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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Detection of Hb H disease caused by a novel mutation and --SEA deletion using capillary electrophoresis.

BACKGROUND: Hb H disease is a serious type of α-thalassemia which cause moderate anemia while misdiagnosis by routine genetic analysis in a rare or novel Hb H disease. METHODS: The study was done on three patients and one fetus in a suspected Hb H disease family. Hb analysis was carried out using capillary electrophoresis (CE), and hematological analysis was conducted with an automated cell counter. Common α- and ß-thalassemia mutations were detected by routine genetic analysis (gap-PCR and RDB-PCR). Novel mutation diagnostic methods were based on DNA sequencing. RESULTS: Capillary electrophoresis revealed clinical feature of classic Hb H disease in the proband, and hematology analysis showed moderate anemia (Hb 87 g/L). But routine genetic analysis was found that it was only a heterozygote for the --SEA deletion. DNA sequencing of α-globin genes (α1 and α2) identified the breakpoints between nts 34162 and 34171 at α2 gene, named CD 90-93 (-AGCTTCGG) mutation. The genotype of proband and fetus was the same --SEA /-αCD90-93 . His father was homozygous for the novel mutation (-αCD90-93 /-αCD90-93 ), and his mother was heterozygote for the --SEA deletion. CONCLUSIONS: Our study for the first time described the novel mutation CD 90-93 (-AGCTTCGG). CE is a way to avoid misdiagnosis of rare or novel Hb H disease.

Hb H Disease Results from Compound Heterozygosity of - -SEA and -αMAL3.5 in a Chinese Family.

The α+-thal deletion of 3.557 kb (NG_000006.1: g.32745_36301del, -αMAL3.5), involving the entire α2-globin gene, was identified in a Chinese family by multiplex ligation-dependent probe amplification (MLPA) followed by gap-polymerase chain reaction (gap-PCR) and sequencing. The proband, a compound heterozygote for this mutant gene and the Southeast Asian (- -SEA; NG_000006.1: g.26264_45564del19301) deletion, had a phenotype of Hb H disease [hemoglobin (Hb) 7.6 g/dL, mean corpuscular volume (MCV) 60.0 fL, Hb H (ß4) 0.7%, Hb Bart's (γ4) 2.4% and Hb A2 1.1%]; one of her sisters with same genotype showed a similar phenotype. Another two family members, who were carriers of this mutant gene, had a hematological phenotype of a silent α-thal. The 5' and 3' breakpoints of this deletion are located at the Y2 and Y1 boxes, respectively, therefore, it probably originated from an unequal crossover between these two homologous boxes. This mutation constitutes an additional heterogeneous defect causing α-thal in the Chinese population and would be valuable for elucidating the arrangement in the human α-globin gene cluster.

First Report of a Case with Nondeletional Hb H Disease Caused by IVS-I-116 (A>G) of the α2-Globin Gene.

In this report, we describe a prenatal case with cardiomegaly and increased middle cerebral artery-peak systolic velocity (MCA-PSV) at 22 weeks' gestation. Fetal blood sampling revealed moderate anemia (Hb 7.4 g/dL) and increased Hb Bart's (γ4) level (28.2%), indicative of Hb H (ß4) disease. Molecular analysis of the family members revealed that the pregnant woman carried a heterozygous IVS-I-116 (A>G) (HBA2: c.96-2A>G) mutation of α2-globin gene, and the fetus was a compound heterozygote for IVS-I-116 and the Southeast Asian (- -SEA) deletion. This is the first reported case of nondeletional Hb H disease caused by the IVS-I-116 (A>G) mutation associated with fetal anemia identified by ultrasound.

Novel α-Globin Splice Site Mutation (HBA2: c.96-5C>A) in Combination with Three-Gene Deletion Hb H Disease.

The choice of acceptor splice site during exon-exon splicing by the spliceosome is determined by a variety of factors. We report here a family with a novel acceptor splice site variant within intron 1 of the α-globin gene that provides some in vivo insight into the rules governing RNA splicing in homo sapiens. A 2-year-old female with Hb H disease, was found to have not only three α-globin genes deleted (- -FIL/-α3.7) but also a HBA2: c.96-5C>A variant on her remaining α-globin gene. The HBA2: c.96-5C>A variant was in cis with -α3.7 and mRNA studies indicate that this variant creates a new acceptor splice site which is used in approximately 35.0% of α-globin mRNA transcripts. The reduced levels of normal mRNA transcript predicts a more severe Hb H disease than expected for the three-gene deletion Hb H disease with a phenotype similar to nondeletional Hb H disease. We propose that this variant be called Hb Beach Haven (HBA2: c.96-5C>A).

Results of Coexistence of ß-Thalassemia Minor in Hb H Disease Patients.

The aim of this study was to determine the hematological characteristics in a large group of Hb H (ß4) patients with or without a coexisting ß-thalassemia (ß-thal), identified by a thalassemia screening program in mainland China. A total of 361 patients with Hb H disease were found, including 343 with deletional types and 18 with nondeletional types. ß-Thalassemia was found in 28 (7.8%) out of the 361 Hb H cases, and all of the 28 cases had the deletional type of Hb H disease. Lower hemoglobin (Hb) levels were detected in cases with the nondeletional type compared to those in cases with the deletional type. ß-Thalassemia significantly increases the Hb levels in Hb H cases. The Hb H and Hb Bart's (γ4) fractions were visible in 270 (85.7%) and 122 (38.7%) out of 315 deletional type cases, respectively, while no Hb H or Hb Bart's fractions were detectable in 28 deletional type cases with ß-thal. Therefore, the diagnosis of Hb H disease in a ß-thal carrier is challenging. Molecular analysis of α- and ß-globin genes is imperative in all cases with a ß-thal trait.