Edmond S. K. Ma

BCL11A is a major HbF quantitative trait locus in three different populations with β-hemoglobinopathies ☆

Increased HbF levels or F-cell (HbF containing erythrocyte) numbers can ameliorate the disease severity of beta-thalassemia major and sickle cell anemia. Recent genome-wide association studies reported that single nucleotide polymorphisms (SNPs) in BCL11A gene on chromosome 2p16.1 were correlated with F-cells among healthy northern Europeans, and HbF among Sardinians with beta-thalassemias. In this study, we showed that SNPs in BCL11A were associated with F-cell numbers in Chinese with beta-thalassemia trait, and with HbF levels in Thais with either beta-thalassemia or HbE trait and in African Americans with sickle cell anemia. Taken together, the data suggest that the functional motifs responsible for modulating F-cells and HbF levels reside within a 3 kb region in the second intron of BCL11A.

Establishment of the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL mRNA.

Serial quantitation of BCR-ABL mRNA levels is an important indicator of therapeutic response for patients with chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia, but there is substantial variation in the real-time quantitative polymerase chain reaction methodologies used by different testing laboratories. To help improve the comparability of results between centers we sought to develop accredited reference reagents that are directly linked to the BCR-ABL international scale. After assessment of candidate cell lines, a reference material panel comprising 4 different dilution levels of freeze-dried preparations of K562 cells diluted in HL60 cells was prepared. After performance evaluation, the materials were assigned fixed percent BCR-ABL/control gene values according to the International Scale. A recommendation that the 4 materials be established as the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL translocation by real-time quantitative polymerase chain reaction was approved by the Expert Committee on Biological Standardization of the World Health Organization in November 2009. We consider that the development of these reagents is a significant milestone in the standardization of this clinically important test, but because they are a limited resource we suggest that their availability is restricted to manufacturers of secondary reference materials.

A 3-bp deletion in the HBS1L-MYB intergenic region on chromosome 6q23 is associated with HbF expression

Fetal hemoglobin (HbF) is regulated as a multigenic trait. By genome-wide association study, we confirmed that HBS1L-MYB intergenic polymorphisms (HMIP) and BCL11A polymorphisms are highly associated with HbF in Chinese β-thalassemia heterozygotes. In this population, the variance in HbF resulting from the HMIP is 13.5%; that resulting from the BCL11A polymorphism is 6.4%. To identify the functional variant in HMIP, we used 1000 Genomes Project data, single nucleotide polymorphism imputation, comparisons of association results across populations, potential transcription factor binding sites, and analysis of phylogenetic conservation. Based on these studies, a hitherto unreported association between HbF expression and a 3-bp deletion, between 135 460 326 and 135 460 328 bp on chromosome 6q23 was found. This 3-bp deletion is in complete linkage disequilibrium with rs9399137, which is the single nucleotide polymorphism in HMIP most significantly associated with HbF among Chinese, Europeans, and Africans. Chromatin immunoprecipitation assays confirmed erythropoiesis-related transcription factors binding to this region in K562 cells. Based on transient expression of a luciferase reporter plasmid, the DNA fragment encompassing the 3-bp deletion polymorphism has enhancer-like activity that is further augmented by the introduction of the 3-bp deletion. This 3-bp deletion polymorphism is probably the most significant functional motif accounting for HMIP modulation of HbF in all 3 populations.

Desmopressin does not decrease blood loss and transfusion requirements in patients undergoing hepatectomy

PurposeTo determine the effects of desmopressin on coagulation and blood loss in patients undergoing elective partial hepatectomy.MethodsA randomized, controlled and double-blind study on 59 patients who received either 0.3 μg·kg−1 of desmopressin or an equal volume of normal saline (control) infused intravenously over 20 min after induction of general anesthesia.ResultsThere was an increase in plasma levels of factors VIII and von Willebrand after the infusion of study drug in both groups (P < 0.001). The activated partial thromboplastin time was shortened in Group D whereas prothrombin time was prolonged in Group C; (P = 0.02). A large range of intraoperative blood loss (400–7128 mL) was observed, with no significant differences between groups. There were no changes in plasma electrolyte levels or osmolality. Transfusion requirements were similar in both groups.ConclusionDesmopressin did not reduce intraoperative blood loss or transfusion requirements during hepatectomy despite raising clotting factor levels and improving tests of hemostasis.RésuméObjectifDéterminer les effets de la desmopressine sur la coagulation et les pertes sanguines pendant une hépatectomie partielle réglée.MéthodeIl s’agit d’une étude randomisée, contrôlée et à double insu auprès de 59 patients qui ont reçu, soit 0,3 μg·kg−1 de desmopressine ou un volume égale de soluté normal (témoin) administré par voie intraveineuse 20 min au moins après l’induction de l’anesthésie générale.RésultatsOn a noté une augmentation des niveaux plasmatiques des facteurs VIII et von Willebrand à la suite de la perfusion du médicament expérimenté chez les patients des deux groupes (P < 0,001). Le temps de céphaline activé a été plus court dans le groupe D tandis que le temps de prothrombine a été plus long dans le groupe T ; (P = t0,02). D’importants écarts ont été observés pour les pertes sanguines peropératoires (400–7128 mL), mais sans différence significative intergroupe. Aucune modification des niveaux plasmatiques d’électrolyte ou d’osmolalité n’a été notée. Les besoins de transfusion ont été comparables dans les deux groupes.ConclusionLa desmopressine ne réduit pas les pertes sanguines ou les besoins de transfusion peropératoires pendant l’hépatectomie, malgré l’élévation des niveaux de facteurs de coagulation et l’amélioration de l’hémostase.

Establishment and Characterization of HKESC-1, a New Cancer Cell Line from Human Esophageal Squamous Cell Carcinoma

The establishment of an esophageal cancer cell line can facilitate the search for molecular mechanisms involved in esophageal carcinogenesis. A new human cancer cell line, HKESC-1, was established from a primary moderately-differentiated squamous cell carcinoma of the esophagus from a 47-year-old Hong Kong Chinese man. The pathological characteristics (morphology, immunohistochemical, and electron microscopic studies), the tumorigenecity in nude mice, the cytogenetic features, the DNA ploidy, and telomerase activity of the cell line were investigated. The HKESC-1 cells have been maintained continuously in vitro for more than 16 months and passaged over 96 times. HKESC-1 cells grow as a monolayer, with a doubling time of 46 hours. The HKESC-1 cells are of a squamous epithelial origin, as shown by their immunopositivity with the anti-cytokeratin antibodies and ultrastructural demonstration of tonofilaments and desmosomes. The HKESC-1 cells possess characteristics of malignancy because they are highly tumorigenic in nude mice and have strong telomerase activity. The HKESC-1 cells had an aneuploid DNA content, as demonstrated by flow cytometric analysis. Cytogenetic analysis revealed hyperdiploidy of greater than 50 in 80% of analyzable metaphases. Chromosome gains and losses were common, and loss of the Y chromosome was a consistent numerical aberration. Additionally, many structural chromosomal abnormalities were encountered, with frequent breakpoints at 1p32, 7p22, 7q34, and 20q13. This newly established cell line serves as a useful model for studying the molecular pathogenesis, and testing new therapeutic reagents for esophageal squamous cell carcinoma.

Establishment, characterization, karyotyping, and comparative genomic hybridization analysis of HKESC-2 and HKESC-3: two newly established human esophageal squamous cell carcinoma cell lines

The establishment of esophageal cancer cell lines can facilitate the search for molecular mechanisms underlying its pathogenesis. Two novel human esophageal squamous cell carcinoma (ESCC) cell lines, HKESC-2 and HKESC-3, were established from a moderately differentiated ESCC of a 46-year-old Chinese woman and a well-differentiated ESCC of a 74-year-old Chinese man, both from Hong Kong. The pathological characteristics (morphological, immunohistochemical, and electron microscopic studies), tumorigenicity in nude mice, cytogenetic features, and DNA ploidy of the two cell lines were investigated. The two cell lines have been maintained in vitro for more than 17 months and passaged over 85 times for HKESC-2 and 58 times for HKESC-3. Both grew as monolayers, with a doubling time of 24 hours for HKESC-2 and 48 h for HKESC-3. Their squamous epithelial nature was authenticated by their strong immunopositivity with the anti-cytokeratin antibodies and the ultrastructural demonstration of tonofilaments and desmosomes. They are tumorigenic in nude mice and had DNA aneuploidy. G-banding cytogenetic analysis showed hyperdiploidy in HKESC-2 and near-tetraploidy in HKESC-3. Frequent breakpoints were noted at 1p22, 1p32, and 9q34 in HKESC-2 and at 1p31, 3p25, 3p14, 6q16, 6q21, 8p21, 9q34, 13q32, and 17q25 in HKESC-3. Comparative genomic hybridization analysis found that chromosomal gains were at 3q24-qter, 5q21-qter, 8q11-qter, 13q21-q31, 17q11-qter, 19, 22q22 for HKESC-2 and at 3q13-qter, 5p, 6p, 9q21-qter, 10q21-q22, 12q15-pter, 14q24-qter, 16, 17q24-qter, 20 for HKESC-3. Chromosomal losses were at 3p13-pter, 18q12-qter for HKESC-3. These two newly established cell lines will be useful tools in the study of the molecular pathogenesis and biological behavior of ESCC cells and for testing new therapeutic reagents for ESCC in the future.

Prevalence and specificity of clinically significant red cell alloantibodies in Chinese women during pregnancy – a review of cases from 1997 to 2001

Summary.  Guidelines for the prevention and management of red cell alloantibodies during pregnancy, related to anti‐D in particular, are well established in Caucasian populations. However, because of the racial difference of the blood group distribution, applicability to Chinese is unknown as a result of insufficient data on the prevalence and their outcome. In a retrospective review of 28 303 (21 327 Chinese) antenatal attendances from 1997 to 2001, 213 (0·79%) women were found to have a total of 230 irregular antibodies. About 137 (0·64%) were ethnic Chinese, and a total of 160 irregular antibodies were identified in their blood samples. About 58 of these Chinese women (0·27%) were found to have 66 clinically significant antibodies. There was only one case of anti‐D detected in an Rh(D)‐negative subject. Our study shows the overall prevalence of clinically significant antibodies in Chinese women, which was not different from that of the Western population. However, the specificities of the antibodies differ with the commonest antibodies encountered; these being anti‐Mi (57·6%), anti‐E (19·7%), anti‐S (10·6%) and anti‐c (7·6%). Neonatal jaundice was observed in 37 babies and 10 of them required phototherapy. The findings support the previous recommendation that routine antenatal antibody screening for Chinese women may not be worthwhile except in Rh(D)‐negative subjects or those with an antecedent history of haemolytic disease of the newborn (HDN). The relative high incidence of anti‐Mi in the present study and the local population, in general, may warrant a large‐scale prospective study of pregnancy outcome in these subjects, especially in the light of the previous case reports of HDN because of anti‐Mi.

Increased Alpha 7 Nicotinic Acetylcholine Receptor Protein Levels in Alzheimer’s Disease Patients

We compared the intact α7 nicotinic acetylcholine receptor (α7nAChR) protein levels in the peripheral blood leukocytes in 15 Alzheimer’s disease (AD) patients and 13 normal elderly control subjects. Demographic data and Mini-Mental State Examination (MMSE) scores were obtained. Western blot analysis for α7nAChR protein levels in peripheral blood leukocytes was performed. There were no significant differences in sex and age between the AD and control groups. The mean MMSE score of the AD subjects was significantly lower than that of the normal control subjects (15.4 ± 5.5 vs. 28.5 ± 1.9 respectively; p < 0.001). The median value of normalized α7nAChR protein levels (optical density, arbitrary unit) of the AD group was significantly higher than that of the normal control group (0.6923 vs. 0.4803 respectively; p = 0.045, Mann-Whitney U test). The normalized α7nAChR protein levels showed a significant inverse correlation with the MMSE scores (Spearman rho = –0.45; p = 0.016; n = 28). Receiver Operating Characteristic curve analyses showed that the area under curve was 0.72 (95% CI 0.52– 0.87). If the cut-off of the α7nAChR protein level was >0.312, the sensitivity, specificity, positive predictive value and negative predictive value would be 80, 39, 60 and 63%, respectively. These findings showed that the α7nAChR protein levels would be a potentially useful diagnostic marker for AD.

Variation and heritability of Hb F and F-cells among β-thalassemia heterozygotes in Hong Kong

Enhanced fetal hemoglobin (Hb F) production can partially compensate for the lack of adult hemoglobin (Hb A) in patients with β‐thalassemia major or intermedia, and ameliorate the clinical severity of these diseases. To further elucidate factors governing Hb F levels, we evaluated demographic, clinical, laboratory, and genetic characteristics in 241 unrelated adult β‐thalassemia carriers in Hong Kong. They had wide variations in Hb F and F‐cell numbers skewing toward higher levels. Individuals who coinherited the Xmn IT‐allele in the Gγ‐globin gene promoter had higher Hb F and more F‐cells compared with those lacking the Xmn I T‐allele. However, both groups exhibited a similarly wide spread of Hb F and F‐cells. The correlation of Hb F and F‐cells corresponded well to both linear and exponential models, suggesting multiple mechanisms for Hb F augmentation. The heritabilities of Hb F and F‐cells were calculated in 66 families (111 parents who were β‐thalassemia carriers and 82 asymptomatic offspring) to be 0.7 to 0.9. The Xmn I polymorphism accounted for 9% of the Hb F and 13% of the F‐cell heritabilities. These results suggest that these family members are well suited for genome wide association studies that will identify genetic loci regulating Hb F production, and likely novel pharmacological targets for reactivating Hb F production in adults. Am. J. Hematol., 2008.

The spectrum of acute lymphoblastic leukemia with mature B-cell phenotype.

We showed heterogeneous disease spectrum among 15 acute lymphoblastic leukemia (ALL) cases with mature B-cell phenotype diagnosed over the past 7 years at our institution. Besides those with typical L3 morphology and 8q24 (c-myc) translocation (n=6), there were cases showing L1 or L2 morphology without 8q24 translocation (n=6), unusually large L3 blasts in hyperdiploid clone (n=2) and blastoid variant of mantle cell lymphoma (n=1). The expression of CD5 and cyclin D1 may need to be routinely determined on ALL cases with mature B-cell phenotype and non-L3 morphology to facilitate timely diagnosis of blastoid MCL and institution of suitable management.