ALBERT

Description: Introduction: Although routine tissue iron monitoring using magnetic resonance imaging (MRI) has become a standard clinical management of both transfusion dependent and non-transfusion dependent thalassemias (TDT & NTDT) in several developed countries since this module can provide a better organ-directed iron measurement and related to clinical outcome including morbidity and mortality secondary to iron overload (IOL). However, accessibility of this monitoring remains limited in several developing countries including Thailand, where thalassemia and hemoglobinopathies are highly prevalent. Earlier in 2016, we have demonstrated that although not so perfectly crafted, a cross-sectional measurement of serum ferritin (SF) can be used for determination of IOL as a predictive marker; in NTDT, MRI for liver iron concentration (LIC) should be performed in those with SF 〉300 ug/L and for TDT, the SF cut off of 〉2,500 and 〉3,500 ug/L are useful to predict patients with severe LIC (〉15 mgFe/g dw) and cardiac siderosis (T2*〈 20 ms) (Ekwattanakit S. et.al., EHA 2016). In this study, we performed a further analysis to evaluate whether a serial measurement of SF and its trend can provide a better prediction. Objectives: To evaluate the clinical utility of serial SF trend compared with a cross-sectional SF cut-off for early detection of IOL in a real-life practice in thalassemic patients in order to select the most vulnerable patients for further MRI evaluation in a resource limited setting. Methods: In this prospective study, total 968 standard MRI for LIC and cardiac T2*were performed at Siriraj hospital during 2009-2014 and paired clinical data including serial SF measurements were collected from 301 thalassemia patients; NTDT (N=76; 109 LIC and 95 cardiac T2* results) and TDT (N=155; 478 LIC and 474 cardiac T2*). In addition, 71 patients were NTDT with regular blood transfusion later on in their life, mainly Hb E/β thalassemia (218 LIC and 210 cardiac T2*). These patients were evaluated for IOL using SF every 4-12 weeks during their follow up. Median follow up time was 96 months. Receiver operating characteristic (ROC) analysis was performed using different SF cut-off levels (1000, 1500 and 2000ug/L) and percentage of serial SF measurements that above each these cut-off levels (50 and 75%) during different durations before MRI (1, 2, or 3 years priori) for predicting liver IOL (LIC 〉5 in NTDT and severe liver siderosis; LIC 〉 15 mgFe/g dw) and cardiac IOL (T2*1000 ug/L over 75% of serial measurements in 1-year period prior to MRI can predict LIC 〉 5 mgFe/g dw with the AUC of 0.59 with PPV 86.7% and NPV 51.1% and serial SF 〉1500 ug/l over 50% of measurement in a year prior predict LIC 〉15 mgFe/g dw (AUC of 0.72, PPV 75% and NPV 89.7%). Only 1 NTDT had cardiac IO. These newly identified criteria do not provide a more sensitive predictor of LIC results than our previous SF cut-off. In TDT population, the majority of patients were HbE/β thal (78%) and cardiac iron overload was detected mainly in patients older than 15 yrs (81/83; 97.6%). The best predictor for LIC〉15 mgFe/g dw was SF 〉2000 ug/L over 75% of serial measurement durations in 3-year period (AUC 0.778, PPV 50.8%, NPV 95.5%). However this cut-off during 1 year priori also provided a similar prediction (AUC 0.769, PPV 51%, NPV 93.6%). This cut-off value also provided the best prediction for cardiac T2* 〈 20 ms in all age group (AUC 0.754, PPV 31.5%, NPV 97.4%) and a higher sensitivity and specificity when it was applied in patients 〉15 yrs of age (AUC 0.764, PPV 41.9%, NPV 96.3%). In NTDT with regular transfusion, all cardiac IOL (N=19) occurred after 15 years of age and again the same criteria was the good predictive cut-off for cardiac IO (AUC 0.788, PPV 19%, NPV 100%) and severe LIC 〉15 mgFe/g dw (AUC 0.728, PPV 52.8%, NPV 87.3%). Conclusions: In a resource limited setting for MRI evaluation, a serial measurement of SF and its values in thalassemic patients who received regular blood transfusion above the cut-off of 2000 ug/L over 75% of measurement in one year priori could be used as a predictive marker for selecting the most vulnerable TDT for MRI evaluation since this criteria is strongly associated with severe liver (LIC 〉 15 mgFe/g dw) and cardiac siderosis (T2*

Description: Severe combined immunodeficiency (SCID), characterized by blocking in T-cell development and/or functions with variable degree of simultaneous association of B-cell or natural killer (NK) cell dysfunction, is caused by heterogeneous genetic defects. Approximately 80% of western SCID patients resulted from mutations of common cytokine receptor γ (IL2RG), interleukin-7 receptor α (IL7RA), CD3δ (CD3D) and Janus kinase 3 (JAK3). Identification of molecular defects in SCID patients is important for proper diagnosis, management and appropriate genetic counseling including prenatal diagnosis. There was a limited data on clinical phenotype and genotype of SCID in Asia. Recently, we have characterized 5 unrelated individuals presenting with serious and life-threatening bacterial infections through our on-going Thailand’s SCID registry. Majority of cases had distinctive immunological profiles of T−, B+, NK+ SCID. Therefore, we firstly analyzed IL7RA and CD3D which have previously been shown to cause such phenotype by direct genomic sequencing of all exons, exonintron boundaries and 5′–3′UTR. Interestingly, only one patient (2 year old girl with generalized BCGosis) has been identified with a novel adenine insertion at an adenine tract located between nucleotide 444–450 of IL7RA encoded CD127, resulting in a frameshift and premature stop codon. Further analyses in fractionated peripheral mononuclear cells in the patient revealed a marked reduction in a full-length IL7RA cDNA and considerable decrease in mRNA expression in her parents who were both carriers of the mutation. This suggested that the mutation hampered mRNA expression, possibly, due to non-sense mediated decay mechanism (NMD). Using double staining flow cytometric analysis by conjugated CD127-PE and CD3-FITC antibodies, we demonstrated that there was a significant reduction of CD127 positive-T cells in the patient (8%) compared to normal control (69.38 ± 12.39, n = 7) confirming an in vivo reduction of CD127 expression on the patient’s T cells. Finally, the truncated form of CD127 in the patient was identified as expected by 2-D gel electrophoresis and immunoblotting assay after the total protein extracted from peripheral mononuclear cells was immunoprecipitated using anti CD127. We further analyzed the CD3D in other 4 cases by direct genomic sequencing, however we could not identified any mutation in such gene. Therefore we continued to determine the IL2RG in these cases with the same strategy. To our surprise, two patients (5 month- and 6 month-old boys) were found to be hemizygotes for a recurrent 678 C→T mutation (R222C) which has been previously reported from European SCID patients. This result indicated that this mutation was associated with a mutation ’hot spot’ due to hydrolytic deamination of 5′methyl-cytosine. Moreover, an unusual genotype-phenotype correlation in our patients also supported previous studies that this mutation might cause a ’leaky’ phenotype since typical cases with IL2RG mutations usually have T−, B+, NK− immunophenotype. Our study provided, for the first time, the molecular study of SCID in Southeast Asia and analysis of further cases will provide more insights on molecular basis of important and essential proteins involved in developing and controlling human immune system.

Description: Key Points KLF1 mutations cause severe congenital hemolytic anemia associated with a deficiency of red cell pyruvate kinase. A severe KLF1 deficiency causes hereditary persistence of embryonic globin synthesis.