Mouse inbred strains with inherited predisposition and resistance to lung cancer provide an essential tool for the dissection of the genetics of this complex disease. We have previously mapped a major locus (Pulmonary adenoma susceptibility 1, Pas1) affecting inherited predisposition to lung cancer in mice on chromosome 6, near Kras2. Appropriate crosses that include susceptible mice (Pas1(s)) provide a model system for identifying loci that can modify the lung cancer predisposition phenotype caused by Pas1. Using this approach we have mapped the Pulmonary adenoma resistance 1 (Par1) locus that behaves like a modifier locus of Pas1. More recently, we mapped additional lung tumor resistance loci (Par2, and Par4), and a locus specifically involved with lung tumor progression (Papg1). The mapping of Pas1 in mice stimulated us to test the possible association of genetic markers located in the homologous human region (12p12) with risk and prognosis of lung adenocarcinomas in man. In the Italian population, we carried out an association study by genotyping lung adenocarcinoma patients and healthy controls for genetic markers located in the putative region of interest. Homozygosity of the A2 allele at a Kras2/RsaI polymorphism, and allele 2 at a VNTR polymorphism in the PTHLH gene showed borderline statistically significant associations with lung cancer risk. Furthermore, the same alleles were significantly associated with tumor prognosis. Studies on association were then performed in the Japanese and in European populations. In the Japanese population, the KRAS2/RsaI marker was significantly associated with prognosis of lung adenocarcinoma, whereas the European study did not confirm this association. Our results may provide evidence for the existence of the human PAS1 locus, suggesting that the mouse model of inherited predisposition to lung tumorigenesis is predictive of a human genetic mechanism of susceptibility to lung cancer.

BACKGROUND

Pseudohypoparathyroidism (PHP) is a genetic disorder characterized by resistance to the peripheral action of PTH due to maternally inherited heterozygous inactivating mutations in the coding sequence of Gsα or intronic regions of GNAS leading to aberrant splice variants (PHP1A), or methylation defects at GNAS (PHP1B). Brachydactyly is a clinical feature associated with both PHP1A and PHP1B, although it is more frequent in PHP1A patients. Loss-of-function mutations in PTHLH, the gene coding for parathyroid hormone related protein (PTHrP) were previously described in some patients with brachydactyly. Primary failure of tooth eruption (PFE) is related to some syndromes involving skeletal development, but it is also known as a nonsyndromic autosomal dominant condition. Previous studies showed that familial nonsyndromic PFE is caused by heterozygous mutations in the gene encoding the G protein-coupled receptor (PTH1R) for PTH and PTHrP. Thus, we hypothesized that PTHrP resistance could result in failure of tooth eruption (FTE) and/or brachydactyly in PHP.

METHODS

Nineteen patients with a molecular diagnosis of PHP underwent dental panoramic radiography (DPR), hand radiography and had their PTHrP levels measured. Patients with alterations at DPR were submitted to clinical dental evaluation.

RESULTS

Nine patients had FTE and 7 patients had brachydactyly; 4 patients presented both features and none of them presented high PTHrP levels. Fourteen patients had PTHrP levels within the normal range and only one patient had slightly elevated PTHrP levels. Additionally, three novel GNAS mutations were described.

CONCLUSIONS

We described the dental abnormalities in a large series of PHP patients that were followed in a single tertiary center. No relationship between plasma PTHrP levels and failure of tooth eruption, dental manifestations of PHP or brachydactyly was found. It is important that doctors pay attention to dental manifestations of the disease in order to refer patients to a proper care with dentists.

Purpose: Parathyroid hormone-related protein (PTHrP) is required for normal mammary gland development and biology. A PTHLH gene polymorphism is associated with breast cancer risk, and PTHrP promotes growth of osteolytic breast cancer bone metastases. Accordingly, current dogma holds that PTHrP is upregulated in malignant primary breast tumors, but solid evidence for this assumption is missing.Experimental Design: We used quantitative IHC to measure PTHrP in normal and malignant breast epithelia, and correlated PTHrP levels in primary breast cancer with clinical outcome.Results: PTHrP levels were markedly downregulated in malignant compared with normal breast epithelia. Moreover, low levels of nuclear localized PTHrP in cancer cells correlated with unfavorable clinical outcome in a test and a validation cohort of breast cancer treated at different institutions totaling nearly 800 cases. PTHrP mRNA levels in tumors of a third cohort of 737 patients corroborated this association, also after multivariable adjustment for standard clinicopathologic parameters. Breast cancer PTHrP levels correlated strongly with transcription factors Stat5a/b, which are established markers of favorable prognosis and key mediators of prolactin signaling. Prolactin stimulated PTHrP transcript and protein in breast cancer cell lines in vitro and in vivo, effects mediated by Stat5 through the P2 gene promoter, producing transcript AT6 encoding the PTHrP 1-173 isoform. Low levels of AT6, but not two alternative transcripts, correlated with poor clinical outcome.Conclusions: This study overturns the prevailing view that PTHrP is upregulated in primary breast cancers and identifies a direct prolactin-Stat5-PTHrP axis that is progressively lost in more aggressive tumors. Clin Cancer Res; 1-12. ©2018 AACR.

Osteoporosis is a bone disease that commonly results in a 30% incidence of fracture in hens used to produce eggs for human consumption. One of the causes of osteoporosis is the lack of mechanical strain placed on weight-bearing bones. In conventionally-caged hens, there is inadequate space for chickens to exercise and induce mechanical strain on their bones. One approach is to encourage mechanical stress on bones by the addition of perches to conventional cages. Our study focuses on the molecular mechanism of bone remodeling in end-of-lay hens (71 weeks) with access to perches. We examined bone-specific transcripts that are actively involved during development and remodeling. Using real-time quantitative PCR, we examined seven transcripts (COL2A1 (collagen, type II, alpha 1), RANKL (receptor activator of nuclear factor kappa-B ligand), OPG (osteoprotegerin), PTHLH (PTH-like hormone), PTH1R (PTH/PTHLH type-1 receptor), PTH3R (PTH/PTHLH type-3 receptor), and SOX9 (Sry-related high mobility group box)) in phalange, tibia and femur. Our results indicate that the only significant effect was a difference among bones for COL2A1 (femur>> phalange). Therefore, we conclude that access to a perch did not alter transcript expression. Furthermore, because hens have been used as a model for human bone metabolism and osteoporosis, the results indicate that bone remodeling due to mechanical loading in chickens may be a product of different pathways than those involved in the mammalian model.

Parathyroid hormone like hormone (PTHLH) signaling is essential for the proper formation of bone and its elevation or disruption has been directly implicated in several different skeletal dysplasias. We report a patient with a 2.802 Mb deletion upstream of the PTHLH coding sequence who presents with multiple fractures, metaphyseal changes, and overall features consistent with hyperparathyroid like disease. Analysis of the deleted region revealed the loss of putative regulatory regions adjacent to PTHLH and the possible gain of a limb enhancer. Furthermore, PTHLH expression appeared to be mis-regulated in fibroblasts derived from the patient. Altogether, we find that the disruption of the regulatory landscape of PTHLH likely results in its inappropriate expression and this novel clinical presentation.

In this work, we propose for the first time the use of anodic aluminum oxide (AAO) nanoporous membranes for in situ monitoring of parathyroid hormone-like hormone (PTHLH) secretion in cultured human cells. The biosensing system is based on the nanochannels blockage upon immunocomplex formation, which is electrically monitored through the voltammetric oxidation of Prussian blue nanoparticles (PBNPs). Models evaluated include a neuroblastoma cell line (SK-N-AS) and immortalized keratinocytes (HaCaT) as a control of high PTHLH production. The effect of total number of seeded cells and incubation time on the secreted PTHLH levels is assessed, finding that secreted PTHLH levels range from approximately 60 to 400 ng/mL. Moreover, our methodology is also applied to analyse PTHLH production following PTHLH gene knockdown upon transient cell transfection with a specific silencing RNA (siRNA). Given that inhibition of PTHLH secretion reduces cell proliferation, survival and invasiveness in a number of tumors, our system provides a powerful tool for the preclinical evaluation of therapies that regulate PTHLH production. This nanoporous membrane - based sensing technology might be useful to monitor the active secretion of other proteins as well, thus contributing to characterize their regulation and function.

The aim of the present study was to identify the differentially expressed genes (DEGs) that are induced by the silencing of transforming growth factor-β-activated kinase 1 (TAK1) in bladder cancer cells and to analyze the potential biological effects. Dataset GSE52452 from mutant fibroblast growth factor receptor 3 (FGFR3) bladder cancer cells transfected with control siRNA or TAK1-specific siRNA was downloaded from Gene Expression Omnibus. The DEGs between the two groups were identified using Limma package following data pre-processing by Affy in Bioconductor. Enrichment analysis of DEGs was performed using the Database for Annotation, Visualization and Integrated Discovery, followed by functional annotation using TRANSFAC, TSGene and TAG databases. Integrated networks were constructed by Cytoscape and sub-networks were extracted employing BioNet, followed by enrichment analysis of DEGs in the sub-network. A total of 43 downregulated and 21 upregulated genes were obtained. The downregulated genes were enriched in five pathways, including NOD-like receptor signaling pathway and functions related to cellular response. The upregulated genes were associated with cellular developmental processes. Transcription factor EGR1 and 9 tumor-associated genes were screened from the DEGs. Among the DEGs, 10 hub nodes may represent important roles in the complex metabolic network, including EGFR, CYP3A5, MAP3K7, GSTA1, PTHLH, ALDH1A1, KCND2, EGR1, ARRB1 and ITPR1. Additionally, EGFR was correlated with ERBB2, GRB2 and PIK3R1, and these were enriched in ErbB signaling pathway and various cancer-associated pathways. Silencing TAK1 may decrease cellular response to chemical stimulus via downregulating CYP3A5, MAP3K7, GSTA1, ALDH1A1, ARRB1 and ITPR1; increase cancer cell development via upregulating EGFR, EGR1 and PTHLH; and regulate cancer metastasis through EGFR, ERBB2, GRB2 and PIK3R1.

Exercise benefits the musculoskeletal system and reduces the effects of cancer. The effects of exercise are multifactorial, where metabolic changes and tissue adaptation influence outcomes. Mechanical signals, a principal component of exercise, are anabolic to the musculoskeletal system and restrict cancer progression. We examined the mechanisms through which cancer cells sense and respond to low-magnitude mechanical signals introduced in the form of vibration. Low-magnitude, high-frequency vibration was applied to human breast cancer cells in the form of low-intensity vibration (LIV). LIV decreased matrix invasion and impaired secretion of osteolytic factors PTHLH, IL-11, and RANKL. Furthermore, paracrine signals from mechanically stimulated cancer cells, reduced osteoclast differentiation and resorptive capacity. Disconnecting the nucleus by knockdown of SUN1 and SUN2 impaired LIV-mediated suppression of invasion and osteolytic factor secretion. LIV increased cell stiffness; an effect dependent on the LINC complex. These data show that mechanical vibration reduces the metastatic potential of human breast cancer cells, where the nucleus serves as a mechanosensory apparatus to alter cell structure and intercellular signaling.

Identifying the signals of artificial selection can contribute to further shaping economically important traits. Here, a chicken 600k SNP-array was employed to detect the signals of artificial selection using 331 individuals from 9 breeds, including Jingfen (JF), Jinghong (JH), Araucanas (AR), White Leghorn (WL), Pekin-Bantam (PB), Shamo (SH), Gallus-Gallus-Spadiceus (GA), Rheinlander (RH) and Vorwerkhuhn (VO). Per the population genetic structure, 9 breeds were combined into 5 breed-pools, and a 'two-step' strategy was used to reveal the signals of artificial selection. GA, which has little artificial selection, was defined as the reference population, and a total of 204, 155, 305 and 323 potential artificial selection signals were identified in AR_VO, PB, RH_WL and JH_JF, respectively. We also found signals derived from standing and de-novo genetic variations have contributed to adaptive evolution during artificial selection. Further enrichment analysis suggests that the genomic regions of artificial selection signals harbour genes, including THSR, PTHLH and PMCH, responsible for economic traits, such as fertility, growth and immunization. Overall, this study found a series of genes that contribute to the improvement of chicken breeds and revealed the genetic mechanisms of adaptive evolution, which can be used as fundamental information in future chicken functional genomics study.

Esophageal squamous cell carcinoma (ESCC) is the predominant subtype of esophageal cancer with a particularly high prevalence in certain geographical regions and a poor prognosis with a 5-year survival rate of 15-25%. Despite numerous studies characterizing the genetic and transcriptomic landscape of ESCC, there are currently no effective targeted therapies. In this study, we used an unbiased screening approach to uncover novel molecular precision oncology targets for ESCC and identified the bromodomain and extraterminal (BET) family member bromodomain testis-specific protein (BRDT) to be uniquely expressed in a subgroup of ESCC. Experimental studies revealed that BRDT expression promotes migration but is dispensable for cell proliferation. Further mechanistic insight was gained through transcriptome analyses, which revealed that BRDT controls the expression of a subset of ΔNp63 target genes. Epigenome and genome-wide occupancy studies, combined with genome-wide chromatin interaction studies, revealed that BRDT colocalizes and interacts with ΔNp63 to drive a unique transcriptional program and modulate cell phenotype. Our data demonstrate that these genomic regions are enriched for super-enhancers that loop to critical ΔNp63 target genes related to the squamous phenotype such as KRT14, FAT2, and PTHLH. Interestingly, BET proteolysis-targeting chimera, MZ1, reversed the activation of these genes. Importantly, we observed a preferential degradation of BRDT by MZ1 compared with BRD2, BRD3, and BRD4. Taken together, these findings reveal a previously unknown function of BRDT in ESCC and provide a proof-of-concept that BRDT may represent a novel therapeutic target in cancer.

Background: Oral leukoplakia is the most common oral mucosal disease. A proportion of such cases can progress to oral squamous cell carcinoma (OSCC). The mechanism of oral leukoplakia malignant transformation is still unclear. In this study, we analyzed the expression of parathyroid hormone-like hormone (PTHLH) in oral leukoplakia and the effect on prognosis, so as to find reliable molecular markers that can predict oral leukoplakia malignant transformation.

Methods: We measured PTHrP which is coded by PTHLH in oral leukoplakia tissues of 79 cases (30 cases progressed to OSCC and 49 did not) and analyzed the clinical outcomes. Then, PTHLH expression was reduced using lentivirus-mediated small hairpin RNA (shRNA) interference to determine the biological role of PTHLH in DOK cells.

Results: PTHrP was found to be highly expressed in 38% of tissues of oral leukoplakia. There was weak or no PTHrP expression in 25 patients, moderate expression in 24 patients, and strong in 30 patients with oral leukoplakia. The expression level was associated with the degree of atypical hyperplasia and poor prognosis. The cell proliferation, invasion, migration, cell cloning, and cell cycle were affected after reducing PTHLH expression.

Conclusion: Our data suggest that either PTHLH or PTHrP plays a key role in the malignant transformation of oral leukoplakia and might be a reliable biomarker for predicting the carcinogenesis of oral leukoplakia.

Keywords: PTHLH; malignant transformation; oral leukoplakia.

Parathyroid hormone (PTH)-related protein (PTHrP) (gene name Pthlh) was discovered as the factor responsible for the humoral hypercalcemia of malignancy. It shares such sequence similarity with PTH in the amino-terminal region that the two are equally able to act through a single G protein-coupled receptor, PTH1R. A number of biological activities are ascribed to domains of PTHrP beyond the amino-terminal domain. PTH functions as a circulating hormone, but PTHrP is generated locally in many tissues including bone, where it acts as a paracrine factor on osteoblasts and osteocytes. The present study compares how PTH and PTHrP influence cyclic AMP (cAMP) formation through adenylyl cyclase, the first event in cell activation through PTH1R. Brief exposure to full length PTHrP(1-141) in several osteoblastic cell culture systems was followed by sustained adenylyl cyclase activity for more than an hour after ligand washout. This effect was dose-dependent and was not found with shorter PTHrP or PTH peptides even though they were fully able to activate adenylyl cyclase with acute treatment. The persistent activation response to PTHrP(1-141) was seen also with later events in the cAMP/PKA pathway, including persistent activation of CRE-luciferase and sustained regulation of several CREB-responsive mRNAs, up to 24 h after the initial exposure. Pharmacologic blockade of endocytosis prevented the persistent activation of cAMP and gene responses. We conclude that full length PTHrP, the likely local physiological effector in bone, differs in intracellular action to PTH by undergoing endosomal translocation to induce a prolonged adenylyl cyclase activation in its target cells.

Genetic heterogeneity denotes the situation when different genetic architectures underlying diverse populations result in the same phenotype. In this study, we explore the genetic background underlying differences in the incidence of hoof disorders between Braunvieh and Fleckvieh cattle in the context of genetic heterogeneity between the breeds. Despite potentially higher power of testing due to twice as large sample size, none of the SNPs was significantly associated with the total number of hoof disorders in Fleckvieh, while 15 SNPs were significant in Braunvieh. The most promising candidate genes in Braunvieh were as follows: CBLB on BTA1, which causes arthritis in rats; CAV2 on BTA4, which affects skeletal muscles in mice; PTHLH on BTA5, which causes disease phenotypes related to the skeleton in humans, mice, and zebrafish; and SORCS2 on BTA6, which causes decreased susceptibility to injury in mice. Some of the significant SNPs (BTA1, BTA4, BTA5, BTA13, and BTA16) revealed allelic heterogeneity-i.e., different allele frequencies between Fleckvieh and Braunvieh. Some of the significant regions (BTA1, BTA5, BTA13, and BTA16) correlated to inter-breed differences in linkage disequilibrium (LD) structure and may thus represent false-positive heterogeneity. However, positions on BTA6 (SORCS2), BTA14, and BTA24 mark Braunvieh-specific regions. We hypothesize that the observed genetic heterogeneity of hoof disorders is a by-product of different selection goals defined for the analyzed breeds-toward dairy production in Braunvieh and toward beef production in Fleckvieh. Based on the current dataset, it is not possible to unequivocally confirm or exclude the hypothesis of genetic heterogeneity in the susceptibility to hoof disorders between Fleckvieh and Braunvieh. The main reason for the problem is that the potential heterogeneity was explored through SNP-phenotype associations and not through causal mutations, due to a limited SNP density offered by the SNP-chip. The rationale against genetic heterogeneity comprises a limited power of detection of true associations as well as differences in the length of LD blocks and in linkage phase between breeds. On the other hand, different selection goals defined for the analyzed breeds accompanied by no systematic, genome-wide differences in LD structure between the breeds favor the heterogeneity hypothesis at some smaller genomic regions.

Keywords: Braunvieh; Fleckvieh; GWAS; genetic heterogeneity; health traits; linkage disequilibrium; principal components.

Chondrocytes in the resting zone of the postnatal growth plate are characterized by slow cell cycle progression, and encompass a population of parathyroid hormone-related protein (PTHrP)-expressing skeletal stem cells that contribute to the formation of columnar chondrocytes. However, how these chondrocytes are maintained in the resting zone remains undefined. We undertook a genetic pulse-chase approach to isolate slow cycling, label-retaining chondrocytes (LRCs) using a chondrocyte-specific doxycycline-controllable Tet-Off system regulating expression of histone 2B-linked GFP. Comparative RNA-seq analysis identified significant enrichment of inhibitors and activators for Wnt signaling in LRCs and non-LRCs, respectively. Activation of Wnt/β-catenin signaling in PTHrP⁺ resting chondrocytes using Pthlh-creER and Apc-floxed allele impaired their ability to form columnar chondrocytes. Therefore, slow-cycling chondrocytes are maintained in a Wnt-inhibitory environment within the resting zone, unraveling a novel mechanism regulating maintenance and differentiation of PTHrP⁺ skeletal stem cells of the postnatal growth plate.

Keywords: bone; cartilage; chondrocyte; developmental biology; growth plate; mouse; skeletal stem cells; wnt signaling.

Bone metastasis frequently occurs in advanced-stage prostate cancer (PCa) patients. Understanding the mechanisms that promote PCa-mediated bone destruction is important for the identification of therapeutic targets against this lethal disease. We found that forkhead box A2 (FOXA2) is expressed in a subset of PCa bone metastasis specimens. To determine the functional role of FOXA2 in PCa metastasis, we knocked down the expression of FOXA2 in PCa PC3 cells, which can grow in bones and elicit an osteolytic reaction. The PC3/FOXA2-knockdown cells generated fewer bone lesions following intra-tibial injection compared to control cells. Further, we found that FOXA2 knockdown decreased the expression of PTHLH, which encodes PTHrP, a well-established factor that regulates bone remodeling. These results indicate that FOXA2 is involved in PCa bone metastasis.

Keywords: FOXA2; PTHrP; bone; prostate cancer.

The chondrogenic differentiation of mesenchymal stem cells (MSCs) is mediated by transcription factors and small noncoding RNAs such as microRNAs (miRNAs). Each miRNA is initially transcribed as a long transcript, which matures to produce -5p and -3p strands. It is widely believed that the mature and functional miRNA from any given pre-miRNA, usually the -5p strand, is functional, while the opposing -3p strand is degraded. However, recent cartilage literature started to show functional -3p strands for a few miRNAs. This study aimed at examining both -5p and -3p strands of two key miRNAs miR-140 and miR-145, known to be involved in the chondrogenic differentiation of MSCs. The level (copy number) of both -5p and -3p strands of miR-145 and miR-140 along the time line of MSC chondrogenic differentiation was determined by polymerase chain reaction. The gene expression profiles of several genes related to MSC chondrogenesis were compared with these miRNA profiles along the same timeline. While miR-145-3p is declining in step with miR-145-5p in pellet cultures during the process, the -3p strand is only 1-2% of the total miR-145 products. In contrast, the mature -3p and -5p products of miR-140 are found to increase with near-equal molar expression throughout chondrogenic differentiation. Numerous genes are expressed by cartilage progenitor cells during development. One such target gene, Sox9, is a regulatory target of the dominant miR-145-5p, consistent with the data. Further experimental validations are warranted to confirm that ACAN, FOXO1, and RUNX3 as direct targets of miR-145-5p in the context of MSC chondrogenesis. Similarly, TRSP1 and ACAN are worth further validation as direct targets of miR-145-3p. For miR-140, SOX4 shall be further validated as a direct target of miR-140-5p, while KLF4, PTHLH, and WNT5A can be validated as direct targets of miR-140-3p.

Osteoporosis is a systemic skeletal disorder characterized by reduced bone mineral density (BMD) and disrupted bone architecture, predisposing the patient to increased fracture risk. Evidence from early genetic epidemiological studies has indicated a major role for genetics in the development of osteoporosis and the variation in BMD. In this study, we focused on two key genes in the endochondral ossification pathway, IBSP and PTHLH. Over 9,000 postmenopausal Han Chinese women were recruited, and 54 SNPs were genotyped. Two significant SNPs within IBSP, rs1054627 and rs17013181, were associated with BMD and postmenopausal osteoporosis by the two-stage strategy, and rs17013181 was also significantly associated with serum IBSP levels. Moreover, one haplotype (rs12425376-rs10843047-rs42294) covering the 5' end of PTHLH was associated with postmenopausal osteoporosis. Our results provide evidence for the association of these two key endochondral ossification pathway genes with BMD and osteoporosis in postmenopausal Han Chinese women. Combined with previous findings, we provide evidence that a particular SNP in IBSP has an allele-specific effect on mRNA levels, which would, in turn, reflect serum IBSP levels.

An 18-year-old female tertiary student was referred to a lipid clinic with hypertriglyceridaemia discovered after presentation with acute pancreatitis. The patient's only medication was l-thyroxine for treatment of hypothyroidism. She was overweight, normotensive, with unremarkable facies. However, she had hypermobile hand joints and brachydactyly resulting in loss of left 3-5 and right 4 and 5 knuckle definitions. Radiography revealed shortening of metacarpals 3-5 on the left and 4 and 5 on the right. Her mother had similar skeletal changes, consistent with a dominant mode of inheritance. Abnormally short digits involving the metacarpals, classified as brachydactyly type E, can be isolated or occur as part of a syndrome. Turner syndrome, Albright hereditary osteodystrophy, hypertension with brachydactyly, chromosome 2q37 microdeletion and PTHLH mutations were excluded following clinical, biochemical and genetic testing. No specific treatment was required. Genetic testing for isolated and syndromic forms of brachydactyly facilitates family screening and prepregnancy counselling.

Determining the cellular content of the nervous system in terms of cell types and the rules of their connectivity represents a fundamental challenge to the neurosciences. The recent advent of high-throughput techniques, such as single-cell RNA-sequencing has allowed for greater resolution in the identification of cell types and/or states. Although most of the current neuronal classification schemes comprise discrete clusters, several recent studies have suggested that, perhaps especially, within the striatum, neuronal populations exist in continua, with regards to both their molecular and electrophysiological properties. Whether these continua are stable properties, established during development, or if they reflect acute differences in activity-dependent regulation of critical genes is currently unknown. We set out to determine whether gradient-like molecular differences in the recently described Pthlh-expressing inhibitory interneuron population, which contains the Pvalb-expressing cells, correlate with differences in morphological and connectivity properties. We show that morphology and long-range inputs correlate with a spatially organized molecular and electrophysiological gradient of Pthlh-interneurons, suggesting that the processing of different types of information (by distinct anatomical striatal regions) has different computational requirements.

Pseudohypoparathyroidism (PHP) is a rare disease whose phenotypic features are rather difficult to identify in some cases. Thus, although these patients may present with the Albright's hereditary osteodystrophy (AHO) phenotype, which is characterized by small stature, obesity with a rounded face, subcutaneous ossifications, mental retardation and brachydactyly, its manifestations are somewhat variable. Indeed, some of them present with a complete phenotype, whereas others show only subtle manifestations. In addition, the features of the AHO phenotype are not specific to it and a similar phenotype is also commonly observed in other syndromes. Brachydactyly type E (BDE) is the most specific and objective feature of the AHO phenotype, and several genes have been associated with syndromic BDE in the past few years. Moreover, these syndromes have a skeletal and endocrinological phenotype that overlaps with AHO/PHP. In light of the above, we have developed an algorithm to aid in genetic testing of patients with clinical features of AHO but with no causative molecular defect at the GNAS locus. Starting with the feature of brachydactyly, this algorithm allows the differential diagnosis to be broadened and, with the addition of other clinical features, can guide genetic testing.

We reviewed our series of patients (n = 23) with a clinical diagnosis of AHO and with brachydactyly type E or similar pattern, who were negative for GNAS anomalies, and classify them according to the diagnosis algorithm to finally propose and analyse the most probable gene(s) in each case.

A review of the clinical data for our series of patients, and subsequent analysis of the candidate gene(s), allowed detection of the underlying molecular defect in 12 out of 23 patients: five patients harboured a mutation in PRKAR1A, one in PDE4D, four in TRPS1 and two in PTHLH.

This study confirmed that the screening of other genes implicated in syndromes with BDE and AHO or a similar phenotype is very helpful for establishing a correct genetic diagnosis for those patients who have been misdiagnosed with "AHO-like phenotype" with an unknown genetic cause, and also for better describing the characteristic and differential features of these less common syndromes.

Brachydactyly type E, which can be an isolated finding or part of a syndrome in combination with other clinical anomalies, involves metacarpals and metatarsals with or without short phalanges. Herein we report two unrelated Turkish females who presented with brachydactyly type E and vitamin D deficiency in the absence of marked alterations in serum calcium, phosphate, and parathyroid hormone. After excluding disease-causing variants in two candidate genes, PTHLH and PDE4D, we identified different pathogenic variants in TRPS1, the gene mutated in patients with tricho-rhino-phalangeal syndrome (TRPS). In one of the patients, who displayed severe brachydactyly and short stature, we identified a novel heterozygous missense pathogenic variant in exon 6 (c.2783A>G, p.Tyr928Cys), located within the GATA DNA-binding domain. The second patient, who had relatively milder brachydactyly and was of normal height, carried a heterozygous nonsense pathogenic variant in exon 4 (c. 1870C>T, p.Arg624Ter), which has been previously described. Both pathogenic variants segregated in affected family members. The patients additionally showed sparse hair and a bulbous nose, consistent with the clinical features of TRPS. Our findings, in addition to identifying the genetic cause of brachydactyly in two unrelated kindreds, emphasize the role of pathogenic TRPS1 variants in the development of brachydactyly type E and highlight the GATA DNA-binding region of TRPS1 protein with respect to phenotype-genotype correlation.

Recently, we showed that parathyroid hormone-like hormone (PTHLH), a cytokine-like polyprotein, is critical for extracellular matrix (ECM) deposition through the activation of hepatic stellate cells (HSCs). Here, we show that N-terminal PTHLH is secreted into the supernatant of injured hepatocytes, its expression is positively correlated with liver fibrosis severity based on mice liver biopsies, and it is primarily expressed in the cytoplasm of hepatocytes along the fibrous septa of fibrotic livers. PTHLH overexpression in mice was achieved through adeno-associated virus-mediated gene delivery (AAV9-PTHLH), and liver fibrosis was induced with carbon tetrachloride (CCl₄). We observed that AAV9-PTHLH induced spontaneous development of liver fibrosis and increased sensitivity to CCl₄. PTHLH increased Hedgehog (Hh) pathway activation in a PTH1R-dependent manner, and the effect of PTHLH was primarily mediated by protein kinase C (PKC) θ. PTHLH-mediated PTH1R-PKC θ pathway activation is a key event in the profibrotic Hh-dependent activation of HSCs.

Background: Cleidocranial dysplasia (CCD) is a genetic disorder with an autosomal dominant inheritance pattern. CCD characterized by abnormal clavicles, patent sutures and fontenelles, supernumerary teeth and short stature. Approximately 60-70% of CCD patients have mutations in the RUNX2 gene. The RUNX2 gene is an essential transcription factor for chondrocyte maturation, osteoblast differentiation and bone formation. Runx2 regulates mesenchymal cell proliferation in sutures and suture closure by inducing the signaling pathways of the genes of Fgf, Pthlh, hedgehog and Wnt. Material and Methods: We summarized molecular genetics aspects of CCD. Result: Approximately 94% of CCD patients have dental anomalies, the most common of which are supernumerary tooth. Dental anomalies are not determined solely by gene mutations of RUNX2, but are also affected by modifier genes, environmental factors, epigenetic factors and copy number variations. Conclusion: a definite diagnosis of CCD should include the patient's clinical history, symptoms and signs, as well as genetic analyses.