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WHAT HAPPENS IN THE INTESTINAL MUCOSA?

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 .

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WHAT HAPPENS IN THE INTESTINAL MUCOSA?

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  1. A NEW LABORATORY FRIENDLY PLATFORM TO DETECT THE CELIAC DISEASE ASSOCIATED HLA-DQ2 AND DQ8 HAPLOTYPE D. Bozzato1, F. Navaglia1, E. Rossi1, M. Gramegna2, M. Pelloso1, R. Favero3, E. Greco1, A. Padoan1, S. Moz1, P. Fogar1, C-F. Zambon1, D. Basso1, M. Plebani1. 1Department of Laboratory Medicine and 3Blood Transfusion Service, University of Padova, Italy2Sentinel 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. B2 A2 B3 B1 A1 B1 B2 B3 B9 A DQ DR chromosomal location: 6p21.3 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. Gluten APC

  4. General population 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. CD CD DQ2 DQ8 DQA1*05 or DQA1*03 DQA1*0201 DQB1*0302 DQB1*02 Trans Cis/Trans Cis

  5. CELIAC DISEASE RISK Risk HIGH RISK LOW RISK NO RISK Megiorni et al. Human Immunology 2009;70:55-59

  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. 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. PERFORMANCE IMPROVEMENT STAT-NAT is a ready-to-use product to minimized analytical variables UNIVERSAL STAT-NAT technology yields very good performances in all the most diffused molecular biology techniques FEATURES AND BENEFITS STAT-NAT DNA MIX

  8. TOTAL DNA STUDIED = 76 (typed with Olerup SSP) 23 bearing only one risk allele 7 absence of risk alleles 30 HLA-DQ2 or DQ2 and DQ8 (including 6 DQB1*02 homozygotes) 16 HLA-DQ8 • Haplotypes: • A1*0201 B1*02 • A1*05 B1*02 • A1*03 B1*0302 • A1*05 B1*02/*02 • Haplotypes: • A1*03 B1*0302 • A1*03 B1*0302/*02 • Alleles: • A1*0201 or *03 or *05 • B1*02 or * 0302 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. HLA-DQA1 46 alleles HLA-DQB1 158 alleles 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.

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

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

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

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

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

  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. CONCLUSIONS 2 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

  17. PROPOSED DIAGNOSTIC ALGORITHM SUBJECTS WITH STRONG SUSPICION OF CELIAC DISEASE SUBJECTS BELONGING TO GROUPS AT RISK Anti-TTG + IgA Anti-TTG + IgA SEROLOGY NEGATIVE +POSITIVE BIOPSY SEROLOGY POSITIVE +POSITIVE BIOPSY SEROLOGY POSITIVE +NEGATIVE BIOPSY NEGATIVE SEROLOGY POSITIVE SEROLOGY EXCLUSION OF OTHERCAUSES OF FLAT MUCOSA CELIAC DISEASE:GLUTEN-FREE DIET DETERMINATIONHLA-DQ2-DQ8 DETERMINATION HLA-DQ2-DQ8 INTESTINAL BIOPSY DQ2 AND / OR DQ8 POSITIVEMONITORING ANTI-TTG AND REPETITION BIOPSY OR TRIAL WITH GLUTEN-FREE DIETTO VERIFY THE CLINICAL- ANTIBODYRESPONSE DQ2 AND / OR DQ8 NEGATIVEANTI-TTG FALSE POSITIVESEARCH FOR OTHER CAUSES DETERMINATIONHLA-DQ2-DQ8 HISTOLOGY POSITIVE(TYPE 3A-3C) HISTOLOGY NEGATIVEOR TYPE 1-2 IF POSITIVE: SUBJECTS AT RISK,PERIODICALLY REPEAT ANTI-TTG IF NEGATIVE: RISK-FREE, NOT REPEAT MORE TESTS CELIAC DISEASE:GLUTEN-FREE DIET DETERMINATIONHLA-DQ2/DQ8 DQ2 AND / OR DQ8 POSITIVE:BE CONFIRMED WITH GLUTEN FREE DIET AND CHALLENGE (IF THE LESION TYPES 1-2MONITORING) DQ2 AND / OR DQ8 NEGATIVE:LOW PROBABILITY OF CELIAC DISEASESEARCH FOR OTHER CAUSES 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

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