X-inactivation analysis of embryonic lethality in Ocrlwt/−;Inpp5b−/− mice

Mutations in the human OCRL gene, which encodes a phosphatidylinositol(4,5)bisphosphate 5-phosphatase, result in the X-linked oculocerebrorenal syndrome of Lowe. Mice with a targeted disruption of Ocrl have no phenotypic abnormalities. Targeted disruption of its closest paralog, Inpp5b, causes male infertility in the 129S6 background. Mice with disruptions of both genes are lost in utero prior to 9.5-10.5 dpc, indicating that there is a functional overlap between the two paralogs early in development. We analyzed the pattern of X-inactivation in four tissues of distinct embryonic origin from Ocrlwt/−;Inpp5b−/− females to explore the timing and tissue distribution of the functional overlap. X-inactivation was strongly skewed against the disrupted Ocrl− allele being on the active X chromosome in all four tissues tested, indicating that there is early selection against cell lineages lacking both Ocrl and Inpp5b. Extraembryonic tissue was also involved in the lethality because there were never any live-born Ocrlwt/−;Inpp5b−/− females when the functional Ocrlwt allele was on the paternal X chromosome, which is preferentially inactivated in trophoblast-derived extraembryonic tissues. Live-born Ocrlwt/−;Inpp5b−/− females were found when the functional Ocrlwt allele was maternal, although in fewer numbers than expected. The importance of the extraembryonic tissues in the early embryonic lethality of embryos lacking both Ocrl and Inpp5b is reinforced by the successful isolation of a viable 40,XX Ocrl−/−;Inpp5b−/− embryonic stem cell from the inner cell mass of a 3.5-dpc blastocyst prior to implantation. These results indicate a functional overlap of Ocrl and Inpp5b in most cell lineages, especially in extraembryonic tissues

Species-specific difference in expression and splice-site choice in Inpp5b, an inositol polyphosphate 5-phosphatase paralogous to the enzyme deficient in Lowe Syndrome

The oculocerebrorenal syndrome of Lowe (OCRL; MIM #309000) is an X-linked human disorder characterized by congenital cataracts, mental retardation, and renal proximal tubular dysfunction caused by loss-of-function mutations in the OCRL gene that encodes Ocrl, a type II phosphatidylinositol bisphosphate (PtdIns4,5P2) 5-phosphatase. In contrast, mice with complete loss-of-function of the highly homologous ortholog Ocrl have no detectable renal, ophthalmological, or central nervous system abnormalities. We inferred that the disparate phenotype between Ocrl-deficient humans and mice was likely due to differences in how the two species compensate for loss of the Ocrl enzyme. We therefore turned our attention to Inpp5b, another type II PtdIns4,5P2 5-phosphatase encoded by Inpp5b in mice and INPP5B in humans, as potential compensating genes in the two species, because Inpp5b/INPP5B are the most highly conserved paralogs to Ocrl/OCRL in the respective genomes of both species and Inpp5b demonstrates functional overlap with Ocrl in mice in vivo. We used in silico sequence analysis, reverse-transcription PCR, quantitative PCR, and transient transfection assays of promoter function to define splice-site usage and the function of an internal promoter in mouse Inpp5b versus human INPP5B. We found mouse Inpp5b and human INPP5B differ in their transcription, splicing, and primary amino acid sequence. These observations form the foundation for analyzing the functional basis for the difference in how Inpp5b and INPP5B compensate for loss of Ocrl function and, by providing insight into the cellular roles of Ocrl and Inpp5b, aid in the development of a model system in which to study Lowe syndrome

Genetic Testing Before Anticoagulation? A Systematic Review of Pharmacogenetic Dosing of Warfarin

Genotype-guided initial warfarin dosing may reduce over-anticoagulation and serious bleeding compared to a one-dose-fits-all dosing method. The objective of this review was to investigate the safety and efficacy of genotype-guided dosing of warfarin in reducing the occurrence of serious bleeding events and over-anticoagulation. The authors searched PubMed, EMBASE and International Pharmaceutical Abstracts through January 23, 2009, without language restrictions. Selected articles were randomized trials comparing pharmacogenetic dosing of warfarin versus a “standard” dose control algorithm in adult patients taking warfarin for the first time. Two reviewers independently extracted data and assessed study quality using a validated instrument. The primary outcomes were major bleeding and time spent within the therapeutic range International Normalized Ratio (INR). Secondary outcomes included minor bleeding, thrombotic events and other measures of anticoagulation quality. Three of 2,014 studies (423 patients) met the inclusion and exclusion criteria. Differences in study quality, dosing algorithms, length of follow-up and outcome measures limited meta-analysis. Summary estimates revealed no statistically significant difference in bleeding rates or time within the therapeutic range INR. The highest quality study found no significant difference in primary or secondary outcomes, although there was a trend towards more rapid achievement of a stable dose (14.1 vs. 19.6 days, p = 0.07) in the pharmocogenetic arm. We did not find sufficient evidence to support the use of pharmacogenetics to guide warfarin therapy. Additional clinical trials are needed to define the optimal approach to use warfarin pharmacogenetics in clinical practice

Expansion of the Parkinson disease-associated SNCA-Rep1 allele upregulates human alpha-synuclein in transgenic mouse brain.

Alpha-synuclein (SNCA) gene has been implicated in the development of rare forms of familial Parkinson disease (PD). Recently, it was shown that an increase in SNCA copy numbers leads to elevated levels of wild-type SNCA-mRNA and protein and is sufficient to cause early-onset, familial PD. A critical question concerning the molecular pathogenesis of PD is what contributory role, if any, is played by the SNCA gene in sporadic PD. The expansion of SNCA-Rep1, an upstream, polymorphic microsatellite of the SNCA gene, is associated with elevated risk for sporadic PD. However, whether SNCA-Rep1 is the causal variant and the underlying mechanism with which its effect is mediated by remained elusive. We report here the effects of three distinct SNCA-Rep1 variants in the brains of 72 mice transgenic for the entire human SNCA locus. Human SNCA-mRNA and protein levels were increased 1.7- and 1.25-fold, respectively, in homozygotes for the expanded, PD risk-conferring allele compared with homozygotes for the shorter, protective allele. When adjusting for the total SNCA-protein concentration (endogenous mouse and transgenic human) expressed in each brain, the expanded risk allele contributed 2.6-fold more to the SNCA steady-state than the shorter allele. Furthermore, targeted deletion of Rep1 resulted in the lowest human SNCA-mRNA and protein concentrations in murine brain. In contrast, the Rep1 effect was not observed in blood lysates from the same mice. These results demonstrate that Rep1 regulates human SNCA expression by enhancing its transcription in the adult nervous system and suggest that homozygosity for the expanded Rep1 allele may mimic locus multiplication, thereby elevating PD risk

Quality improvement program decreases mortality after cardiac surgery

ObjectiveThis study investigated the effects of a quality improvement program and goal-oriented, multidisciplinary protocols on mortality after cardiac surgery.MethodsPatients were divided into two groups: those undergoing surgery (coronary artery bypass grafting, isolated valve surgery, or coronary artery bypass grafting and valve surgery) after establishment of the multidisciplinary quality improvement program (January 2005–December 2006, n = 922) and those undergoing surgery before institution of the program (January 2002–December 2003, n = 1289). Logistic regression and propensity score analysis were used to adjust for imbalances in patients' preoperative characteristics.ResultsOperative mortality was lower in the quality improvement group (2.6% vs 5.0%, P < .01). Unadjusted odds ratio was 0.5 (95% confidence interval 0.3–0.8, P < .01); propensity score–adjusted odds ratio was 0.6 (95% confidence interval 0.4–0.99, P = .04). In multivariable analysis, diabetes (P < .01), chronic renal insufficiency (P = .05), previous cardiovascular operation (P = .04), congestive heart failure (P < .01), unstable angina (P < .01), age older than 75 years (P < .01), prolonged pump time (P < .01), and prolonged operation (P = .05) emerged as independent predictors of higher mortality after cardiac surgery, whereas quality improvement program (P < .01) and male sex (P = .03) were associated with lower mortality. Mortality decline was less pronounced in patients with than without diabetes (P = .04).ConclusionApplication of goal-directed, multidisciplinary protocols and a quality improvement program were associated with lower mortality after cardiac surgery. This decline was less prominent in patients with diabetes, and focused quality improvement protocols may be required for this subset of patients

Renal cell carcinoma in tuberous sclerosis complex

Renal cell carcinoma (RCC) occurs in 2% to 4% of patients with tuberous sclerosis complex (TSC). Previous reports have noted a variety of histologic appearances in these cancers, but the full spectrum of morphologic and molecular features has not been fully elucidated. We encountered 46 renal epithelial neoplasms from 19 TSC patients and analyzed their clinical, pathologic, and molecular features, enabling separation of these 46 tumors into 3 groups. The largest subset of tumors (n=24) had a distinct morphologic, immunologic, and molecular profile, including prominent papillary architecture and uniformly deficient succinate dehydrogenase subunit B (SDHB) expression prompting the novel term "TSC-associated papillary RCC (PRCC)." The second group (n=15) were morphologically similar to a hybrid oncocytic/chromophobe tumor (HOCT), whereas the last 7 renal epithelial neoplasms of group 3 remained unclassifiable. The TSC-associated PRCCs had prominent papillary architecture lined by clear cells with delicate eosinophilic cytoplasmic thread-like strands that occasionally appeared more prominent and aggregated to form eosinophilic globules. All 24 (100%) of these tumors were International Society of Urological Pathology (ISUP) nucleolar grade 2 or 3 with mostly basally located nuclei. Tumor cells from 17 of 24 TSC-associated PRCCs showed strong, diffuse labeling for carbonic anhydrase IX (100%), CK7 (94%), vimentin (88%), and CD10 (83%) and were uniformly negative for SDHB, TFE3, and AMACR. Gains of chromosomes 7 and 17 were found in 2 tumors, whereas chromosome 3p deletion and TFE3 translocations were not detected. In this study, we reported a sizable cohort of renal tumors seen in TSC and were able to identify them as different morphotypes, which may help to expand the morphologic spectrum of TSC-associated RCC

Associations of common breast cancer susceptibility alleles with risk of breast cancer subtypes in BRCA1 and BRCA2 mutation carriers

Peer reviewedPublisher PD

Is tolerance of faith helpful in English school policy? Reification, complexity and values education

Government policies for teachers and schools in England promote values including tolerance of faiths and beliefs alongside law keeping, democracy and respect. Tolerance of faiths has been highlighted as a key value but complexities around tolerance make interpretations and applications of the policy difficult. Policy documents in this area are inevitably interpreted through the context of events and concerns and with the education accountability culture as a driving motivation. In addition, insights from leading scholarly treatments of tolerance raise further difficulties of conceptual clarity and moral worth. One treatment critiques tolerance discourses as sustaining unequal power relationships. Another posits tolerance as reciprocal respect necessary in a democracy. A key claim in the article is that teachers and school leaders are left to resolve difficulties in translating tolerance policy into practice with the threat of sanction through inspection and associated processes. The article identifies for the first time an additional specific danger that the context of this policy simplifies complex factors by compressing concerns about a number of issues into single category of the value of tolerance of religion. While tolerance of religion is necessary in plural liberal democracies, emphasizing religion contributes to a reification that religion is the determining identity criteria of concern which may have the unintended consequence of polarising interests and communities

ACMG recommendations for reporting of incidental findings in clinical exome and genome sequencing

In clinical exome and genome sequencing, there is potential for the recognition and reporting of incidental or secondary findings unrelated to the indication for ordering the sequencing but of medical value for patient care. The American College of Medical Genetics and Genomics (ACMG) recently published a policy statement on clinical sequencing, which emphasized the importance of disclosing the possibility of such results in pretest patient discussions, clinical testing, and reporting of results. The ACMG appointed a Working Group on Incidental Findings in Clinical Exome and Genome Sequencing to make recommendations about responsible management of incidental findings when patients undergo exome or genome sequencing. This Working Group conducted a year-long consensus process, including review by outside experts, and produced recommendations that have been approved by the ACMG Board. Specific and detailed recommendations, and the background and rationale for these recommendations, are described herein. We recommend that laboratories performing clinical sequencing seek and report mutations of the specified classes or types in the genes listed here. This evaluation and reporting should be performed for all clinical germline (constitutional) exome and genome sequencing, including the ‘normal’ of tumor-normal subtractive analyses in all subjects, irrespective of age, but excluding fetal samples. We recognize that there are insufficient data on clinical utility to fully support these recommendations and we encourage the creation of an ongoing process for updating these recommendations at least annually as further data are collected