Attawut Chaibunruang

Fine‐tiling array CGH to improve diagnostics for α‐ and β‐thalassemia rearrangements

Implementation of multiplex ligation‐dependent probe amplification (MLPA) for thalassemia causing deletions has lead to the detection of new rearrangements. Knowledge of the exact breakpoint sequences should give more insight into the molecular mechanisms underlying these rearrangements, and would facilitate the design of gap‐PCRs. We have designed a custom fine‐tiling array with oligonucleotides covering the complete globin gene clusters. We hybridized 27 DNA samples containing newly identified deletions and nine positive controls. We designed specific primers to amplify relatively short fragments containing the breakpoint sequence and analyzed these by direct sequencing. Results from nine positive controls showed that array comparative genomic hybridization (aCGH) is suitable to detect small and large rearrangements. We were able to locate all breakpoints to a region of approximately 2 kb. We designed breakpoint primers for 22 cases and amplification was successful in 19 cases. For 12 of these, the exact locations of the breakpoints were determined. Seven of these deletions have not been reported before. aCGH is a valuable tool for high‐resolution breakpoint characterization. The combination of MLPA and aCGH has lead to relatively cheap and easy to perform PCR assays, which might be of use for laboratories as an alternative for MLPA in populations where only a limited number of specific deletions occur with high frequency. Hum Mutat 33:272–280, 2012.

Interactions of hemoglobin Lepore (δβ hybrid hemoglobin) with various hemoglobinopathies: A molecular and hematological characteristics and differential diagnosis

Hemoglobin (Hb) Lepore is a variant consisting of two alpha-globin and two deltabeta-globin chains. In heterozygote, it is associated with clinical findings of thalassemia minor but interactions with other hemoglobinopathies can lead to various clinical phenotypes. Using a combination of Hb-HPLC, Hb-capillary electrophoresis and DNA analyses, we have identified 14 patients with Hb Lepore-Hollandia including eight heterozygotes, two double heterozygotes with alpha(+)-thalassemia, two compound heterozygotes with Hb E (initially diagnosed as Hb E-beta-thalassemia) and two previously undescribed conditions of double heterozygote for Hb Lepore/Hb Constant Spring and Hb Lepore/alpha(0)-thalassemia, both associated with higher levels of Hb F and lower levels of Hb Lepore. Hematological and molecular features of these patients are presented along with those observed in four other Thai individuals encountered with heterozygous Hb Lepore-Washington-Boston. Haplotype analysis of the beta-globin gene cluster showed that all Hb Lepore-Hollandia genes were associated with a single haplotype not described previously in other populations, (- + - + + - +) whereas the four Hb Lepore-Washington-Boston genes were associated with haplotypes (+ - - - - + -/+) (N=1) and (+ - - - - - +) (N=3), data indicating multiple origins of these two variants. Hb Lepore may not be uncommon in the Thai and other Asian populations and both hematological and molecular studies are required for accurate diagnosis. To facilitate rapid epidemiological, diagnostic screening and differentiation of the two Hb Lepore defects, a simple assay based on multiplex PCR has been developed.

Molecular and hematological studies in a large cohort of α0-thalassemia in northeast Thailand: Data from a single referral center

α(0)-thalassemia is the most severe form of α-thalassemia alleles found among Southeast Asian and Chinese populations and can cause a fatal condition known as hemoglobin Barts hydrops fetalis and hemoglobin H disease. In order to provide the molecular epidemiological characteristic of α(0)-thalassemia in northeast Thailand, a total of 12,525 blood specimens referred to our center at Khon Kaen University in northeast Thailand during October 2008 to January 2012 were studied. Hematological parameters were recorded and DNA deletions causing α(0)-thalassemia were examined by PCR related techniques. Among 12,525 samples examined, α(0)-thalassemia alleles were identified in 1,873 (15.0%) samples, including 1855 (14.8%) cases with Southeast Asian (--(SEA)) deletion and 18 cases (0.2%) with THAI deletion (--(THAI)). As many as twenty genotypes were encountered. Hb profiles and hematological parameters were comparatively presented. Data on prevalence, molecular features and phenotypic expression of α(0)-thalassemia should prove useful in a carrier screening and a prevention and control program of this common genetic disorder in the region.

Genetic heterogeneity of hemoglobin AEBart's disease: A large cohort data from a single referral center in northeast Thailand

AEBarts disease is a thalassemia intermedia usually characterized by the interaction of α(0)-thalassemia with either deletional or non-deletional α(+)-thalassemia in Hb E heterozygote. Genotypic and phenotypic features are heterogeneous. We studied the hematologic and molecular characteristics of this disease in a cohort of 173 Thai patients encountered at our center in northeast Thailand. Hemoglobin and DNA analyses identified patients with deletional AEBarts disease (n=84), Hb Constant Spring AEBarts disease (n=81), Hb Paksé-AEBarts disease (n=5), AEBarts disease with codon 30 mutation (n=1) and two hitherto un-described forms of AEBarts disease due to interaction of Hb E heterozygote and α(0)-thalassemia with the -α(16.6)kb deletional α(+)-thalassemia (n=1) and Hb Q-Thailand (n=1). Different phenotypic expression of these AEBarts diseases with low Hb, Hct and MCV and increased RDW values with marked reduction in Hb E levels were observed. It was found that all these forms of AEBarts disease showed similar thalassemia intermedia phenotypes but those with non-deletional forms were relatively more anemic. Our data confirm that in such area with high prevalence of hemoglobinopathies such as Southeast Asia, identification of rare thalassemia alleles in a thalassemia intermedia patient should not be ignored. Careful consideration of different phenotypic expression may help in providing presumptive diagnosis of this disease where access to molecular testing is limited. However, molecular diagnostic is useful for predicting the clinical outcome and improving genetic counseling of these complex hemoglobinopathies.

A spurious haemoglobin A1c result associated with double heterozygote for haemoglobin Raleigh (β1[NA1]Val → Ala) and α+-thalassaemia

Background Accurate measurement of haemoglobin A1c (HbA1c) is useful for long-term glycaemic control in patients with diabetes. Many Hb variants can interfere with HbA1c measurement and cause inaccurate results. Methods The subject was a 31-year-old Thai man who was discovered because of an unexpected HbA1c result; other diabetic parameters were within the normal range. Abnormal Hb was investigated using automated high-pressure liquid chromatography (HPLC) and a capillary electrophoresis system. Mutation analysis was done by cDNA sequencing, polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) and multiplex allele-specific PCR assays. Results Evaluation of HbA1c by cation-exchange HPLC showed a value of 34.9% (reference interval, 4.0–6.0%), but a value of only 4.0% (reference value, 4.8–5.9%) was found with a turbidimetric immunoassay. Haematological analysis revealed a mild anaemia but other parameters were within the normal range. Hb-HPLC analysis demonstrated an unknown Hb variant (47.0%) separating from HbA (46.7%), but capillary electrophoresis identified no abnormal peaks. Mutation analysis identified the Hb Raleigh (β1[NA1]Val → Ala [G T G → G C G]) mutation in combination with an α +-thalassaemia, a hitherto undescribed association. The Hb Raleigh mutation could be detected by PCR–RFLP or a multiplex allele-specific PCR assay. Conclusions Hb Raleigh can cause falsely increased HbA1c values on cation-exchange HPLC. Definitive diagnosis of this variant using combined Hb and DNA analyses is therefore essential.

FIRST DESCRIPTION OF A Hb A2 VARIANT IN THAILAND. IDENTIFICATION OF Hb A2-MELBOURNE (δ43(CD2)Glu→Lys) IN THAI INDIVIDUALS

We report the molecular and hematological identifications of a Hb A2 variant found for the first time in Thailand. The subjects were two unrelated Thai women who had normal hematological features. In the first case, hemoglobin (Hb) high performance liquid chromatography (HPLC) analysis identified, in addition to Hb A, Hb A2 (1.6%) and a minor Hb variant (0.9%) separating after Hb A2. Analysis using capillary electrophoresis (CE) demonstrated 1.3% Hb A2 and 1.0% minor variant in zone 1. DNA analysis showed a single nucleotide mutation at codon 43 of the δ-globin gene (HBD:c.130G>A) causing a glutamic acid to lysine substitution corresponding to Hb A2-Melbourne, originally documented in an Italian subject, but not previously described in Thailand. The mutation could be confirmed by allele specific polymerase chain reaction (PCR) assay. Further screening using this technique in our series identified another case of a Thai woman with a double heterozygosity for Hb A2-Melbourne and α+-thalassemia (α+-thal). A β-globin gene haplotype associated with this Thai Hb A2-Melbourne gene was also established.

A Newly Modified Hemoglobin H Inclusion Test as a Secondary Screening for α⁰-Thalassemia in Southeast Asian Populations

Screening for α⁰-thalassemia is usually associated with a high false-positive rate, leading to an unnecessary PCR workload for accurate diagnosis. We have developed a modified Hb H inclusion test for use as a secondary screening. This test was performed on young red blood cell enriched fractions using dextran sedimentation. The study was performed in 100 subjects positive on initial screening. Confirmatory tests included Hb analysis and a multiplex PCR assay to identify α⁰-thalassemia deletions. A modified Hb H inclusion test was positive in 31 cases, 30 of whom were α⁰-thalassemia carriers (97%). The remaining case (3.0%) was homozygous for α+-thalassemia. The remaining 69 cases with a negative Hb H inclusion test included normal subjects, α+-thalassemia carriers and β-thalassemia carriers. Two of them (2/69, 3.0%) were found to be double heterozygotes for β⁰-thalassemia and α⁰-thalassemia. The overall sensitivity and specificity of the modified Hb H inclusion test for screening of α⁰-thalassemia were 94.0 and 99.0%, respectively. Therefore, we recommend the use of this test in combination with Hb analysis to exclude cases with αβ-thalassemia. This should lead to a significant reduction in the number of cases referred for PCR analysis of α⁰-thalassemia by about 50.0%.

Molecular Characteristics of Hb New York [β113(G15)Val→Glu, HBB: c.341T>A] in Thailand

Abstract Hb New York or Hb Kaohsiung [β113(G15)Val→Glu (GTG>GAG), HBB: c.341T>A] has been considered a rare β hemoglobin (Hb) variant found originally in an Iranian woman and later in diverse populations but its genetic origin has not been elucidated. Here we report molecular and hematological descriptions of this variant found in the Thai population. Among 5643 subjects referred for hemoglobinopathy investigation during January 2015 to September 2017, 183 (3.2%) were found to carry several Hb variants, including β chain variants (n = 135, 2.4%), α chain variants (n = 33, 0.6%), Hb Lepore-Hollandia (NG_000007.3: g.63290_70702del) and Hb Lepore-Boston-Washington (NG_000007.3: g.63632_71046del) (δβ hybrid Hb) (n = 12, 0.2%) and δ chain variants (n = 3, 0.05%). Of patients with β chain variants, six with normal high performance liquid chromatography (HPLC) patterns, had an abnormal Hb in zone 11 of capillary electrophoresis (CE), the amounts of which ranged from 29.6-45.4% with normal levels of Hb A2 and Hb F. DNA analysis identified a heterozygous Hb New York mutation in all cases. Further screening of α-thalassemia (α-thal) identified coinheritance of α+- and α0-thal in two of them who had reduced levels of Hb New York. Haplotype analysis suggested that the Thai Hb New York was likely associated with a single β-globin haplotype [+ – – – – + +], indicating that it was of the same origin. Hematological findings and simple DNA assay based on allele-specific polymerase chain reaction (PCR) for rapid detection of Hb New York are presented.