RESUMO
OBJECTIVES: Accurate quantification of hemoglobin (Hb) A2 is vital for diagnosing ß-thalassemia carriers. This study aimed to assess the precision and diagnostic utility of HbA2 measurements using the new high-performance liquid chromatography (HPLC) method, Premier Resolution, in comparison to capillary electrophoresis (CE). METHODS: We analyzed 418 samples, previously identified as A2A by CE, using Premier Resolution-HPLC. We compared the results, established correlations, and determined an optimal HbA2 cutoff value for ß-thalassemia screening. Additionally, we prospectively evaluated the chosen cutoff value in 632 samples. Mutations in the ß- and α-globin genes were identified using polymerase chain reaction (PCR) techniques and DNA sequencing. RESULTS: HbA2 levels were consistently higher with Premier Resolution, yet there was a significant correlation with CE in all samples (bias, -0.33; r, 0.991), ß-thalassemia (bias, -0.27; r, 0.927), and non-ß-thalassemia carriers (bias, -0.36; r, 0.928). An HbA2 cutoff value of ≥4.0â¯% for ß-thalassemia screening achieved 100â¯% sensitivity and 99.6â¯% specificity. Further validation yielded sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of 97.3â¯, 99.8, 97.3, 99.8, and 99.7â¯%, respectively. We also identified a rare ß-Hb variant, Hb La Desirade [HBB:c.389C>T], associated with ß-thalassemia and co-inherited with a single α-globin gene. CONCLUSIONS: The Premier Resolution HPLC is a reliable and accurate method for routine ß-thalassemia carrier screening, aligning with existing CE methods.
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Hemoglobina Falciforme , Talassemia beta , Humanos , Talassemia beta/diagnóstico , Talassemia beta/genética , Hemoglobina A/análise , Reprodutibilidade dos Testes , Hemoglobina A2/genética , Hemoglobina A2/análise , Mutação , alfa-Globinas/genéticaRESUMO
Hemoglobin Shaare Zedek (Hb SZ) is a rare structural α-Hb variant. Characterizing its genotype-phenotype relationship and genetic origin enhances diagnostic and clinical management insights. We studied a proband and six family members using high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), PCR, and sequencing to analyze α- and ß-globin genes and α-globin haplotypes. Pathogenicity predictions and a rapid diagnostic method were developed. The proband, his father, grandfather, and aunt had Hb migrating to the HbH-zone on CE and elevated fetal hemoglobin (HbF) on HPLC. Direct sequencing identified an A to G mutation at codon 56 of the α2-globin gene, characteristic of Hb SZ. Additionally, the proband carried a ß-globin gene mutation [HBB.52A>T]. Mild thalassemia-like changes were observed in the proband, whereas individuals with only the Hb SZ variant did not exhibit these changes. Pathogenicity predictions indicated that Hb SZ is benign. The variant can be identified using restriction fragment length polymorphism (RFLP) and allele-specific PCR. The Thai variant of Hb SZ is associated with the haplotype [- - M - - - -]. Hb SZ is a non-pathological variant that minimally affects red blood cell parameters, even when it coexists with ß0-thalassemia. HPLC and CE systems cannot distinguish it from other Hbs, necessitating DNA analysis for accurate diagnosis.
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Hemoglobinas Anormais , Globinas beta , Talassemia beta , Humanos , Hemoglobinas Anormais/genética , Masculino , Talassemia beta/genética , Talassemia beta/diagnóstico , Globinas beta/genética , Feminino , Linhagem , Haplótipos , Mutação , Cromatografia Líquida de Alta Pressão , alfa-Globinas/genética , Adulto , Eletroforese CapilarRESUMO
Ellagic acid (EA) is a phenolic phytochemical found in many plants and their fruits. Vaginal epithelial cells are the first line of defense against pathogen invasion in the female reproductive tract and express antimicrobial peptides, including hBD2 and SLPI. This study investigated the in vitro effects of EA (1) on vaginal innate immunity using human vaginal epithelial cells, and (2) on HPV16 pseudovirus infection. Vaginal cells were cultured in the presence or absence of EA, and the expression of hBD2 and SLPI was determined at both transcriptional and translational levels. In addition, secretion of various cytokines and chemokines was measured. Cytotoxicity of EA was determined by CellTiter-blue and MTT assays. To investigate the ability of EA to inhibit HPV16 infection, EA was used to treat HEK-293FT cells in pre-attachment and adsorption steps. We found significant increases in both hBD2 mRNA (mean 2.9-fold at 12.5 µM EA, p < 0.001) and protein (mean 7.1-fold at 12.5 µM EA, p = 0.002) in response to EA. SLPI mRNA also increased significantly (mean 1.4-fold at 25 µM EA, p = 0.01), but SLPI protein did not. Secretion of IL-2 but not of other cytokines/chemokines was induced by EA in a dose-dependent manner. EA was not cytotoxic. At the pre-attachment step, EA at CC20 and CC50 showed a slight trend towards inhibiting HPV16 pseudovirus, but this was not significant. In summary, vaginal epithelial cells can respond to EA by producing innate immune factors, and at tested concentrations, EA is not cytotoxic. Thus, plant-derived EA could be useful as an immunomodulatory agent to improve vaginal health.
Assuntos
Ácido Elágico , Papillomavirus Humano 16 , Imunidade Inata , Infecções por Papillomavirus , Vagina , Humanos , Feminino , Ácido Elágico/farmacologia , Imunidade Inata/efeitos dos fármacos , Vagina/virologia , Vagina/imunologia , Vagina/efeitos dos fármacos , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/tratamento farmacológico , Citocinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Células Epiteliais/metabolismo , Células Epiteliais/imunologia , beta-Defensinas/metabolismo , Células HEK293RESUMO
OBJECTIVES: Ellagic acid (EA) has a wide range of biological effects. The purpose of this study was to investigate the in vitro effects of EA on HIV-1 replication, viral enzyme activity and cytokine secretion by infected cells. METHODS: The anti-HIV-1 activity of EA in solution was determined in vitro using the infection of TZM-bl cells by the nano luciferase-secreting R5-tropic JRCSF strain of HIV-1, which allows for the quantification of viral growth by measuring nano luciferase in the culture supernatants. The effect of EA on the cytokine secretion of TZM-bl cells was determined by a multiplexed bead array after 48 h of HIV-1 exposure. The antiviral effect of EA in the gel formulation (Ellagel), as would be used for vaginal application, was investigated by the inhibition of infection of UC87.CD4.CCR5 cells with R5-tropic pBaLEnv-recombinant HIV-1. RESULTS: EA in solutions of up to 100 µM was not toxic to TZM-bl cells. EA added either 1 h before or 4 h after HIV-1 exposure suppressed the replication of R5-tropic HIV-1 in TZM-bl cells in a dose-dependent manner, with up to 69% inhibition at 50 µM. EA-containing solutions also exhibited a dose-dependent inhibitory effect on HIV-1 replication in U87 cells. When EA was formulated as a gel, Ellagel containing 25 µM and 50 µM EA inhibited HIV-1 replication in U87 cells by 56% and 84%, respectively. In assays of specific HIV-1 enzyme activity, Ellagel inhibited HIV-1 integrase but not protease. EA did not significantly modulate cytokine secretion. CONCLUSIONS: We conclude that EA either in solution or in a gel form inhibits HIV infection without adverse effects on target cells. Thus, gel containing EA can be tested as a new microbicide against HIV infection.
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Anti-Infecciosos , Infecções por HIV , HIV-1 , Feminino , Humanos , Infecções por HIV/tratamento farmacológico , Ácido Elágico/farmacologia , Anti-Infecciosos/farmacologia , Citocinas/farmacologiaRESUMO
Interaction of structural hemoglobin (Hb) variants with α- or ß-globin defects are occasional in Southeast Asia. Herein we provide the first description of Hb Athens-Georgia (Hb A-Ga) in association with deletional Hb H disease, a novel combination previously undescribed in the population. Hematological, Hb and DNA analysis, and ß-globin haplotype analyses were performed in seven participants from one ethnic Thai family. Hemoglobin analysis by capillary electrophoresis revealed an abnormal Hb fraction in the proband, his father and grandmother (I-2). DNA sequencing revealed that the G > A substitution at codon 40 of the ß-globin gene was identical to the Hb A-Ga (HBB:c.122G > A). Interestingly, α-thal-1 (SEA deletion) and α-thal-2 (-α3.7 deletion) were identified in the proband resulting in Hb H disease, while α-thal-1 was identified in the father, and no α-thal was observed in I-2. Hematological analysis indicated that the proband (ßA-Ga/ßA, -SEA/-α3.7) had moderate anemia and was markedly hypochromic with microcytic red blood cells (RBCs). The father (ßA-Ga/ßA, -SEA/αα) presented mild microcytic anemia, while normal hematology was observed in the I-2 who was heterozygous for Hb Athens-Georgia (ßA-Ga/ßA, αα/αα). The relative level of Hb A-Ga was distinctly reduced according to the degree of α-globin defects. The developed allele-specific PCR method can successfully be used for confirmation of Hb A-Ga. The Thai Hb A-Ga allele associated with a ß-haplotype [+ - - - - - +]. These findings were in accordance with the previous conclusion that this variant is a non-pathological ß-Hb variant.
Assuntos
Hemoglobinas Anormais/genética , alfa-Globinas/genética , Talassemia alfa/diagnóstico , Talassemia alfa/genética , Sequência de Bases , Eletroforese Capilar , Família , Feminino , Haplótipos/genética , Humanos , Masculino , Mutação/genética , Linhagem , Tailândia , Adulto JovemRESUMO
Hb H diseases with the clinical features of thalassemia are found in many parts of the world, including Southeast Asia and southern China. There are limitations in molecular data from the population of Thailand, which includes multiple ethnic groups. Here, we characterized the molecular basis of the disease among a large cohort from this region. A total of 479 unrelated Thai patients with Hb H disease were studied. Mutations of the α-globin gene were characterized by conventional gap-PCR and rare genotypes were identified by MLPA analysis and direct DNA sequencing. The molecular characterization showed five common Hb H genotypes (472/479; 98.54%), including three deletional types (-SEA/-α3.7; n = 312), (-SEA/-α4.2; n = 26), (-THAI/-α3.7; n = 1) and two non-deletional types (-SEA/αCSα; n = 131), (-SEA/αPakséα; n = 2). Herein, we firstly report a rare genotype of Hb H disease with (-SA/-α3.7; n = 1) that has not been documented in Thailand, and rare genotypes related to (-SEA/-α16.6; n = 1), and (-SEA/αQSα; n = 3) as well. The remaining two cases could not be characterized. The hematological parameters demonstrated that the clinical phenotype of non-deletional Hb H diseases is more severe than the deletional type of α+-thalassemia. The molecular spectrum of α-thalassemia is useful for prevention and thalassemia control and genetic counseling for couples at risk in this region.
Assuntos
Deleção de Genes , Talassemia alfa/genética , Sequência de Bases , Humanos , Reação em Cadeia da Polimerase , Tailândia , alfa-Globinas/genética , Talassemia alfa/diagnósticoRESUMO
Elevated Hb A2 level (≥4.0%) is considered to be reliable parameter to identify ß-thalassemia (ß-thal) carriers. However, some ß-thal carriers have been misdiagnosed as their Hb A2 levels are below 4.0%. In addition, coinheritance of α-thalassemia (α-thal) and ß-thal might affect Hb A2 levels. Therefore, the aim of this study was to investigate the mutations of ß- and α-globin genes in individuals with borderline Hb A2 levels in Thailand. Three hundred samples from individuals with Hb A2 levels of 3.5-3.9% were collected for molecular diagnosis of ß-globin gene mutations. In addition, the α0-thal, α+-thal, Hb Constant Spring (Hb CS, HBA2: c.427T>C), and Hb Paksé (HBA2: c.429A>T) diagnostics were also performed. Sixteen samples (5.33%) had ß-globin gene mutations, and codon 41/42 (-TTCT) (HBB: c.126_129delCTTT) was the most prevalent mutation. Ninety-eight samples (32.67%) had α-globin gene mutations including four Hb H (ß4)-Hb CS disease, two Hb H disease, 13 heterozygous α0-thal, 11 homozygous α+-thal, two α+-thal/Hb CS, one α+-thal/Hb Paksé, 61 heterozygous α+-thal, and four Hb CS. Furthermore, seven cases of ß-thal carriers coinheriting α-thal were observed, and five of them carried Hb H disease. High prevalence of both α- and ß-thal in subjects with borderline Hb A2 levels suggested that molecular diagnosis of α- and ß-thal should be performed, especially in a high prevalence area of thalasssemia carriers, for accurate diagnosis and genetic counseling to prevent and control new severe thalassemia cases. Moreover, ß-thal carriers who coinherited Hb H disease might have reduced Hb A2 levels, leading to a misdiagnosis of ß-thal in analysis programs.
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Hemoglobina A2/genética , Mutação , alfa-Globinas/genética , Talassemia alfa/genética , Globinas beta/genética , Talassemia beta/genética , Alelos , Índices de Eritrócitos , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Fenótipo , Prevalência , Talassemia alfa/complicações , Talassemia alfa/diagnóstico , Talassemia alfa/epidemiologia , Talassemia beta/complicações , Talassemia beta/diagnóstico , Talassemia beta/epidemiologiaRESUMO
Hb E [ß26(B8)GluâLys, GAG>AAG, HBB: c.79G>A] is an inherited thalassemic ß-globin variant that favors the Hb E-ß-thalassemia (ß-thal) syndrome when interacting with the ß-thal gene. However, hemoglobin (Hb) variants carrying Hb E in combination with another variant on the same ß gene are rare. We recently studied a 29-year-old pregnant woman, initially diagnosed as a ß-thal carrier. Hemoglobin and DNA analysis were performed by high performance liquid chromatography (HPLC) and DNA sequencing. Hematological data revealed no anemia or altered red blood cell (RBC) parameters. Hemoglobin HPLC showed Hb A and Hb A2 but no Hb E or abnormal Hb peaks, with a markedly elevated Hb A2 level (6.4%) reaching the accepted range (4.0-10.0%) for ß-thal trait. DNA analysis identified a GAG>AAG transition at codon 26 of the ß-globin gene that is responsible for Hb E, and an AAG>AAC mutation at codon 65 in cis on the ß-globin chain resulting in a lysine to asparagine substitution. These two mutations led to the formation of a novel variant, namely Hb E-Myanmar, ß26(B8)GluâLys and ß65(E9)LysâAsn, HBB: c.[79G>A;198G>C]. Moreover, a heterozygous α-thalassemia-2 (α-thal-2) [-α3.7 (rightward)] deletion was also observed. Hb E-Myanmar is a doubly substituted ß-globin variant, which has not been previously described. This variant did not have any clinical or hematological abnormalities, and the genetic mechanism resulting in this variant is discussed. The new simultaneous allele-specific polymerase chain reaction (ASPCR) was developed for rapid detection of these two mutations within the same ß-globin chain.
Assuntos
Alelos , Substituição de Aminoácidos , Hemoglobina E/genética , Mutação , Globinas beta/genética , Talassemia beta/diagnóstico , Talassemia beta/genética , Adulto , Cromatografia Líquida de Alta Pressão , Análise Mutacional de DNA , Índices de Eritrócitos , Feminino , Variação Genética , Genótipo , Heterozigoto , Humanos , Gravidez , Análise de Sequência de DNA , Talassemia beta/sangueRESUMO
Hb Q-Thailand [α74(EF3)AspâHis (α1), GAC>CAC, HBA1: c.223G>C] is an abnormal hemoglobin (Hb) frequently found in Thailand and Southeast Asian countries. The association of the αQ-Thailand allele with other globin gene disorders has important implications in diagnosis. Here, we report how to diagnose the coinheritance of Hb Q-Thailand with ß-thalassemia (ß-thal)/Hb E disease in four Thai samples from high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) testing results. Understanding of the HPLC chromatogram and CE electropherogram patterns of this complex mutation is important for interpretation of testing results and providing genetic counseling.
Assuntos
Hemoglobina E/genética , Hemoglobinopatias/genética , Hemoglobinas Anormais/genética , Cromatografia Líquida de Alta Pressão , Eletroforese Capilar , Hemoglobinopatias/diagnóstico , TailândiaRESUMO
BACKGROUND: Ellagic acid (EA) found in various fruits such as pomegranates, blackberries, raspberries, strawberries, and walnuts has different pharmacological functions including antioxidant, antitumor, antiallergic, anti-inflammatory, antibacterial, and antiviral activities. It is not known, however, if EA could enhance mucosal innate immunity. Our goal was to determine the effects of EA on the expression of innate immune mediators produced by oral epithelial cells. METHODS: Culture of primary human gingival epithelial cells (HGEs) was performed in duplicate, and after the primary HGEs had been treated with EA at a concentration ranging from 12.5 to 100 µM for 18 h the cells and supernatants were harvested. The expression of innate immune mediators including human ß-defensin 2 (hBD2), secretory leukocyte protease inhibitor (SLPI), and various cytokines and chemokines was measured at both transcriptional and translational levels by using quantitative real-time PCR, ELISA, and Luminex assay. RESULTS: In the presence of EA, the expression of hBD2-and SLPI mRNA was 3.7-folds and 2.6-folds greater than untreated controls, respectively, and consistent with their secreted protein levels. For cytokines and chemokines, increased expression of RANTES, IL-2, and IL-1ß was found in response to EA. In contrast, EA decreased the expression of IL-6, IL-8, and TNF-α. CONCLUSIONS: This study demonstrated that oral innate immunity is affected by EA found in fruits. Thus, it may play some roles in mucosal innate immunity. The potential of EA for modulating the innate immune mediators may lead to developing a new topical agent to treat and/or prevent immune-mediated oral diseases.
Assuntos
Ácido Elágico/farmacologia , Gengiva/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Técnicas de Cultura de Células , Células Cultivadas , Quimiocina CCL20/efeitos dos fármacos , Quimiocina CCL5/efeitos dos fármacos , Quimiocina CXCL5/efeitos dos fármacos , Quimiocinas/efeitos dos fármacos , Citocinas/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Gengiva/citologia , Humanos , Imunidade Inata/imunologia , Interleucina-1beta/efeitos dos fármacos , Interleucina-2/análise , Interleucina-6/análise , Interleucina-8/efeitos dos fármacos , Fosfoproteínas/efeitos dos fármacos , Proteínas Ribossômicas/efeitos dos fármacos , Inibidor Secretado de Peptidases Leucocitárias/efeitos dos fármacos , Fator de Necrose Tumoral alfa/efeitos dos fármacos , beta-Defensinas/efeitos dos fármacosRESUMO
BACKGROUND: Vaginal epithelial cells (VECs) produce antimicrobial peptides including human ß-defensin 2 (hBD2) and secretory leukocyte protease inhibitor (SLPI), as well as cytokines and chemokines that play vital roles in mucosal innate immunity of the female reproductive tract. Houttuynia cordata Thunb (H. cordata), a herbal plant found in Asia, possesses various activities including antimicrobial activity and anti-inflammation. As inflammation and infection are commonly found in female reproductive tract, we aimed to investigate the effects of H. cordata water extract in modulating innate immune factors produced by VECs. METHODS: Primary human VECs were cultured and treated with H. cordata at a concentration ranging from 25-200 µg/ml for 6 or 18 h. After treatment, the cells and culture supernatants were harvested. The expression of hBD2 and SLPI mRNA was evaluated by quantitative real-time reverse transcription PCR. Levels of secreted hBD2 and SLPI as well as cytokines and chemokines in the supernatants were measured by ELISA and Luminex assay, respectively. Cytotoxicity of the extract on VECs was assessed by CellTiter-Blue Cell Viability Assay. RESULTS: H. cordata did not cause measurable toxicity on VECs after exposure for 18 h. The expression of hBD2 and SLPI mRNA as well as the secreted hBD2 protein were increased in response to H. cordata exposure for 18 h when compared to the untreated controls. However, treatment with the extract for 6 h had only slight effects on the mRNA expression of hBD2 and SLPI. The secretion of IL-2 and IL-6 proteins by VECs was also increased, while the secretion of CCL5 was decreased after treatment with the extract for 18 h. Treatment with H. cordata extract had some effects on the secretion of IL-4, IL-8, CCL2, and TNF-α, but not statistically significant. CONCLUSIONS: H. cordata water extract modulates the expression of antimicrobial peptides and cytokines produced by VECs, which play an important role in the mucosal innate immunity in the female reproductive tract. Our findings suggest that H. cordata may have immunomodulatory effects on the vaginal mucosa. Further studies should be performed in vivo to determine if it can enhance mucosal immune defenses against microbial pathogens.
Assuntos
Células Epiteliais , Houttuynia/química , Extratos Vegetais/farmacologia , Vagina , Células Cultivadas , Citocinas/análise , Citocinas/genética , Citocinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Feminino , Humanos , Extratos Vegetais/química , Vagina/efeitos dos fármacos , Vagina/imunologia , Vagina/metabolismoRESUMO
BACKGROUND: Excessive expression of hemoglobin F (HbF) is a characteristic feature and important diagnostic marker of ß0-thalassemia/HbE disease. However, some patients may exhibit low-HbF levels, leading to misdiagnosis and precluding genetic counseling. The genetic factors influencing these differences in HbF expression in this atypical disease are not completely understood. AIMS: To investigate determinants contributing to the non-elevation of HbF expression in ß0-thalassemia/HbE disease. METHODS: We studied 231 patients with ß0-thalassemia/HbE confirmed by DNA analysis; classified them into the low-HbF (n = 62) and high-HbF (n = 169) groups; analyzed hematological parameters and hemoglobin levels in both groups; and characterized mutations in ß- and α-globin genes and genetic variants in γ-globin promoters. RESULTS: Both groups showed similar rates of type ß0-thalassemia mutations but significantly different proportions of α-globin mutations: approximately 88.7% (95% confidence interval [CI] = 66.8-115.5) and 39.1% (95% CI = 30.2-49.7) in the low- and high-HbF groups, respectively. The results revealed single-nucleotide polymorphisms (SNPs) at -158 (C>T) in the Gγ-globin promoters and novel SNPs at the 5' untranslated region position 25 (G>A) in Aγ-globin promoters. The distribution of CC genotypes of the Gγ-globin promoter in the low-HbF group was significantly higher than that in the high-HbF group. CONCLUSIONS: Cases with HbE predominance with low-HbF levels and undetectable HbA may not be as conclusive as those with homozygous HbE until DNA analysis is performed. Concomitant inheritance of α-thalassemia is an important inherent factor modifying HbF expression in a typical ß0-thalassemia/HbE, and SNPs with the CC genotype in the Gγ-globin promoter may indicate unelevated HbF expression in patients with this disease.
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Background and aims: We aimed to evaluate the efficiency of identification and quantification of hemoglobin (Hb) Constant Spring (CS) and Hb Paksé by capillary electrophoresis (CE). Materials and methods: Blood samples collected from 2057 patients were used for identifying and quantifying Hb by CE. Molecular analysis of α- and ß-thalassemia, Hb CS, and Hb Paksé was performed. Results: Hb CS and Hb Paksé were identified in 573 samples (27.86%) with diverse genotypes. Thirty-eight samples (6.6%) showed no Hb CS peak. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of Hb CS by CE were 93.37, 95.96, 89.92, 97.40, and 95.24%, respectively. The amount of Hb CS in those carrying Hb CS was 0.2-6.5% which showed an increasing trend according to the number of defective α-globin genes, in contrast to Hb A2 levels, which decreased. Hb CS level ≥1.0% accurately excluded heterozygotes and that of ≥2.0% could identify homozygotes. Conclusion: CE has the high potential for identifying and quantifying Hb CS and Hb Paksé, especially in an area with a high prevalence of thalassemia. Hb CS levels can be used as a potential marker to distinguish the genotype of individuals carrying Hb CS.
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BACKGROUND: An increasing number of α-hemoglobin (Hb) variants is causing various clinical symptoms; therefore, accurate identification of these Hb variants is important. OBJECTIVE: This study aimed to describe the molecular and hematological characteristics of novel Hb Doi-Saket that gives rise to a typical α+-thalassemia phenotype in carriers with and without other hemoglobinopathies. MATERIALS AND METHODS: Biological samples from a proband and his family members were analyzed. Hematological profiles were analyzed using a standard automated cell counter. Hb was analyzed by capillary electrophoresis and high-performance liquid chromatography. Mutations and globin haplotype were identified by DNA analysis. Novel diagnostic tools based on allele-specific polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism were developed. RESULTS: Hb analysis showed a major abnormal Hb fraction, moving slower than HbA, and a minor Hb fraction alongside HbA2 in the proband, his father, and son. DNA analysis of the α-globin gene identified the -α3.7 deletion and in cis the C > A mutation on codon 9 of the α2α1 gene, corresponding to Hb Doi-Saket [α9(A7) Asn > Lys]. This mutation could be identified using newly developed allele-specific PCR-based assays. The Hb Doi-Saket al.lele was significantly associated with haplotype [- + M + + 0 -]. Interaction of αDoi-Saket with ßE globin chains led to a new Hb variant (HbE Doi-Saket). Phenotypic expression was clinically silent in heterozygotes and might present slight microcytosis. CONCLUSIONS: Hb Doi-Saket emphasizes a great diversity present in α-globin gene. The mutation in this family from Thailand was linked to -α3.7 and caused mild microcytosis in the carriers. The combination of this variant with deletions in α genes might cause a severe clinical phenotype. Different methods of separation can provide useful information in diagnosis, and a complete molecular approach is needed for confirmation before considering patient management.
The Hb Doi-Saket is a novel α-globin variant mutation occurring in the α2-globin gene in cis to the -α3.7 kb chromosome.The carrier of Hb Doi-Saket may present slight microcytosis and have severe clinical entities when it interacts with deletions in α-globin genes.Hb analysis with the HPLC system could completely separate Hb Doi-Saket and its derivative from other Hbs.
Assuntos
Hemoglobina E , Hemoglobinas Anormais , Talassemia alfa , Humanos , Hemoglobina E/genética , Tailândia , População do Sudeste Asiático , Talassemia alfa/diagnóstico , Talassemia alfa/genética , Hemoglobinas Anormais/genética , Mutação , DNA , alfa-Globinas/genéticaRESUMO
BACKGROUND: Low HbF expression in HbE-ß+-thalassemia may lead to misdiagnosis of HbE heterozygosity. We aimed to characterize the ß- and α-globin genes and the modifying factors related to HbF expression in patients with an Hb phenotype similar to that of HbE heterozygotes. Furthermore, screening tools for differentiating HbE-ß+-thalassemia from HbE heterozygotes have been investigated. PARTICIPANTS AND METHODS: A total of 2133 participants with HbE and HbA with varying HbF levels were recruited. Polymerase chain reaction-based DNA analysis and sequencing were performed to characterize ß- and α-globin genes. DNA polymorphism at position -158 nt 5' to Gγ-globin was performed by XmnI restriction digestion. Receiver operating characteristic (ROC) curves were constructed using the area under the curve (AUC). Cutoff values of HbA2, HbE, and HbF levels for the differentiation of HbE-ß+-thalassemia from HbE heterozygotes were determined. RESULTS: Five ß+-thalassemia mutations trans to ßE-gene (ß-87(C>A), ß-31(A>G), ß-28(A>G), ß19(A>G), and ß126(T>G)) were identified in 79 patients. Among these, 54 presented with low HbF levels, and 25 presented with high HbF levels. ROC curve analysis revealed an excellent AUC of 1.000 (95% confidence interval:1.000-1.000) for HbE levels, and a cut-off point of ≥35.0% had 100.0% sensitivity, specificity, and Youden's index for differentiating HbE-ß+-thalassemia from HbE heterozygotes. The proportion of α-thalassemia mutations was 46.3 and 8.0% among HbE-ß+-thalassemia patients with low and high HbF levels, respectively. Two rare α-thalassemia mutations (Cap +14(C>G) and initiation codon (ATG>-TG)) of α2-globin genes were identified. The genotype and allele of the polymorphism at -158 nt 5' to Gγ-globin was found to be negatively associated with HbF expression. CONCLUSIONS: HbE-ß+-thalassemia cannot be disregarded until appropriate DNA analysis is performed, and the detection of α-thalassemia mutations should always be performed under these conditions. An HbE level ≥35.0% may indicate screening of samples for DNA analysis for HbE-ß+-thalassemia diagnosis.
HbE-ß+-thalassemia displays a wide range of HbF expression, which may lead to the misdiagnosis of HbE heterozygosity in patients whose Hb analysis shows HbE and HbA. α-Thalassemia may be a major factor associated with decreased secondary activation of HbF expression in the disease.HbE may be a potential indicator for effectively differentiating HbE-ß+-thalassemia from HbE heterozygotes.The high proportion and heterogeneity of α-thalassemia mutations found in patients with HbE-ß+-thalassemia evoke a complex thalassemia syndrome, requiring complete DNA analysis.
Assuntos
Hemoglobina E , Talassemia alfa , Talassemia beta , Humanos , Heterozigoto , gama-Globinas/genética , Hemoglobina E/genética , Hemoglobina E/análise , Hemoglobina E/metabolismo , Hemoglobina Fetal/genética , Hemoglobina Fetal/análise , Hemoglobina Fetal/metabolismo , Talassemia beta/diagnóstico , Talassemia beta/genética , Fenótipo , DNA , alfa-Globinas/genéticaRESUMO
The interactions of δ-globin variants with α- and ß-thalassemia or other hemoglobinopathies cause complex thalassemic syndromes and potential diagnostic problems. Understanding the molecular basis and phenotypic expression is crucial. Four unrelated Thai subjects with second hemoglobin (Hb) A2 fractions were studied. A standard automated cell counter was used to acquire initial hematological data. Hb analysis was carried out by capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) assays. Globin gene mutations and haplotype were identified by appropriate DNA analysis. An allele-specific polymerase chain reaction method was developed to provide a simple molecular diagnostic test. Hb analysis revealed a Hb A2 variant in all cases. DNA analysis of the δ-globin gene identified the Hb A2-Melbourne [δ43(CD2)Glu > Lys] variant in combination with Hb E in three cases. Analysis of the remaining case identified a novel δ-Hb variant, namely Hb A2-Mae Phrik [δ52(D3)GAT > GGT; Asp > Gly], found in association with Hb E and α+-thalassemia, indicative of the as yet undescribed combination of triple heterozygosity of globin gene defects. An allele-specific PCR-based assay was successfully developed to identify this variant. The ß-haplotype of the Hb A2 Mae-Phrik allele was strongly associated with haplotype [+ − − − − ± +]. This study advanced our understanding of the phenotypic expression of known and novel δ-Hb variants coinherited with other globin gene defects, routinely causing problems with diagnosis. Therefore, knowledge and recognition of this Hb variant and molecular assessments are crucial to improving diagnosis.