SOX9 keeps growth plates and articular cartilage healthy by inhibiting chondrocyte dedifferentiation/osteoblastic redifferentiation.

Cartilage is essential throughout vertebrate life. It starts developing in embryos when osteochondroprogenitor cells commit to chondrogenesis, activate a pancartilaginous program to form cartilaginous skeletal primordia, and also embrace a growth-plate program to drive skeletal growth or an articular program to build permanent joint cartilage. Various forms of cartilage malformation and degeneration diseases afflict humans, but underlying mechanisms are still incompletely understood and treatment options suboptimal. The transcription factor SOX9 is required for embryonic chondrogenesis, but its postnatal roles remain unclear, despite evidence that it is down-regulated in osteoarthritis and heterozygously inactivated in campomelic dysplasia, a severe skeletal dysplasia characterized postnatally by small stature and kyphoscoliosis. Using conditional knockout mice and high-throughput sequencing assays, we show here that SOX9 is required postnatally to prevent growth-plate closure and preosteoarthritic deterioration of articular cartilage. Its deficiency prompts growth-plate chondrocytes at all stages to swiftly reach a terminal/dedifferentiated stage marked by expression of chondrocyte-specific (Mgp) and progenitor-specific (Nt5e and Sox4) genes. Up-regulation of osteogenic genes (Runx2, Sp7, and Postn) and overt osteoblastogenesis quickly ensue. SOX9 deficiency does not perturb the articular program, except in load-bearing regions, where it also provokes chondrocyte-to-osteoblast conversion via a progenitor stage. Pathway analyses support roles for SOX9 in controlling TGFß and BMP signaling activities during this cell lineage transition. Altogether, these findings deepen our current understanding of the cellular and molecular mechanisms that specifically ensure lifelong growth-plate and articular cartilage vigor by identifying osteogenic plasticity of growth-plate and articular chondrocytes and a SOX9-countered chondrocyte dedifferentiation/osteoblast redifferentiation process.

Pathogenic variant in EPHB4 results in central conducting lymphatic anomaly.

Central conducting lymphatic anomaly (CCLA) is one of the complex lymphatic anomalies characterized by dilated lymphatic channels, lymphatic channel dysmotility and distal obstruction affecting lymphatic drainage. We performed whole exome sequencing (WES) of DNA from a four-generation pedigree and examined the consequences of the variant by transfection of mammalian cells and morpholino and rescue studies in zebrafish. WES revealed a heterozygous mutation in EPHB4 (RefSeq NM_004444.4; c.2334 + 1G>C) and RNA-Seq demonstrated that the EPHB4 mutation destroys the normal donor site, which leads to the use of a cryptic splice donor that results in retention of the intervening 12-bp intron sequence. Transient co-expression of the wild-type and mutant EPHB4 proteins showed reduced phosphorylation of tyrosine, consistent with a loss-of-function effect. Zebrafish ephb4a morpholino resulted in vessel misbranching and deformities in the lymphatic vessel development, indicative of possible differentiation defects in lymphatic vessels, mimicking the lymphatic presentations of the patients. Immunoblot analysis using zebrafish lysates demonstrated over-activation of mTORC1 as a consequence of reduced EPHB4 signaling. Strikingly, drugs that inhibit mTOR signaling or RAS-MAPK signaling effectively rescued the misbranching phenotype in a comparable manner. Moreover, knock-in of EPHB4 mutation in HEK293T cells also induced mTORC1 activity. Our data demonstrate the pathogenicity of the identified EPHB4 mutation as a novel cause of CCLA and suggesting that ERK inhibitors may have therapeutic benefits in such patients with complex lymphatic anomalies.

Metabolic Insight into Glioma Heterogeneity: Mapping Whole Exome Sequencing to In Vivo Imaging with Stereotactic Localization and Deep Learning.

Intratumoral heterogeneity (ITH) complicates the diagnosis and treatment of glioma, partly due to the diverse metabolic profiles driven by underlying genomic alterations. While multiparametric imaging enhances the characterization of ITH by capturing both spatial and functional variations, it falls short in directly assessing the metabolic activities that underpin these phenotypic differences. This gap stems from the challenge of integrating easily accessible, colocated pathology and detailed genomic data with metabolic insights. This study presents a multifaceted approach combining stereotactic biopsy with standard clinical open-craniotomy for sample collection, voxel-wise analysis of MR images, regression-based GAM, and whole-exome sequencing. This work aims to demonstrate the potential of machine learning algorithms to predict variations in cellular and molecular tumor characteristics. This retrospective study enrolled ten treatment-naïve patients with radiologically confirmed glioma. Each patient underwent a multiparametric MR scan (T1W, T1W-CE, T2W, T2W-FLAIR, DWI) prior to surgery. During standard craniotomy, at least 1 stereotactic biopsy was collected from each patient, with screenshots of the sample locations saved for spatial registration to pre-surgical MR data. Whole-exome sequencing was performed on flash-frozen tumor samples, prioritizing the signatures of five glioma-related genes: IDH1, TP53, EGFR, PIK3CA, and NF1. Regression was implemented with a GAM using a univariate shape function for each predictor. Standard receiver operating characteristic (ROC) analyses were used to evaluate detection, with AUC (area under curve) calculated for each gene target and MR contrast combination. Mean AUC for five gene targets and 31 MR contrast combinations was 0.75 ± 0.11; individual AUCs were as high as 0.96 for both IDH1 and TP53 with T2W-FLAIR and ADC, and 0.99 for EGFR with T2W and ADC. These results suggest the possibility of predicting exome-wide mutation events from noninvasive, in vivo imaging by combining stereotactic localization of glioma samples and a semi-parametric deep learning method. The genomic alterations identified, particularly in IDH1, TP53, EGFR, PIK3CA, and NF1, are known to play pivotal roles in metabolic pathways driving glioma heterogeneity. Our methodology, therefore, indirectly sheds light on the metabolic landscape of glioma through the lens of these critical genomic markers, suggesting a complex interplay between tumor genomics and metabolism. This approach holds potential for refining targeted therapy by better addressing the genomic heterogeneity of glioma tumors.

Mechanism of KMT5B haploinsufficiency in neurodevelopment in humans and mice.

Pathogenic variants in KMT5B, a lysine methyltransferase, are associated with global developmental delay, macrocephaly, autism, and congenital anomalies (OMIM# 617788). Given the relatively recent discovery of this disorder, it has not been fully characterized. Deep phenotyping of the largest (n = 43) patient cohort to date identified that hypotonia and congenital heart defects are prominent features that were previously not associated with this syndrome. Both missense variants and putative loss-of-function variants resulted in slow growth in patient-derived cell lines. KMT5B homozygous knockout mice were smaller in size than their wild-type littermates but did not have significantly smaller brains, suggesting relative macrocephaly, also noted as a prominent clinical feature. RNA sequencing of patient lymphoblasts and Kmt5b haploinsufficient mouse brains identified differentially expressed pathways associated with nervous system development and function including axon guidance signaling. Overall, we identified additional pathogenic variants and clinical features in KMT5B-related neurodevelopmental disorder and provide insights into the molecular mechanisms of the disorder using multiple model systems.

Genomic profiling informs diagnoses and treatment in vascular anomalies.

Vascular anomalies are malformations or tumors of the blood or lymphatic vasculature and can be life-threatening. Although molecularly targeted therapies can be life-saving, identification of the molecular etiology is often impeded by lack of accessibility to affected tissue samples, mosaicism or insufficient sequencing depth. In a cohort of 356 participants with vascular anomalies, including 104 with primary complex lymphatic anomalies (pCLAs), DNA from CD31+ cells isolated from lymphatic fluid or cell-free DNA from lymphatic fluid or plasma underwent ultra-deep sequencing thereby uncovering pathogenic somatic variants down to a variant allele fraction of 0.15%. A molecular diagnosis, including previously undescribed genetic causes, was obtained in 41% of participants with pCLAs and 72% of participants with other vascular malformations, leading to a new medical therapy for 63% (43/69) of participants and resulting in improvement in 63% (35/55) of participants on therapy. Taken together, these data support the development of liquid biopsy-based diagnostic techniques to identify previously undescribed genotype-phenotype associations and guide medical therapy in individuals with vascular anomalies.

Single-cell atlas of craniogenesis uncovers SOXC-dependent, highly proliferative, and myofibroblast-like osteodermal progenitors.

The mammalian skull vault is essential to shape the head and protect the brain, but the cellular and molecular events underlying its development remain incompletely understood. Single-cell transcriptomic profiling from early to late mouse embryonic stages provides a detailed atlas of cranial lineages. It distinguishes various populations of progenitors and reveals a high expression of SOXC genes (encoding the SOX4, SOX11, and SOX12 transcription factors) early in development in actively proliferating and myofibroblast-like osteodermal progenitors. SOXC inactivation in these cells causes severe skull and skin underdevelopment due to the limited expansion of cell populations before and upon lineage commitment. SOXC genes enhance the expression of gene signatures conferring dynamic cellular and molecular properties, including actin cytoskeleton assembly, chromatin remodeling, and signaling pathway induction and responsiveness. These findings shed light onto craniogenic mechanisms and SOXC functions and suggest that similar mechanisms could decisively control many developmental, adult, pathological, and regenerative processes.

Autophagy mitigates ethanol-induced mitochondrial dysfunction and oxidative stress in esophageal keratinocytes.

During alcohol consumption, the esophageal mucosa is directly exposed to high concentrations of ethanol (EtOH). We therefore investigated the response of normal human esophageal epithelial cell lines EPC1, EPC2 and EPC3 to acute EtOH exposure. While these cells were able to tolerate 2% EtOH for 8 h in both three-dimensional organoids and monolayer culture conditions, RNA sequencing suggested that EtOH induced mitochondrial dysfunction. With EtOH treatment, EPC1 and EPC2 cells also demonstrated decreased mitochondrial ATPB protein expression by immunofluorescence and swollen mitochondria lacking intact cristae by transmission electron microscopy. Mitochondrial membrane potential (ΔΨm) was decreased in a subset of EPC1 and EPC2 cells stained with ΔΨm-sensitive dye MitoTracker Deep Red. In EPC2, EtOH decreased ATP level while impairing mitochondrial respiration and electron transportation chain functions, as determined by ATP fluorometric assay, respirometry, and liquid chromatography-mass spectrometry. Additionally, EPC2 cells demonstrated enhanced oxidative stress by flow cytometry for mitochondrial superoxide (MitoSOX), which was antagonized by the mitochondria-specific antioxidant MitoCP. Concurrently, EPC1 and EPC2 cells underwent autophagy following EtOH exposure, as evidenced by flow cytometry for Cyto-ID, which detects autophagic vesicles, and immunoblots demonstrating induction of the lipidated and cleaved form of LC3B and downregulation of SQSTM1/p62. In EPC1 and EPC2, pharmacological inhibition of autophagy flux by chloroquine increased mitochondrial oxidative stress while decreasing cell viability. In EPC2, autophagy induction was coupled with phosphorylation of AMP activated protein kinase (AMPK), a cellular energy sensor responding to low ATP levels, and dephosphorylation of downstream substrates of mechanistic Target of Rapamycin Complex (mTORC)-1 signaling. Pharmacological AMPK activation by AICAR decreased EtOH-induced reduction of ΔΨm and ATP in EPC2. Taken together, acute EtOH exposure leads to mitochondrial dysfunction and oxidative stress in esophageal keratinocytes, where the AMPK-mTORC1 axis may serve as a regulatory mechanism to activate autophagy to provide cytoprotection against EtOH-induced cell injury.

LinkedSV for detection of mosaic structural variants from linked-read exome and genome sequencing data.

Linked-read sequencing provides long-range information on short-read sequencing data by barcoding reads originating from the same DNA molecule, and can improve detection and breakpoint identification for structural variants (SVs). Here we present LinkedSV for SV detection on linked-read sequencing data. LinkedSV considers barcode overlapping and enriched fragment endpoints as signals to detect large SVs, while it leverages read depth, paired-end signals and local assembly to detect small SVs. Benchmarking studies demonstrate that LinkedSV outperforms existing tools, especially on exome data and on somatic SVs with low variant allele frequencies. We demonstrate clinical cases where LinkedSV identifies disease-causal SVs from linked-read exome sequencing data missed by conventional exome sequencing, and show examples where LinkedSV identifies SVs missed by high-coverage long-read sequencing. In summary, LinkedSV can detect SVs missed by conventional short-read and long-read sequencing approaches, and may resolve negative cases from clinical genome/exome sequencing studies.

Genetic risk for Alzheimer's disease and functional brain connectivity in children and adolescents.

Research suggested accumulation of tau proteins might lead to the degeneration of functional networks. Studies investigating the impact of genetic risk for Alzheimer's disease (AD) on early brain connections might shed light on mechanisms leading to AD development later in life. Here, we aim to investigate whether the polygenic risk score for Alzheimer's disease (AD-PRS) influences the connectivity among regions susceptible to tau pathology during childhood and adolescence. Participants were youth, aged 6-14 years, and recruited in Porto Alegre (discovery sample, n = 332) and São Paulo (replication sample, n = 304), Brazil. Subjects underwent genotyping and 6-min resting state funcional magnetic resonance imaging. Connections between the local maxima of tau pathology networks were used as dependent variables. The AD-PRS was associated with the connectivity between the right precuneus and the right superior temporal gyrus (discovery sample: ß = 0.180, padjusted = 0.036; replication sample: ß = 0.202, p = 0.031). This connectivity was also associated with inhibitory control (ß = 0.157, padjusted = 0.035) and moderated the association between the AD-PRS and both immediate and delayed recall. These findings suggest the AD-PRS may affect brain connectivity in youth, which might impact memory performance and inhibitory control in early life.

Exome Sequencing Reveals Mutations in AIRE as a Cause of Isolated Hypoparathyroidism.

Context: Most cases of autosomal recessive hypoparathyroidism (HYPO) are caused by loss-of-function mutations in GCM2 or PTH. Objective: The objective of this study was to identify the underlying genetic basis for isolated HYPO in a kindred in which 3 of 10 siblings were affected. Subjects: We studied the parents and the three adult affected subjects, each of whom was diagnosed with HYPO in the first decade of life. Methods: We collected clinical and biochemical data and performed whole exome sequencing analysis on DNA from the three affected subjects after negative genetic testing for known causes of HYPO. Results: Whole exome sequencing followed by Sanger sequencing revealed that all three affected subjects were compound heterozygous for two previously reported mutations, c.967_979delCTGTCCCCTCCGC:p.(L323SfsX51) and c.995+(3_5)delGAGinsTAT, in AIRE, which encodes the autoimmune regulator protein that is defective in autoimmune polyglandular syndrome type 1 (APS-1). Each parent carries one mutation, and all of the children of the patients are either heterozygous for one mutation or wild type. The affected sister developed premature ovarian failure, but the two affected brothers have no other features of APS-1 despite elevated serum levels of anti-interferon-α antibodies. Conclusions: Our findings indicate that biallelic mutations in AIRE can cause isolated HYPO as well as syndromic APS-1. The presence of antibodies to interferon-α provides a highly sensitive indicator for loss of AIRE function and represents a useful marker for isolated HYPO due to AIRE mutations.