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Comparative analysis of the sea urchin genome has broad implications for the primitive state of deuterostome host defense and the genetic underpinnings of immunity in vertebrates. The sea urchin has an unprecedented complexity of innate immune recognition receptors relative to other animal species yet characterized. These receptor genes include a vast repertoire of 222 Toll-like receptors, a superfamily of more than 200 NACHT domain-leucine-rich repeat proteins (similar to nucleotide-binding and oligomerization domain (NOD) and NALP proteins of vertebrates), and a large family of scavenger receptor cysteine-rich proteins. More typical numbers of genes encode other immune recognition factors. Homologs of important immune and hematopoietic regulators, many of which have previously been identified only from chordates, as well as genes that are critical in adaptive immunity of jawed vertebrates, also are present. The findings serve to underscore the dynamic utilization of receptors and the complexity of immune recognition that may be basal for deuterostomes and predicts features of the ancestral bilaterian form.

The laboratory rat (Rattus norvegicus) is an indispensable tool in experimental medicine and drug development, having made inestimable contributions to human health. We report here the genome sequence of the Brown Norway (BN) rat strain. The sequence represents a high-quality 'draft' covering over 90% of the genome. The BN rat sequence is the third complete mammalian genome to be deciphered, and three-way comparisons with the human and mouse genomes resolve details of mammalian evolution. This first comprehensive analysis includes genes and proteins and their relation to human disease, repeated sequences, comparative genome-wide studies of mammalian orthologous chromosomal regions and rearrangement breakpoints, reconstruction of ancestral karyotypes and the events leading to existing species, rates of variation, and lineage-specific and lineage-independent evolutionary events such as expansion of gene families, orthology relations and protein evolution.

Prader-Willi Syndrome (PWS) is caused by the absence of paternally expressed, maternally silenced genes at 15q11-q13. We report four individuals with truncating mutations on the paternal allele of MAGEL2, a gene within the PWS domain. The first subject was ascertained by whole genome sequencing analysis for PWS features. Three additional subjects were identified by reviewing results of exome sequencing of 1248 cases in a clinical laboratory. All four subjects had autism spectrum disorder (ASD), intellectual disability (ID), and a varying degree of clinical and behavioral features of PWS. These findings suggest MAGEL2 is a novel gene causing complex ASDs, and MAGEL2loss of function can contribute to several aspects of the PWS phenotype.

SummaryRecently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources—potentially useful for the development of new therapeutic approaches. Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC) lines. We then utilized zinc-finger nucleases (ZFNs), designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology-directed repair (HDR). We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other. The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells.

Analysis of ␤-tubulin alleles from nine paclitaxel-resistant Chinese hamster ovary cell lines revealed an unexpected cluster of mutations affecting Leu-215, Leu-217, and Leu-228. Six of the mutant alleles encode a His, Arg, or Phe substitution at Leu-215; another mutant allele has an Arg substitution at Leu-217; and the final two mutant alleles have substitutions of His or Phe at Leu-228. Using plasmids that allow tetracycline regulated expression, the L215H, L217R, and L228F mutations were introduced into a hemagglutinin antigen-tagged ␤-tubulin cDNA and transfected into wild-type Chinese hamster ovary cells. In all three cases, low to moderate expression of the transfected mutant gene conferred paclitaxel resistance. Higher levels of expression caused disruption of microtubule assembly, cell cycle arrest at mitosis, and failure to proliferate. Consistent with reduced microtubule stability, cells expressing mutant hemagglutinin ␤-tubulin had fewer acetylated microtubules than nonexpressing cells in the same population. These data, together with previous studies showing that the paclitaxel-resistant mutant cell lines have less stable microtubules, indicate that the leucine cluster represents an important structural motif for microtubule assembly.Paclitaxel is the prototype for a novel class of agents that inhibit cells in mitosis by promoting and stabilizing microtubule assembly. Early studies with this compound demonstrated that it binds to microtubules in a 1:1 stoichiometry with tubulin heterodimers (1) and inhibits microtubule disassembly. It is also able to induce microtubule assembly both in vitro and in vivo and induces microtubule bundle formation in treated cells (2, 3). Recent interest in this and related compounds has been fueled by clinical studies demonstrating remarkable activity of paclitaxel against a number of malignant diseases (reviewed in Ref. 4). Although still in clinical trials, the demonstrated activity of paclitaxel in phase II studies has led to FDA approval for its use in refractory cases of breast and ovarian cancer. As more patients are treated with this drug, clinical resistance is expected to become an increasingly significant problem.The mechanisms by which tumor cells acquire resistance to paclitaxel are not fully understood. Cell culture studies have shown that paclitaxel is a substrate for the multidrug resistance pump (gP170), 1 and cells selected for high levels of resistance to the drug have increased gP170 (reviewed in Ref. 5). Nevertheless, it has yet to be demonstrated that this mechanism is significant in paclitaxel refractory tumors. Indeed, the remarkable efficacy of paclitaxel in early clinical studies of patients who were pretreated with Adriamycin, a well known substrate for gP170, argues that the multidrug resistance (mdr) phenotype may not be as clinically prevalent as had initially been anticipated (4).Additional mechanisms of resistance to paclitaxel have been reported. For example, several laboratories have provided evidence that changes in the expression of sp...

Precision medicine, taking account of human individuality in genes, environment, and lifestyle for early disease diagnosis and individualized therapy, has shown great promise to transform medical care. Nontargeted metabolomics, with the ability to detect broad classes of biochemicals, can provide a comprehensive functional phenotype integrating clinical phenotypes with genetic and nongenetic factors. To test the application of metabolomics in individual diagnosis, we conducted a metabolomics analysis on plasma samples collected from 80 volunteers of normal health with complete medical records and three-generation pedigrees. Using a broadspectrum metabolomics platform consisting of liquid chromatography and GC coupled with MS, we profiled nearly 600 metabolites covering 72 biochemical pathways in all major branches of biosynthesis, catabolism, gut microbiome activities, and xenobiotics. Statistical analysis revealed a considerable range of variation and potential metabolic abnormalities across the individuals in this cohort. Examination of the convergence of metabolomics profiles with whole-exon sequences (WESs) provided an effective approach to assess and interpret clinical significance of genetic mutations, as shown in a number of cases, including fructose intolerance, xanthinuria, and carnitine deficiency. Metabolic abnormalities consistent with early indications of diabetes, liver dysfunction, and disruption of gut microbiome homeostasis were identified in several volunteers. Additionally, diverse metabolic responses to medications among the volunteers may assist to identify therapeutic effects and sensitivity to toxicity. The results of this study demonstrate that metabolomics could be an effective approach to complement next generation sequencing (NGS) for disease risk analysis, disease monitoring, and drug management in our goal toward precision care.metabolomics | whole-exome sequencing | functional phenotyping | gene penetrance | disease assessment T he rapid progression of next generation sequencing (NGS) technology in recent years has significantly reduced the cost and time required to query a patient's genome accurately. The ability to comprehensively survey genetic variations and their associations with diseases is currently central to personalized medicine and can potentially transform clinical diagnosis and disease management. Indeed, whole-genome sequencing and whole-exome sequencing (WES) have been used successfully to investigate both common and rare diseases (1-7), as well as to provide guidance for drug treatment (8). Despite these successes, significant limitations remain on applying NGS in clinical settings for patient care (9-11). One of the key challenges is the proper interpretation of NGS data. It is known that human exome sequencing identifies ∼10,000 nonsynonymous single-nucleotide variants (12). The computational algorithms and databases for predicting and prioritizing functional pathogenic variants are not yet fully effective. More importantly, the impact of nongenetic factors, such ...

Mutations in gene RASA1 have been historically associated with capillary malformation-arteriovenous malformation, but sporadic reports of lymphatic involvement have yet to be investigated in detail. To investigate the impact of RASA1 mutations in the lymphatic system, we performed investigational near-infrared fluorescence lymphatic imaging and confirmatory radiographic lymphangiography in a Parkes-Weber syndrome (PKWS) patient with suspected RASA1 mutations and correlated the lymphatic abnormalities against that imaged in an inducible Rasa1 knockout mouse. Whole-exome sequencing (WES) analysis and validation by Sanger sequencing of DNA from the patient and unaffected biological parents enabled us to identify an early-frameshift deletion in RASA1 that was shared with the father, who possessed a capillary stain but otherwise no overt disease phenotype. Abnormal lymphatic vasculature was imaged in both affected and unaffected legs of the PKWS subject that transported injected indocyanine green dye to the inguinal lymph node and drained atypically into the abdomen and into dermal lymphocele-like vesicles on the groin. Dermal lymphatic hyperplasia and dilated vessels were observed in Rasa1-deficient mice, with subsequent development of chylous ascites. WES analyses did not identify potential gene modifiers that could explain the variability of penetrance between father and son. Nonetheless, we conclude that the RASA1 mutation is responsible for the aberrant lymphatic architecture and functional abnormalities, as visualized in the PKWS subject and in the animal model. Our unique method to combine investigatory near-infrared fluorescence lymphatic imaging and WES for accurate phenoptyping and unbiased genotyping allows the study of molecular mechanisms of lymphatic involvement of hemovascular disorders.CM-AVM | lymphatics | near-infrared fluorescence imaging | indocyanine green imaging T he gene RASA1 encodes for protein p120 Ras GTPaseactivating protein (p120 RasGAP or RASA1) that regulates Ras activation and blood vessel growth in humans. Germ-line mutations in RASA1 cause the autosomal dominant vascular disorder capillary malformation-arteriovenous malformation (CM-AVM), with high penetrance and variable expressivity [Online Mendelian Inheritance in Man (OMIM) no. 608354] (1-5). CMs are observed in all patients with RASA1 mutations and present as single or multiple small pink cutaneous lesions. Fast-flow lesions that include AVMs, arteriovenous fistulas, and Parkes-Weber syndrome (PKWS) (OMIM no. 608355) are observed in one-third of cases. PKWS caused by RASA1 mutation typically presents with symmetrical overgrowth of the involved extremity with large cutaneous CM and diffuse microshunting causing cardiac volume overload. Recent reports of upper-and lower-extremity lymphedema (6, 7), together with the finding that a small number of CM-AVM patients develop chylothorax and chylous ascites (5), indicate that lymphatic malformation may be an additional phenotype associated with mutations in RASA1. In mice, deletion of ...