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 antigluten 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
B2 A2 B3
B1 A1
DQ
B1 B2 B3
B9 A
DR
chromosomal location: 6p21.3
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
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.
CD
CD
DQ8 (HLA-DQA1*03 - DQB1*0302) is present in the
remaining 5-10% of CD patients.
DQ2
DQ8
DQA1*05 or
DQA1*03
DQA1*0201
DQB1*0302
DQB1*02
Cis
Trans
Cis/Trans
CELIAC DISEASE RISK
Risk
Classical
and
frequent
CDassociated
haplotypes
DQ2 and DQ8
1:7
(A1*05 - B1*02 / A1*03 - B1*0302)
Homozygous DQ2
1:10
(A1*05 - B1*02/*02)
DQ8 and DQ2 b-chain
HIGH RISK
1:24
(A1*03 - B1*0302/*02)
Homozygous DQ2 b-chain
Other and
less
frequent
CDassociated
haplotypes
1:26
(A1*0201- B1*02/*02)
Heterozygous DQ2
1:35
(A1*05 - B1*02/X)
DQ8
1:89
LOW RISK
(A1*03 - B1*0302/X)
Heterozygous DQ2 b-chain
(B1*02/X)
1:210
Only A1*05
1:1842
Other alleles
1:2518
Megiorni et al. Human Immunology 2009;70:55-59
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
EASY
STAT-NAT is freeze-dried and
guarantees long term storage at
room temperature.
STAT-NAT contains all the
reaction components. The
enzyme (Hot Start Polymerase) is
already included.
UNIVERSAL
PERFORMANCE IMPROVEMENT
STAT-NAT technology yields very
good performances in all the
most diffused molecular biology
techniques
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
16 HLA-DQ8
23 bearing only
one risk allele
7 absence of
risk alleles
homozygotes)
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)
ASSAY DESIGN
HLA-DQA1
46 alleles
HLA-DQB1
158 alleles
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.
METHODS 1
Sample
collection
collection in
EDTA tube
Sample
extraction
MagNa Pure System
(Roche)
Amplification
STAT- NAT
DNA Mix
(Sentinel CH)
ABI Prism 7900HT
(Applied Biosystem)
METHODS 2
ADD
80-100 ng DNA
ADD
SIX SPECIFIC MIX (specific
primers and probes) for:
1) DQA1*0201
1
2
3
4
5
6
2) DQA1*03
3) DQA1*05
LYOPHILIZED PCR
AMPLIFICATION
COCKTAIL which includes
a hot start polymerase,
buffers and dNTPs
4) DQB1*02
5) DQB1*0302
6) Homozygous for DQB1*02
All mixes included an internal
amplification control.
RESULTS 1
A complete agreement with the reference method (Olerup
SSP HLA typing) was found for all 76 DNA samples
MIX 1
DQA1*0201
26.20
(±0.34)
MIX 2
MIX 3
DQA1*03
DQA1*05
present
present
absent
absent
25.38
(±0.53)
Ct mean (±SE)
present
absent
23.86
(±0.15)
MIX 4
MIX 5
MIX 6
Homozygous
DQB1*02
DQB1*0302
DQB1*02
present
present
absent
27.89 (±0.71)
heterozygosis
homozygosis
absent
28.22 (±0.31)
27.89 (±0.71)
Internal
control
Ct mean (±SE)
always
present
27.1 (±0.85)
RESULTS 2
Our method
DQA1
UK NEQAS
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
Mix1
(*0201)
Mix2
(*03)
Mix3
(*05)
Our method
DQB1
Mix4
(*02)
Mix5
(*0302)
positive
Mix6
(homozy
gous*02)
positive
positive
positive
positive
positive
positive
positive
positive
positive
positive
positive
CD haplotypes
Negative
Heterozygous
DQ2 b-chain
(B1*02/X)
Negative
Homozygous DQ2
(A1*05–B1*02/*02)
positive
positive
positive
DQ8
(A1*03-B1*0302)
positive
Negative
Empty boxes indicate absence of amplification
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
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
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 OTHER
CAUSES OF FLAT
MUCOSA
CELIAC DISEASE:
GLUTEN-FREE
DIET
DETERMINATION
HLA-DQ2-DQ8
DETERMINATION
HLA-DQ2-DQ8
INTESTINAL
BIOPSY
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
POSITIVE
MONITORING ANTI-TTG
AND REPETITION BIOPSY
OR TRIAL WITH GLUTENFREE DIET
TO VERIFY THE CLINICALANTIBODYRESPONSE
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
IF
NEGATIVE:
RISK-FREE,
NOT REPEAT
MORE TESTS
IF POSITIVE:
SUBJECTS AT
RISK,
PERIODICALLY
REPEAT ANTITTG
IF POSITIVE AND HISTOLOGY
NORMAL MONITORING;
IF POSITIVE AND TYPE 1-2:
DECIDED CASE BY CASE
HISTOLOGY
POSITIVE
(TYPE 3A-3C)
CELIAC
DISEASE:
GLUTENFREE DIET
HISTOLOGY
NEGATIVE
OR TYPE 1-2
DETERMINA
TION
HLADQ2/DQ8
IF NEGATIVE:
PROBABLY ANTI-TTG FALSE
POSITIVE AND POSSIBLE
REMOTE CONTROL