Clinical Features Analysis And Genetic Study Of 140 Cases Of Porokeratosis

BackgroundPorokeratosis(PK,MIM 175800)is a heterogeneous group of keratinization disorders that exhibit an autosomal dominant mode of inheritance.PK is also a skin-specific autoinflammatory disease which was often inherited and linked to ultraviolet light exposure and immunosuppression.As a histological hallmark that unifies all subtypes of PK,cornoid lamella(CL)is a vertical ’column ’ of porokeratosis.PK is currently classified according to the clinical manifestations,such as number,size,morphology,and distribution of the histological lesions.Porokeratosis can be classified as:classic porokeratosis of Mibelli(PM),disseminated superficial actinic porokeratosis(DSAP),disseminated porokeratosis(DSP),linear porokeratosis(LP),punctate porokeratosis(PPt),and porokeratosis palmaris et plantaris disseminate(PPPD).Additionally,there are more than 10 other rarer types of PK.In addition to the heterogeneity in clinical manifestations,genetic heterogeneity is also observed in PK.At least seven linkage loci(12q23.2-24.1,15q25.1-26.1,18p11.3,1p31.3-p31.1,16q24.1-24.3)have been reported for three forms of PK which include DSAP,DSP,porokeratosis palmaris et plantaris disseminata(PPPD).Up to date,seven genes(SSH1,SART3,SLC17A9,MVK,PMVK,MVD,FDPS)were reported as the causal genes.However,the following problems still exist in previous studies:1.The pathogenic genes previously reported can only explain some PK cases:1)MVK can explain 29%(25/87)PK family patients and 20%(13/65)sporadic patients;2.2)since zhang et al.reported MVK,PMVK,MVD,and FDPS genes in 2015,the above four genes can explain 96%of PK patients in families and 67%of PK patients with sporadic PK.2.The correlation between other genes in the mevalonate pathway and PK remains to be further studied:since MVK was reported to be related to PK onset in 2012,4 genes in the pathway have been found to be related to PK onset.There are 12 related genes in this pathway,and whether all of them are related to PK disease remains to be further studied.3.PK has strong genetic heterogeneity and many pathogenic genes.Sanger sequencing method has many disadvantages in detection.4.SSH1,SART3 and SLC17A9 have been reported to be related to PK,but there are doubts:since SSH1 and SART3 were reported to be related to PK in 2004,multiple teams have examined this gene,but no pathogenic mutations have been found.Some experts believe that SSH1 may not be the disease-causing gene of DSAP.By 2015,domestic scholars believe that only MVK is the disease-causing gene of DSAP.The correlation between SSH1,SART3,SLC17A9 and PK remains to be further studied.Based on the above background,this study was carried out to solve the following problems:1.Summarize the clinical characteristics of the above patients,find their pathogenic variation,and summarize the relationship between genotype and phenotype;2.2.Verify whether other genes in the mevalonate pathway are related to PK pathogenesis;3.The correlation between SSH1,SART3,SLC17A9 genes and PK should be verified.Part Ⅰ Clinical features analysis of 140 probands of porokeratosis1.Objective:We retrospectively analyzed the disease characteristics,clinical manifestations and clinical subtypes of PK patients to identify the associations between genotype and phenotype by summarizing the clinical characteristics of PK.2.Methods:The clinical data of 140 cases(64 families and 76 sporadic PK patients),all the PK patients collected in our hospital from 2003 to 2017.The clinical characteristics such as gender,age,onset age and subtypes were collected and analyzed.3.Results:This study investigated 64 families and 76 sporadic cases with different subtypes of porokeratosis and 380 healthy controls in the Chinese population.112 patients with DSAP/DSP,nine patients with PM,seven patients with giant plaque of porokeratosis ptychotropica(PPt),four patients with LP,five patients with hyperkeratotic porokeratosis(HPM)and three patients with genital PK are included in the cases.The age of onset varied from birth to 76 years(mean age 31.2 years).4.Conclusion:based on the above clinical characteristics,the incidence of PK patients in males is higher than that in females,and the incidence ratio of males to females is about 1.64:1.The age onset is from birth to 60 years old.DSAP\DSP was the most common PK subtype,accounting for 80%of the total number of patients.Different clinical subtypes and age of onset have some associations.Part Ⅱ Genetic mutations analysis of 140 porokeratosis by High-throughput target region sequencing1.Objectives:1)Identified the pathogenic genes or new variants in PK;2)Verify whether all 12 genes in the mevalonate pathway participate in the occurrence of PK;3)Verify whether the previously reported 3 genes(SSH1,SART3,SLC17A9)are the pathogenic genes PK;4)Obeserve the correlation between genotypes and phenotypes.2.Materials:A total of 380 healthy controls and 140 PK patients(64 families and 76 sporadic PK patients)were selected in this study.3.Methods:We here performed a new high throughput target region sequencing method to study the 15 established candidate genes(including 3 reported genes SSH1,SART3 and SLC1 7A9 and the 12 genes in the mevalonate pathway(ACAT1,ACAT2,HMGCR,HMGCS1,HMGCS2,MVK,PMVK,MVD,FPDS,IDI1,IDI2,GGPS1)in a Chinese cohort of 140 probands diagnosed with porokeratosis and matched 380 healthy controls.To identify the 15 established PK genes mutations of all subjects in this study,we first applied multiplexed PCR-based amplification using the Fluidigm Access-ArrayTM technology(Fluidigm,SanFrancisco,California)followed by barcoding and next-generation resequencing on the Miseq system(Illumina,San Diego,California).4.Results:As a result,125 rare variants(113 SNVs and 12 frameshift variants)located in exome or splice region of the 15 genes.66 functional variants(54 SNVs and 12 frameshift)were identified in 125 pedigrees.These SNVs were found in 11 genes,including ACAT1,ACAT2,HMGCR,MVK,PMVK,MVD,FPDS,IDI1,SART3,SLC17A9 and SSH1.Eight probands carried more than one SNVs in two or three genes.After Sanger sequencing validation,46 out of 66 SNVs were confirmed to be true SNVs in 111 pedigrees.These mutations include 29 missense(63%),nine frameshift(19.6%),four obligatory splice-site(8.7%)and three stop-gained mutations(6.5%).20 non-pathogenic SNVs were excluded from the validation analysis.For the 111 pedigrees,all patients carried the same mutations with the probands.And no mutation was detected in any of the healthy family members and 380 unrelated ethnically matched healthy controls.The 46 different type mutations(including)site in MVK,PMVK,MVD,FDPS,HMGCR,SSH1 and SLC1 7A9.In total,26 pedigrees carried 21 MVK mutations.11 of them are novel.Elven MVD mutations were identified in 64 pedigrees.Three of them are novel.Two reported MVD mutations(c.746T>C and c.875A>G)were identified in 50 unrelated patients,accounting for 78%of all patients with MVD mutations.Nine pedigrees carried three PMVK mutations.Two of the mutations are novel.The missense PMVK mutation(c.100G>A,p.Gly34Arg)and the stop-gained mutation(c.412C>T,p.Arg138Ter)were the hot spots mutations.Besides,we found that one sporadic PK patient(S50)has both the mutation PMVK c.100G>A and the mutation MVD c.746T>C and one family PK patient(proband of F28)has both the mutation PMVK c.100G>A and the mutation MVK c.604G>A.Interestingly,we identified seven mutations in FDPS,six of them are novel.At the same time,we first discovered a HMGCR mutation c.539G>A in a familial PK patient.Besides,2 novel SSH1 mutations were identified in one DSAP and one DSP patient,and one SLC17A9 mutation(c.C25T)was found in 2 PK probands.In summary,111(79%)of the 140 PK patients were detected pathogenic mutations in seven different genes in the mevalonate pathway.No mutation was found in the remaining 29(21%)PK patients.After analyzing between gene mutations and various clinical features in our study,several associations of genotype and phenotype were observed.First,the mean age of onset in our patients was 31.2 years.In patients with MVD mutations,the age of onset has a large span,varied from birth to 76 years.The onset age of the patients with MVK mutations is usually during adolescence.However,the FDPS patients have the oldest average age of onset,48.14 years old.Second,in this PK cohort,42.8%of patients carried mutations in MVD gene with the highest incidence.Third,giant plaque-type porokeratosis ptychotropica(PPt)was a unique phenotype associated with MVK mutations.This genotype-phenotype correlation is similar with that Zhang et al.reported.While,this clinical characteristic was observed only in 30%(6/20)of patients with MVK mutations,which was different with Zhang’s report Fourth,in this cohort,one novel missense mutation c.908A>C(p.His303Pro)of MVK was identified in a sporadic case with genital PK.Another novel missense mutation c.200T>C(p.Met67Thr)of FDPS was identified in a sporadic case combine DPS with genital PK.However,genital PK was only associated with PMVK mutations in the past report.Fifth,mutation c.746A/G(p.Phe249Ser)of MVD was the most common one.Sixth,two of the LP patients carried PMVK and MVD mutations,this result is in keeping with Zhang and Lihi Atzmony ’s reports.Except that,we identified six mutations of FDPS in eight DSAP/DSP patients.Interestingly,the lesions harboring mutations in FDPS mutations tended to be with the highest number and minimum diameter than those carrying other gene mutations.5.Conclusions:In summary,111(79%)of the 140 PK patients were detected pathogenic mutations in five different genes in the mevalonate pathway,SSH1 and SLC17A9.It’s worth noting that,we first found causative mutation in HMGCR.It implicated that all the genes in mevalonate pathway genes might participate in the pathogenesis of PK.Beside,another 2 SSH1 mutations were found in our study,which implicated that SSH1 might be involved in the pathogenesis of porokeratosis.But in this study,we did not found SART3 mutation.Part III Analysis of SSH1 expression in patients with SSH1 gene defectObjective:In 2004,zhang et.al found that SHH1 has been suggested to be responsible for DSAP.However,the result was not confirmed in other previous studies.The pathogenicity of this gene is controversial,and some scholars speculated that SSH1 may not be the pathogenic gene of PK.However,in this study,2 new mutations of SSH1 gene were found in 1 DSAP family and 1 sporadic case,all of which were predicted as harmful mutations by functional prediction.In this study,the expression of SSH1 protein in skin lesions in patients with SSH1 gene mutation was examined by immunohistochemistry to further clarify whether SSH1 is related to PK.Methods:We performed immunohistochemical analysis of healthy control,pairwise lesional tissues(LTs)and neighboring normal-appearing skin(NNS)from the SSH1-deficient patient,using antibodies against keratin 1,keratin 14,involucrin and SSH1.Results:As a differentiation marker of keratinocytes in the spinous layer,keratin 1 was undetectable in the cornoid lamella and the granular layer.Keratin 14 labeling the immature keratinocytes was observed in the cornoid lamella,indicating that cells in the cornoid lamella were not fully differentiated.Involucrin,which is synthesized in the stratum spinosum and concentrated in the stratum granulosum,was absent in the cornoid lamella and the granular layer.SSH1 locates mainly in the stratum basale of the epidermis.It was also undetectable in the cornoid lamella and the granular layer.Conclusions:After performed immunohistochemical analysis,we identified that contrast with healthy control SSH1 locates mainly in the stratum basale in LTs and NNS rather than the whole parts of the epidermis.We suspect that the damage mutation might influence the expression of SSH1 and broken its pivotal role in epidermal cell morphogenesis in the DSAP skin.And the findings suggest that SSH1 might be involved in the pathogenesis of porokeratosis.