Genomic Contributors to Esophageal Atresia and Tracheoesophageal Fistula: A 12 Year Retrospective Review.

OBJECTIVE: To evaluate genetic testing utilization and diagnostic yield in infants with esophageal atresia (EA)/tracheoesophageal fistula (TEF) over the past 12 years to inform future practices and individualize prognostication and management. STUDY DESIGN: A retrospective cohort study was performed for all infants with EA or EA/TEF hospitalized between January 2011 and January 2023 at a quaternary children's hospital. For each infant, demographic information, prenatal and postnatal history, and genetic testing were reviewed. RESULTS: There were 212 infants who were classified as follows: 1) complex/syndromic with EA/TEF plus an additional major anatomic anomaly (n = 114, of which 74 met VACTERL criteria); 2) isolated/nonsyndromic EA/TEF (n = 88) and 3) isolated/nonsyndromic EA (n = 10). A range of genetic tests were sent with varying diagnostic rates including karyotype analysis in 12 (all with complex/syndromic phenotypes and all positive), chromosomal microarray analysis in 189 (114 of whom were complex/syndromic with an overall diagnostic rate of 3/189), single gene testing for CHD7 in 18 (4 positive), and exome analysis in 37 complex/syndromic patients (8 positive). CONCLUSIONS: EA/TEF with and without additional anomalies is genetically heterogeneous with a broad range of associated phenotypes. While the genetic etiology of EA/TEF with or without VACTERL remains largely unknown, genome wide testing (exome or genome) including copy number analysis is recommended over chromosomal microarray testing. We anticipate that expanded genetic/genomic testing modalities such as RNA sequencing and tissue specific molecular testing are needed in this cohort to improve our understanding of the genomic contributors to EA/TEF.

Surgical neonates: A retrospective review of procedures and postoperative outcomes at a quaternary children's hospital.

INTRODUCTION: Neonates have a high incidence of respiratory and cardiac perioperative events. Disease severity and indications for surgical intervention often dovetail with an overall complex clinical course and predispose these infants to adverse long-term neurodevelopmental outcomes and increased length of stay. Our aims were to describe severe and nonsevere early postoperative complications to establish a baseline of care outcomes and to identify subgroups of surgical neonates and procedures for future prospective studies. METHODS: Electronic health record data were examined retrospectively for a cohort of patients who had general anesthesia from January 26, 2015 to August 31, 2018. Inclusion criteria were full-term infants with postmenstrual age less than 44 weeks or premature infants less than 60 weeks postmenstrual age undergoing nonimaging, noncardiac surgery. Severe postoperative complications were defined as mortality, reintubation, positive blood culture, and surgical site infection. Nonsevere early postoperative outcomes were defined as hypoglycemia, hyperglycemia, hypothermia, hyperthermia, and readmission within 30 days. RESULTS: About 2569 procedures were performed in 1842 neonates of which 10.9% were emergency surgeries. There were 120 postoperative severe complications and 965 nonsevere postoperative outcomes. Overall, 30-day mortality was 1.8% for the first procedure performed, with higher mortality seen on subgroup analysis for patients who underwent exploratory laparotomy (10.3%) and congenital lung lesion resection (4.9%). Postoperative areas for improvement included hyperglycemia (13.9%) and hypothermia (7.9%). DISCUSSION: The mortality rate in our study was comparable to other studies of neonatal surgery despite a high rate of emergency surgery and a high prevalence of prematurity in our cohort. The early outcomes data identified areas for improvement, including prevention of postoperative glucose and temperature derangements. CONCLUSIONS: Neonates in this cohort were at risk for severe and nonsevere adverse postoperative outcomes. Future studies are suggested to improve mortality and adverse event rates.

High rate of extreme thrombocytosis indicates bone marrow hyperactivity and splenic dysfunction among congenital diaphragmatic hernia patients.

Pediatric extreme thrombocytosis (EXT, platelet count > 1000 x 103/µL) is rare. In a single center retrospective analysis of hospitalized children with EXT, infants with congenital diaphragmatic hernia (CDH) were overrepresented. In general pediatric patients, EXT is usually secondary to infection or inflammation, but most of the 14 CDH patients with EXT had no identifiable inciting factor. Instead, there was evidence that splenic dysfunction and bone marrow hyperactivity underlied EXT in CDH patients. None were associated with bleeding or thrombosis. Our findings identify mechanisms underlying EXT, and aid clinical interpretation and management of EXT in the pediatric population.

High-content live-cell imaging assay used to establish mechanism of trastuzumab emtansine (T-DM1)--mediated inhibition of platelet production.

Proplatelet production represents a terminal stage of megakaryocyte development during which long, branching processes composed of platelet-sized swellings are extended and released into the surrounding culture. Whereas the cytoskeletal mechanics driving these transformations have been the focus of many studies, significant limitations in our ability to quantify the rate and extent of proplatelet production have restricted the field to qualitative analyses of a limited number of cells over short intervals. A novel high-content, quantitative, live-cell imaging assay using the IncuCyte system (Essen BioScience) was therefore developed to measure the rate and extent of megakaryocyte maturation and proplatelet production under live culture conditions for extended periods of time. As proof of concept, we used this system in the present study to establish a mechanism by which trastuzumab emtansine (T-DM1), an Ab-drug conjugate currently in clinical development for cancer, affects platelet production. High-content analysis of primary cell cultures revealed that T-DM1 is taken up by mouse megakaryocytes, inhibits megakaryocyte differentiation, and disrupts proplatelet formation by inducing abnormal tubulin organization and suppressing microtubule dynamic instability. Defining the pathways by which therapeutics such as T-DM1 affect megakaryocyte differentiation and proplatelet production may yield strategies to manage drug-induced thrombocytopenias.

MKL1 and MKL2 play redundant and crucial roles in megakaryocyte maturation and platelet formation.

Serum response factor and its transcriptional cofactor MKL1 are critical for megakaryocyte maturation and platelet formation. We show that MKL2, a homologue of MKL1, is expressed in megakaryocytes and plays a role in megakaryocyte maturation. Using a megakaryocyte-specific Mkl2 knockout (KO) mouse on the conventional Mkl1 KO background to produce double KO (DKO) megakaryocytes and platelets, a critical role for MKL2 is revealed. The decrease in megakaryocyte ploidy and platelet counts of DKO mice is more severe than in Mkl1 KO mice. Platelet dysfunction in DKO mice is revealed by prolonged bleeding times and ineffective platelet activation in vitro in response to adenosine 5'-diphosphate. Electron microscopy and immunofluorescence of DKO megakaryocytes and platelets indicate abnormal cytoskeletal and membrane organization with decreased granule complexity. Surprisingly, the DKO mice have a more extreme thrombocytopenia than mice lacking serum response factor (SRF) expression in the megakaryocyte compartment. Comparison of gene expression reveals approximately 4400 genes whose expression is differentially affected in DKO compared with megakaryocytes deficient in SRF, strongly suggesting that MKL1 and MKL2 have both SRF-dependent and SRF-independent activity in megakaryocytopoiesis.

Leveraging Novel Clinical Decision Support to Improve Preferred Language Documentation in a Neonatal Intensive Care Unit.

BACKGROUND: Recognition of the patient and family's diverse backgrounds and language preference is critical for communicating effectively. In our hospital's electronic health record, a patient or family's identified language for communication is documented in a discrete field known as "preferred language." This field serves as an interdepartmental method to identify patients with a non-English preferred language, creating a bolded banner for non-English speakers easily identifiable by health care professionals. Despite the importance of documenting preferred language to facilitate equitable care, this field is often blank. OBJECTIVES: Using the Institute for Healthcare Improvement's Model for Improvement, our team sought to increase preferred language documentation within the neonatal intensive care unit (NICU) from a baseline of 74% in September 2021 to above 90% within 6 months. METHODS: A multidisciplinary team was assembled to address preferred language documentation. Our team incorporated guidance regarding preferred language documentation into a novel clinical decision support (CDS) tool aimed at addressing various safety and quality measures within the NICU. Our primary outcome metric was documentation of family's preferred language. Process measures included overall compliance with the CDS tool. A secondary outcome was the assessment of preferred language documentation accuracy. RESULTS: The average rate of preferred language documentation increased from a baseline of 74 to 92% within 6 months and is currently sustained at 96%. Moreover, our follow-up assessments found that 100% of a random sample of contacted patients (n = 88) had their language preferences documented correctly. Overall compliance with the CDS tool remained at 85% throughout the project. CONCLUSION: Using a quality improvement framework coupled with a CDS initiative, our team was able to successfully and accurately improve preferred language documentation in our NICU. Future projects include strategies for more equitable care for patients with non-English preferences such as improved interpreter usage and discharge instructions in their preferred language.

Treatment of haemoptysis in pulmonary atresia with tranexamic acid.

We report the case of a young woman with continuing haemoptysis, pulmonary atresia, previous shunt surgery, and pulmonary hypertension. She was not suitable for further surgery or for therapeutic embolisation of bronchial vessels. Treatment with tranexamic acid resolved the haemoptysis.

Neonatal Carotid Artery and Internal Jugular Vein Management Practices at Extracorporeal Membrane Oxygenation Decannulation: No Standard Approach.

BACKGROUND: There are currently no commonly accepted standardized guidelines for management of cervical vessels at neonatal extracorporeal membrane oxygenation (ECMO) decannulation. This study investigates neonatal ECMO decannulation practices regarding management of the carotid artery and internal jugular vein, use of post-repair anticoagulation, and follow-up imaging. METHODS: A survey was distributed to the 37 institutions in the Children's Hospitals Neonatal Consortium. Respondents reported their standard approach to carotid artery and internal jugular vein management (ligation or repair) at ECMO decannulation by their pediatric surgery and cardiothoracic (CT) surgery teams as well as post-repair anticoagulation practices and follow-up imaging protocols. RESULTS: The response rate was 95%. Pediatric surgeons performed most neonatal respiratory ECMO cannulations (88%) and decannulations (85%), while all neonatal cardiac ECMO cannulations and decannulations were performed by CT surgeons. Pediatric surgeons overwhelmingly ligate both vessels (90%) while CT surgeons typically repair both vessels at decannulation (83%). Of the responding centers that repair, 28% (7) have a standard anticoagulation protocol after neck vessel repair. While 52% (13) of centers routinely image cervical vessel patency at least once post repair, most do not subsequently repeat neck vessel imaging. CONCLUSIONS: Significant practice differences exist between pediatric and CT surgeons regarding the approach to cervical vessels at neonatal ECMO decannulation. For those centers that do repair the vessels there is little uniformity in post-repair anticoagulation or imaging protocols. There is a need to develop standardized cervical vessel management guidelines for neonatal ECMO patients and to study their impact on both short- and long-term outcomes. LEVEL OF EVIDENCE: IV.

Incidence of hippocampal metastases in non-small-cell lung cancer.

OBJECTIVES: Patients with locally advanced non-small-cell lung cancer (LA-NSCLC) develop brain metastases in 25-50% of cases during the course of their disease. Data on the incidence of metastases occurring in the hippocampus/perihippocampal zones are limited. This is important when considering hippocampal-sparing brain radiation (HS-BR), a method that could potentially reduce the neurocognitive impact of such treatment. The aim of this study was to assess the incidence of hippocampal/perihippocampal metastases in a cohort of patients with advanced NSCLC treated at our institution. METHODS: This retrospective cohort study included NSCLC patients discussed at our institutional lung cancer multidisciplinary meeting between 2000 and 2016. MRI and contrast-enhanced CT (ceCT) brain images were reviewed to assess the incidence of hippocampal/perihippocampal metastases including metastases within the hippocampal subgranular zone and a 5 mm margin (hippocampal avoidance region) defined as per the RTOG 0933 study. RESULTS: Of 2146 patients reviewed, 357 (16.6%) had brain metastases. A total of 335 patients had available MRI/ceCT brain images for review. Thirty (9%) patients had brain metastases in the hippocampal avoidance region, 8 (2.4%) with hippocampal metastases and 22 (6.6%) with perihippocampal metastases. Univariate analyses did not show an association between developing metastases in the hippocampal avoidance region and age (P = 0.75), gender (P = 0.91) and tumour type (P = 0.298). CONCLUSION: The incidence of metastases in the hippocampal avoidance region in our large cohort of patients was 9%. With low rates of metastases in this region, HS-BR can be considered a feasible option in the management of patients with advanced NSCLC.

Extreme thrombocytosis is associated with critical illness and young age, but not increased thrombotic risk, in hospitalized pediatric patients.

BACKGROUND: Extreme thrombocytosis (EXT, platelet count > 1000 × 103 /µL) is an uncommon but potentially clinically significant finding. Primary EXT in the setting of myeloproliferative disorders is linked to thrombotic and/or bleeding complications more frequently than secondary EXT, which typically occurs in reaction to infection, inflammation, or iron deficiency. However, comorbidities have been reported in adults with secondary EXT. Clinical implications of EXT in children are not well defined, as prior studies targeted small and/or specialized pediatric populations. OBJECTIVES: Our objectives were to determine etiologies and sequelae of EXT in a hospitalized general pediatric patient population. PATIENTS AND METHODS: We retrospectively analyzed EXT cases from a single-center pediatric cohort of ~80 000 patients over 8 years. RESULTS: Virtually all cases (99.8%) were secondary in nature, and most were multifactorial. Many cases of EXT occurred in children under 2 years old (47%) and/or during critical illness (55%). No thrombotic or bleeding events directly resulted from EXT, confirming a paucity of clinical complications associated with EXT in pediatric patients. There were indications that neonatal hematopoiesis and individual genetic variation influenced some cases, in addition to certain diagnoses (eg, sickle cell anemia) and clinical contexts (eg, asplenia). CONCLUSION: Our findings confirm that thrombotic events related to EXT are rare in pediatric patients, which can inform the use of empiric anti-platelet therapy.

Cross-ethnic meta-analysis identifies association of the GPX3-TNIP1 locus with amyotrophic lateral sclerosis.

Cross-ethnic genetic studies can leverage power from differences in disease epidemiology and population-specific genetic architecture. In particular, the differences in linkage disequilibrium and allele frequency patterns across ethnic groups may increase gene-mapping resolution. Here we use cross-ethnic genetic data in sporadic amyotrophic lateral sclerosis (ALS), an adult-onset, rapidly progressing neurodegenerative disease. We report analyses of novel genome-wide association study data of 1,234 ALS cases and 2,850 controls. We find a significant association of rs10463311 spanning GPX3-TNIP1 with ALS (p = 1.3 × 10-8), with replication support from two independent Australian samples (combined 576 cases and 683 controls, p = 1.7 × 10-3). Both GPX3 and TNIP1 interact with other known ALS genes (SOD1 and OPTN, respectively). In addition, GGNBP2 was identified using gene-based analysis and summary statistics-based Mendelian randomization analysis, although further replication is needed to confirm this result. Our results increase our understanding of genetic aetiology of ALS.Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease. Here, Wray and colleagues identify association of the GPX3-TNIP1 locus with ALS using cross-ethnic meta-analyses.

Targeted assessment of lower motor neuron burden is associated with survival in amyotrophic lateral sclerosis.

Estimating survival in amyotrophic lateral sclerosis (ALS) is challenging due to heterogeneity in clinical features of disease and a lack of suitable markers that predict survival. Our aim was to determine whether scoring of upper or lower motor neuron weakness is associated with survival. With this objective, 161 ALS subjects were recruited from two tertiary referral centres. Scoring of upper (UMN) and lower motor neuron (LMN) signs was performed, in addition to a brief questionnaire. Subjects were then followed until the censorship date. Univariate analysis was performed to identify variables associated with survival to either non-invasive ventilation (NIV) or death, which were then further characterized using Cox regression. Results showed that factors associated with reduced survival included older age, bulbar and respiratory involvement and shorter diagnostic delay (all p < 0.05). Whole body LMN score was strongly associated with time to NIV or death (p ≤0.001) whereas UMN scores were poorly associated with survival. In conclusion, our results suggest that, early in disease assessment and in the context of other factors (age, bulbar, respiratory status), the burden of LMN weakness provides an accurate estimate of outcome. Such a scoring system could predict prognosis, and thereby aid in selection of patients for clinical trials.

Exposing asymmetric gray matter vulnerability in amyotrophic lateral sclerosis.

Limb weakness in amyotrophic lateral sclerosis (ALS) is typically asymmetric. Previous studies have identified an effect of limb dominance on onset and spread of weakness, however relative atrophy of dominant and non-dominant brain regions has not been investigated. Our objective was to use voxel-based morphometry (VBM) to explore gray matter (GM) asymmetry in ALS, in the context of limb dominance. 30 ALS subjects were matched with 17 healthy controls. All subjects were right-handed. Each underwent a structural MRI sequence, from which GM segmentations were generated. Patterns of GM atrophy were assessed in ALS subjects with first weakness in a right-sided limb (n = 15) or left-sided limb (n = 15). Within each group, a voxelwise comparison was also performed between native and mirror GM images, to identify regions of hemispheric GM asymmetry. Subjects with ALS showed disproportionate atrophy of the dominant (left) motor cortex hand area, irrespective of the side of first limb weakness (p < 0.01). Asymmetric atrophy of the left somatosensory cortex and temporal gyri was only observed in ALS subjects with right-sided onset of limb weakness. Our VBM protocol, contrasting native and mirror images, was able to more sensitively detect asymmetric GM pathology in a small cohort, compared with standard methods. These findings indicate particular vulnerability of dominant upper limb representation in ALS, supporting previous clinical studies, and with implications for cortical organisation and selective vulnerability.

Study of motor asymmetry in ALS indicates an effect of limb dominance on onset and spread of weakness, and an important role for upper motor neurons.

In amyotrophic lateral sclerosis (ALS), onset and spread of upper motor neuron (UMN) and lower motor neuron (LMN) dysfunction is typically asymmetric. Our aim was to investigate the relationship between limb dominance and the onset and spread of clinical UMN and LMN dysfunction in ALS. We studied 138 ALS subjects with unilateral and concordant limb dominance, from two tertiary centres. A questionnaire was used to determine the pattern of disease onset and spread. The clinical severity of UMN and LMN signs in each limb was quantified using a validated scoring system. Results showed that onset of weakness was more likely to occur in the dominant upper limb (p = 0.02). In subjects with initial weakness in a non-dominant limb, spread of weakness was more likely to be to the other limb on that side (p = 0.008). The relative distribution of upper limb UMN signs was affected by whether weakness first occurred on the dominant or non-dominant side (p = 0.03). These findings support limb dominance as a significant factor underlying onset and spread of ALS, with UMN processes playing an important role. The effect of limb dominance on the presentation of ALS may reflect underlying neuronal vulnerabilities, which become exposed by the disease.

Transforming growth factor-ß superfamily ligand trap ACE-536 corrects anemia by promoting late-stage erythropoiesis.

Erythropoietin (EPO) stimulates proliferation of early-stage erythrocyte precursors and is widely used for the treatment of chronic anemia. However, several types of EPO-resistant anemia are characterized by defects in late-stage erythropoiesis, which is EPO independent. Here we investigated regulation of erythropoiesis using a ligand-trapping fusion protein (ACE-536) containing the extracellular domain of human activin receptor type IIB (ActRIIB) modified to reduce activin binding. ACE-536, or its mouse version RAP-536, produced rapid and robust increases in erythrocyte numbers in multiple species under basal conditions and reduced or prevented anemia in murine models. Unlike EPO, RAP-536 promoted maturation of late-stage erythroid precursors in vivo. Cotreatment with ACE-536 and EPO produced a synergistic erythropoietic response. ACE-536 bound growth differentiation factor-11 (GDF11) and potently inhibited GDF11-mediated Smad2/3 signaling. GDF11 inhibited erythroid maturation in mice in vivo and ex vivo. Expression of GDF11 and ActRIIB in erythroid precursors decreased progressively with maturation, suggesting an inhibitory role for GDF11 in late-stage erythroid differentiation. RAP-536 treatment also reduced Smad2/3 activation, anemia, erythroid hyperplasia and ineffective erythropoiesis in a mouse model of myelodysplastic syndromes (MDS). These findings implicate transforming growth factor-ß (TGF-ß) superfamily signaling in erythroid maturation and identify ACE-536 as a new potential treatment for anemia, including that caused by ineffective erythropoiesis.

T granules in human platelets function in TLR9 organization and signaling.

Human and murine platelets (PLTs) variably express toll-like receptors (TLRs), which link the innate and adaptive immune responses during infectious inflammation and atherosclerotic vascular disease. In this paper, we show that the TLR9 transcript is specifically up-regulated during pro-PLT production and is distributed to a novel electron-dense tubular system-related compartment we have named the T granule. TLR9 colocalizes with protein disulfide isomerase and is associated with either VAMP 7 or VAMP 8, which regulates its distribution in PLTs on contact activation (spreading). Preincubation of PLTs with type IV collagen specifically increased TLR9 and CD62P surface expression and augmented oligodeoxynucleotide (ODN) sequestration and PLT clumping upon addition of bacterial/viral ODNs. Collectively, this paper (a) tracks TLR9 to a new intracellular compartment in PLTs and (b) describes a novel mechanism of TLR9 organization and signaling in human PLTs.

Cytoskeletal mechanics of proplatelet maturation and platelet release.

Megakaryocytes generate platelets by remodeling their cytoplasm into long proplatelet extensions, which serve as assembly lines for platelet production. Although the mechanics of proplatelet elongation have been studied, the terminal steps of proplatelet maturation and platelet release remain poorly understood. To elucidate this process, released proplatelets were isolated, and their conversion into individual platelets was assessed. This enabled us to (a) define and quantify the different stages in platelet maturation, (b) identify a new intermediate stage in platelet production, the preplatelet, (c) delineate the cytoskeletal mechanics involved in preplatelet/proplatelet interconversion, and (d) model proplatelet fission and platelet release. Preplatelets are anucleate discoid particles 2-10 µm across that have the capacity to convert reversibly into elongated proplatelets by twisting microtubule-based forces that can be visualized in proplatelets expressing GFP-ß1-tubulin. The release of platelets from the ends of proplatelets occurs at an increasing rate in time during culture, as larger proplatelets undergo successive fission, and is potentiated by shear.

The superficial temporal fat pad and its ramifications for temporalis muscle construction in facial approximation.

The construction of the facial muscles (particularly those of mastication) is generally thought to enhance the accuracy of facial approximation methods because they increase attention paid to face anatomy. However, the lack of consideration for non-muscular structures of the face when using these "anatomical" methods ironically forces one of the two large masticatory muscles to be exaggerated beyond reality. To demonstrate and resolve this issue the temporal region of nineteen caucasoid human cadavers (10 females, 9 males; mean age=84 years, s=9 years, range=58-97 years) were investigated. Soft tissue depths were measured at regular intervals across the temporal fossa in 10 cadavers, and the thickness of the muscle and fat components quantified in nine other cadavers. The measurements indicated that the temporalis muscle generally accounts for <50% of the total soft tissue depth, and does not fill the entirety of the fossa (as generally known in the anatomical literature, but not as followed in facial approximation practice). In addition, a soft tissue bulge was consistently observed in the anteroinferior portion of the temporal fossa (as also evident in younger individuals), and during dissection, this bulge was found to closely correspond to the superficial temporal fat pad (STFP). Thus, the facial surface does not follow a simple undulating curve of the temporalis muscle as currently undertaken in facial approximation methods. New metric-based facial approximation guidelines are presented to facilitate accurate construction of the STFP and the temporalis muscle for future facial approximation casework. This study warrants further investigations of the temporalis muscle and the STFP in younger age groups and demonstrates that untested facial approximation guidelines, including those propounded to be anatomical, should be cautiously regarded.