α-Melanocyte-stimulating hormone alleviates pathological cardiac remodeling via melanocortin 5 receptor.

α-Melanocyte-stimulating hormone (α-MSH) regulates diverse physiological functions by activating melanocortin receptors (MC-R). However, the role of α-MSH and its possible target receptors in the heart remain completely unknown. Here we investigate whether α-MSH could be involved in pathological cardiac remodeling. We found that α-MSH was highly expressed in the mouse heart with reduced ventricular levels after transverse aortic constriction (TAC). Administration of a stable α-MSH analog protected mice against TAC-induced cardiac hypertrophy and systolic dysfunction. In vitro experiments revealed that MC5-R in cardiomyocytes mediates the anti-hypertrophic signaling of α-MSH. Silencing of MC5-R in cardiomyocytes induced hypertrophy and fibrosis markers in vitro and aggravated TAC-induced cardiac hypertrophy and fibrosis in vivo. Conversely, pharmacological activation of MC5-R improved systolic function and reduced cardiac fibrosis in TAC-operated mice. In conclusion, α-MSH is expressed in the heart and protects against pathological cardiac remodeling by activating MC5-R in cardiomyocytes. These results suggest that analogs of naturally occurring α-MSH, that have been recently approved for clinical use and have agonistic activity at MC5-R, may be of benefit in treating heart failure.

Apelin regulates skeletal muscle adaptation to exercise in a high-intensity interval training model.

Plasma apelin levels are reduced in aging and muscle wasting conditions. We aimed to investigate the significance of apelin signaling in cardiac and skeletal muscle responses to physiological stress. Apelin knockout (KO) and wild-type (WT) mice were subjected to high-intensity interval training (HIIT) by treadmill running. The effects of apelin on energy metabolism were studied in primary mouse skeletal muscle myotubes and cardiomyocytes. Apelin increased mitochondrial ATP production and mitochondrial coupling efficiency in myotubes and promoted the expression of mitochondrial genes both in primary myotubes and cardiomyocytes. HIIT induced mild concentric cardiac hypertrophy in WT mice, whereas eccentric growth was observed in the left ventricles of apelin KO mice. HIIT did not affect myofiber size in skeletal muscles of WT mice but decreased the myofiber size in apelin KO mice. The decrease in myofiber size resulted from a fiber type switch toward smaller slow-twitch type I fibers. The increased proportion of slow-twitch type I fibers in apelin KO mice was associated with upregulation of myosin heavy chain slow isoform expression, accompanied with upregulated expression of genes related to fatty acid transport and downregulated expression of genes related to glucose metabolism. Mechanistically, skeletal muscles of apelin KO mice showed defective induction of insulin-like growth factor-1 signaling in response to HIIT. In conclusion, apelin is required for proper skeletal and cardiac muscle adaptation to high-intensity exercise. Promoting apelinergic signaling may have benefits in aging- or disease-related muscle wasting conditions.NEW & NOTEWORTHY Apelin levels decline with age. This study demonstrates that in trained mice, apelin deficiency results in a switch from fast type II myofibers to slow oxidative type I myofibers. This is associated with a concomitant change in gene expression profile toward fatty acid utilization, indicating an aged-muscle phenotype in exercised apelin-deficient mice. These data are of importance in the design of exercise programs for aging individuals and could offer therapeutic target to maintain muscle mass.

MiR-185-5p regulates the development of myocardial fibrosis.

BACKGROUND: Cardiac fibrosis stiffens the ventricular wall, predisposes to cardiac arrhythmias and contributes to the development of heart failure. In the present study, our aim was to identify novel miRNAs that regulate the development of cardiac fibrosis and could serve as potential therapeutic targets for myocardial fibrosis. METHODS AND RESULTS: Analysis for cardiac samples from sudden cardiac death victims with extensive myocardial fibrosis as the primary cause of death identified dysregulation of miR-185-5p. Analysis of resident cardiac cells from mice subjected to experimental cardiac fibrosis model showed induction of miR-185-5p expression specifically in cardiac fibroblasts. In vitro, augmenting miR-185-5p induced collagen production and profibrotic activation in cardiac fibroblasts, whereas inhibition of miR-185-5p attenuated collagen production. In vivo, targeting miR-185-5p in mice abolished pressure overload induced cardiac interstitial fibrosis. Mechanistically, miR-185-5p targets apelin receptor and inhibits the anti-fibrotic effects of apelin. Finally, analysis of left ventricular tissue from patients with severe cardiomyopathy showed an increase in miR-185-5p expression together with pro-fibrotic TGF-ß1 and collagen I. CONCLUSIONS: Our data show that miR-185-5p targets apelin receptor and promotes myocardial fibrosis.

Genome-wide meta-analysis identifies genetic locus on chromosome 9 associated with Modic changes.

BACKGROUND: Low back pain (LBP) is a common disabling condition. Lumbar disc degeneration (LDD) may be a contributing factor for LBP. Modic change (MC), a distinct phenotype of LDD, is presented as a pathological bone marrow signal change adjacent to vertebral endplate on MRI. It is strongly associated with LBP and has heritability around 30%. Our objective was to identify genetic loci associated with MC using a genome-wide meta-analysis. METHODS: Presence of MC was evaluated in lumbar MRI in the Northern Finland Birth Cohort 1966 (n=1182) and TwinsUK (n=647). Genome-wide association analyses were carried out using linear regression model. Inverse-variance weighting approach was used in the meta-analysis. RESULTS: A locus associated with MC (p<5e-8) was found on chromosome 9 with the lead SNP rs1934268 in an intron of the PTPRD gene. It is located in the binding region of BCL11A, SPI1 and PBX3 transcription factors. The SNP was nominally associated with LBP in TwinsUK (p=0.001) but not associated in the UK Biobank (p=0.914). Suggestive signals (p<1e-5) were identified near XKR4, SCIN, MGMT, DLG2, ZNF184 and OPRK1. CONCLUSION: PTPRD is a novel candidate gene for MC that may act via the development of cartilage or nervous system; further work is needed to define the mechanisms underlying the pathways leading to development of MC. This is the first genome-wide meta-analysis of MC, and the results pave the way for further studies on the genetic factors underlying the various features of spine degeneration and LBP.

Whole-exome sequencing suggests multiallelic inheritance for childhood-onset Ménière's disease.

The genetic background of Ménière's disease (MD) was studied in one patient with childhood-onset MD and his grandfather affected with middle age-onset MD. Whole-exome sequencing was performed and the data were compared to 76 exomes from unrelated subjects without MD. Thirteen rare inner ear expressed variants with pathogenic estimations were observed in the case of childhood-onset MD. These variants were in genes involved in the formation of cell membranes or the cytoskeleton and in genes participating in cell death or gene-regulation pathways. His grandfather shared two of the variants: p.Y273N in HMX2 and p.L229F in TMEM55B. HMX2 p.Y273N was considered the more likely candidate for MD, as the gene is known to affect both hearing and vestibular function. The variant in the HMX2 gene may affect inner ear development and structural integrity and thus might predispose to the onset of MD. As there was a significant difference in onset between the patients, an accumulation of defects in several pathways is probably responsible for the exceptionally early onset of the disease, and the genetic etiology of childhood-onset MD is most likely multifactorial. This is the first molecular genetic study of childhood-onset MD.

The interplay of matrix metalloproteinase-8, transforming growth factor-ß1 and vascular endothelial growth factor-C cooperatively contributes to the aggressiveness of oral tongue squamous cell carcinoma.

BACKGROUND: Matrix metalloproteinase-8 (MMP-8) has oncosuppressive properties in various cancers. We attempted to assess MMP-8 function in oral tongue squamous cell carcinoma (OTSCC). METHODS: MMP-8 overexpressing OTSCC cells were used to study the effect of MMP-8 on proliferation, apoptosis, migration, invasion and gene and protein expression. Moreover, MMP-8 functions were assessed in the orthotopic mouse tongue cancer model and by immunohistochemistry in patient samples. RESULTS: MMP-8 reduced the invasion and migration of OTSCC cells and decreased the expression of MMP-1, cathepsin-K and vascular endothelial growth factor-C (VEGF-C). VEGF-C was induced by transforming growth factor-ß1 (TGF-ß1) in control cells, but not in MMP-8 overexpressing cells. In human OTSCC samples, low MMP-8 in combination with high VEGF-C was an independent predictor of poor cancer-specific survival. TGF-ß1 treatment also restored the migration of MMP-8 overexpressing cells to the level of control cells. In mouse tongue cancer, MMP-8 did not inhibit metastasis, possibly because it was eliminated in the peripheral carcinoma cells. CONCLUSIONS: The suppressive effects of MMP-8 in OTSCC may be mediated through interference of TGF-ß1 and VEGF-C function and altered proteinase expression. Together, low MMP-8 and high VEGF-C expression have strong independent prognostic value in OTSCC.

Intrauterine growth restriction and placental gene expression in severe preeclampsia, comparing early-onset and late-onset forms.

OBJECTIVE: To evaluate placental gene expression in severe early- or late-onset preeclampsia with intrauterine growth restriction compared to controls. STUDY DESIGN: Chorionic villus sampling was conducted after cesarean section from the placentas of five women with early- or late-onset severe preeclampsia and five controls for each preeclampsia group. Microarray analysis was performed to identify gene expression differences between the groups. RESULTS: Pathway analysis showed over-representation of gene ontology (GO) biological process terms related to inflammatory and immune response pathways, platelet development, vascular development, female pregnancy and reproduction in early-onset preeclampsia. Pathways related to immunity, complement and coagulation cascade were overrepresented in the hypergeometric test for the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Ten genes (ABI3BP, C7, HLA-G, IL2RB, KRBOX1, LRRC15, METTL7B, MPP5, RFLNB and SLC20A) had a ≥±1 fold expression difference in severe early-onset preeclampsia group compared to early controls. There were 362 genes that had a ≥±1 fold expression difference in severe early-onset preeclampsia group compared to late-onset preeclampsia group including ABI3BP, C7, HLA-G and IL2RB. CONCLUSION: There are significant differences in placental gene expression between severe early- and late-onset preeclampsia when both are associated with intrauterine growth restriction. ABI3BP, C7, HLA-G and IL2RB might contribute to the development of early form of severe preeclampsia.

Genetic variants associated with cardiac hypertrophy-related sudden cardiac death and cardiovascular outcomes in a Finnish population.

BACKGROUND: Hypertrophic cardiomyopathy is a common cause of non-ischaemic sudden cardiac death (SCD). Left ventricular hypertrophy (LVH) without cardiomyopathy-related myocardial disarray is a common autopsy finding and is often associated with prior hypertension in SCD subjects. Our aim was to investigate novel rare gene variants among SCD subjects with presumably hypertension-related LVH and myocardial fibrosis at autopsy. METHODS: Whole exome sequencing was used to study rare variants (minor allele frequency<0.005) estimated to be deleterious in 96 non-ischaemic SCD subjects with presumably hypertension-related LVH and myocardial fibrosis. Associations of the identified variants with cardiac disease endpoints were replicated in the Finnish national genetic study (FinnGen) dataset. RESULTS: 18 variants were estimated likely to affect protein function and 14 of these were associated with cardiomyopathies, heart failure, conduction abnormalities, hypertension and/or cardiac arrest in Finnish population (FinnGen). Three of the variants were classified as pathogenic or likely pathogenic. These include the splice site variant NM_000449.3:c.234-1G>A in regulatory factor X5 and frameshift variants NM_000449.3:c.234-1G>A in dehydrogenase/reductase 7C and NM_015873.3:c.1164del in villin like. CONCLUSIONS: We identified rare deleterious variants associated with LVH in SCD subjects. Several of the identified rare variants associated with cardiovascular endpoints including heart failure, cardiomyopathies, cardiac arrest and hypertension in general population.

Novel Genetic Variants Associated with Primary Myocardial Fibrosis in Sudden Cardiac Death Victims.

Myocardial fibrosis is a common finding in victims of sudden cardiac death (SCD). Whole exome sequencing was performed in 127 victims of SCD with primary myocardial fibrosis as the only pathological finding. These cases are derived from the Fingesture study which has collected data from autopsy-verified SCD victims in Northern Finland. A computational approach was used to identify protein interactions in cardiomyocytes. Associations of the identified variants with cardiac disease endpoints were investigated in the Finnish national genetic study (FinnGen) dataset. We identified 21 missense and one nonsense variant. Four variants were estimated to affect protein function, significantly associated with SCD/primary myocardial fibrosis (Fingesture) and associated with cardiac diseases in Finnish population (FinnGen). These variants locate in cartilage acidic protein 1 (CRATC1), calpain 1 (CAPN1), unc-45 myosin chaperone A (UNC45A) and unc-45 myosin chaperone B (UNC45B). The variants identified contribute to function of extracellular matrix and cardiomyocytes.

The genetic basis of endometriosis and comorbidity with other pain and inflammatory conditions.

Endometriosis is a common condition associated with debilitating pelvic pain and infertility. A genome-wide association study meta-analysis, including 60,674 cases and 701,926 controls of European and East Asian descent, identified 42 genome-wide significant loci comprising 49 distinct association signals. Effect sizes were largest for stage 3/4 disease, driven by ovarian endometriosis. Identified signals explained up to 5.01% of disease variance and regulated expression or methylation of genes in endometrium and blood, many of which were associated with pain perception/maintenance (SRP14/BMF, GDAP1, MLLT10, BSN and NGF). We observed significant genetic correlations between endometriosis and 11 pain conditions, including migraine, back and multisite chronic pain (MCP), as well as inflammatory conditions, including asthma and osteoarthritis. Multitrait genetic analyses identified substantial sharing of variants associated with endometriosis and MCP/migraine. Targeted investigations of genetically regulated mechanisms shared between endometriosis and other pain conditions are needed to aid the development of new treatments and facilitate early symptomatic intervention.

New Genetic Variants in CYP2B6 and SLC6A Support the Role of Oxidative Stress in Familial Ménière's Disease.

The objective was to study the genetic etiology of Ménière's disease (MD) using next-generation sequencing in three families with three cases of MD. Whole exome sequencing was used to identify rare genetic variants co-segregating with MD in Finnish families. In silico estimations and population databases were used to estimate the frequency and pathogenicity of the variants. Variants were validated and genotyped from additional family members using capillary sequencing. A geneMANIA analysis was conducted to investigate the functional pathways and protein interactions of candidate genes. Seven rare variants were identified to co-segregate with MD in the three families: one variant in the CYP2B6 gene in family I, one variant in GUSB and EPB42 in family II, and one variant in each of the SLC6A, ASPM, KNTC1, and OVCH1 genes in family III. Four of these genes were linked to the same co-expression network with previous familial MD candidate genes. Dysfunction of CYP2B6 and SLC6A could predispose to MD via the oxidative stress pathway. Identification of ASPM and KNTC1 as candidate genes for MD suggests dysregulation of mitotic spindle formation in familial MD. The genetic etiology of familial MD is heterogenic. Our findings suggest a role for genes acting on oxidative stress and mitotic spindle formation in MD but also highlight the genetic complexity of MD.

NRF3 Decreases during Melanoma Carcinogenesis and Is an Independent Prognostic Marker in Melanoma.

The prognostic significance of the major redox regulator, nuclear factor erythroid-2-related factor 2 (NRF2), is recognized in many cancers, but the role of NRF3 is not studied. Analysis from the Gene Expression Omnibus datasets showed that NRF3 mRNA levels increased from benign to dysplastic naevi (p = 0.04). We characterized the immunohistochemical expression of NRF3 in 81 naevi, 67 primary skin melanomas, and 51 lymph node metastases. The immunohistochemical expression of cytoplasmic NRF3 decreased from benign to dysplastic naevi (p < 0.001) and further to primary melanomas (p < 0.001). High cytoplasmic NRF3 protein expression in pigment cells of the primary melanomas associated with worse melanoma-specific survival in multivariate analysis, specifically in the subgroup of patients with the lymph node metastases at the time of diagnosis (hazard ratio 3.179; 95% confidence interval 1.065-9.493; p = 0.038). Intriguingly, we did not observe associations between NRF3 and the traditional prognostic factors such as Breslow thickness, ulceration, or stage. Together, this data represents the primary description about the role of NRF3 in pigment tumours that is worthy of further explorations.

Extracellular matrix proteins produced by stromal cells in idiopathic pulmonary fibrosis and lung adenocarcinoma.

Idiopathic pulmonary fibrosis (IPF) and lung cancer share common risk factors, epigenetic and genetic alterations, the activation of similar signaling pathways and poor survival. The aim of this study was to examine the gene expression profiles of stromal cells from patients with IPF and lung adenocarcinoma (ADC) as well as from normal lung. The gene expression levels of cultured stromal cells derived from non-smoking patients with ADC from the tumor (n = 4) and the corresponding normal lung (n = 4) as well as from patients with IPF (n = 4) were investigated with Affymetrix microarrays. The expression of collagen type IV alpha 1 chain, periostin as well as matrix metalloproteinase-1 and -3 in stromal cells and lung tissues were examined with quantitative real-time reverse transcriptase polymerase chain reaction and immunohistochemistry, respectively. Twenty genes were similarly up- or down-regulated in IPF and ADC compared to control, while most of the altered genes in IPF and ADC were differently expressed, including several extracellular matrix genes. Collagen type IV alpha 1 chain as well as matrix metalloproteinases-1 and -3 were differentially expressed in IPF compared to ADC. Periostin was up-regulated in both IPF and ADC in comparison to control. All studied factors were localized by immunohistochemistry in stromal cells within fibroblast foci in IPF and stroma of ADC. Despite the similarities found in gene expressions of IPF and ADC, several differences were also detected, suggesting that the molecular changes occurring in these two lung illnesses are somewhat different.

Genetic Variants Associated With Sudden Cardiac Death in Victims With Single Vessel Coronary Artery Disease and Left Ventricular Hypertrophy With or Without Fibrosis.

Objective: Cardiac hypertrophy with varying degrees of myocardial fibrosis is commonly associated with coronary artery disease (CAD) related sudden cardiac death (SCD), especially in young victims among whom patterns of coronary artery lesions do not entirely appear to explain the cause of SCD. Our aim was to study the genetic background of hypertrophy, with or without fibrosis, among ischemic SCD victims with single vessel CAD. Methods: The study population was derived from the Fingesture study, consisting of all autopsy-verified SCDs in Northern Finland between the years 1998 and 2017 (n = 5,869). We carried out targeted next-generation sequencing using a panel of 174 genes associated with myocardial structure and ion channel function in 95 ischemic-SCD victims (mean age 63.6 ± 10.3 years; 88.4% males) with single-vessel CAD in the absence of previously diagnosed CAD and cardiac hypertrophy with or without myocardial fibrosis at autopsy. Results: A total of 42 rare variants were detected in 43 subjects (45.3% of the study subjects). Five variants in eight subjects (8.4%) were classified as pathogenic or likely pathogenic. We observed 37 variants of uncertain significance in 39 subjects (40.6%). Variants were detected in myocardial structure protein coding genes, associated with arrhythmogenic right ventricular, dilated, hypertrophic and left ventricular non-compaction cardiomyopathies. Also, variants were detected in ryanodine receptor 2 (RYR2), a gene associated with both cardiomyopathies and catecholaminergic polymorphic ventricular tachycardias. Conclusions: Rare variants associated with cardiomyopathies, in the absence of anatomic evidence of the specific inherited cardiomyopathies, were common findings among CAD-related SCD victims with single vessel disease and myocardial hypertrophy found at autopsies, suggesting that these variants may modulate the risk for fatal arrhythmias and SCD in ischemic disease.

A single genetic locus associated with pediatric fractures: A genome-wide association study on 3,230 patients.

The understanding of the biological and environmental risk factors of fractures in pediatrics is limited. Previous studies have reported that fractures involve heritable traits, but the genetic factors contributing to the risk of fractures remain elusive. Furthermore, genetic influences specific to immature bone have not been thoroughly studied. Therefore, the aim of the present study was to identify genetic variations that are associated with fractures in early childhood. The present study used a prospective Northern Finland Birth Cohort (year 1986; n=9,432). The study population was comprised of 3,230 cohort members with available genotype data. A total of 48 members of the cohort (1.5%) had in-hospital treated bone fractures during their first 6 years of life. Furthermore, individuals without fracture (n=3,182) were used as controls. A genome-wide association study (GWAS) was performed using a frequentist association test. In the GWAS analysis, a linear regression model was fitted to test for additive effects of single-nucleotide polymorphisms (SNPs; genotype dosage) adjusting for sex and performing population stratification using genotypic principal components. Using the GWAS analysis, the present study identified one locus with a significant association with fractures during childhood on chromosome 10 (rs112635931) and six loci with a suggested implication. The lead SNP rs112635931 was located near proline- and serine-rich 2 (PROSER2) antisense RNA 1 (PROSER2-AS1) and PROSER2, thus suggesting that these may be novel candidate genes associated with the risk of pediatric fractures.

Exome Sequencing Reveals a Phenotype Modifying Variant in ZNF528 in Primary Osteoporosis With a COL1A2 Deletion.

We studied a family with severe primary osteoporosis carrying a heterozygous p.Arg8Phefs*14 deletion in COL1A2, leading to haploinsufficiency. Three affected individuals carried the mutation and presented nearly identical spinal fractures but lacked other typical features of either osteogenesis imperfecta or Ehlers-Danlos syndrome. Although mutations leading to haploinsufficiency in COL1A2 are rare, mutations in COL1A1 that lead to less protein typically result in a milder phenotype. We hypothesized that other genetic factors may contribute to the severe phenotype in this family. We performed whole-exome sequencing in five family members and identified in all three affected individuals a rare nonsense variant (c.1282C > T/p.Arg428*, rs150257846) in ZNF528. We studied the effect of the variant using qPCR and Western blot and its subcellular localization with immunofluorescence. Our results indicate production of a truncated ZNF528 protein that locates in the cell nucleus as per the wild-type protein. ChIP and RNA sequencing analyses on ZNF528 and ZNF528-c.1282C > T indicated that ZNF528 binding sites are linked to pathways and genes regulating bone morphology. Compared with the wild type, ZNF528-c.1282C > T showed a global shift in genomic binding profile and pathway enrichment, possibly contributing to the pathophysiology of primary osteoporosis. We identified five putative target genes for ZNF528 and showed that the expression of these genes is altered in patient cells. In conclusion, the variant leads to expression of truncated ZNF528 and a global change of its genomic occupancy, which in turn may lead to altered expression of target genes. ZNF528 is a novel candidate gene for bone disorders and may function as a transcriptional regulator in pathways affecting bone morphology and contribute to the phenotype of primary osteoporosis in this family together with the COL1A2 deletion. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Prognostic significance of Twist, ZEB1 and Slug in peripheral T-cell lymphomas.

ABSTRACT Objectives: To investigate the protein expression of the epithelial-mesenchymal transition-inducing transcription factors (TFs) Twist, ZEB1 and Slug in peripheral T-cell lymphomas (PTCL) and their correlation with clinical parameters. Methods: The expression of these TFs was studied in 53 diagnostic biopsy specimens of several different PTCL subtypes with immunohistochemistry. Patient data were retrospectively collected from patient records and a statistical analysis was performed. Results: All three TFs were widely expressed. ZEB1 and Slug had correlations with clinical outcome. In all PTCL cases, high nuclear ZEB1 percentage correlated with a favorable progression-free survival (PFS) (3-year PFS: 70% vs. 34%; P = 0.010) and strong nuclear Slug intensity correlated with an unfavorable PFS (3-year PFS: 17% vs. 62%; P = 0.036). Discussion: The correlations between PFS and ZEB1 or Slug protein expression have not previously been established in PTCLs. The impact of ZEB1 and Slug expression on prognosis differed from our findings in DLBCL and the impact of ZEB1 expression was in line with current studies on mycosis fungoides and sézary syndrome. The findings may be explained by the roles these TFs play in hematopoiesis. Conclusion: ZEB1 and Slug may have potential clinical value for evaluating prognosis in PTCLs. The study size was small and heterogenous, and larger studies are warranted.

NRF1 and NRF2 mRNA and Protein Expression Decrease Early during Melanoma Carcinogenesis: An Insight into Survival and MicroRNAs.

The prognostic significance of the major redox regulator nuclear factor erythroid-2-related factor (NRF2) is recognized in many cancers, but the role of NRF1 is not generally well understood in cancer. Our aim was to investigate these redox transcription factors in conjunction with redox-related microRNAs in naevi and melanoma. We characterized the immunohistochemical expression of NRF1 and NRF2 in 99 naevi, 88 primary skin melanomas, and 67 lymph node metastases. In addition, NRF1 and NRF2 mRNA and miR-23B, miR-93, miR-144, miR-212, miR-340, miR-383, and miR-510 levels were analysed with real-time qPCR from 54 paraffin-embedded naevi and melanoma samples. The immunohistochemical expression of nuclear NRF1 decreased from benign to dysplastic naevi (p < 0.001) and to primary melanoma (p < 0.001) and from primary melanoma to metastatic lesions (p = 0.012). Also, NRF1 mRNA levels decreased from benign naevi to dysplastic naevi (p = 0.034). Similarly, immunopositivity of NRF2 decreased from benign to dysplastic naevi (p = 0.02) and to primary lesions (p = 0.018). NRF2 mRNA decreased from benign to dysplastic naevi and primary melanomas (p = 0.012). Analysis from the Gene Expression Omnibus datasets supported the mRNA findings. High nuclear immunohistochemical NRF1 expression in pigment cells associated with a worse survival (p = 0.048) in patients with N0 disease at the time of diagnosis, and high nuclear NRF2 expression in pigment cells associated with a worse survival (p = 0.033) in patients with M0 disease at the time of diagnosis. In multivariate analysis, neither of these variables exceeded the prognostic power of Breslow. The levels of miR-144 and miR-212 associated positively with ulceration (p = 0.012 and p = 0.027, respectively) while miR-510 levels associated positively with lymph node metastases at the time of diagnosis (p = 0.004). Furthermore, the miRNAs correlated negatively with the immunohistochemical expression of NRF1 and NRF2 but positively with their respective mRNA. Together, this data sheds new light about NFE2L family factors in pigment tumors and suggests that these factors are worth for further explorations.

Rare Copy Number Variants in Array-Based Comparative Genomic Hybridization in Early-Onset Skeletal Fragility.

Early-onset osteoporosis is characterized by low bone mineral density (BMD) and fractures since childhood or young adulthood. Several monogenic forms have been identified but the contributing genes remain inadequately characterized. In search for novel variants and novel candidate loci, we screened a cohort of 70 young subjects with mild to severe skeletal fragility for rare copy-number variants (CNVs). Our study cohort included 15 subjects with primary osteoporosis before age 30 years and 55 subjects with a pathological fracture history and low or normal BMD before age 16 years. A custom-made high-resolution comparative genomic hybridization array with enriched probe density in >1,150 genes important for bone metabolism and ciliary function was used to search for CNVs. We identified altogether 14 rare CNVs. Seven intronic aberrations were classified as likely benign. Five CNVs of unknown clinical significance affected coding regions of genes not previously associated with skeletal fragility (ETV1-DGKB, AGBL2, ATM, RPS6KL1-PGF, and SCN4A). Finally, two CNVs were pathogenic and likely pathogenic, respectively: a 4 kb deletion involving exons 1-4 of COL1A2 (NM_000089.3) and a 12.5 kb duplication of exon 3 in PLS3 (NM_005032.6). Although both genes have been linked to monogenic forms of osteoporosis, COL1A2 deletions are rare and PLS3 duplications have not been described previously. Both CNVs were identified in subjects with significant osteoporosis and segregated with osteoporosis within the families. Our study expands the number of pathogenic CNVs in monogenic skeletal fragility and shows the validity of targeted CNV screening to potentially pinpoint novel candidate loci in early-onset osteoporosis.

Whole exome sequencing in Finnish families identifies new candidate genes for osteoarthritis.

INTRODUCTION: Osteoarthritis (OA) is the most common degenerative joint disease and one of the major causes of disability worldwide. It is a multifactorial disorder with a significant genetic component. The heritability of OA has been estimated to be 60% for hip OA and 39% for knee OA. Genetic factors behind OA are still largely unknown. Studying families with strong history of OA, facilitates examining the co-segregation of genetic variation and OA. The aim of this study was to identify new, rare genetic factors and novel candidate genes for OA. METHODS: Eight patients from three Finnish families with hip and knee OA were studied using whole exome sequencing. We focused on rare exonic variants with predicted pathogenicity and variants located in active promoter or strong enhancer regions. Expression of identified candidate genes were studied in bone and cartilage tissues and the observed variants were investigated using bioinformatic analyses. RESULTS: Two rare variants co-segregated with OA in two families. In Family 8 a missense variant (c.628C>G, p.Arg210Gly) was observed in the OLIG3 gene that encodes a transcription factor known to be associated with rheumatoid arthritis and inflammatory polyarthritis. The Arg210Gly variant was estimated to be pathogenic by Polyphen-2 and Mutation taster and the locus is conserved among mammals. In Family 12 the observed variant (c.-127G>T) was located in the transcription start site of the FIP1L1 gene. FIP1L1 participates in the regulation of polyadenylation. The c.-127G>T is located in the transcription start site and may alter the DNA-binding of transcription factors. Both, OLIG3 and FIP1L1 were observed in human bone and cartilage. CONCLUSION: The identified variants revealed novel candidate genes for OA. OLIG3 and FIP1L1 have specific roles in transcription and may effect expression of other genes. Identified variants in these genes may thus have a role in the regulatory events leading to OA.