Epidemiological Survey of Hemoglobinopathies Based on Next-Generation Sequencing Platform in Hunan Province, China / 生物医学与环境科学（英文）

OBJECTIVE@#This study was aimed at investigating the carrier rate of, and molecular variation in, α- and β-globin gene mutations in Hunan Province.@*METHODS@#We recruited 25,946 individuals attending premarital screening from 42 districts and counties in all 14 cities of Hunan Province. Hematological screening was performed, and molecular parameters were assessed.@*RESULTS@#The overall carrier rate of thalassemia was 7.1%, including 4.83% for α-thalassemia, 2.15% for β-thalassemia, and 0.12% for both α- and β-thalassemia. The highest carrier rate of thalassemia was in Yongzhou (14.57%). The most abundant genotype of α-thalassemia and β-thalassemia was -α 3.7/αα (50.23%) and β IVS-II-654/β N (28.23%), respectively. Four α-globin mutations [CD108 (ACC>AAC), CAP +29 (G>C), Hb Agrinio and Hb Cervantes] and six β-globin mutations [CAP +8 (C>T), IVS-II-848 (C>T), -56 (G>C), beta nt-77 (G>C), codon 20/21 (-TGGA) and Hb Knossos] had not previously been identified in China. Furthermore, this study provides the first report of the carrier rates of abnormal hemoglobin variants and α-globin triplication in Hunan Province, which were 0.49% and 1.99%, respectively.@*CONCLUSION@#Our study demonstrates the high complexity and diversity of thalassemia gene mutations in the Hunan population. The results should facilitate genetic counselling and the prevention of severe thalassemia in this region.

Detección de variantes de hemoglobina en pacientes examinados por hemoglobina A1c / Detection of hemoglobin variants in patients tested for Hemoglobin A1c

Resumen Justificación: La genética en las variantes de hemoglobina en Costa Rica es resultado del cruce de caracteres autóctonos indígenas con poblaciones inmigrantes de europeos, africanos y otros, desde el periodo de la Conquista, que contribuyeron a la mezcla genética presente en la población de Costa Rica. Las hemoglobinopatías mayormente distribuidas en la población humana son: hemoglobina S, C, D y E, siendo la hemoglobina S la más frecuente y la que presenta consecuencias más graves. Objetivo: Detectar variantes de hemoglobina en pacientes examinados por hemoglobina A1c, en la sección de Química Clínica del laboratorio de la Clínica de Filadelfia de la Caja Costarricense de Seguro Social, en el Cantón de Carrillo, Guanacaste, Costa Rica, durante el período de enero a octubre de 2018. Métodos: Se analizaron 2775 muestras sanguíneas de pacientes de los nueve equipos básicos de salud que conforman el Área de Salud de Carrillo, y que además requieren estudio por hemoglobina glicosilada en el período de enero a octubre de 2018. El análisis se realizó en el Laboratorio del Área de Salud de Carrillo. Las muestras fueron recolectadas en tubos vacutainer con EDTA y analizadas en el equipo automatizado TOSOH HLC-723GX, utilizando la metodología HPLC cromatografía de intercambio catiónico con la separación y cuantificación de las diferentes fracciones de hemoglobina. Los datos se analizaron en plantilla de Microsoft Excel. Resultados: En 2775 pacientes examinados por hemoglobina A1c, 167 (6,0 %) fueron portadores de variantes de hemoglobina, con una frecuencia de 1/17, en donde el 97 % correspondió a heterocigotos para hemoglobina un 3% a heterocigotos para hemoglobina C y ninguno para la variante D. La presencia de variantes se observó en los 9 equipos básicos de atención integral en salud del área. La distribución de portadores por equipo básico de atención en el Área de Salud de Carrillo varió de un 4,0 % a un 9,3 %. Conclusiones: Un 6 % de las muestras analizadas presentó variantes de hemoglobina, siendo la hemoglobina S la predominante. Esta característica presente en la población del cantón de Carrillo merece atención a nivel de salud pública; la metodología existente a nivel de área permite estudiar a un grupo de población (costo efectivo) en riesgo que precisa vigilancia y asesoramiento genético, con el fin de concienciar a la población respecto al problema, reducir la incidencia de la enfermedad y prolongar la supervivencia de los afectados.

Abstract Background: The genetics in hemoglobin variants in Costa Rica are a result of the crossing of autochthonous indigenous characters with European, African and other immigrant populations. All of these contributed to the genetic mixture that is currently present in Costa Rica's population. The most distributed hemoglobinopathies in the human population are: hemoglobin S, C, D, and E, with hemoglobin S being the most frequent and having the most serious consequences. Objective: Detection of hemoglobin variants in patients who were examined for hemoglobin A1c in the Clinical Chemistry section of the Filadelfia Clinic Laboratory, from January to October 2018. The clinic is in Canton of Carrillo, Guanacaste (Costa Rica), and it is part of the social security system. Methods: 2775 blood samples and their respective data were analyzed from patients of the nine basic health teams that make up the Carrillo Health Area and required a study for glycosylated hemoglobin from January to October 2018. The analysis was performed in the Carrillo Health Area Laboratory. The samples were collected in vacutainer tubes containing EDTA and analyzed in the TOSOH HLC-723GX automated equipment, using the HPLC cation exchange chromatography methodology with the separation and quantification of the different hemoglobin fractions. The data was then analyzed in a Microsoft Excel template. Results: In the 2775 patients examined for hemoglobin A1c, 167 (6.0%) were found to be carriers of hemoglobin variants, with a frequency of 1/17, where 97% corresponded to heterozygotes for hemoglobin S, 3% heterozygous for hemoglobin C, and none for variant D. The presence of variants was observed in the 9 basic teams of integral health care of the area, and the distribution varied from 4% to 9,3% between them. Conclusions: A total of 6% of the samples analyzed showed a hemoglobin variant, being hemoglobin S the most predominant. This characteristic present in the population of Canton of Carrillo deserves attention at the public health level. The existing methodology at the level area allows professionals to study a population group at risk that deserves surveillance and genetic counseling, in order to raise awareness about the problem, reduce the incidence of the disease, and prolong the survival of those affected by it.

Effect of double heterozygous hemoglobin on the results of different glycosylated hemoglobin detection systems / 临床检验杂志

Objective@#To investigate the effects of glycosylated hemoglobin A1c (HbA1c) from the patients with double heterozygotes Hb Q-H and Hb J-Bangkok combined with β-thalassemia on the results of different HbA1c detection systems. @*Methods@#Blood samples from 20 healthy adults and 20 patients with type 2 diabetes mellitus (T2DM) were collected to assess the results of five glycosylated hemoglobin detection systems. Blood samples from one Hb Q-H patient and one Hb J-Bangkok patient with β-thalassemia were also collected, and they were performed hemoglobin capillary electrophoresis with Capillarys2 and globin gene analysis by gap-PCR, PCR-RDB and DNA sequencing. The levels of HbA1c in all samples were detected by BioRad VARIANT Ⅱ (VⅡ), BioRad VARIANT ⅡTurbo2.0 (V Ⅱ-T2.0), Capillarys 2 Flex Piercing (C2FP), Primus Ultra2 (Ultra2) and Roche PPI 800 (PPI 800) glycosy lated hemoglobin detection instruments, respectively. For the samples with double heterozygotes, the levels of HbA1c were detected for 3 times each sample, and the results were preserved and analyzed. @*Results@#The genotype of the Hb Q-H sample was --α QT /--SEA;β N /β N , and HbA1 CD74 G＞C mutation occurred in globin α1 chain, forming Hb Q-Thailand hemoglobin variant without normal α-globin peptide chain. The genotype of Hb J-Bangkok combined with β-thalassemia was αα/αα;βCD56/βCD41-42, and the point mutation of GGC＞GAC occurred at codon 56 of globin β-chain, forming Hb J-Bangkok hemoglobin variant without normal β-globin peptide chain. For the Hb Q-H sample, HbA1c results were reported by 3 of 5 HbA1c detection systems. The chromatograms of VⅡ and VⅡ-T2.0 detection systems were obviously different from normal chromatograms, and HbA1c results were not reported. However, the chromatograms of the C2FP system were similar to normal chromatograms, and the result of HbA1c was 3.7%. The Ultra2 system and PPI system reported the HbA1c results, 5.3% and 5.7%, respectively, without abnormal alarm. For the Hb J-Bangkok with β-thalassemia sample, HbA1c results were also reported by 3 of 5 HbA1c detection systems. The chromatograms of VⅡ and Sebia detection systems were obviously different from normal chromatograms, and HbA1c results were not reported. However, the chromatograms of VⅡ-T2.0 system were different from normal chromatograms, and a P4 peak (84.9%) was found. The HbA1c result was reported as 4.7%. The Ultra2 system and PPI system reported the HbA1c results, 4.7% and 3.8%, respectively, without abnormal alarm. @*Conclusion@#The samples from the Hb Q-H patient and the Hb J-Bangkok patient with β-thalassemia do not contain normal HbA, and there should be no HbA1c results. The chromatograms of VⅡ and VⅡ-T systems are obviously abnormal, indicating that the results can not be reported. The C2FP system is interfered obviously by Hb Q-H, but reports the HbA1c results, while it does not report the HbA1c results of Hb J-Bangkok combined with β-thalassemia. Both of Hb Q-H and Hb J-Bangkok have obvious interference to PPI and Ultra2 detection systems.

Effects of common hemoglobin variants on 4 ion exchange high performance liquid chromatography methods based HbA1c measurements / 中华检验医学杂志

Objective To evaluate effects of 7 common hemoglobin variants on HbA 1c measurements using 4 ion exchange high performance liquid chromatography methods .Methods Ninety five samples with hemoglobin variants were collected from January 2017 to February 2018 during HbA1c measurements in laboratary medicine of peking university shenzhen hospital .Samples with 7 common hemoglobin variants were measured using Sebia Capillary 2 Flex Piercing, Bio-Rad D-10, Arkray HA8180V, Tosoh G8, and MQ6000 Plus, respectively.Effects of 7 common hemoglobin variants on HbA 1c measurements by the 4 methods were analyzed using Capillary 2 Flex Piercing as a comparative method .All statistical analyses were carried out using SPSS software version 19.0 .Mean bias were calculated for samples with hemoglobin variants , box plot was established to display bias distribution .Results Hb New York showed no interference on the 4 HPLC mechods although Hb New York could not be detected .D-10 could detect 6 Hb variants, and showed clinically significant interference for Hb J-Bangkok, Hb G-Coushatta, and Hb G-Taipei.HA-8180V fast mode yielded no HbA1c values for Hb J-Bangkok, Hb G-Coushatta, and Hb G-Taipei.Hb E, Hb Q-Thailand, and Hb G-Honolulu produced significant negative biases for HA-8180V.G8 standard mode could detect 1 Hb variant, and showed significant negative biases for six Hb variants .MQ6000 Plus could separate six Hb variants , only Hb G-Coushatta and Hb G-Taipei produced significant negative biases for the system . Conclusions Some common hemoglobin variants can interfere with HbA 1c determination by the most popular methods in South China , which may lead to erroneous HbA 1c values.

Anemia ferropénica y variantes de hemoglobina en niños de Caracas / Irion deficiency anemia and hemoglobin variants in children from Caracas

La anemia es un problema de salud publica que afecta a poblaciones tanto en paises desarrollados como en vias de desarrollo. Dentro de los factores mas frecuentemente relacionados al desarrollo de anemia se encuentra la deficiencia de hierro. En nuestro pais se ha reportado en diferentes publicaciones una frecuencia de aproximadamente un 50% de anemia en ninos. Objetivo: Evaluar la prevalencia de anemia ferropenica y variantes de hemoglobina en una poblacion infantil de la Parroquia San Juan de Caracas. Metods: Poblacion. Se estudiaron 336 individuos, con edades comprendidas entre 5 y 13 anos estudiantes de la Unidad Educativa Agustin Zamora Quintana de la ciudad de Caracas. Se conto con el consentimiento informado del padre o representante del nino; a cada uno se le tomo una muestra de sangre en tubo con EDTA y una en tubo seco. Métodos: Se realizo hematologia completa, electroforesis de hemoglobina a pH alcalino, cromatografia liquida de alta resolucion y dinamica de hierro (hierro serico, capacidad de fijacion de hierro y ferritina). Resultados: Se obtuvo un 0,87% de individuos anemicos de los cuales 0,59% tenian deficiencia de hierro. El 2,97% de los ninos presento hemoglobinas anormales (8 A/S, 2 A/C). Conclusión: el porcentaje de individuos anemicos o con deficiencias de hierro fue considerablemente bajo con respecto a lo reportado por diferentes publicaciones en nuestro pais, mientras que la frecuencia de hemoglobinopatias halladas se corresponde con otros estudios realizados en la poblacion venezolana.

Anemia is a public health problem that affects populations in developed and developing countries. The most common factor related to the development of anemia is iron deficiency. In our country there have been several publications reporting a frequency of approximately 50% of anemia in children. Aim: To evaluate the prevalence of iron deficiency anemia and hemoglobin variants in children from “Parroquia San Juan. Caracas”. Methods: Population: We studied 336 individuals, with ages between 5 and 13 years, all students of the Agustin Zamora Quintana Middle School in Caracas. An informed consent was obtained from the parents or guardians. A blood sample with EDTA tube and another with no anticoagulant were obtained from every child. Complete blood count, hemoglobin electrophoresis at alkaline pH, high resolution liquid chromatography and dynamic iron studies (iron in serum, iron binding capacity and ferritin) were performed. Results: Among the studied population, 0.87% individuals were anemic, among which 0.59% had iron deficiency. 2.97% of the children presented abnormal hemoglobin (8 A / S, 2 A / C). Conclusion: The percentage of anemic individuals or with iron deficiency was significantly low compared to the percentage reported by several publications in our country, while the frequency of hemoglobin variants is in agreement with other studies in the Venezuelan population.

Hemoglobin F level in different hemoglobin variants

BACKGROUND: Fetal hemoglobin (HbF) levels in different hemoglobin variants in Osogbo, Nigeria, were estimated using two principal methods of estimation using existing information for HbF concentration and distribution of various hemoglobin variants in the area, as well as diagnosed cases of thalassemia. Two hundred and sixty samples collected from HbSS, HbSC, HbAA, HbAS, and HbAC subjects were analyzed. HbF level and hemoglobin type were determined in this study. METHODS: The hemoglobin type was determined using cellulose acetate electrophoresis at an alka-line pH, and HbF was determined by the acid elution and alkaline denaturation methods. RESULTS: The mean+/-SD of HbF in the respective hemoglobin variants was as follows: HbSS, 2.09+/-1.94%; HbSC, 0.85+/-0.54%; HbAA, 0.69+/-0.46%; HbAS, 0.52+/-0.31%; and HbAC, 0.57+/-0.26%. The mean HbF level across the hemoglobin variants was statistically significant (P<0.05). Investigating the sex distribution of the HbF level in the studied population revealed a slightly higher mean HbF level in females than in their male counterparts. CONCLUSION: Within the study population, the HbF level was found to be highest in HbSS and lowest in HbAS. The two methods of estimating HbF are equally reliable, since there was no significant difference between the results obtained from the two methods.

Detection of Hb variants and hemoglobinopathies in Indian population using HPLC: Report of 2600 cases.

Background: Inherited abnormalities of hemoglobin synthesis include a myriad of disorders ranging from thalassemia syndromes to structurally abnormal hemoglobin variants. Identification of these disorders is immensely important epidemiologically and aid in prevention of more serious hemoglobin disorders. Aims: High performance liquid chromatography (HPLC) forms an important tool for accurate and speedy diagnosis of various hemoglobin disorders. About 2600 cases have been studied for identification of various hemoglobin disorders in Indian population. Material and Methods: The study was performed on BIORAD VARIANT using beta thalassemia short program. Results and conclusion: Abnormal hemoglobin fractions on HPLC were seen in 327 of the 2,600 cases displayed. Of this, the beta thalassemia trait was the predominant abnormality with a total of 232 cases (8.9%). There were 15(0.6%) cases of beta thalassemia major and 16 of thalassemia intermedia. The rest comprised of Hb D Punjab (13 cases; 0.5%), Elevated Hb F (25 cases; 0.9%), Hb E (seven cases including two Hb E homozygous and five Hb E heterozygous), Double heterozygous Hb E-beta thal trait (six cases), Hb Q India (five cases), Double heterozygous Hb Q India -beta thal trait (two cases), Hb S (total cases three including one Hb S homozygous; two Hb S -beta thal trait) and one case each of Hb J Meerut, Hb D-Iran and Hb Lepore trait. Detection of this abnormal hemoglobin, particularly keeping in mind a high prevalence of Hb A2, will help in prevention of more serious hemoglobinopathies including beta thalassemia major. HPLC forms a rapid and accurate tool in early detection and management of various hemoglobin disorders.

Application of diagnostic methods and molecular diagnosis of hemoglobin disorders in Khuzestan province of Iran.

BACKGROUND: The hemoglobinopathies refer to a diverse group of inherited disorders characterized by a reduced synthesis of one or more globin chains (thalassemias) or the synthesis of structurally abnormal hemoglobin (Hb). The thalassemias often coexist with a variety of structural Hb variants giving rise to complex genotypes and an extremely wide spectrum of clinical and hematological phenotypes. Hematological and biochemical investigations and family studies provide essential clues to the different interactions and are fundamental to DNA diagnostics of the Hb disorders. Although DNA diagnostics have made a major impact on our understanding and detection of the hemoglobinopathies, DNA mutation testing should never be considered a shortcut or the test of first choice in the workup of a hemoglobinopathy. MATERIALS AND METHODS: A careful three-tier approach involving: (1) Full blood count (2) Special hematological tests, followed by (3) DNA mutation analysis, provides the most effective way in which to detect primary gene mutations as well as gene-gene interactions that can influence the overall phenotype. With the exception of a few rare deletions and rearrangements, the molecular lesions causing hemoglobinopathies are all identifiable by PCR-based techniques. Furthermore, each at-risk ethnic group has its own combination of common Hb variants and thalassemia mutations. In Iran, there are many different forms of α and β thalassemia. Increasingly, different Hb variants are being detected and their effects per se or in combination with the thalassemias, provide additional diagnostic challenges. RESULTS: We did step-by-step diagnosis workup in 800 patients with hemoglobinopathies who referred to Research center of Thalassemia and Hemoglobinopathies in Shafa Hospital of Ahwaz Joundishapour University of medical sciences, respectively. We detected 173 patients as iron deficiency anemia (IDA) and 627 individuals as thalassemic patients by use of different indices. We have successfully detected 75% (472/627) of the β-thalassemia mutations by using amplification refractory mutation system (ARMS) technique and 19% (130/627) of the β-thalassemia mutations by using Gap-PCR technique and 6% (25/627) as Hb variants by Hb electrophoresis technique. We did prenatal diagnosis (PND) for 176 couples which had background of thalassemia in first pregnancy. Result of PND diagnosis in the first trimester was 35% (62/176) affected fetus with β-thalassemia major and sickle cell disease that led to termination of the pregnancy. CONCLUSION: Almost all hemoglobinopathies can be detected with the current PCR-based assays with the exception of a few rare deletions. However, the molecular diagnostic service is still under development to try and meet the demands of the population it serves. In the short term, the current generation of instruments such as the capillary electrophoresis systems, has greatly simplified DNA sequence analysis.

Utilization of different methodologies for the characterization of Hb Hasharon heterozygotes

Hb Hasharon has an electrophoretic mobility similar to that of Hb S in cellulose acetate and a mobility between Hb S and C at acid pH. In high-performance liquid chromatography, Hb Hasharon shows a distinct chromatographic profile and retention time. The origin of this variant is a mutation in codon 47 (GAC --> CAC) of the alpha2-globin gene, resulting in the replacement of asparagine by histidine during the translation process. Ten blood samples from individuals suspected of being Hb Hasharon carriers were analyzed. In addition to classic laboratory tests and high-performance liquid chromatography, molecular analysis by polymerase chain reaction with restriction fragment length polymorphism designed in the laboratory was performed to confirm this mutation. The study of these cases showed that a combination of classical and molecular methodologies is necessary in the diagnosis of hemoglobinopathies for a correct hemoglobin mutant identification. The accurate identification of hemoglobin variants is essential for genetic counseling and choice of therapy.