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2000 Oxford University Press

Human Molecular Genetics, 2000, Vol. 9, No. 10 15331542

A candidate gene for psoriasis near HLA-C, HCR (Pg8),


is highly polymorphic with a disease-associated
susceptibility allele
Kati Asumalahti, Tarja Laitinen, Raija Itkonen-Vatjus1, Marja-Liisa Lokki2, Sari Suomela3,
Erna Snellman4, Ulpu Saarialho-Kere3 and Juha Kere5,+
Department of Medical Genetics, Haartman Institute, University of Helsinki, 00014 Helsinki, Finland, 1Department of
Dermatology, Oulu University Central Hospital, 90401 Oulu, Finland, 2Finnish Red Cross Blood Transfusion Service,
Department of Tissue Typing, 00310 Helsinki, Finland, 3Department of Dermatology, Helsinki University Central
Hospital, 00250 Helsinki, Finland, 4Department of Dermatology, Central Hospital of Pijt-Hme, 15850 Lahti, Finland
and 5Finnish Genome Center, University of Helsinki, 00014 Helsinki, Finland
Received 15 February 2000; Revised and Accepted 11 April 2000

A susceptibility gene for psoriasis, a chronic skin


disorder, resides in chromosome 6p near the HLA-C
locus. Sequencing of the region has allowed the
identification of a new gene, HCR . We found that HCR
is highly polymorphic with at least 12 coding variants. An association study of the new HCR polymorphisms and the previously suggested susceptibility
alleles HLA-Cw*0602 and corneodesmosin allele 5
(CD*5) with psoriasis revealed a specific HCR variant
associated with psoriasis susceptibility. However,
the HLA-Cw*0602 allele was rarer in controls and
associated with a stronger relative risk. Association
analysis did not support CD*5 as a psoriasis susceptibility allele in our sample of patients (n = 100) and
population-matched controls (n = 93) from an
isolated population. We found HCR to be overexpressed in keratinocytes of psoriatic lesions
compared with paired samples of healthy skin. Our
results suggest a potential role for HCR in the pathogenesis of psoriasis.
INTRODUCTION
Psoriasis is a chronic skin disorder characterized by keratinocyte hyperproliferation, angiogenesis and inflammatory leukocyte infiltrates (1). It shows familial clustering, and in recent
years, several candidate loci for psoriasis have been proposed
(PSORS15) (25). The oldest and still strongest evidence
exists for a susceptibility locus in the HLA region (PSORS1,
OMIM#177900); in the earlier studies for the HLA-A and
HLA-B loci (6,7) and later for the HLA-C locus (8,9). By DNAbased genotyping, the HLA-Cw*0602 allele has been found in
many populations to be the best marker for the major risk allele
of psoriasis. The proportion of patients who are HLA-Cw*0602
positive (5480%), is four to five times higher than in population-based controls (1020%) (10,11). Also linkage results
+To

DDBJ/EMBL/GenBank accession no. AF216493

from genome-wide scans place a major psoriasis locus on


chromosome 6p21.3 (12,13). Recently, the susceptibility
region for psoriasis has been tentatively narrowed to an
interval of a few hundred kilobases either centromeric or telomeric to HLA-C (14,15).
A typical feature of psoriasis is lymphocyte, macrophage
and neutrophil infiltration in the skin lesions. The HLA class I
antigens (HLA-A, -B, -C) have an important role in immune
response as antigen presenting molecules to CD8+ T
lymphocytes (16). Current knowledge of the pathogenesis of
psoriasis suggests, however, that CD4+ T lymphocytes may
have a more important role in the disease process than CD8+
cells (17). The class I antigens can also inhibit NK cell-mediated cytotoxicity through killer cell-inhibitory receptors
(KIRs), but the significance of NK cells in psoriasis has
remained contradictory (1,18). Therefore, the psoriasis susceptibility gene may not be HLA-C, but instead another gene in
linkage disequilibrium (LD) with the HLA-C locus. Indeed, the
corneodesmosin gene allele 5 (CD*5) has been found to be
linked with psoriasis by the transmission disequilibrium test
and CD has been suggested to be a prime functional candidate
for the psoriasis susceptibility gene (1921).
The HLA region has been completely sequenced, and most if
not all of its genes have been predicted by sequence analysis
(22). In the immediate neighborhood of HLA-C, there are only
four known functional genes: HLA-B, OTF3, TCF19 and CD
(Fig. 1). We determined the structure of a new gene predicted
from the genomic sequence. This gene was first called Pg8 (for
putative gene 8, a suggested tricohyalin homolog) (23) and
more recently HCR (for -helix coiled coil rod homolog) (15)
(GenBank accession no. AB029331). Due to its genomic position between HLA-C and CD and from preliminary data on its
expression in keratinocytes, HCR must be considered as an
interesting candidate for a psoriasis susceptibility gene.
We screened the HCR gene for sequence variations and
found that it is highly polymorphic with at least 12 coding variants. The HCR polymorphisms were analyzed together with
HLA-Cw*0602 and CD*5 for association with psoriasis in

whom correspondence should be addressed. Tel: +358 9 191 26538; Fax: +358 9 191 26789; Email: juha.kere@helsinki.fi

1534 Human Molecular Genetics, 2000, Vol. 9, No. 10

Figure 1. A map of the HLA region between HLA-B and CD. Filled bars indicate genes and open bars indicate pseudogenes.

patients and population-matched controls from a Finnish


subisolate. We found a specific HCR variant associated with
psoriasis susceptibility, whereas association analysis did not
support CD*5 as a psoriasis susceptibility gene. We found
HCR to be overexpressed in keratinocytes at psoriatic lesions.
Our results suggest a potential role for HCR in the pathogenesis of psoriasis.
RESULTS
Gene structure and expression pattern
We predicted the exonintron structure of the HCR gene from
the sequence of the genomic clone Y24c027 (GenBank accession no. AC004195) by using two different gene prediction

programs, FGENES and GENSCAN. To verify the coding


sequence, we designed primers specific for the predicted
exons; these were then used in RTPCR with keratinocyte
RNA, and the amplified products sequenced. Compared with
the verified cDNA sequence, FGENES had predicted the intragenic exon structure exactly right, whereas GENSCAN had
merged exons 12 and 13 into one exon. The gene consists of 16
exons, varying in size from 46 to 304 bp (Fig. 2). The coding
sequence of HCR was confirmed to be 2271 bp, encoding a
protein of 757 amino acids (GenBank accession no.
AF216493). HCR was shown to be expressed by RTPCR at
variable levels in all human tissues tested, most abundantly in
heart, liver, skeletal muscle, kidney and pancreas, and more
weakly in lung and placenta (Fig. 3).
Detection of HCR polymorphisms
To detect sequence variations, we screened all exons in five
psoriasis patients and one control by parallel SSCP analysis
and direct sequencing of PCR products. Two patients were
chosen among the HLA-Cw*0602-positive and three among
the HLA-Cw*0602-negative to enrich for different variants.
Altogether, 18 polymorphisms were found in the coding region
distributed in eight exons (Table 1). All sequence variants were
single nucleotide polymorphisms (SNPs). Eleven SNPs were
predicted to cause an amino acid change, nine of them nonconservative, and one SNP introduced a stop codon. Exons 2
and 8 were the most polymorphic, with five and four SNPs,

Table 1. The SNPs of the HCR gene coding sequence with the corresponding amino acid changes
Exon

Base number (coding sequence) Base number (AC004195)

Polymorphism

Amino acid change

Exon 2

+249

+10696

G A

Arg Gln (R76Q)a

+251

+10698

C T

TrpArg (W77R)a

+269

+10716

C T

TrpArg (W83R)a

+421

+10868

C T

no change

+436

+10883

G C

Arg Ser (R138S)a

Exon 4

+715

+14628

C G

no change

+769

+14682

A C

GluAsp (E249D)

Exon 8

+1193

+16946

T C

TrpArg (W391R)a

+1194

+16947

G A

TrpSTOP codon

+1193 and +1194

+16946 and +16947

TC and GA

TrpGln (W391Q)a

+1219

+16972

C T

no change

+1229

+16982

T C

no change

Exon 12

+1667

+20442

T G

Cys Gly (C549G)

Exon 13

+1824

+20695

G A

Arg Gln (R601Q)a

Exon 14

+1855

+21999

G A

no change

+1861

+22005

G T

GlnHis (Q613H)a

+2119b

+22390

G A

no change

+2122

+22393

A T

no change

+2271

+22788

G C

Cys Ser (C750S)a

Exon 15

Exon 16

The sequence of the clone Y24c027 (GenBank accession no. AC004195) has been used as the genomic reference.
aNon-conservative amino acid change.
bIn the Finnish samples in position +2119 only base A was detected.

Human Molecular Genetics, 2000, Vol. 9, No. 10 1535

Figure 2. The coding sequence and structure of the HCR gene. Exon boundaries are indicated with black arrows below the sequence. The 16 exons are illustrated
with black rectangles with the exon number above them. Exons with polymorphism are marked with asterisks.

respectively. We did not screen introns systematically, but


found seven non-coding SNPs in the small introns 3, 7, 10 and
12 that were included in amplicons containing flanking exons.
The Mendelian segregation of each SNP was confirmed in 22
families.
Patients and controls
Altogether, 142 psoriasis patients and 210 family members
(total of 100 families) were recruited from the central-eastern

part of Finland (the Kainuu province) during January 1996. In


addition, we recruited 93 population-based controls. Sample
collection was approved by the Ethical Review Boards of the
Kainuu Central Hospital and the Department of Medical
Genetics, University of Helsinki, and all samples were used
with informed consent.
The clinical characteristics of the study subjects are shown in
Table 2. The subgroup with familial psoriasis consists of
patients (n = 71) who have at least one affected first-degree

1536 Human Molecular Genetics, 2000, Vol. 9, No. 10

Association analyses

Figure 3. The expression of HCR mRNA in different tissues studied by RT


PCR. The upper panel shows the expression of HCR (amplicon size 929 bp)
and the lower panel the expression of the GAPDH control housekeeping gene
(amplicon size 226 bp). The tissues are (from the left) heart, placenta, lung,
liver, skeletal muscle, kidney, pancreas (MTC panel I, Clontech) and keratinocyte (separate experiment).

family member. Fifty-three of familial psoriasis patients had


developed psoriasis before the age of 40 (type I psoriasis).

All unrelated psoriasis patients (n = 100, one from each family)


and 93 population-based controls were included in an association study. HLA-Cw*0602 was genotyped using the PCRSSP
method, and CD*5 using PCR and restriction digestion. HCR
SNPs were screened using SSCP, direct sequencing or restriction digestion of PCR products.
Thirty-seven of the patients (37%) and eight of the controls
(9%) were HLA-Cw*0602-positive (P = 0.0000031). Two
HCR SNPs in exon 2 associated significantly with psoriasis
(Table 3). Both SNPs changed an amino acid from tryptophan
to arginine, and in all instances they occurred together; we call
this the ArgArg allele (HCR*ArgArg). Forty-two percent of
the patients (42/100) compared with 19% of the controls (18/93)
had HCR*ArgArg, showing significant excess of HCR*ArgArg
among patients (P = 0.00068, Table 4). Both HLA-Cw*0602
and HCR*ArgArg associations were even stronger among
type I psoriasis patients (52 versus 9%, P = 0.00000015 and 61
versus 19%, P = 0.0000099, respectively).

Table 2. Clinical characteristics of patients diagnosed with familial or sporadic psoriasis


Clinical characteristics

All (n = 142)

Familial (n = 71)

Sporadic (n = 71)

77 (54%)

40 (56%)

37 (52%)

Gender
Male
Age at the time of the study
median (years)

47

47

47

range (years)

583

683

580

median (years)

30

30

30

range (years)

380

380

375

Age at onset <40 years

94 (66%)

53 (75%)

42 (59%)

>40 years

36 (25%)

11 (16%)

25 (35%)a

unknown

12 (9%)

8 (11%)

4 (6%)

median

3.35

3.2

3.5

range

018.6

015.0

018.6

Age at the time of clinical diagnosis

PASI-score at the time of study

Type of skin lesionsb


plaque

121 (85%)

58 (82%)

63 (89%)

guttate

14 (10%)

8 (11%)

6 (9%)

inversa

9 (6%)

3 (4%)

6 (9%)

unknown

2 (1%)

1 (1%)

1 (1%)

Nail lesions

63 (44%)

27 (38%)

36 (51%)

systemic treatment (metotrexate/acitretin)

29 (20%)

9 (13%)

20 (28%)c

UV-therapy

79 (56%)

29 (41%)

50 (70%)d

hospitalization

46 (32%)

22 (31%)

24 (34%)

Anamnestic information ofb

One patient from each family (n = 100) was used for the association study.
aP < 0.02.
bAn individual can have more than one option.
cP = 0.02.
dP = 0.0004.

Human Molecular Genetics, 2000, Vol. 9, No. 10 1537

Table 3. Allele frequencies of the HCR gene in psoriasis patients compared with population-based controls
Patients (n = 100)

Controls (n = 93)

P2

Base

Polymorphism

+249

GA

9 (9%)

9 (10%)

+251

CT

42 (42%)

18 (19%)

0.00068

+269

CT

42 (42%)

18 (19%)

0.00068

+421

CT

18 (18%)

31 (33%)

0.014

+436

GC

81 (81%)

85 (91%)

+715

CG

43 (43%)

40 (43%)

+769

AC

57 (57%)

53 (57%)

+1193 a

TC

9 (18%)

11 (22%)

+1194 a

GA

1 (2%)

1 (2%)

+1219 a

CT

3 (6%)

5 (10%)

+1229 a

TC

40 (80%)

34 (68%)

+1667

TG

96 (96%)

90 (97%)

+1824

GA

13 (13%)

14 (15%)

+1855

GA

49 (49%)

44 (47%)

+1861

GT

4 (4%)

3 (3%)

+2122

AT

22 (22%)

29 (31%)

+2271

GC

88 (88%)

87 (94%)

aPatients

n = 50, controls n = 50.

All the HLA-Cw*0602-positive patients (37/37) were also


HCR*ArgArg positive. In the control group, HLA-Cw*0602
was also always found in association with HCR*ArgArg (8/8).
Among the HLA-Cw*0602-negative patients, 8% (5/63) had
HCR*ArgArg, which did not differ significantly from the
proportion of HLA-Cw*0602-negative HCR*ArgArg-positive
controls (10/85, 12%). These results suggested that HLACw*0602 and HCR*ArgArg are in strong linkage disequilibrium among both patients and controls.
We next considered two CD SNPs that together form the
allele CD*5 (+619 T, +1243 C) and that have been reported to
associate with psoriasis (1921). In our material, CD*5 was
the major allele in both the patient and control groups. Eightysix percent (86/100) of patients and 85% (79/93) of controls
had CD*5 and thus we could not detect any disease association
(Table 4). We did not observe any significant deviations from
the HardyWeinberg equilibrium for HCR or CD gene.
Haplotype analysis
We genotyped all family members in 30 psoriasis families to
study an eight-marker haplotype including HLA-Cw*0602,
five HCR SNPs representing different exons (+269, +715,
+1667, +2122, +2271) and two CD SNPs (+619, +1243). Two

additional CD SNPs at +1215 and +1236 were genotyped to


differentiate between the CD2 and CD3 haplotypes (21) in all
HLA-Cw*0602-positive chromosomes. Sixty-nine eightmarker haplotypes could be determined unambiguously and 15
of the chromosomes were HLA-Cw*0602-positive. Altogether
17 different haplotypes were detected.
Among the HLA-Cw*0602-positive chromosomes, a single
five-marker haplotype was seen for HCR (Table 5). All 13
HLA-Cw*0602-positive patient chromosomes had the same
HCR susceptibility haplotype, but for the CD gene, the haplotype was broken into three different haplotypes. None of the
HLA-Cw*0602-negative haplotypes associated with psoriasis
by chi-squared analysis.
Tissue in situ hybridization
We examined HCR expression in paired biopsies from
psoriasis lesions and normal-looking skin from six psoriasis
patients and from three unaffected individuals by in situ
hybridization (Fig. 4). HCR expression appeared strong in
keratinocytes of psoriatic lesions. The samples from healthyappearing skin of psoriasis patients and from unaffected individuals remained negative even after 40 days of exposure.

1538 Human Molecular Genetics, 2000, Vol. 9, No. 10

Figure 4. Expression of HCR mRNA in human skin. (A) Dark-field image of a biopsy from psoriatic skin hybridized with an antisense cRNA probe for HCR
displaying abundant signal in keratinocytes. The patient had been treated with only topical corticosteroids. (B) A parallel section from the same tissue block hybridized with a sense cRNA probe for HCR is devoid of signal. (C) A bright-field image of HCR mRNA-positive keratinocytes shown in (A). (D) Dark-field image of
a parallel in situ hybridization experiment, performed on a paired biopsy of normal looking skin from the same individual as in (A), is negative for HCR mRNA in
the epidermis. All samples were counterstained with hematoxylin and eosin.

These results suggested upregulation of HCR in psoriatic


keratinocytes.
DISCUSSION
PSORS1 at 6p21.3 has been the most consistently observed
susceptibility locus for psoriasis in both linkage and association studies. Our data suggest that HCR (Pg8) has many characteristics of a psoriasis susceptibility gene located in the HLA
region and, specifically, it emerges as a better candidate than
CD. This conclusion is based on several lines of evidence.
The region of interest for a psoriasis susceptibility gene has
been narrowed down to a few hundred kilobase interval around
the HLA-C locus (14,15). The role of HLA-C as a causative
gene in the pathogenesis of psoriasis is unclear. Of the nonHLA genes in the region, CD has been suggested to be involved
in skin desquamation, making it a tempting candidate for a
psoriasis susceptibility gene. CD was reported to carry an
HLA-C-independent effect and CD*5 was suggested as a risk
allele (1921). HCR localizes in the most promising interval
for a psoriasis susceptibility gene, between HLA-C and CD,
but we are not aware of any studies assessing its role in
psoriasis directly. The OTF3 and TCF19 genes, located very
close to HCR, are most likely to have housekeeping functions

and no polymorphism has been described in these genes so far


(24).
We verified the structure of HCR and found that its amino
acid sequence shows little homology with known proteins; the
strongest homologies are with various myosins. The corresponding porcine gene in the MHC region has been cloned
(GenBank accession no. AJ251914). The secondary structure
of the HCR protein is predicted to consist mainly of -helical
coils and it may be a nuclear protein with a leucine zipper
motif. The predictions do not allow specific suggestions
beyond speculations about the physiological role of HCR.
We found that HCR is highly polymorphic, including 11
predicted amino acid substitutions and one truncated variant.
Of these variants one, named HCR*ArgArg, is associated
with psoriasis [relative risk (RR) 2.1, 95% confidence interval
(CI) 1.72.6, P = 0.00068]. It was present in all psoriasis-associated HLA-Cw*0602 chromosomes, but its frequency was
higher than that of HLA-Cw*0602 among control chromosomes; thus HLA-Cw*0602 associated with psoriasis even
more strongly (RR 4.3, 95% CI 3.15.0, P = 0.0000031).
HCR*ArgArg was also detected in HLA-Cw*0602-negative
chromosomes, suggesting that it might be an older variant than
HLA-Cw*0602. The proposed different age of the two variants
might explain the difference in the relative risk and strength of

Human Molecular Genetics, 2000, Vol. 9, No. 10 1539

Table 4. Frequency of the HLA-Cw*0602, HCR*ArgArg and CD*5 alleles among all psoriasis and type
I psoriasis patients compared with controls
Locus

All psoriasis patients (n = 100)

Type I patients (n = 31)

Controls (n = 93)

HLA-Cw*0602

37 (37%)a

16 (52%) b

8 (9%)

HCR*ArgArg

42

(42%)c

(61%) d

CD*5

86 (86%)

19

18 (19%)

25 (81%)

79 (85%)

aP

= 0.0000031, RR 4.3 (95% CI, 3.15.0).


= 0.00000015.
cP = 0.00068, RR 2.2 (95% CI, 1.72.6).
dP = 0.0000099.
bP

Table 5. Extended haplotypes in HLA-Cw*0602-positive chromosomes


Haplotype
HLA-C

Number of chromosomes
HCR

CD

Psoriasis (n = 13) Control (n = 2)

+269

+715 +1667 +2122 +2271

+619 +1215 +1236 +1243

0602

0602

0602

0602

A single HCR haplotype was seen in all HLA-Cw*0602-positive chromosomes (dark gray). For CD, the haplotype was broken into
three different haplotypes in the risk chromosomes. For HCR, base T at +269 represents the ArgArg variant. For CD, bases T at
+619 and C at +1243 together represent CD*5 (light gray).

association. However, we cannot exclude the possibility that


HCR*ArgArg together with HLA-Cw*0602 forms the risk
allele. Further association studies of both genes in larger sets of
psoriasis patients and controls from different populations are
obviously needed to verify and refine these suggestions.
We found the previously suggested psoriasis susceptibility
allele CD*5 at a high frequency (8586%) among both
psoriasis patients and controls. This observation argues
strongly against CD*5 as a major psoriasis susceptibility
allele, because the prevalence of psoriasis is 12% in the population studied; a strong effect of such a common variant should
cause a much higher disease incidence. Furthermore, our
haplotype analysis showed that a single susceptibility haplotype was intact across the HLA-C and HCR genes, but split into
three haplotypes in CD. When intragenic SNP haplotypes for
both HCR and CD were determined among HLA-Cw*0602positive chromosomes, a single five-marker haplotype was
detected for HCR, whereas three different four-marker haplotypes were present for CD, including non-CD*5 haplotypes.
Thus, we conclude that HCR is a better candidate than CD for
a psoriasis susceptibility gene by genetic association analysis.
HCR was expressed at high levels in the keratinocytes in
psoriatic skin lesions whereas in paired samples from normal
appearing skin it was barely detectable. At least one gene,
amphiregulin, has been found to be overexpressed in psoriatic
skin lesions, and transgenic mice with overexpression of
amphiregulin in the skin develop lesions similar to psoriasis
(25). It will be informative to study similarities and differences
in the expression of both genes in the psoriatic process.

In conclusion, we have studied HCR, a new highly polymorphic gene near HLA-C, which by position, genetic association
results and expression pattern in normal and psoriatic skin
must be considered a candidate gene for psoriasis susceptibility. Its primary sequence suggests -helical rod structure but
gives little additional insight to its physiological role. Further
association and functional studies with HCR are highly
warranted to find out its role in the pathogenesis of psoriasis.
MATERIAL AND METHODS
Gene structure
Exonintron structure of the HCR (Pg8) gene was predicted
from a genomic clone (GenBank accession no. AC004195)
using two different gene prediction programs GENSCAN
(http://ccr-081.mit.edu/GENSCAN.html ) and FGENES (http://
dot.imgen.bcm.tmc.edu:9331/gene-finder/gf.html ). To amplify
the HCR cDNA, RNA was extracted (RNeasy Mini Kit,
Qiagen, Valencia, CA) from a primary keratinocyte cell line
(54A III/IV) and RTPCR [M-MLV Reverse Transcriptase,
Promega (Madison, WI) and Random Hexamer primers,
Research Genetics (Huntsville, AL)] was carried out using the
manufacturers protocol. The coding regions of the HCR gene
were identified from the cDNA by direct sequencing using the
primer pairs: CCA CCT GGC TCT CAG ACA TT and GTG
CAG CCT CTG AAC CTC TT (exons 1 and 2); AGC AGG
CTG AGG TGA TCGT and CCC TTC AGC TGC TTA ACA
GAG (exons 26); ATT CCC TGG AGC CTG AGT TT and
CCT CCT GCT GGA TGA GGC (exons 611); GGT CAC

1540 Human Molecular Genetics, 2000, Vol. 9, No. 10

AGA TGT GAG CCT TG and GCT GGA GCA TCT GTC
AAG GT (exons 1116). The primers were designed using the
Primer3 program (26). A rough starting point for transcription
was detected by PCR amplification using alternative forward
primers designed to stepwise upstream and to find the site
where no PCR product was obtained from cDNA. Genomic
DNA was used as a positive control and all amplified products
were sequenced. The end of transcription was detected by
comparing several ESTs with the sequence obtained from
keratinocyte cDNA and genomic DNA. The open reading
frame was detected with the program DNAWorks.
PCR assays were carried out in a 50 l volume containing
5 l cDNA, 1 PCR buffer (10 mM TrisHCl, 50 mM KCl,
0.1% Triton X-100), 440 M dNTPs, 2.2 mM MgCl2, 1 M
primer mix and 2 U of DNA polymerase (DyNAzyme EXT,
Finnzymes, Espoo, Finland). The samples were denatured for
3 min at 94C, followed by 32 cycles each of 60 s at 94C and
180 s at 68C. Purification of the PCR products was performed
using a gel extraction kit (Qiagen). Sequencing was performed
by dye-terminator chemistry in both directions using an ABI
373A sequencer.
Patient and control samples
We informed people in Kainuu about the study through the
local psoriasis patient organization, health care centers and the
dermatological clinic of Kainuu Central Hospital. The Kainuu
population represents a late settled region of Finland (27). At
the end of the 16th century, Kainuu was inhabited by only a
few hundred unrelated founders. The population remained
small until rapid expansion occurred at the end of the 19th
century, leading to the present population of ~100 000. Based
on our genealogical study, 65% of the patients grandparents
originated in Kainuu proper, and most of the remaining grandparents originated in the neighboring, often southern municipalities. Our sample set represents 510% of all psoriasis
patients in the region.
Based on self-reported psoriasis, probands were selected for
an interview and a clinical examination was performed by a
senior dermatologist (R. Itkonen-Vatjus). The diagnosis was
confirmed for 142 patients accepted into the study. To allow
haplotyping, 210 unaffected family members were also
recruited from 100 families. Both the probands and their
family members donated blood samples and filled out a health
questionnaire. All participants gave written, informed consent
for access to their medical records to verify disease history,
and the Ministry of Social and Health Affairs granted permission to access the patients medical records. As populationbased controls, we used DNA samples from healthy individuals from the same recruitment area. DNA was extracted from
venous blood using a standard non-enzymic method. The study
protocol was approved by the Ethical Review Board of the
Kainuu Central Hospital and the Department of Medical
Genetics, University of Helsinki.
Screening for polymorphisms
Five psoriasis patients and one control sample were used for
screening sequence variations with 12 different primer pairs
covering all exons: CCC TCC CAC TTT CAA GCTC and
TTC CAG TGA GGA AGG GTC AC (exon 1); CAC CTG
CAC TAA CCT GTC TTTG and TTT CTA CCC CTG CAT

TCA CC (exon 2); CTT CTT TCC GCA GCT GTC CT and
TCC CTA AGT CTG CAC ACA GAT (exons 3 and 4); TAC
AGA GGG GCT GCT TTC CT and GCT GAG GGT GAG
GGG TCT (exon 5); AAA GAT GCC ACC TCC TTC CT and
GAG GGA ATA CCG GGA GAA AA (exon 6); CTG CCC
AGC TCT CTC TCCT and CTC CAT CCC TGA TAC CTG
CT (exons 7 and 8); GGA TCA GTG ACT TGT GCC CT and
GTG GCT CGC AGT TGT CCT AC (exon 9); TTT CTC CCT
GCT TTT TCC CT and CTC ATC CTC TCC ACC CTC TG
(exons 10 and 11); TCC TTT TAG GGG AGG CAG AG and
AAG GCC CTA TCC ACC CTG (exons 12 and 13); TGC
CTT GGC CTC TCT GTA GT and TCT GCC CTC CTG TCT
CCT AC (exon 14); GCT CTA TCC GGG CTA GGT TT and
CCC TTG TCC CTT TGT GCTT (exon 15); TGG TGC TCA
TCT GCT GTC TT and CTT TCC CTC CAA CTG TCA GC
(exon 16).
PCR assays were carried out in 50 l vol containing 50 ng of
genomic DNA, 1 PCR buffer (10 mM TrisHCl, 50 mM KCl,
0.1% Triton X-100), 100 M dNTPs, 1 mM MgCl2, 0.12 M
primer mix, 1% DMSO and 2 U of DNA polymerase
(DyNAzyme II, Finnzymes). The samples were denatured for
5 min at 96C, followed by 3538 cycles each of 30 s at 96C,
120 s at 5764C and 120 s at 72C. Elongation was performed
for 5 min at 72C. Purified (PCR purification kit, Qiagen) PCR
products were sequenced using an ABI 373A sequencer and
dye-terminator chemistry.
Association analyses
DNA samples were genotyped for HLA-Cw*0602 using the
Class I SSP ARMSPCR method (28) and for the HCR
sequence variations using SSCP, sequencing and restriction
digestion. The primers and PCR protocols used were as
described above. The six fully sequenced samples were used as
references, but we also sequenced all variant band patterns
observed in SSCP to confirm the polymorphisms. SSCP gel
was prepared in a 50 ml volume containing 1012.5 ml of 2
MDE gel solution (FMC BioProducts, Rockland, ME), 6 ml of
5 TBE, 100 l of 10% APS and 40 l of TEMED, and the
samples were electrophoresed for 1720 h at 24 W. In exon 8,
the SNPs were identified using direct sequencing in 50 patients
and 50 controls. In addition, seven polymorphisms were verified using altered restriction site recognizing enzymes. Digestions were performed in 20 l reactions containing 10 l of
PCR product and 2.5 U of either PstI (+249), AvaII (+269),
Tsp509I (+421), BsmFI (+715), MslI (+1667), SfaNI (+2122)
or MwoI (+2271) and the appropriate buffer provided by the
manufacturer (New England Biolabs, Beverly, MA).
The SNPs of CD*5 were amplified with primer pairs S5/S6
(+619) and S15/S16 (+1243) and analyzed with restriction site
recognizing enzymes (MnlI for +619 and HphI for +1243) as
described previously (29). The SNPs at +1215 and +1236 were
detected by sequencing the primer pair S15/S16 product.
Statistical significance of the differences in the allele
frequencies between patients and controls was calculated using
the chi-square test. Relative risk was calculated using the
formula [a/(a + c)]/[b/(b + d)], where a is the number of
patients with the risk allele; c the number of patients without
the risk allele; b and d are the equivalent values in the controls,
respectively.

Human Molecular Genetics, 2000, Vol. 9, No. 10 1541

Haplotype classification
The haplotypes were constructed manually. For each family,
the chromosome was classified as trait-associated if it occurred
in any affected family member and as a control if it occurred
only in unaffected family members. Each chromosome was
counted only once per family.
Expression analyses
The tissue expression of the HCR gene was studied by PCR
amplification (Human Multiple Tissue cDNA panel I, Clontech, Palo Alto, CA) using the primer pair GGT CAC AGA
TGT GAG CCT TG and GCT GGA GCA TCT GTC AAG GT
(exons 1115). GAPDH was used as a housekeeping control
gene and was amplified using the primer pair GAA GGT GAA
GGT CGG AGT CA and CTT CTA CCA CTA CCC TAA AG
(PE Biosystems, Foster City, CA). The PCR amplifications for
both genes were performed in 50 l reactions using 35 cycles.
Skin specimens for in situ hybridization were obtained from
the Department of Dermatology, University of Helsinki. The
following subgroups of histological sections were examined:
three samples of normal skin from different parts of the body,
paired biopsies from psoriasis plaques and normal looking skin
from six psoriasis patients. For in situ hybridization, a probe
was generated by PCR amplification of the keratinocyte cell
line 54A III/IV by RTPCR with random hexamer primers as
described above as well as primers ATT TAG GTG ACA CTA
TAC att ccc tgg agc ctg agt tt and TAA TAC GAC TCA CTA
TAc ctc ctg ctg gat gag gc, introducing T7 and Sp6 RNA
polymerase promoter sequences at opposite ends of the 628 bp
gene-specific product. In vitro transcribed antisense and sense
RNA probes were labeled with [35S]UTP. In situ hybridization
was performed at 50C overnight on formalin-fixed paraffinembedded specimens using 4 104 c.p.m./ml of labeled probe
with subsequent washing under stringent conditions, including
treatment of RNase A (30,31). Following autoradiography for
2040 days, the photographic emulsion was developed and the
slides were stained with hematoxylin and eosin for microscopy.

5.

6.

7.

8.
9.

10.

11.

12.

13.

14.

15.

16.

ACKNOWLEDGEMENTS

17.

The authors wish to thank Ms Liisa Rajasalo and Ms Pivikki


Pajunen and their staff in the Kainuu Central Hospital for their
invaluable help in the recruitment of the patients. We also wish
to thank Mr Vesa Ollikainen for his valuable advice
concerning the statistical analysis. This study was supported
by the Academy of Finland, the Sigrid Juselius Foundation and
Helsinki University Research Funds.

18.

19.

20.

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