Using Targeted Sequencing of Paralogous Sequences for Noninvasive Detection of Selected Fetal Aneuploidies.

BACKGROUND: Current methods for noninvasive prenatal testing (NIPT) ascertain fetal aneuploidies using either direct counting measures of DNA fragments from specific genomic regions or relative measures of single nucleotide polymorphism frequencies. Alternatively, the ratios of paralogous sequence pairs were predicted to reflect fetal aneuploidy. We developed a NIPT assay that uses paralog sequences to enable noninvasive detection of fetal trisomy 21 (T21) and trisomy 18 (T18) using cell-free DNA (cfDNA) from maternal plasma. METHODS: A total of 1060 primer pairs were designed to determine fetal aneuploidy status, fetal sex, and fetal fraction. Each library was prepared from cfDNA by coamplifying all 1060 target pairs together in a single reaction well. Products were measured using massively parallel sequencing and deviations from expected paralog ratios were determined based on the read depth from each paralog. RESULTS: We evaluated this assay in a blinded set of 480 cfDNA samples with fetal aneuploidy status determined by the MaterniT21® PLUS assay. Samples were sequenced (mean = 2.3 million reads) with 432 samples returning a result. Using the MaterniT21 PLUS assay for paired plasma aliquots from the same individuals as a reference, all 385 euploid samples, all 31 T21 samples, and 14 of 16 T18 samples were detected with no false positive results observed. CONCLUSIONS: This study introduces a novel NIPT aneuploidy detection approach using targeted sequencing of paralog motifs and establishes proof-of-concept for a potentially low-cost, highly scalable method for the identification of selected fetal aneuploidies with performance and nonreportable rate similar to other published methods.

Studies of the H60a locus in C57BL/6 and 129/Sv mouse strains identify the H60a 3'UTR as a regulator of H60a expression.

The minor histocompatibility antigen 60 (H60a) is expressed in BALB/C and 129/Sv but not in C57BL/6 strains of mice. We recently found that IFNγ down-regulates H60a, but the mechanism of regulation is not known. To better understand the regulation of H60a, we examined the genomic locus of H60a in 129/Sv and C57BL/6 strains. We found that the upstream regulatory region of H60a was present and functional in both strains. Interestingly, IFNγ can down-regulate H60a transcripts in cell lines from 129/Sv but not C57BL/6 strains of mice, suggesting that IFNγ-dependent regulation of H60a proceeds through cis elements other than the conserved promoter region. We determined that the regulation of H60a by IFNγ proceeds through the 3'UTR of H60a, which is present in 129/Sv, but not C57BL/6 cells. We also found that the H60a 3'UTR and microRNAs can contribute to the level of constitutive expression of H60a in tumor cell lines. We conclude that in 129/Sv strain mice, H60a can be regulated by its 3'UTR through IFNγ and unknown microRNAs. Since H60a mediates NK cell target recognition, our studies identify a cis element that can regulate virus and tumor surveillance.