Molecular Cloning And Characterization Of Mtsarg1-β Mutation Detection Of Polycystic Kidney Disease Gene 1 (PKD1)

Most human diseases are related to gene variation and finding gene variation has great significance to the diagnosis,prevention and treatment of diseases.Genetic diseases include gene disease and chromosome disease. The aim of clinical genetics research is to diagnose and prevent genetic disease.The manuscript is to study the genetic basis of male infertility related to spermatogenic gene and autosomal dominant polycystic kidney disease beginning from the direction of clinical genetics.Considerable male infertility is related to spermatogenesis and spermatogenesis is controlled by many genes.In part 1,Mtsarg1-β,a novel transcript variant of Mtsarg1(also called Spata3,Spermatogenesis associated 3),was cloned and the characterization of Mtsarg1-βand Mtsarg1 were analyzed.In addition,some patients with polycystic kidney disease(PKD)ask for genetic consultation in our clinic and genetic diagnosis of patients before they have baby are demanded.In part 2,mutation detection of polycystic kidney disease gene 1(PKD1)in PKD patients were performed firstly in our institute using PCR-DHPLC or SSCP and DNA sequencing methods.A new nonsense mutation,a missense mutation and a polymorphism were identified in PKD patients.Two new polymorphisms were identified in mormal control.Part1:Molecular cloning and characterization of Mtsarg1-βGenetic factor play an important role in male spermatogenesis. Spermatogenic disfunction caused by genetic deficiency including gene mutation and chromosome abnormality accounts for above 30%male infertility and considerable male infertility has unidentified genetic variation.The proliferation and differentiation of spermatogenic cells are controlled by many factors.The regulation of gene in spermatogenic cells plays a pivotal role in spermatogenesis.Spermatogenesis is accompanied by testicular germ cell apoptosis and the apoptosis is also controlled by many genes.The findings of new genes related to spermatogenesis or testicular germ cell apoptosis are important for further understanding physiological and pathological mechanism of spermatogenesis and for the preventing,diagnosing and treating male infertility and testicular tumors.In our previous studies,24 expressed sequence tags(ESTs)of spermatogenic cell apoptosis-related genes have been cloned using suppression subtractive hybridization(SSH)method by establishment of a mouse cryptorchid model by Hong Jiang.Mouse testis and spermatogenesis cell apoptosis related gene 1(Mtsarg1,GenBank accession number:AF399971,2002;redesinated:Spata3,Mus musculus spermatogenesis associated 3,2004)cDNA sequence with 1103 bp in length had been cloned previously by the bioinformatics and experiment technique from mouse testis cDNA library,using one of ESTs(GenBank accession number:BE644538)as an electronic probe by Junjiang Fu.In the present study,in cloning Mtsarg1 we identified a novel transcript variant of Mtsarg1 with 887 bp in length,designated as Mtsarg1-β(Spata3 variant 4,GenBank accession number:EU259321,EF546784,2007)by reverse transcription-polymerase chain reaction(RT-PCR),T-A cloning and sequencing and analysed their characterization.Mtsarg1-βwhich has high similarity with Mtsarg1 contains an entire open reading frame of 417 bp encoding a protein consisting of 138 amino acids.Mtsarg1-βprotein is a non-secretory protein with a theoretic molecular mass around 14.79 kD and an isoelectric point of 9.74,which shares the 100 N-terminal amino acids with Mtsarg1 followed by 38 amino acids differing from Mtsarg1.Multi-tissue RT-PCR results and Northern blot analysis for adult DBA/2 mice showed that Mtsarg1-βand Mtsarg1 mRNAs were specifically expressed in testis at high level.RT-PCR results also showed that Mtsarg1-βand Mtsarg1 mRNAs were not expressed in mouse GC-1 spermatogonia.In situ hybridization revealed that both Mtsarg1 and probably Mtsarg1-βmRNAs were mainly expressed in mouse spermatocytes.Subcellular localization analysis suggested that Mtsarg1 protein was mainly localized in nucleus while Mtsarg1-βprotein was mainly localized in cytoplasm.Prokaryotic expression system pET21-a(+) was used to express Mtsarg1 encoding product.All these results indicate that Mtsarg1 and Mtsarg1-βmay play an important role in mouse testicular function and in spermatocyte development.It is only a primary reseach due to time limited and the further research on functions of Mtsarg1-βand Mtsarg1 will be carried out in future.Part2:Mutation detection of polyeystie kidney disease gene 1(PKD1)Objective Gene diagnosis was carried out in patients with polycystic kidney disease(PKD)to find the cause of PKD by mutation detection of polycystic kidney disease gene 1(PKD1)which is the common disease-causing gene of autosomal dominant polycystic kidney disease (ADPKD).Methods Polymerase chain reaction(PCR)was performed to amplify 12 exons(35～46 exons)of PKD1 3' single copy region in 12 PKD patients and related relatives using corresponding primers and patient's genomic DNA as template.Mutation detection of the PCR products were proceeded by using denaturing high-performance liquid chromatography(DHPLC)or single strand conformation polymorphism(SSCP)analyse.DNA sequencing was carried out on PCR products with abnormal peak shape afterwards.Results PKD1 mutations were found in 3 patients out of 7 patients screend by PCR-DHPLC and DNA sequencing.A new nonsense mutation, C11901A in exon 42 of PKD1,was identified to cause serine in position 3897 turn to a stop codon in patient 1.A missense mutation,C10737T,was detected in exon 35 which caused threonine in position 3509 turn to methionine in patients 2.The missense mutation was previously reported in PKD patients.A samesense mutation,G11470C,were detected in exon 39 in patient 3.Two kinds of samesense mutation,G11824A and C11860T in exon 42,were found in normal control.No mutation in 5 patients was detected by PCR-SSCP and DNA sequencing.Conclusion PKD1 mutation were detected successfully by PCR-DHPLC or SSCP and DNA sequencing.A new nonsense mutation,a missense mutation and a polymorphism were identified in polycystic kidney disease patients.Two new polymorphisms were identified in mormal control.The nonsense mutation in patient can cause that polycystin-1(PC-1)encoded by PKD1 gene change to a truncated protein which is short of 406 amino acids presenting in PC-1.The same mutation has not been detected in 60 normal control.It suggests that the mutation which has only been detected in patient is mostly related to polycystic kidney disease.It can not been excluded that there is other mutaton exsisting in other region of PKD1 gene which was not detected yet.