The molecular characterization of Beta globin gene in thalassemia patients reveals rare and a novel mutations in Pakistani population.

INTRODUCTION A multicentre study (including four cities in Pakistan) aimed to investigate the frequency and spectrum of alpha and beta thalassemia genetic mutations and XmnI polymorphism of the Gamma Globin gene. METHODS One hundred and sixty one beta thalassemia patients, identified on the ground of haematological parameters, were screened for mutations of the Alpha (HBA2 and HBA1) and Beta (HBB) Globin genes as well as Gamma (HBG2) Globin gene, -158 Gγ XmnI polymorphism, using a combination of multiplex GAP polymerase chain reaction (PCR), Sanger sequencing and restriction fragment length polymerase (RFLP) based PCR. RESULTS Mutations of at least one HBB gene was identified in 157 of 161 patients screened. Among 16 identified mutations in the beta gene, HBB:c.27_28insG (p. Ser10Valfs*14) was the most prevalent. α(-3.7) and α(-4.2) deletions were co-inherited with beta thalassemia mutations. Rare mutations such as HBB:c.-138C > T and HBB:c.315 + 1G > A were also identified. One novel variant (HBB:c.-148T > A), two rare mutations [HBB:c.332T > C (p.Leu111Pro); HBB:c.92G > C (p.Arg31Thr] and a novel association, HBB:c.[92G > C (p.Arg31Thr)] and [-92C > G], were reported for the first time in our study. HBG2:c.-211C > T base-pair substitution (historically described as -158 GγXmnI polymorphism) was present in 36% of the patients. CONCLUSION Heterogeneity in clinical and haematological parameters in TM, show that monogenic disorders can present with a wide spectrum of disease severity. Our studies identified rare and novel mutations that will be useful in the prevention of highly prevalent disease of thalassemia in Pakistan following nationwide awareness campaign.

[1]  Jin Ai Mary Anne Tan,et al.  Molecular basis of transfusion dependent beta-thalassemia major patients in Sabah , 2013, Journal of Human Genetics.

[2]  Jana Marie Schwarz,et al.  MutationTaster2: mutation prediction for the deep-sequencing age , 2014, Nature Methods.

[3]  S. Baig,et al.  Spectrum of beta-thalassemia mutations in various regions of Punjab and Islamabad, Pakistan: establishment of prenatal diagnosis. , 2006, Haematologica.

[4]  D. Mohanty,et al.  Molecular pathogenesis and clinical variability of β‐thalassemia syndromes among Indians , 2001 .

[5]  Annie Robert,et al.  Validation of a Recombinant DNA Construct (μLCR and Full-Length β-Globin Gene) for Quantification of Human β-Globin Expression: Application to Mutations in the Promoter, Intronic, and 5′- and 3′-Untranslated Regions of the Human β-Globin Gene , 2002 .

[6]  Subash Gupta,et al.  Molecular pathogenesis and clinical variability of homozygous β0-thalassemia in populations of Jammu region of J&K state (India) , 2006, Hematology.

[7]  J. Clegg,et al.  Rapid detection of α‐thalassaemia deletions and α‐globin gene triplication by multiplex polymerase chain reactions , 2000, British journal of haematology.

[8]  S. Mahmood,et al.  Awareness among parents of β-thalassemia major patients, regarding prenatal diagnosis and premarital screening. , 2012, Journal of the College of Physicians and Surgeons--Pakistan : JCPSP.

[9]  C. Vulpe,et al.  Ferroportin 1 (SCL40A1) variant associated with iron overload in African-Americans. , 2003, Blood cells, molecules & diseases.

[10]  D. Mohanty,et al.  Impact of beta globin gene mutations on the clinical phenotype of beta thalassemia in India. , 2004, Blood cells, molecules & diseases.

[11]  R. Saxena,et al.  Relation of Xmn-1 Polymorphism & Five Common Indian Mutations of Thalassaemia with Phenotypic Presentation in b-Thalassaemia , 2006 .

[12]  H. Siala,et al.  Association between clinical expression and molecular heterogeneity in β-thalassemia Tunisian patients , 2013, Molecular Biology Reports.

[13]  Nidhi Sharma,et al.  Evaluation of the genetic basis of phenotypic heterogeneity in north Indian patients with Thalassemia major , 2010, European journal of haematology.

[14]  T. Moatter,et al.  Prenatal screening for β-thalassemia major reveals new and rare mutations in the Pakistani population , 2012, International Journal of Hematology.

[15]  Mohammad Ahmadifard,et al.  A comprehensive molecular characterization of beta thalassemia in a highly heterogeneous population. , 2011, Blood cells, molecules & diseases.

[16]  T. Huisman,et al.  Beta-thalassemia, HB S-beta-thalassemia and sickle cell anemia among Tunisians. , 1991, Hemoglobin.

[17]  P. Giordano,et al.  A Confidential Inquiry Estimating the Number of Patients Affected with Sickle Cell Disease and Thalassemia Major Confirms the Need for a Prevention Strategy in The Netherlands , 2004, Hemoglobin.

[18]  R. Blackwell,et al.  Hemoglobin J Singapore: alpha 78 Asn--Asp; alpha 79 Ala--Gly. , 1972, Biochimica et biophysica acta.

[19]  W. Pan,et al.  The non-iron-deficiency-related difference in hemoglobin concentration distribution between blacks and whites and between men and women. , 1991, American journal of epidemiology.

[20]  W. Moo-Penn,et al.  The presence of hemoglobin S and C Harlem in an individual in the United States. , 1975, Blood.

[21]  Y. Hattori,et al.  Codon 4 ACT→ACA, codon 5 CCT→TCT, and codon 6 GAG→TAG mutations in cis position: A form of thalassemia trait , 1997, American journal of hematology.

[22]  S. Garn.  Lower hematocrit levels in blacks are not due to diet or socioeconomic factors. , 1981, Pediatrics.

[23]  H. Galehdari,et al.  Identification of IVS-I (-1) (G > C) or Hb Monroe as a report on the beta-globin gene with a beta-thalassemia minor phenotype in south of Iran. , 2013, Archives of Iranian medicine.

[24]  D. Higgs,et al.  Frequency and clinical significance of erythrocyte genetic abnormalities in Omanis. , 1993, Journal of medical genetics.

[25]  T. Huisman,et al.  Binding of nuclear factors to the proximal and distal CACCC motifs of the beta-globin gene promoter: implications for the -101 (C-->T) 'silent' beta-thalassemia mutation. , 1994, Acta haematologica.

[26]  C. Brownie,et al.  Prevalences of anemia and iron deficiency anemia in Black and White women in the United States estimated by two methods. , 1983, American journal of public health.

[27]  M. Petrou,et al.  Screening extended families for genetic hemoglobin disorders in Pakistan. , 2002, The New England journal of medicine.

[28]  Z. Rahimi,et al.  The Xmn1 polymorphic site 5′ to the Gγ gene and its correlation to the Gγ:Aγ ratio, age at first blood transfusion and clinical features in β-Thalassemia patients from Western Iran , 2009, Molecular Biology Reports.

[29]  P. Chakrabarti,et al.  Spectrum of β-thalassemia mutations in North Indian states: A β-thalassemia trait with two mutations in cis , 2005 .

[30]  B. Modell,et al.  The Clinical Approach to Thalassaemia , 1984 .

[31]  P. Heller,et al.  Hemoglobin Arlington Park. A new hemoglobin variant with two amino acid substitutions in the beta chain. , 1977, Hemoglobin.

[32]  M. Petrou,et al.  Molecular genetics of beta-thalassaemia in Pakistan: a basis for prenatal diagnosis. , 1996, British journal of haematology.

[33]  M. El-Hazmi,et al.  Extensive Polymorphisms in Saudi Beta Thalassaemia Patients , 2013 .

[34]  D. Weatherall,et al.  The spectrum of β‐thalassaemia mutations on the Indian subcontinent: the basis for prenatal diagnosis , 1991, British journal of haematology.

[35]  S. Limborska,et al.  IVS-I-1 (G-->C) in combination with -42 (C-->G) in the promoter region of the beta-globin gene in patients from Tajikistan. , 1993, Hemoglobin.

[36]  T. Shamsi,et al.  Molecular epidemiology of β-thalassemia in Pakistan: Far reaching implications , 2012, Indian journal of human genetics.

[37]  S. Bakhtiar,et al.  &bgr;-Thalassemia in Pakistan: A Pilot Program on Prenatal Diagnosis in Multan , 2012, Journal of pediatric hematology/oncology.

[38]  R. Yip,et al.  Hematocrit values in white, black, and American Indian children with comparable iron status. Evidence to support uniform diagnostic criteria for anemia among all races. , 1984, American journal of diseases of children.

[39]  J. Old,et al.  Screening and genetic diagnosis of haemoglobin disorders. , 2003, Blood reviews.

[40]  B. George,et al.  Analysis of β globin mutations in the Indian population: presence of rare and novel mutations and region‐wise heterogeneity , 2008, Clinical genetics.

[41]  T. Shamsi,et al.  Molecular epidemiology of β-thalassemia in Pakistan: far reaching implications. , 2011, International journal of molecular epidemiology and genetics.