The Functional but not Nonfunctional LILRA3 Contributes to Sex Bias in Susceptibility and Severity of ACPA-Positive Rheumatoid Arthritis

Transcription

1 The Functional but not Nonfunctional LILRA3 Contributes to Sex Bias in Susceptibility and Severity of ACPA-Positive Rheumatoid Arthritis Yan Du Peking University People s Hospital Beijing CHINA

2 Contents Background Study plan Methods Results Conclusions

3 Background Nomination: Leukocyte immunoglobulin (Ig)-like receptors [LILRs, also known as Ig-like transcripts (ILTs)] are a family of inhibitory and activating receptors. Classification: Activating receptors: LILRA1-2,4-6 Inhibitory receptors: LILRB1-5 Secretory receptor: LILRA3

4 Structure: The ligand for LILRA3, LILRB1and LILRB2 is MHC class I for other LILRs remains unknown LILRB family contain 2-4 ITIMs while LILRA family lack any signaling domains of their own

5 Genetics: Encoded within the leukocyte receptor complex in 19q13.4, adjacent to Killer cell Immunoglobulin-like Receptor (KIR) family. LILRB1, LILRA2,and LILRA3 - demonstrated to be associate with Autoimmune diseases. LILRB1 associated with HLA-DRB1 shared epitope negative RA.

6 Genes and Immunity (2005) 6, Association of multiple Sclerosis with ILT6 deficiency ILT6 = LILRA3

7 Genes and Immunity (2009) 10, Multiple Sclerosis associates with LILRA3 deletion in Spanish Patients

8 Genes and Immunity (2009) 10,

9 In German Caucasians ILT6 = LILRA3

10 The American Journal of Human Genetics 82, , LILRA3 in Northeast Asians

11 The American Journal of Human Genetics 82, ,

12

13

14

15 The Journal of Rheumatology 2010; 37:8

16 Questions LILRA3 deletion & RA in Chinese populations? LILRA3 genetics and Clinical characteristics of RA patients? LILRA3 genetics and LILRA3 mrna level?

17 Study Plan 1) The distribution of LILRA3 deletion and LILRA3 single nucleotide polymorphism in RA in Han population? 2) LILRA3 deletion and joint destruction in RA? 3) Expression of LILRA3 in mrna level

18 Methods LILRA3 deletion: PCR-SSP LILRA3 SNP rs : Taqman genotyping Measurement of joint destruction: Modified Sharp-van der Heijde score (SHSs) on hands LILRA3 mrna: Realtime-PCR

19 Genes and Immunity (2009) 10,

20 Statistical Analysis The Hardy-Weinberg equilibrium (HWE) test was performed for each polymorphism, using Pearson's goodness-of-fit chi-square test. Chi-square tests were also performed for comparisons of allelic and genotypic frequency differences. The OR and 95% CI (confidence interval)were calculated using logistic regression, adjusting for age and sex. The linkage disequilibrium (LD) and haplotype were calculated using Haploview version 4.2 ( The Unpaired T test was applied for the analysis of radiological scores between two genotypic groups (recessive model). Association of LILRA3 expression with genotypic variants was analyzed using Kruskal Wallis test. All statistical analyses were conducted using program SPSS 16.0 (SPSS Inc., Chicago, IL, USA). The P < 0.05 was considered statistically significant.

21 Results Part 1: Association between LILRA3 deletion, LILRA3 polymorphism and RA in Chinese Han population Part 2: Joint destruction and RA genetics Part 3: Expression of LILRA3 in RA patients and Healthy Controls

22 Part 1 Association between LILRA3 deletion and RA in Chinese Han population Table1 Demographic characteristic of the study cohorts Characteristic North China South China No. of patients No. of controls Female sex (patients, %) Female sex (controls, %) Age of patients (Mean ± SD years) 54.7± ±13.2 Age of controls (Mean ± SD years) 38.3± ±11.9 Onset age (Mean ± SD years) 46.1± ±12.6 Disease duration (Mean ± SD years) 8.7± ±6.3 ACPA positive (%) SHS (Mean ±SEM) 78.4±63.8 -

23 Electrophoretic separation of the three different genotypes of the LILRA3 deletion Allelic discrimination of rs103294

24 Hardy-Weinberg equilibrium (HWE) Both variants (the 6.7-kb deletion and SNP rs103249) were in Hardy- Weinberg equilibrium (HWE) (P > 0.05) in controls in two independent cohorts.

25 Table 2 Association analysis of LILRA3 and rs with RA in two independent cohorts, adjusting for sex and age. Controls RA P value OR (95% CI) N-Han (Discovery) LILRA3 n=1658 n=1618 Allelic (76.1) 792 (23.9) 2355 (72.8) 881 (27.2) Genotypic +/- and -/ (93.7) 1455 (89.9) +/+ 105 (6.3) 163 (10.1) rs n=1636 n=1618 Allelic T 2525 (77.2) 2401 (74.2) C 747 (22.8) 835 (25.8) Genotypic TC and TT 1543 (94.3) 1483 (91.7) CC 93 (5.7) 135 (8.3) S-Han (Replication) LILRA3 n=549 n=575 Allelic (74.6) 279 (25.4) 778 (67.7) 372 (32.3) Genotypic +/- and -/- 516 (94.0) 512 (89.0) +/+ 33 (6.0) 63 (11.0) rs n=380 n=465 Allelic T 579 (76.2) 631 (67.8) C 181 (23.8) 299 (32.2) Genotypic TC and TT 356 (93.7) 407 (87.5) CC 24 (6.3 ) 58 (12.5) Han Chinese (Combined)* LILRA3 n=2207 n=2193 Allelic (75.7) 1071 (24.3) 3133 (71.4) 1253 (28.6) Genotypic +/- and -/ (93.7) 1967 (89.7) +/+ 138 (6.3) 226 (10.3) rs n=2016 n=2083 Allelic T 3104 (77.0) 3032 (72.8) C 928 (23.0) 1134 (27.2) Genotypic TC and TT 1899 (94.2) 1890 (90.7) CC 117 (5.8) 193 (9.3) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) RA: rheumatoid arthritis; OR (95% CI): odds ratio (95% confidence interval); (-): 6.7kb-deletion; (+): non-deletion; N-Han: Northern Han Chinese; S-Han: Southern Han Chinese; *mata analysis.

26 Haplotype analysis D R 2 Table 3. Frequencies of two-marker haplotypes, results of association statistics, and odds ratios (95% confidence intervals) in RA patients and controls. Haplotype RA (%) CON (%) χ2 P OR (95%CI) C-Non-del ( ) T-del ( ) T-Non-del ( )

27 Table 4 RA association of LILRA3 stratified by sex, adjusting for age Controls RA P-value OR (95% CI) N-Han female n=1226 n=1101 Allelic (75.5) 600 (24.5) 1632 (74.1) 570 (25.9) Genotypic +/-and -/ (92.9) 1001(90.9) +/+ 87 (7.1) 100 (9.1) S-Han female n=422 n=457 Allelic (75.1) 616 (67.4) (24.9) 298 (32.6) Genotypic +/- and -/- 396 (93.8) 405 (88.6) +/+ 26 (6.2) 52 (11.4) Total female n=1648 n=1558 Allelic (75.4) 2248 (72.1) (24.6) 868 (27.9) Genotypic +/- and -/ (93.1) 1406 (90.2) +/+ 113 (6.9) 152 (9.8) N-Han male n=411 n=267 Allelic (78.1) 377 (70.6) (21.9) 157 (29.4) Genotypic +/- and -/- 396 (96.4) 234 (87.6) +/+ 15 (3.6) 33 (12.4) S-Han male n=126 n=105 Allelic (72.6) 144 (68.6) + 69 (27.4) 66 (31.4) Genotypic +/- and -/- 119 (94.4) 95 (90.5) +/+ 7 (5.6) 10 (9.5) Total male n=537 n=372 Allelic (76.8) 521 (70.0) (23.2) 223 (30.0) Genotypic +/- and -/- 515 (95.9) 329 (88.4) +/+ 22 (4.1) 43 (11.6) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( )

28 Table 5 RA association of LILRA3 stratified by ACPA status, adjusting for sex and age Groups No. LILRA3 +/- and -/- +/+ P value OR (95%CI) +/+ vs. +/- and -/- All Controls (93.7) 138 (6.3) ACPA-positive (89.6) 102 (10.4) ( ) ACPA- negative (91.7) 19 (8.3) ( ) Female Controls (93.1) 113 (6.9) ACPA- positive (90.8) 71 (9.2) ( ) ACPA- negative (90.6) 17 (9.4) ( ) Male Controls (95.9) 22 (4.1) ACPA- positive (85.2) 31 (14.8) ( ) ACPA- negative (95.3) 2 (4.7) ( ) RA: rheumatoid arthritis; OR (95% CI): odds ratio (95% confidence interval); (-): 6.7kb-deletion; (+): non-deletion; ACPA: anti-citrullinated proteins antibodies; N-Han: Northern Han Chinese; S-Han: Southern Han Chinese.

29 Part 2 LILRA3 genetics and RA joint destruction Table 6. Sharp score levels in ACPA positive RA patients, grouped by disease duration and LILRA3 risk genotype carriage Subgroup (n) T<2 years Sharp scores, (Median±SEM) P (T-test) P (Mann Whitney test) LILRA3 +/+ (8) 61.9± LILRA3 -/- and +/- (58) 30.9±4.1 2 T 10years LILRA3 +/+ (8) 40.5±15.6± LILRA3 -/- and +/- (170) 68.0±3.7 10years<T LILRA3 +/+ (8) 140.0± LILRA3 -/- and +/- (129) 124.5±6.4

30 Figure1 Sharp score levels in anti-ccp positive RA patients, grouped by disease duration and LILRA3 risk genotype carriage

31 Part 3 Figure 2 Expression of LILRA3 in RA patients in mrna level

32 Conclusions Our study provides the first evidence that the functional LILRA3 as a genetic risk factor for RA susceptibility, especially in males. The functional LILRA3 appears to highly predispose to ACPA-positive RA and conferred a higher risk to increase disease severity in early RA patients.

33 Author Contributions Study conception and design. Du, Li, Guo. Acquisition of data. Du, Cui, Liu, Yang, Wu, Liu, Liu, Xu, Zhu, Sun, Zhang, Li, Guo. Analysis and interpretation of data. Du, Guo. Manuscript preparation. Du, Li, Guo. Statistical analysis. Du, Guo.

34 Acknowlegement We thank the Northern Han Chinese (N-Han) study group members and staff for recruiting patients and healthy controls and the technical assistance from Department of Rheumatology and Immunology, People s Hospital, Peking University, Beijing, China. We also thank the Southern Han Chinese (S-Han) study group members and staff for recruiting patients and controls from Department of Rheumatology, No.1 Hospital of Anhui Medical University, Anhui, and from Department of Rheumatology, Gulou Hospital, Nanjing University, Jiangsu. We wish to thank the patients and healthy volunteers for their cooperation and for giving consent to participate in this study.

35