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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., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Genome-wide association studies (GWAS) have repeatedly identified genetic variants associated with bone mineral density (BMD) and osteoporotic fracture in non-coding regions of C7ORF76, a poorly studied gene of unknown function. The aim of the present study was to elucidate the causality and molecular mechanisms underlying the association. We re-sequenced the genomic region in two extreme BMD groups from the BARCOS cohort of postmenopausal women to search for functionally relevant variants. Eight selected variants were tested for association in the complete cohort and 2 of them (rs4342521 and rs10085588) were found significantly associated with lumbar spine BMD and nominally associated with osteoporotic fracture. cis-eQTL analyses of these 2 SNPs, together with SNP rs4727338 (GWAS lead SNP in Estrada et al., Nat Genet. 44:491-501, 2012), performed in human primary osteoblasts, disclosed a statistically significant influence on the expression of the proximal neighbouring gene SLC25A13 and a tendency on the distal SHFM1. We then studied the functionality of a putative upstream regulatory element (UPE), containing rs10085588. Luciferase reporter assays showed transactivation capability with a strong allele-dependent effect. Finally, 4C-seq experiments in osteoblastic cell lines showed that the UPE interacted with different tissue-specific enhancers and a lncRNA (LOC100506136) in the region. In summary, this study is the first one to analyse in depth the functionality of C7ORF76 genomic region. We provide functional regulatory evidence for the rs10085588, which may be a causal SNP within the 7q21.3 GWAS signal for osteoporosis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Osteoporosis is a common disease of bone possessing a strong genetic component. Cytochrome P450 aromatase, which is encoded by the CYP19A1 gene, converts androgens to estradiol. Considerable evidence suggests that extragonadal estrogens play an important role in determining bone mineral density (BMD) in postmenopausal women, and, among them, those synthesized in bone cells may also be important for the determination of bone phenotype. Therefore, CYP19A1 is an excellent candidate gene for osteoporosis. Since a region upstream of exon I.3, including exon I.6, was identified as containing repressor elements of promoter pII, we conducted a search for SNPs in this region of CYP19A1. Two SNPs [Aro1(rs4775936) and Aro2] located in exon I.6 and promoter I.6, respectively, were identified and their association with BMD analyzed in a cohort of 256 Spanish postmenopausal women. Aro1(rs4775936), but not Aro2, was associated with lumbar spine BMD (P = 0.029). Homozygotes AA (16% of the women) exhibited significantly higher lumbar spine BMD, compared with GG or GA individuals. Therefore, this study describes the Aro1 polymorphism which lies within a regulatory region and which may be a functional polymorphism, partially responsible for the bone phenotype it is associated with.

Osteoporosis is a common disease with a strong genetic component. We previously described two polymorphic sites in the COL1A1 gene promoter, -1997 G/T and -1663indelT, which have been associated with bone mineral density (BMD), a surrogate trait for osteoporosis. Here, we explore the molecular mechanisms underlying this association by performing transient transfections in MG-63 cells of constructs bearing different COL1A1 promoter regions, containing different alleles or haplotypes of the polymorphic sites. These promoter regions drove the transcription of a luciferase reporter gene. The main differences in transcriptional activity relied on an inhibitory region localized to the -1284 to -254 interval. Regarding the polymorphisms, reproducible differences were observed between the alleles of each of them: the G allele at -1997 showed a higher transcriptional activity than the T allele, as did the 7T allele of -1663 as compared with 8T. Accordingly, the T-8T haplotype was the weakest transcriber. A functional interaction was found between the -1997 and -1663 polymorphisms, in that the difference in transcriptional activity between the 7T and 8T alleles was dependent on the allele at -1997. This different transcriptional activity of the two -1663indelT alleles correlated with different binding capacities of the corresponding oligonucleotides to osteoblast nuclear proteins. Supershift assays allowed us to identify one of these proteins as the architectural transcription factor Nmp4/CIZ, a protein known to be an inhibitor of BMP/Smad signalling.