Study On The Homozygous Deletion And Point Mutation Of P16 Gene Exons Of Keratinocytes In Psoriasis

Psoriasis is a chronic, inflammatory, proliferative skin disorder characterized by plaques,papules and scales .whose cause and pathogenesis are not clear.Its fundamental cytokinetic characteristic is hyperproliferation of epidermal cells.The cause of hyperproliferation is unknown.At present it's found that the cell cycle of psoriatic epidermal cells is shortened greatly and the number of proliferating cells increases. In normal epidermal skin, the growth fraction,which means the ratio of proliferating cells to the general cells,is 60%.The epidermal growth fraction of active psoriatic lesion is about 1.Therefore the molecular mechanism of cell cycle modulation may participate in genesis of psoriasis.Study on cell cycle reveals the transitions from Gl phase to S phase and from G2 phase to M phase are the most important checkpoints in the modulation of cell cycle.The cyclin dependent kinase4(CDK4),CDK6,cyclinE and cyclinD(the role of cyclin Dl is stronger) play an important role in the modulation of the transition from G1 phase to S phase ,The p16 protein found and isolated in 1993 is one cyclin dependent kinase inhibitor(CKI).The amino terminus of pl6 protein has a gyral structure which is homologous to cyclinD1. Therefore pl6 protein competes with cyclinDl to bind cyclin dependent kinase 4 or 6 whose activities will be inhibited.Then the retinoblastoma tumor suppressor protein(pRb) can't be phosphorylated.The pRb without phosphorylation inhibits the expression of transcription factors like the family of E2F .which is essential to synthesisof DNA.Thus the cell cycle stops at the G1/S phase and the proliferation can't continue.The sequence of p16 gene encoding pl6 protein was determined in 1994.The p16 gene is located in the 9p21 of human chromosome ,and its length is 8.5kb,including 3 exons and 2 introns.The 5' terminus has exonl(126bp),the middle exon2 (307bp),and the 3' terminus exton3(11bp).The exon2 represents the main part of coding area, and the change of pl6 gene occurs mainly there.When homozygous deletion and point mutation of pl6 gene occur,p16 protein may be expressed abnormally or not expressed at all. Then more CDK4/CDK6 can bind cyclinD1,which in turn stimulates cell division and leads to cell proliferation out of control.Many studies show homozygous deletion and point mutation of pl6 gene occurs in high frequency in various tumours.But the relationship between its homozygous deletion or mutation and psoriasis has not been reported.We analysed homozygous deletion and point mutation of pl6 gene exons in keratinocytes of psoriasis lesions and non-lesions.The objective is to investigate the mechanism and clinical meaning of p16 gene in psoriasis and contributes to psoriasis diagnose and treatment.As the exonl(E1) and exon2(E2) include the 97% coding base pairs, just like the majority of scholars ,we only detect the change of E1 and E2. Materials and Methods32 patients with psoriasis vulgaris,18 male and 14 female,age from 12to 62(35.1 ± 9.7),12 with familial history ,29 in progressive phase were chosen.Keratinocytes were cultured primarily from epidermis of the lesions and non-lesions taken respectively by the apparatus of epidermis transplanting for treating vitiligo,and genome DNA obtained by phenol extraction and ethanol precipitation following proteinase digestion.The E1 and E2 of pl6 gene were amplified by Polymerase Chain Reaction (PCR) to analyze their homozygous deletion. Single Strand Conformation Polymorphism(SSCP) was used to detect point mutation of E1 and E2. Results1. In keratinocyte samples from psoriatic lesion skin, the homozygous deletion of p16 gene E1 was identified in 2 cases (6.25%),which were in progressive phase.No homozygous, deletion of E1 was detected in non-lesion skin keratinocytes.Between the two groups there were no significant differences in homozygous deletion frequency ofE1(P>0.05).2. In keratinocyte samples from psoriatic lesion skin, the homozygous deletion of p16 gene E2 was identified in 1 cases (3.13%),which were in progressive phase.No homozygous deletion of E2 was detected in non-lesion skin keratinocytes.Between the two groups there were no significant differences in homozygous deletion frequency of E2(P>0.05).3.In keratinocyte samples from lesion skin,no mutations of E1 was detected and abnormal strands of mobilityshift of E2 were found in 5(15.63%),which were in progressive phase.No mobilityshift of E1 and E2 were detected in non-lesion skin keratinocytes.The E2 mobilityshift frequency was significantly higher in psoriatic lesion samples than that in psoriatic non-lesion skin (P<0.05).4.The E2 mobilityshift frequency was 16.67%(2/12) and 15%(3/20) in lesion skin keratinocytes with and without familial history respectively. Between the two groups there were no significant differences.5. The E2 mobilityshift frequency was 17.24% (5/29) in lesion skin keratinocytes in progressive phase.No E2 mobilityshift was found in lesion skin keratinocytes in resting phase. Between the two groups there were no significant differences.Conclusions1.The point mutations of pl6 gene E2 occurred only in psoriatic lesion skin keratinocytes in progressive phase and its frequency of occurence was significantly higher than that in non-lesion skin keratinocytes. Due to small samples,there was no significant difference in E2 point mutation between the progressive pahse and resting phase.It's suggested that the point mutations of pl6 gene E2 may relate to hyperproliferation of psoriatic lesion skin keratinocytes and the genesis of psoriasis.2.There was no significant difference in the frequency of point mutation of p16 gene E2 in psoriatic lesion skin keratinocytes between patients with and without familial history,which suggested that E2 may not play an important role in the psoriasis heredity.3.No point mutation of p16 gene E1 was found in psoriatic lesion and non-lesion skin keratinocytes,which suggested that E1 point mutation may not relate to the genesis and development of psoriasis.4. The homozygous deletion of p16 gene E1 and E2 was found in psoriatic lesion skin keratinocytes.But their frequency of occurrence has no significant difference from that of non-lesion skin keratinocytes.which suggested that homozygous deletion of E1 and E2 may not be the main cause of genesis and development of psorasis.