Complex Interaction of Hb Q-Thailand (HBA1: c.223G>C) with β-Thalassemia/Hb E (HBB: c.79G>A) Disease

Hb Q-Thailand [α74(EF3)Asp→His (α1), GAC>CAC, HBA1: c.223G>C] is an abnormal hemoglobin (Hb) frequently found in Thailand and Southeast Asian countries. The association of the α^Q-Thailand allele with other globin gene disorders has important implications in diagnosis. Here, we report how to diagnose the coinheritance of Hb Q-Thailand with β-thalassemia (β-thal)/Hb E disease in four Thai samples from high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) testing results. Understanding of the HPLC chromatogram and CE electropherogram patterns of this complex mutation is important for interpretation of testing results and providing genetic counseling.     

Molecular Analysis of β-Thalassemia Patients: First Identification of Mutations HBB:c.93-2A>G and HBB:c.114G>A in Brazil

The various clinical phenotypes in β-thalassemias have stimulated the study of genetic factors that could modify the manifestations of these diseases. We examined 21 patients with β-thalassemia (β-thal) in order to identify some genetic modifying factors: β-thalassemia mutations, HBG2:g.–158C>T polymorphism, α-globin gene deletions and (AT)xNz(AT)y motif within the hypersensitive site 2-locus control region (HS2-LCR). In the 42 alleles analyzed, the most frequent mutations observed were HBB:c.92+6T>C (30.9%), HBB:c.118C>T (16.7%), HBB:c.93-21G>A (11.9%) and HBB:c.92+1G>A (4.8%); this finding is in accordance with previous data of the Brazilian population. The other genetic factors analyzed showed no relation with the severity of the disease. For the first time in Brazil, we report HBB:c.93-2A>G and HBB:c.114G>A mutations on the β-globin gene, both in a heterozygous state. This is also the first study to analyze the HS2-LCR in β-thalassemic individuals in the Brazilian population.     

Hb Midnapore [β53(D4)Ala→Val; HBB: c.161C>T]: A Novel Hemoglobin Variant with a Structural Abnormality Associated with IVS-I-5 (G>C) (HBB: c.92+5G>C) Found in a Bengali Indian Family

We describe a novel C>T substitution at codon 53 of the HBB gene (HBB: c.161C>T). The proband was a transfusion-dependent β-thalassemia major (β-TM) patient. DNA was extracted and subsequently, DNA sequencing was done to detect the mutations on the HBB gene. Capillary zone electrophoresis (CZE) revealed the presence of an unknown peak. She inherited this mutation from her grandmother through her mother. This mutation exists in cis with the common β⁰ mutation IVS-I-5 (G>C) (HBB: c.92+5G>C). The proband is homozygous for HBB: c.92+5G>C and needs monthly transfusions. On the other hand, her grandmother, mother and sister all possess this novel mutation cis with the heterozygous HBB: c.92+5G>C. They are carriers not thalassemic. This mutation produces the substitution β53(D4)Ala→Val; HBB: c.161C>T, a new structural hemoglobin (Hb) variant. As this variant was identified in a Bengali family from Paschim Midnapore district of West Bengal, India, it has been designated as Hb Midnapore. This variant has now been reported to the HbVar database.     

Severe α-Thalassemia Intermedia Due to a Compound Heterozygosity for the Highly Unstable Hb Adana (HBA2: c.179G>A) and a Novel Codon 24 (HBA2: c.75T>A) Mutation

We report a novel mutation at codon 24 of the α2-globin gene (HBA2: c.75T?>?A) found in a Sundanese family. This novel mutation was detected during prenatal diagnosis. The couple already had a 7-year-old boy who exhibited clinically severe α-thalassemia intermedia (α-TI), and he was found to be a compound heterozygote for the novel mutation at codon 24 and the previously described Hb Adana (HBA2: c.179G?>?A) at codon 59 of the α2-globin gene. The father was a carrier of the novel point mutation and showed normal hemoglobin (Hb) and a low mean corpuscular volume (MCV) and mean corpuscular Hb (MCH) value.     

A Unique Interaction of IVS-I-1 (G>A) (HBA2: c.95+1G>A) with Hb Adana (HBA2: c.179G>A) Presenting as Transfusion-Dependent α-Thalassemia

Abstract

Nondeletional α-globin mutations are known to cause more serious clinical effects than deletional ones. A rare IVS-I-1 (G>A) (HBA2: c.95+1G>A) donor splice site mutation interferes with normal splicing of pre mRNA and results in activation of a cryptic splice site as well as a frameshift mutation. Hb Adana [HBA2: c.179G>A (or HBA1)] is a highly unstable variant hemoglobin (Hb) resulting from a mutation at codon 59 on the HBA2 or HBA1 gene, recognized to cause severe α-thalassemia (α-thal) syndromes. We report a unique case of compound heterozygosity for these two mutations in a 9-year-old boy who presented with a Hb level of 5.3?g/dL and hepatomegaly at the age of 15?months. He required regular blood transfusions in view of a Hb level of <7.0?g/dL and failure to thrive. He had thalassemic red cell indices and peripheral blood film. The Hb electrophoresis only showed a raised Hb F level (3.3%) and a pre run peak but the Hb H inclusion test was negative. His father had thalassemic red cell indices but a normal Hb level. His mother had almost normal Hb levels and red cell indices. Hb Adana involving the HBA2 gene was detected by mutiplex amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) in the proband and his father. DNA sequencing of the HBA2 gene confirmed the IVS-I-1 mutation in the proband and his mother. This case highlighted the unique interaction of the IVS-I-1 mutation with Hb Adana in a young Malay boy presenting with transfusion-dependent α-thal.     

Severe Thalassemia Caused by Hb Zunyi [β147(HC3)Stop→Gln; HBB: c.442T>C)] on the β-Globin Gene

Hemoglobinopathies are caused by genetic defects on the globin genes. To date, more than 900 β-globin variants have been recorded worldwide. These gene alterations often cause either a decrease in β-globin synthesis or completely block synthesis, leading to a hemoglobinopathy. While most of these causative mutations are inherited, de novo mutations are quite rare. Here, we investigated three hemoglobinopathy cases. These patients developed severe hemolytic anemia at 3-5?months of age and were transfusion-dependent. In patient 1, a novel β variant, Hb Zunyi [β147(HC3)Stop→Gln; HBB: c.442T>C] was identified. This de novo mutation results in a stop codon substitution to a glutamine residue at codon 147 of the β-globin gene, and leads to severe thalassemia. In patient 2, we discovered the rare Hb Southampton mutation [β106(G8)Leu→Pro; HBB: c.320T>C], while in patient 3, the rare Hb Alesha mutation [β67(E11)Val→Met (GTG>ATG); HBB: c.202G>A] was detected. The identification of the novel β variant, Hb Zunyi, has added to the human globin database and will shed light on future diagnosis of hemoglobinopathy/thalassemia and genetic counseling.     

Two new hemoglobin variants: Hb Aix-Les-Bains [β5(A2)Pro→Leu; HBB:c.17 C>T] and Hb Dubai [α122(H5)His→Leu (α2); HBA2:c.368 A>T

We report two new hemoglobin (Hb) variants; one causing an impairment of the N-terminal glycation of the β-globin chain and the other a hematological phenotype of α-thalassemia (α-thal). The first variant is Hb Aix-les-Bains [β5(A2)Pro→Leu] and the second Hb Dubai [α122(H5)His→Leu (α2)]. These two new Hb variants were detected by chromatographic and electrophoretic methods and characterized by molecular studies. Hb Dubai gives an α-thalassemic phenotype and should be routinely detected for preventing severe Hb H disease in couples at-risk for α-thal.     

A New δ Chain Variant,Hb A2-Tunis [δ46(CD5)Gly → Glu; HBD: c.140G>A], Observed in a Tunisian Family in Association with a Compound Heterozygosity for Hb C [β6(A3)Glu → Lys; HBB: c.19G>A] β0-Thalassemia [IVS-I-1 (β143, G>A); HBB: c.92+1G>A]

We describe a new δ-globin variant, Hb A₂-Tunis [δ46(CD5)Gly?→?Glu; HBD: c.140G>A]. This hemoglobin (Hb) variant displayed a faster electrophoretic mobility than normal Hb A₂ and was expressed at 3.2%. The molecular defect was characterized by DNA sequencing analysis. Hb A₂-Tunis was found in a carrier of a β⁰-thalassemia (β⁰-thal) [IVS I-1 (β143, G>A); HBB: c.92?+?1G>A] and Hb C [β6(A3)Glu?→?Lys; HBB: c.19G>A], presenting with a normal Hb A₂ level. Phenotype and genotype investigations revealed that the patient has a total Hb A₂ level of 7.1% that was expected for a β-thalassemia (β-thal) minor carrier.     

New Insights on β-Thalassemia in the Palestinian Population of Gaza: High Frequency and Milder Phenotype Among Homozygous IVS-I-1 (HBB: c.92+1G>A) Patients with High Levels of Hb F

β-Thalassemia (β-thal) is a very common disease in the Palestinian population of the Gaza Strip. We studied their mutation frequency and clinical features. Thirteen different mutations were identified. The most common mutation was IVS-I-1 (G>A) (HBB: c.92+1G>A), which was prevalent in 31.5% of the thalassemia alleles studied. The IVS-I-110 (G>A) (HBB: c.93?21G>A) mutation was found in 25.0% of the alleles. Homozygotes for the IVS-I-1 mutation had higher mean hemoglobin (Hb) levels, required less blood transfusions, and lower transferrin saturation than the homozygotes for the IVS-I-110 mutation. This milder phenotype was, most likely, the result of the persistent production of Hb F; it was 9-fold higher in absolute terms (g/dL) and 7.7-fold higher in relative terms (percentage of total Hb). About half of our IVS-I-1 patients carried the XmnI polymorphism, which is known to be associated with elevated Hb F levels.     

Molecular analysis of β-thalassemia patients: first identification of mutations HBB:c.93-2A>G and HBB:c.114G>A in Brazil

The various clinical phenotypes in β-thalassemias have stimulated the study of genetic factors that could modify the manifestations of these diseases. We examined 21 patients with β-thalassemia (β-thal) in order to identify some genetic modifying factors: β-thalassemia mutations, HBG2:g.-158C>T polymorphism, α-globin gene deletions and (AT)xNz(AT)y motif within the hypersensitive site 2-locus control region (HS2-LCR). In the 42 alleles analyzed, the most frequent mutations observed were HBB:c.92+6T>C (30.9%), HBB:c.118C>T (16.7%), HBB:c.93-21G>A (11.9%) and HBB:c.92+1G>A (4.8%); this finding is in accordance with previous data of the Brazilian population. The other genetic factors analyzed showed no relation with the severity of the disease. For the first time in Brazil, we report HBB:c.93-2A>G and HBB:c.114G>A mutations on the β-globin gene, both in a heterozygous state. This is also the first study to analyze the HS2-LCR in β-thalassemic individuals in the Brazilian population.     

Hb Nouakchott [α114(GH2)Pro→Leu; HBA1: c.344C>T], A Second and Third Case Described in Two Unrelated Dutch Families

We report two families, members of which are carriers of a hemoglobin (Hb) variant previously described as Hb Nouakchott [α114(GH2)Pro→Leu; HBA1: c.344C>T; p.Pro115Leu]. In the first family of Dutch origin, the proband, a 32-year-old male and his 65-year-old father, were both carriers of Hb Nouakchott. Of the second family we tested, only the proband, a 56-year-old Dutch female was a Hb Nouakchott carrier. Hematological analyses of these cases showed the anomaly behaves as a silent Hb variant without clinical consequences. The Hb variant remained unnoticed using high performance liquid chromatography (HPLC), while an additional peak was detected by capillary electrophoresis (CE). These independent findings of Hb Nouakchott indicate that this Hb variant might not be very rare, but simply remains under diagnosed depending on the Hb separation technique used.     

Compound Heterozygosity for Silent Cap +1570 (T>C) (HBB: c*96T>C), Codon 39 (C>T) (HBB: c.118C>T) and the Presence of αααanti–3.7/αα in Greece. A Case Presentation

Abstract

The rare point mutation Cap +1570 (T>C) (HBB: c*96T>C) has been reported in families of Czech, Greek, Turkish and Italian origin. The mutation contributes to a reduction of the β-globin chain synthesis, and in heterozygous carriers, it causes a silent phenotype, while in compound heterozygosity with severe β-thalassemia (β-thal) mutations, it leads to a non transfusion dependent β-thal intermedia (β-TI) state. We report a case of compound heterozygosity for codon 39 (C>T) (HBB: c.118C>T) and Cap +1570, in addition to the presence of ααα^anti–3.7/αα.     

Hb Tianshui (HBB: C.119A > G) in Compound Heterozygosity with Hb S (HBB: C.20A > T) from Odisha,India

We describe here a rare β-globin gene variant, Hb Tianshui [β39(C5)Glu→Arg; HBB: c.119A?>?G], detected during routine screening in Odisha, India. This is the second report of Hb Tianshui and the first to describe the cation exchange high performance liquid chromatography (HPLC) and DNA studies of two cases of this variant. Both cases had coinherited Hb S (HBB: c.20A?>?T) but none presented with typical symptoms of sickle cell disease. One of the cases was heterozygous for a common α-thalassemia (α-thal) allele (?α^3.7) (rightward) (NG_000006.1: g.34164_37967del3804) and marginally raised Hb F percentage, while the other Hb S/Hb Tianshui case was completely benign and healthy. An atypical Asian Indian haplotype [+???+???+] could be assigned to the Hb Tianshui variant. Hb Tianshui seems to mimic a few other Hb variants in cation exchange HPLC. However, we report two specific patterns in the chromatograms that are characteristic to Hb Tianshui. Combining an alkaline electrophoresis result with cation exchange HPLC at screening would be preferred to detect this rare variant, especially in regions with considerable frequency of Hb E [β26(B8)Glu→Lys; HBB: c.79G?>?A] or Hb S.     

Hb Hornchurch [β43(CD2)Glu→Lys; HBB: c.130G>A] Compromises the Molecular Diagnosis of β-Thalassemia in a Chinese Family

The combination of β-thalassemia (β-thal) and a hemoglobin (Hb) variant is not uncommon in regions with a high prevalence of thalassemia. Although most of the β-globin chain variants will not aggravate the β-thal, some can compromise the accurate molecular diagnosis. In this study, we present a rare case of coinheritance of β-thal and Hb Hornchurch [β43(CD2)Glu→Lys; HBB: c.130G>A], that compromises the molecular diagnosis of homozygous β-thal.     

Characterization of the IVS-II-821 (A>C) (HBB: c.316-30A>C) Mutation in a β-Thalassemia Phenotype in Iran

β-Thalassemia (β-thal) is the most frequently observed hereditary blood disorder that results from genetic defects causing deficient synthesis of hemoglobin (Hb) polypeptide chains. Detecting thalassemia mutations are necessary for prenatal diagnosis (PND) programs leading a better quality of life for the patients, as well as a reduction in the cost of their medical care. There are more than 900 different genomic mutations of the β-globin gene described in the human hemoglobin variant (HbVar) database. In this study, we identified a mid-intronic mutation at IVS-II-821 (A>C) (HBB: c.316-30A>C) position in the HBB gene of an Iranian proband and two of her siblings that was associated with β-thal clinical features. Direct DNA sequence analysis was performed by mutation scanning of the β-globin gene. Based on the observed β-thal phenotype and bioinformatics analysis results, we concluded that this β-globin gene mutation was associated with a mild phenotype of β-thal through activating potential splice sites by creating exonic splicing enhancers (ESEs), exon-identity element (EIE) and exonic splicing regulatory sequences (ESRs) sites.     

A new β chain hemoglobin variant with increased oxygen affinity: Hb Santa Giusta Sardegna [β93(F9)Cys→Trp; HBB c.282T>G

During a screening program for the identification of β-thalassemia (β-thal) carriers in Sardinia, Italy, we identified two subjects with increased hemoglobin (Hb) levels and an abnormal Hb variant. The same variant was detected in a family member. DNA sequencing revealed a TGT > TGG mutation at codon 93 of the β-globin gene. Structural analysis demonstrated that the cystine residue at position 93 of the β chain was substituted by tryptophan. Since this amino acid substitution had not yet been reported, we designated this variant Hb Santa Giusta Sardegna for the place of birth of the subjects. This amino acid substitution occurs at the tyrosine pocket of the β chain as well as at the α1β2/α2β1 contact of the quaternary structure of the molecule. The presence of this Hb in the hemolysate causes an increased oxygen affinity, a slightly reduced Bohr effect and a reduced heme-heme interaction (n(50), Hill's constant) in comparison with those of Hb A.     

Hb Alcorn County: A β-Globin Variant [β40(C6)Arg→Thr; HBB: c.122G>C (p.Arg41Thr)] with Increased Oxygen Affinity

Abstract

We describe Hb Alcorn County, a heterozygous hemoglobin (Hb) variant, in a 6-month-old Hispanic male and his mother. DNA sequencing demonstrated a mutation on the HBB gene [β40(C6)Arg→Thr; HBB: c.122G>C (p.Arg41Thr)], predictive of a substitution of arginine to threonine at position 40 of the β-globin protein. This amino acid substitution involves the α1β2 contact and occurs at the same position as Hb Austin [β40(C6)Arg→Ser; HBB: c.[123G>C or 123G>T] (p.Arg41Ser)] and Hb Athens-GA [β40(C6)Arg→Lys; HBB: c.122G>A (p.Arg41Lys)], both of which show increased oxygen affinity.     

Hb Agenogi [β90(F6)Glu→Lys (GAG>AAG) HBB: c.271G>A)] in a Pregnant Thai Woman

Hb Agenogi [β90(F6)Glu→Lys (GAG>AAG) HBB: c.271G>A)] is a very rare β-globin chain variant. We report for the first time this hemoglobinopathy in a pregnant 20-year-old Thai woman. She was seen by an obstetrician at her 14th week of gestation. She was pale and had an inflammatory lesion of her lower left leg. The hemoglobin (Hb) analysis by high performance liquid chromatography (HPLC) and low pressure liquid chromatography (LPLC) showed a peak of abnormal Hb at the C window. On capillary electrophoresis (CE), the abnormal Hb peak was observed at electrophoretic zone 4 that corresponded to the Hb E (HBB: c.79G>A) peak. Direct DNA sequencing revealed a GAG>AAG mutation at codon 90 of the β-globin gene. Thus, even though Hb Agenogi is very rare, it can be found in Thai people. The knowledge and understanding of this hemoglobinopathy will be used to assist in diagnosis, management and counseling for patients.     

Compound Heterozygote for a Novel Elongated C-Terminal β-Globin Variant (HBB: c.364delG) and Hb E (HBB: c.79G>A) with Heterozygous α-Thalassemia-2

This study reports the case of 2-year-old Northeastern Thai girl with β-thalassemia (β-thal) disease who has required regular blood transfusions since she was 8?months old. Hemoglobin (Hb) analysis by high performance liquid chromatography (HPLC) separated Hb A₂/E (16.5%), Hb F (22.7%), Hb A (51.8%) and an abnormal peak (Hb X) found at a retention time (RT) of 5.05?min. (C-window) with 2.8%. Multiplex gap-polymerase chain reaction (gap-PCR) revealed heterozygous α-thalassemia-2 (α-thal-2) (–α^3.7/αα; NG_000006.1: g.34164_37967 del3804). This patient was suspected of having a β-globin chain variant and Hb E (HBB: c.79G>A) according to the high Hb F level and disease presentations. Surprisingly, Hb Mahasarakham (the geographic origin of the proband), a novel single nucleotide deletion (–G) at the first nucleotide of codon 121 (HBB: c.364delG), was identified by direct DNA sequencing and secondary confirmation by PCR-restriction fragment length polymorphism (PCR-RFLP). This novel mutation causes a frameshift mutation and added 10 more residues to the β-globin chain that was elongated to 156 amino acids. Molecular basis of this novel mutation in the heterozygous state is required to confirm the mode of inheritance.