Recent research, published in nature genetics, identifies novel genetic influences on osteoporosis, with the potential to empower future research on osteoporosis and potentially lead to new drug targets.
Osteoporosis is a condition that develops slowly over several years and leads to increased bone fragility. Fragile bones are more prone to fracture, with hip, wrist and spine bones being particularly vulnerable to breakage. Whilst there are many factors that affect the chances of an individual developing osteoporosis such as low body weight, certain medical conditions and age, the disease is highly heritable – you are much more likely to develop osteoporosis if your family has a history of the condition. By assessing the genetic determinants of bone mineral density (BMD), the most clinically relevant factor for diagnosis, the researchers were able to create an atlas of genetic influences on osteoporosis.
GWAS identifies gene loci linked to bone mineral density and fracture risk
Using UK Biobank data, the researchers carried out a genome-wide association study (GWAS), with the aim of identifying the common genetic variants associated with estimated bone mineral density (eBMD). The study found 518 genome-wide significant loci, with 301 of these being novel. Additionally, they undertook a GWAS aimed at identifying genetic loci linked with fracture risk. The results revealed 13 loci associated with risk of fracture. As might be expected, genetic variation associated with risk of fracture was also found to be associated with BMD i.e. alleles associated with low eBMD increased fracture risk.
Causal genes linked to genetic loci
Genetic associations in humans, however, rarely result in improved clinical care, usually due to a lack of identification of causal genes at the associated loci. With this in mind, the researchers tested the DNA locations associated with BMD and fracture risk, to see which features of the DNA linked to genes that are known to influence bone biology in humans. This resulted in a set of ‘Target Genes’ that are known to have an effect on bone density and strength in humans – prioritising genes at associated loci for functional testing.
Using mouse models for in-depth analysis
The study initially used mouse models to successfully test the validity of the identified Target Genes, with outlier phenotypes more frequent in mice with disruptions to 126 Target Genes compared with 526 unselected knockout lines. The in vivo and in vitro data produced thus far in the study converged to identify DAAM2 as a highly credible and novel osteoporosis gene.
After discovering that inducing a double strand break in the DAAM2 gene severely impairs mineralization in human bone cells, the researchers used mice obtained from the Wellcome Trust/Sanger Institute (generated as part of the IMPC, using ES cells) for in-depth characterisation of DAAM2. Mice with both versions of their DAAM2 gene disrupted (Daam2tm1a/tm1a) exhibited reduced vertebral bone mineral content accompanied by a small reduction in femur length. Perhaps most significantly, the mutant mice also had markedly reduced bone strength. The increased cortical porosity observed in both male and female Daam2tm1a/tm1a mutants, along with the abnormal bone composition and structure exhibited, allowed the researchers to conclude that this decreased bone strength in Daam2tm1a/tm1a mice was not simply a result of abnormal bone turnover. If DAAM2 proves to be a viable drug target – it could result in a complementary therapeutic strategy for the prevention and treatment of osteoporosis.
The goal of the IMPC is to knock out and phenotype all 20,000 or so genes in the mouse genome, potentially providing major insights into unexplored areas of the mammalian genome. To order IMPC mice, click on the shopping cart on the top part of the gene page of interest or scroll down to “Order Mouse and ES Cells”.
Details on more phenotype associations for DAAM2 can be found on the IMPC Daam2 gene page. Various phenotype association data can be found such as that shown below.
Guest post by Basel Al-Barghouthi (ASHG talk: Wed Oct 17, 9:15am – 9:30am in Room 6D)
The sequencing of the human genome and other scientific advances have led to approaches that have revolutionized our ability to understand genetic contributions to disease. One example, genome-wide association studies (GWASs), have successfully identified thousands of associations for hundreds of complex diseases. In the case of osteoporosis, a complex disease characterized by reduced bone strength and increased incidence of fracture, human GWASs have focused on dissecting the genetic basis of bone mineral density (BMD). BMD, however, does not explain all of the phenotypic variance in bone strength and there are many other aspects of bone that influence its strength, such as bone size. Unfortunately, bone strength cannot be measured directly in humans. Therefore, we are performing GWAS for over 60 traits related to bone strength in mice, in order to bridge existing gaps in our knowledge.
In this project, we used the Diversity Outbred (DO), an outbred mouse population derived from eight genetically diverse mouse strains to perform a GWAS for size measurements of mouse femurs. The DO is particularly well-suited for high-resolution GWAS. In the DO, we measured bone size and identified a strong association influencing the width of femurs on Chromosome (Chr.) 1. To identify the gene responsible, we queried the Chr. 1 locus for associated genetic variants that potentially impacted protein activity; however, none were found, suggesting the locus was due to a genetic variant influencing gene expression. We then scanned the locus for variants affecting gene expression. Of all genes within the locus, Quiescin Sulfhydryl Oxidase 1 (Qsox1) was the only one whose expression in bone was regulated by the same variants associated with femoral width in a manner consistent with it being causal. The genetic data suggested that decreased Qsox1 levels would lead to wider bones.
Using CRISPR/Cas9 genome-editing, we tested this prediction by generating Qsox1 mutant mice that completely lacked active QSOX1 protein. Consistent with the genetic data, we observed significantly wider femurs in the absence of QSOX1. We also determined that the bones were wider specifically due to increased formation of bone along the left and right sides of the femur.
These data identify Qsox1 as a genetic determinant of bone size and highlight the power of the DO for the genetic analysis of complex traits, which can be particularly useful for traits that are difficult to measure in humans.
Guest post by Jennifer Zieba
Osteogenesis Imperfecta (OI) is the most common genetic bone dysplasia that is phenotypically and genetically complex. It is characterized by bone deformities and fractures caused by low bone mass and impaired bone quality. Roughly 85-90% of cases are dominantly inherited and result from mutations in genes encoding type I collagen (COL1A1 and COL1A2), the major protein of the bone matrix. 10-15% of OI cases are recessively inherited and the majority of those result from mutations in members of the prolyl-3-hydroxylation complex including Cartilage Associated Protein (CRTAP) involved in collagen posttranslational modification.
OI patients are at an increased risk of fracture throughout their lifetimes and anecdotal evidence suggests successful fracture recovery. However, non-union has been reported in 24% of fractures and 52% of osteotomies and many stabilization techniques result in additional surgery due to re-fracture. Re-fractures typically go unreported making the frequency of re-fractures in OI patients unknown. Thus, there is an unmet need to better understand the mechanisms by which OI affects fracture healing. Assessing fracture healing in human patients is a difficult task as neither X-Ray nor CT analysis provide accurate information concerning fracture callus composition, remodeling rate, or the final bone composition. Mouse models for OI have been proven to accurately reflect OI pathogenesis and phenotype. Furthermore, using mice as models for fracture healing allow us to observe in greater detail the lengthy process of fracture healing in a smaller time frame with more informative in vivo techniques such as histochemistry, uCT analysis and biomechanical testing of the fracture tissue at several timepoints. It is our hypothesis that OI fractures undergo suboptimal healing and that this process results in ultimately weaker bone leading to the increased possibility of re-fracture and we are using two murine models to assess this hypothesis.
Using an open tibial fracture model, we show a decrease in callus size in both Col1a1G610c/+ and Crtap–/– OI mouse models post-fracture indicating delayed healing and decreased cartilage content indicating decreased callus cell proliferation. Additionally, fracture calluses in both models exhibited a significant decrease in polar moment of inertia (pMOI) indicating a decrease in resistance to torsional stress supporting a potential functional deficit in newly healed bone. This data provides valuable insight into the effect of the ECM on fracture healing, a poorly understood mechanism. Most importantly, we performed biomechanical testing via three-point-bending of fully healed Crtap–/– tibia to determine the mechanical strength of the fracture site. In wild type bone, the healed fracture site resulted in stronger bone when compared to the unfractured tibia. However, Crtap–/– healed fractured tibia are mechanically weaker than the contralateral unfractured bone. This implies the possibility that OI fractures do not heal properly and may be a prime location for re-fracture. These data may support aggressive prevention of primary fractures as well as a need for therapies during fracture healing to decrease incidence of refracture and deformity in OI patients.
Guest post by John Morris (ASHG talk: Wed Oct 17, 9:00am – 9:15am in Room 6D)
Osteoporosis is a common, aging-related disease characterized by decreased bone strength and, consequently, increased fracture risk. Bone mineral density (BMD), a non-invasive measurement, is the most clinically relevant risk factor for diagnosing osteoporosis and is highly heritable (i.e. determined by genetics). To understand the genetic determinants of osteoporosis, we performed a genome-wide association study (GWAS) in 426,824 UK Biobank participants to identify regions of the genome associated with BMD estimated from quantitative heel ultrasound (eBMD). This approach is unbiased in that it systematically tests millions of single nucleotide polymorphisms (SNPs) in the human genome—sites of common, uncommon, or rare genetic variation in the general population—for association with eBMD measurements. BMD-associated SNPs can then be used to identify novel bone genes, but such genes would require further study in human cells or animal models to understand their function. Therefore, we collaborated with the Origins of Bone and Cartilage Disease (OBCD, www.boneandcartilage.com) to examine genes in knockout mice. Such genes, when validated by knockout mouse skeletal phenotyping, represent strong candidates for developing new therapies to prevent and treat osteoporosis.
Our eBMD GWAS identified 518 significant regions of the genome, 301 of which were novel findings. Next, to identify target genes, we performed statistical fine-mapping and integrative bone cell functional genomics data analyses. First, by leveraging SNP association summary statistics and SNP-by-SNP correlations, we can identify a subset of plausibly causal SNPs. Then, by intersecting this list of plausibly causal SNPs with genomic characteristics that indicate function (e.g. coding SNPs, osteoblast open chromatin, osteoblast 3D contacts with gene promoters), we can identify a list of target genes likely to be functional in bone cells. These orthogonal approaches resulted in a list of 515 target genes, identified by plausibly causal and putatively functional SNPs, that we found were strongly enriched for known bone genes and osteoporosis drug targets. We sought to examine the effects of as many of these genes as possible in knockout mice and found the OBCD had skeletal phenotyping data on 126. Importantly, the OBCD receives all knockout mouse lines for skeletal phenotyping at random from the International Mouse Phenotyping Consortium (IMPC), therefore it is not known beforehand if a given knockout mouse has a skeletal phenotype. These 126 genes were found to be enriched for outlier skeletal phenotypes, providing strong evidence that our target genes are disease-relevant, and we focused on one such gene in further detail: disheveled-associated activator of morphogenesis 2 (DAAM2).
Mice with hypomorphic Daam2 alleles were found to have increased cortical porosity and markedly reduced bone strength, even though all other cortical bone parameters, including BMD, were normal. We performed further analyses on DAAM2, such as CRISPR-Cas9 mediated knockouts in human osteoblast cell lines, revealing a decreased ability of this crucial bone-forming cell to mineralize. We concluded that DAAM2 is a novel risk gene for osteoporosis meriting further study and highlighted five other strong candidates for follow-up: CBX1, WAC, DSCC1, RGCC, and YWHAE. In summary, we have generated an atlas of genetic influences on osteoporosis in humans and mice, more fully describing its genetic architecture. Human and mouse genetics identified DAAM2 and other genes previously unknown to function in bone biology. We expect the genes identified here to include new drug targets for the treatment of osteoporosis, where novel therapeutic options are a health priority.
An article published in the British Medical Journal reports findings from a large international collaboration that identified 15 variations in the genome that are related to the risk of suffering bone fractures, which are a major healthcare problem affecting more than 9 million persons worldwide every year. The study provides evidence against a causal effect of several proposed clinical risk factors for fractures, including diabetes, rheumatoid arthritis, vitamin D, as well as others. These findings strongly suggest that treatments aimed at increasing bone strength are more likely to be successful in preventing fractures than widespread supplementation of calcium and vitamin D or targeting other risk factors that were not found to mediate the disease.
The study sample was comprised of 185,057 cases of bone fractures and 377,201 controls who were part of the Genetic Factors for Osteoporosis (“GEFOS”) Consortium, the UKBiobank Study and the 23andMe biotech company.
This first genome-wide association study (GWAS) of fracture risk provides insight into the biologic mechanisms leading to fractures. Most importantly all of the identified genomic regions found to be associated with fracture have also been previously found to be associated with variation in bone mineral density (BMD), one of the most important risk factors for fracture. Based on this finding, the study team performed an additional analysis called “Mendelian Randomization,” that uses genetic information to determine causal relations between risk factors and disease outcomes. The Mendelian Randomization analysis determined that only two examined factors – bone mineral density (BMD) and muscle strength – play a potentially causal role in the risk of suffering osteoporotic fracture. One of the most important findings was that the genetic factors that lead to lowered vitamin D levels do not increase risk of fracture, meaning that vitamin D supplementation is not likely to prevent fractures in the general population. Although vitamin D supplementation is part of clinical guidelines, recent randomized control trials have failed to consistently demonstrate a beneficial effect.
According to Dr. Kiel, “Among the clinical risk factors for fracture assessed in the study, only BMD showed a major causal effect on fracture. The genetic factors contributing to fractures are also the same ones that affect BMD. Knowing one’s genetic risk for fracture at an early age could be a useful piece of information to persons wanting to maintain their bone health as they age. Also the study identified novel genetic variants that could be used to target future drug therapies to prevent fracture.
Scientists from the University of Cambridge have identified a potential therapeutic target in the devastating genetic disease Hutchinson-Gilford Progeria Syndrome (HGPS), which is characterised by premature ageing. The research utilises IMPC resources through the use of mouse phenotypic data.
The paper is published in Nature Communications, with preclinical data showing that chemical inhibition or genetic deregulation of the enzyme N-acetyltransferase 10 (NAT10) leads to significant health and lifespan gains in a mouse model of HGPS.
HGPS is a rare condition: patients have an average life expectancy of around 15 years, suffering a variety of symptoms including short stature, low body weight, hair loss, skin thickening, problems with fat storage, osteoporosis, and cardiovascular disease, typically dying of a heart attack.
The disease arises from specific mutations in the gene for the protein Lamin A, which lead to production of a shorter, dysfunctional protein that accumulates in cells, specifically in the membranes surrounding the nucleus. This causes disorganisation of chromatin (the ‘packaging’ around DNA), deregulated transcription, accumulation of DNA damage and defective cell proliferation.
By screening candidate molecules for an effect on nuclear membranes in human HGPS patient-derived cells in vitro, the authors have previously identified a small molecule called remodelin as an effective ameliorative agent. They then identified which component of the cells was being affected by remodelin: an enzyme with a variety of cell functions, called NAT10.
Their aim in the new study was to take these findings into a mouse model with the same genetic defect as HGPS patients, to see whether inhibiting NAT10 – either chemically by administration of remodelin or genetically by engineering reduced production of NAT10 – could ameliorate the disease. The results show that these approaches indeed significantly improved the health of the diseased mice, increased their lifespan, and reduced the effects of the HGPS mutation across a variety of measures in body tissues and at the cellular level.
The research was led by Dr Gabriel Balmus from the Wellcome Trust/ Cancer Research UK Gurdon Institute and Dr Delphine Larrieu from the Cambridge Institute for Medical Research, University of Cambridge; and Dr David Adams from the Wellcome Sanger Institute.
Read our new study led by postdocs Delphine Larrieu, now group leader @TheCIMR and @GabrielBalmus that identifies a new therapeutic target for treatment of premature ageing syndrome HGPS, https://t.co/XCheXRt6Fu https://t.co/Js8ec6gkdi
— Steve Jackson Lab (@SPJacksonGroup) April 27, 2018
Senior author Professor Steve Jackson commented: “We’re very excited by the possibility that drugs targeting NAT10 may, in future, be tested on people suffering from HGPS. I like to describe this approach as a ‘re-balancing towards the healthy state’.
“We first studied the cell biology to understand how the disease affects cells, and then used those findings to identify ways to re-balance the defect at the whole-organism level. Our findings in mice suggest a therapeutic approach to HGPS and other premature ageing diseases.”
Research article: https://www.nature.com/articles/s41467-018-03770-3.pdf
Nat10 gene on the IMPC website: http://www.mousephenotype.org/data/genes/MGI:2138939
A recent study in the journal Nature Medicine utilises IMPC-generated mouse lines as a basis for their discovery. A summary of this research article, and how it may help human patients with osteoporosis, is given in a guest post below. This investigation is a great example of how IMPC resources can be used by the research community and exemplifies how mouse phenomics may help in the development of treatments for human disease.
Guest post by Maureen Salamon
A molecule promoting blood vessel growth in bone can create an environment suitable for bone-building, representing a potential target for new drugs to treat osteoporosis and fractures, according to new research by Weill Cornell Medicine scientists.
The findings, published in Nature Medicine, show that a substance best known for spurring nerve growth, called SLIT3, both reversed the bone-weakening effects of osteoporosis and helped fractures heal when administered in mice. The multi-center research effort could fuel drug development efforts targeting the SLIT3 pathway in humans, enabling a new approach for blood vessel-directed therapy to treat bone loss, persistent fractures and fragile bones.
Existing drugs for osteoporosis work in one of two ways: Either they block the cells that destroy bone or they promote bone formation by cells called osteoblasts. “But only those promoting new bone formation will help you actually heal a bone fracture,” said co-senior study author Dr. Matthew Greenblatt, an assistant professor of pathology and laboratory medicine at Weill Cornell Medicine. “Our findings have potentially demonstrated a third category: drugs that target blood vessel formation within bone, prompting new bone to form.”
Osteoporosis, which causes bones to thin and become brittle, leads to nearly 9 million fractures worldwide each year, or one every three seconds, according to the International Osteoporosis Foundation. Women are disproportionately affected, and the risk increases with age. One in two women and one in five men will have an osteoporotic fracture during their lifetimes, and these fractures kill as many women each year as breast cancer.
“Osteoporosis and skeletal fractures due to osteoporosis are both common and deadly,” Greenblatt said.
To counteract that trend, Greenblatt has been investigating the cellular causes of osteoporosis in an effort to promote bone growth. Prior research using mice genetically engineered to lack an adaptor protein known as SHN3 showed that its absence conferred high bone mass. Building on that discovery, Greenblatt and his team decided to examine the resulting changes in bone blood vessels. “We used those mice as a means to find the signals coming from osteoblasts to control the specific type of blood vessels present in bone,” he said.
The researchers were surprised to find that osteoblasts secreted unchanged amounts of almost all known factors promoting blood vessel growth, but SLIT3 levels rose significantly. And when the mice were genetically altered to delete SLIT3, they exhibited low bone mass.
“We next asked if we could use SLIT3 to treat mice with skeletal disease, especially osteoporosis and fracture healing,” Dr. Greenblatt said. “When we gave the rodents SLIT3, it reversed their osteoporosis and made their fractures heal faster and stronger.
“To my knowledge, this is the first example that we can develop a drug to treat bone disease in mice not by targeting the bone-forming cells,” he said, “but instead by targeting special types of blood vessels that exist in bone.”
Further research is needed to determine the best way to deliver SLIT3 to the bone in humans. SLIT3-pathway drugs could also be used in combination with existing drugs to improve patient outcomes.
“Only a small fraction of patients who’ve had a hip fracture and really require medication to prevent additional fractures get the drugs they need. Many people aren’t aware of how debilitating and deadly these kinds of fractures are,” Greenblatt said. “Having a totally new category of bone drugs that work on different sets of cells could open up new opportunities for treatment.”
In addition to benefiting seniors with osteoporosis, Greenblatt hopes his research will also help patients with bone injuries that aren’t healing properly, such as those who’ve undergone orthopedic surgery or have fragile bones due to genetic diseases.
“Some of those people’s fractures don’t heal because they can’t grow the right type of blood vessels at the site of the fracture,” he said. “That’s what we think SLIT3 will do: help with that growth and promote healing.”
Research article: Targeting skeletal endothelium to ameliorate bone loss
Maureen Salamon is a freelance writer for Weill Cornell Medicine. This article was originally published in the Cornell Chronicle and is re-published with copyright permission.
Age-related memory loss may be reversed by boosting blood levels of osteocalcin, a hormone produced by bone cells, according to mouse studies led by Columbia University Medical Center (CUMC) researchers. The research team also identified a receptor for osteocalcin in the brain, paving the way for a novel approach to treating age-related cognitive decline. The paper was published in the Journal of Experimental Medicine.
“In previous studies, we found that osteocalcin plays multiple roles in the body, including a role in memory,” said study leader Gerard Karsenty, MD, PhD, Paul A. Marks Professor and Chair, Department of Genetics & Development, and Professor of Medicine at CUMC. “We also observed that the hormone declines precipitously in humans during early adulthood. That raised an important question: Could memory loss be reversed by restoring this hormone back to youthful levels? The answer, at least in mice, is yes, suggesting that we’ve opened a new avenue of research into the regulation of behavior by peripheral hormones.”
Karsenty’s group, in collaboration with the laboratory of Eric Kandel, MD, University Professor and Kavli Professor of Brain Science at Columbia University and a key contributor to this study, conducted several experiments to evaluate osteocalcin’s role in age-related memory loss. In one experiment, aged mice were given continuous infusions of osteocalcin over a two-month period. The infusions greatly improved the animals’ performance on two different memory tests, reaching levels seen only in young mice.
The same improvements were seen when blood plasma from young mice, which is rich in osteocalcin, was injected into aged mice. In contrast, there was no memory improvement when plasma from young, osteocalcin-deficient mice was given to aged mice. But adding osteocalcin to this plasma before injecting it into the aged mice resulted in memory improvement. The researchers also used anti-osteocalcin antibodies to deplete the hormone from the plasma of young mice, reducing their performance on memory tests.
The researchers then determined that osteocalcin binds to a receptor called Gpr158 that is abundant in neurons of the CA3 region of the hippocampus, the brain’s memory center. This was confirmed by inactivating hippocampal Gpr158 in mice, and subsequently giving them infusions of osteocalcin, which failed to improve their performance on memory tests.
The researchers did not observe any toxic effects from giving the mice osteocalcin. “It’s a natural part of our body, so it should be safe,” said Dr. Karsenty. “But of course, we need to more research to translate our findings into clinical use for humans.”
In previous research, Dr. Karsenty found that osteocalcin injections also rejuvenate the muscles of older mice, allowing them to match the running speeds and distances of young mice.
“Our laboratory’s long-term interest in the biology of memory and our recent work on age-related memory loss made this a natural collaboration with the Karsenty laboratory, with its background work on osteocalcin,” said Eric Kandel, MD, co-director of the Mortimer B. Zuckerman Mind Brain Behavior Institute at Columbia and a senior investigator at the Howard Hughes Medical Institute.
Research article: Gpr158 mediates osteocalcin’s regulation of cognition