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whom correspondence should be addressed. Tel: +358 9 191 26538; Fax: +358 9 191 26789; Email: juha.kere@helsinki.fi
Figure 1. A map of the HLA region between HLA-B and CD. Filled bars indicate genes and open bars indicate pseudogenes.
Table 1. The SNPs of the HCR gene coding sequence with the corresponding amino acid changes
Exon
Polymorphism
Exon 2
+249
+10696
G 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
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
TC and GA
TrpGln (W391Q)a
+1219
+16972
C T
no change
+1229
+16982
T C
no change
Exon 12
+1667
+20442
T G
Exon 13
+1824
+20695
G 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
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.
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.
Association analyses
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
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
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%)
29 (20%)
9 (13%)
20 (28%)c
UV-therapy
79 (56%)
29 (41%)
50 (70%)d
hospitalization
46 (32%)
22 (31%)
24 (34%)
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.
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
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.
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
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
Number of chromosomes
HCR
CD
+269
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).
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
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.
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.
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ACKNOWLEDGEMENTS
17.
18.
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20.
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