Can Liao

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.

A novel mutation of −50 (G→A) in the direct repeat element of the β-globin gene identified in a patient with severe β-thalassemia

Dear Editor, β-Thalassemia is a heterogeneous genetic disease associated with defective expression of the β-chain of human hemoglobin. So far, more than 200 mutations have been defined and affect almost every known stage of β-globin gene expression resulting in a reduction (β) or complete absence (β) of β-chain synthesis from the affected allele [1, 2]. In southern China, the carrier rate of β-thalassemia in Guangdong province was 2.54% [3], with five common mutations accounting for 90% of all β-thalassemia individuals. Identification of new mutations and update of the mutation spectrum of thalassemia in one specific ethnic population is always needed for improving genetic counseling and prenatal diagnosis. We here describe a novel promoter mutation of −50 (G→A) within the conserved direct repeat element (DRE) leading to β-thalassemia major when associated with a β-thalassemia mutation. A 3-year-old Chinese boy with his parents was seen at our genetics clinic. The boy suffered from β-thalassemia major and had been transfused every 2 months since the age of 6 months. Molecular analyses performed by reverse dot blots (RDB) at other clinics revealed that he was a heterozygote for the common mutation of codons (CDs) 41–42 (−TTCT) of the β-globin gene, but the other mutational allele was not determined. At this referral, the family was planning for another pregnancy and counseled about prenatal diagnosis. Blood samples were obtained from all three family members. Hematological investigations showed that both of the parents had a classical β-thalassemia trait and the son manifested with severe β-thalassemia (Table 1). Determination of the β-thalassemia mutations was performed using the polymerase chain reaction (PCR)–RDB method which can simultaneously detect 17 types of β-globin gene mutations found in Chinese population. This confirmed the heterozygous presence of the CDs 41–42 (−TTCT) mutation in the son and the father, but the mutation was not found in the mother. Gap PCR was also undertaken in the three family members to exclude the coinheritance of αthalassemia, and this did not reveal any of the three common Chinese α-globin gene deletion mutations (−α, −α, –). To further investigate the underlying mutation in this family, the β-globin gene was amplified by PCR from DNA of the mother, and the product was analyzed by direct nucleotide sequencing using the BigDyeTM Terminator Cycle Sequencing Kit and the ABI PRISMTM 310 genetic analyzer (Applied BioSystems). Direct sequencing of the β-globin gene showed the normal sequence except for the heterozygous G→A mutation at position −50 relative to the Cap site (Fig. 1). This so far undescribed mutation was also revealed in the son by DNA sequencing. Thus the patient was confirmed to be a compound heterozygote with a genotype of −50 (G→A)/CDs 41–42 (−TTCT). In order to evaluate the functional effect of this novel mutation, two other family members were investigated. A semiquantitative reverse transcription PCR assay was used to measure β globin mRNA in the reticulocytes from three heterozygous subjects (the mother, grandmother, and an aunt) and eight normal individuals. These analyses showed a mildly reduced β-globin mRNA level (23.4 %) in heterozygotes compared with that in normal subjects. Ann Hematol (2009) 88:1149–1150 DOI 10.1007/s00277-009-0732-8

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.

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

First detection of the codons 41–43 (−CTTTG,+A) β-thalassemia mutation in a Chinese patient

Dear Editor, The β-thalassemias are the most common monogenic diseases and are characterized by hypochromic and microcytic anemia, which results from the absence (β) or reduction (β) of the synthesis of β-globin chains. In thalassemic individuals, various mutations have been defined that affect transcription, translation, or ribonucleic acid (RNA) processing. Up to now, more than 200 βthalassemia mutations have been characterized worldwide, including 30 types that have been found in Chinese subjects [1–2]. Data on spectrum of mutations in any region can serve as a valuable tool for population genetic studies and can also help to plan strategies for prenatal diagnosis. In this letter, we report a novel β-thalassemia mutation due to a five-nucleotide deletion (−CTTTG) plus a one-nucleotide insertion (+A) at codons 41–43. The couple was from Guangzhou City, Guangdong Province, in Southern China. Both of them were tested positive for the β-thalassemia trait at the mother’s 17 weeks of gestation by our prenatal thalassemia screening program, with the following hematological findings: red blood cells (RBC) 5.50×10/L, hemoglobin (Hb) 12.2 g/L, mean corpuscular volume 70.1 fL, Hb A 94.8%, and Hb A2 5.2% in the father and RBC 4.47×10 / L, Hb 10.6 g/L, MCV 72.1 fL, Hb A 93.6%, and Hb A2 6.4% in the mother. This was their first pregnancy. Reverse dot blots (RDB) were employed to detect the 18 known types of Chinese β-thalassemia mutations. This analysis identified an IVS-II-654 (C→T) mutation in the father but failed to identify any of the known mutations in the mother. Gap polymerase chain reaction (PCR) was also undertaken in the couple to exclude the coinheritance of α-thalassemia, and this did not reveal any of the three common Chinese α-globin gene deletion mutations (−, −α, and −α). Considering the unidentified mutation existing in one of the parents, cordocentesis was performed instead of amniocentesis for prenatal diagnosis at 18 weeks gestation. The cord blood was analyzed by automated high-performance liquid chromatography (HPLC) and showed that the Hb A level was 0% (Fig. 1), indicating that the fetus had homozygous βthalassemia [3–5]. The RDB analysis revealed that the fetus had inherited the paternal β-thalassemia mutation allele. The β-globin gene was amplified 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 disclosed that the mother and the fetus were heterozygous for a codons 41–43 (−CTTTG, +A) mutation (Fig. 2). This mutation causes a shift in the reading frame of the β-globin coding sequence and, consequently, a premature translation termination due to the creation of a stop codon at position 59. The control of messenger RNA (mRNA) stability plays key roles in both Ann Hematol (2008) 87:775–776 DOI 10.1007/s00277-008-0481-0

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.

Diversity in clinical presentation of hemoglobin H disease induced by the SEA deletion and the hemoglobin Quong Sze

Dear Editor, α-Thalassemia is commonly found in Southeast Asia and southern China. It results from defects in the α-globin genes and is characterized by absent or decreased rate of α-globin synthesis. Hemoglobin (Hb) H disease is the severest form of α-thalassemia compatible with postnatal life. The hallmark of this disease is the presence of excess β-globin chains forming tetramers (Hb H). Hb H is unstable and precipitates in the red cells, causing premature removal of the red cells from the circulation and splenomegaly. Genetically, there are two types of this disease [1]. The most common is the “deletional Hb H disease,” in which three out of four α-globin genes are taken away by deletions. The second type is the “nondeletional Hb H disease” which is a combination of deletional and nondeletional defects, that is, a deletion removes both α-globin genes on one allele, plus a point mutation, involving either the α2or α1-globin gene on the other allele. Hb H disease patients with nondeletional defects, especially those causing hyperunstable α-globin variants, are usually assumed to be more anemic, more symptomatic, more prone to have significant hepatosplenomegaly, and more likely to require transfusions [2, 3]. However, during our experience of hemoglobinopathy screenings for the past 15 years in Guangdong, a southern province of China, we found that the phenotypes of Hb H disease with a similar genotype are more variable than what have been expected. In this report, patients with Hb H disease induced by the common SEA deletion and the rare Hb Quong Sze but diverse clinical presentation are described.

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.