Background: Vanishing white matter (VWM) is a leukodystrophy, caused by recessive mutations in eukaryotic initiation factor 2B (eIF2B)‐subunit genes (EIF2B1–EIF2B5); 80% are missense mutations. Clinical severity is highly variable, with a strong, unexplained genotype–phenotype correlation.Materials and Methods: With information from a recent natural history study, we severity‐graded 97 missense mutations. Using in silico modeling, we created a new human eIF2B model structure, onto which we mapped the missense mutations. Mutated residues were assessed for location in subunits, eIF2B complex, and functional domains, and for information on biochemical activity.Results: Over 50% of mutations have (ultra‐)severe phenotypic effects. About 60% affect the ε‐subunit, containing the catalytic domain, mostly with (ultra‐)severe effects. About 55% affect subunit cores, with variable clinical severity. About 36% affect subunit interfaces, mostly with severe effects. Very few mutations occur on the external eIf2B surface, perhaps because they have minor functional effects and are tolerated. One external surface mutation affects eIF2B‐substrate interaction and is associated with ultra‐severe phenotype.Conclusion: Mutations that lead to (ultra‐)severe disease mostly affect amino acids with pivotal roles in complex formation and function of eIF2B. Therapies for VWM are emerging and reliable mutation‐based phenotype prediction is required for propensity score matching for trials and in the future for individualized therapy decisions.

Fibrodysplasia ossificans progressiva (FOP) is a catastrophic, ultra-rare disease of heterotopic ossification caused by genetic defects in the ACVR1 gene. The mutant ACVR1 receptor, when triggered by an inflammatory process, leads to heterotopic ossification of the muscles and ligaments. Activin A has been discovered as the main osteogenic ligand of the FOP ACVR1 receptor. However, the source of Activin A itself and the trigger of its production in FOP individuals have remained elusive. We used primary dermal fibroblasts from five FOP patients to investigate Activin A production and how this is influenced by inflammatory cytokines in FOP. FOP fibroblasts showed elevated Activin A production compared to healthy controls, both in standard culture and osteogenic transdifferentiation conditions. We discovered TGFβ1 to be an FOP-specific stimulant of Activin A, shown by the upregulation of the INHBA gene and protein expression. Activin A and TGFβ1 were both induced by BMP4 in FOP and control fibroblasts. Treatment with TNFα and IL6 produced negligible levels of Activin A and TGFβ1 in both cell groups. We present for the first time TGFβ1 as a triggering factor of Activin A production in FOP. As TGFβ1 can promote the induction of the main driver of FOP, TGFβ1 could also be considered a possible therapeutic target in FOP treatment.

(1) Mesenchymal stem cells (MSCs) are a valuable cell model to study the bone pathology of Osteogenesis Imperfecta (OI), a rare genetic collagen-related disorder characterized by bone fragility and skeletal dysplasia. We aimed to generate a novel OI induced mesenchymal stem cell (iMSC) model from induced pluripotent stem cells (iPSCs) derived from human dermal fibroblasts. For the first time, OI iMSCs generation was based on an intermediate neural crest cell (iNCC) stage. (2) Skin fibroblasts from healthy individuals and OI patients were reprogrammed into iPSCs and subsequently differentiated into iMSCs via iNCCs. (3) Successful generation of iPSCs from acquired fibroblasts was confirmed with changes in cell morphology, expression of iPSC markers SOX2, NANOG, and OCT4 and three germ-layer tests. Following differentiation into iNCCs, cells presented increased iNCC markers including P75NTR, TFAP2A, and HNK-1 and decreased iPSC markers, shown to reach the iNCC stage. Induction into iMSCs was confirmed by the presence of CD73, CD105, and CD90 markers, low expression of the hematopoietic, and reduced expression of the iNCC markers. iMSCs were trilineage differentiation-competent, confirmed using molecular analyses and staining for cell-type-specific osteoblast, adipocyte, and chondrocyte markers. (4) In the current study, we have developed a multipotent in vitro iMSC model of OI patients and healthy controls able to differentiate into osteoblast-like cells.

Osteogenesis imperfecta (OI) is a syndromic disorder of bone fragility with high variation in its clinical presentation. Equally variable is molecular aetiology; recessive forms are caused by approximately 20 different genes, many of which are directly implicated in collagen type I biosynthesis. Biallelic variants in prolyl 3-hydroxylase 1 (P3H1) are known to cause severe OI by affecting the competence of the prolyl 3-hydroxylation—cartilage associated protein—peptidyl-prolyl cis-trans isomerase B (P3H1-CRTAP-CyPB) complex, which acts on the Pro986 residue of collagen type I α 1 (COL1A1) and Pro707 collagen type I α 2 (COL1A2) chains. The investigation of an OI cohort of 146 patients in Vietnam identified 14 families with P3H1 variants. The c.1170+5G>C variant was found to be very prevalent (12/14) and accounted for 10.3% of the Vietnamese OI cohort. New P3H1 variants were also identified in this population. Interestingly, the c.1170+5G>C variants were found in families with the severe clinical Sillence types 2 and 3 but also the milder types 1 and 4. This is the first time that OI type 1 is reported in patients with P3H1 variants expanding the clinical spectrum. Patients with a homozygous c.1170+5G>C variant shared severe progressively deforming OI type 3: bowed long bones, deformities of ribcage, long phalanges and hands, bluish sclera, brachycephaly, and early intrauterine fractures. Although it remains unclear if the c.1170+5G>C variant constitutes a founder mutation in the Vietnamese population, its prevalence makes it valuable for the molecular diagnosis of OI in patients of the Kinh ethnicity. Our study provides insight into the clinical and genetic variation of P3H1-related OI in the Vietnamese population.

Key Points: Myofibroblasts and injury repair–related cell types were exclusively observed in polycystic kidney disease and enriched within the cyst microenvironment. Cyst-associated gene signature of 45 genes with decreased expression further away from the cysts was largely related to inflammation. Communication in low-inflamed cystic microdomains related to cellular signaling, morphogenesis, and inflammation in polycystic kidney disease. Background: Changes in the cyst microenvironment in polycystic kidney disease (PKD) may drive progressive cyst formation. Bulk-cell and single-cell RNA sequencing have advanced our understanding of altered signaling; however, the lack of spatial information has limited our insights into local gene expression and cellular communication near cysts. Methods: We used wild-type and Pkd1 -deficient mouse kidneys to generate 10× Genomics Visium Spatial Gene Expression datasets. Using our single-cell mouse kidney atlas and single-cell sequencing data for spot deconvolution, we enhanced resolution and estimated enriched cell types. We analyzed spatial gene expression patterns and used a cyst-centered analysis to identify cyst-associated gene signature. Cell communication near cysts was investigated, identifying key ligand-receptors. Prioritized key factors were validated in tissues. Results: We observed enrichment of fibroblasts, injury repair–related cell types, and diverse immune populations in PKD. Injury repair–related cells were exclusively observed in PKD, predominantly localized within immune cell–dense regions near cysts. These cells collectively contributed to the altered gene expression profile in PKD, including cyst-associated signature genes related to inflammatory processes. Analysis of cellular communication in less-inflamed regions around cysts revealed the involvement of multiple cell types. Key ligand-receptor interactions were associated with cytokine signaling, fibrosis, cellular development, and repair. These included Angpt2 , C3 , Csf1 , Cxcl12 , Il34 , Gas6 , Il16 , Mdk , Mif , Ptn , Sfrp2 , Spp1 , Sdc1 , Tnc , Tnfsf12 , and Wnt5a . In addition, extracellular matrix (ECM) proteins implicated in immune response, ECM remodeling, cell adhesion, and cell signaling were identified, such as Adam9 , Adam10 , Col1a1, Col3a1, Col4a2, Lamb2, Lamc1, Efnb1 , Efnb2 , Thbs1, Thbs2 , and Vcam1 . Immunohistochemistry confirmed expression of Syndecan-1-Collagen IV, Midkine-Integrin β 1, CSF-1, Pleiotrophin, and Tenascin-C in cystic kidneys. Conclusions: Spatial transcriptomics in PKD revealed enrichment of (myo)fibroblasts, immune, and injury repair–related cells near cysts, creating a (pro)inflammatory and (pro)fibrotic niche. Key ligand-receptor and ECM interactions were identified and validated.

The inherited bone disease Osteogenesis Imperfecta (OI) caused by genetic mutation can lead to brittle bones that fracture easily and serious skeletal deformities due to collagen defects. To understand this disease, in vitro models of 2D monolayer fibroblast cultures obtained from subdermal skin biopsies were developed by worldwide research groups, and nowadays were extended to 3D models to further mimic this disease. The models allow for studying of the collagen formation and possible abnormalities in extracellular matrix (ECM) and will enable studies of the mechanisms underlying defects in the quality and quantity of collagen fibres in OI skin fibroblasts when compared to the healthy cells. The most common methods for measuring the components in fibroblasts cultures are collagen immunostaining, which is time consuming and prohibits time-lapse measurements, or second harmonic generation microscopy (SHG) which only enables visualization of collagen fibres but not fibroblasts. Here by our label-free higher harmonic generation microscopy with 1050nm / 1070nm laser, we have extended SHG to with third harmonic generation microscopy (THG) to visualize fibroblasts cell morphology and their nuclei, also with multiphoton autofluorescence (MAF) signal from the cytoplasm of the fibroblasts. Our techniques allow for the visualisation of the 3D cultures in real-time, resulting in the possibility of making time-lapse videos. For both OI patient and control fibroblast cultures, fibroblasts and their nuclei were clearly visualised by forward THG signal; the collagen fibres by the forward SHG channel, and the cytoplasm signal with the backward autofluorescence channel. First results on the comparison between cultures of OI patients and controls reveal differences in fibroblasts orientation, collagen orientation and density.