The CD3 ζ chain (CD247), a gene involved in T-cell signaling, has been shown to associate with blood pressure in human genetic studies. To test the functional role of CD247 in hypertension and renal ...disease, zinc-finger nucleases targeting CD247 were injected into Dahl salt-sensitive (SS/JrHsdMcwi) embryos. The resulting 11-bp frameshift deletion in exon 1 of CD247 led to a predicted premature stop codon. Western blotting confirmed the absence of CD247 protein in the thymus, and flow cytometry (n=5-9 per group) demonstrated that the mutant rats (CD247(-/-)) have a >99% reduction in circulating CD3(+) T cells compared with littermate controls (CD247(+/+)). Studies were performed on age-matched, littermate male, CD247(+/+) and CD247(-/-) rats fed a 4.0% NaCl diet for 3 weeks. The infiltration of CD3(+) T cells into the kidney after high salt was significantly blunted in CD247(-/-) (1.4±0.4×10(5) cells per kidney) when compared with that in the CD247(+/+) (8.7±2.0×10(5) cells per kidney). Accompanying the reduced infiltration of T cells, mean arterial blood pressure was significantly lower in CD247(-/-) than in CD247(+/+) (134±1 versus 151±2 mm Hg). As an index of kidney disease, urinary albumin and protein excretion rates were significantly reduced in CD247(-/-) (17±1 and 62±2 mg/d, respectively) when compared with that in CD247(+/+) (49±3 and 121±5 mg/d, respectively). Glomerular and renal tubular damage were also attenuated in the CD247(-/-). These studies demonstrate that functional T cells are required for the full development of Dahl salt-sensitive hypertension and indicate that the association between CD247 and hypertension in humans may be related to altered immune cell function.
Exosomes, endosome-derived membrane microvesicles, contain specific RNA transcripts that are thought to be involved in cell-cell communication. These RNA transcripts have great potential as disease ...biomarkers. To characterize exosomal RNA profiles systemically, we performed RNA sequencing analysis using three human plasma samples and evaluated the efficacies of small RNA library preparation protocols from three manufacturers. In all we evaluated 14 libraries (7 replicates).
From the 14 size-selected sequencing libraries, we obtained a total of 101.8 million raw single-end reads, an average of about 7.27 million reads per library. Sequence analysis showed that there was a diverse collection of the exosomal RNA species among which microRNAs (miRNAs) were the most abundant, making up over 42.32% of all raw reads and 76.20% of all mappable reads. At the current read depth, 593 miRNAs were detectable. The five most common miRNAs (miR-99a-5p, miR-128, miR-124-3p, miR-22-3p, and miR-99b-5p) collectively accounted for 48.99% of all mappable miRNA sequences. MiRNA target gene enrichment analysis suggested that the highly abundant miRNAs may play an important role in biological functions such as protein phosphorylation, RNA splicing, chromosomal abnormality, and angiogenesis. From the unknown RNA sequences, we predicted 185 potential miRNA candidates. Furthermore, we detected significant fractions of other RNA species including ribosomal RNA (9.16% of all mappable counts), long non-coding RNA (3.36%), piwi-interacting RNA (1.31%), transfer RNA (1.24%), small nuclear RNA (0.18%), and small nucleolar RNA (0.01%); fragments of coding sequence (1.36%), 5' untranslated region (0.21%), and 3' untranslated region (0.54%) were also present. In addition to the RNA composition of the libraries, we found that the three tested commercial kits generated a sufficient number of DNA fragments for sequencing but each had significant bias toward capturing specific RNAs.
This study demonstrated that a wide variety of RNA species are embedded in the circulating vesicles. To our knowledge, this is the first report that applied deep sequencing to discover and characterize profiles of plasma-derived exosomal RNAs. Further characterization of these extracellular RNAs in diverse human populations will provide reference profiles and open new doors for the development of blood-based biomarkers for human diseases.
The toolbox of rat genetics currently lacks the ability to introduce site-directed, heritable mutations into the genome to create knockout animals. By using engineered zinc-finger nucleases (ZFNs) ...designed to target an integrated reporter and two endogenous rat genes, Immunoglobulin M (IgM) and Rab38, we demonstrate that a single injection of DNA or messenger RNA encoding ZFNs into the one-cell rat embryo leads to a high frequency of animals carrying 25 to 100% disruption at the target locus. These mutations are faithfully and efficiently transmitted through the germline. Our data demonstrate the feasibility of targeted gene disruption in multiple rat strains within 4 months time, paving the way to a humanized monoclonal antibody platform and additional human disease models.
Many patients remain without a diagnosis despite extensive medical evaluation. The Undiagnosed Diseases Network (UDN) was established to apply a multidisciplinary model in the evaluation of the most ...challenging cases and to identify the biologic characteristics of newly discovered diseases. The UDN, which is funded by the National Institutes of Health, was formed in 2014 as a network of seven clinical sites, two sequencing cores, and a coordinating center. Later, a central biorepository, a metabolomics core, and a model organisms screening center were added.
We evaluated patients who were referred to the UDN over a period of 20 months. The patients were required to have an undiagnosed condition despite thorough evaluation by a health care provider. We determined the rate of diagnosis among patients who subsequently had a complete evaluation, and we observed the effect of diagnosis on medical care.
A total of 1519 patients (53% female) were referred to the UDN, of whom 601 (40%) were accepted for evaluation. Of the accepted patients, 192 (32%) had previously undergone exome sequencing. Symptoms were neurologic in 40% of the applicants, musculoskeletal in 10%, immunologic in 7%, gastrointestinal in 7%, and rheumatologic in 6%. Of the 382 patients who had a complete evaluation, 132 received a diagnosis, yielding a rate of diagnosis of 35%. A total of 15 diagnoses (11%) were made by clinical review alone, and 98 (74%) were made by exome or genome sequencing. Of the diagnoses, 21% led to recommendations regarding changes in therapy, 37% led to changes in diagnostic testing, and 36% led to variant-specific genetic counseling. We defined 31 new syndromes.
The UDN established a diagnosis in 132 of the 382 patients who had a complete evaluation, yielding a rate of diagnosis of 35%. (Funded by the National Institutes of Health Common Fund.).
Antithrombin III, encoded by SerpinC1, is a major anti-coagulation molecule in vivo and has anti-inflammatory effects. We found that patients with low antithrombin III activities presented a higher ...risk of developing acute kidney injury after cardiac surgery. To study this further, we generated SerpinC1 heterozygous knockout rats and followed the development of acute kidney injury in a model of modest renal ischemia/reperfusion injury. Renal injury, assessed by serum creatinine and renal tubular injury scores after 24 h of reperfusion, was significantly exacerbated in SerpinC1+/− rats compared to wild-type littermates. Concomitantly, renal oxidative stress, tubular apoptosis, and macrophage infiltration following this injury were significantly aggravated in SerpinC1+/− rats. However, significant thrombosis was not found in the kidneys of any group of rats. Antithrombin III is reported to stimulate the production of prostaglandin I2, a known regulator of renal cortical blood flow, in addition to having anti-inflammatory effects and to protect against renal failure. Prostaglandin F1α, an assayable metabolite of prostaglandin I2, was increased in the kidneys of the wild-type rats at 3 h after reperfusion. The increase of prostaglandin F1α was significantly blunted in SerpinC1+/− rats, which preceded increased tubular injury and oxidative stress. Thus, our study found a novel role of SerpinC1 insufficiency in increasing the severity of renal ischemia/reperfusion injury.
The Rat Genome Database (RGD, http://rgd.mcw.edu) provides the most comprehensive data repository and informatics platform related to the laboratory rat, one of the most important model organisms for ...disease studies. RGD maintains and updates datasets for genomic elements such as genes, transcripts and increasingly in recent years, sequence variations, as well as map positions for multiple assemblies and sequence information. Functional annotations for genomic elements are curated from published literature, submitted by researchers and integrated from other public resources. Complementing the genomic data catalogs are those associated with phenotypes and disease, including strains, QTL and experimental phenotype measurements across hundreds of strains. Data are submitted by researchers, acquired through bulk data pipelines or curated from published literature. Innovative software tools provide users with an integrated platform to query, mine, display and analyze valuable genomic and phenomic datasets for discovery and enhancement of their own research. This update highlights recent developments that reflect an increasing focus on: (i) genomic variation, (ii) phenotypes and diseases, (iii) data related to the environment and experimental conditions and (iv) datasets and software tools that allow the user to explore and analyze the interactions among these and their impact on disease.
Hypertension and renal damage in Dahl SS rats are associated with increased infiltrating immune cells in the kidney. To examine the role of infiltrating immune cells in this disease process, a zinc ...finger nuclease targeting bases 672-706 of recombination-activating gene 1 (Rag1) was injected into the pronucleus of Dahl SS (SS/JrHsdMcwi) strain embryos and implanted in pseudopregnant females. This strategy yielded a rat strain with a 13-base frame-shift mutation in the target region of Rag1 and a deletion of immunoreactive Rag1 protein in the thymus. Flow cytometry demonstrated that the Rag1-null mutant rats have a significant reduction in T and B lymphocytes in the circulation and spleen. Studies were performed on SS and Rag1-null rats fed a 4.0% NaCl diet for 3 wk. The infiltration of T cells into the kidney following high-salt intake was significantly blunted in the Rag1-null rats (1.7 ± 0.6 × 10(5) cells/kidney) compared with the Dahl SS (5.6 ± 0.9 × 10(5) cells/kidney). Accompanying the reduction in infiltration of immune cells in the kidney, mean arterial blood pressure and urinary albumin excretion rate were significantly lower in Rag1-null mutants (158 ± 3 mmHg and 60 ± 16 mg/day, respectively) than in SS rats (180 ± 11 mmHg and 251 ± 37 mg/day). Finally, a histological analysis revealed that the glomerular and tubular damage in the kidneys of the SS rats fed a high-salt diet was also attenuated in the Rag1 mutants. These studies demonstrate the importance of renal infiltration of immune cells in the pathogenesis of hypertension and renal damage in Dahl SS rats.
Response to a drug often differs widely among individual patients. This variability is frequently observed not only with respect to effective responses but also with adverse drug reactions. Matching ...patients to the drugs that are most likely to be effective and least likely to cause harm is the goal of effective therapeutics. Pharmacogenomics (PGx) holds the promise of precision medicine through elucidating the genetic determinants responsible for pharmacological outcomes and using them to guide drug selection and dosing. Here we survey the US landscape of research programs in PGx implementation, review current advances and clinical applications of PGx, summarize the obstacles that have hindered PGx implementation, and identify the critical knowledge gaps and possible studies needed to help to address them.
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