Alpha-thalassemia Carriers Research Articles

Spectrum of alpha-thalassemia mutations according to immigration, ethnicity in Bursa, Turkey and first observing of SEA double gene deletion

Background: Thalassemia syndromes are common in the Mediterranean region, including Turkey. Our aim is investigating the genetic mutation diversity of alpha thalassemia cases in Bursa and to investigate the mutation diversity in relation to the cases' immigration status and ethnic origins.
 Materials and Methods:
 Retrospective analysis was performed on 66 cases aged 1-18 years who were diagnosed as alpha thalassemia carriers by genetic mutation analysis. The patients' complete blood count, ferritin levels, hemoglobin electrophoresis, genetic mutations, immigration status, origin, whether they received iron therapy prior to the diagnosis were all evaluated.
 Results: Of the 66 cases, 53% were female and 47% were male. The most common genetic mutation was the 3.7% deletion with a rate of 51.5%, followed by the heterozygous mutation with a deletion of 20.5 with a rate of 21.20%. SEA double gene deletion homozygous mutation 1.5% and, heterozygous mutation 1.5%. Eleven different genotypes of alpha thalassemia were discovered. While 3.7 deletion heterozygous mutation was most common in cases immigrating from Bulgaria, Syria, and Azerbaijan, FIL deletion mutation was most common in cases immigrating from Georgia, and alpha-2 polyA-2 heterozygous mutation was found in one case immigrating from Greece.
 Conclusion: The most common mutation was 3.7 deletion and SEA double gene deletion was discovered, which had not been discovered in previous studies. With this study, we added to the literature the genetic mutation diversity in Bursa, which sees a lot of immigration.

Hemoglobin H hastalığında genetik danışmanlık önemli midir?

Alpha thalassemia is a genetic disease characterized by insufficient expression or definite absence of the α-globin chain. Three large deletions (thal-1; 26.5 kb or MedII, 20.5 kb and 17.4 kb or MedI) and two small deletions (thal-2; 4.2 kb and 3.7 kb) have been characterized in our country. In addition, two different PolyA mutations (PA1: AATAAA>AATAAG and PA2: AATAAA>AATGA) on the α2-globin gene (αα/αPAα), 5nt deletion (αα/α5ntα), and unstable Hb variant (CD 59; GGC→GAC) synthesized by the α1-globin gene (αα/ααCD59) have been reported. More than ten different combinations of α-thal-1 and α-thal-2 (--/-α) or HbH genotypes with point mutations (--/αPAα or --/ααCD59) were determined. In this study, which was carried out in Çukurova region, it is aimed to emphasize the importance of giving genetic counseling to families with alpha thalassemia carriers and to determine genotype combinations. DNA was isolated from blood samples taken from 5 children and their families who were admitted to Çukurova University Balcalı Hospital and diagnosed with severe anemia (Hb

5610480 TRANSFORMATION OF THALASSAEMIA GENETIC TESTING BY THE IMPLEMENTATION OF NGS-BASED THALASSEMIA ASSAY

Background: The haemoglobinopathies, are the most common monogenic diseases worldwide, and have been recognized as a growing global health challenge. Thalassemia is a serious health problem in Cyprus where 12 % of the population are carriers of beta-thalassaemia and about 19% are carriers of alpha-thalassaemia[1]. Because of the high incidence of the disease and the heavy impact on the health care system, the Cyprus National Prevention Programme was initiated and is successfully running since 1978. The traditional strategy for thalassaemia molecular diagnosis was designed either to identify only the most common mutations, or to target the main causing genes of thalassaemia, HBA, and HBB by conventional molecular techniques such as PCR, Real-Time PCR, MLPA, Sanger sequencing, and Mini sequencing. The complex analysis with the use of multiple different techniques to obtain a result, can cause prolonged turn-around times and costly processes and may even not be conclusive. Aim: The aim is to replace the laborious conventional molecular testing with a comprehensive analysis of the thalassaemias by the establishment of an NGS-based test for the simultaneous genetic analysis of all variants on both HBA and HBB gene clusters. Methods: A commercial CE-IVD NGS Thalassemia kit, that detects all sequence variants in HBA1, HBA2, and HBB in a single run was evaluated. The assay detects SNVs, and Indels and also uses two independent methods in parallel to detect CNVs. For the initial evaluation, 25 DNA samples with known genotypes, previously characterised in our lab, were used. For the validation study, 100 DNA samples were analysed using the NGS Thalassemia kit in parallel with the conventional molecular methods. The results obtained were assessed and compared. Results: The initial evaluation of the NGS thalassemia assay demonstrated the strength of the assay to accurately detect all variations in the analysed genes. Moreover, the assay also detected variations that were not previously identifiable by our conventional multi-method strategy, Thus, we demonstrated the ability of the assay for complete analysis of thalassaemia. The assay was successfully validated: the results obtained by the NGS assay were in complete concordance with results from parallel analysis by our standard strategy. Hence, the NGS Thalassemia kit was successfully implemented in our daily routine practice. Summary/conclusion: The implementation of the NGS Thalassemia kit allows for comprehensive analysis for the haemoglobinopathies in a single run. It is very efficient compared to our traditional previously conducted thalassemia testing, yielding results of unprecedented quality, as well as saving time on three levels - for the physician, for the lab, and most importantly for the patient. Reference 1. Kountouris, P., et al, Scientific reports, 2016.6: p. 26371.

5558131 GENETICS OF IRANIAN ALPHA THALASSEMIA PATIENTS; COMPREHENSIVE ORIGINAL STUDY

Background: Alpha thalassemia is the most prevalent monogenic gene disorder in the world and especially in Mediterranean countries. In the current hematological phenotype of patients with different genotypes, and also the effect of missense mutations on the protein function and stability were evaluated in a large cohort study. Methods: One thousand five hundred and and sixty subjects were participated in the study and divided into two groups; 259 normal subjects and 1301 alpha thalassemia carriers. Genomic DNA was extracted and analyzed using ARMS PCR, Multiplex Gap and direct sequencing. The effects of single nucleotide change on the protein function and stability were predicted by free available databases of human polymorphisms. Results: Sixty-three different genotypes were seen in the patients. The more prevalent was heterozygote form of -α3.7 (41.4%) followed by -α3.7 homozygote(11.6%) and - -MED(3.8%). The significant differences were seen in mean hemoglobin level [F=20.5, p<0.001] between the Alpha globin genotypes, when adjusted for gender. Moreover, 28 different mutations were found in our study. A significant relationship was seen between ethnicity and the alpha globin mutation frequency X2 (df;8) = 38.36, p <0.0001). Conclusion: Different genotypes could be displayed as different phenotypes. The mutation frequency distributions in our region is different from other parts of Iran. Significant differences are seen in the spectrum of mutation frequency between various ethnicity. Finally, some missense mutations maybe did not considerably effect on the proteins and could be a neutral mutations. Keywords: Alpha Thalassemia, Genotype, Phenotype, Southwest of Iran

Thalassemia, a human blood disorder.

A group of inherited blood defects is known as Thalassemia is among the world's most prevalent hemoglobinopathies. Thalassemias are of two types such as Alpha and Beta Thalassemia. The cause of these defects is gene mutations leading to low levels and/or malfunctioning α and β globin proteins, respectively. In some cases, one of these proteins may be completely absent. α and β globin chains form a globin fold or pocket for heme (Fe++) attachment to carry oxygen. Genes for alpha and beta-globin proteins are present in the form of a cluster on chromosome 16 and 11, respectively. Different globin genes are used at different stages in the life course. During embryonic and fetal developmental stages, γ globin proteins partner with α globin and are later replaced by β globin protein. Globin chain imbalances result in hemolysis and impede erythropoiesis. Individuals showing mild symptoms include carriers of alpha thalassemia or the people bearing alpha or beta-thalassemia trait. Alpha thalassemia causes conditions like hemolytic anemia or fatal hydrops fetalis depending upon the severity of the disease. Beta thalassemia major results in hemolytic anemia, growth retardation, and skeletal aberrations in early childhood. Children affected by this disorder need regular blood transfusions throughout their lives. Patients that depend on blood transfusion usually develop iron overload that causes other complications in the body systems like renal or hepatic impairment therefore, thalassemias are now categorized as a syndrome. The only cure for Thalassemias would be a bone marrow transplant, or gene therapy with currently no significant success rate. A thorough understanding of the molecular basis of this syndrome may provide novel insights and ideas for its treatment, as scientists have still been unable to find a permanent cure for this deadly disease after more than 87 years since it is first described in 1925.

Individual Red Blood Cell Fetal Hemoglobin Concentrations Determined By Imaging Flow Cytometry and the Number of F Cells Are Very Good Predictors of Clinical Response to Increased Level of HbF and Are Associated with Hemolysis, Tricuspid Regurgitant Jet Velocity, and Sickle Retinopathy in Adult Sickle Cell Disease Patients

Sickle cell disease (SCD) patients with similar total fetal hemoglobin (HbF) levels may present with different severity, which is attributed to the heterogeneous distribution of HbF among red blood cells (RBCs) with varying percentages of circulating HbF-containing RBCs (% F cells). Steinberg et al. (2014) proposed that an individual HbF concentration per cell (iHbF) above 10pg inhibits the polymerization of HbS, so the development of precise methods to quantify iHbF and the percentage of F cells may allow clinicians to use those parameters as targets to monitor patient treatment. We devised a novel procedure to very accurately measure and quantify iHbF using imaging flow cytometry (Amnis Imagestream) and investigated its correlations with clinical manifestations. Samples from 54 adult SCD patients (22 [41%] female, 39 [72%] on hydroxyurea) were used to acquire images from RBCs labeled with fluorescent anti-HbF antibody and were analyzed using IDEAS software v 6.2. Along with the hematimetric data and HPLC quantification of total HbF of each sample, we modified the formulas by Maier-Redelsperger et al. (1994) and Horiuchi et al. (1995), with minimization of interference from autofluorescence of non-F cells, correction for area and fluorescence intensity, and analyzed RBC subpopulations with varying levels of HbF expression. Statistical analysis was performed using R considering a p value <0.05 to be statistically significant. We did not find associations of iHbF with alpha thalassemia carrier state or beta globin cluster haplotypes Benin or Bantu, the most common in our population. Perpendicular cutoff correlation studies defined two patient populations: one group with lower mean iHbF (9.9pg) and lower % F cells (28.2%) and another group with a higher mean iHbF (15.8pg) and higher mean % F cells (72.5%). The latter group had a lower frequency of vaso-occlusive crises (p=0.011). That was further validated with our finding that the cutoffs of % F cells>30% and iHbF>10pg were strongly associated with lower absolute reticulocyte counts (p<0.001), LDH (p=0.002), and indirect bilirubin (p<0.001). The association between hemolysis and iHbF was further explored regarding pulmonary hypertension, showing that a value of iHbF>10pg was associated with a lower median tricuspid regurgitant jet velocity than in patients with iHbF<10pg (2.72m/s vs. 2.29m/s, p=0.046). We also found an association of a mean iHbF<8pg with the presence of sickle retinopathy (p=0.002). Patients with retinopathy had a lower mean iHbF and % F cells than those without it (7.08pg vs. 8.75pg, p=0.032; 48.5% vs. 71.0%, p=0.043, respectively), suggesting increments in iHbF and % F cells may be protective against sickle retinopathy, a complication not usually associated with high hemolytic rates. Our results suggest that a percentage of F cells above 30% along with individual RBC HbF concentration of 10pg may be protective against complications in SCD patients and, therefore, should be explored as goals for hydroxyurea therapy in this population. In addition, the data presented here suggest further studies on the efficacy of HbF induction therapy against the development of sickle retinopathy may be performed in a large population of sickle cell disease patients.

Mutations in Thalassemia Carrier Couples: The Importance of Prenatal Diagnostic Tests.

Thalassemia carrier couples play an important role in increasing thalassemia patients. The study of thalassemia genotypes in carrier couples is also effective in improving genetic counseling for them. The aim of this study was to investigate the prevalence of thalassemia mutations and genotypes in couples. This cross-sectional study was performed on 241 couples who were suspected of thalassemia from April 2018 to March 2020 in Lorestan province. Statistical analysis of data was performed using SPSS software 16.0 (SPSS Inc., Chicago, IL, USA). Online tools such as www.ithanet.eu/db/ithagenes and http://globin.bx.psu.edu/ hbvar/menu.html were also used to match patients' mutations with known cases. IVSII-1 (G>A), CD36-37 (-T), IVSI-110 (G>A), --Med, and α3.7 were the most common mutations in the beta and alpha genes, respectively. IVSII-1 (G>A) β0/β (26.1%), CD36-37 (-T) β0/β (21.1%), and IVSI-110 (G>A) β0/β (10.3%) genotypes were the most common in women. The frequency of these genotypes in men were 24.8%, 28.6%, and 12.8%, respectively. Among alpha thalassemia carriers, the α3.7α/α α genotype had the highest frequency among women (3.7%) and men (5.3%). Alpha and beta-thalassemia were 15 and 13 times higher in related women and 18 and 9 times higher in related men than non-related ones, respectively. This difference was statistically significant (p < 0.001). In addition, 12.8% of fetuses were thalassemia major, 31.9% beta thalassemia minor, and 10.3% normal. Thalassemia screening in related couples plays an important role in reducing thalassemia major infants.

Screening of Some Indicators for Alpha-Thalassemia in Fujian Province of Southern China.

BackgroundCarrier screening is the most effective means of controlling the prevalence of alpha-thalassemia. However, due to the differences in ethnic populations and genotypes, the distribution of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and hemoglobin A2 (HbA2) varies in different regions. This study aimed to examine screening efficiency of these indicators in different genotypes of alpha-thalassemia in Fujian Province, China.MethodsThe data of 13,294 subjects collected from May 2016 to December 2019 were reviewed. The participants were categorized as alpha-thalassemia group and negative-for-alpha-thalassemia group based on the results of the genetic analysis. The distribution of MCV, MCH, and HbA2 in different groups was analysed statistically. And the screening efficiency of different indicators and schemes was compared in different genotypes. The positive criteria of MCV < 80fL, MCH < 27pg, and Hb A2< 2.5% were applied.ResultsAmong the 13,294 subjects, 2658 were alpha-thalassemia carriers. The genotypes of –SEA/αα and -α3.7/αα are the most prevalent with 63.9% and 21.9% in Fujian Province, China. There were significant differences in the distribution of the three indicators in different groups. The detection rate of the three indicators combined screening was 92.6%.ConclusionThe distribution of the three indicators overlapped partly between alpha-thalassemia group and negative-for-alpha-thalassemia group. They showed significant differences in the median comparison of seven common genotypes. Combined screening with MCV, MCH and HbA2 improved the detection rate of alpha-thalassemia. The results of this study provide a data basis for clinical laboratories and a reliable reference for clinical consultation.

P–539 The use of expanded carrier screening of gamete donors

Abstract Study question What are the sperm and egg donor rejection rates after expanded carrier screening (ECS)? Summary answer Using an ECS panel looking at 46/47 genes, 17.6% of donors were rejected. What is known already The use of ECS is becoming commonplace in assisted reproductive technology, including testing of egg and sperm donors. Most national guidelines recommend rejection of donors if they are carriers of a genetic disease. If the use of ECS increases, there will be a decline in the number of donors available. Study design, size, duration A review of the current preconception ECS panels available to donors was carried out through an online search. The genetic testing results of donors from Cryos International were analysed to determine how many were rejected on the basis of the ECS. Participants/materials, setting, methods Data on gamete donors and their carrier status was provided by Cryos International, who screen donors using their own bespoke ECS panel. The ECS panels identified through the review were compared to the Cryos International panel and data. Main results and the role of chance A total of 16 companies and 42 associated ECS panels were reviewed. There were a total of 2673 unique disorders covered by the panels examined, with a mean of 329 disorders screened. None of these disorders were common to all panels. Cryos International screen 46 disorders in males and 47 in females. From 883 candidate donors, 17.6% (155/883) were rejected based on their ECS result. Carriers of alpha-thalassaemia represented the largest proportion of those rejected (19.4%, 30/155), then spinal muscular atrophy (15.5%, 24/155) and cystic fibrosis (14.8%, 23/155). Limitations, reasons for caution Panel information was found on company web sites and may not have been accurate. Wider implications of the findings: This study highlights the need for consistent EU regulations and guidelines which allow genetic matching of gamete donors to recipients, preventing the need to reject donors who are known carriers. A larger ECS panel would be most beneficial, however, this would not be viable without matching of donors and recipients. Trial registration number Not applicable

The use of expanded carrier screening of gamete donors.

STUDY QUESTIONWhat are the sperm and egg donor rejection rates after expanded carrier screening (ECS)?SUMMARY ANSWERUsing an ECS panel looking at 46/47 genes, 17.6% of donors were rejected.WHAT IS KNOWN ALREADYThe use of ECS is becoming commonplace in assisted reproductive technology, including testing of egg and sperm donors. Most national guidelines recommend rejection of donors if they are carriers of a genetic disease. If the use of ECS increases, there will be a decline in the number of donors available.STUDY DESIGN, SIZE, DURATIONA review of the current preconception ECS panels available to donors was carried out through an online search. The genetic testing results of donors from Cryos International were analysed to determine how many were rejected on the basis of the ECS.PARTICIPANTS/MATERIALS, SETTING, METHODSData on gamete donors and their carrier status was provided by Cryos International, who screen donors using their own bespoke ECS panel. The ECS panels identified through the review were compared to the Cryos International panel and data.MAIN RESULTS AND THE ROLE OF CHANCEA total of 16 companies and 42 associated ECS panels were reviewed. There were a total of 2673 unique disorders covered by the panels examined, with a mean of 329 disorders screened. None of these disorders were common to all panels. Cryos International screen 46 disorders in males and 47 in females. From 883 candidate donors, 17.6% (155/883) were rejected based on their ECS result. Carriers of alpha-thalassaemia represented the largest proportion of those rejected (19.4%, 30/155), then spinal muscular atrophy (15.5%, 24/155) and cystic fibrosis (14.8%, 23/155).LIMITATIONS, REASONS FOR CAUTIONPanel information was found on company websites and may not have been accurate.WIDER IMPLICATIONS OF THE FINDINGSThis study highlights the need for consistent EU regulations and guidelines that allow genetic matching of gamete donors to their recipients, preventing the need to reject donors who are known carriers. A larger ECS panel would be most beneficial; however, this would not be viable without matching of donors and recipients.STUDY FUNDING/COMPETING INTEREST(S)No specific funding was obtained. J.C.H. is the founder of Global Women Connected, a platform to discuss women’s health issues and the Embryology and PGD Academy, who deliver education in clinical embryology. She has been paid to give a lecture by Cryos in 2019. A-B.S. is an employee of Cryos International.TRIAL REGISTRATION NUMBERN/A

Interaction of Hematological Analysis and α-globin Genotypes among Eligible Blood Donors

Introduction: Alpha thalassaemia is one of the haemoglobin disorders in which the carriers of alpha thalassaemia may have normal haemoglobin level and are eligible to donate blood which may bring complications. This study is to investigate the interaction of haematological parameter with α-globin genotypes among eligible blood donors. Materials &amp; Methods: A cohort study with 270 eligible blood donors were analysed for red cell indices. Alpha-globin (α-globin) genotyping was performed for seven deletions, six point mutations and two triplications. Statistical analyses were performed to compare the α-globin genotypes with haematological data. Results: High prevalence of α-thalassaemia carriers (7.7%, 21/270) was found among blood donors. All of them did not show anaemic pictures with a normal Hb level (&gt;12 gm/dl). Five genotypes were identified consisting of 249 αα/αα (92.2%), nine -α 3.7/αα (3.3%), nine-- SEA/αα (3.3%), two -α 4.2/αα (0.7%) and one ααCS/αα (0.4%). Different α-globin genotypes showed a significant difference in RBC, MCV, MCH, MCHC, RDW, and Hct/Hb ratio (p&lt;0.05) due to the different extent of α-globin chain reduction. Conclusion: Our study concluded that by using Hb level alone is not sufficient to screen for the eligibility of blood donors. Full blood count (FBC) screening with borderline MCV and MCH levels might be able to rule out silent α-thalassemia carriers. FBC and molecular characterisation should be incorporated together to properly rule out α-thalassaemia carriers.&gt;&lt;0.05) due to the different extent of α-globin chain reduction. Conclusion: Our study concluded that by using Hb level alone is not sufficient to screen for the eligibility of blood donors. Full blood count (FBC) screening with borderline MCV and MCH levels might be able to rule out silent α-thalassemia carriers. FBC and molecular characterisation should be incorporated together to properly rule out α-thalassaemia carriers.

Prevalence of Anemia Among Jazan University Students.

PurposeAnemia is one of the most common disorders affecting the population in both low-income and developing countries. This study aimed to determine the prevalence of anemia and iron deficiency in apparently normal male and female students of Jazan University, Saudi Arabia.Material and MethodsThis cross-sectional study was conducted at the Faculty of Applied Medical Sciences, Jazan University, Gizan, Saudi Arabia. One hundred thirty-four (85 males and 49 females) healthy students with no outward signs of illness were recruited. A specific questionnaire related to sociodemographic and health characteristics was designed and validated. The collected samples were analyzed for complete blood count, serum iron, and serum ferritin.ResultsIn the male group, 81 participants (95.3%) were nonanemic with normal hemoglobin (14.99 ± 1.11g/dl), whereas only 4 males were anemic (hemoglobin <13.0 g/dl). On the other hand, anemia was more prevalent in female students, as 67.35% had hemoglobin values less than 12.0 g/dl. In addition, 26.54% of the anemic females had low serum iron and serum ferritin, indicating iron-deficiency anemia. Although the majority of male participants had normal hemoglobin levels, 37.6% of them had low mean cell volume (MCV), low mean cell hemoglobin (MCH), low mean cell hemoglobin concentration (MCHC), and high red blood cell (RBC) counts, suggesting thalassemia.ConclusionAnemia, in particular iron-deficiency anemia, was found to be highly prevalent in females, whereas more than one-third of the male participants might be carriers of alpha or beta thalassemia. Additionally, low dietary iron intake and irregular meal consumption could be two of the possible causes of anemia in the study population. Conducting this study on a large scale of male and female students from different faculties of Jazan University will provide in-depth details about the prevalence and types of anemia among them.

Deletional Alpha-Thalassemia Alleles in Amazon Blood Donors.

Alpha-thalassemia is highly prevalent in the plural society of Brazil and is a public health problem. There is limited knowledge on its accurate frequency and distribution in the Amazon region. Knowing the frequency of thalassemia and the prevalence of responsible mutations is, therefore, an important step in the understanding and control program. Hematological and molecular data, in addition to serum iron and serum ferritin, from 989 unrelated first-time blood donors from Amazonas Hemotherapy and Hematology Foundation (FHEMOAM) were collected. In this study, the subjects were screened for −α3.7/4.2/20.5, −SEA, −FIL, and −MED deletions. Alpha-thalassemia screening was carried out between 2016 and 2017 among 714 (72.1%) male and 275 (27.9%) female donors. The aims of this analysis were to describe the distribution of various alpha-thalassemia alleles by gender, along with their genotypic interactions, and to illustrate the hematological changes associated with each phenotype. Amongst the patients, 5.35% (n = 53) were diagnosed with deletion –α−3.7 and only one donor with α−4.2 deletion. From the individuals with –α−3.7, 85.8% (n = 46) were heterozygous and 14.20% (n = 7) were homozygous. The frequency of the –α−3.7 deletion was higher in male (5.89%) than in female (4.0%). There is no significant difference in the distribution of –α−3.7 by gender (p = 0.217). The –α20.5, −SEA, and −MED deletions were not found. All subjects were analyzed for serum iron and serum ferritin, with 1.04% being iron deficient (n = 5) and none with very high levels of stored iron (>220 µg/dL). Alpha-thalassemia-23.7kb deletion was the most common allele detected in Manaus blood donors, which is a consistent result, once it is the most common type of α-thalassemia found throughout the world. As expected, the mean of hematological data was significantly lower in alpha-thalassemia carriers (p < 0.001), mainly homozygous genotype. Leukocytes and platelet count did not differ significantly. Due to the small number of individuals with iron deficiency found among blood donors, the differential diagnosis between the two types of anemia was not possible, even because minor changes were found among hematological parameters with iron deficiency and α-thalassemia. Despite this, the study showed the values of hematological parameters, especially MCV and MCH, are lower in donors with iron deficiency, especially when associated with α-thalassemia, and therefore, it may be useful to discriminate different types of microcytic anaemia. In conclusion, we believed screening for thalassemia trait should be included as part of a standard blood testing before blood donation. It should be noted that this was the first study to perform the screening for alpha deletions in blood donors from the Manaus region, and further studies are required to look at the effects of donated thalassemic blood.

Alpha-Thalassemia Carrier due to –α3.7 Deletion: Not So Silent

Background/Objective: Alpha-thalassemia is one of the most prevalent genetic diseases, with the –α^3.7 deletion being the most common mutation. Molecular studies have suggested mechanisms to explain the mild phenotype of “silent carrier” heterozygotes. However, the correlation between the clinical laboratory picture and the –α^3.7 heterozygous state remains unclear, thus we chose to investigate. Methods: We analyzed the medical files of 192 children evaluated for microcytosis at our tertiary center between 2007 and 2017 and diagnosed as heterozygotes for the –α^3.7 deletion. Additional α-thalassemia mutations, iron deficiency anemia, and β-thalassemia were ruled out. Laboratory parameters were compared to age- and sex-matched reference values. Results: The –α^3.7 carriers had significantly lower Hb and mean corpuscular volume (MCV) than the reference population, and significantly higher red blood cell counts across all age groups. The greatest reduction in Hb level appeared among male adolescents, while MCV was consistently 2 SDs lower than normal in most patients older than 2 years. Conclusion: Heterozygosity for the –α^3.7 deletion was associated with clinically significant microcytosis and mild anemia in our pediatric population. In the absence of iron deficiency and β-thalassemia, this finding provides a diagnosis for mild microcytic anemia, making additional investigations of microcytosis unnecessary.

573: A non-invasive prenatal test for Alpha Thalassemia Hb Barts and Hb Constant Spring

Hb Barts and Hb Constant Spring are the two most frequent severe forms of alpha thalassemia. The UNITYTM Carrier screen is a Lab Developed Test that performs reflex alpha thalassemia NIPT when the mother is determined to be an alpha-thalassemia carrier. This study analyzes the ability of the UNITYTM NIPT to exclude inheritance of Hb Barts and detect maternal inheritance of Hb Constant Spring. The UNITYTM Alpha Thalassemia NIPT was performed on simulated cell-free DNA samples. The assay is a multiplex PCR reaction that employs multiple technologies to evaluate the fetal genotype. Polymorphic alleles contained within the SEA deletion region are assayed to exclude inheritance of double deletions and therefore Hb Barts. Quantitative counting templates are used to accurately measure the number of DNA molecules present in the reaction. Relative mutation dosage is performed to determine the maternal and paternal inheritance of the Constant Spring allele. Gap-PCR is used to detect paternal inheritance of an SEA deletion. A likelihood ratio is used to combine these measurements and determine the Hb Constant Spring status of the fetus. Simulated cell-free DNA from pregnant mothers was prepared by shearing and combining genomic DNA samples. Hb Barts exclusion was tested using SEA carrier DNA mixed with healthy or Hb Barts DNA. Hb Barts was excluded from all healthy DNA samples and was not excluded from all affected samples (n=96). Maternal inheritance of the Constant Spring variant was tested on Constant Spring carrier DNA mixed with healthy SEA carrier DNA. All calls were correct at 5%, 10% and 20% fetal fractions, two 5% samples resulted in no-calls (n=12). These results analytically demonstrate the ability of the UNITYTM alpha thalassemia NIPT to identify healthy fetuses in carrier mothers. The UNITYTM alpha thalassemia carrier screen with reflex NIPT will enable detection of both maternal carrier status and fetal disease risk from a single blood tube. This removes the need for paternal DNA and decreases the turn around time to identify high-risk fetuses.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

PB2401 GENETICS OF IRANIAN ALPHA THALASSEMIA PATIENTS; COMPREHENSIVE ORIGINAL STUDY

Please indicate where the abstract has been published before: The full paper was published in Biochemical genetics journalBackground:Alpha thalassemia is the most prevalent monogenic gene disorder in the world and especially in Mediterranean countries. In the current hematological phenotype of patients with different genotypes, and also the effect of missense mutations on the protein function and stability were evaluated in a large cohort study.Aims:To evaluate the genetics of large populations of Iranian alpha thalassemia patientsMethods:One thousand five hundred and sixty subjects were participated in the study and divided into two groups; 259 normal subjects and 1301 alpha thalassemia carriers. Genomic DNA was extracted and analyzed using ARMS PCR, Multiplex Gap, and direct sequencing. The effects of single nucleotide change on the protein function and stability were predicted by free available databases of human polymorphisms.Results:Sixty‐three different genotypes were seen in the patients. The more prevalent was heterozygote form of ‐α3.7 (41.4%) followed by ‐α3.7 homozygote(11.6%) and ‐ ‐MED(3.8%). The significant differences were seen in mean hemoglobin level [F = 20.5, p &lt; 0.001] between the Alpha globin genotypes when adjusted for gender. Moreover, 28 different mutations were found in our study. A significant relationship was seen between ethnicity and the alpha globin mutation frequency X2 (df;8) = 38.36, p &lt;0.0001).Summary/Conclusion:Different genotypes could be displayed as different phenotypes. The mutation frequency distributions in our region are different from other parts of Iran. Significant differences are seen in the spectrum of mutation frequency between various ethnicity. Finally, some missense mutations maybe did not considerably effect on the proteins and could be a neutral mutation.image

Hemoglobinopathy Evaluation in Routine Carrier Testing: Results to Inform Guidelines and Protocols [5O

INTRODUCTION: ACOG guidelines recommend red blood cell indices followed by hemoglobin electrophoresis to assess carrier status for hemoglobinopathies. However, data is limited on outcomes of these recommendations, and the role of DNA testing. METHODS: Our database was queried for abnormal hemoglobin evaluations from 42,166 consecutive samples of mixed ethnicities. Hemoglobin evaluation consisted of red blood indices and, high-performance liquid chromatography followed by, if necessary, hemoglobin electrophoresis. A subset (n= 2,863) also had DNA genotyping. Abnormal reports were evaluated individually, and test metrics and interpretations were tabulated. RESULTS: Of the 42,166 samples, 2,278 (5.4%) had abnormal hemoglobin evaluation results. Of the 2,278 abnormal results, the most common interpretation (32%) was 'possible alpha thalassemia trait', followed by 'sickle cell trait' (22%), 'possible beta thalassemia trait' (10%), 'elevated hemoglobin F' (10%), and 'hemoglobin C trait' (5%). Of the 2,863 samples that had genotyping, 146 (5.1%) had abnormal molecular results. Genotyping confirmed 100% (5/5) of samples with abnormal hemoglobin evaluation indicating 'consistent with alpha thalassemia trait' and 41% (17/41) indicating 'possible alpha thalassemia trait'. Sixteen percent (14/89) of samples with abnormal molecular results, and normal hemoglobin evaluation for alpha thalassemia, were silent alpha thalassemia carriers. CONCLUSION: Our study estimates a 5% positivity rate for hemoglobin evaluation. However, some results are non-specific and require molecular follow up. Forty-one percent of hemoglobin evaluation results interpreted as 'possible alpha thalassemia trait' were confirmed as genetic carriers. Concurrent DNA testing provides information and results more quickly to patients, and better identifies those patients truly at risk to be carriers of alpha thalassemia.

Genetics of Iranian Alpha-Thalassemia Patients: A Comprehensive Original Study.

Alpha thalassemia is the most prevalent monogenic gene disorder in the world, especially in Mediterranean countries. In the current hematological phenotype of patients with different genotypes, the effects of missense mutations on the protein function and also stability were evaluated in a large cohort study. A total of 1,560 subjects were enrolled in the study and divided into two groups: 259 normal subjects; and 1301 alpha-thalassemia carriers. Genomic DNA was extracted and analyzed using ARMS PCR, Multiplex Gap, and direct sequencing. The effects of single nucleotide change on the protein function and stability were predicted by freely available databases of human polymorphisms. Sixty-three different genotypes were seen in the patients. The more prevalent was heterozygote form of -α3.7 (41.4%) followed by -α3.7 homozygote (11.6%) and -MED (3.8%). The significant differences were seen in mean hemoglobin level [F = 20.5, p < 0.001] between the Alpha-globin genotypes, when adjusted for gender. Moreover, 28 different mutations were found in our study. A significant relationship was seen between ethnicity and the alpha-globin mutation frequency χ2 (df;8) = 38.36, p < 0.0001). Different genotypes could display as different phenotypes. The mutation frequency distributions in our region are different from those of other parts of Iran. Significant differences are seen in the spectrum of mutation frequency among various ethnicities. Finally, some missense mutations might not have considerable effect on the proteins, and they could be neutral mutations.

Changes in HbA2 and HbF in alpha thalassemia carriers with KLF1 mutation

α-thalassemia carriers are common in Mediterranean regions, particularly in the Sardinian population. Their haematological phenotype is characterized by reduced MCV and/or MCH with normal or slightly reduced HbA2 levels and normal HbF. Krüppel-like factor 1 (KLF1) is a pleiotropic erythroid transcription factor that is essential for haematopoiesis. Mutations in the KLF1 gene trigger a series of benign human red blood phenotypes, such as an increase in HbA2 and HBF. Recently, it has been found that KLF1 mutations were a frequent cause of borderline HbA2 levels in a group of Sardinian subjects. Here, we found that KLF1 mutations modulate the phenotype in a cohort of α-thalassemia carriers.

Mild to Severe Anemia of Undiagnosed Etiology Diagnosed by Genetic Study

We report a case of a child with beta thalassemia major, whose mother is a carrier of beta thalassemia and father is having hereditary persistence of fetal hemoglobin. Gene study revealed compound heterozygous for codon 8/9+G and IVS-1-5 G&gt;C point mutation. Another four cases of anemia not responding to iron diagnosed to have alpha thalassemia carrier are also reported here.J Nepal Paediatr Soc 2016;36(2):213-215.