Molecular Analysis of Turkish β-Thalassemia Heterozygotes with Normal Hb A2 Levels

Four parents of three unrelated families who are obligatory β-thalassemia heterozygotes and two parents with Hb Knossos are presented. In these subjects, although the red blood cell counts and red cell indices were compatible with β-thalassemia trait, the Hb A₂ values were between 1.9–2.9 % of the total hemoglobin. Examination of the δ-globin gene by Southern blot, restriction endonuclease analysis, and by direct sequencing of amplified DNA revealed the presence of the (δ?) -7.2 kb Corfu type deletion, the (δ⁺) codon 27 (G→T) and (δ?) IVS-I-2 (T→C) mutations in trans or in cis with a severe β-thalassemia allele, and the (δ?) codon 59 (-A) deletion in cis with the β^Knossos allele.     

δ-Thalassemia with Complete Absence of Hb A2 in a Chinese Family

A Chinese family with δ-thalassemia (δ-thal) was found, in which the daughter is homozygous for δ-thal (HBD: c.-127T>C) with complete deficiency of Hb A₂ and the mother is a heterozygote with low level of Hb A₂. The father, however, is a heterozygote with a normal Hb A₂ value due to coinheritance of a β-thalassemia (β-thal). Although no abnormal clinical or hematological findings were noted in the individuals with δ-thal, one should keep in mind that β-thal can be missed during routine preliminary screening when β-thal and δ-thal coexist in a subject.     

A Novel Variant with Positive Natural Selection Influenced Hb A2 Levels in Chinese Individuals with β-Thalassemia

β-Thalassemia (β-thal) is the most common inherited hemolytic anemia worldwide. Elevated Hb A₂ is a mark of β-thal carriers. The aim of this study was to identify the pathogenic variants associated with the Hb A₂ levels. One thousand and thirty β-thal carriers were recruited for this study. Using positive natural expression quantitative trait loci (eQTL) analysis, a significant variant was selected. Genotyping for the rs231841 polymorphism was performed by the Sequenom MassARRAY IPLEX platform. All genetic association analyses were performed with the PLINK program. The linear regression analysis showed that rs231841 in the intron region of the potassium voltage-gated channel subfamily Q member 1 (KCNQ1) gene on chromosome 11p15 was significantly associated with Hb A₂ levels. The presence of the C allele was associated with elevated Hb A₂ levels. Our results suggest that rs231841 on the KCNQ1 gene with positive natural selection is related to Hb A₂ levels in Chinese β-thal carriers, and KCNQ1 is probably associated with the expression of the β-like globin gene cluster.     

High Hb A2 β-Thalassemia Due to a 468 bp Deletion in a Patient with Hb S/β-Thalassemia

We describe a case of Hb S/β-thalassemia (thal) involving a 468 bp deletion that removes the β-globin gene promoter but leaves the coding regions intact. This is the second report of this deletion, and our family study establishes that this deletion causes β⁰-thal with unusually high levels of Hb A₂ and Hb F. As with other genotypes involving deletions of the 5′ region of the β-globin gene, our patient had a mild form of Hb S/β-thal.     

Hb A2/E Levels Found in Co-Inheritance with the α-Thalassemia-1 – –SEA/ Type Deletion and Either Hb E or β-Thalassemia

The α-thalassemia-1 (α-thal-1) Southeast Asian (– –^SEA) type deletion, β-thalassemia (β-thal) and Hb E [β26(B8)Glu→Lys, GAG>AAG] are the most common genetic disorders in Southeast Asian populations. Mean corpuscular volume (MCV) <80.0 fL with normal hemoglobin (Hb) is used for screening α- and β-thal, and a Hb E level of less than 25.0% is used for predicting α-thal-1 in Hb E trait. Thus, levels of Hb, MCV and Hb A₂/E were reviewed and compared between the SEA type deletion co-inherited with β-thal trait (n = 61), with Hb E trait (n = 102) or homozygous Hb E (n = 13) and β-thal trait (n = 636), Hb E trait (n = 544) or homozygous Hb E (n = 83), respectively. When comparing the values of all three analyzed hematological parameters, only the – –^SEA/β^E values were shown to be significantly lower than those of Hb E trait. Furthermore, at a cut-off value of Hb A₂/E of 21.54%, 95.0% of the – –^SEA/β^E had Hb A₂/E levels lower than this cut-off value, while 94.0% of Hb E trait had Hb A₂/E at higher levels. Accordingly, the Hb A₂/E level at 21.54% is the best indicator for predicting co-inheritance of the α-thal-1 – –^SEA/ deletion and Hb E trait.     

Association of β-Thalassemia and Hb Q-Thailand Resulting in a Normal Hb A2 Value

We describe a Chinese woman who was assumed to be a Hb Q-Thailand heterozygote, but was later also shown to have β-thalassemia (β-thal) with a normal amount of Hb A₂.     

Hb A2-Tianhe (HBD: c.323G>A): First Report in a Chinese Family with Normal Hb A2-β-Thalassemia Trait

Coinheritance of δ-globin variants along with β-globin gene defects can interfere with correct diagnosis of β-thalassemia (β-thal) trait. In this report, we present the coinheritance of a δ-globin variant, Hb A₂-Tianhe [δ107(G9)Gly→Asp; HBD: c.323G>A] and a heterozygous β-thal in a Chinese individual with microcytosis, hypochromia and a normal Hb A₂ level.     

Complex Interaction of Hb Q-Thailand with α0- and β0-Thalassemia in a Chinese Family

Hb Q-Thailand [α74(EF3)Asp→His (α1); HBA1: c.223?G>C] is an abnormal hemoglobin (Hb), variant found mainly in China 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 a hitherto undescribed condition of patients with a double heterozygosity for Hb Q-Thailand with α⁰-thalassemia (α⁰-thal) and in combination with β⁰-thalassemia (β⁰-thal) in a Chinese family. Our study will provide some clinical manifestations, laboratory diagnosis and genetic counseling for complex hemoglobinopathies.     

Hb A2 Hong Kong – A Novel δ-Globin Variant in a Chinese Family Masks the Diagnosis of β-Thalassemia Trait

A 42-year-old Chinese woman (FP) was the mother of a patient with β-thalassemia major (β-TM) due to a compound heterozygosity for β⁰-thalassemia (β⁰-thal) mutations. She was also found to have a low Hb A₂ level of 1.6% by high performance liquid chromatography (HPLC) despite being a heterozygous carrier of the codons 41/42 (–TCTT) (HBB:c.126_129delCTTT) β⁰-thal mutation. Doubling the amount of hemolysate loaded for chromatography revealed a widened Hb A₂ peak and raised the level to 4.1%, consistent with β-thal trait. Direct nucleotide sequencing detected a novel δ-globin gene mutation at codon 29 (HBD:c.89G>A), which leads to a glycine to aspartic acid substitution. A homologous mutation at codon 29 in the β-globin gene [Hb Lufkin or β29(B11)Gly→Asp] has been reported in Black families. This report highlights the importance of genotype-phenotype correlation and the potential pitfall of relying on Hb A₂ level for phenotypic diagnosis of β⁰-thal trait.     

A β-Thalassemia Trait with Two Mutations in Cis in a Chinese Family

Abstract

A female of Chinese origin carried the codon 43 (G>T) (HBB: c.130G?>?T) and codons 71/72 (+A) (HBB: c.216_217insA) mutations of the β-globin gene in cis, identified during prenatal thalassemia screening. The double in cis mutations were inherited from her mother. Both of the two carriers behave as a traditional heterozygote for β-thalassemia (β-thal) with microcytosis and a high Hb A₂ level. This case report indicates that the possibility of multiple mutations in cis in a fetus with thalassemia trait has to be considered in a prenatal screening program.     

Molecular Analysis of γ-Globin Promoters,HS-111 and 3′HS1, in β-thalassemia Intermedia Patients Associated with High Levels of Hb F

The nucleotide (nt) variations in the promoter region of the γ-globin genes, HS-111 and 3′HS1 regions, were studied in Iranian patients with β-thalassemia intermedia (β-TI), β-thalassemia major (β-TM) and healthy individuals. Of the five nt variations at the 5′ end of the ^Aγ-globin gene, ?369 (C>G), ?611 (?T) and ?603/604 (GA>AG) were found in all samples, whereas ?588 (A>G) and ?AAGC at ?222 to ?225 were found at different frequencies in the studied groups. Therefore, the ?369, ?611 and ?603/604 variations were considered common mutations in this population, and the difference with respect to the ?AAGC deletion was not significant.

However, the A allele of the ?588 variation and [+] allele of the XmnI polymorphism were more frequent in β-TI patients, especially those who had the IVS-II-1(G>A)/IVS-II-1(G>A) genotype. The + allele of XmnI also had complete correlation with the A allele of ?588 variation. The HS-111 (?21 A) variation also showed association with β-TI patients who had high levels of Hb F. Bearing in mind that the ?588 variation lies within the postulated adult-specific silencer region and that the majority of β-TI patients had allele A, then it can be envisaged that this allele could have a role in altering the repressor function at this region. Therefore, the A allele of ?588, [+] allele of XmnI and HS-111 (?21 A) variation are useful genetic markers to differentiate between β-TM and β-TI patients. However, these nt changes alone may not be the only elements raising the level of Hb F, other regulatory and modifying factors also play a role in Hb F production.     

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

Abstract

The capillary electrophoresis (CE) system allows the quantification of Hb Bart’s (γ4) and Hb H (β4) that is used for screening of Hb H disease. However, Hb Bart’s hydrops fetalis and Hb H are not always codetected in patients with Hb H disease. In this study, 35 samples were analyzed for the α⁰-thalassemia (α⁰-thal) [–?–^SEA (Southeast Asian) and –?–^THAI (Thailand)] deletions and the α⁺-thal [–α^3.7 (rightward) and –α^4.2 (leftward)] type deletions using real time-polymerase chain reaction (real time-PCR) with SYBR Green1 and high-resolution melting (HRM) analysis and conventional gap-PCR techniques, respectively. Results showed that 28 of 29 (96.6%) samples with the Hb A₂-Hb H phenotype on CE electrophoregrams presented the genotype of –?–^SEA/–α^3.7, while the –?–^SEA/–α^4.2 made up the remainder. The –?–^SEA/–α^3.7 genotype was also found in all six samples (100.0%) with Hb A₂-Hb Bart’s on CE electrophoregrams. Thus, for genetic counseling, prevention and control programs of Hb Bart’s hydrops fetalis and Hb H disease, α-thal genotype analysis is required.     

Genetic Variants at BCL11A and HBS1L-MYB loci Influence Hb F Levels in Chinese Zhuang β-Thalassemia Intermedia Patients

Increased Hb F levels can ameliorate the symptoms of β-thalassemia (β-thal). Due to the genetic heterogenicity of β-thal, the relationship between genetic variants in modifier genes and Hb F level has been studied in different populations. The Chinese Zhuang has the second largest population in China and has 6.78% prevalence of β-thal. However, the effects of these single nucleotide polymorphism (SNP) variants on the Hb F levels of β-thal intermedia (β-TI) patients in this population have not been reported. To explore the association between modifier loci (β-globin gene cluster, HBS1L-MYB intergenic region and BCL11A) and Hb F levels in Chinese Zhuang β-TI patients, 96 unrelated β-TI patients (50 males and 46 females) with different Hb F levels were recruited and genotyped by mass spectrometry. A total of 13 SNPs were confirmed to be in a significant relationship with Hb F levels in this population. Of these, high-risk genotypes of six Hb F-associated SNPs, rs9376090, rs7776054, rs9399137, rs9389268, rs9402685 in the HBS1L-MYB intergenic region and rs189984760 in the BCL11A locus, showed association with high Hb F levels, especially for SNPs in linkage disequilibrium. One novel Hb F-associated SNP, rs189984760, was identified in our study. Our findings will be of valuable reference for correlation between modifier genes and Hb F in Chinese Zhuang populations and may lead to better understand the modifying mechanisms for β-thal.     

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.     

Hb J Baltimore (β 16 (A13) Gly ↣ Asp) in Association with β-Thalassemia in a Sicilian Family

Study of the Hpa I polymorphism 3' to the β-globin gene in the Greek population revealed absence of the site in 238 β^S chromosomes, in contrast to a much larger sample of chromosomes carrying the β^A gene, where this site was consistently positive. Subsequent haplotype analysis of the β-globin gene cluster in 82 β^S chromosomes demonstrated that 79 (96%) belonged to haplotype #19, while the three exceptions (all Hpa I negative) could be explained by a δ-β recombination event. Haplotype #19 was never encountered in a parallel study of the 83 β^A chromosomes. Comparison of the above results with similar surveys in other parts of the world and consideration of various historical events suggest that the β^S mutation was introduced into Greece over the last few centuries by the Saracen raids and/or by settlements of North African slaves brought in by the Arabs, Franks, Venetians, or Ottoman Turks, who have occupied the country over the last millennium.     

Diagnosis of Compound Heterozygous Hb Tak/β-Thalassemia and HbD-Punjab/β-Thalassemia by HbA<Subscript>2</Subscript> Levels on Capillary Electrophoresis

A misdiagnosis of β-thalassemia carrier in samples with Hb Tak and HbD-Punjab, the β-variants, can be a cause of inappropriate genetic counseling thus having a new case of β-thalassemia major. A capillary electrophoresis (CE) is very efficient in separating and quantifying HbA₂. In this study, HbA₂ levels of samples which were doubted for compound heterozygous Hb Tak/β-thalassemia or heterozygous HbD-Punjab/β-thalassemia were measured and compared between CE and high performance liquid chromatography (HPLC). The molecular confirmation for Hb Tak, HbD-Punjab and β-thalassemia codons 17 (A > T), 41/42 (-TCTT), 71/72 (+A) and IVSI-nt1 (G > T) mutations and 3.4 kb deletion were also performed. Based on DNA analysis, 3 cases were diagnosed as compound heterozygous Hb Tak/β-thalassemia and one for HbD-Punjab/β-thalassemia. The elevated HbA₂ levels were found in all 4 samples with rages of 4.6–7.3% on CE while those were not found on HPLC. Thus, the elevated HbA₂ measured by CE can be used as a screening parameter for differentiating the homozygote of Hb Tak and HbD-Punjab from the compound heterozygote of these hemoglobinopathies and β-thalassemia.     

High Ascitic Fluid Leptin Levels in Patients with Decompensated Liver Cirrhosis and Sterile Ascites: Relationship with TNF-α Levels

Leptin is an adipocyte-derived hormone involved in the homeostasis of body composition. An imbalance in leptin regulation has been observed in patients with liver cirrhosis. We aimed to assess serum and ascitic leptin levels in a group of patients with decompensated liver cirrhosis and to evaluate the relationship of these levels with tumor necrosis factor alpha (TNF-alpha). We assessed both serum and ascitic fluid leptin levels in a series of 16 consecutive patients with liver cirrhosis. We calculated the body mass index (BMI) and assessed body fat (BF) of all patients by means of bioelectric impedence analysis. Leptin levels were analyzed in relationship to biochemical indexes, TNF-alpha levels, and body composition. None of the patients had spontaneous bacterial peritonitis. Both serum and ascites leptin levels were correlated with BMI and BF. On average, ascitic fluid leptin levels (13.1 +/- 10.9 ng/ml) were twice as high as serum levels (7.0 +/- 6.4 ng/ml), and the ascitic fluid/serum ratio of leptin was > 1 in all patients. Serum and ascites leptin levels were positively correlated (rS = 0.675, P = 0.009), while no correlation was observed between leptin and TNF-alpha levels, both in serum and in ascites. Serum and ascites TNF-alpha were not correlated. The ascitic fluid leptin levels of cirrhotic patients with sterile ascites are on average two times higher than circulating levels of this hormone. Noteworthily, they correlate significantly with body composition. These findings seem to suggest that in patients with decompensated liver cirrhosis, intraabdominal production of leptin may contribute to the metabolic picture.     

Hb Lepore-Leiden: A New δ/β Rearrangement Associated with a β-Thalassemia Minor Phenotype

The Lepore hemoglobins (Hbs) are a group of structural defects resulting from different recombination events between the δ- and β-globin genes. They may come with different β-thalassemia (β-thal) minor-like phenotypes in the carrier and with variably severe phenotypes in the rare homozygote, and in the common compound heterozygote with β-thal. The most seriously affected patients are those of Yugoslavian origin presenting with severe transfusion-dependent hemolytic anemia, dyserythropoiesis, hepatosplenomegaly and skeletal malformations. Because of genetic risk, couples where both partners are carriers of these combinations may require prognosis and prenatal diagnosis. In these cases, recognition of the defect must be done with particular care. We report a case of Hb Lepore induced by a yet unknown crossover event found in a 24-year-old Turkish male and compare the novel mutation with those previously reported.