Nerea Alonso

Mutations of SQSTM1 Are Associated With Severity and Clinical Outcome in Paget Disease of Bone

Paget disease of bone (PDB) is a common disorder characterized by increased bone turnover at one of more sites throughout the skeleton. Genetic factors play an important role in the pathogenesis of PDB, and the most important predisposing gene is SQSTM1, which is mutated in about 10% of patients. Here we investigated the relationship between SQSTM1 mutation status, disease severity, and clinical outcome in 737 patients who took part in a randomized study of two different management strategies for the disease. Mutations of SQSTM1 were detected in 80 of 737 (10.9%) patients. Mutation carriers had an earlier age at diagnosis (59.4 ±11.5 versus 65.0 ± 10.4 years, p < .0001) and a greater number of affected bones (3.2 ± 1.2 versus 2.1 ± 1.2, p < .001) and more commonly required orthopedic surgery (26.2% versus 16.1%, p = .024) and bisphosphonate therapy (86.3% versus 75.2%, p = .01) than those without mutations. Quality of life, as assessed by the short‐form‐36 (SF36) physical summary score, was significantly reduced in carriers (34.0 ± 11.3 versus 37.1 ± 11.4, p = .036). During the study, fractures were more common in carriers (12.5% versus 5.3%, p = .011), although most of these occurred in unaffected bone. This study demonstrates that SQSTM1 mutations are strongly associated with disease severity and complications of PDB. Genetic testing for SQSTM1 mutations may be of value in identifying individuals at risk of developing severe disease, but further studies will be required to determine if a program of genetic testing and early intervention in these individuals would be cost‐effective or be of benefit in preventing these complications.

Common susceptibility alleles and SQSTM1 mutations predict disease extent and severity in a multinational study of patients with Paget's disease

Pagets disease of bone (PDB) has a strong genetic component. Here, we investigated possible associations between genetic variants that predispose to PDB and disease severity. Allelic variants identified as predictors of PDB from genome‐wide association studies were analyzed in 1940 PDB patients from the United Kingdom, Italy, Western Australia, and Spain. A cumulative risk allele score was constructed by adding the variants together and relating this to markers of disease severity, alone and in combination with SQSTM1 mutations. In SQSTM1‐negative patients, risk allele scores in the highest tertile were associated with a 27% increase in disease extent compared with the lowest tertile (p < 0.00001) with intermediate values in the middle tertile (20% increase; p = 0.0007). The effects were similar for disease severity score, which was 15% (p = 0.01) and 25% (p < 0.00001) higher in the middle and upper tertiles, respectively. Risk allele score remained a significant predictor of extent and severity when SQSTM‐positive individuals were included, with an effect size approximately one‐third of that observed with SQSTM1 mutations. A genetic risk score was developed by combining information from both markers, which identified subgroups of individuals with low, medium, and high levels of severity with a specificity of 70% and sensitivity of 55%. Risk allele scores and SQSTM1 mutations both predict extent and severity of PDB. It is possible that with further refinement, genetic profiling may be of clinical value in identifying individuals at high risk of severe disease who might benefit from enhanced surveillance and early intervention.

A genome-wide copy number association study of osteoporotic fractures points to the 6p25.1 locus

Background Osteoporosis is a systemic skeletal disease characterised by reduced bone mineral density and increased susceptibility to fracture; these traits are highly heritable. Both common and rare copy number variants (CNVs) potentially affect the function of genes and may influence disease risk. Aim To identify CNVs associated with osteoporotic bone fracture risk. Method We performed a genome-wide CNV association study in 5178 individuals from a prospective cohort in the Netherlands, including 809 osteoporotic fracture cases, and performed in silico lookups and de novo genotyping to replicate in several independent studies. Results A rare (population prevalence 0.14%, 95% CI 0.03% to 0.24%) 210 kb deletion located on chromosome 6p25.1 was associated with the risk of fracture (OR 32.58, 95% CI 3.95 to 1488.89; p=8.69×10−5). We performed an in silico meta-analysis in four studies with CNV microarray data and the association with fracture risk was replicated (OR 3.11, 95% CI 1.01 to 8.22; p=0.02). The prevalence of this deletion showed geographic diversity, being absent in additional samples from Australia, Canada, Poland, Iceland, Denmark, and Sweden, but present in the Netherlands (0.34%), Spain (0.33%), USA (0.23%), England (0.15%), Scotland (0.10%), and Ireland (0.06%), with insufficient evidence for association with fracture risk. Conclusions These results suggest that deletions in the 6p25.1 locus may predispose to higher risk of fracture in a subset of populations of European origin; larger and geographically restricted studies will be needed to confirm this regional association. This is a first step towards the evaluation of the role of rare CNVs in osteoporosis.

Assessment of Gene-by-Sex Interaction Effect on Bone Mineral Density

Sexual dimorphism in various bone phenotypes, including bone mineral density (BMD), is widely observed; however, the extent to which genes explain these sex differences is unclear. To identify variants with different effects by sex, we examined gene‐by‐sex autosomal interactions genome‐wide, and performed expression quantitative trait loci (eQTL) analysis and bioinformatics network analysis. We conducted an autosomal genome‐wide meta‐analysis of gene‐by‐sex interaction on lumbar spine (LS) and femoral neck (FN) BMD in 25,353 individuals from 8 cohorts. In a second stage, we followed up the 12 top single‐nucleotide polymorphisms (SNPs; p < 1 × 10−5) in an additional set of 24,763 individuals. Gene‐by‐sex interaction and sex‐specific effects were examined in these 12 SNPs. We detected one novel genome‐wide significant interaction associated with LS‐BMD at the Chr3p26.1‐p25.1 locus, near the GRM7 gene (male effect = 0.02 and p = 3.0 × 10−5; female effect = −0.007 and p = 3.3 × 10−2), and 11 suggestive loci associated with either FN‐ or LS‐BMD in discovery cohorts. However, there was no evidence for genome‐wide significant (p < 5 × 10−8) gene‐by‐sex interaction in the joint analysis of discovery and replication cohorts. Despite the large collaborative effort, no genome‐wide significant evidence for gene‐by‐sex interaction was found to influence BMD variation in this screen of autosomal markers. If they exist, gene‐by‐sex interactions for BMD probably have weak effects, accounting for less than 0.08% of the variation in these traits per implicated SNP.

Genome-wide association study for radiographic vertebral fractures: A potential role for the 16q24 BMD locus

Vertebral fracture risk is a heritable complex trait. The aim of this study was to identify genetic susceptibility factors for osteoporotic vertebral fractures applying a genome-wide association study (GWAS) approach. The GWAS discovery was based on the Rotterdam Study, a population-based study of elderly Dutch individuals aged >55years; and comprising 329 cases and 2,666 controls with radiographic scoring (McCloskey-Kanis) and genetic data. Replication of one top-associated SNP was pursued by de-novo genotyping of 15 independent studies across Europe, the United States, Australia and one Asian study. Radiographic vertebral fracture assessment was performed using McCloskey-Kanis or Genant semi-quantitative definitions. SNPs were analyzed in relation to vertebral fracture using logistic regression models corrected for age and sex. Fixed effects inverse variance and Han-Eskin alternative random effects meta-analyses were applied. Genome-wide significance was set at P<5x10-8. In the discovery, a SNP (rs11645938) on chromosome 16q24 was associated with the risk for vertebral fractures at p=4.6 x 10-8. However, the association was not significant across 5,720 cases and 21,791 controls from 14 studies. Fixed-effects meta analyses summary estimate was 1.06 (95% CI: 0.98-1.14; P=0.17), displaying high degree of heterogeneity (I2=57%; Qhet p= 0.0006). Under Han-Eskin alternative random effects model the summary effect was significant (P=0.0005). The SNP maps to a region previously found associated with lumbar spine bone mineral density (LS-BMD) in two large meta-analyses from the GEFOS consortium. A false positive association in the GWAS discovery cannot be excluded, yet, the low-powered setting of the discovery and replication settings (appropriate to identify risk effect size > 1.25) may still be consistent with an effect size <1.10, more of the type expected in complex traits. Larger effort in studies with standardized phenotype definitions are needed to confirm or reject the involvement of this locus on the risk for vertebral fractures.

ATYPICAL FEMORAL FRACTURE IN OSTEOPOROSIS PSEUDOGLIOMA SYNDROME ASSOCIATED WITH TWO NOVEL COMPOUND HETEROZYGOUS MUTATIONS IN LRP5

Osteoporosis pseudoglioma syndrome (OPPG) is a rare autosomal recessive condition of congenital blindness and severe childhood osteoporosis with skeletal fragility, caused by loss‐of‐function mutations in the low‐density lipoprotein receptor‐related protein 5 (LRP5) gene. We report the first case of atypical (subtrochanteric) femoral fracture (AFF) in OPPG, occurring in a 38‐year‐old man within the context of relatively low bone turnover and trabecular osteoporosis on bone histology. We identify two novel LRP5 mutations: R752W is associated with low bone mineral density (BMD), as demonstrated by the heterozygous carriage identified in his 57‐year‐old mother; however, the combination of this R752W mutation with another novel W79R mutation, causes a severe case of compound heterozygous OPPG. We undertake 3D homology modeling of the four extracellular YWTD β‐propeller/EGF‐like domains (E1–E4) of LRP5, and show that both novel mutations destabilize the β‐propeller domains that are critical for protein and ligand binding to regulate Wnt signaling and osteoblast function. Although AFFs have been reported in other rare bone diseases, this is the first in a genetic condition of primary osteoblast dysfunction. The relatively low bone turnover observed, and knowledge of LRP5 function, implicates impaired bone remodeling in the pathogenesis of AFF.

Unveiling the mysteries of the genetics of osteoporosis

AbstractIntroductionOsteoporosis is a common disease characterised by low bone mineral density and an increased risk of fragility fractures. Methods We conducted a literature review of relevant studies relating to the genetics of osteoporosis. Results Family studies have revealed that bone density and fractures have a strong heritable component but environmental factors also play an important role. This makes identification of the causative genetic variants challenging. Linkage analysis has been successful in identifying the genes responsible for rare inherited diseases associated with abnormalities of bone mass but has been of limited value in osteoporosis. In contrast, genome-wide association studies in large cohort studies have identified 56 loci with robust evidence of association with bone density and 14 loci that predispose to fractures. Although the effect size of the implicated variants is small, many of the loci contain genes known to be involved in regulating bone cell activity through the RANK and Wnt signalling pathways, whereas others contain novel genes not previously implicated in bone metabolism. In a few instances, whole genome and exome sequencing have been successfully used to identify rare variants of large effect size that influence susceptibility to osteoporosis.ConclusionA future challenge will be to conduct fine mapping and functional analysis of the loci implicated in osteoporosis in order to identify the causal genetic variants and examine the mechanisms by which they influence bone cell function and bone mass. Ultimately this may lead to the identification of biomarkers for susceptibility to osteoporosis and fractures or new therapeutic targets.

Novel Genetic Variants Associated With Increased Vertebral Volumetric BMD, Reduced Vertebral Fracture Risk, and Increased Expression of SLC1A3 and EPHB2.

Genome‐wide association studies (GWASs) have revealed numerous loci for areal bone mineral density (aBMD). We completed the first GWAS meta‐analysis (n = 15,275) of lumbar spine volumetric BMD (vBMD) measured by quantitative computed tomography (QCT), allowing for examination of the trabecular bone compartment. SNPs that were significantly associated with vBMD were also examined in two GWAS meta‐analyses to determine associations with morphometric vertebral fracture (n = 21,701) and clinical vertebral fracture (n = 5893). Expression quantitative trait locus (eQTL) analyses of iliac crest biopsies were performed in 84 postmenopausal women, and murine osteoblast expression of genes implicated by eQTL or by proximity to vBMD‐associated SNPs was examined. We identified significant vBMD associations with five loci, including: 1p36.12, containing WNT4 and ZBTB40; 8q24, containing TNFRSF11B; and 13q14, containing AKAP11 and TNFSF11. Two loci (5p13 and 1p36.12) also contained associations with radiographic and clinical vertebral fracture, respectively. In 5p13, rs2468531 (minor allele frequency [MAF] = 3%) was associated with higher vBMD (β = 0.22, p = 1.9 × 10–8) and decreased risk of radiographic vertebral fracture (odds ratio [OR] = 0.75; false discovery rate [FDR] p = 0.01). In 1p36.12, rs12742784 (MAF = 21%) was associated with higher vBMD (β = 0.09, p = 1.2 × 10–10) and decreased risk of clinical vertebral fracture (OR = 0.82; FDR p = 7.4 × 10–4). Both SNPs are noncoding and were associated with increased mRNA expression levels in human bone biopsies: rs2468531 with SLC1A3 (β = 0.28, FDR p = 0.01, involved in glutamate signaling and osteogenic response to mechanical loading) and rs12742784 with EPHB2 (β = 0.12, FDR p = 1.7 × 10–3, functions in bone‐related ephrin signaling). Both genes are expressed in murine osteoblasts. This is the first study to link SLC1A3 and EPHB2 to clinically relevant vertebral osteoporosis phenotypes. These results may help elucidate vertebral bone biology and novel approaches to reducing vertebral fracture incidence.

Clinical and Genetic Advances in Paget’s Disease of Bone: a Review

Paget’s disease of bone (PDB) is the second most common metabolic bone disorder, after osteoporosis. It is characterised by focal areas of increased and disorganised bone turnover, coupled with increased bone formation. This disease usually appears in the late stages of life, being slightly more frequent in men than in women. It has been reported worldwide, but primarily affects individuals of British descent. Majority of PDB patients are asymptomatic, but clinical manifestations include pain, bone deformity and complications, like pathological fractures and deafness. The causes of the disease are poorly understood and it is considered as a complex trait, combining genetic predisposition with environmental factors. Linkage analysis identified SQSTM1, at chromosome 5q35, as directly related to the disease. A number of mutations in this gene have been reported, pP392L being the most common variant among different populations. Most of these variants affect the ubiquitin-associated (UBA) domain of the protein, which is involved in autophagy processes. Genome-wide association studies enlarged the number of loci associated with PDB, and further fine-mapping studies, combined with functional analysis, identified OPTN and RIN3 as causal genes for Paget’s disease. A combination of risk alleles identified by genome-wide association studies led to the development of a score to predict disease severity, which could improve the management of the disease. Further studies need to be conducted to elucidate other important aspects of the trait, such as its focal nature and the epidemiological changes found in some populations. In this review, we summarize the clinical characteristics of the disease and the latest genetic advances to identify susceptibility genes. We also list current available treatments and prospective options.

Identification of a novel locus on chromosome 2q13, which predisposes to clinical vertebral fractures independently of bone density

Objectives To identify genetic determinants of susceptibility to clinical vertebral fractures, which is an important complication of osteoporosis. Methods Here we conduct a genome-wide association study in 1553 postmenopausal women with clinical vertebral fractures and 4340 controls, with a two-stage replication involving 1028 cases and 3762 controls. Potentially causal variants were identified using expression quantitative trait loci (eQTL) data from transiliac bone biopsies and bioinformatic studies. Results A locus tagged by rs10190845 was identified on chromosome 2q13, which was significantly associated with clinical vertebral fracture (P=1.04×10−9) with a large effect size (OR 1.74, 95% CI 1.06 to 2.6). Bioinformatic analysis of this locus identified several potentially functional SNPs that are associated with expression of the positional candidate genes TTL (tubulin tyrosine ligase) and SLC20A1 (solute carrier family 20 member 1). Three other suggestive loci were identified on chromosomes 1p31, 11q12 and 15q11. All these loci were novel and had not previously been associated with bone mineral density or clinical fractures. Conclusion We have identified a novel genetic variant that is associated with clinical vertebral fractures by mechanisms that are independent of BMD. Further studies are now in progress to validate this association and evaluate the underlying mechanism.