WHAT HAPPENS IN THE INTESTINAL MUCOSA?
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Transcript of WHAT HAPPENS IN THE INTESTINAL MUCOSA?
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, Italy 2Sentinel CH, Milano, Italy.
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.
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
B2 A2 B3 B1 A1 B2B1 B3 B9 A
DQ DRchromosomal location: 6p21.3
General population
CD
DQA1*05 or
DQB1*02
DQA1*0201
Cis Trans Cis/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.
CELIAC DISEASE RISKClassical
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
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)
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
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)
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
METHODS 1Sample
extractionSample
collection
STAT- NAT DNA Mix
(Sentinel CH)ABI Prism 7900HT
(Applied Biosystem)collection in EDTA tube
MagNa Pure System (Roche)
Amplification
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
1 2 3 4 5 6
80-100 ng DNA ADD
ADD
METHODS 2
All mixes included an internal amplification control.
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
Internalcontrol
alwayspresent
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
RESULTS 2
UK NEQAS
Our methodDQA1
Mix1(*0201)
Mix2(*03)
Mix3(*05)
Mix4(*02)
Mix5(*0302)
Mix6(homozygous*02)
positive positive
positive positive positive
positive positive positive
positive positive positive
positive positive positive
positive 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 methodDQB1
Empty boxes indicate absence of amplification
CD haplotypes
Negative
Negative
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
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
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 OTHERCAUSES OF FLAT
MUCOSA
CELIAC DISEASE:GLUTEN-FREE
DIET DETERMINATIONDETERMINATION
HLA-DQ2-DQ8HLA-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 DISEASESEARCH 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 DIETTO VERIFY THE CLINICAL-
ANTIBODYRESPONSE
DETERMINATIONDETERMINATIONHLA-DQ2-DQ8HLA-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
DETERMINADETERMINATIONTION
HLA-DQ2/HLA-DQ2/DQ8DQ8
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