Genetic HLA Study of Kurds in Iraq, Iran and Tbilisi (Caucasus, Georgia): Relatedness and Medical Implications

Kurds from Iraq (Dohuk and Erbil Area, North Iraq) have been analyzed for HLA genes. Their HLA genetic profile has been compared with that of other Kurd groups from Iran and Tbilisi (Georgia, Caucasus) and also Worldwide populations. A total of 7,746 HLA chromosomes have been used. Genetic distances, NJ dendrograms and correspondence analyses have been carried out. Haplotype HLA-B*52—DRB1*15 is present in all three analyzed Kurd populations. HLA-A*02-B*51-DRB1*11 is present in Iraq and Georgia Kurds. Haplotypes common to Iran and Iraq Kurds are HLA DRB1*11—DQB1*03, HLA DRB1*03—DQB1*02 and others in a lower frequency. Our HLA study conclusions are that Kurds most probably belong to an ancient Mediterranean / Middle East / Caucasian genetic substratum and that present results and those previously obtained by us in Kurds may be useful for Medicine in future Kurd transplantation programs, HLA Epidemiology (HLA linked diseases) and Pharmacogenomics (HLA-associated drug side effects) and also for Anthropology. It is discussed that one of the most ancient Kurd ancestor groups is in Hurrians (2,000 years BC).


Introduction
HLA is the most polymorphic genetic system described in man. It contains several linked loci which encode for cell surface proteins that have an important function in activating immune response after antigenic presentation. New allele variants are frequently being described (i.e.: 1,883 HLA-DRB1 alleles have been recorded by June 2016) [1]. HLA gene frequencies have both a large degree of variability among populations and a striking geographical correlation. These frequencies are useful to infer genetic background and ethnical constitution of modern human groups and also for inferring migrations of ancient ones [2]. In addition, certain combinations of contiguous alleles between HLA neighboring loci show a characteristic frequency due to the robust linkage disequilibrium among them or are distinctive in many extant populations [3]. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Also, HLA allele frequencies are unique for studying the origins of relatively homogeneous groups, like the Kurd people living in Iraq. Other HLA gene characteristics are their link to disease and to different responses to drug treatments in patients according to different HLA alleles. Certain HLA alleles affect drug response to treatment in about sixteen different diseases including AIDS [4]. This is important for personalized drug treatment design (including ethnic groups with specific certain high allele frequencies), particularly if other already obtained Kurd HLA results are also included (from Georgia and Iran, in present study) and samples are further increased.
On the other hand, Kurd people live in different countries in the Near East such as Syria, Armenia, Turkmenistan, Kazakhstan, Turkey, Iraq and Iran, the so called Kurdistan ("land of Kurds") ( Fig 1, Table 1). Kurdistan is a region placed South Caucasus and North of ancient Mesopotamia. According to genetic studies (like HLA) in Turkish and Kurdish populations, a Anatolian-Mediterranean source for both populations was put forwards; it may be possible that Kurds are initially coming from ancient Hurrians, reviewed in [5,6]. Studies performed with mtDNA and Y-chr have also been done for Kurds, however there is no firm conclusion to infer that most Kurd people have originated either from Middle East and/or from Central Asia [7,8]. Most probably, Kurd people gene pool majority may be composed of an admixture of  North Mesopotamian (Caucasus) and Near East peoples; Central Asia gene input is not discarded [5,9,10,11,12,13]. Kurds have mainly been defined by their ancestry, language and cultural uses. Estimations of Kurds number are nowadays between 23 to 41 million people; see Table 1 for numbers and country distribution. In the present paper, a population of Kurds living in North Iraq (Dohuk and Erbil area, North Mosul, Fig 1) has been studied in order to: 1) Determine the HLA class I (A, B and C) and class II (DRB1 and DQB1) allelic Kurd lineages (hereafter "alleles" for simplicity) and specific HLA haplotypes by using standard DNA based techniques, 2) Compare Iraq Kurd HLA profile with those of Central Asia, Siberian, Mediterranean and other World ethnic groups ( Table 2) with specific computer programs in order to find out bases of HLA and disease linkage and origins of Kurd people using genetic distances comparisons, Neighbour Joining (NJ) trees and correspondence analyses, 3) obtaining Kurd HLA profile that may be used for preventive HLA Pharmacogenomics and a virtual regional future transplant waiting list among population, and finally 4) Kurd HLA profiles from Tbilisi (Georgia-Caucasus), Iran and Iraq are also compared among themselves.

Material and Methods
Population sample 209 healthy unrelated blood donor volunteers from the cities of Dohuk and Erbil and their area, North Iraq (Fig 1) were class I and class II typed. Unrelatedness and other sample parameters were checked by Drs. R. Al-Qadi and Shadallah Fareq Salih. Erbil is the capital of Iraq Kurdish Autonomous Region placed 175 km southeast Dohuk. The city of Dohuk is located in Kurdistan region in the North of Iraq, 70 km North Mosul and 60 km far from both Sirian and Turkish borders (36˚51 0 00@N 42˚59 0 00@E); Dohuk is historically and continuously inhabited by Kurds since All subjects in the study and their grandparents were born in the same area. We compared our data with those of worldwide populations (see Table 2), obtaining genetic distances, relatedness trees and correspondence analyses. Comparisons were done with 7,746 HLA chromosomes.

Statistical analysis
Statistical analysis was done with Arlequin v2.0 software [17,18,19] HLA allele typing data from Iran Kurds [13] have been converted to low resolution typing data in order to be able to carry out all analyses with as many populations as possible, while relatedness resolution is satisfactory given the very polymorphic HLA system [1].

HLA allele frequencies found in Kurd Iraq population: comparison with other Populations
The expected and observed allele frequency values for HLA-A, -B, -C, -DRB1 and -DQB1 shows that the population is in Hardy-Weinberg equilibrium. Table 3 depicts HLA allele frequencies found in the sampled population. Sixteen different HLA-A, twenty-seven different HLA-B and thirteen different HLA-C alleles were founds in class I. Only seven HLA-A alleles, nine HLA-B alleles and seven HLA-C alleles had frequencies higher than 4% (- . Twelve different HLA-DRB1 alleles and five different HLA-DQB1 alleles were found. Only eight HLA-DRB1 alleles and four HLA-DQB1 alleles had frequencies higher than 4% (- DRB1 alleles were used to compare our three Kurd samples with other populations in NJ analysis. It was not possible to perform this study with HLA class I allele frequencies due to the lack of class I studies in many worldwide available populations (Table 2).
NJ relatedness dendrogram based on HLA-DRB1 analysis separates populations in two differentiated clusters: A and B (Fig 2). Cluster A groups North and South Mediterraneans (Europeans and Africans) and Middle East populations (included Iraq Kurds, Iran Kurds [13] and Georgia Kurds [5] (Fig 2).
Correspondence analysis based on HLA-DRB1 allele frequencies (Fig 3) shows similar results to those of Fig 2. Two clusters are clearly defined according to first dimension that explains most of the variability among populations. The first one groups together Siberian and Oriental populations (left, Fig 3)

Conclusions and Final Remarks
Kurds are currently living in Kurdistan, a region encompassing different parts of several Middle East countries (Fig 1, Table 1); in addition they have moved to live in Middle East and European cities. We had previously studied Kurds in Tbilisi (Georgia, [5]) and also in Iran [13] for HLA allele frequencies. Our conclusions were that their HLA profile showed that Kurds form part of Mediterranean stock of people and also had Caucasus genetic traits (Svan, Georgians, [13,35,44]). Also, it is worth mentioning that Lak population (East Caucasus Area) may be close to both Lur and Kurd populations, and Lak name could be considered composed of Lur and Kurd words [52].
In the present paper, we have analyzed HLA genes in Kurds living in North Iraq (Erbil and Dohuk areas). Comparison with other populations place them as a Middle East population, close to Kurds from Tbilisi-Georgia, Palestinians, Armenians and Kurds from Iran (Fig 2). Correspondence analysis (Fig 3, right side) shows that Kurds (living in Iran, Tbilisi-Georgia and Iraq) somewhat divide bidimensional representation analysis in western Mediterraneans (upper part) and Eastern Mediterraneans (lower part). In both analyses (Figs 2 and 3), Kurds are also close to Caucasian (Svan, Georgian) populations. Conclusion is that Kurds are genetically close to surrounding Caucasian and Mediterranean populations and that have remained settled down in Kurdistan since ancient times; supporting historical evidence is reviewed in our previous work [5,6] and Ref [15]. HLA genetic similarity have been reported between Turks (whose genes belong to old Anatolian stock) and Kurds [5,6,13]. Kurds and Turks speak languages that are included in different families [53]. However, Kurd HLA genetic studies include them into Mediterranean stock together with Turks [5,13]. Other genetic studies based on Y-Chr in Kurds from Turkey, Georgia and Iran identify the dominant presence of haplogroups originated in Middle East (Anatolia or Mesopotamia) that show a close association with Jews, Lebanese and Turkish genes [7,8,12]. Also, Iranian populations are close to Kurds [54,55]. This again shows that languages and genes do not correlate because languages may be imposed by a genetic (but powerful) minority. This is the case of Turks: Anatolian people were settled down there since ancient prehistoric times, but a minority of people (Turks) coming from Central Asia imposed language in historical times [5,6].
Thus Middle East peoples from Mediterranean border and Kurds seem originally to belong to a similar ethnic group according to HLA autosomic and Y chromosomes genes results. Kurds have always lived in the mountains being "autochthonous" (6000 BC). Hurrians, whose language was Caucasian (and not Indo-European) may be Kurds ancient genetic background, reviewed in Refs [5,6]. By 1200 BC, Medes and others invaded Hurrian area. Kurdish historians consider that Kurds come from Medes, reviewed in [5]. "Kuru" was the first name of Kurds given by Assyrians (1000 BC) to groups living at Mt. Azu, Kurdistan. Kurds are also mentioned by early classical historians like Polybius (133 BC) and Strabo (48 AD), Kurds were named as "the Mountains People" under Persian, Greek and Roman Anatolian Peninsula rule [5].
In summary, all three Kurd populations studied in the present paper are genetically close together and to other Mediterranean and Caucasus populations according to HLA genes. This study may also help for future transplantation programs in their area and Kurd HLA Epidemiology and Pharmacogenomics.