Gene Mutation And RNA-Seq Analysis Of Progeroid Symdrome In A Chinese Han Ethnic Family

Part 1 The clinical features and disease related genes in progeroid patientsObjective We want to analyse the clinical features of the progeroid children by collected their clinical and biochemical data, and to find the related gene of this progeroid family by genetic analysis and gene detection of three progeroid children and their relatives.Methods The clinical and imaging data of the three progeroid children were collected. The peripheral blood were collected for blood routine test, blood biochemistry and autoantibodies test, chromosome G banding analysis, and DNA extraction for exon sequencing analysis of LMNA gene.Results All the three progeroid children showed typical progeroid phenotype, and simultaneously they showed increase in serum lipids, alkaline phosphatase, serum phosphorus and platelet. The imaging of the children showed micromandible, pyriform thorax, absence of clavicle, spinal deformity and so on. The chromosome G banding did not make any difference. All these features met to most of the HGPS patients. LMNA gene sequencing showed a heterozygous mutation in exon 9 (R527C) in parents but a homozygous mutations (R527C) in all 3 children in this family.Conclusion According to the clinical features and gene detection, the children were diagnosed with HGPS; This progeroid pedigree is caused by a homozygous mutation (R527C) in LMNA gene.Part2 RNA-seq analysis of the white blood cells in these HGPS patientsObjective To analyze the different expressed genes and the pathogenic molecular mechanism by RNA-Seq.Methods The peripheral blood of the three progeria patients was obtained, and the white blood cells were separated. Also the peripheral blood of four healthy children of the same age was obtained, and the white blood cells of these 4 healthy children were mixed together as a normal control group. All white blood cells were sent to BGI-Shenzhen for RNA extraction and RNA-Seq analysis.Results All RNA samples and the results of RNA-seq were qualified. There were 712,1175,1215 genes differentially expressed in three progeroid children(according to the age from young to old) compared with healthy controls(P< 0.05). The GO and KEGG pathway analysis of the different expressed genes showed that the genes related to mitosis progress, cell cycle pathway and the rheumatoid pathway and so on were activated.Conclusion The different expressed genes in this research were found in accordance with those obtained by other gene microarray analysis. Most of genes expressed differently in some pathways such as cell cycle and the rheumatoid in HGPS patients by RNA-Seq analysis.Part 3 Validation of the different expressed genes by qPCR and immunohi-stochemistry in the progeroid patientsObjective To detect the different expressed genes by qPCR and immunohistochemical method in progeria children.Methods The peripheral blood of three progeria patients and 11 healthy children of the same age was obtained, and their RNA were extracted, and the key genes expressed in rheumatic pathway such as CD80, FOS, IL8 and CXCL1 were detected by qPCR method. The skin lesions of the proband and 5 systemic lupus erythematosus patients,12 systemic scleroderma patients,9 localized scleroderma patients and 8 healthy persons skin tissue were collected and the expression of FOS in all groups were detected by immunohistochemical method.Results The qPCR results showed that the expression of CD80, FOS, IL8, CXCL1 in peripheral white blood cell of progeria patients increased and they confirmed to the results of RNA-Seq. The differences had statistical significance (P<0.05),except for CD80. Immunohistochemical analysis showed that the FOS expressed in progeria patients, systemic scleroderma and localized scleroderma patients and the difference had no statistical significance(P>0.05), but the expression of all three groups were higher than that of systemic lupus erythematosus group and the healthy group, and the difference had statistical significance(P<0.05).Conclusion The qPCR and immunohistochemical results are consistent to that of RNA-Seq analysis; The activation of rheumatoid pathway may be related to skin sclerosis in progeria patients.