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

Adenoviral-mediated placental gene transfer of IGF-1 corrects placental insufficiency via enhanced placental glucose transport mechanisms.

Previous work in our laboratory demonstrated that over-expression of human insulin-like growth factor -1 (hIGF-1) in the placenta corrects fetal weight deficits in mouse, rat, and rabbit models of intrauterine growth restriction without changes in placental weight. The underlying mechanisms of this effect have not been elucidated. To investigate the effect of intra-placental IGF-1 over-expression on placental function we examined glucose transporter expression and localization in both a mouse model of IUGR and a model of human trophoblast, the BeWo Choriocarcinoma cell line.At gestational day 18, animals were divided into four groups; sham-operated controls, uterine artery branch ligation (UABL), UABL+Ad-hIGF-1 (10(8) PFU), UABL+Ad-LacZ (10(8) PFU). At gestational day 20, pups and placentas were harvested by C-section. For human studies, BeWo choriocarcinoma cells were grown in F12 complete medium +10%FBS. Cells were incubated in serum-free control media ± Ad-IGF-1 or Ad-LacZ for 48 hours. MOIs of 10∶1 and 100∶1 were utilized. The RNA, protein expression and localization of glucose transporters GLUT1, 3, 8, and 9 were analyzed by RT-PCR, Western blot and immunohistochemistry.In both the mouse placenta and BeWo, GLUT1 regulation was linked to altered protein localization. GLUT3, localized to the mouse fetal endothelial cells, was reduced in placental insufficiency but maintained with Ad-I GF-1 treatment. Interestingly, GLUT8 expression was reduced in the UABL placenta but up-regulated following Ad-IGF-1 in both mouse and human systems. GLUT9 expression in the mouse was increased by Ad-IGF-1 but this was not reflected in the BeWo, where Ad-IGF-1 caused moderate membrane relocalization.Enhanced GLUT isoform transporter expression and relocalization to the membrane may be an important mechanism in Ad-hIGF-1mediated correction of placental insufficiency

Resection of bilateral bronchopulmonary sequestrations via a single left-sided muscle-sparing thoracotomy

At 3 months of age asymptomatic bilateral bronchopulmonary sequestrations were both resected through a left muscle-sparing thoracotomy utilizing traction on the systemic arteries supplying the contralateral bronchopulmonary sequestration facilitating resection through the left chest

Localization of GLUT8 in human placenta.

<p>A) Rabbit IgG control demonstrating no staining in human placenta. B) GLUT8 is expressed in the syncytiotrophoblast (closed arrows) and the villous vessel endothelium (black arrowheads) in normal, term, human placenta.</p

Summary of GLUT1, 3, 8 and 9 mRNA expression in control, UABL, Ad-hIGF-1 and Ad-LacZ treated mouse placentas.

<p>ANOVA, Post hoc Tukeys test * p<0.05, n>3 for each treatment</p

Representative micrographs of GLUT1 (A,B,C), GLUT3 (D,E,F), GLUT8 (G,H,I), GLUT9a (J,K,L) and GLUT9b (M,N,O) in mouse placental labyrinth in the 3 treatment groups, Sham, UABL and Ad-hIGF-1 respectively.

<p>100× magnification. ST identifies syncytiotrophoblast, * identifies a fetal villous vessel, # identifies a maternal blood sinus. Micrographs are representative of 3 biological replicates for each treatment group.</p

GLUT expression levels in BeWo cells.

<p>A:Representative Western blots of GLUT1, 3, and 8 in BeWo cells treated with control media (C), Ad-hIGF-1 MOI 10∶1 (10), Ad-hIGF-1 MOI 100∶1 (100) for 48 hours. B: Summary of GLUT1, 3, 8 protein expression. ANOVA, Tukeys posthoc test, *p<0.05, ** p<0.01, n>4 replicates for each treatment.</p

GLUT8 mRNA expression is significantly increased following exposure of BeWo cells to Ad-IGF-1 at an MOI of 100∶1 for 48 hours.

<p>ANOVA, Tukeys posthoc test ***p<0.005, n = 6 replicates</p

Incidence of Complications in Twin-twin Transfusion Syndrome after Selective Fetoscopic Laser Photocoagulation: A Single-center Experience

Objective The purpose of this study was to evaluate the incidence of complications after selective fetoscopic laser photocoagulation for twin-twin transfusion syndrome (TTTS). Study Design One hundred fifty-two cases of TTTS were treated with selective fetoscopic laser photocoagulation from 2005-2008. Complications were TTTS recurrence, amniotic band syndrome, iatrogenic monoamnionicity, and twin anemia-polycythemia sequence. Data were placed in the following categories: no complications; early complications ≤7 days; late complications \u3e7 days; both early and late complications. Results The incidence of early, late, and both early and late complications was 31%, 39%, and 10%. Complications included 2 cases (1.3%) of monoamnionicity, 3 cases (2.0%) of recurrent TTTS, 3 cases (2.0%) of twin anemia-polycythemia sequence, and 5 cases (3.3%) of amniotic band syndrome. Cases with TTTS with early complications had a lower number of superficial arteriovenous vascular anastomoses and 1 or both fetus survival (70.2% vs 96.7%; P \u3c .001), compared with no complications. Fetal survival was 238 of 307 cases (77.5%), with 1 or both twins surviving in 134 of 152 (88%) of pregnancies. Conclusion The incidence of early, late, and both early and late complications was 31%, 39%, and 10%, respectively. Close postoperative surveillance is important