Screening for functional regulatory variants in open chromatin using GenIE-ATAC.

Understanding the effects of genetic variation in gene regulatory elements is crucial to interpreting genome function. This is particularly pertinent for the hundreds of thousands of disease-associated variants identified by GWAS, which frequently sit within gene regulatory elements but whose functional effects are often unknown. Current methods are limited in their scalability and ability to assay regulatory variants in their endogenous context, independently of other tightly linked variants. Here, we present a new medium-throughput screening system: genome engineering based interrogation of enhancers assay for transposase accessible chromatin (GenIE-ATAC), that measures the effect of individual variants on chromatin accessibility in their endogenous genomic and chromatin context. We employ this assay to screen for the effects of regulatory variants in human induced pluripotent stem cells, validating a subset of causal variants, and extend our software package (rgenie) to analyse these new data. We demonstrate that this methodology can be used to understand the impact of defined deletions and point mutations within transcription factor binding sites. We thus establish GenIE-ATAC as a method to screen for the effect of gene regulatory element variation, allowing identification and prioritisation of causal variants from GWAS for functional follow-up and understanding the mechanisms of regulatory element function.

The next-generation Open Targets Platform: reimagined, redesigned, rebuilt.

The Open Targets Platform (https://platform.opentargets.org/) is an open source resource to systematically assist drug target identification and prioritisation using publicly available data. Since our last update, we have reimagined, redesigned, and rebuilt the Platform in order to streamline data integration and harmonisation, expand the ways in which users can explore the data, and improve the user experience. The gene-disease causal evidence has been enhanced and expanded to better capture disease causality across rare, common, and somatic diseases. For target and drug annotations, we have incorporated new features that help assess target safety and tractability, including genetic constraint, PROTACtability assessments, and AlphaFold structure predictions. We have also introduced new machine learning applications for knowledge extraction from the published literature, clinical trial information, and drug labels. The new technologies and frameworks introduced since the last update will ease the introduction of new features and the creation of separate instances of the Platform adapted to user requirements. Our new Community forum, expanded training materials, and outreach programme support our users in a range of use cases.

Open Targets Platform: supporting systematic drug-target identification and prioritisation.

The Open Targets Platform (https://www.targetvalidation.org/) provides users with a queryable knowledgebase and user interface to aid systematic target identification and prioritisation for drug discovery based upon underlying evidence. It is publicly available and the underlying code is open source. Since our last update two years ago, we have had 10 releases to maintain and continuously improve evidence for target-disease relationships from 20 different data sources. In addition, we have integrated new evidence from key datasets, including prioritised targets identified from genome-wide CRISPR knockout screens in 300 cancer models (Project Score), and GWAS/UK BioBank statistical genetic analysis evidence from the Open Targets Genetics Portal. We have evolved our evidence scoring framework to improve target identification. To aid the prioritisation of targets and inform on the potential impact of modulating a given target, we have added evaluation of post-marketing adverse drug reactions and new curated information on target tractability and safety. We have also developed the user interface and backend technologies to improve performance and usability. In this article, we describe the latest enhancements to the Platform, to address the fundamental challenge that developing effective and safe drugs is difficult and expensive.

Open Targets Genetics: systematic identification of trait-associated genes using large-scale genetics and functional genomics.

Open Targets Genetics (https://genetics.opentargets.org) is an open-access integrative resource that aggregates human GWAS and functional genomics data including gene expression, protein abundance, chromatin interaction and conformation data from a wide range of cell types and tissues to make robust connections between GWAS-associated loci, variants and likely causal genes. This enables systematic identification and prioritisation of likely causal variants and genes across all published trait-associated loci. In this paper, we describe the public resources we aggregate, the technology and analyses we use, and the functionality that the portal offers. Open Targets Genetics can be searched by variant, gene or study/phenotype. It offers tools that enable users to prioritise causal variants and genes at disease-associated loci and access systematic cross-disease and disease-molecular trait colocalization analysis across 92 cell types and tissues including the eQTL Catalogue. Data visualizations such as Manhattan-like plots, regional plots, credible sets overlap between studies and PheWAS plots enable users to explore GWAS signals in depth. The integrated data is made available through the web portal, for bulk download and via a GraphQL API, and the software is open source. Applications of this integrated data include identification of novel targets for drug discovery and drug repurposing.

Artificial intelligence for dementia genetics and omics.

Genetics and omics studies of Alzheimer's disease and other dementia subtypes enhance our understanding of underlying mechanisms and pathways that can be targeted. We identified key remaining challenges: First, can we enhance genetic studies to address missing heritability? Can we identify reproducible omics signatures that differentiate between dementia subtypes? Can high-dimensional omics data identify improved biomarkers? How can genetics inform our understanding of causal status of dementia risk factors? And which biological processes are altered by dementia-related genetic variation? Artificial intelligence (AI) and machine learning approaches give us powerful new tools in helping us to tackle these challenges, and we review possible solutions and examples of best practice. However, their limitations also need to be considered, as well as the need for coordinated multidisciplinary research and diverse deeply phenotyped cohorts. Ultimately AI approaches improve our ability to interrogate genetics and omics data for precision dementia medicine. HIGHLIGHTS: We have identified five key challenges in dementia genetics and omics studies. AI can enable detection of undiscovered patterns in dementia genetics and omics data. Enhanced and more diverse genetics and omics datasets are still needed. Multidisciplinary collaborative efforts using AI can boost dementia research.

Screening for functional transcriptional and splicing regulatory variants with GenIE.

Genome-wide association studies (GWAS) have identified numerous genetic loci underlying human diseases, but a fundamental challenge remains to accurately identify the underlying causal genes and variants. Here, we describe an arrayed CRISPR screening method, Genome engineering-based Interrogation of Enhancers (GenIE), which assesses the effects of defined alleles on transcription or splicing when introduced in their endogenous genomic locations. We use this sensitive assay to validate the activity of transcriptional enhancers and splice regulatory elements in human induced pluripotent stem cells (hiPSCs), and develop a software package (rgenie) to analyse the data. We screen the 99% credible set of Alzheimer's disease (AD) GWAS variants identified at the clusterin (CLU) locus to identify a subset of likely causal variants, and employ GenIE to understand the impact of specific mutations on splicing efficiency. We thus establish GenIE as an efficient tool to rapidly screen for the role of transcribed variants on gene expression.

Whole-Genome Sequencing Coupled to Imputation Discovers Genetic Signals for Anthropometric Traits.

Deep sequence-based imputation can enhance the discovery power of genome-wide association studies by assessing previously unexplored variation across the common- and low-frequency spectra. We applied a hybrid whole-genome sequencing (WGS) and deep imputation approach to examine the broader allelic architecture of 12 anthropometric traits associated with height, body mass, and fat distribution in up to 267,616 individuals. We report 106 genome-wide significant signals that have not been previously identified, including 9 low-frequency variants pointing to functional candidates. Of the 106 signals, 6 are in genomic regions that have not been implicated with related traits before, 28 are independent signals at previously reported regions, and 72 represent previously reported signals for a different anthropometric trait. 71% of signals reside within genes and fine mapping resolves 23 signals to one or two likely causal variants. We confirm genetic overlap between human monogenic and polygenic anthropometric traits and find signal enrichment in cis expression QTLs in relevant tissues. Our results highlight the potential of WGS strategies to enhance biologically relevant discoveries across the frequency spectrum.

Very low-depth whole-genome sequencing in complex trait association studies.

MOTIVATION: Very low-depth sequencing has been proposed as a cost-effective approach to capture low-frequency and rare variation in complex trait association studies. However, a full characterization of the genotype quality and association power for very low-depth sequencing designs is still lacking. RESULTS: We perform cohort-wide whole-genome sequencing (WGS) at low depth in 1239 individuals (990 at 1× depth and 249 at 4× depth) from an isolated population, and establish a robust pipeline for calling and imputing very low-depth WGS genotypes from standard bioinformatics tools. Using genotyping chip, whole-exome sequencing (75× depth) and high-depth (22×) WGS data in the same samples, we examine in detail the sensitivity of this approach, and show that imputed 1× WGS recapitulates 95.2% of variants found by imputed GWAS with an average minor allele concordance of 97% for common and low-frequency variants. In our study, 1× further allowed the discovery of 140 844 true low-frequency variants with 73% genotype concordance when compared to high-depth WGS data. Finally, using association results for 57 quantitative traits, we show that very low-depth WGS is an efficient alternative to imputed GWAS chip designs, allowing the discovery of up to twice as many true association signals than the classical imputed GWAS design. AVAILABILITY AND IMPLEMENTATION: The HELIC genotype and WGS datasets have been deposited to the European Genome-phenome Archive (https://www.ebi.ac.uk/ega/home): EGAD00010000518; EGAD00010000522; EGAD00010000610; EGAD00001001636, EGAD00001001637. The peakplotter software is available at https://github.com/wtsi-team144/peakplotter, the transformPhenotype app can be downloaded at https://github.com/wtsi-team144/transformPhenotype. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

CTP synthase 1 deficiency in humans reveals its central role in lymphocyte proliferation.

Lymphocyte functions triggered by antigen recognition and co-stimulation signals are associated with a rapid and intense cell division, and hence with metabolism adaptation. The nucleotide cytidine 5' triphosphate (CTP) is a precursor required for the metabolism of DNA, RNA and phospholipids. CTP originates from two sources: a salvage pathway and a de novo synthesis pathway that depends on two enzymes, the CTP synthases (or synthetases) 1 and 2 (CTPS1 with CTPS2); the respective roles of these two enzymes are not known. CTP synthase activity is a potentially important step for DNA synthesis in lymphocytes. Here we report the identification of a loss-of-function homozygous mutation (rs145092287) in CTPS1 in humans that causes a novel and life-threatening immunodeficiency, characterized by an impaired capacity of activated T and B cells to proliferate in response to antigen receptor-mediated activation. In contrast, proximal and distal T-cell receptor (TCR) signalling events and responses were only weakly affected by the absence of CTPS1. Activated CTPS1-deficient cells had decreased levels of CTP. Normal T-cell proliferation was restored in CTPS1-deficient cells by expressing wild-type CTPS1 or by addition of exogenous CTP or its nucleoside precursor, cytidine. CTPS1 expression was found to be low in resting T cells, but rapidly upregulated following TCR activation. These results highlight a key and specific role of CTPS1 in the immune system by its capacity to sustain the proliferation of activated lymphocytes during the immune response. CTPS1 may therefore represent a therapeutic target of immunosuppressive drugs that could specifically dampen lymphocyte activation.

Joubert Syndrome in French Canadians and Identification of Mutations in CEP104.

Joubert syndrome (JBTS) is a primarily autosomal-recessive disorder characterized by a distinctive mid-hindbrain and cerebellar malformation, oculomotor apraxia, irregular breathing, developmental delay, and ataxia. JBTS is a genetically heterogeneous ciliopathy. We sought to characterize the genetic landscape associated with JBTS in the French Canadian (FC) population. We studied 43 FC JBTS subjects from 35 families by combining targeted and exome sequencing. We identified pathogenic (n = 32 families) or possibly pathogenic (n = 2 families) variants in genes previously associated with JBTS in all of these subjects, except for one. In the latter case, we found a homozygous splice-site mutation (c.735+2T>C) in CEP104. Interestingly, we identified two additional non-FC JBTS subjects with mutations in CEP104; one of these subjects harbors a maternally inherited nonsense mutation (c.496C>T [p.Arg166*]) and a de novo splice-site mutation (c.2572-2A>G), whereas the other bears a homozygous frameshift mutation (c.1328_1329insT [p.Tyr444fs*3]) in CEP104. Previous studies have shown that CEP104 moves from the mother centriole to the tip of the primary cilium during ciliogenesis. Knockdown of CEP104 in retinal pigment epithelial (RPE1) cells resulted in severe defects in ciliogenesis. These observations suggest that CEP104 acts early during cilia formation by regulating the conversion of the mother centriole into the cilia basal body. We conclude that disruption of CEP104 causes JBTS. Our study also reveals that the cause of JBTS has been elucidated in the great majority of our FC subjects (33/35 [94%] families), even though JBTS shows substantial locus and allelic heterogeneity in this population.

A family segregating lethal neonatal coenzyme Q10 deficiency caused by mutations in COQ9.

Primary CoQ10 deficiency is a clinically and genetically heterogeneous, autosomal recessive disorder resulting from mutations in genes involved in the synthesis of coenzyme Q10 (CoQ10). To date, mutations in nine proteins required for the biosynthesis of CoQ10 cause CoQ10 deficiency with varying clinical presentations. In 2009 the first patient with mutations in COQ9 was reported in an infant with a neonatal-onset, primary CoQ10 deficiency with multi-system disease. Here we describe four siblings with a previously undiagnosed lethal disorder characterized by oligohydramnios and intrauterine growth restriction, variable cardiomyopathy, anemia, and renal anomalies. The first and third pregnancy resulted in live born babies with abnormal tone who developed severe, treatment unresponsive lactic acidosis after birth and died hours later. Autopsy on one of the siblings demonstrated brain changes suggestive of the subacute necrotizing encephalopathy of Leigh disease. Whole-exome sequencing (WES) revealed the siblings shared compound heterozygous mutations in the COQ9 gene with both variants predicted to affect splicing. RT-PCR on RNA from patient fibroblasts revealed that the c.521 + 2 T > C variant resulted in splicing out of exons 4-5 and the c.711 + 3G > C variant spliced out exon 6, resulting in undetectable levels of COQ9 protein in patient fibroblasts. The biochemical profile of patient fibroblasts demonstrated a drastic reduction in CoQ10 levels. An additional peak on the chromatogram may represent accumulation of demethoxy coenzyme Q (DMQ), which was shown previously to accumulate as a result of a defect in COQ9. This family expands our understanding of this rare metabolic disease and highlights the prenatal onset, clinical variability, severity, and biochemical profile associated with COQ9-related CoQ10 deficiencies.

Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma.

Glioblastoma multiforme (GBM) is a lethal brain tumour in adults and children. However, DNA copy number and gene expression signatures indicate differences between adult and paediatric cases. To explore the genetic events underlying this distinction, we sequenced the exomes of 48 paediatric GBM samples. Somatic mutations in the H3.3-ATRX-DAXX chromatin remodelling pathway were identified in 44% of tumours (21/48). Recurrent mutations in H3F3A, which encodes the replication-independent histone 3 variant H3.3, were observed in 31% of tumours, and led to amino acid substitutions at two critical positions within the histone tail (K27M, G34R/G34V) involved in key regulatory post-translational modifications. Mutations in ATRX (α-thalassaemia/mental retardation syndrome X-linked) and DAXX (death-domain associated protein), encoding two subunits of a chromatin remodelling complex required for H3.3 incorporation at pericentric heterochromatin and telomeres, were identified in 31% of samples overall, and in 100% of tumours harbouring a G34R or G34V H3.3 mutation. Somatic TP53 mutations were identified in 54% of all cases, and in 86% of samples with H3F3A and/or ATRX mutations. Screening of a large cohort of gliomas of various grades and histologies (n = 784) showed H3F3A mutations to be specific to GBM and highly prevalent in children and young adults. Furthermore, the presence of H3F3A/ATRX-DAXX/TP53 mutations was strongly associated with alternative lengthening of telomeres and specific gene expression profiles. This is, to our knowledge, the first report to highlight recurrent mutations in a regulatory histone in humans, and our data suggest that defects of the chromatin architecture underlie paediatric and young adult GBM pathogenesis.

Clonal selection drives genetic divergence of metastatic medulloblastoma.

Medulloblastoma, the most common malignant paediatric brain tumour, arises in the cerebellum and disseminates through the cerebrospinal fluid in the leptomeningeal space to coat the brain and spinal cord. Dissemination, a marker of poor prognosis, is found in up to 40% of children at diagnosis and in most children at the time of recurrence. Affected children therefore are treated with radiation to the entire developing brain and spinal cord, followed by high-dose chemotherapy, with the ensuing deleterious effects on the developing nervous system. The mechanisms of dissemination through the cerebrospinal fluid are poorly studied, and medulloblastoma metastases have been assumed to be biologically similar to the primary tumour. Here we show that in both mouse and human medulloblastoma, the metastases from an individual are extremely similar to each other but are divergent from the matched primary tumour. Clonal genetic events in the metastases can be demonstrated in a restricted subclone of the primary tumour, suggesting that only rare cells within the primary tumour have the ability to metastasize. Failure to account for the bicompartmental nature of metastatic medulloblastoma could be a major barrier to the development of effective targeted therapies.

An N-terminal formyl methionine on COX 1 is required for the assembly of cytochrome c oxidase.

Protein synthesis in mitochondria is initiated by formylmethionyl-tRNA(Met) (fMet-tRNA(Met)), which requires the activity of the enzyme MTFMT to formylate the methionyl group. We investigated the molecular consequences of mutations in MTFMT in patients with Leigh syndrome or cardiomyopathy. All patients studied were compound heterozygotes. Levels of MTFMT in patient fibroblasts were almost undetectable by immunoblot analysis, and BN-PAGE analysis showed a combined oxidative phosphorylation (OXPHOS) assembly defect involving complexes I, IV and V. The synthesis of only a subset of mitochondrial polypeptides (ND5, ND4, ND1, COXII) was decreased, whereas all others were translated at normal or even increased rates. Expression of the wild-type cDNA rescued the biochemical phenotype when MTFMT was expressed near control levels, but overexpression produced a dominant-negative phenotype, completely abrogating assembly of the OXPHOS complexes, suggesting that MTFMT activity must be tightly regulated. fMet-tRNA(Met) was almost undetectable in control cells and absent in patient cells by high-resolution northern blot analysis, but accumulated in cells overexpressing MTFMT. Newly synthesized COXI was under-represented in complex IV immunoprecipitates from patient fibroblasts, and two-dimensional BN-PAGE analysis of newly synthesized mitochondrial translation products showed an accumulation of free COXI. Quantitative mass spectrophotometry of an N-terminal COXI peptide showed that the ratio of formylated to unmodified N-termini in the assembled complex IV was ∼350:1 in controls and 4:1 in patient cells. These results show that mitochondrial protein synthesis can occur with inefficient formylation of methionyl-tRNA(Met), but that assembly of complex IV is impaired if the COXI N-terminus is not formylated.

Mutations in CSPP1, encoding a core centrosomal protein, cause a range of ciliopathy phenotypes in humans.

Ciliopathies are characterized by a pattern of multisystem involvement that is consistent with the developmental role of the primary cilium. Within this biological module, mutations in genes that encode components of the cilium and its anchoring structure, the basal body, are the major contributors to both disease causality and modification. However, despite rapid advances in this field, the majority of the genes that drive ciliopathies and the mechanisms that govern the pronounced phenotypic variability of this group of disorders remain poorly understood. Here, we show that mutations in CSPP1, which encodes a core centrosomal protein, are disease causing on the basis of the independent identification of two homozygous truncating mutations in three consanguineous families (one Arab and two Hutterite) affected by variable ciliopathy phenotypes ranging from Joubert syndrome to the more severe Meckel-Gruber syndrome with perinatal lethality and occipital encephalocele. Consistent with the recently described role of CSPP1 in ciliogenesis, we show that mutant fibroblasts from one affected individual have severely impaired ciliogenesis with concomitant defects in sonic hedgehog (SHH) signaling. Our results expand the list of centrosomal proteins implicated in human ciliopathies.

FORGE Canada Consortium: outcomes of a 2-year national rare-disease gene-discovery project.

Inherited monogenic disease has an enormous impact on the well-being of children and their families. Over half of the children living with one of these conditions are without a molecular diagnosis because of the rarity of the disease, the marked clinical heterogeneity, and the reality that there are thousands of rare diseases for which causative mutations have yet to be identified. It is in this context that in 2010 a Canadian consortium was formed to rapidly identify mutations causing a wide spectrum of pediatric-onset rare diseases by using whole-exome sequencing. The FORGE (Finding of Rare Disease Genes) Canada Consortium brought together clinicians and scientists from 21 genetics centers and three science and technology innovation centers from across Canada. From nation-wide requests for proposals, 264 disorders were selected for study from the 371 submitted; disease-causing variants (including in 67 genes not previously associated with human disease; 41 of these have been genetically or functionally validated, and 26 are currently under study) were identified for 146 disorders over a 2-year period. Here, we present our experience with four strategies employed for gene discovery and discuss FORGE's impact in a number of realms, from clinical diagnostics to the broadening of the phenotypic spectrum of many diseases to the biological insight gained into both disease states and normal human development. Lastly, on the basis of this experience, we discuss the way forward for rare-disease genetic discovery both in Canada and internationally.

Expansion of the clinical phenotype of the distal 10q26.3 deletion syndrome to include ataxia and hyperemia of the hands and feet.

Distal deletion of the long arm of chromosome 10 is associated with a dysmorphic craniofacial appearance, microcephaly, behavioral issues, developmental delay, intellectual disability, and ocular, urogenital, and limb abnormalities. Herein, we present clinical, molecular, and cytogenetic investigations of four patients, including two siblings, with nearly identical terminal deletions of 10q26.3, all of whom have an atypical presentation of this syndrome. Their prominent features include ataxia, mild-to-moderate intellectual disability, and hyperemia of the hands and feet, and they do not display many of the other features commonly associated with deletions of this region. These results point to a novel gene locus associated with ataxia and highlight the variability of the clinical presentation of patients with deletions of this region.

A novel multisystem disease associated with recessive mutations in the tyrosyl-tRNA synthetase (YARS) gene.

Aminoacyl-tRNA synthetases (ARSs) are a group of ubiquitously expressed enzymes that are best known for their function in the first step of protein translation but have been increasingly associated with secondary functions including transcription and translation control and extracellular signaling. Mutations in numerous ARSs have been linked to a growing number of both autosomal dominant and autosomal recessive human diseases. The tyrosyl-tRNA synthetase (YARS) links the amino acid tyrosine to its cognate tRNA. We report two siblings who presented with failure to thrive (FTT), hypertriglyceridemia, developmental delay, liver dysfunction, lung cysts, and abnormal subcortical white matter. Using exome sequencing the siblings were found to harbor bi-allelic pathogenic-appearing variants within the YARS gene (NM_003680.3):c.638C>T p.(Pro213Leu) and c.1573G>A p.(Gly525Arg). These YARS variants occur in the catalytic domain and the C-terminal domain, respectively. Mutations in YARS have been previously associated with an autosomal dominant form of Charcot-Marie-Tooth (CMT); our findings suggest the disease spectrum associated with YARS dysregulation is broader than peripheral neuropathy. © 2016 Wiley Periodicals, Inc.

Mutations in FLNC are Associated with Familial Restrictive Cardiomyopathy.

Individuals affected by restrictive cardiomyopathy (RCM) often develop heart failure at young ages resulting in early heart transplantation. Familial forms are mainly caused by mutations in sarcomere proteins and demonstrate a common genetic etiology with other inherited cardiomyopathies. Using next-generation sequencing, we identified two novel missense variants (p.S1624L; p.I2160F) in filamin-C (FLNC), an actin-cross-linking protein mainly expressed in heart and skeletal muscle, segregating in two families with autosomal-dominant RCM. Affected individuals presented with heart failure due to severe diastolic dysfunction requiring heart transplantation in some cases. Histopathology of heart tissue from patients of both families showed cytoplasmic inclusions suggesting protein aggregates, which were filamin-C specific for the p.S1624L by immunohistochemistry. Cytoplasmic aggregates were also observed in transfected myoblast cell lines expressing this mutant filamin-C indicating further evidence for its pathogenicity. Thus, FLNC is a disease gene for autosomal-dominant RCM and broadens the phenotype spectrum of filaminopathies.

Metaphyseal dysplasia with maxillary hypoplasia and brachydactyly is caused by a duplication in RUNX2.

Metaphyseal dysplasia with maxillary hypoplasia and brachydactyly (MDMHB) is an autosomal-dominant bone dysplasia characterized by metaphyseal flaring of long bones, enlargement of the medial halves of the clavicles, maxillary hypoplasia, variable brachydactyly, and dystrophic teeth. We performed genome-wide SNP genotyping in five affected and four unaffected members of an extended family with MDMHB. Analysis for copy-number variations revealed that a 105 kb duplication within RUNX2 segregated with the MDMHB phenotype in a region with maximum linkage. Real-time PCR for copy-number variation in genomic DNA in eight samples, as well as sequence analysis of fibroblast cDNA from one subject with MDMHB confirmed that affected family members were heterozygous for the presence of an intragenic duplication encompassing exons 3 to 5 of RUNX2. These three exons code for the Q/A domain and the functionally essential DNA-binding runt domain of RUNX2. Transfection studies with murine Runx2 cDNA showed that cellular levels of mutated RUNX2 were markedly higher than those of wild-type RUNX2, suggesting that the RUNX2 duplication found in individuals with MDMHB leads to a gain of function. Until now, only loss-of-function mutations have been detected in RUNX2; the present report associates an apparent gain-of-function alteration of RUNX2 function with a distinct rare disease.