Identification And Pathogenesis Analysis Of The Pathogenic Gene For Multiple Pilomatricomas

Pilomatricoma（PM）,also known as calcifying epithelioma,is a rare benign skin appendage tumor with an incidence of 0.12%.The tumor often occurs as a single lesion in the skin of head and neck,exhibiting as solid and movable intradermal or subcutaneous nodules,which grow slowly and have no symptoms.Pilomatricoma was reported derived from the dermis or the primary epithelial germ cells,during the differentiation into hair matrix,the cells aggregated into pilomatricomas due to the abnormal proliferation and blocked apoptosis.Pilomatricoma consists of islands of epithelial cells histologically that contain anucleated cells in the center surrounded by basophilic cells,and partial calcification.Sporadic pilomatricoms commonly have somatic mutations in the exon 3 of CTNNB1,the mutations increased the stability ofβ-catenin,leading to the accumulation ofβ-catenin in nucleus and excessive cell proliferation,which in turn leads to tumorigenesis,but causative genes from germline and the underlying pathophysiology are unclear.Here,we collected and identified a family of four generations with autosomal dominant multiple pilomatricomas,32 members including 8 patients participated in the study.The tumors appeared around the age of 20,the basophils cells and shadow cells were observed in the sections of the tumors,the nucleus gradually disappears in the transition cells,and the cell boundary is clear.IHC shows that the level of Bcl-2 and CD34change with the cell gradient.PCR and Sanger sequencing was conducted for CTNNB1 in both somatic and genomic DNA,and no mutation was found.A missense variant in PLCD1（c.1186G>A）which leads to the replacement of a glutamic acid residue with arginine at codon 396（p.E396K）was identified by GES in the DNA of the proband.RFLP assay showed that the mutation segregated with the pilomatricomas phenotype in this family,and no mutation was found in 350 unrelated Han individuals.In silico missense prediction by CADD（24.5）,Mutation Taster（0.999）,and Provean（-3.62）showed that the p.E396K mutation may be deleterious to the function of phospholipase.PLCδ1,a member of phospholipase C family,is responsible for hydrolyzing PIP₂ into two second messengers,IP₃ and DAG,which transduce important signals involved in a variety of cellular responses.First,the effect of the mutation on phospholipase activity was detected,studies on the activity of prokaryotic and eukaryotic PLCδ1 shows the p.E396K mutant upregulated the activity of PLCδ1,displayed a stronger capacity in hydrolyzing PIP2 and enhanced production of IP₃and DAG.Aa the second messenger,IP3 and DAG activate PKC directly,PKD/ERK1/2 as the downstream signal of PKC is essential for the development and maintenance of epidermis,the abnormality of this cascade will lead to the imbalance of cell proliferation and differentiation.Our study showed the p.E396K mutation can enhance the phosphorylation of PKD and ERK1/2 caused by extracellular calcium,while the inhibitor of PKC（G?6983）or PKD（kb NB 142-70）could inhibit the phosphorylation of PKD/ERK1/2 and ERK1/2,respectively,indicating the p.E396K could activate the PKC/PKD/ERK1/2 cascade.The effect of the activation of the cascade on proliferation was further examined,Q-PCR showed expressing the p.E396K mutant downregulates the differentiation genes（KRT10,FLG,LOR,IVL）and upregulates the proliferation genes（p63,CCND1）in vitro.MTT experiments further found that cells expressing the mutant have stronger viability than WT,while the inhibitor of PKD or ERK1/2 can effectively extenuate the proliferation promoted by the mutant.Calcium accumulation is the typical feature of pilomatricoma,the activation of PLCδ1induce the depletion of PIP2,while the activity of the TRPV6 calcium channel located at the membrane depends on PIP2.The effect of the mutants on TRPV6 electrophysiological activity was checked by patch-clamp,the TRPV6 closed faster in cells expressing the mutant than WT ones,and the mutant also inhibited the open of TRPV6,the influx of calcium was significantly weakened,the accumulation of Ca²⁺may be the cause of the calcification of pilomatricomas.To verify the data above in vivo,Plcd1 ^E396K/E396K knock-in mice were introduced in the study,Q-PCR shows the mutant downregulates the differentiation genesand upregulates the proliferation genes.Meanwhile,MTT experiments demonstrated that the primary keratinocytes of Plcd1^E396K/E396K mice have stronger viability than WT.Spontaneously developed tumors were observed in the skin of 54.5%（6/11）Plcd1^E396K/E396K mice by 8months of age,the tumors can reach 1cm in diameter by 12 months of age.The pathological analysis revealed that the tissue contained a lot of cells with shrunken nucleus,shadow cells,and obvious calcium accumulation in the sections,the expression patterns of bcl-2 and CD34 were similar as the human pilomatricoma,the Plcd1^E396K/E396K mice successfully mimic the human pilomatricoma-like phenotype.These data from the model animals prove that the mutation of PLCD1 may be the cause of pilomatricoma.Taken together,we identified the mutation c.1186G>A（p.E396K）of PLCD1 by genetic analysis in a large multiple pilomatricoma family,the variant could increase the activity of PLCδ1 and promote proliferation by activating PKC/PKD/ERK1/2 cascade.The mutation could also influence the cellular calcium absorption by inhibiting TRPV6channel.The skin of Plcd1^E396K/E396K mice not only showed stronger viability but also developed tumors spontaneously,successfully simulating the phenotype of human pilomatricoma.The study unveils the first germline genetic cause PLCD1 and the possible pathological mechanism of familial multiple pilomatricomas,which will benefit the follow-up research and treatment of pilomatricoma.