Discovery of estrogen receptor α targeting caged hypoxia-responsive PROTACs with an inherent bicyclic skeleton for breast cancer treatment.

In view of the potential off-target effects of antitumor drugs, including proteolysis targeting chimera (PROTAC), certain toxic effects may be caused in normal tissues. Herein, based on the characteristics of the tumor microenvironment, we reported the first estrogen receptor α (ERα) targeting hypoxia-responsive PROTACs in order to improve their safety in breast cancer treatment by introducing two hypoxia-activated groups, nitroimidazole and nitrobenzene, into the ER ligand or E3 ligand of an active PROTAC, which has certain cytotoxicity in normal cells. Bioactivity studies showed that these hypoxia-responsive PROTACs exhibited excellent hypoxic responsiveness and ERα degradation activity under hypoxic conditions, and thus improved the toxic effects of the active PROTAC in normal cells. It is expected that our caged compounds provide a new strategy for precise functional control of PROTAC drugs for breast cancer treatment.

Prediction of microvascular invasion in hepatocellular carcinoma with expert-inspiration and skeleton sharing deep learning.

BACKGROUND AND AIMS: Radiological prediction of microvascular invasion (MVI) of hepatocellular carcinoma (HCC) is essential but few models were clinically implemented because of limited interpretability and generalizability. METHODS: Based on 2096 patients in three independent HCC cohorts, we established and validated an MVI predicting model. First, we used data from the primary cohort to train a 3D-ResNet network for MVI prediction and then optimised the model with "expert-inspired training" for model construction. Second, we implemented the model to the other two cohorts using three implementation strategies, the original model implementation, data sharing model implementation and skeleton sharing model implementation, the latter two of which used part of the cohorts' data for fine-tuning. The areas under the receiver operating characteristic curve (AUCs) were calculated to compare the performances of different models. RESULTS: For the MVI predicting model, the AUC of the expert-inspired model was 0.83 (95% CI: 0.77-0.88) compared to 0.54 (95% CI: 0.46-0.62) of model before expert-inspiring. Taking this model as an original model, AUC on the second cohort was 0.76 (95% CI: 0.67-0.84). The AUC was improved to 0.83 (95% CI: 0.77-0.90) with the data-sharing model, and further improved to 0.85 (95% CI: 0.79-0.92) with the skeleton sharing model. The trend that the skeleton sharing model had an advantage in performance was similar in the third cohort. CONCLUSIONS: We established an expert-inspired model with better predictive performance and interpretability than the traditional constructed model. Skeleton sharing process is superior to data sharing and direct model implementation in model implementation.

Congenital lipodystrophy induces severe osteosclerosis.

Berardinelli-Seip congenital generalized lipodystrophy is associated with increased bone mass suggesting that fat tissue regulates the skeleton. Because there is little mechanistic information regarding this issue, we generated "fat-free" (FF) mice completely lacking visible visceral, subcutaneous and brown fat. Due to robust osteoblastic activity, trabecular and cortical bone volume is markedly enhanced in these animals. FF mice, like Berardinelli-Seip patients, are diabetic but normalization of glucose tolerance and significant reduction in circulating insulin fails to alter their skeletal phenotype. Importantly, the skeletal phenotype of FF mice is completely rescued by transplantation of adipocyte precursors or white or brown fat depots, indicating that adipocyte derived products regulate bone mass. Confirming such is the case, transplantation of fat derived from adiponectin and leptin double knockout mice, unlike that obtained from their WT counterparts, fails to normalize FF bone. These observations suggest a paucity of leptin and adiponectin may contribute to the increased bone mass of Berardinelli-Seip patients.

An intrafamilial phenotypic variability in Ellis-Van Creveld syndrome due to a novel 27 bps deletion mutation.

Ellis-van Creveld (EvC) syndrome is an autosomal recessive disease, characterized by ectodermal, skeletal, and cardiac anomalies. We report intrafamilial phenotypic variability in three new EvC syndrome cases. Affected males in this study showed only ectodermal abnormalities, whereas an affected female showed the classical presentation of EvC Syndrome, including bilateral postaxial polydactyly of hands and feet, and congenital heart defects. Whole exome sequencing was performed to identify the causative variant, followed by validation and segregation analysis using Sanger sequencing. A homozygous deletion variant (c.731_757del) was identified in exon 6 of the EVC gene (NM_153717.2). The identified variant is considered to be the most likely candidate variant for the EvC syndrome in the family based on previous reports validating the role of EVC variants in the EvC syndrome. The disease correctly segregated in the family members, as all affected members were homozygous, and obligate carriers were heterozygous. Our family is remarkable in highlighting the variable expressivity of the EvC phenotype within the same family, due to a homozygous deletion mutation in the EVC gene. The variable expressivity might be due to the hypomorphic nature of mutation, or the presence of additional variants in modifier genes or in the regulatory regions of the EVC/EVC2 genes.

Clinical, radiological and computational studies on two novel GNPTG variants causing mucolipidosis III gamma phenotypes with varying severity.

Mucolipidosis III gamma (ML III γ) is a slowly progressive disorder that affects multiple parts of the body such as the skeleton, joints, and connective tissue structures. It is caused by pathogenic variants in the GNPTG gene that provides instructions for producing the γ subunit of GlcNAc-1-phosphotransferase. In this study we aim to characterize clinical findings and biological insights on two novel GNPTG variants causing ML III γ phenotypes with varying severity. We report on two siblings with ML III γ bearing the previously undescribed c.477C > G (p.Y159*) nonsense variant in a homozygous state as well as a patient with ML III γ bearing the novel c.110 + 19_111-17del variant in a homozygous state. These variants were revealed by whole-exome sequencing and Sanger sequencing, respectively. Their parents, who are heterozygotes for the same mutation, are healthy. The clinical and radiographic presentation of ML III γ in our patients who had c.477C > G (p.Y159*) variant is consistent with a relatively severe form of the disease, which is further supported by a working three-dimensional model of the GlcNAc-1-phosphotransferase γ subunit. On the other hand, it is seen that our patient who carries the c.110 + 19_111-17del variant has a milder phenotype. Our findings help broaden the spectrum of GNPTG variants causing ML III γ and offer structural and mechanistic insights into loss of GlcNAc-1-phosphotransferase γ subunit function.

[MRI of the axial skeleton for evaluation of prostate cancer extent in french Guyana]. / IRM du squelette axial pour le bilan du cancer de prostate en Guyane française.

INTRODUCTION: Spread evaluation of Prostate Cancer (PC) in French Guyana is bothered by the lack of bone scintigraphy. The availability of 4 MRI allows to develop alternatives using Axial Skeleton MRI (AS MRI). We report the related results. MATERIAL: AS MRI was done in patients with diagnosis of high risk PC: PSA>=10ng/ml and/or Gleason Score>=7 (predominant Gleason grade 4) and/or clinical T2b and/or T2b/T3 MRI and/or >50% positive biopsies. AS MRI was including spine, pelvis and skull assessement.The results were systematically compared to the clinical, biological and biopsy features. RESULTS: Amongst 163 AS MRI performed, 30 were positive and 133 negative. Of these 133 patients, 60 were submited to radical prostatectomy with or without lymphadenectomy. In these 133 patients with negative AS MRI, median PSA was 11ng/ml (1-51) and 27 (20,3%) had PSA>20ng/ml. In patients with positive AS MRI, only 1 had PSA<20ng/ml and predominant Gleason grade 3. CONCLUSION: Our study shows that AS MRI assessement is especially usefull in patients who are most likely to have bone metastasis that is to say those with PSA>20ng/ml and/or predominant Gleason grade 4. LEVEL OF EVIDENCE: 3.

Antxr1, Which is a Target of Runx2, Regulates Chondrocyte Proliferation and Apoptosis.

Antxr1/Tem8 is highly expressed in tumor endothelial cells and is a receptor for anthrax toxin. Mutation of Antxr1 causes GAPO syndrome, which is characterized by growth retardation, alopecia, pseudo-anodontia, and optic atrophy. However, the mechanism underlying the growth retardation remains to be clarified. Runx2 is essential for osteoblast differentiation and chondrocyte maturation and regulates chondrocyte proliferation through Ihh induction. In the search of Runx2 target genes in chondrocytes, we found that Antxr1 expression is upregulated by Runx2. Antxr1 was highly expressed in cartilaginous tissues and was directly regulated by Runx2. In skeletal development, the process of endochondral ossification proceeded similarly in wild-type and Antxr1-/- mice. However, the limbs of Antxr1-/- mice were shorter than those of wild-type mice from embryonic day 16.5 due to the reduced chondrocyte proliferation. Chondrocyte-specific Antxr1 transgenic mice exhibited shortened limbs, although the process of endochondral ossification proceeded as in wild-type mice. BrdU-uptake and apoptosis were both increased in chondrocytes, and the apoptosis-high regions were mineralized. These findings indicated that Antxr1, of which the expression is regulated by Runx2, plays an important role in chondrocyte proliferation and that overexpression of Antxr1 causes chondrocyte apoptosis accompanied by matrix mineralization.

An emerging ribosomopathy affecting the skeleton due to biallelic variations in NEPRO.

Cartilage hair hypoplasia (CHH), anauxetic dysplasia 1, and anauxetic dysplasia 2 are rare metaphyseal dysplasias caused by biallelic pathogenic variants in RMRP and POP1, which encode the components of RNAse-MRP endoribonuclease complex (RMRP) in ribosomal biogenesis pathway. Nucleolus and neural progenitor protein (NEPRO), encoded by NEPRO (C3orf17), is known to interact with multiple protein subunits of RMRP. We ascertained a 6-year-old girl with skeletal dysplasia and some features of CHH. RMRP and POP1 did not harbor any causative variant in the proband. Parents-child trio exomes revealed a candidate biallelic variant, c.435G>C, p.(Leu145Phe) in NEPRO. Two families with four affected individuals with skeletal dysplasia and a homozygous missense variant, c.280C>T, p.(Arg94Cys) in NEPRO, were identified from literature and their published phenotype was compared in detail to the phenotype of the child we described. All the five affected individuals have severe short stature, brachydactyly, skin laxity, joint hypermobility, and joint dislocations. They also have short metacarpals, broad middle phalanges, and metaphyseal irregularities. Protein modeling and stability prediction showed that the mutant protein has decreased stability. Both the reported variants are in the same domain of the protein. Our report delineates the clinical and radiological characteristics of an emerging ribosomopathy caused by biallelic variants in NEPRO.

Reconsidering osteoarthritis as a skeletal indicator of age at death.

OBJECTIVES: Osteoarthritis (OA) generally increases with age, but it is a complex, multifactorial disease. This study investigated whether obesity, physical activity, and antemortem trauma preclude the use of OA for skeletal age estimation. MATERIALS AND METHODS: The temporomandibular joint (TMJ) and all appendicular joints were scored for skeletal indicators of OA in 408 modern European-Americans (Bass Collection, TN). Binomial generalized linear models (GLMs) assessed the contributions of self-reported demographic data to OA, including: age; body mass index (BMI); and metabolic values for physical activities. Repeated resampling tested whether observed mean OA scores for joints with trauma consistently exceeded mean scores for unaffected joints. Single-variable GLM probit models were generated for OA presence/absence data in relevant joints. RESULTS: Age was the only statistically significant predictor of OA in most multivariable GLMs. Occupation and age were both significant predictors of male hand OA; BMI was the only significant predictor of female ankle OA. Trauma significantly affected OA in most joints. Age cut-offs calculated from the single-variable probit models (representing ages of transition to "OA present") ranged from 29.7 to 77.3 years (90%) and 32.7 to 96.6 years (95%), but were problematic for the male TMJ. DISCUSSION: Ankle OA should not be used to age females; TMJ OA should not be used to age males. For other joints, using OA to inform age estimates appears valid (in absence of antemortem trauma). While skeletal evidence of OA is not a primary age indicator, its presence can refine age ranges and provide essential age data in fragmentary cases.

Heads, Shoulders, Elbows, Knees, and Toes: Modular Gdf5 Enhancers Control Different Joints in the Vertebrate Skeleton.

Synovial joints are crucial for support and locomotion in vertebrates, and are the frequent site of serious skeletal defects and degenerative diseases in humans. Growth and differentiation factor 5 (Gdf5) is one of the earliest markers of joint formation, is required for normal joint development in both mice and humans, and has been genetically linked to risk of common osteoarthritis in Eurasian populations. Here, we systematically survey the mouse Gdf5 gene for regulatory elements controlling expression in synovial joints. We identify separate regions of the locus that control expression in axial tissues, in proximal versus distal joints in the limbs, and in remarkably specific sub-sets of composite joints like the elbow. Predicted transcription factor binding sites within Gdf5 regulatory enhancers are required for expression in particular joints. The multiple enhancers that control Gdf5 expression in different joints are distributed over a hundred kilobases of DNA, including regions both upstream and downstream of Gdf5 coding exons. Functional rescue tests in mice confirm that the large flanking regions are required to restore normal joint formation and patterning. Orthologs of these enhancers are located throughout the large genomic region previously associated with common osteoarthritis risk in humans. The large array of modular enhancers for Gdf5 provide a new foundation for studying the spatial specificity of joint patterning in vertebrates, as well as new candidates for regulatory regions that may also influence osteoarthritis risk in human populations.

Bone and bone marrow disruption by endocrine-active substances.

Bone is a multifaceted dynamic tissue, involved in mobility, mineral metabolism, and mesenchymal or stromal and hematopoietic progenitor or stem cells breading. Recently, an endocrine role has been attributed to bone due to its ability to produce at least two hormones (osteocalcin and fibroblast growth factor 23) and to participate directly or indirectly in leptin, insulin, estrogens, and serotonin signaling; regulation; and action. Also, bearing in mind the enormous amounts of substances secreted by the different bone marrow cell types, it becomes understandable the contribution of bone tissue to systemic homeostasis. Besides, bone is a well-known estrogen-responsive tissue, reacting to environmental influences. Thus, it has been coined as a critical target of environmental xenoestrogens, known as endocrine-disrupting chemicals (EDCs). The exposure to EDCs results to disruption or imbalance of the systemic hormonal regulation of the skeleton including bone modeling and remodeling, local hormones, and cytokine or chemokine release. The present report highlights the harmful EDCs effects on bone tissue and provides up-to-date information of xenoestrogen action on proliferation, maturation, and homing of bone marrow inhabitants.

IFT52 mutations destabilize anterograde complex assembly, disrupt ciliogenesis and result in short rib polydactyly syndrome.

The short-rib polydactyly syndromes (SRPS) encompass a radiographically and genetically heterogeneous group of skeletal ciliopathies that are characterized by a long narrow chest, short extremities, and variable occurrence of polydactyly. Radiographic abnormalities include undermineralization of the calvarium, shortened and bowed appendicular bones, trident shaped acetabula and polydactyly. In a case of SRPS we identified compound heterozygosity for mutations in IFT52, which encodes a component of the anterograde intraflagellar transport complex. The IFT52 mutant cells synthesized a significantly reduced amount of IFT52 protein, leading to reduced synthesis of IFT74, IFT81, IFT88 and ARL13B, other key anterograde complex members. Ciliogenesis was also disrupted in the mutant cells, with a 60% reduction in the presence of cilia on mutant cells and loss of cilia length regulation for the cells with cilia. These data demonstrate that IFT52 is essential for anterograde complex integrity and for the biosynthesis and maintenance of cilia. The data identify a new locus for SRPS and show that IFT52 mutations result in a ciliopathy with primary effects on the skeleton.

[The peculiar biochemical properties of the support structures in the human tissues]. / Osobennosti biomekhanicheskikh kachestv opornykh struktur tkanei cheloveka.

This article is focused on the methodological aspects of the evaluation of the elastic and mechanical strength characteristics of the tissues that form the flexible fibrous and osseous human skeleton. The samples of dura mater and spongy bone (ribs) were subjected to the quantitative analysis for the determination of their bioelemental composition. The study has demonstrated that the force needed to brake dura mater or a rib correlates with the content of biological elements in these tissue, as well as with the age and sex of the human subjects serving as teir sources.

SOFT syndrome caused by compound heterozygous mutations of POC1A and its skeletal manifestation.

SOFT syndrome (MIM614813) is an extremely rare primordial dwarfism characterized by short stature, onychodysplasia, facial dysmorphism and hypotrichosis, which is caused by biallelic mutations in the POC1A gene. Only 19 patients with mutation-confirmed SOFT syndrome have been reported to date, all of whom carried homozygous variants that were strongly associated with consanguineous marriages. We report an 8.5-year-old boy with SOFT syndrome showing primordial dwarfism, no effect of growth-hormone therapy and skeletal dysplasia. This is the first report of compound heterozygous variants in POC1A, one previously reported and the other novel. A characteristic skeletal manifestation is reported.

Tibial hemimelia associated with GLI3 truncation.

Tibial hemimelia is a rare, debilitating and often sporadic congenital deficiency. In syndromic cases, mutations of a Sonic hedgehog (SHH) enhancer have been identified. Here we describe an ~5 kb deletion within the SHH repressor GLI3 in two patients with bilateral tibial hemimelia. This deletion results in a truncated GLI3 protein that lacks a DNA-binding domain and cannot repress hedgehog signaling. These findings strengthen the concept that tibial hemimelia arises because of failure to restrict SHH activity to the posterior aspect of the limb bud.

Identification of a novel mutation confirms the implication of IFT172 (BBS20) in Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS; MIM 209900) is a recessive heterogeneous ciliopathy characterized by retinitis pigmentosa (RP), postaxial polydactyly, obesity, hypogonadism, cognitive impairment and kidney dysfunction. So far, 20 BBS genes have been identified, with the last reported ones being found in one or very few families. Whole-exome sequencing was performed in a consanguineous family in which two affected children presented typical BBS features (retinitis pigmentosa, postaxial polydactyly, obesity, hypogonadism and cognitive impairment) without any mutation identified in known BBS genes at the time of the study. We identified a homozygous splice-site mutation (NM_015662.2: c.4428+3A>G) in both affected siblings in the last reported BBS gene, namely, Intraflagellar Transport 172 Homolog (IFT172). Familial mutation segregation was consistent with autosomal recessive inheritance. IFT172 mutations were initially reported in Jeune and Mainzer-Saldino syndromes. Recently, mutations have also been found in isolated RP and Bardet-Biedl-like ciliopathy. This is the second report of IFT172 mutations in BBS patients validating IFT172 as the twentieth BBS gene (BBS20). Moreover, another IFT gene, IFT27, was already associated with BBS, confirming the implication of IFT genes in the pathogenesis of BBS.

Exome sequencing reveals two novel compound heterozygous XYLT1 mutations in a Polish patient with Desbuquois dysplasia type 2 and growth hormone deficiency.

Desbuquois dysplasia type 2 (DBQD2) is a rare recessively inherited skeletal genetic disorder characterized by severe prenatal and postnatal growth retardation, generalized joint laxity with dislocation of large joints and facial dysmorphism. The condition was recently described to result from autosomal recessive mutations in XYLT1, encoding the enzyme xylosyltransferase-1. In this paper, we report on a Polish patient with DBQD2 who presented with severe short stature of prenatal onset, joint laxity, psychomotor retardation and multiple radiological abnormalities including short metacarpals, advanced bone age and exaggerated trochanters. Endocrinological examinations revealed that sleep-induced growth hormone (GH) release and GH peak in clonidine- and glucagon-induced provocative tests as well as insulin-like growth factor 1 (IGF-1) and IGF-binding protein-3 levels were all markedly decreased, confirming deficiency of GH secretion. Bone age, unlikely to GH deficiency, was significantly advanced. To establish the diagnosis at a molecular level, we performed whole-exome sequencing and bioinformatic analysis in the index patient, which revealed compound heterozygous XYLT1 mutations: c.595C>T(p.Gln199*) and c.1651C>T(p.Arg551Cys), both of which are novel. Sanger sequencing showed that the former mutation was inherited from the healthy mother, whereas the latter one most probably occurred de novo. Our study describes the first case of DBQD2 resulting from compound heterozygous XYLT1 mutation, expands the mutational spectrum of the disease and provides evidence that the severe growth retardation and microsomia observed in DBQD2 patients may result not only from the skeletal dysplasia itself but also from GH and IGF-1 deficiency.

A relatively mild skeletal ciliopathy phenotype consistent with cranioectodermal dysplasia is associated with a homozygous nonsynonymous mutation in WDR35.

Ciliopathies are a class of clinically and genetically heterogeneous disorders characterized by deficits of the primary cilium, an important organelle for cellular signaling and development. Here we report on a patient from a consanguineous family presenting with renal cysts, short stature, distinctive facial features, missing teeth, brachydactyly, narrow chest, and abnormal ribs. His phenotype resembled a skeletal ciliopathy and the initial clinical differential diagnosis included Jeune thoracic dystrophy and cranioectodermal dysplasia. Due to the presence of parental consanguinity, a homozygous recessive mutation was the suspected cause and homozygosity mapping was used to direct candidate gene sequencing. WDR35, an intraflagellar transport protein previously associated with cranioectodermal dysplasia, the more severe short rib polydactyly syndrome type V and recently Ellis van Creveld syndrome, is present within a region of homozygosity and sequencing of all coding exons identified a novel homozygous nonsynonymous variant, p.Trp1153Cys. This variant affects a highly conserved tryptophan residue, is predicted to be deleterious, and is the most distal mutation yet reported in WDR35. This case expands the spectrum of phenotypes caused by WDR35 mutations, which we review herein.

Skeletal manifestations of tuberculosis in modern human remains.

Paleopathologists study the presence of diseases in the past and as such have a vast knowledge of skeletal changes associated with different conditions. Tuberculosis is one of the most studied diseases and still remains a major health problem today. Its manifestations in past populations have been extensively described, but less is known about its bony involvement in the post-antibiotic era. The aim of this study was to assess the frequency and manifestations of skeletal lesions in the post-antibiotic era in a South African sample and compare it to that found before the introduction of antibiotics. Skeletons of 205 individuals from modern skeletal collections and who are known to have died from TB were assessed. It was found that 39.2% of all individuals dying in the post-antibiotic era showed skeletal changes that could be associated with TB, while another 27.5% showed nonspecific changes. The highest incidences were found in individuals who died after 1985, when co-infection with HIV and drug resistance became common. While, as expected, vertebral and rib changes were the most common, the number of individuals who showed changes to the skull, and specifically intracranially, was surprising. These could most probably be associated with TB meningitis, although this specific cause of death was noted in only a few individuals. It seems that individuals may be living longer as a result of long-term antibiotic use, leaving more time for lesions to develop. Clin. Anat. 29:854-861, 2016. © 2016 Wiley Periodicals, Inc.