Clinical Evaluation Of Laporoscopic Surgery For Children With Persistent Cloaca And Whole-exome Sequencing Screen In Human Persistent Cloaca

Part 1:Clinical Evaluation of Laporoscopic Surgery for Children with Persistent CloacaBackgroundPersistent cloaca is the most severe form of anorectal malformation (ARM) encountered in girls. The incidence is approximately 1-2 in 50000 live births. The main goal of surgical reconstruction is achieving fecal and urinary continence and normal sexual function in adult life. The repair of persistent cloaca represents a serious technical challenge. Laparoscopy assisted anorectoplasty (LAARP) has been proven to be a feasible and safe approach for high type ARM, including rectovesical fistula, rectoprostate fistula. However, there were a few reports in the literature describing the laparoscopy approach for management of cloacal malformation.ObjectiveThe current study was done to describe the technique and evaluate outcomes of laparoscopic approach for children with persistent cloaca.MethodsTwenty and six patients with high type persistent cloaca treated in the Capital Pediatric Institution between November 2005 and February 2015 were reviewed. LAARP, vaginoplasty and urethroplasty were performed in 17 patients, Abdominoperineal pull through (APPT), vaginoplasty and urethroplasty were carried out in the other 9 patients. In LP group,12 patients underwent conventional LAARP and 5 patients underwent single-incision LAARP. The clinical data including sacral ratio (SR), the length of common channel, intraoperative blood loss, operation time, postoperative hospital stay, postoperative complications and bowel function were evaluated.ResultsLAARP, vaginoplasty and urethroplasty were successfully performed in all patients of the LAARP group without conversion. There was no significant difference in patients’ SR value, the length of common channel and age at operation between the LAARP group and the APPT group. The mean operative time of the LAARP group was significantly shorter than that of APPT group (124.1±4.9 min VS 131.8±3.2 min, P<0.01). The intraoperative blood loss was significantly less in the LAARP group (10.5±2.2 ml VS 16.3 ±2.7ml, P<0.01). The median follow-up period was 4.0±2.8 years of the LAARP group and 6.0±2.3 years of the APPT group. The wound infections (9.1% vs 0%) were more common in ARRT patients. The rates of voluntary bowel movement, soiling, constipation (grade 1,2& 3) were similar in both groups, however, the overall bowel function after LAARP is better than that of ARRT according to the Krickenbeck classification (10.7±0.2 VS 9.7±1.2, P=0.047).ConclusionsThe LAARP provides a viable option as a surgical management of persistent cloaca. Compared with APPT, this approach has some advantages including minimal surgical trauma,cosmetic appearance and excellent visualization of the rectal fistula and gynecologic anatomy.Part 2:Whole-exome Sequencing Screen in Human Persistent CloacaBackgroundPersistent cloaca is a severe birth defect encountered in pediatric surgical practice. The incidence is approximately 1-2 in 50000 live births.49.4 percent of PC patients have associated congenital anomalies affecting urogenital system, cardiovascular system, skeleton and the gastrointestinal tract. The etiology of PC is unknown. Genetic factors have been considered important contributing factors in the pathogenesis of PC. However, the evidence on human genetic studies and epidemiological researches is very limited. WES methodology enables the researchers to get the whole exome sequence from the total human genome, which give a new way for the advanced research or clinical application. Combined with a large number of the exome data provided by the public databases, WES is conducive to better explain the relationship between the genotype and phenotype, particularly suitable for genetic risk factors screening of the complex congenital diseases.ObjectiveThe aim of this study is to identify the genetic mutations associated with human persistent cloaca (PC) in Chinese population.MethodsFour trio families (PC01, PC02, PC03, PC04) and eight unrelated patients (PC05, PC06, PC07, PC08, PC09, PC 10, PC11, PC 12) were selected for whole exome sequencing, their clinical date was collected. DNA was extracted from peripheral blood. Whole exome sequencing was performed using CG Black Bird sequencing platform. After high confident SNP or InDel were identified, gene-based and filter-based annotation were made (dbSNP,1000 Genome Project, ExAC, SIFT/PolyPhen2 score). Inheritance analysis were performed for de novo mutation and inherited recessive mutation. Based on the previous researches, KEGG and GO analysis were carried out to the possible pathogenic mutations.ResultsCompound heterozygous mutations in DNAH2 (p.Ser312Thr and p.Arg573Cys) and TULP4 (p.Arg490Trp and p.Pro1270Leu)were identified in a trio family, which has a girl with persistent cloaca and her mother with the history of recurrent early pregnancy loss. DNAH2 and TULP4 are involved in ciliary intraflagellar transport (IFT), a process that is essential for the generation and maintenance of mammalian primary cilia.In case share mutation,34 variants were identified in 14 genes (PDE4DIP, OR2T27, ANKRD36, ZNF717, HLA-DQB2, gprin2, OR4C3, OR9G1, HYDIN, MAP2K3, KCNJ18, NPEPPS, KIR2DL1 and KIR3DL1). Among them, KIR2DL1 was related with placentation and embryonic development, we considered KIR2DL1 gene as a candidate gene. A rare heterozygous non synonymous mutation (p.Thr91Lys/c.272C>A) was found in the KIR2DL1 gene. The mutation was identified in 3 cases (PC03, PC09, PC 10).ConclusionsWe utilized whole-exome sequencing to identify two compound heterozygous mutations in DNAH2 (p.Ser312Thr and p.Arg573Cys) and TULP4 (p.Arg490Trp and p.Pro1270Leu), and a rare heterozygous missense mutations (p.Thr91Lys/c.272C>A) in the KIR2DL1 gene. Experimental model systems will be critical for elucidating the function of DNAH2, TULP4 and KIR2DL1, and the developmental pathways impacted by these mutations.