Genetic variation in WNT16 and its association with bone mineral density, fractures and osteoporosis in children with bone fragility

Several genome-wide association studies (GWAS), GWAS meta-analyses, and mouse studies have demonstrated that wingless-related integration site 16 (WNT16) gene is associated with bone mineral density (BMD), cortical bone thickness, bone strength and fracture risk. Practically no data exist regarding the significance of WNT16 in childhood-onset osteoporosis and related fractures. We hypothesized that pathogenic variants and genetic variations in WNT16 could explain skeletal fragility in affected children. We screened the WNT16 gene by Sanger sequencing in three pediatric cohorts: 35 with primary osteoporosis, 59 with multiple fractures, and in 95 healthy controls. Altogether, we identified 12 variants in WNT16. Of them one was a rare 5'UTR variant rs1386898215 in genome aggregate and medical trans-omic databases (GnomAD, TOPMED; minor allele frequency (MAF) 0.00 and 0.000008, respectively). One variant rs1554366753, overrepresented in children with osteoporosis (MAF = 0.06 vs healthy controls MAF = 0.01), was significantly associated with lower BMD. This variant was found associated with increased WNT16 gene expression at mRNA level in fibroblast cultures. None of the other identified variants were rare (MAF < 0.001) or deemed pathogenic by predictor programs. WNT16 may play a role in childhood osteoporosis but genetic WNT16 variation is not a common cause of skeletal fragility in childhood.Peer reviewe

A Novel Osteochondrodysplasia With Empty Sella Associates With a TBX2 Variant

Skeletal dysplasias comprise a heterogenous group of developmental disorders of skeletal and cartilaginous tissues. Several different forms have been described and the full spectrum of their clinical manifestations and underlying genetic causes are still incompletely understood. We report a three-generation Finnish family with an unusual, autosomal dominant form of osteochondrodysplasia and an empty sella. Affected individuals (age range 24-44 years) exhibit unusual codfish-shaped vertebrae, severe early-onset and debilitating osteoarthritis and an empty sella without endocrine abnormalities. Clinical characteristics also include mild dysmorphic features, reduced sitting height ratio, and obesity. Whole-exome sequencing excluded known skeletal dysplasias and identified a novel heterozygous missense mutation c.899C>T (p.Thr300Met) in TBX2, confirmed by Sanger sequencing. TBX2 is important for development of the skeleton and the brain and three prior reports have described variations in TBX2 in patients portraying a complex phenotype with vertebral anomalies, craniofacial dysmorphism and endocrine dysfunctions. Our mutation lies near a previously reported disease-causing variant and is predicted pathogenic with deleterious effects on protein function. Our findings expand the current spectrum of skeletal dysplasias, support the association of TBX2 mutations with skeletal dysplasia and suggest a role for TBX2 in development of the spinal and craniofacial structures and the pituitary gland.Peer reviewe

A novel MYT1L mutation in a patient with severe early-onset obesity and intellectual disability

The genetic background of severe early-onset obesity is still incompletely understood. Deletions at 2p25.3 associate with early-onset obesity and variable intellectual disability. Myelin-transcriptor-factor-1-like (MYT1L) gene in this locus has been proposed a candidate gene for obesity. We report on a 13-year-old boy presenting with overweight already at 1 year of age (body mass index [BMI] Z-score +2.3) and obesity at 2 years of age (BMI Z-score +3.8). The patient had hyperphagia and delayed neurological, cognitive and motor development. He also had speech delay, strabismus, hyperactivity and intellectual disability. Brain MRI was normal. The parents and sister had normal BMI. Whole-genome sequencing identified in the index patient a novel de novo frameshift deletion that introduces a premature termination of translation NM_015025.2(MYT1L): c.2215_2224delACGCGCTGCC, p.(Thr739Alafs*7) in MYT1L. The frameshift variant was confirmed by Sanger sequencing. Our finding supports the association of MYT1L mutations with early-onset syndromic obesity. The identification of novel monogenic forms of childhood-onset obesity will provide insights to the involved genetic and biologic pathways.Peer reviewe

Altered MicroRNA Profile in Osteoporosis Caused by Impaired WNT Signaling

Context: WNT signaling is fundamental to bone health, and its aberrant activation leads to skeletal pathologies. The heterozygous missense mutation p.C218G in WNT1, a key WNT pathway ligand, leads to severe early-onset and progressive osteoporosis with multiple peripheral and spinal fractures. Despite the severe skeletal manifestations, conventional bone turnover markers are normal in mutation-positive patients. Objective: This study sought to explore the circulating microRNA (miRNA) pattern in patients with impaired WNT signaling. Design and Setting: A cross-sectional cohort study at a university hospital. Participants: Altogether, 12 mutation-positive (MP) subjects (median age, 39 years; range, 11 to 76 years) and 12 mutation-negative (MN) subjects (35 years; range, 9 to 59 years) from two Finnish families with WNT1 osteoporosis due to the heterozygous p.C218G WNT1 mutation. Methods and Main Outcome Measure: Serum samples were screened for 192 miRNAs using quantitative polymerase chain reaction. Findings were compared between WNT1 MP and MN subjects. Results: The pattern of circulating miRNAs was significantly different in the MP subjects compared with the MN subjects, with two upregulated (miR-18a-3p and miR-223-3p) and six downregulated miRNAs (miR-22-3p, miR-31-5p, miR-34a-5p, miR-143-5p, miR-423-5p, and miR-423-3p). Three of these (miR-22-3p, miR-34a-5p, and miR-31-5p) are known inhibitors of WNT signaling: miR-22-3p and miR-34a-5p target WNT1 messenger RNA, and miR-31-5p is predicted to bind to WNT1 3'UTR. Conclusions: The circulating miRNA pattern reflects WNT1 mutation status. The findings suggest that the WNT1 mutation disrupts feedback regulation between these miRNAs and WNT1, providing insights into the pathogenesis of WNT-related bone disorders. These miRNAs may have potential in the diagnosis and treatment of osteoporosis.Peer reviewe

Lipocalin-2 is associated with FGF23 in WNT1 and PLS3 osteoporosis

BackgroundThe pathogenic mechanisms of early-onset osteoporosis caused by WNT1 and PLS3 mutations are incompletely understood and diagnostic biomarkers of these disorders are limited. Recently, lipocalin-2 has been recognized as an osteokine involved in bone development and homeostasis. However, the role of lipocalin-2 in WNT1 and PLS3 osteoporosis is unknown. ObjectiveWe aimed to investigate if plasma lipocalin-2 could be utilized as a biomarker for WNT1 and PLS3 osteoporosis and to evaluate the association between lipocalin-2 and other parameters of bone metabolism. MethodsWe measured plasma lipocalin-2 in 17 WNT1 and 14 PLS3 mutation-positive patients and compared them to those of 34 mutation-negative (MN) healthy subjects. We investigated possible associations between lipocalin-2 and several bone biomarkers including collagen type I cross-linked C-telopeptide (CTX), alkaline phosphatase (ALP), type I procollagen intact N-terminal propeptide (PINP), intact and C-terminal fibroblast growth factor 23 (FGF23), dickkopf-1 (DKK1) and sclerostin as well as parameters of iron metabolism (iron, transferrin, transferrin saturation, soluble transferrin receptor and ferritin). ResultsWe found no differences in plasma lipocalin-2 levels in WNT1 or PLS3 patients compared with MN subjects. However, lipocalin-2 was associated with C-terminal FGF23 in WNT1 patients (r=0.62; p=0.008) and PLS3 patients (r=0.63, p=0.017), and with intact FGF23 in PLS3 patients (r=0.80; pPeer reviewe

New Insights Into Monogenic Causes of Osteoporosis

Osteoporosis, characterized by deteriorated bone microarchitecture and low bone mineral density, is a chronic skeletal disease with high worldwide prevalence. Osteoporosis related to aging is the most common form and causes significant morbidity and mortality. Rare, monogenic forms of osteoporosis have their onset usually in childhood or young adulthood and have specific phenotypic features and clinical course depending on the underlying cause. The most common form is osteogenesis imperfecta linked to mutations in COL1A1 and COL1A2, the two genes encoding type I collagen. However, in the past years, remarkable advancements in bone research have expanded our understanding of the intricacies behind bone metabolism and identified novel molecular mechanisms contributing to skeletal health and disease. Especially high-throughput sequencing techniques have made family-based studies an efficient way to identify single genes causative of rare monogenic forms of osteoporosis and these have yielded several novel genes that encode proteins partaking in type I collagen modification or regulating bone cell function directly. New forms of monogenic osteoporosis, such as autosomal dominant osteoporosis caused by WNT1 mutations or X-linked osteoporosis due to PLS3 mutations, have revealed previously unidentified bone-regulating proteins and clarified specific roles of bone cells, expanded our understanding of possible inheritance mechanisms and paces of disease progression, and highlighted the potential of monogenic bone diseases to extend beyond the skeletal tissue. The novel gene discoveries have introduced new challenges to the classification and diagnosis of monogenic osteoporosis, but also provided promising new molecular targets for development of pharmacotherapies. In this article we give an overview of the recent discoveries in the area of monogenic forms of osteoporosis, describing the key cellular mechanisms leading to skeletal fragility, the major recent research findings and the essential challenges and avenues in future diagnostics and treatments

Is sleep apnea underdiagnosed in adult patients with osteogenesis imperfecta? -a single-center cross-sectional study

BackgroundPatients with Osteogenesis imperfecta (OI) suffer from increased bone fracture tendency generally caused by a mutation in genes coding for type I collagen. OI is also characterized by numerous co-morbidities, and recent data from questionnaire studies suggest that these may include increased risk for sleep apnea, a finding that lacks clinical evidence from cohort studies. In this cross-sectional study, 25 adults with OI underwent clinical otorhinolaryngology examination as well as overnight polysomnography to address the question. The participants were aged between 19 and 77years, and ten of them had mild clinical OI phenotype, seven had a moderately severe phenotype, and eight had a severe phenotype.ResultsWe found obstructive sleep apnea (apnea hypopnea index 5/h) in as many as 52% of the OI patients in the cohort. Unexpectedly, however, no correlation was present between sleep apnea and daytime sleepiness, experienced bodily pain, severity of OI, Mallampati score, or neck circumference.ConclusionsSeeing that the usual predictors showed no association with occurrence of sleep apnea, we conclude that obstructive sleep apnea may easily be left as an undetected disorder in individuals with OI. Recurrent nocturnal hypoxia due to episodes of apneas can even affect bone metabolism, thereby further aggravating bone fragility in patients with OI.Peer reviewe

Biomarkers in WNT1 and PLS3 Osteoporosis : Altered Concentrations of DKK1 and FGF23

Recent advancements in genetic research have uncovered new forms of monogenic osteoporosis, expanding our understanding of the molecular pathways regulating bone health. Despite active research, knowledge on the pathomechanisms, disease-specific biomarkers, and optimal treatment in these disorders is still limited. Mutations in WNT1, encoding a WNT/beta-catenin pathway ligand WNT1, and PLS3, encoding X chromosomally inherited plastin 3 (PLS3), both result in early-onset osteoporosis with prevalent fractures and disrupted bone metabolism. However, despite marked skeletal pathology, conventional bone markers are usually normal in both diseases. Our study aimed to identify novel bone markers in PLS3 and WNT1 osteoporosis that could offer diagnostic potential and shed light on the mechanisms behind these skeletal pathologies. We measured several parameters of bone metabolism, including serum dickkopf-1 (DKK1), sclerostin, and intact and C-terminal fibroblast growth factor 23 (FGF23) concentrations in 17 WNT1 and 14 PLS3 mutation-positive subjects. Findings were compared with 34 healthy mutation-negative subjects from the same families. Results confirmed normal concentrations of conventional metabolic bone markers in both groups. DKK1 concentrations were significantly elevated in PLS3 mutation-positive subjects compared with WNT1 mutation-positive subjects (p <.001) or the mutation-negative subjects (p = .002). Similar differences were not seen in WNT1 subjects. Sclerostin concentrations did not differ between any groups. Both intact and C-terminal FGF23 were significantly elevated in WNT1 mutation-positive subjects (p = .039 and p = .027, respectively) and normal in PLS3 subjects. Our results indicate a link between PLS3 and DKK1 and WNT1 and FGF23 in bone metabolism. The normal sclerostin and DKK1 levels in patients with impaired WNT signaling suggest another parallel regulatory mechanism. These findings provide novel information on the molecular networks in bone. Extended studies are needed to investigate whether these biomarkers offer diagnostic value or potential as treatment targets in osteoporosis. (c) 2020 American Society for Bone and Mineral Research.Peer reviewe

Defective WNT signaling associates with bone marrow fibrosis-a cross-sectional cohort study in a family with WNT1 osteoporosis

This study explores bone marrow function in patients with defective WNT1 signaling. Bone marrow samples showed increased reticulin and altered granulopoiesis while overall hematopoiesis was normal. Findings did not associate with severity of osteoporosis. These observations provide new insight into the role of WNT signaling in bone marrow homeostasis. WNT signaling regulates bone homeostasis and survival and self-renewal of hematopoietic stem cells. Aberrant activation may lead to osteoporosis and bone marrow pathology. We aimed to explore bone marrow findings in a large family with early-onset osteoporosis due to a heterozygous WNT1 mutation. We analyzed peripheral blood samples, and bone marrow aspirates and biopsies from 10 subjects with WNT1 mutation p.C218G. One subject was previously diagnosed with idiopathic myelofibrosis and others had no previously diagnosed hematologic disorders. The findings were correlated with the skeletal phenotype, as evaluated by number of peripheral and spinal fractures and bone mineral density. Peripheral blood samples showed no abnormalities in cell counts, morphology or distributions but mild increase in platelet count. Bone marrow aspirates (from 8/10 subjects) showed mild decrease in bone marrow iron storages in 6 and variation in cell distributions in 5 subjects. Bone marrow biopsies (from 6/10 subjects) showed increased bone marrow reticulin (grade MF-2 in the myelofibrosis subject and grade MF-1 in 4 others), and an increase in overall, and a shift towards early-phase, granulopoiesis. The bone marrow findings did not associate with the severity of skeletal phenotype. Defective WNT signaling associates with a mild increase in bone marrow reticulin and may predispose to myelofibrosis, while overall hematopoiesis and peripheral blood values are unaltered in individuals with a WNT1 mutation. In this family with WNT1 osteoporosis, bone marrow findings were not related to the severity of osteoporosis.Peer reviewe

Early-Onset Osteoporosis

Publisher Copyright: © 2021, The Author(s).Osteoporosis is a skeletal disorder with enhanced bone fragility, usually affecting the elderly. It is very rare in children and young adults and the definition is not only based on a low BMD (a Z-score < − 2.0 in growing children and a Z-score ≤ − 2.0 or a T-score ≤ − 2.5 in young adults) but also on the occurrence of fragility fractures and/or the existence of underlying chronic diseases or secondary factors such as use of glucocorticoids. In the absence of a known chronic disease, fragility fractures and low BMD should prompt extensive screening for secondary causes, which can be found in up to 90% of cases. When fragility fractures occur in childhood or young adulthood without an evident secondary cause, investigations should explore the possibility of an underlying monogenetic bone disease, where bone fragility is caused by a single variant in a gene that has a major role in the skeleton. Several monogenic forms relate to type I collagen, but other forms also exist. Loss-of-function variants in LRP5 and WNT1 may lead to early-onset osteoporosis. The X-chromosomal osteoporosis caused by PLS3 gene mutations affects especially males. Another recently discovered form relates to disturbed sphingolipid metabolism due to SGMS2 mutations, underscoring the complexity of molecular pathology in monogenic early-onset osteoporosis. Management of young patients consists of treatment of secondary factors, optimizing lifestyle factors including calcium and vitamin D and physical exercise. Treatment with bone-active medication should be discussed on a personalized basis, considering the severity of osteoporosis and underlying disease versus the absence of evidence on anti-fracture efficacy and potential harmful effects in pregnancy.Peer reviewe