The overall goal of this research is to provide a flexible prenatal genetic testing kit that can be expanded to detect any inheritable trait as early as 5, and up to 20, weeks of gestation, from a safe, noninvasive Pap smear. Studies show that perinatal Pap tests pose no risk to mother or fetus, and capture trophoblast-like cells that migrate from the placenta into the reproductive tract. Trophoblast retrieval and isolation from the cervix (TRIC) efficiently isolates hundreds of fetal cells without limitations due to early gestational age, maternal obesity, or uteroplacental insufficiency disorders. In a recent Science Translational Medicine report, we isolated sufficient genomic DNA from intact fetal cells obtained by TRIC at 5-19 weeks of gestation (n=20) to definitively distinguish maternal and 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 maternal serum, which has a fetal fraction of only 4-10% at week 10 of gestation, DNA obtained by TRIC had a fetal fraction of 85-100%, capable of providing nucleotide-specific haplotyping. TRIC will be commercialized to identify single gene and chromosome number disorders prenatally from Pap smears, initially through a custom multiplex PCR platform to simultaneously amplify SNPs and STRs, as well disease-specific loci, for genotyping by NGS. We will incorporate the locus for the sickle cell anemia (SCA) point mutation, which will be expanded to other diseases in Phase II. We will accomplish four milestones. 1. Primers will be designed and tested with human genomic DNA to amplify STR, SNP and SCA loci, sequencing PCR products by NGS to optimize their amplification and co-amplification in singleplex and multiplex PCR. 2. DNA from fetal and maternal cells isolated by TRIC (N=50), as well as the corresponding newborn bloodspots (reference), will be isolated and compared by targeted NGS of the optimized multiplex PCR products. We expect amplicons to be generated for each set of primers. 3. STR and SNP haplotypes will be identified, based on read distributions in the NGS data, to determine whether fetal DNA differs from maternal DNA, and is identical to the corresponding newborn bloodspot DNA. 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 diagnostics and outcompete existing prenatal testing technologies. Our initial commercial product will to enable management of high risk pregnancies, and provide valuable information to physicians and patients in the process of establishing families. Specifically, this initial product will benefit pregnancies at risk of having a child with SCA.
Public Health Relevance Statement: 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.
Project Terms: African American; Amniocentesis; base; Bioinformatics; Biopsy; Blood; cell free fetal DNA; Cells; Cervix Uteri; Child; Chorion; Chromosomes; Clinical; clinical diagnostics; clinically relevant; Communities; Computer Analysis; Conceptions; Conceptus; Custom; Data; design; Development; Disease; Disease Management; DNA; DNA sequencing; Family; Feasibility Studies; fetal; fetal diagnosis; Fetus; fetus at risk; fetus cell; First Pregnancy Trimester; flexibility; Future; Gene Mutation; Genes; Genetic; Genetic Fingerprintings; Genetic screening method; Genome; Genomic DNA; Genotype; Gestational Age; Goals; Growth; Haplotypes; Health Benefit; Healthcare; Hemoglobin; High-Risk Pregnancy; Human; Inborn Genetic Diseases; Industry; industry partner; Inherited; interest; Intervention; Legal patent; Libraries; maternal obesity; maternal serum; Medical; Mendelian disorder; Methods; Mothers; Newborn Infant; next generation sequencing; Nucleotides; Other Genetics; Pap smear; Patients; Perinatal; perinatal health; Phase; Physicians; Placenta; Point Mutation; Population; Positioning Attribute; Pregnancy; Pregnancy Complications; prenatal; prenatal disorder; prenatal testing; Process; Provider; Public Health; Publishing; Recovery; Reporting; reproductive; reproductive tract; Research; Resolution; Retrieval; Risk; screening; Second Pregnancy Trimester; Services; Short Tandem Repeat; Sickle Cell Anemia; Single Nucleotide Polymorphism; Site; Spottings; targeted sequencing; Technology; Terminal Repeat Sequences; Testing; tool; trait; translational medicine; Translational Research; trophoblast; Validation; Villous; Woman; Work
