Hb Disorders Research Articles

A Novel β-Thalassemia Mutation [IVS-I-6 (T>G), HBB: c.92+6T>G] in a Chinese Family

β-Thalassemia (β-thal) is one of the most common inherited hemoglobin (Hb) disorders in southern China. Up to now, the mutation spectrum of β-thal has been increasingly broadened through various molecular methods. In this study, a 34-year-old female displaying microcytic, hypochromic anemia was first detected with a novel IVS-I-6 (T>G) (HBB: c.92+6T>G) mutation by Sanger sequencing. Pedigree analysis performed on her family showed that her mother and her daughter, who had abnormal hematological indices, also carried this mutation, while her other family members with normal hematological phenotypes, were not detected to carry any mutation. Based on the observed symptoms in this Chinese family, we concluded that this novel mutation was associated with a mild β-thal phenotype.

Pattern of Thalassemia and Hemoglobinopathies in Bangladesh: A Single Center Study at the International Center for Diarrhoeal Disease Research, Bangladesh

Inherited hemoglobin (Hb) disorders exist primarily in the low- or middle-income countries of the tropical belt stretching from sub-Saharan Africa, through the Mediterranean region and the middle E...

Iron status and inherited haemoglobin disorders modify the effects of micronutrient powders on linear growth and morbidity among young Lao children in a double-blind randomised trial.

Some studies found that providing micronutrient powder (MNP) causes adverse health outcomes, but modifying factors are unknown. We aimed to investigate whether Fe status and inherited Hb disorders (IHbD) modify the impact of MNP on growth and diarrhoea among young Lao children. In a double-blind controlled trial, 1704 children of age 6–23 months were randomised to daily MNP (with 6 mg Fe plus fourteen micronutrients) or placebo for about 36 weeks. IHbD, and baseline and final Hb, Fe status and anthropometrics were assessed. Caregivers provided weekly morbidity reports. At enrolment, 55·6 % were anaemic; only 39·3 % had no sign of clinically significant IHbD. MNP had no overall impact on growth and longitudinal diarrhoea prevalence. Baseline Hb modified the effect of MNP on length-for-age (LAZ) (P for interaction = 0·082). Among children who were initially non-anaemic, the final mean LAZ in the MNP group was slightly lower (–1·93 (95 % CI –1·88, –1·97)) v. placebo (–1·88 (95 % CI –1·83, –1·92)), and the opposite occurred among initially anaemic children (final mean LAZ –1·90 (95 % CI –1·86, –1·94) in MNP v. –1·92 (95 % CI –1·88, –1·96) in placebo). IHbD modified the effect on diarrhoea prevalence (P = 0·095). Among children with IHbD, the MNP group had higher diarrhoea prevalence (1·37 (95 % CI 1·17, 1·59) v. 1·21 (95 % CI 1·04, 1·41)), while it was lower among children without IHbD who received MNP (1·15 (95 % CI 0·95, 1·39) v. 1·37 (95 % CI 1·13, 1·64)). In conclusion, there was a small adverse effect of MNP on growth among non-anaemic children and on diarrhoea prevalence among children with IHbD.

Multimicronutrient Biomarkers Are Related to Anemia during Infancy in Indonesia: A Repeated Cross-Sectional Study

ABSTRACTBackgroundAnemia during infancy in Indonesia is common, with iron deficiency (ID) assumed to be the major cause. Other micronutrients besides iron may have a role in determining hemoglobin (Hb) but have not yet been explored in Indonesia.ObjectiveWe investigated 7 micronutrient biomarkers and selected nonnutritional factors as potential predictors of Hb and anemia at ages 6, 9, and 12 mo in a cohort of Indonesian infants at risk of coexisting micronutrient deficiencies.MethodsApparently healthy breastfed infants were randomly selected from birth registries at 6 mo (n = 230) and followed-up at 9 mo (n = 202) and 12 mo (n = 190). Hb, serum micronutrient biomarkers—iron [as ferritin and serum soluble transferrin receptor (sTfR)], zinc, selenium, folate, vitamin A [as retinol-binding protein (RBP)], vitamin B-12, and vitamin D (as 25-hydroxyvitamin D) (adjusted for inflammation, where appropriate)—and maternal sociodemographic status, health, BMI, heminthiasis, and selected Hb genetic disorders were measured. Multivariate analysis examined relations between micronutrient biomarkers and nonnutritional factors (except helminthiasis and genetic Hb disorders) with Hb and anemia at 6 and 12 mo.ResultsID (based on ferritin) was a predictor of lower Hb and anemia at both 6 and 12 mo of age (P < 0.02). Additional predictors at 6 mo were tertiary education and higher maternal Hb for higher Hb, sex (being male) and inflammation (P < 0.05) for both lower Hb and anemia, and greater maternal height (P = 0.036) for anemia only. At 12 mo, a significant biomarker predictor besides ID was RBP (P = 0.035) for Hb.ConclusionID was a major contributor to lower Hb and anemia, although RBP was also associated.

Mutational analysis of thalassemia in transfusion-dependent beta-thalassemia patients from central India.

BACKGROUND:Thalassemia and hemoglobin (Hb) disorders are the most common genetic disorders among humans. These disorders entail huge morbidity, economic, and psychological burden on the families of the affected. Genetic counseling and prenatal diagnosis are the steps, which helps to reduce this burden. At present, there is paucity of data on the mutational spectrum of thalassemia from the central Indian region.METHODS:Blood samples were collected from 62 transfusion-dependent patients, demographic and relevant data were collected and screened for the two rare mutations − 88 (C-T) and CAP + 1 (A-G) using amplification refractory mutation system-polymerase chain reaction (PCR) and GAP PCR technique. PCR was performed for rare Hb disorders such as Hb Lepore and δ β chain disorder by GAP PCR in addition to five common Indian beta-thalassemia mutations IVS1-5 (G-C), IVS1-1 (G-T), Cd41/42 (−TCTT), Cd8/9 (+G), 619 bp deletion.RESULTS:Overall 93.5% of the mutations could be identified. Among the abnormal Hb, sickle cell and HbE were found at 4% and 3% of all the loci studied. We also reported two loci with Hb δ β and one locus with Hb Lepore in the present samples. IVS I-5 (G–C) was the common mutation (46%) followed by IVS I-1 (G–T) (12%) and 619 bp (9%).CONCLUSION:The identification of the genotypes helps to define the severity of the phenotype, plan therapy and form the basis of the comprehensive diagnostic database that would be useful not only for genetic counseling but prenatal diagnosis as well, contributing to the current focus of the National Policy to prevent and control hemoglobinopathies.

A Novel α-Thalassemia Screening Scheme in Neonates with Higher Sensitivity and Specificity Using a Combination of Hb Bart's Quantity and Red Blood Cell Indices

Background: Besides detecting hemoglobin (Hb) variants such as sickle cell disease for early preventive measure, newborn screening (NBS) program using Hb profile could identify other forms of thalassemia (thal) and Hb disorders. Due to continuous expression of fetal Hb (γ globin chains), the presence of Hb Bart's (γ4) at birth caused by unbalanced α to β like-globin synthesis have been used to screen for α-thal. So far, this NBS approach appears to be the only laboratory evaluation of α-thal screening for both carriers and Hb H disease since soon after birth Hb Bart's level would decline and we can only use DNA analysis to identify α-thal traits. Neonatal screening of αº-thal due to two missing α-globin genes (–/αα or -α/-α) from normal four copies and Hb H disease (3 α-globin genes lost; –/-α) could be achieved by using the percentage of Hb Bart's cutoffs from isoelectric focusing (Jindatanmanusan P et al. 2014) or capillary electrophoresis (Suksangpleng T et al. ASH 2017). However this approach has a major limitation to screen for α+-thal trait both deletional (-α/αα) and non-deletional (αTα/αα) types due to rather low sensitivity and specificity.Objective: To develop a novel scheme for early detection of all types of α-thal using a combination of neonatal hematological parameters and Hb profiles at birthMethods: After informed consent approved by the local ethical committee, 0.3-ml EDTA whole blood samples and dried blood spot were collected within 48 hrs after birth in newborn delivered at Siriraj Hospital by heel prick puncture. We measured the complete blood count (CBC) by using Unicel® DxHTM800 Coulter® Cellular Analysis System (Beckman Coulter, Brea, USA). The gDNA was also extracted for PCR-based molecular analysis to detect > 98% of α- and β-globin mutations mostly found in Thailand. Neonatal Hb profile was measured in qualitative and quantitative analysis using Capillarys 2 NEONAT FAST® (SEBIA, Évry, France). All data from each genotype groups were statistically analyzed using PASW statistics 18 Software (SPSS Inc, 2015).Results: Total 732 healthy Thai neonates consisted of normal α-globin alleles (αα/αα; n=505, 69%), α+-thal carrier (-α/αα; n=139, 19%), non-deletional α-thal carrier (αTα/αα, n=33, 4.5%), α°-thal carrier (–/αα) and homozygous α+-thal (-α/-α; n=43, 5.9%), compound heterozygous α+-thal with non-deletional type (-α/αTα; n=8, 1%), and Hb H disease (–/-α; n=4, 0.6%). This population had normal β-globin genotype (ββ; n=533, 72.8%), Hb E carrier (βEβ; n=183, 25%) and homozygous Hb E (βEβE; n=16, 2.2%). Interestingly, red cell indices (MCV and MCH) and Hb Bart's levels were significantly altered due to different α-globin genotypes but not by Hb E coinheritance, gender, gestational age or history of neonatal jaundice. These suggested that the variation of red cell indices at birth is largely dependent on the number of expressed α-globin genes. Therefore we hypothesized that by using a combination of red cell indices and Hb Bart's level, we could improve the efficiency of α-thal screening at birth. Indeed, we have identified several cutoffs of Hb Bart's, MCV and MCH by using a ROC analysis that could differentiate individuals with different defective α-globin genes (data not shown). We have developed a range of cutoffs and in combination using either MCV or MCH as a first screening tool followed by Hb Bart's levels, the sensitivity and specificity of α-thal screening could significantly increase (Figure 1A and B). Moreover, we previously showed the 30% sensitivity and 94% specificity to detect α+-thal trait by using Hb Bart's cutoff alone. Interestingly, our approach can increase both sensitivity and specificity up to >90%. This was also true for other types of α-genotypes (Table 1). This novel approach has a significant impact on screening for both deletional and non-deletional α+-thal traits (-α and αTα).Conclusion: For the first time, this novel combined screening using either MCV or MCH together with Hb Bart's could be used to detect all types of α-thal with a very high sensitivity (>85%) and specificity (100%). This approach would be useful to identify individuals with α-thal as a part of our national program for prevention and control of thalassemia disease currently operating in Thailand. Moreover our recommended screening flow described herein would be useful for other Southeast Asian countries where more than 30% of population is affected by α-thal. [Display omitted] DisclosuresViprakasit:Agios: Consultancy, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Protagonist Therapeutics: Consultancy, Research Funding.

Knowledge, Cultural, and Structural Barriers to Thalassemia Screening in Migrant Populations in Thailand

BACKGROUNDThalassemia syndromes are inherited hemoglobin (Hb) disorders that most severely affect Southeast Asia (SEA). As a result of population migration, thalassemia has grown into a global health issue, posing challenges to health services in many host countries. However, little attention has been paid to mass migration within SEA and its implications for regional prevention and control of Hb disorders. Thailand, with one of the highest burdens of thalassemia in the world, has a successful national prevention and control program, but ~4 million migrants living in Thailand, largely from neighboring countries, are excluded. Lack of thalassemia screening and diagnosis for migrants may undermine national efforts to prevent new births with severe thalassemia. Strategies for thalassemia screening in migrants are needed. Therefore, we sought to characterize the burden of disease, as well as the knowledge, attitudes, and practices (KAP) of migrants surrounding thalassemia in Thailand. METHODSWe surveyed Myanmar and Cambodian migrant workers (age 18-49 years) presenting for health registration and Thai subjects presenting for outpatient care at Laem Chabang Hospital (Chonburi, Thailand). Demographic and KAP surveys were developed and assessed for face and ethnographic validity. Demographic surveys were completed by all subjects. Only subjects with awareness of thalassemia completed the 31-question KAP survey. We offered thalassemia testing to all migrants and compared frequency of Hb variants in our migrant cohort to a Thai cohort. Statistical analysis was performed using SAS 9.4 (SAS Systems, Cary, NC). The study was approved by ethical review boards at Mahidol and Duke Universities. RESULTSMedian age was similar in the three nationalities (Table 1). Migrants were more likely to be married (p=0.003). Only 33.9% of migrants had entered secondary education, compared to 87.6% of Thai. ~30% of migrants did not speak any Thai. Fewer migrants attended doctor visits and used health insurance compared to Thai (both p<0.0001), but migrants showed a stronger preference for health education from doctors (p=0.01). Interestingly, migrants reported residing in Thailand for a median of 5 years, and the majority had never moved within Thailand. Nonetheless, 24.5% of migrants were still unregistered, and unregistered status was associated with a shorter residence in Thailand (p=0.01) and fewer doctor visits (p=0.0003).Only 4.1% of migrants had awareness of thalassemia, compared to 79.6% of Thai; a large knowledge gap also existed for awareness of anemia (Table 1). Female gender (OR 2.12 [95% CI 1.05-4.29]; p=0.03), secondary education (OR 2.69 [95% CI 1.08-6.71]; p=0.03), and awareness of anemia (OR 11.07 [95% CI 4.82-25.44]; p<0.0001) predicted thalassemia awareness in Thai, whereas only anemia awareness (OR 4.89 [95% CI 4.89-72.84]; p<0.0001) and length of residence in Thailand (p=0.0002) predicted awareness in migrants.Among subjects aware of thalassemia, the KAP survey identified important misconceptions in both migrant and Thai populations that may lead to stigmatization of thalassemia carriers and decreased screening uptake. For example, 45.4% and 62.7% of migrants believe that thalassemia is an infectious disease and is rare in Asia, respectively. Though migrants had a lower median knowledge score than Thai (p=0.002), their median attitude score was similar (p=0.21). Both groups generally held positive attitudes towards thalassemia testing, but migrants held particularly negative attitudes towards abortion.Of those migrants offered thalassemia testing who had available blood samples (n=286), 96.9% consented; 3.1% declined. Interestingly, α- and β-globin variants and Hb E were more common in our migrant cohort than in a Thai cohort characterized separately (Table 2). DISCUSSIONWe conclude that the burden of thalassemia in migrant populations is at least as high as in the Thai population and that thalassemia screening is highly acceptable to migrants. However, a tremendous gap in awareness of thalassemia exists between Thai and migrants, suggesting that public education targeted to migrant populations is desperately needed. Other issues identified, including language barriers, lack of insurance, low healthcare utilization, and negative attitudes toward abortion, merit further consideration when designing future health programs for migrants in Thailand and globally. [Display omitted] DisclosuresTelen:Pfizer, Inc.: Consultancy, Research Funding. Viprakasit:F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Protagonist Therapeutics: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Agios: Consultancy, Research Funding.

Psychological Factors Associated with Episodic Chelation Adherence in Thalassemia

β-Thalassemia major (β-TM) is a life-long genetic hemoglobin (Hb) disorder requiring intensive treatment regimens, including frequent blood transfusions and daily chelation therapy. Understanding psychosocial correlates of chelation adherence is important for developing interventions to improve adherence. This study investigated within-participant correlates of oral chelation adherence on a daily (episodic) basis. Thirty-seven adult participants with β-TM were recruited from clinics at two hospitals (22 males, 9 females, mean age 34.5 years, range 19–54 years). A structured interview was used to assess behavioral and psychological situational variables related to an adherent and a nonadherent episode for each participant. Positive outcome expectancies and higher self-efficacy were both significantly associated with adherent episodes. Behavioral variables, including difficulty in accessing medication, location, and whether alone or with others, were also associated with nonadherent episodes. Findings suggested that situational psychological factors are important for chelation adherence. Adherence interventions should consider focusing on potentially modifiable situational variables (psychological and behavioral).

Hb Moscva [β24(B6)Gly→Asp (GGT>GAT), HBB: c.74G>A]: An Unstable Hemoglobin Newly Detected as a De Novo Mutation in a Mauritanian Patient

Unstable hemoglobins (Hbs) are a group of Hb disorders that could be the origin of chronic hemolytic anemia. Most of these disorders are caused by point mutations taking place in the globin genes and affecting the stability of the Hb molecule. They are inherited as autosomal dominant diseases and described worldwide. Herein we report a new observation of an unstable variant in the Mauritanian population. The patient was a young girl of Mauritanian origin. She presented with chronic hemolytic anemia with an unknown etiology after being referred to several medical centers. Laboratory investigations based on routine analyses, capillary electrophoresis (CE), cation exchange high performance liquid chromatography (HPLC) and DNA sequencing revealed an abnormal unstable Hb known as Hb Moscva [β24(B6)Gly→Asp (GGT>GAT), HBB: c.74G>A] that occurred as a de novo mutation newly detected in an African girl of Mauritanian origin.

Prevalence of hemoglobin variants and hemoglobinopathies using cation-exchange high-performance liquid chromatography in central reference laboratory of India: A report of 65779 cases.

CONTEXT:Hemoglobinopathies constitute the world's most common genetically inherited red blood cell disorder. Screening and accurate identification of hemoglobin (Hb) variants have become increasingly important in antenatal diagnosis and prevention of Hb disorders.AIM:The aim of this study was to screen and identify Hb fractions prevalent in the Central Reference Laboratory of India.MATERIALS AND METHODS:A total of 65,779 cases were screened for hemoglobinopathies on the bio-rad variant high-performance liquid chromatography (HPLC) system by beta-thalassemia short program. The retention times, proportion of the hemoglobin (%) and the peak characteristics for all hemoglobin fractions were recorded. Molecular analysis of the beta-globin gene was carried out by DNA sequencing on eight cases.RESULTS:Total number of abnormal Hb fractions on cation exchange-HPLC (CE-HPLC) was seen in 12,131 (18.44%) cases. Beta-thalassemia trait was the predominant genetic Hb disorder accounting for 7377 cases (11.21%) of the total cases. This was followed by sickle cell trait (2.01%), sickle cell disease (1.59%), beta-thalassemia syndrome (0.80%), HbE trait (0.79%), and borderline HbA2 (0.51%). Molecular characterization of eight rare cases of hemoglobin variants by beta-globin gene sequencing identified three cases of Hb Beth Israel, two cases of Hb Hofu trait, and one case each of Hb J Cambridge, Hb Mizunami, and Hb Sherwood Forest.CONCLUSION:Superior resolution, rapid assay time, and accurate quantification make CE-HPLC suitable for the routine investigation of hemoglobinopathies.

A Novel β-Thalassemia Insertion/Frameshift Mutation Between Codons 77/78 (p.Leu78Profs*13 or HBB: c.235_236insC) Observed in a Family in Bangladesh

β-Thalassemia (β-thal) is one of the most common inherited hemoglobin (Hb) disorders, worldwide. A 28-year-old female and her husband came to Dhaka Shishu (Children) Hospital, Bangladesh for prenatal diagnosis for thalassemia mutations. We identified and characterized a novel β-thalassemia (β-thal) mutation due to an insertion of cytosine between codons 77 and 78 (p.Leu78Profs*13) found in mother in a heterozygous state. This mutation caused an insertion in the normal reading frame of the β-globin coding sequence and the new stop codon being the amino acid 90 (HBB: c.235_236insC) in exon 2 that leads to a β0-thal phenotype.

Innovative PCR without DNA extraction for African sickle cell disease diagnosis

ABSTRACTObjectives: Hemoglobin (Hb) disorders consist of thalassemia and Hb structural variants, of which the major forms are associated with severe anemia and/or vascular occlusion. Current diagnostic techniques are highly accurate and mostly based on isoelectric focusing, high-performance liquid chromatography or mass spectrometry, which often require advanced laboratory equipment. In sub-Saharan Africa, the Hb disorders are mainly associated to the pathological variants hemoglobin S (HbS) and HbC. Unfortunately, until now, it is not easy to get a diagnosis of these disorders in this area. In this study, we tested the performance of a new molecular diagnostic tests on qualified samples.Methods: The Human Hb S/C Lamp assay is a new polymerase chain reaction test able to detect HbS, HbC and HbA alleles without DNA extraction, directly on fresh or frozen blood samples, or on dried blood spots (DBS). In this study, we compared the genotyping of 248 blood samples (56 whole blood and 192 DBS) with this LAMP assay to the routine diagnostic methods performed in the genetics lab at the university hospital of Liège.Results: Our results show that the LAMP method can detect HbS and HbC with an accuracy of 100%. Moreover, this test can be used for the neonatal screening because we did not observe any interference with fetal Hb.Discussion: To our knowledge, this method is the only molecular assay that can be performed directly on dried blood cards without DNA extraction, lowering handling, turnaround time and costs.

HbA2 and Fetal haemoglobin in the diagnosis of Thalassemia and Hemoglobinopathies

Background: Hemoglobin (Hb) disorders which include hemoglobinopathies and Thalassemia affect 7% of the world population. Capillary electrophoresis is useful for screening and follow up of Hb disorders . Aim: To evaluate HbA2 and HbF (fetalhemoglobin) in the diagnosis of Thalassemia and hemoglobinopathies. Material and Methods : 100 consecutive Capillary Hemoglobin electrophoresis done as a part of screening programme for Hb disorders from Jan2016 to june 2016 was included in the study. Children <1 yr of age and individuals with recent blood transfusion were excluded . Hb, RBC count and MCV were recorded. Results : Of the 100 Hb electrophoresis performed, 57 normal and 43 abnormal patterns were seen. Among the abnormal Hb patterns, β thalassemia trait (βTT) was the most common constituting 58.2% followed by Sickle cell (HbS) trait(11.7%), HbE trait(9.3%) and HbE/ β Thalassemia (7.0%). The HbA2 levels in normal, βTT, Sickle cell trait , HbE trait and HbE/ β thalassemia were 2.12%(SD 0.5), 4.9%(SD 0.62), 3.28%(SD 0.43), 3.98%(SD 0.67) respectively . The difference in HbA2 were significant (p value <0.0001).The difference in HbA2 levels between HbE trait and HbE disease was also significant (p value 0.036).Based on the HbF levels Sickle cell hemoglobinopathy was further classified. Conclusion: The difference in HbA2 levels in normal subjects, Thalassemia and hemoglobinopathies are statistically significant. The percentage of HbF in sickle cell gives information about coexisting hemoglobin disorder. In HbEhemoglobinopathy, HbA2 along with HbF identifies a specific group HbE/ β Thalassemia which often needs clinical intervention. DOI: 10.21276/APALM.1303

Comparison of MicroRNAs Mediated in Reactivation of the γ-Globin in β-Thalassemia Patients, Responders and Non-Responders to Hydroxyurea

Drug induction of Hb F seems to be an ideal therapy for patients with hemoglobin (Hb) disorders, and many efforts have been made to reveal the mechanism behind it. Thus, we examined in vivo expression of some microRNAs (miRNAs) that are thought to be involved in this process. Among β-thalassemia (β-thal) patients who were undergoing hydroxyurea (HU) therapy in the past 3 months and five healthy individuals, five responders and five non-responders, were also included in the study. Erythroid progenitors were isolated by magnetic activated cell sorting (MACS) and miRNA expression analyzed using reverse transcription-polymerase chain reaction (RT-PCR). We showed that γ-globin, miR-210 and miR-486-3p had higher levels in the responders than the non-responders group. Moreover, miR-150 and miR-320 had higher levels in the healthy group than both non-responders and responders groups, but the expression of miR-96 did not show any significant difference between the study groups. To the best of our knowledge, this is the first study proposing that ‘induction of cellular hypoxic condition by Hb F inducing agents’ could be the milestone of possible mechanisms that explain why responders are able to reactivate γ-globin genes and subsequently, more production of Hb F, in response to these agents in comparison to non-responders. However, further investigations need to be performed to verify this hypothesis.

Detection of Hemoglobin (Hb) Variants By HPLC Screening in Cord Blood Units (CBU) Donated to the National Cord Blood Program (NCBP)

Purpose. To evaluate the frequency and types of Hb abnormalities in CBU collected by the NCBP at the New York Blood Center.Background and Methods. Established in 1992, NCBP has collected and tested over 76,000 ethnically diverse CBU and manages an inventory of over 60,000 clinical grade CB products, including the first FDA-licensed (HEMACORD®). NCBP has released over 5,600 CBU for transplantation worldwide. To prevent transplants from donors affected with clinically significant hemoglobinopathy (HP), current Good Tissue Practices (cGTP) require Hb screening of all CBU before they are accepted to the public CB inventories. Clinically significant Hb disorders are caused by either structurally abnormal Hb variants (e.g., Hb S, Hb C, Hb E) or by decreased or absent production of α- or β-globin chains (thalassemia). Evaluation of NCBP CBU for possible HP includes: a) detailed family history for HP; b) complete blood count (CBC) and RBC indices, particularly mean corpuscular volume (MCV); c) screening using High-Performance Liquid Chromatography (HPLC) and d) confirmatory diagnostic testing using molecular methods (Hemoglobinopathy Reference Laboratory, Oakland, CA). To date, 76,312 CBU have been screened at the NCBP with the Bio-Rad Variant HPLC system using the Sickle Cell Short Program, a 3-min assay, specifically designed and FDA approved to provide a qualitative result for the presence of Hbs A, F, S, C, D and/or E in the neonate.Results.The HPLC chromatographic patterns were normal (Hb FA, where F is fetal and A is adult Hb) in 73,163 CBU (95.87%), and revealed an abnormal Hb in the remaining 3,149 CBU (4.13%), the majority of which were not clinically significant.The most frequent abnormal Hb phenotypes were, sickle cell trait (FAS): 1,755 CBU (57%), Hb E trait (FAE): 562 (18%) and Hb C trait (FAC): 506 (17%). Among the 3,149 CBU with abnormal Hb, 87 carried two genes for Hb disorders of clinical relevance, of which 54 were homozygous for sickle hemoglobin (Hb FS), 6 for Hb C (Hb FC) and 19 were double heterozygotes (Hb FSC). The HPLC results of all CBU with Hb traits and homozygous patterns were confirmed on repeat testing. In addition, all homozygous HPLC results were confirmed by molecular testing.Thalassemia and other abnormalities. Hb Barts (γ4) elutes "Fast" in the HPLC chromatogram. Its normally low concentration in CB increases when γ-chains replace non-functional or deleted α-globin chains in α-thalassemia (α-thal). Testing of all 76,312 NCBP CBU showed levels of Hb Barts from 0.5% to 34% of total Hb: 69.3% of the samples had Hb Barts level below 2.5%, 29.9% between 2.6 and 6.0% and 0.8% greater than 6.1%.Accordingly, for presumptive identification of α- or β-thalassemia, HPLC results must be interpreted together with red cell indices and family history. Of 788 CBU samples submitted for molecular (DNA) analyses as "possible thalassemia carriers", one was confirmed as Hb H disease (three α-globin genes deleted), 270 had α-thal trait (two α-globin gene deletions) and 517 had one or no α-globin gene deletion. The 270 CBU with α-thal trait had Hb Barts levels between 4.0% (above 6% in most cases) and 18.9% of total Hb. Further, 258 (95.6%) of the CBU with α-thal trait had MCV below 105 fL (range: 79.9 - 104.8 fL); only 12 cases (4.4%) had MCV >105 fL, all of which had levels of Hb Barts > 5%. The Hb H disease case had Fast Hb 34.2% (by HPLC) and MCV was 76.1 fL.β-thalassemia (β-thal): 4 CBU had β-thal major and in those, only Hb F (no Hb A) was seen in the HPLC result. Among 26 CBU found to have β-thal trait by the reference lab’s DNA analysis, 16 (61.5%) were tested because of positive family history and 10 (38.5%) because of MCV <105 fL (seven CBU had both, low MCV and family history of thalassemia). Another CBU with a still-uncharacterized β-globin gene mutation had MCV <105 fL.HPLC patterns disclosing "Unknown Hbs" as "traits" in 5 CBU, were confirmed by molecular testing.Conclusions.HPLC is a simple to perform, accurate, comprehensive, inexpensive and fast screening method for HP in cord blood.All homozygous Hb detected by HPLC were confirmed by the reference lab. This is particularly relevant for sickle cell disease, as it is the most common HP in ethnic minority donors.Screening with HPLC, when used in combination with CBC and family history, is a useful tool to identify possible α- and β-thalassemia carriers in CBU.Molecular DNA testing is diagnostic in confirming and identifying globin gene deletions and mutations. DisclosuresNo relevant conflicts of interest to declare.

Coinheritance of a Rare Nucleotide Substitution on the β-Globin Gene and Other Known Mutations in the Globin Clusters: Management in Genetic Counseling

A large number of methods for DNA analysis are available to identify defects in globin genes associated with hemoglobin (Hb) disorders. In this study, we report a rare nucleotide (nt) substitution on the β-globin gene, nt 781 in the second intron [IVS-II-781 (C > G); HBB: c.316-70C > G], identified in four patients. This nt substitution was previously described only as a personal communication to the HbVar database and indicated as a β0 or β+ mutation. The purpose of this study was to evaluate the clinical implication of this nt change, particularly when coinherited with severe β-thalassemia (β-thal), in order to be able to conduct appropriate genetic counseling. Genetic studies were performed on two subjects, one carried Hb S [β6(A3)Glu→Val; HBB: c.20A > T], and the other carried IVS-I-110 (G > A) (HBB: c.93-21G > A). All these subjects showed this new β nt substitution in association with Hb A2' (or Hb B2) [δ16(A13)Gly→Arg; HBD: c.49G > C]. Another 16 samples, carrying the same δ variant as the probands, were processed by β-globin gene sequencing in order to better understand the correlation between this Hb variant and the rare nt substitution reported in this study. The present investigation emphasizes the importance of sharing the observed nt changes in the globin gene cluster, especially in the case of new or rare undefined mutations, in order to facilitate the determination of their phenotypic expression, the possible interactions with known molecular defects and to formulate appropriate genetic counseling for at-risk couples.

Evaluation of α-Globin Gene Mutations Among Different Ethnic Groups in Khuzestan Province, Southwest Iran

α-Thalassemia (α-thal) is one of the most common inherited hemoglobin (Hb) disorders in the world. In addition to large deletions, over 50 different α-thal point mutations were detected around the world, thus, patients showed different phenotypes with regard to genotype. This study evaluated the genetic frequency of α-thal in Khuzestan Province, Southwest Iran, to help implement premarital and prenatal screening programs. The study was conducted on couples proposing to get married and parents who were referred to the genetic center of Shafa Hospital, Ahvaz, Iran, for prenatal diagnosis (PND) in 2012. Genomic DNA was purified by the salting-out method and tested using multiplex gap-polymerase chain reaction (gap-PCR), amplification refractory mutation system-PCR (ARMS-PCR), reverse hybridization test strips and DNA sequencing. Overall, 11 mutations were found on the α-globin genes. Based on gene frequency, the most common mutant allele was –α3.7 (rightward) (71.3%) followed by the two gene deletion – –MED (9.7%). Other common mutations were αcodon 19α (GCG>GC–, α2) (8.4%), the polyadenylation (polyA1) site αpolyA1α (AATAAA>AATAAG) (2.8%), and α–5 ntα (–TGAGG) (2.0%). In addition, an extremely rare mutation at αcodon 21α [Hb Fontainebleau, HBA2: c.64G > C (or HBA1)] was also found. The results of this study are critical for correct diagnosis of α-thal carriers, premarriage counseling and PND. This study suggests that the distribution of mutations on the α-globin genes differs among the ethnic groups in Khuzestan Province as well as in other provinces.

Prospective Evaluation of Fetal Haemoglobin Induction in Maternal Erythrocytes: A Preliminary Analysis of a Cohort of 345 Parturients

Introduction. The mechanisms of haemoglobin (Hb) subtype switch remain largely unknown even though it has a therapeutic potential (Bauer et al., 2012). In fact, reactivation of fetal haemoglobin (HbF) production in adult erythrocytes (F-cells) is protective in many Hb disorders. Pregnancy may represent the only physiological condition where HbF production increases transiently (Ibrahim et al, 2009; Yamada et al., 2012). Moreover, flow cytometry (FC), by intracellular staining using anti-HbF antibodies, has improved the evaluation of maternal F-cells (Corcoran et al., 2014; Chen et al., 2000). In this study, the objectives are: 1) to document HbF expression prospectively peri- and post-partum and confirm its maternal origin; 2) to assess potential causes of HbF modulation throughout pregnancy (i.e. β-hCG, EPO) and; 3) to identify gene differences between HbF-positive and HbF-negative pregnant women.Methods. In this observational, single institution, prospective study, 879 pregnant women were screened. A total of 345 participants were included in the study: the first consecutive 176 women with negative total HbF expression (<1%), and the first consecutive 169 women with positive total HbF expression (≥1%). Hb studies were conducted with high performance liquid chromatography (HPLC), and FC was performed if total HbF expression was ≥1%. Other assessments included DNA sampling, complete blood counts, β-hCG and EPO levels, and qualitative data (i.e. to account for other factors such as tobacco, progesterone use, etc.). Analyses were conducted at each trimester except DNA sampling, which was done during the first visit only. HbF-positive women had an additional post-partum visit to assess changes of HbF expression and account for hereditary persistence of HbF. Descriptive statistics and Pearson correlations were planned for this analysis.Results. At the time of analysis, 93 HbF-positive and 147 HbF-negative women had completed follow-up. Results revealed that, among all pregnant women screened, 22.07% (n = 194) had positive HbF expression. Of this percentage, 90.72% (n = 176) filled the inclusion criteria. Of all women with positive HbF expression during 1 or more of the first 3 visits, 72.04% showed a post-partum decrease of HbF below 1%. Furthermore, FC analyses in HbF-positive women confirmed the maternal origin of HbF in all cases and revealed an average of 13.28% (sd = 5.38%) F-cells when total HbF was ≥ 1%. Only modest correlations could be found so far between HbF levels and β-hCG (ρ = 0.25) or EPO (ρ = 0.07). Comparative DNA analyses of HbF-positive and HbF-negative women are currently underway and will be presented at the meeting.Conclusions. To our knowledge, this represents the largest prospective study evaluating HbF expression throughout pregnancy, as well as the first to evaluate post-partum HbF dynamics and the genetic background of HbF-negative and HbF-positive pregnant women. Preliminary data shows that: 1) a significant percentage of pregnant women have a transient elevation of HbF which cannot be explained by fetomaternal hemorrhage nor hereditary persistence of HbF; 2) the proportion of F-cells in women remains constant peri- and post-partum when HbF ≥ 1%; 3) a large proportion of maternal erythrocytes express HbF at low levels and; 4) HbF modulation during pregnancy is only modestly correlated to β-hCG and EPO. These data may lead to the study of a new paradigm of Hb subtype switch as well as HbF-induction therapeutics for the benefit of Hb disorders. DisclosuresNo relevant conflicts of interest to declare.

The Homozygous Hemoglobin EE Genotype and Chronic Inflammation Are Associated with High Serum Ferritin and Soluble Transferrin Receptor Concentrations among Women in Rural Cambodia

Background: Ferritin and soluble transferrin receptor (sTfR) concentrations are commonly used to assess iron deficiency (ID); however, they are influenced by multiple factors.Objectives: We assessed associations between numerous variables and both ferritin and sTfR concentrations in Cambodian women and compared ID prevalence through the use of study-generated correction factors (CFs) for ferritin with those from a published meta-analysis.Methods: Venous blood from 450 women (aged 18–45 y) was assessed for hemoglobin (Hb), ferritin, sTfR, retinol binding protein, folate, vitamin B-12, C-reactive protein, α-1 acid glycoprotein (AGP), and genetic Hb disorders. Linear regression was used to calculate geometric mean ratios (95% CIs) for ferritin and sTfR concentrations.Results: The variant Hb EE genotype was associated with 50% (14%, 96%) and 51% (37%, 66%) higher geometric mean ferritin and sTfR concentrations, respectively, than was the normal Hb AA genotype; a 1-g/L increase in AGP was associated with 99% (50%, 162%) and 48% (33%, 64%) higher concentrations in the same variables, respectively. ID prevalence in nonpregnant women (n = 420) was 2% (n = 9) with the use of ferritin <15 μg/L and 18% (n = 79) with the use of sTfR >8.3 mg/L as criteria. ID prevalence with the use of sTfR was higher in women with the Hb EE genotype (n = 17; 55%) than in those with the Hb AA genotype (n = 20; 10%); and in women with the Hb AA genotype and chronic inflammation (n = 10; 18%) than in that group of women without chronic inflammation (n = 10; 7%) (P < 0.05). No differences in ID prevalence were found with the use of ferritin between women with Hb EE and AA genotypes (P = 1.0) or by chronic inflammation status (P = 0.32). There were no differences in mean ferritin concentrations among all 450 women when study-generated CFs were compared with those from the meta-analysis (P = 0.87).Conclusions: Compared with sTfR, ferritin concentrations appear to reflect more accurately true ID in rural Cambodian women. The CFs from a published meta-analysis were appropriate for use in this population with a high prevalence of Hb disorders and inflammation.

The Prevalence of α-Thalassemia and Its Relation to Plasmodium falciparum Infection in Patients Presenting to Clinics in Two Distinct Ecological Zones in Ghana

Thalassemia and sickle cell disease constitute the most monogenic hemoglobin (Hb) disorders worldwide. Clinical symptoms of α+-thalassemia (α+-thal) are related to inadequate Hb production and accumulation of β- and/or γ-globin subunits. The association of thalassemia with malaria remains contentious, though from its distribution it appears to have offered some protection against the disease. Data on the prevalence of thalassemia in Ghana and its link with malaria is scanty and restricted. It was an objective of this cross-sectional study to determine the prevalence of thalassemia in areas representing two of Ghana’s distinct ecological zones. The relationship between thalassemia and Plasmodium falciparium (P. falciparum) infection was also ascertained. Overall, 277 patients presenting to health facilities in the study areas were recruited to participate. Tests were carried out to determine the presence of α+-thal, sickle cell and malaria parasites in the blood samples of participants. The outcome of this study showed an α+-thal frequency of 19.9% for heterozygotes (−α/αα) and 6.8% for homozygotes (−α/−α). Plasmodium falciparum was detected in 17.7% of the overall study population and 14.9% in those with α+-thal. No association was observed between those with α+-thal and the study sites (p > 0.05). A test of the Hardy-Weinberg law yielded no significant difference (p < 0.001). Findings from this study suggest a modest distribution of α+-thal in Ghana with no bias to the ecological zones. Although the prevalence and parasite density were relatively low in those with the disorder, no association was found between them.