Whole genome sequencing reveals epistasis effects within RET for Hirschsprung disease.

Common variants in RET and NRG1 have been associated with Hirschsprung disease (HSCR), a congenital disorder characterised by incomplete innervation of distal gut, in East Asian (EA) populations. However, the allelic effects so far identified do not fully explain its heritability, suggesting the presence of epistasis, where effect of one genetic variant differs depending on other (modifier) variants. Few instances of epistasis have been documented in complex diseases due to modelling complexity and data challenges. We proposed four epistasis models to comprehensively capture epistasis for HSCR between and within RET and NRG1 loci using whole genome sequencing (WGS) data in EA samples. 65 variants within the Topologically Associating Domain (TAD) of RET demonstrated significant epistasis with the lead enhancer variant (RET+3; rs2435357). These epistatic variants formed two linkage disequilibrium (LD) clusters represented by rs2506026 and rs2506028 that differed in minor allele frequency and the best-supported epistatic model. Intriguingly, rs2506028 is in high LD with one cis-regulatory variant (rs2506030) highlighted previously, suggesting that detected epistasis might be mediated through synergistic effects on transcription regulation of RET. Our findings demonstrated the advantages of WGS data for detecting epistasis, and support the presence of interactive effects of regulatory variants in RET for HSCR.

Whole-genome analysis of noncoding genetic variations identifies multiscale regulatory element perturbations associated with Hirschsprung disease.

It is widely recognized that noncoding genetic variants play important roles in many human diseases, but there are multiple challenges that hinder the identification of functional disease-associated noncoding variants. The number of noncoding variants can be many times that of coding variants; many of them are not functional but in linkage disequilibrium with the functional ones; different variants can have epistatic effects; different variants can affect the same genes or pathways in different individuals; and some variants are related to each other not by affecting the same gene but by affecting the binding of the same upstream regulator. To overcome these difficulties, we propose a novel analysis framework that considers convergent impacts of different genetic variants on protein binding, which provides multiscale information about disease-associated perturbations of regulatory elements, genes, and pathways. Applying it to our whole-genome sequencing data of 918 short-segment Hirschsprung disease patients and matched controls, we identify various novel genes not detected by standard single-variant and region-based tests, functionally centering on neural crest migration and development. Our framework also identifies upstream regulators whose binding is influenced by the noncoding variants. Using human neural crest cells, we confirm cell stage-specific regulatory roles of three top novel regulatory elements on our list, respectively in the RET, RASGEF1A, and PIK3C2B loci. In the PIK3C2B regulatory element, we further show that a noncoding variant found only in the patients affects the binding of the gliogenesis regulator NFIA, with a corresponding up-regulation of multiple genes in the same topologically associating domain.

Oropharyngeal Oxygen Concentration Is Dependent on the Oxygen Mask System and Sampling Location.

BACKGROUND: Numerous oxygen delivery systems are used to treat hypoxemia. It is unknown if FIO2 at the lips predicts oropharyngeal FIO2 for various oxygen mask systems. We tested whether FIO2 measurements differed between the lips and oropharynx, and whether this difference depends on the mask system. METHODS: Ten healthy volunteers had one sampling catheter positioned at the lips and another catheter in the oropharynx. FIO2 was sampled at each location while the subjects breathed normal tidal volumes with oxygen at 15 L/min via 4 delivery devices: a simple mask, a non-rebreather mask, a face mask with a diffuser that concentrates and directs O2 toward the mouth and nose (mask with diffuser), and a closed mask with a Jackson-Rees circuit. Data were analyzed by using a linear mixed model to account for subject crossover in the repeated measures design. RESULTS: FIO2 levels differed significantly for the 4 delivery mask systems (P < .001) and by sampling catheter location (P < .001). Differences in mean FIO2 between the lips and the oropharynx were observed for the mask with diffuser (difference 0.30, 95% CI 0.25-0.36; P < .001), and non-rebreather mask (difference 0.09, 95% CI 0.04-0.15; P = .001). The mean FIO2 at the oropharynx was highest for the closed mask (0.97, 95% CI 0.92-1.00), followed by the non-rebreather mask (0.76, 95% CI 0.72-0.81), simple mask (0.62, 95% CI 0.58-0.67), and the mask with diffuser (0.51, 95% CI 0.46-0.56). At the lips, the mean FIO2 was highest for the closed mask (0.97, 95% CI 0.92-1.00), followed by the non-rebreather mask (0.86, 95% CI 0.81- 0.90), OxyMask (0.81, 95% CI 0.76-0.86), and simple mask (0.67, 95% CI 0.62-0.71). CONCLUSIONS: With high oxygen flows and normal tidal volume breathing, FIO2 measurements obtained at the oropharynx or at the lips depended on the device used, with the mask with diffuser showing the most significant discrepancies. FIO2 measures at the oropharynx and the lips were only consistent for the closed mask system. (ClinicalTrials.gov registration NCT02523586.).

Identification of Genes Associated With Hirschsprung Disease, Based on Whole-Genome Sequence Analysis, and Potential Effects on Enteric Nervous System Development.

BACKGROUND & AIMS: Hirschsprung disease, or congenital aganglionosis, is believed to be oligogenic-that is, caused by multiple genetic factors. We performed whole-genome sequence analyses of patients with Hirschsprung disease to identify genetic factors that contribute to disease development and analyzed the functional effects of these variants. METHODS: We performed whole-genome sequence analyses of 443 patients with short-segment disease, recruited from hospitals in China and Vietnam, and 493 ethnically matched individuals without Hirschsprung disease (controls). We performed genome-wide association analyses and gene-based rare-variant burden tests to identify rare and common disease-associated variants and study their interactions. We obtained induced pluripotent stem cell (iPSC) lines from 4 patients with Hirschsprung disease and 2 control individuals, and we used these to generate enteric neural crest cells for transcriptomic analyses. We assessed the neuronal lineage differentiation capability of iPSC-derived enteric neural crest cells using an in vitro differentiation assay. RESULTS: We identified 4 susceptibility loci, including 1 in the phospholipase D1 gene (PLD1) (P = 7.4 × 10-7). The patients had a significant excess of rare protein-altering variants in genes previously associated with Hirschsprung disease and in the ß-secretase 2 gene (BACE2) (P = 2.9 × 10-6). The epistatic effects of common and rare variants across these loci provided a sensitized background that increased risk for the disease. In studies of the iPSCs, we observed common and distinct pathways associated with variants in RET that affect risk. In functional assays, we found variants in BACE2 to protect enteric neurons from apoptosis. We propose that alterations in BACE1 signaling via amyloid ß precursor protein and BACE2 contribute to pathogenesis of Hirschsprung disease. CONCLUSIONS: In whole-genome sequence analyses of patients with Hirschsprung disease, we identified rare and common variants associated with disease risk. Using iPSC cells, we discovered some functional effects of these variants.

Blood Type O is not associated with increased blood loss in extensive spine surgery.

STUDY OBJECTIVE: To investigate whether Type O blood group status is associated with increased intraoperative blood loss and requirement of blood transfusion in extensive spine surgery. DESIGN: Retrospective comparative study. SETTING: University-affiliated, non-profit teaching hospital. MEASUREMENTS: Data from 1,050 ASA physical status 1, 2, 3, 4, and 5 patients who underwent spine surgeries involving 4 or more vertebral levels were analyzed. Patients with Type O blood were matched to similar patients with other blood types using propensity scores, which were estimated via demographic and morphometric data, medical history variables, and extent of surgery. Intraoperative estimated blood loss (EBL) was compared among matched patients using a linear regression model; intraoperative transfusion requirement in volume of red blood cells, fresh frozen plasma, platelet, cryoprecipitate, cell salvaged blood, volume of intraoperative infusion of hetastarch, 5% albumin, crystalloids, and hospital length of hospital (LOS) were compared using Wilcoxon rank-sum tests. MAIN RESULTS: Intraoperative EBL and requirement of blood product transfusion were similar in patients with Type O blood group and those with other blood groups. CONCLUSION: There was no association between Type O blood and increased intraoperative blood loss or blood transfusion requirement during extensive spine surgery, with similar hospital LOS in Type O and non-O patients.

Preoperative angiotensin-converting enzyme inhibitor use is not associated with increased postoperative pain and opioid use.

AIM/OBJECTIVES/BACKGROUND: Angiotensin-converting enzyme inhibitors (ACEIs) increase potent proinflammatory and pain mediators in local tissues. Consistent with these observations, animal and human studies demonstrate that ACEIs have hyperalgesic and proinflammatory properties. However, there is no information in literature whether or not the use of ACEIs is associated with increased postoperative pain. Specifically, we tested the primary hypothesis that use of ACEIs is independently associated with increased opioid requirements and pain scores during the initial 72 hours after surgery. METHODS: Data from 9993 patients undergoing colorectal resection, hysterectomy, nephrectomy, or open prostatectomy were obtained from the Cleveland Clinic Perioperative Health Documentation System. A propensity-matching procedure was used to pair ACEI users to similar nonusers. Corresponding estimates and Bonferroni-adjusted 95% confidence intervals for the effect of ACEIs on each outcome were also estimated. The exact matching procedure, based on type of surgery and propensity score, identified 1038 matched pairs. The final analyzed subsample size was 212. RESULTS AND CONCLUSIONS: The adjusted difference in mean 72-hour postoperative using a time-weighted average pain score was estimated at +0.17 [-0.40, +0.74] units on the verbal response scale. This was not statistically significant (P=0.50). Opioid use was estimated by the percent difference in mean 72-hour total postoperative intravenous morphine equivalent dose at -8.1% [-46%, +56%], which was not statistically significant (P=0.72). In conclusion, after controlling for all available factors, we found no significant difference that postoperative pain-as defined by either pain scores or opioid requirements-differed between patients taking ACEIs and patients not taking ACEIs.

The influence of RGD-bearing hydrogels on the re-expression of contractile vascular smooth muscle cell phenotype.

This study reports on the ability of poly(ethylene glycol) diacrylate (PEGDA) hydrogel scaffolds with pendant integrin-binding GRGDSP peptides (RGD-gels) to support the re-differentiation of cultured vascular smooth muscle cells (SMCs) toward a contractile phenotype. Human coronary artery SMCs were seeded on RGD-gels, hydrogels with other extracellular matrix derived peptides, fibronectin (FN) and laminin (LN). Differentiation was induced on RGD-gels with low serum medium containing soluble heparin, and the differentiation status was monitored by mRNA expression, protein expression, and intracellular protein organization of the contractile smooth muscle markers, smooth muscle alpha-actin, calponin, and SM-22alpha. RGD-gels supported a rapid induction (2.7- to 25-fold up-regulation) of SMC marker gene mRNA, with expression levels that were equivalent to FN and LN controls. Marker protein levels mirrored the changes in mRNA expression, with levels on RGD-gels indistinguishable from FN and LN controls. Furthermore, these markers co-localized in stress fibers within SMCs on RGD-gels suggesting the recapitulation of a contractile apparatus within the cells. These results indicate that SMCs cultured on RGD-bearing hydrogels can re-differentiate toward a contractile phenotype suggesting this material is an excellent candidate for further development as a bioactive scaffold that regulates SMC phenotype.

Maxillomandibular relationship in TMD patients before and after short-term flat plane bite plate therapy.

This study assessed the maxillomandibular relationship in temporomandibular disorders (TMD) patients, before and after short-term, flat plane bite plate therapy. It was of interest to determine the incidence and degree of mandibular deviation in a group of TMD patients and whether the mandible would shift to the midline and consequently affect reported symptoms. Seventeen female and three male subjects (age range 19-60) were included in the study. Thirteen subjects were diagnosed with myofascial pain while seven were diagnosed as exhibiting disk displacement with reduction (Research Diagnostic Criteria). After taking impressions for these subjects, casts were fabricated and mounted. Maxillomandibular relationship was evaluated by the Denar Centric Check system (Anaheim, CA). The maxillary and mandibular labial frena were used as a reference to evaluate mandibular shift. Symptom questionnaires were used to assess temporomandibular joint pain and clicking. All subjects exhibited deviation (12 subjects to the right and 8 subjects to the left) prior to bite plate therapy. After flat plane bite plate therapy, the mandibular position of all subjects shifted toward the labial frenum midline position. Based on the Binomial test, the shift was significant (p < 0.001). Measurements on the Centric Check system showed a significant movement of both condyles in the anterio-posterior plane as well as the vertical plane. There was also significant reduction in TMJ pain and clicking (p < 0.01). The results support the hypothesis that the balanced position of the mandible is with frena aligned. When occlusal obstructions are eliminated, the mandible will drift to this position.