Genome‐scale sequencing creates vast amounts of genomic data, increasing the challenge of clinical sequence variant interpretation. The demand for high‐quality interpretation requires multiple ...specialties to join forces to accelerate the interpretation of sequence variant pathogenicity. With over 600 international members including clinicians, researchers, and laboratory diagnosticians, the Clinical Genome Resource (ClinGen), funded by the National Institutes of Health, is forming expert groups to systematically evaluate variants in clinically relevant genes. Here, we describe the first ClinGen variant curation expert panels (VCEPs), development of consistent and streamlined processes for establishing new VCEPs, and creation of standard operating procedures for VCEPs to define application of the ACMG/AMP guidelines for sequence variant interpretation in specific genes or diseases. Additionally, ClinGen has created user interfaces to enhance reliability of curation and a Sequence Variant Interpretation Working Group (SVI WG) to harmonize guideline specifications and ensure consistency between groups. The expansion of VCEPs represents the primary mechanism by which curation of a substantial fraction of genomic variants can be accelerated and ultimately undertaken systematically and comprehensively. We welcome groups to utilize our resources and become involved in our effort to create a publicly accessible, centralized resource for clinically relevant genes and variants.
ClinGen is organizing Variant Curation Expert Panels (VCEPs) to develop specifications to the ACMG/AMP guidelines for genes or diseases of interest, interpret variants according to these guidelines, and publish the expert interpretations through the publicly available ClinVar database. A stepwise process was iteratively developed for ClinGen VCEPs to apply to submit variant assertions to ClinVar at the Expert Panel level of review. Other groups that wish to assemble as VCEPs are encouraged, though not required, to follow these steps.
Next-generation deep sequencing of small RNAs has unveiled the complexity of the microRNA (miRNA) transcriptome, which is in large part due to the diversity of miRNA sequence variants ("isomiRs"). ...Changes to a miRNA's seed sequence (nucleotides 2-8), including shifted start positions, can redirect targeting to a dramatically different set of RNAs and alter biological function. We performed deep sequencing of small RNA from mouse insulinoma (MIN6) cells (widely used as a surrogate for the study of pancreatic beta cells) and developed a bioinformatic analysis pipeline to profile isomiR diversity. Additionally, we applied the pipeline to recently published small RNA-seq data from primary human beta cells and whole islets and compared the miRNA profiles with that of MIN6. We found that: (1) the miRNA expression profile in MIN6 cells is highly correlated with those of primary human beta cells and whole islets; (2) miRNA loci can generate multiple highly expressed isomiRs with different 5'-start positions (5'-isomiRs); (3) isomiRs with shifted start positions (5'-shifted isomiRs) are highly expressed, and can be as abundant as their unshifted counterparts (5'-reference miRNAs). Finally, we identified 10 beta cell miRNA families as candidate regulatory hubs in a type 2 diabetes (T2D) gene network. The most significant candidate hub was miR-29, which we demonstrated regulates the mRNA levels of several genes critical to beta cell function and implicated in T2D. Three of the candidate miRNA hubs were novel 5'-shifted isomiRs: miR-375+1, miR-375-1 and miR-183-5p+1. We showed by in silico target prediction and in vitro transfection studies that both miR-375+1 and miR-375-1 are likely to target an overlapping, but distinct suite of beta cell genes compared to canonical miR-375. In summary, this study characterizes the isomiR profile in beta cells for the first time, and also highlights the potential functional relevance of 5'-shifted isomiRs to T2D.
MicroRNAs (miRNAs) have emerged as biomarkers of metabolic status, etiological factors in complex disease, and promising drug targets. Recent reports suggest that miRNAs are critical regulators of ...pathways underlying the pathophysiology of type 2 diabetes. In this study, we demonstrate by deep sequencing and real-time quantitative PCR that hepatic levels of Foxa2 mRNA and miR-29 are elevated in a mouse model of diet-induced insulin resistance. We also show that Foxa2 and miR-29 are significantly upregulated in the livers of Zucker diabetic fatty (fa/fa) rats and that the levels of both returned to normal upon treatment with the insulin-sensitizing agent pioglitazone. We present evidence that miR-29 expression in human hepatoma cells is controlled in part by FOXA2, which is known to play a critical role in hepatic energy homeostasis. Moreover, we demonstrate that miR-29 fine-tunes FOXA2-mediated activation of key lipid metabolism genes, including PPARGC1A, HMGCS2, and ABHD5. These results suggest that miR-29 is an important regulatory factor in normal metabolism and may represent a novel therapeutic target in type 2 diabetes and related metabolic syndromes.
Recent advances in sequencing technology have helped unveil the unexpected complexity and diversity of small RNAs. A critical step in small RNA library preparation for sequencing is the ligation of ...adapter sequences to both the 5' and 3' ends of small RNAs. Studies have shown that adapter ligation introduces a significant but widely unappreciated bias in the results of high-throughput small RNA sequencing. We show that due to this bias the two widely used Illumina library preparation protocols produce strikingly different microRNA (miRNA) expression profiles in the same batch of cells. There are 102 highly expressed miRNAs that are >5-fold differentially detected and some miRNAs, such as miR-24-3p, are over 30-fold differentially detected. While some level of bias in library preparation is not surprising, the apparent massive differential bias between these two widely used adapter sets is not well appreciated. In an attempt to mitigate this bias, the new Bioo Scientific NEXTflex V2 protocol utilizes a pool of adapters with random nucleotides at the ligation boundary. We show that this protocol is able to detect robustly several miRNAs that evade capture by the Illumina-based methods. While these analyses do not indicate a definitive gold standard for small RNA library preparation, the results of the NEXTflex protocol do correlate best with RT-qPCR. As increasingly more laboratories seek to study small RNAs, researchers should be aware of the extent to which the results may differ with different protocols, and should make an informed decision about the protocol that best fits their study.
MicroRNAs (miRNAs) are important regulators and potential therapeutic targets of metabolic disease. In this study we show by in vivo administration of locked nucleic acid (LNA) inhibitors that ...suppression of endogenous miR-29 lowers plasma cholesterol levels by ~40%, commensurate with the effect of statins, and reduces fatty acid content in the liver by ~20%. Whole transcriptome sequencing of the liver reveals 883 genes dysregulated (612 down, 271 up) by inhibition of miR-29. The set of 612 down-regulated genes are most significantly over-represented in lipid synthesis pathways. Among the up-regulated genes are the anti-lipogenic deacetylase sirtuin 1 (Sirt1) and the anti-lipogenic transcription factor aryl hydrocarbon receptor (Ahr), the latter of which we demonstrate is a direct target of miR-29. In vitro radiolabeled acetate incorporation assays confirm that pharmacologic inhibition of miR-29 significantly reduces de novo cholesterol and fatty acid synthesis. Our findings indicate that miR-29 controls hepatic lipogenic programs, likely in part through regulation of Ahr and Sirt1, and therefore may represent a candidate therapeutic target for metabolic disorders such as dyslipidemia.
Although embryonic brain development and neurodegeneration have received considerable attention, the events that govern postnatal brain maturation are less understood. Here, we identify the miR-29 ...family to be strikingly induced during the late stages of brain maturation. Brain maturation is associated with a transient, postnatal period of de novo non-CG (CH) DNA methylation mediated by DNMT3A. We examine whether an important function of miR-29 during brain maturation is to restrict the period of CH methylation via its targeting of Dnmt3a. Deletion of miR-29 in the brain, or knockin mutations preventing miR-29 to specifically target Dnmt3a, result in increased DNMT3A expression, higher CH methylation, and repression of genes associated with neuronal activity and neuropsychiatric disorders. These mouse models also develop neurological deficits and premature lethality. Our results identify an essential role for miR-29 in restricting CH methylation in the brain and illustrate the importance of CH methylation regulation for normal brain maturation.
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•miR-29 is markedly upregulated during postnatal brain maturation•miR-29 targets Dnmt3a to restrict its expression in vivo in the maturing brain•miR-29 regulates Dnmt3a and mCH to control the expression of synaptic genes•Failure of miR-29 to restrict Dnmt3a leads to neurobehavioral deficits
Non-canonical CH methylation mediated by DNMT3A during neuronal maturation has been recently shown to be critical for brain homeostasis. Swahari et al. identify a key player that is critical for regulating CH methylation: the microRNA miR-29. Failure of miR-29 to regulate the expression of Dnmt3a leads to severe neurobehavioral consequences.
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited disease characterized by ventricular arrhythmias and progressive ventricular dysfunction. Genetic testing is recommended, and a ...pathogenic variant in an ARVC-associated gene is a major criterion for diagnosis according to the 2010 Task Force Criteria. As incorrect attribution of a gene to ARVC can contribute to misdiagnosis, we assembled an international multidisciplinary ARVC Clinical Genome Resource Gene Curation Expert Panel to reappraise all reported ARVC genes.
Following a comprehensive literature search, six 2-member teams conducted blinded independent curation of reported ARVC genes using the semiquantitative Clinical Genome Resource framework.
Of 26 reported ARVC genes, only 6 (
,
,
,
,
, and
) had strong evidence and were classified as definitive for ARVC causation. There was moderate evidence for 2 genes,
and
. The remaining 18 genes had limited or no evidence.
was refuted as an ARVC gene since clinical data and model systems exhibited a catecholaminergic polymorphic ventricular tachycardia phenotype. In ClinVar, only 5 pathogenic/likely pathogenic variants (1.1%) in limited evidence genes had been reported in ARVC cases in contrast to 450 desmosome gene variants (97.4%).
Using the Clinical Genome Resource approach to gene-disease curation, only 8 genes (
,
,
,
,
,
,
, and
) had definitive or moderate evidence for ARVC, and these genes accounted for nearly all pathogenic/likely pathogenic ARVC variants in ClinVar. Therefore, only pathogenic/likely pathogenic variants in these 8 genes should yield a major criterion for ARVC diagnosis. Pathogenic/likely pathogenic variants identified in other genes in a patient should prompt further phenotyping as variants in many of these genes are associated with other cardiovascular conditions.
In 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published consensus standardized guidelines for sequence-level variant ...classification in Mendelian disorders. To increase accuracy and consistency, the Clinical Genome Resource Familial Hypercholesterolemia (FH) Variant Curation Expert Panel was tasked with optimizing the existing ACMG/AMP framework for disease-specific classification in FH. In this study, we provide consensus recommendations for the most common FH-associated gene, LDLR, where >2300 unique FH-associated variants have been identified.
The multidisciplinary FH Variant Curation Expert Panel met in person and through frequent emails and conference calls to develop LDLR-specific modifications of ACMG/AMP guidelines. Through iteration, pilot testing, debate, and commentary, consensus among experts was reached.
The consensus LDLR variant modifications to existing ACMG/AMP guidelines include (1) alteration of population frequency thresholds, (2) delineation of loss-of-function variant types, (3) functional study criteria specifications, (4) cosegregation criteria specifications, and (5) specific use and thresholds for in silico prediction tools, among others.
Establishment of these guidelines as the new standard in the clinical laboratory setting will result in a more evidence-based, harmonized method for LDLR variant classification worldwide, thereby improving the care of patients with FH.
Renalase, a key mediator of cross-talk between kidneys and sympathetic nervous system, exerts protective roles in various cardiovascular/renal disease states. However, molecular mechanisms ...underpinning renalase gene expression remain incompletely understood. Here, we sought to identify the key molecular regulators of renalase under basal/catecholamine-excess conditions.
Identification of the core promoter domain of renalase was carried out by promoter-reporter assays in N2a/HEK-293/H9c2 cells. Computational analysis of the renalase core promoter domain, over-expression of cyclic-AMP-response-element-binding-protein (CREB)/dominant negative mutant of CREB, ChIP assays were performed to determine the role of CREB in transcription regulation. Role of the miR-29b-mediated-suppression of renalase was validated in-vivo by using locked-nucleic-acid-inhibitors of miR-29. qRT-PCR and Western-blot analyses measured the expression of renalase, CREB, miR-29b and normalization controls in cell lysates/ tissue samples under basal/epinephrine-treated conditions.
CREB, a downstream effector in epinephrine signaling, activated renalase expression via its binding to the renalase-promoter. Physiological doses of epinephrine and isoproterenol enhanced renalase-promoter activity and endogenous renalase protein level while propranolol diminished the promoter activity and endogenous renalase protein level indicating a potential role of beta-adrenergic receptor in renalase gene regulation. Multiple animal models (acute exercise, genetically hypertensive/stroke-prone mice/rat) displayed directionally-concordant expression of CREB and renalase. Administration of miR-29b inhibitor in mice upregulated endogenous renalase expression. Moreover, epinephrine treatment down-regulated miR-29b promoter-activity/transcript levels.
This study provides evidence for renalase gene regulation by concomitant transcriptional activation via CREB and post-transcriptional attenuation via miR-29b under excess epinephrine conditions. These findings have implications for disease states with dysregulated catecholamines.
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•Regulation of renalase gene, a novel cardiovascular modulator, is partially understood.•CREB governs renalase expression under basal and epinephrine-induced conditions.•miR-29b modulates renalase expression in vivo.•Epinephrine down-regulates miR-29b and activates renalase expression.•CREB-miR-29b cross-talk modulates renalase expression in excess catecholamine conditions.
Plasma membrane (PM) free cholesterol (FC) is emerging as an important modulator of signal transduction. Here, we show that hepatocyte-specific knockout (HSKO) of the cellular FC exporter, ...ATP-binding cassette transporter A1 (ABCA1), leads to decreased PM FC content and defective trafficking of lysosomal FC to the PM. Compared with controls, chow-fed HSKO mice had reduced hepatic (1) insulin-stimulated Akt phosphorylation, (2) activation of the lipogenic transcription factor Sterol Regulatory Element Binding Protein (SREBP)-1c, and (3) lipogenic gene expression. Consequently, Western-type diet-fed HSKO mice were protected from steatosis. Surprisingly, HSKO mice had intact glucose metabolism; they showed normal gluconeogenic gene suppression in response to re-feeding and normal glucose and insulin tolerance. We conclude that: (1) ABCA1 maintains optimal hepatocyte PM FC, through intracellular FC trafficking, for efficient insulin signaling; and (2) hepatocyte ABCA1 deletion produces a form of selective insulin resistance so that lipogenesis is suppressed but glucose metabolism remains normal.
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•Plasma membrane free cholesterol is decreased in the absence of hepatocyte ABCA1•Insulin signaling and lipogenesis are attenuated in the absence of hepatocyte ABCA1•Glucose metabolism is responsive to insulin in the absence of hepatocyte ABCA1•Hepatocyte-specific Abca1 deletion protects against high-fat-diet-induced steatosis
Key et al. find that deletion of hepatocyte ABCA1 leads to decreased lysosomal free-cholesterol trafficking to the plasma membrane and attenuated cholesterol biosynthesis, resulting in decreased insulin signaling to mTORC1 and attenuated lipogenesis. However, gluconeogenesis remains responsive. Mice lacking hepatocyte ABCA1 are resistant to high-fat-diet-induced hepatosteatosis.
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