Hb H disease associated with compound heterozygosity for --SEA deletion and a novel alpha globin chain variant (HBA2:c.175C>A) on the distal histidine in a Chinese family.

OBJECTIVES: In clinical practice, the majority of α-thalassaemia cases arise from deletions of the α-globin genes. However, a subset of cases is attributed to rare haemoglobin variants, which can manifest with borderline or normal screening results, potentially leading to missed diagnoses in clinical practice. METHODS: Blood samples were collected from family members and underwent haematological, DNA and RNA analysis. RESULTS: The five-month-old proband presented a haematological phenotype consistent with Hb H disease. The mother's haematology profile was consistent with an α-thalassaemia carrier, while the father exhibited a borderline reduction in MCV and MCH. MALDI-TOF identified an abnormal α-chain in the proband. DNA analysis revealed a novel α-globin variant (HBA2:c.175C>A, α58His>Asn, Hb DG-Nancheng) affecting the distal histidine in the family. The father and the mother had α-genotype of --SEA/αα and αDG-Nanchengα/αα, respectively; while the proband inherited both mutant alleles (--SEA/αDG-Nanchengα). Sequencing of cDNA from HBA2 gene identified an equal ratio of normal and mutant alleles. CONCLUSION: This rare case highlighted the importance of identifying rare haemoglobin variant during prenatal screening. The clinical and genetic data provides useful information on the pathogenicity of this variant and further insight into the role of distal histidine residue of α-globin.

Comparing three cardiothoracic ratio measurement techniques and creating multivariable scoring system to predict Bart's hydrops fetalis at 17-22 weeks' gestation.

To assess the diagnostic performance of three cardiothoracic (CT) ratio techniques, including diameter, circumference, and area, for predicting hemoglobin (Hb) Bart's disease between 17 and 22 weeks' gestation, and to create a multivariable scoring system using multiple ultrasound markers. Before invasive testing, three CT ratio techniques and other ultrasound markers were obtained in 151 singleton pregnancies at risk of Hb Bart's disease. CT diameter ratio demonstrated the highest sensitivity among the other techniques. Significant predictors included CT diameter ratio > 0.5, middle cerebral artery-peak systolic velocity (MCA-PSV) > 1.5 multiples of the median, and placental thickness > 3 cm. MCA-PSV exhibited the highest sensitivity (97.8%) in predicting affected fetuses. A multivariable scoring achieved excellent sensitivity (100%) and specificity (84.9%) for disease prediction. CT diameter ratio exhibited slightly outperforming the other techniques. Increased MCA-PSV was the most valuable ultrasound marker. Multivariable scoring surpassed single-parameter analysis in predictive capabilities.

First Family Case of Hemoglobinopathy Titusville in China with Falsely Low SpO2 and Unmeasurable SaO2.

BACKGROUND: Normal hemoglobin is a tetrameric structure, consisting of two alpha-globin chains and two nonalpha (beta, gamma, delta) chains. Hemoglobinopathies occur when the presence of gene mutations affect the molecular structure or expression of the globin chains. METHODS: We reported the case of a 9-year-old Chinese girl who presented with abnormal low oxygen saturation values on pulse oximetry and no oximetry results were obtained during blood gas analysis (BGA). RESULTS: High-performance liquid chromatography (HPLC) and capillary electrophoresis demonstrated that the presence of a low oxygen affinity hemoglobin variant, characterized as hemoglobin Titusville, was proven by gene sequencing. The patient's mother and aunt also carry the hemoglobin variant, representing the first Chinese family case reported. CONCLUSIONS: Hemoglobin Titusville is a rare genetic hemoglobin structural defect. early diagnosis can help patients and clinicians avoid unnecessary anxiety and costly or excessive clinical investigations.

Abnormal hemoglobin anti-Lepore Hong Kong compound with ß0-thalassemia ameliorate thalassemia severity when co-inherited with α-thalassemia.

Abnormal hemoglobin anti-Lepore Hong Kong is a rare ßδ fusion variants resulting from non-homologous crossover during meiosis. Anti-Lepore Hong Kong is known to consistently exhibit significantly increased level of HbA2. In this study, we used multiplex ligation-dependent probe amplification (MLPA) and single molecular real-time (SMRT) sequencing, as well as Sanger sequencing, to identify variants in five unrelated families with abnormal elevated HbA2 level. All probands in these five families were found to be heterozygous for anti-Lepore Hong Kong. Among them, two families showed co-occurrence of ß0-thalassemia and α-thalassemia (-SEA/ or αCSα/). Heterozygotes for anti-Lepore Hong Kong displayed an average HbA2 level of 17.7% and behaved normal. However, when combined with ß0-thalassemia and α-thalassemia, the probands exhibited higher HbA2 level (30.2-40.8%) and behaved with ß-thalassemia trait. Furthermore, determination of the α/ß-mRNA ratio revealed a slight downregulation of ß-globin, similar to that of ß-thalassemia minor. Our study is the first to identify compound heterozygotes for anti-Lepore Hong Kong, ß0-thalassemia and α-thalassemia, provide valuable information for prenatal counseling.

Iron overload in anaemia with underlying haemoglobin constant spring in an antenatal mother in primary care.

This is the case of a gravida 3 para 1 woman in her late 20s with underlying haemoglobin constant spring who visited a healthcare clinic for an antenatal check-up. Towards the end of her second trimester, she experienced lethargy. During her antenatal booking, she was diagnosed with mild asymptomatic anaemia, high serum ferritin, T saturation of 88% and abnormal liver function tests. She was referred to a hospital where an MRI scan revealed over 2 g of iron deposits in her liver, leading to a revised diagnosis of iron overload. Treatment included deferoxamine and expectant management throughout her antenatal period, and her delivery was uncomplicated. While iron deficiency anaemia is common in pregnancy, it is crucial not to overlook iron deposition and the distinction from acute fatty liver during pregnancy to prevent treatment delays.

Hb Hebei [α20 (B1) His→Leu; HBA2:C.62A > T]: A Novel Hemoglobin Variant Found during Measurement of Glycated Hemoglobin.

We report a novel hemoglobin (Hb) variant found in a 34-year-old Chinese male during a routine measurement of glycated hemoglobin. The variant resulted in a P3 peak of 27.5% of the total Hb on high performance liquid chromatography (HPLC) with a glycated hemoglobin mode. However, no abnormal Hb peaks were observed in capillary electrophoresis (CE) with 3.1% Hb A2 and 96.9% Hb A. The amino acid substitution was determined by Sanger sequencing as α20 (B1) His→Leu; the corresponding DNA mutation was identified as CAC > CTC at the first position of codon 20 of the α-chain. This is the first description of the mutation, and we have named it Hb Hebei for the region of origin of the proband.

Fetal hemodynamic changes and mitochondrial dysfunction in myocardium and brain tissues in response to anemia: a lesson from hemoglobin Bart's disease.

OBJECTIVE: Whether or not the effects of anemia in the early phase, while the fetuses attempts to increase cardiac output to meet oxygen requirement in peripheral organs, is detrimental to the fetal developing vital organs is little-known. The objective of this is to compare prenatal cardiovascular changes and post-abortal cellular damages in the myocardium as a pumping organ and the brain as a perfused organ between anemic fetuses (using fetal Hb Bart's disease as a study model) in pre-hydropic phase and non-anemic fetuses. METHODS: Fetuses affected by Hb Bart's disease and non-anemic fetuses at 16-22 weeks were recruited to undergo comprehensive fetal echocardiography. Cord blood analysis was used to confirm the definite diagnosis of fetal Hb Bart's disease and normal fetuses. Fetal cardiac and brain tissues were collected shortly after pregnancy termination for the determination of oxidative stress and mitochondrial function, including mitochondrial ROS production and mitochondrial membrane changes. RESULTS: A total of 18 fetuses affected by Hb Bart's disease and 13 non-anemic fetuses were recruited. The clinical characteristics of both groups were comparable. The affected fetuses showed a significant increase in cardiac dimensions, cardiac function, cardiac output and brain circulation without deteriorating cardiac contractility and preload. However, in the affected fetuses, mitochondrial dysfunction was clearly demonstrated in brain tissues and in the myocardium, as indicated by a significant increase in the membrane potential change (p-value < 0.001), and a significant increase in ROS production in brain tissues, with a trend to increase in myocardium. The findings indicated cellular damage in spite of good clinical compensation. CONCLUSION: The new insight is that, in response to fetal anemia, fetal heart increases in size (dilatation) and function to increase cardiac output and blood flow velocity to provide adequate tissue perfusion, especially brain circulation. However, the myocardium and brain showed a significant increase in mitochondrial dysfunction, suggesting cellular damage secondary to anemic hypoxia. The compensatory increase in circulation could not completely prevent subtle brain and heart damage.

Hb Mizuho Case Report; Early Genomic Testing Facilitates a Life Changing Diagnosis.

Unstable variant hemoglobinopathies are an uncommon cause of hemolysis in the pediatric patient and may cause a delay in diagnosis if there is not a high index of suspicion. Hemoglobin (Hb) Mizuho is a rare unstable hemoglobinopathy caused by a pathogenic variant of the HBB gene with a severe phenotype. Here we report on the first known case of Hb Mizuho in Australia, presenting with features of acute and chronic hemolysis. The morphological features on blood film review, in conjunction with biochemical findings and other clinical features, did not immediately suggest an alternative diagnosis and a Next Generation Sequencing gene analysis approach was taken to investigate genes associated with red blood cell disorders and atypical uremic syndrome. The HBB Mizuho variant was detected and established the diagnosis. This report highlights the challenge of diagnosing Hb Mizuho on conventional testing and the need for early genomic testing to clarify a diagnosis.

Identification of a Novel Variant c.163delG in HBB Gene Resulting in a Beta Null Phenotype in a Proband with Thalassemia Intermedia.

A 21-year-old patient presented with a previous medical history of pallor, mild icterus, increased fatigue, low hemoglobin, and abnormal hemoglobin variant analysis with more than 70 transfusions. He was referred for genetic analysis to identify the pathogenic variations in the ß-globin gene. Sanger's sequencing of the proband and his family revealed the presence of a novel frame shift variant HBB:c.163delG in a compound heterozygous state with hemoglobin E (HbE) (HBB:c.79G > A) variant. The father and the sibling of the patient were found to be normal for the HBB gene. Mother was found to be heterozygous for HbE (HBB:c.79G > A) variant. In silico analysis by Mutalyzer predicted that c.163delG variant generated a premature stop codon after seven codons, leading to a truncated protein. FoldX protein stability analysis showed a positive ΔΔG value of 45.27 kcal/mol suggesting a decrease in protein stability. HBB:c.79G > A is a known variant coding for HbE variant, which results in the reduced synthesis of ß-globin chain and shows mild thalassemia. Combined effect of HBB:c.163delG and HBB:c.79G > A variants in the proband might have led to the reduced synthesis of ß-globin chains resulting in a thalassemia intermedia type of clinical manifestation.

Diagnosis and screening of abnormal hemoglobins.

Hemoglobin (Hb) abnormalities, such as thalassemia and structural Hb variants, are among the most prevalent inherited diseases and are associated with significant mortality and morbidity worldwide. However, there were not comprehensive reviews focusing on different clinical analytical techniques, research methods and artificial intelligence (AI) used in clinical screening and research on hemoglobinopathies. Hence the review offers a comprehensive summary of recent advancements and breakthroughs in the detection of aberrant Hbs, research methods and AI uses as well as the present restrictions anddifficulties in hemoglobinopathies. Recent advances in cation exchange high performance liquid chromatography (HPLC), capillary zone electrophoresis (CZE), isoelectric focusing (IEF), flow cytometry, mass spectrometry (MS) and polymerase chain reaction (PCR) etc have allowed for the definitive detection by using advanced AIand portable point of care tests (POCT) integrating with smartphone microscopic classification, machine learning (ML) model, complete blood counts (CBC), imaging-based method, speedy immunoassay, and electrochemical-, microfluidic- and sensing-related platforms. In addition, to confirm and validate unidentified and novel Hbs, highly specialized genetic based techniques like PCR, reverse transcribed (RT)-PCR, DNA microarray, sequencing of genomic DNA, and sequencing of RT-PCR amplified globin cDNA of the gene of interest have been used. Hence, adequate utilization and improvement of available diagnostic and screening technologies are important for the control and management of hemoglobinopathies.

Extreme Reticulocytosis After Splenectomy in a Patient With Hemoglobin Mizuho.

BACKGROUND: Patients with Hb Mizuho may be splenectomized at a young age to decrease their need for blood transfusions. OBSERVATIONS: Transfusion-dependency decreased dramatically in a 4-year-old white boy with Hb Mizuho after splenectomy. Surprisingly, he developed reticulocytosis (>1000×10 9 /L) with a peak reticulocyte percentage of 49%, and erythrocyte abnormalities, including Heinz bodies, Howell-Jolly bodies, and basophilic stippling. Manual reticulocyte counting and flow cytometric measurement with anti-CD71 antibodies supported a truly elevated reticulocyte count. CONCLUSIONS: We propose possible explanations for the extreme reticulocytosis that arose postsplenectomy and compare the reticulocyte count in the present case with previously published cases.

First report of a patient with homozygous hemoglobin Ernz: Evidence to support a non-pathogenic variant.

Hemoglobin Ernz (Hb Ernz) is a missense variant in ß-globin caused by a Threonine to Asparagine substitution at the 123rd amino acid position and HBB c.371C > A in gene level. Hb Ernz has been classified as Uncertain Significance (VUS) by ACMG due to limited reports and the absence of any homozygote genotypes. In our study, we found eight cases of Hb Ernz by DNA sequencing of the ß-globin gene during >20 years of Thalassemia Screening in individuals with borderline hematological parameters who were possible carriers of thalassemia or their spouses. We also report the first homozygote variant of Hb Ernz. Our findings suggest that the changes in hematological parameters observed in individuals with Hb Ernz are likely due to α-globin gene mutations rather than Hb Ernz itself. These findings support the reclassification of Hb Ernz as a benign variant in variant classification.

The first Chinese with Hb Chile leading to chronic anemia and methemoglobinemia: a case report.

BACKGROUND: Hemoglobin (Hb) Chile [ß28(B10) Leu > Met; HBB: c.85 C > A] is a rare hemoglobin variant caused by a missense mutation in the HBB gene. Only one case of Hb Chile has been reported worldwide so far. It is an unstable hemoglobin, characterized by cyanosis associated with chronic methemoglobinemia and hemolytic anemia induced by sulfonamides or methylene blue. CASE PRESENTATION: A 9-year-3-month-old girl had mild anemia of unknown etiology for more than 6 years. She had a slight pallor without other symptoms or signs. The complete blood count revealed normocytic normochromic anemia with a sometimes-elevated reticulocyte count, and the bone marrow cytology showed marked erythroid hyperplasia, but the tests related to hemolysis were normal. Therefore, the whole exome sequencing was performed and showed a heterozygous mutation for HBB: c.85 C > A. With asymptomatic methemoglobinemia confirmed later, she was eventually diagnosed with Hb Chile. CONCLUSIONS: This is the first report of Hb Chile in China and the second worldwide. This case shows that Hb Chile is clinically heterogeneous and difficult to diagnose and expands our understanding on the clinical and hematological traits of the disease.

A New Hemoglobin Variant: Hb Tangshan [HBA1: c.239C > T, CD79(GCG > GTG)(Ala > Val)] Detected by MALDI-TOF MS.

In this report we decribed a new α-chain variant found during the measurement of hemoglobin A1c (Hb A1c) using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS). MALDI-TOF MS analysis detected an α-chain variant with a mass of 15,155 Da. However, this Hb variant was not detected during Hb A1c measurement by cation-exchange high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) methods. Sanger sequencing validated the presence of a heterozygous missense mutation [HBA1: c.239C > T, CD79(GCG > GTG)(Ala > Val)]. The observed 28 Da mass difference exactly matches the theoretical mass difference (28 Da) resulting from the substitution of alanine (89.079) with valine (117.133). As this represents the initial documentation of the mutation, we named it Hb Tangshan after the proband's residence.

Direct cord blood LAMP colorimetric phenol red assay for detecting α0-thalassemia (SEA deletion); the validation and post-natal screening in Thailand.

Post-natal or newborn screening for thalassemia and hemoglobinopathies is useful for genetic counseling and managing thalassemia in children. We characterized thalassemia genotypes in newborns from the eastern part of Thailand. The results demonstrated a high heterogeneity of thalassemia and hemoglobinopathies with seventeen genotypes. We focused on α0- thalassemia (Southeast Asian [SEA] deletion) in this study. We developed and validated the loop-mediated isothermal amplification (LAMP) colorimetric assay for detecting α0- thalassemia (SEA deletion) using simple direct cord blood sampling compared to genomic DNA. A total of 160 cord blood samples were evaluated with the LAMP assay. The sensitivity and specificity of the LAMP colorimetric assay for α0-thalassemia (SEA deletion) using direct cord blood showed 100% (6/6 x 100) and 98.05% (151/154 x 100) whereas, genomic DNA showed 100% (6/6 x 100) and 100% (154/154 x 100), respectively. Moreover, we demonstrated other simple screening tools for α0-thalassemia with %Hb Bart's, MCV, and MCH values and found that these parameters were not diagnostic in our samples. The direct cord blood with colorimetric LAMP assay is simple, rapid, and does not require a post-LAMP step compared to conventional PCR. These techniques could be applied in post-natal or large population screening for α0-thalassemia (SEA deletion). Finally, this could support early prevention of complications, early management, genetic counseling for α-thalassemia disease in children, or a long-term prevention and control program of severe thalassemia in Thailand.

Single-tube multiplex real-time PCR with EvaGreen and high-resolution melting analysis for diagnosis of α0-thalassemia--SEA,--THAI, and--CR type deletions.

Regions with a high prevalence of α-thalassemia (α-thal) require simple, rapid, and accurate tests for carrier screening and prenatal diagnosis. Diagnosis of multiple deletions in a single tube is necessary to clearly identify individuals with α0-thalassemia in the routine setting, especially in at-risk couples. Therefore, we aimed to develop a single-tube multiplex real-time PCR with EvaGreen and high-resolution melting (HRM) analysis for the identification of α0-thalassemia Southeast Asian (SEA), Thai and Chiang Rai (CR) type deletions. The results of the HRM analysis indicated that the amplified fragments from α0-thal--CR,--THAI,--SEA, and the wild-type α-globin gene had specific peak heights at mean melting temperature (Tm) values of 85.40°C, 86.50°C, 87.65°C, and 91.04°C, respectively. The frequencies of α0-thal--SEA,--THAI,--CR obtained from routine testing in 2,135 samples were 17.89%, 0.19% and 0.19%, respectively. This method would be useful for preventing Hb Bart's hydrops fetalis. Detection of multiple deletions in a single run is cost-effective, highly accurate and timesaving. This technique could enable wider α-thalassemia diagnosis in high prevalence areas and served as an example for thalassemia routine setting.

False HbA1cValue due to a Rare Variant of Hemoglobin J-Cubujuqui.

BACKGROUND: Hemoglobin (Hb) J-Cubujuqui is a rare Hb variant, and reports about it are very limited. There are no descriptions that it affects the results of glycated Hb. METHODS: In this study, we describe a rare variant discovered during newborn screening. Both high-performance liquid chromatography (HPLC) and capillary electrophoresis for hemoglobin analysis displayed abnormal peaks. The Hb variant was confirmed by Sanger sequencing. RESULTS: The pedigree study shows the variant was inherited from the newborn's father. His fasting blood glucose (FBG) level was 5.5 mmol/L. HbA1c measured by HPLC was falsely low in her father (2.41%), whereas that measured by immunoassay was normal (5.11%). Sanger sequencing revealed a heterozygous mutation (CGT˃AGT) at amino acid position 141 of the α1 gene, corresponding to Hb J-Cubujuqui [α1 141(HC3) Arg→Ser (CGT˃AGT); HBA1:c.424C˃A (or HBA2)]. CONCLUSIONS: This is the first report that Hb J-Cubujuqui interferes with the measurement of HbA1cand prompts clinicians to pay attention to the accuracy of glycated Hb results.

Hemoglobin ß-Globin Variants In Hispanic Patients: An Institutional Experience From Dallas, Texas.

Hemoglobinopathies are the most common single-gene disorders in humans. There are 1,424 variants of human hemoglobin described with 951 involving the ß-globin gene. Ancestry and geography play a significant role in the incidence and nature of hemoglobinopathies, with African, Asian, and Mediterranean populations and their descendants being amongst the most affected. Investigation of variants in individuals of Hispanic descent is needed to reflect the changing demographics of the United States. Hemoglobin ß-globin evaluation through gel electrophoresis, high-performance liquid chromatography, and HBB gene sequencing was performed on patients from Texas hospitals between 2010 and 2015 and demographic parameters (age, sex, ethnicity) was subsequently analyzed. A total of 846 patients underwent hemoglobinopathy evaluation. A ß chain variant was detected in 628 of the 846 total patients. Hispanic patients represented 37% (314/846 patients), which were equally distributed between females (50%; 156/314) and males (50%; 156/314). A ß-globin chain variant was found in 67% of Hispanic patients with a distribution across 10 variants seen in greater than 1% of patients. For hemoglobin variants, an understanding of the regional and ethnic prevalence will improve patient care through more effective screening and identification of the variant, early diagnosis, and appropriate treatment if necessary, and better genetic counseling.

Hemoglobin profile and molecular characteristics of the complex interaction of hemoglobin Doi-Saket [α9(A7) asn > lys, HBA2:c.30C > a], a novel α2α1 hybrid globin variant, with hemoglobin E [ß26(B8) Glu > lys, HBB:c.79G > A] and deletional α+-thalassemia in a Thai family.

BACKGROUND: An increasing number of α-hemoglobin (Hb) variants is causing various clinical symptoms; therefore, accurate identification of these Hb variants is important. OBJECTIVE: This study aimed to describe the molecular and hematological characteristics of novel Hb Doi-Saket that gives rise to a typical α+-thalassemia phenotype in carriers with and without other hemoglobinopathies. MATERIALS AND METHODS: Biological samples from a proband and his family members were analyzed. Hematological profiles were analyzed using a standard automated cell counter. Hb was analyzed by capillary electrophoresis and high-performance liquid chromatography. Mutations and globin haplotype were identified by DNA analysis. Novel diagnostic tools based on allele-specific polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism were developed. RESULTS: Hb analysis showed a major abnormal Hb fraction, moving slower than HbA, and a minor Hb fraction alongside HbA2 in the proband, his father, and son. DNA analysis of the α-globin gene identified the -α3.7 deletion and in cis the C > A mutation on codon 9 of the α2α1 gene, corresponding to Hb Doi-Saket [α9(A7) Asn > Lys]. This mutation could be identified using newly developed allele-specific PCR-based assays. The Hb Doi-Saket al.lele was significantly associated with haplotype [- + M + + 0 -]. Interaction of αDoi-Saket with ßE globin chains led to a new Hb variant (HbE Doi-Saket). Phenotypic expression was clinically silent in heterozygotes and might present slight microcytosis. CONCLUSIONS: Hb Doi-Saket emphasizes a great diversity present in α-globin gene. The mutation in this family from Thailand was linked to -α3.7 and caused mild microcytosis in the carriers. The combination of this variant with deletions in α genes might cause a severe clinical phenotype. Different methods of separation can provide useful information in diagnosis, and a complete molecular approach is needed for confirmation before considering patient management.

The Hb Doi-Saket is a novel α-globin variant mutation occurring in the α2-globin gene in cis to the -α^3.7 kb chromosome.The carrier of Hb Doi-Saket may present slight microcytosis and have severe clinical entities when it interacts with deletions in α-globin genes.Hb analysis with the HPLC system could completely separate Hb Doi-Saket and its derivative from other Hbs.