Unrecognised actionability for breast cancer risk variants identified in a national-level review of Australian familial cancer centres.

Breast cancer remains a significant global health challenge. In Australia, the adoption of publicly-funded multigene panel testing for eligible cancer patients has increased accessibility to personalised care, yet has also highlighted the increasing prevalence of variants of uncertain significance (VUS), complicating clinical decision-making. This project aimed to explore the spectrum and actionability of breast cancer VUS in Australian familial cancer centers (FCCs). Leveraging data from 11 FCCs participating in the Inherited Cancer Connect database, we retrieved VUS results from 1472 patients. Through ClinVar crosschecks and application of gene-specific ACMG/AMP guidelines, we showed the potential for reclassification of 4% of unique VUS as pathogenic or likely pathogenic, and 80% as benign or likely benign. Surveys conducted with FCCs and diagnostic laboratories described current practices and challenges in variant reclassifications, highlighting resource constraints preventing periodic VUS review and notifications from the laboratories to the FCCs. Our study suggests there are benefits to routine VUS review and reclassification, particularly in publicly-funded healthcare systems. Future research should focus on assessing the clinical impact and cost-effectiveness of implementing routine variant review practices, alongside efforts to enhance communication between FCCs and laboratories.

Evidence-based recommendations for gene-specific ACMG/AMP variant classification from the ClinGen ENIGMA BRCA1 and BRCA2 Variant Curation Expert Panel.

The ENIGMA research consortium develops and applies methods to determine clinical significance of variants in hereditary breast and ovarian cancer genes. An ENIGMA BRCA1/2 classification sub-group, formed in 2015 as a ClinGen external expert panel, evolved into a ClinGen internal Variant Curation Expert Panel (VCEP) to align with Food and Drug Administration recognized processes for ClinVar contributions. The VCEP reviewed American College of Medical Genetics and Genomics/Association of Molecular Pathology (ACMG/AMP) classification criteria for relevance to interpreting BRCA1 and BRCA2 variants. Statistical methods were used to calibrate evidence strength for different data types. Pilot specifications were tested on 40 variants and documentation revised for clarity and ease of use. The original criterion descriptions for 13 evidence codes were considered non-applicable or overlapping with other criteria. Scenario of use was extended or re-purposed for eight codes. Extensive analysis and/or data review informed specification descriptions and weights for all codes. Specifications were applied to pilot variants with pre-existing ClinVar classification as follows: 13 uncertain significance or conflicting, 14 pathogenic and/or likely pathogenic, and 13 benign and/or likely benign. Review resolved classification for 11/13 uncertain significance or conflicting variants and retained or improved confidence in classification for the remaining variants. Alignment of pre-existing ENIGMA research classification processes with ACMG/AMP classification guidelines highlighted several gaps in the research processes and the baseline ACMG/AMP criteria. Calibration of evidence strength was key to justify utility and strength of different data types for gene-specific application. The gene-specific criteria demonstrated value for improving ACMG/AMP-aligned classification of BRCA1 and BRCA2 variants.

Diagnostic yield of candidate genes in an Australian corneal dystrophy cohort.

Corneal dystrophies describe a clinically and genetically heterogeneous group of inherited disorders. The International Classification of Corneal Dystrophies (IC3D) lists 22 types of corneal dystrophy, 17 of which have been demonstrated to result from pathogenic variants in 19 identified genes. In this study, we investigated the diagnostic yield of genetic testing in a well-characterised cohort of 58 individuals from 44 families with different types of corneal dystrophy. Individuals diagnosed solely with Fuchs endothelial corneal dystrophy were excluded. Clinical details were obtained from the treating ophthalmologist. Participants and their family members were tested using a gene candidate and exome sequencing approach. We identified a likely molecular diagnosis in 70.5% families (31/44). The detection rate was significantly higher among probands with a family history of corneal dystrophy (15/16, 93.8%) than those without (16/28, 57.1%, p = .015), and among those who had undergone corneal graft surgery (9/9, 100.0%) compared to those who had not (22/35, 62.9%, p = .041). We identified eight novel variants in five genes and identified five families with syndromes associated with corneal dystrophies. Our findings highlight the genetic heterogeneity of corneal dystrophies and the clinical utility of genetic testing in reaching an accurate clinical diagnosis.

DNA methylation heterogeneity defines a disease spectrum in Ewing sarcoma.

Developmental tumors in children and young adults carry few genetic alterations, yet they have diverse clinical presentation. Focusing on Ewing sarcoma, we sought to establish the prevalence and characteristics of epigenetic heterogeneity in genetically homogeneous cancers. We performed genome-scale DNA methylation sequencing for a large cohort of Ewing sarcoma tumors and analyzed epigenetic heterogeneity on three levels: between cancers, between tumors, and within tumors. We observed consistent DNA hypomethylation at enhancers regulated by the disease-defining EWS-FLI1 fusion protein, thus establishing epigenomic enhancer reprogramming as a ubiquitous and characteristic feature of Ewing sarcoma. DNA methylation differences between tumors identified a continuous disease spectrum underlying Ewing sarcoma, which reflected the strength of an EWS-FLI1 regulatory signature and a continuum between mesenchymal and stem cell signatures. There was substantial epigenetic heterogeneity within tumors, particularly in patients with metastatic disease. In summary, our study provides a comprehensive assessment of epigenetic heterogeneity in Ewing sarcoma and thereby highlights the importance of considering nongenetic aspects of tumor heterogeneity in the context of cancer biology and personalized medicine.

Genome-wide association study using extreme truncate selection identifies novel genes affecting bone mineral density and fracture risk.

Osteoporotic fracture is a major cause of morbidity and mortality worldwide. Low bone mineral density (BMD) is a major predisposing factor to fracture and is known to be highly heritable. Site-, gender-, and age-specific genetic effects on BMD are thought to be significant, but have largely not been considered in the design of genome-wide association studies (GWAS) of BMD to date. We report here a GWAS using a novel study design focusing on women of a specific age (postmenopausal women, age 55-85 years), with either extreme high or low hip BMD (age- and gender-adjusted BMD z-scores of +1.5 to +4.0, n = 1055, or -4.0 to -1.5, n = 900), with replication in cohorts of women drawn from the general population (n = 20,898). The study replicates 21 of 26 known BMD-associated genes. Additionally, we report suggestive association of a further six new genetic associations in or around the genes CLCN7, GALNT3, IBSP, LTBP3, RSPO3, and SOX4, with replication in two independent datasets. A novel mouse model with a loss-of-function mutation in GALNT3 is also reported, which has high bone mass, supporting the involvement of this gene in BMD determination. In addition to identifying further genes associated with BMD, this study confirms the efficiency of extreme-truncate selection designs for quantitative trait association studies.

Association of variants at 1q32 and STAT3 with ankylosing spondylitis suggests genetic overlap with Crohn's disease.

Ankylosing spondylitis (AS) is a common inflammatory arthritic condition. Overt inflammatory bowel disease (IBD) occurs in about 10% of AS patients, and in addition 70% of AS cases may have subclinical terminal ileitis. Spondyloarthritis is also common in IBD patients. We therefore tested Crohn's disease susceptibility genes for association with AS, aiming to identify pleiotropic genetic associations with both diseases. Genotyping was carried out using Sequenom and Applied Biosystems TaqMan and OpenArray technologies on 53 markers selected from 30 Crohn's disease associated genomic regions. We tested genotypes in a population of unrelated individual cases (n = 2,773) and controls (n = 2,215) of white European ancestry for association with AS. Statistical analysis was carried out using a Cochran-Armitage test for trend in PLINK. Strong association was detected at chr1q32 near KIF21B (rs11584383, P = 1.6 × 10(-10), odds ratio (OR) = 0.74, 95% CI:0.68-0.82). Association with disease was also detected for 2 variants within STAT3 (rs6503695, P = 4.6 × 10(-4). OR = 0.86 (95% CI:0.79-0.93); rs744166, P = 2.6 × 10(-5), OR = 0.84 (95% CI:0.77-0.91)). Association was confirmed for IL23R (rs11465804, P = 1.2 × 10(-5), OR = 0.65 (95% CI:0.54-0.79)), and further associations were detected for IL12B (rs10045431, P = 5.2 × 10(-5), OR = 0.83 (95% CI:0.76-0.91)), CDKAL1 (rs6908425, P = 1.1 × 10(-4), OR = 0.82 (95% CI:0.74-0.91)), LRRK2/MUC19 (rs11175593, P = 9.9 × 10(-5), OR = 1.92 (95% CI: 1.38-2.67)), and chr13q14 (rs3764147, P = 5.9 × 10(-4), OR = 1.19 (95% CI: 1.08-1.31)). Excluding cases with clinical IBD did not significantly affect these findings. This study identifies chr1q32 and STAT3 as ankylosing spondylitis susceptibility loci. It also further confirms association for IL23R and detects suggestive association with another 4 loci. STAT3 is a key signaling molecule within the Th17 lymphocyte differentiation pathway and further enhances the case for a major role of this T-lymphocyte subset in ankylosing spondylitis. Finally these findings suggest common aetiopathogenic pathways for AS and Crohn's disease and further highlight the involvement of common risk variants across multiple diseases.