1. A NEW LABORATORY FRIENDLY PLATFORM TO DETECT THE CELIAC DISEASE ASSOCIATED HLA-DQ2 AND DQ8 HAPLOTYPE D. Bozzato 1, F. Navaglia 1, E. Rossi 1, M. Gramegna 2, M. Pelloso 1, R. Favero 3, E. Greco 1, A. Padoan 1, S. Moz 1, P. Fogar 1, C-F. Zambon 1, D. Basso 1, M. Plebani 1. 1 Department of Laboratory Medicine and 3 Blood Transfusion Service, University of Padova, Italy 2 Sentinel CH, Milano, Italy.

2. WHAT HAPPENS IN THE INTESTINAL MUCOSA? The development of anti- gluten T-cell response in the intestine is specific to people with celiac disease (CD). The immune response to gluten takes place in two compartments, the lamina propria (CD4+) and the epithelium (CD8+). Tissue transglutaminase acts on selected glutamines within the glutamine/proline rich gluten peptides.

3. HLA AND CELIAC DISEASE Specific alleles at HLA-DQA1 and DQB1 loci appear to be necessary, although not sufficient, for the phenotypic expression of the disease. HLA-DQA1 and DQB1 alleles encode the α and β chains, respectively, of the heterodimer which presents gluten peptides and triggers the immume response. APC Gluten B2A2B3B1A1B2B1B3B9A DQDR chromosomal location : 6p21.3

4. General population CD DQA1*05 or DQB1*02 DQA1*0201 Cis TransCis/Trans DQB1*0302 DQA1*03 DQ2 DQ8 CD DQ2 (HLA-DQA1*05/*0201 - DQB1*02) is present in 90-95% of CD patients. Among them individuals homozygous for HLA-DQB1*02 have the highest risk for CD. DQ8 (HLA-DQA1*03 - DQB1*0302) is present in the remaining 5-10% of CD patients.

5. CELIAC DISEASE RISK Classical and frequent CD- associated haplotypes DQ2 and DQ8 (A1*05 - B1*02 / A1*03 - B1*0302) 1:7 Homozygous DQ2 (A1*05 - B1*02/*02) 1:10 DQ8 and DQ2  -chain (A1*03 - B1*0302/*02) 1:24 Other and less frequent CD- associated haplotypes Homozygous DQ2  -chain (A1*0201- B1*02/*02) 1:26 Heterozygous DQ2 (A1*05 - B1*02/X) 1:35 DQ8 (A1*03 - B1*0302/X) 1:89 Heterozygous DQ2  -chain ( B1*02/X) 1:210 Megiorni et al. Human Immunology 2009;70:55-59 Risk Only A1*05 1:1842 Other alleles 1:2518 HIGH RISK LOW RISK NO RISK

6. AIMS To develop a real-time PCR method to detect celiac disease associated HLA-DQA1 and HLA-DQB1 alleles To implement this new assay using a laboratory friendly platform with components stable at room temperature (STAT-NAT DNA Mix - Sentinel, CH)

7. FEATURES AND BENEFITS STAT-NAT DNA MIX ROOM TEMPERATURE STORAGE STAT-NAT is freeze-dried and guarantees long term storage at room temperature. EASY STAT-NAT contains all the reaction components. The enzyme (Hot Start Polymerase) is already included. UNIVERSAL STAT-NAT technology yields very good performances in all the most diffused molecular biology techniques PERFORMANCE IMPROVEMENT STAT-NAT is a ready-to-use product to minimized analytical variables

8. TOTAL DNA STUDIED = 76 (typed with Olerup SSP) 30 HLA-DQ2 or DQ2 and DQ8 (including 6 DQB1*02 homozygotes) Haplotypes: A1*0201 B1*02 A1*05 B1*02 A1*03 B1*0302 A1*05 B1*02/*02 16 HLA-DQ8 Haplotypes: A1*03 B1*0302 A1*03 B1*0302/*02 23 bearing only one risk allele Alleles: A1*0201 or *03 or *05 B1*02 or * 0302 7 absence of risk alleles EXTERNAL QUALITY ASSESSMENT Six DNA samples of 2011 UK NEQAS for H&I pilot scheme 8 (HLA & disease typing or HLA-DR/DQ/DP only)

9. ASSAY DESIGN Specific sequence of primers and taqman (hydrolysis) MGB probes for DQA1*0201, DQA1*03, DQA1*05, DQB1*02 and DQB1*0302 were designed using sequence information from the IMGT/HLA database (http://www.ebi.ac.uk/imgt/hla/align.html). To determine the homozygous state for DQB1*02, specific sequence primers and taqman MGB probes which covered the majority of HLA-DQB1 alleles other than DQ2, were used. HLA-DQA1 HLA-DQB1 46 alleles 158 alleles

10. METHODS 1 Sample extraction Sample collection STAT- NAT DNA Mix (Sentinel CH) ABI Prism 7900HT (Applied Biosystem) collection in EDTA tube MagNa Pure System (Roche) Amplification

11. LYOPHILIZED PCR AMPLIFICATION COCKTAIL which includes a hot start polymerase, buffers and dNTPs SIX SPECIFIC MIX (specific primers and probes) for: 1) DQA1*0201 2) DQA1*03 3) DQA1*05 4) DQB1*02 5) DQB1*0302 6) Homozygous for DQB1*02 1234 5 6 80-100 ng DNA ADD METHODS 2 All mixes included an internal amplification control.

12. RESULTS 1 26.20 (±0.34) 25.38 (±0.53) 23.86 (±0.15) MIX 1 MIX 2 MIX 3 Ct mean (±SE) A complete agreement with the reference method (Olerup SSP HLA typing) was found for all 76 DNA samples DQA1*0201 absent present DQA1*03 present absent DQA1*05 present absent

13. Internal control always present 28.22 (±0.31) 27.89 (±0.71) DQB1*0302 present absent heterozygosis homozygosis Homozygous DQB1*02 27.89 (±0.71) 27.1 (±0.85) MIX 4 MIX 5 MIX 6 Ct mean (±SE) DQB1*02 absent present

14. RESULTS 2 UK NEQAS Our method DQA1 Mix1 (*0201) Mix2 (*03) Mix3 (*05) Mix4 (*02) Mix5 (*0302) Mix6 (homozy gous*02) positive 801/11 DQA1*0102/*0201 DQB1*0303/*0602 802/11 DQA1*0102/*0201 DQB1*0202/*0604 803/11 DQA1*0201/*0505 DQB1*0301/*0303 804/11 DQA1*0201/*0501 DQB1*0201/*0202 805/11 DQA1*0102/*0301 DQB1*0302/*0602 806/11 DQA1*0103/*0505 DQB1*0301/*0603 Heterozygous DQ2  -chain (B1*02/X) Negative Homozygous DQ2 (A1*05–B1*02/*02) DQ8 (A1*03-B1*0302) Our method DQB1 Empty boxes indicate absence of amplification CD haplotypes Negative

15. CONCLUSIONS 1 Our new real-time PCR method to detect celiac disease associated HLA-DQA1 and HLA-DQB1 alleles was shown to be:  specific and reproducible  in agreement with the reference method for all analyzed DNA  in agreement for all UK NEQAS samples By using a close tube system, this method reduces the risk of cross contamination

16. STAT-NAT DNA Mix using lyophilized and ready to use reagents reduces analytical variations and allows rapid preparation of the amplification mix STAT-NAT DNA Mix allows to develop a laboratory friendly high throughput platform CONCLUSIONS 2

17. PROPOSED DIAGNOSTIC ALGORITHM SUBJECTS WITH STRONG SUSPICION OF CELIAC DISEASE Anti-TTG + IgA SEROLOGY NEGATIVE + POSITIVE BIOPSY SEROLOGY POSITIVE + POSITIVE BIOPSY SEROLOGY POSITIVE + NEGATIVE BIOPSY EXCLUSION OF OTHER CAUSES OF FLAT MUCOSA CELIAC DISEASE: GLUTEN-FREE DIET DETERMINATION HLA-DQ2-DQ8 DQ2 AND / OR DQ8 POSITIVE: BE CONFIRMED WITH GLUTEN FREE DIET AND CHALLENGE (IF THE LESION TYPES 1-2 MONITORING) DQ2 AND / OR DQ8 NEGATIVE: LOW PROBABILITY OF CELIAC DISEASE SEARCH FOR OTHER CAUSES DQ2 AND / OR DQ8 NEGATIVE ANTI-TTG FALSE POSITIVE SEARCH FOR OTHER CAUSES DQ2 AND / OR DQ8 POSITIVE MONITORING ANTI-TTG AND REPETITION BIOPSY OR TRIAL WITH GLUTEN- FREE DIET TO VERIFY THE CLINICAL- ANTIBODYRESPONSE DETERMINATION HLA-DQ2-DQ8 SUBJECTS BELONGING TO GROUPS AT RISK Anti-TTG + IgA NEGATIVE SEROLOGY POSITIVE SEROLOGY DETERMINATION HLA-DQ2-DQ8 INTESTINAL BIOPSY HISTOLOGY POSITIVE (TYPE 3A-3C) HISTOLOGY NEGATIVE OR TYPE 1-2 CELIAC DISEASE: GLUTEN- FREE DIET DETERMINA TION HLA- DQ2/DQ8 IF POSITIVE AND HISTOLOGY NORMAL MONITORING; IF POSITIVE AND TYPE 1-2: DECIDED CASE BY CASE IF NEGATIVE: PROBABLY ANTI-TTG FALSE POSITIVE AND POSSIBLE REMOTE CONTROL IF NEGATIVE: RISK-FREE, NOT REPEAT MORE TESTS IF POSITIVE: SUBJECTS AT RISK, PERIODICALLY REPEAT ANTI- TTG