Harnessing Next-Generation Sequencing as a Timely and Accurate Second-Tier Screening Test for Newborn Screening of Inborn Errors of Metabolism.

In this study, we evaluated the implementation of a second-tier genetic screening test using an amplicon-based next-generation sequencing (NGS) panel in our laboratory during the period of 1 September 2021 to 31 August 2022 for the newborn screening (NBS) of six conditions for inborn errors of metabolism: citrullinemia type II (MIM #605814), systemic primary carnitine deficiency (MIM #212140), glutaric acidemia type I (MIM #231670), beta-ketothiolase deficiency (#203750), holocarboxylase synthetase deficiency (MIM #253270) and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency (MIM # 246450). The custom-designed NGS panel can detect sequence variants in the relevant genes and also specifically screen for the presence of the hotspot variant IVS16ins3kb of SLC25A13 by the copy number variant calling algorithm. Genetic second-tier tests were performed for 1.8% of a total of 22,883 NBS samples. The false positive rate for these six conditions after the NGS second-tier test was only 0.017%, and two cases of citrullinemia type II would have been missed as false negatives if only biochemical first-tier testing was performed. The confirmed true positive cases were citrullinemia type II (n = 2) and systemic primary carnitine deficiency (n = 1). The false positives were later confirmed to be carrier of citrullinemia type II (n = 2), carrier of glutaric acidemia type I (n = 1) and carrier of systemic primary carnitine deficiency (n = 1). There were no false negatives reported. The incorporation of a second-tier genetic screening test by NGS greatly enhanced our program's performance with 5-working days turn-around time maintained as before. In addition, early genetic information is available at the time of recall to facilitate better clinical management and genetic counseling.

MMP14 cleaves PTH1R in the chondrocyte-derived osteoblast lineage, curbing signaling intensity for proper bone anabolism.

Bone homeostasis is regulated by hormones such as parathyroid hormone (PTH). While PTH can stimulate osteo-progenitor expansion and bone synthesis, how the PTH-signaling intensity in progenitors is controlled is unclear. Endochondral bone osteoblasts arise from perichondrium-derived osteoprogenitors and hypertrophic chondrocytes (HC). We found, via single-cell transcriptomics, that HC-descendent cells activate membrane-type 1 metalloproteinase 14 (MMP14) and the PTH pathway as they transition to osteoblasts in neonatal and adult mice. Unlike Mmp14 global knockouts, postnatal day 10 (p10) HC lineage-specific Mmp14 null mutants (Mmp14ΔHC) produce more bone. Mechanistically, MMP14 cleaves the extracellular domain of PTH1R, dampening PTH signaling, and consistent with the implied regulatory role, in Mmp14ΔHC mutants, PTH signaling is enhanced. We found that HC-derived osteoblasts contribute ~50% of osteogenesis promoted by treatment with PTH 1-34, and this response was amplified in Mmp14ΔHC. MMP14 control of PTH signaling likely applies also to both HC- and non-HC-derived osteoblasts because their transcriptomes are highly similar. Our study identifies a novel paradigm of MMP14 activity-mediated modulation of PTH signaling in the osteoblast lineage, contributing new insights into bone metabolism with therapeutic significance for bone-wasting diseases.

IRX3 and IRX5 Inhibit Adipogenic Differentiation of Hypertrophic Chondrocytes and Promote Osteogenesis.

Maintaining the correct proportions of different cell types in the bone marrow is critical for bone function. Hypertrophic chondrocytes (HCs) and osteoblasts are a lineage continuum with a minor contribution to adipocytes, but the regulatory network is unclear. Mutations in transcription factors, IRX3 and IRX5, result in skeletal patterning defects in humans and mice. We found coexpression of Irx3 and Irx5 in late-stage HCs and osteoblasts in cortical and trabecular bone. Irx3 and Irx5 null mutants display severe bone deficiency in newborn and adult stages. Quantitative analyses of bone with different combinations of functional alleles of Irx3 and Irx5 suggest these two factors function in a dosage-dependent manner. In Irx3 and Irx5 nulls, the amount of bone marrow adipocytes was increased. In Irx5 nulls, lineage tracing revealed that removal of Irx3 specifically in HCs exacerbated reduction of HC-derived osteoblasts and increased the frequency of HC-derived marrow adipocytes. ß-catenin loss of function and gain of function specifically in HCs affects the expression of Irx3 and Irx5, suggesting IRX3 and IRX5 function downstream of WNT signaling. Our study shows that IRX3 and IRX5 regulate fate decisions in the transition of HCs to osteoblasts and to marrow adipocytes, implicating their potential roles in human skeletal homeostasis and disorders.

The extended chondrocyte lineage: implications for skeletal homeostasis and disorders.

Endochondral bone formation relies on a finely controlled sequence of chondrocyte proliferation, maturation and hypertrophy that establishes the growth plate which, combined with the deposition of bone upon the cartilage template, mediates longitudinal skeletal growth. Recent lineage studies support a chondrocyte-osteoblast differentiation continuum and the presence of skeletal stem cells within cartilage. The biological significance of the lineage extension and the mechanisms controlling the process are unclear. In this review, we describe recent work on the extended chondrocyte-osteoblast lineage and its contribution to the development, growth and repair of bone and to bone disorders that provides insight into the process and the molecular controls involved. The implications for skeletal homeostasis are discussed.

Inhibiting the integrated stress response pathway prevents aberrant chondrocyte differentiation thereby alleviating chondrodysplasia.

The integrated stress response (ISR) is activated by diverse forms of cellular stress, including endoplasmic reticulum (ER) stress, and is associated with diseases. However, the molecular mechanism(s) whereby the ISR impacts on differentiation is incompletely understood. Here, we exploited a mouse model of Metaphyseal Chondrodysplasia type Schmid (MCDS) to provide insight into the impact of the ISR on cell fate. We show the protein kinase RNA-like ER kinase (PERK) pathway that mediates preferential synthesis of ATF4 and CHOP, dominates in causing dysplasia by reverting chondrocyte differentiation via ATF4-directed transactivation of Sox9. Chondrocyte survival is enabled, cell autonomously, by CHOP and dual CHOP-ATF4 transactivation of Fgf21. Treatment of mutant mice with a chemical inhibitor of PERK signaling prevents the differentiation defects and ameliorates chondrodysplasia. By preventing aberrant differentiation, titrated inhibition of the ISR emerges as a rationale therapeutic strategy for stress-induced skeletal disorders.

Synergistic co-regulation and competition by a SOX9-GLI-FOXA phasic transcriptional network coordinate chondrocyte differentiation transitions.

The growth plate mediates bone growth where SOX9 and GLI factors control chondrocyte proliferation, differentiation and entry into hypertrophy. FOXA factors regulate hypertrophic chondrocyte maturation. How these factors integrate into a Gene Regulatory Network (GRN) controlling these differentiation transitions is incompletely understood. We adopted a genome-wide whole tissue approach to establish a Growth Plate Differential Gene Expression Library (GP-DGEL) for fractionated proliferating, pre-hypertrophic, early and late hypertrophic chondrocytes, as an overarching resource for discovery of pathways and disease candidates. De novo motif discovery revealed the enrichment of SOX9 and GLI binding sites in the genes preferentially expressed in proliferating and prehypertrophic chondrocytes, suggesting the potential cooperation between SOX9 and GLI proteins. We integrated the analyses of the transcriptome, SOX9, GLI1 and GLI3 ChIP-seq datasets, with functional validation by transactivation assays and mouse mutants. We identified new SOX9 targets and showed SOX9-GLI directly and cooperatively regulate many genes such as Trps1, Sox9, Sox5, Sox6, Col2a1, Ptch1, Gli1 and Gli2. Further, FOXA2 competes with SOX9 for the transactivation of target genes. The data support a model of SOX9-GLI-FOXA phasic GRN in chondrocyte development. Together, SOX9-GLI auto-regulate and cooperate to activate and repress genes in proliferating chondrocytes. Upon hypertrophy, FOXA competes with SOX9, and control toward terminal differentiation passes to FOXA, RUNX, AP1 and MEF2 factors.

Activating the unfolded protein response in osteocytes causes hyperostosis consistent with craniodiaphyseal dysplasia.

Bone remodeling is a balanced process between bone synthesis and degradation, maintaining homeostasis and a constant bone mass in adult life. Imbalance will lead to conditions such as osteoporosis or hyperostosis. Osteoblasts build bone, becoming embedded in bone matrix as mature osteocytes. Osteocytes have a role in sensing and translating mechanical loads into biochemical signals, regulating the differentiation and activity of osteoblasts residing at the bone surface through the secretion of Sclerostin (SOST), an inhibitor of WNT signaling. Excessive mechanical load can lead to activation of cellular stress responses altering cell behavior and differentiation. The unfolded protein response (UPR) is a shared pathway utilized by cells to cope with stress stimuli. We showed that in a transgenic mouse model, activation of the UPR in early differentiating osteocytes delays maturation, maintaining active bone synthesis. In addition, expression of SOST is delayed or suppressed; resulting in active WNT signaling and enhanced periosteal bone formation, and the combined outcome is generalized hyperostosis. A clear relationship between the activation of the unfolded protein response was established and the onset of hyperostosis that can be suppressed with a chemical chaperone, sodium 4-phenobutyrate (4-PBA). As the phenotype is highly consistent with craniodiaphyseal dysplasia (CDD; OMIM 122860), we propose activation of the UPR could be part of the disease mechanism for CDD patients as these patients are heterozygous for SOST mutations that impair protein folding and secretion. Thus, therapeutic agents ameliorating protein folding or the UPR can be considered as a potential therapeutic treatment.

Fate of growth plate hypertrophic chondrocytes: death or lineage extension?

The vertebrate growth plate is an essential tissue that mediates and controls bone growth. It forms through a multistep differentiation process in which chondrocytes differentiate, proliferate, stop dividing and undergo hypertrophy, which entails a 20-fold increase in size. Hypertrophic chondrocytes are specialized cells considered to be the end state of the chondrocyte differentiation pathway, and are essential for bone growth. They are characterized by expression of type X collagen encoded by the Col10a1 gene, and synthesis of a calcified cartilage matrix. Whether hypertrophy marks a transition preceding osteogenesis, or it is the terminal differentiation stage of chondrocytes with cell death as the ultimate fate has been the subject of debate for over a century. In this review, we revisit this debate in the light of new findings arising from genetic-mediated lineage tracing studies showing that hypertrophic chondrocytes can survive at the chondro-osseous junction and further make the transition to become osteoblasts and osteocytes. The contribution of chondrocytes to the osteoblast lineage has important implications in bone development, disease and repair.

Hypertrophic chondrocytes can become osteoblasts and osteocytes in endochondral bone formation.

According to current dogma, chondrocytes and osteoblasts are considered independent lineages derived from a common osteochondroprogenitor. In endochondral bone formation, chondrocytes undergo a series of differentiation steps to form the growth plate, and it generally is accepted that death is the ultimate fate of terminally differentiated hypertrophic chondrocytes (HCs). Osteoblasts, accompanying vascular invasion, lay down endochondral bone to replace cartilage. However, whether an HC can become an osteoblast and contribute to the full osteogenic lineage has been the subject of a century-long debate. Here we use a cell-specific tamoxifen-inducible genetic recombination approach to track the fate of murine HCs and show that they can survive the cartilage-to-bone transition and become osteogenic cells in fetal and postnatal endochondral bones and persist into adulthood. This discovery of a chondrocyte-to-osteoblast lineage continuum revises concepts of the ontogeny of osteoblasts, with implications for the control of bone homeostasis and the interpretation of the underlying pathological bases of bone disorders.

The chondrocytic journey in endochondral bone growth and skeletal dysplasia.

The endochondral bones of the skeleton develop from a cartilage template and grow via a process involving a cascade of chondrocyte differentiation steps culminating in formation of a growth plate and the replacement of cartilage by bone. This process of endochondral ossification, driven by the generation of chondrocytes and their subsequent proliferation, differentiation, and production of extracellular matrix constitute a journey, deviation from which inevitably disrupts bone growth and development, and is the basis of human skeletal dysplasias with a wide range of phenotypic severity, from perinatal lethality to progressively deforming. This highly coordinated journey of chondrocyte specification and fate determination is controlled by a myriad of intrinsic and extrinsic factors. SOX9 is the master transcription factor that, in concert with varying partners along the way, directs the different phases of the journey from mesenchymal condensation, chondrogenesis, differentiation, proliferation, and maturation. Extracellular signals, including bone morphogenetic proteins, wingless-related MMTV integration site (WNT), fibroblast growth factor, Indian hedgehog, and parathyroid hormone-related peptide, are all indispensable for growth plate chondrocytes to align and organize into the appropriate columnar architecture and controls their maturation and transition to hypertrophy. Chondrocyte hypertrophy, marked by dramatic volume increase in phases, is controlled by transcription factors SOX9, Runt-related transcription factor, and FOXA2. Hypertrophic chondrocytes mediate the cartilage to bone transition and concomitantly face a live-or-die situation, a subject of much debate. We review recent insights into the coordination of the phases of the chondrocyte journey, and highlight the need for a systems level understanding of the regulatory networks that will facilitate the development of therapeutic approaches for skeletal dysplasia.

Decreased c-Jun expression correlates with impaired spinal motoneuron regeneration in aged mice following sciatic nerve crush.

Post-injury nerve regeneration of the peripheral nervous system declines with age, but the mechanisms underlying the weakened axonal regeneration are not well understood. Increased synthesis and activity of the AP-1 transcription factor c-Jun have been implicated in efficient motor axonal regeneration. In the present study, we evaluated the hypothesis that the impaired regenerative capacity in the aged is associated with impaired induction of c-Jun. In non-manipulated young adult or aged mice, no c-Jun and its phosphorylated form were detected in the ventral horn of the spinal cord. Following nerve crush, significant c-Jun and phosphorylated c-Jun occurred in the injured motoneurons of young adult mice, but not in aged animals. In accord with the immunohistochemistry, Western blots also showed that sciatic nerve crush induced c-Jun and its phosphorylation expression in the ventral horn of young adult but not in aged mice. Changes in c-Jun mRNA level detected by in situ hybridization are congruent with that in c-Jun protein content, showing an increase at 5 days after crush in young adult but not aged. Moreover, compared with young adult mice, aged mice showed impaired motor axonal regeneration. These results demonstrate that the impaired motor axonal regeneration seen in aged mice is correlated with impaired c-Jun expression and phosphorylation following injury. These data provide a neurobiological explanation for the poor outcome associated with nerve repair in the aged.

In vivo cellular adaptation to ER stress: survival strategies with double-edged consequences.

Disturbances to the balance of protein synthesis, folding and secretion in the endoplasmic reticulum (ER) induce stress and thereby the ER stress signaling (ERSS) response, which alleviates this stress. In this Commentary, we review the emerging idea that ER stress caused by abnormal physiological conditions and/or mutations in genes that encode client proteins of the ER is a key factor underlying different developmental processes and the pathology of diverse diseases, including diabetes, neurodegeneration and skeletal dysplasias. Recent studies in mouse models indicate that the effect of ERSS in vivo and the nature of the cellular strategies induced to ameliorate pathological ER stress are crucial factors in determining cell fate and clinical disease features. Importantly, ERSS can affect cellular proliferation and the differentiation program; cells that survive the stress can become 'reprogrammed' or dysfunctional. These cell-autonomous adaptation strategies can generate a spectrum of context-dependent cellular consequences, ranging from recovery to death. Secondary effects can include altered cell-extracellular-matrix interactions and non-cell-autonomous alteration of paracrine signaling, which contribute to the final phenotypic outcome. Recent reports showing that ER stress can be alleviated by chemical compounds suggest the potential for novel therapeutic approaches.

The developmental roles of the extracellular matrix: beyond structure to regulation.

Cells in multicellular organisms are surrounded by a complex three-dimensional macromolecular extracellular matrix (ECM). This matrix, traditionally thought to serve a structural function providing support and strength to cells within tissues, is increasingly being recognized as having pleiotropic effects in development and growth. Elucidation of the role that the ECM plays in developmental processes has been significantly advanced by studying the phenotypic and developmental consequences of specific genetic alterations of ECM components in the mouse. These studies have revealed the enormous contribution of the ECM to the regulation of key processes in morphogenesis and organogenesis, such as cell adhesion, proliferation, specification, migration, survival, and differentiation. The ECM interacts with signaling molecules and morphogens thereby modulating their activities. This review considers these advances in our understanding of the function of ECM proteins during development, extending beyond their structural capacity, to embrace their new roles in intercellular signaling.

COL10A1 nonsense and frame-shift mutations have a gain-of-function effect on the growth plate in human and mouse metaphyseal chondrodysplasia type Schmid.

Missense, nonsense and frame-shift mutations in the collagen X gene (COL10A1) result in metaphyseal chondrodysplasia type Schmid (MCDS). Complete degradation of mutant COL10A1 mRNA by nonsense-mediated decay in human MCDS cartilage implicates haploinsufficiency in the pathogenesis for nonsense mutations in vivo. However, the mechanism is unclear in situations where the mutant mRNA persist. We show that nonsense/frame-shift mutations can elicit a gain-of-function effect, affecting chondrocyte differentiation in the growth plate. In an MCDS proband, heterozygous for a p.Y663X nonsense mutation, the growth plate cartilage contained 64% wild-type and 36% mutant mRNA and the hypertrophic zone was disorganized and expanded. The in vitro translated mutant collagen X chains, which are truncated, were misfolded, unable to assemble into trimers and interfered with the assembly of normal alpha1(X) chains into trimers. Unlike Col10a1 null mutants, transgenic mice (FCdel) bearing the mouse equivalent of a human MCDS p.P620fsX621 mutation, displayed typical characteristics of MCDS with disproportionate shortening of limbs and early onset coxa vara. In FCdel mice, the degree of expansion of the hypertrophic zones was transgene-dosage dependent, being most severe in mice homozygous for the transgene. Chondrocytes in the lower region of the expanded hypertrophic zone expressed markers uncharacteristic of hypertrophic chondrocytes, indicating that differentiation was disrupted. Misfolded FCdel alpha1(X) chains were retained within the endoplasmic reticulum of hypertrophic chondrocytes, activating the unfolded protein response. Our findings provide strong in vivo evidence for a gain-of-function effect that is linked to the activation of endoplasmic reticulum-stress response and altered chondrocyte differentiation, as a possible molecular pathogenesis for MCDS.

Surviving endoplasmic reticulum stress is coupled to altered chondrocyte differentiation and function.

In protein folding and secretion disorders, activation of endoplasmic reticulum (ER) stress signaling (ERSS) protects cells, alleviating stress that would otherwise trigger apoptosis. Whether the stress-surviving cells resume normal function is not known. We studied the in vivo impact of ER stress in terminally differentiating hypertrophic chondrocytes (HCs) during endochondral bone formation. In transgenic mice expressing mutant collagen X as a consequence of a 13-base pair deletion in Col10a1 (13del), misfolded alpha1(X) chains accumulate in HCs and elicit ERSS. Histological and gene expression analyses showed that these chondrocytes survived ER stress, but terminal differentiation is interrupted, and endochondral bone formation is delayed, producing a chondrodysplasia phenotype. This altered differentiation involves cell-cycle re-entry, the re-expression of genes characteristic of a prehypertrophic-like state, and is cell-autonomous. Concomitantly, expression of Col10a1 and 13del mRNAs are reduced, and ER stress is alleviated. ERSS, abnormal chondrocyte differentiation, and altered growth plate architecture also occur in mice expressing mutant collagen II and aggrecan. Alteration of the differentiation program in chondrocytes expressing unfolded or misfolded proteins may be part of an adaptive response that facilitates survival and recovery from the ensuing ER stress. However, the altered differentiation disrupts the highly coordinated events of endochondral ossification culminating in chondrodysplasia.