SEA-type Deletion Research Articles

Detection of SEA-type α-thalassemia in embryo biopsies by digital PCR

ObjectiveAccurate and efficient pre-implantation genetic diagnosis (PGD) based on the analysis of single or oligo-cells is needed for timely identification of embryos that are affected by deleterious genetic traits in in vitro fertilization (IVF) clinics. Polymerase chain reaction (PCR) is the backbone of modern genetic diagnoses, and a spectrum of PCR-based techniques have been used to detect various thalassemia mutations in prenatal diagnosis (PND) and PGD. Among thalassemias, SEA-type α-thalassemia is the most common variety found in Asia, and can lead to Bart's hydrops fetalis and serious maternal complications. Materials and methodsTo formulate an efficient digital PCR for clinical diagnosis of SEA-type α-thalassemia in cultured embryos, we conducted a pilot study to detect the α-globin and SEA-type deletion alleles in blastomere biopsies with a highly sensitive microfluidics-based digital PCR method. Genomic DNA from embryo biopsy samples were extracted, and crude DNA extracts were first amplified by a conventional PCR procedure followed by a nested PCR reaction with primers and probes that are designed for digital PCR amplification. ResultsAnalysis of microfluidics-based PCR reactions showed that robust signals for normal α-globin and SEA-type deletion alleles, together with an internal control gene, can be routinely generated using crude embryo biopsies after a 106-fold dilution of primary PCR products. ConclusionThe SEA-type deletion in cultured embryos can be sensitively diagnosed with the digital PCR procedure in clinics. The adoption of this robust PGD method could prevent the implantation of IVF embryos that are destined to develop Bart's hydrops fetalis in a timely manner. The results also help inform future development of a standard digital PCR procedure for cost-effective PGD of α-thalassemia in a standard IVF clinic.

Treatment of alpha(0)-thalassemia (--SEA/--SEA) via serial fetal and post-natal transfusions: Can early fetal intervention improve outcomes?

Objective and importanceHomozygous Southeast Asian alpha-thalassemia mutation (--SEA/--SEA) results in deletion of all alpha-globin genes (alpha(0)-thalassemia). Since all alpha-globin chains are absent, hemoglobin F cannot be synthesized, and hemoglobin Bart's becomes the dominant fetal hemoglobin. Hemoglobin Bart's is a γ tetramer with a very high oxygen affinity, thus oxygen delivery to the tissues is poor. Clinical manifestations include severe fetal anemia, hydrops fetalis, fetal demise, and high risk of neurodevelopmental impairment in the rare survivors.Clinical presentationA 39-year-old Vietnamese woman presented to our center at 28 0/7 weeks' gestation with fetal alpha(0)-thalassemia (--SEA/--SEA type deletion) and ultrasound markers suggestive of severe fetal anemia.InterventionThe fetus was treated with four intrauterine transfusions followed by post-natal chronic transfusions. Formal neurodevelopmental testing (Battelle Developmental Inventory, Second Edition) was performed at 18 months of age, and the developmental quotient was 93 (32nd percentile) with all subdomains noted within normal limits, indicating overall intact neurodevelopment.ConclusionWe posit that earlier diagnosis and fetal treatment, prior to clinical findings suggestive of fetal anemia, may improve long-term outcomes by enhancing oxygen delivery to the tissues of the developing fetus.

Variable Genotype-Phenotype Correlations in Patients With a Rare Nondeletional α-thalassemia; Hb Pak Num Po (HBA1

Transfusion-dependent Hb H disease is rarely reported. In the majority of patients, it is caused mainly by α(0)-thalassemia from deletions of 2 linked α-globin genes and nondeletional mutations. Previously, we had described 2 unrelated Thai patients with this condition because of compound heterozygosity of SEA-type deletion (--SEA/) and a novel nucleotide mutation: a thymine insertion at codon 131 of the α1 gene, namely, Hb Pak Num Po (Hb PNP, αα(PNP)). We herein describe the identification of 4 additional patients with Hb PNP with a broader genotype/phenotype spectrum and provide an overview of clinical management approaches including stem-cell transplantation.

Detection of alpha(0)‐thalassemia South‐East Asian‐type deletion by droplet digital PCR

The α(0)-thalassemia South-East Asian (SEA)-type deletion is the most common genetic disorder in the Asian population. Couples who are both carriers have a 25% chance of conceiving Bart's hydrops fetalis. Therefore, results from carrier screening and prenatal diagnosis frequently need to be available rapidly. The aim of this study was to implement a droplet digital polymerase chain reaction (ddPCR) for diagnosis of α(0)-thalassemia SEA-type deletion. The wild-type α-globin gene allele and α(0)-thalassemia SEA allele were quantified in DNA samples of 20 normal individuals, 15 samples with α(0)-thalassemia SEA trait, and 8 samples with Bart's hydrops fetalis using the ddPCR. The DNA copy number of wild-type α-globin gene allele and α(0)-thalassemia SEA allele was then calculated using the Quantasoft analysis software. The mean ± standard deviation (SD) ratio of wild-type α-globin gene allele and α(0)-thalassemia SEA allele among normal individuals, samples with α(0)-thalassemia SEA trait, and Bart's hydrops fetalis were clearly distinguished with levels of 1.78±0.49, 0.85±0.14, and 0.03±0.03, respectively. The ddPCR may be one alternative technology available for routine clinical diagnosis of α(0)-thalassemia SEA-type deletion and prenatal diagnosis of Bart's hydrops fetalis.

Hematological Characterization of Compound Heterozygous Hemoglobin Hope/E Patients with and without á-Thalassemia-1 SEA Type Deletion

Hematological Characterization of Compound Heterozygous Hemoglobin Hope/E Patients with and without á-Thalassemia-1 SEA Type Deletion

Detection of Common Deletional Alpha-Thalassemia Spectrum by Molecular Technique in Kelantan, Northeastern Malaysia

Thalassemia is a hereditary blood disorder that results from genetic defects causing deficient synthesis of hemoglobin polypeptide chains. Although thalassemia mostly affects developing countries, there is limited knowledge of its accurate frequency and distribution in these regions. Knowing the prevalence of thalassemia and the frequency of responsible mutations is therefore an important step in the prevention and control program as well as treatment strategies. This study was performed to determine the prevalence and to study the spectrum of gene deletions that are responsible in α-thalassemia in Kelantan, located in northeastern Malaysia. A total 400 first-time blood donors from multiple areas of donation centre were chosen randomly. The presence of three types of α-thalassemia gene deletion in southeast Asian population which were -SEAdeletion, -α 3.7 rightward deletion, and -α 4.2 leftward deletion was detected by using multiplex PCR method. 37 (9.25%) of blood donors were confirmed to have α-thalassemia deletion types. 34 (8%) were heterozygous for α3.7 deletion, 1 (0.25%) was heterozygous for α4.2 deletion, and 2 (0.5%) were heterozygous for SEA type deletion. Alpha-thalassemia-2 with 3.7 deletion was the most common determinant detected in Kelantan Malay compared to other ethnic groups. It has been noted that alpha-thalassemia-2 with 3.7 deletion is the most common type of α-thalassemia throughout the world.

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/βE 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/βE 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.

Comparison of capillary electrophoregram among heterozygous Hb Hope, Hb Hope/α-thalassemia-1 SEA type deletion and Hb Hope/β0-thalassemia

Hemoglobin (Hb) A(2) is artifactually elevated in cases of heterozygous Hb Hope when measured by capillary electrophoresis (CE). However, there is no report of HbA(2) levels and capillary electrophoregrams for associations of heterozygote of Hb Hope with α-thalassemia nor β-thalassemia. Levels of HbA(0), HbA(2) and Hb Hope in 16 heterozygous Hb Hope, 3 Hb Hope/α-thalassemia-1 SEA type deletion and 2 Hb Hope/β(0)-thalassemia were measured by CE. Electrophoregram and the levels of those were compared within these three groups. Artifactually elevated HbA(2) levels (≥4%) were found in both groups of heterozygous Hb Hope and Hb Hope/α-thalassemia-1 SEA type deletion. Manual corrections were performed by adjusting baselines, and results showed that means of HbA(2) in both groups decreased from 4.47% and 4.03% to 1.93% and 1.77%, respectively. The highest levels of HbA(2) and Hb Hope were observed in samples with Hb Hope/β(0)-thalassemia. Moreover, HbA(0) was not observed in these cases. The elevation of HbA(2) in patients with heterozygous Hb Hope and with Hb Hope/α-thalassemia-1 SEA type deletion measured by CE leads to incorrect β-thalassemia trait diagnosis. However, using CE electrophoregram together with levels of HbA(0), HbA(2) and Hb Hope would be a more accurate and precise method for diagnosis of Hb Hope/β(0)-thalassemia.

Interference of Hemoglobin Hope on β-Thalassemia Diagnosis by the Capillary Electrophoresis Method

The β-chain hemoglobin (Hb) variants interfere with the diagnosis of β-thalassemia trait using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). We analyzed the effect of Hb Hope, a β-chain Hb variant frequently found in the Thai population, on β-thalassemia trait diagnosis. HPLC and CE were used to quantify the level of HbA(2) in 11 whole blood samples containing Hb Hope. The levels of Hb Hope detected by both methods were similar. An elevated HbA(2) level was found in all samples analyzed by the CE method, while 1 was increased when analyzed by HPLC, which was a compound heterozygous of Hb Hope and α-thalassemia-1 SEA-type deletion. Of 11 samples, 6 had mean corpuscular volumes within the reference range. All samples showed negative results for molecular analysis of β(0)-thalassemia codon 17, 41/42, and 71/72 mutations and β-thalassemia 3.5-kb deletion. Therefore, Hb Hope interfered with the diagnosis of β-thalassemia trait analyzed by CE but not by HPLC.

Hematological features and molecular lesions of hemoglobin gene disorders in Taiwanese patients

Hemoglobin (Hb) gene disorders are one of the most common inherited diseases in Taiwan, which include alpha-thalassemia, beta-thalassemia, and Hb variants. In this study, we collected and analyzed mutations found in 930 patients with Hb gene disorders except Hb Bart's Hydrops and beta-thalassemia major. The patients included 650 cases of alpha-thalassemia, 225 cases of beta-thalassemia, 9 cases of alpha-thalassemia combined with beta-thalassemia, and 46 cases of Hb variants or Hb variants combined with alpha-thalassemia or beta-thalassemia. The most common type of alpha0-thalassemia and alpha++-thalassemia mutations in our study were the SEA type deletion and the alpha3.7 deletion, respectively; the most common beta-thalassemia mutation was the IVS-2 nt 654 C-->T mutation; and the most common Hb variant was the HbE. We compared the relationships between genotype and hematological phenotypes of various Hb gene disorders and found that different genotypes of alpha0-thalassemia have similar hematological features. In conclusion, the results of our study provide data of the complex interaction of thalassemias and Hb variants which might be useful for other researchers in this field.

Homozygous ?-thalassemia associated with hypospadias: SEA-type deletion does not affect expression of the -14 gene and loss of the ?1-globin gene on 16p13.3 is compensated by its duplicate ?2 on chromosome 10

Homozygous ?-thalassemia associated with hypospadias: SEA-type deletion does not affect expression of the -14 gene and loss of the ?1-globin gene on 16p13.3 is compensated by its duplicate ?2 on chromosome 10

Non-radioactive southern hybridization for early diagnosis of ?-thalassemia with Southeast Asian-type deletion in Taiwan

Alpha-thalassemia has been estimated to account for over 60% of hydrops fetalis cases in Taiwan. The most common genotypic lesion found in alpha-thalassemia-1 cases in Taiwan is deletion of a large segment of the alpha-globin gene cluster, termed the Southeast Asian-type deletion (-SEA/; further referred to as SEA-type deletion). Seven chorionic villus samples (CVS) from pregnancies of couples both heterozygous for SEA-type deletion were studied. Non-radioactive Southern-blot hybridization using the dig-alkaline phosphatase detection system was developed to fulfill this purpose. The results were compared with corresponding polymerase chain reaction (PCR) data to elucidate the effectiveness of these two protocols in the diagnosis of the SEA-type deletion. The data showed that of the seven CVS, three demonstrated a distinctive band pattern, indicating their homozygous status of SEA-type deletion, whereas two showed heterozygous patterns, and the other two were free of the deletion. Homozygosity of the deletion was confirmed by Southern-blot hybridization performed on DNA samples extracted from the abortus tissue. However, two of the three cases with SEA-type deletion showed heterozygous PCR results. Maternal cell contamination could be responsible for the artifacts in the PCR results, but the influence due to the contamination is minimal in non-radioactive Southern-blot hybridization. We concluded that PCR is suitable for screening of carrier adults with SEA-type deletion, and non-radioactive Southern hybridization is ideal for early prenatal diagnosis of the SEA-type deletion.