Carpenter syndrome type 2 (CRPT2) is a rare autosomal recessive disease mainly characterized by craniosynostosis and polysyndactyly. CRPT2 is the rarer subtype of Carpenter syndrome (CRPTS) and is caused by biallelic variants in the multiple epidermal growth factor-like domains 8 gene (MEGF8). Due to its rarity and phenotypic overlap with other craniosynostosis syndromes, definitive molecular diagnosis of CRPT2 can be challenging. Here, we describe a proband with CRPT2 carrying compound heterozygous variants in MEGF8: one de novo variant disrupting splicing and a maternally inherited missense variant. To resolve these variants, we leveraged long-read genome sequencing to phase the missense variant alleles and RNA sequencing to determine splicing impact. This case highlights the diagnostic value of using sequencing methods beyond the more conventional short-read exome sequencing. Our findings expand the spectrum of MEGF8 variants causing CRPT2 and underscore the utility of emerging sequencing technologies in elucidating complex or previously unresolved genotypes. Expanding access to such technologies is anticipated to accelerate rare disease diagnosis and deepen our understanding of the genetic mechanisms underlying these conditions.

The primary cilium is a signal transduction organelle whose dysfunction clinically causes ciliopathies in humans. RAB23 is a small GTPase known to regulate the Hedgehog signalling pathway and ciliary trafficking. Mutations of RAB23 in humans lead to Carpenter syndrome (CS), an autosomal recessive disorder clinically characterized by craniosynostosis, polysyndactyly, skeletal defects, obesity, and intellectual disability. Although the clinical features of CS bear some resemblance to those of ciliopathies, the exact relationship between the pathological manifestations of CS and the ciliary function of RAB23 remains ambiguous. Besides, the in vivo ciliary functions of RAB23 remain poorly characterised. Here, we demonstrate in vivo and in vitro Rab23 loss-of-function mutants modelling CS, including Rab23 conditional knockout (CKO) mouse mutants, CS patient-derived induced pluripotent stem cells (iPSCs), and zebrafish morphants. The Rab23-CKO mutants exhibit multiple developmental and phenotypical traits recapitulating the clinical features of human ciliopathies and CS, indicating a causal link between the loss of Rab23 and ciliopathy. In line with the ciliopathy-like phenotypes, all three different vertebrate mutant models consistently show a perturbation of primary cilia formation, intriguingly, in a context-dependent manner. Rab23-CKO mutants reveal cell-type specific ciliary abnormalities in chondrocytes, mouse embryonic fibroblasts, neural progenitor cells and neocortical neurons, but not in epithelial cells, cerebellar granule cells and hippocampus neurons. A profound reduction in ciliation frequency was observed specifically in neurons differentiated from CS patient iPSCs, whereas the patients' fibroblasts, iPSCs and neural progenitor cells maintained normal ciliation percentages but shortened cilia length. Furthermore, Rab23-KO neural progenitor cells show perturbed ciliation and desensitized to primary cilium-dependent activation of the Hedgehog signaling pathway. Collectively, these findings indicate that the absence of RAB23 causes dysfunctional primary cilia in a cell-type distinctive manner, which underlies the pathological manifestations of CS. Our findings present the first in vivo evidence validating the unique context-specific function of RAB23 in the primary cilium. Through the use of patient-derived iPSCs differentiated cells, we present direct evidence of primary cilia anomalies in CS, thereby confirming CS as a ciliopathy disorder.

SEC24D is a key component of the Coat Protein Complex II, which plays a critical role in the selective sorting and transport of cargo proteins from the endoplasmic reticulum. This function is particularly essential for the secretion of extracellular matrix proteins, including collagens. Biallelic pathogenic variants in SEC24D have been associated with Cole-Carpenter Syndrome 2, a rare skeletal dysplasia characterized by craniofacial abnormalities and recurrent fractures. We reported a 12-year-old male patient presenting with recurrent bone fractures, severe skeletal deformities, limb shortening, craniofacial dysmorphism and pseudoarthrosis, a feature not previously reported in this condition. Whole-exome sequencing identified a novel homozygous synonymous variant in SEC24D (c.2361C>T; p.Asn787=), located 16 bases upstream of the donor splice site of intron 18. Functional analyses revealed markedly reduced SEC24D expression and aberrant exon 18 skipping, supported by RNA-seq, qPCR, and Western blot. This case provided the first functional evidence for a synonymous variant in SEC24D causing disease via splicing disruption and expands both the phenotypic and genotypic spectrum of Cole-Carpenter Syndrome 2.

Receptor-type E3 ubiquitin ligases are membrane-spanning assemblies that enable extracellular signals to directly control ubiquitylation in the cytoplasm. Despite playing widespread roles in tissue patterning and homeostasis, metabolism, and immunity, their structures and mechanisms remain poorly understood. Using cryo-electron microscopy, integrated with biophysical and functional studies, we visualized an E3 complex composed of two transmembrane proteins, MEGF8 and MOSMO, and the intracellular RING-family protein MGRN1. This MEGF8-MOSMO-MGRN1 (MMM) complex regulates left-right patterning of the body axis and the development of multiple organs, partly by attenuating signaling through the Hedgehog pathway. We find that the MMM complex functions like a fishing pole: a long, flexible helix attached to a membrane platform suspends an activated and precisely oriented RING domain-like a fishhook-to ubiquitylate the cytoplasmic surfaces of target receptors. Our structure explains how mutations in MEGF8 cause multi-organ birth defects in humans and defines a paradigm for receptor regulation by ubiquitylation.

The ZC4H2 gene is the site of congenital mutations linked to neurodevelopmental and musculoskeletal pathologies collectively termed ZARD (ZC4H2-Associated Rare Disorders). ZC4H2 consists of a coiled coil and a single novel zinc finger with four cysteines and two histidines, from which the protein obtains its name. Alpha Fold 3 confidently predicts a structure for the zinc finger but also for similarly sized random sequences, providing equivocal information on its folding status. We show using synthetic peptide fragments that the zinc finger of ZC4H2 is genuine and folds upon binding a zinc ion with picomolar affinity. NMR pH titration of histidines and UV-Vis of a cobalt complex of the peptide indicate its four cysteines coordinate zinc, while two histidines do not participate in binding. The experimental NMR structure of the zinc finger has a novel structural motif similar to RANBP2 zinc fingers, in which two orthogonal hairpins each contribute two cysteines to coordinate zinc. Most of the nine ZARD mutations that occur in the ZC4H2 zinc finger are likely to perturb this structure. While the ZC4H2 zinc finger shares the folding motif and cysteine-ligand spacing of the RANBP2 family, it is missing key substrate-binding residues. Unlike the NZF branch of the RANBP2 family, the ZC4H2 zinc finger does not bind ubiquitin. Since the ZC4H2 zinc finger occurs in a single copy, it is also unlikely to bind DNA. Based on sequence homology to the VAB-23 protein, the ZC4H2 zinc finger may bind RNA of a currently undetermined sequence or have alternative functions.