Molecular heterogeniety of β-Thalassemia in Karak District, Khyber Pakhtunkhwa, Pakistan

Shoaib ur Rehman1, Jabbar Khan2*, Raaza Malja Khan3, Maimoona Azam4 and Zeeshan Mutahir5 1Department of Biotechnology, university of science and technology Bannu, Pakistan. 2Gomal university Dera Ismail Khan, Pakistan. 3Mayo hospital Lahore, Punjab, Pakistan. 4Women and Children Hospital Bannu, KP, Pakistan. 5Institute of biochemistry and biotechnology, University of the Punjab, Lahore, Pakistan Article Information Received: March 23, 2020 Revised: October 29, 2020 Accepted 10 November 2020 Available online 09 February 2021

B eta-thalassemia (β-thal) is an autosomal recessive genetic disorders, characterized by mutation in β-globulin gene clusters positioned at the short arm (p) of chromosome number 11 band 11p15.4 -1p15.5. The disease mainly results from a variety of molecular defects including point mutations, insertion of stop codons in mRNA encoding the β-thal genes (Chen et al., 2010). The remarkable clinical features associated with heterozygous β-thal carriers include hypochromia, microcytosis, increased Hb A 2 and an unbalanced α/β-globin chain synthesis ratio (Laiska et al., 2016). More than 400 different mutations have recently been detected disturbing the diverse levels of β-globin gene expression and causing β-thal in which 35 mutations are predominant in Pakistan * Corresponding author: sjabbarkhan@yahoo.com 0030-9923/2021/0001-0001 $ 9.00/0 Copyright 2021 Zoological Society of Pakistan (Jalilian et al., 2017). Despite the fact that these mutations are not equally distributed, but have a geographical specificity and racial origin, each is identified by the presence of some common mutations and variable numbers of rare ones (Hussain et al., 2017). Globally, β-thal is one of the most important autosomal single gene disorders and in around more than 60 countries it can be found with a carrier population of up to 150 million (Kountouris et al., 2014). In Pakistan, the carrier rate in different ethnic groups has been estimated to be around 5.0-7.0%. Over 9000 children inheriting homozygous β-thal are born every year (Khan et al., 2018). The present study was aimed to assess the prevalence of the six common β-thal mutations in the district Karak of KP Province, their inheritance pattern and its relation to consanguinity. Moreover, this study can support public health efforts such as carrier screening, genetic counseling and prenatal diagnosis to control affected births by screening the population for O n l i n e

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the presence of common selected mutations through the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) technique.

Materials and methods
The current study was undertaken in the Molecular Laboratory of the Department of Biotechnology, University of Science and Technology Bannu, KP, Pakistan. Different demographic parameters including sex, age, clinical symptoms and family history of β-thal with important pathological parameters were collected on a study-designed proforma (Arif et al., 2008). A written consent was obtained from patients and their parents. In total, 200 blood samples were collected from 110 unrelated families having at least one affected blood transfusiondependent β-thal [Cooley's anemia or β-thal major (β-TM)] child (Ahmed, 2016). The study was permitted by the Ethnic Board of the Thalassemia Center and Bannu University review board. The participating patients in the study were blood transfusion-dependent and diagnosed as β-TM patients through peripheral blood morphology and Hb electrophoresis Laboratory tests (Shakeel et al., 2016;Ahmed et al., 2000). Most of the families were inbred and patients were born due to consanguineous marriages. Socioeconomic status of the non-consanguineous families was more satisfactory than consanguineous (Woods et al., 2006). The genomic DNA from the collected blood samples was isolated using standard protocol (Sambrook, 1987). Screening the samples was done using tetra primer based allele specific PCR (Newton et al., 1989). Two PCR reactions were done either with allele specific primer or control primers (Table I). To perform PCR reaction, 100 ng genomic DNA, 200 µM of each dNTP, 1 unit of Taq polymerase, 10 pmol of each primer i.e. two primers for control fragment and two primers for each of the mutant or control allele and 1x Taq reaction buffer were used in 20 µ reaction volume. The reaction was done through 27 cycles, consisting of denaturation at 94°C for one minute, annealing at 65°C for one minutes and extension at 72°C for 1 minute and 30 seconds. Denaturation during the first cycle was done at 95°C for 6 minutes while the final extension was done at 72°C for 10 minutes. Gel electrophoresis of PCR product was done on 3% agarose gel, containing ethidium bromide for visualization. Hind III digest was used as a marker. β-globin genotypes were assigned on the basis of presence or absence of allele specific bands. The previously characterized samples and dH 2 O were used as positive and negative controls respectively.

Discussion
Approximately 270 million carriers of hemoglobinopathies including 80 million β-thal have been estimated worldwide. Reviewing the previous reports, it can be concluded that 300,000-400,000 children with abnormal HbS including 23,000 β-thal are born every year (Shakeel et al., 2016). In Pakistan, β-thal is very common with an estimated inheritance rate of 5.0-7.0%, and over 9000 homozygous β-thal cases are born every year (Ansari et al., 2011;Ahmed et al., 2000). About 35 β-thal mutations have been reported in the country (Woods et al., 2006). In every population or ethnic group, usually four to seven mutations are reported to be common, comprising nearly 90.0% of all β-thal cases (Woods et al., 2006). The frequency of homozygosity in Pakistan relates to certain aspects, the notable of which are frequent firstcousin marriages, increase in annual birth rates, contact with mutagens and migration of highly conserved and consanguineous Afghani population to Pakistan (Khan et al., 2015(Khan et al., , 2019. Moreover, the increase in annual birth rate in Pakistan accelerates the number of homozygous thalassemic patients. From the Indian subcontinent, nearly 40 β-thal mutations have been identified, of which five, i.e. FSC 8/9 (+G), IVS-I-5 (G>C), codons 41/42 (-TCTT), IVS-I-1 (G>T) and FSC 5 (-CT), are predominant (Khan et al., 2018;Arif et al., 2008). It has been reported that the existing treatment strategies (i.e. chelation therapy, bone marrow transplantation, blood transfusion) may not eliminate the disease due to its high treatment cost and required conditions (Ahmed, 2016;Ahmed et al., 2000). The reasons for the numerous birth rate of β-thal patients and carriers in developing countries include lack of public awareness and lack of facilities in the form of thalassemia centers, molecular diagnosis, premarital screening and prenatal screening (Ahmed, 2016;Ahmed et al., 2000). This study reports four common mutations in the local population, which are in agreement with previous findings (Ansari et al., 2011;Khan et al., 2015Khan et al., , 2019. Most Pashtuns prefer inter-family marriages. Interestingly, IVS-I-5(G>C) was reported as the second most common mutation in the region, which is contrary with the what has previously been reported (Khan et al., 2015(Khan et al., , 2019. The above contradiction can be correlated to regional distribution of the Pathan tribes containing a large number of small tribal groups, unequal sample size and sample sites. The other reason for contradiction could be that the area is comparatively closer to the Afghan border and a large population has migrated here during the Russian invasion of Afghanistan.

Conclusion
Consanguinity and lack of awareness and non-

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availability of health facilities have contributed to the high occurrence of β-thal and miserable life of the patients. All the patients must be molecularly characterized before first transfusion.

Statement of conflict of interest
The authors have declared no conflict of interest.