Development Of Techniques For Prenatal Genetic Diagnosis Of Common Aneuploidy And Monogenic Diseases

Prenatal diagnosis including preimplantation genetic diagnosis (PGD) can be used to avoid delivering of fetuses with severe genetic defects. In China, the techniques of prenatal diagnosis hasn't been fully developed so far. Pregnant women must wait a long time for their results of full karyotype analysis and we do not have suitable techniques for rapid prenatal diagnosis for common aneuploidy. The molecular pathogenic mechanism underlying most of fetal malformations remains to be discovered. In this study, we have conducted some exploring as follows:Part 1:Development of multiple quantitative fluorescent PCR for rapid diagnosis of commom aneuploidy and its clinical applicationTo establish a multiple quantitative fluorescent polymerase chain reaction (QF-PCR) method for trisomy 21 and trisomy 18 test and its potential clinical application.20 cases of trisomy 21,3 cases of trisomy 18 and 40 cases normal controls were used to establish multiple QF-PCR. Several short tandem repeat markers (STR-marker) were chosen from chromosome 21 and 18. The established methods were used for 165 cases of prenatal diagnosis (including 30 cases for hearing loss gene test) and for 4 cases newborn infants with digestive tract obstruction. Among 169 cases, we identified 4 cases of trisomy 21,1 case of trisomy 18. The results were concordant with those of karyotyping.22 cases fetuses with structure malformation were only performed karyotyping analysis.1cases of 45, X, and 1case of 47, XXY were identified.In conclusion, QF-PCR testing is an efficient, rapid and accurate technique for the detection of trisomy 21 and trisomy 18. It should be offered to pregnant women who were identified as having a high risk by serum screening and because of advanced maternal age. It can also be used for pregnant women who were undertaken other prenatal diagnosis. QF-PCR testing combines with karyotype analysis can provide better service for clinical demanding of prenatal diagnosisPart 2:Study of molecular mechanism of some kinds of fetal malformationMeckel-Gruber syndrome (MKS) is an autosomal recessive monogenic lethal disease, characterized by occipital encephalocele, renal cystic dysplasia, hepatic ductal proliferation, fibrosis and polydactyly. MKS is genetically heterogeneous with six loci and five identified genes. We ascertained a Chinese family with four affected fetuses. Three of them had typical characteristics of MKS excepted for polydactyly. We screened the candidate gene of MKS3 by cDNA direct sequencing in this family. We found a c.1645C>T mutation, changed the 549 amino acid of arginine to cysteine (R549C). The R549C mutation was confirmed in two siblings of the proband. All of family members were detected for this mutation and found the couple and one of their parents carried heterozygous R549C mutation. This mutation was not present in 200 normal controls,We also screened the hot mutated exons of FGFR3 gene for five fetuses with short limbs malformations. One fetus carried mutation of c.1108G>T (G370C) and another of c.1138G>A(G380R).They were both de novo mutations. These families have low recurrent risk. For one fetus, we also screened FGFR3, SLC26A2 and TRIP11 gene and found three benign single nucleotide polymorphisms (SNP).Part 3:The establishment of preimplantation genetic diagnosis (PGD) for Meckel-Gruber syndromeThe couple had consecutively got four affected MKS fetuses and they want PGD service to give birth to a healthy baby. We did some basic research for the purpose of providing the PGD service clinically. Multiple displacement amplification (MDA) was used to amplify the whole-genome DNA directly from a single cell of acquired from a heterozygote. MDA products were used for genotyping by real time PCR and for STR markers. The success rate were 85.7% with allele drop out (ADO) of 30.7% in real time PCR and were 80% with ADO of 16.7% in D8S270 genotyping. This protocol of PGD could be used for clinical application after modification.