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 trophoblastlike 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 519 weeks of gestation (n=20) to definitively distinguish maternal and fetal DNA by targeted nextgeneration sequencing (NGS) of 59 short terminal repeats (STRs) and 94 single nucleotide polymorphisms (SNPs). Compared to massively parallel sequencing of cellfree fetal DNA from maternal serum, which has a fetal fraction of only 410% at week 10 of gestation, DNA obtained by TRIC had a fetal fraction of 85100%, capable of providing nucleotidespecific haplotyping. TRIC will be commercialized to identify single gene and chromosome number disorders in a prenatal test from Pap smears. We will develop a custom multiplex PCR platform to simultaneously amplify SNPs and STRs to identify fetal DNA, as well as loci across Chromosome 21 (Chr21) to detect trisomy 21, Down syndrome. This platform will be expanded to other chromosome number diseases in Phase II. We will accomplish four milestones. 1. Primers will be designed and tested with human genomic DNA to amplify STRs, SNPs and loci across Chr21 and Chr1 (reference), sequencing PCR products by NGS to optimize their amplification and coamplification in singleplex and multiplex PCR. 2. DNA isolated from fetal and maternal cells isolated by TRIC (N=50), as well as the corresponding newborn bloodspots (reference), will be compared by targeted NGS. We expect amplicons to be generated for each set of primers. 3. STR and SNP haplotypeswill 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 results for Chr1 and Chr21 will be compared to determine their relative ploidy. 4. DNA from patients carrying a 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 identities for fetal and maternal DNA, identical fetal and newborn haplotypes, and concordance between Chr21 ploidy of fetal and newborn DNA. It should be possible to detect Trisomy 21 and mosaicism, if present. With an estimated annual market potential of $1 billion, the envisioned technology will fill an existing gap in clinical diagnostics by providing an early, safe approach for prenatal genetic analysis. Our initial commercial product will enable management of high risk pregnancies, and provide valuable information to physicians and patients in the process of establishing families, specifically impacting pregnancies at risk of having a child with Trisomy 21.
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 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.
Project Terms: advanced maternal age; Amniocentesis; Aneuploidy; base; Bioinformatics; Biopsy; Birth; Blood; Cells; Cervix Uteri; Child; Chorion; Chromosome abnormality; Chromosomes; Chromosomes, Human, Pair 21; Clinical; clinical diagnostics; clinically relevant; Communities; Computer Analysis; Conceptions; Conceptus; Congenital chromosomal disease; Custom; Data; design; Development; Diagnosis; Diagnostic; Disease; Disease Management; DNA; DNA sequencing; Down Syndrome; Family; Feasibility Studies; fetal; fetal diagnosis; Fetus; fetus at risk; fetus cell; First Pregnancy Trimester; flexibility; Future; Gene Mutation; Genes; Genetic; genetic analysis; Genetic screening method; Genome; Genomic DNA; Genotype; Gestational Age; Goals; Growth; Haplotypes; Health Benefit; Healthcare; Hereditary Disease; High-Risk Pregnancy; Human; Industry; industry partner; Inherited; interest; Intervention; Legal patent; Libraries; Massive Parallel Sequencing; maternal obesity; maternal serum; Medical; Mendelian disorder; Methods; Mosaicism; Mothers; Newborn Infant; next generation sequencing; Nucleotides; Other Genetics; Pap smear; Pathology Report; Patients; Perinatal; perinatal health; Personal Satisfaction; Phase; Physicians; Placenta; Ploidies; Positioning Attribute; Pregnancy; Pregnancy Complications; prenatal; prenatal testing; Process; Provider; Public Health; Publishing; Recovery; Reporting; reproductive; reproductive tract; Research; Resolution; Retrieval; Risk; screening; Second Pregnancy Trimester; Services; Short Tandem Repeat; Single Nucleotide Polymorphism; Site; Spottings; targeted sequencing; Technology; Terminal Repeat Sequences; Testing; tool; trait; translational medicine; Translational Research; trophoblast; Validation; Villous; Woman; Work
