Candidate Genes in Cutaneous Lupus Erythematosus

Selection of candidate genes for analysis in complex multifactorial traits such as CLE draws on multiple sources of information. In the case of LE, these include family linkage studies in human and animal models of lupus, immunohistochemical analysis of skin samples, and studies of alleles identified as possible candidates in other (related)

autoimmune diseases. For example, at least 10 loci have been found to predispose to LE in New Zealand lupus-prone mice (Kono et al. 1994), in addition to the previously mentioned loci that are linked to human lupus. In the murine model, genetic susceptibility seems to confer different levels of pathogenesis, including separate loci for autoantibody production, specific organ destruction, and mortality. Only one locus, the MHC (H-2 in mice), is linked to all three levels of disease.

Immunohistochemical analysis may be used to localize the expression of specific proteins within the skin of patients with lupus, perhaps implying abnormal regulation of the underlying genes that encode these proteins. For example, increased kera-tinocyte expression of intercellular adhesion molecule-1 (ICAM-1) has been demonstrated in evolving ultraviolet (UV)-induced lesions of CLE and PLE, but not in healthy controls (Nyberg et al. 1999). Thus, ICAM-1, its ligand, and upstream regulators may be deemed possible candidates to study (Norris et al. 1992).

Association in other autoimmune disorders is reported for various loci encoding cytokines, cytokine receptors, antioxidant enzymes, and adhesion molecules in rheumatoid arthritis, dermatomyositis, and SLE. It is therefore possible that the polymorphisms at these loci may be candidates for the related autoimmune CLE.

Finally, a candidate gene may be inferred from knowing the therapeutic action of drugs used to treat the disease. For example, thalidomide, which inhibits tumor necrosis factor (TNF)-a, is particularly effective for treating SCLE and DLE (Wallace 1997). This suggests that TNF (or possibly one of its common polymorphisms) may contribute to the pathogenesis of CLE.

The Major Histocompatibility Complex

From such studies, a variety of candidate gene loci have emerged that may prove important in the pathogenesis of CLE. The most important of these loci to date are found within the MHC. The human MHC (Fig. 15.1) is a diverse genetic region that plays a crucial role in control of the immune response and that has recently been sequenced by the MHC Sequencing Consortium (1999). The human MHC genes, clustered together in a segment of chromosome 6 (6p21.3), are organized into three regions (I, II, and III) and encode three major classes of proteins: class I human leukocyte antigens (HLAs) (including the classical antigens A, B, and Cw), HLA class II antigens (DP, DQ, and DR), and class III molecules, including complement components, TNF (a and P), and heat shock proteins.

HLA Genes and Cutaneous Lupus Erythematosus

In several serologic studies of the class I and class II HLA regions in patients with SCLE (mainly Caucasian), the HLA A1, B8, DR3, DQ2, DRw52, C4null ancestral hap-lotype has been consistently identified as a susceptibility haplotype for SCLE, particularly in patients seropositive for anti-Ro/SSA (Bielsa et al. 1991,Herrero et al. 1988, Johansson-Stephansson et al. 1989, Provost et al. 1988, Sontheimer et al. 1981,1982, Vazquez-Doval et al. 1992, Watson et al. 1991), confirmed by our recent analysis of HLA genes in 36 patients with SCLE (Millard et al. 2001b). This ancestral haplotype is associated with susceptibility to a variety of other autoimmune diseases, including insulin-dependent diabetes mellitus, dermatitis herpetiformis, and myasthenia gravis (Price et al. 1999).

Fig. 15.1. Abbreviated gene map of the human MHC at 6p21.3 illustrating the major loci to which the text refers. The illustrated class II loci are, themselves, composed of multiple genes that contribute to the class II antigen structure. HSP, heat shock protein; TNF, tumor necrosis factor

Class I

Class I

Centromeric

I HLA-DRB

C4A&C4B

Telomeric

Watson et al. (Provost and Watson 1993, Watson et al. 1991) found that this association was only present in SCLE, SLE, and Sjögren's syndrome in the presence of anti-Ro/SSA, suggesting that the immunogenetic association is primarily with the production of antibody. There is substantial evidence to support the pathogenic role of the anti-Ro/SSA antibody in SCLE (Ben-Chetrit 1993), where circulating anti-Ro/SSA binds keratinocytes expressing surface Ro/SSA antigen, leading to the destruction of basal keratinocytes (Norris 1993) (Fig. 15.2). In addition to controlling the presence or absence of the anti-Ro/SSA response, the MHC may also determine the level of the response; Harley et al. (Harley et al. 1986) found that possession of both HLA DQ1 and DQ2 led to the highest titers of anti-Ro/SSA. This MHC specificity suggests that class II HLA antigens may determine whether the individual can present fragments of Ro/SSA antigen to their lymphocytes.

The Ro/SSA 60-kDa protein (Ro60 or SSA2) is the major component of the Ro ribonucleoprotein (RNP) complex [in stable association with a human cytoplasmic RNA (hY RNA) and the La/SSB protein, Fig. 15.3], to which an immune response is a specific feature of several autoimmune diseases, including SCLE and SLE. We recently characterized the Ro60 gene structure (Fig. 15.4) and examined whether any observed sequence alterations were associated with serum anti-Ro/SSA antibody in SCLE and could therefore be of pathogenic significance (Millard et al. 2002). Het-eroduplex analysis of polymerase chain reaction (PCR) products from patients and controls spanning all Ro60 exons (1 to 8) revealed a common bandshift in the PCR products spanning exon 7. Sequencing of the corresponding PCR products demon-

UV Light

Activated Cytotoxic Lymphocytes

E-Selectin & ICAM-1

Fig. 15.2. Probable pathogenic mechanism of photosensitive skin lesions in LE.Ultraviolet radiation (UVR) stimulates the cell surface expression of nuclear antigens, including Ro/SSA, on the surface of keratinocytes and induces E-selectin and intercellular adhesion molecule-1 (ICAM-1) expression on dermal endothelial cells, which leads to the margination and local migration of lymphocytes. Norris (Norris 1993) proposed this model of photosensitive LE, whereby circulating anti-Ro/SSA antibody binds keratinocytes that express this surface Ro/SSA antigen, leading to antibody-dependent cellular cytotoxicity by the infiltrating cytotoxic lymphocytes and the subsequent destruction of basal keratinocytes. Casciola-Rosen and Rosen (Casciola-Rosen and Rosen 1997) have added to this model by describing the expression of Ro/SSA antigen in "surface blebs" of UVB-irradiated apoptotic keratinocytes, which may contribute to induction of autoimmunity to Ro/SSA. IL-1, interleukin-1; TNF, tumor necrosis factor

Activated Cytotoxic Lymphocytes

E-Selectin & ICAM-1

Fig. 15.2. Probable pathogenic mechanism of photosensitive skin lesions in LE.Ultraviolet radiation (UVR) stimulates the cell surface expression of nuclear antigens, including Ro/SSA, on the surface of keratinocytes and induces E-selectin and intercellular adhesion molecule-1 (ICAM-1) expression on dermal endothelial cells, which leads to the margination and local migration of lymphocytes. Norris (Norris 1993) proposed this model of photosensitive LE, whereby circulating anti-Ro/SSA antibody binds keratinocytes that express this surface Ro/SSA antigen, leading to antibody-dependent cellular cytotoxicity by the infiltrating cytotoxic lymphocytes and the subsequent destruction of basal keratinocytes. Casciola-Rosen and Rosen (Casciola-Rosen and Rosen 1997) have added to this model by describing the expression of Ro/SSA antigen in "surface blebs" of UVB-irradiated apoptotic keratinocytes, which may contribute to induction of autoimmunity to Ro/SSA. IL-1, interleukin-1; TNF, tumor necrosis factor

Fig. 15.3. Organization of the Ro/SSA RNP complex (Gordon et al. 1994). hY RNA, human cytoplasmic RNA

Ro60

hY RNA

Ro60

strafed an A>G substitution at nucleotide position 1318-7, within the consensus acceptor splice site of exon 7 (GenBank XM001901). The allele frequencies were major allele A (0.71) and minor allele G (0.29) in 72 control chromosomes, with no significant differences among patients with SCLE (anti-Ro/SSA positive), patients with DLE (anti-Ro/SSA negative), and healthy controls, suggesting no relationship between this nucleotide substitution and generation of anti-Ro/SSA.

Fig. 15.4. Structural organization of the Ro60 gene. The coding region of the gene contains 8 exons spanning more than 16 kB of genomic DNA and ranging in size from 114 to greater than 580 bp. Exons are depicted by the vertical black shaded areas, and the start of the 5' and 3' untranslated regions are in white. The exons are numbered above; introns are represented by horizontal lines

A relatively small number of studies have examined HLA associations in Caucasian patients with DLE.The major associations include the A1,B8,DR3 and also the B7, DR2 haplotypes (Fowler et al. 1985, Knop et al. 1990, Millard et al. 1977), although other studies have found no HLA associations with DLE (Bielsa et al. 1991, Tongio et al. 1982). Our own data indicate a significant association with the previously mentioned haplotypes and DLE (Millard et al. 2001b). Finally, the photosensitive "butterfly rash" of lupus (acute CLE) rarely occurs outside the context of active SLE, so little effort has been made to determine any specific HLA associations (Sontheimer and Provost 1997).

Complement Genes

The MHC also includes genes for various complement components (C2, C4A, C4B, and factor B) in the class III region (Meo et al. 1977), between class I and class II, which have been implicated in the pathogenesis of CLE. Inherited deficiencies of components C2 and C4 are associated with DLE (Braathen et al. 1986, Provost et al. 1983), SCLE (Callen et al. 1987, Levy et al. 1979, Provost et al. 1983), and the presence of anti-Ro/SSA (Meyer et al. 1985, Provost et al. 1983). In addition, lupus profundus has been reported in patients with partial deficiency of both C4 allotypes (Burrows et al. 1991, Nousari et al. 1999). The proposed basis for these associations includes either a failure to clear immune complexes and apoptotic cells or linkage disequilibrium with the real disease-predisposing locus (Levy et al. 1979, Sullivan 1998).

TNF Genes

TNF is encoded by a class III gene of the MHC (Carroll et al. 1987, MHC Sequencing Consortium 1999). TNF has been implicated in the pathogenesis of CLE following UVR (Kock 1990,Norris 1993) (Fig. 15.2) since it stimulates expression of the Ro antigen on the keratinocyte surface. The rare -308A polymorphic form of TNF (TNF2)

(Abraham et al. 1999) is associated with increased UVB-induced TNF production in keratinocytes (Silverberg et al. 1999) and has demonstrated a strong association with SCLE (Werth and Sullivan 1999). TNF -308A does, however, lie on the A1, B8, DR3 extended haplotype in Caucasians (Wilson et al. 1993) and may therefore demonstrate association only because of linkage disequilibrium. However, a recent analysis in patients with SLE suggests that both TNF -308A and HLA-DR3 contribute independently to lupus susceptibility (Rood et al. 2000).

Heat Shock Protein Genes

The three heat shock protein 70 (Hsp70) genes are located within the class III MHC region, and linkage disequilibrium with other HLA genes has led several authors to investigate the role of Hsp70 genes in susceptibility to autoimmune and allergic disease (Aron et al. 1999). The activation of Hsp gene expression (the "stress response") is a cellular mechanism that protects cells against stresses such as heat, UVR, cytokines, and chemicals (Muramatsu et al. 1992, Stephanou et al. 1997). Ghoreishi et al. (Ghoreishi et al. 1993) used immunohistologic analysis to demonstrate diffusely increased Hsp70 expression in the lesional skin of patients with SLE vs controls and DLE samples. Furukawa et al. (Furukawa et al. 1993) observed increased binding of anti-Ro/SSA antibodies to keratinocytes after incubation with a prostaglandin stressor that is known to induce Hsp formation, suggesting that heat shock proteins may be involved in the expression of Ro antigen at the cell surface. Various polymorphisms exist for the Hsp70 genes (Bolla et al. 1998, Esaki et al. 1999), although none have yet been examined for association in CLE.

Candidate Loci Outside the MHC

Several genetic regions outside the MHC seem to confer susceptibility to cutaneous forms of LE or demonstrate association or linkage with the anti-Ro/SSA response, including loci encoding cytokines, cytokine receptors, molecules involved in antigen recognition, and antioxidant enzymes, all of which are plausible candidates and are summarized in Table 15.1.

IL-1 Gene Cluster

The primary cytokine IL-1 is a major proinflammatory cytokine, encoded by a gene cluster at band 2q13, comprising IL-1a and IL-10 (synthesized by keratinocytes and Langerhans' cells, respectively) and the IL-1 receptor antagonist gene (IL-1RN), which has been associated with photosensitivity in LE and with DLE by two separate teams in case-control studies (Blakemore et al. 1994, Suzuki et al. 1997). In our association analysis, we found that IL1B +3954 T was significantly less frequent in patients with SCLE (28%) compared with controls (47%; P=0.039), although this was lost on correction for multiple testing (Millard et al. 2001c).

IL-10

Linkage in human lupus has been shown for band 1q31 (Moser et al. 1998), within which lies the gene encoding IL-10 (Eskdale et al. 1997, Kim et al. 1992). IL-10 is expressed by UVB-stimulated keratinocytes and is chemotactic for CD8+ Tcells. Itpro-motes an inflammatory response through its effects on B cells in lupus,whose produc-

Table 15.1. Genes outside the MHCa that seem to confer susceptibility to cutaneous forms of lupus erythematosus (LE) or demonstrate association or linkage with the anti-Ro/SSA response

Gene

Locus

Evidence

1. Cytokine genes

Interleukin-1 gene

2q13

Association of an IL-1 RA

cluster (IL-1A, B, and RA)

allele with DLE and with

photosensitivity in SLE.

Interleukin-10 (IL-10)

1q31

Linkage of 1q31 to human LE.

Association of three IL-10

SNPs with anti-Ro produc-

tion in SLE.

2. Adhesion molecule/

receptor genes

Intercellular adhesion

19p13.3-p13.2

Increased keratinocyte

molecule-1 (ICAM1)

expression of ICAM-1 in

CLE.

E-selectin (SELE)

1q23-25

Up-regulated endothelial

expression in the skin of

patients with cutaneous LE vs

controls.

Fc gamma receptor II

1q23

Linkage of 1q23 to human

(FCGR2A)

systemic LE. Necessary for

ADCC (Fig. 15.4).

T-cell receptor (TCR)

7q35

Association of two RFLPs, for

C|31 and C|32

TCRs Cb1 and Cb2, with anti-

Ro/SSA production in SLE.

3. Antioxidant enzyme

genes

Glutathione S-trans-

1p13

Association of GSTM1 null

ferase M1 (GSTM1)

status and anti-Ro/SSA pro-

duction in SLE.

4. Apoptosis genes

Fas (TNFRSF6)

10q24.1

Association of an SNP with

photosensitivity in SLE

a Genes within the MHC are illustrated in Fig. 15.1.

ADCC, antibody-dependent cellular cytotoxicity; CLE, cutaneous lupus erythematosus; DLE, discoid LE; MHC, major histocompatibility complex; RA, receptor antagonist; RFLP, restriction fragment length polymorphism; SLE, systemic LE; SNP, single nucleotide polymorphism.

tion of immunoglobulin is largely IL-10 dependent (Llorente et al. 1995). It also up-reg-ulates the expression of ICAM-1 and E-selectin on human dermal endothelial cells (Palmetshofer et al. 1994). Three functional single nucleotide polymorphisms (SNPs) of the IL-10 gene promoter were reported by Turner et al. (Turner et al. 1997), including -1082 G/A, -819 C/T, and -592 C/A, who showed that they influenced in vitro production of IL-10 by mononuclear cells. Comparing the overall haplotype combinations at these three loci, significant distortion was demonstrated between anti-Ro/SSA-pos-itive and anti-Ro/SSA-negative SLE cases (P=0.005), with overrepresentation of the GCC and ACC haplotypes in the former group (Lazarus et al. 1997). This suggests that

IL-10 may be important in generation of the immune response to Ro and, therefore, potentially relevant to SCLE, although our initial study found no association with SCLE or DLE for either the -819 or-1082 SNPs (Millard et al. 2001c).

T-Cell Receptor Cp Gene

Genes encoding the T-cell receptor (TCR) have demonstrated an association with the generation of anti-Ro/SSA. Frank et al. (Frank et al. 1990) reported an association between a set of restriction fragment length polymorphisms (RFLPs) for TCRs Cßl and Cß2 (Bgl II 9.8-kb and Kpn I 1.75-kb) that was present in 76% of patients with SLE and anti-Ro/SSA but in only 41% of patients with SLE without anti-Ro/SSA (P=0.002). The same group found that certain HLA DQ types, in combination with the TCR Cß RFLPs, increased the strength of this association (Scofield et al. 1994). This molecular specificity was cited as evidence that the breaking of tolerance to the Ro proteins in lupus requires the association of a class II HLA molecule with a fragment of Ro peptide and a particular TCR form.

Glutathione S-Transferase

The antioxidant enzymes encoded by the glutathione S-transferase (GST) genes are widely expressed in mammalian tissues. Ollier et al. (Ollier et al. 1996) examined the role of the GSTM1 null polymorphisms in the production of anti-Ro/SSA and anti-La/SSB in SLE using PCR to identify GSTM1 [chromosome band 1p13 (Pearson et al. 1993)] null homozygotes. A significant association was demonstrated between the Ro+ve/La-ve phenotype in SLE and GSTM1 null status, implicating oxidant stress in the loss of immunologic tolerance to Ro/SSA antigen.

Adhesion Molecules

ICAM-1 and E-selectin are potential candidate genes for CLE. ICAM-1 is usually expressed at very low levels on keratinocytes, but increased keratinocyte expression has been demonstrated in evolving lesions of CLE and PLE (Nyberg et al. 1999, Nor-ris et al. 1992). Two functional polymorphisms of the ICAM-1 gene (ICAM1) were reported by Vora et al. (Vora et al. 1994), including G/R at codon 241 and K/E at codon 469, which therefore represent plausible candidates for CLE. The endothelial adhesion molecule E-selectin is also up-regulated in the skin of patients with CLE and patients with PLE compared with controls (Nyberg et al. 1999); elevated serum levels of soluble E-selectin have also been described in patients with DLE (Nyberg and Stephansson 1999). The E-selectin gene at band 1q23-25 (Collins et al. 1991) contains an A/C SNP at position 561, coding for serine or arginine at codon 128 (Wenzel et al. 1994a), which, so far, has demonstrated clinical association with atheromatous vascular disease (Wenzel et al. 1994b).

Fcy Receptor II

The Fcy receptor II genes, at chromosome band 1q23 (Qiu et al. 1990), lie within an area that has demonstrated strong linkage to SLE in humans, with an lod score of 3.37 (Moser et al. 1998). The Fc receptor is required by cytotoxic cells to initiate antibody-dependent cellular cytotoxicity of keratinocytes, which is up-regulated in CLE (Furukawa et al. 1999), and possibly directed against the Ro/SSA antigen (Norris 1993). The FcyRIIa gene (FCGR2A) possesses an SNP (G/A) coding for arginine or histidine at codon 131 (R/H131) in the EC2 domain of FcyRIIa, which alters the ability of the receptor to bind immunoglobulin G (Clark et al. 1989, Warmerdam et al. 1990).

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