Structural Chromosomal Rearrangements Require Nucleotide-Level Resolution: Lessons from Next-Generation Sequencing in Prenatal Diagnosis.

In this exciting era of "next-gen cytogenetics," integrating genomic sequencing into the prenatal diagnostic setting is possible within an actionable time frame and can provide precise delineation of balanced chromosomal rearrangements at the nucleotide level. Given the increased risk of congenital abnormalities in newborns with de novo balanced chromosomal rearrangements, comprehensive interpretation of breakpoints could substantially improve prediction of phenotypic outcomes and support perinatal medical care. Herein, we present and evaluate sequencing results of balanced chromosomal rearrangements in ten prenatal subjects with respect to the location of regulatory chromatin domains (topologically associated domains [TADs]). The genomic material from all subjects was interpreted to be "normal" by microarray analyses, and their rearrangements would not have been detected by cell-free DNA (cfDNA) screening. The findings of our systematic approach correlate with phenotypes of both pregnancies with untoward outcomes (5/10) and with healthy newborns (3/10). Two pregnancies, one with a chromosomal aberration predicted to be of unknown clinical significance and another one predicted to be likely benign, were terminated prior to phenotype-genotype correlation (2/10). We demonstrate that the clinical interpretation of structural rearrangements should not be limited to interruption, deletion, or duplication of specific genes and should also incorporate regulatory domains of the human genome with critical ramifications for the control of gene expression. As detailed in this study, our molecular approach to both detecting and interpreting the breakpoints of structural rearrangements yields unparalleled information in comparison to other commonly used first-tier diagnostic methods, such as non-invasive cfDNA screening and microarray analysis, to provide improved genetic counseling for phenotypic outcome in the prenatal setting.

Clinical, pathologic, cytogenetic, and molecular profiling in self-identified black women with uterine leiomyomata.

Black women are disproportionately affected by uterine leiomyomata (UL), or fibroids, compared to other racial groups, having a greater lifetime risk of developing UL and an earlier age of diagnosis. In order to elucidate molecular and genetic mechanisms responsible for the increased prevalence and morbidity associated with UL in black women, clinical, pathologic, cytogenetic, and select molecular profiling (MED12 mutation analysis) of 75 self-reported black women undergoing surgical treatment for UL was performed. Our observations are broadly representative of previous cytogenetic studies of UL: karyotypically abnormal tumors were detected in 30.7% of women and 17.4% of analyzed tumors. No notable association was observed between race and increased occurrence of cytogenetic abnormalities that might contribute to any population-specific morbidity or prevalence rate. Our data on MED12 mutation analyses (73.2% of tumors harbored a MED12 mutation) provide additional support for a significant role of MED12 in tumorigenesis. Although the effect of MED12-mediated tumorigenesis appears significant irrespective of race, other genetic events such as the distribution of karyotypic abnormalities appear differently in black women. This case series indicates that presently recognized genetic and molecular characteristics of UL do not appear to explain the increased prevalence and morbidity of UL in black women.

Immiscible phase nucleic acid purification eliminates PCR inhibitors with a single pass of paramagnetic particles through a hydrophobic liquid.

Extraction and purification of nucleic acids from complex biological samples for PCR are critical steps because inhibitors must be removed that can affect reaction efficiency and the accuracy of results. This preanalytical processing generally involves capturing nucleic acids on microparticles that are then washed with a series of buffers to desorb and dilute out interfering substances. We have developed a novel purification method that replaces multiple wash steps with a single pass of paramagnetic particles (PMPs) though an immiscible hydrophobic liquid. Only two aqueous solutions are required: a lysis buffer, in which nucleic acids are captured on PMPs, and an elution buffer, in which they are released for amplification. The PMPs containing the nucleic acids are magnetically transported through a channel containing liquid wax that connects the lysis chamber to the elution chamber in a specially designed cartridge. Transporting PMPs through the immiscible phase yielded DNA and RNA as pure as that obtained after extensive wash steps required by comparable purification methods. Our immiscible-phase process has been applied to targets in whole blood, plasma, and urine and will enable the development of faster and simpler purification systems.