Identification And Functional Analysis Of Causative Gene In An Autosomal Recessive Family With Lipid Deposition And Joint Contracture

Rare diseases are also known as "orphan diseases".According to the definition of the world health organization(WHO),the percentage of patients suffering from rare diseases is 0.65‰?1‰ of the total population.Worldwide,5000?6000 rare diseases have been identified,accounting for about 10%of human diseases.About 80%of rare diseases are caused by genetic defects.The disease usually progresses rapidly and has a high mortality rate,while less than 1%of rare diseases can be effectively treated with drugs.Our research group collected a consanguineous family in Shandong province,including 3 normal people and 1 patient.The patient was hospitalized for distal myasthenia.Lipid infiltration was found in the muscle tissue section.He was initially diagnosed as lipid deposition with joint contracture disease.Blood samples of four family members were collected and DNA was extracted for whole exome sequencing.Considering the particularity of inbreeding families,variants are heterozygous in parents and homozygous in patient were listed as candidate.We firstly found out the frequency in ExAC,1000 genomes,and dbSNP database,variations with frequency more than 1%is considered to be polymorphic.MASP2(c.A464G,p.H155R,ExAC_EAS:0.0282)and PDSS1(c.T407g,p.F136C,ExAC_EAS:0.0221)were excluded for excessive frequency.Subsequently,we analyzed the conservativeness of amino acid changed by variants,and used bioinformatics software including PolyPhen2,Mutation Taster and SIFT to predict the pathogenicity of missense mutations.It was found that histidine of MASP2 and phenylalanine of PDSS1 were not conserved in the process of evolution.Finally,the STR linkage analysis was used for co-separation verification,and only C1ORF50(c.415-1G>C)co-segregates with disease in this pedigree.According to the analysis,the mutation in splicing site of the fourth intron will result in the detention of the fourth intron in mature mRNA,and the protein will be reduced from 199aa to 154aa.RNA was extracted from muscle of the patient and reversed transcription PCR confirmed that the mutation did lead to abnormal splicing of the mRNA.The background of C1ORF50(Chromosome 1 Open Reading Frame 50)is almost blank,so we firstly detected the expression profile of CIORF50,C1ORF50 is highly expressed in liver.Subsequently,immunofluorescence experiments showed that C1ORF50 was expressed in the cytoplasm of HeLa cells and Chang liver cells,and this expression was not organelle specific.Combined with functional analysis,we speculated that it may regulate energy metabolism by affecting mitochondrial function.To verify the pathogenicity of C1ORF50 and further explore gene function,we constructed A U022252 knockout mouse model by CRISPR/Cas9 system,preliminary data show that the knockout mice have decreased motility.But there are no significant pathological changes in gastrocnemius of KO mice.By transcriptome analysis of cell lines with high expression of C1ORF50,we found that after overexpression of C1ORF50,the level of FASN,ACSL3,ACSL4,ALDH2 was decreased,and we verified this trend by qPCR in both vivo and vitro,suggesting that C1ORF50 may have an effect on fatty acid metabolism.Based on the above,we aim to reveal the pathogenesis of the disease in this pedigree,and the potential functions of C1ORF50 in the process of neurogenesis,lipid metabolism,energy metabolism,motor control and others.