks of gestation. The α0-thalassemia gene types were detected by gap polymerase chain reaction (Gap-PCR). All results were finally confirmed by DNA analysis after delivery or termination of pregnancy. RESULTS: An invasive prenatal α-thalassemia diagnosis was performed in 3155 cases at risk for Hb Bart's hydrops fetalis syndrome at our hospital from 2002 to 2016. CVS was performed in 1559 cases (49.4%), amniocentesis in 1240 cases (39.3%) and cordocentesis in 356 cases (11.3%). In total, 786 fetuses were diagnosed as Hb Bart's hydrops fetalis syndrome. Among these cases, the α-thalassemia genotype was --SEA/--SEA in 784 cases and --SEA/--THAI in 2 cases. All affected pregnancies were terminated in time. CONCLUSIONS: This extensive experience suggests that carrier screening, molecular diagnostics, genetic counselling, and prenatal diagnosis are effective measures to prevent Hb Bart's hydrops fetalis syndrome.


Assuntos
Hemoglobinas Anormais/efeitos adversos , Hidropisia Fetal/diagnóstico , Diagnóstico Pré-Natal/métodos , Talassemia alfa/diagnóstico , Feminino , Humanos , Hidropisia Fetal/patologia , Gravidez , Estudos Retrospectivos , Fatores de Tempo , Talassemia alfa/patologia
20.
Ann Clin Lab Sci ; 48(2): 231-235, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29678852

RESUMO

Hemoglobin Bart's hydrops fetalis (homozygous α0-thalassemia) is the most severe form of thalassemia in the Southeast Asian population. Fetuses with this disorder almost always die in utero or shortly after birth. Screening of α0-thalassemia carrier is therefore crucial. Currently, diagnosis of α0-thalassemia genes is done by DNA-based analysis which relies on DNA extraction. We have developed a simple screening format based on whole blood PCR assay. The method was validated on 198 specimens and the results show 100% concordance with a conventional gap-PCR on DNA specimens. The protocol could also be applied to amniotic fluid specimens in prenatal diagnostic testing. The assay developed should facilitate carrier screening and prenatal diagnosis of Hb Bart's hydrops fetalis syndrome in the region.


Assuntos
Reação em Cadeia da Polimerase/métodos , Talassemia alfa/diagnóstico , Talassemia alfa/genética , Hemoglobinas Anormais/genética , Homozigoto , Humanos , Diagnóstico Pré-Natal
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