4.1.1 Association of SNP variants within PARK2-PACRG gene regulatory region with leprosy susceptibility sharing chromosomal region 6q26

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1 4.1 GENETIC VARIATIONS IN PARK2 AND PACRG GENE REGULATORY REGIONS AND THEIR INTERACTION WITH IMPORTANT IMMUNO-REGULATORY GENES IN THE OUTCOME OF LEPROSY PARK2 and PACRG gene regulatory region was saturated with 96 SNPs with approximately 1 SNP per Kb for PARK2 gene regulatory region to perform a population based case-control study in an Indian population group. To rule out population stratification in the studied group which confounds a disease association study, the MDS (multi-dimensional scaling) analysis based on IBS (identity by state) pair-wise distances for 226 SNPs from chromosome 1, 3, 4, 5, 6, 7, 9, 11, 16, 19, 21, 22 and X, not associated with the disease was carried out, resulting in a compact cluster suggesting the population under study to be homogeneous without any substructures (Aggarwal et al. 211; Ali et al. 212). Locus wise F ST was also calculated for the SNPs associated with Leprosy in the Indian population. All the polymorphisms showed a very low locus-wise F ST value, indicating that the patients and controls belonged to the same population group. Thus, these results indicated that the selected region of 6q25-26 did not show any population differentiation, suggesting that the studied cases and controls belonged to a homogenous population group Association of SNP variants within PARK2-PACRG gene regulatory region with leprosy susceptibility sharing chromosomal region 6q26 In this study a total of 2551 samples were screened. These comprised of two independent cohorts of North Indian population, recruited at different time intervals; and a geographically independent East Indian population. Screening of 96 SNPs, distributed in the selected PARK2- PACRG, 6p25-26 region with approximately 1 SNP per Kb for PARK2 gene regulatory region, was carried out in 829 patients and 1476 controls of North India, using MassArray-SEQUENOM platform. The selected region included PARK2 and PACRG overlapping genomic region. Each constituent gene was evaluated independently and together to ascertain an association with leprosy susceptibility. A total of 11 SNPs out of the 96 SNPs studied showed a consistent and strong Page 78

2 association with susceptibility to leprosy in the North Indian population. Ten out of 11 SNPs were located in the regulatory region of the PARK2 gene and a single SNP within the regulatory region of the PACRG gene (Figure 4.1). Further, to replicate and confirm these results in a geographically independent population of India, a case-control study (93 patients and 153 controls) on Orissa (East Indian) population was carried out. The result showed consistent association of 11 SNPs in two different cohorts of the Indian population. (Table 4.1) Page 79

3 Figure 4.1: A partial map of Chromosome-6q26, expanded to show the position and distribution of 11 significant SNPs (shown with rs numbers) in the regulatory region of the PARK2 and PACRG genes is shown in Figure 1a along with the log P values for the 11 significantly associated SNPs in the combined Indian population (Figure 1b). Page 8

4 Table 4.1: Allele and Genotype frequencies for 11 significant SNPs within PARK2 and PACRG gene regulatory region in two different cohorts of patients with Leprosy. North Indian Orissa Combined Logistic p Value SNPs Gene Details Risk P a P b value P c value P d Unadjusted Adjusted corrected Model Allele value P e value P f value OR(95%CI) rs PARK2 Intron 1 Minor C CTvs ( ) rs PARK2 (-258)+PACRG(5' near gene) Minor C CAvs ( ) rs PACRG Intron 2 Minor C CTvs ( ) rs PACRG Intron 2 Minor A GAvs ( ) rs PACRG Intron 2 Minor C GCvs ( ) rs PACRG Intron 2 Minor G CGvs ( ) rs PACRG Intron 2 Minor T CTvs ( ) rs PACRG Intron 2 Minor G CGvs ( ) rs PACRG Intron 2 Minor A CAvs ( ) rs PACRG Intron 2 Minor C TCvs ( ) rs PACRG Intron 2 Minor A GAvs ( ) P a and P b value for 2 X 2 chi test for overall allelic frequencies comparison of samples from North Indian and samples from Orissa. p c value for 2 X 2 chi test for overall allelic frequencies comparison of combined samples. P d corrected P-values after bonferroni correction Bonferroni correction of 32 SNPs was applied for multiples testing. Out of total 96 SNPs tested 64 were in 14 bin set (r 2 >.8). P e and P f values for genotypic model by logistic regression for combines samples before and after adjustment for sex as a covariate respectively. Page 81

5 4.1.2 Detailed results of chosen SNPs and their significant association with Leprosy: Following are the detailed results of the SNPs located in PARK2 and PACRG gene regulatory region out of the 96 SNPs selected to be studied in the Indian population 15 SNPs were selected from the PARK2 gene region, 5 SNPs from the overlapping regulatory region of the PARK2 and PACRG gene and remaining 76 SNPs were located in the PACRG intronic region Table 4.2. Table 4.2. Association statistics of SNPs in PARK2 and PACRG gene in total leprosy patients and its subtypes. SNP ID Gene Cont Pat PB MB P(3X2 test) a rs GA PARK2:Exon 1571 (.98) 16 (.1) 895 (.99) (.99) (.98) 7 (.1).9757,.267,.357 G ,.268,.358 rs TG PARK2:Exon 1587 (.99) ,.596,.59 T ,.454,.446 rs GA PARK2:Exon 1345 (.84) 224 (.14) 18 (.1) 744 (.82) 15 (.16) 1 (.1) 37 (.83) 7 (.15) (.81) 8 (.17) 6 (.1).25,.634,.24 G ,.572,.84 rs PARK2:Exon Page 82

6 CA 1582 (.99) ,.236,.228 C ,.236,.228 rs AG PARK2:Exon AG Homo rs TC PARK2:Intron 155 (.94) 8 (.5) (.93) 6 (.6) (.94) 26 (.5) 425 (.92) 34 (.7) ,.6,.137 T ,.647,.44 rs GC PARK2:Intron 112 (.7) 415 (.26) 49 (.3) 626 (.69) 253 (.28) 21 (.2) 36 (.68) 125 (.28) 13 (.2) 32 (.7) 128 (.28) 8 (.1).3561,.79,.252 G ,.563,.77 rs CG PARK2:Intron 194 (.68) 44 (.27) 52 (.3) 626 (.69) 254 (.28) 24 (.2) 37 (.69) 124 (.27) 13 (.2) 319 (.69) 13 (.28) 11 (.2).6817,.932,.62 C ,.856,.655 rs28314 PARK2:Intron Page 83

7 AC 197 (.69) 438 (.27) 43 (.2) 626 (.69) 253 (.27) 25 (.2) 37 (.69) 124 (.27) 13 (.2) 319 (.69) 129 (.28) 12 (.2).9898,.969,.985 A ,.838,.984 rs TC PARK2:Intron 864 (.54) 64 (.38) 11 (.6) 423 (.46) 391 (.43) 9 (.9) 213 (.47) 188 (.42) 43 (.9) 21 (.45) 23 (.44) 47 (.1) T ,.19,.11.36,.49,.22 rs GA PARK2:Intron 458 (.29) 773 (.49) 346 (.21) 235 (.26) 436 (.48) 232 (.25) 14 (.23) 232 (.52) 17 (.24) 131 (.28) 24 (.44) 125 (.27).671,.67,.52 G ,.4,.121 rs TC PARK2:Intron 1484 (.93) 96 (.6) 839 (.92) 63 (.6) 41 (.92) 34 (.7) 429 (.93) 29 (.6) ,.318,.972 T ,.374,.815 rs AG PARK2:Intron 1284 (.8) 282 (.17) 22 (.1) 747 (.82) 148 (.16) (.81) 78 (.17) (.83) 7 (.15) 5 (.1).4564,.717,.38 A ,.612,.16 Page 84

8 rs PARK2:Intron A Homo rs AC PARK2:Intron;PACRG:5'UTR 12 (.64) 495 (.31) 72 (.4) 61 (.66) 276 (.3) 27 (.2) 283 (.63) 15 (.33) 11 (.2) 318 (.69) 126 (.27) 16 (.3).1356,.113,.141 A ,.615,.45 rs AC PARK2:Promoter;PACRG:5'UTR 865 (.54) 61 (.38) 114 (.7) 424 (.47) 385 (.42) 93 (.1) 213 (.47) 188 (.42) 43 (.9) 211 (.46) 197 (.43) 5 (.1) A ,.25,.12.45,.62,.22 rs PARK2:Promoter;PACRG:5'UTR C Homo rs PARK2:Promoter;PACRG:5'UTR A Homo Page 85

9 rs AG 777 (.48) 672 (.42) 138 (.8) 472 (.52) 362 (.4) 69 (.7) 219 (.49) 189 (.42) 36 (.8) 253 (.55) 173 (.37) 33 (.7).2551,.926,.63 A ,.784,.24 rs AC 87 (.54) 59 (.37) 125 (.7) 533 (.59) 39 (.34) 61 (.6) 267 (.6) 15 (.33) 27 (.6) 266 (.57) 159 (.34) 34 (.7).1244,.114,.58 A ,.33,.282 rs TC 1117 (.7) 418 (.26) 5 (.3) 621 (.69) 256 (.28) 23 (.2) 34 (.68) 127 (.28) 13 (.2) 317 (.69) 129 (.28) 1 (.2).489,.637,.439 T ,.528,.998 rs TC 897 (.56) 574 (.36) 115 (.7) 443 (.49) 375 (.41) 85 (.9) 219 (.49) 186 (.41) 39 (.8) 224 (.48) 189 (.41) 46 (.1).1,.24,.76 T ,.87,.14 rs CA 1119 (.7) 417 (.26) 51 (.3) 623 (.69) 256 (.28) 23 (.2) 36 (.68) 125 (.28) 13 (.2) 317 (.69) 131 (.28) 1 (.2).3742,.714,.355 C ,.643,.923 Page 86

10 rs (.64) 612 (.67) 287 (.64) 325 (.7) GT 49 (.3) 265 (.29) 146 (.32) 119 (.25) 7 (.4) 27 (.2) 11 (.2) 16 (.3).1198,.157,.59 G ,.537,.19 rs (.54) 424 (.46) 213 (.47) 211 (.45) GA 598 (.37) 389 (.43) 189 (.42) 2 (.43) 12 (.7) 91 (.1) 42 (.9) 49 (.1).5,.36,.19 G ,.1,.36 rs CT 1578 (.99) (.98) 1 (.1) 441 (.99) (.98) 7 (.1).199,.914,.63 C ,.914,.63 rs (.33) 242 (.26) 122 (.27) 12 (.26) GA 74 (.46) 451 (.49) 221 (.49) 23 (.5) 321 (.2) 211 (.23) 11 (.22) 11 (.23).39,.79,.14 G ,.33,.45 rs (.8) 737 (.81) 36 (.81) 377 (.81) GA 292 (.18) 155 (.17) 79 (.17) 76 (.16) (.1) 5 (.1) 7 (.1).2185,.638,.17 Page 87

11 G ,.99,.921 rs TG 626 (.39) 718 (.45) 232 (.14) 391 (.43) 48 (.45) 13 (.11) 195 (.44) 24 (.46) 44 (.9) 196 (.42) 24 (.44) 59 (.12).39,.24,.417 T ,.13,.18 rs AG 129 (.81) 286 (.18) (.81) 153 (.16) 11 (.1) 36 (.81) 8 (.18) (.82) 73 (.15) 7 (.1).333,.92,.15 A ,.854,.837 rs TA 1549 (.97) 36 (.2) 876 (.96) 27 (.2) (.97) 11 (.2) 443 (.96) 16 (.3) 1.228,.798,.61 T ,.799,.54 rs CT 618 (.38) 74 (.46) 228 (.14) 369 (.4) 432 (.47) 13 (.11) 175 (.39) 214 (.48) 55 (.12) 194 (.42) 218 (.47) 48 (.1).15,.555,.79 C ,.57,.47 rs TA 152 (.94) 81 (.5) 844 (.93) 59 (.6) 418 (.94) 26 (.5) 426 (.92) 33 (.7) ,.625,.212 Page 88

12 T ,.69,.75 rs GC 156 (.94) 79 (.4) (.93) 59 (.6) (.94) 26 (.5) 426 (.92) 33 (.7) 1.266,.577,.169 G ,.69,.57 rs A Homo rs GC 667 (.42) 721 (.45) 194 (.12) 326 (.36) 424 (.46) 154 (.17) 162 (.36) 25 (.46) 77 (.17) 164 (.35) 219 (.47) 77 (.16).57,.89,.9 G ,.32,.22 rs TC 68 (.38) 755 (.47) 222 (.14) 365 (.4) 433 (.47) 15 (.11) 173 (.39) 214 (.48) 56 (.12) 192 (.41) 219 (.47) 49 (.1).211,.76,.13 T ,.576,.62 rs TC 688 (.43) 558 (.35) 329 (.36) 327 (.36) 162 (.36) 156 (.35) 167 (.36) 171 (.37) Page 89

13 333 (.21) 246 (.27) 126 (.28) 12 (.26) T ,.21,.12.48,.11,.89 rs CG 629 (.39) 737 (.46) 21 (.13) 397 (.44) 48 (.45) 97 (.1) 196 (.44) 26 (.46) 41 (.9) 21 (.43) 22 (.44) 56 (.12).58,.45,.326 C ,.21,.164 rs GA 1126 (.71) 47 (.25) 48 (.3) 628 (.69) 254 (.28) 2 (.2) 35 (.68) 126 (.28) 12 (.2) 323 (.7) 128 (.27) 8 (.1).238,.57,.243 G ,.464,.871 rs TC 613 (.38) 735 (.46) 236 (.14) 363 (.4) 433 (.47) 17 (.11) 173 (.38) 214 (.48) 57 (.12) 19 (.41) 219 (.47) 5 (.1).15,.531,.87 T ,.527,.64 rs TC 1124 (.7) 414 (.26) 47 (.2) 627 (.69) 25 (.27) 23 (.2) 35 (.69) 124 (.28) 13 (.2) 322 (.7) 126 (.27) 1 (.2).589,.716,.589 T ,.51,.949 rs (.43) 328 (.36) 162 (.36) 166 (.36) Page 9

14 CG 72 (.44) 191 (.12) 423 (.46) 152 (.16) 27 (.46) 75 (.16) 216 (.47) 77 (.16) C ,.48,.37.24,.1,.75 rs (.67) 618 (.68) 289 (.65) 329 (.71) TC 446 (.28) 262 (.28) 147 (.33) 115 (.25) 52 (.3) 24 (.2) 8 (.1) 16 (.3).635,.65,.31 T ,.645,.229 rs (.54) 422 (.46) 212 (.47) 21 (.45) CT 597 (.37) 39 (.43) 189 (.42) 21 (.43) 12 (.7) 9 (.9) 43 (.9) 47 (.1).5,.28,.27 C ,.77,.61 rs (.7) 625 (.69) 37 (.69) 318 (.69) TC 43 (.25) 24 (.26) 12 (.27) 12 (.26) 59 (.3) 36 (.3) 17 (.3) 19 (.4).751,.791,.848 T ,.535,.557 rs47965 AG 152 (.94) 84 (.5) (.93) 6 (.6) (.93) 26 (.5) (.92) 34 (.7) 1.322,.797,.21 A ,.545,.74 rs Page 91

15 CG 675 (.42) 715 (.45) 189 (.11) 324 (.35) 426 (.47) 152 (.16) 162 (.36) 25 (.46) 77 (.17) 162 (.35) 221 (.48) 75 (.16).19,.41,.47 C ,.14,.1 rs TC 197 (.69) 437 (.27) 52 (.3) 624 (.69) 253 (.27) 27 (.2) 37 (.69) 123 (.27) 14 (.3) 317 (.68) 13 (.28) 13 (.2).97,.99,.861 T ,.971,.943 rs GC 659 (.41) 77 (.44) 216 (.13) 43 (.44) 45 (.44) 95 (.1) 199 (.44) 22 (.45) 42 (.9) 24 (.44) 23 (.44) 53 (.11).58,.58,.393 G ,.4,.178 rs GT 1469 (.92) 18 (.6) (.91) 69 (.7) 11 (.1) 44 (.9) 36 (.8) 4 42 (.91) 33 (.7) 7 (.1).291,.569,.232 G ,.262,.182 rs AT 658 (.41) 713 (.44) 215 (.13) 4 (.44) 49 (.45) 95 (.1) 196 (.44) 26 (.46) 42 (.9) 24 (.44) 23 (.44) 53 (.11).69,.69,.391 A ,.62,.171 Page 92

16 rs GC 1114 (.7) 423 (.26) 5 (.3) 624 (.69) 255 (.28) 23 (.2) 35 (.68) 125 (.28) 14 (.3) 319 (.69) 13 (.28) 9 (.1).511,.818,.343 G ,.594,.817 rs93566 AG 189 (.68) 437 (.27) 54 (.3) 611 (.67) 267 (.29) 25 (.2) 3 (.67) 13 (.29) 14 (.3) 311 (.67) 137 (.29) 11 (.2).442,.782,.398 A ,.74,.958 rs TC 657 (.41) 78 (.44) 216 (.13) 399 (.44) 42 (.44) 12 (.11) 196 (.44) 22 (.45) 45 (.1) 23 (.44) 2 (.43) 57 (.12).177,.139,.569 T ,.88,.283 rs CT 143 (.65) 476 (.3) 65 (.4) 619 (.68) 259 (.28) 26 (.2) 291 (.65) 143 (.32) 1 (.2) 328 (.71) 116 (.25) 16 (.3).188,.153,.9 C ,.63,.36 rs AG 1319 (.83) 25 (.15) (.82) 144 (.15) 1 (.1) 363 (.81) 76 (.17) (.83) 68 (.14) 6 (.1).554,.711,.4 Page 93

17 A ,.417,.919 rs CA 874 (.55) 598 (.37) 113 (.7) 426 (.47) 389 (.43) 89 (.9) 213 (.47) 189 (.42) 42 (.9) 213 (.46) 2 (.43) 47 (.1) C ,.19,.18.49,.48,.35 rs CT 191 (.68) 442 (.27) 55 (.3) 623 (.68) 256 (.28) 25 (.2) 35 (.68) 125 (.28) 14 (.3) 318 (.69) 131 (.28) 11 (.2).63,.945,.513 C ,.916,.595 rs TC 467 (.29) 794 (.5) 322 (.2) 315 (.34) 42 (.46) 168 (.18) 156 (.35) 211 (.47) 76 (.17) 159 (.34) 29 (.45) 92 (.2).21,.52,.99 T ,.18,.146 rs CT 197 (.69) 436 (.27) 54 (.3) 623 (.68) 256 (.28) 25 (.2) 35 (.68) 125 (.28) 14 (.3) 318 (.69) 131 (.28) 11 (.2).641,.936,.527 C ,.949,.717 rs AG 477 (.3) 792 (.49) 319 (.35) 42 (.46) 158 (.35) 211 (.47) 161 (.35) 29 (.45) Page 94

18 317 (.19) 165 (.18) 75 (.16) 9 (.19).27,.64,.117 A ,.22,.15 rs TC 893 (.56) 575 (.36) 113 (.7) 426 (.47) 386 (.42) 89 (.9) 215 (.48) 188 (.42) 41 (.9) 211 (.46) 198 (.43) 48 (.1) T ,.91,.25.6,.24,.4 rs GA 876 (.55) 598 (.37) 113 (.7) 429 (.47) 386 (.42) 89 (.9) 217 (.48) 185 (.41) 42 (.9) 212 (.46) 21 (.43) 47 (.1).42,.38,.13 G ,.1,.25 rs CT 156 (.66) 464 (.29) 68 (.4) 623 (.68) 254 (.28) 27 (.2) 292 (.65) 14 (.31) 12 (.2) 331 (.71) 114 (.24) 15 (.3).193,.24,.83 C ,.775,.24 rs GA 474 (.29) 793 (.5) 319 (.2) 314 (.34) 422 (.46) 168 (.18) 155 (.34) 213 (.47) 76 (.17) 159 (.34) 29 (.45) 92 (.2).44,.94,.132 G ,.33,.197 rs (.52) 499 (.55) 233 (.52) 266 (.57) Page 95

19 TC 63 (.39) 123 (.7) 342 (.37) 63 (.6) 175 (.39) 36 (.8) 167 (.36) 27 (.5).427,.968,.11 T ,.89,.31 rs CA 157 (.66) 46 (.29) 69 (.4) 624 (.69) 251 (.27) 28 (.3) 293 (.65) 139 (.31) 12 (.2) 331 (.72) 112 (.24) 16 (.3).211,.222,.86 C ,.737,.28 rs AG 117 (.69) 43 (.27) 47 (.2) 646 (.71) 233 (.25) 24 (.2) 34 (.68) 128 (.28) 11 (.2) 342 (.74) 15 (.22) 13 (.2).6685,.688,.168 A ,.783,.93 rs AG 1511 (.95) 75 (.4) 848 (.93) 52 (.5) 418 (.94) 25 (.5) 43 (.93) 27 (.5) ,.653,.78 A ,.36,.71 rs CT 564 (.35) 744 (.46) 279 (.17) 278 (.3) 46 (.5) 166 (.18) 134 (.3) 23 (.51) 8 (.18) 144 (.31) 23 (.5) 86 (.18).5,.95,.243 C ,.121,.147 rs Page 96

20 CT 179 (.68) 462 (.29) 44 (.2) 649 (.72) 221 (.24) 3 (.3) 35 (.68) 125 (.28) 14 (.3) 344 (.75) 96 (.21) 16 (.3).42,.856,.26 C ,.933,.17 rs GT 591 (.37) 727 (.45) 269 (.16) 29 (.32) 45 (.49) 161 (.17) 141 (.31) 224 (.5) 78 (.17) 149 (.32) 226 (.49) 83 (.18).39,.11,.181 G ,.13,.11 rs GA 559 (.35) 752 (.47) 272 (.17) 274 (.3) 46 (.51) 166 (.18) 134 (.3) 229 (.51) 8 (.18) 14 (.3) 231 (.5) 86 (.18).473,.135,.174 G ,.112,.88 rs AG 614 (.38) 79 (.44) 259 (.16) 29 (.32) 452 (.5) 158 (.17) 141 (.31) 226 (.51) 76 (.17) 149 (.32) 226 (.49) 82 (.17).42,.23,.54 A ,.36,.34 rs AG 657 (.41) 678 (.43) 24 (.15) 35 (.34) 449 (.5) 142 (.15) 153 (.34) 223 (.5) 67 (.15) 152 (.33) 226 (.49) 75 (.16).54,.13,.68 A ,.55,.9 Page 97

21 rs TC 178 (.67) 467 (.29) 42 (.2) 647 (.71) 229 (.25) 26 (.2) 34 (.68) 128 (.28) 11 (.2) 343 (.74) 11 (.22) 15 (.3).967,.954,.69 T ,.764,.26 rs CG 49 (.31) 736 (.46) 353 (.22) 34 (.33) 438 (.48) 161 (.17) 149 (.33) 221 (.49) 74 (.16) 155 (.33) 217 (.47) 87 (.18).25,.34,.248 C ,.29,.99 rs CT 1467 (.92) 118 (.7) 831 (.91) 69 (.7) 41 (.92) 33 (.7) 421 (.91) 36 (.7) ,.89,.125 C ,.893,.325 rs69291 GA 1474 (.92) 92 (.5) 19 (.1) 87 (.89) 77 (.8) 14 (.1) 397 (.9) 37 (.8) 7 (.1) 41 (.89) 4 (.8) 7 (.1).22,.113,.64 G ,.32,.2 rs AT 772 (.48) 64 (.4) 173 (.1) 38 (.42) 429 (.47) 94 (.1) 194 (.43) 22 (.45) 47 (.1) 186 (.4) 227 (.49) 47 (.1).2,.131,.23 A ,.192,.3 Page 98

22 rs TC 79 (.5) 628 (.39) 161 (.1) 379 (.42) 426 (.47) 95 (.1) 193 (.43) 23 (.45) 47 (.1) 186 (.4) 223 (.48) 48 (.1).45,.47,.12 T ,.5,.56 rs TC 42 (.26) 724 (.45) 432 (.27) 276 (.3) 448 (.49) 179 (.19) 131 (.29) 229 (.51) 84 (.18) 145 (.31) 219 (.47) 95 (.2).11,.13,.84 T ,.28,.18 rs GC 488 (.3) 735 (.46) 355 (.22) 36 (.33) 438 (.48) 159 (.17) 149 (.33) 223 (.5) 72 (.16) 157 (.34) 215 (.46) 87 (.18).13,.16,.194 G ,.18,.67 rs TG 487 (.3) 717 (.45) 372 (.23) 214 (.23) 449 (.49) 238 (.26) 99 (.22) 231 (.52) 113 (.25) 115 (.25) 218 (.47) 125 (.27).71,.19,.42 T ,.58,.11 rs GC 1517 (.95) 64 (.4) 853 (.94) 49 (.5) 42 (.94) 24 (.5) 433 (.94) 25 (.5) ,.35,.387 Page 99

23 G ,.326,.16 rs TC 423 (.26) 731 (.46) 426 (.26) 277 (.3) 438 (.48) 183 (.2) 132 (.29) 226 (.51) 85 (.19) 145 (.31) 212 (.46) 98 (.21).81,.39,.25 T ,.44,.51 rs PACRG:Exon C Homo rs PACRG:Exon Homo C a P- value for comparison of frequencies between total leprosy patients and controls; paucibacillary (PB) patients and controls; multibacillary (MB) patients and controls in North Delhi samples Details of the Significant SNPs of PARK2 and PACRG gene regulatory region An earlier preliminary study involving PARK2 and PACRG gene from our laboratory had shown non-involvement of the 6 SNPs in the Indian population (Malhotra et al. 26) within this shared region of the genes with Leprosy; contrary to what was observed in Vietnamese and Brazilian population (Mira et al. 24). It was therefore pertinent to re-visit the region with sufficiently saturated number of SNPs. The purpose was to unravel any difference in LD structures and the heterogeneity in association in-between population groups. The region was saturated with the 96 SNPs (Table 3.1). Eleven of the studied 96 SNPs showed a consistent and strong association with susceptibility towards leprosy both in the North Indian and the East Indian-Orissa Page 1

24 population groups. Ten out of 11 SNPs were located in the regulatory region of the PARK2 gene and a single SNP within the regulatory region of the PACRG gene (Figure 4.1). The observation was made for the 11 SNPs on 235 samples (829 leprosy patients and 1476 controls) from northern India and in a geographically unrelated Indian population of 246 individuals from Orissa in Eastern India with a consistent association for SNP rs , located 6.67kb upstream of PACRG gene, rs (-258) within the core promoter region of PARK2 gene and SNPs rs (-324), rs , rs479648, rs , rs186765, rs , rs , rs , rs935543, located within 63.8kb upstream region of the PARK2 gene. A combined analysis of the North Indian and East Indian (Orissa) population groups confirmed the strong association for these 11 SNPs: rs (+CT vs., OR=1.37, 95% CI= , p=1.4e-4); rs (-258) (+CA vs., OR=1.36, 95% CI= ), p=2.1e- 4); rs (+CT vs., OR=1.35, 95% CI= , p=3.1e-4), rs (+GA vs., OR=1.36, 95% CI= , p=1.7e-4), rs ( vs. GC+, OR=1.46, 95% CI= , p=1.e-3), rs (+CG vs., OR=1.35, 95% CI= , p=4.3e-4), rs (+CT vs., OR=1.37, 95% CI= , p=1.6e-4), rs ( vs. CG+, OR=1.49, 95% CI= , p=7.2e-4), rs (+CA vs., OR=1.37, 95% CI= , p=1.2e-4), rs (+TC vs., OR=1.44, 95% CI= , p=1.e-5), rs (+GA vs., OR=1.36, 95% CI= , p=2.e-4) (Table 4.2). The association of all 11 SNPs, involving the minor allele for the risk, was strong even after adjustment with sex as a covariate and the Bonferroni correction for multiple testing. In addition, analysis after dividing the patients in two known subtypes of the disease, i.e., pauci-bacillary (PB) and multi-bacillary (MB) both within North Indian and East Indian-Orissa population, showed a strong association of all the 11 SNPs with PB and MB form of the leprosy (Table 4.3) with a power of 98.55%, MAF=.27 and OR 1.44 in the combined analysis. However, the association with the MB sub-type in comparison to the PB form of the disease showed higher significance values but with considerable low power of association, due to small sample size. Page 11

25 Table 4.3: Association statistics of SNPs within PARK2 and PACRG gene regulatory region in different cohorts of leprosy samples GENOTYPE CONT PAT North India Orissa Population Combined P a value rs _t/c PARK2 Intron 1 TC 744 (.52) 574 (.4) 17 (.7) 367 (.45) 358 (.44) 89 (.1).94 CONT 12 (.78) 3 (.19) 3 (.2) PAT 56 (.62) 33 (.36) 1 (.1) P b value.13 CONT PAT P c value 864 (.54) 64 (.38) 11 (.7) 423 (.47) 391 (.43) 9 (.1) Logistic P d Value Adjusted P e value OR (95%CI).22 Referance.15.75( ) (.44-.8) T (%) ( ) +CTvs ( ) rs _a/c (-258) AC 745 (.52) 571 (.4) 111 (.7) PACRG(5' near gene)+park2(intron_1) 368 (.45) 352 (.43) 92 (.11) (.78) 3 (.19) 3 (.2) 56 (.62) 33 (.36) 1 (.1) (.55) 61 (.38) 114 (.7) 424 (.47) 385 (.43) 93 (.1).3 Referance.24.76( ) (.44-.8) A (%) ( ) +CAvs ( ) rs _t/c PACRG Intron 2 77 (.53) 385 (.47) 127 (.83) 58 (.64) 897 (.57) 443 (.49).11 Referance Page 12

26 TC 551 (.38) 112 (.7) 344 (.42) 84 (.1) (.15) 3 (.2) 31 (.34) 1 (.1) (.36) 115 (.7) 375 (.42) 85 (.9).15.75( ) (.49-.9) T (%) ( ) +CTvs ( ) rs _g/a PACRG Intron 2 GA 745 (.52) 571 (.39) 117 (.8) 368 (.45) 356 (.43) 9 (.11) (.8) 27 (.17) 3 (.2) 56 (.62) 33 (.36) 1 (.1) (.55) 598 (.38) 12 (.8) 424 (.47) 389 (.43) 91 (.1).52 Referance.11.75( ) ( ) G (%) ( ) +GAvs ( ) rs479648_g/c PACRG Intron 2 GC 565 (.39) 676 (.47) 188 (.13) 284 (.34) 382 (.46) 148 (.18) (.66) 45 (.29) 6 (.3) 42 (.46) 42 (.46) 6 (.6) (.42) 721 (.46) 194 (.12) 326 (.36) 424 (.47) 154 (.17).6 Referance.42.83( ) ( ) G (%) (.7-.89) +GCvs ( ) rs _c/g PACRG Intron (.41) 285 (.35) 12 (.67) 43 (.48) 689 (.44) 328 (.36).16 Referance Page 13

27 CG 658 (.46) 185 (.12) 382 (.46) 147 (.18) (.28) 6 (.3) 41 (.46) 5 (.5) (.44) 191 (.12) 423 (.47) 152 (.17).1.79( ) ( ) C (%) ( ) +CGvs ( ) rs186765_c/t PACRG Intron 2 CT 742 (.51) 572 (.4) 116 (.8) 366 (.45) 357 (.44) 89 (.11) (.8) 25 (.16) 4 (.2) 56 (.62) 33 (.36) 1 (.1) (.55) 597 (.38) 12 (.8) 422 (.47) 39 (.43) 9 (.1).52 Referance.92.74( ) ( ) C (%) ( ) +CTvs ( ) rs _c/g PACRG Intron 2 CG 574 (.4) 67 (.47) 183 (.12) 283 (.34) 384 (.47) 147 (.18) (.66) 45 (.29) 6 (.3) 41 (.46) 42 (.47) 5 (.5) (.43) 715 (.45) 189 (.12) 324 (.36) 426 (.47) 152 (.17).21 Referance.18.8( ) ( ) C (%) ( ) +CGvs ( ) rs _c/a PACRG Intron (.52) 369 (.45) 123 (.8) 57 (.63) 874 (.55) 426 (.47).29 Referance Page 14

28 CA 572 (.39) 11 (.7) 357 (.43) 88 (.1) (.17) 3 (.2) 32 (.35) 1 (.1) (.38) 113 (.7) 389 (.43) 89 (.1).1.74( ) ( ) C (%) ( ) +CAvs ( ) rs _t/c PACRG Intron 2 TC 77 (.53) 548 (.38) 11 (.7) 37 (.45) 356 (.43) 88 (.1) (.8) 27 (.17) 3 (.2) 56 (.64) 3 (.34) 1 (.1) (.57) 575 (.36) 113 (.7) 426 (.47) 386 (.43) 89 (.1).35 Referance.11.71( ) ( ) T (%) ( ) +TCvs ( ) rs935543_g/a PACRG Intron 2 GA 753 (.52) 571 (.39) 11 (.7) 373 (45.8) 353 (.43) 88 (1.8) (.8) 27 (.17) 3 (2.5) 56 (.62) 33 (.36) 1 (.1) (.55) 598 (.38) 113 (.7) 429 (.48) 386 (.43) 89 (.1).44 Referance.17.75(.63-.9) ( ) G (%) ( ) +GAvs ( ) a P- value for comparison of frequencies between North Delhi leprosy patients and controls b P- value for comparison of frequencies between East India-Orissa leprosy patients and controls c P- value for comparison of frequencies between total leprosy patients and controls d P and e P- value for genotypic model by logistic regression for combines samples before and after adjustment for sex as a covariate. Bonferroni correction of 32 SNPs was applied for multiples testing. Out of total 96 SNPs tested 64 were in 14 bin set (r 2 >.8) Page 15

29 Table 4.4: Analysis of SNPs within PARK2 and PACRG gene in association with the subtypes of leprosy compared to healthy controls in two different cohorts of Indian population GENOTYPE CONT North India Orissa Population Combined PAT (PB) PAT (MB) rs _t/c PARK2 Intron 1 TC 744 (.52) 574 (.4) 17 (.7) 194 (.46) 179 (.43) 43 (.1) 173 (.43) 179 (.44) 46 (.11) T (%) CTvs P a value (PB, MB).57,.2.18,.44 CONT 12 (.78) 3 (.19) 3 (.1) PAT (PB) 19 (.67) 9 (.32) PAT (MB) 37 (.59) 24 (.38) 1 (.1) P b value (PB, MB).27,.14.37,.14 CONT PAT PB MB P c value 864 (.54) 64 (.38) 11 (.7) 423 (.47) 391 (.43) 9 (.1) 213 (.48) 188 (.42) 43 (.1) 21 (.46) 23 (.44) 47 (.1) ,.2,.11,.37,.5,.22 Logistic P d Value.22,.2,.12.15,.4,.37.86,.18,.31.14,.12,.6 Adjusted P e value.16,.49,.4 OR(95%CI) Referance.75( ),.79( ),.72(.58-.9).59(.44-.8),.63( ),.56( ).77( ),.79( ),.74( ) 1.37( ), 1.31( ), 1.44( ) rs _a/c (-258) PACRG (5' near gene)+park2 (Intron_1) AC 745 (.52) 571 (.4) 111 (.7) 194 (.46) 179 (.43) 43 (.1) 174 (.43) 173 (.43) 49 (.12) A (%) ,.18.22, (.78) 3 (.19) 3 (.1) 19 (.67) 9 (.32) 37 (.59) 24 (.38) 1 (.1) ,.14.37, (.55) 61 (.38) 114 (.7) 424 (.47) 385 (.43) 93 (.1) 213 (.48) 188 (.42) 43 (.1) 211 (.46) 197 (.43) 5 (.11) ,.25,.13.45,.63,.23.3,.26,.13.24,.35,.86.79,.28,.16 Referance.76( ),.78( ),.74( ).6(.44-.8),.65( ),.55(.38-.8).77( ),.8( ),.74( ) Page 16

30 +CAvs.21,.12,.11.25,.51, ( ), 1,31( ), 1.41( ) rs _t/c PACRG Intron 2 TC 565 (.39) 676 (.47) 188 (.13) 146 (.35) 195 (.46) 75 (.18) 185 (.46) 167 (.42) 45 (.11) T (%) CTvs.116,.14.4, (.83) 23 (.15) 3 (.1) (.67) 9 (.32) 39 (.62) 22 (.35) 1 (.1).76,.38.14, (.57) 574 (.36) 115 (.7) 443 (.49) 375 (.42) 85 (.9) 219 (.49) 186 (.42) 39 (.9) 224 (.49) 189 (.41) 46 (.1) ,.25,.76.23,.87,.15.11,.25,.79.15,.12,.14.9,.1,.13.31,.69,.33.28,.3,.15 Referance.75( ),.75(.6-.94),.75(.6-.94).66(.49-.9),.72( ),.62(.43-.9).79( ),.8( ),.77(.65-.9) 1.35( ), 1.33( ), 1.36( ) rs _g/a PACRG Intron 2 GA 745 (.51) 571 (.39) 117 (.8) 194 (.46) 18 (.43) 42 (.1) 174 (.43) 176 (.44) 48 (.12) G (%) GAvs.125,.42.41, (.8) 27 (.17) 3 (.1) 19 (.67) 9 (.32) (.59) 24 (.38) 1 (.1).17,.45.26, (.55) 598 (.38) 12 (.8) 424 (.47) 389 (.43) 91 (.1) 213 (.48) 189 (.43) 42 (.1) 211 (.46) 2 (.44) 49 (.11) ,.36,.2.1,.11,.37.52,.36,.21.11,.25,.44.36,.68,.53.17,.12,.83.21,.5,.55 Referance.75( ),.77( ),.72(.58-.9).64( ),.7( ),.59( ).78( ),.81( ),.75( ) 1.36( ), 1.31( ), 1.42( ) rs479648_g/c PACRG Intron 2 Page 17

31 Page (.39) 146 (.35) 138 (.34) 12 (.66) 16 (.57) 26 (.41) 667 (.42) 326 (.36) 162 (.37) 164 (.36).6,.93,.94 Referance GC 676 (.47) 195 (.46) 187 (.46) 45 (.29) 1 (.35) 32 (.51) 721 (.46) 424 (.47) 25 (.46) 219 (.48).42,.182,.69.83( ),.85( ),.8( ) 188 (.13) 75 (.18) 73 (.18).29,.2 6 (.3) 2 (.7) 4 (.6).55, (.12) 154 (.17) 77 (.17) 77 (.17).57,.89,.9.14,.22,.28.61( ),.61( ),.61( ) G (%) , , ,.32,.23.79(.7-.89),.8( ),.79( ) +GCvs.28,.32,.13.17,.114, ( ), 1.26( ), 1.31( ) rs _c/g PACRG Intron (.41) 146 (.35) 139 (.34) 12 (.67) 16 (.57) 27 (.44) 689 (.44) 328 (.36) 162 (.37) 166 (36.2%).16,.51,.39 Referance CG 658 (.46) 197 (.47) 185 (.46) 44 (.28) 1 (.35) 31 (.5) 72 (.44) 423 (.47) 27 (.47) 216 (.47).1,.55,.35.79( ),.79(.63-1.),.78( ) 185 (.12) 73 (.17) 74 (.18).19,.68 6 (.3) 2 (.7) 3 (.4).53, (.12) 152 (.17) 75 (.17) 77 (.17).15,.49,.37.57,.15,.13.59( ),.59( ),.59( ) C (%) , , ,.11,.76.77( ),.78(.67-.9),.77(.66-.9) +CGvs.43,.78,.48.31,.28, ( ), 1.34( ), 1.36( ) rs186765_c/t PACRG Intron (.51) 193 (.46) 173 (.43) 123 (.8) 19 (.67) 37 (.59) 865 (.55) 422 (.47) 212 (.48) 21 (.46).52,.29,.29 Referance CT 572 (.4) 18 (.43) 177 (.44) 25 (.16) 9 (.32) 24 (.38) 597 (.38) 39 (.43) 189 (.42) 21 (.44).92,.24,.35.74( ),.77( ),.72( )

32 116 (.8) 43 (.1) 46 (.11) C (%) CTvs.99,.63.3,.14 4 (.2) 1 (.1) ,.2.26, (.8) 9 (.1) 43 (.1) 47 (.1) ,.29,.28.11,.77,.62.45,.5,.11.16,.1,.89.19,.42,.57.65( ),.68(.46-1.),.62( ).78( ),.8( ),.76( ) 1.37( ), 1.32( ), 1.42( ) rs _c/g PACRG Intron 2 CG 574 (.4) 67 (.46) 183 (.12) 146 (.35) 195 (.46) 75 (.18) 137 (.34) 189 (.47) 72 (.18) C (%) CGvs.14,.12.6, (.66) 45 (.29) 6 (.3) 16 (.57) 1 (.35) 2 (.7) 25 (.41) 32 (.53) 3 (.5) ,.37.28, (.43) 715 (.45) 189 (.12) 324 (.36) 426 (.47) 152 (.17) 162 (.37) 25 (.46) 77 (.17) 162 (.35) 221 (.48) 75 (.16) ,.42,.47.38,.14,.11.21,.44,.49.18,.132,.3.57,.1,.19.86,.18,.48.57,.61,.19 Referance.8( ),.83( ),.77( ).59( ),.58(.43-.8),.6( ).78( ),.78( ),.78(.67-.9) 1.33( ), 1.3( ), 1.36( ) rs _c/a PACRG Intron 2 CA 751 (.52) 572 (.39) 11 (.7) 194 (.46) 18 (.43) 42 (.1) 175 (.43) 177 (.44) 46 (.11) C (%) ,.32.21, (.8) 26 (.17) 3 (.1) 19 (.67) 9 (.32) 38 (.61) 23 (.37) 1 (.1) ,.68.23, (.55) 598 (.38) 113 (.7) 426 (.47) 389 (.43) 89 (.1) 213 (.48) 189 (.43) 42 (.1) 213 (.46) 2 (.44) 47 (.1) ,.19,.18.49,.49,.35.29,.2,.19.1,.22,.47.18,.31,.48 Referance.74( ),.77( ),.72(.58-.9).61( ),.65( ),.58(.4-.85).77( ),.79( ),.75( ) Page 19

33 +CAvs.12,.75,.85.17,.37, ( ), 1.33( ), 1.42( ) rs _t/c PACRG Intron 2 TC 77 (.53) 548 (.38) 11 (.7) 196 (.47) 179 (.43) 41 (.9) 174 (.43) 177 (.44) 47 (.11) T (%) TCvs.39,.48.11, (.8) 27 (.17) 3 (.1) 19 (.67) 9 (.32) 37 (.62) 21 (.35) 1 (.1) ,.2.26, (.57) 575 (.36) 113 (.7) 426 (.47) 386 (.43) 89 (.1) 215 (.48) 188 (.42) 41 (.9) 211 (.46) 198 (.43) 48 (.11) ,.92,.26.59,.25,.4.35,.94,.28.11,.69,.81.11,.38,.19.1,.26,.1.17,.11,.87 Referance.71( ),.73( ),.68( ).6( ),.66( ),.55(.38-.8).74( ),.78( ),.72( ) 1.44( ), 1.38( ), 1.51( ) rs935543_g/a PACRG Intron 2 GA 753 (.52) 571 (.39) 11 (.7) 198 (.47) 176 (.42) 42 (.1) 175 (.43) 177 (.44) 46 (.11) G (%) GAvs.115,.29.38, (.8) 27 (.17) 3 (.1) 19 (.67) 9 (.32) 37 (.59) 24 (.38) 1 (.1) ,.45.26, (.55) 598 (.38) 113 (.7) 429 (.48) 386 (.43) 89 (.1) 217 (..49) 185 (.42) 42 (.1) 212 (.46) 21 (.44) 47 (.1) ,.39,.13.74,.1,.25.44,.39,.14.17,.5,.33.2,.38,.43.2,.18,.58.25,.71,.41 Referance.75(.63-.9),.8(.64-1.),.72( ).62( ),.66( ),.58(.4-.84).77( ),.81( ),.74( ) 1.36( ), 1.28( ), 1.44( ) a P- value for comparison of frequencies between North Delhi leprosy patients subtypes (PB,MB) and controls; b P- value for comparison of frequencies between East India-Orissa leprosy patients subtypes (PB,MB) and controls; c P- value for comparison of frequencies between total leprosy patients subtypes (PB,MB) and controls; d P and e P- value for genotypic model by logistic regression for combines samples, PB and MB samples before and after adjustment for sex as a covariate. Bonferroni correction of 32 SNPs was applied for multiples testing. Out of total 96 SNPs tested 64 were in 14 bin set (r 2 >.8) Page 11

34 LD and Bin structure of Studied SNPs of PARK2 and PACRG gene regulatory region in Indian population group Linkage Disequilibrium (LD) analysis of the 96 studied SNPs in regulatory region of PARK2 and PACRG was performed using Haploview v4.2 in controls of North Indian and Orissa, Eastern Indian population and compared with the Vietnamese. Both groups of Indian population, for r 2 cut off value of.8, Bin structure was generated for all the studied 96 SNPs (Figure 4.2) which showed two BINs (one BIN with 8 and another BIN with 3 significantly associated SNPs) where all the 11 significantly associated SNPs were distributed. Exactly the same BIN structure was revealed for both the North Indian and East Indian-Orissa population. In order to draw parity between the studied SNPs for the overlapping regulatory region of PARK2 and PACRG genes in Vietnamese and both groups of Indian populations, detailed information was sought for the Vietnamese samples. Information of 81 SNPs studied in the Vietnamese population (Mira et al. 24) was made available (courtesy Dr. Schurr) and rest of the recently studied SNP Bin structure information (Alter et al. 212) was retrieved from the supplementary files of the publication. A comparison with 96 SNPs studied in Indians showed 36 SNPs common to both Vietnamese and Indian population and 5 significant SNPs exclusive to Indian population and not studied in the Vietnamese. The 5 SNPs were part of the 11 significantly associated SNPs observed in Indian samples; and the remaining 6 SNPs were part of the group of 36 SNPs common between Vietnamese and Indians. This allowed us to generate the BIN structure for 41 SNPs, which included 41 SNPs in Indian population and 36 SNPs for Vietnamese. The 11 significant SNPs observed in this study were distributed in two BINs (of 8 in one BIN and 3 in another BIN) and rest of the 3 non-significant SNPs were distributed in seven other BINs in Indian population (Figure 4.3). However, the BIN structure generated for 36 SNPs in Vietnamese were distributed in five BINs (Figure 4.3). BIN-3 and BIN-4 in Vietnamese contained 15 and 8 SNPs, respectively to add up to 23, where 21 out of 23 SNPs were significantly associated in this population. However, 2 out of these 21 SNPs were observed to be non-significant in Indian population groups studied, and constituted different BIN structures (BIN-3 to BIN-9). BIN-1 in Vietnamese population contained 7 SNPs, including the SNP rs located 6.67kb upstream of PACRG gene, that Page 111

35 was significantly associated both in Vietnamese and Indian population. The other 3 SNPs out of the 6 SNPs located in this bin within the promoter region of PARK2 were non-significant in the Vietnamese population and showed significance in Indian population, where the other significant SNP (rs ) was present in a single BIN (BIN-2). Thus, comparing BIN-1 with 7 SNPs in Vietnamese population with BIN-1 carrying 8 significantly associated SNPs in Indian population; we found that 1 SNP in the BIN in Vietnamese and all the 8 SNPs in Indians showed a significant association with leprosy. However, the functional significance of the 2 common significant SNPs between the two populations (Vietnamese and Indian) did not show any significant difference in expression in in vitro reporter assay Haplotype Analysis of the significantly associated SNPs in the PARK2 and PACRG gene regulatory region In Haplotype analysis (Tables 4.5a-c), using haplostats software, the Haplotype 4 with risk alleles at all the 11 significantly associated SNP positions provided an increased risk (OR=1.36, p=2.46e-6, Freq controls =23%, Freq patients =29%) when compared to other haplotypes, generated for the 11 significantly associated SNPs in the combined Indian population (Table 4.5a). Haplotypes were also generated for the 2 significant BIN structures separately, BIN-1 with 8 SNPs and BIN-2 with 3 SNPs (Figure 4.3), this was done to assay for combination of SNPs in either of the BINs providing more risk towards leprosy susceptibility. Haplotype 3 with risk alleles at all the 8 significantly associated positions provided an increased risk (OR=1.34, p=2.88e-6, Freq controls =23%, Freq patients =29%) in comparison to other haplotypes generated in the combined Indian population (Table 4.5b). Similarly BIN-2 representing the Haplotype of 3 significantly associated SNPs showed Haplotype 2 with risk alleles at all the 3 significantly associated positions providing an increased risk (OR=1.29, p=7.56e-6, Freq controls =34%, Freq patients =4%) in comparison to other haplotypes generated for the 3 significantly associated SNPs in the combined Indian population (Table 4.5c). Page 112

36 Figure 4.2: Bin structure of 96 SNPs studied in PARK2 and PARCG gene region. Page 113

37 Figure 4.3: A schematic lay-out of the BIN comparison for r 2 >.8 in the regulatory region of the PARK2 and PACRG genes in Indian (North and East Indian-Orissa) and Vietnamese population, for 41 SNPs spanning 148Kb region of Chromosome 6q26, where 36 SNPs are common to both Vietnamese and Indian population and 5 significant SNPs (No. 2, 22, 23, 26, 32) are exclusively studied in the Indian population. Physical location of the studied chromosomal region is given in Mb on the top of the graph. Vietnamese population information of Mira et al, 24 was shared by Prof. Schurr and rest of the SNPs, BIN structure information was retrieved from the recently published Alter et al, 212. SNPs in star shape indicate the significantly associated SNPs (in two respective populations-indian and Vietnamese), indicating 11 significantly associated SNPs of Indian population are distributed in the two BINs (BIN 1 with 8 SNPs and BIN 2 with 3 SNPs) and significant SNPs of Vietnamese population are distributed in BIN 1, BIN 2 and BIN3. SNP rs (No.1) and rs (No.9) are commonly significant between both the Indian and Vietnamese population but did not show any significant difference in expression in in vitro reporter assay for the alternative alleles.each SNP is designated by a No. ranging from 1 to 41 according to increasing order of the chromosomal position. -Significant SNPs of Indian population (North and East Indian-Orissa), -Significant SNPs of Vietnamese population, -Non- Significant SNPs of North and East Indian-Orissa population, - Non-Significant SNPs of Vietnamese population, and SNPs (No. 5, 7, 8, 24, 35) studied by us earlier Malhotra et al. Page 114

38 Table 4.5: Haplotype structure, haplotype frequencies, significant p values and odds ratio between patients versus healthy controls. a. Haplotype structure of 11 significantly associated SNPs, b. Haplotype structure of 8 SNPs representing BIN-1 of Indian population, c. Haplotype structure of 3 SNPs representing BIN-2 of Indian population. a. Haplotype rs rs rs rs rs rs rs rs rs rs rs Hap-Score 1 T A T G G C C C C T G E Base NA 1 NA 2 C C T A C G T G A C A.2 8.4E Eff T A T G C G C G C T G E Eff C C C A C G T G A C A E Eff C A C A C C T C A C A NA NA.. NA <NA> NA NA NA p a -val pool.hf control.hf case.hf glm.eff OR.lower OR OR.upper Page 115

39 b. Haplotype rs rs rs rs rs rs rs rs Hap-Score p a -val pool.hf control.hf case.hf glm.eff OR.lower OR OR.upper c. 1 T A T G C C T G E Base NA 1. NA 2 C C T A T A C A E Eff C C C A T A C A E Eff C A C A T A C A NA NA.. NA <NA> NA NA NA Haplotype rs rs rs Hap-Score p a -val pool.hf control.hf case.hf glm.eff OR.lower OR OR.upper 1 G C C E Base NA 1. NA 2 C G G E Eff C C C NA NA.1.1. R Column: Hap-Score shows haplotype score statistic; Base, part of the baseline; Frequencies and disease association of haplotype of SNP alleles was tested using haplo.cc extended application of Haplo.stasts software (v1.4.4) which combines the results of haplo.score, haplo.group and haplo.glm. Haplotype frequency was computed by maximum likelihood estimates of haplotype probabilities with progressive insertion algorithm and haplo.cc computed score statistic to test association between haplotype and traits with adjustment for non-genetic covariates (sex). a p Indicates the haplotype comparison statistics for patients vs controls. Page 116

40 Evolutionary conservation of the SNPs To understand the status of disease associated alleles of significantly associated SNPs in evolutionary perspective, reference sequences from the closest primates (Chimpanzee, Orangutan, Rhesus and Marmoset) were downloaded (The UCSC Genome Browser, created by the Genome Bioinformatics Group of UC Santa Cruz. and compared with the Humans (Figure 4.4). We observed that the most significant SNP positions (rs ) and its 113bp apart SNP (rs935543) were not polymorphic in primates and the ancestral rs T allele in PARK2 regulatory region and its 113 bp apart rs G alleles, providing protection in humans, existed as major frequency alleles. These two SNPs also were found to be a consistent part of the group of SNPs present in the same BIN in all the HapMAP populations but one of the SNP (rs935543) was not studied in Vietnamese (Figure 4.3 & 5.1). Further the comparison of BIN structure generated for the Indian population with four HapMap and Vietnamese population revealed differences in the BINs with similarity between the European and Indian populations as compared to Chinese, Japanese and Vietnamese, explaining the heterogeneity and the reason for non-replication of the associated genomic regions in different populations (Figure 5.1) Bio-informatics analysis Bioinformatics analysis, using Tansfac-AliBaba2 tool showed that the: (i) Protective allele T for rs SNP disrupted the binding of transcription factors, CRE-BP1 (The camp response element binding protein) and AFT (Activating transcription factor); (ii) Risk allele A for rs SNP disrupted the binding of transcription factors, C/EBPalp, C/EBbeta (T/enhancer-binding protein alpha), HNF1 (hepatocyte nuclear transcription factor 1), GATA-1 (Erythroid binding transcription factor). Further confirmation by HaploReg software showed that these affected the binding of the transcription factors too (Table 4.6). Page 117

41 Figure 4.4: Evolutionary conservation of 2 (rs and rs935543) significantly associated variants across 4 species compared with humans. H-Humans, C-Chimpanzee, G-Gibbon, R-Rhesus, G-Gorilla, B-Baboon. Page 118

42 Table 4.6: Describe the allele combination of 4 clones generated for 2 SNPs, located upstream of the PARK2 gene regulatory region and their Bioinformatics prediction result for transcription binding at different alleles by using TRANSFEC and HaploReg database. Transcription factors binding prediction by Tansfac-AliBaba2 tool Result of HeploReg database CLONE Allele combination rs (T/*C) rs (G/*A) rs (T/*C) rs (G/*A) Clone1 rs (t)-rs935543(g) C/EBPalp,HNF-1,GATA-1,C-EBPbeta Sox,XBP-1 Foxa Clone2 rs (*c)-rs935543(g) ATF,CRE-BP1 C/EBPalp,HNF-1,GATA-1,C-EBPbeta Foxa Clone3 rs (t)-rs935543(*a) Sox,XBP-1 Clone4 rs (*c)-rs935543(*a) ATF,CRE-BP1 *Alleles represent the risk allele for the SNP. Page 119

43 Functional relevance of associated SNPs; Luciferase Expression study for the SNPs significantly associated with the Disease Out of 11 significantly associated SNPs with leprosy in Indian population, only one core promoter SNP rs (-258) of PARK2 gene had been analysed functionally and documented in literature (Tan et al. 25; West et al. 22). None of the other SNPs in the region were studied earlier for their functional implication. The 2 SNPs (rs and rs935543), lying within 63.8kb upstream region of PARK2 gene and two more SNPs found significant in both Indian and Vietnamese population, SNP rs located within the 3.5kb upstream region of the PARK2 gene and another SNP rs located 6.67kb upstream of PACRG gene, were chosen to assay their functional role and cloned in the pgl3 promoter bearing luciferase-reporter-expressing-vector. To test the enhancer activity of the SNPs rs and rs935543, the region bearing both the SNPs were cloned in pgl3 promoter vector in 4 allele combinations (Table 4.6). All 4 clones were transfected in 3 different cell lines: HepG2, MCF7 and HeLa. The results showed the lower expression in Clone2, Clone3 and Clone4 compared to Clone1 which contained both protective SNP alleles (Figure 4.5). The expression was lowest in clone 3 with rs (t)- rs935543(a), representing protective allele for SNP rs and risk allele for rs SNP rs located 3.5kb upstream in the PARK2 gene and SNP rs located within the 6.67kb upstream of PACRG gene were cloned in pgl3 promoter vector to test for enhancer activity. Clone1 with rs protective T allele, Clone2 with risk C allele and similarly Clone1 with rs protective T allele and Clone2 risk C allele did not show any significant change in the reporter gene expression in any of the 3 cell lines used. Page 12

44 Figure 4.5: Luciferase expression assay of upstream SNPs of PARK2 gene, (rs (T/*C) and rs (G/*A)): (*) Allele represents risk allele for the SNP. Bar with standard error shows the mean expression value for different Clones, where Clone1 with protective allele combination, rs (t)-rs935543(g); Clone2 with risk and protective allele combination, rs (*c)- rs935543(g); Clone3 with protective and risk allele combination, rs (t)-rs935543(*a) and Clone4 with risk allele combination, rs (*c)-rs935543(*a) in PGL3 promoter vector in three different cell lines (HepG2, MCF7 and HeLa) are represented. P-Values for comparison of mean (one way ANOVA) expression between clones with different allele combination of 2 SNPs is also shown. Page 121

45 4.2 INTERACTION ANALYSIS WITH IMMUNO-REGULATORY GENES Interaction analysis of PARK2 regulatory region SNPs with anti-inflammatory cytokines Genotype interaction analyses was performed between the two major significant SNPs (rs rs935543, 113 bp apart) of the PARK2 regulatory region in association with leprosy and 8 significant SNPs distributed over 4 anti-inflammatory cytokines genes showing a strong association with leprosy susceptibility in North Indian population (Aggarwal et al. 211). The latter included IL-1 promoter polymorphisms, rs18872 (-592C/A), rs18871 (-819C/T) and SNPs, rs located at the boundary of the intron 3, IL-1RB polymorphisms, rs located in the 3 UTR and rs , downstream of the 3 UTR. Two more polymorphisms (synonymous variant rs and rs744751, located downstream of the 3 UTR of TGFBR2, and SNP, rs18797 (promoter polymorphism) of IL-6 were also considered for the interaction analysis. The results revealed that the combined analysis between the PARK2 and IL-1 SNPs with risk genotypes at all the loci rs rs rs18872-rs rs in the North Indian samples provided highly significant risk towards leprosy, increasing the odds (OR, 1.99; 95% CI, ; p = 3.22E-5). Analysis between the PARK2 and IL-1RB SNPs with protective genotype at all the loci rs rs rs rs showed highly significant protection towards the disease (OR,.61; 95% CI, ; p = 1.1E-5). Further analysis of PARK2 region SNPs rs rs with TGFBR2 gene SNPs rs rs reflected that the in presence of the risk genotype TC+ (rs ), GA+ (rs935543) of PARK2 regulatory region SNPs with risk genotypes (rs744751) and protective genotype CT+ (rs222848) of TGFBR2 significant risk was observed towards leprosy (OR, 1.29; 95% CI, ; p = 1.4E-2). The similar analysis with IL-6 SNP rs18797 showed a significant risk towards leprosy (OR, 1.33; 95% CI, ; p = 2.9E-3) too, in presence of the risk genotype combination at all the loci, (Figure 4.6 A, Table 4.7) Page 122

46 Figure 4.6 A: SNP Interaction analysis of PARK2 and PACRG gene regulatory region SNPs rs and rs with the SNPs of anti-inflammatory cytokines gene. Alleles in red colour are the risk alleles for the SNPs. Page 123

47 Table 4.7: Interaction analysis of PARK2 SNPs with SNPs of genes providing risk towards Leprosy No. of Samples PARK2 PARK2 IL-1 IL-1 IL-1 Sig. OR 95% C.I.for EXP(B) Pat Cont rs _t/*c rs935543_g/*a rs18871_c/*t rs _c/*t rs18872_c/*a Lower Upper TOTAL TC+ GA+ CT+ CA+ 3.22E PB TC+ GA+ CT+ CA+ 9.35E MB TC+ GA+ CT+ CA+ 8.2E PARK2 PARK2 TGFBR2 TGFBR2 Sig. OR 95% C.I.for EXP(B) Pat Cont rs _t/*c rs935543_g/*a rs222848_c/*t rs744751_*g/a Lower Upper TOTAL TC+ GA+ CT+ 1.4E PB NS MB TC+ GA+ CT PARK2 PARK2 IL6 Sig. OR 95% C.I.for EXP(B) Pat Cont rs _t/*c rs935543_g/*a rs18797_*g/a Lower Upper TOTAL TC+ GA+ 2.9E PB TC+ GA+ 1.66E MB TC+ GA+ 8.16E PARK2 PARK2 TNF(-38) Promoter TNF intron1 Sig. OR 95% C.I.for EXP(B) Pat Cont rs _t/*c rs935543_g/*a rs18629_*g/a rs1861_*g/a Lower Upper TOTAL TC+ GA+ 2.6E PB TC+ GA+ 2.42E MB TC+ GA PARK2 PARK2 BTNL2-DRA interval BTNL2-DRA interval Sig. OR 95% C.I.for EXP(B) Pat Cont rs _t/*c rs935543_g/*a rs _a/*c rs _c/*t Lower Upper TOTAL TC+ GA+ CA+ CT+ 1.22E PB TC+ GA+ CA+ CT+ 1.89E MB TC+ GA+ CA+ CT+ 1.63E *Allele represents risk allele for the SNP; PB-Paucibacillary; MB-Multipbacillary Page 124

48 4.2.2 Interaction analysis of PARK2 regulatory region SNPs with pro-inflammatory cytokines and the genes spanning 6q21.3 chromosomal region A similar interaction analyses was also carried out between the 2 major significant SNPs in the regulatory region of PARK2 gene (rs rs935543) and 9 SNPs in the pro-inflammatory cytokine genes, spanning the HLA 6q21.3 region, depicting a strong association with Leprosy susceptibility in the North Indian population (Ali et al. 212). SNPs, rs in LTA promoter, rs in LTA 13 kb upstream, rs1861 located in TNF intron1, rs within TNF-LTB downstream and the SNPs in the genes spanning HLA 6q21.3 region including rs in BAT1 promoter, rs in exon 3 of NFKBIL1, rs and rs in the BTNL2-DRA region, were included in the interaction analyses. The ninth SNP at TNF-38 promoter (rs18629) had lost statistical significance after correction but was included in an overall interaction analysis, because of its functional importance (Ali et al. 212, Bayley et al. 21). The interaction results between the PARK2 gene SNPs and TNF gene SNPs showed that in presence of the risk genotype at all the loci rs rs rs18629-rs1861, a highly significant risk was obtained towards leprosy with increased odds (OR, 2.1; 95% CI, ; p = 2.6E-9). Further analysis of PARK2 SNPs with LTA gene SNPs rs rs showed that in presence of protective genotype at all the loci rs rs rs rs provided highly significant protection towards leprosy (OR,.61; 95% CI, ; p = 3.56E-7). Analysis between PARK2 and TNF-LTB region SNP rs also showed a highly significant protection towards the disease (OR,.54; 95% CI,.4-.73; p = 8.93E-5). The interaction analysis carried out between PARK2 SNPs and BAT1 gene promoter SNP rs252354, provided a significant protection towards leprosy (OR,.65; 95% CI, ; p = 4.15E-5). The analyses between PARK2 and NFKBIL1 exon3 SNP, with protective genotypes rs rs rs223365, showed that the combination provide a significant protection towards leprosy (OR,.58; 95% CI, ; p = 1.1E-7) and the combination of risk genotypes of PARK2 with BTNL2-DRA SNPs rs rs rs rs provided a highly significant risk towards leprosy (OR, 5.3; 95% CI, ; p = 1.22E-21) (Figure 4.6 B, Table-4.8). Page 125

49 Figure 4.6 B: SNP Interaction analysis of PARK2 and PACRG gene regulatory region SNPs rs and rs with the SNPs of gene spanning HLA III at 6p21.3 chromosomal region including pro-inflammatory cytokines TNF-alpha and LT-alpha. Alleles in red color are the risk allele for the SNP. Page 126