Research on Circassians and Chechens in Jordan

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Research on Circassians and Chechens in Jordan

Rana Dajani, PhD

 

Introduction

Jordan’s population is comprised of three ethnic groups: Arab, Circassian and Chechan.

Arabs are the main inhabitants of Jordan while the two other ethnic groups have immigrated to Jordan 140 years ago. Circassians have migrated from the Cocas region while Chechans from Chechnya. Both Circassians and Chechans in Jordan have managed to maintain their identity and ethnicity during the last 100 years. One of the main ways they have done this has been through endogamous marriages, the use of the original language and the preservation of certain traditions of their respective cultures.

Dr Rana Dajani initiated a project to study these two ethnic groups.  Dr Dajani created a team of researchers from different fields of expertise from Hashemite University, the University of Jordan, The Jordan University of Science and Technology and the Center for Diabetes, Endocrinology. Our collaborator in the US is the Center of Genomics, University of Pennsylvania and Children Hospital of Philadelphia.

The studies included anthropology, prevalence of complex diseases such as diabetes, CVD and cancer, genes associated with complex diseases, association of diseases and genes with life style such as nutrition, pharmacogenes related to drug metabolism, mitochondrial DNA and forensic DNA as well as effect of environment and epigenetics on complex diseases.

As a community service family trees were built and the community was served by a team of local physicians to address any medical problems.

Research methodology in general

Data were collected in the period between August 2008 and March 2017. IRB approvals have been obtained from local universities.  A pilot-tested structured questionnaire prepared specifically for the study was administered by trained interviewers to collect information on socio-demographic factors, DM, hypertension, hyperlipidemia, smoking habits, and potential risk factors. To ensure that all we sampled were pure Circassians/Chechans, each participant in the study filled out a survey that included pedigree information. The names and ethnicity of parents, grandparents, great grandparents both maternal and paternal and any individual with non Circassian/Chechan heritage for even one person in his/her pedigree was excluded.

Anthropometric measurements including weight, height, hip and waist circumferences, were measured. Body mass index (BMI) was calculated. The blood pressure was measured. For laboratory analysis and all biochemical measurements, two sets of fasting blood samples were drawn for glucose and lipids determination. Lipid parameters [total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triacylglycerol], and glucose were analyzed for all samples. DNA was sampled to be used for genetic studies including GWAS and polymorphisms for Pharmcogenes, mitochondrial DNA, Disease associated genes as well as epigenetics 

Anthropology

Anthropometric measurements including weight, height, hip and waist circumferences, will be measured with the subjects wearing light clothing and no shoes. Waist circumference will be measured to the nearest centimeter using non-stretchable tailors measuring tape at the narrowest point between the umbilicus and the rib cage and hip circumference will be measured at the widest part of the body below the waist. Body mass index (BMI) will be calculated as the ratio of weight (kilograms) to the square of height (meters).

Shape of the head will be measured by taking the following measurements:

  1. Maximum Cranial Length (g-op): distance between glabella and opisthocranion in the midsagittal plane, measured in a straight line.
  2. Maximum Cranial Breadth (eu-eu): maximum width of skull perpendicular to midsagittal plane wherever it is located (euryon).
  3. Bizygomatic Diameter (zy-zy): direct distance between most lateral points on the zygomatic arches. 4. Minimum Frontal Breadth (ft-ft): direct distance between the two frontotemporal.
  4. Nasal Height (n-ns): direct distance from nasion (n) to the midpoint of a line connecting the lowest point of the inferior margin of the nasal notches (ns).
  5. Nasal Breadth (al-al): Maximum breadth of the nasal aperture.
  6. Skin Color : Lightest, medium light, medium, medium dark and darkest
  7. Hair: Crinkly-curly, wavy-straight and straight.
  8. Color and form of the eyes: Black, Blue, Green, blue-green, blue-white, auburn. Round, oval or almond
  9. Shape of the nose 

Diabetes, hypertension and metabolic syndrome.

The prevalences of diabetes (Type 2), hypertension and metabolic syndrome were investigated as cross sectional studies in both populations.

The data showed that the prevalence of diabetes mellitus in the Circassian (9.6%) and Chechan population (10.1%) is similar. There were no significant differences in sociodemographic, anthropometric and clinical characteristics between the two groups. These are less than the prevalence rates reported in Jordan of 17%.

The homogenous, genetically isolated Circassian and Chechan populations sharing the same environmental influences with Arabs suggest a role for genetic risk factors for diabetes. Thus these two populations are suitable for additional genetics studies that may lead to the identification of novel risk factors for type 2 diabetes. This suggests that the ethnicity of these populations may play a role in the predisposition to diabetes onset since all populations share similar environments.

Further genome wide association studies were conducted after the recruitment of a total of 144 subjects including cases and controls from the Chechen population and 140 subjects from the Circassian population and performed meta-analysis. We identified a novel type 2 diabetes locus at chr20p12.2 at genome-wide significance (rs6134031, P=1.12×10-8) and replicated in Wellcome Trust Case Control Consortium (WTCCC). Another locus at chr12q24.31 (top SNP rs4758690, P= 4.2×10-5) is associated with type 2 diabetes at suggestive significance level and it is a robust eQTL for gene MLXIP (P=1.8×10-14) and is significantly associated with methylation level in MLXIP which functions in cellular glucose response. Therefore, in the first GWAS of type 2 diabetes in Jordan subpopulations, we identified novel and unique susceptibility loci which may be further tested in other populations.

In another study, the potential role of rare variants in type 2 diabetes was investigated, by performing copy number variance (CNV) analysis on two ethnic populations of ancient descent, the Circassians and the Chechens, which were genotyped by Illumina single nucleotide polymorphism (SNP) arrays.

It was found a CNV region in protein tyrosine phosphatase receptor type D (PTPRD) with significant association with type 2 diabetes. Additionally, 16 CNV regions were identified that were associated with type 2 diabetes which overlapped with gene exons. Of particular interest, a CNV region in the gene AKNA Domain Containing 1 (AKNAD1) surpassed the experiment-wide significance threshold. This is the first CNV analysis of a complex disease in populations of Jordan. A significant CNV in the gene AKNAD1 which was associated with type 2 diabetes was identified and experimentally validated.

Association of lifestyle, nutrition and disease with genetics

The intake of energy, macro- and micronutrients was investigated as a cross sectional study among 437 Circassians (160 males and 277 females) and 355 Chechans (119 males and 236 females). One 24-hour dietary recall for each participant was collected by face-to-face interview

The intake was studied between normal and diabetic adults. Most of the measured anthropometric and biochemical parameters showed a significant difference between normal individuals and those with impaired fasting glucose or diabetes. Intake of nutrients did not differ in participants with normal blood glucose from those who had impaired fasting glucose or diabetes in the two studied populations. These two populations may need genetic studies to identify the risk factors other than dietary and lifestyle factors for type 2 diabetes.

Furthermore, we analyzed a total of 36 traits including the macro/micronutrient intake and biochemical data with valid values in association with the Genome wide association studies data for 34 diabetic Chechans and 34 diabetic Circassians. We have identified a genetic association with intake of carbohydrate, calorie, vitamin B2 and caffeine in the Circassian population and calorie intake in the Chechan population. Three of these intake traits (carbohydrate, calorie and vitamin B2) were correlated with type 2 diabetes development in Circassians. On the other hand, only calorie intake was correlated with the occurrence of significant SNPs in Chechans, however, none of them were related to type 2 diabetes. The association between macronutrient intake and diabetes development can shed light on causative variants for the pathogenesis of T2D.

Cancer prevalence

The National Cancer Registry data was obtained for the years 1996-2005 to study the Chechan and Circassian cancer cases and compare the incident of cancer cases with the Jordanian population. The rates were calculated based on the number of cancer cases and estimated populations. From the data, it was revealed that in incident of colorectal cancer and in male Arab and Chechans was twice as high as Circassians. Breast cancer was the most prevalent type of cancer compromising one third of all female cancer cases in all groups, higher rates were observed in Chechans and Circassians compared to Arabs. The most widespread type of cancer in male Arabs and Chechans was lung cancer. Cancer is a complex disease caused by several genetic and environmental factors. This study has uncovered the considerable ethnic variations that exist in the cancer incident rates in Jordan.

Clotting factors

Studying the main genetic risk factors in venous thrombosis which are the polymorphism of factor II, factor V Leiden and methylenetetrahydrofolate reductase (MTHR). This was conducted on both ethnic groups. The study of the polymorphism will provide insight on medications, population genetics and biological anthropology.

Factor II G20210A, factor V Leiden and MTHR C677T single nucleotide polymorphisms (SNP) were investigated. These polymorphisms were analysed in 120 unrelated Chechans. Conversely, factor II and factor V and MTHR polymorphisms were investigated in 104 and 72 unrelated Circassian subjects, respectively.

Within the two ethnic groups, no homozygous allele was found in factor II G20210A mutation while the prevalence of the heterozygous allele was 2.5% and 12.5% in Chechans and Circassians, respectively. Factor V G1691A mutation was detected as heterozygous with a prevalence of 18.3% in Chechans and 7.8% in Circassians, no homozygous allele was detected.

When MTHR C677T was investigated a prevalence of 27.5% and 50% was found in Chechans and Circassians, respectively.

These findings could be useful in genome wide association studies and in clinical testing settings. The knowledge of such frequencies in the Middle East region through population-based studies will contribute to a better understanding of the interaction between genetic and environmental risk factors underlying thrombosis. Thus, these two inherited prothrombotic polymorphisms represent interesting tools for population genetics studies. The knowledge of these frequencies in the Middle East region through population-based studies will contribute to a better understanding of the interaction between genetic and environmental risk factors underlying thrombosis. The relationship between venous thrombophilia and these mutations have to be further studied in the Chechan population. 

Family trees

We have built pedigrees of the families of both populations.   .

DNA Databank

This is a bank of DNA from the populations studies to be used for future studies.  This is invaluable since many have deceased dn more and more of the community are marrying outside the population. This is a treasure trove for biology. 

Pharmacogenomics for drugs

Individual responses to the same medication differ significantly among populations, leading to difficulties in developing new treatments for diseases. These inter-patient variabilities in drug reactions result from many factors such as disease determinants, genetic and environmental factors (diet, climate, smoking, alcohol consumption, etc.) and variability in drug target response or idiosyncratic response.

These factors affect drug absorption, distribution, biotransformation, excretion, or combination of these. Thus, understanding the variation in efficacy and toxicity of the same doses of drugs in humans may provide a safer and more efficient drug treatment, where two million patients in the United States suffer from adverse drug reactions (ADR) including 100,000 deaths per year.

In this regard, the field of pharmacogenomics aims to elucidate candidate gene variants involved in drug pharmacokinetics (PKs) or pharmacodynamics (PDs), by identifying single nucleotide polymorphisms (SNPs) or multiple gene profiles which may be associated with drug reactions. Thus, providing information in order to personalize medicine, i.e. give the patient the right medication with the right dose at the right time to get better treatment outcome.

Differences in individual responses to the same medications vary significantly between populations. A number of genes that play an important role in drug responses have been designated as very important pharmacogenes (VIP) by the Pharmacogenomics Knowledge Base (PharmGKB), which are responsible for differences in drug safety, efficacy and the risk associated with adverse drug reactions among ethnic groups. Identifying the polymorphic distribution of VIP in various ethnic groups will assist in personalized medicine for various populations.

We are studying a number of genes related to pharmacological drugs metabolism in the circassian and Chechan population.  We are analyzing the polymorpshiss of these genesso that we can advise on personalized medicine in the future for these two populations.  Polymorphisms in these genes could potentially be significant for the pharmacological, toxicological and physiological effects of the enzymes. We have studied UDP-glucuronosyltransferase enzymes (UGTs), CYP2C8, ABCB1, VDR and TPMT genes.   

Mitochondrial DNA for ethnicity

The Circassian and Chechan population is an ethnic a human mitochondrial DNA haplogroup is a haplogroup defined by differences in human mitochondrial DNA. Haplogroups are used to represent the major branch points on the mitochondrial phylogenetic tree. Understanding the evolutionary path of the female lineage has helped population geneticists trace the matrilineal inheritance of modern humans back to human origins in Africa and the subsequent spread around the globe. We will analyse the mitochondrial DNA of the Circassian and Chechan population.

Forensics

Microsatellites or Short Tandem Repeats (STRs) are highly polymorphic repetitive sequences molecular markers which are usually used as DNA fingerprint in forensic genetics and population studies, and help in identifying individuals according to their inherited genetic material. The main aim was  to determine the allele frequencies and evaluate the forensic efficiency parameters of 21 highly polymorphic autosomal STR loci among Circassian subpopulation in Jordan.  The GlobalFiler™24 loci system (Life Technologies) was used to determine the allele frequencies and forensic efficiency parameters of the 21 autosomal STR loci. Autosomal STR information was collected from whole blood samples of 150 unrelated healthy individuals belonging to the Jordanian population of Circassians. The GlobalFiler™ 24 loci kit PCR System (Life Technologies) was used to amplify the 21 autosomal STRs and the amplified samples were analyzed on the 3130xl Genetic Analyzer using GeneMapper ID-X software (Life Technologies). Finally, the allele frequencies and forensic genetic efficiency parameters of the studied 21 autosomal STR markers were calculated. Circassians showed high number of observed alleles (250) with a predicated number of 1.248E36 PG. Departure from HWE was not significant for all of investigated 21 loci after applying Bonferroni corrected significance level for multiple testing (P=0.002). All of the tested loci exhibited linkage equilibrium pattern (P>0.05). SE33 locus was the highest informative and polymorphic STR marker meanwhile TPOX was the least marker. Allele 8 for TPOX was the most common one cross all of the investigated 21 loci.  The investigated forensic parameters showed high average values in Circassians subpopulation of Jordan: polymorphism information content (PIC) 78%; power of discrimination (PD) 93%; power of exclusion (PE) 62% and observed heterozygosity (OH) 79%. Combined matching probability (CMP) in Circassians was 5.02E-24 or (1 in 19 septillion) and combined discrimination power (CDP) was 0.9999999. Based on the results, it can be confidently concluded that: genetic efficiency of the investigated 21 polymorphic autosomal (STR) markers are suitable for criminal forensic investigations, paternity testing and human identification studies for Jordanian Circassian subpopulation. 

Ancient Human Migration

We have been working with a consortium of scientists studying the migration of humans between Asia and Europe over thousands of years. Our modern DNA data was pooled to analyse and understand the anceicnet migrations. This was the first paper in five years from Jordan to be published in Nature which is the number one journal in the world for science.

Future work, Turkey, Caucasus and Palestine.

Our objectives are to study the Chechan and Circassain population in terms of genetics and effects of the environment.  We aim to do this by comparing two populations that are genetically identical but living in two different environments for the past 150 years.  This situation provides an elegant experimental/ control experiment to study the effect and impact of the environment on the phenotype and epigenetics of these populations.

References:

 List of research articles on Circassians and Chechans

  1. Shishani K, Dajani R and Khader Y Hypertension Risk Assessment in the Largest Ethnic Groups in Jordan. J Immigr Minor Health. 2013 Feb;15(1):43-8
  1. Dajani R et al Diabetes mellitus in genetically isolated populations in Jordan: prevalence, awareness, glycemic control, and associated factors Journal of Diabetes and its complications 2012 May; 26 (3) : 175-80
  1. Dajani, R Khader YS, Hakooz, N, Fatallah R, Quadan F Metabolic syndrome and its components among two ethnic minority groups (Circassians and Chechens) and the original inhabitants of Jordan. Endocrine: Volume 43, Issue 1 (2013), Page 112-119.
  1. Dajani R, Fatallah R, Dajani A, Al-Shboul M, Khader Y Prevalence of coagulation factor II G20210A and coagulation Factor V G1691A (Leiden) polymorphisms in the Chechans, a genetically isolated population in Jordan. Mol Biol Rep. 2012 Sep;39(9):9133-8.
  1. Dajani, R et al Polymorphisms in Factor II and Factor V thromobophila genes among Circassians in Jordan  Journal of thrombosis and thrombolysis Volume 35, Issue 1 (2013), Page 83-89
  1. Dajani, R et al Prevalence of MTHFR C667T single nucleotide polymorphism in genetically isolated populations in Jordan Biochemical Genetics: Volume 51, Issue 9 (2013), Page 780-788
  1. Fatahallah, R and Dajani, R A comparison of population based cancer incidence rates among Circassians, Chechans and Arabs in Jordan (1996-2005). Asian Pac J Cancer Prev. 2013;14(10):6035-40.
  1. Tayyem, R., Dajani, R et al Nutrients Intakes among Normal and Type 2 Diabetic Individuals from Genetically Isolated Populations in Jordan Ethn Dis. 2014 Spring;24(2):200-6
  1. Dajani R et al CNV analysis suggests AKNAD1 associated with Type-2 Diabetes in the Circassian and the Chechan Populations of Jordan Sci Rep. 2015 Aug 21;5:13391. doi: 10.1038/srep13391.
  1. Eitan, Laith N., Ahmad M. Nassar, Rana B. Dajani, Basima A. Almomani, and Nesreen A. Saadeh. “Diabetes Mellitus in Two Genetically Distinct Populations in Jordan. A Comparison between Arabs and Circassians/Chechens Living with Diabetes.” Saudi Medical Journal 38, no. 2 (February 2017): 163–69. https://doi.org/10.15537/smj.2017.2.17910
  1. Dajani, Rana, Jin Li, Zhi Wei, Michael E. March, Qianghua Xia, Yousef Khader, Nancy Hakooz, et al. “Genome-Wide Association Study Identifies Novel Type II Diabetes Risk Loci in Jordan Subpopulations.” PeerJ 5 (2017): e3618. https://doi.org/10.7717/peerj.3618.
  1. Dajani R, Shbeilat S, Arafat A, Sobar S, Bitar H,Tayyem R, Wei Z, Hakonarson  Association of genetic variants with macronutrient intake in Circassian and Chechan populations in relation to diabetes accepted in MetaGene February 2018
  1. Dajani R et al Genes associated with diabetes in Circassians and Chechans accepted in Jordan medical Journal January 2018
  1. De Barros Damgaard, P …. Dajani, R et al Population genomic history of the Euroasian steppe accepted in Nature May 2018
  1. Dajani R Why the right questions make the biggest impact Nature Middle east July 31, 2018. doi:10.1038/nmiddleeast.2018.88
  1. https://www.natureasia.com/en/nmiddleeast/article/10.1038/nmiddleeast.2018.73
  1. Sara Abudahab, Nancy Hakooz, Yazun Jarrar, Mohammad Al Shahhab, Ahmad Saleh, Malek Zihlif, Rana Dajani. Differences in UGT1A1 and UGT1A7 Polymorphisms between Circassian, Chechen and Jordanian-Arab Populations submitted to Molecular reports

 

 

 

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