The availability of both the mouse and human genome sequences allows for the systematic discovery of human gene function through the use of the mouse as a model system. To accelerate the genetic determination of gene function, we have developed a sequence-tagged gene-trap library of >270,000 mouse embryonic stem cell clones representing mutations in ≈60% of mammalian genes. Through the generation and phenotypic analysis of knockout mice from this resource, we are undertaking a functional screen to identify genes regulating physiological parameters such as blood pressure. As part of this screen, mice deficient for the Wnk1 kinase gene were generated and analyzed. Genetic studies in humans have shown that large intronic deletions in WNK1 lead to its overexpression and are responsible for pseudohypoaldosteronism type II, an autosomal dominant disorder characterized by hypertension, increased renal salt reabsorption, and impaired K+ and H+ excretion. Consistent with the human genetic studies, Wnk1 heterozygous mice displayed a significant decrease in blood pressure. Mice homozygous for the Wnk1 mutation died during embryonic development before day 13 of gestation. These results demonstrate that Wnk1 is a regulator of blood pressure critical for development and illustrate the utility of a functional screen driven by a sequence-based mutagenesis approach

Background and Purpose Body mass index (BMI) has been associated with ischemic stroke in older populations, but its association with stroke in younger populations is not known. In light of the current obesity epidemic in the United States, the potential impact of obesity on stroke risk in young adults deserves attention. Methods A population-based case-control study design with 1201 cases and 1154 controls was used to investigate the relationship of obesity and young-onset ischemic stroke. Stroke cases were between the ages of 15 and 49. Logistic regression analysis was used to evaluate the association between BMI and ischemic stroke with and without adjustment for co-morbid conditions associated with stroke. Results In analyses adjusted for age, sex, and ethnicity, obesity (BMI > 30 kg/m2) was associated with an increased stroke risk (odds ratio, 1.57, 95% C.I. = 1.28–1.94), although this increased risk was highly attenuated and not statistically significant after adjustment for smoking, hypertension, and diabetes mellitus. Conclusion These results indicate that obesity is a risk factor for young onset ischemic stroke, and suggest that this association may be partially mediated through hypertension, diabetes mellitus, and/or other variables associated with these conditions.

Summary Introduction The discovery of disease-associated loci through genome-wide association studies (GWAS) is the leading approach to the identification of novel biological pathways for human disease. To date, GWAS have had been limited by relatively small sample sizes and yielded relatively few loci associated with ischemic stroke The National Institute of Neurological Disorders Stroke Genetics Network (NINDS-SiGN) is an international consortium that has taken a systematic approach to phenotyping and produced the largest ischemic stroke GWAS to date. Methods In order to identify genetic loci associated with ischemic stroke, we performed a two-stage genome-wide association study. The first stage consisted of 16,851 cases with state-of-the-art phenotyping and 32,473 stroke-free controls. Cases were aged 16 to 104 years, recruited between 1989 and 2012, and subtyped by centrally trained and certified investigators using the web-based protocol, Causative Classification of Stroke (CCS). We constructed case-control strata by identify samples genotyped on (nearly) identical arrays and of similar genetic ancestral background. Data was cleaned and imputed using dense imputation reference panels generated from whole-genome sequence data. Genome-wide testing was performed within each stratum for each available phenotype, and summary level results were combined using inverse variance-weighted fixed effects meta-analysis. The second stage consisted of in silico look-ups of 1,372 SNPs in 20,941 cases and 364,736 stroke-free controls, with cases previously subtyped using the TOAST classification system according to local standards. The two stages were then jointly analyzed in a final meta-analysis. Findings We identified a novel locus at 1p13.2 near TSPAN2 associated with large artery atherosclerosis (LAA)-related stroke (stage I OR for the G allele at rs12122341 = 1·21, p = 4.50 × 10−8; stage II OR = 1·19, p = 1·30 × 10−9). We also confirmed four loci robustly associated with ischemic stroke and reported in prior studies, including PITX2 and ZFHX3 for cardioembolic stroke, and HDAC9 for LAA stroke. The 12q24 locus near ALDH2, originally associated with all ischemic stroke but not with any specific subtype, exceeded genome-wide significance in the meta-analysis of small artery stroke. Other loci, including NINJ2, were not confirmed. Interpretation Our results identify a novel LAA-stroke susceptibility gene and now indicate that all loci implicated by GWAS to date are subtype specific. Follow-up studies will be necessary to determine whether the locus near TSPAN2 yields a novel therapeutic approach to stroke prevention. Given the subtype-specificity of these associations, the rich phenotyping available in SiGN is likely to prove vital for further genetic discovery in ischemic stroke. Funding National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH).

Normal sensory transduction requires the efficient disposal of acid (H+) generated by neuronal and sensory receptor activity. Multiple highly sensitive transport mechanisms have evolved in prokaryotic and eukaryotic organisms to maintain acidity within strict limits. It is currently assumed that the multiplicity of these processes provides a biological robustness. Here we report that the visual and auditory systems have a specific requirement for H+ disposal mediated by the sodium bicarbonate cotransporter NBC3 (refs. 7,8). Mice lacking NBC3 develop blindness and auditory impairment because of degeneration of sensory receptors in the eye and inner ear as in Usher syndrome. Our results indicate that in certain sensory organs, in which the requirement to transduce specific environmental signals with speed, sensitivity and reliability is paramount, the choice of the H+ disposal mechanism used is limited.

Objective: To compare the performance of formal prognostic instruments vs subjective clinical judgment with regards to predicting functional outcome in patients with spontaneous intracerebral hemorrhage (ICH).Methods: This prospective observational study enrolled 121 ICH patients hospitalized at 5 US tertiary care centers. Within 24 hours of each patient's admission to the hospital, one physician and one nurse on each patient's clinical team were each asked to predict the patient's modified Rankin Scale (mRS) score at 3 months and to indicate whether he or she would recommend comfort measures. The admission ICH score and FUNC score, 2 prognostic scales selected for their common use in neurologic practice, were calculated for each patient. Spearman rank correlation coefficients (r) with respect to patients' actual 3-month mRS for the physician and nursing predictions were compared against the same correlation coefficients for the ICH score and FUNC score. Results:The absolute value of the correlation coefficient for physician predictions with respect to actual outcome (0.75) was higher than that of either the ICH score (0.62, p 5 0.057) or the FUNC score (0.56, p 5 0.01). The nursing predictions of outcome (r 5 0.72) also trended towards an accuracy advantage over the ICH score (p 5 0.09) and FUNC score (p 5 0.03). In an analysis that excluded patients for whom comfort care was recommended, the 65 available attending physician predictions retained greater accuracy (r 5 0.73) than either the ICH score (r 5 0.50, p 5 0.02) or the FUNC score (r 5 0.42, p 5 0.004). Conclusions:Early subjective clinical judgment of physicians correlates more closely with 3-month outcome after ICH than prognostic scales. Neurology ® 2016;86:126-133 GLOSSARY ERICH 5 Ethnic/Racial Variations of Intracerebral Hemorrhage; GCS 5 Glasgow Coma Scale; ICH 5 intracerebral hemorrhage; mRS 5 modified Rankin Scale; NICU 5 neuroscience intensive care unit.Spontaneous intracerebral hemorrhage (ICH) is a devastating stroke subtype, with high rates of mortality and long-term disability.

We conclude that young-onset stroke aggregates in families and that the magnitude of aggregation increases with decreasing proband age.

Background and purpose Although case reports have long identified a temporal association between cocaine use and ischemic stroke, few epidemiological studies have examined the association of cocaine use with ischemic stroke in young adults, by timing, route and frequency of use. Methods A population-based case-control study design with 1,090 cases and 1,154 controls was used to investigate the relationship of cocaine use and young-onset ischemic stroke. Stroke cases were between the ages of 15 and 49. Logistic regression analysis was used to evaluate the association between cocaine use and ischemic stroke with and without adjustment for potential confounders. Results Ever use of cocaine was not associated with stroke with 28% of cases and 26% of controls reporting ever use. In contrast, acute cocaine use in the prior 24 hours was strongly associated with increased risk of stroke (age-sex-race adjusted OR = 6.4, 95% CI=2.2 - 18.6). Among acute users, the smoking route had an adjusted odds ratio of 7.9 (95% CI=1.8 – 35.0), while the inhalation route had an adjusted odds ratio of 3.5 (95% CI=0.7 – 16.9). After additional adjustment for current alcohol, smoking use and hypertension, the odds ratio for acute cocaine use by any route was 5.7 (95% CI =1.7, 19.7). Of the 26 patients with cocaine use within 24 hours of their stroke, 14 reported use within 6 hours of their event. Conclusion Our data are consistent with a causal association between acute cocaine use and risk of early-onset ischemic stroke.

The Rrm2b gene encodes p53R2, a catalytic subunit of ribonucleotide reductase that is required for DNA repair. Embryonic stem (ES) cells containing a retroviral insertion in the Rrm2b locus were used to generate mutant mice. Analysis of kidney RNA from Rrm2b (-/-) mice showed that the retroviral insertion disrupted expression of Rrm2b transcripts. Rrm2b (-/-) pups were represented at the expected Mendelian ratios at 10-12 days of age and grew normally past weaning. Mice failed to thrive after 6 weeks of age and began to die by 8 weeks of age. Phenotyping revealed that Rrm2b (-/-) mice died from a severe glomerular lesion that led to nephrotic syndrome and chronic renal failure. In kidneys of Rrm2b (-/-) mice, podocytes were enlarged and there was evidence of foot process effacement by 6 weeks of age. By 8 weeks of age, progressive podocyte hypertrophy and loss of foot processes was accompanied by hypertrophy of glomerular capillary endothelial cells that was extensive enough to restrict capillary blood flow. Collapsing glomerulopathy with avascular glomeruli was widespread in mice surviving beyond 9 weeks of age. Additional abnormalities in other organ systems were minor or consistent with secondary effects of renal failure. These findings suggest that lack of p53R2, the protein encoded by Rrm2b, has early and relatively selective detrimental effects on the kidney glomerulus that lead to rapid death from progressive renal failure.