A cell type-aware framework for nominating non-coding variants in Mendelian regulatory disorders.

Unsolved Mendelian cases often lack obvious pathogenic coding variants, suggesting potential non-coding etiologies. Here, we present a single cell multi-omic framework integrating embryonic mouse chromatin accessibility, histone modification, and gene expression assays to discover cranial motor neuron (cMN) cis-regulatory elements and subsequently nominate candidate non-coding variants in the congenital cranial dysinnervation disorders (CCDDs), a set of Mendelian disorders altering cMN development. We generate single cell epigenomic profiles for ~86,000 cMNs and related cell types, identifying ~250,000 accessible regulatory elements with cognate gene predictions for ~145,000 putative enhancers. We evaluate enhancer activity for 59 elements using an in vivo transgenic assay and validate 44 (75%), demonstrating that single cell accessibility can be a strong predictor of enhancer activity. Applying our cMN atlas to 899 whole genome sequences from 270 genetically unsolved CCDD pedigrees, we achieve significant reduction in our variant search space and nominate candidate variants predicted to regulate known CCDD disease genes MAFB, PHOX2A, CHN1, and EBF3 - as well as candidates in recurrently mutated enhancers through peak- and gene-centric allelic aggregation. This work delivers non-coding variant discoveries of relevance to CCDDs and a generalizable framework for nominating non-coding variants of potentially high functional impact in other Mendelian disorders.

Noncoding variants alter GATA2 expression in rhombomere 4 motor neurons and cause dominant hereditary congenital facial paresis.

Hereditary congenital facial paresis type 1 (HCFP1) is an autosomal dominant disorder of absent or limited facial movement that maps to chromosome 3q21-q22 and is hypothesized to result from facial branchial motor neuron (FBMN) maldevelopment. In the present study, we report that HCFP1 results from heterozygous duplications within a neuron-specific GATA2 regulatory region that includes two enhancers and one silencer, and from noncoding single-nucleotide variants (SNVs) within the silencer. Some SNVs impair binding of NR2F1 to the silencer in vitro and in vivo and attenuate in vivo enhancer reporter expression in FBMNs. Gata2 and its effector Gata3 are essential for inner-ear efferent neuron (IEE) but not FBMN development. A humanized HCFP1 mouse model extends Gata2 expression, favors the formation of IEEs over FBMNs and is rescued by conditional loss of Gata3. These findings highlight the importance of temporal gene regulation in development and of noncoding variation in rare mendelian disease.

A cell type-aware framework for nominating non-coding variants in Mendelian regulatory disorders.

Unsolved Mendelian cases often lack obvious pathogenic coding variants, suggesting potential non-coding etiologies. Here, we present a single cell multi-omic framework integrating embryonic mouse chromatin accessibility, histone modification, and gene expression assays to discover cranial motor neuron (cMN) cis-regulatory elements and subsequently nominate candidate non-coding variants in the congenital cranial dysinnervation disorders (CCDDs), a set of Mendelian disorders altering cMN development. We generated single cell epigenomic profiles for ~86,000 cMNs and related cell types, identifying ~250,000 accessible regulatory elements with cognate gene predictions for ~145,000 putative enhancers. Seventy-five percent of elements (44 of 59) validated in an in vivo transgenic reporter assay, demonstrating that single cell accessibility is a strong predictor of enhancer activity. Applying our cMN atlas to 899 whole genome sequences from 270 genetically unsolved CCDD pedigrees, we achieved significant reduction in our variant search space and nominated candidate variants predicted to regulate known CCDD disease genes MAFB, PHOX2A, CHN1, and EBF3 - as well as new candidates in recurrently mutated enhancers through peak- and gene-centric allelic aggregation. This work provides novel non-coding variant discoveries of relevance to CCDDs and a generalizable framework for nominating non-coding variants of potentially high functional impact in other Mendelian disorders.

Recurrent Rare Copy Number Variants Increase Risk for Esotropia.

Purpose: To determine whether rare copy number variants (CNVs) increase risk for comitant esotropia. Methods: CNVs were identified in 1614 Caucasian individuals with comitant esotropia and 3922 Caucasian controls from Illumina SNP genotyping using two Hidden Markov model (HMM) algorithms, PennCNV and QuantiSNP, which call CNVs based on logR ratio and B allele frequency. Deletions and duplications greater than 10 kb were included. Common CNVs were excluded. Association testing was performed with 1 million permutations in PLINK. Significant CNVs were confirmed with digital droplet polymerase chain reaction (ddPCR). Whole genome sequencing was performed to determine insertion location and breakpoints. Results: Esotropia patients have similar rates and proportions of CNVs compared with controls but greater total length and average size of both deletions and duplications. Three recurrent rare duplications significantly (P = 1 × 10-6) increase the risk of esotropia: chromosome 2p11.2 (hg19, 2:87428677-87965359), spanning one long noncoding RNA (lncRNA) and two microRNAs (OR 14.16; 95% confidence interval [CI] 5.4-38.1); chromosome 4p15.2 (hg19, 4:25554332-25577184), spanning one lncRNA (OR 11.1; 95% CI 4.6-25.2); chromosome 10q11.22 (hg19, 10:47049547-47703870) spanning seven protein-coding genes, one lncRNA, and four pseudogenes (OR 8.96; 95% CI 5.4-14.9). Overall, 114 cases (7%) and only 28 controls (0.7%) had one of the three rare duplications. No case nor control had more than one of these three duplications. Conclusions: Rare CNVs are a source of genetic variation that contribute to the genetic risk for comitant esotropia, which is likely polygenic. Future research into the functional consequences of these recurrent duplications may shed light on the pathophysiology of esotropia.

Effect of fine-motor-skill activities on surgical simulator performance.

PURPOSE: To determine the effect of fine motor activity and nondominant-hand training on cataract surgical simulator (Eyesi) performance. SETTING: Departments of Ophthalmology, University of Iowa, and Veterans Affairs Health Care Systems, Iowa City, Iowa, USA. DESIGN: Prospective controlled trial. METHODS: Medical students completed a questionnaire and baseline microsurgical dexterity evaluation using the following 3 surgical simulator tasks: navigation, forceps, and bimanual. Participants were randomized to control (16) or intervention (17) consisting of writing, completing a labyrinth, eating, and brushing teeth once per day with their nondominant hand. Participants returned 4 weeks after baseline evaluation for follow-up simulator testing. RESULTS: Of the 33 students, regular video game players had greater baseline scores than nonplayers on navigation (P = .021) and bimanual tasks (P = .089). All participants showed statistically significant improvements in all 3 tasks at follow-up after a single baseline evaluation on the surgical simulator (navigation: P = .004; forceps: P < .001; bimanual: P = .004). Nondominant-hand training with daily activities did not show statistically significant differences for dominant hands or nondominant hands. The intervention group (n = 17) trended toward greater improvement than the control group (n = 16) in navigation (14.78 versus 7.06; P = .445) and bimanual tasks (15.2 versus 6.0; P = .324) at follow-up. CONCLUSIONS: Regular video game play enhanced baseline microsurgical performance measured on the surgical simulator. Simulation performance improved significantly in the intervention group and control group after 1 session on the simulator. Although not statistically significant, training the nondominant hand with daily activities showed a trend toward improved navigation and bimanual performance.

Normothermic cardiopulmonary bypass with beating heart for management of renal cell carcinoma with atrial tumor thrombus.

Renal cell carcinoma is occasionally complicated by the formation of a neoplastic thrombus invading the inferior vena cava. Rarely, the thrombus extends into the vena cava, reaching the right atrium. In these situations, despite the advanced tumor stage, surgical resection continues to offer the best chance for effective treatment. The operation requires a complex surgical approach with mobilization of the liver and use, in most cases, of extracorporeal circulation, which allows removal of the tumor thrombus from the right atrium. Traditionally, the intervention is performed using deep hypothermic circulatory arrest or, less frequently, using moderate hypothermia, aortic cross clamping, and cardioplegic cardiac arrest. These strategies have the downside of causing increased blood loss, coagulopathy, and long operative time and can potentially have a negative impact on survival. We report a different operative approach using normothermic cardiopulmonary bypass, with the expectation of lowering the rate of blood product transfusions, hospital length of stay, and overall incidence of complications.

Longterm outcomes for whole and segmental liver grafts in adult and pediatric liver transplant recipients: a 10-year comparative analysis of 2,988 cases.

BACKGROUND: Data on longterm outcomes after liver transplantation with partial grafts are limited. We compared 10-year outcomes for liver transplant patients who received whole grafts (WLT), split grafts from deceased donors (SLT), and partial grafts from living donors (LDLT). STUDY DESIGN: We conducted a single-center analysis of 2,988 liver transplantations performed between August 1993 and May 2006 with median followup of 5 years. Graft types included 2,717 whole-liver, 181 split-liver, and 90 living-donor partial livers. Split-liver grafts included 109 left lateral and 72 extended right partial livers. Living-donor grafts included 49 left lateral and 41 right partial livers. RESULTS: The 10-year patient survivals for WLT, SLT, and LDLT were 72%, 69%, and 83%, respectively (p=0.11), and those for graft survival were 62%, 55%, and 65%, respectively (p=0.088). There were differences in outcomes between adults and children when compared separately by graft types. In adults, 10-year patient survival was significantly lower for split extended right liver graft compared with adult whole liver and living-donor right liver graft (57% versus 72% versus 75%, respectively, p=0.03). Graft survival for adults was similar for all graft types. Retransplantation, recipient age older than 60 years, donor age older than 45 years, split extended right liver graft, and cold ischemia time>10 hours were predictors of diminished patient survival outcomes. In children, the 10-year patient and graft survivals were similar for all graft types. CONCLUSIONS: Longterm graft survival rates in both adults and children for segmental grafts from deceased and living donors are comparable with those in whole organ liver transplantation. In adults, patient survival was lower for split compared with whole grafts when used in retransplantations and in critically ill recipients. Split graft-to-recipient matching is crucial for optimal organ allocation and best use of a scarce and precious resource.

Optimal utilization of donor grafts with extended criteria: a single-center experience in over 1000 liver transplants.

OBJECTIVE: Severely limited organ resources mandate maximum utilization of donor allografts for orthotopic liver transplantation (OLT). This work aimed to identify factors that impact survival outcomes for extended criteria donors (ECD) and developed an ECD scoring system to facilitate graft-recipient matching and optimize utilization of ECDs. METHODS: Retrospective analysis of over 1000 primary adult OLTs at UCLA. Extended criteria (EC) considered included donor age (>55 years), donor hospital stay (>5 days), cold ischemia time (>10 hours), and warm ischemia time (>40 minutes). One point was assigned for each extended criterion. Cox proportional hazard regression model was used for multivariate analysis. RESULTS: Of 1153 allografts considered in the study, 568 organs exhibited no extended criteria (0 score), while 429, 135 and 21 donor allografts exhibited an EC score of 1, 2 and 3, respectively. Overall 1-year patient survival rates were 88%, 82%, 77% and 48% for recipients with EC scores of 0, 1, 2 and 3 respectively (P < 0.001). Adjusting for recipient age and urgency at the time of transplantation, multivariate analysis identified an ascending mortality risk ratio of 1.4 and 1.8 compared to a score of 0 for an EC score of 1, and 2 (P < 0.01) respectively. In contrast, an EC score of 3 was associated with a mortality risk ratio of 4.5 (P < 0.001). Further, advanced recipient age linearly increased the death hazard ratio, while an urgent recipient status increased the risk ratio of death by 50%. CONCLUSIONS: Extended criteria donors can be scored using readily available parameters. Optimizing perioperative variables and matching ECD allografts to appropriately selected recipients are crucial to maintain acceptable outcomes and represent a preferable alternative to both high waiting list mortality and to a potentially futile transplant that utilizes an ECD for a critically ill recipient.

Adequacy of online medical command communication and emergency medical services documentation of informed refusals.

BACKGROUND: In the out-of-hospital setting, when emergency medical services (EMS) providers respond to a 9-1-1 call and encounter a patient who wishes to refuse medical treatment and/or transport to the hospital, the EMS providers must ensure the patient possesses medical decision-making capacity and obtain an informed refusal. In the city of Cleveland, Ohio, Cleveland EMS completes a nontransport worksheet that prompts the paramedics to evaluate specific patient characteristics that can influence medical decision-making capacity and then discuss the risks of refusing with the patient. Cleveland EMS then contacts an online medical command (OLMC) physician to authorize the refusal. OLMC calls are recorded for review. OBJECTIVES: To assess the ability of EMS to determine medical decision-making capacity and obtain an informed refusal of transport. METHODS: This study was a retrospective review of a cohort of recorded OLMC refusal calls and of the accompanying written documentation by Cleveland EMS. The completeness of the verbal communication between the paramedic and OLMC physician and the written documentation on the nontransport worksheet were measured as surrogate markers of the adequacy of determining medical decision-making capacity and obtaining an informed refusal. RESULTS: One hundred thirty-seven OLMC calls for patient-initiated refusals were reviewed. Vital signs and alertness/orientation were verbally communicated more than 83% of the time. The presence of head injury, presence of alcohol or drug intoxication, and presence of hypoglycemia were verbally communicated less than 31% of the time. Verbal communication stating that the risks of refusing had been discussed with the patient occurred 44.5% of the time. The written documentation of the refusal encounter was more complete, exceeding 95% for vital signs and alertness/orientation, and exceeding 80% for the remaining patient characteristics. The rate of written documentation that the risks of refusing had been discussed with the patient was 48.7%. Discrepancies between the verbal and written paramedic reports were clinically insignificant. CONCLUSIONS: Paramedic and OLMC physician communication for patients refusing out-of-hospital medical treatment and/or transport is inadequate in the Cleveland EMS system. A written nontransport worksheet improves documentation of the refusal encounter but does not ensure that every patient who refuses possesses medical decision-making capacity and the capacity to provide an informed refusal.