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Autosomal recessive Cohen syndrome is a neurodevelopmental disorder characterized by postnatal microcephaly, intellectual disability, and a typical facial gestalt. Genetic variants in VPS13B have been found to cause Cohen syndrome, but have also been linked to autism, retinal disease, primary immunodeficiency, and short stature. While it is well established that loss-of-function mutations of VPS13B cause Cohen syndrome, the relevance of missense variants for the pathomechanism remains unexplained. Here, we investigate their pathogenic effect through a systematic re-evaluation of clinical patient information, comprehensive in silico predictions, and in vitro testing of previously published missense variants. In vitro analysis of 10 subcloned VPS13B missense variants resulted in full-length proteins after transient overexpression. 6/10 VPS13B missense variants show reduced accumulation at the Golgi complex in the steady state. The overexpression of these 6/10 VPS13B missense variants did not rescue the Golgi fragmentation after the RNAi-mediated depletion of endogenous VPS13B . These results thus validate 6/10 missense variants as likely pathogenic according to the classification of the American College of Medical Genetics through the integration of clinical, genetic, in silico , and experimental data. In summary, we state that exact variant classification should be the first step towards elucidating the pathomechanisms of genetically inherited neuronal diseases.

Mesenchymal stromal cells (MSCs) are of high relevance for the regeneration of mesenchymal tissues such as bone and cartilage. The promising role of MSCs in cell-based therapies and tissue engineering appears to be limited due to a decline of their regenerative potential with increasing donor age, their limited availability in human tissues and the need of in vitro expansion prior to treatment. We therefore aimed to determine to which degree in vitro aging and chronological aging may be similar processes or if in vitro culture-related changes at the cellular and molecular level are at least altered as a function of donor age. For that purpose we established MSCs cultures from young (yMSCs) and aged (aMSCs) rats that were cultured for more than 100 passages. These long-term MSCs cultures were non-tumorigenic and exhibited similar surface marker patterns as primary MSCs of passage 2. During in vitro expansion, but not during chronological aging, MSCs progressively lose their progenitor characteristics, e.g., complete loss of osteogenic differentiation potential, diminished adipogenic differentiation, altered cell morphology and increased susceptibility towards senescence. Transcriptome analysis revealed that long-term in vitro MSCs cultivation leads to down-regulation of genes involved in cell differentiation, focal adhesion organization, cytoskeleton turnover and mitochondria function. Accordingly, functional analysis demonstrated altered mitochondrial morphology, decreased antioxidant capacities and elevated ROS levels in long-term cultivated yMSCs as well as aMSCs. Notably, only the MSC migration potential and their antioxidative capacity were altered by in vitro as well as chronological aging. Based on specific differences observed between the impact of chronological and in vitro MSC aging we conclude that both are distinct processes.

Cohen syndrome (CS) is an early-onset pediatric neurodevelopmental disorder characterized by postnatal microcephaly and intellectual disability. An accurate diagnosis for individuals with CS is crucial, particularly for their caretakers and future prospects. CS is predominantly caused by rare homozygous or compound heterozygous pathogenic variants in the vacuolar protein sorting-associated 13B ( ) gene, which disrupt protein translation and lead to a loss of function (LoF) of the encoded VPS13B protein. The widespread incorporation of next-generation sequencing approaches in genetic diagnostics increases the number of individuals carrying mutant alleles. At the same time, it increases the detection of variants of unknown clinical significance, necessitating further functional pathogenicity validation. In this study, we present a family with two CS patients. Within this family, four rare variants were detected: c.710G > C, p.Arg237Pro; c.6804delT, p.Phe2268Leufs*24; c.7304C > T, p.Ala2435Val; and c.10302T > A, p.Tyr3434*. These variants challenge the interpretation of their disease-causing role. Specifically, the variants c.6804delT, p.Phe2268Leufs*24 and c.710G > C, p.Arg237Pro were detected in trans configuration and are considered to be causing CS genetically. The functional characterization of the missense variant c.710G > C, p.Arg237Pro shows diminished localization at the Golgi complex, highlighting its clinical relevance and supporting its classification by the American College of Medical Genetics and Genomics (ACMG) as likely pathogenic, class 4. Overall, we emphasize the need for combining genetic and functional testing of VPS13B missense variants to ensure accurate molecular diagnosis and personalized medical care for CS patients.

Gfi1 is a key molecule in hematopoietic lineage development and mutations in GFI1 cause severe congenital neutropenia (SCN). Neutropenia is associated with low bone mass, but the underlying mechanisms are poorly characterized. Using Gfi1 knock-out mice (Gfi1-ko/ko) as SCN model, we studied the relationship between neutropenia and bone mass upon different pathogen load conditions. Our analysis reveals that Gfi1-ko/ko mice kept under strict specific pathogen free (SPF) conditions demonstrate normal bone mass and survival. However, Gfi1-ko/ko mice with early (nonSPF) or late (SPF+nonSPF) pathogen exposure develop low bone mass. Gfi1-ko/ko mice demonstrate a striking rise of systemic inflammatory markers according to elevated pathogen exposure and reduced bone mass. Elevated inflammatory cytokines include for instance Il-1b, Il-6, and Tnf-alpha that regulate osteoclast development. We conclude that low bone mass, due to low neutrophil counts, is caused by the degree of systemic inflammation promoting osteoclastogenesis.

Left ventricular noncompaction (LVNC) is a ventricular wall anomaly morphologically characterized by numerous, excessively prominent trabeculations and deep intertrabecular recesses. Accumulating data now suggest that LVNC is a distinct phenotype but must not constitute a pathological phenotype. Some individuals fulfill the morphologic criteria of LVNC and are without clinical manifestations. Most importantly, morphologic criteria for LVNC are insufficient to diagnose patients with an associated cardiomyopathy (CMP). Genetic testing has become relevant to establish a diagnosis associated with CMP, congenital heart disease, neuromuscular disease, inborn error of metabolism, or syndromic disorder. Genetic factors play a more decisive role in children than in adults and severe courses of LVNC tend to occur in childhood. We reviewed the current literature and highlight the difficulties in establishing the correct diagnosis for children with LVNC. Novel insights show that the interplay of genetics, morphology, and function determine the outcome in pediatric LVNC.

Stefan Mundlos and colleagues report the identification of mutations in SCYL1BP1 in families with gerodermia osteodysplastica, a disorder characterized by wrinkly skin and osteoporosis. SCYL1BP1 localizes to the Golgi apparatus and interacts with Rab6. Gerodermia osteodysplastica is an autosomal recessive disorder characterized by wrinkly skin and osteoporosis. Here we demonstrate that gerodermia osteodysplastica is caused by loss-of-function mutations in SCYL1BP1 , which is highly expressed in skin and osteoblasts. The protein localizes to the Golgi apparatus and interacts with Rab6, identifying SCYL1BP1 as a golgin. These results associate abnormalities of the secretory pathway with age-related changes in connective tissues.

Myocarditis is an inflammatory disease of the heart. Pediatric myocarditis with the dilated cardiomyopathy (DCM) phenotype may be caused by likely pathogenic or pathogenic genetic variants [(L)P] in cardiomyopathy (CMP) genes. Systematic analysis of immune disorder gene defects has not been performed so far. We analyzed 12 patients with biopsy-proven myocarditis and the DCM phenotype together with their parents using whole-exome sequencing (WES). The WES data were filtered for rare pathogenic variants in CMP (n = 89) and immune disorder genes (n = 631). Twelve children with a median age of 2.9 (1.0–6.8) years had a mean left ventricular ejection fraction of 28% (22–32%) and myocarditis was confirmed by endomyocardial biopsy. Patients with primary immunodeficiency were excluded from the study. Four patients underwent implantation of a ventricular assist device and subsequent heart transplantation. Genetic analysis of the 12 families revealed an (L)P variant in the CMP gene in 8/12 index patients explaining DCM. Screening of recessive immune disorder genes identified a heterozygous (L)P variant in 3/12 index patients. This study supports the genetic impact of CMP genes for pediatric myocarditis with the DCM phenotype. Piloting the idea that additional immune-related genetic defects promote myocarditis suggests that the presence of heterozygous variants in these genes needs further investigation. Altered cilium function might play an additional role in inducing inflammation in the context of CMP.

Bone fragility due to osteopenia, osteoporosis or debilitating focal skeletal dysplasias is a frequent observation in the Mendelian disease Neurofibromatosis type 1 (NF1). To determine the mechanisms underlying bone fragility in NF1 we analyzed two conditional mouse models, Nf1Prx1 (limb knock-out) and Nf1Col1 (osteoblast specific knock-out), as well as cortical bone samples from individuals with NF1. We examined mouse bone tissue with micro-computed tomography, qualitative and quantitative histology, mechanical tensile analysis, small-angle X-ray scattering (SAXS), energy dispersive X-ray spectroscopy (EDX), and scanning acoustic microscopy (SAM). In cortical bone of Nf1Prx1 mice we detected ectopic blood vessels that were associated with diaphyseal mineralization defects. Defective mineral binding in the proximity of blood vessels was most likely due to impaired bone collagen formation, as these areas were completely devoid of acidic matrix proteins and contained thin collagen fibers. Additionally, we found significantly reduced mechanical strength of the bone material, which was partially caused by increased osteocyte volume. Consistent with these observations, bone samples from individuals with NF1 and tibial dysplasia showed increased osteocyte lacuna volume. Reduced mechanical properties were associated with diminished matrix stiffness, as determined by SAM. In line with these observations, bone tissue from individuals with NF1 and tibial dysplasia showed heterogeneous mineralization and reduced collagen fiber thickness and packaging. Collectively, the data indicate that bone fragility in NF1 tibial dysplasia is partly due to an increased osteocyte-related micro-porosity, hypomineralization, a generalized defect of organic matrix formation, exacerbated in the regions of tensional and bending force integration, and finally persistence of ectopic blood vessels associated with localized macro-porotic bone lesions.

Neurofibromatosis type I (NF1, MIM#162200) is a relatively frequent genetic condition, which predisposes to tumor formation. Apart from tumors, individuals with NF1 often exhibit endocrine abnormalities such as precocious puberty (2,5-5% of NF1 patients) and some cases of hypertension (16% of NF1 patients). Several cases of adrenal cortex adenomas have been described in NF1 individuals supporting the notion that neurofibromin might play a role in adrenal cortex homeostasis. However, no experimental data were available to prove this hypothesis. We analysed Nf1Prx1 mice and one case of adrenal cortical hyperplasia in a NF1patient. In Nf1Prx1 mice Nf1 is inactivated in the developing limbs, head mesenchyme as well as in the adrenal gland cortex, but not the adrenal medulla or brain. We show that adrenal gland size is increased in NF1Prx1 mice. Nf1Prx1 female mice showed corticosterone and aldosterone overproduction. Molecular analysis of Nf1 deficient adrenals revealed deregulation of multiple proteins, including steroidogenic acute regulatory protein (StAR), a vital mitochondrial factor promoting transfer of cholesterol into steroid making mitochondria. This was associated with a marked upregulation of MAPK pathway and a female specific increase of cAMP concentration in murine adrenal lysates. Complementarily, we characterized a patient with neurofibromatosis type I with macronodular adrenal hyperplasia with ACTH-independent cortisol overproduction. Comparison of normal control tissue- and adrenal hyperplasia- derived genomic DNA revealed loss of heterozygosity (LOH) of the wild type NF1 allele, showing that biallelic NF1 gene inactivation occurred in the hyperplastic adrenal gland. Our data suggest that biallelic loss of Nf1 induces autonomous adrenal hyper-activity. We conclude that Nf1 is involved in the regulation of adrenal cortex function in mice and humans.

Background Neurofibromatosis type 1 (NF1) is a frequent genetic disease characterized by multiple benign tumours with increased risk for malignancy. There is currently no biomarker for tumour load in NF1 patients. Methods In situ hybridization and quantitative real-time polymerase reaction were applied to investigate expression of cartilage-specific genes in mice bearing conditional inactivation of NF1 in the developing limbs. These mice do not develop tumours but recapitulate aspects of NF1 bone dysplasia, including deregulation of cartilage differentiation. It has been recently shown that NF1 tumours require for their growth the master regulator of cartilage differentiation SOX9. We thus hypothesized that some of the cartilage-specific genes deregulated in an Nf1Prx1 mouse model might prove to be relevant biomarkers of NF1 tumours. We tested this hypothesis by analyzing expression of the SOX9 target gene product melanoma-inhibitory activity/cd-rap (MIA) in tumour and serum samples of NF1 patients. Results Increased expression of Mia was found in Nf1 -deficient cartilage in mice. In humans, MIA was expressed in all NF1-related tumours and its serum levels were significantly higher in NF1 patients than in healthy controls. Among NF1 patients, MIA serum levels were significantly higher in those with plexiform neurofibromas and in those with large number of cutaneous (> 1,000) or subcutaneous (> 100) neurofibromas than in patients without such tumours. Most notably, MIA serum levels correlated significantly with internal tumour burden. Conclusions MIA is a potential serum biomarker of tumour load in NF1 patients which could be useful in following the disease course and monitoring the efficacy of therapies.