Theoverallgoalofthisresearchistoprovideaflexibleprenatalgenetictestingkitthatcanbeexpandedtodetect anyinheritabletraitasearlyas5,andupto20,weeksofgestation,fromasafe,noninvasivePapsmear.Studies showthatperinatalPaptestsposenorisktomotherorfetus,andcapturetrophoblast-likecellsthatmigratefrom the placenta into the reproductive tract. Trophoblast retrieval and isolation from the cervix (TRIC) efficiently isolateshundredsoffetalcellswithoutlimitationsduetoearlygestationalage,maternalobesity,oruteroplacental insufficiencydisorders.InarecentScienceTranslationalMedicinereport,weisolatedsufficientgenomicDNA fromintactfetalcellsobtainedbyTRICat5-19weeksofgestation(n=20)todefinitivelydistinguishmaternaland fetal DNA by targeted next-generation sequencing (NGS) of 59 short terminal repeats (STRs) and 94 single nucleotide polymorphisms (SNPs). Compared to massively parallel sequencing of cell-free fetal DNA from maternalserum,whichhasafetalfractionofonly4-10%atweek10ofgestation,DNAobtainedbyTRIChada fetalfractionof85-100%,capableofprovidingnucleotide-specifichaplotyping.TRICwillbecommercializedto identifysinglegeneandchromosomenumberdisordersinaprenataltestfromPapsmears.Wewilldevelopa custommultiplexPCRplatformtosimultaneouslyamplifySNPsandSTRstoidentifyfetalDNA,aswellasloci acrossChromosome21(Chr21)todetecttrisomy21,Downsyndrome.Thisplatformwillbeexpandedtoother chromosomenumberdiseasesinPhaseII.Wewillaccomplishfourmilestones.1.Primerswillbedesignedand testedwithhumangenomicDNAtoamplifySTRs,SNPsandlociacrossChr21andChr1(reference),sequencing PCRproductsbyNGStooptimizetheiramplificationandco-amplificationinsingle-plexandmultiplexPCR.2. DNA isolated from fetal and maternal cells isolated by TRIC (N=50), as well as the corresponding newborn bloodspots(reference),willbecomparedbytargetedNGS.Weexpectampliconstobegeneratedforeachset of primers. 3. STR and SNP haplotypes will be identified, based on read distributions in the NGS data, to determine the proportion of fetal and maternal DNA, and correspondence to newborn bloodspot DNA. NGS resultsforChr1andChr21willbecomparedtodeterminetheirrelativeploidy.4.DNAfrompatientscarryinga fetus at risk for Trisomy 21 (N=50) will be analyzed by targeted NGS to compare STR, SNP and sequences across Chr21 and Chr1 in fetal, maternal and newborn bloodspot DNA. We expect to demonstrate unique identitiesforfetalandmaternalDNA,identicalfetalandnewbornhaplotypes,andconcordancebetweenChr21 ploidyoffetalandnewbornDNA.ItshouldbepossibletodetectTrisomy21andmosaicism,ifpresent.Withan estimated annual market potential of $1 billion, the envisioned technology will fill an existing gap in clinical diagnosticsbyprovidinganearly,safeapproachforprenatalgeneticanalysis.Ourinitialcommercialproductwill enablemanagementofhighriskpregnancies,andprovidevaluableinformationtophysiciansandpatientsinthe processofestablishingfamilies,specificallyimpactingpregnanciesatriskofhavingachildwithTrisomy21.
This research provides major public health benefits by leveraging a safe, noninvasive method to capture fetal cells that migrate into the reproductive tract from a Pap smear for development of genetic tests to identify women carrying a fetus with a chromosomal disorder such as Down Syndrome. We will build a DNA sequencing kit that can be commercialized to detect trisomy 21 in fetuses as early as 5 weeks of pregnancy. Advances emerging from the proposed research will generate new clinical tools for managing pregnancy complications to benefit the well-being of mothers and their babies.
| Drewlo, Sascha; Armant, D Randall (2017) Quo vadis, trophoblast? Exploring the new ways of an old cell lineage. Placenta 60 Suppl 1:S27-S31 |
