1. Genetic Variations of CD55 and Pathogenesis of Pandemic A(H1N1)2009 Influenza ZHOU JIE, Jane August 19, 2014

2. Pandemic A(H1N1)2009 influenza  First report on spring 2009 by US CDC. WHO signaled the 1 st influence pandemic in this centaury.  A(H1N1)pdm09 virus derived from a reassortment of avian H1N1, classical swine H1N1 and human seasonal H3N2 viruses.  people under 65 years of age infected preferentially.  high rate of severe infection in younger patients. As of Oct 2010, at least 299 severe infection and 83 death in HK.  1/3 of the severe cases don’t have predisposing comorbidities, including chronic heart or pulmonary diseases and metabolic disorders.  WHO identified the studies of role of genetic factors on susceptibility to severe influenza as a priority. Aim: Host genetic variation may affect the disease severity of A(H1N1)pdm09 infection. Introduction

3. Genome-wide association study (GWAS)in a pilot cohort SNP allele allelic distribution p value mild cases (%) severe cases (%) rs6691942C0.48080.841.333E-04 rs4844592T0.42310.765.476E-04 rs2564978T0.42310.765.476E-04 rs1507758G0.42310.765.476E-04 rs10864231T0.42310.765.476E-04 rs6700168C0.57690.245.476E-04 Selected genetic association result for CD55 SNPs in GWAS.

4. CD55, a complement regulatory protein. CD55

5. rs2564978 rs4844592 rs6691942 rs6700168 rs10864231 Rs1507758 Exon 1 2 3 4 5 6 78 9 10 11 12 13 14 Linkage disequilibrium (LD) pattern of CD55 variants

6. Genotypic distribution and logistic regression analysis of rs2564978 in study participants. Severe patientsMild controls (n = 177)(n = 248) Genotypic distribution, n (%) TT94 (53.1)94 (37.9) CT60 (33.9)109 (44.0) CC23 (13.0)45 (18.1) Univariate regression analysis OR (95% CI)1.85 (1.15-2.78) P value0.0019 Multivariate regression analysis OR (95% CI)1.75 (1.13-2.70) P value0.0110

7. Allele-specific effect of rs2564978 on CD55 expression in peripheral blood monocytes 0 0.1 0.2 0.3 0.4 T/T C/T C/C Genotype Relative CD55 mRNA levels p=0.003 p=0.038 P=0.0003 CC CT TT Genotype Log 10 (MEFL) 5 435 43

8. expression Quantitative Trait Loci (eQTL) eQTL, expression-related variations, mapped by paralleled genome-wide analysis of gene expression and genetic variations in LCLs and various human tissues, populations and in response to different stimuli. Interpret association signal, prioritize causal gene(s), provide immediate insight about disease pathogenesis… http://eqtl.uchicago.edu/Home.html

9. Luciferase assay of CD55 promoter variants p=0.0006 Luc Relative Luc Activity CI -C---Ins--- CD -C---Del--- TI -T---Ins--- TD -T---Del--- 0 10 20 30 40 p=0.0002 rs2564978 C>Trs3841376 indel

10. Complement dependent cytolysis (CDC) assays in respiratory epithelial cell lines A549 α-CD55iso-ctrl PBS Treatment Cell lysis (%) p<0.00001 p=0.0008 Cell lysis (%) p=0.0001 p=0.0004 α-CD55iso-ctrl PBS Treatment BEAS-2B

11. Influenza infection enhances CD55 expression 0.00 0.01 0.02 0.03 0.04 0.05 0.06 Normalized CD55 mRNA levels p=0.0193 NS 7hpi 24hpi mock H1N1pdm A B 0 10 20 30 40 50 12hpi 24hpi 32hpi mock H1N1pdm CD55 levels (pg/ml)

12. Summary Genetic association study: rs2564978 T/T genotype associated with H1N1pdm severe infection. CD55 expression assay: rs2564978 T/T genotype carriers showing the lower expression in human peripheral blood monocytes. Luciferase assay: T/T genotype and rs3841376 del displaying lower transcriptional activity. The indel dictating CD55 transcriptional regulation. CD55 protecting A549 and BEAS-2B cells from complement attack. Upon flu infection, CD55 (membrane and soluble form) was up-regulated. Journal of infectious Diseases. ZHOU. J 2012

13. On-going Study To define the role of CD55 in influenza virus-infected CD55-KO mice. 0 20 40 60 80 100 120 067891011121314151617 CD55KO Balb/c Survival Rate (%) Day(s) post inoculation

14. Acknowledgement Professor Kwok-Yung Yuen 袁国勇教授 My team: CHU Hin 朱轩 CHENG Zhongshan (Sam) 程中山 LI Cun 李存 WONG Ho Yin (Bosco) Health and Medical Research Fund (HMRF), HKSAR & Providence Foundation Limited in memory of the late Dr Lui Hac Minh

15. jiezhou@hku.hk