Search

Skeletal dysplasias comprise a large spectrum of mostly monogenic disorders affecting bone growth, patterning, and homeostasis, and ranging in severity from lethal to mild phenotypes. This study aimed to underpin the genetic cause of skeletal dysplasia in three unrelated families with variable skeletal manifestations. The six affected individuals from three families had severe short stature with extreme shortening of forelimbs, short long-bones, and metatarsals, and brachydactyly (family 1); mild short stature, platyspondyly, and metaphyseal irregularities (family 2); or a prenatally lethal skeletal dysplasia with kidney features suggestive of a ciliopathy (family 3). Genetic studies by whole genome, whole exome, and ciliome panel sequencing identified in all affected individuals biallelic missense variants in KIF24, which encodes a kinesin family member controlling ciliogenesis. In families 1 and 3, with the more severe phenotype, the affected subjects harbored homozygous variants (c.1457A>G; p.(Ile486Val) and c.1565A>G; p.(Asn522Ser), respectively) in the motor domain which plays a crucial role in KIF24 function. In family 2, compound heterozygous variants (c.1697C>T; p.(Ser566Phe)/c.1811C>T; p.(Thr604Met)) were found C-terminal to the motor domain, in agreement with a genotype-phenotype correlation. In vitro experiments performed on amnioblasts of one affected fetus from family 3 showed that primary cilia assembly was severely impaired, and that cytokinesis was also affected. In conclusion, our study describes novel forms of skeletal dysplasia associated with biallelic variants in KIF24. To our knowledge this is the first report implicating KIF24 variants as the cause of a skeletal dysplasia, thereby extending the genetic heterogeneity and the phenotypic spectrum of rare bone disorders and underscoring the wide range of monogenetic skeletal ciliopathies. (c) 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).

Context Phosphate homeostasis and its modifiers in early childhood are inadequately characterized. Objective To determine physiological plasma phosphate concentration and modifying factors in healthy infants at 12 to 24 months of age. Design This study included 525 healthy infants (53% girls), who participated in a randomized vitamin D intervention trial and received daily vitamin D3 supplementation of either 10 or 30 μg from age 2 weeks to 24 months. Biochemical parameters were measured at 12 and 24 months. Dietary phosphate intake was determined at 12 months. Main Outcome Measures Plasma phosphate concentrations at 12 and 24 months of age. Results Mean (SD) phosphate concentration decreased from 12 months (1.9 ± 0.15 mmol/L) to 24 months (1.6 ± 0.17 mmol/L) of age (P 0.472 for all). Plasma iron was associated positively with plasma phosphate at both time points (B, 0.006 and 0.005; 95% CI, 0.004-0.009 and 0.002-0.008; P Conclusion Plasma phosphate concentration decreased from age 12 to 24 months. In infants and toddlers, the strongest plasma phosphate modifiers were sex, iron, and creatinine, whereas vitamin D supplementation did not modify phosphate concentrations.

ContextSingle nucleotide polymorphisms (SNPs) of the vitamin D binding protein encoding the GC (group component) gene affect 25-hydroxyvitamin D (25OHD) concentrations, but their influence on vitamin D status and response to vitamin D supplementation in infants is unknown.ObjectiveTo study GC genotype–related differences in 25OHD concentrations and the response to supplementation during a vitamin D intervention study in infants.DesignIn this randomized controlled trial, healthy term infants received vitamin D3 (10 or 30 μg/d) from 2 weeks to 24 months of age. GC SNPs rs2282679, rs4588, rs7041, and rs1155563 were genotyped. rs4588/7041 diplotype and haplotypes of rs2282679, rs4588, and rs7041 (Haplo3SNP) and of all four SNPs (Haplo4SNP) were determined.Main Outcome Measures25OHD measured in cord blood at birth and at 12 and 24 months during intervention.ResultsA total of 913 infants were included. Minor allele homozygosity of all studied GC SNPs, their combined haplotypes, and rs4588/rs7041 diplotype 2/2 were associated with lower 25OHD concentrations at all time points in one or both intervention groups [analysis of covariance (ANCOVA) P < 0.043], with the exception of rs7041, which did not affect 25OHD at birth. In the high-dose supplementation group receiving 30 μg/d vitamin D3, but not in those receiving 10 µg/d, genotype of rs2282679, rs4588, and rs7041; diplotype; and Haplo3SNP significantly affected intervention response (repeated measurement ANCOVA Pinteraction < 0.019). Minor allele homozygotes had lower 25OHD concentrations and smaller increases in 25OHD throughout the intervention.ConclusionsIn infants, vitamin D binding protein genotype affects 25OHD concentration and efficiency of high-dose vitamin D3 supplementation.

Pathological variants in SGMS2, encoding sphingomyelin synthase 2 (SMS2), result in a rare autosomal dominant skeletal disorder with cranial doughnut lesions. The disease manifests as early-onset osteoporosis or a more severe skeletal dysplasia with low bone mineral density, frequent fractures, long-bone deformities, and multiple sclerotic cranial lesions. The exact underlying molecular features and skeletal consequences, however, remain elusive. This study investigated bone tissue characteristics in two adult males with a heterozygous SGMS2 mutation p.Arg50* and significant bone fragility. Transiliac bone biopsy samples from both (patient 1: 61 years; patient 2: 29 years) were analyzed by bone histomorphometry, confocal laser scanning microscopy, and quantitative backscattered electron imaging (qBEI). Bone histomorphometry portrayed largely normal values for structural and turnover parameters, but in both patient 1 and patient 2, respectively, osteoid thickness (-1.80 SD, -1.37 SD) and mineralizing surface (-1.03 SD, -2.73 SD) were reduced and osteoid surface increased (+9.03 SD, +0.98 SD), leading to elevated mineralization lag time (+8.16 SD, +4.10 SD). qBEI showed low and heterogeneous matrix mineralization (CaPeak -2.41 SD, -3.72 SD; CaWidth +7.47 SD, +4.41 SD) with a chaotic arrangement of collagenous fibrils under polarized light. Last, osteocyte lacunae appeared abnormally large and round in shape and the canalicular network severely disturbed with short-spanned canaliculi lacking any orderliness or continuity. Taken together, these data underline a central role for functional SMS2 in bone matrix organization and mineralization, lacunocanalicular network, and in maintaining skeletal strength and integrity. These data bring new knowledge on changes in bone histology resulting from abnormal sphingomyelin metabolism and aid en route to better understanding of sphingolipid-related skeletal disorders. (c) 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

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.

Osteogenesis imperfecta (OI) and other decreased bone density disorders comprise a heterogeneous group of heritable diseases with skeletal fragility. Recently, it was discovered that mutations in SGMS2, encoding sphingomyelin synthetase 2, result in aberrant sphingomyelin metabolism and lead to a novel form of OI termed osteoporosis with calvarial doughnut lesions (OP-CDL) with moderate to severe skeletal fragility and variable cranial hyperostotic lesions. This study describes a Japanese family with the skeletal phenotype of OP-CDL. The affected individuals have moderately severe, childhood-onset skeletal fragility with multiple long-bone fractures, scoliosis and bone deformities. In addition, they exhibit multiple CDLs or calvarial bumps with central radiolucency and peripheral radiopacity. However, SGMS2 sequencing was normal. Instead, whole-exome sequencing identified a novel IFITM5 missense mutation c.143A>G (p.N48S) (classified as a VUS by ACMG). IFITM5 encodes an osteoblast-restricted protein BRIL and a recurrent c.-14C>T mutation in its 5' UTR region results in OI type V, a distinctive subtype of OI associated with hyperplastic callus formation and ossification of the interosseous membranes. The patients described here have a phenotype clearly different from OI type V and with hyperostotic cranial lesions, feature previously unreported in association with IFITM5. Our findings expand the genetic spectrum of OP-CDL, indicate diverse phenotypic consequences of pathogenic IFITM5 variants, and imply an important role for BRIL in cranial skeletogenesis.

Epiphyseal chondrodysplasia, Miura type (ECDM) is a skeletal dysplasia with tall stature and distinctive skeletal features caused by heterozygous NPR2 pathogenic variants. Only four families have been reported. We present a family with five affected individuals (mother, three sons, and daughter). The mother's phenotype was relatively mild: borderline tall stature and elongated halluces operated during childhood. The children were remarkably more severely affected with tall stature, scoliosis, and elongated toes and fingers leading to suspicion of Marfan syndrome. Progressive valgus deformities (at the hips, knees, and ankles) were the main complaints and necessitated orthopedic investigations and surgery. Radiographs showed coxa valga, scoliosis, multiple pseudoepiphyses of the fingers and toes with uneven elongation of the digits and ankle valgus. The two older brothers underwent osteotomies and guided growth for axial deformities and arthrodesis for elongated halluces. Genetic testing confirmed the clinical diagnosis of ECDM: all affected individuals had a heterozygous c.2647G>A (p.Val883Met) NPR2 variant in a highly conserved region in the carboxyl-terminal guanylyl cyclase domain. This two-generation family elucidates the clinical and radiological variability of the disease. These rare cases are important to gain further understanding of the fundamental processes of growth regulation.

Summary Altogether 95 children with primary bone fragility were screened for variants in PLS3, the gene underlying X-linked osteoporosis. Two children with multiple peripheral and spinal fractures and low BMD had novel disease-causing PLS3 variants. Children with milder phenotypes had no pathogenic variants. PLS3 screening is indicated in childhood-onset primary osteoporosis. Introduction The study aimed to determine the role of pathogenic PLS3 variants in children’s bone fragility and to elucidate the associated phenotypic features. Methods Two cohorts of children with bone fragility were screened for variants in PLS3, the gene underlying X-linked osteoporosis. Cohort I comprised 31 patients with childhood-onset primary osteoporosis of unknown etiology. Cohort II comprised 64 children who had sustained multiple fractures but were otherwise healthy. Clinical and radiological data were reviewed. Peripheral blood DNA was Sanger sequenced for coding exons and flanking intronic regions of PLS3. Results In two patients of cohort I, where other common genetic causes had been excluded, we identified two novel disease-causing PLS3 variants. Patient 1 was a male with bilateral femoral fractures at 10 years, low BMD (Z-score −4.1; 18 years), and multiple vertebral compression fractures. He had a novel nonsense variant in PLS3. Patient 2 was a girl with multiple long bone and vertebral fractures and low BMD (Z-score −6.6 at 6 years). She had a de novo missense variant in PLS3; whole exome sequencing and array-CGH identified no other genetic causes. Iliac crest bone biopsies confirmed low-turnover osteoporosis in both patients. In cohort II, no pathogenic PLS3 variants were identified in any of the subjects. Conclusions Two novel disease-causing variants in PLS3 were identified in a boy and a girl with multiple peripheral and spinal fractures and very low BMD while no pathogenic variants were identified in children with less severe skeletal fragility. PLS3 screening is warranted in male and female patients with childhood-onset primary osteoporosis., published version, peerReviewed

Vitamin D is important for normal skeletal homeostasis, especially in growing children. There are no previous genome-wide association (GWA) studies exploring genetic factors that influence vitamin D metabolism in early childhood. We performed a GWA study on serum 25-hydroxyvitamin D (25(OH)D) and response to supplementation in 761 healthy term-born Finnish 24-month-old children, who participated in a randomized clinical trial comparing effects of 10 μg and 30 μg of daily vitamin D supplementation from age 2 weeks to 24 months. Using the Illumina Infinium Global Screening Array, which has been optimized for imputation, a total of 686085 markers were genotyped across the genome. Serum 25(OH)D was measured at the end of the intervention at 24 months of age. Skeletal parameters reflecting bone strength were determined at the distal tibia at 24 months using peripheral quantitative computed tomography (pQCT) (data available for 648 children). For 25(OH)D, two strong GWA signals were identified, localizing to GC (Vitamin D binding protein) and CYP2R1 (Vitamin D 25-hydroxylase) genes. The GWA locus comprising the GC gene also associated with response to supplementation. Further evidence for the importance of these two genes was obtained by comparing association signals to gene expression data from the Genotype-Tissue Expression project and performing colocalization analyses. Through the identification of haplotypes associated with low or high 25(OH)D concentrations we used a Mendelian randomization approach to show that haplotypes associating with low 25(OH)D were also associated with low pQCT parameters in the 24-month-old children. In this first GWA study on 25(OH)D in this age group we show that already at the age of 24 months genetic variation influences 25(OH)D concentrations and determines response to supplementation, with genome-wide significant associations with GC and CYP2R1. Also, the dual association between haplotypes, 25(OH)D and pQCT parameters gives support for vertical pleiotropy mediated by 25(OH)D., Author summary The effect of vitamin D continues to be highly debated in various health outcomes, including bone health. In this first study of children this young we searched for genes that modify vitamin D metabolism in early childhood using a genome-wide analysis of almost 700,000 genetic variants in a cohort of 761 healthy children participating in a vitamin D intervention study. We show that genetic variation in the genes coding for Vitamin D binding protein (GC) and Vitamin D 25-hydroxylase (CYP2R1) are important determinants for serum 25-hydroxyvitamin D concentration in 2-year-old children. Genetic variants within the GC gene also affect how the child responds to vitamin D supplementation. Moreover, our findings suggest that in 2-year-old children vitamin D concentration, even when within the normal range, influences bone strength as children with genetic constellations associating with lower vitamin D concentration and poorer response to vitamin D supplementation also have weaker bones.

RMRP was the first non-coding nuclear RNA gene implicated in a disease. Its mutations cause cartilage-hair hypoplasia (CHH), an autosomal recessive skeletal dysplasia with growth failure, immunodeficiency, and a high risk for malignancies. This study aimed to gain further insight into the role of RNA Component of Mitochondrial RNA Processing Endoribonuclease (RMRP) in cellular physiology and disease pathogenesis. We combined transcriptome analysis with single-cell analysis using fibroblasts from CHH patients and healthy controls. To directly assess cell cycle progression, we followed CHH fibroblasts by pulse-labeling and time-lapse microscopy. Transcriptome analysis identified 35 significantly upregulated and 130 downregulated genes in CHH fibroblasts. The downregulated genes were significantly connected to the cell cycle. Multiple other pathways, involving regulation of apoptosis, bone and cartilage formation, and lymphocyte function, were also affected, as well as PI3K-Akt signaling. Cell-cycle studies indicated that the CHH cells were delayed specifically in the passage from G2 phase to mitosis. Our findings expand the mechanistic understanding of CHH, indicate possible pathways for therapeutic intervention and add to the limited understanding of the functions of RMRP.

Mechanisms leading to osteoporosis are incompletely understood. Genetic disorders with skeletal fragility provide insight into metabolic pathways contributing to bone strength. We evaluated 6 families with rare skeletal phenotypes and osteoporosis by next-generation sequencing. In all the families, we identified a heterozygous variant in SGMS2, a gene prominently expressed in cortical bone and encoding the plasma membrane–resident sphingomyelin synthase SMS2. Four unrelated families shared the same nonsense variant, c.148C>T (p.Arg50*), whereas the other families had a missense variant, c.185T>G (p.Ile62Ser) or c.191T>G (p.Met64Arg). Subjects with p.Arg50* presented with childhood-onset osteoporosis with or without cranial sclerosis. Patients with p.Ile62Ser or p.Met64Arg had a more severe presentation, with neonatal fractures, severe short stature, and spondylometaphyseal dysplasia. Several subjects had experienced peripheral facial nerve palsy or other neurological manifestations. Bone biopsies showed markedly altered bone material characteristics, including defective bone mineralization. Osteoclast formation and function in vitro was normal. While the p.Arg50* mutation yielded a catalytically inactive enzyme, p.Ile62Ser and p.Met64Arg each enhanced the rate of de novo sphingomyelin production by blocking export of a functional enzyme from the endoplasmic reticulum. SGMS2 pathogenic variants underlie a spectrum of skeletal conditions, ranging from isolated osteoporosis to complex skeletal dysplasia, suggesting a critical role for plasma membrane–bound sphingomyelin metabolism in skeletal homeostasis., The identification of 6 families with childhood-onset osteoporosis with mutations in SGMS2 suggests a critical role for plasma membrane–bound sphingomyelin metabolism in skeletal homeostasis.

Spondyloocular syndrome is an autosomal-recessive disorder with spinal compression fractures, osteoporosis, and cataract. Mutations in XYLT2, encoding isoform of xylosyltransferase, were recently identified as the cause of the syndrome. We report on 4 patients, 2 unrelated patients and 2 siblings, with spondyloocular syndrome and novel mutations in XYLT2. Exome sequencing revealed a homozygous nonsense mutation, NM_022167.3(XYLT2): c.2188C>T, resulting in a premature stop codon (p.Arg730*) in a female patient. The patient presents visual impairment, generalized osteoporosis, short stature with short trunk, spinal compression fractures, and increased intervertebral disc space and hearing loss. We extended our XYLT2 analysis to a cohort of 22 patients with generalized osteoporosis, mostly from consanguineous families. In this cohort, we found by Sanger sequencing 2 siblings and 1 single patient who were homozygous for missense mutations in the XYLT2 gene (p.Arg563Gly and p.Leu605Pro). The patients had osteoporosis, compression fractures, cataracts, and hearing loss. Bisphosphonate treatment in 1 patient resulted in almost complete normalization of vertebral structures by adolescence, whereas treatment response in the others was variable. This report together with a previous study shows that mutations in the XYLT2 gene result in a variable phenotype dominated by spinal osteoporosis, cataract, and hearing loss. © 2016 American Society for Bone and Mineral Research.

WNT proteins comprise a 19-member glycoprotein family that act in several developmental and regenerative processes. In bone, WNT proteins regulate osteoblast differentiation and maintain bone health by activating the canonical WNT/β-catenin pathway. We reported a heterozygous missense mutation c.652T>G (p.C218G) in WNT1 exon 4 as the cause for severe early-onset, autosomal dominant osteoporosis. The initial study concerned a large Finnish family with 10 affected adults. Here we report clinical findings of the WNT1 osteoporosis in 8 children and young adults (median age 14 years; range 10 to 30 years) in two families, all with the p.C218G mutation in WNT1. Clinical assessments showed no apparent dysmorphia or features similar to typical osteogenesis imperfecta (OI). Biochemistry revealed no changes in parameters of calcium metabolism and bone turnover markers. Fracture frequencies varied, but all subjects had sustained at least one fracture and 4 had a pathological fracture history. Plain radiographs showed osteopenic appearance, loss in vertebral height, and thin diaphyses of the long bones. Bone densitometry showed the BMD to be below normal median in all subjects and the bone mass deficit seemed to be more severe in older participants. Bone histomorphometry revealed a low turnover osteoporosis in 2 subjects at ages 14 and 16 years. These findings are congruent with earlier findings in adult patients and indicate that WNT1 osteoporosis causes significant skeletal changes already in early childhood and impairs bone mass gain during pubertal years. Genetic testing of children or close relatives of affected individuals is recommended for appropriate preventive measures. © 2016 American Society for Bone and Mineral Research.

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.

Genetic factors play an important role in the development of osteoporosis. Several monogenic forms of osteoporosis have been recognized, most recently an X-chromosomal form resulting from mutations in the gene encoding plastin 3 (PLS3). PLS3 is a protein involved in actin bundle formation in the cytoskeleton. We present a large family with early onset osteoporosis and X-linked inheritance. Phenotyping was performed on 19 family members and whole-exome sequencing on 7 family members (5 with a diagnosis of early onset osteoporosis and 2 with normal bone parameters). Osteoporosis had its onset in childhood and was characterized by recurrent peripheral fractures, low bone mineral density (BMD), vertebral compression fractures, and significant height loss in adulthood. Males were in general more severely affected than females. Bone histomorphometry findings in 4 males and 1 female showed severe trabecular osteoporosis, low amount of osteoid, and decreased mineral apposition rate, indicating impaired bone formation; resorption parameters were increased in some. All affected subjects shared a single base substitution (c.73-24T > A) in intron 2 of PLS3 on Xq23. The mutation, confirmed by Sanger sequencing, segregated according to the skeletal phenotype. The mutation introduces a new acceptor splice site with a predicted splice score of 0.99 and, thereby, as confirmed by cDNA sequencing, induces the insertion of 22 bases between exons 2 and 3, causing a frameshift and premature termination of mRNA translation (p.Asp25Alafs*17). The mutation affects the first N-terminal calcium-binding EF-hand domain and abolishes all calcium- and actin-binding domains of the protein. Our results confirm the role of PLS3 mutations in early onset osteoporosis. The mechanism whereby PLS3 affects bone health is unclear, but it may be linked to osteocyte dendrite function and skeletal mechanosensing. Future studies are needed to elucidate the role of PLS3 in osteoporosis and to define optimal treatment. © 2014 American Society for Bone and Mineral Research.

Context: Only a few genetic causes for childhood obesity have been identified to date. Copy number variants (CNVs) are known to contribute to obesity, both syndromic (15q11.2 deletions, Prader-Willi syndrome) and nonsyndromic (16p11.2 deletions) obesity. Objective: To study the contribution of CNVs to early-onset obesity and evaluate the expression of candidate genes in subcutaneous adipose tissue. Design and Setting: A case-control study in a tertiary academic center. Participants: CNV analysis was performed on 90 subjects with early-onset obesity and 67 normalweight controls. Subcutaneous adipose tissue from body mass index-discordant siblings was used for the gene expression analyses. Main Outcome Measures: We used custom high-density array comparative genomic hybridization with exon resolution in 1989 genes, including all known obesity loci. The expression of candidate genes was assessed using microarray analysis of messenger RNA from subcutaneous adipose tissue. Results: We identified rare CNVs in 17 subjects (19%) with obesity and 2 controls (3%). In three cases (3%), the identified variant involved a known syndromic lesion (22q11.21 duplication, 1q21.1 deletion, and 16p11.2 deletion, respectively), although the others were not known. Seven CNVs in 10 families were inherited and segregated with obesity. Expression analysis of 37 candidate genes showed discordant expression for 10 genes (PCM1, EFEMP1, MAMLD1, ACP6, BAZ2B, SORBS1, KLF15, MACROD2, ATR, and MBD5). Conclusions: Rare CNVs contribute possibly pathogenic alleles to a substantial fraction of children with early-onset obesity. The involved genes might provide insights into pathogenic mechanisms and involved cellular pathways. These findings highlight the importance of CNV screening in children with early-onset obesity.

This report identifies human skeletal diseases associated with mutations in WNT1. In 10 family members with dominantly inherited, early-onset osteoporosis, we identified a heterozygous missense mutation in WNT1, c.652T→G (p.Cys218Gly). In a separate family with 2 siblings affected by recessive osteogenesis imperfecta, we identified a homozygous nonsense mutation, c.884C→A, p.Ser295*. In vitro, aberrant forms of the WNT1 protein showed impaired capacity to induce canonical WNT signaling, their target genes, and mineralization. In mice, Wnt1 was clearly expressed in bone marrow, especially in B-cell lineage and hematopoietic progenitors; lineage tracing identified the expression of the gene in a subset of osteocytes, suggesting the presence of altered cross-talk in WNT signaling between the hematopoietic and osteoblastic lineage cells in these diseases.

Background Cartilage-hair hypoplasia (CHH) is an autosomal recessive chondrodysplasia caused by RMRP (RNA component of mitochondrial RNA processing endoribonuclease) gene mutations. Manifestations include short stature, variable immunodeficiency, anaemia and increased risk of malignancies, all of which have been described also in telomere biology disorders. RMRP interacts with the telomerase RT (TERT) subunit, but the influence of RMRP mutations on telomere length is unknown. We measured relative telomere length (RTL) in patients with CHH, their first-degree relatives and healthy controls and correlated RTL with clinical and laboratory features. Methods The study cohort included 48 patients with CHH with homozygous (n=36) or compound heterozygous RMRP mutations (median age 38.2 years, range 6.0-70.8 years), 86 relatives (74 with a heterozygous RMRP mutation) and 94 unrelated healthy controls. We extracted DNA from peripheral blood, sequenced the RMRP gene and measured RTL by qPCR. Results Compared with age-matched and sex-matched healthy controls, median RTL was significantly shorter in patients with CHH (n=40 pairs, 1.05 vs 1.21, p=0.017), but not in mutation carriers (n=48 pairs, 1.16 vs 1.10, p=0.224). RTL correlated significantly with age in RMRP mutation carriers (r=-0.482, p

BACKGROUND: Vitamin D binding protein (DBP) binds vitamin D and its plasma metabolites, including 25-hydroxyvitamin D (25(OH)D), in the circulation. Only a small fraction circulates free (free 25(OH)D). Genetic variation of the GC gene, encoding DBP, has been associated with 25(OH)D concentrations. The roles of DBP and free 25(OH)D concentrations in the biological actions of vitamin D remain unclear. METHODS: We assessed the relationship between GC gene variants rs4588, rs7041, and rs705124, and serum total 25(OH)D, free and bioavailable 25(OH)D, and serum DBP and parathyroid hormone (PTH) concentrations in 622 Caucasian females (421) and males (201) aged 37–47 years. Concentrations of 25(OH)D, DBP, and PTH were measured from fasting blood samples. Dietary intakes of vitamin D and Ca were evaluated using 1-month food use frequency data, which were collected by a validated Food Frequency Questionnaire on vitamin D and calcium intakes. The subjects filled in the questionnaire covering overall health, medications, use of vitamin D and calcium supplements, and holidays in sunny locations. Three SNPs in the GC gene were genotyped: rs4588, rs7041, and rs705124. The SNPs rs4588 and rs7041 combine to form six common diplotypes. Free and bioavailable 25(OH)D were calculated by using specific binding coefficients. Differences among the diplo- and haplotypes of the GC gene in measures of 25(OH)D, DBP, and PTH were tested by analysis of covariance (ANCOVA) using appropriate covariates. RESULTS: We found significant variation among the SNPs rs4588 and rs7041 variants in DBP, total, free, and bioavailable 25(OH)D, and PTH. DBP concentration was lowest in genotype GC2/2 in both diplotypes and haplotypes (p = 0.039 and 0.039, respectively). The lowest 25(OH)D concentrations were found in diplotype variants GC1S/2, GC1S/F,and GC2/2 (p = 0.033), but free and bioavailable 25(OH)D concentrations were highest in the GC2/2 variant after corrected with a genotype-specific binding coefficient (p

OBJECTIVE Although obesity is a risk factor for vitamin D insufficiency, its impact on vitamin D-binding protein (DBP) concentration, and thereby possibly also on free 25OHD, is less well known. Our aim was to compare total and free serum 25OHD, and DBP concentrations between obese and normal-weight young adults at baseline and their responses to cholecalciferol supplementation. DESIGN A 12-week randomized, double-blinded clinical trial. PATIENTS Obese subjects N = 18 (BMI = 38, 67% men) with severe childhood-onset obesity and 24 normal-weight subjects (BMI = 23, 46% men), age between 15 and 25 years, were randomized into two groups to receive either placebo or cholecalciferol 50 μg (2000 IU) daily. MEASUREMENTS At baseline, 6-week and 12-week blood samples and anthropometric measurements were collected; baseline body composition was assessed by dual-energy X-ray absorptiometry. RESULTS At baseline, obese subjects had, compared with normal-weight, lower total and free serum 25OHD (49 vs 62 nmol/l, P = 0·041; 2·8 vs 4·7 pg/ml, P = 0·001), without differences in DBP concentrations (309 vs 346 μg/ml, P = 0·212). Cholecalciferol 50 μg per day increased both total and free 25OHD (ancova P < 0·001 and P = 0·021). The response of total 25OHD to supplementation was inferior in the obese compared with normal-weight subjects (P = 0·027). On the contrary, the change in free 25OHD concentration was similar in groups (P = 0·487). CONCLUSIONS Obese young adults exhibit lower total and free 25OHD concentration, which is not directly explained by differences in DBP status. The response of free 25OHD to supplementation did not differ between obese and normal-weight subjects.

Parathyroid hormone-related protein (PTHrP) has been shown to have anabolic effects in women with postmenopausal osteoporosis. PTHrP promotes the recruitment of osteogenic cells and prevents apoptotic death of osteoblasts and osteocytes. The receptor responsible for the effects of PTHrP is the common PTH/PTHrP receptor (PTH1R). Glucocorticoids (GC) are commonly used as drugs to treat inflammatory diseases. Long-term GC treatments are often associated with bone loss which can lead to GC-induced osteoporosis. The aim of this work was to study the effects of the glucocorticoid dexamethasone (Dex) on the expression of PTHrP and PTH1R in adult human mesenchymal stem cells, the progenitor cells of osteoblasts. Adult human mesenchymal stem cells (hMSC) were cultured and differentiated by standard methods. The expression of PTHrP and PTH1R mRNA was assayed by real-time qPCR. The PTHrP release into the culture media was measured by an immunoradiometric assay. Treatment with Dex (10 nM) resulted in an 80% drop in the PTHrP release within 6 h. A 24 h Dex treatment also reduced the expression of PTHrP mRNA by up to 90%. The expression of PTH1R receptor mRNA was simultaneously increased up to 20-fold by 10 nM Dex. The effects of Dex on PTHrP and PTH1R were dose-dependent and experiments with the GC-receptor antagonist mifepristone showed an involvement of GC-receptors in these effects. In addition to the Dex-induced effects on PTHrP and PTH1R, Dex also increased mineralization and the expression of the osteoblast markers Runx2 and alkaline phosphatase. In our studies, we show that dexamethasone decreases the expression of PTHrP and increases the expression of the PTH1R receptor. This could have an impact on PTHrP-mediated anabolic actions on bone and could also affect the responsiveness of circulating PTH. The results indicate that glucocorticoids affect the signalling pathway of PTHrP by regulating both PTHrP and PTH1R expression and these mechanisms could be involved in glucocorticoid-induced osteoporosis.

The second messenger molecule cyclic adenosine monophosphate (cAMP) plays an important role in the hormonal regulation of bone metabolism. cAMP is inactivated by the cyclic nucleotide phosphodiesterases (PDEs), a superfamily of enzymes divided into 11 known families designated PDE 1–11. The aim of this study was to investigate the effect of PDE7 and PDE8 inhibition on the gene expression and differentiation of human osteoblasts. Osteoblasts differentiated from human mesenchymal stem cells (hMSC) were cultured and treated with short interfering RNAs (siRNAs) generated from PDE7 and PDE8 PCR products. Total RNA was isolated from the cells, and gene expression was assayed with cDNA microarray and quantitative real-time PCR. bALP measurements were assayed during differentiation, and mineralization was determined by quantitative Alizarin red S staining. PDE7 and PDE8 inhibition by RNA interference decreased the gene expression of PDE7A by 60–70%, PDE7B by 40–50%, and PDE8A by 30%. PDE7 silencing increased the expression of β-catenin, osteocalcin, caspase-8, and cAMP-responsive element-binding protein 5 (CREB-5) genes and decreased the expression of the 1, 25-dihydroxyvitamin D3 receptor gene. PDE8A silencing increased the expression of anti-apoptotic genes, but decreased the expression of osteoglycin (osteoinductive factor) and bone morphogenetic protein 1 (BMP-1). PDE7 silencing increased bALP and mineralization up to three-fold compared to controls. Treatment with the PDE7-selective PDE inhibitor BRL-50481 had similar effects on mineralization as the gene silencing. The PDE7 silencing also increased forskolin stimulated cAMP response, but had no effect on the proliferation rate. Furthermore, osteocalcin expression was increased by PDE7 silencing by a mechanism dependent on protein kinase A. Our results show that specific gene silencing with the RNAi method is a useful tool for inhibiting the gene expression of specific PDEs and that PDE7 silencing upregulates several osteogenic genes and increases mineralization. PDE7 may play an important role in the regulation of osteoblastic differentiation.

Parathyroid hormone-related peptide (PTHrP) has been shown to have anabolic effects on bone in women with postmenopausal osteoporosis. On the cellular level PTHrP promotes the recruitment of osteogenic cells and prevents apoptotic death of osteoblasts and osteocytes. The calcium concentration is considerably higher in the vicinity of resorbing osteoclasts than in the plasma. Therefore the osteoblasts are likely to be confronted by elevated extracellular calcium concentrations in the areas of resorptive activity. The present study was designed to assess the possibility that extracellular calcium could regulate PTHrP expression in osteoblastic cells. Adult human mesenchymal stem cells (hMSC) were cultured and differentiated by standard methods. The PTHrP release into the culture media was measured by an immunoradiometric assay and the expression of PTHrP, osteocalcin and Runx2 mRNA was assayed by real-time PCR. Increasing the extracellular calcium from 1 mM to 5 mM for 24 h resulted in a 4-6-fold increase in the PTHrP release. PTHrP mRNA was also increased by elevated calcium levels. The effect of calcium stimulation on PTHrP release could be seen within 60 min of treatment. The extracellular calcium sensing receptor (CaR) agonist neomycin mimicked the effects of calcium and the MEK/MAPK inhibitor PD98059 abolished the effect of calcium and neomycin. High extracellular calcium increased the mineralization of hMSC and the expression of osteocalcin, but this effect was not mimicked by neomycin. Our results show that in hMSC, elevated extracellular calcium levels increases both released PTHrP and PTHrP mRNA expression. The effect of calcium on PTHrP can be mimicked by activation of the CaR and can be diminished by inhibition of the MAPK signalling pathway.

Spondyloocular syndrome is an autosomal-recessive disorder with spinal compression fractures, osteoporosis, and cataract. Mutations in XYLT2, encoding isoform of xylosyltransferase, were recently identified as the cause of the syndrome. We report on 4 patients, 2 unrelated patients and 2 siblings, with spondyloocular syndrome and novel mutations in XYLT2. Exome sequencing revealed a homozygous nonsense mutation, NM_022167.3(XYLT2): c.2188CT, resulting in a premature stop codon (p.Arg730*) in a female patient. The patient presents visual impairment, generalized osteoporosis, short stature with short trunk, spinal compression fractures, and increased intervertebral disc space and hearing loss. We extended our XYLT2 analysis to a cohort of 22 patients with generalized osteoporosis, mostly from consanguineous families. In this cohort, we found by Sanger sequencing 2 siblings and 1 single patient who were homozygous for missense mutations in the XYLT2 gene (p.Arg563Gly and p.Leu605Pro). The patients had osteoporosis, compression fractures, cataracts, and hearing loss. Bisphosphonate treatment in 1 patient resulted in almost complete normalization of vertebral structures by adolescence, whereas treatment response in the others was variable. This report together with a previous study shows that mutations in the XYLT2 gene result in a variable phenotype dominated by spinal osteoporosis, cataract, and hearing loss. © 2016 American Society for Bone and Mineral Research.

Background The salivary alpha-amylase locus (AMY1) is located in a highly polymorphic multi allelic copy number variable chromosomal region. A recent report identified an association between AMY1 copy numbers and BMI in common obesity. The present study investigated the relationship between AMY1 copy number, BMI and serum amylase in childhood-onset obesity. Patients Sixty-one subjects with a history of childhood-onset obesity (mean age 19.1 years, 54% males) and 71 matched controls (19.8 yrs, 45% males) were included. All anthropometric measures were greater in the obese; their mean BMI was 40 kg/m(2) (range 25-62 kg/m(2)) compared with 23 kg/m(2) in the controls (15-32 kg/m(2)). Results Mean AMY1 copy numbers did not differ between the obese and control subjects, but gender differences were observed; obese men showed the highest and obese women the lowest number of AMY1 copies (p=0.045). Further, only in affected females, AMY1 copy number correlated significantly with whole body fat percent (r=-0.512, p=0.013) and BMI (r=-0.416, p=0.025). Finally, a clear linear association between AMY1 copy number and serum salivary amylase was observed in all subgroups but again differences existed between obese males and females. Conclusions In conclusion, our findings suggest that AMY1 copy number differences play a role in childhood-onset obesity but the effect differs between males and females. Further studies in larger cohorts are needed to confirm these observations.

Nutrition influences peak bone mass development in early adulthood. The effect of high dietary phosphate intake on the growing skeleton of 1-month-old male rats (n = 30) was assessed in an 8-week intervention. High dietary phosphate intake increased bone remodeling and impaired bone material properties, diminishing bone mechanical strength. Introduction: High dietary phosphate intake is typical in the Western diet. Abundant phosphate intake enhances parathyroid secretion and bone metabolism. To study the influence of high dietary phosphate intake on growing bone homeostasis and structure, we submitted growing rats to experimental diets that varied in their phosphate content. Materials and Methods: One-month-old intact male rats (n = 30) were fed a control diet (Ca:P 1:1) or an experimental diet of either Ca:P 1:2 or Ca:P 1:3 for 8 weeks. At the beginning and the end of the study period, the right femurs were measured using DXA. Double labeling with tetracycline injection was performed 12 and 2 days before death. After death, hind legs were cut loose. Left femurs were processed for histomorphometry. Right femurs were measured with pQCT. Mechanical testing was performed on the right femoral neck and tibial shaft. Six right tibias were analyzed with μCT. Serum PTH, calcium, and phosphate contents were analyzed. Results: High-phosphate intake impaired growth of the animal, limited bone longitudinal growth, and restricted femur BMC and BMD build-up. Osteoclast number, osteoblast perimeter, and mineral apposition rate were increased, and trabecular area and width were decreased. Phosphate decreased femur midshaft total bone BMD, cortical bone BMD, and mean cortical thickness. High-phosphate diet reduced femoral neck and tibial shaft ultimate strength and tibia stiffness and toughness. In addition, serum PTH increased. Conclusions: High dietary phosphate intake reduced growth, skeletal material, and structural properties and decreased bone strength in growing male rats. Adequate calcium could not overcome this.

The first genetic linkage map for silver birch, Betula pendula Roth, was constructed by using a pseudo-testcross mapping strategy and integration of linkage data from 3 unrelated 2-generation pedigrees. The map is based on the genetic inheritance and segregation of 82 amplified fragment length polyhmorphisms and 19 microsatellite markers, and was constructed by simultaneously comparing the performance of CRI-MAP and OUTMAP packages. The analysis revealed 16 linkage groups, and the total map coverage is 1561 cM (Kosambi units). Average map distance between adjacent markers is 15.5 cM. Linkage groups range between 6 and 18 loci and from 81.2 to 326.5 cM; the remaining 9 linkage groups consist of 2 or 3 loci ranging from 6.3 to 42.4 cM. The uncertainty of the map is illustrated with sensitivity analysis. This initial map can serve as a basis for developing a more detailed genetic map.Key words: Betula pendula, linkage map, microsatellite, AFLP, CRI-MAP, OUTMAP.