Kerstin Kutsche

The neurodevelopmental, jaw, eye, and digital syndrome shows high clinical variability and is caused by heterozygous FBXW11 missense variants. FBXW11 encodes an F-box protein containing a WD40 domain that forms a seven-bladed β-propeller. FBXW11 is part of the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complex, which mediates the ubiquitination and proteasomal degradation of numerous substrates, thereby regulating diverse cellular processes and pathways. Here, we report a de novo FBXW11 missense variant, NM_033644.3:c.1100 C > T; p.(Ser367Phe), in an infant with developmental delay and severe skeletal phenotype characterized by multisuture craniosynostosis with turricephaly and postaxial polysyndactyly. We studied the functional consequences of the reported FBXW11 variants p.(Gly229Arg), p.(Arg350Trp), and p.(Glu431Lys), as well as p.(Ser367Phe), all located within the WD40 substrate-binding domain, by ectopic expression of HA-tagged wild-type and variant FBXW11 β-isoforms. Subcellular localization studies revealed FBXW11 ^WT mainly in the nucleus. HA-FBXW11 ^E431K showed either nuclear or nucleo-cytoplasmic distribution, whereas FBXW11 ^G229R and FBXW11 ^S367F were excluded from the nucleus. Using liquid chromatography-tandem mass spectrometry and independent co-immunoprecipitations, we identified variant-specific and shared alterations in FBXW11 interaction networks. Decreased complex formation with the SCF components was observed for FBXW11 ^G229R . Both FBXW11 ^G229R and FBXW11 ^S367F showed reduced binding to the nuclear substrate SUN2. Enriched complex formation of FBXW11 ^G229R and FBXW11 ^S367F with the chaperonin-containing TCP-1 (CCT) subunits suggests that both FBXW11 protein variants are trapped by CCT, potentially delaying or arresting folding of FBXW11’s β-propeller. Together, our data show that FBXW11 missense variants differentially affect binding to specific substrates and/or interaction partners, providing a molecular explanation for the clinical variability.

The T-type voltage-gated calcium channel Ca_V3.3 is expressed in GABAergic neurons of the thalamic reticular nucleus (TRN), where its pacemaking activity controls sleep spindle rhythmogenesis during the non-rapid eye movement (NREM) phase of natural sleep. Previously, we established CACNA1I, the gene coding for Ca_V3.3, as a disease gene for neurodevelopmental disease with or without epilepsy. Here we report three newly identified activation-gate-modifying heterozygous missense variants of CACNA1I, found in four unrelated patients with neurodevelopmental disease with or without seizures. One of these variants, p.(Met1425Val), is an amino-acid substitution at the same position as previously published variant p.(Met1425Ile). Notably, the other two variants studied here are also a pair of two different substitutions of the same amino acid: p.(Ala398Val) and p.(Ala398Glu). By using site-directed mutagenesis, voltage-clamp electrophysiology, computational modelling of neuronal excitability, and structure modelling, we found that the two substitutions of M1425 both result in a gain of channel function including left-shifted voltage-dependence of activation and inactivation, slowed inactivation and deactivation kinetics, and increased neuronal excitability. Remarkably, the two substitutions of A398 show opposite effects on channel function. While substitution A398E leads to a gain of channel function, A398V results in decreased current density, accelerated gating kinetics, and a decreased neuronal excitability. The lack of seizures in the two independent p.(Ala398Val) patients correlates with the absence of increased neuronal excitability in this variant. This is the first report of a gate-modifying Ca_V3.3 channel variant with partial loss-of-function effects associated with developmental delay and intellectual disability without seizures. Our study corroborates the role of Ca_V3.3 dysfunction in the etiology of neurodevelopmental disorders. Moreover, our data suggest that substantial gain-of-function of Ca_V3.3 leads to the development of seizures, whereas both gain- and loss-of-function variants of CACNA1I can cause neurodevelopmental disease.

BACKGROUND: Heterozygous pathogenic variants in the central region (exon 23–34) of FBN2 cause a hereditary connective tissue disorder named congenital contractural arachnodactyly, which presents with obligatory skeletal features but rarely with vascular manifestations. Scarce data exist on the association between FBN2 variants and aortic disease. This study aimed to investigate whether the location of FBN2 variants correlates with distinct clinical features, including aortic disease. METHODS: In this case-controlled cohort study, we ascertained clinical features, sequenced 62 (candidate) disease genes, and classified variants according to the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines in 392 patients with suspected connective tissue or thoracic aortic diseases. We summarized our results and published data and compared clinical manifestations between patients with variants outside and within the central region of FBN2 . RESULTS: Heterozygous FBN2 variants outside the central region were identified in 10 patients from 5 families. Two variants were of uncertain significance, 1 was likely pathogenic, and 2 were pathogenic. A total of 60% of these patients had thoracic aortic disease, but only 20% were diagnosed with congenital contractural arachnodactyly according to an established clinical scoring system. Combined data from the literature and this study revealed that patients with FBN2 variants outside the central region presented with aortic dilatation (55.0% versus 9.9%; P P =0.011) compared with those with central region variants. CONCLUSIONS: Our results suggest that heterozygous FBN2 variants outside the central region predispose individuals to thoracic aortic disease and are less associated with the typical clinical presentation of congenital contractural arachnodactyly than pathogenic variants in the FBN2 central region.

Biallelic variants in NUP107 cause isolated or syndromic steroid-resistant nephrotic syndrome (SRNS), characterised by proteinuria, hypoalbuminaemia and focal segmental glomerulosclerosis that progresses to end-stage renal disease. Patients with syndromic SRNS have microcephaly, developmental delay or intellectual disability and short stature. Simplified gyration is observed in some individuals. We report on a 2-year-old girl with novel biallelic NUP107 variants, c.2606G>T; p.(Gly869Val) and c.1576+1G>A, proteinuria and a severe neurodevelopmental disorder with microcephaly, developmental delay, early-onset seizures, sensorineural hearing loss and brain structural anomalies, including simplified gyral pattern and hypoplasia of the corpus callosum, pons, brainstem and cerebellum. NUP107 is part of the NUP107-160 complex, which, together with other proteins termed nucleoporins, forms the nuclear pore complex (NPC). The NPC regulates nucleocytoplasmic transport and other cellular processes. In patient-derived fibroblasts, we identified aberrantly spliced NUP107 mRNAs with a frameshift and premature stop codon leading to non-sense-mediated mRNA decay, reduced levels of NUP107 transcripts, reduced NUP107 and NUP133 proteins, and a reduced NPC number. In addition, an abnormal nucleolar morphology was found in patient-derived cells. Our functional data support the conclusion that the NUP107 variants underlie the patient’s phenotype, thereby broadening the clinical spectrum associated with NUP107 variants to include abnormal brain development.