Abstract
Inflammatory diseases encompass a variety of medical conditions. In this chapter
autoimmune diseases and allergic disorders will be our focus. The autoimmune diseases
include organ-specific autoimmunities, such as type I diabetes mellitus and autoimmune
thyroiditis (AITD) and organ non-specific disorders such as systemic lupus
erythematosus (SLE). All of them seem to share aspects of aberrant immunologic
tolerance toward self-antigens. Asthma and atopic diathesis are among the allergies.
Crohn disease and SLE are relatively rare with their prevalence from 10 to 50 per 100,000,
and rheumatoid arthritis, psoriasis, AITD and asthma are commoner with their prevalence
being 500 per 100,000 or much higher. The difference among ethnic groups is not
prominent for rheumatoid arthritis, psoriasis, AITD or asthma, but Crohn disease and
SLE affect some ethnic populations more than others. Although all of these disorders
have some environmental components, asthma and atopy seem to be affected by
environmental factors most, as is suggested by the significant increase in their incidence
for the last several decades with changes in various environmental factors especially in
developed countries. For the last 10 years, multiple linkage studies revealed many
disease-linked loci throughout the genome with various consistencies. As some
pathophysiological studies of inflammatory immune system related disorders have
implicated, some linkage studies suggested that loci were involved in multiple disorders.
(Figure 1) It is of particular interest that some of the identified genes or loci were shared
by multiple diseases. (Table 1) In the following sections, reports on identification of
disease-associated genes or markers will be summarized for individual diseases (CTLA4,
CARD15, DLG5, SLC22A4/A5, PDCD1, RUNX1, SLC9A3R1/NAT9, PADI4,
ADAM33, DPP10, PHF11 and GPRA) initially, and then genets that have been reported
for multiple disorders will be discussed.
1. Introduction
There are many medical disorders or phenotypes that have a genetic contribution. Of note
infectious diseases that had been believed to represent environmental factor-oriented
disorders are now known to be influenced by genetic factors. [1] It would not be an
exaggeration to say that almost all medical conditions are affected by genetic factors.
Although any disorder with genetic components is a potential target of SNP-based
genetic evaluation, current technologies for SNPs seem to be oriented toward
investigations of so-called common disorders with multiple susceptibility genes.
Therefore, this chapter will focus on recent progress in gene-identification research to
identify disease-susceptible genes using multiple SNPs for autoimmune or allergic
disorders and characterize their approaches and findings so that insight into the future
direction of high-throughput genetics in the inflammatory diseases can be obtained. Since
the strategy to evaluate genetic contribution of HLA locus is different from other regions
due to the extreme variations in HLA genes, this topic will not be discussed much,
although the majority of inflammatory disorders are linked to the HLA locus somehow
and SNPs seem to be beneficial tools for understanding the complicated genetic factors in
the region. [2]
2. Identification of disease-associated genes or genetic markers.
Here reports on 12 genes will be introduced. A report on CTLA4 in the 1990s was based
on a candidate approach. The rest of the reports were based on the whole genome survey
at its beginning. The majority of them identified susceptible gene(s) with or without
functionally relevant variant(s) to a disease by positional cloning strategy. In those cases a
locus was identified by a whole genome linkage survey(s) using microsatellites, followed
by detailed evaluations of the locus with an increased number of genetic markers,
including tens to hundreds of SNPs. In this strategy, the locus of linkage was usually
narrowed by analyzing family-based samples that were originally used for the initial
linkage study, and then the associated variants were identified in the narrowed segment
which were validated by additional familial sample set(s) and/or sporadic case-control
samples. A minor number of reports were based on the strategy where SNP-based
case-control association tests were used from the initial step of the investigation.
2.1 Autoimmune thyroiditis and type I diabetes mellitus and other multiple autoimmune
disease-associated gene; CTLA4
Autoimmune thyroiditis is manifested with increased or decreased activity of thyroid
hormone production from the thyroid gland and is classified mainly into Grave’s disease
and Hashimoto thyroiditis, both of which are characterized by tissue-specific
autoimmune reactions with tissue specific autoantibody production. About five to ten %
of the population is affected worldwide. Familial aggregation is documented and the
contribution of HLA gene has been well studied. [3]
Cytotoxic T lymphocyte-associated 4 (CTLA4) gene was mapped to 2q33 clustering with
CD28 gene. Both CTLA4 and CD28 function as members of the same regulatory
pathway of T lymphocytes. CD28 signal activates T cells and CTLA4 acts as a negative
regulator of T cells. CTLA4 is also known to have splice variants; one is a
transmembranous form and the other produces soluble molecules [4]. The two peptide
isoforms function differently, and their expression is physiologically regulated and also
related to disease phenotypes. Even before its splice variants were reported, its function
made the CTLA4 gene a strong candidate for susceptibility to autoimmune disorders
including AITD, a microsatellite marker that was investigated and reported for
association with AITD [5]. Since the initial report on the association, many association
studies have been carried out and repeatedly demonstrated an association between AITD
and its subtypes (Grave’s disease and Hashimoto thyroiditis), such as three
polymorphisms in the gene; a SNP in the 5’ flank region（C-318T), another SNP in the
coding region (A49G(THR17ALA)) and the microsatellite (3’(AT)n) with original
association. The studies encompassed multiple ethnic groups, e.g. Caucasian, Tunisian,
Asian, African American, with a sample size of up to three hundred. [3] However, it is not
yet conclusive whether CTLA4 is the only susceptible gene in the locus or which
polymorphism(s) in the CTLA4 gene conveys its genetic predisposition because no
comprehensive analysis of linkage disequilibrium of the locus or haplotypic dissection of
the association was reported, and also because no associated polymorphism was shown to
be functional. Although the function of CTLA4 is relevant to susceptibility to AITD and a
fraction of the splice variants were different between AITD subjects and healthy
controls[6], a functional link still needs to be shown between the associated
polymorphisms and molecular function. AITD is believed to be a polygenic disease and
CTLA4 should be one of multiple susceptible genes. The genetic strength of the CTLA4
locus varies among the reports; the odds ratio of the locus ranges between 1.4 and 3 [7].
AITD often develops in conjunction with other autoimmune diseases, including type I
diabetes mellitus (IDDM), rheumatoid arthritis, and systemic lupus erythematosus.
Besides the association with AITD, multiple autoimmune diseases, including celiac
disease, rheumatoid arthritis, Addison’s disease, autoimmune hepatitis, SLR, and
myasthenia gravis have been reported to be associated with polymorphisms in the
CTLA4 gene [8] [9] [7,10]. Particularly, a linkage study on IDDM suggested 2q33 as one
of the susceptible loci (IDDM12) [11,12], and the following family-based association
studies [11] [13] supported the relation between the IDDM and CTLA4 gene. The
association between IDDM and CTLA4 was also evidenced in multiple ethnic groups,
although all the reports were not consistent with each other.
Since the identification of an association between CTLA4 and AITD was originally
reported in 1995 when the human genome project was in its infancy and data on SNPs or
linkage disequilibrium were very limited, the association is now under reassessment with
multiple SNPs and linkage disequilibrium to identify the origin of the association and
functional explanation of the association.
2.2 Inflammatory bowel disease-associated genes; CARD15, DLG5 and SLC22A4 or
SLC22A5
Inflammatory bowel disease (IBD) is characterized by chronic relapsing intestinal
inflammation with autoimmune involvement. The incidence is about 100 to 200 per
100,000 and there is an ethnic and geographic variation in the incidence. European
populations are more likely to be affected and the prevalence decreases for Jewish,
non-Jewish Caucasian, African-American, Hispanic, and Asian populations. It is divided
into Crohn disease and uncreative colitis. IBD is known to occur with other autoimmune
diseases, including psoriasis, multiple sclerosis and spondyloarthropathies. The genetic
component has been well characterized by multiple familial studies and multiple genes
are believed to be a pathogenesis of IBD.[14] Since the first systematic genome search for
IBD susceptibility loci reported in 1996, studies to identify inflammatory bowel disease
genetics has progressed to the identification of three susceptible genes. [15] [16] [17] [18]
All three genes were identified by a similar strategy. Initially linkage analyses identified
loci, then the loci were evaluated by the method of positional cloning, and finally
susceptible gene(s) and/or variant(s) were identified.
(a) Caspase recruitment domain-containing protein 15 (CARD15)
CARD15 was cloned as a homolog of apoptosis-related molecules of CARD4 (NOD1).
Expression and functional analysis suggested that CARD15 acts as an intracellular
receptor for bacterial products in monocytes and transmits signals via activation of NFkB.
[19] Besides the association with IBD, CARD15 was reported to have mutations
associated with Blau syndrome, a rare autosomal dominant disorder characterized by
arthritis, uveitis and a skin rash with camptodactyly. [20] [21]
IBD1 in 16q12, a linkage locus for Crohn disease, was initially reported in 1996 [22] and
supportive reports followed. [23] [24] The locus was searched for a candidate gene and
CARD15 was identified. Genetic variants were searched for in the gene and a frameshift
variant (1007fs or 3020insC) was identified, which was associated with Crohn disease by
case-control association test and TDT. [15] The study used 235 families. They also
identified mutually independent two missense SNPs in the CARD15 (R702W and
G908R) gene that were also associated with Crohn disease. All three Crohn-associated
variants causing alteration in peptide sequence were relatively rare with population
frequency less than 5%. At the same time, another research group reported that identical
variants were associated with Crohn disease by investigating the loci with TDT (416
families) and case-control linkage disequilibrium mapping using a combination of
microsatellite markers and eleven SNPs. [16] [25] After the two reports, additional
polymorphisms in the introns of the gene were also found to be associated with Crohn
disease [26], and the relative risk of these variants in multiple studies ranged from 1.5 to
17.6 as genotypic relative risks. [27] Since this locus was suggested for presence of a
psoriatic arthritis-susceptible gene by a linkage study[28], the initial three variants were
evaluated for association with the arthritis phenotype of psoriasis and they were
positively associated with the phenotype with a relative risk between 1.27 and 4.47. [29]
Since Crohn disease and psoriasis sometimes affect the same individuals [30] and
because Crohn disease and psoriasis as well as Blau syndrome share autoimmune
pathogenesis and clinical features, e.g., arthritis, [31] the findings on genetic variants in
the CARD15 gene seem to implicate CARD15 as a pleiotropic autoimmune and/or
inflammatory gene.
Although the accumulation of all these positive data was reliable, the positive results
were only obtained from studies using samples from Caucasian origins or a Jewish
population. In Asian populations, the three variants associated with Crohn disease in
Caucasians were very rare. None of them exist in Koreans[32], only the R702Q variant is
in Japanese [33] and only 1007fs was barely detected in Han (China) [34]. Although some
polymorphisms were common between Caucasians and Asians, they were not associated
with the disease in either population. Moreover, no additional disease-susceptible
variants were discovered in Japanese samples, suggesting that the contribution of
CARD15 variants was not major in Asians. [33] In the case of CARD15, evaluation of
genes with SNPs has been more extensively done than CTLA4. Since the two initial
reports of functional variants in CARD15, more variants have been identified as
associating with IBD, and those variants were independently associated with Crohn
disease and its associated phenotypes. It seems true at least in European descendants that
there are multiple functional variants in the CARD15 gene associated with Crohn disease,
that the origin of the susceptible variants was mutually independent and that the variants
also affect susceptibility to other autoimmune disorders. Asian analyses did not deny the
findings in Europeans but did show that every Crohn-susceptible variant was highly
specific to Caucasians and that no susceptible variant was present in Asians. It will be
necessary to investigate the mechanism of how the variants produce disease susceptibility
by biological analysis and origin of the variants by genetic dissection.
(b) DLG5 and SLC22A4/SLC22A5
Following the success of identification of CARD15 and subsequent validating studies,
two genes were reported to be associated with inflammatory bowel disease and Crohn
disease at the same time from two independent research groups. One was DLG5 in 10q23
and the other was a segment containing two OCTN cation transporter genes in 5q31. Both
groups identified the gene or the narrow segment by investigating a linkage-positive
locus with linkage disequilibrium mapping with multiple SNPs.
(b)-1 DLG5
The locus 10q23 was one of the linkage-suggested loci of a genome-wide linkage scan of
IBD. [35] The locus was narrowed by denser allocation of microsatellite markers to 5 cM
in two steps with the subsequent finding of a significantly associated microsatellite. By
evaluating the narrowed interval with a transmission disequilibrium test using 37 publicly
available SNPs, a significantly associated SNP was identified. [17] In the vicinity of the
associated SNP, two genes with possible pathophysiological relevance to inflammation in
bowels were found; KCNMA1, encoding a potassium-gated calcium channel and DLG5,
a member of the membrane-associated guanylate kinase gene family. The segment was
further evaluated for LD, and a haplotype block extending 85 kb was identified as the
origin of the association that contained only the DLG5 gene. All the coding exons and
exon-intron boundaries of the DLG5 gene were sequenced, and the SNPs of the DLG5
gene were characterized. There were 33 SNPs consisting of four common haplotypes.
The association was initially found in TDT (422 affected sib-pairs) and subsequently
validated by another case-control sample set. Two SNPs tagging common haplotypes
(R30Q and DLG5_e27) were associated with IBD (OR=1.62 and 1.35) (538 cases vs. 548
controls). The population frequency of the commoner SNPs was 0.09 and 0.04. Besides
the relatively common SNPs, five rarer SNPs with amino acid substitution (P1371Q,
S121G, E514Q, R957H and P979L) that did not represent any common haplotypes were
also identified in the affected individuals. Their frequency was less than 0.5 %. The
authors further evaluated for possible gene-gene interaction between CARD15 and
DLG5 and observed a disproportional distribution of genotypes in the Crohn disease
subgroup, although the association was both observed with Crohn disease and ulcerative
colitis with some variations depending on the tested sample sets. Although the finding of
association between IBD and genetic variants in DLG5 gene was observed in the multiple
sample sets, the samples sets were again limited to European descendants. Therefore,
further association tests in other ethnic groups are required. Because the physiological
function of DLG5 is still unclear except for its suggested role in cell-cell contact[36], its
function has to be identified along with a mechanism for alteration of activity by its
variants.
(b)-2 SLC22A4 or SLC22A5
SLC22A4 and SLC22A5 were reported to be associated with Crohn disease at the same
time as DLG5. The report was very interesting from the viewpoint that autoimmune
inflammatory disorders share common genetic background because the genes were
located in the middle of the cytokine cluster of 5q31 where whole genome linkage
searches had indicated the presence of susceptible genes for IBD and asthma/allergic
disorders [37-41]. Also SLC22A4 was reported to have a functional variant associated
with rheumatoid arthritis (RA) only several months before. [42] Although the report of
association with RA preceded the one with Crohn disease, the finding of Crohn disease is
discussed first and the report on RA will be dealt with later with other RA-related genes.
The chromosome 5q31 cytokine cluster includes multiple T helper 2-type cytokines (the
interleukin [IL] genes IL3, IL4, IL5, IL9, and IL13) as well as interferon regulatory
factor-1 (IRF1), colony-stimulating factor-2 (CSF2) and T-cell transcription factor-7
(TCF7) (Figure 2). Several studies have suggested a genetic linkage or association of
atopy, high serum immunoglobulin E levels, bronchial hyper-responsiveness, and asthma
in this region.[40] The whole genome linkage study identified IBD5 as a IBD-linked
locus [43],
and subsequent hierarchical strategy analyzing trios using denser
microsatellites narrowed the locus down to 1cM with 2 loci. There were 11 known genes
in the 680 kb sequence of one of the two loci. The region was initially evaluated with 16
newly identified SNPs in gene structure, and the flanking segments and association peak
were validated close to SLC22A4 and SLC22A5 in the center of the region. Then, the
region was very closely evaluated for SNPs; 651 SNPs were identified, and 301 of them
were genotyped to construct haplotype structures. The data and TDT-based simulation
evaluation of the data indicated a 250kb segment most likely containing susceptible
variant(s). Although the segment is close to multiple known immune system genes,
[38,44] genes encoding two organic cation transporters, SLC22A4 and SLC22A5, are
located in the center of the region. SLC22A5 transports carnitine and has been shown to
be responsible for primary carnitine deficiency disorders in humans and a mouse
model.[45] SLC22A4 is located next to the SLC22A5 gene and known to be in the same
group of organic cation transporters as SLC22A5, but no precise function was known.
[38]
The region containing five known genes was successively studied by resequencing, and
an additional 10 SNPs were identified. An association with Crohn disease was observed
in the case-control samples (370 vs. 246) that had showed a linkage in the locus as well as
replication samples in haplotypes consisting of two SNPs in SLC22A4 and SLC22A5
genes. (OR ～1.6) The one SNP substitutes 503rd L to F with non-conservative effects on
the tertiary structure of SLC22A4, and the other disrupts the heat shock element in 5’
UTR of SLC22A5. (Figure 3) Pharmacological assays revealed that the polymorphic
amino acid substitution of SLC22A4 affected the transporting function of the molecule
with several changes in Vmax and Km for some potential transport compounds. The
allelic difference of 5’ UTR SNP in SLC22A5 was observed in the binding of nuclear
factors and in vitro transcription assay. [18] It was not concluded which or both of the
genes or SNPs was responsible for the disease susceptibility.
2.3 Systemic lupus erythematosus; PDCD1 and RUNX1
Systemic lupus erythematosus (SLE) is a disease of unclear etiology, characterized by a
pathogenic autoantibody-related reaction to various organs and tissues. Its prevalence is
about 15 to 50 per 100,000 worldwide with some variation among ethnic groups. Women
in child-bearing age are predominantly affected and familial studies have suggested
genetic factor involvement. [46]
A whole genome linkage scan for SLE-associated genes identified multiple loci. [47,48]
Among them a locus named SLEB2 in 2q37 contained a strong candidate gene,
programmed cell death 1 (PDCD1). The PDCD1 is known to regulate peripheral
tolerance in T and B cells, and mice lacking the counterpart develop SLE-like
phenotypes. The PDCD1 gene was sequenced with family members in whom the
SLEB2 locus was detected and seven SNPs were identified. They constructed five
haplotypes, one of which was transmitted more frequently in affected individuals and
confirmed the positive result of the linkage study. Three representative SNPs
constructing the linkage-responsible haplotype were tested for association with 2510
samples from three European origin familial sample sets, one Mexican family, one
African-American family and sporadic sample sets. Except for the African-American
set, all sets were consistent with each other and a SNP in intron 4 was the most strongly
associated. (Figure 4) The odds ratios for positive samples ranged from 2.2 to 5.3. The
associated SNP is located in an enhancer-like structure in intron4 of PDCD1 and
disrupts the DNA-binding site for RUNX1, a transcription factor responsible for acute
myelogenous leukemia. [49] It still has to be revealed how the allelic difference affects
the function of PDCD1. After this report, the SLE-associated SNP was investigated for
association with RA and insulin-dependent diabetes mellitus as a target for a
polymorphism approach. Association was inconclusive for RA [48] and positive for
insulin-dependent diabetes mellitus. （OR 1.92）[50] Disruption of RUNX1-binding
sites by a SNP was reported to be related to two other autoimmune disorders, psoriasis
[51] and rheumatoid arthritis [42], by two independent research groups, less than a year
after the publication of the association between SLE and PDCD1. The findings on the
RUNX1-binding site between SLC9A3R1 and NAT9 being associated with psoriasis are
summarized first, and then the association between rheumatoid arthritis and SLC22A4
and its RUNX1 binding site, followed by a discussion of the RUNX1-related aspects of
three independent studies for SLE, psoriasis and rheumatoid arthritis.
2.4 Psoriasis; SLC9A3R1 or NAT9 with RUNX1
Psoriasis is one of the most common skin diseases, affecting up to 1 to 2% of the world
population. [51]Chronic inflammation causes erythematosus papules covered by scale.
About 20 to 30% of psoriasis patients have joint symptoms, a small part of which
coincides with rheumatoid arthritis.
The locus 17q was detected as a psoriasis susceptible locus by whole-genome linkage
surveys, and the finding was confirmed by multiple family sets. [52,53] [54] In the locus
an association with psoriasis was reported. [55] The same research group further
investigated the region of 8Mb with 123 genetic markers (28 microsatellites and 95
SNPs) in two steps. Initially 78 genetic markers, including 28 microsatellite markers and
50 SNPs were genotyped using the TDT method for the 242 nuclear families. The
evaluation narrowed the causative region down to 200kb. Another 47 SNPs including 16
SNPs newly discovered by sequencing the region were genotyped for lineage
disequilibrium mapping. There were two associated LD segments observed by TDT. The
first LD segment contains solute carrier family 9, isoform 3, regulatory factor 1 and an
N-acetyl transferase, named NAT9. The second segment lies 6 Mb away from the first
and contains a RAPTOR gene.
The polymorphisms and genes in the first LD segment were investigated and reported as a
psoriasis susceptible variant. [51]
The LD region extends 20 kb that contains 15 SNPs. The authors divided the region into
multiple short segments constructed by consecutive 5 SNPs and haplotypes were phased
for each segment and tested for transmission disequilibrium (242 families). Five SNPs in
the region were indicated as the origin of association, and they were further genotyped
using sporadic cases and controls. All five SNPs were located in non-coding regions
(intron or 3’UTR or 3’flanking region) (Figure 4). The result of the case-control
association test of the haplotypes constructed by the five SNPs was consistent with TDT.
The frequency of susceptible haplotypes in the general population was about 38% and the
odds ratio was 1.45. Among the five SNPs, a SNP between the 3’ ends of SLC9A3R1 and
NAT9 was located in a RUNX1-binding sequence. Further biological assays including
electrophoretic mobility shift assays and reporter assays indicated that RUNX1 binds to
the sequence with allelic difference in affinity and expression in vitro.
2.5 Rheumatoid Arthritis; PADI4, SLC22A4 and RUNX1
Rheumatoid arthritis (RA) is a chronic inflammatory disorder, which characteristically
has joint involvement. The prevalence is about 0.8% with some variation among ethnic
groups. Its genetic contribution was well documented by multiple family studies, and
multiple whole-genome sib-pair linkage studies have been reported with limited
consistency among them.
Two reports on RA-susceptible genes were issued from a group based on a
high-throughput SNP genotyping facility that adopts case-control LD mapping on a large
scale as an initial survey method without using subjects that were used for preceding
linkage studies. [56] [57,58] One of them identified functionally relevant polymorphisms
of peptidylarginine deiminase 4, an enzyme that catalyzes the post-translational
citrullination of proteins, as a rheumatoid arthritis gene. (55,[59] The other reported that
SLC22A4 was in conjunction with RUNX1 as RA-associated genes [42], both of which
were reported by other research groups to be associated with different autoimmune
diseases. [18,60]
(a) PADI4
As a part of a large-scale hypothesis-free survey of genomes, strongly associated SNPs
were identified by a case-control association (830 vs. 736) study in the Japanese
population. The region in 1q36 turned out to be 3.1Mb and 9.8Mb centromeric from
linkage-indicated markers reported by Cornelis et al. [61] using European sibpairs and by
Shiozawa et al. [62] using Japanese sibpairs, respectively. The region of 450Kb was
evaluated for LD with 119 SNPs, and three LD blocks were identified. The region with
three LD blocks is a cluster of 5 genes encoding peptidylarginine deiminase (PADI). It
was of particular interest that the function of PADI to modify proteins post-translationally
was known to be closely linked to the most RA-specific autoantibodies. [63] Although all
of the 5 PADIs are potentially relevant to RA-pathogenesis, the strongest association was
limited to SNPs in PADI type4 in the central LD block. Seventeen SNPs in the PADI4
gene constructed two common haplotypes (>25%) with two other rare haplotypes (5%).
The highest odds ratio was 2.0.
The association’s peak was observed in SNPs in absolute LD including four coding SNPs
with three amino acid substitutions. The authors demonstrated that the associated coding
SNPs affected stability of transcripts in vitro, suggesting an allelic difference in the
function of the enzyme in vivo. Clinical data of anti-citrullinated antibody, one of RAspecific autoantibodies whose antigen target seems to produce PADI enzymes, were also
correlated with genotypes of the associated SNPs, consistent with data of an in vitro
stability assay of transcripts. Although a genetic association test was performed only for
one ethnic group, the finding was well supported by the fact that the hypothesis-free
survey identified a functionally relevant gene. In addition, the associated variants were
functional, and they were linked to clinical phenotype. Moreover, the linked clinical
phenotype was closely associated with the gene's function. Further validation of the
association by different ethnic samples and other types of genetic studies, such as TDT, is
a pressing requirement.
(b) SLC22A4 and RUNX1
The research group from Japan, the same as PADI4, reported that two genes associated
with rheumatoid arthritis. One gene, SLC22A4, was identified by the same strategy as
PADI4 and the other gene, RUNX1, was identified as a transcriptional regulator of
SLC22A4 binding on a DNA sequence containing an associated SNP of SLC22A4, which
was the same as the case of PDCD1 in SLE and SLC9A3R1/NAT9 in psoriasis. [42]
Although SLC22A4 and RUNX1 were not so specifically relevant to rheumatoid arthritis
as PADI4, the findings turned out to be very encouraging because SLC22A4 was reported
to be associated with Crohn disease [18] and RUNX1 was with SLE and psoriasis as
described above. [49,51]
An associated SNP was identified by the hypothesis-free LD mapping approach in 5q31,
which has not been indicated for rheumatoid arthritis susceptible genes by previous
linkage studies but for allergic disorders and IBD. The chromosome 2.4Mb long 5q31
cytokine cluster region (Figure 2) was evaluated; 115 SNPs and six LD blocks were
identified. An association was found in a segment of 240kb (819 vs. 656) containing RIL,
SLC22A4, SLC22A5 and IRF1, which was similar to the segment identified by the
hierarchical approach to narrow down the IBD5 loci for Crohn disease, even though the
former was from Asian data and the latter was from Caucasians. [38] Because within the
associated LD block, more-associated SNPs clustered in SLC22A4 and SLC22A5, those
two genes were further investigated for causative variants, and an organic cation
transporter encoded by SLC22A4 was found to be expressed in RA-relevant tissues and
cells and a SNP in intron 1 of SLC22A4 was observed to affect transcription of SLC22A4
by the altering binding affinity of RUNX1 by in vitro assays as seen in the case of
psoriasis. [51] (Figure 3) The odds ratio was 1.98 for the most associated SNP, and
haplotype phasing revealed three common haplotypes in SLC22A4 and three rarer ones.
Since the difference in regulation of expression of SLC22A4 by RUNX1 was associated
with rheumatoid arthritis, it is a straightforward hypothesis that polymorphism(s) in
RUNX1 were also associated with rheumatoid arthritis. The authors identified a
preliminary association with RUNX1 that has to be confirmed, but the finding is
consistent with the role of RUNX proteins in autoimmune susceptibility.
2.6 Asthma and atopy; ADAM33, DPP10, PHF11 and GPRA
Asthma is a chronic inflammatory disorder of airways with increased responsiveness to
various stimuli. It is common and about 5% of the population is affected. Atopy is one of
the biggest risk factor of asthmas and is characterized by type I allergic reactions against
various antigens. Both genetic predisposition and environmental exposure to allergens
play key roles in the development of these diseases. [64]
To identify asthma-associated genes, whole-genome multiple linkage studies have been
carried out with some consistency in their results. The suggested loci were investigated
by further linkage analysis with denser markers, different sample sets and different ethnic
groups. SNPs were used to narrow down the loci, sometimes in combination with
microsatellite markers for linkage analysis and sometimes solely for linkage
disequilibrium mapping. [64] Since the report of the identification of ADAM33 [65],
three more genes were reported. [66] [67,68] The strategy was similar for all four, so I
outline the four on asthma using the SNPs.
(a) ADAM33
A whole genome linkage survey using 460 affected sib-pairs from two Caucasian
sampling groups identified a locus linked with asthma, bronchial hyperresponsiveness
and elevated serum IgE. Forty genes were identified in the linked 2.5 Mb region (20q13).
Using case subjects from the sib-pair survey, case-control association tests were carried
out for all 135 SNPs, which showed the strongest association in ADAM33, a
metalloprotease with diverse functions (130 vs. 217). The associated LD block extended
185 kb including three genes. Between the two sampling groups there was a minor
difference in LD. By testing the association for two sampling groups and for a combined
sample set, the SNPs in ADAM33 showed positive for association. The case-control
association for five SNPs in ADAM33 was also validated by TDT.
The odds ration in the most significantly associated SNP was 1.95. [69] Confirmation of
genetic studies from other ethnic groups are awaited for validation. [64]
(b) DPP10, PHF11 and GPRA
A locus in 2q14 where DPP10 was identified had been suggested by a linkage study.
Therefore, multiple studies have investigated this by target approach, but they have
reached no conclusions. [70] Allen et al. reported DPP10 as an asthma-atopy-associated
gene by positional cloning. [66] They analyzed the locus with a dense microsatellite
marker map by TDT using three Caucasian 244 familial sample sets that indicated an
association in the locus. Subsequently, they targeted 200kb around the microsatellite
marker with the most significant association as a tentative LD extension. The 105SNPs
were discovered in the segment and 4 LD blocks were identified. Association was
detected in both asthma and elevated IgE, but the finding was not simple. The LD blocks
were next to each other and distinct. The association of allergic phenotypes was observed
in an independent sample set. However, their haplotype structure was different from the
original samples. Overall, it is likely that susceptibility-determinant(s) are located in this
segment, but the relation between susceptible variant(s) and allergy-related phenotypes
seems to be complicated. It is yet to be investigated how the variants produce the
susceptibility, in vitro assays of DPP10, a solitary gene in this associated region,
suggested polymorphism affects transcription of DPP10 encoding a homolog of
dipeptidyl peptidase.
PHF11 was reported by a positional cloning of a linkage locus of atopy and elevated IgE
(13q14).[64] Association with IgE was evaluated by QTL method (80 families) in a
targeted region of 700 kb where 53 common polymorphisms (49 SNPs and 4 ins/del)
were identified. The association was identified in an 8.1kb segment with 3 SNPs. They
were located in intron or 3’UTR of PHF11, a putative transcription regulator. This
association was ascertained by additional Caucasian multiple sample sets (237 families),
and asthma and atopy were also associated in independent case-controlled association
tests (total 271 vs. 28) . The odds ratio of SNPs in the region to asthma and related clinical
phenotypes ranged from 2.2 to 4.
GPRA is a G protein-coupled receptor of unknown function. It was identified in a
linkage-implicated locus (7q14) [71] by hierarchical approach of haplotype pattern
mining. [67] A twenty cM region was narrowed down to 3.5cM by the TDT of 86 genome
scan families and additional trios (874 individuals) using 76 microsatellites. Then ten
more microsatellites were incorporated into the analysis, the targeted segment of 301 kb
was identified and evaluated with 5 microsatellites and 13 SNPs, and subsequently a 47kb
segment was identified as associated. The LD was evaluated for 133kb around the
associated 47kb with 51 SNPs and investigated with two additional ethnic groups. The
range of the associated block was similar to the initial result. Haplotypes were estimated
for each group and phylogenetic analysis of them revealed only minor differences among
them. Within the 133kb segment, GPRA was identified. Isoform distribution of GPRA
was observed between bronchial biopsies from asthmatics and healthy controls. For the
further validation of GPRA as an asthma susceptible gene, a connection between genetic
variants in GPRA and the difference of isoform distribution need to be documented.
RR1.4
3. Overlaps of loci or genes by multiple disorders
As already mentioned above, some of the identified disease-associated genes were
reported to be linked to multiple disorders. CTLA4, CARD15, RUNX1 and
SLC22A4/A5 are among them. (Figure 5) The first two genes, CTLA4 and CARD15,
were initially associated with one disease and then additional associations with another
disorder followed by targeting the gene as an association candidate. On the other hand, in
case of RUNX1 and SLC22A4/A5, three (RUNX1) or two (SLC22A4/A5) distinct
autoimmune disorders were reported to be associated with the gene by mutually
independent approaches. Here the three reports on RUNX1 and the two reports on
SLC22A4/A5 are summarized to focus on overlaps by multiple diseases.
3.1 RUNX1
RUNX1 is expressed in all hematopoietic lineages and is known to regulate the
expression of various genes specific for hematopoiesis and myeloid differentiation, such
as macrophage colony-stimulating factor receptor, interleukin 3, meyloperoxydate and
TCR beta gene. [72] RUNX1 is one of the genes whose abnormality is most frequently
found in leukemia. The main abnormality is translocation. [73] [74] As previously
mentioned, SLE, psoriasis and rheumatoid arthritis were reported to have an associated
SNP that disrupts the RUNX1 binding sequence in PDCD1, the intergenic region between
SLC9A3R1, NAT9, and SLC22A4, respectively. Table 2 summarizes the pertinent
findings on RUNX1. For all the diseases, EMSA assay was performed for allelic
difference of RUNX1 binding, and a reporter assay was done for psoriasis and
rheumatoid arthritis. A SNP in RUNX1 was evaluated for association with RA but not for
the other two. The base substitution of SLE and psoriasis was in the middle of the
sequence and disease-susceptibility decreases affinity of RUNX1 binding. In the case of
rheumatoid arthritis, the SNP was at the end of the binding sequence, and a susceptible
allele increased binding affinity. This difference might be explained by the fact that
RUNX1 regulates gene expression bidirectionally. In other words, in case of SLE and
psoriasis, disease-susceptible alleles decrease RUNX1 binding with subsequent
down-regulation of expression of disease-susceptible genes (PDCD1 or
SLC9A3R1/NAT9). In case of rheumatoid arthritis, disease-susceptible alleles bind
strongly with RUNX1, but this time RUNX1 works as a suppressor of expression,
therefore, expression of SLC22A4 is decreased. Detailed mechanisms of gene-gene
interaction and the role of polymorphisms needs to be confirmed by further
investigations.
3.2 SLC22A4/A5
SLC22A4 and SLC22A5 encode organic cation transporters. SLC22A5 is known to
transport carnitin, and its mutation causes a familial carnitine metabolic disorder. [75]
SLC22A4 was known to share some transporting activity with SLC22A5, but its function
is unclear. Until association with rheumatoid arthritis and Crohn disease was reported, no
inflammatory or immunologic function was expected for either of them. The LD segment
reported to be associated with rheumatoid arthritis and Crohn disease was similar even
though the former was a report on Asian samples and the latter on Caucasians. However,
the associated SNPs were distinct between the two studies. (Figure 3) The intronic SNPs
reported in rheumatoid arthritis (Asians) were not evaluated by Crohn’s group
(Caucasians). The coding SNP reported by Crohn disease was evaluated and found to be
very rare, if found at all, in the rheumatoid arthritis group (unpublished data). Although
the encounter of two autoimmune inflammatory disorders on a gene with unknown
function is very attractive, the difference of associated SNPs should be carefully
evaluated based on the difference between diseases and also between haplotype
compositions of the studied ethnic groups.
4. Perspectives
Following multiple whole genome sib-pair linkage surveys, multiple reports on
identification of common inflammatory disease-susceptible genes are being issued using
SNPs as primary markers. Although each report described above was considerably
convincing, all of the genes still have to be validated further regarding some points.
Validation is divided into two types. One is validation of association itself by additional
association tests; the other is validation of association by biological assays.
When associations are to be confirmed by additional association tests, several points
should be considered. As described above, some studies lacked confirmatory association
tests, and some reports performed repeating association tests, but the result was difficult
to interpret due to the difference in haplotype composition. When multiple ethnic groups
were tested, the results from all the ethnic groups were not always uniform. Therefore,
although repeating association tests with different sample set(s) is mandatory, it would be
better to confirm with samples from the same or close ethnic groups and from remotely
different groups. This is the case for diseases without major difference in prevalence
among ethnic groups, such as rheumatoid arthritis, as well as for diseases with differences,
like Crohn disease. With or without variations among ethnic groups, the association test’s
method could be altered. A case-control association test with large sample size and TDT
with familial samples with limited sample size would be mutually complementary.
Another lesson from the reports is that responsible variants could be heterogeneous, and
that makes identification of associated genetic markers difficult because there are
multiple susceptibility-responsible variants and each variant is rare throughout the
population, as observed in Crohn disease.
When the function of the identified gene is the issue of validation, there are two points to
be considered. The first point is that the function of the molecule encoded by identified
susceptible genes should be relevant to the disease. Although this did not matter when
functionally relevant genes were targeted, the whole genome survey looks at associated
genes without known functions or genes and unclear existence. The physiological
functions of these novel genes should be well investigated. The second point is that
susceptibility responsible variants should be functional to affect activity of encoded
molecules in a context consistent with the pathology of the disease.
In order to understand complex genetic mechanisms of inflammatory and autoimmune
diseases, the interaction among multiple genes needs to be revealed. Although gene-gene
interaction was believed to be present, its analysis was not realistic, because no good
target genes were available. As introduced in this chapter, multiple genes seem to be
candidates for gene-gene interaction analyses. Actually, the interaction between PADI4
and HLA was discussed by Suzuki et al. [76], SLC22A4 and RUNX1 by Tokuhiro et al.,
[42] rheumatoid arthritis, DLG5 and CARD15 by Stoll et al. [17] and SLC22A4/A5 and
CADR15 by Peltekova et al. [18] for Crohn disease. Although none of these reports
concluded gene-gene interaction, this was mainly due to limitation in sample size. Future
investigation will untangle the web of multiple genes.
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