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