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BACKGROUND: Recent studies suggested that genetic variants associated with monogenic bone disorders were involved in the pathogenesis of atypical femoral fractures (AFF). Here, we aim to identify rare genetic variants by whole exome sequencing in genes involved in monogenic rare skeletal diseases in 12 women with AFF and 4 controls without any fracture. RESULTS: Out of 33 genetic variants identified in women with AFF, eleven (33.3%) were found in genes belonging to the Wnt pathway (LRP5, LRP6, DAAM2, WNT1, and WNT3A). One of them was rated as pathogenic (p.Pro582His in DAAM2), while all others were rated as variants of uncertain significance according to ClinVar and ACMG criteria. CONCLUSIONS: Osteoporosis, rare bone diseases, and AFFs may share the same genes, thus making it even more difficult to identify unique risk factors.
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Secuenciación del Exoma , Fracturas del Fémur , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Humanos , Femenino , Fracturas del Fémur/genética , Fracturas del Fémur/patología , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Persona de Mediana Edad , Anciano , Predisposición Genética a la Enfermedad , Proteína Wnt1/genética , Proteína Wnt3A/genética , Vía de Señalización Wnt/genética , Osteoporosis/genética , Osteoporosis/patología , Enfermedades Óseas/genética , Estudios de Casos y ControlesRESUMEN
BACKGROUND: The human lineage has undergone a postcranial skeleton gracilization (i.e. lower bone mass and strength relative to body size) compared to other primates and archaic populations such as the Neanderthals. This gracilization has been traditionally explained by differences in the mechanical load that our ancestors exercised. However, there is growing evidence that gracilization could also be genetically influenced. RESULTS: We have analyzed the LRP5 gene, which is known to be associated with high bone mineral density conditions, from an evolutionary and functional point of view. Taking advantage of the published genomes of archaic Homo populations, our results suggest that this gene has a complex evolutionary history both between archaic and living humans and within living human populations. In particular, we identified the presence of different selective pressures in archaics and extant modern humans, as well as evidence of positive selection in the African and South East Asian populations from the 1000 Genomes Project. Furthermore, we observed a very limited evidence of archaic introgression in this gene (only at three haplotypes of East Asian ancestry out of the 1000 Genomes), compatible with a general erasing of the fingerprint of archaic introgression due to functional differences in archaics compared to extant modern humans. In agreement with this hypothesis, we observed private mutations in the archaic genomes that we experimentally validated as putatively increasing bone mineral density. In particular, four of five archaic missense mutations affecting the first ß-propeller of LRP5 displayed enhanced Wnt pathway activation, of which two also displayed reduced negative regulation. CONCLUSIONS: In summary, these data suggest a genetic component contributing to the understanding of skeletal differences between extant modern humans and archaic Homo populations.
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Evolución Molecular , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad , Hombre de Neandertal , Humanos , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Animales , Hombre de Neandertal/genética , Selección Genética/genética , Hominidae/genética , Haplotipos/genética , Densidad Ósea/genética , Genoma Humano/genéticaRESUMEN
BACKGROUND: TRAF7-related cardiac, facial, and digital anomalies with developmental delay (CAFDADD), a multisystemic neurodevelopmental disorder caused by germline missense variants in the TRAF7 gene, exhibits heterogeneous clinical presentations. METHODS: We present a detailed description of 11 new TRAF7-related CAFDADD cases, featuring eight distinct variants, including a novel one. RESULTS: Phenotypic analysis and a comprehensive review of the 58 previously reported cases outline consistent clinical presentations, emphasizing dysmorphic features, developmental delay, endocrine manifestations, and cardiac defects. In this enlarged collection, novelties include a wider range of cognitive dysfunction, with some individuals exhibiting normal development despite early psychomotor delay. Communication challenges, particularly in expressive language, are prevalent, necessitating alternative communication methods. Autistic traits, notably rigidity, are observed in the cohort. Also, worth highlighting are hearing loss, sleep disturbances, and endocrine anomalies, including growth deficiency. Cardiac defects, frequently severe, pose early-life complications. Facial features, including arched eyebrows, contribute to the distinct gestalt. A novel missense variant, p.(Arg653Leu), further underscores the complex relationship between germline TRAF7 variants and somatic changes linked to meningiomas. CONCLUSIONS: Our comprehensive analysis expands the phenotypic spectrum, emphasizing the need for oncological evaluations and proposing an evidence-based schedule for clinical management. This study contributes to a better understanding of TRAF7-related CAFDADD, offering insights for improved diagnosis, intervention, and patient care.
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Discapacidades del Desarrollo , Cardiopatías Congénitas , Fenotipo , Humanos , Discapacidades del Desarrollo/genética , Masculino , Femenino , Niño , Preescolar , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/fisiopatología , Lactante , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/genética , Mutación Missense , AdolescenteRESUMEN
Schaaf-Yang syndrome (SYS) is an ultra-rare neurodevelopmental disorder caused by truncating mutations in MAGEL2 Heterologous expression of wild-type (WT) or a truncated (p.Gln638*) C-terminal HA-tagged MAGEL2 revealed a shift from a primarily cytoplasmic to a more nuclear localisation for the truncated protein variant. We now extend this analysis to six additional SYS mutations on a N-terminal FLAG-tagged MAGEL2. Our results replicate and extend our previous findings, showing that all the truncated MAGEL2 proteins consistently display a predominant nuclear localisation, irrespective of the C-terminal or N-terminal position and the chemistry of the tag. The variants associated with arthrogryposis multiplex congenita display a more pronounced nuclear retention phenotype, suggesting a correlation between clinical severity and the degree of nuclear mislocalisation. These results point to a neomorphic effect of truncated MAGEL2, which might contribute to the pathogenesis of SYS.
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Núcleo Celular , Proteínas de Neoplasias , Humanos , Núcleo Celular/metabolismo , Núcleo Celular/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Mutación , Fenotipo , Artrogriposis/genética , Artrogriposis/patología , Citoplasma/metabolismo , Citoplasma/genética , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/patología , Transporte de Proteínas , Células HEK293 , Hipopituitarismo , Facies , Péptidos y Proteínas de Señalización Intracelular , Proteínas Intrínsecamente Desordenadas , Trastornos de Impronta , Discapacidades del Desarrollo , Trastornos de los CromosomasRESUMEN
BACKGROUND: Schaaf-Yang syndrome (SYS) is caused by truncating mutations in MAGEL2, mapping to the Prader-Willi region (15q11-q13), with an observed phenotype partially overlapping that of Prader-Willi syndrome. MAGEL2 plays a role in retrograde transport and protein recycling regulation. Our aim is to contribute to the characterisation of SYS pathophysiology at clinical, genetic and molecular levels. METHODS: We performed an extensive phenotypic and mutational revision of previously reported patients with SYS. We analysed the secretion levels of amyloid-ß 1-40 peptide (Aß1-40) and performed targeted metabolomic and transcriptomic profiles in fibroblasts of patients with SYS (n=7) compared with controls (n=11). We also transfected cell lines with vectors encoding wild-type (WT) or mutated MAGEL2 to assess stability and subcellular localisation of the truncated protein. RESULTS: Functional studies show significantly decreased levels of secreted Aß1-40 and intracellular glutamine in SYS fibroblasts compared with WT. We also identified 132 differentially expressed genes, including non-coding RNAs (ncRNAs) such as HOTAIR, and many of them related to developmental processes and mitotic mechanisms. The truncated form of MAGEL2 displayed a stability similar to the WT but it was significantly switched to the nucleus, compared with a mainly cytoplasmic distribution of the WT MAGEL2. Based on the updated knowledge, we offer guidelines for the clinical management of patients with SYS. CONCLUSION: A truncated MAGEL2 protein is stable and localises mainly in the nucleus, where it might exert a pathogenic neomorphic effect. Aß1-40 secretion levels and HOTAIR mRNA levels might be promising biomarkers for SYS. Our findings may improve SYS understanding and clinical management.
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Síndrome de Prader-Willi , Humanos , Síndrome de Prader-Willi/genética , Fenotipo , Mutación , Proteínas/genética , BiomarcadoresRESUMEN
High bone mass (HBM) disorders are a clinically and genetically heterogeneous subgroup of rare skeletal dysplasias. Here we present a case of a previously unreported familial skeletal dysplasia characterized by HBM and lucent bone lesions that we aimed to clinically characterize and genetically investigate. For phenotyping, we reviewed past clinical records and imaging tests, and performed physical examination (PE), bone densitometry, and mineral panels in affected individuals, including a male proband, his son and daughter, in addition to unaffected controls, including the proband's wife and brother. Affected individuals also underwent impact microindentation (IMI). In an effort to elucidate the disorder's molecular etiology, whole exome sequencing (WES) was performed in all individuals to filter for rare variants present only in affected ones. The cases displayed a unique skeletal phenotype with a mix of sclerotic features and lucent bone lesions, and high IMI values. Bone mineral density was very elevated in the proband and his daughter. The proband's daughter also exhibited idiopathic scoliosis (IS), in addition to mild thrombocytopenia and mild structural thyroid abnormalities, which were the only extra-skeletal abnormalities identified. WES analysis yielded 5 rare putative pathogenic variants in affected members in genes that are associated with bone metabolism including: SEM4AD, TBX18, PTCH1, PTK7, and ADGRE5. The PTK7 variant appeared as possibly implicated in the development of IS while the TBX18 and SEMA4D variants stood out as the strongest candidates for the lucent bone lesions and HBM, respectively, given their high predicted pathogenicity and putative role in bone biology. Variant functionality should be addressed in the future to assess their implication in skeletal metabolism as it is the first time that mutations in TBX18 and SEMA4D have been associated to bone developmental lesions and mineral metabolism in a clinical setting.
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Enfermedades Óseas , Osteocondrodisplasias , Moléculas de Adhesión Celular , Humanos , Masculino , Mutación/genética , Linaje , Fenotipo , Proteínas Tirosina Quinasas Receptoras/genética , Secuenciación del ExomaRESUMEN
Osteoporosis is the most common bone disease, characterized by a low bone mineral density (BMD) and increased risk of fracture. At the other end of the BMD spectrum, some individuals present strong, fracture-resistant, bones. Both osteoporosis and high BMD are heritable and their genetic architecture encompasses polygenic inheritance of common variants and some cases of monogenic highly penetrant variants in causal genes. We have investigated the genetics of high BMD in a family segregating this trait in an apparently Mendelian dominant pattern. We searched for rare causal variants by whole-exome sequencing in three affected and three nonaffected family members. Using this approach, we have identified 38 rare coding variants present in the proband and absent in the three individuals with normal BMD. Although we have found four variants shared by the three affected members of the family, we have not been able to relate any of these to the high-BMD phenotype. In contrast, we have identified missense variants in two genes, VAV3 and ADGRE5, each shared by two of out of three affected members, whose loss of function fits with the phenotype of the family. In particular, the proband, a woman displaying the highest BMD (sum Z-score = 7), carries both variants, whereas the other two affected members carry one each. VAV3 encodes a guanine-nucleotide-exchange factor with an important role in osteoclast activation and function. Although no previous cases of VAV3 mutations have been reported in humans, Vav3 knockout (KO) mice display dense bones, similarly to the high-BMD phenotype present in our family. The ADGRE5 gene encodes an adhesion G protein-coupled receptor expressed in osteoclasts whose KO mouse displays increased trabecular bone volume. Combined, these mouse and human data highlight VAV3 and ADGRE5 as novel putative high-BMD genes with additive effects, and potential therapeutic targets for osteoporosis. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
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Collagen VI-related disorders are the second most common congenital muscular dystrophies for which no treatments are presently available. They are mostly caused by dominant-negative pathogenic variants in the genes encoding α chains of collagen VI, a heteromeric network forming collagen; for example, the c.877G>A; p.Gly293Arg COL6A1 variant, which alters the proper association of the tetramers to form microfibrils. We tested the potential of CRISPR/Cas9-based genome editing to silence or correct (using a donor template) a mutant allele in the dermal fibroblasts of four individuals bearing the c.877G>A pathogenic variant. Evaluation of gene-edited cells by next-generation sequencing revealed that correction of the mutant allele by homologous-directed repair occurred at a frequency lower than 1%. However, the presence of frameshift variants and others that provoked the silencing of the mutant allele were found in >40% of reads, with no effects on the wild-type allele. This was confirmed by droplet digital PCR with allele-specific probes, which revealed a reduction in the expression of the mutant allele. Finally, immunofluorescence analyses revealed a recovery in the collagen VI extracellular matrix. In summary, we demonstrate that CRISPR/Cas9 gene-edition can specifically reverse the pathogenic effects of a dominant negative variant in COL6A1.
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Sistemas CRISPR-Cas , Colágeno Tipo VI , Alelos , Sistemas CRISPR-Cas/genética , Colágeno Tipo VI/genética , Colágeno Tipo VI/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Humanos , MutaciónRESUMEN
Background: Chiari malformation type 1 (C1M) is a neurological disease characterized by herniation of the cerebellar tonsils below the foramen magnum. Cranial bone constriction is suspected to be its main cause. To date, genes related to bone development (e.g. DKK1 or COL1A2) have been associated with C1M, while some bone diseases (e.g. Paget) have been found to cosegregate with C1M. Nevertheless, the association between bone mineral density (BMD) and C1M has not been investigated, yet. Here, we systematically investigate the association between C1M and BMD, and between bone related genes and C1M. Methods: We have recruited a small cohort of C1M patients (12 unrelated patients) in whom we have performed targeted sequencing of an in-house bone-related gene panel and BMD determination through non-invasive DXA. Results: In the search for association between the bone related genes and C1M we have found variants in more than one C1M patient in WNT16, CRTAP, MYO7A and NOTCH2. These genes have been either associated with craniofacial development in different ways, or previously associated with C1M (MYO7A). Regarding the potential link between BMD and C1M, we have found three osteoporotic patients and one patient who had high BMD, very close to the HBM phenotype values, although most patients had normal BMD. Conclusions: Variants in bone related genes have been repeatedly found in some C1M cases. The relationship of bone genes with C1M deserves further study, to get a clearer estimate of their contribution to its etiology. No direct correlation between BMD and C1M was observed.
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The Wnt pathway is involved in several processes essential for bone development and homeostasis. For proper functioning, the Wnt pathway is tightly regulated by numerous extracellular elements that act by both activating and inhibiting the pathway at different moments. This review aims to describe, summarize and update the findings regarding the extracellular modulators of the Wnt pathway, including co-receptors, ligands and inhibitors, in relation to bone homeostasis, with an emphasis on the animal models generated, the diseases associated with each gene and the bone processes in which each member is involved. The precise knowledge of all these elements will help us to identify possible targets that can be used as a therapeutic target for the treatment of bone diseases such as osteoporosis.
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Enfermedades Óseas/patología , Huesos/fisiología , Homeostasis , Osteogénesis , Vía de Señalización Wnt , Animales , Enfermedades Óseas/metabolismo , Huesos/metabolismo , HumanosRESUMEN
Atypical femoral fractures (AFF) are rare fragility fractures in the subtrocantheric or diaphysis femoral region associated with long-term bisphosphonate (BP) treatment. The etiology of AFF is still unclear even though a genetic basis is suggested. We performed whole exome sequencing (WES) analysis of 12 patients receiving BPs for at least 5 years who sustained AFFs and 4 controls, also long-term treated with BPs but without any fracture. After filtration and prioritization of rare variants predicted to be damaging and present in genes shared among at least two patients, a total of 272 variants in 132 genes were identified. Twelve of these genes were known to be involved in bone metabolism and/or AFF, highlighting DAAM2 and LRP5, both involved in the Wnt pathway, as the most representative. Afterwards, we intersected all mutated genes with a list of 34 genes obtained from a previous study of three sisters with BP-related AFF, identifying nine genes. One of these (MEX3D) harbored damaging variants in two AFF patients from the present study and one shared among the three sisters. Gene interaction analysis using the AFFNET web suggested a complex network among bone-related genes as well as with other mutated genes. BinGO biological function analysis highlighted cytoskeleton and cilium organization. In conclusion, several genes and their interactions could provide genetic susceptibility to AFF, that along with BPs treatment and in some cases with glucocorticoids may trigger this so feared complication.
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Conservadores de la Densidad Ósea/efectos adversos , Difosfonatos/efectos adversos , Fracturas del Fémur/etiología , Fracturas del Fémur/patología , Regulación de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes , Osteoporosis Posmenopáusica/tratamiento farmacológico , Anciano , Estudios de Casos y Controles , Femenino , Fracturas del Fémur/genética , Perfilación de la Expresión Génica , Humanos , Osteoporosis Posmenopáusica/patologíaRESUMEN
Although the etiology of obsessive-compulsive disorder (OCD) is largely unknown, it is accepted that OCD is a complex disorder. There is a known bi-directional interaction between the gut microbiome and brain activity. Several authors have reported associations between changes in gut microbiota and neuropsychiatric disorders, including depression or autism. Furthermore, a pediatric-onset neuropsychiatric OCD-related syndrome occurs after streptococcal infection, which might indicate that exposure to certain microbes could be involved in OCD susceptibility. However, only one study has investigated the microbiome of OCD patients to date. We performed 16S ribosomal RNA gene-based metagenomic sequencing to analyze the stool and oropharyngeal microbiome composition of 32 OCD cases and 32 age and gender matched controls. We estimated different α- and ß-diversity measures and performed LEfSe and Wilcoxon tests to assess differences in bacterial distribution. OCD stool samples showed a trend towards lower bacterial α-diversity, as well as an increase of the relative abundance of Rikenellaceae, particularly of the genus Alistipes, and lower relative abundance of Prevotellaceae, and two genera within the Lachnospiraceae: Agathobacer and Coprococcus. However, we did not observe a different Bacteroidetes to Firmicutes ratio between OCD cases and controls. Analysis of the oropharyngeal microbiome composition showed a lower Fusobacteria to Actinobacteria ratio in OCD cases. In conclusion, we observed an imbalance in the gut and oropharyngeal microbiomes of OCD cases, including, in stool, an increase of bacteria from the Rikenellaceae family, associated with gut inflammation, and a decrease of bacteria from the Coprococcus genus, associated with DOPAC synthesis.
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Eje Cerebro-Intestino/inmunología , Microbioma Gastrointestinal/inmunología , Trastorno Obsesivo Compulsivo/microbiología , Orofaringe/microbiología , Adulto , Estudios de Casos y Controles , ADN Bacteriano/aislamiento & purificación , Heces/microbiología , Femenino , Microbioma Gastrointestinal/genética , Voluntarios Sanos , Humanos , Masculino , Metagenoma , Metagenómica , Persona de Mediana Edad , Trastorno Obsesivo Compulsivo/inmunología , ARN Ribosómico 16S/genéticaRESUMEN
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.
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Estudio de Asociación del Genoma Completo/métodos , Enfermedades Musculoesqueléticas/genética , Animales , Animales Modificados Genéticamente , Enfermedades Óseas/genética , Enfermedades Óseas/metabolismo , Enfermedades Óseas/patología , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo/tendencias , Humanos , Modelos Animales , Herencia Multifactorial/genética , Enfermedades Musculoesqueléticas/metabolismo , Enfermedades Musculoesqueléticas/patología , Fenotipo , Sitios de Carácter Cuantitativo , Integración de Sistemas , Estudios de Validación como AsuntoRESUMEN
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.
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Densidad Ósea , Enfermedades Óseas/genética , Enfermedades Óseas/patología , Genes , Mutación , Animales , Enfermedades Óseas/terapia , Estudio de Asociación del Genoma Completo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , FenotipoRESUMEN
The transition from a transcriptionally active state (GV) to a transcriptionally inactive state (mature MII oocytes) is required for the acquisition of oocyte developmental competence. We hypothesize that the expression of specific genes at the in vivo matured (MII) stage could be modulated by posttranscriptional mechanisms, particularly regulation of alternative splicing (AS). In this study, we examined the transcriptional activity of GV oocytes after ovarian stimulation followed by oocyte pick-up and the landscape of alternatively spliced isoforms in human MII oocytes. Individual oocytes were processed and analyzed for transcriptional activity (GV), gene expression (GV and MII), and AS signatures (GV and MII) on HTA 2.0 microarrays. Samples were grouped according to maturation stage, and then subgrouped according to women's age and antral follicular count (AFC); array results were validated by quantitative polymerase chain reaction. Differentially expressed genes between GV and MII oocytes clustered mainly in biological processes related to mitochondrial metabolism. Interestingly, 16 genes that were related to the regulation of transcription and mitochondrial translation showed differences in alternatively spliced isoform profiles despite not being differentially expressed between groups. Altogether, our results contribute to our understanding of the role of AS in oocyte developmental competence acquisition.
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Oocitos , Oogénesis , Femenino , Humanos , Mitocondrias/fisiología , Oocitos/metabolismo , Oogénesis/genética , Inducción de la Ovulación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
Osteoporosis is the most common metabolic bone disorder and nitrogen-containing bisphosphonates (BP) are a first line treatment for it. Yet, atypical femoral fractures (AFF), a rare adverse effect, may appear after prolonged BP administration. Given the low incidence of AFF, an underlying genetic cause that increases the susceptibility to these fractures is suspected. Previous studies uncovered rare CYP1A1 mutations in osteoporosis patients who suffered AFF after long-term BP treatment. CYP1A1 is involved in drug metabolism and steroid catabolism, making it an interesting candidate. However, a functional validation for the AFF-associated CYP1A1 mutations was lacking. Here we tested the enzymatic activity of four such CYP1A1 variants, by transfecting them into Saos-2 cells. We also tested the effect of commonly used BPs on the enzymatic activity of the CYP1A1 forms. We demonstrated that the p.Arg98Trp and p.Arg136His CYP1A1 variants have a significant negative effect on enzymatic activity. Moreover, all the BP treatments decreased CYP1A1 activity, although no specific interaction with CYP1A1 variants was found. Our results provide functional support to the hypothesis that an additive effect between CYP1A1 heterozygous mutations p.Arg98Trp and p.Arg136His, other rare mutations and long-term BP exposure might generate susceptibility to AFF.
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Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Fracturas del Fémur/genética , Fracturas del Fémur/metabolismo , Secuencia de Aminoácidos , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/genética , Citocromo P-450 CYP1A1/química , Difosfonatos/uso terapéutico , Fracturas del Fémur/enzimología , Humanos , Incidencia , Mutagénesis Sitio-Dirigida , Mutación Missense , Filogenia , Alineación de SecuenciaRESUMEN
We present a Turkish family with two cousins (OC15 and OC15b) affected with syndromic developmental delay, microcephaly, and trigonocephaly but with some phenotypic traits distinct between them. OC15 showed asymmetrical skeletal defects and syndactyly, while OC15b presented with a more severe microcephaly and semilobal holoprosencephaly. All four progenitors were related and OC15 parents were consanguineous. Whole Exome Sequencing (WES) analysis was performed on patient OC15 as a singleton and on the OC15b trio. Selected variants were validated by Sanger sequencing. We did not identify any shared variant that could be associated with the disease. Instead, each patient presented a de novo heterozygous variant in a different gene. OC15 carried a nonsense mutation (p.Arg95*) in PORCN, which is a gene responsible for Goltz-Gorlin syndrome, while OC15b carried an indel mutation in ZIC2 leading to the substitution of three residues by a proline (p.His404_Ser406delinsPro). Autosomal dominant mutations in ZIC2 have been associated with holoprosencephaly 5. Both variants are absent in the general population and are predicted to be pathogenic. These two de novo heterozygous variants identified in the two patients seem to explain the major phenotypic alterations of each particular case, instead of a homozygous variant that would be expected by the underlying consanguinity.
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Aciltransferasas/genética , Consanguinidad , Proteínas de la Membrana/genética , Mutación , Proteínas Nucleares/genética , Factores de Transcripción/genética , Niño , Preescolar , Análisis Mutacional de ADN , Facies , Femenino , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Masculino , Linaje , Fenotipo , Radiografía , TurquíaRESUMEN
SOST encodes the sclerostin protein, which acts as a key extracellular inhibitor of the canonical Wnt pathway in bone, playing a crucial role in skeletal development and bone homeostasis. The objective of this work was to assess the functionality of two variants previously identified (the rare variant rs570754792 and the missense variant p.Val10Ile) and to investigate the physical interactors of the SOST proximal promoter region in bone cells. Through a promoter luciferase reporter assay we show that the minor allele of rs570754792, a variant located in the extended TATA box motif, displays a significant decrease in promoter activity. Likewise, through western blot studies of extracellular and intracellular sclerostin, we observe a reduced expression of the p.Val10Ile mutant protein. Finally, using a circular chromosome conformation capture assay (4C-seq) in 3 bone cell types (MSC, hFOB, Saos-2), we have detected physical interactions between the SOST proximal promoter and the ECR5 enhancer, several additional enhancers located between EVT4 and MEOX1 and a distant region containing exon 18 of DHX8. In conclusion, SOST presents functional regulatory and missense variants that affect its expression and displays physical contacts with far reaching genomic sequences, which may play a role in its regulation within bone cells.
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Proteínas Adaptadoras Transductoras de Señales/genética , Genómica , Alelos , Línea Celular , Elementos de Facilitación Genéticos , Genes Reporteros , Humanos , Mutación Missense , Osteoblastos/citología , Osteoblastos/metabolismo , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , TATA Box/genéticaRESUMEN
The DKK1 gene encodes an extracellular inhibitor of the Wnt pathway with an important role in bone tissue development, bone homeostasis, and different critical aspects of bone biology. Several BMD genome-wide association studies (GWASs) have consistently found association with SNPs in the DKK1 genomic region. For these reasons, it is important to assess the functionality of coding and regulatory variants in the gene. Here, we have studied the functionality of putative regulatory variants, previously found associated with BMD in different studies by others and ourselves, and also six missense variants present in the general population. Using a Wnt-pathway-specific luciferase reporter assay, we have determined that the variants p.Ala41Thr, p.Tyr74Phe, p.Arg120Leu, and p.Ser157Ile display a reduced DKK1 inhibitory capacity as compared with WT. This result agrees with the high-bone-mass (HBM) phenotype of two women from our cohort who carried mutations p.Tyr74Phe or p.Arg120Leu. On the other hand, by means of a circularized chromosome conformation capture- (4C-) sequencing experiment, we have detected that the region containing 24 BMD-GWA variants, located 350-kb downstream of DKK1, interacts both with DKK1 and the LNCAROD (LncRNA-activating regulator of DKK1, AKA LINC0148) in osteoblastic cells. In conclusion, we have shown that some rare coding variants are partial loss-of-function mutations that may lead to a HBM phenotype, whereas the common SNPs associated with BMD in GWASs belong to a putative long-range regulatory region, through a yet unknown mechanism involving LNCAROD. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
RESUMEN
BACKGROUND: Pathogenic variants of the lysine acetyltransferase 6A or KAT6A gene are associated with a newly identified neurodevelopmental disorder characterized mainly by intellectual disability of variable severity and speech delay, hypotonia, and heart and eye malformations. Although loss of function (LoF) mutations were initially reported as causing this disorder, missense mutations, to date always involving serine residues, have recently been associated with a form of the disorder without cardiac involvement. RESULTS: In this study we present five new patients, four with truncating mutations and one with a missense change and the only one not presenting with cardiac anomalies. The missense change [p.(Gly359Ser)], also predicted to affect splicing by in silico tools, was functionally tested in the patient's lymphocyte RNA revealing a splicing effect for this allele that would lead to a frameshift and premature truncation. CONCLUSIONS: An extensive revision of the clinical features of these five patients revealed high concordance with the 80 cases previously reported, including developmental delay with speech delay, feeding difficulties, hypotonia, a high bulbous nose, and recurrent infections. Other features present in some of these five patients, such as cryptorchidism in males, syndactyly, and trigonocephaly, expand the clinical spectrum of this syndrome.