α‐thalassemia carrier identification by DNA analysis in the screening for thalassemia

Galanello, Renzo; Sollaino, Carla; Paglietti, E.; Barella, Susanna; Perra, Cristian; Doneddu, Ilaria; Pirroni, M. G.; Maccioni, Liliana; Cao, Antonio

doi:10.1002/(sici)1096-8652(199812)59:4<273::aid-ajh2>3.3.co;2-v

Cited by 10 publications

(11 citation statements)

References 16 publications

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“…19 The HBG2:g.-158C>T (rs7482144) polymorphism was determined as previously described, while other SNPs were directly genotyped using TaqMan SNP genotyping assay (Applied Biosystems, Warrington, UK).…”

Section: Marker Selection and Genotypingsupporting

confidence: 69%

A genetic score for the prediction of beta-thalassemia severity

et al. 2014

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Clinical and hematologic characteristics of beta(β)-thalassemia are determined by several factors resulting in a wide spectrum of severity. Phenotype modulators are: HBB mutations, HBA defects and fetal hemoglobin production modulators (HBG2:g.-158C>T polymorphism, HBS1L-MYB intergenic region and the BCL11A). We characterized 54 genetic variants at these five loci robustly associated with the amelioration of beta-thalassemia phenotype, to build a predictive score of severity using a representative cohort of 890 β-thalassemic patients. Using Cox proportional hazard analysis on a training set, we assessed the effect of these loci on the age at which patient started regular transfusions, built a Thalassemia Severity Score, and validated it on a testing set. Discriminatory power of the model was high (C-index=0.705; R(2)=0.343) and the validation conducted on the testing set confirmed its predictive accuracy with transfusion-free survival probability (P<0.001) and with transfusion dependency status (Area Under the Receiver Operating Characteristic Curve=0.774; P<0.001). Finally, an automatized on-line calculation of the score was made available at http://tss.unica.it. Besides the accurate assessment of genetic predictors effect, the present results could be helpful in the management of patients, both as a predictive score for screening and a standardized scale of severity to overcome the major-intermedia dichotomy and support clinical decisions

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Section: Marker Selection and Genotypingsupporting

confidence: 69%

A genetic score for the prediction of beta-thalassemia severity

et al. 2014

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Section: Design and Methodsmentioning

confidence: 99%

“…Alpha globin and bilirubin UDP-glucuronosyltransferase (UGT1A1) gene genotyping was carried out as previously described. 7,8 The site directed mutation in K332Q in the KLF1 cDNA was obtained with the QuickChange Mutagenesis Kit (Stratagene, La Jolla, CA, USA).Band shift, supershift, Western blots and transactivation analysisCompound heterozygosity for KLF1 mutations associated with remarkable increase of fetal hemoglobin and red cell protoporphyrin ABSTRACT were performed as previously described. 9 The intensities of the KLF1 shifted bands were determined with the ImageQuant software after gel autoradiography on phosphoscreen and acquisition with PhosphoImager Storm 840 (GE Healthcare).…”

mentioning

confidence: 99%

Compound heterozygosity for KLF1 mutations associated with remarkable increase of fetal hemoglobin and red cell protoporphyrin

Satta¹,

Perseu²,

Moi³

et al. 2011

Haematologica

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Brief Reporthaematologica | 2011; 96 (5) 767 IntroductionPersistent expression of fetal hemoglobin (HbF) is of great clinical relevance given its role in the amelioration of the phenotype of beta-thalassemia and sickle cell anemia. Several studies have identified genes and genetic variants controlling HbF levels in adults (HBG1/HBG2, HBS1-MYB and BCL11A) able to improve the severity of the two major beta-hemoglobinopathies, beta thalassemia and sickle cell anemia. [1][2][3][4] Recently a nonsense mutation in the KLF1 gene, which ablates the DNA binding domain of this key erythroid transcriptional regulator, has been reported in a large Maltese family with hereditary persistence of HbF (HPFH). 5,6 Haploinsufficiency of KLF1 expression has been considered to be responsible for HPFH.In the Sardinian family described here, we found a marked increase of HbF only in compound heterozygotes for two KLF1 mutations and we did not confirm the KLF1 haploinsufficiency as a cause of HPFH. Moreover, we report, for the first time in humans, very high levels of zinc protoporphyrin associated with KLF1 mutations. Design and MethodsWe studied a Sardinian family with HPFH. Blood samples were obtained after informed consent. Hematologic and biochemical analyses were performed according to standard procedures. Zinc protoporphyrin in RBC was determined with ZPP hematofluorometer (AVIV Biomedical, Lakewood, NJ, USA) and blood protoporphyrin IX with a fluorometric method.Genomic DNA was obtained from peripheral blood by standard methods.Mutation analysis was performed by PCR amplification and DNA sequence analysis of the KLF1 gene using previously described primers.6 Genotyping of individual SNPs in the HBS1L-MYB (rs9399137) and BCL11A (rs11886868) loci was performed using Taqman genotyping assay (Applied Biosystem, Warrington, UK). Alpha globin and bilirubin UDP-glucuronosyltransferase (UGT1A1) gene genotyping was carried out as previously described. 7,8 The site directed mutation in K332Q in the KLF1 cDNA was obtained with the QuickChange Mutagenesis Kit (Stratagene, La Jolla, CA, USA).Band shift, supershift, Western blots and transactivation analysisCompound heterozygosity for KLF1 mutations associated with remarkable increase of fetal hemoglobin and red cell protoporphyrin ABSTRACT were performed as previously described. 9 The intensities of the KLF1 shifted bands were determined with the ImageQuant software after gel autoradiography on phosphoscreen and acquisition with PhosphoImager Storm 840 (GE Healthcare).The study was approved by the institutional review board of the hospital (ASL8 Ethics Commitee).

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“…α-globin genotype was defined by GAP PCR for the deletional defects and the triple α gene arrangement, and by digestion of amplification products with NcoI and HphI restriction enzymes for non-deletional defects. 15 To detect the less common anti-α 4.2 triplication, the Southern blot analysis was carried out.…”

Section: Dna Analysismentioning

confidence: 99%