WNT16 stands up as an essential gene for bone homeostasis. Here, we present new evidence of the functional role of a particular region within WNT16. Performing 4 C chromatin conformation analysis in three osteoblast-related cells (the human fetal osteoblast hFOB 1.19 cell line, Saos 2 osteosarcoma cell line and mesenchymal Stem Cells –MSC-), we identify physical interactions between the proximal part of WNT16 intron 2, shown here to be an active promoter in Saos 2 osteosarcoma cells, and several putative regulatory regions within CPED1. Analysis of previously published RNA-seq data from hFOB cells disclosed low expression of a region located downstream of this promoter. Our results suggest a novel regulatory mechanism of WNT16 in bone, mediated by physical interaction with various enhancer regions within CPED1.

ABSTRACTTRAF7 syndrome, a multisystemic neurodevelopmental disorder caused by germline missense variants in theTRAF7gene, exhibits heterogeneous clinical presentations. We present a detailed description of eleven new TRAF7 syndrome cases, featuring eight distinct variants, including a novel one. Phenotypic analysis and a comprehensive review of the 58 previously reported cases outline consistent clinical presentations, emphasizing dysmorphic features, developmental delay, endocrine manifestations, and cardiac defects. In this enlarged collection, novelties include a wider range of cognitive dysfunction, with some individuals exhibiting normal development despite early psychomotor delay. Communication challenges, particularly in expressive language, are prevalent, necessitating alternative communication methods. Autistic traits, notably rigidity, are observed in the cohort. Also, worth highlighting are hearing loss, sleep disturbances, and endocrine anomalies, including growth deficiency. Cardiac defects, frequently severe, pose early-life complications. Facial features, including arched eyebrows, contribute to the distinct gestalt. A novel missense variant, p.(Arg653Leu), further underscores the complex relationship between germlineTRAF7variants and somatic changes linked to meningiomas. Our comprehensive analysis expands the phenotypic spectrum, emphasizing the need for oncological evaluations and proposing an evidence-based schedule for clinical management. This study contributes to a better understanding of TRAF7 syndrome, offering insights for improved diagnosis, intervention, and patient care.Key MessageTRAF7 syndrome, a complex neurodevelopmental disorder, exhibits variable cognitive outcomes, complex behavioral presentation and clinical complications emphasizing the need for tailored interventions and follow-up.

Abstract Background The human lineage has undergone a postcranial skeleton gracilization (i.e. lower bone mass and strength relative to body size) compared to other primates and archaic populations such as the Neanderthals. This gracilization has been traditionally explained by differences in the mechanical load that our ancestors exercised. However, there is growing evidence that gracilization could also be genetically influenced. Results We have analyzed the LRP5 gene, which is known to be associated with high bone mineral density conditions, from an evolutionary and functional point of view. Taking advantage of the published genomes of archaic Homo populations, our results suggest that this gene has a complex evolutionary history both between archaic and living humans and within living human populations. In particular, we identified the presence of different selective pressures in archaics and extant modern humans, as well as evidence of positive selection in the African and South East Asian populations from the 1000 Genomes Project. Furthermore, we observed a very limited evidence of archaic introgression in this gene (only at three haplotypes of East Asian ancestry out of the 1000 Genomes), compatible with a general erasing of the fingerprint of archaic introgression due to functional differences in archaics compared to extant modern humans. In agreement with this hypothesis, we observed private mutations in the archaic genomes that we experimentally validated as putatively increasing bone mineral density. In particular, four of five archaic missense mutations affecting the first β-propeller of LRP5 displayed enhanced Wnt pathway activation, of which two also displayed reduced negative regulation. Conclusions In summary, these data suggest a genetic component contributing to the understanding of skeletal differences between extant modern humans and archaic Homo populations.

Background: TRAF7 -related cardiac, facial, and digital anomalies with developmental delay (CAFDADD), a multisystemic neurodevelopmental disorder caused by germline missense variants in the TRAF7 gene, exhibits heterogeneous clinical presentations. Methods: We present a detailed description of 11 new TRAF7 -related CAFDADD cases, featuring eight distinct variants, including a novel one. Results: Phenotypic analysis and a comprehensive review of the 58 previously reported cases outline consistent clinical presentations, emphasizing dysmorphic features, developmental delay, endocrine manifestations, and cardiac defects. In this enlarged collection, novelties include a wider range of cognitive dysfunction, with some individuals exhibiting normal development despite early psychomotor delay. Communication challenges, particularly in expressive language, are prevalent, necessitating alternative communication methods. Autistic traits, notably rigidity, are observed in the cohort. Also, worth highlighting are hearing loss, sleep disturbances, and endocrine anomalies, including growth deficiency. Cardiac defects, frequently severe, pose early -life complications. Facial features, including arched eyebrows, contribute to the distinct gestalt. A novel missense variant, p.(Arg653Leu), further underscores the complex relationship between germline TRAF7 variants and somatic changes linked to meningiomas. Conclusions: Our comprehensive analysis expands the phenotypic spectrum, emphasizing the need for oncological evaluations and proposing an evidence -based schedule for clinical management. This study contributes to a better understanding of TRAF7 -related CAFDADD, offering insights for improved diagnosis, intervention, and patient care. (c) 2024 Elsevier Inc. All rights reserved.

Schaaf-Yang syndrome (SYS) is an ultra-rare neurodevelopmental disorder caused by truncating mutations inMAGEL2. Heterologous expression of wild-type (WT) or a truncated (p.Gln638*) C-terminal HA-tagged MAGEL2 revealed a shift from a primarily cytoplasmic to a more nuclear localisation for the truncated protein variant. We now extend this analysis to six additional SYS mutations on a N-terminal FLAG-tagged MAGEL2. Our results replicate and extend our previous findings, showing that all the truncated MAGEL2 proteins consistently display a predominant nuclear localisation, irrespective of the C-terminal or N-terminal position and the chemistry of the tag. The variants associated with arthrogryposis multiplex congenita display a more pronounced nuclear retention phenotype, suggesting a correlation between clinical severity and the degree of nuclear mislocalisation. These results point to a neomorphic effect of truncated MAGEL2, which might contribute to the pathogenesis of SYS.

BACKGROUND:Recent studies suggested that genetic variants associated with monogenic bone disorders were involved in the pathogenesis of atypical femoral fractures (AFF). Here, we aim to identify rare genetic variants by whole exome sequencing in genes involved in monogenic rare skeletal diseases in 12 women with AFF and 4 controls without any fracture. RESULTS:Out of 33 genetic variants identified in women with AFF, eleven (33.3%) were found in genes belonging to the Wnt pathway (LRP5, LRP6, DAAM2, WNT1, and WNT3A). One of them was rated as pathogenic (p.Pro582His in DAAM2), while all others were rated as variants of uncertain significance according to ClinVar and ACMG criteria. CONCLUSIONS:Osteoporosis, rare bone diseases, and AFFs may share the same genes, thus making it even more difficult to identify unique risk factors.

Abstract Background The human lineage has suffered a skeleton gracilization compared to other primates and archaic populations such as the Neanderthals. This gracilization has been traditionally explained by differences in the mechanical load that our ancestors exercised. However, there is growing evidence that gracilization could be also genetically determined. Results We have analyzed the LRP5 gene from an evolutionary and functional point of view, taking advantage of the published genomes of archaic Homo populations. Mutations in LRP5 are involved in high bone mineral density conditions. Our results suggest that this gene has a complex evolutionary history both between archaic and anatomically modern humans and within the anatomically modern human populations. In particular, we identified the presence of different selective pressures in archaics and anatomically modern humans, as well as evidence of positive selection in the African and South East Asian populations from the 1000G. Furthermore, we observed limited evidence of archaic introgression in this gene at haplotypes of East Asian ancestry, compatible with a general clearing of the archaic introgression due to functional differences in archaics compared to anatomically modern humans. In agreement with this hypothesis, we observed private mutations in the archaic genomes that we experimentally validated as putatively increasing high bone mineral density. In particular, four of five archaic missense mutations affecting the first β-propeller of LRP5 displayed enhanced Wnt pathway activation, of which two also displayed reduced negative regulation. Conclusions In summary, these data suggest a genetic component contributing to the understanding of skeletal differences between anatomically modern humans and archaic Homo populations.

BackgroundSchaaf-Yang syndrome (SYS) is caused by truncating mutations inMAGEL2, mapping to the Prader-Willi region (15q11-q13), with an observed phenotype partially overlapping that of Prader-Willi syndrome. MAGEL2 plays a role in retrograde transport and protein recycling regulation. Our aim is to contribute to the characterisation of SYS pathophysiology at clinical, genetic and molecular levels.MethodsWe performed an extensive phenotypic and mutational revision of previously reported patients with SYS. We analysed the secretion levels of amyloid-β 1–40 peptide (Aβ1-40) and performed targeted metabolomic and transcriptomic profiles in fibroblasts of patients with SYS (n=7) compared with controls (n=11). We also transfected cell lines with vectors encoding wild-type (WT) or mutated MAGEL2 to assess stability and subcellular localisation of the truncated protein.ResultsFunctional studies show significantly decreased levels of secreted Aβ1-40and intracellular glutamine in SYS fibroblasts compared with WT. We also identified 132 differentially expressed genes, including non-coding RNAs (ncRNAs) such asHOTAIR, and many of them related to developmental processes and mitotic mechanisms. The truncated form of MAGEL2 displayed a stability similar to the WT but it was significantly switched to the nucleus, compared with a mainly cytoplasmic distribution of the WT MAGEL2. Based on the updated knowledge, we offer guidelines for the clinical management of patients with SYS.ConclusionA truncated MAGEL2 protein is stable and localises mainly in the nucleus, where it might exert a pathogenic neomorphic effect. Aβ1-40secretion levels andHOTAIRmRNA levels might be promising biomarkers for SYS. Our findings may improve SYS understanding and clinical management.

Over the past few years, efforts have been made to determine the variants and genes that may be important to determine bone mineral density (BMD) that, at the same time, are involved in several bone diseases. To achieve this, the approach that has been the most successful of all has been genome-wide association studies (GWAS). In particular, in research on bone biology over 50 different large GWAS or GWAS metanalyses have been published identifying a total of 500 genetic loci associated with different bone parameters such as BMD, bone resistance, and risk of fracture. Although the discovery of associated variants is an essential aspect, the functional validation of such variants is equally important to elucidate their effect, as well as the causal correlation they have with genetic disease. Since it is a much more time consuming and tedious aspect it has become the new challenge of this post-GWAS era. Among the genes that have already been studied several Wnt signaling pathway genes have been included, among them, the SOST gene that plays a crucial role both determining the BMD of the population and monogenic diseases with elevated bone mass giving rise to a new therapy against osteoporosis. In this review well be collecting the main GWAS associated with bone phenotypes, as well as some functional validations undertaken to analyze the associations found in them.

The discovery that the CRISPR/Cas9 system could be used for genome editing purposes represented a major breakthrough in the field. This advancement has notably facilitated the introduction or correction of disease-specific mutations in healthy or disease stem cell lines respectively; therefore, easing disease modeling studies in combination with differentiation protocols. For many years, variability in the genetic background of different stem cell lines has been a major burden to specifically identify phenotypes arising uniquely from the presence of the mutation and not from differences in other genomic regions. Here, we provide a complete protocol to introduce random indels in human wild type pluripotent stem cells using CRISPR/Cas9 in order to generate clonal lines with potential pathogenic alterations in any gene of interest. In this protocol, we use transfection of a ribonucleoprotein complex to diminish the risk of off-target effects, and select clonal lines with promising indels to obtain disease induced pluripotent stem cell lines.