STUDY OF THE GENETIC DIVERSITY OF KILLER CELL IMMUNOGLOBULIN LIKE RECEPTORS (KIRs) IN SOME HUMAN POPULATIONS OF SUB-HIMALAYAN REGION. A THESIS SUBMITTED TO THE UNIVERSITY OF NORTH BENGAL FOR THE AWARD OF DOCTOR OF PHILOSOPHY (Ph.D.) IN ZOOLOGY BY POKHRAJ GUHA SUPERVISOR Prof. Tapas K. Chaudhuri CO-SUPERVISOR Dr. Soumen Bhattacharjee Dr. Biswajit Haldar DEPARTMENT OF ZOOLOGY UNIVERSITY OF NORTH BENGAL JANUARY 2017 DEDICATED TO MY PARENTS AND MY MENTOR PROF. T.K. CHAUDHURI. DECLARATION I declare that the thesis entitled “STUDY OF THE GENETIC DIVERSITY OF KILLER CELL IMMUNOGLOBULIN LIKE RECEPTORS (KIRs) IN SOME HUMAN POPULATIONS OF SUB-HIMALAYAN REGION”, has been prepared by me under the guidance of Prof. T.K. Chaudhuri and Dr. Soumen Bhattacharjee, Department of Zoology, University of North Bengal and Dr. Biswajit Halder, Department of Pathology, North Bengal Medical College and Hospital. No part of the thesis has formed the basis for the award of any degree or fellowship previously. (Pokhraj Guha) Department of Zoology University of North Bengal Raja Rammohunpur, Dist. Darjeeling Dated: 19.01.2017 PREFACE I started my research work in 2009 under the supervision and expert guidance of Prof. T. K. Chaudhuri and co-supervision of Dr. Soumen Bhattacharjee, Department of Zoology, University of North Bengal, and Dr. Biswajit Halder, Department of Pathology, North Bengal Medical College and Hospital, Sushrutnagar, Dist. Darjeeling. My entire work has been documented in this dissertation entitled “STUDY OF THE GENETIC DIVERSITY OF KILLER CELL IMMUNOGLOBULIN LIKE RECEPTORS (KIRS) IN SOME HUMAN POPULATIONS OF SUB-HIMALAYAN REGION”. Killer-cell immunoglobulin-like receptors (KIR) constitute a family of receptor molecules that are present on both natural killer (NK) cells as well as on a subset of T- lymphocytes. The nomenclature of these receptors is based on the number of extracellular immunoglobulin-like domains (2D or 3D) and the character of the cytoplasmic tail (L or S). KIRs with long cytoplasmic tails are involved in inhibitory signaling while those with short cytoplasmic tails transduce activation signals. The members of the KIR family interact with human leukocyte antigen (HLA) expressed on the surface of all nucleated cells. Normal KIR/HLA interaction protect the cell against cytotoxic effects of NK cells, failure of which may result in positive intracellular signals. These resulting signals affect NK cell activity causing the release of cytotoxic enzymes. Thus, KIR-HLA interaction plays a major role in innate immunity. The KIR genes are highly polymorphic and are located on the long arm of chromosome 19, within the Leukocyte Receptor Complex (LRC). KIR gene content and haplotypes vary between ethnic cohorts and individuals within a population. Based on all these unique features, KIR has gained importance parallel to other established markers in the fields of human genetic diversity, in tracing the routes of human migration in different parts of the world and also in disease researches. The sub-Himalayan part of India is inhabited by many tribes and castes each having their own cultural and linguistic ethnicity. In one hand, some populations in this region arose Page | i due to blending of gene pool of different population groups while; on the other hand, some other populations of this region are strictly endogamous with a highly conserved gene pool of their own. In other words, it can be said that the sub-Himalayan part of West Bengal, India, is a melting pot of different ethnicity and is therefore extensively rich in genetic diversity. The present study was designed with the broad objective to study the KIR gene profile in different ethnic populations inhabiting the northern part of West Bengal. In addition, we also aimed to study association of KIR genes with Rheumatoid arthritis, which is one of the most common autoimmune debilitating diseases, affecting nearly 1% of the total world population. This disease is characterized by progressive articular damage leading to joint deformities and disability. Blood samples were collected from the volunteers with their prior informed consent. Individuals with three generations of unrelatedness were selected for the study. RA was diagnosed by the physician and the subjects were recruited from the Department of Pathology, North Bengal Medical College & Hospital, Sushrutnagar, Siliguri and 3gen diagnostic Pvt. Ltd, Siliguri. Blood samples were collected from the participants under appropriate conditions and brought to the Cellular Immunology Laboratory, Department of Zoology, University of North Bengal, where the further experiments were performed. The findings of the study are published in various research journals and are presented and discussed in details in the Results and Discussion part of this dissertation. Page | ii ABSTRACT Killer cells Immunoglobulin-like Receptors (KIR) constitute a family of regulatory receptor molecules found on the surface of NK cells and T cell subsets. They can be either activating or inhibitory in function. These receptors interact with polymorphic Human Leukocyte Antigen (HLA) class I molecules and influence the innate and adaptive immune responses to infection. Variations in gene content and sequence polymorphism are noticeable in the KIR family and these features make the family suitable for distinguishing individuals and populations around the world. The KIR multigene family consists of 14 genes and 2 pseudogenes. These genes are clustered together on chromosome 19q13.4, within the 1 Mb Leukocyte Receptor Complex (LRC). Two distinct KIR haplotypes can be distinguished based on gene contents, namely A and B. The A haplotypes have seven KIR loci, with only one gene coding for an activating receptor i.e. KIR2DS4 gene. On the other hand, the B haplotypes are characterized by presence of loci most of which encode activating receptors. Inspite of variations in gene content, all KIR haplotypes contain the ‘framework genes, namely KIR3DP1, KIR3DL2, KIR3DL3 and KIR2DL4 that are well conserved in virtually all individuals. KIR genotyping has documented substantial variation in the relative frequencies of A and B haplotypes in different populations. These unique characteristics of KIR genes have generated immense interest among the researchers all around the world. As a result, KIR genes are progressively gaining applications in the field of human genetic diversity and for tracing the routes of migration in different parts of the world in a way parallel to other established markers in this field such as HLA, mitochondrial DNA (mtDNA) and Y- chromosome haplogroups. The present study was aimed to estimate the frequencies of 16 KIR genes among five ethnic populations inhabiting northern part of Bengal, namely Rajbanshi, Rabha, Bengali, Gurkha and the Muslims, and to study their phylogenetic relationship among themselves and with other populations around the World. Additionally, the frequencies of the KIR Page | iii genes in patients with Rheumatoid Arthritis were also estimated to study the association of the KIR genes with occurrence of the disease if any. Blood Samples were collected from the volunteers with their prior informed consent. In both the population-based and disease related section of the study, three generation pedigree of the volunteers were taken into account and only unrelated individuals were included in the study. DNA was extracted from each blood sample using the phenol- chloroform extraction procedure. Sixteen KIR genes were analyzed in the sample individuals using PCR-SSP typing. Different freely available statistical software were used to analyze the data. It is evident from the population-based section of our study that the KIR genes demonstrated the highest range of frequencies among the Gurkhas in comparison to other studied populations. In contrast, the lowest range was demonstrated in case of the Muslims. Interestingly, all the five studied populations demonstrated the lowest frequency value for KIR2DL3 gene while the highest frequency value was seen to shuffle between the A haplotype associated genes except that for the activating receptor (i.e. KIR2DL4). Based on the distribution of KIR genes, 76 KIR genotypes were listed from our studied populations, out of which, 3 were AA genotypes while the remaining 73 were Bx genotypes. When compared with published data from other populations around the World, it was found that the Rajbanshis are phylogenetically closer to the East Asians, especially the Tibetan population than that with the Dravidian populations, which are considered native to India. Besides having some genotypes in common with that of the East Asians, the Rajbanshis also share some genotypes with the Dravidian settlers of India, such as the Paravars and Kanikars. There are many theories regarding the origin and migration of the Rajbanshis in the northern part of Bengal. Nevertheless, all of them confirmed the presence of mongoloid elements in the Rajbanshis. Page | iv The Rabha tribe is a small endogamous population belonging to the Indo-mongoloid group of Northeastern region of India. Their KIR gene frequencies ranged between 0.26 and 0.96 corresponding to KIR2DS3 and KIR2DL1 respectively. Phylogenetic assessment revealed that the Rabhas shared the same clade with the East Asians owing to their tribal ethnicity and strict endogamous nature, which helped them to retain their ancestral genotypic pattern. Thus, it is evident from our study that although Indian by origin, the gene pool of the Rabha population has been considerably