[Cases Analysis of Hemoglobin H Disease Caused by HBA2:c.2T>C and HBA2:c.2delT Mutations].

OBJECTIVE: To investigate two cases of rare pathogenic genes, initiation codon mutations in HBA2 gene, combined with Southeast Asian deletion and their family members to understand the relationship of HBA2:c.2T>C and HBA2:c.2delT mutations with clinical phenotype. METHODS: The peripheral blood of family members was obtained for blood cell analysis and capillary electrophoresis hemoglobin analysis. Gap-PCR and reverse dot blotting (RDB) were used to detect common types of mutations in ɑ-thalassaemia gene. Sanger sequencing was used to analyze HBA1 and HBA2 gene sequence. RESULTS: Two proband genotypes were identified as --SEA/αα with HBA2:c.2T>C and --SEA/αα with HBA2:c.2delT. HBA2:c.2T>C/WT and HBA2:c.2delT/WT was detected in family members. They all presented with microcytic hypochromic anemia. CONCLUSION: When HBA2:c.2T>C and HBA2:c.2delT are heterozygous that can lead to static α-thalassemia phenotype, and when combined with mild α-thalassemia, they can lead to the clinical manifestations of hemoglobin H disease. This study provides a basis for genetic counseling.

Severity scoring system to guide transfusion management in pediatric non-deletional HbH.

BACKGROUND: Hemoglobin (Hb) H is generally recognized as mild thalassemia, despite its actual phenotypic diversity. A disease severity scoring system to guide initiation of regular transfusion among severely affected pediatric patients has not previously been reported. METHODS: Patients with HbH were classified into transfusion-dependent thalassemia (TDT) and non-transfusion-dependent thalassemia (NTDT) as a surrogate for disease severity. Alpha-globin genotypes and relevant clinical parameters associated with TDT were identified. Univariate and multiple logistic regression analyses were performed to yield the most suitable severity scoring system. RESULTS: From 246 patients with a median age of 14.3 (interquartile range 9.9-18.4) years initially enrolled into the study, the chance of having severe disease and developing TDT was remarkable only among patients with non-deletional HbH, for whom the scoring system was developed. Univariate and multiple logistic regression analyses resulted in three retained parameters associated with TDT, ß-coefficients of which were used to develop the score. The final scoring system comprised age at diagnosis <2 years (score = 1), spleen size ≥3 cm (score = 1) and Hb at steady-state <7 (score = 4) or 7-8 g/dL (score = 3). A cutoff score ≥4 was associated with severe disease likely requiring regular transfusion (sensitivity 89.3%, specificity 81.4%), given regular transfusion resulted in maintained growth. The scoring system was validated in the second cohort of 77 non-deletional HbH, from which comparable sensitivity and specificity were obtained. CONCLUSION: The newly developed scoring system was practical and helpful to highlight severely affected pediatric non-deletional HbH patients with potential needs of regular transfusion. This can be used as a guide for optimal treatment and disease monitoring in the future.

Detection of hemoglobin H disease by long molecule sequencing.

BACKGROUND: Hemoglobin H (Hb H) disease is a moderate-to-severe form of α-thalassemia (α-thal), and parts of patients may require intermittent transfusion therapy, especially during intercurrent illness. However, rare Hb H diseases remain undetected using routine methods being outside of the testing scope. In this study, we present an approach to detecting Hb H disease by long molecule sequencing (LMS). METHODS: A total of 206 known genotype samples were collected and carried to blind detected by LMS on the PacBio Sequel platform. Circular consensus sequencing reads were aligned to the hg19 reference genome using Free-Bayes finished LMS. LMS accuracy would be compared with routine methods, including Gap-PCR and PCR-Reverse dot blot hybridization (PCR-RDB). RESULTS: The assay could detect carriers of both deletion and point mutations. It had an overall accuracy of 100% when compared with routine methods. In addition, LMS detected six mutations based on routine methods and corrected three case results. Hb H diseases were identified using LMS, whether a common or rare genotype, a deletion or non-deletion genotype. However, two cases of Hb H disease were misdiagnosed using routine methods. CONCLUSIONS: Long molecule sequencing can be suggested as a rapid and reliable assay to detect probable carriers of hemoglobinopathies. LMS accurately identified the common and rare genotypes of Hb H disease.

Human m6A-mRNA and lncRNA epitranscriptomic microarray reveal function of RNA methylation in hemoglobin H-constant spring disease.

The thalassemia of Hemoglobin H-Constant Spring disease (HbH-CS) is the most common type of Thalassemia in non-transfusion thalassemia. Interestingly, the clinical manifestations of the same genotype of thalassemia can be vastly different, likely due to epigenetic regulation. Here, we used microarray technology to reveal the epigenetic regulation of m6A in modifiable diseases and demonstrated a role of BCL2A1 in disease regulation. In this study, we revealed that methylating enzyme writers including METTL16, WTAP, CBLL1, RBM15B, and ZC3H13 displayed low expression and the demethylating enzyme ALKBH5, along with reader proteins including IGF2BP2 and YTHDF3 exhibited high expression. In addition, BCL2A1 was hypo-methylated and showed low expression. We also revealed that the BCL2A1 methylation level and IGF2BP2 expression were negatively correlated. Additionally, the mRNAs expression between ALKBH5 and IGF2BP2 were positively correlated. In HbH-CS, most genes were hypo-methylated. This included BCL2A1, which may play an important role in the process of red blood cell differentiation and development of HbH-CS. Moreover, the mRNA-M6A methylation status may be regulated by the demethylating enzyme ALKBH5 via IGF2BP2.

Telomere shortening correlates with disease severity in hemoglobin H disease patients.

Hemoglobin H (Hb H) disease is the most significant health problem of the α-thalassemia syndromes. The Hb disease patients are categorized based on their genotype to deletional and nondeletional, with the latter genotype presents the more severe clinical symptoms. Since telomere length is an indicator of biological aging and health, we hypothesized that telomere length could reflect Hb H disease's severity. In this study, we recruited 48 deletional and 47 nondeletional Hb H disease patients, along with 109 normal controls, for telomere length assessment. The leukocyte telomere length was assessed by monochromatic multiplex real-time PCR and reported as the telomere to single-copy gene (T/S) ratio. When telomere length was adjusted for age, the analysis of covariance between the control and the two Hb H disease groups revealed no significant difference. However, the telomere shortening rate was more rapid in the nondeletional Hb H disease group than those of the control and deletional Hb H disease groups. Gender analysis found that male patients have a significantly lower T/S ratio than females in the nondeletional group but not in the control and deletional groups. In the two disease groups, the T/S ratio was not influenced by ferritin level or transfusion burden but was positively correlated with the absolute reticulocyte count.

Coinherited Hemoglobin H/Constant Spring Disease and Heterozygous Hemoglobin Tak Causing Severe Hemolytic Anemia in a Thai Boy.

Hemoglobin (Hb) H/Constant Spring disease is a common nondeletional Hb H disease, typically causing a more severe phenotype than the deletional Hb H disease counterpart. Hb Tak, resulting from a dinucleotide insertion (+AC) at codon 146 of beta-globin gene, has an increased oxygen affinity and usually presents with polycythemia. We studied a case of a 4-year-old Thai boy with a severe, early-onset anemia. To our knowledge, he is the first reported patient with Hb H/Constant Spring disease and heterozygous Hb Tak. Trio-whole-exome sequencing does not identify other genetic variants that may contribute to the severity of anemia. The observation suggests that coinherited Hb H/Constant Spring and heterozygous Hb Tak lead to severe hemolytic anemia.

Screening for Iron Deficiency Anemia in Infants in a Thalassemia-endemic Region.

Screening for iron deficiency anemia (IDA) in infants is usually carried out by hemoglobin (Hb) level and mean corpuscular volume (MCV). A coinherited thalassemia carrier may confound the diagnosis of IDA. This study aimed to characterize the hematologic parameters in infants with IDA and in thalassemia carriers, and to study the use of red cell parameters in IDA screening in a thalassemia-endemic area. Healthy infants, 6 to 12 months of age were enrolled. Blood samples were taken for complete blood count, ferritin level, Hb analysis, and polymerase chain reaction for alpha-thalassemia. IDA was defined as Hb <11.0 g/dL and ferritin <12 µg/L. Formulae calculated from red cell parameters to distinguish thalassemia carriers were analyzed. Eighty-five infants, 8.3±2.4 months of age, including 48 (56.5%) male infants were enrolled. Sixteen infants (18.8%) had IDA. There were 25 thalassemia carriers (29.4%), 1 Hb H disease, and 1 homozygous Hb E. Hb levels and MCV in the IDA and thalassemia carrier groups were significantly lower than those in the normal group. Area under the curve of Mentzer index (MCV/red blood cell count <13) to suggest thalassemia carriers was 0.867 (95% confidence interval: 0.784-0.951), and the sensitivity and specificity were 92.6% and 72.4%, respectively. In conclusion, both Hb level and Mentzer index are recommended for screening of IDA and thalassemia carriers in the population.

Genotype-phenotype correlation of HbH disease in northern Iraq.

BACKGROUND: HbH disease results from dysfunction of three, less commonly two, α-globin genes through various combinations of deletion and non-deletion mutations. Characterization of the mutations and the underlying genotypes is fundamental for proper screening and prevention of thalassaemia in any region. The aim of this study was to explore the genetic arrangements of HbH disease and to correlate the genotypes with the clinical phenotypes. METHODS: A total of 44 HbH disease patients were enrolled in this study. They were clinically and haematologically assessed. The patients were tested for 21 common α-globin gene mutations using multiplex PCR and reverse hybridization. According to the genotype, the patients were categorized into two separate sub-groups, deletion and non-deletion types HbH disease. RESULTS: Within the studied HbH disease patients, eight different α-globin gene mutations were detected in nine different genetic arrangements. The --MED and -α3.7 deletions were the two most frequently encountered mutations (37.5 and 35.2% respectively). Patients with deletion genotypes constituted 70.4%. The most common detected genotype was --MED/-α3.7 (59.1%), followed by αpoly-A1α/αpoly-A1α (13.6%). For the first time, coinheritance of two relatively mild mutations (-α3.7/ααAdana) was unpredictably detected in a 1.5 year-old child with Hb of 7.1 g/dL. CONCLUSION: The HbH disease patients' clinical characteristics were variable with no ample difference between the deletion and non-deletion types. These results can be of benefit for the screening and management of thalassaemia in this region.

Analysis of Hb levels and degree of anemia in relation to genotype in 615 patients with hemoglobin H disease.

OBJECTIVES: We analyzed hemoglobin (Hb) levels and degree of anemia in relation to genotype in patients with hemoglobin H (Hb H) disease, thereby providing a scientific basis for the prevention and treatment of Hb H disease in the Guangxi region of China. METHODS: Hb analysis was conducted in 615 patients using high performance liquid chromatography. Seven α-thalassemia and 17 ß-thalassemia genotypes commonly found in the Chinese population were detected by Gap-polymerase chain reaction and reverse dot hybridization. Multiple ligation-dependent probe amplification and sequencing were used to detect α-globin gene. RESULTS: On analyzing the degree of anemia, we found that the proportion of severe and moderate anemia was the highest among cases with - SEA/αCSα genotype, followed by - SEA/αQSα. When Hb H disease was present in combination with ß-thalassemia, the clinical symptoms of most patients were milder than those with simple Hb H disease. CONCLUSION: The clinical manifestations of various types of Hb H disease are heterogeneous; the Hb levels of patients with deletional Hb H are generally higher than those with non-deletional Hb H (P < 0.05). In-depth knowledge of the gene mutation spectrum of thalassemia in Guangxi can provide a basis for genetic counseling of couples and enable prenatal diagnosis.

Hb H Disease Diagnosed During Adolescent Pregnancy.

Hb H disease is a moderate to severe form of α-thalassemia (α-thal). Patients with Hb H disease may become symptomatic, especially during infections and pregnancy, and may require transfusions. Herein, we present a 16-year-old female with Hb H disease who was initially diagnosed during adolescent pregnancy and was found to carry the -α3.7/-(α)20.5 deletions. The relatively mild presentation of this case highlights the milder phenotypic consequences of deletional α mutations. The case describes the screening and management of pregnancy with Hb H disease. Additionally, this case demonstrates that screening of some undiagnosed inherited blood disorders is important during pregnancy.

α-Globin Genotypes Associated with Hb H Disease: A Report from Oman and a Review of the Literature from the Eastern Mediterranean Region.

α-Thalassemia (α-thal) is the most common autosomal recessive hemoglobinopathy. There is a vast diversity and geographical variability in underlying genotypes in Hb H (ß4) patients. Herein, we describe the genotypes found in the largest report of Omani Hb H patients. Moreover, we reviewed and summarized the literature published from the Eastern Mediterranean region. A retrospective review of all genetically confirmed Hb H disease patients diagnosed between 2007 and 2017 at Sultan Qaboos University Hospital, Muscat, Oman, was performed. Hematological parameters and clinical presentations were assessed. Both α-globin genes were screened for deletional and nondeletional mutations using a stepwise diagnostic strategy as described before. A total of 52 patients (27 females and 25 males) with a mean age of 20.6 years (range 0.23-80.0) were molecularly confirmed to carry Hb H disease. The patients had a hemoglobin (Hb) level of 9.3 g/dL (range 5.7-13.0) and mean corpuscular volume (MCV) of 58.4 fL (range 48.2-82.1). A total of eight genotype combinations were identified, with α2 polyadenylation signal mutation (polyA1) (AATAAA>AATAAG (αPA1α/αPA1α), often cited as αT-Saudiα/αT-Saudiα, being the most common (53.8%) followed by -α3.7/- -MED I (28.8%). Our cohort also included patients with combinations of αPA1 with other Hb variants: αPA1α/αPA1α with Hb S (HBB: c.20A>T) trait (n = 2), -α3.7/αPA1α (n = 2) and αcodon 19α (HBA2: c.56delG)/αPA1α (n = 1). Nondeletional Hb H disease due to the αPA1 mutation is the most common in Omanis. Molecular diagnosis is necessary for accurate confirmation of the diagnosis of α-thal, determination of underlying genotypes, follow-up and counseling.

Nondeletional α-Thalassemia: Two New Mutations on the α2 Gene.

About 10.0% of α-thalassemia (α-thal) cases are due to point mutations, small deletions, or insertions of one or more bases on the α genes that can alter mRNA processing at the transcription, translation, or post-translation level; these cases are called nondeletional α-thalassemias (α-thal). Most occur within the domain of the α2 gene without changes in the expression of the α1 gene. We present two new frameshift mutations on the HBA2 gene, associated with a nondeletional α-thal phenotype. The probands were referred to our clinic because of persistent microcytosis and hypochromia. The molecular characterization was performed by automatic sequencing of the α-globin genes. Two new mutations were detected on the HBA2 gene; HBA2: c.85delG, p.(Ala29fs*21), and HBA2: c.268_280delCACAAGCTTCGGG, p.(His90Trpfs*9). These new mutations cause a change of the reading frame, the first on codon 28 and the second from codons 89 to 93. In the first mutation, the result is an altered amino acid sequence and a premature termination codon at position 87, while the elimination of 13 bp generates a protein of 95 residues and in this case, the premature termination codon is at position 96. These types of mutation are among the most damaging changes to the coding of a protein. Not only do they lead to changes in the length of the polypeptide, but they also vary the chemical composition, which would result in a nonfunctional protein. The importance of identifying these new mutations lies in their possible association with α0-thal, which could lead to a severe thalassemia.

Diagnosis and Prenatal Diagnosis in a Chinese Family Carrying the Rare α-Thalassemia Gene HBA2: c.1A>G Mutation.

The aim of this study was to identify the rare thalassemia genotype in a family and perform prenatal diagnosis (PND) on the proband's unborn child. Peripheral blood was collected from the family members for hematology analysis and capillary electrophoresis (CE) analysis. Peripheral blood and cord blood were analyzed by gap-polymerase chain reaction (gap-PCR), reverse dot-blot and Sanger sequencing for genotypes of α-thalassemia (α-thal). A heterozygous mutation, HBA2: c.1A>G, was identified in the proband and his father. Two compound heterozygous variants, HBA2: c.1A>G and the - -SEA (Southeast Asian) deletion, were revealed in the proband's unborn child. The hemoglobin (Hb) CE result of the fetal cord blood indicated the fetus had Hb H disease. We have identified a rare thalassemia mutation (HBA2: c.1A>G) in a Chinese family and enriched the rare α-thal gene pool in the Chinese population. When the patient's phenotype does not match the genotype detected by thalassemia gene detection kits, further investigation of rare genotypes should be conducted to avoid missed diagnosis or misdiagnosis, which can help guide clinical diagnosis, population screening and genetic counseling.

Analysis of Deletional Hb H Diseases in Samples with Hb A2-Hb H and Hb A2-Hb Bart's on Capillary Electrophoresis.

The capillary electrophoresis (CE) system allows the quantification of Hb Bart's (γ4) and Hb H (ß4) that is used for screening of Hb H disease. However, Hb Bart's hydrops fetalis and Hb H are not always codetected in patients with Hb H disease. In this study, 35 samples were analyzed for the α0-thalassemia (α0-thal) [- -SEA (Southeast Asian) and - -THAI (Thailand)] deletions and the α+-thal [-α3.7 (rightward) and -α4.2 (leftward)] type deletions using real time-polymerase chain reaction (real time-PCR) with SYBR Green1 and high-resolution melting (HRM) analysis and conventional gap-PCR techniques, respectively. Results showed that 28 of 29 (96.6%) samples with the Hb A2-Hb H phenotype on CE electrophoregrams presented the genotype of - -SEA/-α3.7, while the - -SEA/-α4.2 made up the remainder. The - -SEA/-α3.7 genotype was also found in all six samples (100.0%) with Hb A2-Hb Bart's on CE electrophoregrams. Thus, for genetic counseling, prevention and control programs of Hb Bart's hydrops fetalis and Hb H disease, α-thal genotype analysis is required.

Combined and differential effects of alpha-thalassemia and HbF-quantitative trait loci in Senegalese hydroxyurea-free children with sickle cell anemia.

BACKGROUND: Our objective was to investigate the combined and differential effects of alpha-thalassemia -3.7 kb deletion and HbF-promoting quantitative trait loci (HbF-QTL) in Senegalese hydroxyurea (HU)-free children and young adults with sickle cell anemia (SCA). PROCEDURE: Steady-state biological parameters and vaso-occlusive crises (VOC) requiring emergency admission were recorded over a 2-year period in 301 children with SCA. The age of the first hospitalized VOC was also recorded. These data were correlated with the alpha-globin and HbF-QTL genotypes. For the latter, three different genetic loci were studied (XmnI, rs7482144; BCL11A, rs1427407; and the HBS1L-MYB region, rs28384513) and a composite score was calculated, ranging from zero (none of these three polymorphisms) to six (all three polymorphisms at the homozygous state). RESULTS: A positive clinical impact of the HbF-QTL score on VOC rate, HbF, leucocytes, and C-reactive protein levels was observed only for patients without alpha-thalassemia deletion. Conversely, combination of homozygous -3.7 kb deletion with three to six HbF-QTL was associated with a higher VOC rate. The age of the first hospitalized VOC was delayed for patients with one or two alpha-thalassemia deletions and at least two HbF-QTL. CONCLUSION: Alpha-thalassemia -3.7 kb deletion and HbF-QTL are modulating factors of SCA clinical severity that interact with each other. They should be studied and interpreted together and not separately, at least in HU-free children.

Identification of a Novel Nonsense Mutation in a Patient with Transfusion-Dependent Hb H Disease.

BACKGROUND: Hb H disease is a form of α-thalassemia. The high clinical variability is influenced by the exact combination of mutations. Here we report on a 29-year-old female patient from Afghanistan who received regular blood transfusions since her childhood. METHODS: For diagnosis we employed Sanger sequencing, multiplex ligation-dependent probe amplification, hemoglobin-electrophoresis, and hematological analysis. RESULTS: Molecular genetic analysis revealed a non-deletional Hb H genotype with two in cis point mutations in HBA1 (c.183G>T;p.Lys61Asn and c.184A>T;p.Lys62*) in addition to the common deletions α4.2 and α3.7 in HBA2. The nonsense-mutation p.Lys62* has not been described before. Hematological data were in accordance with the genetic findings. CONCLUSIONS: We describe here a novel mutation in the HBA1 gene and support evidence for non-deletional type of Hb H leading to transfusion-dependent anemia.

Hb H Disease Caused by Multiple Mutations in the Polyadenylation Signal Site and - -SEA/αα.

Thalassemia is the most common monogenic disease, with the highest incidence in Guangxi Zhuang Autonomous Region, People's Republic of China (PRC). The blood test result was not consistent with α-globin gene testing in one of the patients during daily screening. It was confirmed that there were multiple mutations at the α2-globin gene polyadenylation (polyA) signal site: HBA2: c.*64(T>C), HBA2: c.*68(A>C), HBA2: c.*71(G>A), HBA2: c.*74(C>A), HBA2: c.*82(G>A), HBA2: c.*92(A>G) and HBA2: c.*98(T>C) and compound - -SEA/αα by sequencing of the HBA1 and HBA2 genes of the proband and core family members. After that, we found a further two cases of unrelated patients with this type of mutation. The mutation is not an accidental phenomenon, and likely to occur with a considerable incidence in Guangxi Zhuang Autonomous Region, PRC. We analyzed the hematological manifestations of this type of thalassemia and showed that it was a Hb H (ß4) disease caused by rare mutations. We suggest that it is essential to pay attention to this mutation during future clinical diagnoses and genetic counseling of patients.

Association of classical markers and establishment of the dyslipidemic sub-phenotype of sickle cell anemia.

BACKGROUND: Sickle cell anemia (SCA) patients exhibit sub-phenotypes associated to hemolysis and vaso-occlusion. The disease has a chronic inflammatory nature that has been also associated to alterations in the lipid profile. This study aims to analyze hematological and biochemical parameters to provide knowledge about the SCA sub-phenotypes previously described and suggest a dyslipidemic sub-phenotype. METHODS: A cross-sectional study was conducted from 2013 to 2014, and 99 SCA patients in steady state were enrolled. We assessed correlations and associations with hematological and biochemical data and investigated the co-inheritance of -α3.7Kb-thalassemia (-α3.7Kb-thal). Correlation analyses were performed using Spearman and Pearson coefficient. The median of quantitative variables between two groups was compared using t-test and Mann-Whitney. P-values <0.05 were considered statistically significant. RESULTS: We found significant association of high lactate dehydrogenase levels with decreased red blood cell count and hematocrit as well as high levels of total and indirect bilirubin. SCA patients with low nitric oxide metabolites had high total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol and reduced very low-density cholesterol, triglycerides, direct bilirubin level and reticulocyte counts. In SCA patients with high-density lipoprotein cholesterol greater than 40 mg/dL, we observed increased red blood cell count, hemoglobin, hematocrit, and fetal hemoglobin and decreased nitric oxide metabolites levels. The presence of -α3.7Kb-thal was associated with high red blood cell count and low mean corpuscular volume, mean corpuscular hemoglobin, platelet count and total and indirect bilirubin levels. CONCLUSIONS: Our results provide additional information about the association between biomarkers and co-inheritance of -α3.7Kb-thal in SCA, and suggest the role of dyslipidemia and nitric oxide metabolites in the characterization of this sub-phenotype.