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Neurogenic heterotopic ossifications (NHOs) form in periarticular muscles following severe spinal cord (SCI) and traumatic brain injuries. The pathogenesis of NHO is poorly understood with no effective preventive treatment. The only curative treatment remains surgical resection of pathological NHOs. In a mouse model of SCI-induced NHO that involves a transection of the spinal cord combined with a muscle injury, a differential gene expression analysis revealed that genes involved in inflammation such as interleukin-1β (IL-1β) were overexpressed in muscles developing NHO. Using mice knocked-out for the gene encoding IL-1 receptor (IL1R1) and neutralizing antibodies for IL-1α and IL-1β, we show that IL-1 signaling contributes to NHO development following SCI in mice. Interestingly, other proteins involved in inflammation that were also overexpressed in muscles developing NHO, such as colony-stimulating factor-1, tumor necrosis factor or C-C chemokine ligand-2 did not promote NHO development. Finally using NHO biopsies from SCI and TBI patients, we show that IL-1β is expressed by CD68+ macrophages. IL-1α and IL-1β produced by activated human monocytes promote calcium mineralization and RUNX2 expression in fibro-adipogenic progenitors isolated from muscles surrounding NHOs. Altogether these data suggest that interleukin-1 promotes NHO development in both humans and mice. This article is protected by copyright. All rights reserved.

Lung cancer is the commonest cause of cancer deaths worldwide. Although strongly associated with smoking, predisposition to lung cancer is also heritable, with multiple common risk variants identified. Rarely, dominantly inherited non-small-cell lung cancer (NSCLC) has been reported due to somatic mutations in EGFR/ErbB1 and ERBB2. Germline exome sequencing was performed in a multi-generation family with autosomal dominant NSCLC, including an affected child. Tumour samples were also sequenced. Full-length wild-type (wtErbB3) and mutant ERBB3 (mutErbB3) constructs were transfected into HeLa cells. Protein expression, stability, and subcellular localization were assessed, and cellular proliferation, pAkt/Akt and pERK levels determined. A novel germline variant in ERBB3 (c.1946 T > G: p.Iso649Arg), coding for receptor tyrosine-protein kinase erbB-3 (ErbB3), was identified, with appropriate segregation. There was no loss-of-heterozygosity in tumour samples. Both wtErbB3 and mutErbB3 were stably expressed. MutErbB3-transfected cells demonstrated an increased ratio of the 80 kDa form (which enhances proliferation) compared with the full-length (180 kDa) form. MutErbB3 and wtErbB3 had similar punctate cytoplasmic localization pre- and post-epidermal growth factor stimulation; however, epidermal growth factor receptor (EGFR) levels decreased faster post-stimulation in mutErbB3-transfected cells, suggesting more rapid processing of the mutErbB3/EGFR heterodimer. Cellular proliferation was increased in mutErbB3-transfected cells compared with wtErbB3 transfection. MutErbB3-transfected cells also showed decreased pAkt/tAkt ratios and increased pERK/tERK 30 min post-stimulation compared with wtErbB3 transfection, demonstrating altered signalling pathway activation. Cumulatively, these results support this mutation as tumorogenic. This is the first reported family with a germline ERBB3 mutation causing heritable NSCLC, furthering understanding of the ErbB family pathway in oncogenesis.

Background. Next-generation sequencing (NGS) technology enables profiling of individual tumours and to measure the increasing number of biomarkers relevant to the management of breast cancer patients. The Australian Translational Genomics Centre (ATGC) is a collaboration between the healthcare sector (Metro South Hospital and Health Service), higher education sector (Queensland University of Technology) and a government-run pathology service (Pathology Queensland) to provide genomic profiling of breast cancer patients at the Princess Alexandra Hospital. The program was developed to integrate the ordering, processing and interpretation of large-scale NGS into clinical practice. Methods. Patients were consented for somatic testing and ordering was integrated into the hospital’s electronic ordering system. Samples were derived from fresh tissue biopsies after surgeries (47%) or from FFPE histology sections. No selection criteria were applied during the initial phase and the cohort was representative of newly presenting patients at the hospitals breast cancer clinic. Patients were sequenced using a NATA-accredited ISO15189 program using whole-exome sequencing (WES) combined with a high-coverage spike-in panel of known cancer genes. Clinical reports included the calculation of tumour purity, tumour mutational burden (TMB), the assessment of copy number events and somatic mutations down to 3% allele frequency. Standard molecular testing in Australia includes ER, PR and HER2 status, and additional testing included testing of Tier 1-2 somatic variants in the genes ABCC3, AKT1, CCND1, CCNE1, CDKN2A, ERBB2, ESR1, FGF3, FGFR1, FGFR2, MTOR, NCOA3, NF2, PIK3CA, PIK3R1, PTEN, RB1, RSF1, SF3B1, TP53. Results. Seventy-one patients were tested by WES/panel, and an average of 1.5 clinically significant Tier 1- 2 mutations were detected per patient. In 77% of cases, the molecular profiling could stratify patients to those with either PI3K/Akt/mTOR pathway activation (by PIK3CA activating mutations, AKT, MTOR mutations or PTEN loss) or CDK4/6 activation (by CCND1 expansion or CDKN2A loss). The most frequently observed mutations were PIK3CA activation (40%) and CCND1 copy number expansions (24%). A small proportion (n=3) were found to have mTOR or TCS1/2 mutations reported to have association with a durable response to mTOR inhibitors. Additionally, 11 patients (15%) had a high TMB (TMB, >6.8 mutations/megabase) with 6 having >10 mutations/Mb. Within this high-TMB cohort, 3 were found to be ER-PR-HER-, however, the majority (n=7) were ER+PR+HER2- patients. Calculation of the tumour purity indicated that, despite expert resection of the biopsies to isolate the most tumour dense regions, tumour purity was not significantly enriched. In 58% of cases, the tumour purity was less than 50%, and in 13% of samples, it was less than 25%, indicating that in clinical practice the method of sequencing must be robust, as many samples have significant amounts of contaminating stroma. Conclusion. We demonstrate that integration of WES/panel testing into clinical practice is practical and provides multiplexed testing of current and emerging biomarkers in a significant number of tested patients. While a high proportion of patients had mutations that could stratify them to targeted therapies in the event of metastatic disease, the somatic molecular profiles did not modify first line therapy decision-making. Training of clinical staff for patient consent and the dissemination of findings, the development of a dedicated molecular tumour board, and decision protocols to identify patients of metastatic risk were identified as key developments in this clinical program. The program identified previously unidentified subsets of patients including a subset of ER+ patients with high TMB for which for there are no effective treatments option in Australia. Citation Format: Kate Roberts, Paul J Leo, Jeremy Khoo, Alice Febery, Jonathan Ellis, Mhairi Clout, Lawrie Wheeler, Lisa Anderson, Matthew Brown, Ian Bennett. Establishing whole-exome sequencing for breast cancer patient care [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-07-07.

ObjectiveWe sought to test the hypothesis that Polygenic Risk Scores (PRSs) have strong capacity to discriminate cases of ankylosing spondylitis (AS) from healthy controls and individuals in the community with chronic back pain.MethodsPRSs were developed and validated in individuals of European and East Asian ethnicity, using data from genome-wide association studies in 15 585 AS cases and 20 452 controls. The discriminatory values of PRSs in these populations were compared with other widely used diagnostic tests, including C-reactive protein (CRP), HLA-B27 and sacroiliac MRI.ResultsIn people of European descent, PRS had high discriminatory capacity with area under the curve (AUC) in receiver operator characteristic analysis of 0.924. This was significantly better than for HLA-B27 testing alone (AUC=0.869), MRI (AUC=0.885) or C-reactive protein (AUC=0.700). PRS developed and validated in individuals of East Asian descent performed similarly (AUC=0.948). Assuming a prior probability of AS of 10% such as in patients with chronic back pain under 45 years of age, compared with HLA-B27 testing alone, PRS provides higher positive values for 35% of patients and negative predictive values for 67.5% of patients. For PRS, in people of European descent, the maximum positive predictive value was 78.2% and negative predictive value was 100%, whereas for HLA-B27, these values were 51.9% and 97.9%, respectively.ConclusionsPRS have higher discriminatory capacity for AS than CRP, sacroiliac MRI or HLA-B27 status alone. For optimal performance, PRS should be developed for use in the specific ethnic groups to which they are to be applied.

BackgroundLung cancer is the commonest cause of cancer deaths worldwide. Although strongly associated with smoking, predisposition to lung cancer is also heritable with multiple common risk variants identified. Rarely, dominantly inherited non-small-cell lung cancer (NSCLC) has been reported due to somatic mutations in EGFR/ErbB1 and ERBB2.MethodsGermline exome sequencing was performed in a multi-generation family with autosomal dominant NSCLC, including an affected child. Tumour samples were also sequenced. Full-length wild-type (wtErbB3) and mutant ERBB3 (mutErbB3) constructs were transfected into HeLa cells. Protein expression, stability, and sub-cellular localisation were assessed; and cellular proliferation, pAkt/Akt, and pERK levels were determined.ResultsA novel germline variant in ERBB3 (c.1946T>G: p.Iso649Arg), coding for receptor tyrosineprotein kinase erbB-3 (ErbB3), was identified, with appropriate segregation. There was no loss-of-heterozygosity in tumour samples. Both wtErbB3 and mutErbB3 were stably expressed. MutErbB3-transfected cells demonstrated an increased ratio of the 80kD form (which enhances proliferation) compared to the full-length (180kD) form. MutErbB3 and wtErbB3 had similar punctate cytoplasmic localisation pre- and post-EGF stimulation; however, EGFR levels decreased faster post-stimulation in mutErbB3-transfected cells, suggesting more rapid processing of the mutErbB3/EGFR heterodimer. Cellular proliferation was increased in mutErbB3-transfected cells compared to wtErbB3 transfection. MutErbB3-transfected cells also showed decreased pAkt/tAkt ratios and increased pERK/tERK 30 minutes post-stimulation compared to wtErbB3 transfection, demonstrating altered signalling pathway activation by mutErbB3. Cumulatively, these results support this mutation as tumorogenic.ConclusionsThis is the first reported family with a germline ERBB3 mutation causing heritable NSCLC, furthering understanding of the ErbB family pathway in oncogenesis.

Background Marfan syndrome (MFS) is a dominant monogenic disorder caused by mutations in fibrillin 1 (FBN1). Rarely, compound heterozygosity for FBN1 mutations has been described. Methods A large kindred with MFS was assessed clinically over decades, and genetically using exome and/or Sanger sequencing. Results A previously identified FBN1 missense variant (p.Tyr754Cys) was confirmed in all subjects with MFS. An additional variant (p.Met2273Thr), previously associated with incomplete MFS, was identified in three siblings. These three compound heterozygous individuals had aortic dilatation at early age (all 40 years but not requiring surgical intervention. Another heterozygous (p.Tyr754Cys) sibling did require aortic root repair (28 years). The heterozygous (p.Met2273Thr) mother had aortic dilatation diagnosed at age 68 years but has not required surgical repair. Conclusion Although compound heterozygosity or homozygosity is rare in MFS, it should be considered when there is an unusually severe phenotype in a subset of family members., Marfan syndrome (MFS) is a dominant monogenic disorder caused by mutations in FBN1. Compound heterozygous mutations in FBN1 were identified in a large family with Marfan syndrome. Although compound heterozygosity or homozygosity is rare in MFS, it should be considered when there is an unusually severe phenotype in a subset of family members.

We previously reported exome sequencing in a short-rib thoracic dystrophy (SRTD) cohort, in whom recessive mutations were identified in SRTD-associated genes in 10 of 11 cases. A heterozygous stop mutation in the known SRTD gene WDR60 was identified in the remaining case; no novel candidate gene/s were suggested by homozygous/compound heterozygous analysis. This case was thus considered unsolved. Re-analysis following an analysis pipeline update identified a homozygous mutation in C21orf2 (c.218G > C; p.Arg73Pro). This homozygous variant was previously removed at the quality control stage by the default GATK parameter “in-breeding co-efficient.” C21orf2 was recently associated with both Jeune asphyxiating thoracic dystrophy (JATD) and axial spondylometaphyseal dysplasia (axial SMD); this particular mutation was reported in homozygous and compound heterozygous state in both conditions. Our case has phenotypic features of both JATD and axial SMD; and the extent of thoracic involvement appears more severe than in other C21orf2-positive cases. Identification of a homozygous C21orf2 mutation in this case emphasizes the value of exome sequencing for simultaneously screening known genes and identifying novel genes. Additionally, it highlights the importance of re-interrogating data both as novel gene associations are identified and as analysis pipelines are refined. Finally, the severity of thoracic restriction in this case adds to the phenotypic spectrum attributable to C21orf2 mutations.

Meta-analysis of 36,760 cases and 375,188 controls identifies 54 loci associated with susceptibility to cutaneous melanoma. Further analysis combining nevus count and hair color GWAS results provide insights into the genetic architecture of melanoma.Most genetic susceptibility to cutaneous melanoma remains to be discovered. Meta-analysis genome-wide association study (GWAS) of 36,760 cases of melanoma (67% newly genotyped) and 375,188 controls identified 54 significant (P < 5 x 10(-8)) loci with 68 independent single nucleotide polymorphisms. Analysis of risk estimates across geographical regions and host factors suggests the acral melanoma subtype is uniquely unrelated to pigmentation. Combining this meta-analysis with GWAS of nevus count and hair color, and transcriptome association approaches, uncovered 31 potential secondary loci for a total of 85 cutaneous melanoma susceptibility loci. These findings provide insights into cutaneous melanoma genetic architecture, reinforcing the importance of nevogenesis, pigmentation and telomere maintenance, together with identifying potential new pathways for cutaneous melanoma pathogenesis.

Çalışmada 30 yazar bulunmaktadır. Bu yazarlardan sadece Bursa Uludağ Üniversitesi mensuplarının girişleri yapılmıştır. Ankylosing spondylitis (AS) is a highly heritable immune-mediated arthritis common in Turkish and Iranian populations. Familial Mediterranean Fever (FMF) is an autosomal recessive autoinflammatory disease most common in people of Mediterranean origin. MEFV, an FMF-associated gene, is also a candidate gene for AS. We aimed to identify AS susceptibility loci and also examine the association between MEFV and AS in Turkish and Iranian cohorts. We performed genome-wide association studies in 1001 Turkish AS patients and 1011 Turkish controls, and 479 Iranian AS patients and 830 Iranian controls. Serum IL-1, IL-17 and IL-23 cytokine levels were quantified in Turkish samples. An association of major effect was observed with a novel rare coding variant in MEFV in the Turkish cohort (rs61752717, M694V, OR = 5.3, P = 7.63x10(-12)), Iranian cohort (OR = 2.9, P = 0.042), and combined dataset (OR = 5.1, P = 1.65x10(-13)). 99.6% of Turkish AS cases, and 96% of those carrying MEFV rs61752717 variants, did not have FMF. In Turkish subjects, the association of rs61752717 was particularly strong in HLA-B27-negative cases (OR = 7.8, P = 8.93x10(-15)), but also positive in HLA-B27-positive cases (OR = 4.3, P = 7.69x10(-8)). Serum IL-1, IL-17 and IL-23 levels were higher in AS cases than controls. Among AS cases, serum IL-1 and IL-23 levels were increased in MEFV 694V carriers compared with non-carriers. Our data suggest that FMF and AS have overlapping aetiopathogenic mechanisms. Functionally important MEFV mutations, such as M694V, lead to dysregulated inflammasome function and excessive IL-1 function. As IL-1 inhibition is effective in FMF, AS cases carrying FMF-associated MEFV variants may benefit from such therapy. Author summary Ankylosing spondylitis (AS) is a highly heritable immune-mediated arthritis. To identify new genetic associations with AS, we performed genome-wide association studies in Turkish and Iranian AS patients and controls. We identified a novel rare coding MEFV variant associated with AS. Rare polymorphisms of MEFV, which encodes the protein pyrin, are known to cause Familial Mediterranean Fever (FMF), a monogenic, autosomal recessive, autoinflammatory disease which can be complicated by arthritis. 99.6% of Turkish AS cases, and 96% of those carrying the MEFV variant, did not have FMF, and the association with AS remains excluding cases with FMF. In Turkish subjects, the MEFV variant association was particularly strong in HLA-B27-negative cases, but also positive in HLA-B27-positive cases. This represents the first rare variant association with AS, and has the highest odds ratio for AS of any non-MHC reported hitherto, indicating a major effect on disease pathogenesis. We assessed serum cytokine levels in the cohort, and found that IL-1, IL-17 and IL-23 levels were higher in AS cases. Furthermore, among AS cases, IL-1 and IL-23 levels were increased in MEFV variant carriers compared with non-carriers. This study has therapeutic implications; as IL-1 inhibition is effective in FMF, AS cases carrying FMF-associated MEFV variants may benefit from such therapy. National Health and Medical Research Council (NHMRC) of Australia Pfizer

Fryns syndrome is an autosomal recessive condition characterized by congenital diaphragmatic hernia (CDH), dysmorphic facial features, distal digital hypoplasia, and other associated malformations, and is the most common syndromic form of CDH. No gene has been associated with this condition. Whole-exome sequence data from two siblings and three unrelated individuals with Fryns syndrome were filtered for rare, good quality, coding mutations fitting a recessive inheritance model. Compound heterozygous mutations in PIGN were identified in the siblings, with appropriate parental segregation: a novel STOP mutation (c.1966CT: p.Glu656X) and a rare (minor allele frequency0.001) donor splice site mutation (c.1674+1GC) causing skipping of exon 18 and utilization of a cryptic acceptor site in exon 19. A further novel homozygous STOP mutation in PIGN (c.694AT: p.Lys232X) was detected in one unrelated case. All three variants affected highly conserved bases. The two remaining cases were negative for PIGN mutations. Mutations in PIGN have been reported in cases with multiple congenital anomalies, including one case with syndromic CDH. Fryns syndrome can be caused by recessive mutations in PIGN. Whether PIGN affects other syndromic and non-syndromic forms of CDH warrants investigation.

Objectives Ankylosing spondylitis (AS) is the most prevalent form of spondyloarthritis, with a known genetic association with the HLA-B27 molecule. The aim of this study was to assess the contribution of the HLA-G, HLA-E and HLA-F to AS susceptibility/protection in Portuguese patients with HLA-B27 AS and HLA-B27 unaffected controls. Methods High-resolution typing of HLA-G, HLA-E and HLA-F was performed in 228 patients with HLA-B27 AS and 244 HLA-B27 unaffected controls. Allelic, genotypic and haplotypic frequencies were compared between cohorts. To replicate the results, single nucleotide polymorphisms (SNPs) in HLA-E and HLA-F genes were typed in Australian cohorts. For further confirmation, a group of European-descent patients with AS and unaffected controls were genotyped for Major Histocompatibility Complex SNPs using the Illumina microarray. Results In the Portuguese population, no significant differences were found in HLA-G. For HLA-E, a significant difference was detected for the genotype HLA-E*01:01:01/01:03:01 (p=0.009; pc=0.009; OR=0.51), with a protection effect. For HLA-F, significant differences were detected in the allele HLA-F*01:01:02 (p=0.0049; pc=0.0098; OR=0.60) and corresponding SNP rs2075682 (p=0.0004; pc=0.0008; OR=0.53), suggesting protection and in the genotype HLA-F*01:01:01/01:03:01 (p=0.011; pc=0.043; OR=2.00), suggesting a susceptibility effect. Three G-E-F haplotypes with significant differences were detected but occur in a very small number of individuals. The only significant differences detected in the replication studies were for HLA-E rs1059510 in the Australians and for HLA-F rs1736924 in the European-descent cohorts. Conclusion Our results reveal suggestive AS protective and susceptibility effects from both HLA-E and HLA-F loci, however with population differences. To our knowledge, this is the first study showing association of HLA-F with AS.

BackgroundGiant cell arteritis (GCA) is the most common form of vasculitis affecting elderly people. It is one of the few true ophthalmic emergencies. GCA is a heterogenous disease, symptoms and signs are variable thereby making it challenging to diagnose and often delaying diagnosis. A temporal artery biopsy is the gold standard to test for GCA, and there are currently no specific biochemical markers to categorize or aid diagnosis of the disease. We aimed to identify a less invasive method to confirm the diagnosis of GCA, as well as to ascertain clinically relevant predictive biomarkers by studying the transcriptome of purified peripheral CD4+ and CD8+ T lymphocytes in patients with GCA.Methods and FindingsWe recruited 16 patients with histological evidence of GCA at the Royal Victorian Eye and Ear Hospital (RVEEH), Melbourne, Australia, and aimed to collect blood samples at six time points: acute phase, 2–3 weeks, 6–8 weeks, 3 months, 6 months and 12 months after clinical diagnosis. CD4+ and CD8+ T-cells were positively selected at each time point through magnetic-assisted cell sorting (MACS). RNA was extracted from all 195 collected samples for subsequent RNA sequencing. The expression profiles of patients were compared to those of 16 age-matched controls. Over the 12-month study period, polynomial modelling analyses identified 179 and 4 statistically significant transcripts with altered expression profiles (FDR < 0.05) between cases and controls in CD4+ and CD8+ populations, respectively. In CD8+ cells, we identified two transcripts that remained differentially expressed after 12 months, namely SGTB, associated with neuronal apoptosis, and FCGR3A, which has been found in association with Takayasu arteritis (TA), another large vessel vasculitis. We detected genes that correlate with both symptoms and biochemical markers used in the acute setting for predicting long-term prognosis. 15 genes were shared across 3 phenotypes in CD4 and 16 across CD8 cells. In CD8, IL32 was common to 5 phenotypes: a history of Polymyalgia Rheumatica, both visual disturbance and raised neutrophils at the time of presentation, bilateral blindness and death within 12 months. Altered IL32 gene expression could provide risk evaluation of GCA diagnosis at the time of presentation and give an indication of prognosis, which may influence management.ConclusionsThis is the first longitudinal gene expression study undertaken to identify robust transcriptomic biomarkers of GCA. Our results show cell type-specific transcript expression profiles, novel gene-phenotype associations, and uncover important biological pathways for this disease. These data significantly enhance the current knowledge of relevant biomarkers, their association with clinical prognostic markers, as well as potential candidates for detecting disease activity in whole blood samples. In the acute phase, the gene-phenotype relationships we have identified could provide insight to potential disease severity and as such guide us in initiating appropriate patient management.

Ankylosing spondylitis (AS) susceptibility is strongly correlated with genetic variation within the major histocompatibility complex (MHC), and the class I HLA-B*27 allele confers the major genetic risk factor to AS. Furthermore, strong evidence for additional alleles in the MHC has been observed which affect susceptibility independently from the HLA-B*27 allele, either by direct genotyping in candidate gene studies or through large-scale imputation-driven association studies. We have previously identified variants in both class I and II which affect susceptibility to AS through imputation of classical HLA alleles...

Cross-ethnic genetic studies can leverage power from differences in disease epidemiology and population-specific genetic architecture. In particular, the differences in linkage disequilibrium and allele frequency patterns across ethnic groups may increase gene-mapping resolution. Here we use cross-ethnic genetic data in sporadic amyotrophic lateral sclerosis (ALS), an adult-onset, rapidly progressing neurodegenerative disease. We report analyses of novel genome-wide association study data of 1,234 ALS cases and 2,850 controls. We find a significant association of rs10463311 spanning GPX3-TNIP1 with ALS (p = 1.3 × 10−8), with replication support from two independent Australian samples (combined 576 cases and 683 controls, p = 1.7 × 10−3). Both GPX3 and TNIP1 interact with other known ALS genes (SOD1 and OPTN, respectively). In addition, GGNBP2 was identified using gene-based analysis and summary statistics-based Mendelian randomization analysis, although further replication is needed to confirm this result. Our results increase our understanding of genetic aetiology of ALS., Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease. Here, Wray and colleagues identify association of the GPX3-TNIP1 locus with ALS using cross-ethnic meta-analyses.