Performance of Semiconductor sequencing platform for non-invasive prenatal genetic screening for fetal aneuploidies: results from a multicenter prospective cohort study in a clinical setting

Objectives To validate and evaluate an integrated protocol for non‐invasive prenatal genetic screening (NIPS) for common fetal aneuploidies in a clinical setting, using the semiconductor sequencing technology, Ion Proton. Methods This prospective cohort study included 2505 pregnant women from seven academic genetic laboratories (695 high risk pregnancies in a validation study and 1810 pregnancies with a risk higher than 1/250 without ultrasound anomalies, in a real NIPS clinical setting). Cell free DNA from plasma samples was sequenced using Ion Proton sequencer, and sequencing data were analyzed using the open‐access software WISECONDOR. Performance metrics for detection of trisomies 21, 18 and 13, were calculated based on either fetal karyotype result or clinical data collected at birth. We also evaluated the failure rate and compared three methods of fetal fraction quantification (RASSF1A assay, DEFRAG and SANEFALCON software). Results Sensitivities and specificities were: 98.3% (95%CI: 93.5 ‐ 99.7) and 99.9% (95%CI: 99.4 ‐ 100) for T21, 96.7% (95%CI: 80.9 ‐ 99.8) and 100% (95%CI: 99.6 ‐ 100) for T18, 94.1% (95%CI: 69.2 ‐ 99.7) and 100% (95%CI: 99.6 ‐ 100) for T13. Our failure rate was 1.2% at first and as low as 0.6% after re‐testing some of the failed samples. Fetal fraction estimation by RASSF1A assay was consistent with DEFRAG results, both of which are adequate for routine diagnosis. Conclusions We describe one of the largest studies evaluating the Ion Proton based NIPS and the first clinical study reporting pregnancy outcome in a large set of patients. We demonstrate that this platform is highly efficient in detecting the three most common trisomies. Our protocol is robust and can be easily implemented in any medical genetics laboratory

Mutations in MDH2, Encoding a Krebs Cycle Enzyme, Cause Early-Onset Severe Encephalopathy

MDH2 encodes mitochondrial malate dehydrogenase (MDH), which is essential for the conversion of malate to oxaloacetate as part of the proper functioning of the Krebs cycle. We report bi-allelic pathogenic mutations in MDH2 in three unrelated subjects presenting with early-onset generalized hypotonia, psychomotor delay, refractory epilepsy, and elevated lactate in the blood and cerebrospinal fluid. Functional studies in fibroblasts from affected subjects showed both an apparently complete loss of MDH2 levels and MDH2 enzymatic activity close to null. Metabolomics analyses demonstrated a significant concomitant accumulation of the MDH substrate, malate, and fumarate, its immediate precursor in the Krebs cycle, in affected subjects' fibroblasts. Lentiviral complementation with wild-type MDH2 cDNA restored MDH2 levels and mitochondrial MDH activity. Additionally, introduction of the three missense mutations from the affected subjects into Saccharomyces cerevisiae provided functional evidence to support their pathogenicity. Disruption of the Krebs cycle is a hallmark of cancer, and MDH2 has been recently identified as a novel pheochromocytoma and paraganglioma susceptibility gene. We show that loss-of-function mutations in MDH2 are also associated with severe neurological clinical presentations in children