Dong-Zhi Li

Detection of Hb Constant Spring by a Capillary Electrophoresis Method

Hb Constant Spring [Hb CS; α142, Term→Gln (TAA>CAA in α2)] is the most prevalent nondeletional α-thalassemia (α-thal) anomaly in southern China. In conjunction with α0-thal, it can cause severe Hb H (β4) disease. The present study was done to evaluate the efficiency of two diagnostic methods in detecting Hb CS. Automated high performance liquid chromatography (HPLC) and Sebia Capillarys 2, a capillary electrophoresis method, were applied to blood samples from 21 individuals with Hb CS trait. Of the 21 cases, all (100%) were detected by capillary electrophoresis, whereas only 16 (76.2%) were detected by HPLC. We concluded that the Sebia Capillarys 2 is the preferred method for Hb CS screening.

Newborn screening for α-thalassaemia by a capillary electrophoresis method

Bouva et al. found that the percentage haemoglobin Bart’s (HbBart’s), as depicted by the FAST peak, was only a relative indication for the number of a genes affected in a-thalassaemia. No significant difference was demonstrated between HPLC in 2a/aa and 2a/2a, between 2a/2a and – –/aa or between – –/aa and – –/2a genotypes. It seemed that HPLC was technically not a preferable approach in neonatal screening for a-thalassaemia. Until recently, newborn screening for a-thalassaemia was conducted at our hospital, using a capillary electrophoresis method for quantification of HbBart’s in cord blood, which exactly distinguished HbH disease from other milder a-thalassaemia forms. Over 14 months, 6525 post-delivery cord blood samples were collected. Two milliliters of cord blood with ethylenediaminetetraacetic acid as an anticoagulant was obtained. Haemoglobin analysis was performed within 24 hours using an automated capillary electrophoresis system (CapillaryS 2; Sebia, Paris, France; software version 6.2), in which charged molecules are separated at alkaline pH by their electrophoreticmobility, electrolyte pH and electroosmotic flow. Each peak of the Hbs appears in a specific zone. Normal newborn Hb consists of approximately 70–80% Hb F (a2g2) and 20–30% Hb A (a2b2). The absence or reduced synthesis of the a-globin chain can lead to the production of abnormal HbBart’s in the fetus. In our experience with CapillaryS 2, HbBart’s levels as low as 0.1% could be detected. For samples with HbBart’s present, genomic DNA was extracted and gap-polymerase chain reaction was used to identify the three common deletional defects (–SEA, 2a3.7 and 2a4.2). If no deletional mutations were found, reverse dot-blot was used to simultaneously detect two common mutations (Hb Constant Spring and Hb Quong Sze). Otherwise, direct DNA sequencing was used to identify rare point mutations of a-globin gene. The automated capillary electrophoresis detected various amounts of HbBart’s in 377 (5.8%) samples. The results of genotyping are summarized in Table 1. A recent study showed a decreased rate of morbidity if HbH disease is diagnosed at birth, making a strong case for newborn screening, especially in areas with high a-thalassaemia prevalence. Newborn screening for HbH disease is based on the detection of HbBart’s which is only possible within the newborn period, before g-globin production switches to b-globin. As shown in our study, an elevation of the HbBart’s percentage correlated with the number of affected a genes. Significant differences were found in Bart’s levels between samples with HbH disease and those with two a-globin gene defect. Using an HbBart’s cut-off value of 10%, HbH disease could be differentiated with absolute certainty from other milder a-thalassaemia forms. Our results suggest that analysis of HbBart’s using the capillary electrophoresis system could be an efficient screening approach for HbH disease in newborns. This has also been confirmed in other studies.

The detection of aneuploidy and maternal contamination by QF-PCR in samples undergoing prenatal diagnosis for thalassemia in Southern China

OBJECTIVESnTo apply a simple and low-cost approach, which would easily and accurately detect both the common chromosomal abnormalities and maternal cell contamination (MCC) when invasive prenatal testing is performed for diagnosis of thalassemia.nnnSTUDY DESIGNnQuantitative fluorescence-polymerase chain reaction (QF-PCR) was carried out by amplification of microsatellite markers using fluorescence-labelled primers, followed by quantitative analysis of the allele peaks on a genetic analyser. A multiplex of 11 primer pairs for loci on each of chromosomes 13, 18 and 21 was used.nnnRESULTSnA total of 387 prenatal samples were tested. Five (1.3%) samples showed MCC, including two (0.7%, 2/289) amniotic fluid samples and three (3.1%; 3/98) chorionic villi samples. Of the 379 prenatal samples without MCC, QF-PCR assays detected two (0.5%) cases of trisomy 21, which were confirmed by traditional karyotyping, and missed one case of trisomy 2 mosaicism and one case of monosomy X. The case of trisomy 2 mosaicism was later found to be limited to the placenta, and the case of monosomy X was picked up by ultrasound. There was no clinically significant case that would have been missed if QF-PCR had been used as a stand-alone test instead of karyotyping when invasive prenatal testing was performed for diagnosis of thalassaemia.nnnCONCLUSIONSnThe QF-PCR assay could allow simultaneous detection of aneuploidy and possible MCC in the fetal material. This is especially valuable when PCR-based techniques are used in the DNA analysis for thalassaemia. This strategy may be applied to prenatal diagnosis of other recessive disorders.

Delineation of the molecular basis of borderline hemoglobin A2 in Chinese individuals.

BACKGROUNDnThe gray zone of borderline hemoglobin A2 (Hb A2) may be present in a large section of the population, especially in countries where thalassemia is common. However, very little is currently known of the molecular basis of borderline Hb A2 in Chinese individuals.nnnMETHODnIn this study, we performed a comprehensive analysis of the globin genotypes and KLF1 gene mutations associated with borderline Hb A2 in 165 Chinese subjects.nnnRESULTnFifteen (9.1%) were positive for a molecular defect in the α-,β-globin genes, of whom, α-thalassemia mutations and α-globin gene triplication were found in eleven cases, accounting for about 73.3% of these globin gene defects. Twenty (12.1%) were positive for a molecular defect in the KLF1 gene. Eight different mutations were identified, six of which are here reported for the first time. The most common is the G176AfsX179 mutation, accounting for 50% of the total.nnnCONCLUSIONSnThe molecular characterization of borderline Hb A2 in Chinese individuals is significantly different than in Italian population. Our data is conductive to provision of genetic counseling for Chinese individuals with borderline Hb A2.

Screening for Hb Constant Spring in the Guangdong Province, South China, Using the Sebia Capillary Electrophoresis System

Hb Constant Spring [Hb CS; α142, Term→Gln (TAA>CAA in α2)] is a nondeletional α-thalassemia (α-thal) defect difficult to detect on conventional electrophoresis because of its small amount in heterozygotes. We have found that individuals with an Hb CS trait could efficiently be detected using the Sebia capillarys 2 system. In the present study, we have confirmed this method in a cohort of 23,842 individuals from Guangdong Province (South China). Hb CS was detected in 71 (0.3%) of the cases. The levels of Hb CS in heterozygotes ranged from 0.1–1.0% with an average of 0.6%. We propose the reported 0.3% as a realistic figure for the prevalence of Hb CS in South China.

A novel β-thalassemic allele due to a thirteen nucleotide deletion: codons 54–58 (-T ATG GGC AAC CCT)

Dear Editor, The β-thalassemia is one of the most common monogenetic diseases in the world. It is characterized by reduced or absent synthesis of β-globin chains of hemoglobin. Severe cases of β-thalassemia result in pronounced anemia which needs lifelong blood transfusion. Worldwide control of thalassemia is achieved through population screening, counseling of carriers and prenatal diagnosis of high-risk couples. Over 200 mutations causing β-thalassemia have been described from different parts of the world though relatively small numbers of these mutations can be found for each population. For example, in the Chinese population, five mutations of the 30 known, account for more than 90% of all cases [1]. Delineation of rare mutations is important for prenatal diagnosis in affected families. They may also provide insight into the underlying processes of globin gene regulation and in the studies of genotype– phenotype relationship. Here, we report a novel βthalassemic mutation due to a thirteen nucleotide deletion in the β-globin gene at codons 54–58. The family was from Maoming city of Guangdong province in southern China. They were referred to our center for prenatal diagnosis at the mother’s first trimester of gestation. Their first daughter had been diagnosed with β-thalassemia major at 4 months of age and had been regularly transfused. The child died at the age of 3 years. Blood samples were obtained from the parents, and blood count and morphology showed red blood cell microcytosis, with the following hematological findings: RBC 5.40× 10/L, Hb 11.8 g/L, MCV 72.1 fL, MCH 21.6 pg, Hb A 92.9%, and Hb A2 7.1% in the father, and RBC 4.57×10 / L, Hb 10.8 g/L, MCV 76.1 fL, MCH 22.4 pg, Hb A 94.5%, and Hb A2 5.5% in the mother. No abnormal hemoglobin was present in routine electrophoresis or in high-performance liquid chromatography analyses. β-Globin gene mutation analysis for the parents was performed by using polymerase chain reaction (PCR)-based reverse dot blots (RDB) hybridization technique [2]. After a screening of 17 known mutations in Chinese population, the father was revealed to be a heterozygote for the mutation of codon 17 (A→T), and the mother failed to identify any of the known mutations. The mother was then assumed to have an uncommon β-thalassemic allele. The β-globin gene was amplified from DNA of the mother by PCR and the PCR product was analyzed by direct nucleotide sequencing using the BigDyeTM terminator Cycle Sequencing Kit and the ABI PRISMTM 310 genetic analyzer. Sequence analysis of the entire β-globin gene revealed a novel β-thalassemia mutation in the mother. The β-thalassemic mutation was found to be a deletion of 13 nucleotides (-T ATG GGC AAC CCT) from codon 54 through codon 58 (Fig. 1). Gap-PCR was also undertaken in the couple to exclude the coinheritance of αthalassemia [3], and this did not reveal any of the three common Chinese α-globin gene deletion mutations (−, −α, and −α). The clarification of the mutations in the parents made the prenatal diagnosis easy. At 12 weeks of Ann Hematol (2009) 88:799–801 DOI 10.1007/s00277-008-0658-6

KLF1 Gene Mutations in Chinese Adults with Increased Fetal Hemoglobin

We investigated the Krüppel-like factor 1 (KLF1) gene mutations in Chinese adults with increased Hb F levels (>1.5%) referred to our laboratory for thalassemia screening. Functionally effective KLF1 mutations were identified in five out of 140 samples with an elevated Hb F (1.9-11.4%). Only two different KLF1 mutations were detected. Functional KLF1 mutations were not identified in the matched cohort of 110 samples with normal Hb F values (<1.0%). The KLF1 mutations could be one of the causes of hereditary persistence of fetal hemoglobin (HPFH) in regions where thalassemias are common.

Analysis Of δ-Globin Gene Mutations in the Chinese Population

Although δ-globin gene (HBD MIM#142000) mutations have no immediate physical consequence, it can interfere with the diagnosis of β-thalassemia (β-thal), which can be severe. In the present study, of 40,863 samples referred for thalassemia trait screening, 167 samples with lower than expected Hb A2 levels, in the presence or absence of a second Hb A2 fraction, were selected for our analysis and 152 samples (0.4%) were positive for δ-globin gene mutations. Twenty-one different mutations were detected, and of these 12 have not been previously described. We found that −77 (T>C) was the most common mutation in Chinese followed by −30 (T>C), together accounting for almost 82.3% of the total number of δ-globin gene defects. Since compound heterozygotes for β-thal and a δ-globin gene mutation may have low mean cell volume (MCV) and normal Hb A2 levels, and therefore be overlooked as β-thal heterozygotes, a detailed molecular analysis for both α- and β-thal is necessary, especially when one partner has been identified to have β-thal trait.

Screening for mutations in the α-globin genes leading to abnormal hemoglobin variants with high resolution melting analysis

Abstract Background: α-Thalassemia is one of the most commonly inherited single-gene disorders in southern China. It is important to identify non-deletional α-thalassemia in areas where α-thalassemia is prevalent, since non-deletional HbH disease (--/αTα or --/ααT) is caused by the interaction of a non-deletional α-thalassemia with α-thalassemia-1 trait (--/αα). In this study, we developed an optimized molecular protocol for screening for α-globin gene mutations and validated the feasibility of using it as a rapid detection method. Methods: An approach based on high-resolution melting (HRM) analysis was used. A total of 74 samples, including 54 abnormal α-chain samples and 20 control samples, were tested. Results: All of the 54 samples with point mutations at the exons 1, 2 or 3 of the α-globin genes, including 33 non-deletional α-thalassemia, were successfully detected. Conclusions: HRM has the potential to become an efficient, rapid screening method for non-deletional α-thalassemia.

Detection of Hb Constant Spring [α142, Term→Gln, TAA>CAA (α2)] in Heterozygotes Combined With β-Thalassemia

Hb Constant Spring [Hb CS, α142, Term→Gln, TAA>CAA (α2)] is a nondeletional form of α-thalassemia (α-thal) that is most prevalent in Southern Chinese and Southeast Asian populations. We previously found that Hb CS trait could efficiently be screened using Sebia Capillarys2. In this study, we report that Hb CS heterozygotes combined with β-thal could not be detected by the Sebia Capillarys2 method due to the very small amount of Hb CS.