Influence of HLA-DRB1 Susceptibility Alleles on the Clinical Phenotypes of Systemic Lupus

Erythematosus PatientsShiva Krishna Katkam Msc Biochemistry (PhD Student)

Liza Rajasekhar#, Konda Kumaraswami#, Ramesh Manthri#, Vijay Kumar Kutala*

Departments of Clinical Pharmacology and Therapeutics * and Rheumatology#,

Nizam's Institute of Medical Sciences, Hyderabad, India

SLE-EtiologyThe etiology of SLE remains unknown. But SLE is clearly multifactorial

Immune deficiency

(C1q ,C4,C2)

Hormonal(Estrogen

Testosterone, Prolactin

Vitamin D)

Genetic

Environmental(Infection, Vaccines ,

Drugs, UV)

Candidate genes

PhagocytosisITGAM , FcγR

Autophagy & B cell differentiationPRDM1-ATG5

B cell activation and signaling

IKZF1, BANK1

Lymphocyte development and activation TNIP1, ETS1

Adhesion and endothelial migration

CNTN6

Transcription factorsIRAF8, IFIH1, TYK2, LRRC18-WDFY4,

PXK, JAZF1, UHRF1BP1 and XKR6, EHF, MED1

UbiquitinationTNFAIP3

Interferon regulation and production

SLC15A4, IRAF5

T cell activation and signaling

STAT4

Antigen presentationPTPN22, TNFSF4, HLA-DRB1,

BIN1,SEC61G

Components of the NF-kB pathway

RASSF2 and RNF114

Antigen presentationHuman leukocyte antigen region

Exogenous antigen presentationEndogenous antigen presentation

a) Class I MHC assoc. with peptides from endogenous Ag’sb) Class II MHC assoc with peptides from exogenous Ag’s

Mechanism of HLA-DRβ1 Association with SLE

+

Strong binding of peptide fragment

Loose binding of peptide fragment

Peptide fragments

T cell CD4+ Helper cell

T cell receptor

Antigen presenting cell (Macrophage ,B cell and

Dendritic cell)

MHC class II

DR β chain

DR α chain

DR β chain

MHC class II

MHC class II

Binding groove

Peptide binding groove

HLA-DRB1 gene exonic regionsHLA -DRB1 is highly polymorphic

HLA Class II - DRB Alleles from IMGT/HLA

Gene DRB1 DRB3 DRB4 DRB5 DQB1Alleles 1977 127 53 43 978

Proteins 1440 106 42 39 678Nulls 50 4 5 2 27

MHC class II

HLA-DRB1 and SLE• Altered frequencies of human leukocyte antigen (HLA) alleles are known

to be associated with SLE. • Some HLA-DRB1 alleles are reported to be positively associated with SLE

susceptibility in several ethnic groups studied: DRB1*03:01 and DRB1*15:01 in EuropeansDRB1*15:03 in African-AmericansDRB1*08:02 in Hispanic DRB1*15:01,*09:01,*07:01 and *08:03 in KoreansProtective Role for the HLA-DR6 Alleles DRB1*13:02 and *14:03

in Japanese population

Limited studies representing Indian SLE patients

Objective

What are predisposition HLA-DRB1 and DQB1 alleles in Indian SLE patients??

What are HLA-DRB1-DQB1 Haplotypes in Indian SLE patients??

Associations of DRB1 with SLE of different clinical and phenotypic manifestations

Step IBlood Sample collection from SLE patients * (n=130) and Healthy controls (n=120)

* Satisfying the ACR 1997 update of 1982 SLE classification criteria

Step II DNA isolation using phenol chloroform method

Step IIIPCR-SSP method using Histo Type DR DQB (SSP Kit), BAG Health Care GmbH

HLA-DRB1 and DQB1alleles

Step IV Alleles were typed using Histomatch software

Step VAllele, Genotype and Haplotype carrier frequencies were analyzed by Fisher’s

exact test using 2×2 contingency tables

Step VI Associations of DRB1

alleles with severe SLE phenotypes was explored

Methodology

4%7%

9%

19%

2%2%

5%5%3%

8%7%

27%

3%SLE 2n=260

HLA-DRB1*01HLA-DRB1*03HLA-DRB1*04HLA-DRB1*07HLA-DRB1*08HLA-DRB1*09HLA-DRB1*10HLA-DRB1*11HLA-DRB1*12HLA-DRB1*13HLA-DRB1*14HLA-DRB1*15HLA-DRB1*16

3%10%

10%

9%3%

2%7%

3%3%

7%

15%

25%

3%Healthy controls 2n=240

HLA DRB1 AllelesSLE

2n=260Healthy controls

2n=240 OR 95% CI p value PcHLA-DRB1*01 11(4.2) 6(2.50) 1.72 0.62-4.73 0.33 4.29

HLA-DRB1*03 19(7.3) 23(9.58) 0.74 0.39-1.40 0.42 5.46

HLA-DRB1*04 23(8.8) 25(10.42) 0.83 0.46-1.51 0.64 8.43HLA-DRB1*07 49(18.8) 22(9.17) 2.30 1.34-3.93 0.002 0.02

HLA-DRB1*08 4(1.5) 8(3.33) 0.45 0.13-1.52 0.24 3.20

HLA-DRB1*09 4(1.5) 5(2.08) 0.73 0.19-2.76 0.74 9.66

HLA-DRB1*10 14(5.4) 16(6.67) 0.80 0.38-1.66 0.57 7.49

HLA-DRB1*11 13(5.0) 8(3.33) 1.53 0.62-3.74 0.38 4.95

HLA-DRB1*12 7(2.7) 8(3.33) 0.80 0.28-2.24 0.79 10.33

HLA-DRB1*13 22(8.5) 16(6.67) 1.29 0.66-2.52 0.50 6.52HLA-DRB1*14 18(6.9) 35(14.58) 0.44 0.23-0.79 0.005 0.07

HLA-DRB1*15 69(26.5) 60(25.00) 1.08 0.72-1.61 0.75 9.86

HLA-DRB1*16 7(2.7) 8(3.33) 0.80 0.28-2.24 0.79 10.33

HLA-DRB1 allele carrier frequency in the SLE patients and controls

*P value < 0.05 were considered to be significant

SLE phenotype N(%) DRB1*07/alleles other than DRB1*14 (%) OR 95% CI p value Q

Severe_Constitutional 17(13.1) 6(35.29) 3.8 1.23-11.84 0.0255 0.045Severe_Cutaneous_Lupus_Ever 32(24.6) 16(50.00) 7 2.90-16.86 0.0000016 0.0002

NPSLE_Ever 31(23.8) 11(35.48) 3.8 1.54-9.59 0.0045 0.0145Arthritis_moderate_severe_Eve

r 105(80.8) 30(28.57) 3.2 1.66-6.40 0.00042 0.0033

Myositis_severe 8(6.2) 3(37.50) 4.2 0.90-19.4 0.08358 0.1215Lupus Nephritis 65(50.0) 15(23.08) 2.1 0.95-4.63 0.09314 0.1146

Active_Hemolysis_Ever 7(5.4) 1(14.29) 1.1 0.13-10.37 1 1.0666Severe_Thrombocytopenia_Eve

r 12(9.2) 3(25.00) 2.3 0.56-9.59 0.3697 0.4225

Severe_leucopenia_Ever 7(5.4) 1(14.29) 1.1 0.13-10.37 1 1Severe_Cardiorespiratory_Ever 18(13.8) 6(33.33) 3.5 1.14-10.72 0.03328 0.0532Severe_Gastrointestinal_Ever 8(6.2) 3(37.50) 4.2 0.90-19.40 0.08358 0.1114

Severe_Ophthalmic_Ever 4(3.1) 3(75.00) 21 2.04-215 0.00954 0.0254Antinuclear antibody (+) 121(93.1) 34(28.10) 2.7 1.39-5.34 0.00368 0.01475

Anticardiolipin antibody(+) 27(20.8) 9(33.33) 3.5 1.33-9.19 0.01272 0.02545Anti-dsDNA antibodies (+) 114(87.7) 35(30.70) 3.1 1.58-6.07 0.00077 0.00411Hypocomplementemia(+) 67(51.5) 19(28.36) 2.7 1.29-5.91 0.00982 0.02245

Control 120 15(12.50)

HLA-DRB1 genotype frequency in the SLE patients relative to SLE phenotype

*P value < 0.05 were considered to be significant

HLA-DRB1 genotype frequency in the SLE patients and controlsGenotypes# SLE (n=130) Frequency Healthy controls

(n=120) Frequency OR 95% CI p value

DRB1*01/03 2 1.54 2 1.67 0.92 0.12-6.64 1DRB1*03/07 2 1.54 2 1.67 0.92 0.12-6.64 1DRB1*03/15 6 4.62 5 4.17 1.11 0.33-3.74 1DRB1*04/15 7 5.38 5 4.17 1.30 0.40-4.24 0.77DRB1*07/07 4 3.08 3 2.50 1.23 0.27-5.64 1DRB1*07/12 4 3.08 2 1.67 1.87 0.33-10.41 0.68DRB1*07/13 3 2.31 3 2.50 0.92 0.18-4.65 1DRB1*07/14 3 2.31 3 2.50 0.92 0.18-4.65 1DRB1*07/15 18 13.85 4 3.33 4.66 1.52-14.20 0.003*DRB1*09/15 2 1.54 3 2.50 0.60 0.10-3.71 0.67DRB1*10/13 2 1.54 2 1.67 0.92 0.12-6.64 1DRB1*10/15 4 3.08 6 5.00 0.60 0.1-2.19 0.52DRB1*13/15 2 1.54 3 2.50 0.60 0.10-3.71 0.67DRB1*14/15 7 5.38 12 10.00 0.51 0.19-1.34 0.23DRB1*14/16 2 1.54 2 1.67 0.92 0.12-6.64 1DRB1*15/15 6 4.62 5 4.17 1.11 0.33-3.74 1DRB1*15/16 5 3.85 3 2.50 1.56 0.36-6.67 0.72

*P value < 0.05 were considered to be significant ; #Genotype frequencies >1.5%

HLA-DRB1 EXON 2 Extracellular β domain 1DRB1*01:01:01DRB1*03:01:01DRB1*04:01:01DRB1*07:01:01DRB1*08:01:03DRB1*09:01:02DRB1*10:01:01DRB1*11:01:01DRB1*12:01:01DRB1*13:01:01DRB1*14:01:01DRB1*15:01:01DRB1*16:01:01

DRB1*01:01:01DRB1*03:01:01DRB1*04:01:01DRB1*07:01:01DRB1*08:01:03DRB1*09:01:02DRB1*10:01:01DRB1*11:01:01DRB1*12:01:01DRB1*13:01:01DRB1*14:01:01DRB1*15:01:01DRB1*16:01:01

HLA-DRB1 EXON 3 Extracellular β domain 211&1370,71&74

Group I Group II Group III Group IV Group V Group VI Group VIIA De Dr E Q R a

HLA-DR Alleles A/RR/KAa DE/TA DR/KA/L Q/RRE DRQ QKR/Q QAA(+) (-) (±) (±) (±) (+) (n)

DRB1*0101 QRA (+)DRB1*0301 QKR (+)DRB1*0401 QKA(+)DRB1*0701 DRQ (±)DRB1*0801 DRL (±)DRB1*0901 RRE (±)DRB1*1001 RRA (+)DRB1*1101 DRA(±)DRB1*1201 DRA(±)DRB1*1301 DEA(-)DRB1*1401 RRE(±)DRB1*1501 QAA (n)DRB1*1601 DRA(±)

DR restrictive supertype patterns at position 70, 71, and 74 of DRB1 chains

Protective vs Disease-promoting MHCs

Gly11 Tyr13

Arg71 Gln74

Asp70

Key amino acid residues in HLA-DRβ chain associated SLE risk

0 10 20 30 40 50 60 70 80

0.001

0.01

0.1

1

Pc v

alue

11G 13Y70D

74Q

HLA-DRβ1 amino acid residue

71R

HLA DQB1 Alleles SLE 2n=260

Healthy controls 2n=240 OR 95% CI p value

HLA-DQB1*02 52(20.00) 40(16.67) 1.25 0.79-1.97 0.35

HLA-DQB1*03 58(22.31) 46(19.17) 1.21 0.78-1.86 0.44

HLA-DQB1*04 4(1.54) 5(2.08) 0.73 0.19-2.76 0.74

HLA-DQB1*05 61(23.46) 68(28.33) 0.78 0.51-1.15 0.22

HLA-DQB1*06 85(32.69) 81(33.75) 0.95 0.65-1.38 0.84

HLA-DQB1 allele carrier frequency in the SLE patients and controls

DRB1-DQB1 Haplotype# SLE (n=130) Frequncy Healthy controls (n=120) Frequency OR 95% CI P valueDRB1*03-DQB1*02 18 3.46 24 5.0 0.28 0.12-0.66 0.003DRB1*04-DQB1*03 19 3.65 22 4.6 0.76 0.38-1.49 0.49DRB1*07-DQB1*02 35 6.73 20 4.2 1.84 0.99-3.41 0.06DRB1*07-DQB1*03 25 4.81 9 1.9 2.93 1.30-6.58 0.009DRB1*07-DQB1*05 18 3.46 8 1.7 2.25 0.93-5.38 0.09DRB1*07-DQB1*06 18 3.46 7 1.5 2.59 1.04-6.453 0.05DRB1*10-DQB1*05 18 3.46 18 3.8 0.91 0.44-1.84 0.85DRB1*11-DQB1*03 15 2.88 11 2.3 1.29 0.56-2.93 0.67DRB1*13-DQB1*06 25 4.81 19 4.0 1.26 0.65-2.43 0.51DRB1*14-DQB1*05 19 3.65 35 7.3 0.41 0.22-0.77 0.005DRB1*15-DQB1*06 70 13.46 72 15.0 0.77 0.47-1.28 0.37DRB1*15-DQB1*02 20 3.85 13 2.7 1.49 0.70-3.15 0.35DRB1*15-DQB1*03 20 3.85 11 2.3 1.80 0.82-3.93 0.17DRB1*15-DQB1*05 28 5.38 24 5.0 1.09 0.59-2.02 0.87

HLA-DRB1& DQB1 Haplotype frequency in the SLE patients and controls

*P value < 0.05 were considered to be significant ; #DRB1-DQB1 Haplotype >1.5%

SLE phenotype DRB1 allele Frequency Frequency OR 95% CI p value

Severe Constitutional (+) HLA-DRB1*10 14.71 3.98 4.15 1.30-13.25 0.023

Severe Cutaneous Lupus (+) HLA-DRB1*07 29.69 15.31 2.33 1.20-4.53 0.015

Arthritis(+) HLA-DRB1*01 2.38 12.00 0.17 0.05-0.61 0.008

Myocytis sever(+) HLA-DRB1*13 25.00 7.38 4.18 1.22-14.30 0.035

Anti nuclear antiody (+) HLA-DRB1*11 3.72 22.22 0.13 0.037-0.49 0.007

Discoid lupus (+) HLA-DRB1*07 33.33 13.92 3.09 1.60-5.94 0.0007

Oral ulcers (+) HLA-DRB1*15 18.42 32.88 0.46 0.25-0.82 0.01

HLA-DRB1 genotype frequency in the SLE patients relative to SLE phenotype

Conclusion We have identified a positive association of HLADRB1*07 allele, DRB1*07/15

genotype, DRB1*07-DQB1*03 Haplotypes with south Indian SLE patients.

Further HLADRB1*07 allele showed a significant association with severe forms of

SLE clinical phenotypes like: Constitutional lupus Cutaneous lupusNPSLEDiscoid lupus

Protective role of HLA-DRB1*14 allele, DRB1*14-DQB1*05 haplotype also found in SLE patients.

Thank you………