Eckhard Westphal

Scanning Chromosome 17 for Psoriasis Susceptibility: Lack of Evidence for a Distal 17q Locus

Evidence for a genetically heterogeneous psoriasis susceptibility locus on distal human chromosome 17q has recently been reported [Science 1994;264:1141]. Making use of an independently ascertained collection of 24 multiplex psoriasis kindreds, we have performed a genotyping scan of chromosome 17 using 12 microsatellite markers and analyzed the data using parametric (lod score) as well as novel nonparametric methods. Pairwise lod scores revealed no evidence for linkage to the previously implicated marker D17S784 under any of eight models varying in mode of inheritance, penetrance, and sporadic cases. Homogeneous linkage to D17S784 could be excluded under all four autosomal dominant models tested (Z < - 5.8 at theta = 0.05), and there was no evidence for genetic heterogeneity. All other chromosome 17 markers tested also failed to detect evidence for linkage in any of the kindreds under either a dominant or a recessive model. Although further analysis using affected sib pair methods provided no statistically significant evidence for linkage to any chromosome 17 marker, a cluster of three distal 17q loci displayed a trend towards greater than expected allele-sharing values (observed/expected = 1.10-1.14). These results do not formally confirm the existence of a psoriasis susceptibility locus on the distal long arm of human chromosome 17, but are suggestive of its possible involvement under a polygenic model, warranting its further investigation in familial psoriasis.

CHARACTERIZATION OF SOLUBLE HLA MOLECULES IN SWEAT AND QUANTITATIVE HLA DIFFERENCES IN SERUM OF HEALTHY INDIVIDUALS

Soluble class I molecules were immunoprecipitated from human sweat and serum using the BB7.7 monoclonal antibody (mAb) coupled to immunomagnetic beads. Molecules were analysed biochemically on SDS‐PAGE gels and finally by 1D‐isoelectric‐focusing (IEF). Serum‐ and sweat‐HLA IEF‐band patterns of the same individual were fully identical, showing that HLA excreted in sweat possess polymorphic structures like those in serum. Quantitatively, we used a highly sensitive competitive enzyme‐linked immunosorbent (ELISA) assay to determine soluble class I concentrations. The first group was that of non‐HLA‐A9 and ‐Bw62 sera, which were found to contain HLA levels with a mean concentration of 0.82 ± 0.63 μg/ml (n= 44). However, sera that were HLA‐A23 or −24 (splits of HLA‐A9) contained higher levels, with a mean of 3.2 ± 0.94 μg/ml (n= 20). Similarly, HLA‐Bw62 individuals had a higher mean of 2.05 ± 0.65 μg/ml (n= 10). The difference of the HLA‐A9 group to the first group was statistically highly significant, P < 0.0001, and that of the HLA‐Bw62 to the first was also significant, P < 0.004. Individuals who were both HLA‐A9 and ‐Bw62 (n = 5) did not express significantly higher levels than those who only had one of these specificities. Sweat HLA levels had a mean of 0.42 ± 0.4 μg/ml (n= 10). These results show for the first time that soluble class I peptides are excreted in relatively high concentrations in sweat and possess polymorphic structures identical to those of serum HLA and that serum HLA levels are allotype dependent.

MHC-Related Odors in Humans

Up to now, MHC-related odors have been only described for rodents Yamazaki et al., 1991; Roser et al., 1991). Nevertheless, it has been speculated that a similar phenomenon may also occur in humans (Beauchamp et al., 1985; Boyse et al., 1987). Individual specific body odors do indeed play a role in human self-perception (Porter and Moore, 1981; Lord and Kasprzak, 1989) and recognition of offspring (Porter et al., 1983; Kaitz et al., 1987), but there is no information available on the biological basis of these odors.

Expression of MHC class I and II molecules by cadaver retinal pigment epithelium cells: optimization of post‐mortem HLA typing

The objective of this study was to investigate the expression of MHC antigens by retinal pigment epithelium cells (RPE) after stimulation with interferon‐gamma (IFN‐γ) and to improve the currently practised technique of cadaver HLA typing. A concentration of 100 U/ml IFN‐γ induced expression of class I molecules up to > 90% 3 days after stimulation, whereas 50 U/ml were required for the expression of HLA‐DR to > 90%. A concentration of 750 U/ml induced 35–45% expression of HLA‐DP and <25% HLA‐DQ after 3 days. Cells were serologically typed using the standard lymphocytotoxicity assay 3 days after stimulation with 250 U/ml IFN‐γ. Typing of class I specificities was complemented by one‐dimensional isoelectric focusing (1D‐IEF). We observed high concordance between the results of the RPE typing and the lymphocytotoxicity test on the same donors. Our results show complete typing of class I and II antigens post‐mortem, which, in particular, enables graft matching and improvement of graft survival in recipients of organs removed many hours after death such as the cornea.

Peripheral multifocal chorioretinitis with panuveitis: clinical and immunogenetic characterization in older patients.

Abstract · Background: The etiology of peripheral multifocal chorioretinitis with panuveitis (MCP) is unclear. Characteristic signs of MCP are punched-out, white chorioretinal lesions of the lower fundus periphery, chronic smoldering chorioretinal inflammation, vitritis, and mild inflammation of the anterior chamber. In this retrospective study we investigated clinical and immunogenetic abnormalities in MCP in older patients. · Patients and methods: 20 patients (18 women, 2 men), median age 70.5 years, were investigated clinically by ophthalmologists and were typed for HLA class I antigens using the standard microlymphocytotoxicity test. Typing for HLA-DR antigens was performed by polymerase chain reaction with sequence-specific primers (PCR-SSP). The HLA controls consisted of healthy people (108 for HLA class I, 114 for HLA class II). · Results: MCP was bilateral in 18 patients. Disease-related symptoms were present for 8 months (median) before diagnosis. The main presenting symptoms or findings were glaucoma (in 11 patients), visual loss (7), iritis (5), and vitritis (2). Anterior segment changes were frequently seen: keratitic precipitates (32 eyes), anterior chamber cells (25 eyes), aqueous flare (26 eyes), posterior synechiae (22 eyes), secondary glaucoma (15 eyes), and iris neovascularization (8 eyes). All patients had vitritis and typical chorioretinal fundus lesions. Fourteen patients developed cystoid macular edema (bilateral in seven cases). Subretinal neovascularization occurred in three patients. Although systemic medication was given to 17 patients and surgical treatment was performed in 25 eyes, improvement in vision was found in only 6 eyes, but 18 eyes deteriorated markedly (median 5 lines) during follow-up (median 24.5 months). Immunogenetically significant reduced frequencies of HLA-B7 and HLA-DR1 were found; also HLA-DR15(2) was reduced. However, several alleles were increased in MCP, although not significantly: HLA-A31; HLA-B57, HLA-B62; HLA-Cw3, HLA-Cw6; HLA-DR4, HLA-DR7, and HLA-DR8. · Conclusions: MCP is clinically and immunogenetically open to speculation. The present diagnosis and treatment of MCP are insufficient. Further DNA typing methods should clarify, whether HLA-DQ antigens are associated with the disease.

Post-Mortem HLA Tissue Typing of Retinal Pigment Epithelial Cells

Retinal pigment epithelial cells (RPE) were derived from bulbi of cornea donors and maintained in culture. The timespan between donors death and cell cultivation ranged from 2 to 122 h. The mean numbers of hours was 25.6 (n = 130). After IFN‐y stimulation, cells were serologically typed for class I and class II antigens. Unclear class I allospecificities were verified by one‐dimensional isoelectric focusing. Data from the serological typing of lymphocytes and those of the serological and biochemical typing of RPE from the same donors were compared in 22 cases. There was a discrepancy of less than 5%, whereby either the typing on lymphocytes could not identify some specificities declared as blanks or the RPE typing had failed to clearly define a specificity. Our data show that the strategy adopted here is very successful for tissue typing post mortem, thus increasing the number of available HLA‐matched corneas and consequently reducing the number of corneal graft rejections.

DNA-based HLA class II postmortem typing: evaluation of different techniques for prospective corneal allografting.

Abstract• Background: The objective of this study was to establish DNA-based HLA-DR postmortem tissue typing techniques in order to improve the quality and quanity of fully HLA-typed corneas for prospective allografting. •Methods: Four hundred and thirty-seven cornea donors were investigated. DNA was derived from cultivated retinal pigment epithelial cells by spin column purification or a salting out technique, and from scleral tissue by a very simple boiling method. Donors were typed by hybridization of polymerase chain reaction (PCR) products with sequence-specific oligodesoxynucleotide probes (PCR-SSOP) or by PCR with sequence-specific primers (PCR-SSP). Twenty-two of the donors were pretyped by serology. • Results: We observed high concordance (96%) between the results of DNA-based postmortem typing and the serological lymphocytotoxicity test. Furthermore, the distribution of the HLA-DR specificities that were detected correlated well with the distribution in a control population. • Conclusions: The results of this study demonstrate that both PCR-SSP and PCR-SSOP allow prospective allocation of HLA class II-matched corneas with high accuracy.

Immunoelectron microscopic demonstration of antigenic sites on lymphoid cells using a human monoclonal antibody (Ha6D3)

The reactivity of a human monoclonal antibody directed against human B and T lymphocytes was tested for the first time at the ultrastructural level. The antigenic sites detected by this antibody were localized on the surface membrane of lymphocytes and, to a lesser extent, in the cytoplasm on membranes of the endoplasmic reticulum and perinuclear envelop of some centroblasts and immunoblasts. Ultrastructural demonstration of target antigen detected by human monoclonal antibodies may be important prior to therapeutic application of these antibodies.

Reply to Leder and Hodge

To the Editor:On the basis of human leukocyte antigen (HLA) association studies, workers in the field of psoriasis have long been aware that the HLA complex plays an important role in determining psoriasis susceptibility. The question has always been why many families appear not to show linkage to HLA. We share in the pleasure of Drs. Leder and Hodge (1999xPsoriasis linkage in the HLA region. Leder, RO and Hodge, SE. Am J Hum Genet. 1999; 64: 895Abstract | Full Text | Full Text PDF | PubMed | Scopus (7)See all References1999 [in this issue]) now that the genetics of the HLA region in psoriasis is coming into sharper focus.The general agreement between Leder and Hodges studies (Leder et al. 1998xFamilial psoriasis and HLA-B: unambiguous support for linkage in 97 published families. Leder, RO, Mansbridge, JN, Hallmayer, J, and Hodge, SE. Hum Hered. 1998; 48: 198–211Crossref | PubMed | Scopus (30)See all References1998), our own work (Jenisch et al. 1998xLinkage analysis of HLA markers in familial psoriasis: strong disequilibrium effects provide evidence for a major determinant in the HLA-B/-C region. Jenisch, S, Henseler, T, Nair, RP, Guo, S-W, Westphal, E, Stuart, P, Kronke, M et al. Am J Hum Genet. 1998; 63: 191–199Abstract | Full Text | Full Text PDF | PubMed | Scopus (113)See all References1998 and in pressxSee all Referencesin press), and the recent studies of Trembath et al. (1997xIdentification of a major susceptibility locus on chromosome 6p and evidence for further disease loci revealed by a two stage genome-wide search in psoriasis. Trembath, RC, Clough, RL, Rosbotham, JL, Jones, AB, Camp, RDR, Frodsham, A, Browne, J et al. Hum Mol Genet. 1997; 6: 813–820Crossref | PubMedSee all References1997) and Burden et al. (1998xGenetics of psoriasis: paternal inheritance and a locus on chromosome 6p. Burden, AD, Javed, S, Bailey, M, Hodgins, M, Connor, M, and Tillman, D. J Invest Dermatol. 1998; 110: 958–960Crossref | PubMed | Scopus (108)See all References1998) provides welcome insight into this long-standing puzzle. By optimizing LOD scores over a variety of penetrance functions, assuming Hardy-Weinberg equilibrium, Leder and Hodge (Leder et al. 1998xFamilial psoriasis and HLA-B: unambiguous support for linkage in 97 published families. Leder, RO, Mansbridge, JN, Hallmayer, J, and Hodge, SE. Hum Hered. 1998; 48: 198–211Crossref | PubMed | Scopus (30)See all References1998) found the highest LOD scores for dominant models specifying high disease allele frequency and low penetrance. We reached essentially the same conclusion, following the suggestions of Risch et al. (1989xLinkage and mode of inheritance in complex traits. Risch, N, Claus, E, and Giuffra, L. Prog Clin Biol Res. 1989; 329: 183–188PubMedSee all References1989) for complex-trait data. It is well appreciated that power to detect linkage is diminished when the disease allele frequency is high and the penetrance is low. Given the smaller sample sizes of earlier studies, it is not surprising that linkage to the HLA region was not always apparent.We have reported that linkage to HLA is more readily detected when marker-trait disequilibrium is taken into account, in part because of more-accurate specification of phase (Jenisch et al. 1998xLinkage analysis of HLA markers in familial psoriasis: strong disequilibrium effects provide evidence for a major determinant in the HLA-B/-C region. Jenisch, S, Henseler, T, Nair, RP, Guo, S-W, Westphal, E, Stuart, P, Kronke, M et al. Am J Hum Genet. 1998; 63: 191–199Abstract | Full Text | Full Text PDF | PubMed | Scopus (113)See all References1998). Leder et al. (1998xFamilial psoriasis and HLA-B: unambiguous support for linkage in 97 published families. Leder, RO, Mansbridge, JN, Hallmayer, J, and Hodge, SE. Hum Hered. 1998; 48: 198–211Crossref | PubMed | Scopus (30)See all References1998) and Trembath et al. (1997xIdentification of a major susceptibility locus on chromosome 6p and evidence for further disease loci revealed by a two stage genome-wide search in psoriasis. Trembath, RC, Clough, RL, Rosbotham, JL, Jones, AB, Camp, RDR, Frodsham, A, Browne, J et al. Hum Mol Genet. 1997; 6: 813–820Crossref | PubMedSee all References1997) report similar results. This effect was first pointed out 15 years ago (Clerget-Darpoux 1982xBias of the estimated recombination fraction and LOD score due to an association between a disease gene and a marker gene. Clerget-Darpoux, F. Ann Hum Genet. 1982; 46: 363–372Crossref | PubMedSee all References1982) but has not been widely exploited in the genetic analysis of other common HLA-associated disorders. Even without incorporation of disease-marker haplotype frequencies, Leder et al. (1998xFamilial psoriasis and HLA-B: unambiguous support for linkage in 97 published families. Leder, RO, Mansbridge, JN, Hallmayer, J, and Hodge, SE. Hum Hered. 1998; 48: 198–211Crossref | PubMed | Scopus (30)See all References1998) found strong evidence for linkage to HLA under a dominant model, whereas we did not. Leder et al.s study made use of previously published pedigrees, and concerns regarding ascertainment bias in favor of linkage are inevitable in such a study. However, it is also possible that our sample yielded lower LOD scores because it contained a number of small pedigrees, thereby increasing the number of phase-unknown individuals.We concur with Leder and Hodge (1999xPsoriasis linkage in the HLA region. Leder, RO and Hodge, SE. Am J Hum Genet. 1999; 64: 895Abstract | Full Text | Full Text PDF | PubMed | Scopus (7)See all References1999) that there is now excellent agreement regarding the importance of the HLA region in familial psoriasis and that this locus should now be referred to as PSORS1. We would emphasize that, because the HLA loci yielding the highest LOD scores in familial psoriasis are so similar to those observed in prior case-control association studies, there is unlikely to be any difference between familial and “sporadic” juvenile-onset psoriasis with respect to the involvement of PSORS1. We can also infer that genetic differences between juvenile- and adult-onset psoriasis must exist, because of their different HLA associations (Henseler and Christophers 1985xPsoriasis of early and late onset: characterization of two types of psoriasis vulgaris. Henseler, T and Christophers, E. J Am Acad Dermatol. 1985; 13: 450–456Abstract | Full Text PDF | PubMedSee all References1985). Whether an HLA locus different from PSORS1 is involved in the adult-onset form of this disease remains to be determined.High disease allele frequencies and low penetrance values are likely to be the rule rather than the exception in common multifactorial diseases. We hope that these recent insights into the genetics of the HLA region in psoriasis will be of benefit to other groups studying complex genetic disorders.