| PXE is an autosomal recessive disorder disease caused by mutations of ABCC6gene. The precise incidence of the disease is not clear, the incidence ever reported is about1:70000～1:100000. The skin, eyes, and cardiovascular system can be affected, Theearly skin damage are yellow papules on the neck in flexural areas, it gradually merge intoa flaccid patches, and skin folding occasionally develops. The calcification of Bruch’smembrane form angioid streaks, which can lead to impaired vision, and even loss ofvision. The arterial mineralization can damage cardiovascular system, includingatherosclerosis, intermittent claudication of lower limb, high blood pressure, sometimesearly myocardial infarction can be induced. Additionally, PXE can also give rise togastrointestinal bleeding, abnormal mineralization cause calcium deposition in differentorgans, including liver, kidney, spleen, breast, testicular, tc. he dermatologicalmanifestations are similar, the prognosis with PXE depends on the complication of fundus and cardiovascular system. The involvement of the skin and eye often occurre inadolescence, but can also occur in early childhood. Cardiovascular complications oftenappear relatively late, developping in adulthood.The location of ABCC6gene is on chromosome16p1, including31exons. It is codedby transmembrane protein composed of1503amino acids, which called multi-drugresistance related proteins MRP6. This kind of transmembrane protein expressed mainly inthe liver, kidney and intestine, nevertheless the skin, eye and cardiovascular systemaffected by elastic fibers can only be detected traces of expression. The function ofABCC6gene is as a kind of organic anion output pump transporting an uncertainmolecules from the liver to the peripheral circulation. A recent research by establishing aknockout mice model of ABCC6gene(Abcc6/), have confirmed that PXE is a metabolicdisorder disease, however, the pathogenesis from the ABCC6gene mutations to theperipheral tissue mineralization is not clear. More than90%of patients with PXE can befound ABCC6gene mutations. So far more than800mutations of ABCC6genne havebeen found in PXE patients, including a nonsense mutation of R1141X and a deletionmutation of exon23-29, which account for about40%of all mutations.With the rapid development of molecular genetics technology, the application ofexome sequencing in the medical field is wider and wider. Most of pathogenicitymutations of genetic disease focus on1%of exons area. Exome sequencing is the mostsimple and common strategy for studying the single gene genetic disorders. We collected29cases with PXE from nine hospitals analyzing the clinical and pathological features,bytaking use of the exome sequencing, we can discover the mutations of ABCC6gene with22PXE patients.We predict the pathogenicity to all mutations by software SIFT andPolyPhen-2Prediction. In vivo, mouse and zebrafish models are used to identify thepathogenicity by the group of professor Jiang from Thomas Jefferson university.Objective1. To analyze the clinical and pathological features in29cases with pseudoxanthomaelasticum from China.2. To test pathogenic mutations of ABCC6gene in22Chinese patients with PXE, identify the pathogenicity mutations.Methods:1. The clinical data of29cases with pseudoxanthoma elasticum were retrospectivelysurveyed and the results of histopathology section, elastic fiber staining, and calciumstaining in skin tissues were observed and analyzed.2. PCR was performed to determine the mutation of exon23-29deletion reportedin patients with PXE.3. Exome sequencing was performed to determine the mutations of ABCC6gene in22Chinese patients with PXE.4. The software of SIFT prediction and PolyPhen-2prediction were performed topredict the pathogenicity of the mutations of ABCC6gene.5. By taking use of mouse model, immunofluorescence was performed to determineexpression of mutations sites on the membrane or within the membrane of mice liver cell,verifying the pathogenicity of the mutations of ABCC6gene.6. Zebrafish model was performed to determine whether the carrier can rescueknockdown model of zebrafish, further verifying the pathogenicity of the mutations ofABCC6gene.Results:1.In29cases with PXE,the ratio of male and female was1:8.67, the average age ofonset was （22.6±11.2）. The pathogenetic locations:29cases involved the neck(100%),11cases(37.9%) involved the axillary,5cases (17.2%) involved the abdomenand,4cases(13.8%) involved the groin. By HE staining we can find the curl-like structure in themiddle dermis. By the elastic fiber staining we can find the elastic fibers swelling fracture,curl, and degeneration. Through calcium staining we can find calcium deposition. Fivepatients with PXE had been made an examination of fundus oculi and cardiovascularsystem. Two of them were found angioid streaks in fundus oculi. One patient was foundbleeding in retina and Impaired vision.The examination of cardiovascularsystem werenormal.2. PCR was performed, the mutation of exon23-29deletion was not found in all patients with PXE.3.5newframeshift mutations or deletion mutations were found: C.4254delG,C.30delG, C.196-197inT, C.3586delC，C.196delT. These mutations are pathogenicwhich cause amino acid change or coding is terminated.4. We use the software of SIFT prediction and PolyPhen-2to predict thepathogenicity of the mutations of ABCC6gene and found6definite pathogenic mutations:①C.1814T>C（SIFT：Damaging0，PolyPhen-2：probably/1）②C.373G>A（SIFT：Damaging0.01，PolyPhen-2：probably/0.997）③C.11C>A（SIFT：Damaging0，PolyPhen-2：probably/0.957）④C.1256G>A（SIFT：Damaging0，PolyPhen-2：probably/0.994）⑤C.2843T>C（SIFT：Damaging0，PolyPhen-2：probably/0.988）⑥C.2126A>G（SIFT：Damaging0，PolyPhen-2：probably/0.916）; we found4mutationswith high possibility of pathogenicity:①C.2501T>C（SIFT：Tolerated0.28，PolyPhen-2：Benign/0.047）②C.61C>T（SIFT：Tolerated0.45，PolyPhen-2：benign/0.209）③C.191G>A（SIFT：Tolerated0.21，PolyPhen-2：benign/0.051④C.26C>A（SIFT：Damaging0.02，PolyPhen-2：benign/0.109）;5. By establishing mouse model the method of immunofluorescence, we identifiedone pathogenic mutation expressed within the liver cell membrane: C.1814T>C（SIFT：Damaging0，PolyPhen-2：probably/1）; one probably pathogenic mutation mostly expresswithin the liver cell membrane: C.1256G>A（SIFT：Damaging0，PolyPhen-2：probably/0.994）; other mutations expressed mainly on mice liver cell membrane, thesemutations need to to be further tested and comprehensive analysis to verify thepathogenicity.6. By zebrafish model, we identified eight pathogenic mutations of ABCC6genewhich can not rescue knockdown model of zebrafish:①C.11C>A（SIFT：Damaging0，PolyPhen-2：probably/0.957）②C.1256G>A（SIFT：Damaging0，PolyPhen-2：probably/0.994）③C.26C>A（SIFT：Damaging0.02，PolyPhen-2：benign/0.109）④C.2843T>C（SIFT：Damaging0，PolyPhen-2：probably/0.988）⑤C.2126A>G（SIFT：Damaging0，PolyPhen-2：probably/0.916）⑥C.373G>A（SIFT：Damaging0.01，PolyPhen-2：probably/0.997⑦C.61C>T（SIFT：Tolerated0.45，PolyPhen-2：benign/0.209） ⑧C.2501T>C（SIFT：Tolerated0.28，PolyPhen-2：Benign/0.047）; one mutation canpartialy rescue knockdown model of zebrafish: C.191G>A（SIFT：Tolerated0.21，PolyPhen-2：benign/0.051.Conclusions1. The most of the patients with PXE that we collected are female, the age of onset isyoung. The pathogenetic locations are on the neck and armpits. Through the skinhistopathological examination we can make timely and correct diagnose. It is necessaryand important to make an examination of cardiovascular system and fundus oculi and tofollow up the patients.2. The mutation of exon23-29deletion which are commonly discoved in Europeansand Americans was not found in all patients with PXE that we collected, the large area ofdeletion mutation may not be a common mutation in PXE cases of Chinese Hanpopulation.3. We identified five new pathogenic frameshift mutations of PXE cases in ChineseHan population: C.4254delG, C.30delG, C.196-197inT, C.3586delC, C.196delT.4. We identified nine pathogenic missense mutation of PXE cases in Chinese Hanpopulation by mouse and zebrafish model: C.1814T>C, C.1256G>A, C.11C>A, C.26C>A,C.2843T>C, C.2126A>G, C.373G>A, C.61C>T, C.2501T>C. |
PDF Full Text Request