The Story of a Ship Journey, Malaria, and the HBB Gene IVS-II-745 Mutation: Circassian Immigration to Cyprus

Background  During 19th century, the Circassians were secluded from their lands and forced to migrate to Ottoman Empire properties. Approximately 2,346 Circassians were exiled from Istanbul to Cyprus Island. During the deportation journey, many of Circassian passed away in consequence of malaria and unknown reasons. Overall, 1,351 survivor Circassians managed to reach the island, however, many of them had faced with endemic malaria again in Cyprus. An autosomal recessive hematological disorder thalassemia was the second endemic health condition after malaria, whereas thalassemia carriers show resistance to malaria infections. Materials and Methods  A large Cypriot family with 57 members whose grandparents were supposed to be in that ship journey has been investigated in this study. Polymerase chain reaction (PCR)–amplification refractory mutation system (ARMS) analysis technique was used for genotyping the HHB gene. Results  The human β-globin ( HBB ) gene c.316–106C > G (IVS-II-745) (II-745) heterozygous variation have been detected. Conclusion  Overall, this study is a very good example for a typical natural selection. In this case, one single gene point mutation did not limit survival in the society; natively, it increased their survival changes to form new colonization and the inheritance of the mutation to the next generations.


Introduction
During the 19th century, the autochthonous Circassians (Adyghe, Cherkess) of the Caucasus, who resisted to dominate the Eurasian Basin, were driven away from the mainland number of Circassians directly killed by the Russians was estimated to be more than 500,000. 1 According to the British war historian Allen, the number of Circassians placed in the Ottoman territory was more than 600,000 between the years of 1863 and 1864. 2 Immigration to Istanbul was banned due to epidemic diseases; therefore, shipment was directed to the Balkans. The Circassians, who had settled to Cyprus, were originally from Samsun, prior to Caucasus and Istanbul. Approximately 2,346 Circassians were exiled from Istanbul to Cyprus by three Ottoman flagged Aufdromachi vessels named "Revan-i Ticaret," "Hıfz-i Rahman," and "Eflak." During the deportation journey, many people could not survive due to malaria and unknown reasons. 3 It was suspected that they fell victim to malaria or typhus. In fact, only 1,351 of 2,346 Circassians arrived to Cyprus.
An autosomal recessive hematological disorder, β-thalassemia, was one of the most serious endemic health conditions in Mediterranean region after malaria. 4-6 During 1944 to 1946 Dr. Alan Fawdry was first to report thalassemia in Cyprus 5 and the control of malaria was successful in Cyprus between the years of 1946 and 1950. 7 Haldane suggested that heterozygous carriers for β-thalassemia are less predispose to severe malarial infections, since cells containing reduce or absent synthesis of globin chains are not very conductive to malarial parasite expansion properly within the erythrocytes. 8 Previous studies have revealed that five mutations were detected amongst the Turkish Cypriots 9,10 and Greek Cypriots. 11 The results were generally similar IVS-I-110 (G!A) 74.1%, IVS-I-1 (G!A) 7.3%, IVS-I-6 (T!C) 7.8%, IVS-II-745 (C!G) 6.5%, codon 39 (C!T) 0.9%, unknown 3.4% were the frequency of Turkish Cypriots and IVS-I-110 (G!A) 79.0%, IVS-I-6 (G!A) 6.3%, IVS-I-I (T!C) 6.0%, IVS-II-745 (C!G) 4.1%, codon 39 (C!T) 1.8%, and the other 3.4% were the frequency for Greek Cypriots, respectively (►Table 1). 10,11 The mutation denoted as IVS-II-745 (C!G) was introduced to the Eastern Mediterranean region during Muslim groups movement living in former Turkish territories to Southern Europe as a consequence of the Ottoman Empire falling (starting from 1914 AD), consequently contributing to a racial admixture. 12,13 In addition, the IVS-II-745 (C!G) mutation was detected in less heterogenic regions, for instance Northern, Southern, and Western Anatolia 14-16 (►Table 2). In this study, we aimed to trace the introducer of the mutation in the island and explained natural selection process.

Patients and Genotyping
Venous blood was collected with ethylenediaminetetra-acetic acid (EDTA) tubes from 57 Turkish Cypriot family mem-bers and genomic DNA was isolated using PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific, Waltham, Massachusetts, United States) for sequencing. Medical history was questioned and written informed consent form obtained from all the patients. DNA extraction was conducted under a class-II laminar flow using autoclaved pipets to minimize the risk of contamination in a class-II laminar flow hood. All solutions and equipments were ultraviolet (UV) treated to prevent any potential contamination. Mutation screening was performed in the human β-globin (HBB) gene using polymerase chain reaction (PCR)-amplification refractory mutation system (ARMS) analysis for point mutations as described by Sozuöz et al. 10 Informed consents were obtained from all participants. This study has been approved by the institutional ethics committee (registration number: YDU/2020/77-978).

Studied Patients and Genotyping
Fifty-seven members of the conserved Circassian family members whose grandparents were driven away from their mainland and migrated to Cyprus in early 1860s was screened for common thalassemia-causing mutations using PCR-ARMS. The sequence analysis revealed heterozygous IVS-II-745 (C!G) mutation in 12 family members (II:1, II:3, II:9, II:11, III:1, III:3,  III-5, III:

Discussion
The family data can be used to summarize that this studied family has relations to Caucasia, it further demonstrates that ancestors of the family were at an advantage during the migration to Cyprus, as this mutation protected them from malaria. The example provided in this study represents a very example for natural selection scenario. Overall, this current study indicated that the HBB gene IVS-II-745 (C!G) did not limit survival, in fact it enhanced the survival changes of the migrating family members, allowing them to form new colonization, and inheritance of the mutation to their offspring.