Identification And Investigation On The Novel Gene Mutations From Two Different Genetic Metabolic Diseases

Inherited metabolic disorder refers to a kind of genetic defects that result in metabolism problems.Most of them are single gene genetic disorders caused by the abnormal genes encoding enzymes or receptors that maintain the normal metabolism of the body.Reed syndrome is a rare autosomal dominant inherited disease,patients often have skin leiomyoma,and the risk of kidney cancer is much higher than that of ordinary people.The FH gene plays an important role in Reed syndrome,but there are few reports on how FH mutations cause this disease.In this study,we identified a novel FH mutation c.557G>A(p.S186N)in a family with uterine leiomyoma of Reed syndrome and conducted a series of studies to confirm the pathogenicity of the mutation.Sanger sequencing were performed to detect the gene mutation of the patient with Reed syndrome and a novel FH mutation c.557G>A was identified.Therefore,this study will focus on the pathogenesis of the FH mutation of c.557G>A.Bioinformatics,biochemistry,and biology methods were used to analyze the structure and function of mutant;both p EGFP-FH-WT and p EGFP-FH-MT were constructed;the formation of FH tetramer and the cellular localization of the mutant were analyzed;cell proliferation,fabric acid hydrates activity and extracellular acidification rate value were measured with the stable overexpression cell lines;the impact on cancer related phenotype and signaling pathway were detected by western blot analysis.Results showed that FH mutant exhibited significantly lower fumarase enzyme activity compared with the wild type that might due to the impaired homotetramer formation in the native gel electrophoresis.Interestingly,immunofluorescence study revealed that the overexpressed FH mutant exhibited punctual structures compared with the evenly expressed FH wild type in cytoplasm suggesting that the altered amino acid might resulting in the dysfunctional proteins which were accumulated to reduce its cytotoxicity.Importantly,the cells overexpressing FH mutant exhibited higher proliferation and extracellular acidification rate value(ECAR)which might cause from the unregulated HIF-1? indicating the tumor phenotype.Notably,phospho-m TOR was significantly increased and autophagy was inhibited in FH mutant overexpression cells compared with the wild type.Our work revealed the molecular mechanism causing Reed syndrome by the novel FH mutation c.557G>A(p.S186N)for the first time and provide important information for accurate genetic counseling and clinical diagnosis of the disease.Congenital glycosylation disorder(NGLY1-CDDG)is a rare autosomal recessive genetic disorder.The clinical symptoms of the patients contain drooping eyelids,weak muscles,red eyes and bloodiness.The NGLY1 gene plays an important role in glycosylation.However,there are few studies on congenital glycosylation disorder caused by NGLY1 mutation.Whole exon sequencing and Sanger sequencing were performed to detect the gene mutation of the patient with the disease.It and found that both allele of the patient carried novel NGLY1 mutation c.1231G>A and c.1405G>A,respectively.Therefore,this study focused on the pathogenic mechanism of the two novel mutations.p EGFP-NGLY1-WT and p EGFP-NGLY1-MT were constructed and localization of the mutants was analyzed by immunofluorescence.In order to further explore NGLY1 function,lent virus packaging system,screen of stable knocking down cell line were performed;co-immunoprecipitation(Co-IP)was used to find the interaction partners of NGLY1 protein.to determine whether zebrafish can be used as an animal model to study the disease,sequence alignment,homology modeling and phylogenetic analysis were used to compare the evolutionary conservation of zebrafish Ngly1 from different species;q RT-PCR analysis were performed to detect the spatial and temporal expression of zebrafish ngly1.The results showed that overexpressed NGLY1 mutant exhibited punctual structures compared with the evenly expressed NGLY1 wild type in cytoplasm.Western blot assay confirmed that the stable knocking down NGLY1 HEK293 T cell line was successfully constructed.During embryogenesis,zebrafish ngly1 m RNA was ubiquitously expressed at all developmental stages including 2 cells,24 h,48h and 72 h,but highest expression exhibited in 5d.In adult fish,the ngly1 m RNA was most abundantly expressed in ovary and testis compared with other tissues,however,a lowest expression level of ngly1 gene was observed in liver.These results provide new information about the structural conservation,spatial and temporal expression,and lay the foundation for future functional studies of Ngly1 in a fish model organism.To summarize,the patients with Reed syndrome and congenital glycosylation disorder were recruited as the research object,and whole exon sequencing and Sanger sequencing were performed with the blood of the patients to identify and verify the novel gene mutations.Bioinformatics,biochemistry,molecular biology and cell biology methods were used to study the pathogenicity mechanism of both FH mutation and NGLY1 mutations.Meanwhile,the biological characteristics of zebrafish ngly1 gene was cloned,expressed and identified.Our results will provide theoretical basis for the clinical diagnosis of Reed syndrome and congenital glycosylation disorder.