Prevalence of ß-Thalassemia Mutations among Northeastern Iranian Population and their Impacts on Hematological Indices and Application of Prenatal Diagnosis, a Seven-Years Study
Mohammad Ehsan Jaripour1#, Kourosh Hayatigolkhatmi1#, Vahid Iranmanesh1, Farhad Khadivi Zand1, Zahra Badiei2, Hamid Farhangi2, Ali Ghasemi2, Abdollah Banihashem2, Reza Jafarzadeh Esfehani3 and Ariane Sadr-Nabavi1,3,4*.
1 Iranian Academic Center for Education, Culture and Research, (ACECR), Mashhad, Iran.
2 Department of Pediatric Diseases, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
3 Department of Medical Genetics, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
4 Medical Genetics Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
# These authors contributed equally to this work.
Received: March 13, 2018
Accepted: June 14, 2018
Mediterr J Hematol Infect Dis 2018, 10(1): e2018042 DOI 10.4084/MJHID.2018.042
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Background and Objective: ß-thalassemia results from a diverse range of mutations inside the hemoglobin subunit β (HBB)
gene. In a study of β-thalassemia carriers and some of their at-risk
fetuses in the Khorasan province of Iran, we aimed to recognize the
most common mutations in the region. We also investigated a possible
link between these mutations and some of the relevant hematological
Researches prove that ß-thalassemia is one of the most frequent genetic diseases worldwide, with a range of ethnically and geographically distributed mutations.[1,2] Moreover, the large number of carriers is a warning for the health system and an emphasis on the importance of preventing programs.[3-14] ß-globin genetic mutations distribute among every ethnic group. Identification of these mutations helps authorities for more accurate evaluations and more practical prevention programs.[3-14]
The purpose of this study was to recognize the most common mutations related to ß-thalassemia in the Khorasan province of Iran and to find the possible relation of these mutations with some of the relevant hematological indices. These indices are presented in complete blood count (CBC) and Hb electrophoresis tests (identifying HbA1, HbA2, and HbF). These indices include RBC, Hb, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH).
Material and Methods
Study subjects: The population investigated in this study comprises of 1593 individuals with Fars ethnicity, suspected of possessing a mutated allele for ß-thalassemia and referred to the ACECR diagnostic medical genetics laboratory for molecular diagnosis. Personal consent form to obtain the permission to use the patients’ samples and the relevant data in the research performed by the ACECR was signed by every single individual of this study. The ACECR ethics committee, which functions under the regulations of the national medical ethics committee, approved these consent forms. Clinical criteria for suspicious ß-thalassemia was determined by the hypochromic microcytic anemia, including decreased MCV (<80 fL) and MCH (<27 pg/cell) and unusual findings in Hb electrophoresis, including elevated HbA2 (≥ 3.5 %) or HbF (≥ 1%) and other abnormally high Hb variants. Peripheral blood (PB) samples from subjects were collected in EDTA containing tubes. Furthermore, chorionic villus samples (CVS) were collected from 644 conceptions, whose fetuses were at risk of inheriting two mutated alleles for hemoglobin subunit β (HBB) gene. Samples were collected by a neonatologist and sent to the ACECR medical genetics laboratory for ß-thalassemia prenatal diagnosis (PND). All samples (PB and CVS) were collected since March of 2011 up to January of 2018.
DNA extraction: DNA from selected individuals was extracted using standard salting out method as described by. DNA from CVS was isolated using QiAmp® DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany) as described by the company instructions.
Mutation detection: For each amplification-refractory mutation system (ARMS-PCR) was used to detect ten common β-globin mutations: IVS-I-5, IVS-II-1, IVS-I-110, IVS-I-1, IVS-II-745, IVS-I-6, codon 30, codon 39, codon 819, and codon 16. In cases, where none of the mentioned variants was detected; standard Sanger sequencing of the HBB had been performed to reveal the mutation. Supplementary table 1 shows primer sequences, micro-tube components and the thermal protocol used for the mentioned methods. Moreover, in some emergency cases, reverse dot blot (strip assay) was conducted using Thalassemia StripAssays® kit (ViennaLab, Vienna, Austria). For ß-thalassemia PND cases, we have routinely confirmed the result with two alternative molecular techniques. (See supplementary files)
Statistical analysis: Data was analyzed using the statistical package for social sciences (SPSS) version 22 (IBM Inc, Chicago, Il, USA). Continuous data were checked for normality using the Kolmogorov-Smirnov test. Means and standard deviations (SD) were used to describe continuous variables while frequency and percentage were used to describe categorical variables. Analysis of variance (ANOVA) was used to compare parametric variables, including RBC count, Hb, MCV, MCH, HbA2, and HbA1, between the mutation categories while the Kruskal-Wallis test was used to compare non-parametric variable (HbF) values between mutation groups. Multinomial logistic regression was performed to assess the relationship between the mutation categories and the study parameters considering other mutations than the nine evaluated (mostly reported mutations) as the reference category. The odds ratio (OR) and 95% confidence interval (CI) for OR were presented along with p-value for the regression model. Values of p less than 0.05 were considered statistically significant.
During the years of this study, 644 conception cases on the formerly mentioned ß-thalassemia carrier couples were subjected to PND, using CVS samples as described in the “methods” section.
For 118 cases which were reported to carry no detectable pathogenic variant and 352 cases which were detected as carriers (heterozygous with one pathogenic variant in HBB) and expected to show the ND ß-thalassemia phenotype, pregnancies were followed up till the delivery. Whereas 174 cases, which were diagnosed as homozygous with two pathogenic variants in HBB and expected as TD ß-thalassemia phenotype, have received the proper genetic counseling based on the regional guidelines and prevention programs.[13,14]
Further, the relation of the first nine more commonly reported mutations with the hematological indices was observed as mentioned previously. There was a significant difference in MCV and HbA2 levels between the mutation types (p<0.001) (Table 1). Multinomial logistic regression model revealed that mutation categorized as IVS-II-I was associated with increased risk for higher MCH (p=0.01, OR=1.15, 95% CI for OR= 1.03 and 1.28) and HbA2 (p=0.002, OR= 1.26, 95% CI for OR= 1.09, 1.46) and lower MCV (p<0.001, OR=0.93, 95% CI for OR= 0.90, 0.97) compared to other mutations. Furthermore, the codons 8/9 mutation was found to be associated with significant increase in HbF values compared to other mutations (p=0.04, OR= 1.05, 95% CI for OR= 1.00, 1.09).
Additionally, we investigated the relation of the first nine most commonly reported mutations with the hematological indices as mentioned formerly. The highest and the lowest RBC mean values were reported among Los Angeles and IVS-I-110 carriers respectively (ranges from 5.89 - 5.46 x 106 cells/mcL). The highest and the lowest Hb mean values were reported among IVS-I-5 and -88 carriers respectively (ranges from 13.23 – 11.47 mg/dL). The highest and the lowest MCV mean values were reported among codon 5 and IVS-II-1 carriers respectively (ranges from 69.12 – 64.59 fL). The highest and the lowest MCH mean values were reported among IVS-I-5 and Los Angeles carriers respectively (ranges from 22.46 – 19.77 pg/cell). The highest and the lowest HbA2 mean values were reported among IVS-II-1 and codon 5 carriers respectively (ranges from 4.72 – 3.80% of the total Hb). The highest and the lowest HbF mean values were reported among IVS-I-5 and Los Angeles carriers respectively (ranges from 3.16 – 0.26% of the total Hb). The highest and the lowest HbA1 mean values were reported among IVS-I-110 and IVS-I-5 carriers respectively (ranges from 94.92 – 83.26% of the total Hb). Hence, these reported values while considering the SD, p-value, OR and referring to the relevant guidelines can be helpful in offering a hint to the local clinicians for more accurate referrals. This can also be helpful as a boost for laboratory professionals for a more straightforward ß-thalassemia testing guide in the region. We have also found a significant difference regarding the MCV and the HbA2 levels between the mutation types. Our findings mean that the type of mutation causing ß-thalassemia has a high chance of affecting the MCV value and HbA2 ratio. In addition, moving further into the details of mutations impact on hematological indices we have illustrated that IVS-II-1 was associated with increased risk for higher MCH and HbA2 in comparison to other reported variants. It is also causing a lower MCV compared to other mutations. Also, the codons 8/9 mutation was found to be associated with significant increase in HbF values compared to other mutations. That means that the IVS-II-1 variant has a high chance of increasing the MCH and HbA2 while lowering the MCV when compared to other mutation types. On the other hand, the presence of the codons 8/9 will probably raise the HbF proportion when compared to other mutations. These findings act as a start point for more focused interdisciplinary studies on the genomic and hematologic profile of ß-thalassemia patients to find a more comprehensive map of genotype-phenotype correlation.
As previously discussed, ß-thalassemia is one of the most common genetic disorders worldwide and also in Iran.[3-14] Although there are some treatments available for controlling and recovering the disease such as routine blood transfusion (followed by the iron chelation therapy), bone marrow transplantation and even gene therapy, still genetic counseling and PND are known to be the best available preventive options.[1,2] Authors hope the current study will make a more accurate and useful guide for ß-thalassemia diagnosis and prevention in the region.
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