Development of pre-implantation genetic testing protocol for monogenic disorders (PGT-M) of Hb H disease.

Hb H disease is the most severe form of α-thalassemia compatible with post-natal life. Compound heterozygous α0-thalassemia- SEA deletion/α+-thalassemia- 3.7kb deletion is the commonest cause of Hb H disease in Thailand. Preimplantation genetics testing for monogenic disorders (PGT-M) is an alternative for couples at risk of the disorder to begin a pregnancy with a healthy baby. This study aims to develop a novel PCR protocol for PGT-M of Hb H disease- SEA/-3.7kb using multiplex fluorescent PCR. A novel set of primers for α+-thalassemia- 3.7kb deletion was developed and tested. The PCR protocol for α0-thalassemia- SEA deletion was combined for Hb H disease- SEA/-3.7kb genotyping. The PCR protocols were applied to genomic DNA extracted from subjects with different thalassemia genotypes and on whole genome amplification (WGA) products from clinical PGT-M cycles of the families at risk of Hb Bart's. The results were compared and discussed. The results showed three PCR products from α+-thalassemia- 3.7kb primer set, and three from α0thalassemiaSEA primer set. The results were consistent with the known thalassemia genotypes. The novel -α3.7 primers protocol was also tested on 37 WGA products from clinical PGT-M cycles giving accurate genotyping results and a satisfying amplification efficiency with the ADO rates of 2.7%, 0%, and 0% for HBA2, HBA1, and internal control fragments, respectively. This novel PCR protocol can precisely distinguish Hb H disease- SEA/-3.7kb from other genotypes. Additionally, this is the first PCR protocol for Hb H disease- SEA/-3.7kb which is optimal for PGT-M.

A novel gain-of-function PIP4K2A mutation elevates the expression of ß-globin and aggravates the severity of α-thalassemia.

Haemoglobin H (Hb H) disease (intermediate status of α-thalassemia) shows marked phenotypic variability from asymptomatic to severe anaemia. Apart from the combined ß-thalassemia allele ameliorating clinical severity, reports of genetic modifier genes affecting the phenotype of Hb H disease are scarce which bring inconvenience to precise diagnosis and genetic counselling of the patients. Here, we present a novel mutation (c.948C>A, p.S316R) in the PIP4K2A gene in a female Hb H disease patient who displayed moderate anaemia and a relatively high Hb H level. Haematological analysis in her family members revealed that individuals carrying this mutation have upregulated ß-globin expression, leading to a more imbalanced ß/α-globin ratio and more Hb H inclusion bodies in peripheral red blood cells. According to functional experiments, the mutant PIP4K2A protein exhibits enhanced protein stability, increased kinase activity and a stronger regulatory effect on downstream proteins, suggesting a gain-of-function mutation. Moreover, introduction of the S316R mutation into HUDEP-2 cells increased expression of ß-globin, further inhibiting erythroid differentiation and terminal enucleation. Thus, the S316R mutation is a novel genetic factor associated with ß-globin expression, and the PIP4K2A gene is a new potential modifier gene affecting the α-thalassemia phenotype.

Molecular Basis and Hematologic Phenotype of Hemoglobin H Disease Combined with Two Rare ß-Globin Mutations.

In area where α-thalassemia and ß-thalassemia are prevalent, the coinheritance of hemoglobin H disease (Hb H disease) and ß-thalassemia are not uncommon and could result in complex thalassemia intermedia syndromes. In this study, we investigate the hematological and molecular characteristics of two previously undescribed cases that co-inherited Hb H disease and rare ß-globin gene (HBB) mutations found in Chinese populations. Proband I was a boy with Hb H disease in association with IVS-II-5(G > C) (HBB:c0.315 + 5G > C) mutation. Proband II was a boy with a combination of Hb H and Hb Zengcheng [ß114(G16) Leu > Met; HBB:c.343C > A]. Both of them had mild hypochromic microcytic anemia, and neither had ever received a blood transfusion. In both cases, the level of Hb A2 was within normal range, and no Hb H was detected, but a small amount of Hb Bart's was observed in proband I. Routine DNA analysis detected the deletional Hb H disease in both cases. IVS-II-5(G > C) (HBB:c0.315 + 5G > C) and Hb Zengcheng (HBB:c.343C > A) mutations were found by DNA sequencing of ß-globin gene. The co-inheritance of Hb H disease with rare ß-thalassemia may result in an atypical pattern of Hb H disease, and further investigation of rare genotypes should be conducted to avoid missed diagnosis.

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.

Identification of a Novel Hb H Disease with Glucose-6-Phosphate Dehydrogenase Deficiency Using Whole Genome Sequencing.

With the development of sequencing technology, more and more rare thalassemia types have been found. In this article, we found a novel Hb H disease combined with glucose-6-phosphate dehydrogenase (G6PD) deficiency through whole genome sequencing (WGS), which was verified by Sanger sequencing and polymerase chain reaction (PCR)-reverse dot-blot hybridization, respectively.

A Case of Misdiagnosis Caused by the Coinheritance of Hb G-Siriraj [ß7(A4)Glu→Lys; HBB: c.22G>A] and Hb H Disease.

Despite the fact that most hemoglobin (Hb) variants are clinically and hematologically silent, they can interact with thalassemias, which could sometimes give rise to complicated routine thalassemia diagnostics. Hb G-Siriraj [ß7(A4)Glu→Lys; HBB: c.22G>A] alone is a benign condition, but its coinheritance with α-thalassemia (α-thal) may lead to misdiagnosis. We describe the case of a Chinese woman with an elevated Hb A2 level who was assumed to carry heterozygous ß-thalassemia (ß-thal), but was later shown to be a double heterozygote for Hb G-Siriraj and Hb H disease. This study for the first time described hematological characteristics of a patient with a double heterozygosity for Hb G-Siriraj and Hb H disease. It is of great significance for technicians and clinicians to expand their knowledge as well as to help guide clinical diagnosis, population screening and genetic counseling.

Severe Hb H Disease Caused by Hb Zürich-Albisrieden (HBA1: c.178G>C): Another Case Report.

Hb Zürich-Albisrieden, [α59(E8)Gly→Arg, HBA1: c.178G>C] is a rare and highly unstable α-globin chain variant. The involved mutation has been reported in both HBA1 and HBA2 genes. A few compound heterozygotes of Hb Zürich-Albisrieden and α0-thalassemia have shown that this variant is associated with severe Hb H disease. We describe here another case of Hb Zürich-Albisrieden who presented with transfusion-dependent anemia beginning shortly after birth.

Genetic and non-genetic factors affecting hemoglobin A2 expression in a large cohort of Thai individuals: implication for population screening for thalassemia.

OBJECTIVE: Increased hemoglobin (Hb) A2 level is an important diagnostic marker for ß-thalassemia carrier screening. The level of Hb A2 is also useful for differentiating several thalassemia syndromes. We have examined data bases for reduced Hb A2 expression in a large cohort of Thai subjects. METHODS: A study was done on 1,498 subjects with non-thalassemia and various types of thalassemia and Hb variants to determine the effect of thalassemia genotypes and on 103 women of reproductive age to determine the effect of iron deficiency. Hb analysis was done using capillary electrophoresis, and thalassemia genotypes were defined by DNA analysis. Serum ferritin was measured using chemiluminescent microparticle immunoassay. RESULTS: Subjects were divided into 35 groups based on iron status, Hb, and DNA analysis. Decreased Hb A2 level was observed in those with Hb Q-Thailand, δ-hemoglobinopathies, δß0-thalassemia, Hb Lepore, iron deficiency, α-thalassemia, and especially Hb Constant Spring (Hb CS). While ß-thalassemia carriers with Hb H disease still had elevated Hb A2 levels, most of the ß-thalassemia carriers with Hb H-CS disease had Hb A2 less than 3.5% as a diagnostic cut-off. The lowest Hb A2 level was observed in those with Hb H-CS disease. CONCLUSION: Iron deficiency, Hb CS trait, homozygous Hb CS, and Hb H disease may reduce Hb A2 level, leading possibly to misdiagnosis of ß-thalassemia, especially in carriers with borderline Hb A2. Hb CS showed the strongest effect on Hb A2 expression. Understanding the basis for reduced Hb A2 expression may help reduce the diagnostic pitfalls of ß-thalassemia in the region.

Genetic counseling and prenatal decision for hemoglobin H disease caused by the rare α2 codon 30 (-GAG) (HBA2: c.91_93delGAG) mutation and the SEA deletion: Case series study.

OBJECTIVE: We report a rare mutation on the α2-globin gene, HBA2: c.91_93delGAG and its potential functions. CASE REPORT: We mainly described four patients with hemoglobin (Hb) H disease caused by the rare mutation and the SEA deletion but diversity in clinical presentation. Two had survived to adulthood with normal physical and mental development, except for mild anemia. However, two were children, who had more severe clinical manifestations. One child had developmental disorders of speech and language and mild growth retardation, and the other child suffered from severe hemolytic crises precipitated by infection and received blood transfusion. CONCLUSION: This study is of great significance for clinicians to provide genetic counseling to couples at-risk of having offspring with Hb H disease and let them make the pregnancy decision, particularly reduce the occurrence of severe Hb H disease.

First Report of Nondeletional Hb H Disease Caused by an α2-Globin Gene Mutation: HBA2: c.184A>T.

We report a rare mutation, HBA2: c.184A>T on the α2-globin gene, detected in a Chinese proband who presented with Hb H disease and a mild anemia. This frameshift mutation results in a premature termination of translation at position 61 of the α2-globin gene. Carriers of this mutation showed a borderline microcytic hypochromia. Our study indicates the importance of screening nondeletional α-thalassemia (α-thal) in areas with a particularly high prevalence of thalassemia such as in Southern China, especially for couples with one partner carrying an α0-thal deletion.

Hb Bart's Hydrops Fetalis Syndrome and Hb H Disease Caused by Deletional Chiang Rai (- -CR) α0-Thalassemia in Two Unrelated Thai Families.

α0-Thalassemia (α0-thal) Chiang Rai (- -CR; NC_000016.10: g.144215_188843del) was identified as a novel 44.6 kb deletional type of α-thalassemia (α-thal), removing all α-like globin genes. However, little is known about the deleterious effects of this genetic disorder, particularly when it is combined with other types of thalassemia. We performed molecular analysis of the - -CR deletion using gap-polymerase chain reaction (gap-PCR) in two independent families residing in Phayao and Chiang Mai, Thailand, with an unknown causative mutation for Hb Bart's hydrops fetalis syndrome and Hb H disease. Five out of seven individuals were diagnosed to be heterozygous for the - -CR deletion. Of these, two also carried Hb H disease with compound heterozygosities for - -CR and -α3.7 (rightward) deletions. However, hematological parameters of the - -CR carriers displayed microcytic hypochromic anemia that is comparable to other α0-thal traits. Although the prevalence of - -CR has never been elucidated in a specific population, our study demonstrated that genotyping for - -CR might be considered as an additional investigation for unexplained Hb Bart's hydrops fetal syndrome and Hb H disease.

A Rare Case of Hb H Disease and Systemic Lupus Erythematosus.

Anemia is common in patients with systemic lupus erythematosus (SLE). The association between thalassemia and SLE is rare. In this study, we report the first patient who was found to have a severe hemolytic anemia caused by combination of SLE and Hb H disease. The patient had a more severe presentation in the hematological system. Our case indicates that for a patient who was diagnosed with SLE and developed deterioration in her hematological cell lines, investigation of other possible coexisting causes would be warranted.

Correlation of hepcidin and serum ferritin levels in thalassemia patients at Chiang Mai University Hospital.

Hepcidin is a key iron-regulatory hormone, the production of which is controlled by iron stores, inflammation, hypoxia and erythropoiesis. The regulation of iron by hepcidin is of clinical importance in thalassemia patients in which anemia occurs along with iron overload. The present study aimed to evaluate the correlation between serum hepcidin and ferritin levels in thalassemia patients. This cross-sectional study investigated 64 patients with thalassemia; 16 ß-thalassemia major (BTM), 31 ß-thalassemia/hemoglobin (Hb) E (BE), and 17 Hb H + AE Bart's disease (Hb H + AE Bart's). The levels of serum hepcidin and ferritin, and Hb of the three groups were measured. The median values of serum ferritin and Hb were significantly different among the three groups, whereas serum hepcidin values were not observed to be significantly different. The correlation of the serum hepcidin and ferritin levels was not statistically significant in any of the three groups of thalassemia patients with BTM, BE, or Hb H + AE Bart's (r = -0.141, 0.065 and -0.016, respectively). In conclusion, no statistically significant correlations were observed between serum hepcidin with any variables including serum ferritin, Hb, age, labile plasma iron (LPI), and number of blood transfusion units among the three groups of thalassemia patients. Likely, the regulation of hepcidin in thalassemia patients is affected more by erythropoietic activity than iron storage.

Hb Athens-Georgia (beta 40(C6) Arg > Lys, HBB:c.122G > A) with a single α-globin gene (Hb H disease) in a Thai family: molecular, hematological, and diagnostic aspects.

Interaction of structural hemoglobin (Hb) variants with α- or ß-globin defects are occasional in Southeast Asia. Herein we provide the first description of Hb Athens-Georgia (Hb A-Ga) in association with deletional Hb H disease, a novel combination previously undescribed in the population. Hematological, Hb and DNA analysis, and ß-globin haplotype analyses were performed in seven participants from one ethnic Thai family. Hemoglobin analysis by capillary electrophoresis revealed an abnormal Hb fraction in the proband, his father and grandmother (I-2). DNA sequencing revealed that the G > A substitution at codon 40 of the ß-globin gene was identical to the Hb A-Ga (HBB:c.122G > A). Interestingly, α-thal-1 (SEA deletion) and α-thal-2 (-α3.7 deletion) were identified in the proband resulting in Hb H disease, while α-thal-1 was identified in the father, and no α-thal was observed in I-2. Hematological analysis indicated that the proband (ßA-Ga/ßA, -SEA/-α3.7) had moderate anemia and was markedly hypochromic with microcytic red blood cells (RBCs). The father (ßA-Ga/ßA, -SEA/αα) presented mild microcytic anemia, while normal hematology was observed in the I-2 who was heterozygous for Hb Athens-Georgia (ßA-Ga/ßA, αα/αα). The relative level of Hb A-Ga was distinctly reduced according to the degree of α-globin defects. The developed allele-specific PCR method can successfully be used for confirmation of Hb A-Ga. The Thai Hb A-Ga allele associated with a ß-haplotype [+ - - - - - +]. These findings were in accordance with the previous conclusion that this variant is a non-pathological ß-Hb variant.

Prenatal diagnosis of a rare ß-thalassemia gene －90 (C>T) (HBB: c.-140 C>T) mutation associated with deletional Hb H disease (--SEA /-α4.2 ).

BACKGROUND: Hemoglobin H (Hb H) disease can be caused by compound heterozygosity for two different mutations or from homozygotes for mutations, and conventional genetic methods may lead to misdiagnosis when Hb H disease is combined with a rare ß-thalassemia. METHODS: Hematology parameters and hemoglobin electrophoresis analysis, gap-polymerase chain reaction (gap-PCR) and reverse dot-blot hybridization (RDB-PCR) were employed to identify common α-thalassemia and Hb H disease. Rare ß-thalassemia mutations were detected by DNA sequencing. RESULTS: Hematological analysis and hemoglobin electrophoresis revealed a mild anemia α0 -thalassemia trait (Hb 90 g/L, MCV 71 fL, and MCH 22.7 pg) compound with ß+ -thalassemia trait (MCV 71 fL, MCH 22.7 pg, and HbA2 5.51%) for the pregnant woman. DNA sequencing for the ß-globin gene revealed rare a －90 (C>T) (HBB: c.-140 C>T) mutation for the woman. DNA analysis identified that the fetus inherited the α0 -thalassemia mutation [--SEA (Southeast Asian)] and a rare ß+ -thalassemia mutation －90 (C>T) (HBB: c.-140 C>T) from the mother, and the α+ -thalassemia mutation [-α4.2 (leftward)] from the father. CONCLUSION: We reported a rare －90 (C>T) (HBB: c.-140 C>T) mutation combined with the --SEA /-α4.2 in a family. This finding enriched the mutation spectrum of thalassemia molecular characteristics in China and emphasized the significance in DNA sequencing in mutation screening for the families with thalassemia.

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.

Novel α0-Thalassemia Deletion Identified in an Indian Infant with Hb H Disease.

We report the identification of a large deletion of the α-globin gene cluster, which removed both HBA2 and HBA1 and included the region from HBZ to HBQ1 on chromosome 16 (16p13.3). The α0-thalassemia (α0-thal) deletion was discovered in an Indian family residing in New Zealand. The proband was a 3-month-old female, who presented with a Hb H disease of unknown molecular origin. Routine hematology showed marked hypochromic microcytic anemia, with numerous Hb H inclusion bodies. In the absence of iron deficiency, there was a strong clinical suspicion of α-thal. On initial screening using a multiplex gap polymerase chain reaction (gap-PCR), only the common rightward deletion (-α3.7) was detected. Investigation of the proband's mother and father revealed the mother was heterozygous for the -α3.7 deletion, while none of the seven most common pathogenic α-thal deletions were detected in the father. Multiplex ligation-dependent probe amplification (MLPA) was employed to detect the presence of a novel α0-thal deletion in both the proband and her father. For the proband, the α0-thal deletion in combination with the -α3.7 deletion, eliminated three copies of HBA consistent with a clinical diagnosis of Hb H disease.

Hb Westmead (HBA2: c.369C>G): Hematological Characteristics in Heterozygotes with and without α0-Thalassemia.

Hb Westmead (α122(H5)His>Gln) (HBA2: c.369C>G) is a common α-globin variant causing α-thalassemia (α-thal) in Mainland China. In this study, we report the hematological characteristics in Hb Westmead carriers in a Chinese population. There were 546 individuals carrying Hb Westmead based on their molecular diagnosis: 514 Hb Westmead heterozygotes and 32 compound heterozygotes for Hb Westmead and α0-thal. Compared to common deletional α+-thal, Hb Westmead was associated with higher mean corpuscular hemoglobin (Hb) (MCH) values. Compound heterozygotes for Hb Westmead and α0-thal showed significantly higher Hb, mean corpuscular volume (MCV) and MCH values than subjects with deletional Hb H disease. When compared to α0-thal carriers, compound heterozygotes for Hb Westmead and α0-thal showed similar Hb values, but significantly lower MCV and MCH values. Our results indicate that Hb Westmead is a silent nondeletional α+-thal, with a deficiency of α-globin chain milder than deletional α+-thal, and compound heterozygotes for Hb Westmead/α0-thal have a phenotype similar to simple α0-thal.

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.

Early development of decreased ß-cell insulin secretion in children and adolescents with hemoglobin H disease and its relationship with levels of anemia.

BACKGROUND: Diabetes mellitus (DM) associated with iron overload has been reported among adults with transfusion-dependent thalassemia and those with non-transfusion-dependent thalassemia (NTDT), especially in ß-thalassemia disease. However, little is known about glucose metabolism and how early its dysregulation can develop in α-thalassemia hemoglobin H (Hb H) disease, which is one of the most common types of NTDT worldwide. PROCEDURE: We prospectively calculated glucose metabolism index in 40 patients (aged 10-25 years) with Hb H disease. Glucose metabolism data were compared between patients with deletional versus nondeletional Hb H, and between patients with normal versus abnormal insulin secretion/sensitivity. RESULTS: Despite normal glucose tolerance in all patients, 52.5% had abnormal insulinogenic index indicating decreased ß-cell insulin secretion. Patients with functional hemoglobin < 8 g/dL had significantly higher percentages of abnormal insulinogenic index. There was no significant difference in abnormal insulinogenic index between deletional and nondeletional Hb H. CONCLUSION: Decreased ß-cell insulin secretion is highly prevalent among children and adolescents with Hb H disease, and it is associated with levels of functional anemia at baseline, but not with the type of Hb H disease. This result warrants heightened awareness among hematologists due to potentially increased risk of DM later in life.