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.

A Novel IFITM5 Variant Associated with Phenotype of Osteoporosis with Calvarial Doughnut Lesions: A Case Report.

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.

PLS3 sequencing in childhood-onset primary osteoporosis identifies two novel disease-causing variants.

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.

Body composition and adipokines in patients with juvenile idiopathic arthritis and systemic glucocorticoids.

OBJECTIVES: The aim of this cross-sectional study was to explore body composition, and the relationship of serum adipokines with bone mass and disease activity, in a cohort of JIA patients with at least three months' exposure to systemic glucocorticoids (GC). METHODS: Fifty patients with JIA (34 girls, median age 12.4 years and disease duration 6.3 years) and 88 controls matched for gender and age participated in this study. Bone mineral content (BMC) and areal bone mineral density (BMD) of the lumbar spine and whole body, as well as body composition were assessed with dual-energy x-ray absorptiometry. Fasting serum leptin and adiponectin were measured. RESULTS: Fat and lean mass were similar between patients and controls, but patients had slightly decreased BMD Z-scores. Serum leptin and adiponectin concentrations were similar. Disease activity was low, and no correlation with adipokines was observed. Patients with bone age-corrected lumbar spine BMD Z-score ≤-1.0 ("low BMD") did not show alterations in body composition, GC exposure or current disease activity, but had decreased BMC-to-lean mass ratio (p<0.001) and tendency for increased serum leptin (p=0.064). However, no association of leptin with BMD in multivariate analysis existed in patients or controls. An inverse association between adiponectin and whole body BMD was observed in both groups. CONCLUSIONS: Normal body composition was observed in a JIA cohort with low-dose GC exposure. Patients with "low BMD" tended to have increased serum leptin, but leptin did not associate with BMD. In this cohort with low disease activity, no correlation between adipokines and disease activity was present.

Novel mutations affecting LRP5 splicing in patients with osteoporosis-pseudoglioma syndrome (OPPG).

Osteoporosis-pseudoglioma sydrome (OPPG) is an autosomal recessive disorder with early-onset severe osteoporosis and blindness, caused by biallelic loss-of-function mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) gene. Heterozygous carriers exhibit a milder bone phenotype. Only a few splice mutations in LRP5 have been published. We present clinical and genetic data for four patients with novel LRP5 mutations, three of which affect splicing. Patients were evaluated clinically and by radiography and bone densitometry. Genetic screening of LRP5 was performed on the basis of the clinical diagnosis of OPPG. Splice aberrances were confirmed by cDNA sequencing or exon trapping. The effect of one splice mutation on LRP5 protein function was studied. A novel splice-site mutation c.1584+4A>T abolished the donor splice site of exon 7 and activated a cryptic splice site, which led to an in-frame insertion of 21 amino acids (p.E528_V529ins21). Functional studies revealed severely impaired signal transduction presumably caused by defective intracellular transport of the mutated receptor. Exon trapping was used on two samples to confirm that splice-site mutations c.4112-2A>G and c.1015+1G>T caused splicing-out of exons 20 and 5, respectively. One patient carried a homozygous deletion of exon 4 causing the loss of exons 4 and 5, as demonstrated by cDNA analysis. Our results broaden the spectrum of mutations in LRP5 and provide the first functional data on splice aberrations.

Linkage map of birch, Betula pendula Roth, based on microsatellites and amplified fragment length polymorphisms.

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.