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For neurodevelopmental disorders (NDDs), a molecular diagnosis is key for management, predicting outcome, and counseling. Often, routine DNA-based tests fail to establish a genetic diagnosis in NDDs. Transcriptome analysis (RNA sequencing [RNA-seq]) promises to improve the diagnostic yield but has not been applied to NDDs in routine diagnostics. Here, we explored the diagnostic potential of RNA-seq in 96 individuals including 67 undiagnosed subjects with NDDs. We performed RNA-seq on single individuals' cultured skin fibroblasts, with and without cycloheximide treatment, and used modified OUTRIDER Z scores to detect gene expression outliers and mis-splicing by exonic and intronic outliers. Analysis was performed by a user-friendly web application, and candidate pathogenic transcriptional events were confirmed by secondary assays. We identified intragenic deletions, monoallelic expression, and pseudoexonic insertions but also synonymous and non-synonymous variants with deleterious effects on transcription, increasing the diagnostic yield for NDDs by 13%. We found that cycloheximide treatment and exonic/intronic Z score analysis increased detection and resolution of aberrant splicing. Importantly, in one individual mis-splicing was found in a candidate gene nearly matching the individual's specific phenotype. However, pathogenic splicing occurred in another neuronal-expressed gene and provided a molecular diagnosis, stressing the need to customize RNA-seq. Lastly, our web browser application allowed custom analysis settings that facilitate diagnostic application and ranked pathogenic transcripts as top candidates. Our results demonstrate that RNA-seq is a complementary method in the genomic diagnosis of NDDs and, by providing accessible analysis with improved sensitivity, our transcriptome analysis approach facilitates wider implementation of RNA-seq in routine genome diagnostics., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The redox state of the neural progenitors regulates physiological processes such as neuronal differentiation and dendritic and axonal growth. The relevance of endoplasmic reticulum (ER)-associated oxidoreductases in these processes is largely unexplored. We describe a severe neurological disorder caused by bi-allelic loss-of-function variants in thioredoxin (TRX)-related transmembrane-2 (TMX2); these variants were detected by exome sequencing in 14 affected individuals from ten unrelated families presenting with congenital microcephaly, cortical polymicrogyria, and other migration disorders. TMX2 encodes one of the five TMX proteins of the protein disulfide isomerase family, hitherto not linked to human developmental brain disease. Our mechanistic studies on protein function show that TMX2 localizes to the ER mitochondria-associated membranes (MAMs), is involved in posttranslational modification and protein folding, and undergoes physical interaction with the MAM-associated and ER folding chaperone calnexin and ER calcium pump SERCA2. These interactions are functionally relevant because TMX2-deficient fibroblasts show decreased mitochondrial respiratory reserve capacity and compensatory increased glycolytic activity. Intriguingly, under basal conditions TMX2 occurs in both reduced and oxidized monomeric form, while it forms a stable dimer under treatment with hydrogen peroxide, recently recognized as a signaling molecule in neural morphogenesis and axonal pathfinding. Exogenous expression of the pathogenic TMX2 variants or of variants with an in vitro mutagenized TRX domain induces a constitutive TMX2 polymerization, mimicking an increased oxidative state. Altogether these data uncover TMX2 as a sensor in the MAM-regulated redox signaling pathway and identify it as a key adaptive regulator of neuronal proliferation, migration, and organization in the developing brain., (Copyright © 2019 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Sphingomyelinases generate ceramide from sphingomyelin as a second messenger in intracellular signaling pathways involved in cell proliferation, differentiation, or apoptosis. Children from 12 unrelated families presented with microcephaly, simplified gyral pattern of the cortex, hypomyelination, cerebellar hypoplasia, congenital arthrogryposis, and early fetal/postnatal demise. Genomic analysis revealed bi-allelic loss-of-function variants in SMPD4, coding for the neutral sphingomyelinase-3 (nSMase-3/SMPD4). Overexpression of human Myc-tagged SMPD4 showed localization both to the outer nuclear envelope and the ER and additionally revealed interactions with several nuclear pore complex proteins by proteomics analysis. Fibroblasts from affected individuals showed ER cisternae abnormalities, suspected for increased autophagy, and were more susceptible to apoptosis under stress conditions, while treatment with siSMPD4 caused delayed cell cycle progression. Our data show that SMPD4 links homeostasis of membrane sphingolipids to cell fate by regulating the cross-talk between the ER and the outer nuclear envelope, while its loss reveals a pathogenic mechanism in microcephaly., (Copyright © 2019 American Society of Human Genetics. All rights reserved.)

To date, mutations in 15 actin- or microtubule-associated genes have been associated with the cortical malformation lissencephaly and variable brainstem hypoplasia. During a multicenter review, we recognized a rare lissencephaly variant with a complex brainstem malformation in three unrelated children. We searched our large brain-malformation databases and found another five children with this malformation (as well as one with a less severe variant), analyzed available whole-exome or -genome sequencing data, and tested ciliogenesis in two affected individuals. The brain malformation comprised posterior predominant lissencephaly and midline crossing defects consisting of absent anterior commissure and a striking W-shaped brainstem malformation caused by small or absent pontine crossing fibers. We discovered heterozygous de novo missense variants or an in-frame deletion involving highly conserved zinc-binding residues within the GAR domain of MACF1 in the first eight subjects. We studied cilium formation and found a higher proportion of mutant cells with short cilia than of control cells with short cilia. A ninth child had similar lissencephaly but only subtle brainstem dysplasia associated with a heterozygous de novo missense variant in the spectrin repeat domain of MACF1. Thus, we report variants of the microtubule-binding GAR domain of MACF1 as the cause of a distinctive and most likely pathognomonic brain malformation. A gain-of-function or dominant-negative mechanism appears likely given that many heterozygous mutations leading to protein truncation are included in the ExAC Browser. However, three de novo variants in MACF1 have been observed in large schizophrenia cohorts., (Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)