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Differentiating COVID-19 from other causes of viral pneumonia, like herpes simplex (HSV), can be complicated by shared clinical and laboratory features. Viral pneumonia is mostly diagnosed based on molecular or serological techniques. Serological immunoassay interferences, often attributed to concurrent appearance of heterologous (viral) immunoglobulins, is well-known, but has not been studied in COVID-19 patients. Following false positive HSV immunoglobulin M (IgM) results in our index patient, 25 other COVID-19 patients were tested for HSV-1/2 IgM with the chemiluminescent Liaison assay and Euroimmun enzyme-linked immunosorbent assay. Forty-five percent of COVID-19 patients tested positive for HSV IgM with Liaison. No HSV indices were positive with Euroimmun enzyme-linked immunosorbent assay, suggesting immunoassay interference. Significant correlation between HSV IgM and SARS-CoV-2 IgM/IgG positivity was found. Adding 0.5% polyvinylpyrrolidone, inhibiting non-specific solid-phase adsorption, abolished interference in 22% of false positive cases, suggesting interference caused by solid-phase reactive IgM. Hence, serologic immunoassay results should be interpreted with caution in COVID-19 patients. (c) 2022 Elsevier Inc. All rights reserved.

See Uzquiano and Francis (doi:10.1093/brain/awz048) for a scientific commentary on this article. Mutations in RTTN, which encodes Rotatin, give rise to various brain malformations. Vandervore et al. reveal mitotic failure, aneuploidy, apoptosis and defective ciliogenesis in patient cells. Rotatin binds to myosin subunits in the leading edge of human neurons, which may explain the proliferation and migration defects observed., Recessive mutations in RTTN, encoding the protein rotatin, were originally identified as cause of polymicrogyria, a cortical malformation. With time, a wide variety of other brain malformations has been ascribed to RTTN mutations, including primary microcephaly. Rotatin is a centrosomal protein possibly involved in centriolar elongation and ciliogenesis. However, the function of rotatin in brain development is largely unknown and the molecular disease mechanism underlying cortical malformations has not yet been elucidated. We performed both clinical and cell biological studies, aimed at clarifying rotatin function and pathogenesis. Review of the 23 published and five unpublished clinical cases and genomic mutations, including the effect of novel deep intronic pathogenic mutations on RTTN transcripts, allowed us to extrapolate the core phenotype, consisting of intellectual disability, short stature, microcephaly, lissencephaly, periventricular heterotopia, polymicrogyria and other malformations. We show that the severity of the phenotype is related to residual function of the protein, not only the level of mRNA expression. Skin fibroblasts from eight affected individuals were studied by high resolution immunomicroscopy and flow cytometry, in parallel with in vitro expression of RTTN in HEK293T cells. We demonstrate that rotatin regulates different phases of the cell cycle and is mislocalized in affected individuals. Mutant cells showed consistent and severe mitotic failure with centrosome amplification and multipolar spindle formation, leading to aneuploidy and apoptosis, which could relate to depletion of neuronal progenitors often observed in microcephaly. We confirmed the role of rotatin in functional and structural maintenance of primary cilia and determined that the protein localized not only to the basal body, but also to the axoneme, proving the functional interconnectivity between ciliogenesis and cell cycle progression. Proteomics analysis of both native and exogenous rotatin uncovered that rotatin interacts with the neuronal (non-muscle) myosin heavy chain subunits, motors of nucleokinesis during neuronal migration, and in human induced pluripotent stem cell-derived bipolar mature neurons rotatin localizes at the centrosome in the leading edge. This illustrates the role of rotatin in neuronal migration. These different functions of rotatin explain why RTTN mutations can lead to heterogeneous cerebral malformations, both related to proliferation and migration defects.

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.

Heterozygous gain of function mutations in the ZIC1 gene have been described with syndromic craniosynostosis, variable cerebral or cerebellar abnormalities and mild to moderate developmental delay. Deletion of chromosome 3q25.1 including both adjacent ZIC1 and ZIC4 genes have been described as a cause of variable cerebellar abnormalities including Dandy-Walker malformation. We report two siblings presenting with neonatal microcephaly, agenesis of the corpus callosum, brachycephaly with reduced volume of the posterior fossa, cerebellar and pons hypoplasia, scoliosis and tethered cord (closed neural tube defect). One of the siblings had apparent partial rhombencephalosynapsis. Trio analysis of exome sequencing data revealed a novel heterozygous frameshift mutation in ZIC1 at the end of exon 3 in one sibling and was confirmed by Sanger sequencing in both children. The mutation was not detected in DNA of both parents, which suggests parental gonadal mosaicism. We show that expression of the mutant allele leads to synthesis of a stable abnormal transcript in patient cells, without evidence for nonsense-mediated decay. Craniosynostosis was not present at birth, which explains why ZIC1 mutations were not initially considered. This severe brain malformation indicates that premature closure of sutures can be independent of the abnormal brain development in subjects with pathogenic variants in ZIC1.

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.

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.

ZNF335 plays an essential role in neurogenesis and biallelic variants in ZNF335 have been identified as the cause of severe primary autosomal recessive microcephaly in two unrelated families. We describe herein two additional affected individuals with biallelic ZNF335 variants, one individual with a homozygous c.1399T>C, p.(Cys467Arg) variant, and a second individual with compound heterozygous c.2171_2173delTCT, p.(Phe724del) and c.3998A>G, p.(Glu1333Gly) variants in ZNF335; with the latter variant predicted to affect splicing. Whereas the first case presented with early death and a severe phenotype characterized by anterior agyria with prominent extra-axial spaces, absent basal ganglia, and hypoplasia of the brainstem and cerebellum, the second case had a milder clinical presentation with hypomyelination and otherwise preserved brain structures on MRI. Our findings expand the clinical spectrum of ZNF335 associated microcephaly.

Biallelic mutations in the RTTN gene have been reported in association with microcephaly, short stature, developmental delay and malformations of cortical development. RTTN mutations have previously shown to link aberrant ciliary function with abnormal development and organization of the human cerebral cortex. We here report three individuals from two unrelated families with novel mutations in the RTTN gene. The phenotype consisted of microcephaly, short stature, pachygyria or polymicrogyria, colpocephaly, hypoplasia of the corpus callosum and superior vermis. These findings provide further confirmation of the phenotype related to pathogenic variants in RTTN.

BackgroundCollagens are one of the major constituents of the pial membrane, which plays a crucial role in neuronal migration and cortical lamination during brain development. Type III procollagen, the chains of which are encoded byCOL3A1, is the ligand of the G protein-coupled receptor 56 (GPR56), also known as adhesion G protein-coupled receptor G1. Bi-allelic mutations inGPR56give rise to cobblestone-like malformation, white matter changes and cerebellar dysplasia. This report shows that bi-allelic mutations inCOL3A1are associated with a similar phenotype.MethodsExome analysis was performed in a family consisting of two affected and two non-affected siblings. Brain imaging studies of this family and of two previously reported individuals with bi-allelic mutations inCOL3A1were reviewed. Functional assays were performed on dermal fibroblasts.ResultsExome analysis revealed a novel homozygous variant c.145C>G (p.Pro49Ala) in exon 2 ofCOL3A1. Brain MRI in the affected siblings as well as in the two previously reported individuals with bi-allelicCOL3A1mutations showed a brain phenotype similar to that associated with mutations inGPR56.ConclusionHomozygous or compound heterozygous mutations inCOL3A1are associated with cobblestone-like malformation in all three families reported to date. The variability of the phenotype across patients suggests that genetic alterations in distinct domains of type III procollagen can lead to different outcomes. The presence of cobblestone-like malformation in patients with bi-allelicCOL3A1mutations emphasises the critical role of the type III collagen–GPR56 axis and the pial membrane in the regulation of brain development and cortical lamination.

Collagens are one of the major constituents of the pial membrane, which plays a crucial role in neuronal migration and cortical lamination during brain development. Type III procollagen, the chains of which are encoded byExome analysis was performed in a family consisting of two affected and two non-affected siblings. Brain imaging studies of this family and of two previously reported individuals with bi-allelic mutations inExome analysis revealed a novel homozygous variant c.145CG (p.Pro49Ala) in exon 2 ofHomozygous or compound heterozygous mutations in