Kalaya Tachavanich

Mutations in Krüppel-like factor 1 cause transfusion-dependent hemolytic anemia and persistence of embryonic globin gene expression

In this study, we report on 8 compound heterozygotes for mutations in the key erythroid transcription factor Krüppel-like factor 1 in patients who presented with severe, transfusion-dependent hemolytic anemia. In most cases, the red cells were hypochromic and microcytic, consistent with abnormalities in hemoglobin synthesis. In addition, in many cases, the red cells resembled those seen in patients with membrane defects or enzymopathies, known as chronic nonspherocytic hemolytic anemia (CNSHA). Analysis of RNA and protein in primary erythroid cells from these individuals provided evidence of abnormal globin synthesis, with persistent expression of fetal hemoglobin and, most remarkably, expression of large quantities of embryonic globins in postnatal life. The red cell membranes were abnormal, most notably expressing reduced amounts of CD44 and, consequently, manifesting the rare In(Lu) blood group. Finally, all tested patients showed abnormally low levels of the red cell enzyme pyruvate kinase, a known cause of CNSHA. These patients define a new type of severe, transfusion-dependent CNSHA caused by mutations in a trans-acting factor (Krüppel-like factor 1) and reveal an important pathway regulating embryonic globin gene expression in adult humans.

Lymphocyte subsets and specific T-cell immune response in thalassemia.

Infection is very common in thalassemia and is one of the major causes of death. To date, it is not quite clear why these patients are susceptible to infection. In this study, lymphocyte immunophenotyping for CD3(+) (T-cells), CD3(+)CD4(+) (T-helper/inducer cells), CD3(+)CD8(+) (T-suppressor/cytotoxic cells), CD3(-)CD19(+) (B-cells), and CD3(-)CD16/56(+) (natural killer cells) subsets and expression of the activation antigen CD69 on CD3(+)CD4(+) and CD3(+)CD8(+) T-cells were determined in the whole blood of thalassemia patients, using a three-color flow cytometric technique. Results showed that only splenectomized beta-thalassemia/hemoglobin (Hb) E patients displayed a marked increase in absolute number of all lymphocytes. In addition, splenectomized beta-thalassemia/Hb E showed a significantly lower percentage of CD3(+) cells, with a corresponding increase in CD19(+) cells. These differences, when compared with normal subjects and other thalassemia patients, may be attributed to splenectomy. alpha-thalassemia patients, on the other hand, showed no significant difference from the normal group. While lymphocyte subsets in splenectomized beta-thalassemia/Hb E patients showed an abnormal distribution, T-cell activation in these patients was not different from the activation seen in normal subjects. This implies that thalassemia patients, during the steady state of disease, appear to have normal T-lymphocyte function with only moderate abnormalities of T- and B-lymphocyte subsets.

Prevalence of HFE mutations among the Thai population and correlation with iron loading in haemoglobin E disorder

Abstract:  Co‐inheritance of HFE mutations has a substantial role in iron overload in β‐thalassaemia carriers in north European populations where two HFE mutations, C282Y and H63D, are prevalent. In Thailand, there was little information about the allele frequency of HFE mutations. It is of interest to determine whether such determinants represent a potential risk in developing iron overload as nearly 40% of the Thai population carry either one of thalassaemia or haemoglobinpathy alleles. A total of 380 normal controls from five different regions including Bangkok were screened for the HFE C282Y, H63D and IVS5+1 G→A alleles. In addition, 70 individuals with homozygous haemoglobin E (Hb EE) were also tested and their genotypes were correlated with levels of serum ferritin. H63D is the major HFE mutation found in the Thai population with an average allele frequency of 3% (range 1–5%). One individual was heterozygous for the splice site mutation IVS5 + 1 G → A, and the C282Y allele was not detected. In the Hb EE group, five individuals had iron deficiency (ferritin <12 μg/L) and the remaining 65 individuals had a wide range of serum ferritin levels of 16–700 μg/L. Four individuals with Hb EE were heterozygous for the H63D allele. No significant difference in serum ferritin level was detected in this group with or without the HFE mutation (137.2 ± 78 vs. 116.3 ± 128 μg/L). HFE mutations are relatively uncommon among the Thai population, and the average allele frequency of the ancient H63D mutation is similar to that of other countries in this region. Because of their paucity, it appears that these alleles are less likely to be responsible for high ferritin levels and iron loading in individuals with Hb E related disorders.

Clinical Presentation and Molecular Identification of Four Uncommon Alpha Globin Variants in Thailand

Alpha thalassemia is the most common genetic disease in the world with the prevalence of carriers ranging from 5-50% in several populations. Coinheritance of two defective α-globin genes usually gives rise to a symptomatic condition, hemoglobin (Hb) H disease. Previously, it has been suggested from several studies in different populations that nondeletional Hb H disease (--/αTα or --/ααT) is generally more severe than the deletional type (--/-α). In this report, we describe four rare nondeletional α-thalassemia mutations in Thai individuals, including initiation codon mutation (HBA2:c.1delA), donor splice site mutation (IVSI-1, HBA1:c.95 + 1G>A), Hb Queens Park (HBA1:c.98T>A) [α32(B13)Met>Lys], and Hb Westmead (HBA2:c.369C>G) [α122(H5)His>Gln]. Interactions of the first three mutations with the α⁰-thalassemia resulted in nondeletional Hb H disease; however, their clinical presentations were rather mild and some were detected accidentally. This suggests that a genotype-phenotype correlation of α-thalassemia syndrome might be more heterogeneous and so the type of mutation does not simply imply the prediction of the resulting phenotype. Our data will be of use in future genetic counseling of such conditions that are increasingly identified thanks to the improvement of molecular analysis in routine laboratories.

Flow cytometric quantitation of opsonophagocytosis and intracellular killing of Candida albicans using a whole blood microassay

Flow cytometric assays for quantifying polymorphonuclear leucocyte (PMN) function involve peripheral blood fractionation methods. However, most are of limited use for conditions like pediatrics and thalassemia, as they may require large blood volumes or complicated by red blood cell contamination. In whole blood assay, 500‐μl aliquots of normal or thalassemic whole blood were incubated with bis‐carboxyethyl‐carboxyfluorescein pentaacetoxymethylester (BCECF‐AM)‐labeled Candida albicans cells and phycoerythrin‐conjugated anti‐CD13 monoclonal antibody for PMNs. Phagocytosis was quantitated by gating on CD13 label PMNs and determining the frequency of phagocytosed yeast using flow cytometry. The killing activity was quantitated by estimating the number of viable fluorescence retaining yeast cells liberated following lysis of PMNs. In normal control or thalassemia samples, nonphagocytosed yeast and PMNs with or without phagocytosed yeast were readily distinguished by two‐color dot plot, permitting phagocytosis estimates. Viable and nonviable yeast killed by whole blood PMNs were distinguished by side scatter and fluorescence, permitting killing estimates. The patterns seen using whole blood was similar to that seen with fractionated PMNs. Data from thalassemia patients showed similar opsonophagocytosis but slightly decreased intracellular killing of yeast cells when compared with normal subjects. This assay provides an alternative method to assess PMN function in small blood samples, which could be especially useful in conditions like thalassemia and pediatric patients.

Association of Xmn I Polymorphism and Hemoglobin E Haplotypes on Postnatal Gamma Globin Gene Expression in Homozygous Hemoglobin E

Background and Objectives. To explore the role of cis-regulatory sequences within the β globin gene cluster at chromosome 11 on human γ globin gene expression related to Hb E allele, we analyze baseline hematological data and Hb F values together with β globin haplotypes in homozygous Hb E. Patients and Methods. 80 individuals with molecularly confirmed homozygous Hb E were analyzed for the β globin haplotypes and Xmn I polymorphism using PCR-RFLPs. 74 individuals with complete laboratory data were further studied for association analyses. Results. Eight different β globin haplotypes were found linked to Hb E alleles; three major haplotypes were (a) (III), (b) (V), and (c) (IV) accounting for 94% of Hb E chromosomes. A new haplotype (Th-1) was identified and most likely converted from the major ones. The majority of individuals had Hb F < 5%; only 10.8% of homozygous Hb E had high Hb F (average 10.5%, range 5.8–14.3%). No association was found on a specific haplotype or Xmn I in these individuals with high Hb F, measured by alkaline denaturation. Conclusion. The cis-regulation of γ globin gene expression might not be apparent under a milder condition with lesser globin imbalance such as homozygous Hb E.

The origin of sickle cell disease in Thailand

Sickle cell disease (SCD) is one of the most common monogenic diseases in the world, with serious complications including chronic anaemia, pain crises, acute chest syndrome, stroke, splenic dysfunction, nephropathy, pulmonary hypertension and avascular necrosis.1 The Hb S allele is found to be in linkage disequilibrium with five major haplotypes in the βglobin gene cluster, and haplotype analysis has provided insight into historic migration patterns of the human race. Despite its global geographical distribution, Hb S is rare in Thailand,2 and its origins in this country have never been characterised. We report three cases of Hb S/β0thalassaemia within a family from Chiang Mai, Thailand. A 31yearold Thai woman who recently moved to Cardiff, United Kingdom, was diagnosed with SCD following a pregnancy. She carried a prior diagnosis of thalassaemia trait after presenting for anaemia with easy fatigability at age 15. She reported receiving 5 blood transfusions during her lifetime and being hospitalised at age 20 for severe bone pain. Her family history was significant for two younger sisters with “Hb S disease”. The middle sister presented with anaemia requiring transfusion at age 9 and was diagnosed with SCD at that time. Her reported baseline Hb was 68 g/dL, and her SCD was complicated by recurrent pain crises, requiring intermittent hospitalisations for chest pain, bone pain, and arthralgias, and treatment with morphine. Her hospitalisations for pain decreased in frequency in adulthood, but she required blood transfusion therapy again during her pregnancy at age 25. The youngest sister had a history of neonatal jaundice and an initial episode of anaemia and jaundice at age 3, followed by subsequent episodes of symptomatic anaemia requiring repeated blood transfusion, and frequent chest pain and diffuse arthralgia that improved with transfusion and analgesics. However, she was initially diagnosed with somatoform disorder and was not diagnosed with SCD until age 12, when she presented to a local hospital with sudden onset of leftsided hemiparesis and ataxia, found to have acute ischaemic stroke. Workup of the stroke revealed a stenosis on neck ultrasound and the diagnosis of SCD. She was started on regular transfusion therapy for secondary stroke prevention, with concurrent phlebotomy and iron chelation with deferiprone due to secondary iron overload (serum ferritin 1800 μg/L). She recovered without any residual neurological deficits, and a followup neck ultrasound at age 16 was normal. The three SCD cases and their immediate family members were interviewed, examined and underwent haematological and genetic testing for haemoglobinopathies, including whole sequence analysis for αand βglobin genes, followed by polymerase chain reactionrestriction fragment length polymorphism (PCRRFLP) to confirm αand βglobin mutations. Laboratory findings for the eldest (II1), middle (II2) and youngest (II4) cases were consistent with SCD (Table 1). Molecular analysis revealed that the three cases had the same genotype of Hb S/β0thalassaemia, not Hb S disease (ie homozygous Hb S). Additional investigation identified the father (I1) as a Hb S carrier, while the mother (I2) and brother (II3) were found to be β0thalassaemia carriers (HBB:c.92+1G>T). It was surprising that the two younger cases had notably more severe SCD phenotypes than the eldest case. Therefore, direct genomic sequencing and a gapPCR based assay for αglobin mutations were also performed to investigate potential genetic modifiers of disease severity. Both the father (I1) and the eldest case (II1) were discovered to be heterozygous for Hb Westmead (αWM), a nondeletional α2globin mutation (HBA2:c.369C>G). Finally, in order to identify the origins of this Hb S allele in a Thai family, βglobin gene haplotypes were analysed using the presence (+) or absence (−) of 8 restriction sites for each family member. The βS mutation was found to be located in the ArabIndian haplotype, while the βIVS-I-1 mutation was found on the haplotype IX (Figure 1).