Genome-wide identification of disease-causing copy number variations in 450 individuals with anorectal malformations.

Anorectal malformations (ARM) represent a spectrum of rare malformations originating from a perturbated development of the embryonic hindgut. Approximately 60% occur as a part of a defined genetic syndrome or within the spectrum of additional congenital anomalies. Rare copy number variations (CNVs) have been associated with both syndromic and non-syndromic forms. The present study represents the largest study to date to explore the contribution of CNVs to the expression of ARMs. SNP-array-based molecular karyotyping was applied in 450 individuals with ARM and 4392 healthy controls. CNVs were identified from raw intensity data using PennCNV. Overlapping CNVs between cases and controls were discarded. Remaining CNVs were filtered using a stringent filter algorithm of nine filter steps. Prioritized CNVs were confirmed using qPCR. Filtering prioritized and qPCR confirmed four microscopic chromosomal anomalies and nine submicroscopic CNVs comprising seven microdeletions (del2p13.2, del4p16.2, del7q31.33, del9p24.1, del16q12.1, del18q32, del22q11.21) and two microduplications (dup2p13.2, dup17q12) in 14 individuals (12 singletons and one affected sib-pair). Within these CNVs, based on their embryonic expression data and function, we suggest FOXK2, LPP, and SALL3 as putative candidate genes. Overall, our CNV analysis identified putative microscopic and submicroscopic chromosomal rearrangements in 3% of cases. Functional characterization and re-sequencing of suggested candidate genes is warranted.

Significance of the Extent of Intestinal Resection on the Outcome of a Short-bowel Syndrome in a Porcine Model.

AIM OF THE STUDY: Insufficient data are available to determine the most suitable extent of intestinal resection required to induce short-bowel syndrome (SBS) in pigs. This study aimed to compare the three main SBS-models published. METHODS: A 75%, 90%, or 100% mid-intestinal resection was performed in groups of n = 5 pigs each. Clinical (body weight, stool consistency) and biochemical (serum eletrolytes, citrulline, albumin, prealbumin, and transferrin) parameters were determined daily, functional (D-xylose resorption) and histological (intestinal villus length) parameters were determined after 2 weeks. A t-test and ANOVA were used for statistical analysis. RESULTS: Only in the 100% group, we observed a persistent weight loss (13.6 ± 3.8%) and diarrhea, as well as a decrease in prealbumin-levels (41%) and transferrin levels (33%). Serum electrolytes remained stable in all groups during the observation period. Citrulline stabilized at different levels (100% group 13.9 ± 1.0 µmol/L; 90% group 18.8 ± 1.0 µmol/L; 75% group 26.3 ± 1.4 µmol/L; all p < .05). D-xylose resorption was lowest in the 100%, followed by 90% and 75% group (100% group 32.8 ± 4.9 mg/L; 90% group 50.0 ± 19.6 mg/L; 75% group 57.8 ± 8.8 mg/L; p = .393). Intestinal villus length decreased in all groups (100% group 11.0%; 90% group 14.0%; 75% group 19.1%). CONCLUSIONS: 75% intestinal resection is less suitable as an SBS model, as animals tend to recover remarkably. The 90% model is suitable for longer-term studies, as animals might survive longer due to partial compensation. Due to severe nutritional, biochemical, and physiological derangements, the 100% model can only be used for acute experiments and those immediately followed by small bowel transplantation.

De novo 13q deletions in two patients with mild anorectal malformations as part of VATER/VACTERL and VATER/VACTERL-like association and analysis of EFNB2 in patients with anorectal malformations.

Anorectal malformations (ARMs) comprise a broad spectrum of conditions ranging from mild anal anomalies to complex cloacal malformations. In 40-50% of cases, ARM occurs within the context of defined genetic syndromes or complex multiple congenital anomalies, such as VATER/VACTERL (vertebral defects [V], ARMs [A], cardiac defects [C], tracheoesophageal fistula with or without esophageal atresia [TE], renal malformations [R], and limb defects [L]) association. Here, we report the identification of deletions at chromosome 13q using single nucleotide polymorphism-based array analysis in two patients with mild ARM as part of VATER/VACTERL and VATER/VACTERL-like associations. Both deletions overlap the previously defined critical region for ARM. Heterozygous Efnb2 murine knockout models presenting with mild ARM suggest EFNB2 as an excellent candidate gene in this region. Our patients showed a mild ARM phenotype, closely resembling that of the mouse. We performed a comprehensive mutation analysis of the EFNB2 gene in 331 patients with isolated ARM, or ARM as part of VATER/VACTERL or VATER/VACTERL-like associations. However, we did not identify any disease-causing mutations. Given the convincing argument for EFNB2 as a candidate gene for ARM, analyses of larger samples and screening of functionally relevant non-coding regions of EFNB2 are warranted. In conclusion, our report underlines the association of chromosome 13q deletions with ARM, suggesting that routine molecular diagnostic workup should include the search for these deletions. Despite the negative results of our mutation screening, we still consider EFNB2 an excellent candidate gene for contributing to the development of ARM in humans.

Comparison of LILT and STEP procedures in children with short bowel syndrome -- a systematic review of the literature.

PURPOSE: To compare LILT and STEP, the two principal procedures to lengthen the native bowel in children with a short bowel syndrome (SBS), by discussing the indications and presenting the outcome from published data. METHODS: A review of literature was performed. N=39 publications were reviewed. RESULTS: For LILT and STEP, failure to achieve intestinal autonomy by conservative therapy represents the main indication, and end-stage liver disease the main contraindication. A sufficiently dilated intestinal segment is a common anatomical precondition for both procedures. STEP can be performed on shorter intestinal segments and on intricate segments such as the duodenum, which is technically not feasible for LILT. Both procedures have a similar extent of intestinal lengthening (approximately 70%) and result in improvement of enteral nutrition and reversal of complications of parenteral nutrition. STEP seems to have a lower mortality and overall progression to transplantation. CONCLUSIONS: STEP and LILT are both accepted procedures for non-transplant surgical management of SBS in children. The outcome after STEP seems to be more favourable, but larger series are needed to further assess accurate selection of eligible patients and to estimate effectiveness of procedures. A considerably higher number of cases for evaluation might be accomplished through the widespread use of a centralised registry.

Assisted reproductive techniques and the risk of anorectal malformations: a German case-control study.

BACKGROUND: The use of assisted reproductive techniques (ART) for treatment of infertility is increasing rapidly worldwide. However, various health effects have been reported including a higher risk of congenital malformations. Therefore, we assessed the risk of anorectal malformations (ARM) after in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). METHODS: Data of the German Network for Congenital Uro-REctal malformations (CURE-Net) were compared to nationwide data of the German IVF register and the Federal Statistical Office (DESTATIS). Odds ratios (95% confidence intervals) were determined to quantify associations using multivariable logistic regression accounting for potential confounding or interaction by plurality of births. RESULTS: In total, 295 ARM patients born between 1997 and 2011 in Germany, who were recruited through participating pediatric surgeries from all over Germany and the German self-help organisation SoMA, were included. Controls were all German live-births (n = 10,069,986) born between 1997 and 2010. Overall, 30 cases (10%) and 129,982 controls (1%) were born after IVF or ICSI, which translates to an odds ratio (95% confidence interval) of 8.7 (5.9-12.6) between ART and ARM in bivariate analyses. Separate analyses showed a significantly increased risk for ARM after IVF (OR, 10.9; 95% CI, 6.2-19.0; P < 0.0001) as well as after ICSI (OR, 7.5; 95% CI, 4.6-12.2; P < 0.0001). Furthermore, separate analyses of patients with isolated ARM, ARM with associated anomalies and those with a VATER/VACTERL association showed strong associations with ART (ORs 4.9, 11.9 and 7.9, respectively). After stratification for plurality of birth, the corresponding odds ratios (95% confidence intervals) were 7.7 (4.6-12.7) for singletons and 4.9 (2.4-10.1) for multiple births. CONCLUSIONS: There is a strongly increased risk for ARM among children born after ART. Elevations of risk were seen after both IVF and ICSI. Further, separate analyses of patients with isolated ARM, ARM with associated anomalies and those with a VATER/VACTERL association showed increased risks in each group. An increased risk of ARM was also seen among both singletons and multiple births.

Inheritance of the VATER/VACTERL association.

VATER/VACTERL association refers to the non-random co-occurrence of the following component features: vertebral defects, anal atresia, cardiac malformations, tracheoesophageal atresia, renal abnormalities, and limb defects. Recently, Solomon et al. (Hum Genet 127:731-733, 2010) observed an increased prevalence of component features among first-degree relatives of VATER/VACTERL patients suggesting that in some patients, the disorder may be inherited. To replicate these findings, we investigated 87 VATER/VACTERL patients with the presence of a minimum of three component features and their first-degree relatives (n = 271). No increase in the overall prevalence of component features was observed in first-degree relatives compared to the general population (χ² = 2.68, p = 0.10). Separate analysis for the prevalence of single component features showed a higher prevalence of tracheoesophageal fistula/atresia among first-degree relatives compared to the general population (OR 17.65, 95% CI 2.47-126.05). However, this was based on occurrence in one family only. Our findings suggest that although familial occurrence renders a genetic contribution likely, the overall risk of recurrence among the first-degree relatives of patients with VATER/VACTERL association is probably very low. Since the patients in the present study were young and no offspring could be studied, estimation of the role of de novo mutations in the development of VATER/VACTERL was not possible.

Review of various techniques of small bowel transplantation in pigs.

Because of anatomical and physiological similarities to humans, porcine small bowel transplantation (SBTx) can be used as an appropriate experimental model in the field of surgical research. Various approaches to SBTx have been described in literature. The aim of this work is to present a review of different surgical techniques of SBTx which have been developed using the porcine model. Our analysis of Medline-cited studies dealing with different techniques of SBTx in porcine models was particularly focused on surgical aspects. With regard to graft procurement and enterectomy, the reported techniques vary widely. Arterial reconstruction is mainly conducted by performing the anastomosis between the superior mesenteric artery (SMA) of the donor and SMA or infrarenal aorta of the recipient. Alternatively, an aortic segment of the donor can be anastomosed to the infrarenal aorta of the recipient. Venous anastomosis is frequently performed between the superior mesenteric vein (SMV) of the donor and SMV or the inferior vena cava (IVC) of the recipient. Some studies also report venous anastomosis between the portal vein of the donor and the recipient. Bowel continuity is then restored by end-to-end or end-to-side anastomosis. Remarkable results were generated thanks to improved techniques which include proximal side-to-side ileo-ileal anastomosis with double-barrel ileostomy, or so-called "Paul-Mikulicz-Ileostomy". Most frequently used were jejunostomy and the "Bishop-Koop-Ileostomy"--where the proximal part of the bowel is anastomosed end-to-side to the distal part, which is then exteriorized as an ostomy. Based on the techniques presented in this review, one must select the most suitable surgical technique of porcine SBTx among those various models.

Anastomotic sealing with a fibrin-coated collagen patch in small-diameter bowel.

PURPOSE: The aim of this study was to evaluate the complication rates and inflammatory response in TachoSil™-sealed small-diameter anastomoses with conventional and reduced suture number as a model for neonatal bowel surgery. METHODS: Ileo-ileal anastomoses were performed in 73 rats. In the control group, the anastomosis was accomplished with the conventional technique, using nine interrupted sutures. In the other groups with nine, six, and three interrupted sutures, the anastomotic line was additionally sealed with a fibrin-coated collagen patch (TachoSil™). The rats were sacrificed on days 0, 2, and 10. Clinical and functional parameters included the rates of ileus, insufficiency and death, operating time, adhesions, bursting pressure, and preanastomotic dilatation. The histological examination of the anastomoses concentrated on assessing the inflammatory cell infiltration of the TachoSil™ patch and the intestinal wall. RESULTS: Severe preanastomotic dilatation was observed in additionally sealed ileo-ileal anastomoses with conventional suture number and high complication rates (ileus, perforation, death) occurred in additionally sealed anastomoses with reduced suture number. We found a massive microabscess-forming inflammation in additionally sealed anastomoses. Inflammatory cell infiltration was highest in the collagen layer of the sealing patch (p < 0.05 vs. fibrin layer of the sealing patch and vs. intestinal wall). CONCLUSIONS: As a result of our findings, additional sealing of small-diameter intestinal anastomoses with TachoSil™ cannot be recommended.

Intestinal anastomoses in small children: is there a higher risk?

AIM: The aim of the retrospective study was to evaluate complications and outcome of children in whom an intestinal anastomosis had been performed at age younger than 1 year. PATIENTS AND METHODS: Data were collected both retrospectively and by interviewing the parents. For the retrospective analysis, the underlying disease, biometric data, technical information about the anastomoses, associated illnesses, and complications were evaluated. In the parent interviews, the present quality of life and the state of health of the children were evaluated. RESULTS: From 1997 to 2007, intestinal anatomoses had been performed in 131 children for various reasons. There were no complications in 81.7% of the patients; 17 children received reoperation. In only 5 cases were anastomotic complications the reasons for the reoperation. Only 7.3% of the 77 interviewed families believe that their child has severe or relevant restrictions in daily life. CONCLUSIONS: Among 131 children in whom an intestinal bowel anastomosis procedure was performed at age younger than 1 year, only 5 (3.2%) experienced complications associated with the anastomosis. The children are in good physical condition and have an excellent quality of life.

Immunotargeting of catalase to lung endothelium via anti-angiotensin-converting enzyme antibodies attenuates ischemia-reperfusion injury of the lung in vivo.

Limitation of reactive oxygen species-mediated ischemia-reperfusion (I/R) injury of the lung by vascular immunotargeting of antioxidative enzymes has the potential to become a promising modality for extension of the viability of banked transplantation tissue. The preferential expression of angiotensin-converting enzyme (ACE) in pulmonary capillaries makes it an ideal target for therapy directed toward the pulmonary endothelium. Conjugates of ACE monoclonal antibody (MAb) 9B9 with catalase (9B9-CAT) have been evaluated in vivo for limitation of lung I/R injury in rats. Ischemia of the right lung was induced for 60 min followed by 120 min of reperfusion. Sham-operated animals (sham, n = 6) were compared with ischemia-reperfused untreated animals (I/R, n = 6), I/R animals treated with biotinylated catalase (CAT, n = 6), and I/R rats treated with the conjugates (9B9-CAT, n = 6). The 9B9-CAT accumulation in the pulmonary endothelium of injured lungs was elucidated immunohistochemically. Arterial oxygenation during reperfusion was significantly higher in 9B9-CAT (221 +/- 36 mmHg) and sham (215 +/- 16 mmHg; P < 0.001 for both) compared with I/R (110 +/- 10 mmHg) and CAT (114 +/- 30 mmHg). Wet-dry weight ratio of I/R (6.78 +/- 0.94%) and CAT (6.54 +/- 0.87%) was significantly higher than of sham (4.85 +/- 0.29%; P < 0.05), which did not differ from 9B9-CAT (5.58 +/- 0.80%). The significantly lower degree of lung injury in 9B9-CAT-treated animals compared with I/R rats was also shown by decreased serum levels of endothelin-1 (sham, 18 +/- 9 fmol/mg; I/R, 42 +/- 12 fmol/mg; CAT, 36 +/- 11 fmol/mg; 9B9-CAT, 26 +/- 9 fmol/mg; P < 0.01) and mRNA for inducible nitric oxide synthase (iNOS) [iNOS-GAPDH ratio: sham, 0.15 +/- 0.06 arbitrary units (a.u.); I/R, 0.33 +/- 0.08 a.u.; CAT, 0.26 +/- 0.05 a.u.; 9B9-CAT, 0.14 +/- 0.04 a.u.; P < 0.001]. These results validate immunotargeting by anti-ACE conjugates as a prospective and specific strategy to augment antioxidative defenses of the pulmonary endothelium in vivo.