Association of genetic variation in COL11A1 with adolescent idiopathic scoliosis.

Adolescent idiopathic scoliosis (AIS) is a common and progressive spinal deformity in children that exhibits striking sexual dimorphism, with girls at more than fivefold greater risk of severe disease compared to boys. Despite its medical impact, the molecular mechanisms that drive AIS are largely unknown. We previously defined a female-specific AIS genetic risk locus in an enhancer near the PAX1 gene. Here, we sought to define the roles of PAX1 and newly identified AIS-associated genes in the developmental mechanism of AIS. In a genetic study of 10,519 individuals with AIS and 93,238 unaffected controls, significant association was identified with a variant in COL11A1 encoding collagen (α1) XI (rs3753841; NM_080629.2_c.4004C>T; p.(Pro1335Leu); p=7.07E-11, OR = 1.118). Using CRISPR mutagenesis we generated Pax1 knockout mice (Pax1-/-). In postnatal spines we found that PAX1 and collagen (α1) XI protein both localize within the intervertebral disc-vertebral junction region encompassing the growth plate, with less collagen (α1) XI detected in Pax1-/- spines compared to wild-type. By genetic targeting we found that wild-type Col11a1 expression in costal chondrocytes suppresses expression of Pax1 and of Mmp3, encoding the matrix metalloproteinase 3 enzyme implicated in matrix remodeling. However, the latter suppression was abrogated in the presence of the AIS-associated COL11A1P1335L mutant. Further, we found that either knockdown of the estrogen receptor gene Esr2 or tamoxifen treatment significantly altered Col11a1 and Mmp3 expression in chondrocytes. We propose a new molecular model of AIS pathogenesis wherein genetic variation and estrogen signaling increase disease susceptibility by altering a PAX1-COL11a1-MMP3 signaling axis in spinal chondrocytes.

Adolescent idiopathic scoliosis (AIS) is a twisting deformity of the spine that occurs during periods of rapid growth in children worldwide. Children with severe cases of AIS require surgery to stop it from getting worse, presenting a significant financial burden to health systems and families. Although AIS is known to cluster in families, its genetic causes and its inheritance pattern have remained elusive. Additionally, AIS is known to be more prevalent in females, a bias that has not been explained. Advances in techniques to study the genetics underlying diseases have revealed that certain variations that increase the risk of AIS affect cartilage and connective tissue. In humans, one such variation is near a gene called Pax1, and it is female-specific. The extracellular matrix is a network of proteins and other molecules in the space between cells that help connect tissues together, and it is particularly important in cartilage and other connective tissues. One of the main components of the extracellular matrix is collagen. Yu, Kanshour, Ushiki et al. hypothesized that changes in the extracellular matrix could affect the cartilage and connective tissues of the spine, leading to AIS. To show this, the scientists screened over 100,000 individuals and found that AIS is associated with variants in two genes coding for extracellular matrix proteins. One of these variants was found in a gene called Col11a1, which codes for one of the proteins that makes up collagen. To understand the relationship between Pax1 and Col11a1, Yu, Kanshour, Ushiki et al. genetically modified mice so that they would lack the Pax1 gene. In these mice, the activation of Col11a1 was reduced in the mouse spine. They also found that the form of Col11a1 associated with AIS could not suppress the activation of a gene called Mmp3 in mouse cartilage cells as effectively as unmutated Col11a1. Going one step further, the researchers found that lowering the levels of an estrogen receptor altered the activation patterns of Pax1, Col11a1, and Mmp3 in mouse cartilage cells. These findings suggest a possible mechanism for AIS, particularly in females. The findings of Yu, Kanshour, Ushiki et al. highlight that cartilage cells in the spine are particularly relevant in AIS. The results also point to specific molecules within the extracellular matrix as important for maintaining proper alignment in the spine when children are growing rapidly. This information may guide future therapies aimed at maintaining healthy spinal cells in adolescent children, particularly girls.

Association of genetic variation in COL11A1 with adolescent idiopathic scoliosis.

Adolescent idiopathic scoliosis (AIS) is a common and progressive spinal deformity in children that exhibits striking sexual dimorphism, with girls at more than five-fold greater risk of severe disease compared to boys. Despite its medical impact, the molecular mechanisms that drive AIS are largely unknown. We previously defined a female-specific AIS genetic risk locus in an enhancer near the PAX1 gene. Here we sought to define the roles of PAX1 and newly-identified AIS-associated genes in the developmental mechanism of AIS. In a genetic study of 10,519 individuals with AIS and 93,238 unaffected controls, significant association was identified with a variant in COL11A1 encoding collagen (α1) XI (rs3753841; NM_080629.2_c.4004C>T; p.(Pro1335Leu); P=7.07e-11, OR=1.118). Using CRISPR mutagenesis we generated Pax1 knockout mice (Pax1-/-). In postnatal spines we found that PAX1 and collagen (α1) XI protein both localize within the intervertebral disc (IVD)-vertebral junction region encompassing the growth plate, with less collagen (α1) XI detected in Pax1-/- spines compared to wildtype. By genetic targeting we found that wildtype Col11a1 expression in costal chondrocytes suppresses expression of Pax1 and of Mmp3, encoding the matrix metalloproteinase 3 enzyme implicated in matrix remodeling. However, this suppression was abrogated in the presence of the AIS-associated COL11A1P1335L mutant. Further, we found that either knockdown of the estrogen receptor gene Esr2, or tamoxifen treatment, significantly altered Col11a1 and Mmp3 expression in chondrocytes. We propose a new molecular model of AIS pathogenesis wherein genetic variation and estrogen signaling increase disease susceptibility by altering a Pax1-Col11a1-Mmp3 signaling axis in spinal chondrocytes.

Long-chain polyphosphates inhibit type I interferon signaling and augment LPS-induced cytokine secretion in human leukocytes.

Inorganic polyphosphates are evolutionarily conserved bioactive phosphate polymers found as various chain lengths in all living organisms. In mammals, polyphosphates play a vital role in the regulation of cellular metabolism, coagulation, and inflammation. Long-chain polyphosphates are found along with endotoxins in pathogenic gram-negative bacteria and can participate in bacterial virulence. We aimed to investigate whether exogenously administered polyphosphates modulate human leukocyte function in vitro by treating the cells with 3 different chain lengths of polyphosphates (P14, P100, and P700). The long-chain polyphosphates, P700, had a remarkable capacity to downregulate type I interferon signaling dose dependently in THP1-Dual cells while only a slight elevation could be observed in the NF-κB pathway with the highest dose of P700. P700 treatment decreased lipopolysaccharide-induced IFNß transcription and secretion, reduced STAT1 phosphorylation, and downregulated subsequent interferon-stimulated gene expression in primary human peripheral blood mononuclear cells. P700 also augmented lipopolysaccharide-induced secretion of IL-1α, IL-1ß, IL-4, IL-5, IL-10, and IFNγ. Furthermore, P700 has previously been reported to increase the phosphorylation of several intracellular signaling mediators, such as AKT, mTOR, ERK, p38, GSK3α/ß, HSP27, and JNK pathway components, which was supported by our findings. Taken together, these observations demonstrate the extensive modulatory effects P700 has on cytokine signaling and the inhibitory effects specifically targeted to type I interferon signaling in human leukocytes.

The role of CDHR3 in susceptibility to otitis media.

Otitis media (OM) is common in young children and can cause hearing loss and speech, language, and developmental delays. OM has high heritability; however, little is known about OM-related molecular and genetic processes. CDHR3 was previously identified as a locus for OM susceptibility, but to date, studies have focused on how the CDHR3 p.Cys529Tyr variant increases epithelial binding of rhinovirus-C and risk for lung or sinus pathology. In order to further delineate a role for CDHR3 in OM, we performed the following: exome sequencing using DNA samples from OM-affected individuals from 257 multi-ethnic families; Sanger sequencing, logistic regression and transmission disequilibrium tests for 407 US trios or probands with OM; 16S rRNA sequencing and analysis for middle ear and nasopharyngeal samples; and single-cell RNA sequencing and differential expression analyses for mouse middle ear. From exome sequence data, we identified a novel pathogenic CDHR3 splice variant that co-segregates with OM in US and Finnish families. Additionally, a frameshift and six missense rare or low-frequency variants were identified in Finnish probands. In US probands, the CDHR3 p.Cys529Tyr variant was associated with the absence of middle ear fluid at surgery and also with increased relative abundance of Lysobacter in the nasopharynx and Streptomyces in the middle ear. Consistent with published data on airway epithelial cells and our RNA-sequence data from human middle ear tissues, Cdhr3 expression is restricted to ciliated epithelial cells of the middle ear and is downregulated after acute OM. Overall, these findings suggest a critical role for CDHR3 in OM susceptibility. KEY MESSAGES: • Novel rare or low-frequency CDHR3 variants putatively confer risk for otitis media. • Pathogenic variant CDHR3 c.1653 + 3G > A was found in nine families with otitis media. • CDHR3 p.Cys529Tyr was associated with lack of effusion and bacterial otopathogens. • Cdhr3 expression was limited to ciliated epithelial cells in mouse middle ear. • Cdhr3 was downregulated 3 h after infection of mouse middle ear.

Nasal upregulation of CST1 in dog-sensitised children with severe allergic airway disease.

BACKGROUND: The clinical presentation of children sensitised to dog dander varies from asymptomatic to severe allergic airway disease, but the genetic mechanisms underlying these differences are not clear. The objective of the present study was to investigate nasal transcriptomic profiles associated with dog dander sensitisation in school children and to reveal clinical symptoms related with these profiles. METHODS: RNA was extracted from nasal epithelial cell brushings of children sensitised to dog dander and healthy controls. Blood sample analyses included IgE against dog dander, dog allergen molecules, other airborne and food allergens, basophil activation and white blood cell counts. Clinical history of asthma and rhinitis was recorded, and lung function was assessed (spirometry, methacholine provocation and exhaled nitric oxide fraction). RESULTS: The most overexpressed gene in children sensitised to dog dander compared to healthy controls was CST1, coding for Cystatin 1. A cluster of these children with enhanced CST1 expression showed lower forced expiratory volume in 1 s, increased bronchial hyperreactivity, pronounced eosinophilia and higher basophil allergen threshold sensitivity compared with other children sensitised to dog dander. In addition, multi-sensitisation to lipocalins was more common in this group. CONCLUSIONS: Overexpression of CST1 is associated with more severe allergic airway disease in children sensitised to dog dander. CST1 is thus a possible biomarker of the severity of allergic airway disease and a possible therapeutic target for the future treatment of airborne allergy.

Otitis media susceptibility and shifts in the head and neck microbiome due to SPINK5 variants.

BACKGROUND: Otitis media (OM) susceptibility has significant heritability; however, the role of rare variants in OM is mostly unknown. Our goal is to identify novel rare variants that confer OM susceptibility. METHODS: We performed exome and Sanger sequencing of >1000 DNA samples from 551 multiethnic families with OM and unrelated individuals, RNA-sequencing and microbiome sequencing and analyses of swabs from the outer ear, middle ear, nasopharynx and oral cavity. We also examined protein localisation and gene expression in infected and healthy middle ear tissues. RESULTS: A large, intermarried pedigree that includes 81 OM-affected and 53 unaffected individuals cosegregates two known rare A2ML1 variants, a common FUT2 variant and a rare, novel pathogenic variant c.1682A>G (p.Glu561Gly) within SPINK5 (LOD=4.09). Carriage of the SPINK5 missense variant resulted in increased relative abundance of Microbacteriaceae in the middle ear, along with occurrence of Microbacteriaceae in the outer ear and oral cavity but not the nasopharynx. Eight additional novel SPINK5 variants were identified in 12 families and individuals with OM. A role for SPINK5 in OM susceptibility is further supported by lower RNA counts in variant carriers, strong SPINK5 localisation in outer ear skin, faint localisation to middle ear mucosa and eardrum and increased SPINK5 expression in human cholesteatoma. CONCLUSION: SPINK5 variants confer susceptibility to non-syndromic OM. These variants potentially contribute to middle ear pathology through breakdown of mucosal and epithelial barriers, immunodeficiency such as poor vaccination response, alteration of head and neck microbiota and facilitation of entry of opportunistic pathogens into the middle ear.

Rare variants in dynein heavy chain genes in two individuals with situs inversus and developmental dyslexia: a case report.

BACKGROUND: Developmental dyslexia (DD) is a neurodevelopmental learning disorder with high heritability. A number of candidate susceptibility genes have been identified, some of which are linked to the function of the cilium, an organelle regulating left-right asymmetry development in the embryo. Furthermore, it has been suggested that disrupted left-right asymmetry of the brain may play a role in neurodevelopmental disorders such as DD. However, it is unknown whether there is a common genetic cause to DD and laterality defects or ciliopathies. CASE PRESENTATION: Here, we studied two individuals with co-occurring situs inversus (SI) and DD using whole genome sequencing to identify genetic variants of importance for DD and SI. Individual 1 had primary ciliary dyskinesia (PCD), a rare, autosomal recessive disorder with oto-sino-pulmonary phenotype and SI. We identified two rare nonsynonymous variants in the dynein axonemal heavy chain 5 gene (DNAH5): a previously reported variant c.7502G > C; p.(R2501P), and a novel variant c.12043 T > G; p.(Y4015D). Both variants are predicted to be damaging. Ultrastructural analysis of the cilia revealed a lack of outer dynein arms and normal inner dynein arms. MRI of the brain revealed no significant abnormalities. Individual 2 had non-syndromic SI and DD. In individual 2, one rare variant (c.9110A > G;p.(H3037R)) in the dynein axonemal heavy chain 11 gene (DNAH11), coding for another component of the outer dynein arm, was identified. CONCLUSIONS: We identified the likely genetic cause of SI and PCD in one individual, and a possibly significant heterozygosity in the other, both involving dynein genes. Given the present evidence, it is unclear if the identified variants also predispose to DD and further studies into the association between laterality, ciliopathies and DD are needed.

Novel TMEM173 Mutation and the Role of Disease Modifying Alleles.

Upon binding to pathogen or self-derived cytosolic nucleic acids cyclic GMP-AMP synthase (cGAS) triggers the production of cGAMP that further activates transmembrane protein STING. Upon activation STING translocates from ER via Golgi to vesicles. Monogenic STING gain-of-function mutations cause early-onset type I interferonopathy, with disease presentation ranging from fatal vasculopathy to mild chilblain lupus. Molecular mechanisms underlying the variable phenotype-genotype correlation are presently unclear. Here, we report a novel gain-of-function G207E STING mutation causing a distinct phenotype with alopecia, photosensitivity, thyroid dysfunction, and features of STING-associated vasculopathy with onset in infancy (SAVI), such as livedo reticularis, skin vasculitis, nasal septum perforation, facial erythema, and bacterial infections. Polymorphism in TMEM173 and IFIH1 showed variable penetrance in the affected family, implying contribution to varying phenotype spectrum. The G207E mutation constitutively activates inflammation-related pathways in vitro, and causes aberrant interferon signature and inflammasome activation in patient PBMCs. Treatment with Janus kinase 1 and 2 (JAK1/2) inhibitor baricitinib was beneficiary for a vasculitic ulcer, induced hair regrowth and improved overall well-being in one patient. Protein-protein interactions propose impaired cellular trafficking of G207E mutant. These findings reveal the molecular landscape of STING and propose common polymorphisms in TMEM173 and IFIH1 as likely modifiers of the phenotype.

The human long non-coding RNA gene RMRP has pleiotropic effects and regulates cell-cycle progression at G2.

RMRP was the first non-coding nuclear RNA gene implicated in a disease. Its mutations cause cartilage-hair hypoplasia (CHH), an autosomal recessive skeletal dysplasia with growth failure, immunodeficiency, and a high risk for malignancies. This study aimed to gain further insight into the role of RNA Component of Mitochondrial RNA Processing Endoribonuclease (RMRP) in cellular physiology and disease pathogenesis. We combined transcriptome analysis with single-cell analysis using fibroblasts from CHH patients and healthy controls. To directly assess cell cycle progression, we followed CHH fibroblasts by pulse-labeling and time-lapse microscopy. Transcriptome analysis identified 35 significantly upregulated and 130 downregulated genes in CHH fibroblasts. The downregulated genes were significantly connected to the cell cycle. Multiple other pathways, involving regulation of apoptosis, bone and cartilage formation, and lymphocyte function, were also affected, as well as PI3K-Akt signaling. Cell-cycle studies indicated that the CHH cells were delayed specifically in the passage from G2 phase to mitosis. Our findings expand the mechanistic understanding of CHH, indicate possible pathways for therapeutic intervention and add to the limited understanding of the functions of RMRP.

Pleomorphic Adenoma Gene 1 Is Needed For Timely Zygotic Genome Activation and Early Embryo Development.

Pleomorphic adenoma gene 1 (PLAG1) is a transcription factor involved in cancer and growth. We discovered a de novo DNA motif containing a PLAG1 binding site in the promoters of genes activated during zygotic genome activation (ZGA) in human embryos. This motif was located within an Alu element in a region that was conserved in the murine B1 element. We show that maternally provided Plag1 is needed for timely mouse preimplantation embryo development. Heterozygous mouse embryos lacking maternal Plag1 showed disrupted regulation of 1,089 genes, spent significantly longer time in the 2-cell stage, and started expressing Plag1 ectopically from the paternal allele. The de novo PLAG1 motif was enriched in the promoters of the genes whose activation was delayed in the absence of Plag1. Further, these mouse genes showed a significant overlap with genes upregulated during human ZGA that also contain the motif. By gene ontology, the mouse and human ZGA genes with de novo PLAG1 motifs were involved in ribosome biogenesis and protein synthesis. Collectively, our data suggest that PLAG1 affects embryo development in mice and humans through a conserved DNA motif within Alu/B1 elements located in the promoters of a subset of ZGA genes.

Gain-of-function CEBPE mutation causes noncanonical autoinflammatory inflammasomopathy.

BACKGROUND: CCAAT enhancer-binding protein epsilon (C/EBPε) is a transcription factor involved in late myeloid lineage differentiation and cellular function. The only previously known disorder linked to C/EBPε is autosomal recessive neutrophil-specific granule deficiency leading to severely impaired neutrophil function and early mortality. OBJECTIVE: The aim of this study was to molecularly characterize the effects of C/EBPε transcription factor Arg219His mutation identified in a Finnish family with previously genetically uncharacterized autoinflammatory and immunodeficiency syndrome. METHODS: Genetic analysis, proteomics, genome-wide transcriptional profiling by means of RNA-sequencing, chromatin immunoprecipitation (ChIP) sequencing, and assessment of the inflammasome function of primary macrophages were performed. RESULTS: Studies revealed a novel mechanism of genome-wide gain-of-function that dysregulated transcription of 464 genes. Mechanisms involved dysregulated noncanonical inflammasome activation caused by decreased association with transcriptional repressors, leading to increased chromatin occupancy and considerable changes in transcriptional activity, including increased expression of NLR family, pyrin domain-containing 3 protein (NLRP3) and constitutively expressed caspase-5 in macrophages. CONCLUSION: We describe a novel autoinflammatory disease with defective neutrophil function caused by a homozygous Arg219His mutation in the transcription factor C/EBPε. Mutated C/EBPε acts as a regulator of both the inflammasome and interferome, and the Arg219His mutation causes the first human monogenic neomorphic and noncanonical inflammasomopathy/immunodeficiency. The mechanism, including widely dysregulated transcription, is likely not unique for C/EBPε. Similar multiomics approaches should also be used in studying other transcription factor-associated diseases.

Mlh1 deficiency in normal mouse colon mucosa associates with chromosomally unstable colon cancer.

Colorectal cancer (CRC) genome is unstable and different types of instabilities, such as chromosomal instability (CIN) and microsatellite instability (MSI) are thought to reflect distinct cancer initiating mechanisms. Although 85% of sporadic CRC reveal CIN, 15% reveal mismatch repair (MMR) malfunction and MSI, the hallmarks of Lynch syndrome with inherited heterozygous germline mutations in MMR genes. Our study was designed to comprehensively follow genome-wide expression changes and their implications during colon tumorigenesis. We conducted a long-term feeding experiment in the mouse to address expression changes arising in histologically normal colonic mucosa as putative cancer preceding events, and the effect of inherited predisposition (Mlh1+/-) and Western-style diet (WD) on those. During the 21-month experiment, carcinomas developed mainly in WD-fed mice and were evenly distributed between genotypes. Unexpectedly, the heterozygote (B6.129-Mlh1tm1Rak) mice did not show MSI in their CRCs. Instead, both wildtype and heterozygote CRC mice showed a distinct mRNA expression profile and shortage of several chromosomal segregation gene-specific transcripts (Mlh1, Bub1, Mis18a, Tpx2, Rad9a, Pms2, Cenpe, Ncapd3, Odf2 and Dclre1b) in their colon mucosa, as well as an increased mitotic activity and abundant numbers of unbalanced/atypical mitoses in tumours. Our genome-wide expression profiling experiment demonstrates that cancer preceding changes are already seen in histologically normal colon mucosa and that decreased expressions of Mlh1 and other chromosomal segregation genes may form a field-defect in mucosa, which trigger MMR-proficient, chromosomally unstable CRC.

Unexpectedly High Prevalence of Common Variable Immunodeficiency in Finland.

BACKGROUND: Common variable immunodeficiency (CVID) is the most common primary immunodeficiency. Prevalence varies greatly between countries and studies. Most diagnostic criteria include hypogammaglobulinemia and impaired vaccine response. AIM: To evaluate the minimum prevalence as well as the clinical and immunological phenotypes of CVID in Southern Finland. METHODS: We performed a cross-sectional study to assess all adult CVID patients followed up in three hospital districts in Southern and South-Eastern Finland between April 2007 and August 2015. CVID diagnosis was based, with a minor modification, on the ESID/PAGID criteria for primary CVID. Antipolysaccharide responses to Pneumovax® were defined as impaired only if 50% or more of the serotypes did not reach a level of 0.35 µg/mL after vaccination. We further characterized the patients' B cell phenotypes and complications associated with CVID. RESULTS: In total, 9 patients were excluded due to potential secondary causes before diagnosis. ESID/PAGID criteria were met by 132 patients (males 52%), of whom, 106 had "probable" and 26 "possible CVID." Based on the population statistics in the three hospital districts, the minimum adult prevalence per 100,000 inhabitants in Finland for all CVID ("probable CVID," respectively) patients was 6.9 (5.5). In the highest prevalence district (Helsinki and Uusimaa), the prevalence was 7.7 (6.1). CVID patients suffer from frequent complications. Ten patients died during follow-up. Of probable CVID patients, 73% had more than one clinical phenotype. Intriguingly, gradual B cell loss from peripheral blood during follow-up was seen in as many as 16% of "probable CVID" patients. Patients with possible CVID displayed somewhat milder clinical and laboratory phenotypes than probable CVID patients. We also confirm that large granular lymphocyte lymphoproliferation is a CVID-associated complication. CONCLUSION: The prevalence of CVID in Finland appears the highest recorded, likely reflecting the genetic isolation and potential founder effects in the Finnish population. Studies to discover potential gene variants responsible for the high prevalence in Finland thus seem warranted. Increased awareness of CVID among physicians would likely lead to earlier diagnosis and improved quality of care.

A mouse-to-man candidate gene study identifies association of chronic otitis media with the loci TGIF1 and FBXO11.

Chronic otitis media with effusion (COME) is the most common cause of hearing loss in children, and known to have high heritability. Mutant mouse models have identified Fbxo11, Evi1, Tgif1, and Nisch as potential risk loci. We recruited children aged 10 and under undergoing surgical treatment for COME from 35 hospitals in the UK, and their nuclear family. We performed association testing with the loci FBXO11, EVI1, TGIF1 and NISCH and sought to replicate significant results in a case-control cohort from Finland. We tested 1296 families (3828 individuals), and found strength of association with the T allele at rs881835 (p = 0.006, OR 1.39) and the G allele at rs1962914 (p = 0.007, OR 1.58) at TGIF1, and the A allele at rs10490302 (p = 0.016, OR 1.17) and the G allele at rs2537742 (p = 0.038, OR 1.16) at FBXO11. Results were not replicated. This study supports smaller studies that have also suggested association of otitis media with polymorphism at FBX011, but this is the first study to report association with the locus TGIF1. Both FBX011 and TGIF1 are involved in TGF-ß signalling, suggesting this pathway may be important in the transition from acute to chronic middle ear inflammation, and a potential molecular target.

Estrogen receptor ß, a regulator of androgen receptor signaling in the mouse ventral prostate.

As estrogen receptor ß-/- (ERß-/-) mice age, the ventral prostate (VP) develops increased numbers of hyperplastic, fibroplastic lesions and inflammatory cells. To identify genes involved in these changes, we used RNA sequencing and immunohistochemistry to compare gene expression profiles in the VP of young (2-mo-old) and aging (18-mo-old) ERß-/- mice and their WT littermates. We also treated young and old WT mice with an ERß-selective agonist and evaluated protein expression. The most significant findings were that ERß down-regulates androgen receptor (AR) signaling and up-regulates the tumor suppressor phosphatase and tensin homolog (PTEN). ERß agonist increased expression of the AR corepressor dachshund family (DACH1/2), T-cadherin, stromal caveolin-1, and nuclear PTEN and decreased expression of RAR-related orphan receptor c, Bcl2, inducible nitric oxide synthase, and IL-6. In the ERß-/- mouse VP, RNA sequencing revealed that the following genes were up-regulated more than fivefold: Bcl2, clusterin, the cytokines CXCL16 and -17, and a marker of basal/intermediate cells (prostate stem cell antigen) and cytokeratins 4, 5, and 17. The most down-regulated genes were the following: the antioxidant gene glutathione peroxidase 3; protease inhibitors WAP four-disulfide core domain 3 (WFDC3); the tumor-suppressive genes T-cadherin and caveolin-1; the regulator of transforming growth factor ß signaling SMAD7; and the PTEN ubiquitin ligase NEDD4. The role of ERß in opposing AR signaling, proliferation, and inflammation suggests that ERß-selective agonists may be used to prevent progression of prostate cancer, prevent fibrosis and development of benign prostatic hyperplasia, and treat prostatitis.

Comprehensive mapping of the effects of azacitidine on DNA methylation, repressive/permissive histone marks and gene expression in primary cells from patients with MDS and MDS-related disease.

Azacitidine (Aza) is first-line treatment for patients with high-risk myelodysplastic syndromes (MDS), although its precise mechanism of action is unknown. We performed the first study to globally evaluate the epigenetic effects of Aza on MDS bone marrow progenitor cells assessing gene expression (RNA seq), DNA methylation (Illumina 450k) and the histone modifications H3K18ac and H3K9me3 (ChIP seq). Aza induced a general increase in gene expression with 924 significantly upregulated genes but this increase showed no correlation with changes in DNA methylation or H3K18ac, and only a weak association with changes in H3K9me3. Interestingly, we observed activation of transcripts containing 15 endogenous retroviruses (ERVs) confirming previous cell line studies. DNA methylation decreased moderately in 99% of all genes, with a median ß-value reduction of 0.018; the most pronounced effects seen in heterochromatin. Aza-induced hypomethylation correlated significantly with change in H3K9me3. The pattern of H3K18ac and H3K9me3 displayed large differences between patients and healthy controls without any consistent pattern induced by Aza. We conclude that the marked induction of gene expression only partly could be explained by epigenetic changes, and propose that activation of ERVs may contribute to the clinical effects of Aza in MDS.

Damaging heterozygous mutations in NFKB1 lead to diverse immunologic phenotypes.

BACKGROUND: The nuclear factor κ light-chain enhancer of activated B cells (NF-κB) signaling pathway is a key regulator of immune responses. Accordingly, mutations in several NF-κB pathway genes cause immunodeficiency. OBJECTIVE: We sought to identify the cause of disease in 3 unrelated Finnish kindreds with variable symptoms of immunodeficiency and autoinflammation. METHODS: We applied genetic linkage analysis and next-generation sequencing and functional analyses of NFKB1 and its mutated alleles. RESULTS: In all affected subjects we detected novel heterozygous variants in NFKB1, encoding for p50/p105. Symptoms in variant carriers differed depending on the mutation. Patients harboring a p.I553M variant presented with antibody deficiency, infection susceptibility, and multiorgan autoimmunity. Patients with a p.H67R substitution had antibody deficiency and experienced autoinflammatory episodes, including aphthae, gastrointestinal disease, febrile attacks, and small-vessel vasculitis characteristic of Behçet disease. Patients with a p.R157X stop-gain experienced hyperinflammatory responses to surgery and showed enhanced inflammasome activation. In functional analyses the p.R157X variant caused proteasome-dependent degradation of both the truncated and wild-type proteins, leading to a dramatic loss of p50/p105. The p.H67R variant reduced nuclear entry of p50 and showed decreased transcriptional activity in luciferase reporter assays. The p.I553M mutation in turn showed no change in p50 function but exhibited reduced p105 phosphorylation and stability. Affinity purification mass spectrometry also demonstrated that both missense variants led to altered protein-protein interactions. CONCLUSION: Our findings broaden the scope of phenotypes caused by mutations in NFKB1 and suggest that a subset of autoinflammatory diseases, such as Behçet disease, can be caused by rare monogenic variants in genes of the NF-κB pathway.

Targeted high-throughput sequencing of candidate genes for chronic obstructive pulmonary disease.

BACKGROUND: Reduced lung function in patients with chronic obstructive pulmonary disease (COPD) is likely due to both environmental and genetic factors. We report here a targeted high-throughput DNA sequencing approach to identify new and previously known genetic variants in a set of candidate genes for COPD. METHODS: Exons in 22 genes implicated in lung development as well as 61 genes and 10 genomic regions previously associated with COPD were sequenced using individual DNA samples from 68 cases with moderate or severe COPD and 66 controls matched for age, gender and smoking. Cases and controls were selected from the Obstructive Lung Disease in Northern Sweden (OLIN) studies. RESULTS: In total, 37 genetic variants showed association with COPD (p < 0.05, uncorrected). Several variants previously discovered to be associated with COPD from genetic genome-wide analysis studies were replicated using our sample. Two high-risk variants were followed-up for functional characterization in a large eQTL mapping study of 1,111 human lung specimens. The C allele of a synonymous variant, rs8040868, predicting a p.(S45=) in the gene for cholinergic receptor nicotinic alpha 3 (CHRNA3) was associated with COPD (p = 8.8 x 10-3). This association remained (p = 0.003 and OR = 1.4, 95 % CI 1.1-1.7) when analysing all available cases and controls in OLIN (n = 1,534). The rs8040868 variant is in linkage disequilibrium with rs16969968 previously associated with COPD and altered expression of the CHRNA5 gene. A follow-up analysis for detection of expression quantitative trait loci revealed that rs8040868-C was found to be significantly associated with a decreased expression of the nearby gene cholinergic receptor, nicotinic, alpha 5 (CHRNA5) in lung tissue. CONCLUSION: Our data replicate previous result suggesting CHRNA5 as a candidate gene for COPD and rs8040868 as a risk variant for the development of COPD in the Swedish population.