Heterozygosity for the Novel HBA2: c.*91_*92delTA Polyadenylation Site Variant on the α2-Globin Gene Expanding the Genetic Spectrum of α-Thalassemia in Iran.

There are four copy numbers of α-globin genes (16p13.3) in the human genome and the number of defective α-globin genes dictates the severity of α-thalassemia (α-thal). Mutations that occur in the 3' untranslated region (3'UTR), and especially at the polyadenylation (polyA) sites, affect the translation, stability and export of mRNA. A patient with hypochromic microcytic anemia was referred to the Kariminejad-Najmabadi Pathology & Genetics Center, Tehran, Iran by the health network. Molecular analysis of genomic DNA for the evaluation of mutations on the α- and ß-globin genes was performed. Direct sequencing of the hemoglobin (Hb) subunit α2 (HBA2) gene revealed a two nucleotide deletion between +816 and +817 in the 3'UTR, located at the polyA site, which seems to be a novel pathogenic variant. This novel variant expands the genetic spectrum of α-thal in the 3'UTR of the HBA2 gene.

Identification of Mutations Causing Aberrant Termination and Deficient Splice Donor Site on the HBA1 Gene.

α-Thalassemia (α-thal) is a common genetic disorder in Iran and many parts of the world. Genetic defects on the α-globin gene cluster can result in α-thal that may develop a clinical phenotype varying from almost asymptomatic to a lethal hemolytic anemia. In the present study, four Iranian individuals with hypochromic microcytic anemia, who revealed none of the known mutations responsible for α-thal, were subjected for further investigations. The thalassemic phenotype of these patients resulted from abnormal RNA splicing sites owing to a missense at the splice donor site, a truncated protein or hemoglobin (Hb) variants as a result of two different substitutions on the α1-globin gene. The clinical presentation of mild anemia in these individuals showed the contribution of these novel mutations in α-thal in spite of the dominantly expressed α2-globin gene. This study describes hematological manifestations of subjects carrying some novel mutations comparable to the reported phenotype of α(+)-thal trait.

Mutations on the α2-Globin Gene That May Trigger α(+)-Thalassemia.

In the present study, a total of 11 individuals with hypochromic microcytic anemia who did not reveal the most common α-thalassemia (α-thal) deletions or mutations, were subjected to more investigations by DNA sequencing of the α-globin genes. Seven novel nondeletional α-thal mutations localized on the α2-globin gene in the heterozygous state were identified. These mutations either corrupted regulatory splice sites and consequently affected RNA processing or created unstable hemoglobin (Hb) variants. The mutations described here produced globin gene variants that lead to amino acid changes in critical regions of the globin chain. The clinical presentation of most patients was a persistent mild microcytic anemia similar to an α(+)-thal. In the last decade, numerous α-globin mutations have been observed leading to an α-thal phenotype and these studies have been considered to be important as discussed here.

The value of the red cell distribution width index versus other parameters in the differentiation between iron deficiency anemia and beta thalassemia trait among children in Mosul, Iraq.

BACKGROUND: Iron deficiency anemia (IDA) in children may be confounded with the beta-thalassemia trait (BTT). This study aimed to reevaluate the role of the red blood cell distribution width index (RDWI) in distinguishing BTT from IDA. RESEARCH DESIGN AND METHODS: This cross-sectional study was conducted from June 2018 to February 2019 in two pediatric teaching hospitals in Mosul, Iraq. Two sets of patients with hypochromic microcytic anemia in the age range 6 months-12 years were included. The receiver-operating characteristics curve was used to identify the test best distinguishing BTT from IDA. RESULTS: In the first patient set, 54 (51.92%) had IDA and 50 (48.07%) had BTT. The area under the curve (AUC) for the RDWI was larger than for other parameters at 0.963, suggesting a discriminant tool to detect cases of BTT (p-value < 0.0001) . In the second patient set, 25 (42.85%) IDA cases and 20 (57.14%) BTT cases were analyzed, and the RDWI still had the highest AUC when compared with other parameters (p-value < 0.0001). An RDWI cutoff value suggested to differentiate BTT from IDA was 200.18 and 200.35 respectively for both groups. CONCLUSIONS: RDWI may be a useful screening parameter in differentiating BTT from IDA.

A new case of congenital atransferrinemia with a novel splice site mutation: c.293-63del.

Congenital atransferrinemia is an extremely rare autosomal recessive disorder resulting in the complete absence or extremely reduced amount of transferrin. In this study, we describe the first case of congenital atransferrinemia in Tunisia and the 18th patient in the reported data. The patient was referred to our hospital to explore a severe hypochromic and microcytic anemia. The laboratory evaluation including hematological and biochemical examination was performed in the proband and her parents. All exons of the transferrin gene were PCR amplified. The products were screened for mutations by direct sequencing. Based on laboratory and clinical findings, diagnosis of congenital atransferrinemia was confirmed. DNA sequencing revealed the presence of a novel homozygous deletion (c.293-63del) in the intron 13. This mutation is predicted to generate a higher score cryptic branch point leading to the production of an altered mRNA molecule. The second previously reported missense mutation p.Arg609Trp. Crystallographic structure analyzes demonstrate that the mutation would probably lead to significant conformational change not allowing the expression of transferrin protein. Current molecular characterization of this novel transferrin abnormality puts to the proof the variability in onset, first blood transfusion, and phenotypic expression in atransferrinemic patients.

Application of HbA2 levels and red cell indices-based new model in the differentiation of thalassemia traits from iron deficiency in hypochromic microcytic anemia Cases.

INTRODUCTION: Thalassemia traits and iron deficiency anemia are the most common types of hypochromic microcytic anemia with similar clinical and laboratory features. It is vital to establish a new screening model based on HbA2 levels and red cell indices for the differentiation of TT from IDA in hypochromic microcytic anemia cases. METHOD: The data comprised of the red blood cell indices and HbA2 prenatal diagnostic test results of 810 individuals who were identified to conform to the following criteria: MCV < 80 fl or MCH < 26 pg. We launched a new model consisting mainly of significative red cell indices and HbA2 levels, as well as proposing cutoff values by using decision trees and logistic regression analyses. Next, we evaluated our new method by comparing the sensitivity, specificity, positive, and negative predictive values with those of the previous formulas. RESULTS: We put forward a new model and compared it with 5 efficient formulas. The new model exhibited the highest accuracy (0.918), with its sensitivity and specificity calculated as 0.917 and 0.921, respectively. Our new model's Youden index was 0.838, which is higher than the other formulas' Youden indices. CONCLUSIONS: The new screening model, based on HbA2 levels and red cell indices, is suitable for the screening of thalassemia patients in the hypochromic microcytic anemia group and has the best efficiency in distinguishing TT and IDA.

Prevalence of anemia in schools of the metropolitan region of Curitiba, Brazil.

BACKGROUND: Anemia during childhood is one of the biggest public health problems worldwide, including Brazil. Insufficient or abnormal production of hemoglobin, loss of iron and excessive destruction of red blood cells are the most common causes of anemia. Among the reasons of anemia, iron deficiency accounts for 50% of anemia cases in developing countries. Affected individuals present a wide range of clinical problems, including delayed neuropsychomotor progression, impaired cellular immunity and reduction of intellectual capacity. This study aimed to evaluate the prevalence of anemia in children attending public schools in the metropolitan region of Curitiba, Paraná, Brazil. METHOD: A retrospective study was conducted of 409 children aged 8-12 years old included in an extension project of the Universidade Federal do Paraná. The results of complete blood count and hemoglobin electrophoresis of all children were evaluated. Anemia was considered when the hemoglobin levels were <11.5 g/dL. RESULTS: The prevalence of anemia was found to be 2.2% of the population studied, with hypochromic microcytic anemia being the most common type. Seven children had sickle cell trait and one had ß-thalassemia. CONCLUSION: The prevalence of anemia in this study was considered normal according the World Health Organization classification, which is different from the data found in other Brazilian regions.

Whole-exome sequencing identifies an α-globin cluster triplication resulting in increased clinical severity of ß-thalassemia.

Whole-exome sequencing (WES) has been increasingly useful for the diagnosis of patients with rare causes of anemia, particularly when there is an atypical clinical presentation or targeted genotyping approaches are inconclusive. Here, we describe a 20-yr-old man with a lifelong moderate-to-severe anemia with accompanying splenomegaly who lacked a definitive diagnosis. After a thorough clinical workup and targeted genetic sequencing, we identified a paternally inherited ß-globin mutation (HBB:c.93-21G>A, IVS-I-110:G>A), a known cause of ß-thalassemia minor. As this mutation alone was inconsistent with the severity of the anemia, we performed WES. Although we could not identify any relevant pathogenic single-nucleotide variants (SNVs) or small indels, copy-number variant (CNV) analyses revealed a likely triplication of the entire α-globin cluster, which was subsequently confirmed by multiplex ligation-dependent probe amplification. Treatment and follow-up was redefined according to the diagnosis of ß-thalassemia intermedia resulting from a single ß-thalassemia mutation in combination with an α-globin cluster triplication. Thus, we describe a case where the typical WES-based analysis of SNVs and small indels was unrevealing, but WES-based CNV analysis resulted in a definitive diagnosis that informed clinical decision-making. More generally, this case illustrates the value of performing CNV analysis when WES is otherwise unable to elucidate a clear genetic diagnosis.

A child with severe iron-deficiency anemia and a complex TMPRSS6 genotype.

OBJECTIVES: We report a case of a 7-year-old girl with severe hypochromic microcytic anemia, who was unresponsive to classical iron supplements. We suspected IRIDA, iron-refractory iron-deficiency anemia, a genetic iron metabolism disorder, caused by TMPRSS6 variations. TMPRSS6 encodes matriptase-2, a negative regulator of hepcidin, and its pathological variants are related to normal to high levels of hepcidin. We analyzed the TMPRSS6 gene and we improved clinical management of the patient, selecting the appropriate supplementation therapy. Intervention & Technique: The parenteral iron therapy was started, but the patient was only partially responsive and the anemia persisted. To confirm the diagnosis, the TMPRSS6 gene sequence was analyzed by DNA sequencing and other relevant biochemical parameters were evaluated. RESULTS: The TMPRSS6 sequence analysis showed a complex genotype with a rare heterozygous missense variant, in addition to other common polymorphisms. The serum hepcidin value was normal. We unexpectedly observed a normalization of patient's hemoglobin (Hb) levels only after liposomal iron treatment. DISCUSSION AND CONCLUSION: The proband was symptomatic for IRIDA during a critical phase of growth and development, but we did not find a clearly causative genotype. A long-term result, improving stably patient's Hb levels, was obtained only after liposomal iron supplementation. Children may be at greater risk for iron deficiency and the degree of anemia as well as the response to the iron supplements varies markedly patient to patient. Here, we show the importance of comprehensive study of these patients in order to collect useful information about genotype-phenotype association of genes involved in iron metabolism.

Prevalence of anemia in schools of the metropolitan region of Curitiba, Brazil

ABSTRACT Background: Anemia during childhood is one of the biggest public health problems worldwide, including Brazil. Insufficient or abnormal production of hemoglobin, loss of iron and excessive destruction of red blood cells are the most common causes of anemia. Among the reasons of anemia, iron deficiency accounts for 50% of anemia cases in developing countries. Affected individuals present a wide range of clinical problems, including delayed neuropsychomotor progression, impaired cellular immunity and reduction of intellectual capacity. This study aimed to evaluate the prevalence of anemia in children attending public schools in the metropolitan region of Curitiba, Paraná, Brazil. Method: A retrospective study was conducted of 409 children aged 8-12 years old included in an extension project of the Universidade Federal do Paraná. The results of complete blood count and hemoglobin electrophoresis of all children were evaluated. Anemia was considered when the hemoglobin levels were <11.5 g/dL. Results: The prevalence of anemia was found to be 2.2% of the population studied, with hypochromic microcytic anemia being the most common type. Seven children had sickle cell trait and one had β-thalassemia. Conclusion: The prevalence of anemia in this study was considered normal according the World Health Organization classification, which is different from the data found in other Brazilian regions.

Iron-deficiency anemia in children: from diagnosis to treatment

Iron-deficiency anemia (IDA) is the most frequent hematologic and nutritional disorder in children. The risk factors associated with IDA in children are rapid growth with inadequate dietary iron, low birth weight, premature birth, perinatal bleeding, early cow's milk intake, and breastfeeding beyond 6 months without iron supplementation. Blood loss is also an important cause of IDA. Most children with IDA are asymptomatic and may go undiagnosed. The diagnosis of IDA is confirmed by microcytic hypochromic anemia and a low level of serum ferritin. Monitoring the response to iron supplementation is a reasonable intervention for a clinically stable child with mild anemia and inadequate iron intake. IDA must be differentiated from the anemia that arises from chronic disease and thalassemia. Oral iron is usually recommended as first-line therapy. Parenteral iron is indicated in cases of poor compliance or failure of oral iron, intestinal malabsorption, or chronic bleeding.