Thalassemia Genotypes Research Articles

Difficulties in the diagnosis of HbS/beta thalassemia: Really a mild disease?

BackgroundHbS/b cases having clinical, hematologic and electrophoretic similarities cannot be sufficiently distinguished from sickle cell anemia cases and are misdiagnosed as sickle cell anemia. This study will investigate the congruence between the HPLC thalassemia scanning tests and the laboratory findings compared to the DNA sequence analysis results of the patients diagnosed with SCA between 2016 and 2020. This study also aims to indicate the current status to accurately diagnose sickle cell anemia and HbS/b in the light of hematologic, electrophoretic and molecular studies.MethodsFourteen patients who were diagnosed with SCA in hospitals at different cities in Turkey and followed by the Thalassemia Diagnosis, Treatment and Research Center, Muğla Sıtkı Koçman University were included in this retrospective study. The socio-demographic characteristics, hemogram, hemoglobin variant analysis results and DNA chain analysis results of the patients were taken from the database of the centre and then examined. The informed consents were taken from the patients. The patients were administered a survey containing questions about transfusion history and diagnostic awareness. The Beta-Thalassemia mutations were analysed using a DNA sequencer (Dade Behring, Germany) based on the Sanger method.ResultsAccording to the DNA sequence analysis, the results of these patients diagnosed with SCA in hospitals in different cities of Turkey were the following: of 14 patients, 8 had HbS/b0, and HbS/b+ and one had HbS carrier, and one had Hb-O, and three had SCA. The patient with HbS carrier status also contains three additional mutations, all of which are heterozygous. We discovered that although two of three mutations, which are c.315+16G>C and c.316-185C>T, are previously reported as benign, at least one of the two mentioned mutations, when combined with HbS, causes transfusion-dependent HbS/b.ConclusionsBriefly, HbSS and HbS/b thalassemia genotypes cannot be definitely characterized by electrophoretic and hematologic data, resulting in misdiagnosis. c.315+16G>C and c.316-185C>T are previously reported as benign; at least one of the two mentioned mutations, when combined with HbS, causes transfusion-dependent HbS/b. In undeveloped or some developing countries, molecular diagnosis methods and genetic analyses cannot be used. If mutation analyses could be performed, then such differential diagnosis errors would reduce. However, if mutation analysis cannot be performed, other methods such as HPLC, capillary electrophoresis absolutely be sought to have insight into the parental carriage status.

Comparison of cord blood hematological parameters among normal, \u03b1-thalassemia, and \u03b2-thalassemia fetuses between 17 and 38\xa0weeks of gestation

The aim of this study was to retrospectively compare hematological parameters among normal, α-, and β-thalassemia fetuses between 17 and 38 weeks of gestation. Pregnant women at risk of having fetuses with thalassemia major and underwent cordocentesis for prenatal diagnosis were recruited. Fetal cord blood samples were collected from 249 fetuses for hematological and DNA analysis. Fetuses were divided into subgroups according to thalassemia DNA genotypes. The average and gestational age of subjects were 27.95 ± 5.78 years and 27.78 ± 3.57 weeks, respectively. The distribution of α-thalassemia, β-thalassemia, and normal cases was 67.87%, 19.68%, and 12.45%, respectively. Significant differences in almost all the hematological parameters (HbF, HbA, Hb, HCT, MCV, MCH, MCHC, RDW, and NBRCs) were observed in three groups (P < 0.001, except for RBC, P = 0.446). These differences were also observed in four α-thalassemia subgroups (P < 0.001) and were associated with the number of defected genes. Similarly, in five β-thalassemia genotypes, HbF, HbA, RBC, MCV, MCH and NBRCs were presented differently (P < 0.05). Additionally, the trends in RBC, Hb, and HCT changes in three α-thalassemia subgroups (silent carrier, trait, and major) and β+/β+ fetuses’ MCV, MCH, and RDW levels with gestation age were opposite to those of normal fetuses. We compared the distribution of hematological parameters in fetuses affected by most genotypes of thalassemia, as well as their trends in relation to gestational age for the first time, which is a good reference for future studies and prenatal diagnostic practices. The investigated hematological parameters are also valuable in diagnosing and differentiating thalassemia.

359 Pregnancy outcomes in a contemporary cohort of thalassemia patients

Historically, women with transfusion dependent thalassemia (TDT) were considered very high risk for adverse pregnancy outcomes including death, primarily due to iron induced cardiomyopathy. However, recent advances in chelation therapy, better transfusion protocols and enhanced cardiac monitoring have resulted in markedly improved pregnancy outcomes in women with TDT. The purpose of this study is to evaluate risks of pregnancy in women with TDT in a contemporary cohort. A retrospective chart review was conducted of pregnancies in transfusion dependent women with different beta thalassemia genotypes treated at the Weill Cornell Thalassemia Center from 2005-2020. 15 women with TDT had a total of 25 pregnancies with 20 liveborns. 48% of pregnancies were achieved via ART with 4 multiple gestations but only one live set of twins (1 multifetal reduction, 1 2nd trimester loss of quadruplets, 1 2nd trimester loss of triplets due to maternal death). Pregnancy loss rate was 24% and women with TDT had high rates of hypertension in pregnancy (21%), gestational diabetes (52.4%) and post-partum hemorrhage (23.5%). Ongoing pregnancies had favorable fetal outcomes with mean GA of 38 weeks and mean birthweight of 3129g. Cesarean section rates were high (10/19 deliveries). 40% of women had diagnosis of cardiac disease before conception or during pregnancy (see Table 1). There was 1 major complication in the second pregnancy in a woman with a triplet pregnancy, known cardiomyopathy, and severe myocardial iron overload resulting in maternal death from cardiogenic shock at 18 weeks. Women with TDT who are considering pregnancy should have a thorough assessment preconception and select patients, without evidence of significant myocardial iron overload or cardiomyopathy, may safely pursue pregnancy. While many women with TDT will require ART for conception, multiple gestations should be discouraged due to poor outcomes. A multidisciplinary approach to care in pregnancy is paramount in this high-risk population with majority of patients achieving favorable pregnancy outcomes.

Influence of UGT1A1 promoter polymorphism, α-thalassemia and βs haplotype in bilirubin levels and cholelithiasis in a large sickle cell anemia cohort.

Hyperbilirubinemia in patients with sickle cell anemia (SCA) as a result of enhanced erythrocyte destruction, lead to cholelithiasis development in a subset of patients. Evidence suggests that hyperbilirubinemia may be related to genetic variations, such as the UGT1A1 gene promoter polymorphism, which causes Gilbert syndrome (GS). Here, we aimed to determine the frequencies of UGT1A1 promoter alleles, alpha thalassemia, and βS haplotypes and analyze their association with cholelithiasis and bilirubin levels. The UGT1A1 alleles, -3.7 kb alpha thalassemia deletion and βS haplotypes were determined using DNA sequencing and PCR-based assays in 913 patients with SCA. The mean of total and unconjugated bilirubin and the frequency of cholelithiasis in GS patients were higher when compared to those without this condition, regardless of age (P < 0.05). Cumulative analysis demonstrated an early age-at-onset for cholelithiasis in GS genotypes (P < 0.05). Low fetal hemoglobin (HbF) levels and normal alpha thalassemia genotype were related to cholelithiasis development (P > 0.05). However, not cholelithiasis but total and unconjugated bilirubin levels were associated with βS haplotype. These findings confirm in a large cohort that the UGT1A1 polymorphism influences cholelithiasis and hyperbilirubinemia in SCA. HbF and alpha thalassemia also appear as modulators for cholelithiasis risk.

Clinical value of PCR-flow Fluorescence Hybridization in Prenatal Genetic Diagnosis of Thalassemia

To explore the value of PCR-flow fluorenscence immunmicrobeads assay in prenatal gene diagnosis of thalassemia. A total of 1001 pregnant women and their couples checked in the First Affiliated Hospital of Sun Yat-Sen University from January 2016 to August 2019 were selected. Both pregnant women and their spouses were the carriers of thalassemia gene. Samples such as amniotic fluid, were used to extract genomic DNA at the right time. Parallel detection of α- and β- thalassemia genes to samples should be carried out by PCR-flow cytometric fluorescence hybridization and traditional multiple Gap-PCR and PCR-RDB techniques. The consistency of two methods in gene diagnosis of thalassemia was evaluated by analyzing the results of detection. 389 normal genotypes (38.86%, 389/1001) and 59 abnormal genotypes (61.14%, 612/1001) was cheked out by the two methods, including 416 cases of α-thalassemia, 162 cases of β-thalassemia and 34 cases of αβ- complex thalassemia. The main genotypes of α-thalassemia were --SEA, -α3.7 and -α4.2. The mutation frequency of CD41-42 was the highest among the β-thalassemia genotypes, which followed by IVS-II-654 and CD17. A rare HKαα/--SEA thalassemia genotype was detected. Compared the traditional multiple Gap-PCR and PCR-RDB techniques, the sensitivity, specificity, positive predictive value, negative predictive value and total consistent rate of PCR-flow fluorenscence immunmicrobeads assay were 100%, which showed that the two methods were completely consistent. Guangzhou is a area with high incidence of thalassemia, and the genetic types of thalassemia are complex and diverse. Prenatal diagnosis is the final barrier to the prevention of thalassemia. PCR flow-cytometric fluorescence hybridization, as a simple and fast technique, combined with traditional techniques in parallel contributed to the accuracy of prenatal gene diagnosis of thalassemia.

Analysis of hippocampal subfields in sickle cell disease using ultrahigh field MRI

Sickle cell disease (SCD) is an inherited hemoglobinopathy that causes organ dysfunction, including cerebral vasculopathy and neurological complications. Hippocampal segmentation with newer and advanced 7 Tesla (7T) MRI protocols has revealed atrophy in specific subregions in other neurodegenerative and neuroinflammatory diseases, however, there is limited evidence of hippocampal involvement in SCD. Thus, we explored whether SCD may be also associated with abnormalities in hippocampal subregions. We conducted 7T MRI imaging in individuals with SCD, including the HbSS, HbSC and HbS/beta thalassemia genotypes (n = 53), and healthy race and age-matched controls (n = 47), using a customized head coil. Both T1- and T2-weighted images were used for automatic segmentation of the hippocampal subfields. Individuals with SCD had, on average, significantly smaller volume of the region including the Dentate Gyrus and Cornu Ammonis (CA) 2 and 3 as compared to the control group. Other hippocampal subregions also showed a trend towards smaller volumes in the SCD group. These findings support and extend previous reports of reduced volume in the temporal lobe in SCD patients. Further studies are necessary to investigate the mechanisms that lead to structural changes in the hippocampus subfields and their relationship with cognitive performance in SCD patients.

Comparison of Hematological Parameters and Critical Value Analysis in the Prevention and Control of Thalassaemia in Hainan Li and Han Nationalities

Objective: To compare the distribution of “mean corpuscular hemoglobin”-MCV, “mean corpuscular volume”-MCH, “hemoglobin”-HGB, “hemoglobin A”-HbA and “hemoglobin A2”-HbA2 in α and β thalassemia hematology screening between Li and Han nationality, and analyze the best diagnostic cut-off value. Methods: Select 7816 middle school students from Li nationality area as the research object, collect peripheral blood for blood cell analysis, hemoglobin electrophoresis and thalassaemia gene detection, and compare the difference in hematological parameters of common thalassemia genotype between Li and Han nationalities. Taking the genetic test results as the gold standard, construct the receiver operator characteristic curve (ROC curve) of relevant hematology parameters, calculate the Youden index and take its maximum diagnostic cut-off point as the best critical value. Results: Comparison of hematological parameters of common thalassemia genotypes showed that the average value of MCH and MCV of -α3.7/-α4.2 type in Li nationality was lower than that of Han nationality, and the average value of HbA2 of CD41-42/βN type was higher than that of Han nationality, there was no significant difference among other genotypes. ROC curve analysis shows that the MCH, MCV, and HGB values have poor diagnostic efficiency for thalassaemia, HbA has a slightly better diagnostic efficiency for α thalassaemia, and the optimal cut-off values of HbA for Li and Han nationalities are 96.95% and 97.75%, respectively; HbA2 has better screening efficiency for β-thalassemia, and the optimal cut-off values of HbA2 for Li and Han nationalities are 4.20% and 3.45% respectively. Conclusion: In the prevention and control screening of thalassaemia in the Li and Han nationalities, hemoglobin electrophoresis technology has a better diagnostic efficiency.

English

BACKGROUND Malaria is a global health burden. About 300 - 500 million people suffer from the disease every year, out of whom, about 1 million succumb.1 This study was undertaken, as there has been no such study regarding the possible effect of - thalassemia and ABO blood group in Indian population on falciparum malarial infection. METHODS This is an observational study carried on in all malarial patients admitted in the Department of General Medicine, VSS Medical College &amp; Hospital, Burla, between October 2008 - September 2010. Inclusion criteria: (i) Fever with positive asexual forms of falciparum malarial parasites [thick smear, thin smear, positive quantitative buffy coat (QBC), ICT test]. (ii) WHO criteria for severe falciparum malaria2 . “Controls”: Healthy persons of about same age, sex, ethnicity and locality. Exclusion Criteria: Blood transfusion within 3 months, cases of DM, CKD, hepatitis, SCD, tuberculosis, HIV, chronic liver disease, and COPD. RESULTS 128 cases of malaria, between 15 - 75 years, both sexes, pregnant / non-pregnant were included in the study. For control, the gene frequencies were  /  29 (45.3 %),  3.7 /  27 (42.2 %) and 3.7 / 3.7 8 (12.5 %). For cases, it was found 33 (51.56 %), 25 (39.1 %) and 6 (9.4 %) respectively. In HPLC, HbA0 values of 3.7 / 3.7 (81.83  10) were &gt;  /  (77.11  21.6) &gt;  3.7 / , (64.8 ± 32.42), HbA2 values of  /  (2.1  1.4) &gt;  3.7 /  (1.8 ± 0.8) &gt; 3.7 / 3.7 (1.43  0.27). In HbF, there were nearly same number of cases in all three variants and were negligible in HbS. Anaemia, jaundice, oliguria were the predominant causes of morbidity in alpha thalassaemic patients with severe falciparum malaria. Blood group A patients had significantly higher morbidity than blood group B, AB and O. CONCLUSIONS The percentage of anaemia, coma, convulsion and death was significantly less in homozygous alpha thalassemia cases in comparison to normal alpha thalassemia and heterozygous alpha thalassemia. Above features were also found to be significantly less in blood group O patients, and significantly high in blood group A patients, when compared to other blood groups. Prevalence of heterozygous and homozygous -thalassemia was lower in cases in comparison to controls. MCV was significantly lower in homozygous alpha thalassemia patients in comparison to other genotypes of alpha thalassemia. Anaemia, jaundice, coma, shock, oliguria, being the major co-morbidity conditions, should be detected and treated early. KEYWORDS Severe falciparum Malaria, ABO Blood Group, Homozygous &amp; Heterozygous, -Thalassemia

Diagnostic Work-up of Pulmonary Hypertension in Non Transfusion Dependent Thalassemia Patients: Pathophysiologichal Mechanisms and Clinical Implications

INTRODUCTION: Pulmonary hypertension (PH) is a well documented clinical complication in Non-Transfusion Dependent Thalassemia (NTDT) patients, and it is associated with poor outcome [1]. PH prevalence is extremely variable among studied cohorts, ranging from 4.8% to 59% [2, 3]. Nevertheless, in the majority of the studies, the diagnosis is based on echocardiography, whereas the gold standard for PH definition is right heart catheterization (RHC). Thus, PH real prevalence, its pathophysiological mechanisms and risk factors still need to be elucidated. AIM:The aim of this study is to define the prevalence of PH in a cohort of NTDT patients through the application of the PH diagnostic work-up of the European Society of Cardiology Guidelines (ESC) [4]. We also aimed to investigate the involved pathophysiological mechanisms, to define risk factors, and to identify a potential role for cardiopulmonary exercise testing (CPET) in the risk stratification. MATERIALS AND METHODS: We planned a screening program for all NTDT patients referring to Rare Disease Center, in collaboration with the Dyspnea Lab of the Cardiology Unit, at Fondazione IRCCS Ca' Granda Policlinico in Milan. Following the systematic approach of ESC PH algorithm, together with an echocardiogram and cardiological evaluation, our patients underwent cardiopulmonary exercise testing (CPET) and the dosage of NT-proBNP. Patients were stratified according to PH probability (low vs intermediate-high) and further investigations (including V/Q lung scan and RHC) to confirm PH were performed in those with intermediate-high risk. RESULTS AND DISCUSSION:48 NTDT patients (18 females and 30 males) were consecutively enrolled over a period of 10 months. The mean age at enrollment was 46±12 years (median 45, range 25-72 years) and a wide spectrum of thalassemia genotypes was observed. 38 out of 48 (79%) had a low PH probability according to NYHA class and echocardiogram, thus they were addressed to regular follow-up. 10 patients presented intermediate-high PH probability and underwent further tests. So far, 6 out of 10 intermediate-high risk underwent RHC: PH was confirmed in 5 cases, allowing to identify 2 post-capillary PH, 1 pre-capillary PH (due to pulmonary embolism detected with V/Q scan) and 2 forms of PH due to high cardiac output (CO). The sixth patient showed a condition of high CO but with a mean arterial pulmonary pressure just below the value needed to diagnose PH. Thus, PH was confirmed in 5 out of 48 patients, with a prevalence of 10.4%. The four remaining patients with high PH probability are planned to be tested. Comparing those with high and low PH probability, the first were older (58.4 ±8.9 vs 42.6 ±10.7 years, p=0.0002) and presented higher NT-proBNP (450±442 vs 92±99 ng/mL, p=0.0001). Hemoglobin, erythroblasts, and platelet count were similar. Patients with high PH probability on CPET reached a lower maximal workload (87.9 W ±32.1 vs 126.9 ±41.6W) and lower O2 consumption (1207.7 ±284.5 vs 1594 ±454.8 mL/min), together with worse ventilation efficiency (VE/VCO2 slope 40.9 ±6.9 vs 29.4 ±3.85). CONCLUSIONS: In our cohort, according to these preliminary data, PH prevalence in NTDT is 10.4% and older patients seem to be at higher risk. Thus, cardiac evaluation programs are required, considering the increased life span. RHC is mandatory to confirm the diagnosis, to identify the underlying mechanisms and consequently a proper treatment. CPET may have an essential role in detecting predictive and prognostic parameters of PH and in defining different hemodynamic mechanisms (pre- or post-capillary) to select patients requiring RHC. Together with pulmonary embolism, which needs to be excluded with imaging, high CO due to chronic anemia seems to play an important role. This hypothesis could have significant treatment implications, giving more importance to increase Hb levels, either with blood transfusions or new therapies. Sleiman J, Int J Mol Sci 2018 2 Derchi G, Circulation 2014 3 Aessopos E., Blood 2001 4 Galiè N, Eur Heart J 2016 Disclosures Motta: Sanofi Genzyme:Honoraria.Cappellini:Genzyme/Sanofi:Honoraria, Membership on an entity's Board of Directors or advisory committees;CRISPR Therapeutics, Novartis, Vifor Pharma:Membership on an entity's Board of Directors or advisory committees;BMS:Honoraria.

A Rare Case Presentation of HbE/ β Thalassemia

HbE/β thalassemia (HbE/β thal) genotype accounts for approximately one half of severe β thalassemia cases worldwide. The disorder shows marked clinical variability ranging from mild asymptomatic anemia to life threatening disease. Here, we report a case of a 2-year-old boy from Bihar presenting with severe haemolytic jaundice. Complete haematological profile and Haemoglobin High Performance Liquid Chromatography (Hb HPLC) using Biorad version was done of the patient along with the family was done to arrive at the diagnosis of this rare hemoglobinopathy.

Frequency and genotyping of alpha thalassemia in individuals undergoing premarital screening.

To evaluate the frequency of alpha thalassemia and detect mutations in the alpha genes in individuals undergoing premarital screening. The cross-sectional study was conducted at King Fahad Central Hospital, Jazan, Saudi Arabia, from January 2018 to May 2019, and comprised blood samples of individuals visiting the premarital screening clinic. The samples were analyzed for complete blood counts and haemoglobin electrophoresis. Molecular analysis of samples suspected for alpha thalassemia was done using alpha-globin StripAssay kit. Data was anlaysed using SPSS 20. Of the 3,970 samples analysed, 1,859(46.83%) were from males and 2,111(53.17%) from females. The overall frequency of suspected alpha thalassemia was 4.43% based upon haematological parameters including complete blood count and haemoglobin electrophoresis. Overall, 80 suspected samples were selected for genetic analyses, and, of them, 76 (95%) were positive for deletional and non-deletional mutations of alpha-globin genes, while 4 (5%) were negative for any of the 21 mutations tested. Alpha thalassemia was found to be highly prevalent in the study area.

Pedigree Analysis of Nonhomologous Sequence Recombination of HBA1 and HBA2 Genes

The aim of this study was to investigate a family with nonhomologous sequence recombination of HBA1 and HBA2 genes and provide a favorable basis for genetic counseling and eugenics. Peripheral blood of family members was collected. Hematological parameters were determined by an automated cell counter and hemoglobin (Hb) analysis was performed using high performance liquid chromatography (HPLC). Villus samples were taken for prenatal diagnosis (PND). Gap-polymerase chain reaction (gap-PCR) and reverse dot-blot were used for thalassemia genotyping. DNA sequencing was used to analyze the gene sequence of HBA1 (α1-globin) and HBA2 (α2-globin). The nonhomologous sequence recombination allelic variant of HBA1 and HBA2 genes were identified, namely, a gene conversion on the HBA2 gene called α12 (HBA12). The α12 allele consists primarily of the HBA2 gene sequence except for a segment of the IVS-II in which HBA2-specific sequences have been replaced by HBA1-specific sequences. The following genotypes were observed: – –SEA/αα12 (Southeast Asian deletion), αα/αα12 and αQSα/αα12 (Hb Quong Sze or Hb QS; HBA2: c.377T>C), and all manifested as small cell hypochromic anemia. To find the α12 allele in the Chinese population and clarify the influence of the α12 allele and its common inheritance with abnormal Hb and α-thalassemia (α-thal) on α-globin gene expression can help guide clinical diagnosis and genetic counseling.

Southeast Asian Ovalocytosis and Hemoglobinopathies in Newborns: Prevalence, Molecular, and Hematologic Analyses.

Southeast Asian ovalocytosis (SAO) is an inherited red blood cell (RBC) membrane disorder, whereas hemoglobinopathies are inherited globin gene disorders. In an area where both diseases are prevalent, the interaction between them resulting in variable hematologic parameters can be encountered. However, little is known about the genetic interaction of SAO and thalassemia. We investigated the prevalence of SAO and hemoglobinopathy genotypes among newborns in southern Thailand. This study was carried out on 297 newborns recruited consecutively at Naradhiwas Rajanagarindra Hospital in the south of Thailand. The SAO was identified on blood smear examination and polymerase chain reaction analysis. Thalassemia genotypes were defined. Hematologic parameters and hemoglobin (Hb) profiles were recorded and analyzed. Among 297 newborns, 15 (5.1%) carried SAO, whereas 70 (23.6%) had thalassemia with 15 different thalassemia genotypes. Abnormal Hb including Hb C, Hb Q-Thailand, and Hb D-Punjab were observed in 5 newborns. It was found in the nonthalassemic newborns that RBC count, Hb, and hematocrit of the nonthalassemic newborns with SAO were significantly lower than those without SAO. The same finding was also observed in the thalassemic newborns; RBC count, Hb, and hematocrit of the thalassemic newborns with SAO were significantly lower than those without SAO. However, the mean corpuscular volume, mean corpuscular Hb, and RBC distribution width of the SAO-newborns were significantly higher. Both SAO and hemoglobinopathy genotypes are common in southern Thailand. One should take this into consideration when evaluating neonatal anemia and other hematologic abnormalities. Identification of both genetic defects and long-term monitoring on the clinical outcome of this genetic interaction should be essential to understand the pathogenesis of these common genetic disorders in the region.

Ocular Manifestations of Sickle Cell Disease in Different Genotypes

ABSTRACT Background Sickle cell disease (SCD) is a multisystemic disorder with variable systemic involvement which varies according to genotype. In this study, our aim is to compare ocular complications between HbSS, HbSC, HbS/β+ thalassemia, HbS/β0 thalassemia, SS alpha thalassemia, and S/β0 + alpha thalassemia genotypes. Methods Data of patients included in this study was recruited from the Cooperative Study of Sickle Cell Disease (CSSCD). Patients with major sickle cell hemoglobinopathies (SS, SC, Sβ- thalassemia, SS alpha thalassemia) were eligible for enrollment, after that, a detailed eye exam was performed. We categorized ocular complications into conjunctival sign, iris atrophy, and both proliferative and non-proliferative sickle cell retinopathy. Results A total of 1867 patients were included in this study, with a mean age of 27.7 (± 11.7) years. They were 830 (44.5%) males and 1037 (55.5%) females. The most common genotype was SS with 971 (52%) patients, and the least common form was sickle cell with both alpha and beta thalassemia major with 42 (2.2%) patients. We found a significant difference in the frequency of proliferative sickle cell retinopathy, where SC genotype had the highest frequency and S B0 thalassemia genotype had the lowest frequency. We also found a significant difference in the frequency of conjunctival sign, where SS genotype had the highest frequency and the S B+ thalassemia has the lowest frequency. Conclusion We identified ocular complications for major sickle cell hemoglobinopathies, where we confirmed previous small study’s findings and identified ocular complications of less common hemoglobinopathies.

Effect of high-throughput sequencing for the prevention and control of thalassemia

To assess the value of next generation sequencing (NGS) for the prevention and control of thalassemia. NGS was used to sequence 3083 clinical blood samples suspected for thalassemia during initial screening. Retrospective analysis was conducted on blood samples detected with rare genotypes of thalassemia and abnormal hemoglobin. NGS analysis of the 3083 samples has found 1089 subjects with thalassemia genotypes (alpha-thelassemia genotype: 26.01%, beta-thalassemia genotype: 6.71%, and alpha-compound-beta genotype: 2.59%), which yielded a positive detection rate of 35.32%. Rare alpha-thalassemia genotypes including HBA2 c.123delG, HBA1 c.354_355insATC and Fusion gene, and rare beta-thalassemia genotypes including HBB c.-100G>A and HBB c.316-90A>G, were discovered. In addition, 19 patients were found to have abnormal hemoglobin, mainly including Hb Hamilton, Hb Hekinan II, Hb Shizuoka, Hb Owari, Hb New York, Hb J-Bangkok and Hb Port Phillip. NGS can play a crucial role for improving of the prevention and control of thalassemia and formulating a screening system with better efficacy.

Prevalence and Genotypes Analysis of Thalassemia Among People of Reproductive Age in Yuzhong District of Chongqing Based on Next-Generation Sequencing

To investigate the prevalence and gene distribution of thalassemia among people at reproductive age in yuzhong district, Chongqing. 1000 pre -pregnancy examination couples in yuzhong district were investigated. Peripheral venous blood was extracted and next-generation sequencing was used to screen the thalassemia genes. Among the 1000 pregnant couples, the thalassemia gene carrying rate was 7.45%, the carrying rate of α and β thalassemia genes were 4.60% and 2.10%, respectively. The most common α thalassemia genotypes in αα/-α3.7 (53.26%), αα/--SEA (23.91%), αα/-α4.2 (11.96%); and the most common genotypes in β thalassemia genotypes were mainly Codons17 (A＞T) (26.19%)、Condon41/42 (-TTCT) (26.19%)、IVS-II-654 (C＞T) (14.29%) At the same time, 3 cases of α and β complex thalassemia and 3 pairs of homotypic thalassemia genes were detected, more over, 12 cases of 5 new genes were found. Yuzhong district of Chongqing is a high incidence area of thalassemia, and the diversity of gene mutation types is relatively rich. Screening for thalassemia before pregnancy is of great significance to improve the quality of population.

Update in Laboratory Diagnosis of Thalassemia.

Alpha- and β-thalassemias and abnormal hemoglobin (Hb) are common in tropical countries. These abnormal globin genes in different combinations lead to many thalassemic diseases including three severe thalassemia diseases, i.e., homozygous β-thalassemia, β-thalassemia/Hb E, and Hb Bart’s hydrops fetalis. Laboratory diagnosis of thalassemia requires a number of tests including red blood cell indices and Hb and DNA analyses. Thalassemic red blood cell analysis with an automated hematology analyzer is a primary screening for thalassemia since microcytosis and decreased Hb content of red blood cells are hallmarks of all thalassemic red blood cells. However, these two red blood cell indices cannot discriminate between thalassemia trait and iron deficiency or between α- and β-thalassemic conditions. Today, Hb analysis may be carried out by either automatic high-performance liquid chromatography (HPLC) or capillary zone electrophoresis (CE) system. These two systems give both qualitative and quantitative analysis of Hb components and help to do thalassemia prenatal and postnatal diagnoses within a short period. Both systems have a good correlation, but the interpretation under the CE system should be done with caution because Hb A2 is clearly separated from Hb E. In case of α-thalassemia gene interaction, it can affect the amount of Hb A2/E. Thalassemia genotypes can be characterized by the intensities between alpha-/beta-globin chains or alpha-/beta-mRNA ratios. However, those are presumptive diagnoses. Only DNA analysis can be made for specific thalassemia mutation diagnosis. Various molecular techniques have been used for point mutation detection in β-thalassemia and large-deletion detection in α-thalassemia. All of these techniques have some advantages and disadvantages. Recently, screening for both α- and β-thalassemia genes by next-generation sequencing (NGS) has been introduced. This technique gives an accurate diagnosis of thalassemia that may be misdiagnosed by other conventional techniques. The major limitation for using NGS in the screening of thalassemia is its cost which is still expensive. All service labs highly recommend to select the technique(s) they are most familiar and most economic one for their routine use.

Development of a genomic DNA reference material panel for thalassemia genetic testing.

Thalassemia is one of the most common autosomal recessive inherited diseases worldwide, and it is also highly prevalent and variable in southern China. Various types of genetic testing technologies have been developed for diagnosis and screening of thalassemia. Characterized genomic DNA reference materials (RMs) are necessary for assay development, validation, proficiency testing, and quality assurance. However, there are no publicly available RMs for thalassemia genetic testing as yet. To address the need for the publicly available DNA RMs for thalassemia genetic testing, the National Institutes for Food and Drug Control and the China National GeneBank established 32 new cell lines with three wild-type genotypes and 29 distinct genotypes of thalassemia which account for approximately 90% thalassemia carriers in China. The genomic DNA of 32 cell lines was characterized by four clinical genetic testing laboratories using different genetic testing methods and technology platforms. The genotyping results are concordant among four laboratories. In addition, the results of stability test demonstrated that the genotypes of these DNA samples are not influenced by preanalytical conditions such as long-term exposure to high-temperature (37°C) environment and repeated freeze-thawing. We developed the first national panel of 32 genomic DNA RMs which are renewable and publicly available for the quality assurance of various genetic testing methods and will facilitate research and development in thalassemia genetic testing.

Visual genotyping of thalassemia by using pyrrolidinyl peptide nucleic acid probes immobilized on carboxymethylcellulose-modified paper and enzyme-induced pigmentation.

A simple probe pair was designed for the detection of hemoglobin E (HbE) genotype, a single-point mutation that leads to abnormal red blood cells commonly found in South East Asia. The key to differentiation is the use of a conformationally constrained peptide nucleic acid (PNA) that was immobilized on carboxymethylcellulose-modified paper. This was then used for target DNA binding and visualization by an enzyme-catalyzed pigmentation. The biotinylated target DNA bound to the immobilized probe was visually detected via alkaline phosphatase-linked streptavidin. This enzyme conjugate catalyzed the dephosphorylation of the substrate 5-bromo-4-chloro-3-indolyl phosphate, leading to a series of reactions that generate an intense, dark blue pigment. The test was validated with 100 DNA samples, which shows good discrimination among different genotypes (normal, HbE, and heterozygous) with 100% accuracy when optimal conditions of analysis were applied. The method does not require temperature control and can be performed at ambient temperature. This is an attractive feature for diagnosis in primary care, which accounts for a large part of affected population. Graphical abstract Schematic representation of a paper-based sensor for the detection of the gene Hemoglobin E. The interaction between an immobilized peptide nucleic acid and a DNA target leads to enzymatic pigmentation, allowing simple visual readout with up to 100% accuracy.

A New Indicator Derived From Reticulocyte Hemoglobin Content for Screening Iron Deficiency in an Area Prevalent for Thalassemia.

To establish a new indicator derived from reticulocyte hemoglobin (Ret-He) content and red blood cell (RBC) indices for screening for iron deficiency anemia (IDA) in an area in whch thalassemia is prevalent. Blood specimens from 304 women aged between 18 and 30 years residing in northeast Thailand were collected and measured for RBC and reticulocyte parameters. Iron deficiency was diagnosed when a participant had a serum ferritin level of less than 15 ng per mL. Thalassemia genotypes were defined by hemoglobin (Hb) and DNA analyses. Of the total participants, 25% had iron deficiency (ID) and 50% carried the thalassemia gene. Various mathematical formulas were established and analyzed using the receiver operating characteristic (ROC) curve. The formula derived from Ret-He: (Ret-He/RDW-SD) × 10, was the best predictor for identifying ID among participants (area under the curve [AUC] = 0.812). Further testing of this indicator among individuals with positive thalassemia-screening results revealed stronger performance with an AUC of 0.874. The findings indicate that the formula derived from Ret-He might be applicable for screening ID in areas in which thalassemia is prevalent.