Alpha-globin Gene Triplication Research Articles

A-145 Development of a Novel Algorithm Using Red Blood Cell Indices for Screening of Alpha Globin Gene Deletions

Abstract Background Distinguishing between different causes of microcytic anemia is essential for management of anemic patients. More than 20 different RBC indices have been used to screen thalassemia trait with inconsistent results. Differentiation between iron deficiency anemia (IDA) and alpha-thalassemia is challenging based on RBC indices alone. Moreover, thalassemia and IDA may coexist. A highly sensitive screening algorithm is needed to select clinical cases for alpha globin gene analysis to reduce unnecessary testing and improve pre-test probabilities. Methods A retrospective study was performed to evaluate the reliability of 22 RBC indices (RBC, MCV, RDW, MCH, Mentzer Index, RDW Index, etc) and formulae in differentiating cases with 1–2 alpha globin gene deletions from negative cases, in a cohort of 90 individuals with confirmatory alpha globin gene analysis results. Results The number of patients with each alpha globin gene deletion pattern were: 37 patients with alpha-thalassemia trait, 21 silent carriers (one gene deletion), 31 patients with negative result and 1 patient with triplicated alpha globin gene. The mean RBC count in each group were 4.79 ± 0.65 (trait), 4.45 ± 0.73 (carrier), and 4.21 ± 0.84 (negative) (million/mm). The mean MCV in each group were 71.1 ± 4.8 (trait), 76.1 ± 5.9 (carrier), and 74.2 ± 9.3 (negative) (fL). The mean RDW in each group were 16.5 ± 3.0 (trait), 17.2 ± 3.4 (carrier), and 18.3 ± 4.9 (negative) (%). The mean MCH in each group were 22.0 ± 1.8 (trait), 23.8 ± 2.3 (carrier), and 23.5 ± 3.7 (negative) (pg). None of the RBC indices showed significant difference between carrier and negative groups, while 20 RBC indices showed significant difference (P < 0.05) between trait and negative groups. The sensitivities of the RBC indices in detecting 1–2 alpha globin gene deletions based on published cut-offs ranged between 3.4% and 81.0%, while the specificities ranged between 41.9% and 100%. A novel scoring algorithm was evaluated based on the combination of 12 selected RBC indices and formulae, with each index or formula contributing to a score of 0 or 1. The average value of the negative cases was used as the score cut-off value for each index or formula. The scoring algorithm gave a score distribution between 0 and 12, with lower scores indicating alpha globin gene deletions. It achieved a sensitivity of 96.6% in detecting 1–2 alpha globin gene deletions, with a negative likelihood ratio of 0.15. The negative likelihood ratio of the scoring algorithm outperformed each of the individual RBC indices at similar levels of sensitivities (>95%). Conclusion The novel algorithm is a highly sensitive tool for screening of alpha globin gene deletions, including one gene deletion. It is more effective at identifying negative cases compared to individual RBC indices. Implementation of the algorithm could efficiently reduce 10.0% (9/90) of the test volume of alpha globin gene analysis. A more stringent version of the algorithm that incorporates standard deviation in the cut-off values could potentially achieve nearly 100% sensitivity and reduce 5.6% (5/90) of the alpha globin gene analysis test volume.

Development of a Novel Algorithm Using Red Blood Cell Indices for Screening of Alpha Globin Gene Deletions

Abstract Distinguishing between different causes of microcytic anemia is essential for management of anemic patients. More than 20 different RBC indices have been used to screen thalassemia trait with inconsistent results. Differentiation between iron deficiency anemia (IDA) and alpha-thalassemia appears to be more challenging than differentiation between IDA and beta-thalassemia, based on RBC indices alone. Moreover, thalassemia and IDA may coexist. A highly sensitive screening algorithm is needed to select clinical cases for alpha globin gene analysis to reduce unnecessary testing and improve pre-test probabilities. A retrospective study was performed to evaluate the reliability of 22 RBC indices (RBC, MCV, RDW, MCH, Mentzer Index, RDW Index, etc) and formulae in differentiating cases with 1-2 alpha globin gene deletions from negative cases, in a cohort of 90 individuals with confirmatory alpha globin gene analysis results. The number of patients with each alpha globin gene deletion pattern were: 37 patients with alpha-thalassemia trait, 21 silent carriers (one gene deletion), 31 negative patients and 1 patient with triplicated alpha globin gene. The mean RBC count in each group were 4.79 +/- 0.65 (trait), 4.45 +/- 0.73 (carrier), and 4.21 +/- 0.84 (negative) (million/mm). The mean MCV in each group were 71.1 +/- 4.8 (trait), 76.1 +/- 5.9 (carrier), and 74.2 +/- 9.3 (negative) (fl). The mean RDW in each group were 16.5 +/- 3.0 (trait), 17.2 +/- 3.4 (carrier), and 18.3 +/- 4.9 (negative) (%). The mean MCH in each group were 22.0 +/- 1.8 (trait), 23.8 +/- 2.3 (carrier), and 23.5 +/- 3.7 (negative) (pg). None of the RBC indices showed significant difference between carrier and negative groups, while 20 RBC indices showed significant difference (p<0.05) between trait and negative groups. The sensitivities of the RBC indices in detecting 1-2 alpha globin gene deletions based on published cutoffs ranged between 3.4% and 81.0%, while the specificities ranged between 41.9% and 100%. A novel scoring algorithm based on the combination of 12 RBC indices and formulae achieved a sensitivity of 96.6% in detecting 1-2 alpha globin gene deletions, with a negative likelihood ratio of 0.15. The negative likelihood ratio of the scoring algorithm outperformed each of the 12 RBC indices at similar levels of sensitivities (>95%) based on revised cutoffs. The algorithm is a highly sensitive tool for screening of alpha globin gene deletions, including one gene deletion. It is more effective at identifying negative cases compared to individual RBC indices. Implementation of the algorithm could efficiently reduce 10.0% (9/90) of the test volume of alpha globin gene analysis. A more stringent version of the algorithm could achieve 100% sensitivity and reduce 5.6% (5/90) of the alpha globin gene analysis test volume.

The Use of Next-generation Sequencing in the Diagnosis of Rare Inherited Anaemias: A Joint BSH/EHA Good Practice Paper.

The Use of Next-generation Sequencing in the Diagnosis of Rare Inherited Anaemias: A Joint BSH/EHA Good Practice Paper.

Alpha-globin gene triplication and its effect in beta-thalassemia carrier, sickle cell trait, and healthy individual.

The genotype and phenotype correlation between coinheritance of heterozygous beta‐thalassemia with the alpha‐globin triplication is unclear. In this study we have investigated and reviewed alpha triplication frequency in beta‐thalassemia carriers, sickle cell trait, and healthy individuals and its effect on hematological and phenotypical changes. In this study, 4005 beta‐thalassemia carriers, 455 sickle cell trait, and 2000 healthy individuals were included. Molecular characterization of beta and alpha‐thalassemia was performed. The frequencies of alpha‐globin triplication in beta‐thalassemia carriers, sickle cell trait, and healthy individuals were 67 (1.67%), 4 (0.88%), and 18 (0.9%), respectively. In total, the frequency of alpha‐triplications is approximately 89 (1.39%) in Khuzestan province, South of Iran population. We have compared the average hematological parameters of beta‐thalassemia carriers, sickle cell trait, and healthy individuals with and without alpha gene triplication.This mutation did not show any significant effect on the change of blood indices, neither in healthy individuals nor in sickle cell trait and beta‐thalassemia carriers. Therefore, there is no need to take more notice of anti 3.7 mutation in beta‐thalassemia carriers is opposed with some studies reported that the presence of excess alpha‐globin genes in beta‐thalassemia carriers can lead to the phenotype of beta‐thalassemia intermedia. Therefore, not every individual with triplicated alpha globin coinherited with beta‐thalassemia trait will have a significantly lower Hb than normal, and it is highly likely that none of them will need transfusion.

Coinheritance of Triplicated Alpha-Globin Gene and Beta-Thalassemia Mutations in Adulthood: Ten Years of Referrals in Northern Greece.

Greece is a country of ~11 million people, where hemoglobinopathies are the most common genetic diseases. The reported data describe the clinical phenotype of cases with coinheritance of triplicated α-globin (anti-α3.7 kb) and β-globin gene mutations in Northern Greece, that were referred within the last 10 years, in The Adult Thalassemia Unit of "Hippokration" Hospital, Thessaloniki, Northern Greece. The description of specific genotypes of the β-globin gene mutations in coinheritance with the triplicated α-globin gene (anti-α3.7 kb) and correlation with the hematologic and clinical data in adulthood may be useful in the evaluation of pediatric patients' prognosis and in genetic counseling of couples at risk.

The phenotypic and molecular diversity of hemoglobinopathies in India: A review of 15 years at a referral center

The hemoglobinopathies pose a significant health burden in India. Apart from the β thalassemias and sickle cell disorders, α thalassemias and structural hemoglobin variants are also common. Here we have reviewed the phenotypic and molecular diversity of hemoglobinopathies encountered at a referral center in western India over a period of 15years. Screening for hemoglobinopathies was done using HPLC and cellulose acetate electrophoresis. Molecular characterization was done using Covalent Reverse Dot Blot Hybridization (CRDB), Amplification Refractory Mutation System (ARMS), GAP PCR and direct DNA sequencing. The study includes 31075 individuals who were referred for diagnosis of hemoglobinopathies and prenatal diagnosis. Of these 14423 individuals showed various hemoglobin abnormalities. Beta genotyping in 5615 individuals showed the presence of 49 β thalassemia mutations. 143 β thalassemia heterozygotes had normal or borderline HbA2 levels. We identified three δ gene mutations (HbA2 Pellendri, HbA2 St.George, HbA2 Saurashtra) in β thalassemia heterozygotes leading to normal HbA2 levels. The commonest defects among the raised Hb F determinants were Gγ(Αγδβ)0 Indian inversion and the HPFH-3 Indian deletion. A total of 312 individuals showed the presence of α thalassemia, of which 12.0% had a single α gene deletion (-α/αα). HbH disease was identified in 29 cases with 10 different genotypes. Alpha globin gene triplication was seen in 2.1% of β thalassemia heterozygotes with a thalassemia intermedia phenotype. Seven unusual α chain variants and eight uncommon β chain variants were identified. The repertoire of molecular defects seen in the different globin genes will be valuable for management and control of these disorders both in India as well as in other countries where there is a huge influx of migrant populations from India.

Phenotypes of Hb Okayama Mutation

Introduction:Since its first detection in the year of 1983 very little has been reported about Hb Okayama mutation. Hb Okayama, reported as a silent mutation (Globin Gene Server Hb Var ID: 220, dbSNP rs713040), happened to be detected in the process of HPLC analysis for the measurement of HbA1c in Japanese diabetic patient [1,2,3]. Till date only few Hb Okayama has been reported from Japanese and Austrian ethnicity [2,3,4]. Its phenotypes and co-inheritance with other beta globin gene mutation is not yet known. In this study we report phenotypes of Hb Okayama in heterozygous as well as compound heterozygous state when it is co-existing with a common beta globin gene mutation. To the best of our knowledge no such study has been reported in world literature.Methodology:Two groups of patients- beta carriers requiring blood transfusion; and patients with low MCV, MCH but normal in HPLC were further investigated by molecular analysis. ARMS PCR analysis for beta globin gene mutation detection, GAP PCR for alpha deletion and triplication analysis and DNA sequencing (ABI 3500 Genetic Analyzer) to identify rare beta mutations were carried out.Result:Among around 300 patients subjected to molecular investigation over last three years there were 118 cases of thalassaemia trait (by HPLC and confirmed to carry a heterozygous common beta mutations by ARMS PCR analysis), but were requiring blood transfusion or behaving like intermedia. They were subjected to alpha globin gene triplication analysis by GAP PCR. There were 84 cases found to carry absence of alpha triplication with heterozygous beta mutation. Beta globin gene sequencing analysis of these 84 patients revealed that there were 4 patients carrying Okayama heterozygous mutation [ Figure 1, Figure 2 ] along with a common heterozygous beta gene mutation ( IVS 1-5 G>C mutation). HPLC report of these patients carrying Hb Okayama along with IVS 1-5 mutations in compound heterozygous state showed increased HbA2 values like that of beta trait [Table 1 ]. Another group of patients with low MCV, MCH but Normal in HPLC were subjected to alpha deletion analysis after ruling out low Ferritin level. There were 72 patients showing absence of alpha deletion by GAP PCR analysis and carried low MCV, MCH value with Normal HPLC parameters were also subjected to beta globin gene sequencing which revealed the presence of Hb Okayama mutation among two patients in heterozygous state [Figure- 1, Figure 2], who had HbA2 values in normal or border line range with low MCV, MCH levels [Table-2].Discussion:HbA2 measurement is used as the marker for screening of beta trait. Silent beta-thalassaemia carriers represent normal HbA2 level which makes their identification difficult. Okayama mutation or Hb Okayama is known to be a silent mutation [1-4] with normal HPLC. Hb Okayama is structural beta variant with a change of amino acid ( His > Gln) at Codon 2 (CD 2). Change in the nucleotide sequence at 70603 position from T to A or C (CAT>CAA or CAG) of beta globin gene (NG_000007.3) causes this mutation. The phenotypic associations of Hb Okayama in thalassaemia have very little been known till date. There has been report of very high expression of HbF(70%) value in HPLC resulting from compound heterozygous mutations one of which being silent (Cap+1) [ 5] ; where as there are also reports where the phenotypes of compound heterozygous including a silent mutation showing normal HPLC parameters [ 6 ]. However, in those studies no information about the clinical history and blood transfusion is described. In this study Hb Okayama heterozygous co-inheriting with IVS1-5(G>C) heterozygous mutation showed beta trait like HPLC parameters though all the patients carrying these compound heterozygous mutations required blood transfusion. The silent feature of Hb Okayama was evident in the case of the patients carrying only Hb Okayama mutation who showed absolutely normal HPLC parameters with low MCV, MCH and none of them requiring blood transfusion.Conclusion:Beta globin gene expression analysis to understand the association of Hb Okayama mutation in heterozygous and compound heterozygous states will enable to explain the mechanism of its phenotypes. How this mutation interferes with the expression of HbF or switching of delta globin gene is also to be understood as the HbF levels in HPLC was found to be like beta traits in this study. DisclosuresNo relevant conflicts of interest to declare.

Challenges in prenatal diagnosis of beta thalassaemia: couples with normal HbA2 in one partner.

To undertake β-genotyping in couples having normal/borderline HbA2 levels in one partner to offer the possibility of prenatal diagnosis of thalassaemia. A total of 967 couples were screened for β-thalassaemia. Haematological analysis was carried out on a Sysmex K-1000 analyser. The HbA2 and HbF levels were measured by High Performance Liquid Chromatography-Variant II analyser (Bio-Rad, USA). β-globin gene analysis was done by reverse dot blot hybridization, amplification refractory mutation system or DNA sequencing. Alpha globin gene triplication was determined by Multiplex PCR. In 33 of 967 couples, one partner had a normal/borderline HbA2 level (1-3.5%); however a β-thalassaemia mutation could be identified in 24 of these individuals. Molecular analysis of the β-globin gene revealed the presence of the capsite +1 (A → C) [HBB: c.-50 A → C] mutation in 15 cases (60%), Poly A(T → C) [HBB: c.*110 T → C] mutation in two cases (8%), IVS 1-5 (G → C) [HBB: c. 92 + 5 (G → C)] mutation in four cases (17%) and the CD 15 (G → A) [HBB: c. 47 G → A] mutation, CD 16 (-C) [HBB: c. 51 del C] mutation and CD 30 (G → C) [HBB: c. 93 G → C] mutation in one case each (4%). Alpha gene triplication was found in five cases, while four cases remained uncharacterized. β-genotyping should always be done in a couple if one partner is a β-thalassaemia carrier irrespective of the RBC indices and HbA2 levels of the other partner.

Diverse phenotypes and transfusion requirements due to interaction of β-thalassemias with triplicated α-globin genes.

Co-inheritance of triplicated α-genes can alter the clinical and hematological phenotypes of β-thalassemias. We evaluated the phenotypic diversity and transfusion requirements in β-thalassemia heterozygotes, homozygotes, and normal individuals with associated α-gene triplication. Clinical and hematological evaluation was done and the β-thalassemia mutations characterized by a covalent reverse dot blot hybridization/amplification refractory mutation system. Alpha-globin gene triplication was assessed by multiplex PCR. During the last 2.5 years, 181 β-thalassemia patients and β-thalassemia carriers with an unusual clinical presentation were referred to us for screening for the presence of associated α-globin gene triplication. Twenty-nine of them had associated α-gene triplication (3 β-thalassemia homozygotes or compound heterozygotes and 26 β-thalassemia heterozygotes). One β-thalassemia compound heterozygote [IVS 1-5 (G → C) + CD 41/42 (-CTTT)] was anemic at birth and required blood transfusions unusually early by 6 weeks of age. The second patient (4.5 years) was also clinically severe and became transfusion dependent in spite of having one mild β-thalassemia mutation [Capsite +1 (A → C)]. The third case (3.5 years) who was homozygous for a mild β-gene mutation [-88 (C → T)] with α gene triplication was untransfused. The 26 β-thalassemia heterozygotes with associated triplicated α-genes presented variably, with a β-thalassemia intermedia-like presentation. While screening the family members of all these cases, we found another 10 β-thalassemia heterozygotes and 9 normal individuals with α-globin gene triplication; however, all of them were asymptomatic. Beta-thalassemia carriers, homozygotes, and compound heterozygotes with an unusual presentation should be screened for the possible presence of associated α-globin gene triplication which could influence the clinical and hematological presentation.

Use of HbA2 As a Discriminator for S/Beta Thalassaemia in a Nigerian Setting,

Abstract 4206An integral part of an EU-UNDP funded pilot sickle cell screening project, was the installation, in Abuja (Federal Capital Territory) Nigeria, June 2010, of a High Performance Liquid Chromatography (HPLC) instrument. Prior to the installation, haemoglobinopathy screening was carried out using only unstained paper electrophoresis. Minor bands were difficult to visualise and the proportions of haemoglobins were not measured. Full blood count (FBC) data was also not routinely available. Therefore awareness of both alpha and beta thalassaemia was low as was the implications of coinheritance of beta thalassaemia with haemoglobin (Hb) S.Stratified community surveys were carried out with samples collected as Guthrie card blood spots; children aged less than 6 months, and whole blood; children aged between 6 months and 5 years. Blood spot samples were analysed using newborn sickle cell screening reagents and whole blood samples using beta thalassaemia short reagents, the later allowing accurate Hb A2 and Hb F measurement. Samples were processed on a Biorad™ Classic (instrument and reagents, Biorad, Hercules, CA.). Reporting algorithms were defined for both reagent sets. Using the beta thalassaemia reagents, Hb S/beta thalassaemia was considered when Hb S was predominant, Hb A absent or significantly reduced and the Hb A2>5.Over 10,000 samples were analysed, 410 had sickle cell disease, of which 370 were transported to London for further analysis, 70 of these had an Hb A2>5%, mean and range 5.9(5.1 – 9.1)%. To validate the algorithm 31 samples (Hb A2 3.5 – 7.4%), were selected for beta gene sequencing (Mai et al, 2004) and/or PCR analysis for the 7 common alpha gene deletions (3.7kb, 4.2kb, SEA, MED, THAI, FILL, 20.5kb and triplicated alpha gene locus, anti 3.7kb), (Chong et al, 2000, Wang et al, 2003). Five samples had an Hb A2of 3.5 – 4.9%, 2 with Hb A present (23 and 25%), all were Hb SS, 4 negative for alpha thalassaemia deletions and 1 heterozygous for the alpha 3.7kb deletion. Sixteen samples had an Hb A2 of 5.0 – 5.9%, all were Hb SS, 5 negative for alpha thalassaemia deletions and 11 heterozygous for the alpha 3.7kb deletion. Seven samples had an Hb A2 of 6.0 – 6.9%, 6 were Hb SS, 1 negative for alpha thalassaemia deletions, 1 heterozygous and 4 homozygous for the alpha 3.7kb deletion. One, Hb A2 6.9%, was a compound heterozygote for Hb S/ beta zero codon 106/107(+G) mutation. Three samples had an Hb A2 of 7.0 – 7.4%, review of chromatograms indicated that all showed poor chromatography due to lack of separation between Hb A2 and Hb S or shoulders to the left of the Hb A2 peak. All were Hb SS and heterozygous for the alpha 3.7kb deletion, with 1 also positive for the triplicated alpha globin gene, anti 3.7kb. Four other samples with Hb A2 >7% also showed poor chromatography, but were insufficient for molecular testing.The results indicate that Hb A2values >5% were mainly due to co-existing alpha thalassaemia with 76% of those tested, positive for the 3.7kb deletion. Poor chromatography was also a contributor particularly at the higher Hb A2 levels. During the initial stages of the project air conditioning failure caused major temperature fluctuations on overnight runs, this problem was resolved, improving chromatography. One case of S/beta zero thalassaemia was detected confirming the presence in this population. The results suggest that the Hb A2 may be considered as a discriminator for S/beta thalassaemia screening in this setting, increasing the algorithm Hb A2 level to >6.0% may improve specificity and reduce the number of false positives, this warrants further investigation. Future aims of the project for children over 6 months of age, include initiating routine FBC analysis and testing parental samples as a cost effective means of confirming suspected cases. Disclosures:No relevant conflicts of interest to declare.

Adaptive functional divergence among triplicated alpha-globin genes in rodents.

The functional divergence of duplicated genes is thought to play an important role in the evolution of new developmental and physiological pathways, but the role of positive selection in driving this process remains controversial. The objective of this study was to test whether amino acid differences among triplicated alpha-globin paralogs of the Norway rat (Rattus norvegicus) and the deer mouse (Peromyscus maniculatus) are attributable to a relaxation of purifying selection or to a history of positive selection that has adapted the gene products to new or modified physiological tasks. In each rodent species, the two paralogs at the 5'-end of the alpha-globin gene cluster (HBA-T1 and HBA-T2) are evolving in concert and are therefore identical or nearly identical in sequence. However, in each case, the HBA-T1 and HBA-T2 paralogs are distinguished from the third paralog at the 3'-end of the gene cluster (HBA-T3) by multiple amino acid substitutions. An analysis of genomic sequence data from several rodent species revealed that the HBA-T3 genes of Rattus and Peromyscus originated via independent, lineage-specific duplication events. In the independently derived HBA-T3 genes of both species, a likelihood analysis based on a codon-substitution model revealed that accelerated rates of amino acid substitution are attributable to positive directional selection, not to a relaxation of purifying selection. As a result of functional divergence among the triplicated alpha-globin genes in Rattus and Peromyscus, the red blood cells of both rodent species contain a mixture of functionally distinct alpha-chain hemoglobin isoforms that are predicted to have different oxygen-binding affinities. In P. maniculatus, a species that is able to sustain physiological function under conditions of chronic hypoxia at high altitude, the coexpression of distinct hemoglobin isoforms with graded oxygen affinities is expected to broaden the permissible range of arterial oxygen tensions for pulmonary/tissue oxygen transport.

Coinheritance of the different copy numbers of α-globin gene modifies severity of β-thalassemia/Hb E disease

beta-Thalassemia/Hb E patients show a range of clinical severities, from nearly asymptomatic to transfusion-dependent thalassemia major. This study investigated the clinical heterogeneity and hematologic parameters obtained in the large cohort of 925 Thai beta 0-thalassemia/Hb E patients. Coinheritance of alpha-thalassemia with beta 0-thalassemia/Hb E produces a milder clinical phenotype in contrast to an interaction of alpha-globin gene triplication in severe thalassemia. The mean steady-state Hb was also higher, whereas the mean corpuscular volume and the percentage of Hb F were markedly lower in the former group. This finding demonstrates that the genetic combination leading to the more/less degree of alpha- to non-alpha-globin chains imbalance is indeed the cause of the severe/mild thalassemia phenotype.

α‐Haemoglobin stabilising protein is a quantitative trait gene that modifies the phenotype of β‐thalassaemia

It has been suggested that altered levels or function of alpha-haemoglobin stabilising protein (AHSP), an erythroid-specific protein that binds specifically to free alpha-(haemo)globin, might account for some of the clinical variability in beta-thalassaemia. To assess the variation of AHSP expression, mRNA levels in circulating reticulocytes of 103 healthy individuals were measured by quantitative reverse transcription-polymerase chain reaction. AHSP expression varied up to threefold, and did not correlate with age or sex. A systematic survey of the AHSP locus identified eight sequence variants, of which six were common. Four common variants, including the longer homopolymer (T18) in the putative promoter, are strongly associated with AHSP expression. Reporter assays in K562 cells showed that the activity of the shorter (T15) reporter was relatively lower than that of the T18 reporter. In a study of nine anaemic patients who were heterozygous for beta-thalassaemia and also heterozygous for the triplicated alpha-globin gene (alpha alpha alpha/alpha alpha), frequency of the shorter homopolymer was higher than expected. AHSP expression is variable, with cis control accounting for some of its variance. In some families, the subtle altered levels in AHSP related to the AHSP genotype appears to be a relevant contributory factor in the haematological phenotype.

Jaundice and alpha gene triplication in beta-thalassemia: Association or causation?

There are few studies investigating alpha globin gene triplications in β-thalassemia in Asian Indians and its effect on phenotype, which was the primary aim of this study. Gap-PCR was performed in order to detect common alpha thalassemia determinants (−α3.7, −α4.2 and αααanti 3.7 triplication). Alpha-triplication was detected in 15.4% (10/65) of patients with thalassemia intermedia, 8.8% (4/45) of those with thalassemia minor and in 2.7% (2/74) of healthy controls. The severity of jaundice was higher in thalassemia intermedia cases with alpha-triplication and two of the alpha-triplication cases had a marked increase in serum bilirubin following intercurrent illness. Thus, alpha globin gene triplication is important genetic determinant underlying thalassemia intermedia in North Indians. Patients with α-triplication may develop prominent jaundice with marked increase in serum bilirubin following antecedent aggravating factors.

Clinical phenotype of triplicated α-globin genes and heterozygosity for β0-thalassemia in Chinese subjects

The presence of extra copies of alpha-globin gene has been shown to worsen the degree of anemia in beta-thalassemia heterozygotes. We describe the clinical phenotype of eight Chinese subjects with heterozygosity for both triplicated alpha-globin gene and a beta0-thalassemia allele. They were identified through genotyping of beta-thalassemia intermedia and major patients, and through community-based thalassemia screening program in Hong Kong. Standard molecular techniques were used in the determination of genotype. All subjects in this series showed five copies of alpha-globin genes (alphaalphaalpha/alphaalpha) in association with a beta0-thalassemia allele. Although genotypically identical, six subjects showed a beta-thalassemia intermedia phenotype while two were clinically indistinguishable from beta-thalassemia minor, implying the presence of genetic modifying factors that remained undefined. Triplication of alpha-globin gene and heterozygosity for beta0-thalassemia accounted for 15% of beta-thalassemia intermedia patients at our locality and was associated with a mild clinical phenotype. This genotype was not found among beta-thalassemia major patients. They presented in adulthood and were usually not transfusion dependent. When compared with simple beta-thalassemia heterozygotes, they showed obvious red cell abnormalities (hypochromasia, anisopoikilocytosis, circulating normoblasts), lower hemoglobin (Hb) and higher HbF levels. The presence of triplicated alpha-globin genes should always be considered in apparent beta-thalassemia carriers who were more symptomatic than expected, so that unnecessary investigations for the cause of anemia could be avoided. Finally, triplication of alpha-globin genes should be looked for in families with children affected by beta-thalassemia intermedia in which only one parent showed a picture of beta-thalassemia on Hb analysis.

Genetic analysis of beta-thalassemia intermedia in Israel: diversity of mechanisms and unpredictability of phenotype.

Molecular analysis was performed on 95 Israeli patients with thalassemia intermedia, representing 60 families of Arab (Moslem and Christian), Jewish, Druze, and Samaritan origin. There was a wide range of phenotypic severity, with baseline hemoglobin levels ranging from 5.5 to 10.7. Eighteen thalassemia mutations were found (29 genotypes), which were subdivided into groups, according to the severity of mutations. A consistently mild phenotype (10 families) was caused by compound heterozygosity for a silent mutation, such as -101 C-T or by coexistence of triplicated alpha-globin genes with thalassemia trait. In 39 thalassemia intermedia families, the genotype which was found was one which led to severe thalassemia intermedia, or, in other families, was associated with thalassemia major. Elevated hemoglobin F ameliorated the disease in some patients with a severe genotype. We did not find a beneficial effect of concurrent alpha-thalassemia in any of the families studied. In 11 families, only one beta-thalassemia allele was identified. One was a dominant thalassemia intermedia allele. Three additional families with heterozygous beta-thalassemia had excess alpha-globin genes (5 or 6 total). In 7 of these heterozygotes, no explanation was found for the thalassemia intermedia phenotype. Our results suggest a substantial influence of as yet unknown genetic modifiers. These findings have important implications for prenatal diagnosis and for the genetic counseling of families with thalassemia intermedia.

The triplicated alpha-globin gene locus in beta-thalassaemia heterozygotes: clinical, haematological, biosynthetic and molecular studies.

Excess alpha-globin chains play a major role in the pathophysiology of homozygous beta-thalassaemia. In beta-thalassaemia carriers a minor effect of alpha-globin chain excess is reflected in a minimal or mild anaemia without clinical symptoms. Factors that increase alpha-chain excess in heterozygotes are expected to accentuate the severity of the clinical and haematological phenotype. We report the clinical, haematological, biosynthetic and molecular data in three beta-thalassaemia heterozygotes with the rare interaction of homozygosity for alpha-globin gene triplication, and in 17 heterozygotes with a single additional alpha-globin gene. The three patients homozygous for the alpha-globin gene locus (anti 3.7 kb arrangement) had beta(0)-thalassaemia mutations and a diagnosis of thalassaemia intermedia, preserving haemoglobin levels around 7-8 g/dl. Of the 17 beta-thalassaemia heterozygotes (six children and 11 adults), 16 had severe beta-thalassaemia mutations interacting with an additional alpha-globin gene (13 with alpha alpha alpha anti-3.7 and four with alpha alpha alpha anti-4.2). Compared to simple beta-thalassaemia heterozygotes, they had lower haemoglobin levels and red cell indices, but higher alpha/beta biosynthesis, HbF levels and reticulocytes. Our results suggest that homozygous alpha-gene triplication interacts with a severe beta-thalassaemia mutation to cause an alpha-chain excess equivalent to that observed in homozygous beta-thalassaemia intermedia. In heterozygotes for severe beta-thalassaemia mutations with one additional alpha-globin gene, the alpha-chain excess causes a more pronounced degree of anaemia than is usually seen in simple beta-thalassaemia heterozygotes.

The Importance of the 3′ Untranslated Region for the Expression of the α-Globin Genes

With a reverse transcription-polymerase chain reaction procedure, we have determined the relative quantities of alpha 2- and alpha 1-mRNA in several patients with heterozygosities for alpha 2- or alpha 1-globin gene mutations, in subjects with two forms of alpha-thalassemia-2 (-3.7 kb; -4.2 kb), and in two children with an alpha-globin gene triplication. Mutations in either one of the two genes do not affect the mRNA production, and the alpha 2- to alpha 1-mRNA ratios in our heterozygotes are the same (approximately 2.7) as in normal persons with four alpha-globin genes, while the alpha/alpha X ratios of approximately 1.7 for alpha 2 variants and of approximately 6.2 for alpha 1 variants agree with the theoretic values. The deletion of 3.7 kb (leading to the formation of the alpha 2 alpha 1 hybrid gene) and of 4.2 kb (resulting in the presence of only the alpha 1 gene) causes the alpha 2/alpha 1 ratio to decrease to approximately 1.7, indicating that both are expressed as an alpha 1 gene. Data obtained for an Hb G-Philadelphia heterozygote (alpha alpha/-alpha G) show that the alpha 2 alpha 1 hybrid gene produces approximately 30% less mRNA than an alpha 1-globin gene on a normal chromosome, which may be caused by loss of some sequences 3' to the alpha 2 gene. The same may be the case for the alpha 1-globin gene on the chromosome with the 4.2 kb alpha-thal-2 deletion. These results suggest an important role for sequences located 3' to the terminating codon in regulating transcription. Support for this hypothesis was obtained from data for the two children with an alpha-globin gene triplication; the high alpha 2/alpha 1-mRNA ratio can be explained by assuming that the alpha 1 alpha 2 hybrid gene of the alpha 2(alpha 1 alpha 2)alpha 1 triplication expresses as an alpha 2 gene.

Severe thalassaemia intermedia caused by interaction of homozygosity for α‐globin gene triplication with heterozygosity for β thalassaemia

A 3-year-old child was evaluated for beta-thalassaemia intermedia. Molecular characterization including beta-globin gene sequence analysis revealed heterozygosity for a single beta-thalassaemia mutation, IVSI nt1 (G-->A). In addition the patient was found to be homozygous for alpha-globin gene triplication (alpha alpha alpha anti3.7/alpha alpha alpah anti3.7). The propositus has a significantly more severe phenotype than has been previously reported with this combination of genetic defects. In contrast, four individuals heterozygous for both triplicated alpha and for beta thalassaemia had a phenotype of thalassaemia minor, and a fifth had very mild thalassaemia intermedia.

A Czechoslovakian teenager with Hb E-β∘-thalassemia [IVS-I-1 (G → A)] complicated by the presence of an α-globin gene triplication

We have examined the molecular basis of three inherited hemoglobin (Hb) disorders present in a Czechoslovakian girl with a severe, transfusion-dependent, hemolytic anemia. She is heterozygous for Hb E (on a genetic background specific for Czechoslovakian families), heterozygous for the beta zero-thalassemia (thal) allele IVS-I-1 (G----A), and heterozygous for an alpha-globin gene triplication. The combination of these three undesirable traits results in a severe chain imbalance that is the basis of the serious hemolytic disorder observed in this teenager.