Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Background: Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. // Methods: We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung's disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. // Findings: We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung's disease) from 264 hospitals (89 in high-income countries, 166 in middle-income countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in low-income countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. // Interpretation: Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between low-income, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

A positive feedback loop links opposing functions of P-TEFb/Cdk9 and histone H2B ubiquitylation to regulate transcript elongation in fission yeast.

Transcript elongation by RNA polymerase II (RNAPII) is accompanied by conserved patterns of histone modification. Whereas histone modifications have established roles in transcription initiation, their functions during elongation are not understood. Mono-ubiquitylation of histone H2B (H2Bub1) plays a key role in coordinating co-transcriptional histone modification by promoting site-specific methylation of histone H3. H2Bub1 also regulates gene expression through an unidentified, methylation-independent mechanism. Here we reveal bidirectional communication between H2Bub1 and Cdk9, the ortholog of metazoan positive transcription elongation factor b (P-TEFb), in the fission yeast Schizosaccharomyces pombe. Chemical and classical genetic analyses indicate that lowering Cdk9 activity or preventing phosphorylation of its substrate, the transcription processivity factor Spt5, reduces H2Bub1 in vivo. Conversely, mutations in the H2Bub1 pathway impair Cdk9 recruitment to chromatin and decrease Spt5 phosphorylation. Moreover, an Spt5 phosphorylation-site mutation, combined with deletion of the histone H3 Lys4 methyltransferase Set1, phenocopies morphologic and growth defects due to H2Bub1 loss, suggesting independent, partially redundant roles for Cdk9 and Set1 downstream of H2Bub1. Surprisingly, mutation of the histone H2B ubiquitin-acceptor residue relaxes the Cdk9 activity requirement in vivo, and cdk9 mutations suppress cell-morphology defects in H2Bub1-deficient strains. Genome-wide analyses by chromatin immunoprecipitation also demonstrate opposing effects of Cdk9 and H2Bub1 on distribution of transcribing RNAPII. Therefore, whereas mutual dependence of H2Bub1 and Spt5 phosphorylation indicates positive feedback, mutual suppression by cdk9 and H2Bub1-pathway mutations suggests antagonistic functions that must be kept in balance to regulate elongation. Loss of H2Bub1 disrupts that balance and leads to deranged gene expression and aberrant cell morphologies, revealing a novel function of a conserved, co-transcriptional histone modification

Cdk9 activity towards multiple substrates is required for abnormal morphologies of H2Bub1-deficient cells.

<p>(A) Quantification of abnormal septation patterns in the indicated <i>htb1-FLAG</i> strains (JTB378, JTB351, JTB379, JTB353, JTB380, JTB355). Error bars represent standard deviations from 2 independent experiments; at least 200 cells were counted in each. (B) As in (A) for the indicated <i>cdk9^as</i> strains (KL289, KL291, KL293). Cells were grown in the presence of either DMSO or 3-MB-PP1 as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002822#pgen-1002822-g005" target="_blank">Figure 5B</a>. (C) As in (A) for the indicated <i>htb1-FLAG</i> strains (MS260, MS256, MS261, MS257, MS272, MS259). (D) Flocculation of indicated strains (JS78, JTB67-1, JTB351, JTB353, KL291, KL293, MS259) was quantified as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002822#s4" target="_blank">Materials and Methods</a>. (E) Growth rates in liquid rich medium (YES) were measured for strains analyzed in (D).</p

Opposing effects of H2Bub1 and Cdk9 activity on RNAPII distribution revealed by ChIP–chip.

<p>(A) Average distribution of RNAPII at 540 <i>S. pombe</i> genes, as determined by ChIP-chip in a <i>cdk9-T212A</i> strain (JTB325). Genes were grouped according to total levels of RNAPII enrichment. (B) As in (A) for <i>cdk9-T212A htb1-K119R</i> (JTB326). (C) Average distributions of differences between <i>cdk9-T212A</i> (JTB325) and wild-type (JTB62-1) RNAPII enrichment grouped according to RNAPII enrichment in wild-type cells. (D) As in (C) for differences between <i>cdk9-T212A htb1-K119R</i> and wild-type RNAPII enrichment. (E) As in (C) for differences between <i>cdk9-T212A htb1-K119R</i> and <i>cdk9-T212A</i> RNAPII enrichment. (F) As in (C) for differences between <i>cdk9-T212A htb1-K119R</i> and <i>htb1-K119R</i> RNAPII enrichment. The keys below C-F illustrate the statistical significance of the differences for each group at 50 positions along the average gene. The rows of the key are color-coded according to the graph. Open squares denote p>0.01; light shading denotes 0.01>p>10exp-5; dark shading denotes p<10exp-5 (one-sample t-tests; μ₀ = 0). Note that there is only light shading for the last row (corresponding to the blue curve).</p

Mutual phenotypic suppression in <i>cdk9 htb1-K119R</i> double mutants.

<p>(A) For indicated strains (JS78, LV7, JTB62-1, JTB67-1, MS249, LV193), growth in increasing [3-MB-PP1] is plotted as a percentage of growth in the absence of 3-MB-PP1. Error bars denote standard deviations from 3 independent experiments. (B) Images of DAPI- and calcofluor-stained <i>htb1-K119R</i> (JTB67-1) and <i>cdk9^as htb1-K119R</i> (LV193) cells grown in the absence (top) or presence (middle) of 10 µM 3-MB-PP1 for 7 hr, or after inhibitor washout and return to growth (bottom). (C) Fluorescent images of DAPI/calcofluor-stained wild-type (JS78), <i>cdk9-T212A</i> (HD7-24), <i>brl2Δ</i> (JTB331), <i>brl2Δ cdk9-T212A</i> (JTB335), <i>ubp8Δ</i> (JTB297), <i>ubp8Δ cdk9-T212A</i> (JTB336), <i>htb1-K119R</i> (JTB67-1), <i>cdk9-T212A htb1-K119R</i> (LV252) <i>cdk9-T212E htb1-K119R</i> (LV256), and <i>mcs6-S165A htb1-K119R</i> (LV254) cells. (D) Quantification of abnormal septation patterns in strains of indicated genotypes (JTB62-1, JTB67-1, JTB325, JTB326, JTB331, JTB335, JTB377, JTB333 respectively). Error bars represent standard deviations from 2 independent experiments; at least 200 cells were counted in each. (E) Flocculation of indicated <i>htb1-FLAG</i> strains (JTB62-1, JTB67-1, JTB325, JTB326) was quantified as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002822#s4" target="_blank">Materials and Methods</a>. Error bars denote standard deviations from 2 independent experiments.</p

H2Bub1 enhances Cdk9 recruitment and Spt5 phosphorylation at transcribed genes.

<p>(A,B) Spt5 phosphorylation was measured by ChIP using anti-Spt5-P in <i>spt5-myc</i> (MS265; black bars) and <i>spt5-myc htb1-K119R</i> (LV239; gray bars) strains and quantified at the indicated genes by qPCR. Enrichment is plotted as a percentage of input signal for each primer pair. Positions of PCR primer pairs within coding regions are indicated schematically at top. (C,D) Spt5-myc occupancy was measured by ChIP as in A and B. (E,F) Spt5-P enrichment normalized to total Spt5-myc occupancy. (G,H) Cdk9-myc occupancy was measured by ChIP in <i>cdk9-myc</i> (MS264; black bars) and <i>cdk9-myc htb1-K119R</i> (KL259; gray bars) strains. (I,J) H2Bub1 enrichment was measured by ChIP and normalized to H2B-FLAG occupancy. Error bars denote standard deviations from 3 independent experiments. Asterisks denote a significant difference between wild-type and mutant (“*” p<0.04, “**” p<0.02; unpaired t-test).</p

Loss of H2Bub1 globally alters distribution of RNAPII in gene coding regions.

<p>(A) Average distribution of H2Bub1 at 540 <i>S. pombe</i> genes, as determined by ChIP-chip. Genes were grouped according to total levels of RNAPII enrichment (see key at top). The grey box in the “average gene” representation at bottom denotes the gene coding region; 5′ and 3′ untranslated regions are denoted by thin black lines. The arrow denotes the transcription start site. (B) Average distribution of RNAPII at 540 <i>S. pombe</i> genes, as determined by ChIP-chip in a wild-type strain (JTB62-1). Genes were grouped according to total levels of RNAPII enrichment. (C) As in (B), determined in an <i>htb1-K119R</i> mutant strain (JTB67-1). Gene groupings were created using wild-type RNAPII enrichment values. (D) Average distributions of differences between mutant and wild-type RNAPII enrichment grouped according to RNAPII enrichment in wild-type cells. The key below the graph illustrates the statistical significance of the differences for each group at 50 positions along the average gene. The rows of the key are color-coded according to the graph. Open squares denote p>0.01; light shading denotes 0.01>p>10exp-5; dark shading denotes p<10exp-5 (one-sample t-tests; μ₀ = 0). Note that there is only light shading for the last row (corresponding to the blue curve).</p

Model depicting positive and negative interactions between P-TEFb and H2Bub1 during transcript elongation (see text for details).

<p>Model depicting positive and negative interactions between P-TEFb and H2Bub1 during transcript elongation (see text for details).</p