Clinical interpretation of cell-based non-invasive prenatal testing for monogenic disorders including repeat expansion disorders: potentials and pitfalls.

Introduction: Circulating fetal cells isolated from maternal blood can be used for prenatal testing, representing a safe alternative to invasive testing. The present study investigated the potential of cell-based noninvasive prenatal testing (NIPT) for diagnosing monogenic disorders dependent on the mode of inheritance. Methods: Maternal blood samples were collected from women opting for prenatal diagnostics for specific monogenic disorders (N = 7). Fetal trophoblasts were enriched and stained using magnetic activated cell sorting and isolated by fluorescens activated single-cell sorting. Individual cells were subject to whole genome amplification, and cells of fetal origin were identified by DNA-profiling using short tandem repeat markers. The amplified fetal DNA was input for genetic testing for autosomal dominant-, autosomal recessive-, X-linked and repeat expansion disorders by direct variant analysis and haplotyping. The cell-based NIPT results were compared with those of invasive testing. Results: In two cases at risk of skeletal dysplasia, caused by variants in the FGFR3 gene (autosomal dominant disorders), cell-based NIPT correctly stated an affected fetus, but allelic dropout of the normal alleles were observed in both cases. Cell-based NIPT gave an accurate result in two cases at risk of autosomal recessive disorders, where the parents carried either different diastrophic dysplasia causing variants in the SLC26A2 gene or the same cystic fibrosis disease-causing variant in the CFTR gene. Cell-based NIPT accurately identified an affected male fetus in a pregnancy at risk of Duchenne muscular dystrophy (DMD gene, X-linked recessive disorders). In two cases at risk of the myotonic dystrophy type 1 (DMPK gene, repeat expansion disorder), cell-based NIPT correctly detected an affected and an unaffected fetus, respectively. Discussion: Circulating fetal cells can be used to detect both maternally- and paternally inherited monogenic disorders irrespective of the type of variant, however, the risk of allelic dropout must be considered. We conclude that the clinical interpretation of the cell-based NIPT result thus varies depending on the disorders' mode of inheritance.

Cell-based non-invasive prenatal testing for monogenic disorders: confirmation of unaffected fetuses following preimplantation genetic testing.

PURPOSE: Proof of concept of the use of cell-based non-invasive prenatal testing (cbNIPT) as an alternative to chorionic villus sampling (CVS) following preimplantation genetic testing for monogenic disorders (PGT-M). METHOD: PGT-M was performed by combined testing of short tandem repeat (STR) markers and direct mutation detection, followed by transfer of an unaffected embryo. Patients who opted for follow-up of PGT-M by CVS had blood sampled, from which potential fetal extravillous throphoblast cells were isolated. The cell origin and mutational status were determined by combined testing of STR markers and direct mutation detection using the same setup as during PGT. The cbNIPT results with respect to the mutational status were compared to those of genetic testing of the CVS. RESULTS: Eight patients had blood collected between gestational weeks 10 and 13, from which 33 potential fetal cell samples were isolated. Twenty-seven out of 33 isolated cell samples were successfully tested (82%), of which 24 were of fetal origin (89%). This corresponds to a median of 2.5 successfully tested fetal cell samples per case (range 1-6). All fetal cell samples had a genetic profile identical to that of the transferred embryo confirming a pregnancy with an unaffected fetus, in accordance with the CVS results. CONCLUSION: These findings show that although measures are needed to enhance the test success rate and the number of cells identified, cbNIPT is a promising alternative to CVS. TRIAL REGISTRATION NUMBER: N-20180001.

Genetic disruption of slc4a10 alters the capacity for cellular metabolism and vectorial ion transport in the choroid plexus epithelium.

BACKGROUND: Genetic disruption of slc4a10, which encodes the sodium-dependent chloride/bicarbonate exchanger Ncbe, leads to a major decrease in Na+-dependent HCO3- import into choroid plexus epithelial cells in mice and to a marked reduction in brain intraventricular fluid volume. This suggests that Ncbe functionally is a key element in vectorial Na+ transport and thereby for cerebrospinal fluid secretion in the choroid plexus. However, slc4a10 disruption results in severe changes in expression of Na+,K+-ATPase complexes and other major transport proteins, indicating that profound cellular changes accompany the genetic manipulation. METHODS: A tandem mass tag labeling strategy was chosen for quantitative mass spectrometry. Alterations in the broader patterns of protein expression in the choroid plexus in response to genetic disruption of Ncbe was validated by semi-quantitative immunoblotting, immunohistochemistry and morphometry. RESULTS: The abundance of 601 proteins were found significantly altered in the choroid plexus from Ncbe ko mice relative to Ncbe wt. In addition to a variety of transport proteins, particularly large changes in the abundance of proteins involved in cellular energy metabolism were detected in the Ncbe ko mice. In general, the abundance of rate limiting glycolytic enzymes and several mitochondrial enzymes were reduced following slc4a10 disruption. Surprisingly, this was accompanied by increased ATP levels in choroid plexus cells, indicating that the reduction in capacity for energy metabolism was adaptive to high ATP rather than causal for a decreased capacity for ion and water transport. Ncbe-deficient cells also had a reduced cell area and decreased K+ content. CONCLUSION: Our findings suggest that the lack of effective Na+-entry into the epithelial cells of the choroid plexus leads to a profound change in the cellular phenotype, shifting from a high-rate secretory function towards a more dormant state; similar to what is observed during ageing or Alzheimer's disease.

Choroid plexus epithelial cells express the adhesion protein P-cadherin at cell-cell contacts and syntaxin-4 in the luminal membrane domain.

The choroid plexus epithelial cells (CPECs) belong to a small group of polarized cells, where the Na+-K+-ATPase is expressed in the luminal membrane. The basic polarity of the cells is, therefore, still debated. We investigated the subcellular distribution of an array of proteins known to play fundamental roles either in establishing and maintaining basic cell polarity or in the polarized delivery and recycling of plasma membrane proteins. Immunofluorescence histochemical analysis was applied to determine the subcellular localization of apical and basolateral membrane determinants. Mass spectrometry analysis of CPECs isolated by fluorescence-activated cell sorting was applied to determine the expression of specific forms of the proteins. CPECs mainly express the cell-adhesive P-cadherin, which is localized to the lateral membranes. Proteins belonging to the Crumbs and partitioning defective (Par) protein complexes were all localized to the luminal membrane domain. Par-1 and the Scribble complex were localized to the basolateral membrane domain. Lethal(2) giant larvae homolog 2 (Lgl2) labeling was preferentially observed in the luminal membrane domain. Phosphatidylinositol 3,4,5-trisphosphate (PIP3) was immunolocalized to the basolateral membrane domain, while phosphatidylinositol 4,5-bisphosphate (PIP2) staining was most prominent in the luminal membrane domain along with the PIP3 phosphatase, Pten. The apical target-SNARE syntaxin-3 and the basolateral target-SNARE syntaxin-4 were both localized to the apical membrane domain in CPECs, which lack cellular expression of the clathrin adaptor protein AP-1B for basolateral protein recycling. In conclusion, the CPECs are conventionally polarized, but express P-cadherin at cell-cell contacts, and Lgl2 and syntaxin-4 in the luminal plasma membrane domain.