1. Non-invasive prenatal testing (NIPD)

11. NIPD Fetal Cells in Maternal Blood Trophoblastic Cells in Cervical Samples Free Fetal Nucleic Acids –Prediction of Fetal Rhesus Phenotype in Maternal Plasma –Prediction of Fetal Sex in Maternal Plasma New Clinical Applications –Genetic Disorders (trisomy, single gene disorder) –Obstetric Complications –Fetal RNA in Maternal Plasma

12. Fetal Cells in Maternal Blood In 1969, Walknowska et al. –Fetal lymphocytes in maternal peripheral blood Rarity of circulating fetal cells –several cells per milliliter of maternal whole blood Isolate these cells in sufficient quantities Ten-year multicenter study (1994–2003) by NICHD –male fetal cells in whole maternal blood were detected in 41% of pregnancies with male fetuses –false-positive rate of 11% Fetal cells other than erythrocytes –able to persist in the maternal circulation for years

13. Trophoblastic Cells in Cervical Samples prenatal diagnosis –fetal Rh (D) phenotypes –some single gene defects, Ex:thalassemia and sickle cell anemia marked variation in the proportion –between 4 to 80 percent at least minimally invasive

14. Free Fetal Nucleic Acids breakthrough in NIPD Cell-free fetal (cff) DNA found in 1997 After apoptosis of trophoblastic cells As early as 5 weeks Quantity of cff DNA increases with gestational age –median values of 15.9, 21.5 and 52.0 genome equivalents/ml of blood in the 1,2,3 trimesters cleared rapidly post partum

19. 血型的遺傳一般遵守孟德爾定律孟德爾定律 Rh 血型系統的遺傳 –Rh 是由第一對染色體上一對有兩個等位的基因所控制。 –Rh+ 是顯性， Rh- 是隱性

20. the accuracy of the RhD PCR system 10-ml blood samples from 30 blood donors RhD(+) 30 blood donors RhD(-) 10-ml blood samples from 57 women with singleton pregnancies –12 in the first trimester of pregnancy (7 to 14 weeks) –30 in the second trimester (15 to 23 weeks) –15 in the third trimester (37 to 41 weeks)

22. Real-Time Fluorogenic PCR Analysis Perkin–Elmer Sequence Detector (model 7700, Perkin–Elmer Applied Biosystems, Foster City, Calif.), –a combined thermal cycler and fluorescence detector with the ability to monitor the progress of individual PCR reactions optically. The RhD fluorogenic PCR system consisted of amplification primers RD-A (5'CCTCTCACTGTTGCCTGCATT3') and RD-B (5'AGTGCCTGCGCGAACATT3') and a dual-labeled fluorescent probe,RD-T (5'(FAM)TACGTGAGAAACGCTCATGACAGCAAAGTCT The primers and probe were targeted toward the 3' untranslated region (exon 10) of the RhD gene. The b -globin PCR system consisted of the amplification primers and probe as previously described. The fluorescent probes contained a 3'-blocking phosphate group to prevent extension of the probe during the PCR Combinations of primers and probes were designed with Primer Express software (Perkin– Elmer) Sequence data for the RhD gene were obtained from the GenBank data base (accession number, X63097).

23. Results RhD PCR system complete concordance between the results of RhD PCR genotyping and the serologic results. determine the sensitivity of fluorogenic RhD PCR analysis genomic DNA from an RhD- positive subject was diluted serially both in water and in 1 μg of genomic DNA from an RhD- negative subject. The smaller the amount of DNA, the more amplification cycles were needed

26. paternally inherited fetal alleles determination of fetal gender for sex-linked disorders paternally inherited mutations for autosomal dominant diseases –Myotonic dystrophy –Huntington disease paternally inherited single gene mutations –achondroplasia

27. Further Challenging areas for fetal chromosomal aneuploidies monogenic diseases other than those caused by unique paternally inherited mutations –maternally inherited genotypically identical to the background maternal DNA cannot be established by simply detecting its presence in maternal plasma –autosomal recessive monogenic diseases for couples harboring non-identical mutations Detection of paternal wild-type allele b-thalassemia and cystic fibrosis

32. NIPD of chromosomal aneuploidies Fetal DNA in maternal plasma is cell-free high maternal DNA background detection of molecular signatures are contributed almost completely by the fetus. –Circulating fetal DNA derived from placenta –Differential DNA methylation or expression profiles

34. SERPINB5 chromosome 18 serpin peptidase inhibitor, clade B (ovalbumin), member 5 hypomethylated in placental tissues completely methylated in maternal blood cells

35. determining the ratio between polymorphic alleles of hypomethylated SERPINB5 molecules in the maternal circulation. Heterozygous trisomy 18 fetus, the ratio between the SERPINB5 alleles would be 2:1 or 1:2 1:1 as in a heterozygous euploid fetus

36. it is the first strategy reported for the direct detection of chromosomal aneuploidies by cell-free DNA analysis from maternal plasma

38. Extensive searches for chromosome 21 loci which demonstrate differential methylation between placental tissues and blood cells have been conducted The low abundance of fetal DNA –constraint on the practical feasibility of epigenetic based approaches

40. fetal-specific, placentally expressed mRNA molecules are detectable in maternal plasma polymorphic alleles of placenta-specific 4 (PLAC4) mRNA –a transcript on chromosome 21 –more abundant than fetal DNA RNA–SNP allelic ratio test

42. Results 90% sensitivity and 96% specificity by using the PLAC4 RNA–SNP test alone this is comparable to many of the currently used multi-modality screening tests only informative for heterozygous fetuses A panel of coding SNPs is required to increase the population coverage of those tests

43. Digitizing cell-free fetal DNA analysis interference caused by the high maternal DNA background

45. 90+90+10+10=200 90+90+10+10+10=210

46. Digital relative chromosome dosage (RCD) analysis –The relative amounts of the two amplicons were compared relative mutation dosage (RMD) approach –compare the relative amounts of the maternal mutant and wild-type alleles in maternal plasma to determine the inherited dosage of the mutant allele.

48. massively parallel maternal plasma DNA sequencing

50. Lo and Chiu Nature Reviews Genetics 8, 71–77 (January 2007) | doi:10.1038 / nrg1982