ARTICLE
Psoriasis (PSORS, OMIM #177900) is a chronic inflammatory
skin disorder affecting approximately 2% of the Caucasian population [1].
The disease is characterized by skin lesions showing epidermal hyper proliferation,
abnormal keratinocyte differentiation and infiltration of inflammatory
elements [2]. Despite the established influence of several environmental
factors, epidemiological data and twin studies have demonstrated a genetic
basis for disease susceptibility [3, 4]. Moreover, associations with several
HLA alleles have been repeatedly reported, suggesting the hypothesis that
psoriasis may be a T-cell-mediated, autoimmune disorder [2]. Parametric
and non-parametric linkage analyses have mapped a major psoriasis susceptibility
region to chromosome 6p21 and several non-MHC loci [5]. One of such susceptibility
regions was mapped to chromosome 16 [6], within the region containing
the CARD15 (caspase recruitment domain) gene (previously named NOD2),
which carries alleles associated to inflammatory bowel disease (IBD1,
OMIM #266600) [7-11]). IBD is an autoimmune disease characterized by chronic
relapsing intestinal inflammation, presenting as Crohn's Disease (CD,
OMIM #266600) or Ulcerative Colitis (UC, OMIM 191390). The prevalence
of IBD is increased in individuals with other autoimmune conditions, particularly
ankylosing spondylitis, psoriasis, sclerosing cholangitis, and multiple
sclerosis. In particular, patients with CD have an increased risk (about
seven-fold) to develop psoriasis during their life-time [6]. In this context,
the mapping of a psoriasis susceptibility locus within the region containing
CARD15 gene suggests the possibility that this gene is involved in the
pathogenesis of other inflammatory disorders. In fact, mutations of CARD15
were also found in Blau syndrome (ACUG, OMIM #186580), a rare autosomal
dominant disorder characterized by early-onset granulomatous arthritis,
uveitis, and skin rash with camptodactyly [12]. The possibility that CARD15
is involved in psoriasis has been preliminarily excluded by Nair et
al. [13], who analysed a cohort of psoriatic trios and failed to observe
preferential transmission of the penetrant CARD15 allele 3020insC. In
order to confirm and extend this result, we analysed a dataset of Italian
patients and controls. We examined the distribution of the 3020insC allele
and of two additional variants (G908R and R702W), showing independent
association to CD [7, 9].
Materials and methods
Ninety psoriatic patients and 160 healthy volunteers were recruited
at the Department of Dermatology, "Tor Vergata" University of Rome. All
subjects granted their informed consent for participating in this study.
In all cases consensus diagnosis of the disease was assessed by two expert
dermatologists, based on established clinical criteria [2]. Genomic DNA
was extracted from peripheral blood lymphocytes by standard phenol-chloroform
extraction. PCR amplification of CARD15 exon 11 was carried out using
the following primers: forward, 5'-CTCACCATTGTATCTTCTTTTC; reverse, 5'-GAATGTCAGAATCAGAAGGG.
PCR was performed in a final volume of 50 mul (2 mM MgCl2, 200 mM dNTPs,
0.2 mM of each primer, 200 ng of genomic DNA, and 1 U of AmpliTaq Gold
DNA Polymerase). Cycling was performed with an initial denaturation step
at 94° for 10 min to activate AmpliTaq Gold, followed by 35 cycles
of 94° for 30 sec, 55° for 30 sec and 72° for 1 min, and
a final extension step of 72° for 10 min.
The presence of the 3020insC mutation (+) was
assessed by Denaturing High-Performance Liquid
Chromatography (DHPLC) and confirmed by direct automated sequencing.
The DHPLC conditions were established using the Wave Maker 3.1 software.
The experimental run temperature of 60° C was tested by running a
wild-type PCR product at the calculated temperature ± 2° C.
Heterozygous profiles were identified by visual inspection of the chromatograms
(Fig. 1). The genotyping
of G908R (also known as SNP12) and R702W (SNP8) variants was performed
using the Pyrosequencing technology (PSQ 96 system) [14]. A 130-bp (SNP8)
and a 116-bp (SNP12) fragment were amplified in a 50 mul PCR reaction
containing 50 ng of genomic DNA, 10 pmol of forward biotinylated and reverse
unlabelled primer, 1X PCR Buffer (Perkin-Elmer), 1mM MgCl2,
0.2 nM dNTPs, and 1 Unit of AmpliTaq Gold DNA polymerase.
The PCR primers were the following: SNP8, forward 5'-CTTCCTGGCAGGGCTGTT-3';
SNP8, reverse 5'-GAAGTGCTTGCGGAGGCT-3'; SNP12, forward 5'-CACATATCAGGTACTCACTGACAC-3';
SNP12, reverse 5'-GTGATCACCCAAGGCTTCAG-3'.
Biotinylated PCR products (25 mul) were immobilized on streptavidin-coated
paramagnetic beads by a 30 min incubation in 2X binding-washing buffer
(10 mM Tris-HCl, 2M NaCl, 1 mM EDTA, 0.1% Tween 20, pH 7.6), at 45°
C. Single-stranded DNA was obtained by incubating the immobilized PCR
product in 50 mul of 0.5 M NaOH for 1 min and washing the beads once in
100 mul of annealing buffer 1X (20 mM Tris-Acetate and 5 mM MgAc2, pH
7.6). A total of 10 pmol of detection sequence primer (SNP8: 5'-TCTGAGAAGGCCCTG-3'
and SNP12: 5'-TCACCCACTCTGTTGC-3'), designed with its 3' end immediately
upstream of the splice mutation, was allowed to hybridise to ssDNA in
40 mul of annealing buffer, at 80° C for 2 min, with subsequent cooling
down to room temperature. Pyrosequencing was carried out using the PSQ96
instrument and the SNP Reagent kit containing dATPalphaS, dCTP, dGTP,
dTTP, enzyme mixture (DNA polymerase, ATP sulfurylase, luciferase and
apyrase), and substrate mixture (APS and luciferin) (Fig. 2).
To compare genotypic and allelic frequencies between patients and controls,
a Chi-square test was performed.
Results and discussion
The genotypic and allelic frequencies of the three CARD15 variants are
shown in Table I. No significant
difference in genotypic or allelic frequencies was detected between the
two examined sample groups. None of the subjects included in this study
carried more than a single mutated allele, with the exception of one psoriatic
patient who was found to be homozygous for the SNP R702W and heterozygous
for the 3020insC variant. All together, these results allow us to exclude
an involvement in psoriasis pathogenesis of the most common CD susceptibility
alleles of the CARD15 gene, thereby confirming and extending the results
reported by Nair et al. [13]. Although we cannot definitively exclude
a role of CARD15 protein in the alteration of immune system observed in
psoriasis, our studies reduce the possibility that the clinical concomitance
of psoriasis and CD is due to specific alleles of the CARD15 gene.
CONCLUSION
Acknowledgements
This work was supported by grants from Italian Ministry of Health and
Ministry of Education and University and Research (MIUR Fondi Centro di
Eccellenza). We are grateful to C. Farachi and the Biosense s.r.l. for
the useful support and help in set-up the Pyrosequencing technology.
Article accepted on 18/7/02
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