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Competing Interests: The authors report no competing interests.

Background: RNA sequencing (RNA-seq) is increasingly being used as a complementary tool to DNA sequencing in diagnostics where DNA analysis has been uninformative. RNA-seq enables the identification of aberrant splicing and aberrant gene expression, improving the interpretation of variants of unknown significance (VUSs), and provides the opportunity to scan the transcriptome for aberrant splicing and expression in relevant genes that may be the cause of a patient's phenotype. This work aims to investigate the feasibility of generating new diagnostic candidates in patients without a previously reported VUS using an RNA-seq-centric approach., Methods: We systematically assessed the transcriptomic profiles of 86 patients with suspected Mendelian disorders, 38 of whom had no candidate sequence variant, using RNA from blood samples. Each VUS was visually inspected to search for splicing abnormalities. Once aberrant splicing was identified in cases with VUS, multiple open-source alternative splicing tools were used to investigate if they would identify what was observed in IGV. Expression outliers were detected using OUTRIDER. Diagnoses in cases without a VUS were explored using two separate strategies., Results: RNA-seq allowed us to assess 71% of VUSs, detecting aberrant splicing in 14/48 patients with a VUS. We identified four new diagnoses by detecting novel aberrant splicing events in patients with no candidate sequence variants from prior DNA testing (n = 32) or where the candidate VUS did not affect splicing (n = 23). An additional diagnosis was made through the detection of skewed X-inactivation., Conclusion: This work demonstrates the utility of an RNA-centric approach in identifying novel diagnoses in patients without candidate VUSs. It underscores the utility of blood-based RNA analysis in improving diagnostic yields and highlights optimal approaches for such analyses., (© 2024. The Author(s).)

Objective: In the current exploratory study we estimate comorbidity rates between FDs [fibromyalgia (FM), myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), and irritable bowel syndrome (IBS)]-and IDs-[major depressive disorder (MDD) and generalized anxiety disorder (GAD)] by using self-reported diagnostic criteria., Method: We analyzed data from 107,849 participants (mean age = 49.3 (SD = 13.0), 58.6% women) of the Lifelines Cohort Study. Lifelines is a prospective population-based cohort study in the northeast of the Netherlands. Current IDs were assessed using the Mini-International Neuropsychiatric Interview. Current FM, ME/CFS, and IBS were assessed according to the 2010 American College of Rheumatology criteria, the 1994 Centers for Disease Control and Prevention criteria and the ROME IV criteria, respectively. We estimated tetrachoric correlations between diagnoses and tested for sex differences. Additionally, we estimated the ratio of observed-to-expected frequency for combinations of diagnoses., Results: FDs and IDs are highly comorbid (odds ratios: 3.2-12.6) with associations stronger among men. Participants with at least three disorders/diagnoses were more prevalent than expected by chance., Conclusion: Studies that aim to explain sex differences and the comorbidity of specific combinations of IDs and FDs will be an important contribution to understanding the etiology of these conditions., Competing Interests: Declaration of competing interest Nathaniel S. Thomas, Nathan A. Gillespie, Kenneth S. Kendler, Albertine J. Oldehinkel, Judith G.M. Rosmalen and Hanna M. van Loo declare that they have no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Purpose: The 100,000 Genomes Project diagnosed a quarter of affected participants, but 26% of diagnoses were not on the applied gene panel(s); with many being de novo variants. Assessing biallelic variants without a gene panel is more challenging., Methods: We sought to identify missed biallelic diagnoses using GenePy, which incorporates allele frequency, zygosity, and a user-defined deleterious metric, generating an aggregate GenePy score per gene, per participant. We calculated GenePy scores for 2862 recessive disease genes in 78,216 100,000 Genomes Project participants. For each gene, we ranked participant GenePy scores and scrutinized affected participants without a diagnosis, whose scores ranked among the top 5 for each gene. In cases which participant phenotypes overlapped with the disease gene of interest, we extracted rare variants and applied phase, ClinVar, and ACMG classification., Results: 3184 affected individuals without a molecular diagnosis had a top-5-ranked GenePy score and 682 of 3184 (21%) had phenotypes overlapping with a top-ranking gene. In 122 of 669 (18%) phenotype-matched cases (excluding 13 withdrawn participants), we identified a putative missed diagnosis (2.2% of all undiagnosed participants). A further 334 of 669 (50%) cases have a possible missed diagnosis but require functional validation., Conclusion: Applying GenePy at scale has identified 456 potential diagnoses, demonstrating the value of novel diagnostic strategies., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Contemporary genome-wide association study (GWAS) methods typically do not account for variability in genetic effects throughout development. We applied genomic structural equation modeling to combine developmentally-informative phenotype data and GWAS to create polygenic scores (PGS) for alcohol use frequency that are specific to developmental stage. Longitudinal cohort studies targeted for gene-identification analyses include the Collaborative Study on the Genetics of Alcoholism (adolescence n = 1,118, early adulthood n = 2,762, adulthood n = 5,255), the National Longitudinal Study of Adolescent to Adult Health (adolescence n = 3,089, early adulthood n = 3,993, adulthood n = 5,149), and the Avon Longitudinal Study of Parents and Children (ALSPAC; adolescence n = 5,382, early adulthood n = 3,613). PGS validation analyses were conducted in the COGA sample using an alternate version of the discovery analysis with COGA removed. Results suggest that genetic liability for alcohol use frequency in adolescence may be distinct from genetic liability for alcohol use frequency later in developmental periods. The age-specific PGS predicts an increase of 4 drinking days per year per PGS standard deviation when modeled separately from the common factor PGS in adulthood. The current work was underpowered at all steps of the analysis plan. Though small sample sizes and low statistical power limit the substantive conclusions that can be drawn regarding these research questions, this work provides a foundation for future genetic studies of developmental variability in the genetic underpinnings of alcohol use behaviors and genetically-informed, age-matched phenotype prediction., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Breast cancer is the most common invasive malignancy in the world, with millions of survivors living today. Type 2 diabetes mellitus (T2DM) is also a globally prevalent disease that is a widely studied risk factor for breast cancer. Most breast tumours express the oestrogen receptor and are treated with systemic therapies designed to disrupt oestrogen-dependent signalling. Since the advent of targeted endocrine therapy six decades ago, the mortality from breast cancer has steadily declined; however, during the past decade, an elevated risk of T2DM after breast cancer treatment has been reported, particularly for those who received endocrine therapy. In this Review, we highlight key events in the history of endocrine therapies, beginning with the development of tamoxifen. We also summarize the sequence of reported adverse metabolic effects, which include dyslipidaemia, hepatic steatosis and impaired glucose tolerance. We discuss the limitations of determining a causal role for breast cancer treatments in T2DM development from epidemiological data and describe informative preclinical studies that suggest complex mechanisms through which endocrine therapy might drive T2DM risk and progression. We also reinforce the life-saving benefits of endocrine therapy and highlight the need for better predictive biomarkers of T2DM risk and preventive strategies for the growing population of breast cancer survivors., (© 2023. Springer Nature Limited.)

Genome sequencing is available as a clinical test in the UK through the Genomic Medicine Service (GMS). The GMS analytical strategy predominantly filters genome data on preselected gene panels. Whilst this reduces variants requiring assessment by reporting laboratories, pathogenic variants outside applied panels may be missed, and variants in genes without established disease-gene relationships are largely ignored. This study compares the analysis of a research exome to a GMS clinical genome for the same patients. For the research exome, we applied a panel-agnostic approach filtering for variants with Hi gh P athogenic Po tential (HiPPo) using ClinVar, allele frequency, and in silico prediction tools. We then restricted HiPPo variants to Gene Curation Coalition (GenCC) disease genes. These results were compared with the GMS genome panel-based approach. Twenty-four participants from eight families underwent parallel research exome and GMS genome sequencing. Exome HiPPo analysis identified a similar number of variants as the GMS panel-based approach. GMS genome analysis returned two pathogenic variants and one de novo variant. Exome HiPPo analysis returned the same variants plus an additional pathogenic variant and three further de novo variants in novel genes, where case series are underway. When HiPPo was restricted to GenCC disease genes, statistically fewer variants required assessment to identify more pathogenic variants than reported by the GMS, giving a diagnostic rate per variant assessed of 20% for HiPPo versus 3% for the GMS. With UK plans to sequence 5 million genomes, strategies are needed to optimise genome analysis beyond gene panels whilst minimising the burden of variants requiring clinical assessment.

Background: Current clinical testing methods used to uncover the genetic basis of rare disease have inherent limitations, which can lead to causative pathogenic variants being missed. Within the rare disease arm of the 100 000 Genomes Project (100kGP), families were recruited under the clinical indication 'single autosomal recessive mutation in rare disease'. These participants presented with strong clinical suspicion for a specific autosomal recessive disorder, but only one suspected pathogenic variant had been identified through standard-of-care testing. Whole genome sequencing (WGS) aimed to identify cryptic 'second-hit' variants., Methods: To investigate the 31 families with available data that remained unsolved following formal review within the 100kGP, SVRare was used to aggregate structural variants present in <1% of 100kGP participants. Small variants were assessed using population allele frequency data and SpliceAI. Literature searches and publicly available online tools were used for further annotation of pathogenicity., Results: Using these strategies, 8/31 cases were solved, increasing the overall diagnostic yield of this cohort from 10/41 (24.4%) to 18/41 (43.9%). Exemplar cases include a patient with cystic fibrosis harbouring a novel exonic LINE1 insertion in CFTR and a patient with generalised arterial calcification of infancy with complex interlinked duplications involving exons 2-6 of ENPP1 . Although ambiguous by short-read WGS, the ENPP1 variant structure was resolved using optical genome mapping and RNA analysis., Conclusion: Systematic examination of cryptic variants across a multi-disease cohort successfully identifies additional pathogenic variants. WGS data analysis in autosomal recessive rare disease should consider complex structural and small intronic variants as potentially pathogenic second hits., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.)

Background: Obesity increases breast cancer risk and breast cancer-specific mortality, particularly for people with estrogen receptor (ER)-positive tumors. Body mass index (BMI) is used to define obesity, but it may not be the best predictor of breast cancer risk or prognosis on an individual level. Adult weight gain is an independent indicator of breast cancer risk. Our previous work described a murine model of obesity, ER-positive breast cancer, and weight gain and identified fibroblast growth factor receptor (FGFR) as a potential driver of tumor progression. During adipose tissue expansion, the FGF1 ligand is produced by hypertrophic adipocytes as a stimulus to stromal preadipocytes that proliferate and differentiate to provide additional lipid storage capacity. In breast adipose tissue, FGF1 production may stimulate cancer cell proliferation and tumor progression., Methods: We explored the effects of FGF1 on ER-positive endocrine-sensitive and resistant breast cancer and compared that to the effects of the canonical ER ligand, estradiol. We used untargeted proteomics, specific immunoblot assays, gene expression profiling, and functional metabolic assessments of breast cancer cells. The results were validated in tumors from obese mice and breast cancer datasets from women with obesity., Results: FGF1 stimulated ER phosphorylation independently of estradiol in cells that grow in obese female mice after estrogen deprivation treatment. Phospho- and total proteomic, genomic, and functional analyses of endocrine-sensitive and resistant breast cancer cells show that FGF1 promoted a cellular phenotype characterized by glycolytic metabolism. In endocrine-sensitive but not endocrine-resistant breast cancer cells, mitochondrial metabolism was also regulated by FGF1. Comparison of gene expression profiles indicated that tumors from women with obesity shared hallmarks with endocrine-resistant breast cancer cells., Conclusions: Collectively, our data suggest that one mechanism by which obesity and weight gain promote breast cancer progression is through estrogen-independent ER activation and cancer cell metabolic reprogramming, partly driven by FGF/FGFR. The first-line treatment for many patients with ER-positive breast cancer is inhibition of estrogen synthesis using aromatase inhibitors. In women with obesity who are experiencing weight gain, locally produced FGF1 may activate ER to promote cancer cell metabolic reprogramming and tumor progression independently of estrogen., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Diagnostic testing for primary ciliary dyskinesia (PCD) started in 2013 in Palestine. We aimed to describe the diagnostic, genetic and clinical spectrum of the Palestinian PCD population., Methods: Individuals with symptoms suggestive of PCD were opportunistically considered for diagnostic testing: nasal nitric oxide (nNO) measurement, transmission electron microscopy (TEM) and/or PCD genetic panel or whole-exome testing. Clinical characteristics of those with a positive diagnosis were collected close to testing including forced expiratory volume in 1 s (FEV 1 ) Global Lung Index z-scores and body mass index z-scores., Results: 68 individuals had a definite positive PCD diagnosis, 31 confirmed by genetic and TEM results, 23 by TEM results alone, and 14 by genetic variants alone. 45 individuals from 40 families had 17 clinically actionable variants and four had variants of unknown significance in 14 PCD genes . CCDC39 , DNAH11 and DNAAF11 were the most commonly mutated genes. 100% of variants were homozygous. Patients had a median age of 10.0 years at diagnosis, were highly consanguineous (93%) and 100% were of Arabic descent. Clinical features included persistent wet cough (99%), neonatal respiratory distress (84%) and situs inversus (43%). Lung function at diagnosis was already impaired (FEV 1 z-score median -1.90 (-5.0-1.32)) and growth was mostly within the normal range (z-score mean -0.36 (-3.03-2.57). 19% individuals had finger clubbing., Conclusions: Despite limited local resources in Palestine, detailed geno- and phenotyping forms the basis of one of the largest national PCD populations globally. There was notable familial homozygosity within the context of significant population heterogeneity., Competing Interests: Conflict of interest: N. Rumman received support for the present manuscript from two NIOX MINO devices from Circassia (previously called Aerocrine) to perform nasal nitric oxide testing and AAIR Charity funded whole-exome sequencing testing, and support for attending meetings and/or travel from the European Respiratory Society for a short term research training fellowship (3 months at Southampton University) (STRTF 2014-6816), outside the submitted work. M.R. Fassad received support for the present manuscript from a Wellcome Trust Collaborative Award in Science (210585/Z/18/Z). C. Driessens received support for the present manuscript from NHS England: work for this manuscript was performed while working for the National PCD Diagnostic Service at University Hospital Southampton; this service is commissioned and funded by NHS England. R. Pengelly received support for the present manuscript from Asthma Allergy and Inflammation Research. G. Wheway received support for the present manuscript from Asthma Allergy and Inflammation Research Trust; grants or contracts from UKRI COVID-19 Agile Response Fund, Wessex Medical Research/Rosetrees Trust PhD studentship, and Asthma Allergy and Inflammation Research Trust, outside the submitted work; stock or stock options for Illumina Inc., outside the submitted work; and receipt of equipment, materials, medical writing, gifts or other services from Synthego, outside the submitted work. H.M. Mitchison received support for the present manuscript from Great Ormond Street Children's Charity, NIHR Biomedical Research Centre at Great Ormond Street Hospital, and British Council Newton-Mosharafa Fund and Ministry of Higher Education in Egypt. J.S. Lucas received grants or contracts from NIHR, AAIR Charity, and NHS England, outside the submitted work. The remaining authors have nothing to disclose., (Copyright ©The authors 2023.)

Background: Parental divorce and discord are associated with poorer alcohol-related outcomes for offspring. However, not all children exposed to these stressors develop alcohol problems. Our objective was to test gene-by-environment interaction effects whereby children's genetic risk for alcohol problems modifies the effects of parental divorce and discord to predict alcohol outcomes., Methods: The sample included European (EA; N = 5608, 47% male, M age  ~ 36 years) and African (AA; N = 1714, 46% female, M age  ~ 33 years) ancestry participants from the Collaborative Study on the Genetics of Alcoholism. Outcomes included age at initiation of regular drinking and lifetime DSM-5 alcohol use disorder (AUD). Predictors included parental divorce, parental relationship discord, and offspring alcohol problems polygenic risk scores (PRS ALC ). Mixed effects Cox proportional hazard models were used to examine alcohol initiation and generalized linear mixed effects models were used to examine lifetime AUD. Tests of PRS moderation of the effects of parental divorce/relationship discord on alcohol outcomes were examined on multiplicative and additive scales., Results: Among EA participants, parental divorce, parental discord, and higher PRS ALC were associated with earlier alcohol initiation and greater lifetime AUD risk. Among AA participants, parental divorce was associated with earlier alcohol initiation and discord was associated with earlier initiation and AUD. PRS ALC was not associated with either. Parental divorce/discord and PRS ALC interacted on an additive scale in the EA sample, but no interactions were found in AA participants., Conclusions: Children's genetic risk for alcohol problems modifies the impact of parental divorce/discord, consistent with an additive model of diathesis-stress interaction, with some differences across ancestry., (© 2023 The Authors. Alcohol: Clinical and Experimental Research published by Wiley Periodicals LLC on behalf of Research Society on Alcohol.)

Ciliopathies may be classed as primary or motile depending on the underlying ciliary defect and are usually considered distinct clinical entities. Primary ciliopathies are associated with multisystem syndromes typically affecting the brain, kidney, and eye, as well as other organ systems such as the liver, skeleton, auditory system, and metabolism. Motile ciliopathies are a heterogenous group of disorders with defects in specialised motile ciliated tissues found within the lung, brain, and reproductive system, and are associated with primary ciliary dyskinesia, bronchiectasis, infertility and rarely hydrocephalus. Primary and motile cilia share defined core ultra-structures with an overlapping proteome, and human disease phenotypes can reflect both primary and motile ciliopathies. CEP164 encodes a centrosomal distal appendage protein vital for primary ciliogenesis. Human CEP164 mutations are typically described in patients with nephronophthisis-related primary ciliopathies but have also been implicated in motile ciliary dysfunction. Here we describe a patient with an atypical motile ciliopathy phenotype and biallelic CEP164 variants. This work provides further evidence that CEP164 mutations can contribute to both primary and motile ciliopathy syndromes, supporting their functional and clinical overlap, and informs the investigation and management of CEP164 ciliopathy patients., (© 2022 The Authors. Clinical Genetics published by John Wiley & Sons Ltd.)

Background: Genome sequencing was first offered clinically in the UK through the 100,000 Genomes Project (100KGP). Analysis was restricted to predefined gene panels associated with the patient's phenotype. However, panels rely on clearly characterised phenotypes and risk missing diagnoses outside of the panel(s) applied. We propose a complementary method to rapidly identify pathogenic variants, including those missed by 100KGP methods., Methods: The Loss-of-function Observed/Expected Upper-bound Fraction (LOEUF) score quantifies gene constraint, with low scores correlated with haploinsufficiency. We applied DeNovoLOEUF, a filtering strategy to sequencing data from 13,949 rare disease trios in the 100KGP, by filtering for rare, de novo, loss-of-function variants in disease genes with a LOEUF score < 0.2. We compared our findings with the corresponding patient's diagnostic reports., Results: 324/332 (98%) of the variants identified using DeNovoLOEUF were diagnostic or partially diagnostic (whereby the variant was responsible for some of the phenotype). We identified 39 diagnoses that were "missed" by 100KGP standard analyses, which are now being returned to patients., Conclusion: We have demonstrated a highly specific and rapid method with a 98% positive predictive value that has good concordance with standard analysis, low false-positive rate, and can identify additional diagnoses. Globally, as more patients are being offered genome sequencing, we anticipate that DeNovoLOEUF will rapidly identify new diagnoses and facilitate iterative analyses when new disease genes are discovered., (© 2022. The Author(s).)

We tested whether aspects of the childhood/adolescent home environment mediate genetic risk for alcohol problems within families across generations. Parental relationship discord and parental divorce were the focal environments examined. The sample included participants of European ancestry (N = 4806, 51% female) and African ancestry (N = 1960, 52% female) from the high-risk Collaborative Study on the Genetics of Alcoholism. Alcohol outcomes in the child generation included lifetime criterion counts for DSM-5 Alcohol Use Disorder (AUD), lifetime maximum drinks in 24 h, age at initiation of regular drinking, and age at first alcohol intoxication. Predictors in the parent generation included relationship discord, divorce, alcohol measures parallel to those in the child generation, and polygenic scores for alcohol problems. Parental polygenic scores were partitioned into alleles that were transmitted and non-transmitted to the child. The results from structural equation models were consistent with genetic nurture effects in European ancestry families. Exposure to parental relationship discord and parental divorce mediated, in part, the transmission of genetic risk for alcohol problems from parents to children to predict earlier ages regular drinking (β indirect  = -0.018 [-0.026, -0.011]) and intoxication (β indirect  = -0.015 [-0.023, -0.008]), greater lifetime maximum drinks (β indirect  = 0.006 [0.002, 0.01]) and more lifetime AUD criteria (β indirect  = 0.011 [0.006, 0.016]). In contrast, there was no evidence that parental alleles had indirect effects on offspring alcohol outcomes via parental relationship discord or divorce in the smaller number of families of African ancestry. In conclusion, parents transmit genetic risk for alcohol problems to their children not only directly, but also indirectly via genetically influenced aspects of the home environment. Further investigation of genetic nurture in non-European samples is needed., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

In this study, we test principal component analysis (PCA) of measured confounders as a method to reduce collider bias in polygenic association models. We present results from simulations and application of the method in the Collaborative Study of the Genetics of Alcoholism (COGA) sample with a polygenic score for alcohol problems, DSM-5 alcohol use disorder as the target phenotype, and two collider variables: tobacco use and educational attainment. Simulation results suggest that assumptions regarding the correlation structure and availability of measured confounders are complementary, such that meeting one assumption relaxes the other. Application of the method in COGA shows that PC covariates reduce collider bias when tobacco use is used as the collider variable. Application of this method may improve PRS effect size estimation in some cases by reducing the effect of collider bias, making efficient use of data resources that are available in many studies., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Genomic variants which disrupt splicing are a major cause of rare genetic diseases. However, variants which lie outside of the canonical splice sites are difficult to interpret clinically. Improving the clinical interpretation of non-canonical splicing variants offers a major opportunity to uplift diagnostic yields from whole genome sequencing data., Methods: Here, we examine the landscape of splicing variants in whole-genome sequencing data from 38,688 individuals in the 100,000 Genomes Project and assess the contribution of non-canonical splicing variants to rare genetic diseases. We use a variant-level constraint metric (the mutability-adjusted proportion of singletons) to identify constrained functional variant classes near exon-intron junctions and at putative splicing branchpoints. To identify new diagnoses for individuals with unsolved rare diseases in the 100,000 Genomes Project, we identified individuals with de novo single-nucleotide variants near exon-intron boundaries and at putative splicing branchpoints in known disease genes. We identified candidate diagnostic variants through manual phenotype matching and confirmed new molecular diagnoses through clinical variant interpretation and functional RNA studies., Results: We show that near-splice positions and splicing branchpoints are highly constrained by purifying selection and harbour potentially damaging non-coding variants which are amenable to systematic analysis in sequencing data. From 258 de novo splicing variants in known rare disease genes, we identify 35 new likely diagnoses in probands with an unsolved rare disease. To date, we have confirmed a new diagnosis for six individuals, including four in whom RNA studies were performed., Conclusions: Overall, we demonstrate the clinical value of examining non-canonical splicing variants in individuals with unsolved rare diseases., (© 2022. The Author(s).)

Objective: To examine associations between alcohol use disorder (AUD), its psychiatric comorbidities, and their interactions, with marital outcomes in a diverse high-risk, genetically informative sample., Method: Participants included European ancestry (EA; n = 4,045) and African ancestry (AA; n = 1,550) individuals from the multigenerational Collaborative Study on the Genetics of Alcoholism (COGA) sample (56% female, M age ∼ 41 years). Outcomes were lifetime marriage and divorce. Predictors included lifetime AUD, an alcohol problems polygenic score (PRS), and AUD comorbidities, including conduct or antisocial personality disorder (ASP), cannabis dependence/abuse (CAN), frequent tobacco use (TOB), and major depressive disorder (MDD). Mixed effect Cox models and generalized linear mixed effects models were fit., Results: Among EA participants, those with AUD and CAN were less likely to marry (hazard ratios [HRs] 0.70-0.83, p s < 0.01). Among AA participants, those with AUD and TOB were less likely to marry (HRs 0.66-0.82, p s < 0.05) and those with MDD were more likely to marry (HR = 1.34, p s < 0.01). Among EA participants, AUD, CAN, TOB, and MDD were associated with higher odds of divorce (odds ratios [ORs] 1.59-2.21, p s < 0.01). Among AA participants, no predictors were significantly associated with divorce. Significant random effects indicated genetic and environmental influences on marriage, but only environmental factors on divorce., Conclusions: In a high-risk sample, AUD was associated with reduced likelihood of marriage in EA and AA individuals and increased risk of divorce in EA individuals. These associations were largely independent of comorbidities. Genetic and environmental background factors contributed to marriage, while only environmental background factors contributed to divorce. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Oculocutaneous albinism type 1 (OCA1) is caused by pathogenic variants in the TYR (tyrosinase) gene which encodes the critical and rate-limiting enzyme in melanin synthesis. It is the most common OCA subtype found in Caucasians, accounting for ~50% of cases worldwide. The apparent 'missing heritability' in OCA is well described, with ~25-30% of clinically diagnosed individuals lacking two clearly pathogenic variants. Here we undertook empowered genetic studies in an extensive multigenerational Amish family, alongside a review of previously published literature, a retrospective analysis of in-house datasets, and tyrosinase activity studies. Together this provides irrefutable evidence of the pathogenicity of two common TYR variants, p.(Ser192Tyr) and p.(Arg402Gln) when inherited in cis alongside a pathogenic TYR variant in trans. We also show that homozygosity for the p.(Ser192Tyr)/p.(Arg402Gln) TYR haplotype results in a very mild, but fully penetrant, albinism phenotype. Together these data underscore the importance of including the TYR p.(Ser192Tyr)/p.(Arg402Gln) in cis haplotype as a pathogenic allele causative of OCA, which would likely increase molecular diagnoses in this missing heritability albinism cohort by 25-50%., (© 2022. The Author(s).)

Transcription of SHOX is dependent upon the interaction of the gene with a complex array of flanking regulatory elements. Duplications that contain flanking regulatory elements but not the SHOX gene have been reported in individuals with SHOX haploinsufficiency syndromes, suggesting that alterations to the physical organisation or genomic architecture may affect SHOX transcription. Individuals with tall stature and an additional X or Y chromosome have an extra copy of both the SHOX gene and the entire SHOX regulatory region, so all three copies of SHOX can be expressed fully. However, for a duplication of the SHOX gene that does not include all of the flanking regulatory elements, the potential effect on SHOX expression is difficult to predict. We present nine unpublished individuals with a SHOX whole gene duplication in whom the duplication contains variable amounts of the SHOX regulatory region, and we review 29 similar cases from the literature where phenotypic data were clearly stated. While tall stature was present in a proportion of these cases, we present evidence that SHOX whole gene duplications can also result in a phenotype more typically associated with SHOX haploinsufficiency and are significantly overrepresented in Leri-Weill dyschondrosteosis and idiopathic short stature probands compared to population controls. Although similar-looking duplications do not always produce a consistent phenotype, there may be potential genotype-phenotype correlations regarding the duplication size, regulatory element content, and the breakpoint proximity to the SHOX gene. Although ClinGen does not currently consider SHOX whole gene duplications to be clinically significant, the ClinGen triplosensitivity score does not take into account the context of the duplication, and more is now known about SHOX duplications and the role of flanking elements in SHOX regulation. The evidence presented here suggests that these duplications should not be discounted without considering the extent of the duplication and the patient phenotype, and should be included in diagnostic laboratory reports as variants of uncertain significance. Given the uncertain pathogenicity of these duplications, any reports should encourage the exclusion of all other causes of short stature where possible., (© 2023 S. Karger AG, Basel.)

Risky behaviors, such as substance use and unprotected sex, are associated with various physical and mental health problems. Recent genome-wide association studies indicated that variation in the cell adhesion molecule 2 (CADM2) gene plays a role in risky behaviors and self-control. In this phenome-wide scan for risky behavior, it was tested if underlying common vulnerability could be (partly) explained by pleiotropic effects of this gene and how large the effects were. Single nucleotide polymorphism (SNP)-level and gene-level association tests within four samples (25 and Up, Spit for Science, Netherlands Twin Register, and UK Biobank and meta-analyses over all samples (combined sample of 362,018 participants) were conducted to test associations between CADM2, substance- and sex-related risk behaviors, and various measures related to self-control. We found significant associations between the CADM2 gene, various risky behaviors, and different measures of self-control. The largest effect sizes were found for cannabis use, sensation seeking, and disinhibition. Effect sizes ranged from 0.01% to 0.26% for single top SNPs and from 0.07% to 3.02% for independent top SNPs together, with sufficient power observed only in the larger samples and meta-analyses. In the largest cohort, we found indications that risk-taking proneness mediated the association between CADM2 and latent factors for lifetime smoking and regular alcohol use. This study extends earlier findings that CADM2 plays a role in risky behaviors and self-control. It also provides insight into gene-level effect sizes and demonstrates the feasibility of testing mediation. These findings present a good starting point for investigating biological etiological pathways underlying risky behaviors., (© 2021 The Authors. Addiction Biology published by John Wiley & Sons Ltd on behalf of Society for the Study of Addiction.)

Background: It is estimated that 1-13% of cases of bronchiectasis in adults globally are attributable to primary ciliary dyskinesia (PCD) but many adult patients with bronchiectasis have not been investigated for PCD. PCD is a disorder caused by mutations in genes required for motile cilium structure or function, resulting in impaired mucociliary clearance. Symptoms appear in infancy but diagnosis is often late or missed, often due to the lack of a "gold standard" diagnostic tool and non-specific symptoms. Mutations in > 50 genes account for around 70% of cases, with additional genes, and non-coding, synonymous, missense changes or structural variants (SVs) in known genes presumed to account for the missing heritability., Methods: UK patients with no identified genetic confirmation for the cause of their PCD or bronchiectasis were eligible for whole genome sequencing (WGS) in the Genomics England Ltd 100,000 Genomes Project. 21 PCD probands and 52 non-cystic fibrosis (CF) bronchiectasis probands were recruited in Wessex Genome Medicine Centre (GMC). We carried out analysis of single nucleotide variants (SNVs) and SVs in all families recruited in Wessex GMC., Results: 16/21 probands in the PCD cohort received confirmed (n = 9), probable (n = 4) or possible (n = 3) diagnosis from WGS, although 13/16 of these could have been picked up by current standard of care gene panel testing. In the other cases, SVs were identified which were missed by panel testing. We identified variants in novel PCD candidate genes (IFT140 and PLK4) in 2 probands in the PCD cohort. 3/52 probands in the non-CF bronchiectasis cohort received a confirmed (n = 2) or possible (n = 1) diagnosis of PCD. We identified variants in novel PCD candidate genes (CFAP53 and CEP164) in 2 further probands in the non-CF bronchiectasis cohort., Conclusions: Genetic testing is an important component of diagnosing PCD, especially in cases of atypical disease history. WGS is effective in cases where prior gene panel testing has found no variants or only heterozygous variants. In these cases it may detect SVs and is a powerful tool for novel gene discovery., (© 2021. The Author(s).)

Background: Primary ciliary dyskinesia (PCD) is a heterogeneous inherited disorder caused by mutations in approximately 50 cilia-related genes. PCD genotype-phenotype relationships have mostly arisen from small case series because existing statistical approaches to investigating relationships have been unsuitable for rare diseases., Methods: We applied a topological data analysis (TDA) approach to investigate genotype-phenotype relationships in PCD. Data from separate training and validation cohorts included 396 genetically defined individuals carrying pathogenic variants in PCD genes. To develop the TDA models, 12 clinical and diagnostic variables were included. TDA-driven hypotheses were subsequently tested using traditional statistics., Results: Disease severity at diagnosis, measured by forced expiratory volume in 1 s (FEV 1 ) z-score, was significantly worse in individuals with CCDC39 mutations (compared to other gene mutations) and better in those with DNAH11 mutations; the latter also reported less neonatal respiratory distress. Patients without neonatal respiratory distress had better preserved FEV 1 at diagnosis. Individuals with DNAH5 mutations were phenotypically diverse. Cilia ultrastructure and beat pattern defects correlated closely to specific causative gene groups, confirming these tests can be used to support a genetic diagnosis., Conclusions: This large scale, multi-national study presents PCD as a syndrome with overlapping symptoms and variations in phenotype according to genotype. TDA modelling confirmed genotype-phenotype relationships reported by smaller studies ( e.g. FEV 1 worse with CCDC39 mutation) and identified new relationships, including FEV 1 preservation with DNAH11 mutations and diversity of severity with DNAH5 mutations., Competing Interests: Conflict of interest: B. Rubbo has nothing to disclose. Conflict of interest: M. Legendre has nothing to disclose. Conflict of interest: M.R. Fassad has nothing to disclose. Conflict of interest: E.G. Haarman has nothing to disclose. Conflict of interest: S. Best has nothing to disclose. Conflict of interest: I.C.M. Bon has nothing to disclose. Conflict of interest: J. Brandsma has nothing to disclose. Conflict of interest: P-R. Burgel reports personal fees for lectures and advisory board work from AstraZeneca, Boehringer Ingelheim, Chiesi, Novartis, Teva and Vertex, as well as personal fees for lectures from GSK, Pfizer and Zambon, outside the submitted work. Conflict of interest: G. Carlsson has nothing to disclose. Conflict of interest: S.B. Carr reports non-financial support and other (advisory board, lecture fee, travel, steering committee) from Vertex Pharmaceuticals, other (advisory board) from Profile Pharmaceuticals and other (lectures) from Teva Pharmaceuticals, as well as non-financial support and other (advisory board, travel, accommodation) from Chiesi Pharmaceuticals, outside the submitted work. Conflict of interest: M. Carroll has nothing to disclose. Conflict of interest: M. Edwards has nothing to disclose. Conflict of interest: E. Escudier has nothing to disclose. Conflict of interest: I. Honoré has nothing to disclose. Conflict of interest: D. Hunt has nothing to disclose. Conflict of interest: G. Jouvion has nothing to disclose. Conflict of interest: M.R. Loebinger reports personal fees for advisory board work and consultancy from AstraZeneca, Insmed, Polyphor, Bayer and Griffols, outside the submitted work. Conflict of interest: B. Maitre has nothing to disclose. Conflict of interest: D. Morris-Rosendahl has nothing to disclose. Conflict of interest: J-F. Papon has nothing to disclose. Conflict of interest: C.M. Parsons has nothing to disclose. Conflict of interest: M.P. Patel has nothing to disclose. Conflict of interest: N.S. Thomas has nothing to disclose. Conflict of interest: G. Thouvenin has nothing to disclose. Conflict of interest: W.T. Walker has nothing to disclose. Conflict of interest: R. Wilson has nothing to disclose. Conflict of interest: C. Hogg has nothing to disclose. Conflict of interest: H.M. Mitchison has nothing to disclose. Conflict of interest: J.S. Lucas has nothing to disclose. Conflict of interest: A. Shoemark has nothing to disclose., (Copyright ©The authors 2021. For reproduction rights and permissions contact permissions@ersnet.org.)

Context: Although primary adrenal insufficiency (PAI) in children and young people is often due to congenital adrenal hyperplasia (CAH) or autoimmunity, other genetic causes occur. The relative prevalence of these conditions is poorly understood., Objective: We investigated genetic causes of PAI in children and young people over a 25 year period., Design Setting and Participants: Unpublished and published data were reviewed for 155 young people in the United Kingdom who underwent genetic analysis for PAI of unknown etiology in three major research centers between 1993 and 2018. We pre-excluded those with CAH, autoimmune, or metabolic causes. We obtained additional data from NR0B1 (DAX-1) clinical testing centers., Intervention and Outcome Measurements: Genetic analysis involved a candidate gene approach (1993 onward) or next generation sequencing (NGS; targeted panels, exomes) (2013-2018)., Results: A genetic diagnosis was reached in 103/155 (66.5%) individuals. In 5 children the adrenal insufficiency resolved and no genetic cause was found. Pathogenic variants occurred in 11 genes: MC2R (adrenocorticotropin receptor; 30/155, 19.4%), NR0B1 (DAX-1; 7.7%), CYP11A1 (7.7%), AAAS (7.1%), NNT (6.5%), MRAP (4.5%), TXNRD2 (4.5%), STAR (3.9%), SAMD9 (3.2%), CDKN1C (1.3%), and NR5A1 /steroidogenic factor-1 (SF-1; 0.6%). Additionally, 51 boys had NR0B1 variants identified through clinical testing. Although age at presentation, treatment, ancestral background, and birthweight can provide diagnostic clues, genetic testing was often needed to define the cause., Conclusions: PAI in children and young people often has a genetic basis. Establishing the specific etiology can influence management of this lifelong condition. NGS approaches improve the diagnostic yield when many potential candidate genes are involved., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.)

Ciliopathies are a broad range of inherited developmental and degenerative diseases associated with structural or functional defects in motile or primary non-motile cilia. There are around 200 known ciliopathy disease genes and whilst genetic testing can provide an accurate diagnosis, 24-60% of ciliopathy patients who undergo genetic testing do not receive a genetic diagnosis. This is partly because following current guidelines from the American College of Medical Genetics and the Association for Molecular Pathology, it is difficult to provide a confident clinical diagnosis of disease caused by missense or non-coding variants, which account for more than one-third of cases of disease. Mutations in PRPF31 are the second most common cause of the degenerative retinal ciliopathy autosomal dominant retinitis pigmentosa. Here, we present a high-throughput high-content imaging assay providing quantitative measure of effect of missense variants in PRPF31 which meets the recently published criteria for a baseline standard in vitro test for clinical variant interpretation. This assay utilizes a new PRPF31 +/- human retinal cell line generated using CRISPR gene editing to provide a stable cell line with significantly fewer cilia in which novel missense variants are expressed and characterised. We show that high-content imaging of cells expressing missense variants in a ciliopathy gene on a null background can allow characterisation of variants according to the cilia phenotype. We hope that this will be a useful tool for clinical characterisation of PRPF31 variants of uncertain significance, and can be extended to variant classification in other ciliopathies.

Objective: Cardiovascular disease is one among the major mortality threats throughout the world. Autonomic activity of the nervous system can be examined by heart rate variability (HRV) analysis. Association of sympathetic and parasympathetic activities is directly related to HRV modulation. The aim of the study is to determine variations in HRV parameters among adult/adolescent male and female subjects due to vegetarian and nonvegetarian diet. Method: Ninety undergraduate students in each male and female group (N = 180) volunteered for the study. Based upon food habits, male and female subjects were categorized into four groups. Short-term (5-minute) heart rate recordings were measured from the subjects in a seated position before breakfast with minimum of 12 hours' fasting. Two-way analysis of variance was performed among the time and frequency domain variables. Results: Time domain variables are observed as significant ( p <  0.05) between vegetarian males and females and also ( p <  0.05) between male vegetarian and female nonvegetarians for standard deviation of NN intervals. Frequency domain HRV indices such as low frequency (LF; p =  0.01), high frequency (HF; p =  0.0001), and LF/HF ( p <  0.001) resulted between male and female vegetarians. Significance of LF ( p =  0.02), HF ( p <  0.0001), and LF/HF ( p <  0.01) was measured between male vegetarians and female nonvegetarians. LF ( p =  0.02), HF ( p =  0.04), and LF/HF ( p =  0.002) resulted between nonvegetarian males and females. HF ( p =  0.05) was enumerated between male vegetarians and nonvegetarians. Conclusions: Significant predominance of sympathetic cardiac activity was observed among male nonvegetarian consumers more than female vegetarians. Analysis demonstrates that the gender-based influence of vegetarian and nonvegetarian diet has significant correlation under HRV measurements.

Beeswax is the natural secretion from the wax gland of the genus Apis mellifera and Apis cerana whereas honey is a by-product of flower nectar and the upper aero-digestive tract of the honey bee. The introduction of beeswax and honey to clinical use dates back to the fourteenth century but subsequently it was restricted to the cosmetic and food industry. With the emerging trend of using natural resources as cure for many diseases, beeswax and honey have regained its popularity in medical field. The presented manuscript describes the production, composition, and attempts to highlight the uses of beeswax, honey, and advances especially in oral and maxillofacial surgery.

Background: Among adult college students in the US, cannabis use is common and associated with considerable negative consequences to health, cognition, and academic functioning, underscoring the importance of identifying risk and protective factors. Cannabis use is influenced by genetic factors, but genetic risk is not determinative. Accordingly, it is critical to identify environments that reduce risk among those who are at elevated genetic risk. This study examined the impact of polygenic scores for cannabis initiation, various forms of social activity participation, and peer deviance on recent cannabis use. Our aim was to test whether these environments moderate genetic risk for cannabis use., Methods: Data came from a longitudinal sample of undergraduate college students of European American (EA; N EA = 750) and African American (AA; N AA = 405) ancestry. Generalized estimating equations with a logit link function were used to examine main effects and two-way interactions., Results: Engagement with church activities was associated with lower probability of cannabis use. Peer deviance was associated with higher probability of cannabis use. Engagement with community activities moderated the influence of the polygenic risk score in the EA sample, such that PRS was associated with recent cannabis use among those who never engaged in community activities. This effect did not replicate in AAs, which may have been due to the portability of PRS based on EA discovery samples., Conclusions: Results suggest that community activities may limit the influence of genetic risk, as associations between PRS and cannabis use were only observed among individuals who never engaged in community activities., (Copyright © 2020. Published by Elsevier B.V.)

Pathogenic variants within the CREBBP and EP300 genes account for the majority of individuals with Rubinstein-Taybi syndrome (RSTS). Data are presented from a large cohort of 395 individuals referred for diagnostic testing of CREBBP, and of the 19 CREBBP missense variants classified as likely pathogenic in this study, 17 were within the histone acetyltransferase (HAT) domain, providing evidence that this domain is critical to the normal function of the CREBBP protein (CBP). The data presented here, combined with other published results, suggest that the presence of a missense variant within the CBP HAT domain can be considered as moderate evidence of pathogenicity in the context of official variant interpretation guidelines. Within our study cohort, 129 had a pathogenic or likely pathogenic CREBBP variant and 5 had a variant of uncertain significance (VUS) which warranted familial studies. 147 of the remaining probands were also screened for EP300 and a further 16 pathogenic or likely pathogenic variants were identified, plus one VUS. Therefore, this analysis has provided a molecular diagnosis in at least 145 individuals with RSTS (37%) and identified a wide range of variants (n = 133) of which 103 were novel., (© 2020 Crown copyright. American Journal of Medical Genetics Part A published by Wiley Periodicals, LLC. This article is published with the permission of the Controller of HMSO and the Queen's Printer for Scotland.)

E-cigarettes have dramatically increased in popularity among youth. Coincident with expanded legalization, young adults' use of cannabis (marijuana) has also steadily increased in recent years. Use of tobacco products can increase the chances of later cannabis initiation among youth. However, most longitudinal investigations of tobacco and cannabis use patterns have focused on tobacco cigarettes, included adolescents as opposed to young adults, and have only employed two timepoints. The current study examined prospective associations between e-cigarette and cannabis use in a large, diverse college sample assessed over four timepoints (freshman - senior year; N = 4,670). E-cigarette use and cannabis use were modelled in a four-wave cross-lagged model. The results showed significant bidirectional associations between both substances, even after controlling for time-varying levels of depressive symptoms, alcohol use, and polysubstance use, sensation seeking, demographic variables, concurrent associations and previous levels of use. Moreover, the significance of the predictive path from e-cigarette use to later cannabis use remained unchanged when we ran the same model, but restricted the sample to e-cigarette-only users (i.e., never cigarette smokers), whereas only one prospective path from cannabis to e-cigarette use was significant in this subsample. The current findings suggest that the association of e-cigarette use and cannabis use is likely bidirectional, with stronger support for the link from e-cigarette use to later cannabis use, above and beyond cigarette use. As e-cigarettes gain further hold of the tobacco product market share and cannabis legalization continues to expand, data such as these will be critical for informing regulatory decisions for e-cigarettes and cannabis, particularly involving their accessibility to youth and young adults., Competing Interests: Declaration of Competing Interest Dr. Eissenberg is a paid consultant in litigation against the tobacco industry and the electronic cigarette industry and is named on a patent application for a device that measures the puffing behavior of electronic cigarette users. All remaining authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Pathogenic variants within PAX6 are most often associated with aniridia, but have been linked with other phenotypes such as nystagmus, cataracts and foveal hypoplasia. Data are presented from a large cohort of 434 probands referred for PAX6 diagnostic testing. This analysis identified a wide range of pathogenic variants (n = 145) in 254 probands (including 61 novel variants). Excluding missense variants predicted to affect splicing, all 29 of the remaining missense variants were located within the paired (n = 27) or homeobox (n = 2) domains of the PAX6 protein, providing further evidence that these domains are critical to normal PAX6 function. Genotype-phenotype evidence suggests that while aniridia is associated with most variant types, a much broader clinical spectrum is seen in patients harbouring a missense variant, or a frameshift or run-on variant that results in an elongated or extended PAX6 protein., Competing Interests: Declaration of competing interest There is no conflict of interest to declare., (Crown Copyright © 2020. Published by Elsevier Masson SAS. All rights reserved.)

The stilbene derivative, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), an anion channel blocker is used in the present study to evaluate its modulatory effect on voltage-gated K + current (I K ) in human prostate cancer cell lines (LNCaP and PC-3). Voltage-gated K + (K V ) channels in the plasma membrane are critically involved in the proliferation of tumor cells. Therefore, K V channels are considered as a novel potential target for cancer treatment. The results of the present study show that the external perfusion of DIDS activates I K in a concentration-dependent manner, although the known K + channel blocker TEA failed to block the DIDS activated I K in PC-3 cells. Whereas, in LNCaP cells, the higher concentration of DIDS blocked I K , though this effect was not completely recovered after washout. The difference in function of DIDS might be due to the expression of different Kv channel isoforms in LNCaP and PC-3 cells. Further, the anticancer studies show that treatment of DIDS significantly induced G2/M phase cell cycle arrest and induced moderate and low level of cell death in LNCaP and PC-3 cells respectively. This finding reveals that DIDS modulates I K and exerts cell cycle arrest and cell death in LNCaP and PC-3 cells.

Purpose: Diagnosis of genetic disorders is hampered by large numbers of variants of uncertain significance (VUSs) identified through next-generation sequencing. Many such variants may disrupt normal RNA splicing. We examined effects on splicing of a large cohort of clinically identified variants and compared performance of bioinformatic splicing prediction tools commonly used in diagnostic laboratories., Methods: Two hundred fifty-seven variants (coding and noncoding) were referred for analysis across three laboratories. Blood RNA samples underwent targeted reverse transcription polymerase chain reaction (RT-PCR) analysis with Sanger sequencing of PCR products and agarose gel electrophoresis. Seventeen samples also underwent transcriptome-wide RNA sequencing with targeted splicing analysis based on Sashimi plot visualization. Bioinformatic splicing predictions were obtained using Alamut, HSF 3.1, and SpliceAI software., Results: Eighty-five variants (33%) were associated with abnormal splicing. The most frequent abnormality was upstream exon skipping (39/85 variants), which was most often associated with splice donor region variants. SpliceAI had greatest accuracy in predicting splicing abnormalities (0.91) and outperformed other tools in sensitivity and specificity., Conclusion: Splicing analysis of blood RNA identifies diagnostically important splicing abnormalities and clarifies functional effects of a significant proportion of VUSs. Bioinformatic predictions are improving but still make significant errors. RNA analysis should therefore be routinely considered in genetic disease diagnostics.

Blepharophimosis, Ptosis, and Epicanthus inversus Syndrome (BPES) is caused by autosomal dominant mutations in FOXL2. There are two forms of BPES: type I (with primary ovarian insufficiency (POI)) and type II (without POI). Data are presented from a large cohort of 177 BPES probands. Diagnostic testing identified a wide range of mutations in 119 mutation-positive patients (including 38 novel mutations). Although FOXL2 mutations are distributed throughout the gene, over 50% were frameshift mutations within a hotspot region of the gene that can be detected using a single primer pair to provide a cost-effective and rapid screening method. There was a significant proportion of de novo cases in this study, although in 7% there may be undetected parental mosaicism. There was an excess of female compared to male probands and a highly significant bias in the parental original of inherited mutations, with 20/21 found to be paternal in origin (95%). This could be because BPES in a female is more likely to come to clinical attention and because there is a generalised and more widespread clinical effect on fertility, in addition to the established association with POI. This study demonstrates the importance of cascade screening and provides new information on inheritance and parental mosaicism in BPES which will aid genetic counselling and accurate risk management., (Crown Copyright © 2019. Published by Elsevier Masson SAS. All rights reserved.)

Phytochemicals are ubiquitous in naturally occurring dietary elements that exhibits diverse pharmacological properties over various pathological disorders, including cancer. Voltage gated K + (K V ) channel in the plasma membrane contributes to wide range of cellular processes including cancer progression. Therefore, modulation of K V channel is being considered as a novel potential target for cancer therapy. The whole cell patch clamp technique was used to record the modulatory effect of chrysin, naringenin, caffeic acid, gallic acid, and zingerone on delayed rectifier potassium current (I K ) in human prostate cancer cells LNCaP and PC-3. Among the tested compounds, zingerone blocked I K in a concentration-dependent manner in LNCaP cells and estimated the IC 50 value of 141 μM and E max was 81.3%. Further analysis of K V channel activation kinetics showed that zingerone induces a positive shift in the activation curve in LNCaP cells, whereas the inhibitory effect of gallic acid on I K was significantly less potent than the inhibition caused by zingerone. However, chrysin, naringenin, and caffeic acid did not modulate the K V channel conductance in LNCaP or PC-3 cells. Our findings confirmed that not all the tested phytochemicals to be effective modulators of I K and suggested that I K inhibitory effect of zingerone and gallic acid may be responsible for their anticancer effects in prostate cancer cells.

Piperine is an attractive therapeutic alkaloid from black pepper that exhibits a broad spectrum of pharmacological properties over various pathological disorders including cancer. Voltage gated K + (K V ) channels play an important role in regulating cancer cell proliferation and are considered as potential target for cancer treatment. However, the implication of piperine in K V associated anticancer activities on human prostate cancer cells LNCaP and PC-3 remains unrevealed. The electrophysiological and pharmacological data identifies that both androgen sensitive (LNCaP) and insensitive (PC-3) prostate cancer cells typically expressed voltage gated K + current (I K ). This current was significantly blocked by piperine in a concentration-dependent manner with an IC 50 value 39.91 μM in LNCaP and 49.45 μM in PC-3 cells. Analysis of voltage-dependence of activation kinetics showed that piperine induces a positive shift in the relative activation curve in both the cells. Piperine also depolarized the resting membrane potential by an average of 10.2 mV and 8.3 mV in LNCaP and PC-3 cells, respectively. The anticancer studies showed that, treatment with piperine concentration dependently induced G 1 phase cell cycle arrest and apoptosis in LNCaP and PC-3 cells. These results unravel that the I K inhibition might be responsible for the anticancer effect of piperine on androgen sensitive and insensitive human prostate cancer cells., (Copyright © 2019. Published by Elsevier Inc.)

Troxerutin (TX), a bioflavonoid widely present in various fruits and vegetables, has shown to exhibit numerous pharmacological properties including anti-neoplastic and anti-cancer activities. Nrf2 and NF-κB are the key transcription factors that regulate oxidative stress and inflammation, therefore we assessed whether TX modulate these pathways and its downstream proteins in HuH-7 hepatocarcinoma cells. TX induced apoptotic cellular and nuclear changes were examined by fluorescence staining techniques, agarose gel electrophoresis and flow cytometry. Oxidative stress was determined through biochemical analysis of antioxidant enzymes and lipid peroxidation profile. The protein expressions of NF-κB and Nrf2 pathway regulators, cell proliferation markers and apoptotic pathway mediators were evaluated by performing immunoblotting, immunocytochemistry and molecular docking. Our results revealed that TX inhibits the growth of HuH-7 cells in a concentration and time-dependent manner. TX treated HuH-7 cells exhibited increased heme oxygenase (HO)-1 protein expression, augmented nuclear translocation of Nrf2, and reduced oxidative stress. Furthermore, TX suppressed the expression of IKKβ which subsequently inhibited the nuclear translocation of NF-κB (p65 subunit), and thus downregulated NF-κB mediated inflammatory responses, proliferation and cell survival. Collectively, our results indicate that TX exerts anti-cancer effect in HuH-7 hepatocarcinoma cells possibly through simultaneous regulation of the molecular signalling pathways, Nrf2 and NF-κB., (Copyright © 2018. Published by Elsevier Ltd.)