Alpha-globin Gene Research Articles

Hematologic Parameters Cut-off Assessment of Adult Alpha-Thalassemia Patients in Iran

Background: Thalassemia is one of the most common blood disorders in Iran. Alpha-thalassemia is caused by the deletion of the alpha-globin gene. The frequency of deletions in the alpha-globin gene is associated with microcytosis and hypochromia, making hematological parameters valuable predictive tools in the initial identification of alpha-thalassemia patients. This study aimed to compare hematologic parameters such as Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), red blood cell (RBC) count, and hemoglobin (HGB) levels in silent and minor patients, whose genotypes were genetically characterized, with normal patients to establish cut-off points for these groups. Materials and Methods: The study involved a total of 860 patients with alpha-thalassemia, including 267 cases of silent, 261 cases of minor, and 332 cases of normal alpha-thalassemia. Results: Analysis of blood indices based on sex revealed that the male group had higher values than the female group. Assessment of alpha-thalassemia in minor patients showed that the Cis form (—/αα) had higher microcytosis than the Trans form (–α/–α) in this group. This difference was also observed between α-3.7α/ α-3.7α and (αα)-MED/αα as two different genetic forms in minor patients, with (αα)-MED/αα being in the Cis form. Data indicated that the cut-off value was insignificant in silent patients compared to the normal group. However, minor patients with MCH≤23.7 and MCV≤74.9 had an AUC greater than 0.9 (p-value< 0.01), distinguishing them from the normal group. Conclusion: Comparing hematological parameters in these groups illustrated that MCV and MCH are the best predictor parameters for distinguishing between groups.

Case Report: Hemoglobin Hasharon with Concurrent Alpha Thalassemia in a Patient with Maple Syrup Urine Disease

Abstract Hemoglobin Hasharon (Hb Hasharon) is an unstable hemoglobin variant stemming from a mutation in the alpha globin gene. This mutation involves the replacement of aspartic acid with histidine at position 47 of the alpha globin chain. Initially identified in 1967 in Israel, Hb Hasharon has since been sporadically reported in various populations, including in Italy, Greece, Germany, and among Latin Americans of Mediterranean descent. Only three English articles have reported several cases of the combined alpha thalassemia deletion and Hasharon mutation, all between 1978-1980. Notably, there has been no prior report of Hb Hasharon with concurrent alpha thalassemia in patients with maple syrup urine disease (MSUD). We hereby present the first case of Hb Hasharon with alpha thalassemia in a patient diagnosed with MSUD. This patient, a 16-month-old female, received a prenatal diagnosis of MSUD through amniocentesis, confirming homozygosity for a known pathogenic variant in DBT (c.75_76delAT, p.C26wfsX2). Prompt initiation of formula feeding has effectively controlled her MSUD. The patient was referred for further evaluation due to hemoglobin newborn screen showing hemoglobin A, Hb F and “variant hemoglobin”. Her parents denied any family history of hemoglobinopathy. Her complete blood count was predominantly normal except for mild microcytosis, with a mean corpuscular volume of 69.1 fl (normal range 70.0-86.0 fl). The patient didn’t exhibit neonatal jaundice or any other signs or symptoms of hemolysis. The reticulocyte count was also within normal limits. The hemoglobin electrophoresis results revealed a complex pattern, with 28.8% of an unknown hemoglobin between zone 4 and 5 on capillary zone electrophoresis (CZE) as well as a possible A2 variant in zone 1. High-performance liquid chromatography (HPLC) indicated a 29.1% peak at a retention time of 4.72 minutes, along with several smaller peaks, that were indicative of the degradation of the unstable unknown hemoglobin. Although the zones and retention time aligned with Hb Hasharon, the percentage differed from the expected range of 19-20% in CZE and 18-21% in HPLC for this variant, prompting the team to order alpha and beta globin sequencing tests. Beta globin gene complete sequencing yielded negative results, while the sequencing of alpha globin gene revealed that the patient carries one copy of the -alpha3.7 alpha-globin deletion and one copy of the c.142G>C (p.Asp48His) variant in the alpha2-globin (HBA2) gene, resulting in an -alpha/alpha2alpha1 genotype with Hb Hasharon. While Hb Hasharon is generally not clinically significant, its co-occurrence with alpha-thalassemia can lead to hemolytic anemia. In our patient, clinic and chemical parameters were predominantly within normal ranges, except for mild microcytosis. The potential interaction between MSUD and Hb Hasharon with concurrent alpha thalassemia remains unknown. Continued patient monitoring will enable us to evaluate whether these conditions may influence each other’s clinical and biochemical profiles.

A Rare Case of Homozygous Hemoglobin G-Philadelphia & Alpha Thalassemia

Abstract Introduction/Objective Hemoglobin (Hb) G-Philadelphia (GPh) is an alpha chain gene variant caused by a missense mutation resulting in a change from Glu to Asp at position 68. This change leads to alterations in the Hb structure and oxygen affinity. The mutation is inherited in an autosomal codominant pattern allowing for an asymptomatic carrier state. While the most common alpha chain variant, GPh heterozygosity is still infrequent, estimated at 1:5000 in African Americans. In African Americans, GPh is frequently linked to an alpha thalassemia deletion. On its own it is considered a benign variant that does not result in significant health issues. Homozygous HbGPh frequency is estimated at 1 in 25 million. Methods/Case Report A postpartum woman in her 4th decade of life was found to have a Hb of 10.8 and a mean corpuscular volume of 74. Initial work up included Hb electrophoresis that quantified HbA at 0%, Hb F at 0%, HbA2 at 0%, and 2 Hb variants representing 96.1% and 3.8% of the total globin. The major Hb variant migrated in the D or G position by alkaline and acid gel Hb electrophoresis. The minor variant was consistent with HbG2. Due to this abnormal Hb electrophoresis, a sample was sent out for a Hb evaluation reflexive cascade (HERC). HERC analysis by high-performance liquid chromatography and capillary gel electrophoresis found 99.9% of Hb consistent with the alpha chain variant HbG. Analysis of the alpha globin gene region revealed 2 copies of an alpha globin gene deletion that was interpreted as alpha-thal trait (-a/-a). This result is consistent with homozygous alpha-thal-2 and homozygous HbGPh (alpha^G/thal + alpha^G/thal). Results (if a Case Study enter NA) NA Conclusion Alpha^G/thal + alpha^G/thal presents a unique and complex challenge for diagnosis due to its rarity and because HbG2 is not always visualized by alkaline gel electrophoresis. The presence of a Hb G or D variant, a HbG2 variant, and the lack of both HbA and HbA2 are key electrophoretic findings. Clinically, alpha^G/thal + alpha^G/thal behaves essentially like a homozygous alpha-thal-2 trait with persistent microcytosis and erythrocytosis. Thus, co- inheritance of the alpha-thal trait will be suggested by a thalassemic complete blood count pattern.

Alpha Thalassemia in Istanbul: Distribution of Deletions in Alpha-globin Gene Cluster

Alpha Thalassemia in Istanbul: Distribution of Deletions in Alpha-globin Gene Cluster

Expert consensus on clinical genetic counseling of α-thalassemia gene analysis

α-thalassemia is a type of microcytic hypochromic anemia caused by variants of alpha-globin gene, and is one of the most common monogenic disorders in southern China. The population screening model based on hematologic phenotype has achieved great results in areas with high incidence of thalassemia. However, with the continuous decline of the cost of genetic testing and implementation of screening programs for thalassemia gene carriers, more variants in the alpha-globin gene have been discovered, which also brings great challenges to clinical genetic counseling. From the perspective of alpha-globin genetic analysis, this consensus has discussed the contents of pre- and post-test genetic counseling, with an aim to provide standardized guidance for clinicians.

A rare hemoglobinopathy duo: Hb Adana×Hb SEA in a 1-year-old patient – a case report and a brief literature review

Introduction and importance: Alpha thalassemia, resulting from nondeletional mutations, typically presents a more severe clinical manifestation compared to deletional mutations. Severe outcomes, such as hydrops fetalis, are associated with two specific nondeletional mutations. Therefore, DNA-based investigation is crucial for suspected carriers exhibiting subtle hematological abnormalities to facilitate proper diagnosis and effective family counseling. Case presentation: In this report, the authors describe a phenotypically normal 1-year-old girl with a rare and unique alpha-thalassemia genotype due to the presence of Hb Adana, a nondeletional alpha-chain mutation compounded with Hb SEA, an alpha-globin gene deletion. Clinical discussion: Mutations determine the clinical manifestations of alpha-thalassemia. DNA testing is recommended for suspected carriers with relatively small hematological abnormalities, for precise diagnosis and family counseling. To provide clinicians with a reference for diagnostic assessment, the authors established a genotype-phenotype correlations based on reported cases of Hb Adana following an exhaustive literature review. Being interested in determining which ethnicities and genotypes are associated with a higher risk of complications, including hydrops fetalis and transfusion dependence, the authors formalized a diagnostic evaluation guide and a guide for early screening to improve outcomes. Conclusion: Precise genetic evaluation is important for the diagnosis of alpha thalassemia. Hematologists play a critical role in managing these disorders, understanding genotype-phenotype correlations, and highlighting the significance of genetic counseling for high-risk patients. Extensive studies on these various genophenotypes are required to improve the diagnosis and prognosis of such medical conditions and advocate preventative strategies.

Alpha globin gene alterations modifying the phenotype of homozygous beta thalassaemia.

The phenotype of β-thalassemia varies widely. The primary determinant is the type of beta-globin gene mutation; however, there are secondary and tertiary modifiers also as associated alpha mutations, polymorphisms, as well as coinheritance of mutations affecting other related systems. Co-inheritance of alpha thalassemia mutations is known to ameliorate the severity of HbE-β thalassemia. However, the role of alpha globin gene alterations (deletions and triplication) is not well illustrated in homozygous β-thalassemia. Here we evaluated the role of alpha globin gene alterations in 122 β-thalassemia patients having IVS1-5 (G>C) homozygous mutation. β-thalassemia mutations were detected by ARMS PCR and alpha mutations by GAP-PCR. Gene expression by qRT-PCR. Out of 122 cases, 15 patients had alpha 3.7 triplications (ααα3.7anti), 24 had alpha 3.7kb deletion (-α3.7) mutation and three patients had 4.2kb deletion (-α4.2). Patients were divided into two groups, requiring less than 8 units (NTDT) and more than 8 units (TDT) of blood transfusion per year (≥8U BT/year). The percentage of alpha deletion was significantly (p=0.0042) high in NTDT (42.1%) as compared with TDT (13.2%). Conversely, the proportion of alpha triplication is high in the TDT as compared with NTDT. Even mean serum ferritin level was found to be significantly high in patients having alpha triplication as compared with those having alpha deletions (p=0.0184) and normal alpha gene (p=0.0003). α/β globin ratio was highest in TDT patients with alpha triplication and lowest in NTDT patients with alpha-del. The results show that concurrent inheritance of alpha gene alterations influences the phenotypic severity of homozygous β-thalassemia.

Association of alpha globin gene copy number with exhaled nitric oxide in a cross-sectional study of healthy Black adults

IntroductionThe genetic determinants of fractional exhalation of nitric oxide (FeNO), a marker of lung inflammation, are understudied in Black individuals. Alpha globin (HBA) restricts nitric oxide signalling in arterial endothelial...

A rare case of compound Hemoglobin Lansing/Hemoglobin S coinherited with alpha thalassemia trait (-alpha3.7) presented with spurious hypoxemia and moderate hemolytic anemia

Abstract We present a case of a 27-year-old female at 27 weeks of gestation, who presented at the emergency department for vaginal spotting. She was also found to have low oxygenation on pulse oximetry of approximately 82% at room air. Her SpO2 did not improve with O2 supplement. She had a similar presentation a month before, with mild light-headedness and low oxygenation. However, two repeated air blood gas (ABG) findings were normal for oxygen saturation. Hemoglobinopathy was suspected due to the disparity. She was anemic during her presentation, with hemoglobin 8.3 g/dl, RBC count 3.36 million/ul, MCV 81 fl and MCH 24.7 pg. Additional workup showed increased reticulocyte count, decreased haptoglobin, negative direct Coombs test, negative G6PD test, and no evidence of a PNH clone. Capillary hemoglobin electrophoresis showed HbA 59.8%, HbS 34.5%, HbA2 3.6% and a small peak (2.1%) in the Z8 zone with a trailing edge at HbF window. HPLC revealed an “unknown peak” of 8.2% with a retention time of 1.93 minutes as well as the HbS peak. There was also a tiny additional A2 peak unresolved from the main A2 peak. Alpha globin gene sequencing revealed one copy of the -alpha3.7 alpha-globin deletion and one copy of Hb Lansing variant in the alpha2-globin gene. This is consistent with the compound Hemoglobin Lansing/Hemoglobin S coinherited with alpha thalassemia trait. Hemoglobin Lansing is a rare unstable alpha hemoglobin variant with mutation of c.264C>G resulting with a substitution of His by Gln at codon 87. The mutation was first discovered in the alpha 2 gene. Then the same mutation was found in alpha 1 gene later and named as Hemoglobin Lansing-Ramathibodi. To the best of our knowledge, this is the first reported case of Hemoglobin Lansing with alpha thalassemia trait (-alpha3.7) and HbS. Spuriously low pulse oximetry measurements have previously been reported in Hb Lansing variants. It was speculated to be due to different absorption spectra of Hb Lansing which is under study now. The patient also presented with hemolytic anemia. The exact cause was undetermined yet although it might be related to her hemoglobinopathy. Mild hemolytic anemia was reported in two cases of co-inheritance of Lansing-Ramathibodi and Hb Pakse (a non-deletional alpha thalassemia) but not in the members of the same family with Hb Lansing alone. It was also reported that some unstable alpha chain variants, when associated with another alpha thalassemia defect, present with a more severe phenotype including hemolytic anemia even though essentially asymptomatic in a heterozygous state.

A-145 Development of a Novel Algorithm Using Red Blood Cell Indices for Screening of Alpha Globin Gene Deletions

Abstract Background Distinguishing between different causes of microcytic anemia is essential for management of anemic patients. More than 20 different RBC indices have been used to screen thalassemia trait with inconsistent results. Differentiation between iron deficiency anemia (IDA) and alpha-thalassemia is challenging based on RBC indices alone. Moreover, thalassemia and IDA may coexist. A highly sensitive screening algorithm is needed to select clinical cases for alpha globin gene analysis to reduce unnecessary testing and improve pre-test probabilities. Methods A retrospective study was performed to evaluate the reliability of 22 RBC indices (RBC, MCV, RDW, MCH, Mentzer Index, RDW Index, etc) and formulae in differentiating cases with 1–2 alpha globin gene deletions from negative cases, in a cohort of 90 individuals with confirmatory alpha globin gene analysis results. Results The number of patients with each alpha globin gene deletion pattern were: 37 patients with alpha-thalassemia trait, 21 silent carriers (one gene deletion), 31 patients with negative result and 1 patient with triplicated alpha globin gene. The mean RBC count in each group were 4.79 ± 0.65 (trait), 4.45 ± 0.73 (carrier), and 4.21 ± 0.84 (negative) (million/mm). The mean MCV in each group were 71.1 ± 4.8 (trait), 76.1 ± 5.9 (carrier), and 74.2 ± 9.3 (negative) (fL). The mean RDW in each group were 16.5 ± 3.0 (trait), 17.2 ± 3.4 (carrier), and 18.3 ± 4.9 (negative) (%). The mean MCH in each group were 22.0 ± 1.8 (trait), 23.8 ± 2.3 (carrier), and 23.5 ± 3.7 (negative) (pg). None of the RBC indices showed significant difference between carrier and negative groups, while 20 RBC indices showed significant difference (P < 0.05) between trait and negative groups. The sensitivities of the RBC indices in detecting 1–2 alpha globin gene deletions based on published cut-offs ranged between 3.4% and 81.0%, while the specificities ranged between 41.9% and 100%. A novel scoring algorithm was evaluated based on the combination of 12 selected RBC indices and formulae, with each index or formula contributing to a score of 0 or 1. The average value of the negative cases was used as the score cut-off value for each index or formula. The scoring algorithm gave a score distribution between 0 and 12, with lower scores indicating alpha globin gene deletions. It achieved a sensitivity of 96.6% in detecting 1–2 alpha globin gene deletions, with a negative likelihood ratio of 0.15. The negative likelihood ratio of the scoring algorithm outperformed each of the individual RBC indices at similar levels of sensitivities (>95%). Conclusion The novel algorithm is a highly sensitive tool for screening of alpha globin gene deletions, including one gene deletion. It is more effective at identifying negative cases compared to individual RBC indices. Implementation of the algorithm could efficiently reduce 10.0% (9/90) of the test volume of alpha globin gene analysis. A more stringent version of the algorithm that incorporates standard deviation in the cut-off values could potentially achieve nearly 100% sensitivity and reduce 5.6% (5/90) of the alpha globin gene analysis test volume.

Hemoglobin H Disease and Growth: A Comparative Study of DHbH and NDHbH Patients.

Hemoglobin H disease (HbH), a hemoglobinopathy resulting from abnormal alpha globin genes, is classified into two categories: deletional HbH (DHbH) and non-deletional HbH (NDHbH). The alpha-mutation genotypes exhibit a range of clinical anemias, which differentially impact patient growth. This retrospective study assessed the growth of HbH patients at Siriraj Hospital, Mahidol University. Patients diagnosed with HbH between January 2005 and April 2021 were analyzed using growth standard scores of the Thai Society for Pediatric Endocrinology (2022 version) and BMI-for-age Z scores of the World Health Organization. Growth failure was defined as a patient's height for age exceeding two standard deviations below the mean. Of the 145 HbH patients, 75 (51.7%) had NDHbH, with --SEA/αCSα being the most common genotype (70 patients; 93.3%). The mean baseline hemoglobin level was significantly lower in NDHbH patients than in DHbH patients (8.16 ± 0.93 g/dL vs. 9.51 ± 0.68 g/dL; P < 0.001). Splenomegaly and growth failure prevalences were higher in NDHbH patients (37.3% vs. 0%, with P < 0.001, and 22.7% vs. 8.6%, with P = 0.020, respectively). Multivariable analysis revealed splenomegaly > 3 cm was associated with growth failure (OR = 4.28; 95% CI, 1.19-15.39; P = 0.026). NDHbH patients exhibited lower hemoglobin levels and more pronounced splenomegaly than DHbH patients. Growth failure can occur in both HbH types but appears more prevalent in NDHbH. Close monitoring of growth velocity is essential, and early treatment interventions may be required to prevent growth failure.

Clinical significance of mutational variants in beta and alpha genes in patients with hemoglobinopathies from two large Greek centers: a complex interplay between genotype and phenotype.

Hemoglobinopathies affect patients in the wider Mediterranean area consisting of 4 distinct subgroups: beta thalassemia major (TM), beta thalassemia intermedia (TI), sickle cell disease (SCD) and hemoglobin H disease (alpha thalassemia). The clinical spectrum varies from mild to severe. Complex interactions between genes and environmental factors form the clinical manifestations. There is an unmet need to clarify these multifactorial mechanisms.This is the first Greek study describing mutational alleles (HBB and HBA1/HBA2 gene variants) in 217 patients with hemoglobinopathies of two large centers in Greece (Larissa and Athens) and associating particular genotypes or gene variants with clinical manifestations (transfusion frequency, complications). Thus, the complex interplay between corresponding genotypes and phenotypes was investigated.Our results are in accordance with previous national studies with limited variations, due to regional prevalence of specific gene variants, as expected. It is also a description of the prevalence of hemoglobinopathies in the Greek population. The type and prevalence of beta and alpha globin gene variants differ significantly among countries. We also confirm the well-known observation of many studies that in our beta thalassemic or SCD patients, co-inheritance of variants in the alpha globin genes, leading to absence or reduction of alpha globin synthesis were associated with milder clinical course, whereas the inheritance of additional alpha genes (triplication) led to a more severe clinical phenotype.In cases in whom the genotype and phenotype did not correlate, factors like the function or modification of possible regulatory genes or additional nutritional-environmental effects should be investigated. KEY MESSAGES: • This is the first Greek study, fully molecularly defining the beta and alpha mutational alleles in 217 patients with hemoglobinopathies of two large centers in Greece and correlating particular genotypes or gene variants with clinical manifestations (transfusion frequency, complications). • In the beta thalassemic or SCD patients of our cohort, co-inheritance of variants in the alpha globin genes, leading to absence or reduction of alpha globin synthesis were associated with milder clinical course (confirmation of a well-known previous observation). • The inheritance of additional alpha genes (triplication) led to a more severe clinical phenotype (confirmation of a well known previous observation). • The function or modification of possible regulatory genes should be investigated in cases in whom the genotype and phenotype did not correlate.

Development of a Quantitative Multiplex PCR to Detect Three Common Alpha Thalassemia Deletions

Alpha thalassemia is an autosomal recessive genetic disorder with a high prevalence in the Middle East. The severe form of alpha-thalassemia is incompatible with life and can cause significant obstetric complications in the mother. Therefore, it is important to determine the genotype in parents who have a chance of having a fetus with one of the severe forms of this disease. A total of 112 samples that were previously analyzed for common alpha thalassemia mutations in Iran were used in this study. A new multiplex PCR including quantitative polymerase chain reaction to amplify the homologous regions of the alpha-globin gene cluster and fluorescent gap PCR was designed to identify −α3.7, −α4.2, --MED deletions. The ROC curve was used to determine the optimum cutoff points. Statistical analysis showed that there is a significant difference between the peak height ratios for different genotypes. The peak corresponding to the 297 bp fragment resulting from the amplification of the allele with MED-I deletion was detected in all the samples with this deletion. Different cutoffs for a range of sensitivities and specificities were determined by the ROC curve. The suggested method can identify three common large deletions in the alpha-globin gene cluster. A study with a larger sample size can provide more accurate information about the sensitivity and specificity of this test.

Alpha globin gene copy number and incident ischemic stroke risk among Black Americans.

People with African ancestry have greater stroke risk and greater heritability of stroke risk than people of other ancestries. Given the importance of nitric oxide (NO) in stroke, and recent evidence that alpha globin restricts nitric oxide release from vascular endothelial cells, we hypothesized that alpha globin gene (HBA) deletion would be associated with reduced risk of incident ischemic stroke. We evaluated 8,947 participants self-reporting African ancestry in the national, prospective Reasons for Geographic And Racial Differences in Stroke (REGARDS) cohort. Incident ischemic stroke was defined as non-hemorrhagic stroke with focal neurological deficit lasting ≥ 24 hours confirmed by the medical record or focal or non-focal neurological deficit with positive imaging confirmed with medical records. Genomic DNA was analyzed using droplet digital PCR to determine HBA copy number. Multivariable Cox proportional hazards regression was used to estimate the hazard ratio (HR) of HBA copy number on time to first ischemic stroke. Four-hundred seventy-nine (5.3%) participants had an incident ischemic stroke over a median (IQR) of 11.0 (5.7, 14.0) years' follow-up. HBA copy number ranged from 2 to 6: 368 (4%) -α/-α, 2,480 (28%) -α/αα, 6,014 (67%) αα/αα, 83 (1%) ααα/αα and 2 (<1%) ααα/ααα. The adjusted HR of ischemic stroke with HBA copy number was 1.04; 95%CI 0.89, 1.21; p = 0.66. Although a reduction in HBA copy number is expected to increase endothelial nitric oxide signaling in the human vascular endothelium, HBA copy number was not associated with incident ischemic stroke in this large cohort of Black Americans.

Alpha-Thalassemia in Southern Italy: Characterization of Five New Deletions Removing the Alpha-Globin Gene Cluster.

α-thalassemia is characterized in about 80% of cases by deletions generated by the presence of duplications and interspersed repeated sequences in the α-globin gene cluster. In a project on the molecular basis of α-thalassemia in Southern Italy, we identified six families, showing an absence of the most common deletions, and normal α-globin gene sequences. Multiplex Ligation-dependent Probe Amplification (MLPA), qRT-PCR, and the sequencing of long-range PCR amplicon have been used for the identification and characterization of new deletions. MLPA analysis for the identification of α- and β-globin rearrangement revealed the presence of five new α-thalassemia deletions. The set-up of qRT-PCR allowed us to delimit the extent of the deletions ranging from about 10 kb to more than 250 kb, two of them being of the telomeric type. The long-range PCR generated a specific anomalous fragment in three deletions, and only several unspecific bands in the other two deletions. The sequencing of the anomalous amplicons revealed the breakpoints of two deletions: the --PA, 34 kb long, identified in two families, and the telomeric --AG, 274 kb long. The anomalous fragment containing the breakpoint of the deletion --FG was partially sequenced, and it was not possible to identify the breakpoints due to the presence of several repetitive Alu sequences. The analysis of the breakpoint regions of the --Sciacca and --Puglia, respectively, are about 10 and 165 kb long, and revealed the presence of repeats that most likely impaired the amplification of a specific fragment for the identification of the breakpoint. MLPA, in association with qRT-PCR and long-range PCR, is a good approach for the identification and molecular characterization of rare or new deletions. Breakpoint analysis confirms that Alu sequences play an important role in favoring unequal crossing-over. Southern Italy shows considerable genetic heterogeneity, as expected with its central position in the Mediterranean basin, favoring migratory flows.

Molecular characterization of alpha globin and beta globin genes in patients with hemoglobinopathies in Central Vietnam

Background: Hemoglobinopathy is the most common monogenic disease worldwide. The aims of the current study were: (1) to investigate some hematological characteristics of patients with hemoglobinopathies; and (2) to detect the mutation of α-globin and β-globin genes, as well as the association between genotype and degree of anemia. Materials and method: 251 patients with hemoglobinopathies were examined for the α-globin or β-globin gene mutations. Results: 51% were the carriers, and 49% were thalassemia intermedia or thalassemia major. Hematological characteristics were suitable for α-thalassemia or β-thalassemia. Elevenβ-globin gene mutations were observed. The β0/βA, βE/βA, βE/βE, βE/β+, β+/β+ genotypes were only found in β-thalassemia intermedia individuals; the β0/β0 genotype was limited to β-thalassemia major patients; the β+/β0 and βE/β0 genotypes were seen in both types. Four α-globin gene mutations were observed. All α-thalassemia patients were intermedia, the most common genotype was --SEA/-α3.7. Conclusion: There were differences in anemia degree between β-globin genotypes Key words: hemoglobinopathies, α-globin, β-globin.

Development of a Novel Algorithm Using Red Blood Cell Indices for Screening of Alpha Globin Gene Deletions

Abstract Distinguishing between different causes of microcytic anemia is essential for management of anemic patients. More than 20 different RBC indices have been used to screen thalassemia trait with inconsistent results. Differentiation between iron deficiency anemia (IDA) and alpha-thalassemia appears to be more challenging than differentiation between IDA and beta-thalassemia, based on RBC indices alone. Moreover, thalassemia and IDA may coexist. A highly sensitive screening algorithm is needed to select clinical cases for alpha globin gene analysis to reduce unnecessary testing and improve pre-test probabilities. A retrospective study was performed to evaluate the reliability of 22 RBC indices (RBC, MCV, RDW, MCH, Mentzer Index, RDW Index, etc) and formulae in differentiating cases with 1-2 alpha globin gene deletions from negative cases, in a cohort of 90 individuals with confirmatory alpha globin gene analysis results. The number of patients with each alpha globin gene deletion pattern were: 37 patients with alpha-thalassemia trait, 21 silent carriers (one gene deletion), 31 negative patients and 1 patient with triplicated alpha globin gene. The mean RBC count in each group were 4.79 +/- 0.65 (trait), 4.45 +/- 0.73 (carrier), and 4.21 +/- 0.84 (negative) (million/mm). The mean MCV in each group were 71.1 +/- 4.8 (trait), 76.1 +/- 5.9 (carrier), and 74.2 +/- 9.3 (negative) (fl). The mean RDW in each group were 16.5 +/- 3.0 (trait), 17.2 +/- 3.4 (carrier), and 18.3 +/- 4.9 (negative) (%). The mean MCH in each group were 22.0 +/- 1.8 (trait), 23.8 +/- 2.3 (carrier), and 23.5 +/- 3.7 (negative) (pg). None of the RBC indices showed significant difference between carrier and negative groups, while 20 RBC indices showed significant difference (p&amp;lt;0.05) between trait and negative groups. The sensitivities of the RBC indices in detecting 1-2 alpha globin gene deletions based on published cutoffs ranged between 3.4% and 81.0%, while the specificities ranged between 41.9% and 100%. A novel scoring algorithm based on the combination of 12 RBC indices and formulae achieved a sensitivity of 96.6% in detecting 1-2 alpha globin gene deletions, with a negative likelihood ratio of 0.15. The negative likelihood ratio of the scoring algorithm outperformed each of the 12 RBC indices at similar levels of sensitivities (&amp;gt;95%) based on revised cutoffs. The algorithm is a highly sensitive tool for screening of alpha globin gene deletions, including one gene deletion. It is more effective at identifying negative cases compared to individual RBC indices. Implementation of the algorithm could efficiently reduce 10.0% (9/90) of the test volume of alpha globin gene analysis. A more stringent version of the algorithm could achieve 100% sensitivity and reduce 5.6% (5/90) of the alpha globin gene analysis test volume.

Common and ethnic-specific genetic determinants of hemoglobin concentration between Taiwanese Han Chinese and European Whites: findings from comparative two-stage genome-wide association studies

Human iron nutrition is a result of interplays between genetic and environmental factors. However, there has been scarcity of data on the genetic variants associated with altered iron homeostasis and ethnic-specific associations are further lacking. In this study, we compared between the Taiwanese Han Chinese (HC) and European Whites the genetic determinants of hemoglobin (Hb) concentration, a biochemical parameter that in part reflects the amount of functional iron in the body. Through sex-specific two-stage genome-wide association studies (2S-GWAS), we observed the consistent Hb-association of SNPs in TMPRSS6 (chr 22), ABO (chr 9), and PRKCE (chr 2) across sexes in both ethnic groups. Specific to the Taiwanese HC, the Hb-association of AXIN1, together with other loci near the chr 16 alpha-globin gene cluster, was found novel. On the other hand, majority of the Hb-associated SNPs among Europeans were identified along the chr 6 major histocompatibility complex (MHC) region, which has established roles in immune system control. We report here strong Hb-associations of HFE and members of gene families (SLC17; H2A, H2B, H3, H4, H1; TRIM; ZSCAN, ZKSCAN, ZNF; HLA; BTN, OR), numerous SNPs in/nearby CARMIL1, PRRC2A, PSORS1C1, NOTCH4, TSBP1, C6orf15, and distinct associations with non-coding RNA genes. Our findings provide evidence for both common and ethnic-specific genetic determinants of Hb between East Asians and Caucasians. These will help to further our understanding of the iron and/or erythropoiesis physiology in humans and to identify high risk subgroups for iron imbalances - a primary requirement to meet the goal of precision nutrition for optimal health.

Two large novel alpha-globin gene cluster deletions causing alpha(0)-thalassemia in two Chinese families

IntroductionMonosomy of terminal 16p13.3 is a relatively common subtelomeric abnormality, most affected individuals presented α-thalassemia, some also have mental retardation, developmental abnormalities and/or speech delay and facial dysmorphism, which is termed ATR-16 syndrome. Here, we reported two novel 16p13.3 deletions involving the α-globin gene cluster and multispecies conserved sequences (MCSs), causing only a phenotype of α-thalassemia. MethodsSamples were collected from members of the two families and were subjected to haematological and comprehensive genetic analysis. ResultsThe novel 108 Kb deletion in family A extends from the non-protein coding RNA gene (WASIR2) to the NPRL3 gene, removing MCS-R1 to R3. This deletion should arise de novo because it wasn’t detected in both parents. The novel 336 Kb deletion in family B should extend from telomere to ∼ chr16:336000, removing the entire α-globin gene cluster. Carriers of these two deletions presented with microcytosis and hypochromic red cells, in accordance with a phenotype of α0-thalassemia carrier. ConclusionOur study increases the mutation spectrum of α-thalassemia. MCSs deletion should be considered in clinical practice of thalassemia screening and diagnosis.

Alpha globin gene copy number and hypertension risk among Black Americans.

BackgroundAlpha globin is expressed in the endothelial cells of human resistance arteries where it binds to endothelial nitric oxide synthase and limits release of the vasodilator nitric oxide. Genomic deletion of the alpha globin gene (HBA) is common among Black Americans and could lead to increased endothelial nitric oxide signaling and reduced risk of hypertension.MethodsCommunity-dwelling US adults aged 45 years or older were enrolled and examined from 2003 to 2007, followed by telephone every 6 months, and reexamined from 2013 to 2016. At both visits, trained personnel performed standardized, in-home blood pressure measurements and pill bottle review. Prevalent hypertension was defined as systolic blood pressure ≥ 140mmHg or diastolic blood pressure ≥ 90mmHg or anti-hypertensive medication use. Droplet digital PCR was used to determine HBA copy number. The associations of HBA copy number with prevalent hypertension, resistant hypertension, and incident hypertension were estimated using multivariable regression.ResultsAmong 9,684 Black participants, 7,439 (77%) had hypertension at baseline and 1,044 of those had treatment-resistant hypertension. 1,000 participants were not hypertensive at baseline and participated in a follow up visit; 517 (52%) developed hypertension over median 9.2 years follow-up. Increased HBA copy number was not associated with prevalent hypertension (PR = 1.00; 95%CI 0.98,1.02), resistant hypertension (PR = 0.95; 95%CI 0.86,1.05), or incident hypertension (RR = 0.96; 95%CI 0.86,1.07).ConclusionsThere were no associations between increased HBA copy number and risk of hypertension. These findings suggest that variation in alpha globin gene copy number does not modify the risk of hypertension among Black American adults.