Study Of Clinical,histopathologic And Pathogenic Gene In Patient With Symmetrical Acral Keratoderma

In May 2003.a 24-year-old female presented with lesions on the dorsal of hands and wrists for 3 years.Three years ago,the brown hyperkeratotic lesions started on both wrists,the dorsal of hands and fingers without itch or pain.The lesions developed maceration rapidly after soaking in water for 5 min and recovered gradually after drying.Physical examination showed brown hyperkeratotic patches with rough elevations symmetrically distributed on both wrists,and the dorsa of both hands and fingers.The lesions became grayish whiteafter 3-min water contact,and recovered gradually after drying.Histological examination revealed epidermal basket-weave hyperkeratosis,acanthosis and papillomatous hyperplasia,superficial ectatic capillaries and perivascular infiltration with a few lymphocytes in dermal.The routine laboratory tests such as complete blood count,serum glucose,and liver and renal function were otherwise normal.Temporary improvement was achieved by topical application of clobetasol propionate cream after two weeks,but relapse occurred after discontinuation.To our best knowlege,we could't find a suitable dianosis for these disorders from literature or textbook.We enrolled about 10 cases with these disorders everyyear from May 2003 to August 2008.Although we consulted some professors from Nanjing Institute of Dermatology,Chinese Academy of Sciences and the First Affiliated Hospital of Peking University,we could't make an accurate diagnosis for these disorders.Therefore,we speculated that it is a new disease.In 2008,Jiang YQ et al.1 described a new entity in 11 cases characterized by symmetric keratosis on the acral extremities and rapid immersion upon exposure to water,and they coined the term 'symmetric acral keratoderma' in Chinese literature.They reported 11 cases presented with hyperkeratotic patches on the acral extremities and review the demographic information,clinical features and clinicopathologic features.Among these patients,there were 9 males and 2 females,with an average age and onset age of 35.4 and 26.5 years,respectively.The clinical manifestation was characterized by brown or black hyperkeratotic patches or maculopapular rash distributed on the dorsum of the hands,dorsum of the palms,wrists,elbows,knees,and ankles.Histological examination revealed epidermal basket-weave hyperkeratosis,acanthosis and papillomatous hyperplasia.These disorders can't be diagnosis be the presented disease.They term Symmetric Acral Keratoderma is proposed to name this disorder as a newly described skin disease.Zhu et al.2 reported 16 cases characterized by pigmented symmetrical acrokeratoderma,and pathologically characterized by epidermal hyperkeratosis,acanthosis and papillomatous hyperplasia.The lesions appeared dramatically whitish and mild swelling after soaking in water within 3-6 minutes,but began to regress in 30 minutes after drying.Most of the lesions resolved spontaneously in winter and relapse in the next summer.Up to July 2013,there were 11 case reports with 105 cases in the Chinese literature.These reports described small cases.The reported paper had described the clinical features and histopathologic features of SAK.As a new disease,these studies were not enough.To control SAK,we need to further study the demographic information,clinical features,histopathologic features,ultrastructural features and pathogenesis.To further study a new shin disease--SAK,this study divided into three sections.In the first section,clinical investigation,non-invasive electronic devices Multi Skin Center(?)MC760(Courage&Khazaka,Germany),histopathologic features,electron microscope and immunohistochemistry were used to investigate the demographic information,clinical features,histopathologic features,ultrastructural features and immunophenotype in patients with SAK.In the second section,whole exome sequencing was used to identify the pathagenic gene of family SAK.In the third section,cell biological function was used to investigate the function of the pathagenic gene in patient with family SAK.Chapter 1 The clinical and histopathologic study of Symmetrical acral keratodermaObjectives:To investigate the demographic information,clinical features and histopathologic features of SAK.Methods:SAK outpatients were enrolled in the study from May 2003 to December 2014.The demographic information and clinical features were summarized by retrospective study.Non-invasive electronic devices Multi Skin Center(?)MC760(Courage&Khazaka,Germany)was used to dectected the sebum content,skin hydration and Transepidermal water loss(TEWL).Histopathological examination,transmission electron microscopy and immunohistopathological examination were used to investigate the histopathologic features,ultrastructural features and the expression of KRT1,KRT14,loricrin and involucrin in patients with SAK.Results:Among our 71 cases,the demographic,clinical,histopathologic features and ultrastructural features are summarized as follows:(1)SAK predominantly affects men outnumber women by a ratio of 9.14:1,and patients at onset age of 24.02×9.10 years;(2)40.85%cases are factory workers engaged in 'tie and dye'industries;(3)the lesions are brown to black hyperkeratotic patches or maculopapular rash and become whitish after short-term water,but recover gradually after drying;(4)the lesions are symmetrically distributed on the acral areas,particularly on the wrists and dorsa of hands,but the Palmoplantar is not involved;(5)subjective symptoms are generally absent;the lesions aggravate in summer and resolve spontaneously in winter;(6)14.08%cases are complicated with ichthyosis vulgaris,11.27%cases are complicated with palmar and foot hyperhidrosis,and 11.27%cases have a familial history;(7)the values of sebum content and skin hydration were significance lower than that of healthy volunteers,the value of TEWL was higher than that of healthy volunteers,and the epidermal barrier function of the skin are negatively affected;(8)histopathologic examination reveals epidermal basket-weave hyperkeratosis,acanthosis and papillomatous hyperplasia,superficial ectatic capillaries and perivascular infiltration with a few lymphocytes in dermal,as well as hyperkeratosis around the dilated ducts of the sweat glands in the dermis.(9)Ultrastructurally,the epidermis was thickened by acanthosis and compact stratum corneum.The horny cell layers were remarkably.The keratin filaments were remarkably clumped or aggregated and irregularly distributed in the horny,spinous,granular and basal cell layers.The tonofilaments formed tight clumps or aggregated at the perinuclear cytoplasm.(10)The expression of KRT1 in the lesional skin of SAK was higher than that of healthy skin.The expression of KRT14 in the lesional skin of SAK was higher than that of healthy skin.The expression of loricrin and involucrin in the lesional skin of SAK was higher than that of healthy skin.Conclusion:The classical symptoms of SAK are brown to black hyperkeratotic patches or maculopapular rash on the acral area and the epidermal barrier function of the skin are negatively affected.The classical histopathologic features are epidermal basket-weave hyperkeratosis and acanthosis,as well as the dilated ducts of the sweat glands in the dermis.SAK is predilection on the young and middle-aged patients.The classical ultrastructural features of SAK were epidermal hyperkeratosis and abnormalities of the keratin filaments and tonofilaments.The immuonphenotyping of SAK was overexpression of KRT1,KRT14,loricrin and involucrin.Chapter 2 Exome sequencing identifies the pathogenic gene of family SAKObjectives:To identify the candidate pathogenic gene of SAK by exome sequencing.Methods:(1)A four-generation Chinese family consisting of 11 members with SAK was enrolled in the study.(2)We performed exome capture by Agilent SureSelect Human All Exon51M followed by massively parallel sequencing(Illumina Hiseq 2500 rapid PE90)in two affected individuals and one unaffected individual from a family with DSAP(family 1).(3)Raw image files were processed by Illumina basecalling Software 1.7.Sequence reads in each individual were aligned to human reference genome(NCBI build 36.3,hg19)using SOAPaligner 2.20.The consensus genotypes in target regions were called by SOAPsnp(v 1.03).A consensus genotype with Phred-like quality of at least 20 and at least four coverage depth was considered as the high-confident genotype.The genotypes that were different from the reference were extracted as candidate SNPs.For indels calling,we switched to GATK pipline.(4)The variants were functionally annotated using an in-house pipeline and categorized into missense,nonsense,readthrougth,splice-site mutations and coding indels,synonymous and non-coding mutations.Based on these annotations,variants were filtered first for the nonsysnonymous(NS),splice acceptor and donor site(SS)mutations and coding indels(I),and then filtered against available public databases(dbSNP129,eight HapMap exomes and 1000 Genome variants databases)and the control step by step.At last the variants which were shared between two cases but were neither present in the public databases nor in the control individual of the family were considered to be the candidate variants.Then,we selected possible SAK causal variants using ANNOVAR and GERP to assess these variants for likely functional impact combing with the reported linkage regions of SAK.(5)We sequenced the exons containing possible SAK causal variants in all available individuals in family 1(4 case and 7 controls).(6)We further sequenced all the exons and exon-intron boundaries of the gene containing the variant which was cosegregated with SAK in famiy 1 in additional SAK individuals and family 2.We filtered the mutations against 100 unrelated,ethnically matched controls.(7)We performed immunohistochemistry to explore the expression of TCF4 on lesional and healthy skin.Results:After exome capture,massively parallel sequencing,mapping and filtering step by step,we selected 67 candidate variants which were shared by the two affected individuals but not present in the non-affected individual from the family and available variants databases.The 67 candidate variants were filterting by SIFT and GERP.Tweenty-one candidate variants were predicted harmful by SIFT and 45 were predicted harmful by GERP.Sixteen candidate variants were predicted harmful by both of SIFT and GERP.Among these candidate variants,only one heterozygous variant,found in TCF4,was relate with the development of epidermis.All the 4 affected individuals of the family 1 were heterozygous for TCF4(c.85C>A)mutation which was absent in the 7 unaffected family members in family 1.None of this mutation was detected in 100 unrelated,ethnically matched controls,which provided evidence that this mutation was not polymorphisms.No mutations within TCF were found in family 2 and 28 individuals with SAK patients.The TCF4 protein was expressed on the strtum corneum,granular layer,spinous layer and basal layer of epidermis of healthy skin.The expression of TCF4 protein in the lesional skin of SAK was lower than that of healthy skin.Conclusion:We identified a pathogenic gene TCF4 of SAK by exome sequencing in one SAK family.The expresson of TCF4 protein was lower in the lesional skin of SAK.Chapter 3 Fuctional study of TCF4(c.85C>A)gene mutation of SAK patientObjective:To study the functional study of TCF4(c.85C>A)gene mutation of SAK patient.Methods:(1)Real-time PCR and western blot were used to validated the expression of TCF4 in Hacat cell lines.(2)Construction of mutation-TCF4(c.85C>A)and wild-TCF4 lentiviral vector.Mutation-TCF4(c.85C>A)and wild-TCF4 lentiviral vector were transferred to the Hacat(3)By using the cell proliferation assay,flow cytometry,real-time PCR and western blot,we detected the effect of mutation and wild-TCF4 on the cell proliferation,differentiation and apoptosis of Hacat.Results:(1)We successfully verify the expression of TCF4 gene and protein in the Hacat cell lines.(2)We successfully construct the mutated TCF4(c.85C>A)and wild TCF4 lentiviral vector,and transferred to the Hacat.(3)Both of mutation-TCF4 and wild-TCF4 increased the proliferation of Hacat.The proliferation rate of mutation-TCF4 was lower than that of wild-TCF4.The flow cytometry results suggested that the proportion of cells in S phase increased in the mutation-TCF4 and wild-TCF4.No significant effect of mutation and wild-TCF4 on the apotosis of Hacat.The expression of TCF4mRNA and protein,KRT1mRNA and protein,involucrin mRNA and protein,loricrin mRNA and protein in the mutation and wild-TCF4 group was higher than that of lentiviral vector group.Conclusions:Wild and mutation-TCF4 might increase the process of keratinization and differentiation of keratinocyte.