Analysis of RUNX1 binding site and RAPTOR polymorphisms in psoriasis: no evidence for association despite adequate power and evidence for linkage.

BACKGROUND: A previous study identified two peaks of allelic association between psoriasis and single nucleotide polymorphisms (SNPs) mapping to distal chromosome 17q, including a disease associated SNP that leads to loss of a RUNX1 transcription factor binding site, and additional SNPs in the third intron of the RAPTOR gene. Another study found an association with SNPs in the RAPTOR gene, but not with the RUNX1 binding site polymorphism. METHODS: In an effort to confirm these observations, we genotyped 579 pedigrees containing 1285 affected individuals for three SNPs immediately flanking and including the RUNX1 binding site, and for three SNPs in the RAPTOR gene. RESULTS: Here we report further evidence for linkage to distal chromosome 17q, with a linkage peak mapping 1.7 cM distal to the RUNX1 binding site (logarithm of the odds 2.26 to 2.73, depending upon statistic used). However, we found no evidence for association to individual SNPs or haplotypes in either of the previously identified peaks of association. Power analysis demonstrated 80% power to detect significant association at genotype relative risks of 1.2 (additive and multiplicative models) to 1.5 (dominant and recessive models) for the RUNX1 binding site, and 1.3 to 1.4 for the RAPTOR locus under all models except dominant. CONCLUSIONS: Our data provide no support for the previously identified RUNX1 binding site or for the RAPTOR locus as genetic determinants of psoriasis, despite evidence for linkage of psoriasis to distal chromosome 17q.

Semiquantitative reverse transcription polymerase chain reaction analysis for detection of bcr/abl rearrangement using RNA extracts from bone marrow aspirates compared with glass slide smears after 0, 2 and 4 d of storage.

The polymerase chain reaction (PCR) is an established tool for the detection of specific chromosomal aberrations in different haematological malignancies. Owing to fast degradation of RNA, the immediate processing of samples is thought to have a major influence on the reliability of results. Any delay caused by transport may be an obstacle to reverse transcription PCR (RT-PCR)-based methods in multicentre studies. However, as air-dried bone marrow smears are usually available, we have improved a method to use smears as a source for routine RT-PCR investigations. We studied whether this source of RNA could overcome problems caused by delayed transport of samples. The aim of the present study was (i) to investigate the influence of a storage period of up to 4 d before processing of a specimen by nested bcr/abl RT-PCR, and (ii) to compare bone marrow aspirates with bone marrow smears stored at room temperature in parallel. Bone marrow aspirates and smears were taken from 11 patients with Ph-positive chronic myeloid leukaemia (CML). PCR results were semiquantified using a limiting dilution assay. We observed a loss of sensitivity < 1 log in stored bone marrow aspirates, even after 96 h. Results obtained from air-dried unstained glass slide smears were similar to investigations performed on approximately 1 x 10(5) cells of a bone marrow aspirate. We conclude that a storage period of up to 96 h has little influence on the detection of a bcr/abl fusion transcript in CML at diagnosis. Glass slide smears were equivalent to bone marrow aspirates in 8 out of 11 cases as a source for RT-PCR analysis when nested PCR was performed.

Integration of amplified BCR/ABL fusion genes into the short arm of chromosome 17 as a novel mechanism of disease progression in chronic myeloid leukemia.

We describe the cases of two patients with Philadelphia chromosome-positive chronic myeloid leukemia (CML), in whom the extramedullary blastic phase developed during disease progression. The similar clinical presentations of these patients was accompanied by gain of identical secondary chromosome abnormalities, that is, monosomies 9, 14, and 22, and by a clustered amplification of the BCR/ABL fusion gene. The additional copies of the BCR/ABL fusion gene were integrated into the short arm of structurally abnormal chromosomes 17 in both patients. The conformity of these genetic features in two patients with a rare disease manifestation leads us to the assumption that either the clustered amplification of the BCR/ABL fusion gene or the integration of this cluster into the short arm of chromosome 17 or both are associated with extramedullar disease progression in CML. Furthermore, the insertion of amplified BCR/ABL fusion genes into structurally abnormal chromosomes provides a novel mechanism of disease progression in BCR/ABL-positive CML.

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

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 HLD-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.

Cross-matches on donor cadaver retinal pigment epithelial cells in corneal risk patients.

BACKGROUND: Allografts can be rejected either through the antibody-mediated or cellular pathways. The objective of this study was to look at the extent of antibody formation in patients awaiting re-keratoplasty using cross-matches on cadaver retinal pigment epithelial (RPE) cells. METHODS: Cadaver RPE cells were derived by trypsin digestion from donor eyes (n = 1200). After 3 days of cell cultivation, the cells were adherent and began to lose their pigment. By day 7 most cells were clear and grew as a polygonal monolayer. MHC class I expression by RPE cells was studied by the W6/32 (anti-HLA-A, B, C) monoclonal antibody (MoAb) and that of class II (HLA-DR) by the 136 MoAb. Normal RPE cells express few class I and no detectable class II antigens. For the induction of MHC expression, cells were subsequently stimulated with 250 U/ml of recombinant gamma-interferon for 5 days. Cells were used for tissue typing and also for cross-matches with recipient serum. Cross-matches were subsequently performed and measured by flow cytometry. RESULTS: Both class I and class II antigens were strongly enhanced, as could be shown by immunohistochemical staining. Some 20% of those patients awaiting rekeratoplasty (n = 60) were positive for anti-HLA antibodies. In one case anti-DR3 antibodies were detected in a recipient who had had several rejection episodes after keratoplasty. CONCLUSIONS: RPE cells are not only useful for cadaver post-mortem HLA typing but also for donor-specific cross-matches. The degree of antibody formation after keratoplasty in rejecting patients was, however, low. This may imply that anti-HLA antibodies are not the major cause of corneal graft loss after keratoplasty.

Glass slide smears are a suitable source for RT-PCR-based analysis of chromosomal aberrations in leukaemias.

The polymerase chain reaction (PCR) is a well-established method to detect cytogenetic abnormalities. The handling of fresh specimens is difficult, therefore a method to use smears of blood or bone marrow as a source would be advantageous. Furthermore, such a technique would give the opportunity to investigate retrospectively bone marrow smears in leukaemias without cytogenetic results. The aim of the present study was to investigate the influence of staining procedures and laboratory handling of smears. We chose CML cases as a model. We demonstrated that smears are a suitable source for PCR without loss of information caused by previous routine laboratory handling.