The Identification Of PHKG2 Gene Mutation In A Patient With Glycogen Storage Disease Type ?c And Functional Study Of Novel PHKG2 Gene Mutation

Background Glycogen storage disease type IX?GSD IX?is caused by a deficiency in phosphorylase kinase?Ph K?,which plays a major role in regulating the breakdown of glycogen.Ph K is a hexadecameric enzyme complex that is composed of four copies each of four subunits ?,?,? and ?.It is one of most common forms of glycogen storage disease.GSD IX is genetically and clinically heterogeneous and known to have four subtypes:IXa,IXb,IXc and IXd.Glycogen storage disease type IXc?OMIM 613027?is a rare form of GSD IX.GSD IXc is an autosomal-recessive disease caused by gene PHKG2 mutations which encodes hepatic isoform ? unit of Ph K.Glycogen storage disease type IXc presents infancy onset and is characterized by hepatomegaly,growth retardation,increased serum transaminases,fasting hypoglycemia,lactatemia,acidosis,hypertriglyceridemia,hypercholesterolemia and an increased risk for developing liver fibrosis or cirrhosis in childhood.Patients of GSD IXc are treated with uncooked cornstarch and monitored of blood glucose regularly.The PHKG2 gene on chromosome 16p12.1 has 10 exons spread over 9.5kb.Thirty-one mutation have been reported in the PHKG2 gene that include missense,nonsense,small insertion and deletion and splice site changes.There are a total of 27 patients that have been reported so far,most of cases are from Europe and Americas,3 from Saudi cases and 2 from Japanese cases in Asia.There is no any GSD IXc case report yet in china.Maichele et al.had studied PHKG2 gene mutation in gsd rat on the first time,however,there was no research on the function of PHKG2 gene mutation in vitro at all.To further expand our knowledge of GSD IXc,we collected clinical data of GSD IXc patient,identified two novel PHKG2 Gene mutations in the family,compared case phenotype with genotype.We also evaluated the functional effect of the two novel mutations in gene PHKG2 verified that mutations can cause m RNA and protein changes.Our study provides a new insight on the molecular mechanism of GSD IXc and will benefit future,genetic counseling,prenatal diagnosis and the interaction between Ph K and other substances.Objective:To identificate PHKG2 Gene mutation in a patient with Glycogen Storage Disease type IXc.To investigate the clinical characterization of GSD IXc and the pathogenicity of PHKG2 gene novel mutations.To explore the expression and mutant protein by transient expression system in vitro.Method1.Retrospective analysis of clinical data of GSD IXc patient.2.The mutations of PHKG2 gene were detected by using the whole genome sequencing and conformed by Sanger sequencing.3.Using SIFT,Polyphen-2 online software and the amino acid conserved analysis of12 species to predict the pathogenicity of novel missense mutation.4.Using Py MOL software to predict the pathogenicity of new missense mutation.5.The total RNA was extracted from the patient and the expression of PHKG2 m RNA level was detected by q RT-PCR.6.In this study,we used the expression of enhanced green fluorescent protein plasmid pc DNA3.1-EGFP as empty vector to construct pc DNA3.1-PHKG2-EGFP wild-type vector,then sited-mutagenesis into novel mutations corresponding mutant vector in vitro and transfected into COS-1 cell.We established vitro eukaryotic expression system of PHKG2 gene,and analysis of m RNA expression by q RT-PCR and protein expression by immunofluorescence and Western blotting.Result1.The GSD IXc patient presented at newborn and was characterized by the hepatomegaly,elevated liver enzymes,fasting hypoglycemia and acidosis.2.Two novel mutation were found,one missense c.698T>C?p.Phe233Ser?and one insertion mutation c.957₉58ins GG?p.Gly319 Glyfs X6?.12 different species of the missense mutation F233 S in the amino acid conservative analysis suggested highly conserved.3.Pathogenicity prediction of software: the new missense mutation F233 S was carried out SIFT and Polyphen-2 software,then predictions showed that the mutation of SIFT rate was closed to 0,Polyphen-2 scores was closed to 1.4.The prediction of the three-dimensional structure of new missense mutant protein:F233S changed the connection of the Loop with other Amie acids,and formed an unstable hydrogen bond with M241 of H9.5.The expression of Erna in peripheral blood: the expression of PHKG2 Erna in patient was significantly lower than that of the peers.Compared with the same sex adults,the Erna expression level of the parents was significantly higher than that of the same age group.6.The function of mutation in vitriol: the level of F233 S m RNA was reduced and G319 fs X remained unaltered and the level of F233 S and G319 fs X protein decreased.Conclusions1.It was the first case of GSD IXc that was reported in China,manifested hepatomegaly,elevated liver enzymes,and acidosis.2.Two novel mutations of PHKG2 gene were found.This study enriched the gene mutation profile of GSD IXc.3.Pathogenicity prediction of new mutation by bioinformatics software suggested probably damaging.4.The m RNA expression of PHKG2 gene with peripheral blood of the patient family was analyzed for the first time.The first time using transfection methods to study the function of PHKG2 gene mutations and was used to identify the mutant protein pathogenicity.