Riikka E. Mäkitie

Skeletal Characteristics of WNT1 Osteoporosis in Children and Young Adults.

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.

New Genetic Forms of Childhood-Onset Primary Osteoporosis

Recent developments in genetic technology have given us the opportunity to look at diseases in a new and more detailed way. This Mini Review discusses monogenetic forms of childhood-onset primary osteoporosis, with the main focus on osteoporosis caused by mutations in WNT1 and PLS3, two of the most recently discovered genes underlying early-onset osteoporosis. The importance of WNT1 in the accrual and maintenance of bone mass through activation of canonical WNT signaling was recognized in 2013. WNT1 was shown to be a key ligand for the WNT-signaling pathway, which is of major importance in the regulation of bone formation. More recently, mutations in PLS3, located on the X chromosome, were shown to be the cause of X-linked childhood-onset primary osteoporosis affecting mainly males. The function of PLS3 in bone metabolism is still not completely understood, but it has been speculated to have an important role in mechanosensing by osteocytes and in matrix mineralization. In this new era of genetics, our knowledge on genetic causes of childhood-onset osteoporosis expands constantly. These discoveries bring new possibilities, but also new challenges. Guidelines are needed to implement this new genetic knowledge to clinical patient care and to guide genetic investigations in affected families.

FGF23 gene variation and its association with phosphate homeostasis and bone mineral density in Finnish children and adolescents

Fibroblast growth factor 23 (FGF23), a bone-derived hormone, participates in the hormonal bone-parathyroid-kidney axis, which is modulated by PTH, 1,25-dihydroxyvitamin D, plasma phosphate (Pi), and diet. Inappropriately high serum FGF23, seen in certain genetic and acquired disorders, results in urinary phosphate wasting and impaired bone mineralization. This study investigated the impact of FGF23 gene variation on phosphate homeostasis and bone health. The study included 183 children and adolescents (110 girls) aged 7-19 years (median 13.2years). Urine and blood parameters of calcium and phosphate homeostasis were analyzed. Bone characteristics were quantified by DXA and peripheral quantitative computed tomography (pQCT). Genetic FGF23 variation was assessed by direct sequencing of coding exons and flanking intronic regions. Nine FGF23 polymorphisms were detected; three of them were common: rs3832879 (c.212-37insC), rs7955866 (c.716C>T, p.T239M) and rs11063112 (c.2185A>T). Four different haplotypes and six different diplotypes were observed among these three polymorphisms. The variations in FGF23 significantly associated with plasma PTH and urinary Pi excretion, even after adjusting for relevant covariates. FGF23 variations independently associated with total hip BMD Z-score, but not with other bone outcomes. In instrument analysis, genetic variance in FGF23 was considered a weak instrument as it only induced small variations in circulating FGF23, PTH and Pi concentrations (F statistic less than 10). The observed associations between FGF23 variations and circulating PTH, and Pi excretion and total hip BMD Z-scores suggest that FGF23 polymorphisms may play a role in mineral homeostasis and bone metabolism.

Impaired WNT signaling and the spine—Heterozygous WNT1 mutation causes severe age-related spinal pathology

BACKGROUND WNT signaling plays a major role in bone and cartilage metabolism. Impaired WNT/β-catenin signaling leads to early-onset osteoporosis, but specific features in bone and other tissues remain inadequately characterized. We have identified two large Finnish families with early-onset osteoporosis due to a heterozygous WNT1 mutation c.652T>G, p.C218G. This study evaluated the impact of impaired WNT/β-catenin signaling on spinal structures. METHODS Altogether 18 WNT1 mutation-positive (age range 11-76years, median 49years) and 14 mutation-negative subjects (10-77years, median 43years) underwent magnetic resonance imaging (MRI) of the spine. The images were reviewed for spinal alignment, vertebral compression fractures, intervertebral disc changes and possible endplate deterioration. The findings were correlated with clinical data. RESULTS Vertebral compression fractures were present in 78% (7/9) of those aged over 50years but were not seen in younger mutation-positive subjects. All those with fractures had several severely compressed vertebrae. Altogether spinal compression fractures were present in 39% of those with a WNT1 mutation. Only 14% (2/14) mutation-negative subjects had one mild compressed vertebra each. The mutation-positive subjects had a higher mean spinal deformity index (4.0±7.3 vs 0.0±0.4) and more often increased thoracic kyphosis (Z-score>+2.0 in 33% vs 0%). Further, they had more often Schmorl nodes (61% vs 36%), already in adolescence, and their intervertebral discs were enlarged. CONCLUSION Compromised WNT signaling introduces severe and progressive changes to the spinal structures. Schmorl nodes are prevalent even at an early age and increased thoracic kyphosis and compression fractures become evident after the age of 50years. Therapies targeting the WNT pathway may be an effective way to prevent spinal pathology not only in those harboring a mutation but also in the general population with similar pathology.

Recent Discoveries in Monogenic Disorders of Childhood Bone Fragility

Purpose of ReviewThis review summarizes our current knowledge on primary osteoporosis in children with focus on recent genetic findings.Recent FindingsAdvances in genetic research, particularly next-generation sequencing, have found several genetic loci that associate with monogenic forms of inherited osteoporosis, widening the scope of primary osteoporosis beyond classical osteogenesis imperfecta. New forms of primary osteoporosis, such as those related to WNT1, PLS3, and XYLT2, have identified defects outside the extracellular matrix components and collagen-related pathways, in intracellular cascades directly affecting bone cell function.SummaryPrimary osteoporosis can lead to severe skeletal morbidity, including abnormal longitudinal growth, compromised bone mass gain, and noticeable fracture tendency beginning at childhood. Early diagnosis and timely care are warranted to ensure the best achievable bone health. Future research will most likely broaden the spectrum of primary osteoporosis, hopefully provide more insight into the genetics governing bone health, and offer new targets for treatment.

Osteocyte Protein Expression Is Altered in Low-Turnover Osteoporosis Caused by Mutations in WNT1 and PLS3

Context Osteocytes express proteins that regulate bone remodeling and mineralization. Objective To evaluate the relationship between osteocyte-specific protein expression and bone histology in patients with monogenic osteoporosis due to wingless integration site 1 (WNT1) or plastin 3 (PLS3) mutations. Design and Setting Cross-sectional cohort study at a university hospital. Participants Six patients (four males; ages: 14 to 72 years) with a heterozygous WNT1 mutation and five patients (four males; ages: 9 to 70 years) with a heterozygous/hemizygous PLS3 mutation. Methods and Main Outcome Measures Immunohistochemistry was performed for fibroblast growth factor 23 (FGF23), dentin matrix protein 1 (DMP1), sclerostin, and phosphorylated (phospho-)β-catenin in iliac crest samples and compared with bone histomorphometry. Results FGF23 expression in WNT1 patients was 243% that observed in PLS3 patients (P < 0.01). DMP1, sclerostin, and phospho-β-catenin expression did not differ between groups. Serum phosphate correlated inversely with FGF23 expression (r = -0.79, P = 0.01) and serum ionized calcium correlated inversely with sclerostin expression (r = -0.60, P = 0.05). Phospho-β-catenin expression correlated inversely with DMP1 expression (r = -0.88, P < 0.001), osteoid volume/bone volume (r = -0.68, P = 0.02), and bone formation rate (r = -0.78, P < 0.01). FGF23 expression did not correlate with DMP1 expression, sclerostin expression, or bone histomorphometry. Marrow adiposity was higher in WNT1 than in PLS3 patients (P = 0.04). Conclusions Mutations that disrupt WNT signaling and osteocytic mechanosensing affect osteocyte protein expression. Abnormal osteocyte function may play a role in the pathogenesis of monogenetic forms of osteoporosis.

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

SummaryThis 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.IntroductionWNT 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.MethodsWe 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.ResultsPeripheral 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.ConclusionsDefective 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.

CRTAP variants in early-onset osteoporosis and recurrent fractures.

CRTAP Variants in Early-Onset Osteoporosis and Recurrent Fractures Alice Costantini,* Ilkka Vuorimies, Riikka M€akitie, Mervi K. M€ayr€anp€a€a, Jutta Becker, Minna Pekkinen, Helena Valta, Christian Netzer, Anders K€ampe, Fulya Taylan, Hong Jiao, and Outi M€akitie Department of Molecular Medicine and Surgery and Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden Folkh€alsan Institute of Genetics, University of Helsinki, Helsinki, Finland Children’s Hospital, University of Helsinki and Helsinki University Hospital, Helsinki, Finland Institute of Human Genetics, University of Cologne, Cologne, Germany Department of Biosciences and Nutrition, Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden Clinical Research Centre, Karolinska University Hospital, Huddinge, Sweden Department of Clinical Genetics, Karolinska University Hospital, Stockholm, Sweden

A Wide Spectrum of Autoimmune Manifestations and Other Symptoms Suggesting Immune Dysregulation in Patients With Cartilage-Hair Hypoplasia

Background: Mutations in RMRP, encoding a non-coding RNA molecule, underlie cartilage-hair hypoplasia (CHH), a syndromic immunodeficiency with multiple pathogenetic mechanisms and variable phenotype. Allergy and asthma have been reported in the CHH population and some patients suffer from autoimmune (AI) diseases. Objective: We explored AI and allergic manifestations in a large cohort of Finnish patients with CHH and correlated clinical features with laboratory parameters and autoantibodies. Methods: We collected clinical and laboratory data from patient interviews and hospital records. Serum samples were tested for a range of autoantibodies including celiac, anti-cytokine, and anti-21-hydroxylase antibodies. Nasal cytology samples were analyzed with microscopy. Results: The study cohort included 104 patients with genetically confirmed CHH; their median age was 39.2 years (range 0.6–73.6). Clinical autoimmunity was common (11/104, 10.6%) and included conditions previously undescribed in subjects with CHH (narcolepsy, psoriasis, idiopathic thrombocytopenic purpura, and multifocal motor axonal neuropathy). Patients with autoimmunity more often had recurrent pneumonia, sepsis, high immunoglobulin (Ig) E and/or undetectable IgA levels. The mortality rates were higher in subjects with AI diseases (χ(2)2 = 14.056, p = 0.0002). Several patients demonstrated serum autoantibody positivity without compatible symptoms. We confirmed the high prevalence of asthma (23%) and allergic rhinoconjunctivitis (39%). Gastrointestinal complaints, mostly persistent diarrhea, were also frequently reported (32/104, 31%). Despite the history of allergic rhinitis, no eosinophils were observed in nasal cytology in five tested patients. Conclusions: AI diseases are common in Finnish patients with CHH and are associated with higher mortality, recurrent pneumonia, sepsis, high IgE and/or undetectable IgA levels. Serum positivity for some autoantibodies was not associated with clinical autoimmunity. The high prevalence of persistent diarrhea, asthma, and symptoms of inflammation of nasal mucosa may indicate common pathways of immune dysregulation.

Rare Copy Number Variants in Array-Based Comparative Genomic Hybridization in Early-Onset Skeletal Fragility

Early-onset osteoporosis is characterized by low bone mineral density (BMD) and fractures since childhood or young adulthood. Several monogenic forms have been identified but the contributing genes remain inadequately characterized. In search for novel variants and novel candidate loci, we screened a cohort of 70 young subjects with mild to severe skeletal fragility for rare copy-number variants (CNVs). Our study cohort included 15 subjects with primary osteoporosis before age 30 years and 55 subjects with a pathological fracture history and low or normal BMD before age 16 years. A custom-made high-resolution comparative genomic hybridization array with enriched probe density in >1,150 genes important for bone metabolism and ciliary function was used to search for CNVs. We identified altogether 14 rare CNVs. Seven intronic aberrations were classified as likely benign. Five CNVs of unknown clinical significance affected coding regions of genes not previously associated with skeletal fragility (ETV1-DGKB, AGBL2, ATM, RPS6KL1-PGF, and SCN4A). Finally, two CNVs were pathogenic and likely pathogenic, respectively: a 4 kb deletion involving exons 1–4 of COL1A2 (NM_000089.3) and a 12.5 kb duplication of exon 3 in PLS3 (NM_005032.6). Although both genes have been linked to monogenic forms of osteoporosis, COL1A2 deletions are rare and PLS3 duplications have not been described previously. Both CNVs were identified in subjects with significant osteoporosis and segregated with osteoporosis within the families. Our study expands the number of pathogenic CNVs in monogenic skeletal fragility and shows the validity of targeted CNV screening to potentially pinpoint novel candidate loci in early-onset osteoporosis.