Study On The Pathogenic Mutations Of Two Single-gene Inherited Diseases Of The Locomotion System

Part One:Mutation Screening of ChineseFamilies with HypophosphatemicRicketsBackground:Hypophosphatemic rickets?HR?is a genetically metabolic bone disease caused by abnormal reabsorption of phosphate in the proximal renal tubules and the increase of phosphate excretion.Main clinical manifestations of the disease include bone malformation,short stature,bone softening,tooth damage and painment of bone.At present,the inheritance patterns of HR are X-linked dominant?XLH?,autosomal dominant?ADHR?and autosomal recessive?ARHR?.XLH is the most frequent form of HR,accounting for 80%of familial HR cases,and the prevalence of XLH is 1/20000.The disease-causing gene PHEX was located at chromosome Xp22.11.HR has overlappedphenotypes with other rare bone diseases,so it is easily misdiagnosed.Genetic diagnosis is therefore the goldem standard for the diagnosis of this disease.Objective:We aim to identify the genetic mutations of 7 Chinese families with suspected hypophosphatemia rickets,and to understand the molecular mechanism;to expand the mutation spectrum of PHEX genes and to provide accurate molecular diagnosis of patients.Methods:Seven HR families were recuited in this study and their clinical examinations were collected.Peripheral blood was collected and genomic DNA or blood RNA was extracted using phenol-chloroform method or Trizol method respectively.Candidated pathogenic mutations were identified using whole exome sequencing or panel sequencing,followed by verification by Sanger sequencing.Results:Through PCR-sanger sequencing of PHEX gene,we found three novel mutations and three reported mutations,including a synonymous mutation c.591A>G in proband of family 1;a missense mutation c.980A>G and nonsense mutation c.1078A>T onexon 9 of PHEX gene in family 4;a splicing mutation c.1079+1 G>A in family 5;a small duplication c.1017 1051dup on exon 9 in family 6.By cDNA sequencing of PHEX gene,we found there was a 26bp deletion c.1661₁726del in family 2.Through high-throughput sequencing,a compound heterozygous mutation?c.1336G>A,c.1364T>C?on Exon 13 of SLC34A3 was observed in family 7.Conclusions:In this study,we screened out three novel mutations c.1661₁726del,c.980A>G,c.1017₁051dup on PHEX gene.By high-throughput sequencing we found a compound heterozygous mutation?c.1336G>A,c.1364T>C?on the gene SLC34A3 in family 7,finally diagnosed as hypophosphatemia rickets with hypercalciuria.Part Two:Mutation Screening of Chinese Families with Charcot-Marie-ToothBackground:Charcot-Marie-Tooth?CMT?is a hereditary peripheral neuropathy with high phenotypic and genetic heterogeneity,and the prevalence is approximately 1 in 3000.Current researches indicate that more than 80 genes are involved in the pathogenesis of CMT.The clinical manifestations include progressive muscular atrophy,gait instability,high arch and foot drop with anonset age before 20.Because the clinical phenotype of CMT overlaps with other neurological diseases,it is difficult to perform clinical classification and diagnosis.Therefore developing a mature molecular diagnostic platform would have great clinical guiding significance.Objective:We aim to identify the disease-causing mutations and further clarifythe cause of genetic change from five Chinese Charcot-Marie Tooth families,with a final goal to provide information for molecular diagnosis and genetic counseling of this disease.Methods:Clinical data and diagnostic information of the proband were collected,and the peripheral blood samples of the family members were used for the study.The genomic DNA of the patient was extracted by phenol-chloroform method.High-throughput sequencing platforms?panel sequencing,WES sequencing?was used to screenpathogenic mutations of probands within five families.The canadicate pathogenic sites were verified by Sanger sequencing and restriction enzyme fragment polymorphism,and then the conservation and pathogenicity of the variants were analyzed by bioinformatic tools.Results:We found two novel muations and three reported mutations,the pathogenic variation of family 1 was located in the MFN2 gene c.1190G>C and the mutation in family 2 was located in the HSPB1 gene c.539C>T;family 3 was MPZ gene c.404T>C;family 4 is GJB1 gene c.44G>A;family 5 is KIF1B gene c.2870G>A.Conclusion:This study identifiedtwo novel mutations responsible for hypophosphatemic Rickets,oneof them is c.1190G>C in MFN2 and the other is c.2870G>A in KIF1B,which laid the foundation for prenatal diagnosis and genetic counseling of these two families.