Enhancers, critical determinants of cellular identity, are commonly recognized by correlative chromatin marks and gain-of-function potential, although only loss-of-function studies can demonstrate ...their requirement in the native genomic context. Previously, we identified an erythroid enhancer of human BCL11A, subject to common genetic variation associated with the fetal haemoglobin level, the mouse orthologue of which is necessary for erythroid BCL11A expression. Here we develop pooled clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear to be primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for fetal haemoglobin reinduction. The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements.
Fertility Preservation for Transgender Adolescents Chen, Diane, Ph.D; Simons, Lisa, M.D; Johnson, Emilie K., M.D., M.P.H ...
Journal of adolescent health,
07/2017, Letnik:
61, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Abstract Purpose To describe fertility preservation (FP) utilization by transgender adolescents within a pediatric gender clinic between July 2013 and July 2016. Methods A retrospective chart review ...was conducted to abstract demographic and clinical information among adolescents initiating gender-affirming hormones, including patient age at initial FP consultation, birth-assigned sex, race/ethnicity, and outcome of FP consultation. Results In our sample of 105 transgender adolescents, a total of 13 (seven transgender men and six transgender women) between the age of 14.2 and 20.6 years were seen in formal consultation for FP before initiating hormones. Of these adolescents, four completed sperm cryopreservation and one completed oocyte cryopreservation. Conclusions Rates of FP utilization among transgender youth were low, which is consistent with a recently published report of FP utilization among transgender youth at another pediatric institution. Identified barriers to FP in our sample included cost, invasiveness of procedures, and desire not to delay medical transition.
Anomalous left coronary artery from pulmonary artery (ALCAPA) is associated with high mortality if left uncorrected. We describe long-term outcomes after surgical repair and ventricular function ...changes on long-term follow-up.
A retrospective review of patients who had ALCAPA repair from January 1996 to December 2011 was completed. Ventricular function was assessed by shortening fraction and ejection fraction, left ventricular end-diastolic dimension, and severity of mitral regurgitation. Speckle tracking echocardiography served as a marker of early myocardial dysfunction.
In total, 34 patients underwent ALCAPA repair at median age of 5 months (range, 3 days to 39 years). Surgical interventions included coronary translocation in 31 patients (91%), Takeuchi repair in 2 (6%), and coronary ligation in 1 patient (3%). Concomitant mitral valve repair was performed in 5 patients (15%). No patient required mechanical circulatory support postoperatively. There was no early mortality. At median follow-up of 6 years (range, 1 month to 14 years), there were 5 reoperations: 2 heart transplants (6%), 1 mitral valve replacement (3%), 1 coronary artery bypass graft (3%), and 1 ventricular septal defect closure (3%); there was 1 death 2 years after surgery. Ejection fraction improved from 21% ± 6% to 60% ± 7% (p = 0.008), mean shortening fraction from 25% ± 14% to 38% ± 5% (p = 0.01), mean left ventricular end-diastolic dimension Z-score from 6.0 ± 3.8 to 0.9 ± 0.7 (p < 0.001), and number of patients with moderate-severe mitral regurgitation from 44% (15 of 34) to 5% (1 of 21). All patients had normal ejection fraction and shortening fraction at last follow-up. Speckle tracking echocardiography showed decreased global longitudinal (p = 0.01) and circumferential strain (p = 0.03) for 11 of 14 patients (79%).
Excellent outcomes are achieved with repair of ALCAPA without mechanical circulatory support and with low reintervention rates. Normal ejection fraction and shortening fraction do not accurately convey myocardial dysfunction in these patients.
Identifying and genotyping mice prior to weaning can be useful for mouse colony management. Mice of an undesiredgenotype can be identified prior to weaning and removed from further study, resulting ...in a reduction of housing costs, andlabor time. We hypothesized that a pinna edge biopsy (PEB) performed by removing a portion of its edge with scissors is areliable method for identifying and genotyping mice on postnatal day (PND) 7 consistent with PND 21, weaned mice. Thepinnae of 54 C57BL/NCrl6 mice were biopsied on PND 7, and another 54 were biopsied on PND 21. Nine pinna patternswere tested. The accuracy of pattern identification was assessed on PND 7, 14, 21, 30, and 63. The mean times were comparedfor performing the biopsy on PND 7 and PND 21 mice, and the average time taken to identify the patterns were determined.Weight, milk spot presence, pup rejection, morbidity, and mortality were examined at various time points. During the biopsy,bleeding of the pinna, urination, vocalization, and flinching were assessed. No significant differences were detected in DNAquality, relative DNA quantity, genotyping reliability, or body weight ( P ≥ 0.05) between mice biopsied on PND 7 and PND21. Flinching at the time of PEB was significantly higher in PND 21 mice as compared with PND 7 mice ( P < 0.00001). Pinnapattern identification accuracy for mice biopsied on PND 7 and PND 21 were 96% and 98%, respectively. This study validatesthe use of PEB for simultaneous identification and genotyping of PND 7 mice.
Cas9-mediated, high-throughput, saturating in situ mutagenesis permits fine-mapping of function across genomic segments. Disease- and trait-associated variants identified in genome-wide association ...studies largely cluster at regulatory loci. Here we demonstrate the use of multiple designer nucleases and variant-aware library design to interrogate trait-associated regulatory DNA at high resolution. We developed a computational tool for the creation of saturating-mutagenesis libraries with single or multiple nucleases with incorporation of variants. We applied this methodology to the HBS1L-MYB intergenic region, which is associated with red-blood-cell traits, including fetal hemoglobin levels. This approach identified putative regulatory elements that control MYB expression. Analysis of genomic copy number highlighted potential false-positive regions, thus emphasizing the importance of off-target analysis in the design of saturating-mutagenesis experiments. Together, these data establish a widely applicable high-throughput and high-resolution methodology to identify minimal functional sequences within large disease- and trait-associated regions.
•About half of patients achieve a total oral diet after MVFTT reconstruction following glossectomy.•Resections of subtotal glossectomy or greater have worse total oral achievement.•Forearm-based free ...flaps had superior outcomes but may be surrogate marker for resection extent.•Normal or low BMI, prior radiation, and adjuvant chemoradiation were negative prognostic factors.
Primary surgery followed by adjuvant therapy is the current standard of care in the multidisciplinary management of squamous cell carcinoma (SCC) of the oral tongue. Additionally, salvage glossectomy is used to treat recurrent base of tongue SCC. Microvascular free tissue transfer reconstruction (MVFTT) is utilized to maximize functional outcomes such as swallowing. We sought to identify prognostic factors related to achievement of a total oral diet in patients that underwent glossectomy with MVFTT.
Retrospective review at a tertiary care center from 2010 to 2015.
200 patients (69% male, mean age 60 years) met inclusion criteria. Extent of glossectomy was categorized as partial or hemiglossectomy (39%), tongue base resection with or without hemi-oral glossectomy (23%), composite resection with mandibulectomy (18%), and subtotal or total glossectomy (21%). Flap success rate was 96%. Median follow-up time was 14 months. A total oral diet was achieved by 49% of patients with median time to achievement of 31 days (IQR 9-209). Multivariate analysis identified body mass index < 25 kg/m2, prior radiation therapy, adjuvant chemoradiation, and resection requiring subtotal or total glossectomy or concurrent mandibulectomy as independent risk factors for worse total oral diet achievement.
Swallowing dysfunction represents a significant morbidity following glossectomy in the treatment of SCC. High BMI, smaller resection fields, and absence of prior radiation therapy or adjuvant chemoradiotherapy correlated with improved likelihood of obtaining a total oral diet. Patients should be appropriately counseled of this risk with emphasis placed on aggressive swallow rehabilitation in the post- treatment setting.
Gene editing technologies offer new options for developing novel biomedical research models and for gene and stem cell based therapies. However, applications in many species demand high efficiencies, ...specificity, and a thorough understanding of likely editing outcomes. To date, overall efficiencies, rates of off-targeting and degree of genetic mosaicism have not been well-characterized for most species, limiting our ability to optimize methods. As a model gene for measuring these parameters of the CRISPR/Cas9 application in a primate species (rhesus monkey), we selected the β-hemoglobin gene (HBB), which also has high relevance to the potential application of gene editing and stem-cell technologies for treating human disease. Our data demonstrate an ability to achieve a high efficiency of gene editing in rhesus monkey zygotes, with no detected off-target effects at selected off-target loci. Considerable genetic mosaicism and variation in the fraction of embryonic cells bearing targeted alleles are observed, and the timing of editing events is revealed using a new model. The uses of Cas9-WT protein combined with optimized concentrations of sgRNAs are two likely areas for further refinement to enhance efficiency while limiting unfavorable outcomes that can be exceedingly costly for application of gene editing in primate species.
Importance:
Vocal fold motion impairment (VFMI) due to neuronal injury is a known complication following thoracic aortic repair that can impair pulmonary toilet function and post-operative recovery.
...Objective:
To demonstrate clinical outcomes of patients undergoing inpatient vocal fold medialization for VFMI after aortic surgery.
Design:
A 15-year retrospective chart review (2005-2019) of 259 patients with postoperative VFMI after thoracic aortic surgery registry was conducted. Data included demographics, surgery characteristics, laryngology exam, and postoperative clinical outcomes. Medialization procedures consisted of type 1 thyroplasty and injection laryngoplasty.
Setting:
Tertiary care hospital
Participants:
Two hundred and fifty-nine patients (median age 61, 71% male) with VFMI post-thoracic aortic repair met inclusion criteria; inpatient vocal fold medialization was performed for 203 (78%) patients. One hundred and twenty-six. (49%) received type 1 thyroplasty and 77 (30%) received injection laryngoplasty procedures at a median 7 days (IQR 5-8 days) from extubation.
Main Outcomes:
Primary study outcome measurements consisted of median LOS, median ICU LOS, complications intra- and postoperatively, and pulmonary complications (post-medialization bronchoscopies, pneumonia, tracheostomy, etc.).
Results:
Post-medialization bronchoscopy rates were significantly lower in the medialization (n = 11) versus the non-medialization group (n = 8) (5% vs 14%, P = .02) and significantly higher in the injection laryngoplasty group (n = 77) versus thyroplasty group (n = 126) (10% vs 2%, P = .02). Further analysis revealed no significant difference in overall LOS and pulmonary complications between the techniques.
Conclusion:
Inpatient thyroplasty and injection laryngoplasty are both effective vocal fold medialization techniques after extent I and II aortic repair. Thyroplasty may have a small pulmonary toilet advantage, as measured by need for post-medialization bronchoscopy, compared to injection laryngoplasty.
Neurodevelopmentally impaired (NI) children with chronic sialorrhea are at elevated risk for aspiration and respiratory tract infections. Direct resection or ligation (“DROOL”) of the submandibular ...glands (SMG) with parotid duct ligation are surgical interventions intended to decrease salivary output. The objective of this study is to determine the impact of DROOL surgery on the incidence of nonviral respiratory-related (NVR) post-procedure hospital encounters including emergency department visits and admissions.
Retrospective case series of NVR related outcomes after DROOL surgery in children performed at a single institution, tertiary referral center.
A total of 35 gastrostomy tube-dependent patients (60% male, average age 8.2 SD 6.0 years) with NI underwent DROOL surgery (86% SMG excision). Pre- and post-surgical follow-up time was 3.6 and 3.2 years, respectively. Presurgical and postsurgical NVR hospital encounters occurred in 28 (80%) and 14 (40%) patients, respectively (p < 0.01). Mean (SD) postoperative NVR hospital encounters occurred less frequently when compared to presurgical period (0.4 0.6 vs. 1.0 1.2 per year, p < 0.01) with average change of −0.7 encounters per year (SD 1.4, 95% CI -1.0 to −0.2). Patients with encounters within a year preceding DROOL (OR 4.9, p = 0.04, 95% CI 1.1–22.8), or those with at least 3 preoperative encounters (OR 8.0, p = 0.01, 95% CI 1.6–40.3) were significantly associated with a postsurgical NVR event. Fewer patients used anti-sialorrhea medication postoperatively compared to preoperatively (60% vs. 17%, p < 0.01). No patient developed surgical site complications requiring operative interventions.
DROOL surgery for chronic sialorrhea in patients with NI was associated with decreased hospitalization and ED visits for NVR respiratory events post-procedurally. Sialorrhea may be an actionable source of recurrent respiratory illnesses requiring hospitalizations.
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