Breast Cancer is Increased in Women with Primary Ovarian Insufficiency.

CONTEXT: DNA damage/repair gene variants are associated with both primary ovarian insufficiency (POI) and cancer risk. OBJECTIVE: We hypothesized that a subset of women with POI and family members would have increased risk for cancer. DESIGN: Case-control population-based study using records from 1995-2022. SETTING: Two major Utah academic healthcare systems serving 85% of the state. SUBJECTS: Women with POI (n=613) were identified using ICD codes and reviewed for accuracy. Relatives were linked using the Utah Population Database. INTERVENTION: Cancer diagnoses were identified using the Utah Cancer Registry. MAIN OUTCOME MEASURES: The relative risk of cancer in women with POI and relatives was estimated by comparison to population rates. Whole genome sequencing was performed on a subset of women. RESULTS: Breast cancer was increased in women with POI (OR [95%CI] 2.20 [1.30, 3.47]; p=0.0023) and there was a nominally significant increase in ovarian cancer. Probands with POI were 36.5±4.3 years and 59.5±12.7 years when diagnosed with POI and cancer, respectively. Causal and candidate gene variants for cancer and POI were identified.Among second-degree relatives of these women, there was an increased risk of breast (1.28 [1.08, 1.52]; p=0.0078) and colon cancer (1.50 [1.14, 1.94]; p=0.0036). Prostate cancer was increased in first- (1.64 [1.18, 2.23]; p=0.0026), second- (1.54 [1.32, 1.79]; p<0.001), and third-degree relatives (1.33 [1.20, 1.48]; p<0.001). CONCLUSIONS: Data suggest common genetic risk for POI and reproductive cancers. Tools are needed to predict cancer risk in women with POI and potentially to counsel about risks of hormone replacement therapy.

Self-Adjuvanting Calcium-Phosphate-Coated Microcrystal-Based Vaccines Induce Pyroptosis in Human and Livestock Immune Cells.

Successful vaccines require adjuvants able to activate the innate immune system, eliciting antigen-specific immune responses and B-cell-mediated antibody production. However, unwanted secondary effects and the lack of effectiveness of traditional adjuvants has prompted investigation into novel adjuvants in recent years. Protein-coated microcrystals modified with calcium phosphate (CaP-PCMCs) in which vaccine antigens are co-immobilised within amino acid crystals represent one of these promising self-adjuvanting vaccine delivery systems. CaP-PCMCs has been shown to enhance antigen-specific IgG responses in mouse models; however, the exact mechanism of action of these microcrystals is currently unclear. Here, we set out to investigate this mechanism by studying the interaction between CaP-PCMCs and mammalian immune cells in an in vitro system. Incubation of cells with CaP-PCMCs induced rapid pyroptosis of peripheral blood mononuclear cells and monocyte-derived dendritic cells from cattle, sheep and humans, which was accompanied by the release of interleukin-1ß and the activation of Caspase-1. We show that this pyroptotic event was cell-CaP-PCMCs contact dependent, and neither soluble calcium nor microcrystals without CaP (soluble PCMCs) induced pyroptosis. Our results corroborate CaP-PCMCs as a promising delivery system for vaccine antigens, showing great potential for subunit vaccines where the enhancement or find tuning of adaptive immunity is required.

DIS3 Variants are Associated With Primary Ovarian Insufficiency: Importance of Transcription/Translation in Oogenesis.

CONTEXT: A genetic etiology accounts for the majority of unexplained primary ovarian insufficiency (POI). OBJECTIVE: We hypothesized a genetic cause of POI for a sister pair with primary amenorrhea. DESIGN: The study was an observational study. Subjects were recruited at an academic institution. SUBJECTS: Subjects were sisters with primary amenorrhea caused by POI and their parents. Additional subjects included women with POI analyzed previously (n = 291). Controls were recruited for health in old age or were from the 1000 Genomes Project (total n = 233). INTERVENTION: We performed whole exome sequencing, and data were analyzed using the Pedigree Variant Annotation, Analysis and Search Tool, which identifies genes harboring pathogenic variants in families. We performed functional studies in a Drosophila melanogaster model. MAIN OUTCOME: Genes with rare pathogenic variants were identified. RESULTS: The sisters carried compound heterozygous variants in DIS3. The sisters did not carry additional rare variants that were absent in publicly available datasets. DIS3 knockdown in the ovary of D. melanogaster resulted in lack of oocyte production and severe infertility. CONCLUSIONS: Compound heterozygous variants in highly conserved amino acids in DIS3 and failure of oocyte production in a functional model suggest that mutations in DIS3 cause POI. DIS3 is a 3' to 5' exoribonuclease that is the catalytic subunit of the exosome involved in RNA degradation and metabolism in the nucleus. The findings provide further evidence that mutations in genes important for transcription and translation are associated with POI.

Automated prioritization of sick newborns for whole genome sequencing using clinical natural language processing and machine learning.

BACKGROUND: Rapidly and efficiently identifying critically ill infants for whole genome sequencing (WGS) is a costly and challenging task currently performed by scarce, highly trained experts and is a major bottleneck for application of WGS in the NICU. There is a dire need for automated means to prioritize patients for WGS. METHODS: Institutional databases of electronic health records (EHRs) are logical starting points for identifying patients with undiagnosed Mendelian diseases. We have developed automated means to prioritize patients for rapid and whole genome sequencing (rWGS and WGS) directly from clinical notes. Our approach combines a clinical natural language processing (CNLP) workflow with a machine learning-based prioritization tool named Mendelian Phenotype Search Engine (MPSE). RESULTS: MPSE accurately and robustly identified NICU patients selected for WGS by clinical experts from Rady Children's Hospital in San Diego (AUC 0.86) and the University of Utah (AUC 0.85). In addition to effectively identifying patients for WGS, MPSE scores also strongly prioritize diagnostic cases over non-diagnostic cases, with projected diagnostic yields exceeding 50% throughout the first and second quartiles of score-ranked patients. CONCLUSIONS: Our results indicate that an automated pipeline for selecting acutely ill infants in neonatal intensive care units (NICU) for WGS can meet or exceed diagnostic yields obtained through current selection procedures, which require time-consuming manual review of clinical notes and histories by specialized personnel.

Rapid genome sequencing identifies a novel de novo SNAP25 variant for neonatal congenital myasthenic syndrome.

Congenital myasthenic syndrome (CMS) is a group of 32 disorders involving genetic dysfunction at the neuromuscular junction resulting in skeletal muscle weakness that worsens with physical activity. Precise diagnosis and molecular subtype identification are critical for treatment as medication for one subtype may exacerbate disease in another (Engel et al., Lancet Neurol 14: 420 [2015]; Finsterer, Orphanet J Rare Dis 14: 57 [2019]; Prior and Ghosh, J Child Neurol 36: 610 [2021]). The SNAP25-related CMS subtype (congenital myasthenic syndrome 18, CMS18; MIM #616330) is a rare disorder characterized by muscle fatigability, delayed psychomotor development, and ataxia. Herein, we performed rapid whole-genome sequencing (rWGS) on a critically ill newborn leading to the discovery of an unreported pathogenic de novo SNAP25 c.529C > T; p.Gln177Ter variant. In this report, we present a novel case of CMS18 with complex neonatal consequence. This discovery offers unique insight into the extent of phenotypic severity in CMS18, expands the reported SNAP25 variant phenotype, and paves a foundation for personalized management for CMS18.

A dominant negative ADIPOQ mutation in a diabetic family with renal disease, hypoadiponectinemia, and hyperceramidemia.

Adiponectin, encoded by ADIPOQ, is an insulin-sensitizing, anti-inflammatory, and renoprotective adipokine that activates receptors with intrinsic ceramidase activity. We identified a family harboring a 10-nucleotide deletion mutation in ADIPOQ that cosegregates with diabetes and end-stage renal disease. This mutation introduces a frameshift in exon 3, resulting in a premature termination codon that disrupts translation of adiponectin's globular domain. Subjects with the mutation had dramatically reduced circulating adiponectin and increased long-chain ceramides levels. Functional studies suggest that the mutated protein acts as a dominant negative through its interaction with non-mutated adiponectin, decreasing circulating adiponectin levels, and correlating with metabolic disease.

Predictors of Survival after Vaccination in a Pneumonic Plague Model.

Background: The need for an updated plague vaccine is highlighted by outbreaks in endemic regions together with the pandemic potential of this disease. There is no easily available, approved vaccine. Methods: Here we have used a murine model of pneumonic plague to examine the factors that maximise immunogenicity and contribute to survival following vaccination. We varied vaccine type, as either a genetic fusion of the F1 and V protein antigens or a mixture of these two recombinant antigens, as well as antigen dose-level and formulation in order to correlate immune response to survival. Results: Whilst there was interaction between each of the variables of vaccine type, dose level and formulation and these all contributed to survival, vaccine formulation in protein-coated microcrystals (PCMCs) was the key contributor in inducing antibody titres. From these data, we propose a cut-off in total serum antibody titre to the F1 and V proteins of 100 µg/mL and 200 µg/mL, respectively. At these thresholds, survival is predicted in this murine pneumonic model to be >90%. Within the total titre of antibody to the V antigen, the neutralising antibody component correlated with dose level and was enhanced when the V antigen in free form was formulated in PCMCs. Antibody titre to F1 was limited by fusion to V, but this was compensated for by PCMC formulation. Conclusions: These data will enable clinical assessment of this and other candidate plague vaccines that utilise the same vaccine antigens by identifying a target antibody titre from murine models, which will guide the evaluation of clinical titres as serological surrogate markers of efficacy.

Comprehensive variant calling from whole-genome sequencing identifies a complex inversion that disrupts ZFPM2 in familial congenital diaphragmatic hernia.

BACKGROUND: Genetic disorders contribute to significant morbidity and mortality in critically ill newborns. Despite advances in genome sequencing technologies, a majority of neonatal cases remain unsolved. Complex structural variants (SVs) often elude conventional genome sequencing variant calling pipelines and will explain a portion of these unsolved cases. METHODS: As part of the Utah NeoSeq project, we used a research-based, rapid whole-genome sequencing (WGS) protocol to investigate the genomic etiology for a newborn with a left-sided congenital diaphragmatic hernia (CDH) and cardiac malformations, whose mother also had a history of CDH and atrial septal defect. RESULTS: Using both a novel, alignment-free and traditional alignment-based variant callers, we identified a maternally inherited complex SV on chromosome 8, consisting of an inversion flanked by deletions. This complex inversion, further confirmed using orthogonal molecular techniques, disrupts the ZFPM2 gene, which is associated with both CDH and various congenital heart defects. CONCLUSIONS: Our results demonstrate that complex structural events, which often are unidentifiable or not reported by clinically validated testing procedures, can be discovered and accurately characterized with conventional, short-read sequencing and underscore the utility of WGS as a first-line diagnostic tool.

Clin.iobio: A Collaborative Diagnostic Workflow to Enable Team-Based Precision Genomics.

The primary goal of precision genomics is the identification of causative genetic variants in targeted or whole-genome sequencing data. The ultimate clinical hope is that these findings lead to an efficacious change in treatment for the patient. In current clinical practice, these findings are typically returned by expert analysts as static, text-based reports. Ideally, these reports summarize the quality of the data obtained, integrate known gene-phenotype associations, follow allele segregation and affected status within the sequenced samples, and weigh computational evidence of pathogenicity. These findings are used to prioritize the variant(s) most likely to cause the given patient's phenotypes. In most diagnostic settings, a team of experts contribute to these reports, including bioinformaticians, clinicians, and genetic counselors, among others. However, these experts often do not have the necessary tools to review genomic findings, test genetic hypotheses, or query specific gene and variant information. Additionally, team members often rely on different tools and methods based on their given expertise, resulting in further difficulties in communicating and discussing genomic findings. Here, we present clin.iobio-a web-based solution to collaborative genomic analysis that enables diagnostic team members to focus on their area of expertise within the diagnostic process, while allowing them to easily review and contribute to all steps of the diagnostic process. Clin.iobio integrates tools from the popular iobio genomic visualization suite into a comprehensive diagnostic workflow, encompassing (1) genomic data quality review, (2) dynamic phenotype-driven gene prioritization, (3) variant prioritization using a comprehensive set of knowledge bases and annotations, (4) and an exportable findings summary. In conclusion, clin.iobio is a comprehensive solution to team-based precision genomics, the findings of which stand to inform genomic considerations in clinical practice.

Artificial intelligence enables comprehensive genome interpretation and nomination of candidate diagnoses for rare genetic diseases.

BACKGROUND: Clinical interpretation of genetic variants in the context of the patient's phenotype is becoming the largest component of cost and time expenditure for genome-based diagnosis of rare genetic diseases. Artificial intelligence (AI) holds promise to greatly simplify and speed genome interpretation by integrating predictive methods with the growing knowledge of genetic disease. Here we assess the diagnostic performance of Fabric GEM, a new, AI-based, clinical decision support tool for expediting genome interpretation. METHODS: We benchmarked GEM in a retrospective cohort of 119 probands, mostly NICU infants, diagnosed with rare genetic diseases, who received whole-genome or whole-exome sequencing (WGS, WES). We replicated our analyses in a separate cohort of 60 cases collected from five academic medical centers. For comparison, we also analyzed these cases with current state-of-the-art variant prioritization tools. Included in the comparisons were trio, duo, and singleton cases. Variants underpinning diagnoses spanned diverse modes of inheritance and types, including structural variants (SVs). Patient phenotypes were extracted from clinical notes by two means: manually and using an automated clinical natural language processing (CNLP) tool. Finally, 14 previously unsolved cases were reanalyzed. RESULTS: GEM ranked over 90% of the causal genes among the top or second candidate and prioritized for review a median of 3 candidate genes per case, using either manually curated or CNLP-derived phenotype descriptions. Ranking of trios and duos was unchanged when analyzed as singletons. In 17 of 20 cases with diagnostic SVs, GEM identified the causal SVs as the top candidate and in 19/20 within the top five, irrespective of whether SV calls were provided or inferred ab initio by GEM using its own internal SV detection algorithm. GEM showed similar performance in absence of parental genotypes. Analysis of 14 previously unsolved cases resulted in a novel finding for one case, candidates ultimately not advanced upon manual review for 3 cases, and no new findings for 10 cases. CONCLUSIONS: GEM enabled diagnostic interpretation inclusive of all variant types through automated nomination of a very short list of candidate genes and disorders for final review and reporting. In combination with deep phenotyping by CNLP, GEM enables substantial automation of genetic disease diagnosis, potentially decreasing cost and expediting case review.

Deep whole-genome sequencing of multiple proband tissues and parental blood reveals the complex genetic etiology of congenital diaphragmatic hernias.

The diaphragm is critical for respiration and separation of the thoracic and abdominal cavities, and defects in diaphragm development are the cause of congenital diaphragmatic hernias (CDH), a common and often lethal birth defect. The genetic etiology of CDH is complex. Single-nucleotide variants (SNVs), insertions/deletions (indels), and structural variants (SVs) in more than 150 genes have been associated with CDH, although few genes are recurrently mutated in multiple individuals and mutated genes are incompletely penetrant. This suggests that multiple genetic variants in combination, other not-yet-investigated classes of variants, and/or nongenetic factors contribute to CDH etiology. However, no studies have comprehensively investigated in affected individuals the contribution of all possible classes of variants throughout the genome to CDH etiology. In our study, we used a unique cohort of four individuals with isolated CDH with samples from blood, skin, and diaphragm connective tissue and parental blood and deep whole-genome sequencing to assess germline and somatic de novo and inherited SNVs, indels, and SVs. In each individual we found a different mutational landscape that included germline de novo and inherited SNVs and indels in multiple genes. We also found in two individuals a 343 bp deletion interrupting an annotated enhancer of the CDH-associated gene GATA4, and we hypothesize that this common SV (found in 1%-2% of the population) acts as a sensitizing allele for CDH. Overall, our comprehensive reconstruction of the genetic architecture of four CDH individuals demonstrates that the etiology of CDH is heterogeneous and multifactorial.

The relationship between common risk factors and the pathology of pressure ulcer development: a systematic review.

OBJECTIVE: The aim of this systematic review was to examine the associations and relationship between commonly cited risk factors and the pathology of pressure ulcer (PU) development. METHOD: Using systematic review methodology, original research studies, prospective design and human studies written in English were included. The search was conducted in March 2018, using Ovid, Ovid EMBASE and CINAHL databases. Data were extracted using a pre-designed extraction tool and all included studies were quality appraised using the evidence-based librarianship critical appraisal. RESULTS: A total of 382 records were identified, of which five met the inclusion criteria. The studies were conducted between 1994 and 2017. Most studies were conducted in hospital and geriatric wards. The mean sample size was 96±145.7 participants. Ischaemia, recovery of blood flow and pathological impact of pressure and shear was mainly found as the cited risk factor and PU aetiology. CONCLUSION: This review systematically analysed five papers exploring the relationship between risk factors for PU development and aetiology. It identified many risk factors and underlying pathological mechanisms that interact in the development of PU including ischaemia, stress, recovery of blood flow, tissue hypoxia and the pathological impact of pressure and shear. There are several pathways in which these pathological mechanisms contribute to PU development and identifying these could establish potential ways of preventing or treating the development of PU for patients.

Ankle Fracture Detection Utilizing a Convolutional Neural Network Ensemble Implemented with a Small Sample, De Novo Training, and Multiview Incorporation.

To determine whether we could train convolutional neural network (CNN) models de novo with a small dataset, a total of 596 normal and abnormal ankle cases were collected and processed. Single- and multiview models were created to determine the effect of multiple views. Data augmentation was performed during training. The Inception V3, Resnet, and Xception convolutional neural networks were constructed utilizing the Python programming language with Tensorflow as the framework. Training was performed using single radiographic views. Measured output metrics were accuracy, positive predictive value (PPV), negative predictive value (NPV), sensitivity, and specificity. Model outputs were evaluated using both one and three radiographic views. Ensembles were created from a combination of CNNs after training. A voting method was implemented to consolidate the output from the three views and model ensemble. For single radiographic views, the ensemble of all 5 models produced the best accuracy at 76%. When all three views for a single case were utilized, the ensemble of all models resulted in the best output metrics with an accuracy of 81%. Despite our small dataset size, by utilizing an ensemble of models and 3 views for each case, we achieved an accuracy of 81%, which was in line with the accuracy of other models using a much higher number of cases with pre-trained models and models which implemented manual feature extraction.

Identification of Polycystic Kidney Disease 1 Like 1 Gene Variants in Children With Biliary Atresia Splenic Malformation Syndrome.

Biliary atresia (BA) is the most common cause of end-stage liver disease in children and the primary indication for pediatric liver transplantation, yet underlying etiologies remain unknown. Approximately 10% of infants affected by BA exhibit various laterality defects (heterotaxy) including splenic abnormalities and complex cardiac malformations-a distinctive subgroup commonly referred to as the biliary atresia splenic malformation (BASM) syndrome. We hypothesized that genetic factors linking laterality features with the etiopathogenesis of BA in BASM patients could be identified through whole-exome sequencing (WES) of an affected cohort. DNA specimens from 67 BASM subjects, including 58 patient-parent trios, from the National Institute of Diabetes and Digestive and Kidney Diseases-supported Childhood Liver Disease Research Network (ChiLDReN) underwent WES. Candidate gene variants derived from a prespecified set of 2,016 genes associated with ciliary dysgenesis and/or dysfunction or cholestasis were prioritized according to pathogenicity, population frequency, and mode of inheritance. Five BASM subjects harbored rare and potentially deleterious biallelic variants in polycystic kidney disease 1 like 1 (PKD1L1), a gene associated with ciliary calcium signaling and embryonic laterality determination in fish, mice, and humans. Heterozygous PKD1L1 variants were found in 3 additional subjects. Immunohistochemical analysis of liver from the one BASM subject available revealed decreased PKD1L1 expression in bile duct epithelium when compared to normal livers and livers affected by other noncholestatic diseases. Conclusion: WES identified biallelic and heterozygous PKD1L1 variants of interest in 8 BASM subjects from the ChiLDReN data set; the dual roles for PKD1L1 in laterality determination and ciliary function suggest that PKD1L1 is a biologically plausible, cholangiocyte-expressed candidate gene for the BASM syndrome.

The VAAST Variant Prioritizer (VVP): ultrafast, easy to use whole genome variant prioritization tool.

BACKGROUND: Prioritization of sequence variants for diagnosis and discovery of Mendelian diseases is challenging, especially in large collections of whole genome sequences (WGS). Fast, scalable solutions are needed for discovery research, for clinical applications, and for curation of massive public variant repositories such as dbSNP and gnomAD. In response, we have developed VVP, the VAAST Variant Prioritizer. VVP is ultrafast, scales to even the largest variant repositories and genome collections, and its outputs are designed to simplify clinical interpretation of variants of uncertain significance. RESULTS: We show that scoring the entire contents of dbSNP (> 155 million variants) requires only 95 min using a machine with 4 cpus and 16 GB of RAM, and that a 60X WGS can be processed in less than 5 min. We also demonstrate that VVP can score variants anywhere in the genome, regardless of type, effect, or location. It does so by integrating sequence conservation, the type of sequence change, allele frequencies, variant burden, and zygosity. Finally, we also show that VVP scores are consistently accurate, and easily interpreted, traits not shared by many commonly used tools such as SIFT and CADD. CONCLUSIONS: VVP provides rapid and scalable means to prioritize any sequence variant, anywhere in the genome, and its scores are designed to facilitate variant interpretation using ACMG and NHS guidelines. These traits make it well suited for operation on very large collections of WGS sequences.

Primary Ovarian Insufficiency and Azoospermia in Carriers of a Homozygous PSMC3IP Stop Gain Mutation.

Context: The etiology of primary ovarian insufficiency (POI) remains unknown in most cases. Objective: We sought to identify the genes causing POI. Design: The study was a familial genetic study. Setting: The study was performed at two academic institutions. Patients: We identified a consanguineous Yemeni family in which four daughters had POI. A brother had azoospermia. Intervention: DNA was subjected to whole genome sequencing. Shared regions of homozygosity were identified using Truploidy and prioritized using the Variant Annotation, Analysis, and Search Tool with control data from 387 healthy subjects. Imaging and quantification of protein localization and mitochondrial function were examined in cell lines. Main Outcome: Homozygous recessive gene variants shared by the four sisters. Results: The sisters shared a homozygous stop gain mutation in exon 6 of PSMC3IP (c.489 C>G, p.Tyr163Ter) and a missense variant in exon 1 of CLPP (c.100C>T, p.Pro34Ser). The affected brother also carried the homozygous PSMC3IP mutation. Functional studies demonstrated mitochondrial fragmentation in cells infected with the CLPP mutation. However, no abnormality was found in mitochondrial targeting or respiration. Conclusions: The PSMC3IP mutation provides additional evidence that mutations in meiotic homologous recombination and DNA repair genes result in distinct female and male reproductive phenotypes, including delayed puberty and primary amenorrhea caused by POI (XX gonadal dysgenesis) in females but isolated azoospermia with normal pubertal development in males. The findings also suggest that the N-terminal missense mutation in CLPP does not cause substantial mitochondrial dysfunction or contribute to ovarian insufficiency in an oligogenic manner.

Tracking long-distance atmospheric deposition of trace metal emissions from smelters in the upper Columbia River valley using Pb isotope analysis of lake sediments.

Heavy metal discharge from mining and smelting operations into aquatic ecosystems can cause long-term biological and ecological impacts. The upper Columbia River is highly contaminated with heavy metal wastes from nearby smelting operations in Trail, British Columbia, Canada, and to a lesser extent, Northport (Le Roi smelter), Washington, USA. Airborne emissions from the Trail operations were historically and are currently transported by prevailing winds down the Columbia River canyon, where particulate metals can be deposited into lakes and watersheds. In lakes, sediment cores contain records of past environmental conditions, providing a timeline of fundamental chemical and biological relationships within aquatic ecosystems, including records of airborne metal depositions. We analyzed trace metal concentrations (Ni, Cd, Zn, As, Cu, Sb, Pb, Hg) and Pb isotope compositions of sediment cores from six remote eastern Washington lakes to assess potential sources of atmospheric heavy metal deposition. Sediment cores displayed evidence to support trace metal loading as a direct consequence of smelting operations in Trail. Smelter contamination was detected 144 km downwind of the Trail Smelter. Cd, Sb, Pb (p < 0.001), and to a lesser extent As and Hg (p < 0.05) concentrations were correlated with Pb isotope compositions, suggesting that the Trail operations were likely the main source for these trace metals.

Electronic π-to-π* Excitations of Rhodamine Dyes Exhibit a Time-Dependent Kohn-Sham Theory "Cyanine Problem".

The longest-wavelength π-to-π* electronic excitations of rhodamine-like dyes (RDs) with different group 16 heteroatoms (O, S, Se, Te) have been investigated. Time-dependent Kohn-Sham theory (TDKST) calculations were compared with coupled-cluster (CC) and equations-of-motion (EOM) CC results for π-to-π* singlet and triplet excitations. The RDs exhibit characteristics in the TDKST calculations that are very similar to previously investigated cyanine dyes, in the sense that the singlet energies obtained with nonhybrid functionals are too high compared with the CC results at the SD(T) level. The errors became increasingly larger for functionals with increasing amounts of exact exchange. TDKST with all tested functionals led to severe underestimations of the corresponding triplet excitations and overestimations of the singlet-triplet gaps. Long-range-corrected range-separated exchange and "optimal tuning" of the range separation parameter did not significantly improve the TDKST results. A detailed analysis suggests that the problem is differential electron correlation between the ground and excited states, which is not treated sufficiently by the relatively small integrals over the exchange-correlation response kernel that enter the excitation energy expression. Numerical criteria are suggested that may help identify "cyanine-like" problems in TDKST calculations of excitation spectra.

POLR2C Mutations Are Associated With Primary Ovarian Insufficiency in Women.

CONTEXT: Primary ovarian insufficiency (POI) results from a premature loss of oocytes, causing infertility and early menopause. The etiology of POI remains unknown in a majority of cases. OBJECTIVE: To identify candidate genes in families affected by POI. DESIGN: This was a family-based genetic study. SETTING: The study was performed at two academic institutions. PATIENTS AND OTHER PARTICIPANTS: A family with four generations of women affected by POI (n = 5). Four of these women, three with an associated autoimmune diagnosis, were studied. The controls (n = 387) were recruited for health in old age. INTERVENTION: Whole-genome sequencing was performed. MAIN OUTCOME MEASURE: Candidate genes were identified by comparing gene mutations in three family members and 387 control subjects analyzed simultaneously using the pedigree Variant Annotation, Analysis and Search Tool. Data were also compared with that in publicly available databases. RESULTS: We identified a heterozygous nonsense mutation in a subunit of RNA polymerase II (POLR2C) that synthesizes messenger RNA. A rare sequence variant in POLR2C was also identified in one of 96 women with sporadic POI. POLR2C expression was decreased in the proband compared with women with POI from another cause. Knockdown in an embryonic carcinoma cell line resulted in decreased protein production and impaired cell proliferation. CONCLUSIONS: These data support a role for RNA polymerase II mutations as candidates in the etiology of POI. The current data also support results from genome-wide association studies that hypothesize a role for RNA polymerase II subunits in age at menopause in the population.

Is lipid correction necessary in the stable isotope analysis of fish tissues?

RATIONALE: Stable isotope analysis (SIA) is a powerful tool for examining diet and food-web dynamics. SIA assumes "you are what you eat" relative to carbon (C) and nitrogen (N). However, fractionation of carbon during lipid synthesis violates this assumption; high-lipid tissues do not reflect δ(13) C values of diet and therefore have the potential to skew mixing model results and diet interpretations, making corrections necessary. METHODS: Brook Trout (Salvelinus fontinalis) white muscle and liver samples from several fish species representing the temperate North American cold- and warm-water fish community were corrected for lipids via chemical lipid extraction and mathematical lipid normalization. To assess the accuracy of model-predicted lipid-free δ(13) C values calculated from four normalization models, we compared model-predicted values with those measured after lipid extraction. RESULTS: We found that chemical lipid extraction is unnecessary for Brook Trout white muscle tissue with low initial lipid content. However, in tissues with C:N ratios greater than 3.5, lipid extraction increased δ(13) C values in fish liver by more than 1.0 ‰, indicating that liver lipid content is sufficient to bias δ(13) C values. We also found that lipids were accurately accounted for with mathematical normalization and recommend that tissues with C:N ratios greater than 3.5 be corrected mathematically. CONCLUSIONS: Our findings indicate that mathematical normalization is sufficient to account for bias in δ(13) C values associated with lipid content in fish tissues when C:N ratios are above 3.5. C:N ratios below 3.5 indicate that tissues have insufficient levels of lipid to bias the δ(13) C values. Generally, these findings support the use of more timely and cost-effective processing and analysis methods in future aquatic food-web studies utilizing SIA. Copyright © 2016 John Wiley & Sons, Ltd.