Supachai Ekwattanakit

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.

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.

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.

Worldwide study of hematopoietic allogeneic stem cell transplantation in pyruvate kinase deficiency

Pyruvate kinase deficiency (PKD) is the most frequent glycolytic enzyme defect causing hereditary non-spherocytic hemolytic anemia.[1][1] PKD leads to energy deprivation of the red cell, ultimately resulting in premature red cell death. Premature red cell death causes clinical symptoms of hemolytic

Estimating the burden of α-thalassaemia in Thailand using a comprehensive prevalence database for Southeast Asia

Severe forms of α-thalassaemia, haemoglobin H disease and haemoglobin Bart’s hydrops fetalis, are an important public health concern in Southeast Asia. Yet information on the prevalence, genetic diversity and health burden of α-thalassaemia in the region remains limited. We compiled a geodatabase of α-thalassaemia prevalence and genetic diversity surveys and, using geostatistical modelling methods, generated the first continuous maps of α-thalassaemia mutations in Thailand and sub-national estimates of the number of newborns with severe forms in 2020. We also summarised the current evidence-base for α-thalassaemia prevalence and diversity for the region. We estimate that 3,595 (95% credible interval 1,717 – 6,199) newborns will be born with severe α-thalassaemia in Thailand in 2020, which is considerably higher than previous estimates. Accurate, fine-scale epidemiological data are necessary to guide sustainable national and regional health policies for α-thalassaemia control. Our maps and newborn estimates are an important first step towards this aim. Funding This work was supported by European Union’s Seventh Framework Programme (FP7//2007-2013)/European Research Council [268904 – DIVERSITY]

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).

Interaction between Hb E and Hb Yala (HBB:c.129delT); a novel frameshift beta globin gene mutation, resulting in Hemoglobin E/β0 thalassemia

ABSTRACT Objectives: There are more than 200 known mutations found in patients with β-thalassemia, a possibility to identify an unknown or novel mutation becomes less possible. Here, we report a novel mutation in a patient from Thailand who presented with chronic hemolytic anemia. Methods: A comprehensive hematology and DNA analysis was applied in the index patient and her mother. Results: Hematological and hemoglobin analyses were consistent with the clinical diagnosis of Hb E/β0-thalassemia. However, we could find only Hb E heterozygous mutation using our common polymerase chain reaction-based mutation detection of the β-globin genes. Furthermore, the molecular analysis demonstrated a novel T-deletion at codon 42 of the second exon of the β-globin gene which we named ‘Hb Yala’ according to the origin of this index family. Discussion: This mutation was assumed to generate a truncated β-globin chain terminating at codon 60 with possible unstable variant leading to a ‘null’ or β0-thalassemia. However, the clinical phenotype was surprisingly mild and no other ameliorating genetic factors, including co-inheritance of α-thalassemia and high propensity of Hb F by Xmn I polymorphism, were found. Conclusion: This report has provided evidence that genotype–phenotype correlation in thalassemia syndromes is highly complex and a correct clinical severity classification of thalassemia should be mainly based on clinical evaluation.

A prospective analysis for prevalence of complications in Thai nontransfusion-dependent Hb E/β-thalassemia and α-thalassemia (Hb H disease)

Recently, complications in patients with nontransfusion‐dependent thalassemia (NTDT), in particular those with β‐thalassemia intermedia (β‐TI), were found to be significantly different from those in patients with transfusion dependent thalassemia (TDT), mainly β‐thalassemia major (β–TM). However, this information is rather limited in other forms of NTDT. In this prospective study, adult Thai NTDT patients were interviewed and clinically evaluated for thalassemia related complications. Fifty‐seven NTDT patients (age 18‐74 years), 59.6% Hb E/β‐thalassemia and 40.4% Hb H disease, were recruited; 26.4% were splenectomized. The most common complications were gallstones (68.4%), osteoporosis (26.3%), and pulmonary hypertension (15.8%). Splenectomy was associated with higher rate of gallstones and serious infection (P = .001 and .052, respectively), consistent with a multivariate analysis (RR = 9.5, P = .044, and RR = 15.1, P = .043, respectively). In addition, a higher hemoglobin level was inversely associated with gallstones in both univariate and multivariate analyses (P = .01 and .022, respectively). Serum ferritin was associated with abnormal liver function (P = .002). In contrast to the previous study, the prevalence of thrombosis was less common in our population (1.7%), probably due to differences in transfusion therapy, ethnicity, and underlying genotypes. For the first time, this prospective study provided the current prevalence of NTDT related complications in a Southeast Asian population with a different underlying genetic basis compared with previous studies. Although individual prevalence of each complication might differ from other studies, several important clinical factors such as splenectomy, degree of anemia, and iron overload seem to be determining risks of developing these complications consistently across different ethnicities.