Voravarn S. Tanphaichitr

Novel AE1 mutations in recessive distal renal tubular acidosis. Loss-of-function is rescued by glycophorin A.

The AE1 gene encodes band 3 Cl-/HCO3- exchangers that are expressed both in the erythrocyte and in the acid-secreting, type A intercalated cells of the kidney. Kidney AE1 contributes to urinary acidification by providing the major exit route for HCO3- across the basolateral membrane. Several AE1 mutations cosegregate with dominantly transmitted nonsyndromic renal tubular acidosis (dRTA). However, the modest degree of in vitro hypofunction exhibited by these dRTA-associated mutations fails to explain the disease phenotype in light of the normal urinary acidification associated with the complete loss-of-function exhibited by AE1 mutations linked to dominant spherocytosis. We report here novel AE1 mutations linked to a recessive syndrome of dRTA and hemolytic anemia in which red cell anion transport is normal. Both affected individuals were triply homozygous for two benign mutations M31T and K56E and for the loss-of-function mutation, G701D. AE1 G701D loss-of-function was accompanied by impaired trafficking to the Xenopus oocyte surface. Coexpression with AE1 G701D of the erythroid AE1 chaperonin, glycophorin A, rescued both AE1-mediated Cl- transport and AE1 surface expression in oocytes. The genetic and functional data both suggest that the homozygous AE1 G701D mutation causes recessively transmitted dRTA in this kindred with apparently normal erythroid anion transport.

Molecular basis of β-thalassemia in Thailand: analysis of β-thalassemia mutations using the polymerase chain reaction

Summaryβ-Thalassemia mutations in 71 chromosomes of Thai patients from the northeast, the middle and the south of the country were investigated using dot blot hybridization of PCR (polymerase chain reaction)-amplified DNA with allelespecific oligonucleotide probes. Eight different known molecular defects were detected, at different frequencies. There was an amber mutation in codon 17, a C-T transversion at position 654 of IVS-2, a frameshift mutation between codons 71 and 72, an A-G transition at nucleotide -28 within the TATA box (known as Chinese mutations), a G-T transversion at position 1 of IVS-1 (an Indian mutation), a 4bp deletion in codons 41/42 and a G-C transversion at position 5 of IVS-1 (described as both Chinese and Indian mutations) and a Thai original mutation, an ochre mutation in codon 35. Analysis of the three unknown alleles by DNA sequencing of the cloned DNA fragment amplified by PCR revealed an A-G substitution at the second position of the codon for amino acid 19 (AAC-AGC). The analytic approach used in the present study and the characteristic distribution of mutations in each region of Thailand will prove useful for setting up a prenatal diagnosis program.

Renal tubular function in β-thalassemia

Abstract. Studies of the renal involvement in thalassemic syndromes have been varied and few. This study was designed to define the renal abnormalities associated with β-thalassemia and to correlate the renal findings with clinical parameters. One hundred and four β-thalassemic children with various disease severity were studied. The patients were divided into three groups: 48 with severe anemia [hematocrit (Hct) <25%], 31 on a hypertransfusion program and desferrioxamine treatment, and 25 with moderate anemia (Hct >25%). The results were compared with 15 normal children. Significantly higher levels of proteinuria and low molecular weight proteinuria were found in all patients compared with normal children. Aminoaciduria was detected in one-third of patients. Thalassemic patients had significantly lower morning urine osmolarity, higher urine N-acetyl-β-D-glucoseminidase and malondialdehyde (MDA, an indicator of lipid peroxidation). Patients with severe anemia had significantly higher low-molecular weight proteinuria and MDA, and lower urine osmolarity than those with moderate anemia. Our data confirmed the high frequency of renal abnormalities in β-thalassemia patients and indicated some degree of proximal tubular dysfunction. Severity of the abnormalities correlated with the degree of anemia and were least severe in patients on hypertransfusion and desferrioxamine therapy. This suggested that the damage might be caused by anemia and increased oxidation induced by excess iron deposits.

Malignant lymphoma in Thailand: changes in the frequency of malignant lymphoma determined from a histopathologic and immunophenotypic analysis of 425 cases at Siriraj Hospital

BACKGROUND Analysis of malignant lymphoma in a single institution at different periods of time can determine the changing status of the disease in the region. METHODS To compare with the large series of 1095 lymphoma cases reported between 1957-1971 at Siriraj Hospital, the largest hospital in Thailand, a similar study was performed through histopathologic evaluation of 425 lymphoma cases diagnosed consecutively at the same institution between August 1993 and October 1995. Phenotypic analysis was performed by paraffin section-immunoperoxidase studies. RESULTS A striking increase in lymphoma cases was noted from 73 cases/year in the first series to 189 cases/year in the second series (an increase of 158.9%). Lymphoma occurred in all age groups, with a peak incidence at the seventh decade of life. The male to female ratio decreased from 2:1 in 1957-1971 to 1.3:1 in the more recent series. The incidence of Hodgkins disease (HD) was found to have decreased from 28.9% to 8.5%. There were 36 cases (8.5%) of HD and 389 cases (91.5%) of non-Hodgkins lymphoma (NHL) reported in the second series. The subtypes of HD included 16 cases of mixed cellularity, 13 cases of nodular sclerosis, 6 cases of lymphocyte depletion, and 1 case of lymphocyte predominance. According to the Working Formulation, the 389 NHL cases included low grade (14.1%), intermediate grade (57.3%), high grade (11.3%), and miscellaneous groups (17.2%). They were classified as small lymphocytic (9.5%), follicular (11.1%), diffuse (50.9%), immunoblastic (4.1%), small noncleaved (4.4%), lymphoblastic (2.8%), anaplastic large cell (9.0%), mycosis fungoides (1.8%), hairy cell leukemia (0.3%), true histiocytic (0.5%), and extramedullary plasmacytoma (1.0%). The immunophenotypes of the 359 NHL cases available for paraffin section-immunoperoxidase studies were B-cell (71.0%), T-cell (24.5%), histiocyte (0.6%), and undetermined phenotypes (3.9%). CONCLUSIONS The incidence of malignant lymphoma is increasing in Thailand, with a high frequency of intermediate to high grade NHL of B-cell phenotype reported.

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.

Deferiprone (GPO‐L‐ONE®) monotherapy reduces iron overload in transfusion‐dependent thalassemias: 1‐year results from a multicenter prospective, single arm, open label, dose escalating phase III pediatric study (GPO‐L‐ONE; A001) from Thailand

Accessibility to iron chelators including deferoxamine and deferasirox remains obscured in many developing countries. To provide an alternative, the government pharmaceutical organization of Thailand (GPO) manufactured deferiprone which has similar bioequivalent to the standard product. Seventy‐three pediatric patients with severe β thalassemias, age range 3.2–19 years, were recruited to a 1‐year multicenter prospective, single arm, open label, dose escalating Phase III study of deferiprone to determine its clinical efficacy and safety. Sixty‐four patients (87.6%) completed the study with good compliance (>94%). Average deferiprone dose was 79.1±4.3 mg/kg/day. Overall, mean serum ferritin (SF) levels at 1 year were not significantly changed from baseline. However, 45% of patients (response group) had SF reduced >15% from baseline at 1 year with a median reduction of 1,065 ng ml−1. Baseline SF was the major factor that predicts clinical efficacy; patients with baseline SF>3,500 ng ml−1 had the most significant fall of SF at 1 year. A subgroup analysis by MRI‐T2* confirmed that the response group had higher baseline liver iron and deferiprone could significantly reduce liver iron overload and normalize levels of ALT at 1 year. Although, gastrointestinal irritation (20.5%) was the most common drug‐related adverse events (AEs) followed by transaminitis (16.4%) and neutropenia (6.8%), all patients were well tolerated. There was no mortality and agranulocytosis found in this trial. Monotherapy of deferiprone with appropriate dose adjustment and monitoring for adverse events appeared to be an effective chelation therapy in some patients with good compliance and acceptable safety profiles. Am. J. Hematol. 88:251–260, 2013.

Complex interactions of δβ hybrid haemoglobin (Hb Lepore‐Hollandia) Hb E (β26 G→A) and α+ thalassaemia in a Thai family

Abstract: Haemoglobin Lepore‐Hollandia is an extremely rare condition in which a small deletion gives rise to a δβ hybrid, β‐like globin. There are two single reports of patients from South Pacific Islands and Bangladesh. We describe a family from central Thailand, in which this Hb Lepore‐Hollandia interacts with a common β globin variant (βE resulting from the codon 26, G→A mutation) and α+ thalassaemia (−α3.7). This intriguing interaction caused a troublesome diagnosis, as the two proband brothers were diagnosed as having Hb E/β thalassaemia. Molecular analysis of genomic DNA performed in this study allowed the definitive diagnosis of this complicated interaction. Such studies are required in the diagnosis of thalassaemia and haemoglobinopathies for particular regions like South‐east Asia, where many different genotypes may give rise to haemoglobin disorders.

Homozygous hemoglobin Tak causes symptomatic secondary polycythemia in a Thai boy.

Secondary polycythemia caused by high-oxygen-affinity hemoglobin is rare in children. Most patients with this condition have asymptomatic erythrocytosis. In this article the authors describe a young boy from Thailand with plethora, hypoxemia, and aggravated respiratory distress following a chest infection. Hematological and molecular studies revealed that the boy is homozygous for Hb Tak, an extended &bgr;-globin variant with high oxygen affinity. This report of a patient who is homozygous for high-oxygen-affinity hemoglobin highlights the clinical significance of this hemoglobin disorder, which has been previously reported in several unrelated families from Southeast Asia.

COMPOUND HETEROZYGOSITY FOR α0-THALASSEMIA (−−THAI) AND Hb CONSTANT SPRING CAUSES SEVERE Hb H DISEASE

We report two unrelated cases of Hb H-Constant Spring (Hb H-CS) disease caused by a compound heterozygosity for α0-thalassemia (−−THAI deletion) and Hb CS (α142, TAA→CAA) in Thai patients. Hematological and clinical observations in the probands are more severe than those of deletional type of Hb H disease (−−/–α), owing to an early onset of anemia and recurrent episode of anemic crises. These cases also address the importance of the −−THAI deletion which causes a severe clinical phenotype, and that could be missed by routine screening. We suggest that testing for this mutation should be included in the screening program for the prevention and control of thalassemia in Thailand, and possibly in other countries in Southeast Asia, where α0-thalassemias are highly prevalent.

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.