Phenotype Changes In The Epidermal Scale And Nail Of The Patients With Congenital Ichthyosis

BackgroundThe process of cornification is as well as a process of keratinocyte (KC) differentiation and apoptosis. Both transglutaminase 1 (TGase 1) and involucrin, involved in formation of the thicken cell envelope (CE) are the marker of the differentiated CE. An elastic and permeable epidermal barrier is made up of the intercellular lipids, such as sphingomyelin, ceramide and together with cell envelope to prevent from loss of water and ions in the organism and also to contribute to desquamation. The epidermal TGase 1 can crosslinke-(γ)-glutamyl and lysine to form a precursor of the cell envelope, including involucrin, loricrin, filaggrin and small proline-rich proteins. The TGase 1 is a crucial enzyme to form the cell envelope, since its crosslinking product is stable enough to resist proteolysis. Besides, the TGase 1 can also crosslink the glutamyl residue in human involucrin and the specific epidermalO-hydroxyceramide by an ester bond, involved in formation of the lipid envelope. Hence, the lipid envelope is a crucial constituent to perform the epidermal barrier function.The appearance of the patients with congenital ichthyosis is similar and characterized by a layer of sticky large scale in grey-brownish color over skin with different degrees of hyperkeratosis. The congenital hereditary ways may be mainly autosomal recessive ichthyosis (ARCI)/ lamellar ichthyosis (LI) or autosomal dominant iehthyosis/ichthyosis vulgaris (IV). In 1994 Russell et al found an affected gene or TGase family located on chromosome 14q11 through family linkage analysis of the patients with autosomal recessive lamellar ichthyosis. The gene defect of TGase 1 was found in about 33% patients with ARCI which were called typical lamellar ichthyosis(LI).In 2005 Rice et al compared the structures of nail and epidermal scale under light microscope with phase contrast or the ultra-structure under electron microscope. He found that the cell envelope of skin appendages was not intact in most patients with ARCI as an indicator of defective TGase 1 enzyme activity. The present paper studied the cholesterol, unsaturated fatty acid and phospolipid in the epidermal scale by using histochemistry, SDS-PAGE maps with nail protein extracted by Trizol reagent and the involucrin expression by immunohistochemistry and immunoblotting in both epidermal scale and nail in comparison of the patients with LI with the normal control. To analyse the relationship of genetype and phenotype in LI patients, which can provide research basis of clinical therapy.Material and Methods:1. The trimmed nail and scrubbed epidermal scale samples were collected from 5 cases with lamellar ichthyosis and 5 cases with ichthyosis vulgaris and 10 cases from the normal volunteers. The collected samples were stored under dry and deep-cold condition.2. The trimmed nail was ground into powder and 50 mg nail powder was put into an Eppendorf tube. The SDS-PAGE was carried out after the nail protein was extracted with Trizol reagent and the protein concentration was detected.3. After the target bands in the SDS-PAGE were electro-transferred onto a nitrocellulose membrane, the immunoblotting against involucrin was performed.4. Both epidermal scale slides and frozen sections of detergent-extracted nail were immunostained against involucrin.5. The epidermal scale spread onto a slide was fixed with 10% calcium-formalin. The phospholipid with acetone-nile blue-sulfate method, the unsaturated fatty acid with OSO₄ and the cholesterol with perchloric acid -naphthyl quinon method were demonstrated.Results:1. In the SDS-PAGE map a different pattern between the congenital ichthyosis group and the normal group was found despite of individual difference in band intensity. There were 4 major bands: 63kDa,54kDa,48kDa,38kDa in the control group; while there were 5 major bands in the patients with ARCI: 69 kDa,63kDa,54 kDa,48 kDa,38 kDa. Besides, 5 cases with LI displayed higher OD ratio of 69kDa vervus 63 kDa, P<0.05; 5 cases with IV exhibited higher OD ratio Of 63 kDa vervus 69kDa, P<0.05.2. In the nail immunoblotting a range of bands of 63 kDa-158kDa appeared involucrin immunoreactivity.3. In both slides of epidermal scale and nail frozen section the intensity of immunosiaining against involucrin was lower in the patients with LI than that in the control group, P<0.05.4. In the epidermal scale slides the keratinocyte boundary was not so visible as that in the control group. Besides, the content of cholesterol, unsaturated fatty acid and phopsolipid was less in the patients with LI than that in the control group, P<0.05.Conclusions:1. The immunoblotting displayed the involucrin expression was located in the range of bands with 63 kDa-158 kDa in the nail.2. The involucrin expression was not only down-regulated, but the content of lipids was also decreased in the cases with LI, suggesting that there was a deficiency in differentiation of both cell and lipid envelopes and impaired barrier function in the patients with LI.3. In the nail SDS-PAGE there was one more major band in cases with congenital ichthyosis than the control group. Besides, the OD ratio of 69 kDa versus 63 kDa in cases with LI was higher than the control group, while the OD ratio Of 63 kDa versus 69kDa in cases with IV exhibited higher than the control group. The results suggest that the difference in OD ratio may be a potential marker for cases with LI distinguished from cases with IV.4. Overexpression of of 69 kDa in cases with LI may be associated with defect TGase 1, which may be used as a referred diagnostic marker for screening deficiency in TGase 1 enzyme from non-deficiency in cases with LI.