Validated gene expression biomarker analysis for biopsy-based clinical trials in ulcerative colitis.

BACKGROUND: Accurate and reproducible measurement of expression of pro-inflammatory cytokines in colonic biopsies from patients with ulcerative colitis (UC) is essential for proof-of-concept and mechanism-of-action studies. Few studies have rigorously established the number of biopsies required for accurate and reproducible biomarker measurements. AIM: To validate methods for measuring changes in gene expression in colonic biopsy samples. METHODS: Twelve colonic biopsies were obtained from each of six healthy controls, six patients with inactive UC and seven patients with active UC. Mayo endoscopic scores were used as a clinical reference standard. Quantitative PCR was used to assess mRNA expression of eight known inflammatory genes. The power to detect a reduction in gene expression in active vs. inactive UC was calculated using a linear mixed effect model. RESULTS: mRNA analysis of colonic biopsies is a sensitive and feasible approach for measuring inflammatory gene expression in colonic biopsies. Inflammatory biomarkers correlate with Mayo endoscopic subscores for each colonic region. For most genes, three rectal biopsies from two to four patients are required to detect changes in gene expression corresponding to active vs. inactive UC to achieve a power of 80% with an alpha of 0.05. CONCLUSION: Our data suggest that systematic measurement of inflammatory biomarkers at the mRNA level can be a valuable tool for hypothesis testing, and assessment of clinical activity and response to therapy in ulcerative colitis.

X-linked myotubular myopathy: refinement of the gene to a 280-kb region with new and highly informative microsatellite markers.

We have recently refined the localization of the myotubular myopathy (MTM1) gene to a 430-kb region between DXS304 and DXS1345 in proximal Xq28. We report two new polymorphic microsatellite markers, DXS8377 and DXS7423, that were physically mapped within the critical interval. A recombination event in a family segregating for MTM1 placed the disease gene telomeric to the trinucleotide polymorphism DXS8377. Together with the recent mapping of two microdeletions associated with MTM1, the recombination refines the critical region to 280 kb. A second recombination event was observed distal to the tetranucleotide repeat DXS7423. This recombination has occurred in the off-spring of a female with a more than 67% probability of being a carrier and very likely restricts the MTM1 gene to a 130-kb region. This physical refinement is significant for positional cloning of the disease gene. The highly polymorphic markers and the precise localization of the MTM1 gene will facilitate genetic diagnosis of the disorder.