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 identifysinglegeneandchromosomenumberdisordersprenatallyfromPapsmears,initiallythroughacustom multiplexPCRplatformtosimultaneouslyamplifySNPsandSTRs,aswelldisease-specificloci,forgenotyping byNGS.Wewillincorporatethelocusforthesicklecellanemia(SCA)pointmutation,whichwillbeexpanded tootherdiseasesinPhaseII.Wewillaccomplishfourmilestones.1.Primerswillbedesignedandtestedwith humangenomicDNAtoamplifySTR,SNPandSCAloci,sequencingPCRproductsbyNGStooptimizetheir amplificationandco-amplificationinsingleplexandmultiplexPCR.2.DNAfromfetalandmaternalcellsisolated byTRIC(N=50),aswellasthecorrespondingnewbornbloodspots(reference),willbeisolatedandcompared bytargetedNGSoftheoptimizedmultiplexPCRproducts.Weexpectampliconstobegeneratedforeachsetof primers.3.STRandSNPhaplotypeswillbeidentified,basedonreaddistributionsintheNGSdata,todetermine whetherfetalDNAdiffersfrommaternalDNA,andisidenticaltothecorrespondingnewbornbloodspotDNA. The fetal fraction will also be determined. 4. DNA from patients carrying a fetus at risk for SCA (N=50) will be analyzed by targeted NGS to compare STR, SNP and SCA haplotypes among fetal, maternal and newborn bloodspot DNA. We expect to demonstrate unique identities for fetal and maternal DNA, identical fetal and newborn haplotypes, and concordance between the SCA haplotype of fetal and newborn DNA. With an estimated annual market potential of $1 billion, the envisioned technology will fill an existing gap in clinical diagnosticsandoutcompeteexistingprenataltestingtechnologies.Ourinitialcommercialproductwilltoenable managementofhighriskpregnancies,andprovidevaluableinformationtophysiciansandpatientsintheprocess ofestablishingfamilies.Specifically,thisinitialproductwillbenefitpregnanciesatriskofhavingachildwithSCA.
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 an inherited disorder. We will build a DNA sequencing kit that can be commercialized to determine the genotype of fetuses at risk for sickle cell anemia 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 |
