Common variants increase risk for congenital diaphragmatic hernia within the context of de novo variants.

Congenital diaphragmatic hernia (CDH) is a severe congenital anomaly often accompanied by other structural anomalies and/or neurobehavioral manifestations. Rare de novo protein-coding variants and copy-number variations contribute to CDH in the population. However, most individuals with CDH remain genetically undiagnosed. Here, we perform integrated de novo and common-variant analyses using 1,469 CDH individuals, including 1,064 child-parent trios and 6,133 ancestry-matched, unaffected controls for the genome-wide association study. We identify candidate CDH variants in 15 genes, including eight novel genes, through deleterious de novo variants. We further identify two genomic loci contributing to CDH risk through common variants with similar effect sizes among Europeans and Latinx. Both loci are in putative transcriptional regulatory regions of developmental patterning genes. Estimated heritability in common variants is ∼19%. Strikingly, there is no significant difference in estimated polygenic risk scores between isolated and complex CDH or between individuals harboring deleterious de novo variants and individuals without these variants. The data support a polygenic model as part of the CDH genetic architecture.

Rare and de novo variants in 827 congenital diaphragmatic hernia probands implicate LONP1 as candidate risk gene.

Congenital diaphragmatic hernia (CDH) is a severe congenital anomaly that is often accompanied by other anomalies. Although the role of genetics in the pathogenesis of CDH has been established, only a small number of disease-associated genes have been identified. To further investigate the genetics of CDH, we analyzed de novo coding variants in 827 proband-parent trios and confirmed an overall significant enrichment of damaging de novo variants, especially in constrained genes. We identified LONP1 (lon peptidase 1, mitochondrial) and ALYREF (Aly/REF export factor) as candidate CDH-associated genes on the basis of de novo variants at a false discovery rate below 0.05. We also performed ultra-rare variant association analyses in 748 affected individuals and 11,220 ancestry-matched population control individuals and identified LONP1 as a risk gene contributing to CDH through both de novo and ultra-rare inherited largely heterozygous variants clustered in the core of the domains and segregating with CDH in affected familial individuals. Approximately 3% of our CDH cohort who are heterozygous with ultra-rare predicted damaging variants in LONP1 have a range of clinical phenotypes, including other anomalies in some individuals and higher mortality and requirement for extracorporeal membrane oxygenation. Mice with lung epithelium-specific deletion of Lonp1 die immediately after birth, most likely because of the observed severe reduction of lung growth, a known contributor to the high mortality in humans. Our findings of both de novo and inherited rare variants in the same gene may have implications in the design and analysis for other genetic studies of congenital anomalies.

Catalyst-free reactions of anilines with ß-chloroenones: synthesis of α-chloroenaminones and 1,4-benzodiazepines.

The Michael addition of anilines to ß-chloroenones gives enaminones by the elimination of hydrochloric acid (HCl). These enaminones are transformed into α-chloroenaminones via in situ sp2 C-H functionalization. Anilines that are attached to an electron-donating group react more readily with ß-chloroenone to give the corresponding products in excellent yields. A highly atom-economical method has been developed using dimethyl sulfoxide (DMSO) as a green oxidant and solvent. The desired α-functionalized enaminones are formed in good yields with excellent Z-selectivity. We have established the generality of this reaction with many substrates, and scaled-up reactions have been performed to showcase the practical applications. A catalyst-free double annulation of ß-chloroenones with o-phenylenediamine has also been demonstrated for the synthesis of 1,4-benzodiazepine derivatives in moderate yields under mild reaction conditions.

Iodine-Catalyzed Regioselective Synthesis of Diphenyl-Substituted Carbazoles via [4 + 2] Annulation of ß-Formyl Ketones with Indoles.

The [4 + 2] annulation of ß-formyl ketones with an indole has been developed for the regioselective synthesis of diphenyl-substituted carbazoles in the presence of a catalytic amount of iodine. The 1,4-dicarbonyl compound containing a phenyl group at the α-position of an aldehyde group reacts more readily with indoles to form carbazole derivatives. Using this method, a variety of carbazole derivatives can be readily accessed under mild reaction conditions.

WDR5 is a conserved regulator of protein synthesis gene expression.

WDR5 is a highly-conserved nuclear protein that performs multiple scaffolding functions in the context of chromatin. WDR5 is also a promising target for pharmacological inhibition in cancer, with small molecule inhibitors of an arginine-binding pocket of WDR5 (the 'WIN' site) showing efficacy against a range of cancer cell lines in vitro. Efforts to understand WDR5, or establish the mechanism of action of WIN site inhibitors, however, are stymied by its many functions in the nucleus, and a lack of knowledge of the conserved gene networks-if any-that are under its control. Here, we have performed comparative genomic analyses to identify the conserved sites of WDR5 binding to chromatin, and the conserved genes regulated by WDR5, across a diverse panel of cancer cell lines. We show that a specific cohort of protein synthesis genes (PSGs) are invariantly bound by WDR5, demonstrate that the WIN site anchors WDR5 to chromatin at these sites, and establish that PSGs are bona fide, acute, and persistent targets of WIN site blockade. Together, these data reveal that WDR5 plays a predominant transcriptional role in biomass accumulation and provide further evidence that WIN site inhibitors act to repress gene networks linked to protein synthesis homeostasis.

Acoustic evolution of old Italian violins from Amati to Stradivari.

The shape and design of the modern violin are largely influenced by two makers from Cremona, Italy: The instrument was invented by Andrea Amati and then improved by Antonio Stradivari. Although the construction methods of Amati and Stradivari have been carefully examined, the underlying acoustic qualities which contribute to their popularity are little understood. According to Geminiani, a Baroque violinist, the ideal violin tone should "rival the most perfect human voice." To investigate whether Amati and Stradivari violins produce voice-like features, we recorded the scales of 15 antique Italian violins as well as male and female singers. The frequency response curves are similar between the Andrea Amati violin and human singers, up to ∼4.2 kHz. By linear predictive coding analyses, the first two formants of the Amati exhibit vowel-like qualities (F1/F2 = 503/1,583 Hz), mapping to the central region on the vowel diagram. Its third and fourth formants (F3/F4 = 2,602/3,731 Hz) resemble those produced by male singers. Using F1 to F4 values to estimate the corresponding vocal tract length, we observed that antique Italian violins generally resemble basses/baritones, but Stradivari violins are closer to tenors/altos. Furthermore, the vowel qualities of Stradivari violins show reduced backness and height. The unique formant properties displayed by Stradivari violins may represent the acoustic correlate of their distinctive brilliance perceived by musicians. Our data demonstrate that the pioneering designs of Cremonese violins exhibit voice-like qualities in their acoustic output.

De novo variants in congenital diaphragmatic hernia identify MYRF as a new syndrome and reveal genetic overlaps with other developmental disorders.

Congenital diaphragmatic hernia (CDH) is a severe birth defect that is often accompanied by other congenital anomalies. Previous exome sequencing studies for CDH have supported a role of de novo damaging variants but did not identify any recurrently mutated genes. To investigate further the genetics of CDH, we analyzed de novo coding variants in 362 proband-parent trios including 271 new trios reported in this study. We identified four unrelated individuals with damaging de novo variants in MYRF (P = 5.3x10(-8)), including one likely gene-disrupting (LGD) and three deleterious missense (D-mis) variants. Eight additional individuals with de novo LGD or missense variants were identified from our other genetic studies or from the literature. Common phenotypes of MYRF de novo variant carriers include CDH, congenital heart disease and genitourinary abnormalities, suggesting that it represents a novel syndrome. MYRF is a membrane associated transcriptional factor highly expressed in developing diaphragm and is depleted of LGD variants in the general population. All de novo missense variants aggregated in two functional protein domains. Analyzing the transcriptome of patient-derived diaphragm fibroblast cells suggest that disease associated variants abolish the transcription factor activity. Furthermore, we showed that the remaining genes with damaging variants in CDH significantly overlap with genes implicated in other developmental disorders. Gene expression patterns and patient phenotypes support pleiotropic effects of damaging variants in these genes on CDH and other developmental disorders. Finally, functional enrichment analysis implicates the disruption of regulation of gene expression, kinase activities, intra-cellular signaling, and cytoskeleton organization as pathogenic mechanisms in CDH.

Materials Engineering of Violin Soundboards by Stradivari and Guarneri.

We investigated the material properties of Cremonese soundboards using a wide range of spectroscopic, microscopic, and chemical techniques. We found similar types of spruce in Cremonese soundboards as in modern instruments, but Cremonese spruces exhibit unnatural elemental compositions and oxidation patterns that suggest artificial manipulation. Combining analytical data and historical information, we may deduce the minerals being added and their potential functions-borax and metal sulfates for fungal suppression, table salt for moisture control, alum for molecular crosslinking, and potash or quicklime for alkaline treatment. The overall purpose may have been wood preservation or acoustic tuning. Hemicellulose fragmentation and altered cellulose nanostructures are observed in heavily treated Stradivari specimens, which show diminished second-harmonic generation signals. Guarneri's practice of crosslinking wood fibers via aluminum coordination may also affect mechanical and acoustic properties. Our data suggest that old masters undertook materials engineering experiments to produce soundboards with unique properties.

Likely damaging de novo variants in congenital diaphragmatic hernia patients are associated with worse clinical outcomes.

PURPOSE: Congenital diaphragmatic hernia (CDH) is associated with significant mortality and long-term morbidity in some but not all individuals. We hypothesize monogenic factors that cause CDH are likely to have pleiotropic effects and be associated with worse clinical outcomes. METHODS: We enrolled and prospectively followed 647 newborns with CDH and performed genomic sequencing on 462 trios to identify de novo variants. We grouped cases into those with and without likely damaging (LD) variants and systematically assessed CDH clinical outcomes between the genetic groups. RESULTS: Complex cases with additional congenital anomalies had higher mortality than isolated cases (P = 8 × 10-6). Isolated cases with LD variants had similar mortality to complex cases and much higher mortality than isolated cases without LD (P = 3 × 10-3). The trend was similar with pulmonary hypertension at 1 month. Cases with LD variants had an estimated 12-17 points lower scores on neurodevelopmental assessments at 2 years compared with cases without LD variants, and this difference is similar in isolated and complex cases. CONCLUSION: We found that the LD genetic variants are associated with higher mortality, worse pulmonary hypertension, and worse neurodevelopment outcomes compared with non-LD variants. Our results have important implications for prognosis, potential intervention and long-term follow up for children with CDH.

Chemical distinctions between Stradivari's maple and modern tonewood.

Violins made by Antonio Stradivari are renowned for having been the preferred instruments of many leading violinists for over two centuries. There have been long-standing questions about whether wood used by Stradivari possessed unique properties compared with modern tonewood for violin making. Analyses of maple samples removed from four Stradivari and a Guarneri instrument revealed highly distinct organic and inorganic compositions compared with modern maples. By solid-state 13C NMR spectroscopy, we observed that about one-third of hemicellulose had decomposed after three centuries, accompanied by signs of lignin oxidation. No apparent changes in cellulose were detected by NMR and synchrotron X-ray diffraction. By thermogravimetric analysis, historical maples exhibited reduced equilibrium moisture content. In differential scanning calorimetry measurements, only maples from Stradivari violins, but not his cellos, exhibited unusual thermooxidation patterns distinct from natural wood. Elemental analyses by inductively coupled plasma mass spectrometry suggested that Stradivari's maples were treated with complex mineral preservatives containing Al, Ca, Cu, Na, K, and Zn. This type of chemical seasoning was an unusual practice, unknown to later generations of violin makers. In their current state, maples in Stradivari violins have very different chemical properties compared with their modern counterparts, likely due to the combined effects of aging, chemical treatments, and vibrations. These findings may inspire further chemical experimentation with tonewood processing for instrument making in the 21st century.

Characterization of pediatric golf cart injuries to guide injury prevention efforts.

BACKGROUND: Golf cart injuries represent an increasing source of morbidity and mortality in the United States. Characterization of the circumstances of these injuries can inform injury prevention efforts. METHODS: This study retrospectively reviews a prospective trauma registry at a level-one pediatric trauma center for golf cart-related injuries in patients under 18years of age admitted to the hospital between 2008 and 2016. RESULTS: The 40 identified crashes were associated with 82 hospital days, 17 ICU days, and more than $1 million in hospital charges over the study period. The median hospital stay was 1.5days, and the median hospital charge was $20,489. Severe injuries with an Injury Severity Score of >15 were identified in 25% of patients, and moderate injuries with scores between nine and 15 were identified in an additional 30%. The most common injures were head and neck (60%) and external injuries to the body surface (52.5%). Only a single child was wearing a seatbelt, and the vast majority was not using any safety equipment. Children as young as nine years old were driving golf carts, and child drivers were associated with the cart overturning (p=0.007). CONCLUSIONS: Golf cart crashes were a source of substantial morbidity at a level-one trauma center. Increased safety measures, such as higher hip restraints, seatbelts, and front-wheel breaks could substantially increase the safety of golf carts. Increased regulation of driving age as well as driver education may also reduce these injuries.

ML327 induces apoptosis and sensitizes Ewing sarcoma cells to TNF-related apoptosis-inducing ligand.

Ewing sarcomas are rare mesenchymal-derived bone and soft tissue tumors in children. Afflicted children with distant metastases have poor survival despite aggressive therapeutics. Epithelial-to-mesenchymal transition in epithelial carcinomas is associated with loss of E-cadherin and resistance to apoptosis. ML327 is a novel small molecule that we have previously shown to reverse epithelial-to-mesenchymal transition features in both epithelial and neural crest-derived cancers. Herein, we sought to evaluate the effects of ML327 on mesenchymal-derived Ewing sarcoma cells, hypothesizing that ML327 initiates growth arrest and sensitizes to TNF-related apoptosis-inducing ligand. ML327 induced protein expression changes, increased E-cadherin and decreased vimentin, consistent with partial induction of mesenchymal-to-epithelial transition in multiple Ewing Sarcoma cell lines (SK-N-MC, TC71, and ES-5838). Induction of epithelial features was associated with apoptosis, as demonstrated by PARP and Caspase 3 cleavage by immunoblotting. Cell cycle analysis validated these findings by marked induction of the subG0 cell population. In vitro combination treatment with TRAIL demonstrated additive induction of apoptotic markers. Taken together, these findings establish a rationale for further in vivo trials of ML327 in cells of mesenchymal origin both alone and in combination with TRAIL.

Induced differentiation inhibits sphere formation in neuroblastoma.

Neuroblastoma arises from the neural crest, the precursor cells of the sympathoadrenal axis, and differentiation status is a key prognostic factor used for clinical risk group stratification and treatment strategies. Neuroblastoma tumor-initiating cells have been successfully isolated from patient tumor samples and bone marrow using sphere culture, which is well established to promote growth of neural crest stem cells. However, accurate quantification of sphere-forming frequency of commonly used neuroblastoma cell lines has not been reported. Here, we show that MYCN-amplified neuroblastoma cell lines form spheres more frequently than non-MYCN-amplified cell lines. We also show that sphere formation is directly sensitive to cellular differentiation status. 13-cis-retinoic acid is a clinically used differentiating agent that induces a neuronal phenotype in neuroblastoma cells. Induced differentiation nearly completely blocked sphere formation. Furthermore, sphere formation was specifically FGF-responsive and did not respond to increasing doses of EGF. Taken together, these data suggest that sphere formation is an accurate method of quantifying the stemness phenotype in neuroblastoma.

Silencing gastrin-releasing peptide receptor suppresses key regulators of aerobic glycolysis in neuroblastoma cells.

BACKGROUND: Under normoxic conditions, cancer cells use aerobic glycolysis as opposed to glucose oxidation for energy production; this altered metabolism correlates with poor outcomes in neuroblastoma. Hypoxia-inducible factor-1 alpha (HIF-1α) and pyruvate dehydrogenase kinase 4 (PDK4) regulate aerobic glycolysis, while pyruvate dehydrogenase phosphatase 2 (PDP2) promotes glucose oxidation. Here, we sought to determine whether gastrin-releasing peptide receptor (GRP-R) signaling regulates glucose metabolism. PROCEDURE: Neuroblastoma cell lines, BE(2)-C and SK-N-AS, were used. PCR microararay for glucose metabolism was performed on GRP-R silenced cells. Target protein expression was validated using Western blotting and VEGF ELISA. Cobalt chloride (CoCl2 ) was used to induce chemical hypoxia. Efficacy of targeting PDK regulation in neuroblastoma was assessed using dichloroacetate (DCA) by conducting cell viability assays and Western blotting for apoptotic markers. RESULTS: Silencing GRP-R decreased HIF-1α expression and blocked VEGF expression and secretion in both normoxic and CoCl2 induced hypoxia. PCR array analysis identified that GRP-R silencing reduced PDK4 and increased PDP2 mRNA expression. These findings were validated by Western blotting. CoCl2 induced hypoxia increased VEGF secretion, HIF-1α, and PDK4 expression. PDK4 silencing decreased HIF-1α expression and VEGF expression and secretion. DCA treatment decreased BE(2)-C and SK-N-AS proliferation while promoting cell death. GRP-R silencing and DCA treatment synergistically halted BE(2)-C proliferation. CONCLUSIONS: We report that GRP-R regulates glucose metabolism in neuroblastoma by modulating HIF-1α, PDK4 and PDP2. PDK4 regulates glucose metabolism, in part, via regulation of HIF-1α. Synergistic consequences of GRP-R inhibition and DCA treatment may suggest a novel therapeutic strategy for the treatment of aggressive neuroblastoma.

Whole exome sequencing identifies de novo mutations in GATA6 associated with congenital diaphragmatic hernia.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is a common birth defect affecting 1 in 3000 births. It is characterised by herniation of abdominal viscera through an incompletely formed diaphragm. Although chromosomal anomalies and mutations in several genes have been implicated, the cause for most patients is unknown. METHODS: We used whole exome sequencing in two families with CDH and congenital heart disease, and identified mutations in GATA6 in both. RESULTS: In the first family, we identified a de novo missense mutation (c.1366C>T, p.R456C) in a sporadic CDH patient with tetralogy of Fallot. In the second, a nonsense mutation (c.712G>T, p.G238*) was identified in two siblings with CDH and a large ventricular septal defect. The G238* mutation was inherited from their mother, who was clinically affected with congenital absence of the pericardium, patent ductus arteriosus and intestinal malrotation. Deep sequencing of blood and saliva-derived DNA from the mother suggested somatic mosaicism as an explanation for her milder phenotype, with only approximately 15% mutant alleles. To determine the frequency of GATA6 mutations in CDH, we sequenced the gene in 378 patients with CDH. We identified one additional de novo mutation (c.1071delG, p.V358Cfs34*). CONCLUSIONS: Mutations in GATA6 have been previously associated with pancreatic agenesis and congenital heart disease. We conclude that, in addition to the heart and the pancreas, GATA6 is involved in development of two additional organs, the diaphragm and the pericardium. In addition, we have shown that de novo mutations can contribute to the development of CDH, a common birth defect.

Non-fecalith-induced appendicitis: etiology, imaging, and pathology.

This study aims to document the imaging and pathology findings in non-fecalith-induced appendicitis. We reviewed the imaging and pathologic findings in 40 patients with histologically proven purulent appendicitis seen over a 2-year period. Findings documented were (1) total appendiceal involvement, (2) predominant appendiceal tip involvement, (3) presence of a fecalith, and (4) presence of lymphoid hyperplasia. There were a total of 40 patients, 28 males and 12 females. The age range was 2-18 years with a mean of 11.5 years. Twenty-two (55 %) patients demonstrated classic purulent appendicitis of the whole appendix, 20 (91 %) of these appendices had a fecalith. Eighteen (45 %) patients demonstrated purulent appendicitis confined to or predominately involving the tip of the appendix, and all 18 (100 %) patients demonstrated marked lymphoid hyperplasia. Only two (11 %) of these appendices had a fecalith. Overall, a fecalith was found in only 55 % of our cases, while 45 % demonstrated no fecalith, but rather marked lymphoid hyperplasia. Lymphoid hyperplasia appeared to be the underlying predisposing cause of purulent appendicitis in these cases.

Abnormal changes in both mandibular salivary glands in a dog: Non-mineral radiopaque sialoliths.

A 10-year-old Maltese dog was presented with a firm mass on the left side of his neck. Physical examination confirmed a firm mass in the left and a submandibular swelling in the right cervical region. Sialolithiasis and associated sialocele in both mandibular salivary glands were suspected and bilateral sialoadenectomy was performed. The stones were identified as non-mineral sialoliths.

Changements anormaux dans les deux glandes salivaires mandibulaires chez un chien : sialolithes radiopaques non minéraux. Un chien Maltais âgé de 10 ans a été présenté avec une masse ferme du côté droit du cou. L'examen physique a confirmé une masse ferme dans l'enflure gauche et sous-mandibulaire dans la région cervicale droite. La sialolithiase et une sialocèle connexe dans les deux glandes salivaires mandibulaires ont été suspectées et une sialoadénectomie bilatérale a été réalisée. Les pierres ont été identifiées comme des sialolithes non minéraux.(Traduit par Isabelle Vallières).

Migration, invasion, and metastasis are mediated by P-Rex1 in neuroblastoma.

Neuroblastoma accounts for approximately 15% of pediatric cancer-related deaths despite intensive multimodal therapy. This is due, in part, to high rates of metastatic disease at diagnosis and disease relapse. A better understanding of tumor biology of aggressive, pro-metastatic phenotypes is necessary to develop novel, more effective therapeutics against neuroblastoma. Phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 (P-Rex1) has been found to stimulate migration, invasion, and metastasis in several adult malignancies. However, its role in neuroblastoma is currently unknown. In the present study, we found that P-Rex1 is upregulated in pro-metastatic murine models of neuroblastoma, as well as human neuroblastoma metastases. Correspondingly, silencing of P-Rex1 was associated with decreased migration and invasion in vitro. This was associated with decreased AKT-mTOR and ERK2 activity, dysregulation of Rac, and diminished secretion of matrix metalloproteinases. Furthermore, increased P-Rex1 expression was associated with inferior relapse-free and overall survival via tissue microarray and Kaplan-Meier survival analysis of a publicly available clinical database. Together, these findings suggest that P-Rex1 may be a novel therapeutic target and potential prognostic factor in neuroblastoma.

Social Determinants of Outcomes Disparity among Pediatric Patients with Solid Tumor.

BACKGROUND: Socioeconomic factors have a significant impact on healthcare outcomes. Metrics such as area deprivation index (ADI) are used to quantify the anticipated influence of these factors. Here, we sought to assess the impact of socioeconomic factors on clinical outcomes among pediatric patients with solid tumor in our region. STUDY DESIGN: We identified 3,863 pediatric patients who were diagnosed with a malignant solid tumor in the Texas Cancer Registry between 1995 and 2019. ADI was used to quantify socioeconomic determinants of health. These outcome variables were determined: stage of disease at diagnosis, time between diagnosis and treatment initiation, and overall mortality. Statistical analysis was performed using logistic regression, linear regression, Cox proportional hazards regression, and Kaplan-Meier survival curves. RESULTS: A total of 53.5% of patients were male and the average age at diagnosis was 4.5 years. Forty-seven percent of patients were White, 13.3% were Black, 36.2% were Hispanic, 1.7% were Asian, and other rare minority groups made up 1.8%. On multivariable analysis, increased risk of death was associated with Black race, rare minority race, residence in a border county, and increasing ADI score, with the risk of death at 5 years rising 4% with each increasing ADI point. CONCLUSIONS: Social determinants of health are associated with disparate outcomes among pediatric patients with solid tumor. Our results suggest that patients who are part of racial minority groups and those who reside in socioeconomically disadvantaged neighborhoods or regions near the Texas-Mexico border are at an increased risk of death. This information may be useful in strategizing outreach and expanding resources to improve outcomes in at-risk communities.