Patrick Ho Yu Chung

Identification of a HOXD13 mutation in a VACTERL patient

VACTERL acronym is assigned to a non‐random association of malformations in humans with poorly known etiology. It is comprised of vertebral defects (V), anal atresia (A), cardiac anomaly (C), tracheoesophageal fistula with esophageal atresia (TE), renal dysplasia (R) and limb lesions (L). Here, we report on, for the first time, a female patient with VACTERL association with a 21 base‐pair deletion in the exon 1 triplet repeats of HOXD13, a sonic hedgehog (SHH) downstream target. Our data provide the first piece of clinical evidence of the implication of the SHH pathway in VACTERL. Moreover, HOXD13 may not only be implicated in limb malformations but also in the development of gut and genitourinary structures, as predicted from the mouse models.

Genome-wide association study identifies a susceptibility locus for biliary atresia on 10q24.2

Biliary atresia (BA) is characterized by the progressive fibrosclerosing obliteration of the extrahepatic biliary system during the first few weeks of life. Despite early diagnosis and prompt surgical intervention, the disease progresses to cirrhosis in many patients. The current theory for the pathogenesis of BA proposes that during the perinatal period, a still unknown exogenous factor meets the innate immune system of a genetically predisposed individual and induces an uncontrollable and potentially self-limiting immune response, which becomes manifest in liver fibrosis and atresia of the extrahepatic bile ducts. Genetic factors that could account for the disease, let alone for its high incidence in Chinese, are to be investigated. To identify BA susceptibility loci, we carried out a genome-wide association study (GWAS) using the Affymetrix 5.0 and 500 K marker sets. We genotyped nearly 500 000 single-nucleotide polymorphisms (SNPs) in 200 Chinese BA patients and 481 ethnically matched control subjects. The 10 most BA-associated SNPs from the GWAS were genotyped in an independent set of 124 BA and 90 control subjects. The strongest overall association was found for rs17095355 on 10q24, downstream XPNPEP1, a gene involved in the metabolism of inflammatory mediators. Allelic chi-square test P-value for the meta-analysis of the GWAS and replication results was 6.94 x 10(-9). The identification of putative BA susceptibility loci not only opens new fields of investigation into the mechanisms underlying BA but may also provide new clues for the development of preventive and curative strategies.

Should open Kasai portoenterostomy be performed for biliary atresia in the era of laparoscopy

Kasai portoenterostomy has been the treatment of choice for neonates with biliary atresia since its introduction. With the advance in laparoscopic techniques, a few centers have reported the feasibility of performing laparoscopic Kasai portoenterostomy. However, the outcome of this new technique is not known. Here, we aim to evaluate, as the only referral center for liver transplantation, our experience with patients referred for transplantation after failed Kasai portoenterostomy. A retrospective study was carried out between October 1996 and September 2005. The records of all patients with the diagnosis of biliary atresia were retrieved. The type of procedure and clinical outcome of the patients were noted. Early failure of Kasai enterostomy was defined as the need for liver transplantation within 1-year post-Kasai operation. For the period studied, a total of 72 patients with biliary atresia were identified. Sixty-three of the 72 patients had their Kasai portoenterostomies performed openly while nine patients underwent laparoscopic Kasai portoenterostomy in a center experienced in laparoscopic surgery. Six of these patients were referred for transplantation within 1 year, giving the early failure rate of 66.6%. In comparison, the early failure rate for open Kasai procedure was 38.5%. Regarding post-operative complications, one patient who underwent laparoscopic Kasai procedure also suffered intestinal volvulus after initial surgery and another was found to have internal herniation of the Roux loop. Laparoscopic Kasai portoenterostomy seems to be associated with more post-operative complications and worse early clinical outcome. As a result, we remain guarded about the present-day technique of laparoscopy for biliary atresia.

Genome-wide copy number analysis uncovers a new HSCR gene: NRG3

Hirschsprung disease (HSCR) is a congenital disorder characterized by aganglionosis of the distal intestine. To assess the contribution of copy number variants (CNVs) to HSCR, we analysed the data generated from our previous genome-wide association study on HSCR patients, whereby we identified NRG1 as a new HSCR susceptibility locus. Analysis of 129 Chinese patients and 331 ethnically matched controls showed that HSCR patients have a greater burden of rare CNVs (p = 1.50×10−5), particularly for those encompassing genes (p = 5.00×10−6). Our study identified 246 rare-genic CNVs exclusive to patients. Among those, we detected a NRG3 deletion (p = 1.64×10−3). Subsequent follow-up (96 additional patients and 220 controls) on NRG3 revealed 9 deletions (combined p = 3.36×10−5) and 2 de novo duplications among patients and two deletions among controls. Importantly, NRG3 is a paralog of NRG1. Stratification of patients by presence/absence of HSCR–associated syndromes showed that while syndromic–HSCR patients carried significantly longer CNVs than the non-syndromic or controls (p = 1.50×10−5), non-syndromic patients were enriched in CNV number when compared to controls (p = 4.00×10−6) or the syndromic counterpart. Our results suggest a role for NRG3 in HSCR etiology and provide insights into the relative contribution of structural variants in both syndromic and non-syndromic HSCR. This would be the first genome-wide catalog of copy number variants identified in HSCR.

Fine Mapping of the NRG1 Hirschsprung's Disease Locus

The primary pathology of Hirschsprungs disease (HSCR, colon aganglionosis) is the absence of ganglia in variable lengths of the hindgut, resulting in functional obstruction. HSCR is attributed to a failure of migration of the enteric ganglion precursors along the developing gut. RET is a key regulator of the development of the enteric nervous system (ENS) and the major HSCR-causing gene. Yet the reduced penetrance of RET DNA HSCR-associated variants together with the phenotypic variability suggest the involvement of additional genes in the disease. Through a genome-wide association study, we uncovered a ∼350 kb HSCR-associated region encompassing part of the neuregulin-1 gene (NRG1). To identify the causal NRG1 variants contributing to HSCR, we genotyped 243 SNPs variants on 343 ethnic Chinese HSCR patients and 359 controls. Genotype analysis coupled with imputation narrowed down the HSCR-associated region to 21 kb, with four of the most associated SNPs (rs10088313, rs10094655, rs4624987, and rs3884552) mapping to the NRG1 promoter. We investigated whether there was correlation between the genotype at the rs10088313 locus and the amount of NRG1 expressed in human gut tissues (40 patients and 21 controls) and found differences in expression as a function of genotype. We also found significant differences in NRG1 expression levels between diseased and control individuals bearing the same rs10088313 risk genotype. This indicates that the effects of NRG1 common variants are likely to depend on other alleles or epigenetic factors present in the patients and would account for the variability in the genetic predisposition to HSCR.

MNX1 (HLXB9) mutations in Currarino patients

PURPOSE The combination of partial absence of the sacrum, anorectal anomalies, and presacral mass constitutes Currarino syndrome (CS), which is associated with mutations in MNX1 motor neuron and pancreas homeobox 1 (previously HLXB9). Here, we report on the MNX1 mutations found in a family segregating CS and in 3 sporadic CS patients, as well as on the clinical characteristics of the affected individuals. METHODS MNX1 mutations were identified by direct sequencing the coding regions, intron/exon boundaries of MNX1 in 5 CS Japanese family members and 3 Chinese sporadic cases and their parents. RESULTS There were 2 novel (P18PfsX37, R243W) and 2 previously described (W288G and IVS2 + 1G > A) mutations. These mutations were not found in 198 control individuals and are predicted to impair the functioning of the MNX1 protein. CONCLUSIONS The variability of the CS phenotype among related or unrelated patients bearing the same mutation advocates for differences in the genetic background of each individual and invokes the implication of additional CS susceptibility genes.

Predictors for failure after Kasai operation

AIM OF THE STUDY The outcome of Kasai operation is not uniformly successful. This objective of this study is to identify risk factors that predict early failure of this operation. METHODS A retrospective study was carried out for all patients who received Kasai operation between 1980 and 2012. Patients referred from other centers for liver transplantation because of failed Kasai operation were also recruited. MAIN RESULTS A total of 185 patients were reviewed. A total of 119 (64.3%) patients failed Kasai operation, whereas 86 (46.5%) were considered as cases of early failure (<3 years post-Kasai). With univariate analysis, the presence of associated anomaly (RR: 1.90, 95%CI 1:45-2:36), operation with laparoscopic surgery (RR: 3.14, 95% CI: 2.39-5.42), delayed clearance of jaundice (RR: 1.89, 95% CI: 1.56-2.67), and repeated cholangitis (RR: 2.56, 95% CI: 1.39-3.42) were associated with adverse outcome, whereas the use of adjuvant steroid at post-operative period appeared to be protective (RR: 0.64, 95% CI: 0.29-0.93). With multivariate analysis, repeated cholangitis was an independent risk factor for early failure after Kasai operation (RR: 3.16, 95% CI: 1.83-4.62). Furthermore, it was also considered as a risk factor for failure after 3 year post-Kasai (RR: 2.07, 95% CI: 1.43-3.42). CONCLUSION Patients with above risk factors are prone to fail Kasai operation in an early stage and require more frequent monitoring. Prevention and aggressive management of cholangitis should be considered as measures to avoid disease progression in the early stage. Lastly, performing open Kasai operation and the use of adjuvant steroid are potentially beneficial.

Performing Kasai Portoenterostomy Beyond 60 Days of Life Is Not Necessarily Associated With a Worse Outcome

The introduction of Kasai portoenterostomy has dramatically improved the management and survival of children with biliary atresia. The success rate of this operation worldwide varies with different centers. In this respect, many authors have studied the correlation of a successful outcome with various factors, such as the experience and workload of the surgical center, the use of postoperative steroids, the underlying biliary anatomy, as well as the age of patients at the time of the operation. Indeed, the age of 60 days has been used by clinicians as a critical time beyond which the rate of success of the Kasai operation markedly reduces. Despite this worldwide adoption, clear evidence supporting this critical operative time is still lacking. We undertook a review of our experience in the management of children with biliary atresia and focused specifically on the issue of the timing of operation. We showed that performing the Kasai operation beyond the age of 60 days was not associated with a worse outcome and that a high percentage of patients could still achieve good bile flow with normal bilirubin postoperatively. Thus, we believe that until the age of 100 days, the age of the patients does not play a significant role in determining the success of the Kasai operation.

Comparing open and pneumovesical approach for ureteric reimplantation in pediatric patients—a preliminary review

BACKGROUND/PURPOSE In this article, we are going to report our early experience on laparoscopic pneumovesical approach for ureteric reimplantation and to compare the result with traditional open approach. METHODS A retrospective review of patients who underwent ureteric reimplantation from 2000 to 2007 was carried out. They were divided into open and pneumovesical group according to the surgical approach. Comparison in terms of operative duration, hospital stay, and complication rate was made. RESULTS During the study period, a total of 22 ureteric reimplantation procedures were carried out. Thirteen and nine patients were operated by open and pneumovesical approaches, respectively. The mean age for operation in the 2 groups were similar (P = .62). Although the average operation time is 38.0 minutes longer in the pneumovesical group (P = .049), patients from this group had a hospital stay 2.3 days shorter than the open group (P = .065). Regarding curative success rate, both groups show similar result and were able to achieve more than 95% success rate. No major complications were found in the 2 groups. Cost-effective analysis favors pneumovesical approach in this study. CONCLUSIONS The pneumovesical approach is safe and effective for ureteric reimplantation in children. It offers less postoperative morbidities and shorter hospital stay when compared to traditional open method.

Genome-wide copy number variation study in anorectal malformations

Anorectal malformations (ARMs, congenital obstruction of the anal opening) are among the most common birth defects requiring surgical treatment (2-5/10 000 live-births) and carry significant chronic morbidity. ARMs present either as isolated or as part of the phenotypic spectrum of some chromosomal abnormalities or monogenic syndromes. The etiology is unknown. To assess the genetic contribution to ARMs, we investigated single-nucleotide polymorphisms and copy number variations (CNVs) at genome-wide scale. A total of 363 Han Chinese sporadic ARM patients and 4006 Han Chinese controls were included. Overall, we detected a 1.3-fold significant excess of rare CNVs in patients. Stratification of patients by presence/absence of other congenital anomalies showed that while syndromic ARM patients carried significantly longer rare duplications than controls (P = 0.049), non-syndromic patients were enriched with both rare deletions and duplications when compared with controls (P = 0.00031). Twelve chromosomal aberrations and 114 rare CNVs were observed in patients but not in 868 controls nor 11 943 healthy individuals from the Database of Genomic Variants. Importantly, these aberrations were observed in isolated ARM patients. Gene-based analysis revealed 79 genes interfered by CNVs in patients only. In particular, we identified a de novo DKK4 duplication. DKK4 is a member of the WNT signaling pathway which is involved in the development of the anorectal region. In mice, Wnt disruption results in ARMs. Our data suggest a role for rare CNVs not only in syndromic but also in isolated ARM patients and provide a list of plausible candidate genes for the disorder.