Amy Y.Y. Chan

Single-Tube Multiplex-PCR Screen for Anti-3.7 and Anti-4.2 α-Globin Gene Triplications

The coexistence of α-globin gene triplication (ααα) is an important modulator of the severity of β-thalassemia trait or β-thalassemia intermedia, exacerbating the phenotypic severity of β-thalassemia by causing more globin chain imbalance (1)(2). Typically, the inheritance of a single β-thalassemia allele is associated with mild anemia and hypochromic microcytic red cells. Compared with simple β-heterozygotes, co-inheritance of triplicated or quadruplicated α-globin genes in β-heterozygotes often leads to more significant anemia, splenomegaly, more pronounced red cell abnormalities, the presence of circulating normoblasts, higher hemoglobin F concentrations, and even the presence of inclusion bodies in erythroblasts (3)(4). Because the α- and β-globin gene clusters are physically unlinked and segregate independently, β-thalassemia carriers who also carry triplicated or quadruplicated α-globin genes have a 25% risk of having a similarly affected offspring, although their partners may be entirely normal. Triplicated α-globin genes appear to be ubiquitous and have been found in most populations (2). They result from misalignment and unequal crossover between the homologous X-, Y-, and Z-box segments of the α-globin gene cluster during meiosis (Fig. 1A⇓ ). Generally, two types of triplicated alleles can be generated from an unequal crossover, αααanti3.7 and αααanti4.2. If the crossover occurs between the homologous Z2 and Z1 boxes, also referred to as a “rightward crossover”, this produces a −α3.7 single-gene deletion allele and the reciprocal αααanti3.7 triplicated allele. However, if the crossover occurs between the X2 and X1 boxes (a “leftward crossover”), a −α4.2 single-gene deletion allele and the reciprocal αααanti4.2 triplicated allele are generated (5). A Sri Lankan study of individuals with severe to moderate β-thalassemia revealed a 2% frequency of α-globin gene triplications (6), whereas a preliminary study in Hong Kong suggests that the frequency …

Evaluation of hypermethylated tumor suppressor genes as tumor markers in mouth and throat rinsing fluid, nasopharyngeal swab and peripheral blood of nasopharygeal carcinoma patient

The purpose of our study was to evaluate the frequency of hypermethylated tumor suppressor genes (TSGs) in peripheral blood, mouth and throat (M&T) rinsing fluid and nasopharyngeal (NP) swabs of nasopharyngeal carcinoma (NPC) patients. Six normal NP tissues, 43 M&T rinsing fluid, 37 NP swabs and 43 peripheral blood from healthy non‐smokers and non‐drinkers without a family history of NPC, and 30 NPC tumors and their matched body fluid were analyzed for the presence of hypermethylated p15, p16, Ras association domain family 1 (RASSF1A), E‐cadherin, and death‐associated protein kinase (DAPK) by methylation‐specific PCR. Sequencing analysis was carried out on selected NPC tumors and body fluid samples. Twenty‐nine (97%) tumors displayed methylation in at least 1 of the 5 genes. The methylation frequencies were 80% for p15, 77% for DAPK, 67% for RASSF1A, 53% for E‐cadherin and 33% for p16. The frequency range of aberrant methylated genes in the body fluids were NP swabs (17–63%) and M&T rinsing fluid (17–50%). Methylation was found in <20% of peripheral blood for each respective gene. Methylation was, however, detected in 1 M&T rinsing fluid in which the primary tumor showed methylation free for RASSF1A. Five healthy individuals exhibited methylation for DAPK, or RASSF1A, or p15 in their body fluid samples. All body fluid samples of healthy controls showed methylation free for E‐cadherin and p16. Epigenetic change is found frequently in NPC and the high detection rate in body fluids suggest its potential application in non‐invasive screening of NPC or detection of residual carcinoma after treatment.

A laboratory strategy for genotyping haemoglobin H disease in the Chinese

Background: The thalassaemias are the commonest blood disorders worldwide, with South East Asia and southern China as areas of high prevalence. Accurate diagnosis of these disorders helps in clinical management with improved outcome. Methods: The α-globin genotypes of 100 Chinese patients in Hong Kong with haemoglobin H (Hb H) disease were characterised. Single-tube multiplex gap-PCR was used to detect −−SEA, −α3.7 and −α4.2, while Hb CS, Hb QS and codon 30 (ΔGAG) were identified by single-tube multiplex amplification refractory mutation system (ARMS). Automated direct nucleotide sequencing of the amplified α2- and α1-globin genes was performed to characterise other non-deletional α-thalassaemia determinants. Results: In the 100 cases studied, 99 cases had −−SEA in combination with deletional α+-thalassaemia or non-deletional α-globin gene mutation involving the α2-globin gene. In 70 cases of the deletional form, 43 cases showed the genotype of (−−SEA/−α3.7) and 27 cases of (−−SEA/−α4.2). Three of the 27 cases of (−−SEA/−α4.2) were found to have Hb Q-Thailand linked in-cis with −α4.2. The remaining 30 cases were of non-deletional form with the following genotypes: 11 cases of (−−SEA/αHbCSα), 9 cases of (−−SEA/αHbQSα), 3 cases of (−−SEA/αcd30 (ΔGAG)α), 3 cases of (−−SEA/αcd31α), 2 cases of (−−SEA/αpoly-Aα), 1 case of (−−SEA/αHbWestmeadα) and 1 case of (−−non-SEA/αHbQSα). Conclusions: Based on two rapid diagnostic tests, multiplex gap-PCR and multiplex ARMS, more than 90% of the cases were genetically characterised. This laboratory strategy should be widely applicable for genetic diagnosis of α-thalassaemia.

Hb A2 Hong Kong - A novel δ-globin variant in a chinese family masks the diagnosis of β-thalassemia trait

A 42-year-old Chinese woman (FP) was the mother of a patient with β-thalassemia major (β-TM) due to a compound heterozygosity for β0-thalassemia (β0-thal) mutations. She was also found to have a low Hb A2 level of 1.6% by high performance liquid chromatography (HPLC) despite being a heterozygous carrier of the codons 41/42 (–TCTT) (HBB:c.126_129delCTTT) β0-thal mutation. Doubling the amount of hemolysate loaded for chromatography revealed a widened Hb A2 peak and raised the level to 4.1%, consistent with β-thal trait. Direct nucleotide sequencing detected a novel δ-globin gene mutation at codon 29 (HBD:c.89G>A), which leads to a glycine to aspartic acid substitution. A homologous mutation at codon 29 in the β-globin gene [Hb Lufkin or β29(B11)Gly→Asp] has been reported in Black families. This report highlights the importance of genotype-phenotype correlation and the potential pitfall of relying on Hb A2 level for phenotypic diagnosis of β0-thal trait.

Routine Screening of (--SEA) α-Thalassemia Deletion by an Enzyme-Linked Immunosorbent Assay for Embryonic ζ-Globin Chains

We evaluated an enzyme-linked immunosorbent assay (ELISA) for embryonic ζ-globin chains as a routine screening test for (--SEA) α-thalassemia deletion (SEA deletion). A total of 174 consecutive patient samples with a request for Hb analysis were recruited. The ELISA method was evaluated against a polymerase chain reaction (PCR)-based technique that was taken as the standard. Among 56 simple carriers of SEA deletion diagnosed by PCR and 112 subjects without the SEA deletion, the sensitivity and specificity of the ELISA method was 89.3–96.4 and 98.2–100%, respectively, depending on the cutoff value for optical density that was adopted. The ELISA method was able to detect both subjects with SEA deletion and concurrent β-thalassemia trait in this series, but only 1 out of 4 patients (25%) with Hb H disease. We speculate that incomplete lysis of hypochromic microcytic red cells together with the low red cell count in Hb H disease might account for the false-negative results. We showed that the ELISA method for embryonic ζ-chains was a sensitive method of screening for SEA deletion carriers at our locality, and should be easily adopted in a routine diagnostic laboratory. The method was rapid and also amendable to automation. In areas with a high prevalence of α-thalassemia, improved detection of SEA deletion carriers would ultimately facilitate the identification of pregnancies at risk of hydrops fetalis and its prevention through prenatal diagnosis.

Multiple Minisequencing Screen for Seven Southeast Asian Nondeletional α-Thalassemia Mutations

α-Thalassemia is the most common globin disorder in the world, and the severe forms are especially prevalent among Southeast Asians. It is a disorder of absent or reduced production of α-globin chains resulting from mutations in the α-globin gene cluster on chromosome 16p13.3. Most α-thalassemia mutations involve deletions of one (−α) or both (− −) α-globin genes, whereas point mutations within the α-globin genes (αTα or ααT) are much less frequent. Nonetheless, the number of point mutations that have been described has been steadily increasing, with >40 identified to date (1)(2). The importance of nondeletional α-thalassemia mutations is underscored by the observation that patients with nondeletional hemoglobin (Hb) H disease (αTα/− −) are generally more severely affected and more likely to require transfusions compared with deletional Hb H disease patients (−α/− −) (3). There have also been a few reports of Hb H disease caused by homozygosity or compound heterozygosity of nondeletional mutations involving the α2-globin gene (αTα/αTα) (1)(3). Additionally, nondeletional Hb H disease involving the α2 codon 30 or codon 59 mutation can cause the fatal Hb H hydrops fetalis syndrome, especially if associated with large ζ-α-globin gene deletions (4). In certain regions of Southeast Asia, nondeletional Hb H disease can account for as many as 50% of all Hb H disease patients (3). Several specific and reliable molecular tests have previously been developed to diagnose and screen the most common deletional (5)(6) and nondeletional (7)(8) α-thalassemia mutations. We now describe a multiplex minisequencing assay to detect seven Southeast Asian nondeletional mutations: Hb Constant Spring (α2 codon 142 TAA→CAA), Hb Pakse (α2 codon 142 TAA→TAT), Hb Quong Sze (α2 codon 125 CTG→CCG), α2 codon 0 Δ1bp (−T), α2 …

The lack of association between JAK2 V617F mutation and myelodysplastic syndrome with or without myelofibrosis.

The lack of association between JAK2 V617F mutation and myelodysplastic syndrome with or without myelofibrosis

Myelodysplastic syndrome with myelofibrosis and basophilia : Detection of trisomy 8 in basophils by fluorescence in-situ hybridization

We report a case of myelodysplastic syndrome (MDS) with myelofibrosis and pulmonary tuberculosis who had marked basophilia in the peripheral blood. A clonal karyotypic abnormality characterized by trisomy 8 was demonstrated by cytogenetic analysis. By correlation of cell morphology with results of fluorescence in situ hybridization using a chromosome 8 probe, we demonstrated that the basophils were not reactive but belonged to the neoplastic MDS clone.

Novel Point Mutation of the α2-Globin Gene (HBA2) and a Rare 2.4 kb Deletion of the α1-Globin Gene (HBA1), Identified in Two Chinese Patients with Hb H Disease

Abstract Two Chinese patients with mild and moderate Hb H disease were investigated for rare mutations on the α-globin genes (HBA1, HBA2) in addition to the – –SEA deletion. One patient was a 41-year old man with mild anemia (Hb 11.3 g/dL). Multiplex ligation-dependent probe amplification (MLPA) revealed a rare 2392 bp deletion involving the entire HBA1 gene. Mapping by gap-polymerase chain reaction (gap-PCR) defined the exact breakpoints of this deletion (HBA1: g36859_39252del2392) and confirmed its identity with a recently reported HBA1 deletion found in a Southern Chinese. The other patient was a 53-year old man with moderate anemia (Hb 9.5 g/dL). Automated direct nucleotide (nt) sequencing identified a novel single nt deletion at codon 40 (HBA2: c.123delG). This leads to a frameshift that modifies the C-terminal sequence to (40)Lys-Pro-Thr-Ser-Arg-Thr-Ser-Thr(47)COOH and the introduction of a stop codon TGA 23 nts downstream. These two cases demonstrate the power of MLPA and direct nt sequencing to detect and characterize rare and novel mutations. They also highlight the differential effect of HBA1 and HBA2 gene mutations on an α-thalassemia (α-thal) phenotype due to their different transcriptional activity.

DOUBLE HETEROZYGOSITY FOR Hb NEW YORK [β113 GTG→GAG; VAL→GLU] AND β°-THALASSEMIA MUTATIONS MANIFESTS AS A THALASSEMIA TRAIT

An extended family with three individuals affected by two different forms of double heterozygosity for β-thalassemia and Hb New York is reported. Double heterozygosity of Hb New York [β113 GTG→GAG; VAL→GLU] and β° codon 17 was detected in a fetus following prenatal screening for thalassemia. The father and a paternal aunt were also found to be heterozygous for Hb New York and β° IVSII-654. Both adults had clinical and hematological features consistent with β-thalassemia trait. The affected child was followed up after birth and manifested the typical course of a thalassemia trait, with no signs of organomegaly or overt hemolysis. Observations strongly suggest that double heterozygosity of Hb New York and β° thalassemia has mild, if any, clinical symptoms, and is not an indication of therapeutic abortion when detected antenatally.