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Technical Briefs |
a address for correspondence: Dipto. di Biochim. e Biotecnol. Mediche, Facoltà di Med. e Chirurg., Univ. "Federico II", via S. Pansini 5, 80131 Napoli, Italy, fax +39-81-7463650
1
Dipto. di Biochim. e Biotecnol. Mediche, Facoltà di Med. e Chirurg., and CEINGE-Biotecnol. Avanzate, Univ. "Federico II", via S. Pansini 5, 80131 Napoli, Italy;
2
Ist. policattedra di Biochim. e Chim. Medica, Univ. di Bari, p.zza Giulio Cesare, 70124 Bari, Italy;
Celiac disease (CD) is an autoimmune disorder
associated with a small bowel lesion induced by toxic gliadin
components [1, 2]. In
this condition, an antigen peptide from 
-gliadin, corresponding to
the amino acid sequence between residues 31 and 49, initiates the
cellular immune response, which is mediated by gliadin-specific T
lymphocytes [3]. Antigen recognition by T
lymphocytes in CD mainly occurs if the gliadin-derived peptides are
carried by the HLA class II molecules HLA-DQ2 or HLA-DR4
[4, 5]. Thus, the
genetic susceptibility towards CD derives from inheriting certain HLA
class II alleles encoding for the above-mentioned specific molecules.
Among the CD-HLA associations that have been described so far, the one
caused by the presence of alleles DQA10501/DQB10201 (encoding for the
DQ2 molecule) is present in most cases, whereas the DRB104 alleles
(encoding for the DR4 molecule) occurs almost invariably in the other
cases [5, 6]. In fact,
these two associations characterized >95% of the affected celiac
patients in European populations [7].
The aim of our study was to improve a PCR-based methodology for the rapid typing of the DQA10501, DQB10201, and DRB104 alleles, and thus provide an additive simple tool for the diagnosis of CD.
DNA was extracted from 5 mL of fresh whole blood in EDTA by proteinase K treatment followed by phenol–chloroform extraction (8). Isolated DNA samples were quantified by UV spectrophotometry at 260 nm absorbance and diluted with distilled water to the concentration required for PCR (100 ng/µL). We examined the DNA of three subjects whose HLA serological specificities, previously studied, were: (a) DQ2/DQ5, (b) DQ5/DQ7, and (c) DR4/DR16. The first haplotype showed the DQ2 molecule that is strongly associated with DQA10501, DQB10201 genotype and so represented a positive DNA control for these alleles. The second haplotype lacked the DQ2 and DR4 molecules and so we used it as a negative control for the other alleles. The third haplotype showed the DR4 molecule encoded by DRB104 alleles, thus representing a positive DNA control for these alleles.
Two reaction mixtures were used, one for the amplification of the
DQA10501 and DQB10201 alleles, and the other for the identification
of the DRB104 alleles, with the allele-specific primers described in
Table 1
. The sequences of these primers differed in 2 or 3 nucleotides
from those of the other known alleles at the same HLA loci. To increase
primer specificity, we introduced a mismatch at their 3' end
(9). We used exon 10 of the pyruvate kinase gene
(10) in the first mix and exon 10 of the cystic fibrosis
gene (11) in the second mix (see Table 1
). These two
primers did not match the HLA allelic sequences and so their PCR
products (respectively 238 bp and 491 bp length) represented internal
positive amplification controls. Both PCR reaction mixtures contained
in a final volume of 50 µL: 100 ng of genomic DNA; 200 µmol/L each
of dATP, dCTP, dGTP, and dTTP (Pharmacia Biotech); PCR buffer [83
mmol/L (NH4)2SO4, 335 mmol/L
Tris-HCl pH 8.8, 33.5 mmol/L MgCl2, 50 mmol/L
ß-mercaptoethanol, 100 g/L bovine serum albumin, 34 mmol/L EDTA];
2.5 U of AmpliTaq DNA polymerase (Perkin-Elmer–Roche Molecular
Systems); and the allele-specific primers and the internal positive
control primers.
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The first mix consisted of 0.6 µmol/L DQA10501 primers, 0.4 µmol/L DQB10201 primers, and 0.8 µmol/L internal positive control primers. The second mix contained 0.4 µmol/L DRB104 primers and 0.2 µmol/L internal positive control primers. A DNA thermal cycler (Gene Amp PCR System 9600, Perkin-Elmer) was used for PCR amplification. Double-stranded DNA was denatured initially by heating to 95 °C for 5 min, followed by a 30-cycle profile of 30 s at 94 °C for denaturation, 10 s of annealing at 64 °C, and 20 s of extension at 72 °C. The last PCR cycle was followed by an additional 10 min at 72 °C to complete the final extension.
PCR products were separated by agarose gel electrophoresis. Thirty
microlitres of the PCR reaction mixtures were added to 5 µL of
loading buffer (300 mL/L glycerol stained with bromphenol blue and
xylene cyanol) and loaded in a 3% agarose gel prestained with ethidium
bromide (0.5 µg/mL gel) (Sigma Chemical Co.). Electrophoresis was
performed at 100 V for 40 min in 1x TBE buffer (89 mmol/L Tris base,
89 mmol/L boric acid, 0.2 mmol/L EDTA). PCR products on gel were
visualized by UV illumination as bands of 144 bp (DQA10501), 110 bp
(DQB10201), and 177 bp (DRB104) and then photographed. Fig. 1
shows an example of HLA typing obtained with our procedure on
DNA samples from the three previously HLA-characterized (serologically)
individuals. All three HLA-DQA10501, DQB10201, and DRB104 alleles
appeared as well-separated bands.
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In the diagnostic iter of gastrointestinal malfunction, the finding of the HLA DQA10501, DQB10201, and DRB104 alleles increases the possibility of having CD (7). Various methods have been described for DNA typing at the HLA level, mainly for histocompatibility testing. These procedures involve a combination of PCR amplification with restriction fragment length polymorphism (12), sequencing, oligonucleotide probing, or sequence-specific primers (13)(14)(15)(16), all of which are time consuming and expensive when used for CD diagnosis. We have devised a rapid, accurate, and cost-effective PCR-based method to type, with a high level of resolution, the HLA-DQA10501, DQB10201, and DRB104 alleles. The advantage of our method is that it does not require radioactive material and that, after the same PCR program, the three most frequently CD-associated alleles are visualized on the same gel, within ~2 h from DNA sample collection. We have used this method to examine >100 celiac patients and ~30 patients affected by other gastrointestinal syndromes clinically similar to CD. The results are similar to those obtained in other populations, with other methods (manuscript in preparation). We suggest that this procedure could represent an adjunctive tool in the diagnostic approach to CD; it is suitable for family screening, epidemiological studies, and for diagnosis particularly when histological and immunological patterns are ambiguous.
Acknowledgments
This study was supported by grants from the MURST, Rome, Italy; the Progetto Finalizzato Biotecnologie, CNR (Rome); and from Ricerca Sanitaria Finalizzata (Regione Campania, Italy).
References
-gliadin presented by the celiac disease-associated HLA-DQ (1*0501, ß1*0201) heterodimer. Hum Immunol 1994;39:243-252.
[Web of Science][Medline]
[Order article via Infotrieve]
1*0501, ß1*0201) restricted T cells isolated from small intestinal mucosa of celiac disease patients. J Exp Med 1993;178:187-196.
The following articles in journals at HighWire Press have cited this article:
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  L. Sacchetti, G. Calcagno, A. Ferrajolo, C. Sarrantonio, R. Troncone, M. Micillo, S. Auricchio, and F. Salvatore Discrimination between Celiac and Other Gastrointestinal Disorders in Childhood by Rapid Human Lymphocyte Antigen Typing Clin. Chem., August 1, 1998; 44(8): 1755 - 1757. [Full Text] [PDF]
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