VAMP7 modulates ciliary biogenesis in kidney cells

Epithelial cells elaborate specialized domains that have distinct protein and lipid compositions, including the apical and basolateral surfaces and primary cilia. Maintaining the identity of these domains is required for proper cell function, and requires the efficient and selective SNARE-mediated fusion of vesicles containing newly synthesized and recycling proteins with the proper target membrane. Multiple pathways exist to deliver newly synthesized proteins to the apical surface of kidney cells, and the post-Golgi SNAREs, or VAMPs, involved in these distinct pathways have not been identified. VAMP7 has been implicated in apical protein delivery in other cell types, and we hypothesized that this SNARE would have differential effects on the trafficking of apical proteins known to take distinct routes to the apical surface in kidney cells. VAMP7 expressed in polarized Madin Darby canine kidney cells colocalized primarily with LAMP2-positive compartments, and siRNA-mediated knockdown modulated lysosome size, consistent with the known function of VAMP7 in lysosomal delivery. Surprisingly, VAMP7 knockdown had no effect on apical delivery of numerous cargoes tested, but did decrease the length and frequency of primary cilia. Additionally, VAMP7 knockdown disrupted cystogenesis in cells grown in a three-dimensional basement membrane matrix. The effects of VAMP7 depletion on ciliogenesis and cystogenesis are not directly linked to the disruption of lysosomal function, as cilia lengths and cyst morphology were unaffected in an MDCK lysosomal storage disorder model. Together, our data suggest that VAMP7 plays an essential role in ciliogenesis and lumen formation. To our knowledge, this is the first study implicating an R-SNARE in ciliogenesis and cystogenesis. © 2014 Szalinski et al

Painting a portrait: Analysis of national health survey data for cancer genetic counseling.

BACKGROUND: Despite a growing body of literature describing the geographic and sociodemographic distribution of cancer genetic testing, work focused on these domains in cancer genetic counseling is limited. Research describing the epidemiology of cancer genetic counseling has mainly focused on isolated populations, a single gender (women) and a single condition (hereditary breast and ovarian cancer). Study findings to date are contradictory, making it unclear what, if any, disparities in receipt of cancer genetic counseling exist. METHODS: Utilizing the 2015 National Health Interview Survey (NHIS)-a cross-sectional, in person interview survey collecting self-reported health data for the US population-geographic and sociodemographic factors were compared between those receiving genetic counseling and the national sample. Bivariate analysis and subsequent multivariable logistic regression were performed with stratification by cancer status (affected/unaffected). Reason for (eg, doctor recommended) and focus of (eg, breast/ovarian) genetic counseling were also assessed. To generate nationally representative estimates, all analyses were adjusted for survey weights. RESULTS: An estimated 4.8 million individuals in the United States had cancer genetic counseling. On bivariate analysis, there were significant differences in proportions undergoing genetic counseling by sex, race/ethnicity, insurance, citizenship, education, age, and cancer status (P \u3c 0.01). After adjustment, however, only female sex (Odds Ratio [OR]: 1.78 [95% CI: 1.18-2.67]) remained a significant predictor of genetic counseling among the affected. Among the unaffected, female sex (OR: 1.70 [1.30-2.21]), non-Hispanic black race (OR: 1.44 [1.02-2.05], reference: non-Hispanic white), graduate education (OR: 1.76 [1.03-2.98], reference: less than high school), and age (OR: 1.06 [1.01-1.11]) predicted higher rates of genetic counseling. An estimated 2.1 million individuals have undergone genetic counseling focused on breast/ovarian cancer, 1.3 million on colorectal cancer, and 1.4 million on other cancers. Of those receiving genetic counseling focused on breast/ovarian cancer, 3% were male and 97% female (breast cancer alone-4% male, 96% female); for colorectal cancer, 49% male and 51% female, and for other cancers, 60% male and 40% female. The majority of individuals receiving genetic counseling reported they did so because their doctor recommended it (66%), with smaller proportions describing self (12%), family (10%), or media (5%) influences as the primary reason. CONCLUSION: This is the first study to depict the sociodemographic and geographic distribution of cancer genetic counseling at the national level. Despite perceived disparities in access, cancer genetic counseling in the United States appears to be accessed by individuals of diverse racial/ethnic backgrounds, with various insurance coverage and educational levels, and across a broad range of ages and geographic regions. The only sociodemographic factor that independently predicted receipt of genetic counseling across both the affected and unaffected population was sex. With physician recommendation as the predominant driver for counseling, targeting physician education, and awareness is crucial to utilization

Absence of TGFBR2 mutations in patients with spontaneous spinal CSF leaks and intracranial hypotension.

A heritable connective-tissue-disorder often is suspected in patients with spontaneous spinal CSF leaks and intracranial hypotension, but the nature of the disorder remains unknown in most patients. The aim of this study was to assess the gene encoding TGF-beta receptor-2 (TGFBR2) as a candidate gene for spinal CSF leaks. We searched the TGFBR2 gene for mutations in eight patients with spontaneous spinal CSF leaks who also had other features associated with TGFBR2 mutations, i.e., skeletal features of Marfan syndrome, arterial tortuosity, and(or) thoracic aortic aneurysm. The mean age of these 7 women and 1 man was 38 years (range 14-60 years). We detected no TGFBR2 mutations and conclude that TGFBR2 mutations are not a major factor in spontaneous spinal CSF leaks

Design and Validation of a Whole-Genome Sequencing- Based Assay for Population Health

Introduction: Next-generation sequencing (NGS) is rapidly emerging as a key methodology in the clinical laboratory for a wide array of clinical uses, including heritable disease testing, somatic tumor profiling, and microbiology. Whereas current clinical methods are largely focused on targeted gene panels, the low cost and high throughput of modern sequencing platforms are making it possible to use whole-genome sequencing (WGS) for routine clinical applications. Here we developed a clinical WGS-based lab developed test (LDT) for heritable disease gene testing and pharmacogenomics (PGx) and performed extensive validation across a large cohort of blood and saliva specimens. Methods: DNA was isolated from both whole blood (EDTA) and saliva (Oragene collection) using the Qiagen QIAsymphony DSP Midi Kit. Whole genome libraries were prepared from 300 to 500 ng gDNA with the Illumina DNA PCR-Free Tagmentation kit. Sequencing (30X) was performed on the Illumina NovaSeq 6000. Pharmacogenetic genotyping and variant detection analysis (82 genes) were performed using standard analysis pipelines on the Illumina Dynamic Read Analysis for GENomics (DRAGEN) platform. Interpretation of heritable disease gene variants as well as PGx star allele assignment were performed by experts utilizing the Fabric Genomics interpretation platform as well as a novel in-house developed platform for clinical reporting. Results: WGS was performed on a combined retrospective and prospective validation cohort of 119 whole blood and 69 saliva specimens that were orthogonally tested via single gene or small panel tests at commercial laboratories. Validation was performed across a set of 77 actionable disease genes and resulted in 100% agreement in called pathogenic and likely pathogenic single nucleotide variants, indels, and copy number variants when compared to the reference method. Genotyping from WGS across a panel of PGx genes (CYP2C19, CYP2C9, VKORC1, and CYP4F2) in the 119 whole blood and 69 saliva gDNA samples resulted in 100% concordance with the reference method (MALDI-TOF mass spectrometry). Conclusions: WGS exhibited equivalent performance with targeted gene panel testing across a large cohort of clinical specimens. The comprehensive nature of a WGS backbone for clinical testing has the added benefit of facilitating expanded reanalysis of new actionable genes, rapid redeployment for use in other clinical contexts, as well as for use by researchers in supporting novel genomic discoveries. Given the anticipated upcoming reductions in cost for WGS, a singular clinical genome-based platform will likely represent a viable streamlined option for supporting clinical genetic/genomic testing across the clinical spectrum

Geno4ME: establishment of an equitable whole-genome sequencing-based platform for clinical screening in a large healthcare system

Recent population-wide genetic testing analyses suggest that over 1% of the general population are carriers for a clinically actionable single-gene genetic condition. In addition, approximately 80% of variability in drug efficacy and safety are attributed to an individual’s pharmacogenomics (PGx) profile. Here, we report the establishment of the Genomic Medicine for Everyone (Geno4ME) program across the highly diverse seven-state Providence Health System. Key components include targeted and multi-lingual outreach to traditionally underrepresented groups, a novel e-consent and education platform, and whole genome sequencing with clinical return of results via the Electronic Health Record (EHR) for 78 hereditary disease genes and four clinically relevant pharmacogenomics (PGx) genes. The program provides genetic counseling and pharmacist support for patients and educational resources and peer-to-peer support to assist providers in caring for patients with a positive result. Over the initial months of the study, over 23,000 potential participants were outreached; of this, 1,971 were consented to the study (of which 48.4% are people of color: 15.4% Hispanic, 14.9% Asian, 7.9% more than one race, 7.2% Black, 3.0% other) and 753 have had results returned so far. Fifty-two (7.0%) initial participants were found to have an actionable gene variant in the hereditary disease panel. Additionally, 111 (14.7%) of initial participants were currently taking one of the supported medications with a medical recommendation resulting from PGx genotype. Overall, 20.5% of initial participants had a test result with one or more medical intervention recommendations. Geno4ME plans to enroll up to 5,000 total participants within the next year and plans to expand the proportion of the genome that is included in the clinical deliverable and scheduled re-evaluation of variants for clinical significance. We propose this model as a viable, effective framework for population screening into routine healthcare and the whole-genome sequencing platform as key for continued reanalysis and long-term integration into routine clinical practice. This model specifically addresses issues of recruitment of diverse populations in genomics research and incorporating primary care provider education and genomics fluency into core program delivery

Abatacept in Early Diffuse Cutaneous Systemic Sclerosis - Results of a Phase 2 Investigator-Initiated, Multicenter, Double-Blind Randomized Placebo-Controlled Trial

OBJECTIVES T cells play a key role in the pathogenesis of early systemic sclerosis. This study assessed the safety and efficacy of abatacept in patients with diffuse cutaneous systemic sclerosis (dcSSc). METHODS A 12-month, randomized, double-blind, placebo-controlled trial with participants randomized in a 1:1 ratio to either abatacept 125 mg subcutaneous or matching placebo, stratified by duration of dcSSc. Escape therapy was allowed at six months for worsening disease. The co-primary end points were change in modified Rodnan skin score (mRSS) and safety over 12 months. Treatment differences in longitudinal outcomes were assessed using linear mixed models, with outcomes censored after initiation of escape therapy. Baseline skin tissue was classified into intrinsic gene expression subsets. RESULTS Among 88 participants, the adjusted mean change in mRSS at 12 months was -6.24 units in the abatacept and -4.49 units in the placebo, with adjusted mean treatment difference of -1.75 units (p=0.28). Two secondary outcome measures (HAQ-DI and a composite measure) were clinically and statistically significant favoring abatacept. A larger proportion of placebo subjects required escape therapy relative to abatacept (36% vs. 16%). Decline in mRSS over 12 months was clinically and significantly higher in abatacept vs. placebo for the Inflammatory (p<0.001) and Normal-like skin gene expression subsets (p=0.03). 35 participants in the abatacept versus 40 in the placebo had adverse events (AEs), including two and one deaths, respectively. CONCLUSIONS In this Phase 2 trial, abatacept was well tolerated, but change in mRSS was not statistically significant. Secondary outcome measures, including gene expression subsets, showed some evidence in favor of abatacept. These data should be confirmed in a Phase 3 trial. This article is protected by copyright. All rights reserved

Development and Validation of a Breast Cancer Polygenic Risk Score on the Basis of Genetic Ancestry Composition.

PURPOSE: Polygenic risk scores (PRSs) for breast cancer (BC) risk stratification have been developed primarily in women of European ancestry. Their application to women of non-European ancestry has lagged because of the lack of a formal approach to incorporate genetic ancestry and ancestry-dependent variant frequencies and effect sizes. Here, we propose a multiple-ancestry PRS (MA-PRS) that addresses these issues and may be useful in the development of equitable PRSs across other cancers and common diseases. MATERIALS AND METHODS: Women referred for hereditary cancer testing were divided into consecutive cohorts for development (n = 189,230) and for independent validation (n = 89,126). Individual genetic composition as fractions of three reference ancestries (African, East Asian, and European) was determined from ancestry-informative single-nucleotide polymorphisms. The MA-PRS is a combination of three ancestry-specific PRSs on the basis of genetic ancestral composition. Stratification of risk was evaluated by multivariable logistic regression models controlling for family cancer history. Goodness-of-fit analysis compared expected with observed relative risks by quantiles of the MA-PRS distribution. RESULTS: In independent validation, the MA-PRS was significantly associated with BC risk in the full cohort (odds ratio, 1.43; 95% CI, 1.40 to 1.46; CONCLUSION: The MA-PRS uses genetic ancestral composition to expand the utility of polygenic risk prediction to non-European women. Inclusion of genetic ancestry in polygenic risk prediction presents an opportunity for more personalized treatment decisions for women of varying and mixed ancestries

Correction to: Physician-directed genetic screening to evaluate personal risk for medically actionable disorders: a large multi-center cohort study.

BACKGROUND: The use of proactive genetic screening for disease prevention and early detection is not yet widespread. Professional practice guidelines from the American College of Medical Genetics and Genomics (ACMG) have encouraged reporting pathogenic variants that confer personal risk for actionable monogenic hereditary disorders, but only as secondary findings from exome or genome sequencing. The Centers for Disease Control and Prevention (CDC) recognizes the potential public health impact of three Tier 1 actionable disorders. Here, we report results of a large multi-center cohort study to determine the yield and potential value of screening healthy individuals for variants associated with a broad range of actionable monogenic disorders, outside the context of secondary findings. METHODS: Eligible adults were offered a proactive genetic screening test by health care providers in a variety of clinical settings. The screening panel based on next-generation sequencing contained up to 147 genes associated with monogenic disorders within cancer, cardiovascular, and other important clinical areas. Sequence and intragenic copy number variants classified as pathogenic, likely pathogenic, pathogenic (low penetrance), or increased risk allele were considered clinically significant and reported. Results were analyzed by clinical area and severity/burden of disease using chi-square tests without Yates\u27 correction. RESULTS: Among 10,478 unrelated adults screened, 1619 (15.5%) had results indicating personal risk for an actionable monogenic disorder. In contrast, only 3.1 to 5.2% had clinically reportable variants in genes suggested by the ACMG version 2 secondary findings list to be examined during exome or genome sequencing, and 2% had reportable variants related to CDC Tier 1 conditions. Among patients, 649 (6.2%) were positive for a genotype associated with a disease of high severity/burden, including hereditary cancer syndromes, cardiovascular disorders, or malignant hyperthermia susceptibility. CONCLUSIONS: This is one of the first real-world examples of specialists and primary care providers using genetic screening with a multi-gene panel to identify health risks in their patients. Nearly one in six individuals screened for variants associated with actionable monogenic disorders had clinically significant results. These findings provide a foundation for further studies to assess the role of genetic screening as part of regular medical care