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

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 transfusion-dependent 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 dH2O were used as positive and negative controls respectively.

DNA from 200 individuals was screened for the presence of six β-thal mutations reported in Pakistani population. Only four point mutations; FSC 8/9 (HBB: c.27_28insG), IVS-1-5 (G>C) (HBB: c.92+5G>C), FSC 5 (-CT) (HBB: c.17_18delCT) and codon 15 (G>A) (HBB: c.47G>A) were found to be the most common in 181 patients (Table II). Nineteen patients remained uncharacterized for

Table I. The amplification refractory mutation system-polymerase chain reaction allele specific primers used in this study.

IVS, Intervening sequence; FSC, frameshift codon; Mt, mutant; N, normal.

known 22 mutations reported so far in Pakistani community (Table II). Of the 181 characterized samples, 74 (40.88%) were found to be homozygous for four mutations and 107 (59.12%) were heterozygous for the same mutations. The frame-shift mutation at codon 8/9 (+G) was found to as the most prevalent mutation (49%), followed by IVS-I-5(G>C) (29%), codon 5 (-CT) (14.5%) and codon 15 ((G>A) (7.5%) (Table II). A total of 255 mutant alleles observed for the above mentioned four mutations. Of the 74 homozygous patients 34 (45.9%) had codon 8/9 (+G) mutation, 21(28.3%) possessed IVS-I-5(G>C), and 13 (17.6%) and 6 (8.1%) were homozygous for FSC 5 (–CT) and codon 15 (G>A) respectively (Table II). Similarly, maximum number of patients (57=53.3%) were found with codon 8/9 (+G) mutation out of 107 compound heterozygous thalassemia patients, followed by IVS-I-5(G>C) (29.9%), codon 5 (–CT) (9.4%), and only 6.5% patients with Codon 15 (G>A) mutation.

Moreover, out of 200 patients, 40 (20.0%) were reported to be over 15 years old, 110 (55.0%) patients demonstrated early onset of disease, i.e. <5 years old, and 50 (25.0%) patients were >10 years old. Furthermore, the study also showed 109 (54.5%) patients with poor socioeconomic status, having a monthly income of <20,000 PKR. The parents of 89 (44.5%) patients were first cousins, 84 (42.0%) were related but not first cousins, and 27 (13.5%) were not related. The above mentioned data suggests consanguinity as the main driving force behind the inheritance of β-thalassemia in KP province.

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 first-cousin marriages, increase in annual birth rates, contact with mutagens and migration of highly conserved and consanguineous Afghani population to Pakistan (Khan et al., 2015, 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., 2015, 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, 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.

Consanguinity and lack of awareness and non-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.

The authors have declared no conflict of interest.