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Next Generation Sequencing to study Thalidomide mechanism in Cutaneous Lupus

ABSTRACT: Thalidomide has been shown to be effective in patients with refractory cutaneous lupus erythematosus (CLE). However, its use is still limited by its potential severe side-effects. We conducted an RNA-sequencing study using CLE skin biopsies before and after thalidomide treatment to discover its mechanism of action. Methods: Total mRNA from skin biopsies from 10 CLE patient have been processed by RNA-seq in duplicate using Illumina Hiseq 2000 sequencing platform version 3. Application was stranded mRNA-Seq, paired-end and read length was 75bp. The sequence reads that passed quality filters were analyzed at the transcript isoform level using STAR program, RSEM program and DESeq2. Mapping was done with STAR (version 2.5.2a), quantification by RSEM (1.2.28) and differential analysis by DESeq. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to human genome (hs37d5). Average un-mapped was between 11,40-7,05 %, average unique was 80.48-76,62% and average alignment insert size was 170,00-155,00 in the 20 samples. Conclusions: Plausibles mechanisms of action for thalidomide in lupus cutaneous were discovered using this study; however, in vitro and in vivo experiments would be performed to demostrate them.

ORGANISM(S): Homo sapiens

PROVIDER: GSE162424 | GEO | 2021/12/07

REPOSITORIES: GEO

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Project description:Cutaneous lupus erythematosus (CLE) is an autoimmune disease that localizes to the skin and is known to contain elevated glycosaminoglycans (GAGs) on Hale’s stain of skin biopsy specimens. Recently, different GAG species have been shown to have distinct effects on the recruitment and activation of immune cells and stimulation of cytokine production (Taylor and Gallo, FASEB, 2006; 20: 9-22). Thus, we speculate that the elevated GAGs observed in CLE play a role in the local inflammatory process that produces skin lesions in these patients. In order to further investigate a molecular basis for the elevated expression of these GAGs in CLE skin lesions, we would like to determine the gene expression profiles of GAG synthesis, degradation, and modifier genes in lesional and non-lesional skin samples from CLE patients and compare to those from healthy controls. A microarray approach will give us a broader understanding of the genetic regulation of the expression of various GAG species in CLE skin. We will then be able to target future quantitative gene expression experiments by real-time RT-PCR to the genes that are shown to be involved in CLE. In order to accomplish our goal, we would like to examine the GAG gene expression profiles of DLE, TLE, and SCLE subtypes due to the differences in CS and HA staining that we found among these subtypes. Since HA and CS are elevated in DLE and HA in TLE, but not in SCLE, the SCLE samples will also serve as an internal control. We would like to examine both lesional and non-lesional skin biopsies to determine if CLE skin prior to developing a lesion is different at the genetic level from healthy control skin and how it changes once a lesion does develop. We will separate the dermis from the epidermis of the skin biopsies and extract RNA just from the dermis to enrich for dermal fibroblast RNA. We aim to submit four patient biopsies per subtype as well as four samples from healthy control skin for comparison. This number is necessary in order to account for the biologic variability among different patients. We would submit more samples per subtype but are limited by availability of patients in clinic. Thus, we will have a total of 28 samples to submit for microarray. This study design will allow us to analyze the GAG gene expression profiles among different CLE subtypes and enable us to identify which GAc

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Next Generation Sequencing to study Thalidomide mechanism in Cutaneous Lupus

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