Thalassemia Mutations Research Articles

Molecular Characterization Analysis of Thalassemia and Hemoglobinopathy in Quanzhou, Southeast China: A Large-Scale Retrospective Study.

Background: There are limited reports available on investigations into the molecular spectrum of thalassemia and hemoglobinopathy in Fujian province, Southeast China. Here, we aim to reveal the spectrum of the thalassemia mutation and hemoglobinopathy in Quanzhou prefecture, Fujian province.Methods: We collected data from a total of 17,407 subjects with the thalassemia trait in Quanzhou prefecture. Gap-PCR, DNA reverse dot blot hybridization, and DNA sequencing were utilized for common and rare thalassemia gene testing.Results: In our study, we identified 7,085 subjects who were carrying thalassemia mutations, representing a detection rate of 40.70% (7,085/17,407). Among them, 13 different α-thalassemia gene mutations were detected, with the most common mutation being –SEA (69.01%), followed by –α3.7 (21.34%) and –α4.2 (3.96%). We also discovered 26 β-thalassemia gene mutations, with the mutations of IVS-II-654 (C > T) (36.28%) and CD41/42(–TCTT) (29.16%) being the most prevalent. Besides, a variety of rare thalassemia variants were identified. Among them, the –FIL, βMalay, βIVS–I–130, and βIVS–II–672 mutations were identified in Fujian province for the first time. Additionally, we detected 78 cases of hemoglobinopathies, of which Hb Owari was the first reported case in Fujian province and Hb Miyashiro was the first case identified in the Chinese population.Conclusion: Our study indicates that there is a diverse range of thalassemia mutations, and it also reveals the mutation spectrum of rare thalassemia and hemoglobinopathies in Quanzhou, Fujian province. It provides valuable data for the prevention and control of thalassemia in Southeast China.

English

BACKGROUND Sickle delta beta thalassemia is a rare genetic disorder, with varied symptoms, signs, requiring careful monitoring for potential complications. It is due to sickle mutation and thalassemia mutation occurring together, with sparse data available worldwide. The purpose of this study was to assess the clinical and laboratory profile of possible sickle delta beta thalassemia. METHODS The study design is retrospective analysis of clinical information of those selected patients done in our multi-specialty tertiary care referral hospital situated in Telangana state in south India. The case material was collected from December 2017 to December 2019 (2 years duration). All haemoglobin electrophoresis reports were collected with no prior blood transfusions in preceding 4 months. The information collected was analysed and presented. RESULTS Total 9 patients were diagnosed as possible sickle delta beta thalassemia, with male to female ratio of 5 : 4 and age ranging from 12 years to 45 years of age. The commonest symptoms were joint pain and jaundice in 5 patients and sign was splenomegaly in 2 patients. Ultrasonogram of abdomen showed that 3 patients had gall stones, 1 patient had gall bladder sludge, 1 patient had autosplenectomy and 3 patients had splenomegaly. Mild to moderate anaemia was seen with reticulocytosis, sickling test positive in all patients, with haemoglobin in the range of 5.5 g/dl to 12.7 g/dl. 3 patients had iron over load, 2 patients had hepatopathy, 5 patients had unconjugated hyperbilirubinemia, Acute chest syndrome, hepatic necrosis, and nephropathy was seen in 1 patient each. Haemoglobin electrophoresis showed Hb S was from 46.1 % to 76.4, Hb A from 5.3 % to 34.7 %, Hb F from 4.8 % to 22.7 %, Hb A2 from 1.5 % to 3.3 %. 2 patients were treated with hydroxyurea. 2 patients had mutation analysis elsewhere that was reported as compound heterozygous for β-globulin gene for Hb S (GAG-GTG) and IVS 1 - 5 (G-C). CONCLUSIONS Sickle delta beta thalassemia presents as mild to moderate anaemia haemolysis, splenomegaly with vaso-occlusive crises. Hydroxyurea may help in the treatment. Genetic analysis helps in diagnosis and future therapies. KEYWORDS Haemoglobin S, Haemoglobin A2, Haemoglobin A, Haemoglobin F

SCREENING AND MOLECULAR CHARACTERIZATION OF HAEMOGLOBINOPATHIES USING ARMS (AMPLIFICATION REFRACTORY MUTATION SYSTEM) AND DIRECT DNA SEQUENCING TECHNIQUES AMONG YOUNG IN CENTRAL GUJARAT WESTERN INDIA

Haemoglobinopathies is consider the most common inherited disorders in human and results from genetic mutation in . one or more genes The present study was aimed to characterize the β-thalassemia mutation and haemoglobin variant in youth by ARMS PCR which is an uncomplicated and convenient method for identification of five common mutation from central Gujarat, western India region. This Study included 44 randomly selected haemoglobinopathies carrier student's sample of Anand People's Medicare Society (APMS), Anand for DNA analysis by ARMS PCR from March 2021-April 2021. Identification of five common Indian β thalassemia mutations along with Hb S and HB E were carried out by ARMS PCR method and δβ- thalassemia mutation was characterized by GAP-PCR. The samples which remain uncharacterized were sent to S N gene lab, Surat for DNA sequencing. In our study the most common mutation among five common mutations characterized was IVS-1, nt5 (G→C) in 22 (50%), followed by Codon41/42 (-CTTT) in 5 (11.3%). IVS-1, nt1 (G→T), Codon8/9 (+G) and 619bp del mutation was not identified in any carrier students screened for haemoglobinopathies. Other than these five common mutation Codon -88 (C→T) (2.27%) and Codon 30 (G→A) (2.27%) are also detected. The prevalence of haemoglobinopathies with respect to communities, reflects that SC/ST/OBC are at the highest risk with 50%. Communities like Rajput (22.7%), Patel (18.1%), Brahmin (6.8%) and Muslim (2.2%) are also showing prevalence. The study has included mutation in different communities reflects characterization of mutation of central Gujarat, western India which is significant for rapid and convenient identification of mutation while conducting screening programs and prenatal diagnosis. Rare mutations which are not recognized, need further confirmation for carrier detection followed by prenatal diagnosis.

A Review on Red Blood Disorder: Thalassemia

Thalassemia is classified as major (homozygous), minor (heterozygous) or intermedia (compound heterozygous). It’s diagnosed is based on hematological findings whereas molecular genetic analysis has also been gradually developed. Due to untreatable nature of thalassemia, it is important to adopt preventive measures. In this regard, the community awareness, carrier testing and genetic counselling are important milestones. Region-wise division of thalassemia mutations is diverse in distinct provinces. HBB: c.92+5G > C represents raised incidence in Balochistan and Sindh. HBB: c.27_28insG is most common in KPK and Punjab. Similarly, NG_000007.3: g.71609_72227del619 has its roots previously in India subsequently in Gujratis along with Memon community populating Sindh. Widespread family testing can employ DNA-based genomic analysis in areas where consanguine marriages are usual. An assessed carrier ratio for HBV (3-5%) along with (4-5%) for hepatitis C virus being found in Pakistan due to asymptomatic distribution. Bone marrow transplantation is the only solution to get rid from thalassemia major complications and this facility is accessible ever since 1999 in Pakistan.

Presentation of two new mutations in the 3'untranslated region of the β-globin gene and evaluating the molecular spectrum of thalassemia mutations in the Mediterranean region of Turkey.

Thalassemia is a common genetic disorder. We aimed to present thalassemia mutation data that covers a period of 7 years from the Mediterranean region of Turkey by comparing with hemoglobin indices and to contribute to prenatal diagnosis and genetic counseling studies which should be decided very quickly. In this study, in which a retrospective archive was scanned, the cases were first grouped as α and β thalassemia, and then β thalassemia mutations were examined in a total of 5 groups as UTR-Pro, Codon, IVS, β0, and β+. We have reached the family of the proband that analyzed their Hb indices and genetic mutation. All mutations were statistically compared with Hb indices, HbF, and HbA2. We have identified two new β thalassemia mutations that have the feature of not being defined previously [HBB:C*62 A>G. (3'UTR+1536 A>G) and HBB:C*1 G>A (3'UTR+1475 G>A)]. The most commonly encountered 23 mutations account for 74.7% of all mutations which is unlike the literature. In the β thalassemia group, 73 different mutations were detected. The most common β thalassemia mutation was HBB: c.93-21 G>A (IVS I-110 G>A) with a frequency of 19.72%. A statistically significant difference was found when comparing the mutation groups with Hb indices. We think that it may be useful to evaluate the mutations we have newly identified too together with the Hb indices especially in evaluating the carriers of thalassemia and it will contribute to prenatal diagnosis and genetic counseling studies which should be decided very quickly.

Frequencies of Beta Thalassemia Mutations Show Different Pattern in Bannu Region than Other Parts of Pashtun Population in Khyber Pakhtunkhwa Province Pakistan.

β-Thalassemia arises as result of mutations in HBB gene, influencing the globin production which results in hypochromic and microcytic anaemia. The present study was aimed to investigate the occurrence of six common β-thal mutations, its inheritance pattern, frequency, and consanguinity in parents of Bannu region Khyber Pakhtunkhwa (KP) province, Pakistan. Conducting such studies may impart important information about thalassemia prevention like prenatal diagnosis (PND), carrier screening and genetic counselling which may be helpful in controlling the suspected births. During the study, 250 blood samples were retrieved from different families comprising of one transfusion dependent child and sporadic patients from different areas of Bannu region. The collected blood samples were investigated to see if there is any common mutations which may trigger β-Thalassemia employing amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) approach. Amongst the studied mutation in District Bannu, frame shift codons (FSC) 8/9 (+ G) (HBB: c.27_28insG) was observed to be the most common mutation followed by Codons 41/42 (- TTCT), IVS-I-5(G > C) and FSC 5 (- CT) having frequencies of 42, 26, 19 and 13 respectively. The results obtained by the present study were found different from previous studies demonstrated from other Pashtun regions of KP, showing heterogeneity in frequencies of known mutations. These observations may help in implementing parental meetings about disease recurrence in future, large scale mutation screening, and prenatal diagnosis in the whole Pashtun ethnicity including District Bannu.

Novel mechanism of hereditary pyropoikilocytosis phenotype due to co‐inheritance of β globin and α spectrin mutations

Novel mechanism of hereditary pyropoikilocytosis phenotype due to co‐inheritance of β globin and α spectrin mutations

Alpha thalassemia, but not βS-globin haplotypes, influence sickle cell anemia clinical outcome in a large, single-center Brazilian cohort.

Alpha thalassemia and beta-globin haplotype are considered classical genetic disease modifiers in sickle cell anemia (SCA) causing clinical heterogeneity. Nevertheless, their functional impact on SCA disease emergence and progression remains elusive. To better understand the role of alpha thalassemia and beta-globin haplotype in SCA, we performed a retrospective study evaluating the clinical manifestations of 614 patients. The univariate analysis showed that the presence of alpha-thalassemia -3.7-kb mutation (αα/-α and -α/-α) decreased the risk of stroke development (p = 0.046), priapism (p = 0.033), and cholelithiasis (p = 0.021). Furthermore, the cumulative incidence of stroke (p = 0.023) and cholelithiasis (p = 0.006) was also significantly lower for patients carrying the alpha thalassemia -3.7-kb mutation. No clinical effects were associated with the beta-globin haplotype analysis, which could be explained by the relatively homogeneous haplotype composition in our cohort. Our results reinforce that alpha thalassemia can provide protective functions against hemolysis-related symptoms in SCA. Although, several genetic modifiers can impact the inflammatory state of SCA patients, the alpha thalassemia mutation remains one of the most recurrent genetic aberration and should therefore always be considered first.

Sickle Cell Disease in Jordan: The Experience of a Major Referral Center.

Introduction:Sickle cell disorders are the most frequently encountered hemoglobin variants in Jordan. Both alpha and beta thalassemias are also prevalent in this population. However, studies on the interaction between these hemoglobin disorders are lacking.Aim:To determine the genotypes responsible for Sickle cell disease in Jordan, by retrospectively reviewing the data from a major referral center in the country’s capital.Methods:A total 29,712 peripheral blood samples referred and investigated for hemoglobinopathies over a 10-year period at Princess Iman Center at Amman, Jordan were retrospectively reviewed. In addition to full blood counts, high performance liquid chromatography, those who were identified with sickle cell hemoglobin were studied using polymerase chain reaction and reverse hybridization to determine the various sickle cell disease genotypes.Results:Out of the (29,712) blood samples, 450 were sickle cell trait, while 216 had sickle cell disease. Of the latter: 120 were found to be cases of Sickle cell anemia (Hb SS), 66 were compound heterozygous for Sickle cell and a beta thalassemia mutation (Sickle/β-thalassemia), while 30 had concomitant alpha thalassemia (HbSS/alpha thalassemia). The most frequent genotype associated with sickle/β-thalassemia was HbS/ IVS-110 (G>A), followed by Hb S/IVS-I-6 (T>C), HbS/IVS-II-745 (C>G) and HbS/ IVS-II-1 (G>A). While the most frequent alpha genotype detected in HbSS/α-thalassemia samples was (-α3.7/αα) followed by (-α3.7/-α3.7). Hb SS patients had the severest hematological phenotype compared to those with sickle/β-thalassemia and sickle/ α-thalassemia. Furthermore, within the sickle/β-thalassemia subgroup the least severe hematological phenotype was encountered in HbS/IVS-1-6 (T>C), while the most severe in HbS/IVS-II-1 (G>A) genotype.Conclusion:The most frequent Sickle cell disease genotype in Jordanians is Sickle cell anemia (HbSS), followed by Sickle/β-thalassemia and least frequent is HbSS/alpha thalassemia. The concomitant identified thalassemia mutations were consistent with their spectrum among the Jordanian population.

Alpha thalassemia and alpha-MRE haplotypes in Uruguayan patients with microcytosis and hypochromia without anemia.

Alpha thalassemia is the most common genetic disorder across the world, being the α-3.7 deletion the most frequent mutation. In order to analyze the spectrum and origin of alpha thalassemia mutations in Uruguay, we obtained a sample of 168 unrelated outpatients with normal hemoglobin levels with microcytosis and hypochromia from two cities: Montevideo and Salto. The presence of α-thalassemia mutations was investigated by gap-PCR, restriction endonucleases analysis and HBA2 and HBA1 genes sequencing, whereas the alpha-MRE haplotypes were investigated by sequencing. We found 55 individuals (32.7%) with α-thalassemia mutations, 51(30.4%) carrying the -α3.7 deletion, one with the -α4.2 deletion and three having the rare punctual mutation HBA2:c.-59C>T. Regarding alpha-MRE analysis, we observed a significant higher frequency of haplotype D, characteristic of African populations, in the sample with the -α3.7 deletion. These results show that α-thalassemia mutations are an important determinant of microcytosis and hypochromia in Uruguayan patients with microcytosis and hypochromia without anemia, mainly due to the -α3.7 deletion. The alpha-MRE haplotypes and the α-thalassemia mutations spectrum suggest a predominant, but not exclusive, African origin of these mutations in Uruguay.

Analysis of Gene Mutation Types of Thalassemia in Yulin Childbearing-age Population of Guangxi China

To investigate the genotype distribution of thalassemia in the population of childbearing age in Yulin area. The polymerase reaction (PCR) combined with agargel eletrophoresis and reserve dot bolt hybridization was used to detected the α- and β-thalassemia gene in 31 769 cases of suspected thalassemia population at childbearing-age. A total of 22 254 cases were identified as thalassemia gene detetion or mutation in 31 769 cases with a detecting rate of 70.05%, and the detecting rate of α-thalassemia, β-thalassemia and α-combining β-thalassemia were 45.86% (14 569/31 769), 19.45% (6 178/31 769) and 4.74% (1 507/31 769) respectively. 28 kinds of α-thalassemia gene mutations were detected, the common mutations were as follows: --SEA/αα (28.18%), -α3.7/αα (6.29%), -α4.2/αα (3.66%), αCSα/αα (1.93%) and αWSα/αα (1.89%)，and including two rare gene mutations: -THAI and HKαα. 16 kinds of β-thalassemia gene mutations were detected, the common mutations were as follows: β41-42/βN (9.41%), β-28/βN (3.05%), β-17/βN (2.86%) and β654/βN (2.18%). 93 kinds of α combining β-thalassemia gene mutations were detected, the common mutations were as follows: --SEA/αα (1.05%) and -α3.7/αα (0.56%) combining β41-42/βN. The detection rate of thalassemia gene is high in Yulin caildbearing-age population, and there is diversity in mutation spectrums of thalassemia. The most common genotypes are --SEA/αα in α-thalassemia and β41-42/βN in β-thalassemia. The results are beneficial for the intervention and genetic consultation of thalassemia.

Analysis and Prevention of Gene Mutation Types of Severe Thala- ssemia in Hakka People in Gannan of Jiangxi Province

To investigate the types and proportion of gene mutations of thalassemia in Hakka people in Gannan Area of Jiangxi, and to provide some references for prevention and treatment of thalassemia major, genetic counseling and epidemiological studies. 81 cases Hakka patients with severe thalassemia admitted treated in First Affiliated Hospital of Gannan Medical College from January 2009 to June 2019 were enrolled. The deletion type of α-thalassemia was detected by Gap-PCR. The point mutations of α-thalassemia and β-thalassemia were detected by PCR-RDB. The thalassemia gene was detected and analyzed in the patients with anemia, and the frequency of gene mutation was calculated. Among 81 Hakka patients with thalassemia major, 4 β-thalassemia (homozygote) genotypes were detected out, including: CD41-42（TTCT）(19 cases), β-IVS-II-654 (C→T) (9 cases), -28M (A→G) (1 case), CD17 (A→T) (1 case); 12 β-thalassemithalassemia (heterozygote) genotypes were detected out, including: CD41-42(-TTCT)/β-IVS-II-654(C→T) (15 cases, 29.41%), β-IVS-II-654(C→T)/β-28M(A→G) (13 cases，25.49%) ； CD41-42(-TTCT)/β-28M(A→G) (9 cases，17.65%); β-IVS-II-654(C→T) /CD27/28(+C) (3 cases， 5.88%) ； CD41-42(-TTCT)/CD27/28(+C)(3 case，5.88%)；β-28M(A→G)/CD17(A→T) (2 cases，3.92%)；CD41-42(-TTCT)/CD17(A→T), CD41-42(-TTCT)/Βe, β-IVS-II-654(C→T)/β-29、βCD17（A→T）/CD71-72(+a), βCD71-72/β-28M(A→G), β-28M(A→G) /β-IVS-II-654(C→T)(1 cases，1.96%). There were 3 cases of β homozygous thalassemia with α-thalassemia gene and 5 cases of β heterozygotes thalassemia with α-thalassemia gene. The incidence rate of thalassemia in Hakka people in Gannan Area of Jiangxi is relatively high. The distribution of gene mutation types is as follows: the genotype of CD41-42 (-TTCT) is the main genotype of β-thalassemia (homozygous); the major genotypes of β- thalassemia (heterozygotes) are CD41-42 (-TTCT)/β-IVS-II-654 (C→T) and β-IVS-II-654 (C→T) /β-28M (A→G); CD41-42 (-TTCT) gene is dominant in β-complex α-thalassemia.

Clinical Features of Pregnant Women with Thalassemia in Non Endemic Area

To investigate the clinical features of pregnant women with thalassemia in non endemic area, and to prevent the births of babies with intermedia or major thalassemia. Two hundred and thirty-five pregnants women with thalassemia diagnosed from March 2015 to April 2016 in our hospital were enrolled and retrospectively analysed. The blood routine and hemoglobin electrophoresis were performed respectively by XN-9000 automatic blood cell analyzer and HYDRASYS hemoglobin electrophoresis apparatus. The three commonest deletion of α-thalassemia, the three non-deletion α-thalassemia and 21 known β-thalassemia mutation were all detected by fluorescence melting curve analysis. Among 235 pregnant women of thalassemia, the majority were β-thalassemia, which were followed by α-thalassemia and composite thalassemia. Most pregnant women showed a mild anemia, and suffered from microcytic anemia, but less suffered from iron deficiency anemia. The ratio of second-child pregnant women was increased, and the ratio was close to one third both in α-thalassemia and β-thalassemia patients, and 75% patients were composite thalassemia. HbF was found to be more in native pregnant women with β-thalassemia. Hemoglobin isomer was easy to found in the pregnant with α-thalassemia, and they were all non native. The genotype of --sea were found majority in both native and non native pregnant women with thalassemia. The genotype of IVS-II-654 made up a large majority(55.38%) in native pregnant with β-thalassemia, as well as one of whose parents was native pregnant women. The genotypes of CD41-42,IVS-II-654 and CD17 were found to be a large majority in non native pregnant women, each of them accounted for 30%. More pregnant women with thalassemia are founded to be in non endemic area, and shows their own unique clinical features. It is certainly to detect thalassemia mutation in their spouse and their babies, to prevent the births of babies with intermedia or major thalassemia.

Development and clinical application of a preimplantation genetic testing for monogenic disease (PGT-M) for beta thalassemia in Vietnam

PurposeThe purpose of this research is to study the clinical outcomes using a next-generation sequencing-based protocol allowing for simultaneous testing of mutations in the beta thalassemia (HBB) gene, including single nucleotide polymorphism (SNP) markers for PGT-M along with low-pass whole genome analysis of chromosome aneuploidies for PGT-A.MethodsA combined PGT-M (thalassemia) plus PGT-A system was developed for patients undergoing IVF in Vietnam. Here we developed a system for testing numerous thalassemia mutations plus SNP-based testing for backup mutation analysis and contamination control using next-generation sequencing (NGS). Low -pass next-generation sequencing was used to assess aneuploidy in some of the clinical PGT cases. Patients underwent IVF followed by embryo biopsy at the blastocyst stage for combined PGT-A/M.ResultsTwo cases have completed the entire process including transfer of embryos, while a further nine cases have completed the IVF and PGT-M/A analysis but have not completed embryo transfer. In the two cases with embryo transfer, both patients achieved pregnancy with an unaffected, euploid embryo confirmed through prenatal diagnosis. In the further nine cases, 39 embryos were biopsied and all passed QC for amplification. There were 8 unaffected embryos, 31 carrier embryos, and 11 affected embryos. A subset of 24 embryos also had PGT-A analysis with 22 euploid embryos and 2 aneuploid embryos.ConclusionsHere we report the development and clinical application of a combined PGT-M for HBB and PGT-A for gross chromosome aneuploidies from 11 patients with detailed laboratory findings along with 2 cases that have completed embryo transfer.

Analysis of New Pediatric Thalassemia Diagnoses between 2010-2019 in a University System and Comparisons with Statewide Demography of Minnesota

Introduction Thalassemias are common inherited hematologic disorders worldwide, and with increasing migration, its prevalence evolves in different countries. However, community prevalence across most of the United States is unknown. Minnesota, particularly Minneapolis/St. Paul, has a relatively large community of immigrant families, with focal ethnic clusters of Karen and Hmong individuals from Southeast Asia, Liberia, and neighboring countries in Western Sub-Saharan Africa. Statewide data show that these ethnic minority populations have grown in the past decade; it is unknown whether thalassemia prevalence mirrors these migration trends. This report aims to quantify the demography and disease characteristics of children with thalassemia mutations seen at University of Minnesota/M Health Fairview (MHF) diagnosed between January 1, 2010 and December 31, 2019. Methods An institutional review board-approved electronic medical record (EMR) review was undertaken. Patients born on or after January 1, 2001 were included if they received medical care within the MHF system and thalassemia status could be confirmed. The prevalence by thalassemia type, demography, language, treatment details, and specialist involvement were detailed. A patient's ethnicity determination was defaulted to "American" if the ethnicity and/or immigration country of origin going back at least two generations could not be confirmed via EMR. The trends of new diagnoses over the decade were reviewed and compared to available state demography data trends. Descriptive statistical analyses were performed for the full group and by thalassemia type. Results A total of 404 patients met inclusion criteria, representing 48 country or region-specific ethnicities, 17 known countries of birth, and 30 separate languages. The most prevalent immigrant ethnicities were Karen (15%), Hmong (10%), and Vietnamese (5%), representing the 14th, 2nd, and 6th-largest immigrant communities in the state, respectively (www.mncompass.org). In Minnesota, the Asian population grew by 32% (69,800 people) between 2010-2018 (Minnesota State Demographic Center, https://mn.gov/admin/demography/data-by-topic/age-race-ethnicity/). The overall number of thalassemia diagnoses also increased over the decade, with Karen patients representing the largest proportional increase over the available time frame (Figure 1). Including both trait and disease, alpha thalassemias were more frequently diagnosed than beta thalassemias (63% versus 37%). Of the alpha thalassemias, 79% were suspected or confirmed 1-2 gene deletions based on newborn screening. Eleven (2.7%) had hemoglobin (Hb) H disease and 2 (0.5%) had alpha thalassemia major. Beta thalassemia intermedia, beta thalassemia major, and HbE were collectively 5.4% of all patients, 59% of whom were transfusion-dependent. Genetic confirmation was performed for 10.4% of all thalassemias. Those with alpha thalassemias were more ethnically diverse. Aside from those categorized as "American", those with beta thalassemias were more likely to be Karen (25%), while those with alpha thalassemias were most likely to be Hmong (14.4%). Seven had chelation prescribed during the time frame and 5 underwent bone marrow transplant. Only 16% were seen by a hematologist in the MHF system, though the yearly trends of hematology care did not match the increased diagnostic rate (Figure 2). Conclusions The incidence of pediatric thalassemia diagnoses have increased over the past decade at MHF, somewhat reflecting the statewide demographic trends. However, most patients are never being seen by hematology. A caveat to the data described is the fact that MHF is 1 of 2 pediatric hospitals locally, with the other having the historically larger thalassemia patient population. Thus, while these data offer crucial insights into the potential frequency of thalassemia mutations in our state, they also are likely to significantly under-represent the local prevalence. Also, the data do not reflect the prevalence in youths diagnosed prior to 2010 or the adult prevalence, for which institutional investigations are currently ongoing. These data illuminate the need to ensure adequate educational resources and financial support for these primarily non-English-speaking communities to improve awareness for families and medical providers and offset substantial medical costs for life-saving therapies. Disclosures No relevant conflicts of interest to declare.

Pattern of Sickle Cell Disorders: A Study from a Tertiary Care Center in Eastern Part of India

Introduction:Sickle cell disorders were originally found in the African regions, Arabian Peninsula and parts of India. However, in today's age of globalization patients with homozygous or compound heterozygous Sickle cell disorders can be found all over the world. The objective of our study was to assess the distribution and clinical presentation of patients with Sickle homozygous or heterozygous diseases in the Eastern part of India. Methods:Patients who attended the Thalassemia Clinic in our tertiary care center, between 1st January 2018 to 31st May 2020 (2 years and 4 months) were retrospectively analysed and the ones with a component of Sickle haemoglobin(HbS), either in the form of Sickle cell anemia/homozygous Sickle cell disorders(SCA) or compound heterozygous diseases, like Sickle cell/β thalassemia(HbS/β), Sickle cell/Delta thalassemia(HbS/D), Sickle cell Haemoglobin/E thalassemia(HbS/E), were included in the study. People having Sickle cell trait (HbS trait), have also been included. Thorough history of painful crises, blood transfusions, family history and treatment history was elicited and every patient was clinically examined. The patients were diagnosed by High Pressure Liquid Chromatography (HPLC) or Thalassemia Mutation analysis by Polymerase Chain reaction (PCR). Results:A total of 95 patients with a component of HbS were considered as our study cohort, with HbS/β thalassemia patients being the majority (53.7%), followed by SCA (30.5%). Age of the study cohort ranged between 2-50 years age. HbS/β thalassemia patients presented at a later age (median 17.5 years) than SCA patients (median 12 years). Their demographic distribution is depicted in Table 1. The most common clinical presentation was painful crisis (32,33.7%), be it abdominal pain (11,11.6%) or bone pain (13,13.7%). Other presenting complaints were pallor (26,27.4%), jaundice (12,12.6%) and fever (4,4.2%). Some rarer presenting manifestations were fatigue (4,4.2%), splenic infarction (1,1%), convulsions (1,1%), Raynaud's phenomenon (1,1%), headache (1,1%) or itchy skin lesions (1,1%). Few patients (4,4.2%) had recurrent pregnancy loss, and one patient was diagnosed incidentally during an antenatal check-up. Most patients had more than one complaint. Very occasionally patients required hospital admission, the reasons being, chest pain, fever, convulsions or abdominal pain. HbS trait was diagnosed incidentally during evaluation for other illnesses, most commonly during evaluation of pallor (3,60%): one patient was later diagnosed with iron deficiency anemia. Most patients who attended our center were from within the state or neighbouring states. The patients were treated with Hydroxyurea, with/without blood transfusions, chelation therapy with Deferasirox as required and Folic acid supplementation. People with HbS Trait continued to receive Folate supplementation. Discussion and Conclusions:This study highlights the varied distribution of HbS among the population attending a tertiary care center, irrespective of a specific area-based population. Till date most studies conducted in India have highlighted the prevalence of Sickle cell disorders among specific focused populations. HbS/β thalassemia was the most common sickle cell disorder in our study. This is in contrast to most findings in published literature from other countries, where SCA is the commonest. Only one other study conducted in eastern India, has depicted a finding similar to ours. The median age of disease presentation was at a later age in our study, in contrast to findings in published literature from other countries. There is a variation in the severity of disease manifestation in our study cohort. The most common painful crisis was bone pain, followed by abdominal pain. Pallor was also one of the commonest presenting symptoms. Stroke, a common manifestation of SCA in other countries, was rare in our study cohort. Disclosures No relevant conflicts of interest to declare.

Clinically relevant updates of the HbVar database of human hemoglobin variants and thalassemia mutations.

HbVar (http://globin.bx.psu.edu/hbvar) is a widely-used locus-specific database (LSDB) launched 20 years ago by a multi-center academic effort to provide timely information on the numerous genomic variants leading to hemoglobin variants and all types of thalassemia and hemoglobinopathies. Here, we report several advances for the database. We made clinically relevant updates of HbVar, implemented as additional querying options in the HbVar query page, allowing the user to explore the clinical phenotype of compound heterozygous patients. We also made significant improvements to the HbVar front page, making comparative data querying, analysis and output more user-friendly. We continued to expand and enrich the regular data content, involving 1820 variants, 230 of which are new entries. We also increased the querying potential and expanded the usefulness of HbVar database in the clinical setting. These several additions, expansions and updates should improve the utility of HbVar both for the globin research community and in a clinical setting.

Prenatal Screening and Diagnosis of ß-Thalassemia in India: Is ARMS-PCR Enough?

Accurate and timely prenatal diagnosis of thalassemia is cornerstone to the success of thalassemia control; currently parents are screened for ß-thalassemia mutations by ARMS-PCR and subsequently chorionic villus sampling is done. We did an audit to ascertain whether the present design is adequate and determined the role of sequencing for pre-natal diagnosis of beta-thalassemia. This was a retrospective analysis of prenatal testing data collected over 10years, (2010-2019). ARMS-PCR was done to identify the beta-globin mutation followed by CVS wherever indicated. Data was classified into 3 groups:-5 most commonly occurring mutations (group 1), less common mutations (group 2) and mutations not detected (group 3). Total number of cases studied were 2128. Mean age of the cohort was 29.30years (range 18-48years). Approximately 90% individuals had one of the 5 common mutations in decreasing order of frequency: IVS 1-5 G>C (1297/2128); Codon 26G>A/HbE (451/2128); codon 30G>C (69/2128); codon 15G>A (61/2128); FS 41-42-CTTT (48/2128). Undetected mutations amounted to 7.3% (156/2128). Mean haemoglobin was highest in the group 2 (12.46g/dl) followed by the group 1 (11.20g/dl) and least in group 3 (10.99g/dl). MCV, MCH and MCHC showed similar trends. ANOVA on all these parameters, except RDW, within groups and for individual mutations, were statistically significant (p < 0.001). The hemogram-HPLC-ARMS-PCR-CVS approach is a cost-effective and established method but tends to miss out a considerable number of thalassemia mutations (~7%), emphasizing the role of sequencing in difficult cases. This needs to be addressed while formulating guidelines for thalassemia screening in future.

Prenatal Diagnosis and Screening of Thalassemia Mutations in Bangladesh: Presence of Rare Mutations

Thalassemia is the most common congenital monogenic disorder in Bangladesh. Prenatal diagnosis (PND) and pregnation termination of an affected child is one of the best options to reduce the burden of thalassemic children. This article reports the results of DNA analyses of chorionic villus sampling (CVS) and amniocentesis of fetuses of mothers who came to the thalassemia center of Dhaka Shishu (Children) Hospital, Dhaka, Bangladesh. DNA analysis was done by real-time polymerase chain reaction (qPCR) and Sanger sequencing. Maternal contamination was ruled out by variable number of tandem repeats (VNTRs). A total of 232 samples were analyzed. Hb E (HBB: c.79G>A)/β-thalassemia (Hb E/β-thal) was the most common type of thalassemia seen in 32 samples (13.79%) followed by β-thal major (β-TM) in 10 cases (4.31%). Molecular characterization of the most predominant mutation was IVS-I-5 (G>A) (HBB: c.92+5G>C). The analysis also revealed five rare mutations: IVS-II-654 (C>T) (HBB: c.316-197C>T), IVS-II-1 (G>A) (HBB: c.315+5G>A), codon 44 (–C) (HBB: c.135delC), −86 (C>A) (HBB: c.-136C>A) and codons 14/15 (+G) (HBB: c.45_46insG), which have not been reported previously in Bangladesh. This study provides important information for PND and will help in the development of similar diagnostic programs for other DNA centers in Bangladesh.

Thalassemia and erythroid transcription factor KLF1 mutations associated with borderline hemoglobin A2 in the Thai population.

IntroductionElevated hemoglobin (Hb) A2 is an important diagnostic marker for β-thalassemia carriers. However, diagnosis of cases with borderline Hb A2 may be problematic. We described the molecular characteristics found in a large cohort of Thai subjects with borderline Hb A2.Material and methodsExamination was done on 21,657 Thai subjects investigated for thalassemia at Khon Kaen University, Thailand. A total of 202 subjects with borderline Hb A2 (3.5–4.0%) were selectively recruited and hematological parameters were recorded. DNA variants in α-, β-, δ-globin, and Krüppel-like factor 1 (KLF1) genes were examined using PCR.ResultsAmong 202 subjects, DNA analysis identified carriers of α+-thalassemia (n = 48; 23.8%), β-thalassemia (n = 22; 10.9%) and KLF1 mutations (n = 48; 23.8%). No molecular defect was observed in the remaining 84 (41.5%) subjects. Interaction of KLF1 and α-thalassemia was observed in 10 cases. Of the 22 β-thalassemia carriers, five β+-thalassemia mutations were identified with lower MCV and higher Hb A2. Seven KLF1 mutations were detected in 10 genotypes in subjects with higher MCV and Hb F. No β0-thalassemia, α-globin gene triplication or δ-globin gene mutation was detected.ConclusionsA large proportion of subjects with borderline Hb A2 are not β-thalassemia carriers and for those with β-thalassemia, only mild β+-thalassemia mutations were detected. Evaluation of the patients using Hb A2, Hb F and MCV values will help in selecting cases for further molecular analysis. The results should explain the unusual phenotype of the cases and facilitate a thalassemia screening program in the region.