The Feasibility Study Of Preimplantation Genetic Diagnosis For DMD By Multiple Displacement Amplification Combined Haplotyping

Preimplantation genetic diagnosis (PGD) is a new genetic diagnosis technology, developing with assisted reproductive technology. PGD is performed before pregnancy, and then embryos without genetic disorders are transferred to mother's uterus. So it prevents the occurrence of inherited diseases. Duchenne muscular dystrophy (DMD) is an X-linked recessive and neuromuscular inherited disease which has a high frequency, with high attack rate and mortality rate. There is no utility therapy process up to now, so the main method to prevent birth of the DMD patients is prenatal diagnosis. PGD of DMD can not only prevent birth of the DMD patients, but also greatly help to reduce the physical and mental pressure caused by elective abortion and termination of gravidity of the abnormal fetus among pregnant women. So PGD is very significant in the precaution of DMD. But the stuff in PGD usually only has one or two cells, which is limited and prevents the application of PGD of DMD, and sexing using FISH would induce discarding of normal male embryos. So the establishment of a feasible single cell amplification and diagnosis technology is a key step in carrying out PGD of DMD in clinical practice.Objective:To study the feasibility of multiple displacement amplification (MDA) combined haplotyping in DMD-PGD, and to establish an efficient and safe PGD of DMD.Method:8 STRs with high heterozygosity and 3 sexing markers were chosen, and MDA-PGH was preformed at 12 couples without DMD family history. After whole genome amplification of single blastomeres by MDA, haplotyping was taken. High- and low-risk haplotype were supposed randomly, and then performed genetic analysis on the embryos.Result:19 embryos were obtained, including 11 embryos with 2PN, 1 embryo with 1PN,2 embryos with 3PN, and 5 embryos with OPN.51 blastomeres were obtained, in which 1 bastomere was contaminated in MDA,6 MDA failed, MDA from single blastomeres success rate was 86.3%(44/51). All 44 blastomeres with success MDA were haplotyped, with 100%(44/44) success rate of haplotyping and 86.3%(44/51) success rate of MDA-PGH. In 33 blastomeres from embryos with 2PN,28 MDA succeeded, with 84.9%(28/33) success rate of MDA-PGH, the average ADO and AF were 25.3% and 3.2%, respectively. On the hypothesis that the certain haplotype of mothers had the mutation of deletion of exon3-11 and exon44-45 of DMD, the haplotyping results of 25 blastomeres showed that embryos were normal male or female carriers and the other 3 were haploid. In 18 blastomeres from embryos with 1PN,3PN and OPN, 16 MDA succeeded, with 88.9%(16/18) success rate of MDA-PGH.Conclusion:1. In this research we got 86.3%success rate of MDA, and 100% success rate of haplotyping, which confirmed the feasibility of MDA combined haplotyping in clinical practice of PGD.2. For PGH of DMD,100% success rate of haplotyping could be got when 4 or 8 STRs were chosen on the basis of mutation, but at least 2 STRs should be fully informative in every haplotype.3. Haplotyping could identify DMD female carrier and normal male, bypassing the discarding of normal male embryos following sexing by FISH.