The Study Of Gene Diagnosis Of Monogenic Diseases And It's Application

As its large varieties of kinds and high incidence rates, monogenicdisease not only severely affected the patients' health, but also brought aheavy mental and economic burden to their families and the society. By genediagnosis techniques of genetic conditions, we can perform gene diagnosis ofdifferent growth stage individual. Not only genetic condition patients andpresymptomatic patients could be diagnosed after birth, but also prenatal fetuscould be diagnosed (prenatal diagnosis,PND), even preimplantation embryocould be diagnosed (Preimplantation genetic diagnosis,PGD). Therefore, wecan take therapeutic measure and provide the suggestion of marriage andfertility for patients and presymptomatic patients in time. According to theresult of prenatal gene diagnosis of high risk pregnancy, pregnancy ofabnormal embryo could be terminated selectively and to infant of geneticcondition could be avoided. And that PGD screens out the normal embryos ofwoman to transfer before pregnancy, so the healthy infant was born, whichavoided physical and mental trauma to the gravida that caused by repeatedselective abortion. For couples, at risk of transmitting a genetic condition,have one more choice preventive measure.To totally avoid the birth of infants with monogenic diseases, we aim toconsummate and plent available general gene diagnosis and prenatal genediagnosis techniques in our institute, and to set up a technic platform of singlecell PCR diagnosis of monogenic diseases such as achondroplasia andβ-thalassemia, basis of our existent gene diagnostic techniques and the matureassitant reproductive techniques, and then to use these techniques in clinictreatment further, to develop PGD of monogenic diseases and to lay down afoundation for PGD of numerous monogenic diseases. Increasing thepreventive methods of genetic conditions continuously, not only used todiagnosis of genetic condition patients and presymptomatic patients after birth,but also used to preventing the transmission of genetic conditions from theprenatal level and even from the preimplantation level and decrease the birthof genetic condition patients.1. Gene diagnosis and prenatal gene diagnosis of hemophilia A by acombination of St14 (DXS52) VNTR polymorphism and (CA)n repeatpolymorphism within FⅧ geneThe huge gene structure of hemophilia A and the complexity of its genedefect had brought certain degree of difficulty to its direct gene diagnosis. Soa linkage analysis method based on the (CA)n repetition and VNTRpolymorphism in and out the hemophilia A gene will become a quick, easyand efficient gene diagnosis method for HA. We performed the indirect genediagnosis of the family with HA using the linkage analysis of the above 2tandem repeat polymorphic loci. The results indicated that using one of theabove 2 polymorphic loci to perform linkage analysis of 9 pedigrees with HA,the diagnosis rate was 66.7%;while using these 2 loci together could improvethe diagnosis rate to 88.9%. We also performed the prenatal gene diagnosis of4 pedigrees with HA. When we analyse locus St14, the recombination couldbe detected by using (CA)n polymorphism. For this way, we could avoid aprobable prenatal misdiagnosis only using VNTR polymorphic linkageanalysis at locus St14. The results indicated that using the above 2polymorphic loci together could make quick and accurate gene diagnosis andprenatal gene diagnosis to 90% pedigrees with HA too.2. Rapid gene diagnosis and prenatal gene diagnosis in families withDMD/BMD by linkage analysisSince the dystrophin gene of Duchenne/Becker muscular dystrophy(DMD/BMD) has a huge gene structure, involving 79 exons. 60% mutationsare deletion or insertion mutations and 40% are non-deletion or microdeletionmutations. PCR, using 9 pairs of primers, could detect 80% patients withdeletion mutation, and so there still left more than 50% patients withDMD/BMD couldn't be diagnosed. To consummate the diagnosis system ofDMD/BMD, we established the linkage analysis technique to quickly detectcarrier and perform prenatal gene diagnosis in the families with DMD/BMD.Using PCR and polyacrylamide gel silver staining method to analyse thepolymorphism of the (CA)n short tandem repeat (STR) in 3' end of DMDgene and in the introns 45, 50 of DMD gene, 10 pedigrees with DMD wereperformed the linkage analysis, the diagnosis rate was 70%;3 pedigrees withDMD were also performed the prenatal gene diagnosis. These resultsindicated that STR polymorphic linkage analysis was specially suitable todetect patients with DMD/BMD which were not found by common deletiondetecting method, and to screen carriers in families in which the patient hasdied. It also could be used to the gene diagnosis of DMD/BMD patients withdeletion type. The linkage analysis method is a benefit supplement of thedeletion type detection method, which is a kind of quick, effective, accurateand easily to be populated gene diagnosis method,it can improve effectivelythe diagnosis rates of gene diagnosis and prenatal gene diagosis in pedigreeswith DMD/BMD.3. The establishing of diagnostic technique platform of β-thalassemia bysingle cell duplex-nested PCRTo develop the clinic PGD of β-thalassemia, its diagnostic techniqueplatform at single cell level should be set up firstly to know the efficiency ofsingle cell PCR amplification and diagnosis. We chose a strategy of detectingsingle cell specimen through duplex-nested PCR together with amplificationrefractory mutation system (ARMS) technique to make sure the genotype ofcomprehensive β-thalassemia mutation spectrum in Chinese population. Westudied the optimizaiton, accuracy and reliability of this technique bydefinition the genotype of five common mutation loci of β-globin gene in thecarrier with CD41-42 mutation and that of β-globin gene of normal people'ssingle lymphocyte and single blastomere. TaKaRa EX Taq was the mostefficient Taq DNA ploymerase during different Taq DNA ploymerases;primer pair R1+F1 at final concertration of 0.25μM and R2+F2 at 0.3μM werethe most efficient ones in amplification;the amplification efficient rate ofneutralization buffer-1 (200mM Tricine) was obviously higher than that ofneutralization buffer-2 (900mM Tris-HCl, PH 8.3/300mM KCl/200mM HCl)(P<0.05);predenaturation at 98℃ before the single cell PCR componentbeing added could not improve the PCR amplification rate;the amplificationsuccess rate of 88 single lymphocytes was 95.5% (84/88), the incidence ofADO was 4.8% (4/84), and the diagnosis accurate rate was 95.2% (80/84);theamplification success rate of 34 single blastomeres was 88.2% (30/34);thepseudopositive amplification rate of 17 tubes of blank with washing cellsolution was 0. These results indicated that the single cell gene diagnosistechnique set up by us which was suitable to detect the mutations at lociCD41-42, CD17, nt.-28, CD71-72 and IVS-Ⅱ654 and was efficient, simpleand quick, and had broad detecting mutation spectum and high accuracy,which laid a solid foundation for our advance developing of clinical PGD ofβ-thalassemia.4. The establishing of diagnostic technique platform of achondroplasia bysingle cell nested PCRTo study the reliability of diagnosing achondroplasia (ACH) at the singlecell level and to lay down a foundation of clinical PGD of ACH, The highfrequently mutation region G380R of FGFR3 gene was amplified bynested-PCR with single lymphocyte and single blastomere. The products ofPCR were digested by restriction enzyme Bfm I, then the digested productswere detected by 10% polyacrylamide gel electrophoresis (PAGE). Resultsindicated that the amplification success rate, allele dropout rate and correctdiagnosis rate of single lymphocyte's PCR were 90.4%, 8.2% and 91.8%,respectively, and the amplification success rate of single blastomere's PCRwas 75.4%. This indicated that our diagnostic technique platform ofachondroplasia by single cell PCR was comparatively stable and reliable,which laid a solid foundation for our further developing PGD of ACH.5. The preimplantation genetic diagnosis (PGD) of achondroplasia (ACH)Based on our setted diagnosis technique platform of achondroplasia bysingle cell nested PCR, we accomplished one PGD cycle of a couples whosesterility was caused by male factors such as severely oligozoospermia andasthenospermia, and the male is a ACH patient. We biopsied 1-2 singleblastomere from 6-8 cell cleavage stage intracytoplasmic sperm injection(ICSI) embryo, and using nested PCR amplified the high frequently mutationregion G380R of FGFR3 gene of the single blastomere. The products of PCRwere digested by restriction enzyme Bfm I, then the digested products weredetected by 10% PAGE to see whether the embryo inherted the mutation ofthe patient and to screen out normal embryo transfer. The results of detectingthe single blastomere from 11 embryos showed that 3 were normal, 5 wereabnormal and 3 turned no results. There were 8 embryos' single blastomereamplified, and the amplification success rate was 72.7% (8/11);3 embryos'not amplified;2 embryos' showed preferential amplification (PA), and the PArate was 25%(2/8);and the 7 tubes of blank with washing cell solution werenot contaminated. We transferred 3 unaffected embryos to the uterus. Butunfortunately, blood hCG was negative after 2 weeks of transferringindicating no pregnancy. The repeated detection of single blastomere ofuntransferred embryos verified the PGD results, and further indicated that ourPGD technique of ACH was relatively stable and reliable.According to the above, the establishing of new gene diagnosis andprenatal gene diagnosis methods of DMD/BMD and hemophilia A could getthe results of gene diagnosis more quickly and more accurately, and it also canimprove the diagnosis rates of the genetic conditions. The establishing of newdiagnostic technique platforms of monogenic genetic conditions based onsingle cell PCR has a wide application prospect in clinical PGD of geneticconditions, and can provide an advanced method to prevent the geneticconditions more actively and more initiatively. The clinical application ofPGD of ACH has accumulated experience for us to developing clinical PGDof other monogenic genetic conditions further. Gene diagnosis being drivenfrom gene diagnosis level after birth to prenatal gene diagnosis level and topreimplantation gene diagnosis level plent the prevention method of geneticconditions further. The spreading and application of these diagnosistechniques will play an important role in decreasing the birth of geneticcondition infants.