Study On Whole-genome DNA Methylation In Skin Lesions From Patients With Psoriasis Vulgaris

Psoriasis is a common, chronic, and relapsing inflammatory skin disease, which is incurable. Clinically, psoriasis vulgaris is the commonest subtype of psoriasis, mainly affecting young people and the lesions are often distributed at the exposed parts of bodies, such as scalp and the extensor aspect of extremities, which could badly destroy the patients'appearances and images, therefore resulting in mental burden. As a result, studies on the pathogenesis of psoriasis have always been a hot and challenging issue. Up to now, the pathogenesis of psoriasis has not been interpreted. It is believed that psoriasis is a T lymphocyte mediated autoimmune disease together with inner and outer factors impacting on susceptible individuals. A large body of studies proposed that epigenetic mechanisms play key roles in the pathogenesis of autoimmune disease, such as systemic lupus erythematosus (SLE). However, studies on epigenetics related to psoriasis are less reported. Our previous studies showed that abnormal DNA methylation status existed in the PBMCs and skin tissues from patients with psoriasis vulgaris, indicating that DNA methylation may be a substaintial mechanism responsible for psoriasis.DNA methylation, the first and most widely-studied epigenetic modification, exists in many kinds of organisms as a natural DNA modification style. DNA methylation refers to the addition of a methyl group to5'-carbon position of cytosine residues within CpG pairs, which is catalyzed by DNA methyltransferases (DNMTs). In genetics, the regions clustered with CG dinucleotides are named as CpG islands, which are located in promoter region in unmethylated status. In general, DNA hypermethylation of genes results in silencing of gene expression, while DNA hypomethylation leads to over-expression of gene expression.Many studies showed that DNA methylation plays significant roles in many aspects such as gene expression and cellular function. In recent twenty years, a lot of sequencing techniques for precise DNA methylation profile have came into practice, such as methylation specific restriction endonuclease digestion, bisulfite modification sequencing, DNA methylation specific PCR, methylation specific single nucleotide primer extension and pyrosequencing. However, those methods are not suitable for whole genome DNA methylation sequencing in high-throughput and low-price way. In addition, they are not sensitive to low-extent DNA methylaiton (10%-20%hypermethylation).Since the invention of first generation sequencing Sanger sequencing in1977, DNA sequencing has came into new era after thirty-year rapid revolution and development. At present, second generation sequencing, characterized by high-throughput and low-price, has matured and formed commercial scales. Besides, the third generation sequencing---single molecule sequencing---represents better performance. The advent of second generation sequencing (next generation sequencing, NGS) satisfies the requirement of large-scale genome sequencing such as deep sequencing and repetitive sequencing. The most notable feature is high-throughput, which means the completion of hundreds of thousands, even millions of a species of genome sequencing in a short time, making depth sequencing and detailed analysis possible, therefore, it is also known as deep sequencing.Methylated DNA immunoprecipitation sequencing (MeDIP-seq) is a technique which uses5'methyl cytosine antibody to co-immunoprecipitate methylated DNA, enriching specific purified DNA fragments followed by NGS, in order to find out differentially methylated regions and compare the DNA methylation patterns between various cells, tissues and diseased samples.Therefore, we expect to uncover the roles of DNA methylation patterns and changes in the pathogenesis of psoriasis through making comparison between psoriatic involved lesions, uninvolved lesions and normal skin tissues using MeDIP-seq. Part I Methylated DNA sequencing and validation in skin lesions and non-skin lesions from patients with psoriasis vulgarisSection one Methylated DNA sequencing in skin lesions and non-skin lesions from patients with psoriasis vulgarisObjective Investigation of genomic DNA methylation status between involved lesions, uninvolved lesions from patients with psoriasis vulgaris and normal skin tissues, uncovering the roles of DNA methylation in the pathogenesis of patients with psoriasis vulgaris.Methods Genomic DNA was isolated from skin biopsy specimens of four involved lesions, uninvolved lesions from patients with psoriasis vulgaris and four normal skin tissues and explore the DNA methylation pattern by MeDIP-seq.Results Through MeDIP-seq, DNA methylation profile of whole genome with sufficient depth and resolution was obtained. In total,63,194DMRs had aberrant DNA methylation modification and the amount of raw data was20.10Gb. Compared to normal skin tissues, DNA hypermethylation was represented in the skin lesions from patients with psoriasis vulgaris, and the number of hypermethylated DMRs (40,357) was much more than that of hypomethylated DMRs (22,837).Conclusion DNA hypermethylation status was obversed in skin tissues from patients with psoriasis vulgaris. DNA hypermethylation may play important roles in the pathogenesis and development of psoriasis vulgaris. Section two Validation of the methylated DNA sequencing resultsObjective To validate the results of methylated DNA sequencing in skin lesions and non-skin lesions from patients with psoriasis vulgaris.Methods Ten patients (including skin lesions and non-skin lesions) and ten normal controls were enrolled, and bisulfite sequencing was applied to validate the PDCD5and TIMP2result of methylated DNA sequencing.Results Compared to normal control, hypermethylation of gene PDCD5was observed in both skin lesions and non-skin lesions (0.806+0.058vs0.485±0.059, p=0.000;0.705±0.042vs0.495±0.059, p=0.000). In contrast to non-skin lesions, hypermethylation of gene PDCD5was also found in skin lesions from patients with psoriasis vulgaris (0.806±0.058vs0.705±0.042, p=0.014).There was no significant difference of DNA methylation level in the all sixteen CpG loci of TIMP2DMRs regions (-1500bp～-1130bp). However, six continuous clusted CpG loci (-1399,-1406,-1420,-1434,-1443,-1454) showed significant differences. Compared to skin lesions, hypomethylation of gene TIMP2was observed in non-skin lesions from patients with psoriasis vulgaris (0.354±0.08vs0.664±0.04, p=0.000). Compared to normal control, hypomethylation of gene TIMP2was also observed in non-skin lesions from patients with psoriasis vulgaris (0.354±0.08vs0.557±0.14, p=0.011).Correlation analysis showed that methylation levels of gene PDCD5and TIMP2were positively correlated with PASI scores of patients with psoriasis vulgaris (PDCD5:r=0.717, p=0.020; TIMP2:r=0.763, p=0.010).Conclusions Results of bisulfite sequencing of PDCD5and TIMP2are in accordance with the results of MeDIP-Seq, both of which are positively correlated with PASI scores of patients with psoriasis vulgaris. Part II Gene expression of PDCD5and TIMP2in skin lesions and non-skin lesions from patients with psoriasis vulgaris and correlation analysis between DNA methylation and gene expressionObjective To investigate gene expression of PDCD5and TIMP2in skin lesions and non-skin lesions from patients with psoriasis and analyze the correlation between DNA methylation and gene expression.Methods Total RNA was isolated from skin biopsy specimens of thirty patients with psoirasis vulgaris (including involved and uninvolved lesions) and twenty normal skin tissues using Trizol reagent, and mRNA expression of PDCD5and TIMP2was detected by Real-time PCR. And correlation between DNA methylation and gene expression was analyzed.Results mRNA expression of PDCD5was significantly down-regulated in skin lesions from patients with psoriasis vulgaris when compared to normal skin tissues (2.201±0.78vs2.914±1.08, p=0.013), and decreased trend of PDCD5mRNA expression was also found in uninvolved skin lesions from patients with psoriasis vulgaris but without significant meanings (2.774±1.03vs2.914±1.08, p=0.659). Compared to non-skin lesions, mRNA expression of PDCD5was significantly down-regulated in skin lesions from patients with psoriasis vulgaris (2.201±0.78vs2.774±1.03, p=0.025).Compared to normal controls, mRNA expression of TIMP2was down-regulated in skin lesions and up-regulated in non-skin lesions from patients with psoriasis vulgaris, both of which had no significant difference (1.340±0.69vs1.512±0.84, p=0.866;1.917±1.09vs1.512±0.84, p=0.156). mRNA expression of TIMP2was significantly up-regulated in uninvolved skin lesions from patients with psoriasis vulgaris (1.917±1.09vs1.340±0.69, p=0.023) when compared to skin lesions.Correlation analysis showed that methylation levels of gene PDCD5and TIMP2was negatively correlated with their mRNA expression (PDCD5:r=-0.709, p=0.022; TIMP2:r=-0.766, p=0.01).Conclusion Abberant mRNA expression of PDCD5and TIMP2are observed in involved/uninvolved skin lesions from patients with psoriasis vulgaris, both of which are negativly correlated with DNA methylation.