In vitro gastrointestinal gas monitoring with carbon nanotube sensors.

In vitro simulators of the human gastrointestinal (GI) tract are remarkable technological platforms for studying the impact of food on the gut microbiota, enabling continuous and real-time monitoring of key biomarkers. However, comprehensive real-time monitoring of gaseous biomarkers in these systems is required with a cost-effective approach, which has been challenging to perform experimentally to date. In this work, we demonstrate the integration and in-line use of carbon nanotube (CNT)-based chemiresitive gas sensors coated with a thin polydimethylsiloxane (PDMS) membrane for the continuous monitoring of gases within the Simulator of the Human Microbial Ecosystem (SHIME). The findings demonstrate the ability of the gas sensor to continuously monitor the different phases of gas production in this harsh, anaerobic, highly humid, and acidic environment for a long exposure time (16 h) without saturation. This establishes our sensor platform as an effective tool for real-time monitoring of gaseous biomarkers in in vitro systems like SHIME.

TGF-ß and BMP Signaling Pathways in Skeletal Dysplasia with Short and Tall Stature.

The transforming growth factor ß (TGF-ß) and bone morphogenetic protein (BMP) signaling pathways play a pivotal role in bone development and skeletal health. More than 30 different types of skeletal dysplasia are now known to be caused by pathogenic variants in genes that belong to the TGF-ß superfamily and/or regulate TGF-ß/BMP bioavailability. This review describes the latest advances in skeletal dysplasia that is due to impaired TGF-ß/BMP signaling and results in short stature (acromelic dysplasia and cardiospondylocarpofacial syndrome) or tall stature (Marfan syndrome). We thoroughly describe the clinical features of the patients, the underlying genetic findings, and the pathomolecular mechanisms leading to disease, which have been investigated mainly using patient-derived skin fibroblasts and mouse models. Although no pharmacological treatment is yet available for skeletal dysplasia due to impaired TGF-ß/BMP signaling, in recent years advances in the use of drugs targeting TGF-ß have been made, and we also discuss these advances.

Mosaic Deletions of Known Genes Explain Skeletal Dysplasias With High and Low Bone Mass.

Mosaicism, a state in which an individual has two or more genetically distinct populations of cells in the body, can be difficult to detect because of either mild or atypical clinical presentation and limitations in the commonly used detection methods. Knowledge of the role of mosaicism is limited in many skeletal disorders, including osteopathia striata with cranial sclerosis (OSCS) and cleidocranial dysplasia (CCD). We used whole-genome sequencing (WGS) with coverage >40× to identify the genetic causes of disease in two clinically diagnosed patients. In a female patient with OSCS, we identified a mosaic 7-nucleotide frameshift deletion in exon 2 of AMER1, NM_152424.4:c.855_861del:p.(His285Glnfs*7), affecting 8.3% of the WGS reads. In a male patient with CCD, approximately 34% of the WGS reads harbored a 3710-basepair mosaic deletion, NC_000006.11:g.45514471_45518181del, starting in intron 8 of RUNX2 and terminating in the 3' untranslated region. Droplet digital polymerase chain reaction was used to validate these deletions and quantify the absolute level of mosaicism in each patient. Although constitutional variants in AMER1 and RUNX2 are a known cause of OSCS and CCD, respectively, the mosaic changes here reported have not been described previously. Our study indicates that mosaicism should be considered in unsolved cases of skeletal dysplasia and should be investigated with comprehensive and sensitive detection methods. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Early-Onset Osteoporosis: Rare Monogenic Forms Elucidate the Complexity of Disease Pathogenesis Beyond Type I Collagen.

Early-onset osteoporosis (EOOP), characterized by low bone mineral density (BMD) and fractures, affects children, premenopausal women and men aged <50 years. EOOP may be secondary to a chronic illness, long-term medication, nutritional deficiencies, etc. If no such cause is identified, EOOP is regarded primary and may then be related to rare variants in genes playing a pivotal role in bone homeostasis. If the cause remains unknown, EOOP is considered idiopathic. The scope of this review is to guide through clinical and genetic diagnostics of EOOP, summarize the present knowledge on rare monogenic forms of EOOP, and describe how analysis of bone biopsy samples can lead to a better understanding of the disease pathogenesis. The diagnostic pathway of EOOP is often complicated and extensive assessments may be needed to reliably exclude secondary causes. Due to the genetic heterogeneity and overlapping features in the various genetic forms of EOOP and other bone fragility disorders, the genetic diagnosis usually requires the use of next-generation sequencing to investigate several genes simultaneously. Recent discoveries have elucidated the complexity of disease pathogenesis both regarding genetic architecture and bone tissue-level pathology. Two rare monogenic forms of EOOP are due to defects in genes partaking in the canonical WNT pathway: LRP5 and WNT1. Variants in the genes encoding plastin-3 (PLS3) and sphingomyelin synthase 2 (SGMS2) have also been found in children and young adults with skeletal fragility. The molecular mechanisms leading from gene defects to clinical manifestations are often not fully understood. Detailed analysis of patient-derived transiliac bone biopsies gives valuable information to understand disease pathogenesis, distinguishes EOOP from other bone fragility disorders, and guides in patient management, but is not widely available in clinical settings. Despite the great advances in this field, EOOP remains an insufficiently explored entity and further research is needed to optimize diagnostic and therapeutic approaches. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

An ARHGAP25 variant links aberrant Rac1 function to early-onset skeletal fragility.

Ras homologous guanosine triphosphatases (RhoGTPases) control several cellular functions, including cytoskeletal actin remodeling and cell migration. Their activities are downregulated by GTPase-activating proteins (GAPs). Although RhoGTPases are implicated in bone remodeling and osteoclast and osteoblast function, their significance in human bone health and disease remains elusive. Here, we report defective RhoGTPase regulation as a cause of severe, early-onset, autosomal-dominant skeletal fragility in a three-generation Finnish family. Affected individuals (n = 13) presented with multiple low-energy peripheral and vertebral fractures despite normal bone mineral density (BMD). Bone histomorphometry suggested reduced bone volume, low surface area covered by osteoblasts and osteoclasts, and low bone turnover. Exome sequencing identified a novel heterozygous missense variant c.652G>A (p.G218R) in ARHGAP25, encoding a GAP for Rho-family GTPase Rac1. Variants in the ARHGAP25 5' untranslated region (UTR) also associated with BMD and fracture risk in the general population, across multiple genomewide association study (GWAS) meta-analyses (lead variant rs10048745). ARHGAP25 messenger RNA (mRNA) was expressed in macrophage colony-stimulating factor (M-CSF)-stimulated human monocytes and mouse osteoblasts, indicating a possible role for ARHGAP25 in osteoclast and osteoblast differentiation and activity. Studies on subject-derived osteoclasts from peripheral blood mononuclear cells did not reveal robust defects in mature osteoclast formation or resorptive activity. However, analysis of osteosarcoma cells overexpressing the ARHGAP25 G218R-mutant, combined with structural modeling, confirmed that the mutant protein had decreased GAP-activity against Rac1, resulting in elevated Rac1 activity, increased cell spreading, and membrane ruffling. Our findings indicate that mutated ARHGAP25 causes aberrant Rac1 function and consequently abnormal bone metabolism, highlighting the importance of RhoGAP signaling in bone metabolism in familial forms of skeletal fragility and in the general population, and expanding our understanding of the molecular pathways underlying skeletal fragility. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Oligogenic Inheritance of Monoallelic TRIP11, FKBP10, NEK1, TBX5, and NBAS Variants Leading to a Phenotype Similar to Odontochondrodysplasia.

Skeletal dysplasias are often well characterized, and only a minority of the cases remain unsolved after a thorough analysis of pathogenic variants in over 400 genes that are presently known to cause monogenic skeletal diseases. Here, we describe an 11-year-old Finnish girl, born to unrelated healthy parents, who had severe short stature and a phenotype similar to odontochondrodysplasia (ODCD), a monogenic skeletal dysplasia caused by biallelic TRIP11 variants. The family had previously lost a fetus due to severe skeletal dysplasia. Exome sequencing and bioinformatic analysis revealed an oligogenic inheritance of a heterozygous nonsense mutation in TRIP11 and four likely pathogenic missense variants in FKBP10, TBX5, NEK1, and NBAS in the index patient. Interestingly, all these genes except TBX5 are known to cause skeletal dysplasia in an autosomal recessive manner. In contrast, the fetus was found homozygous for the TRIP11 mutation, and achondrogenesis type IA diagnosis was, thus, molecularly confirmed, indicating two different skeletal dysplasia forms in the family. To the best of our knowledge, this is the first report of an oligogenic inheritance model of a skeletal dysplasia in a Finnish family. Our findings may have implications for genetic counseling and for understanding the yet unsolved cases of rare skeletal dysplasias.

Novel RPL13 Variants and Variable Clinical Expressivity in a Human Ribosomopathy With Spondyloepimetaphyseal Dysplasia.

Spondyloepimetaphyseal dysplasias (SEMDs) are a heterogeneous group of disorders with variable growth failure and skeletal impairments affecting the spine and long bone epiphyses and metaphyses. Here we report on four unrelated families with SEMD in which we identified two monoallelic missense variants and one monoallelic splice site variant in RPL13, encoding the ribosomal protein eL13. In two out of four families, we observed autosomal dominant inheritance with incomplete penetrance and variable clinical expressivity; the phenotypes of the mutation-positive subjects ranged from normal height with or without hip dysplasia to severe SEMD with severe short stature and marked skeletal dysplasia. In vitro studies on patient-derived dermal fibroblasts harboring RPL13 missense mutations demonstrated normal eL13 expression, with proper subcellular localization but reduced colocalization with eL28 (p < 0.001). Cellular functional defects in fibroblasts from mutation-positive subjects indicated a significant increase in the ratio of 60S subunits to 80S ribosomes (p = 0.007) and attenuated global translation (p = 0.017). In line with the human phenotype, our rpl13 mutant zebrafish model, generated by CRISPR-Cas9 editing, showed cartilage deformities at embryonic and juvenile stages. These findings extend the genetic spectrum of RPL13 mutations causing this novel human ribosomopathy with variable skeletal features. Our study underscores for the first time incomplete penetrance and broad phenotypic variability in SEMD-RPL13 type and confirms impaired ribosomal function. Furthermore, the newly generated rpl13 mutant zebrafish model corroborates the role of eL13 in skeletogenesis. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)..

Novel form of rhizomelic skeletal dysplasia associated with a homozygous variant in GNPNAT1.

BACKGROUND: Studies exploring molecular mechanisms underlying congenital skeletal disorders have revealed novel regulators of skeletal homeostasis and shown protein glycosylation to play an important role. OBJECTIVE: To identify the genetic cause of rhizomelic skeletal dysplasia in a consanguineous Pakistani family. METHODS: Clinical investigations were carried out for four affected individuals in the recruited family. Whole genome sequencing (WGS) was completed using DNA from two affected and two unaffected individuals from the family. Sequencing data were processed, filtered and analysed. In silico analyses were performed to predict the effects of the candidate variant on the protein structure and function. Small interfering RNAs (siRNAs) were used to study the effect of Gnpnat1 gene knockdown in primary rat chondrocytes. RESULTS: The patients presented with short stature due to extreme shortening of the proximal segments of the limbs. Radiographs of one individual showed hip dysplasia and severe platyspondyly. WGS data analyses identified a homozygous missense variant c.226G>A; p.(Glu76Lys) in GNPNAT1, segregating with the disease. Glucosamine 6-phosphate N-acetyltransferase, encoded by the highly conserved gene GNPNAT1, is one of the enzymes required for synthesis of uridine diphosphate N-acetylglucosamine, which participates in protein glycosylation. Knockdown of Gnpnat1 by siRNAs decreased cellular proliferation and expression of chondrocyte differentiation markers collagen type 2 and alkaline phosphatase, indicating that Gnpnat1 is important for growth plate chondrocyte proliferation and differentiation. CONCLUSIONS: This study describes a novel severe skeletal dysplasia associated with a biallelic, variant in GNPNAT1. Our data suggest that GNPNAT1 is important for growth plate chondrogenesis.

'Metaphyseal dysplasia without hypotrichosis' can present with late-onset extraskeletal manifestations.

BACKGROUND: Metaphyseal dysplasia without hypotrichosis (MDWH) is a rare form of chondrodysplasia with no extraskeletal manifestations. MDWH is caused by RMRP mutations, but it is differentiated from the allelic condition cartilage-hair hypoplasia (CHH), which in addition to chondrodysplasia is characterised by thin hair, immunodeficiency and increased risk of malignancy. The long-term outcome of MDWH remains unknown. OBJECTIVE: We diagnosed severe agranulocytosis in a subject with RMRP mutations and normal hair. Based on this observation, we hypothesised that MDWH may, similar to CHH, associate with immune deficiency and malignancy. METHODS: We collected clinical and laboratory data for a cohort of 80 patients with RMRP mutations followed for over 30 years and analysed outcome data for those with features consistent with MDWH. RESULTS: In our cohort, we identified 10 patients with skeletal but no extraskeletal features during preschool age. Eight of these patients developed malignancy or clinically significant immunodeficiency during follow-up. Two of them died during chemotherapy for malignancy. At the time of the first extraskeletal manifestation, patients were school aged, 20, 43 and 50 years old. Laboratory signs of immunodeficiency (impaired lymphocyte proliferative responses) were demonstrated in four patients before the onset of symptoms. The patient outside this cohort, who had RMRP mutations, skeletal dysplasia, normal hair and severe agranulocytosis at 18 years of age, underwent haematopoietic stem cell transplantation. CONCLUSIONS: MDWH can present with severe late-onset extraskeletal manifestations and thus should be reclassified and managed as CHH.

Novel fibronectin mutations and expansion of the phenotype in spondylometaphyseal dysplasia with "corner fractures".

Heterozygous pathogenic variants in the FN1 gene, encoding fibronectin (FN), have recently been shown to be associated with a skeletal disorder in some individuals affected by spondylometaphyseal dysplasia with "corner fractures" (SMD-CF). The most striking feature characterizing SMD-CF is irregularly shaped metaphyses giving the appearance of "corner fractures". An array of secondary features, including developmental coxa vara, ovoid vertebral bodies and severe scoliosis, may also be present. FN is an important extracellular matrix component for bone and cartilage development. Here we report five patients affected by this subtype of SMD-CF caused by five novel FN1 missense mutations: p.Cys123Tyr, p.Cys169Tyr, p.Cys213Tyr, p.Cys231Trp and p.Cys258Tyr. All individuals shared a substitution of a cysteine residue, disrupting disulfide bonds in the FN type-I assembly domains located in the N-terminal assembly region. The abnormal metaphyseal ossification and "corner fracture" appearances were the most remarkable clinical feature in these patients. In addition, generalized skeletal fragility with low-trauma bilateral femoral fractures was identified in one patient. Interestingly, the distal femoral changes in this patient healed with skeletal maturation. Our report expands the phenotypic and genetic spectrum of the FN1-related SMD-CF and emphasizes the importance of FN in bone formation and possibly also in the maintenance of bone strength.

Expansion of the clinical spectrum of frontometaphyseal dysplasia 2 caused by the recurrent mutation p.Pro485Leu in MAP3K7.

Frontometaphyseal dysplasia 2 (FMD2) is a skeletal dysplasia with supraorbital hyperostosis combined with undermodeling of the bones, joint contractures and some extraskeletal features. It is caused by heterozygous mutations in MAP3K7, encoding the Mitogen-Activated Protein 3-Kinase 7. MAP3K7 is activated by TGF-ß and plays an important role in osteogenesis. Less than 20 patients with FMD2 and MAP3K7 mutations have been described thus far. The majority of the patients harbor a recurrent missense mutation, NM_003188.3: c.1454C > T [NP_003179.1: p.(Pro485Leu)], which leads to a more severe phenotype than mutations in other domains. Here we describe an additional patient with FMD2 caused by the recurrent c.1454C > T MAP3K7 mutation, identified as a de novo variant by whole-genome sequencing. The 17-year-old boy has the characteristic skeletal and facial features of FMD2. However, some novel features were also observed, including growth retardation and spina bifida occulta. In line with other patients harboring the same mutation he also showed keloid scars and had no intellectual disability. This report expands the clinical spectrum of FMD2 caused by the recurrent c.1454C > T [p.(Pro485Leu)] mutation in MAP3K7.

Autosomal Recessive Osteogenesis Imperfecta Caused by a Novel Homozygous COL1A2 Mutation.

Osteogenesis imperfecta (OI) is a skeletal dysplasia characterized by brittle bones and extraskeletal manifestations. The disease phenotype varies greatly. Most commonly, OI arises from monoallelic mutations in one of the two genes encoding type I collagen, COL1A1 and COL1A2 and is inherited as an autosomal dominant trait. Here, we describe a consanguineous family with autosomal recessive OI caused by a novel homozygous glycine substitution in COL1A2, NM_000089.3: c.604G>A, p.(Gly202Ser), detected by whole-genome sequencing. The index patient is a 31-year-old Greek woman with severe skeletal fragility. She had mild short stature, low bone mineral density of the lumbar spine and blue sclerae. She had sustained multiple long bone and vertebral fractures since childhood and had been treated with bisphosphonates for several years. She also had an affected sister with similar clinical manifestations. Interestingly, the parents and one sister, all carriers of the COL1A2 glycine mutation, did not have manifestations of OI. In summary, we report on autosomal recessive OI caused by a homozygous glycine-to-serine substitution in COL1A2, leading to severe skeletal fragility. The mutation carriers lacked OI manifestations. This family further expands the complex genetic spectrum of OI and underscores the importance of genetic evaluation for correct genetic counselling.

High bone mass due to novel LRP5 and AMER1 mutations.

WNT signaling is a key regulator of bone metabolism and its increased or decreased activity leads to skeletal disorders. Here we describe two patients with high bone mass (HBM) caused by novel mutations in two different WNT pathway components. The first patient is a 53-year-old male with HBM. He was diagnosed at adult age based on significantly increased bone mineral density (BMD). He has undergone several surgeries due to excessive bone in ear canals, bilateral jaw exostoses and mandibular tori. Radiographs show severe cortical thickening of cranial and long bones. Sanger sequencing identified a novel heterozygous mutation c.592A>T (p.N198Y) in LRP5 (Low-density lipoprotein receptor-related protein 5). The second patient, an adolescent female, was diagnosed with skeletal dysplasia in early childhood. She had macrocephaly (head circumference +6.0 SD), facial dysmorphism, delayed motor development, laryngomalasia and epilepsy. Radiographic findings were consistent with osteopathia striata with cranial sclerosis. A novel heterozygous frameshift mutation c.655del (p.E219Rfs*63) in AMER1 (APC Membrane Recruiting Protein 1) was identified. Although both mutations are predicted to lead to increased WNT signaling with a consequent increase in bone formation, the resulting phenotypes are different; cranial sclerosis versus macrocephaly, long bone cortical thickening versus vertical striations and discordant neurological development. This report underscores the diversity of genotypes and phenotypes of HBM and facilitates their differential diagnosis.

PLS3 Deletions Lead to Severe Spinal Osteoporosis and Disturbed Bone Matrix Mineralization.

Mutations in the PLS3 gene, encoding Plastin 3, were described in 2013 as a cause for X-linked primary bone fragility in children. The specific role of PLS3 in bone metabolism remains inadequately understood. Here we describe for the first time PLS3 deletions as the underlying cause for childhood-onset primary osteoporosis in 3 boys from 2 families. We carried out thorough clinical, radiological, and bone tissue analyses to explore the consequences of these deletions and to further elucidate the role of PLS3 in bone homeostasis. In family 1, the 2 affected brothers had a deletion of exons 4-16 (NM_005032) in PLS3, inherited from their healthy mother. In family 2, the index patient had a deletion involving the entire PLS3 gene (exons 1-16), inherited from his mother who had osteoporosis. The 3 patients presented in early childhood with severe spinal compression fractures involving all vertebral bodies. The 2 brothers in family 1 also displayed subtle dysmorphic facial features and both had developed a myopathic gait. Extensive analyses of a transiliac bone biopsy from 1 patient showed a prominent increase in osteoid volume, osteoid thickness, and in mineralizing lag time. Results from quantitative backscattered electron imaging and Raman microspectroscopy showed a significant hypomineralization of the bone. Together our results indicate that PLS3 deletions lead to severe childhood-onset osteoporosis resulting from defective bone matrix mineralization, suggesting a specific role for PLS3 in the mineralization process. © 2017 American Society for Bone and Mineral Research.