Hematologic features of Beta-gloBin gene mutation type ( βo) witH Homozygous Beta tHalassemia

β-Tthalassemia is common genetic disorders in Turkey that characterized by the reduced synthesis (β + ) or absence (β o ) of the β-globin chains in the HbA molecule. In this study, we aimed to determine the effect of the mutation type of the β-globin gene on hematological values in homozygous β-thalassemia. This retro - spective study was undertaken by Prenatal Diagnosis Centres of Cukurova University Medical Biochemistry at adana. We evaluated 60 homozygous by implementing dNa sequencing analysis for mutations undetectable by conventional methods. 30 patients with β o [FSC 44/ C-A] mutations and the other 30 patients with β o [(IVS-II-1(G>a), Cd39 (C>t), Cd8 (-aa) Cd 39 C> T and CD36/37 (–T)] mutations, totally 60 patients were included in the study. Erythrocyte indices, hbF, hba 2 levels were compared between the two groups. FSC 44/(-C) mutations were detected in patients. Hb, Hct, MCV in this group values were statistically lower than in patients with other detected mutations (P < 0.05). Between the two groups, there is no statistically differ - ent rBC, MCh, MChC, hbF, hba 2 levels ( P ˃ 0.05). For the first time in this study, it was found that the Hb, Hct and MCV value of the persons who carried the FSC 44/(-C) mutation were significantly lower than the persons who carrying other mutations. Between the two groups, there was no statistical difference in RBC, MCh, MChC, hbF and hba 2 levels


Hematologic features of Beta-gloBin gene mutation type (βo) witH Homozygous Beta tHalassemia
Gülüzar ÖzBolat  , aBdUllah tUlI Cukurova University, Faculty of Medicine department of Medical Biochemistry, adana, turkey;  e-mail: guluzarozbolat@gmail.com β-Tthalassemia is common genetic disorders in Turkey that characterized by the reduced synthesis (β + ) or absence (β o ) of the β-globin chains in the HbA molecule.In this study, we aimed to determine the effect of the mutation type of the β-globin gene on hematological values in homozygous β-thalassemia.This retrospective study was undertaken by Prenatal Diagnosis Centres of Cukurova University Medical Biochemistry at adana.We evaluated 60 homozygous by implementing dNa sequencing analysis for mutations undetectable by conventional methods.30 patients with β o [FSC 44/ C-A] mutations and the other 30 patients with β o [(IVS-II-1(G>a), Cd39 (C>t), Cd8 (-aa) Cd39 C> T and CD36/37 (-T)] mutations, totally 60 patients were included in the study.Erythrocyte indices, hbF, hba 2 levels were compared between the two groups.FSC 44/(-C) mutations were detected in patients.Hb, Hct, MCV in this group values were statistically lower than in patients with other detected mutations (P < 0.05).Between the two groups, there is no statistically different rBC, MCh, MChC, hbF, hba 2 levels (P ˃ 0.05).For the first time in this study, it was found that the Hb, Hct and MCV value of the persons who carried the FSC 44/(-C) mutation were significantly lower than the persons who carrying other mutations.Between the two groups, there was no statistical difference in RBC, MCh, MChC, hbF and hba 2 levels.Awareness of FSC/44 mutation, which may have a heterogeneous clinical presentation, is required.We herein present the hematologic findings of a Turkish population carrying this mutation.This will also help to make a diagnosis.K e y w o r d s: Homozygous β-thalassemia, FSC 44/(-C), erythrocyte indices, DNA sequence analysis.

H
emoglobinopathies are among the most common inherited diseases around the world.They are divided into two main groups: thalassemia syndromes and the structural hemoglobin variants [1,2].Thalassemia is a severe genetic blood disorder brought about by a mutation in the globin gene [3].Beta thalassemia is a group of inherited autosomal recessive disease characteri zed by the presence of the defective synthesis chain β-globin part of the hemoglobin molecule [4].They are characterized by the reduced synthesis (β + ) or absence (β o ) of the β-globin chains in the HbA molecule [5].
Three clinical and hematological condi tions of increasing severity are recognized, the β-thalassemia carrier state, thalassemia major (ho mozygous) and thalassemia minor (heterozygous).The β-thalassemia carrier state, which results from © 2018 Özbolat G., Tuli A. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.heterozygosity for β-thalassemia, is defined by spe cific hematological features [5].Individuals with beta thalassemia minor usually do not have any symptoms (asymptomatic) and people often are una ware that they have the condition [6].Persons with thalassemia major usually come to medical attention within the first two years of life.These patients re quire lifelong RBC transfusions at regular intervals to survive [7].
The β-thalassemia is widespread throu ghout the Mediterranean Region, in Africa, the Middle East, the Indian subcontinent, and Burma, Southeast Asia and Indonesia [8].It estimates that 4.5% of the worldwide population carries β-thalassemia mutants.The first β-thalassemia study for Turkey was pub lished in 1985 [9].

R E T R A C
T E D ent mutations described in the literature.More than 200 different molecular defects are defined and 95% are caused by β-globin gene point mutations [10][11][12].Heterogeneity of β-thalassemia is associated with more than 40 different mutations in Turkey [13].So β-thalassemia is a major public health concern in Turkey; throughout the country, the gene frequency is expected to be 2.1%.But in certain regions, this figure increases to 10% [14].Traditional hemato logical methods contributing to the identification of candidate carriers involve a primary screen based on a complete blood count (CBC), hemoglobin electro phoresis for Hb fractionation and initial quantifica tion of HbA 2 and HbF levels [15].The key compo nents of the CBC include: Hb, red blood cell (RBC) number, mean corpuscular volume (MCV), and red cell distribution width (RDW) [16].There is now much different polymerase chain reaction (PCR)based techniques that can be used to diagnose the globin gene mutations.Direct mutation detection with Amplification Refractory Mutation System-PCR (ARMS-PCR) and Restriction endonuclease Analysis of PCR fragments (PCR-RFLP) was per formed by using amplified DNA from amniotic cells samples, while mutations in the parents were deter mined in advance [17].DNA sequencing is one of the most widely used methods for analyzing DNA and has been successfully used to detect any muta tion in the sequence being analyzed [18].
In this study, we aimed to determine the effect of the mutation type of the β-globin gene on the he matological values in homozygous β-thalassemia.We evaluated 60 patients by implementing DNA sequencing analysis for mutations undetectable by conventional methods.

Materials and Methods
The study was designed retrospectively among the β-thalassemia patients.A retrospective chart re view was conducted for subjects seen at the Depart ment of Biochemistry between 2008 and 2017.The study was performed in compliance with the national regulations on human experimentation and approved by the institutional committee.
Study participants.This retrospective study was conducted by Prenatal Diagnosis Centres of Cukurova University Medical Biochemistry at Adana.The medical files of 60 patients diagnosed with β-thalassemia were systematically reviewed in the study.DNA sequence analysis was performed for mutation scanning of the β-globin gene.
design.Clinical data were provided by a re view of medical records.The results of hematologi cal values were provided by the patient's registration system.We evaluated 60 patients by implementing DNA sequencing analysis for mutations undetectab le by conventional methods.30 patients with β o [FSC 44/ C-A] mutation and the other 30 patients with β o [(IVS-II-1(G>A), CD39 (C>T), CD8 (-AA) CD39 C> T and CD36/37 (-T)] mutations, totally 60 patients were included in the study.The common muta tions were screened for first by restriction fragment length polymorphism (RFLP) and amplification re fractory mutation systempolymerase chain reaction (ARMS-PCR) for each patient.Then any remaining uncharac teristic samples were analyzed by DNA se quencing to identify thalassemia mutations.eryth rocyte indices, HbF, HbA 2 levels were compared between the two groups.
Statistical analysis.Data are presented as de scriptive statistics including means.Data were ex pressed as a mean ± standard deviation for quanti tative variables, with ANOVA tests.P < 0.05 was considered to be statistically significant.

Result and Dıscussıon
In this study were originally investigated using a twostep diagnostic strategy in which the common mutations were screened for first by restriction frag ment length polymorphism (RFLP) and amplifica tion refractory mutation systempolymerase chain reaction (ARMS-PCR) for each patient.Then any re maining uncharacteristic samples were analyzed by DNA sequencing to identify thalassemia mutations.Subsequently, 30 patients with β o [FSC 44/ C-A] mutation and the other 30 patients with β o [(IVS-II-1(G>A), CD39 (C>T), CD8 (-AA) CD39 C> T and CD36/37 (-T)] mutations, totally 60 patients were in cluded in the study.DNA mutations sequence analyses were detected in 30 patients.The hematological values are shown in Table .FSC 44/(-C) mutations were detected in patients.Hb, Hct, MCV values were statistically lower in this group than in patients with other detected mutations (P < 0.05).Between the two groups, there was no statistical difference in RBC, MCH, MCHC, HbF, HbA 2 levels (P ˃ 0.05).
β-Thalassemia is extremely heterogeneous in terms of both of genotype and phenotype, depen ding on the nature of β-gene mutation and the extent of impairment in β-globin chain production.To date, more than 350 β-thalassemia mutations have been reported in the IthaGenes database, 40 of which have are useful for identifying carriers of the thalasse mia.When biochemical tests are not exhaustive, it is necessary to study the molecular globin genes [18].Seve ral studies have been carried out since the 1980s to identify β-globin gene mutations and the rate of finding new mutations significantly increased after the invention of PCR technique that can be used to diagnose the globin gene mutations, including the amplification refractory mutation system (ARMS), denaturing gradient gel electrophoresis (DGGe) and gap-PCR.Today DNA sequencing is one of the most widely used methods for analyzing DNA and has been successfully used to detect any mutation in the sequence being analyzed [2023].

R E T R A C T E D
In this study, we evaluated 60 patients by im plementing DNA sequencing analysis of the muta tions undetectable by conventional methods.We aimed to determine the effect of the mutation type (β o ) in the β-globin gene on the hematological pa rameters in β-thalassemia patients.FSC 44/(-C) mutation results or has resulted from a single base deletion (C) at codon 44 of HBB gene and creates a β o allele [24].FSC 44/(-C) mutations detected in patients.Hb, Hct, MCV values were statistically lower than, with other detected mutations (P < 0.05).Between the two groups, there was no statistical dif ference in RBC, MCH, MCHC, HbF, HbA 2 levels (P ˃ 0.05).
For the first time in this study, it was found that the Hb, Hct, and MCV values of the persons who carried the FSC 44/(-C) mutation were significantly lower than the persons who carrying other mutations and between two groups there was no significant difference in RBC, MCH, MCHC, HbF, HbA 2 levels (P ˃ 0.05).Awareness of FSC/44 mutation, which may have a heterogeneous clinical presentation, is required.Prevention of homozygous β-thalassemia, the clinically severe subtype, is possible with prena tal diagnosis and simply by detecting carriers.This will also help to make a diagnosis.
Hematological values in βthalassemia patients depending on mutation type in the hBB gene