SUMMARY The extent to which low-frequency (minor allele frequency [MAF] between 1–5%) and rare (MAF ≤ 1%) variants contribute to complex traits and disease in the general population is largely unknown. Bone mineral density (BMD) is highly heritable, is a major predictor of osteoporotic fractures and has been previously associated with common genetic variants1–8, and rare, population-specific, coding variants9. Here we identify novel non-coding genetic variants with large effects on BMD (ntotal = 53,236) and fracture (ntotal = 508,253) in individuals of European ancestry from the general population. Associations for BMD were derived from whole-genome sequencing (n=2,882 from UK10K), whole-exome sequencing (n= 3,549), deep imputation of genotyped samples using a combined UK10K/1000Genomes reference panel (n=26,534), and de-novo replication genotyping (n= 20,271). We identified a low-frequency non-coding variant near a novel locus, EN1, with an effect size 4-fold larger than the mean of previously reported common variants for lumbar spine BMD8 (rs11692564[T], MAF = 1.7%, replication effect size = +0.20 standard deviations [SD], Pmeta = 2×10−14), which was also associated with a decreased risk of fracture (OR = 0.85; P = 2×10−11; ncases = 98,742 and ncontrols = 409,511). Using an En1Cre/flox mouse model, we observed that conditional loss of En1 results in low bone mass, likely as a consequence of high bone turn-over. We also identified a novel low-frequency non-coding variant with large effects on BMD near WNT16 (rs148771817[T], MAF = 1.1%, replication effect size = +0.39 SD, Pmeta = 1×10−11). In general, there was an excess of association signals arising from deleterious coding and conserved non-coding variants. These findings provide evidence that low-frequency non-coding variants have large effects on BMD and fracture, thereby providing rationale for whole-genome sequencing and improved imputation reference panels to study the genetic architecture of complex traits and disease in the general population.

Genetic disorders of the skeleton encompass a diverse group of bone diseases differing in clinical characteristics, severity, incidence and molecular etiology. Of particular interest are the monogenic rare bone mass disorders, with the underlying genetic defect contributing to either low or high bone mass phenotype. Extensive, deep phenotyping coupled with high-throughput, cost-effective genotyping is crucial in the characterization and diagnosis of affected individuals. Massive parallel sequencing efforts have been instrumental in the discovery of novel causal genes that merit functional validation using in vitro and ex vivo cell-based techniques, and in vivo models, mainly mice and zebrafish. These translational models also serve as an excellent platform for therapeutic discovery, bridging the gap between basic science research and the clinic. Altogether, genetic studies of monogenic rare bone mass disorders have broadened our knowledge on molecular signaling pathways coordinating bone development and metabolism, disease inheritance patterns, development of new and improved bone biomarkers, and identification of novel drug targets. In this comprehensive review we describe approaches to further enhance the innovative processes taking discoveries from clinic to bench, and then back to clinic in rare bone mass disorders. We highlight the importance of cross laboratory collaboration to perform functional validation in multiple model systems after identification of a novel disease gene. We describe the monogenic forms of rare low and high rare bone mass disorders known to date, provide a roadmap to unravel the genetic determinants of monogenic rare bone mass disorders using proper phenotyping and genotyping methods, and describe different genetic validation approaches paving the way for future treatments.

The availability of large human datasets for genome-wide association studies (GWAS) and the advancement of sequencing technologies have boosted the identification of genetic variants in complex and rare diseases in the skeletal field. Yet, interpreting results from human association studies remains a challenge. To bridge the gap between genetic association and causality, a systematic functional investigation is necessary. Multiple unknowns exist for putative causal genes, including cellular localization of the molecular function. Intermediate traits (“endophenotypes”), e.g. molecular quantitative trait loci (molQTLs), are needed to identify mechanisms of underlying associations. Furthermore, index variants often reside in non-coding regions of the genome, therefore challenging for interpretation. Knowledge of non-coding variance (e.g. ncRNAs), repetitive sequences, and regulatory interactions between enhancers and their target genes is central for understanding causal genes in skeletal conditions. Animal models with deep skeletal phenotyping and cell culture models have already facilitated fine mapping of some association signals, elucidated gene mechanisms, and revealed disease-relevant biology. However, to accelerate research towards bridging the current gap between association and causality in skeletal diseases, alternative in vivo platforms need to be used and developed in parallel with the current -omics and traditional in vivo resources. Therefore, we argue that as a field we need to establish resource-sharing standards to collectively address complex research questions. These standards will promote data integration from various -omics technologies and functional dissection of human complex traits. In this mission statement, we review the current available resources and as a group propose a consensus to facilitate resource sharing using existing and future resources. Such coordination efforts will maximize the acquisition of knowledge from different approaches and thus reduce redundancy and duplication of resources. These measures will help to understand the pathogenesis of osteoporosis and other skeletal diseases towards defining new and more efficient therapeutic targets.

The Ehlers-Danlos syndromes (EDS) are a collection of rare hereditary connective tissue disorders with heterogeneous phenotypes, usually diagnosed following clinical examination and confirmatory genetic testing. Diagnosis of the commonest subtype, hypermobile Ehlers-Danlos Syndrome (hEDS), relies solely on a clinical diagnosis

Osteoporosis and fractures are complex conditions influenced by an interplay of genetic and environmental factors. The aim of the study was to investigate three biochemical parameters including total serum calcium, total serum alkaline phosphatase (sALP) and albumin in relation to bone mineral density (BMD) at the lumbar spine and femoral neck (FN), and with all-type of low-trauma fractures in Maltese postmenopausal women. Levels were also correlated with age and physical activity. A case-control study of 1045 women was performed. Women who suffered a fracture were classified as cases whereas women without a fracture history were included as controls subdivided into normal, osteopenic, or osteoporotic according to their BMD measurements. Blood specimens were collected following good standard practice and testing was performed by spectrophotometry. Calcium and sALP levels were weakly correlated with FN BMD levels (calcium: r = -0.111, p = 0.002; sALP: r = 0.089, p = 0.013). Fracture cases had the lowest serum levels of calcium, sALP and albumin relative to all other control groups, which decreased with increasing age, possibly increasing fracture risk. Biochemical levels were lowest in women who sustained a hip fracture and more than one fracture. Biochemical parameters decreased with reduced physical activity; however, this was most evident for fracture cases. Reduced physical activity was associated with lower BMD levels at the hip, and to a lower extent at the spine. In conclusion, results suggest that levels of serum calcium and albumin could be indicative of fracture risk, whereas calcium levels and to lower extent sALP levels could be indicators of hip BMD.

Acute myeloid leukemia is the most common form of acute leukemia in adults, constituting about 80% of cases. Although remarkable progress has been made in the therapeutic scenario for patients with acute myeloid leukemia, research and development of new and effective anticancer agents to improve patient outcome and minimize toxicity is needed. In this study, the antitumor activity of axolotl (AXO) Ambystoma mexicanum crude extract was assessed in vitro on the human acute myeloid leukemia HL-60 cell line. The anticancer activity was evaluated in terms of ability to influence proliferative activity, cell viability, cell cycle arrest, and differentiation. Moreover, gene expression analysis was performed to evaluate the genes involved in the regulation of these processes. The AXO crude extract exhibited antiproliferative but not cytotoxic activities on HL-60 cells, with cell cycle arrest in the G0/G1 phase. Furthermore, the AXO-treated HL-60 cells showed an increase in both the percentage of nitroblue tetrazolium positive cells and the expression of CD11b, whereas the proportion of CD14-positive cells did not change, suggesting that extract is able to induce differentiation toward the granulocytic lineage. Finally, the treatment with AXO extract caused upregulation of CEBPA, CEBPB, CEBPE, SPI1, CDKN1A, and CDKN2C, and downregulation of c-MYC. Our data clearly show the potential anticancer activity of Ambystoma mexicanum on HL-60 cells and suggest that it could help develop promising therapeutic agents for the treatment of acute myeloid leukemia.

Monogenic high bone mass (HBM) disorders are characterized by an increased amount of bone in general, or at specific sites in the skeleton. Here, we describe 59 HBM disorders with 50 known disease-causing genes from the literature, and we provide an overview of the signaling pathways and mechanisms involved in the pathogenesis of these disorders. Based on this, we classify the known HBM genes into HBM (sub)groups according to uniform Gene Ontology (GO) terminology. This classification system may aid in hypothesis generation, for both wet lab experimental design and clinical genetic screening strategies. We discuss how functional genomics canThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Background: Osteoporosis is a skeletal disease with a strong genetic background. The study aimed to identify the genetic determinants of early-onset familial osteoporosis and low bone mineral density (BMD) in a two-generation Maltese family. Methods: Fifteen relatives aged between 28–74 years were recruited. Whole genome sequencing was conducted on 12 relatives and shortlisted variants were genotyped in the Malta Osteoporotic Fracture Study (MOFS) for replication. Results: Sequential variant filtering following a dominant inheritance pattern identified rare missense variants within SELP, TGF-β2 and ADAMTS20, all of which were predicted to be likely pathogenic and participate in osteoimmunology. TGF-β2 c.1136C>T was identified in five individuals from the MOFS in heterozygosity, four of whom had osteopenia/osteoporosis at the lumbar spine and hip, and/or had sustained a low-trauma fracture. Heterozygosity for the ADAMTS20 c.4090A>T was accompanied by lower total hip BMD (p = 0.018) and lower total serum calcium levels in MOFS (p < 0.01), recapitulating the findings from the family. Women carrying at least one copy of the alternative allele (TC/CC) for SELP c.2177T>C exhibited a tendency for lower lumbar spine BMD and/or wrist fracture history relative to women with TT genotype. Conclusions: Our findings suggest that the identified variants, alone or in combination, could be causal factors of familial osteoporosis and low BMD, requiring replication in larger collections.