Path to Facilitate the Prediction of Functional Amino Acid Substitutions in Red Blood Cell Disorders – A Computational Approach

A major area of effort in current genomics is to distinguish mutations that are functionally neutral from those that contribute to disease. Single Nucleotide Polymorphisms (SNPs) are amino acid substitutions that currently account for approximately half of the known gene lesions responsible for human inherited diseases. As a result, the prediction of non-synonymous SNPs (nsSNPs) that affect protein functions and relate to disease is an important task.In this study, we performed a comprehensive analysis of deleterious SNPs at both functional and structural level in the respective genes associated with red blood cell metabolism disorders using bioinformatics tools. We analyzed the variants in Glucose-6-phosphate dehydrogenase (G6PD) and isoforms of Pyruvate Kinase (PKLR & PKM2) genes responsible for major red blood cell disorders. Deleterious nsSNPs were categorized based on empirical rule and support vector machine based methods to predict the impact on protein functions. Furthermore, we modeled mutant proteins and compared them with the native protein for evaluation of protein structure stability.We argue here that bioinformatics tools can play an important role in addressing the complexity of the underlying genetic basis of Red Blood Cell disorders. Based on our investigation, we report here the potential candidate SNPs, for future studies in human Red Blood Cell disorders. Current study also demonstrates the presence of other deleterious mutations and also endorses with in vivo experimental studies. Our approach will present the application of computational tools in understanding functional variation from the perspective of structure, expression, evolution and phenotype

Cytogenetic and molecular monitoring of residual disease in chronic myeloid leukaemia

For patients with chronic myeloid leukaemia, methods for monitoring response to treatment have changed considerably in recent years. In the 1980s, the principal approach was repeated examination of bone marrow metaphases for the presence of the Ph chromosome in patients treated by interferon-alpha (IFN-alpha) or allogeneic stem cell transplantation. The use of fluorescence in situ hybridisation (FISH) techniques to detect the BCR-ABL fusion gene in Ph-positive leukaemia cells increased the sensitivity of cytogenetic studies to some degree. In the last 10 years, the reverse-transcriptase polymerase chain reaction (RT-PCR) has proved extremely valuable for assessing and monitoring minimal residual disease in patients who achieve Ph negativity after treatment with IFN-alpha or with the new Abl tyrosine kinase inhibitor imatinib mesylate or after allogeneic stem cell transplantation (SCT). Results are consistent with the notion that the majority of long-term survivors after allogeneic SCT are probably 'cured'; for other patients monitored serially in complete cytogenetic remission, rising numbers of BCR-ABL transcripts detected by RT-PCR can indicate the need for further therap

Mild clinical expression of S-beta thalassemia in a Brazilian patient with the beta(+) IVS-I-6 (T -> C) mutation

We report on an eight-year-old Brazilian girl with S-beta(+) thalassemia. The patient had a steady 10.1 g/dl hemoglobin with 57% HbS. Direct sequence analysis of beta-globin gene showed her to be heterozygous for the IVS-I-6 (T-->C) mutation. This beta(+) thalassemia mutation, sometimes referred to as the Portuguese type, was found to be associated with the C-->T polymorphism at codon 2. In combination with the beta(S) gene, this mutation results in very mild sickle cell disease symptoms.21443143

Mild Clinical Expression Of S-β Thalassemia In A Brazilian Patient With The β+ Ivs-i-6 (t→c) Mutation

We report on an eight-year-old Brazilian girl with S-β+ thalassemia. The patient had a steady 10.1 g/dl hemoglobin with 57% HbS. Direct sequence analysis of β-globin gene showed her to be heterozygous for the IVS-I-6 (T→C) mutation. This β+ thalassemia mutation, sometimes referred to as the Portuguese type, was found to be associated with the C→T polymorphism at codon 2. In combination with the β(s) gene, this mutation results in very mild sickle cell disease symptoms.214431433Antonarakis, S.E., Orkin, S.H., Cheng, T.C., Scott, A.F., Sexton, J.P., Trusko, S., Charache, S., Kazazian Jr., H.H., β Thalassemia in American blacks: Novel mutations in the "TATA" box and an acceptor splice site (1984) Proc. Natl. Acad. Sci. USA, 81, pp. 1154-1158Atweh, G., Forget, B.G., Identification of a β-thalassemia mutation associated with a novel haplotype of RFLPs (1986) Am. J. Hum. Genet., 38, pp. 855-859Baysal, E., Huisman, T.H.J., Detection of common deletional α-thalassemia-2 determinants by PCR (1994) Am. J. Hematol., 46, pp. 208-213Bunn, H.F., Forget, B.G., (1986) Hemoglobin: Molecular, Genetic and Clinical Aspects, , W.B. Saunders Company, PhiladelphiaMartins, C.S.B., Ramalho, A.S., Sonati, M.F., Gonçalves, M.S., Costa, F.F., Molecular characterisation of β thalassaemia heterozygotes in Brazil (1993) J. Med. Genet., 30, pp. 797-798Orkin, S.H., Kazazian Jr., H.H., Antonarakis, S.E., Goff, S.C., Boehm, C.D., Sexton, J.P., Waber, P.G., Giardina, P.J.V., Linkage of β-thalassemia mutations and β-globin gene polymorphisms with DNA polymorphisms in human β-globin gene cluster (1982) Nature, 296, pp. 627-631Treisman, R., Orkin, S.H., Maniatis, T., Specific transcription and RNA splicing defects in five cloned β-thalassaemia genes (1983) Nature, 302, pp. 591-596Weatherall, D.J., Clegg, J.B., (1981) The Thalassaemia Syndromes. 3rd Edn., , Blackwell Scientific Publications, OxfordZago, M.A., Costa, F.F., Freitas, T.C., Bottura, C., Clinical, hematological and genetic features of sickle cell anemia and sickle cell-beta thalassemia in a Brazilian population (1980) Clin. Genet., 18, pp. 58-6

High-level, erythroid-specific expression of the human alpha-globin gene in transgenic mice and the production of human hemoglobin in murine erythrocytes.

Using the dominant control region (DCR) sequences that flank the beta-globin gene locus, we have been able to achieve high-level expression of the human alpha-globin gene in transgenic mice. Expression in fetal liver and blood is copy number dependent and at levels comparable to that of the endogenous mouse alpha-globin genes. Transgenic fetuses with high-copy numbers of the transgene suffer severe anemia and die before birth. Using a construct with both the human alpha- and beta-globin genes and the beta-globin DCR, live mice with low-copy numbers were obtained. Both human globin genes are expressed at high levels in adult red cells to give human hemoglobin HbA in amounts equal to or greater than endogenous mouse hemoglobin. Expression of HbA in murine red cells is not accompanied by any increase in mean corpuscular volume (MCV) or mean corpuscular hemoglobin concentration (MCHC). However, these transgenic mice tend to have an increased number of reticulocytes in peripheral blood; consistent with some degree of hemolysis. Metabolic labeling experiments showed balanced mouse globin synthesis, but imbalanced human globin synthesis, with an alpha/beta biosynthetic ratio of approximately 0.6. Thus, these mice have mild anemia. These results are discussed with relation to the coordinate regulation of alpha- and beta-globin synthesis in erythroid tissues

Rationale for the recommendations for harmonizing current methodology for detecting BCR-ABL transcripts in patients with chronic myeloid leukaemia

Molecular monitoring for patients with chronic myeloid leukaemia (CML) has become an important practice in the era of imatinib therapy. For successful widespread introduction into the mainstream patient monitoring schedule, many procedural aspects of the complex real-time quantitative polymerase chain reaction (RQ-PCR) technique for measuring BCR-ABL transcripts require optimization. Recommendations for harmonizing the differing methodologies have recently been proposed. These recommendations were designed to maximize reliability of analysis for clinical decision making and proposed the adoption of an International Scale of measurement. The purpose of this review is to present the evidence and supporting data for specific recommendations. These recommendations include use of the same source of cells, either blood or marrow, for analysis; for validation of equal PCR amplification efficiencies of cDNA and standards when using a plasmid to construct standard curves and for ensuring ongoing high-level performance by undertaking a quality assurance programme. Clinicians must know the measurement reliability of an RQ-PCR assay to be able to determine the significance of a change in BCR-ABL level. An assay with poor precision limits the clinical usefulness of results. International harmonization should establish RQ-PCR measurement of BCR-ABL as the best method for monitoring treatment response for patients with CML

A third instance of the high oxygen affinity variant, Hb Heathrow [beta 103(G5)Phe- greater than Leu]: identification of the mutation by mass spectrometry and by DNA analysis.

Hb. Heathrow [beta 103(G5)Phe- greater than Leu] was identified in an Englishman with a life-long history of polycythemia, his father had been similarly affected. A hemoglobin variant was suspected from the high oxygen affinity of the patient's blood. The Hb Heathrow abnormal beta chain was resolved from the normal beta chain by high performance liquid chromatography, and the abnormal peptide and the amino acid replacement were identified by mass spectrometry. The corresponding base change (C- greater than G at codon 103) was demonstrated by sequence analysis of the polymerase chain reaction amplified exon 2 of the genomic beta-globin gene. This is only the third known instance of Hb Heathrow