Contributions
Abstract: PB1820
Type: Publication Only
Background
Aplastic anemia (AA) is a rare and life threatening hematopoietic disease characterized by peripheral blood pancytopenia accompanied with trilineage bone marrow (BM) aplasia. AA may develop at any stage of lifeAccording to epidemiological studies, the estimated annual incidence of AA is two per million in Western countries. The incidence rate is 2-3 times higher in Asian population but the cause for this is still not clear. In Pakistan, AA is the second most common serious non-malignant blood disorder after thalassemia. AA has been reported predominantly in young adult male population.
Aims
To gain insight into the genetics and pathophysiology of AA in the Pakistani population, we investigated samples from AA patients reporting at the Armed Forces Bone Marrow Transplant Centre, Rawalpindi, Pakistan. This is the first study where examination of socio-etiological data, cytokine profiling, HLA allelic association and mutations in TERT and TERC genes were simultaneously studied to dissect a high incidence of AA cases in Pakistani population.
Methods
Genomic DNA (gDNA) was extracted from whole blood and peripheral blood mononuclear cells (PBMCs) of AA patients using the DNeasy Blood and Tissue kit (Qiagen, Valencia, CA, USA).For mutation analysis, polymerase-chain-reaction (PCR) amplification of TERT and TERC genes was performed.Purified PCR products were subjected to direct sequencing with the BigDye Terminator v3.1 Cycle Sequencing kit (ThermoFisher Scientific, Waltham, MA, USA) and the 3130xl Genetic Analyzer (ThermoFisher Scientific). Serum cytokine levels were measured by Magnetic Luminex Screening Assay using the Human Premixed Multi-Analyte kit. HLA typing was carried out in 74 of our patients by PCR with sets of sequence specific primers (SSP) or serological technique.The descriptive statistics were performed using SPSS version 17.0. GraphPad PRISM software version 6 was used for analysis of variance using Krusk-Wallis test to compare serum levels of cytokines, growth factors and hormones between different patient groups and control subjects. A P value < 0.05 was considered statistically significant.
Results
Epidemiological data revealed 2.75-fold higher frequency of AA among males. A single peak of disease onset was observed between age 10-29 years followed by a steady decline. AA was strongly associated with lower socioeconomic profile, rural residence and high rate of consanguineous marriages. Serum G-CSF and TPO levels were significantly elevated in AA patients, compared to healthy controls (P<0.001), while there was no statistical significance in other nine cytokine levels screened. Allele frequencies of DRB1*15 (56.8%) and DQB1*06 (70.3%) were predominantly high in AA patients. Ten mutations were found in TERT and TERC genes, including two novel mutations (Val526Ala and Val777Met) in exon 3 and 7 of TERT gene.
Conclusion
Despite specific features of the AA cohort, this study suggests that epidemiologic and etiologic factors as well as host genetic predisposition exclusively or cooperatively trigger AA in Pakistan.
Session topic: 11. Bone marrow failure syndromes incl. PNH – Biology & Translational Research
Keyword(s): Aplastic anemia, Cytokine, Telomere length
Abstract: PB1820
Type: Publication Only
Background
Aplastic anemia (AA) is a rare and life threatening hematopoietic disease characterized by peripheral blood pancytopenia accompanied with trilineage bone marrow (BM) aplasia. AA may develop at any stage of lifeAccording to epidemiological studies, the estimated annual incidence of AA is two per million in Western countries. The incidence rate is 2-3 times higher in Asian population but the cause for this is still not clear. In Pakistan, AA is the second most common serious non-malignant blood disorder after thalassemia. AA has been reported predominantly in young adult male population.
Aims
To gain insight into the genetics and pathophysiology of AA in the Pakistani population, we investigated samples from AA patients reporting at the Armed Forces Bone Marrow Transplant Centre, Rawalpindi, Pakistan. This is the first study where examination of socio-etiological data, cytokine profiling, HLA allelic association and mutations in TERT and TERC genes were simultaneously studied to dissect a high incidence of AA cases in Pakistani population.
Methods
Genomic DNA (gDNA) was extracted from whole blood and peripheral blood mononuclear cells (PBMCs) of AA patients using the DNeasy Blood and Tissue kit (Qiagen, Valencia, CA, USA).For mutation analysis, polymerase-chain-reaction (PCR) amplification of TERT and TERC genes was performed.Purified PCR products were subjected to direct sequencing with the BigDye Terminator v3.1 Cycle Sequencing kit (ThermoFisher Scientific, Waltham, MA, USA) and the 3130xl Genetic Analyzer (ThermoFisher Scientific). Serum cytokine levels were measured by Magnetic Luminex Screening Assay using the Human Premixed Multi-Analyte kit. HLA typing was carried out in 74 of our patients by PCR with sets of sequence specific primers (SSP) or serological technique.The descriptive statistics were performed using SPSS version 17.0. GraphPad PRISM software version 6 was used for analysis of variance using Krusk-Wallis test to compare serum levels of cytokines, growth factors and hormones between different patient groups and control subjects. A P value < 0.05 was considered statistically significant.
Results
Epidemiological data revealed 2.75-fold higher frequency of AA among males. A single peak of disease onset was observed between age 10-29 years followed by a steady decline. AA was strongly associated with lower socioeconomic profile, rural residence and high rate of consanguineous marriages. Serum G-CSF and TPO levels were significantly elevated in AA patients, compared to healthy controls (P<0.001), while there was no statistical significance in other nine cytokine levels screened. Allele frequencies of DRB1*15 (56.8%) and DQB1*06 (70.3%) were predominantly high in AA patients. Ten mutations were found in TERT and TERC genes, including two novel mutations (Val526Ala and Val777Met) in exon 3 and 7 of TERT gene.
Conclusion
Despite specific features of the AA cohort, this study suggests that epidemiologic and etiologic factors as well as host genetic predisposition exclusively or cooperatively trigger AA in Pakistan.
Session topic: 11. Bone marrow failure syndromes incl. PNH – Biology & Translational Research
Keyword(s): Aplastic anemia, Cytokine, Telomere length