Nonimmune hydrops fetalis (NIHF) is a rare disorder with a high perinatal mortality of at least 50%. One cause of NIHF is generalized lymphatic dysplasia (GLD), a rare form of primary lymphedema of the extremities and systemic involvement including chylothoraces and pericardial effusions. An autosomal recessive form of GLD has been described, caused by variants in the PIEZO1 gene. It has been reported clinically to cause NIHF and childhood onset of facial and limb lymphedema, most of which were diagnosed postnatally. We present a case of a woman with recurrent pregnancies affected by NIHF because of novel compound heterozygous variants in the PIEZO1 gene diagnosed prenatally using exome sequencing (ES). Two variants in PIEZO1 (c.3206G>A and c.6208A>C) were identified that were inherited from the father and mother, and are predicted to cause a nonsense and missense change, respectively, in the PIEZO1 subunits. Ultrasound demonstrated severe bilateral pleural effusions, whole body edema and polyhydramnios. Histopathology revealed an increased number of lymphatic channels, many of which showed failure of luminal canalization. Sanger sequencing confirmed the same variants in a prior fetal demise. We provide phenotypic correlation with ultrasound and autopsy finding, review PIEZO1 variants as a cause of GLD and discuss the uses of prenatal ES to date.

Focal amplifications (FA) can mediate targeted therapy resistance in cancer. Understanding the structure and dynamics of FAs is critical for designing treatments that overcome plasticity-mediated resistance. We developed a melanoma model of dual MAPK inhibitor (MAPKi) resistance that bears BRAFV600 amplifications through either extrachromosomal DNA (ecDNA)/double minutes (DM) or intrachromosomal homogenously staining regions (HSR). Cells harboring BRAFV600E FAs displayed mode switching between DMs and HSRs, from both de novo genetic changes and selection of preexisting subpopulations. Plasticity is not exclusive to ecDNAs, as cells harboring HSRs exhibit drug addiction–driven structural loss of BRAF amplicons upon dose reduction. FA mechanisms can couple with kinase domain duplications and alternative splicing to enhance resistance. Drug-responsive amplicon plasticity is observed in the clinic and can involve other MAPK pathway genes, such as RAF1 and NRAS. BRAF FA-mediated dual MAPKi–resistant cells are more sensitive to proferroptotic drugs, extending the spectrum of ferroptosis sensitivity in MAPKi resistance beyond cases of dedifferentiation. Significance: Understanding the structure and dynamics of oncogene amplifications is critical for overcoming tumor relapse. BRAF amplifications are highly plastic under MAPKi dosage challenges in melanoma, through involvement of de novo genomic alterations, even in the HSR mode. Moreover, BRAF FA-driven, dual MAPKi–resistant cells extend the spectrum of resistance-linked ferroptosis sensitivity. This article is highlighted in the In This Issue feature, p. 873

RNA polymerase II interacts with various other complexes and factors to ensure correct initiation, elongation, and termination of mRNA transcription. One of these proteins is SR-related CTD-associated factor 4 (SCAF4), which is important for correct usage of polyA sites for mRNA termination. Using exome sequencing and international matchmaking, we identified nine likely pathogenic germline variants in SCAF4 including two splice-site and seven truncating variants, all residing in the N-terminal two thirds of the protein. Eight of these variants occurred de novo, and one was inherited. Affected individuals demonstrated a variable neurodevelopmental disorder characterized by mild intellectual disability, seizures, behavioral abnormalities, and various skeletal and structural anomalies. Paired-end RNA sequencing on blood lymphocytes of SCAF4-deficient individuals revealed a broad deregulation of more than 9,000 genes and significant differential splicing of more than 2,900 genes, indicating an important role of SCAF4 in mRNA processing. Knockdown of the SCAF4 ortholog CG4266 in the model organism Drosophila melanogaster resulted in impaired locomotor function, learning, and short-term memory. Furthermore, we observed an increased number of active zones in larval neuromuscular junctions, representing large glutamatergic synapses. These observations indicate a role of CG4266 in nervous system development and function and support the implication of SCAF4 in neurodevelopmental phenotypes. In summary, our data show that heterozygous, likely gene-disrupting variants in SCAF4 are causative for a variable neurodevelopmental disorder associated with impaired mRNA processing.

Objective. Mutational analysis by next-generation sequencing (NGS) obtained by peripheral blood NGS has been of clinical interest to use as a minimally invasive screening tool. Our study evaluates the correlation between NGS results on peripheral blood and bone marrow in hematolymphoid disease. Method. We evaluated patients who had NGS for presumed hematologic malignancy performed on peripheral blood and bone marrow within a 1-year interval of each other. We excluded cases in which chemotherapy or bone marrow transplant occurred in the interval between the two tests. The concordance across peripheral blood and bone marrow NGS results was assessed by kappa coefficient analysis. Results. A total of 163 patients were studied. Concordance of peripheral blood and bone marrow NGS found in 150 patients (92.0%) with a kappa coefficient of 0.794 (kappa standard error 0.054) and P value for testing kappa <0.0001. Myeloid neoplasms showed concordant results in 77/78 cases (98.7%) with a kappa coefficient of 0.916. Lymphoid neoplasms showed concordant results in 26/31 cases (83.9%) with a kappa coefficient of 0.599. Nonneoplastic cases showed concordant results in 47/54 cases (87.0%) with a kappa coefficient of 0.743. Conclusion. Peripheral blood NGS is a reliable tool for mutational analysis and provides a less invasive method for screening and monitoring of the molecular profile.