De novo heterozygous variants in the brain-specific transcription factor Neuronal Differentiation Factor 2 (NEUROD2) have been recently associated with early-onset epileptic encephalopathy and developmental delay. Here, we report an adolescent with developmental delay without seizures who was found to have a novel de novo heterozygous NEUROD2 missense variant, p.(Leu163Pro). Functional testing using an in vivo assay of neuronal differentiation in Xenopus laevis tadpoles demonstrated that the patient variant of NEUROD2 displays minimal protein activity, strongly suggesting a loss of function effect. In contrast, a second rare NEUROD2 variant, p.(Ala235Thr), identified in an adolescent with developmental delay but lacking parental studies for inheritance, showed normal in vivo NEUROD2 activity. We thus provide clinical, genetic, and functional evidence that NEUROD2 variants can lead to developmental delay without accompanying early-onset seizures, and demonstrate how functional testing can complement genetic data when determining variant pathogenicity.

Dysregulated innate immune responses contribute to multisystem inflammatory syndrome in children (MIS-C), characterized by gastrointestinal, mucocutaneous, and/or cardiovascular injury occurring weeks after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure. To investigate innate immune functions, we stimulated ex vivo peripheral blood cells from MIS-C patients with agonists of Toll-like receptors (TLR), key innate immune response initiators. We found severely dampened cytokine responses and elevated gene expression of negative regulators of TLR signaling. Increased plasma levels of zonulin, a gut leakage marker, were also detected. These effects were also observed in fully convalescent children months after MIS-C recovery. When we investigated the genetic background of patients in relation to TLR responsiveness, we found that cells from MIS-C children carrying rare heterozygous variants of lysosomal trafficking regulator (LYST) were less refractory to TLR stimulation and exhibited lysosomal and mitochondrial abnormalities with altered energy metabolism. Moreover, these rare LYST variant heterozygous carriers tended to exhibit unfavorable clinical laboratory indicators of inflammation, including more profound lymphopenia. The results of our observational study have several implications. First, TLR hyporesponsiveness may be associated with hyperinflammation and/or excessive or prolonged stimulation with gut-originated TLR ligands. Second, TLR hyporesponsiveness during MIS-C may be protective, since LYST variant heterozygous carriers exhibited reduced TLR hyporesponsiveness and unfavorable clinical laboratory indicators of inflammation. Thus, links may exist between genetic background, ability to establish a refractory immune state, and MIS-C clinical spectrum. Third, the possibility exists that prolonged TLR hyporesponsiveness is one of the mechanisms driving long coronavirus disease (COVID), which highlights the need to monitor long-term consequences of MIS-C.

PURPOSE: We sought to delineate a multisystem disorder caused by recessive cysteine-rich with epidermal growth factor-like domains 1 (CRELD1) gene variants. METHODS: The impact of CRELD1 variants was characterized through an international collaboration utilizing next-generation DNA sequencing, gene knockdown, and protein overexpression in Xenopus tropicalis, and in vitro analysis of patient immune cells. RESULTS: Biallelic variants in CRELD1 were found in 18 participants from 14 families. Affected individuals displayed an array of phenotypes involving developmental delay, early-onset epilepsy, and hypotonia, with about half demonstrating cardiac arrhythmias and some experiencing recurrent infections. Most harbored a frameshift in trans with a missense allele, with 1 recurrent variant, p.(Cys192Tyr), identified in 10 families. X tropicalis tadpoles with creld1 knockdown displayed developmental defects along with increased susceptibility to induced seizures compared with controls. Additionally, human CRELD1 harboring missense variants from affected individuals had reduced protein function, indicated by a diminished ability to induce craniofacial defects when overexpressed in X tropicalis. Finally, baseline analyses of peripheral blood mononuclear cells showed similar proportions of immune cell subtypes in patients compared with healthy donors. CONCLUSION: This patient cohort, combined with experimental data, provide evidence of a multisystem clinical syndrome mediated by recessive variants in CRELD1.

Purpose

Dominant variants in the retinoic acid receptor beta (RARB) gene underlie a syndromic form of microphthalmia, known as MCOPS12, which is associated with other birth anomalies and global developmental delay with spasticity and/or dystonia. Here, we report 25 affected individuals with 17 novel pathogenic or likely pathogenic variants in RARB. This study aims to characterize the functional impact of these variants and describe the clinical spectrum of MCOPS12.

Methods

We used in vitro transcriptional assays and in silico structural analysis to assess the functional relevance of RARB variants in affecting the normal response to retinoids.

Results

We found that all RARB variants tested in our assays exhibited either a gain-of-function or a loss-of-function activity. Loss-of-function variants disrupted RARB function through a dominant-negative effect, possibly by disrupting ligand binding and/or coactivators' recruitment. By reviewing clinical data from 52 affected individuals, we found that disruption of RARB is associated with a more variable phenotype than initially suspected, with the absence in some individuals of cardinal features of MCOPS12, such as developmental eye anomaly or motor impairment.

Conclusion

Our study indicates that pathogenic variants in RARB are functionally heterogeneous and associated with extensive clinical heterogeneity.