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Background: Genomic sequencing technology allows for identification of reproductive couples with an increased chance, as compared with that in the general population, of having a child with an autosomal recessive or X-linked genetic condition., Methods: We investigated the feasibility, acceptability, and outcomes of a nationwide, couple-based genetic carrier screening program in Australia as part of the Mackenzie's Mission project. Health care providers offered screening to persons before pregnancy or early in pregnancy. The results obtained from testing at least 1281 genes were provided to the reproductive couples. We aimed to ascertain the psychosocial effects on participants, the acceptability of screening to all participants, and the reproductive choices of persons identified as having an increased chance of having a child with a condition for which we screened., Results: Among 10,038 reproductive couples enrolled in the study, 9107 (90.7%) completed screening, and 175 (1.9%) were newly identified as having an increased chance of having a child with a genetic condition for which we screened. These conditions involved pathogenic variants in 90 different genes; 74.3% of the conditions were autosomal recessive. Three months after receiving the results, 76.6% of the couples with a newly identified increased chance had used or planned to use reproductive interventions to avoid having an affected child. Those newly identified as having an increased chance had greater anxiety than those with a low chance. The median level of decisional regret was low in all result groups, and 98.9% of participants perceived screening to be acceptable., Conclusions: Couple-based reproductive genetic carrier screening was largely acceptable to participants and was used to inform reproductive decision making. The delivery of screening to a diverse and geographically dispersed population was feasible. (Funded by the Medical Research Future Fund of the Australian government; ClinicalTrials.gov number, NCT04157595.)., (Copyright © 2024 Massachusetts Medical Society.)

Human cytomegalovirus (HCMV) is carried lifelong by ∼80 % of adults worldwide, generating distinct disease syndromes in transplant recipients, people with HIV (PWH) and neonates. Amino acids 15-23 encoded by the HCMV gene UL40 match positions 3-11 of HLA-A and HLA-C, and constitute a "signal peptide" able to stabilise cell surface HLA-E as a restriction element and a ligand of NKG2A and NKG2C. We present next generation sequencing of UL40 amplified from 15 Australian renal transplant recipients (RTR), six healthy adults and four neonates, and 21 Indonesian PWH. We found no groupwise associations between the presence of multiple sequences and HCMV burden (highest in PWH) or HCMV-associated symptoms in neonates. Homology between UL40 and corresponding HLA-C and HLA-A peptides in 11 RTR revealed perfect matches with HLA-C in three individuals, all carrying HCMV encoding only VMAPRTLIL - a peptide previously associated with viremia. However indices of the burden of HCMV did not segregate in our cohort., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 American Society for Histocompatibility and Immunogenetics. All rights reserved.)

Reproductive genetic carrier screening (RGCS) provides people with information about their chance of having children with autosomal recessive or X-linked genetic conditions, enabling informed reproductive decision-making. RGCS is recommended to be offered to all couples during preconception or in early pregnancy. However, cost and a lack of awareness may prevent access. To address this, the Australian Government funded Mackenzie’s Mission—the Australian Reproductive Genetic Carrier Screening Project. Mackenzie’s Mission aims to assess the acceptability and feasibility of an easily accessible RGCS program, provided free of charge to the participant. In study Phase 1, implementation needs were mapped, and key study elements were developed. In Phase 2, RGCS is being offered by healthcare providers educated by the study team. Reproductive couples who provide consent are screened for over 1200 genes associated with >750 serious, childhood-onset genetic conditions. Those with an increased chance result are provided comprehensive genetic counseling support. Reproductive couples, recruiting healthcare providers, and study team members are also invited to complete surveys and/or interviews. In Phase 3, a mixed-methods analysis will be undertaken to assess the program outcomes, psychosocial implications and implementation considerations alongside an ongoing bioethical analysis and a health economic evaluation. Findings will inform the implementation of an ethically robust RGCS program.

Around 80% of adults worldwide carry human cytomegaloviris (HCMV). The HCMV gene UL18 is a homolog of HLA class I genes and encodes a protein with high affinity for the NK and T-cell cytotoxicity inhibitor LIR-1. UL18 was deep sequenced from blood, saliva or urine from Indonesian people with HIV (PWH) ( n = 28), Australian renal transplant recipients (RTR) ( n = 21), healthy adults ( n = 7) and neonates ( n = 4). 95% of samples contained more than one variant of HCMV UL18 , as defined by carriage of nonsynonymous variations. When aligned with immunological markers of the host's burden of HCMV, the S318N variation associated with high levels of antibody reactive with HCMV lysate in PWH over 12 months on antiretroviral therapy. The A107T variation associated with HCMV antibody levels and inflammatory biomarkers in PWH at early timepoints. Variants D32G, D248N, V250A and E252D aligned with elevated HCMV antibody levels in RTR, while M191K, E196Q and F165L were associated with HCMV-reactive T-cells and proportions of Vδ2 - γδ T-cells-populations linked with high burdens of HCMV. We conclude that UL18 is a highly variable gene, where variation may alter the persistent burden of HCMV and/or the host response to that burden.

Background: Beta-ureidopropionase deficiency, caused by variants in UPB1, has been reported in association with various neurodevelopmental phenotypes including intellectual disability, seizures and autism., Aim: We aimed to reassess the relationship between variants in UPB1 and a clinical phenotype., Methods: Literature review, calculation of carrier frequencies from population databases, long-term follow-up of a previously published case and reporting of additional cases., Results: Fifty-three published cases were identified, and two additional cases are reported here. Of these, 14 were asymptomatic and four had transient neurological features; clinical features in the remainder were variable and included non-neurological presentations. Several of the variants previously reported as pathogenic are present in population databases at frequencies higher than expected for a rare condition. In particular, the variant most frequently reported as pathogenic, p.Arg326Gln, is very common among East Asians, with a carrier frequency of 1 in 19 and 1 in 907 being homozygous for the variant in gnomAD v2.1.1., Conclusion: Pending the availability of further evidence, UPB1 should be considered a 'gene of uncertain clinical significance'. Caution should be used in ascribing clinical significance to biochemical features of beta-ureidopropionase deficiency and/or UPB1 variants in patients with neurodevelopmental phenotypes. UPB1 is not currently suitable for inclusion in gene panels for reproductive genetic carrier screening., Synopsis: The relationship between beta-ureidopropionase deficiency due to UPB1 variants and clinical phenotypes is uncertain., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Human cytomegalovirus (HCMV) is a beta-herpesvirus carried by ~80% of adults worldwide. Acute infections are often asymptomatic in healthy individuals but generate diverse syndromes in neonates, renal transplant recipients (RTR), and people with HIV (PWH). The HCMV gene UL111a encodes a homolog of human interleukin-10 (IL-10) that interacts with the human IL-10 receptor. Deep sequencing technologies were used to sequence UL111a directly from 59 clinical samples from Indonesian PWH and Australian RTR, healthy adults, and neonates. Overall, 93% of samples contained more than one variant of HCMV, as defined by at least one nonsynonymous variation. Carriage of these variants differed between neonates and adults, Australians and Indonesians, and between saliva and blood leukocytes. The variant alleles of N41D and S71Y occurred together in Australian RTR and were associated with higher T-cell responses to HCMV pp65. The variant P122S was associated with lower levels of antibodies reactive with a lysate of HCMV-infected fibroblasts. L174F was associated with increased levels of antibodies reactive with HCMV lysate, immediate-early 1 (IE-1), and glycoprotein B (gB) in Australian RTR and Indonesians PWH, suggesting a higher viral burden. We conclude that variants of UL111a are common in all populations and may influence systemic responses to HCMV.

Human cytomegalovirus (HCMV) is a beta-herpesvirus carried by ∼80% of the world's population. Acute infections are asymptomatic in healthy individuals but generate diverse syndromes in neonates, solid organ transplant recipients, and HIV-infected individuals. The HCMV gene US28 encodes a homolog of a human chemokine receptor that is able to bind several chemokines and HIV gp120. Deep sequencing technologies were used to sequence US28 directly from 60 clinical samples from Indonesian HIV patients and Australian renal transplant recipients, healthy adults, and neonates. Molecular modeling approaches were used to predict whether nine nonsynonymous mutations in US28 may alter protein binding to a panel of six chemokines and two variants of HIV gp120. Ninety-two percent of samples contained more than one variant of HCMV, as defined by at least one nonsynonymous mutation. Carriage of these variants differed between neonates and adults, Australian and Indonesian samples, and saliva samples and blood leukocytes. Two nonsynonymous mutations (N170D and R267K) were associated with increased levels of immediate early protein 1 (IE-1) and glycoprotein B (gB) HCMV-reactive antibodies, suggesting a higher viral burden. Seven of the nine mutations were predicted to alter binding of at least one ligand. Overall, HCMV variants are common in all populations and have the potential to affect US28 interactions with human chemokines and/or gp120 and alter responses to the virus. The findings relied on deep sequencing technologies applied directly to clinical samples, so the variants exist in vivo . IMPORTANCE Human cytomegalovirus (HCMV) is a common viral pathogen of solid organ transplant recipients, neonates, and HIV-infected individuals. HCMV encodes homologs of several host genes with the potential to influence viral persistence and/or pathogenesis. Here, we present deep sequencing of an HCMV chemokine receptor homolog, US28, acquired directly from clinical specimens. Carriage of these variants differed between patient groups and was associated with different levels of circulating HCMV-reactive antibodies. These features are consistent with a role for US28 in HCMV persistence and pathogenesis. This was supported by in silico analyses of the variant sequences demonstrating altered ligand-binding profiles. The data delineate a novel approach to understanding the pathogenesis of HCMV and may impact the development of an effective vaccine.

The aim of this longitudinal microbiome study was to investigate the effects of a commercially available veterinary synbiotic product (Blackmore's® Paw DigestiCare 60™) on the fecal microbiome of healthy dogs using 16S rRNA gene microbial profiling. Fifteen healthy, privately-owned dogs participated in a 2-week trial administration of the product. Fecal samples were collected at different time points, including baseline (prior to treatment), during administration and after discontinuation of product. Large intra- and inter-individual variation was observed throughout the study, but microbiome composition at higher phylogenetic levels, alpha and beta diversity were not significantly altered after 2 weeks of probiotic administration, suggesting an absence of probiotic impact on microbial diversity. Administration of the synbiotic preparation did, however, result in transient increases in probiotic species from Enterococacceae and Streptococacceae families as well as an increase in Fusobacteria; with the fecal microbiota partially reverting to its baseline state 3-weeks after cessation of probiotic administration., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ciaravolo, Martínez-López, Allcock, Woodward and Mansfield.)

Chikungunya virus (CHIKV), Ross River virus (RRV), o'nyong nyong virus (ONNV), Mayaro virus (MAYV) and Getah virus (GETV) represent arthritogenic alphaviruses belonging to the Semliki Forest virus antigenic complex. Antibodies raised against one of these viruses can cross-react with other serogroup members, suggesting that, for instance, a CHIKV vaccine (deemed commercially viable) might provide cross-protection against antigenically related alphaviruses. Herein we use human alphavirus isolates (including a new human RRV isolate) and wild-type mice to explore whether infection with one virus leads to cross-protection against viremia after challenge with other members of the antigenic complex. Persistently infected Rag1 -/- mice were also used to assess the cross-protective capacity of convalescent CHIKV serum. We also assessed the ability of a recombinant poxvirus-based CHIKV vaccine and a commercially available formalin-fixed, whole-virus GETV vaccine to induce cross-protective responses. Although cross-protection and/or cross-reactivity were clearly evident, they were not universal and were often suboptimal. Even for the more closely related viruses (e.g., CHIKV and ONNV, or RRV and GETV), vaccine-mediated neutralization and/or protection against the intended homologous target was significantly more effective than cross-neutralization and/or cross-protection against the heterologous virus. Effective vaccine-mediated cross-protection would thus likely require a higher dose and/or more vaccinations, which is likely to be unattractive to regulators and vaccine manufacturers.

Objective: To develop, test, and iterate a comprehensive neuromuscular targeted gene panel in a national referral center., Methods: We designed two iterations of a comprehensive targeted gene panel for neuromuscular disorders. Version 1 included 336 genes, which was increased to 464 genes in Version 2. Both panels used TargetSeq TM probe-based hybridization for target enrichment followed by Ion Torrent sequencing. Targeted high-coverage sequencing and analysis was performed on 2249 neurology patients from Australia and New Zealand (1054 Version 1, 1195 Version 2) from 2012 to 2015. No selection criteria were used other than referral from a suitable medical specialist (e.g., neurologist or clinical geneticist). Patients were classified into 15 clinical categories based on the clinical diagnosis from the referring clinician., Results: Six hundred and sixty-five patients received a genetic diagnosis (30%). Diagnosed patients were significantly younger that undiagnosed patients (26.4 and 32.5 years, respectively; P = 4.6326E-9). The diagnostic success varied markedly between disease categories. Pathogenic variants in 10 genes explained 38% of the disease burden. Unexpected phenotypic expansions were discovered in multiple cases. Triage of unsolved cases for research exome testing led to the discovery of six new disease genes., Interpretation: A comprehensive targeted diagnostic panel was an effective method for neuromuscular disease diagnosis within the context of an Australasian referral center. Use of smaller disease-specific panels would have precluded diagnosis in many patients and increased cost. Analysis through a centralized laboratory facilitated detection of recurrent, but under-recognized pathogenic variants., (© 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association.)

Analysis of specific somatic copy number alterations (SCNAs) using multiplex ligation-dependent probe amplification (MLPA) is used routinely as a prognostic test for uveal melanoma (UM). This technique requires relatively large amounts of input DNA, unattainable from many small fine-needle aspirate biopsy specimens. Herein, we compared the use of MLPA with whole-genome amplification (WGA) combined with low-pass whole-genome sequencing (LP-WGS) for detection of SCNA profiles in UM biopsy specimens. DNA was extracted from 21 formalin-fixed, paraffin-embedded UM samples and SCNAs were assessed using MLPA and WGA followed by LP-WGS. Cohen's κ was used to assess the concordance of copy number calls of each individual chromosome arm for each patient. MLPA and WGA/LP-WGS detection of SCNAs in chromosomes 1p, 3, 6, and 8 were compared and found to be highly concordant with a Cohen's κ of 0.856 (bias-corrected and accelerated 95% CI, 0.770-0.934). Only 13 of 147 (8.8%) chromosomal arms investigated resulted in discordant calls, predominantly SCNAs detected by WGA/LP-WGS but not MLPA. These results indicate that LP-WGS might be a suitable alternative or adjunct to MLPA for the detection of SCNAs associated with prognosis of UM, for cases with limiting tissue or DNA yields., (Copyright © 2020 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

The implementation and popularity of next generation sequencing (NGS) has led to the development of various rapid whole mitochondrial genome sequencing techniques. We summarise an efficient and cost-effective NGS approach for mitochondrial genomic DNA in humans using the Ion Torrent platform, and further discuss our bioinformatics pipeline for streamlined variant calling. Ion 316 chips were utilised with the Ion Torrent semi-conductor platform Personal Genome Machine (PGM) to perform tandem sequencing of mitochondrial genomes from the core pedigree (n = 315) of the Norfolk Island Health Study. Key improvements from commercial methods focus on the initial PCR step, which currently requires extensive optimisation to ensure the accurate and reproducible elongation of each section of the complete mitochondrial genome. Dual-platform barcodes were incorporated into our protocol thereby extending its potential application onto Illumina-based systems. Our bioinformatics pipeline consists of a modified version of GATK best practices tailored for mitochondrial genomic data. When compared with current commercial methods, our method, termed high throughput mitochondrial genome sequencing (HTMGS), allows high multiplexing of samples and the use of alternate library preparation reagents at a lower cost per sample (~1.7 times) when compared to current commercial methodologies. Our HTMGS methodology also provides robust mitochondrial sequencing data (>450X average coverage) that can be applied and modified to suit various study designs. On average, we were able to identify ~30 variants per sample with 572 variants observed across 315 samples. We have developed a high throughput sequencing and analysis method targeting complete mitochondrial genomes; with the potential to be platform agnostic with analysis options that adhere to current best practices., Competing Interests: The authors have declared that no competing interests exist.

Background: The Djallonke sheep is well adapted to harsh environmental conditions, and is relatively resistant to Haemonchosis and resilient to animal trypanosomiasis. The larger Sahelian sheep, which cohabit the same region, is less well adapted to these disease challenges. Haemonchosis and Trypanosomiasis collectively cost the worldwide animal industry billions of dollars in production losses annually., Results: Here, we separately sequenced and then pooled according to breed the genomes from five unrelated individuals from each of the Djallonke and Sahelian sheep breeds (sourced from Ghana), at greater than 22-fold combined coverage for each breed. A total of approximately 404 million (97%) and 343 million (97%) sequence reads from the Djallonke and Sahelian breeds respectively, were successfully mapped to the sheep reference genome Oar v3.1. We identified approximately 11.1 million and 10.9 million single nucleotide polymorphisms (SNPs) in the Djallonke and Sahelian breeds, with approximately 15 and 16% respectively of these not previously reported in sheep. Multiple regions of reduced heterozygosity were also found; 70 co-localised within genomic regions harbouring genes that mediate disease resistance, immune response and adaptation in sheep or cattle. Thirty- three of the regions of reduced heterozygosity co-localised with previously reported genes for resistance to haemonchosis and trypanosomiasis., Conclusions: Our analyses suggest that these regions of reduced heterozygosity may be signatures of selection for these economically important diseases.

Neoantigens present unique and specific targets for personalized cancer immunotherapy strategies. Given the low mutational burden yet immunotherapy responsiveness of malignant mesothelioma (MM) when compared to other carcinogen-induced malignancies, identifying candidate neoantigens and T cells that recognize them has been a challenge. We used pleural effusions to gain access to MM tumor cells as well as immune cells in order to characterize the tumor-immune interface in MM. We characterized the landscape of potential neoantigens from SNVs identified in 27 MM patients and performed whole transcriptome sequencing of cell populations from 18 patient-matched pleural effusions. IFNγ ELISpot was performed to detect a CD8+ T cell responses to predicted neoantigens in one patient. We detected a median of 68 (range 7-258) predicted neoantigens across the samples. Wild-type non-binding to mutant binding predicted neoantigens increased risk of death in a model adjusting for age, sex, smoking status, histology and treatment (HR: 33.22, CI: 2.55-433.02, p = .007). Gene expression analysis indicated a dynamic immune environment within the pleural effusions. TCR clonotypes increased with predicted neoantigen burden. A strong activated CD8+ T-cell response was identified for a predicted neoantigen produced by a spontaneous mutation in the ROBO3 gene. Despite the challenges associated with the identification of bonafide neoantigens, there is growing evidence that these molecular changes can provide an actionable target for personalized therapeutics in difficult to treat cancers. Our findings support the existence of candidate neoantigens in MM despite the low mutation burden of the tumor, and may present improved treatment opportunities for patients., (© 2019 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Malignant pleural mesothelioma (MPM) is an incurable cancer of the pleura that can be difficult to diagnose. Biomarkers for an easier and/or earlier diagnosis are needed. Approximately 90% of MPM patients develop a pleural effusion (PE). PEs are ideal sources of biomarkers as the fluid would almost always require drainage for diagnostic and/or therapeutic reasons. However, differentiating MPM PE from PE caused by other diseases can be challenging. MicroRNAs are popular biomarkers given their stable expression in tissue and fluid. MicroRNAs have been analysed in PE cytology samples for the diagnosis of MPM but have not been measured in frozen/fresh PE. We hypothesise that microRNAs expressed in PE are biomarkers for MPM. TaqMan OpenArray was used to analyse over 700 microRNAs in PE cells and supernatants from 26 MPMs and 21 other PE-causing diseases. In PE cells, combining miR-143, miR-210, and miR-200c could differentiate MPM with an area under the curve (AUC) of 0.92. The three-microRNA signature could also discriminate MPM from a further 40 adenocarcinomas with an AUC of 0.9887. These results suggest that the expression of miR-143, miR-210, and miR-200c in PE cells might provide a signature for diagnosing MPM., Competing Interests: No potential conflicts of interest were disclosed.

Background: PIK3CA pathways are the most frequently mutated oncogenic pathway in head and neck squamous cell carcinoma (HNSCC), including virally driven HNCs. PIK3CA is involved in the PI3K-PTEN-mTOR signalling pathway. PIK3CA has been implicated in HNSCC progression and PIK3CA mutations may serve as predictive biomarkers for therapy selection. Circulating tumour DNA (ctDNA) derived from necrotic and apoptotic tumour cells are thought to harbour tumour-specific genetic alterations. As such, the detection of PIK3CA alterations detected by ctDNA holds promise as a potential biomarker in HNSCC., Methods: Blood samples from treatment naïve HNSCC patients ( n = 29) were interrogated for a commonly mutated PIK3CA hotspot mutation using low cost allele-specific Plex-PCR TM technology., Results: In this pilot, cross sectional study, PIK3CA E545K mutation was detected in the plasma samples of 9/29 HNSCC patients using the Plex-PCR TM technology., Conclusion: The results of this pilot study support the notion of using allele-specific technologies for cost-effective testing of ctDNA, and further assert the potential utility of ctDNA in HNSCC.

Background: Carcinoma of unknown primary (CUP) is a metastatic epithelial malignancy in the absence of an identifiable primary tumour. Prognosis for patients with CUP is poor because treatment options are generally limited to broad spectrum chemotherapy. A shift towards personalised cancer management based on mutation profiling offers the possibility of new treatment paradigms. This study has explored whether actionable, oncogenic driver mutations are present in CUP that have potential to better inform treatment decisions., Methods: Carcinoma of unknown primary cases (n = 21) were selected and DNA was isolated from formalin-fixed paraffin embedded sections prior to amplification and sequencing. Two distinct yet complementary targeted gene panels were used to assess variants in up to 76 known cancer-related genes for the identification of biologically relevant and actionable mutations., Results: Variants were detected in 17/21 cases (81%) of which 11 (52%) were potentially actionable with drugs currently approved for use in known primary cancer types or undergoing clinical trials. The most common variants detected were in TP53 (47%), KRAS (12%), MET (12%) and MYC (12%). Differences at the molecular level were seen between common CUP histological subtypes. CUP adenocarcinomas and poorly differentiated carcinomas harboured the highest frequency of variants in genes involved in signal transduction pathways (e.g. MET, EGFR, HRAS, KRAS, and BRAF). In contrast, squamous cell carcinoma exhibited a higher frequency of variants in cell cycle control and DNA repair genes (e.g. TP53, CDKN2A and MLH1)., Conclusion: Taken together, mutations in biologically relevant genes were detected in the vast majority of CUP tumours, of which half provided a potentially novel treatment option not generally considered in CUP.

Objectives: Malignant mesothelioma (MM) is an asbestos related tumour affecting cells of serosal cavities. More than 70% of MM patients develop pleural effusions which contain tumour cells, representing a readily accessible source of malignant cells for genetic analysis. Although common somatic mutations and losses have been identified in solid MM tumours, the characterization of tumour cells within pleural effusions could provide novel insights but is little studied., Materials and Methods: DNA and RNA were extracted from cells from short term cultures of 27 human MM pleural effusion samples. Whole exome and transcriptome sequencing was performed using the Ion Torrent platform. Somatic mutations were identified using VarScan2 and SomaticSniper. Copy number alterations were identified using ExomeCNV in R. Significant copy number alterations were identified across all samples using GISTIC2.0. The association between tumour intrinsic properties and survival was analyzed using the Cox proportional hazards regression model., Results: We identified BAP1, CDKN2A and NF2 alterations in the cells from MM pleural effusions at a higher frequency than what is typically seen in MM tumours from surgical series. The median mutation rate was 1.09 mutations/Mb. TRAF7 and LATS2 alterations were also identified at a high frequency (66% and 59% respectively). Novel regions of interest were identified, including alterations in FGFR3, and the regions 19p13.3, 8p23.1 and 1p36.32., Conclusion: Short term cultures of tumour cells from MM pleural effusions offer an accessible alternative to surgical tumour biopsies in the study of MM genomics and reveal novel mutations of interest. Pleural effusion tumour cells provide an opportunity for the monitoring of tumour dynamics, treatment response and the clonal evolution of MM tumours., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Bipolar disorder (BD) is a complex psychiatric condition with high heritability, the genetic architecture of which likely comprises both common variants of small effect and rare variants of higher penetrance, the latter of which are largely unknown. Extended families with high density of illness provide an opportunity to map novel risk genes or consolidate evidence for existing candidates, by identifying genes carrying pathogenic rare variants. We performed whole-exome sequencing (WES) in 15 BD families (117 subjects, of whom 72 were affected), augmented with copy number variant (CNV) microarray data, to examine contributions of multiple classes of rare genetic variants within a familial context. Linkage analysis and haplotype reconstruction using WES-derived genotypes enabled exclusion of false-positive single-nucleotide variants (SNVs), CNV inheritance estimation, de novo variant identification and candidate gene prioritization. We found that rare predicted pathogenic variants shared among ≥3 affected relatives were overrepresented in postsynaptic density (PSD) genes (P = 0.002), with no enrichment in unaffected relatives. Genome-wide burden of likely gene-disruptive variants was no different in affected vs. unaffected relatives (P = 0.24), but correlated significantly with age of onset (P = 0.017), suggesting that a high disruptive variant burden may expedite symptom onset. The number of de novo variants was no different in affected vs. unaffected offspring (P = 0.89). We observed heterogeneity within and between families, with the most likely genetic model involving alleles of modest effect and reduced penetrance: a possible exception being a truncating X-linked mutation in IRS4 within a family-specific linkage peak. Genetic approaches combining WES, CNV and linkage analyses in extended families are promising strategies for gene discovery.

Premise of the Study: Microsatellite markers were developed for population genetic analysis in the rare shrub Styphelia longissima (Ericaceae)., Methods and Results: We generated ca. 2.5 million sequence reads using a Personal Genome Machine semiconductor sequencer. Using the QDD pipeline, we designed primers for >12,000 sequences with PCR product lengths of 80-480 bp. From these, 30 primer pairs were selected and screened using PCR; of these, 16 loci were found to be polymorphic, four loci were monomorphic, and 10 loci did not amplify reliably for S. longissima . For a sample of 57 plants from the only known population, the number of alleles observed for these 16 loci ranged from two to 21 and expected heterozygosity ranged from 0.49 to 0.91. These markers were also amplified in Astroloma xerophyllum , a closely related species., Conclusions: These markers will be used to characterize population genetic variation, spatial genetic structure, mating system parameters, and dispersal to aid in the management and conservation of the rare shrub S. longissima .

The standard paradigm for microbiological testing is dependent on the presentation of a patient to a clinician. Tests are then requested based on differential diagnoses using the patient's symptoms as a guide. The era of high-throughput genomic methods has the potential to replace this model for the first time with what could be referred to as "hypothesis-free testing." This approach utilizes one of a variety of methodologies to obtain a sequence from potentially any nucleic acid in a clinical sample, without prior knowledge of its content. We discuss the advantages of such an approach and the challenges in making this a reality., (© Crown copyright 2017.)

Premise of the Study: Microsatellites were developed for the future assessment of population genetic structure, mating system, and dispersal of the perennial kangaroo paw, Anigozanthos manglesii (Haemodoraceae), and related species., Methods and Results: Using a Personal Genome Machine (PGM) semiconductor sequencer, ca. 4.03 million sequence reads were generated. QDD pipeline software was used to identify 190,000 microsatellite-containing regions and priming sites. From these, 90 were chosen and screened using PCR, and 15 polymorphic markers identified. These sites amplified di-, tri-, and pentanucleotide repeats with one to 20 alleles per locus. Primers were also amplified across congeners A. bicolor , A. flavidus , A. gabrielae , A. humilis , A. preissii , A. pulcherrimus , A. rufus , and A. viridis to assess cross-species transferability., Conclusions: These markers provide a resource for population genetic studies in A. manglesii and other species within the genus.

Background: Malignant mesothelioma (MM) is an aggressive cancer of the pleural and peritoneal cavities caused by exposure to asbestos. Asbestos-induced mesotheliomas in wild-type mice have been used extensively as a preclinical model because they are phenotypically identical to their human counterpart. However, it is not known if the genetic lesions in these mice tumours are similar to in the human disease, a prerequisite for any new preclinical studies that target genetic abnormalities., Methods: We performed whole exome sequencing of fifteen asbestos-induced murine MM tumour cell lines from BALB/c, CBA and C57BL/6 mouse strains and compared the somatic mutations and copy number variations with those recurrently reported in human MM. We then catalogued and characterised the mutational landscape of the wild-type murine MM tumours. Quantitative RT-PCR was used to interrogate the expression of key MM genes of interest in the mRNA., Results: Consistent with human MM tumours, we identified homozygous loss of the tumour suppressor Cdkn2a in 14/15 tumours. One tumour retained the first exon of both of the p16INK4a and p19ARF isoforms though this tumour also contained genetic amplification of Myc resulting in increased expression of the c-Myc proto-oncogene in the mRNA. There were no chromosomal losses in either the Bap1 or Nf2 regions. One tumour harbored homozygous loss of Trp53 in the DNA. Mutation rates were similar in tumours generated in the CBA and C57BL/6 strains when compared to human MM. Interestingly, all BALB/c tumour lines displayed high mutational loads, consistent with the known mutator phenotype of the host strain. The Wnt, MAPK and Jak-STAT signaling pathways were found to be the most commonly affected biological pathways. Mutations and copy number deletions also occurred in the Hedgehog and Hippo pathways., Conclusions: These data suggest that in the wild-type murine model asbestos causes mesotheliomas in a similar way to in human MM. This further supports the notion that the murine model of MM represents a genuine homologue of the human disease, something uncommon in cancer, and is thus a valuable tool to provide insight into MM tumour development and to aide the search for novel therapeutic strategies.

Mining of mineral resources produces substantial volumes of crushed rock based wastes that are characterised by poor physical structure and hydrology, unstable geochemistry and potentially toxic chemical conditions. Recycling of these substrates is desirable and can be achieved by blending waste with native soil to form a 'novel substrate' which may be used in future landscape restoration. However, these post-mining substrate based 'soils' are likely to contain significant abiotic constraints for both plant and microbial growth. Effective use of these novel substrates for ecosystem restoration will depend on the efficacy of stored topsoil as a potential microbial inoculum as well as the subsequent generation of key microbial soil functions originally apparent in local pristine sites. Here, using both marker gene and shotgun metagenome sequencing, we show that topsoil storage and the blending of soil and waste substrates to form planting substrates gives rise to variable bacterial and archaeal phylogenetic composition but a high degree of metabolic conservation at the community metagenome level. Our data indicates that whilst low phylogenetic conservation is apparent across substrate blends we observe high functional redundancy in relation to key soil microbial pathways, allowing the potential for functional recovery of key belowground pathways under targeted management.