Intestinal dysbiosis and bacterial enteroinvasion in a murine model of Hirschsprung’s disease

Background/purpose

Hirschsprung’s disease (HSCR), characterized by the absence of ganglia in the distal colon, results in functional obstruction. Despite surgical resection of the aganglionic segment, around 40% of patients suffer recurrent life threatening Hirschsprung’s-associated enterocolitis (HAEC). The aim of this study was to investigate whether gut microbiota and intestinal immunity changes contribute to the HAEC risk in an HSCR model.

Methods

Mice with neural crest conditional deletion of Endothelin receptor B (EdnrB) and their littermate controls were used (EdnrB-null and EdnrB-het). Bacterial DNA was prepared from cecal contents of P16-18 and P21-24 animals and pyrosequencing employed for microbiome analysis. Ileal tissue was isolated and secretory phospholipase A₂ (sPLA₂) expression and activity determined. Enteroinvasion of Escherichia coli into ileal explants was measured using an ex vivo organ culture system.

Results

EdnrB-het and EdnrB-nulls displayed similar flora, sPLA₂ expression and activity at P16-18. However, by P21-24, EdnrB-hets demonstrated increased Lactobacillus and decreased Bacteroides and Clostridium, while EdnrB-nulls exhibited reciprocal changes. EdnrB-nulls also showed reduced sPLA₂ expression and luminal activity at this stage. Functionally, EdnrB-nulls were more susceptible to enteroinvasion with E. coli ex vivo and released less sPLA₂ than EdnrB-hets.

Conclusions

Initially, EdnrB-het and EdnrB-nulls contain similar cecal flora but then undergo reciprocal changes. EdnrB-nulls display dysbiosis, demonstrate impaired mucosal defense, decreased luminal sPLA₂ and increased enteroinvasion of E. coli just prior to robust colonic inflammation and death. These findings suggest a role for the intestinal microbiome in the development of HAEC.     

The research on screening differentially expressed genes in Hirschsprung’s disease by using Microarray

Objective

To study the differential expression of genes between Hirschsprung’s disease (HSCR) and normal tissue by using microarray for exploring the mechanism of HSCR development and establishing the gene expression profiles of HSCR.

Methods

Colon tissues (aganglionic and normal segments) of 4 patients with HSCR were detected by the Agilent SurePrint G3 Human GE 8x60K Microarrays. RT-PCR was used to verify the results of Microarray test. Then, immunohistochemistry was used to demonstrate the expression of HAND2 in the myenteric plexus of the colon from 46 patients with HSCR to further explore the relationship between HAND2 and development of HSCR.

Results

A total of 12,125 meaningful expressed genes were screened out. 4 pairs of specimens had 622 differentially expressed genes, 584 (93.89%) of which were up-regulated while 38(6.11%) were down-regulated. 6 of the 622 genes were tested by RT-PCR, which were consistent with the results detected by Microarray. The average optical density of positive expression of HAND2 in myenteric plexus was compared between the aganglionic, transitional, dilated, normal segments and control group. The average optical density in the aganglionic segments was obviously reduced. Statistical analyzed data showed that it has significant deviation (P<0.01).

Conclusion

1. A set of differentially expressed genes between aganglionic and normal segments of HSCR was obtained. Our data may provide significant information to research the pathogenesis of HSCR. 2. Reduced protein expression of HAND2 in the myenteric plexus of the aganglionic would suggest that HAND2 was involved in the pathogenesis of HSCR.     

New variations of the EDNRB gene and its association with sporadic Hirschsprung's disease in Korea

Background/Purpose

The endothelin receptor B (EDNRB) signaling pathway, which is the second major susceptible gene for Hirschsprung's disease (HSCR), is crucial for the development of the enteric nervous system. The allele frequency of polymorphisms was mostly tested in the American and European population, but the data of an ethnically diverse, non-Caucasian population are unclear. To further investigate the variants and haplotypes of the EDNRB gene, this study examined sequence variations in Korean patients with sporadic HSCR.

Methods

All 8 exons and intron/exon boundaries of the EDNRB gene in 18 Korean patients with sporadic HSCR and 84 healthy individuals were screened using PCR amplification and direct sequencing.

Results

A total of 8 different nucleotide substitutions were identified. Of these, 4 were new variants (promoter-116C>T; 5′UTR-121G>T; IVS4+62C>A; IVS5+121G>C) and the others were previously described variants. The distribution of variations was even different from that reported for Chinese and Japanese subjects as well as other ethnic groups. This study also analyzed the haplotypes for an association between the variants identified with HSCR.

Conclusions

This study identified additional sequence variants of the EDNRB gene, but the estimated EDNRB haplotypes did not show any disease risk.     

Biological characteristics of pediatric renal cell carcinoma associated with Xp11.2 translocations/TFE3 gene fusions

Purpose

To investigate the clinical features of pediatric Xp11.2 translocation renal cell carcinoma (RCC).

Methods

A retrospective review of 22 cases over 35 years.

Results

Xp11.2 translocation RCCs were identified in 13 boys and 9 girls with a median age of 10.5 years (range: 2.5–16 years). RCC presented with hematuria in 17, abdominal mass in 1, abdominal masses with hematuria in 2, abdominal pain with hematuria in 1, and as an incidental finding in 1 patient. Ten patients were classified stage I, 10 were stage III, and two were stage IV. Of the 10 patients with stage I RCCs, 3 patients with tumor measuring less than 7 cm had nephron-sparing surgery (NSS) and 17 patients underwent simple nephrectomy. A 15-cm tumor was incompletely removed in one patient and another patient with a 25-cm × 18-cm × 15-cm tumor had gross residual. Of the 15 patients followed up between 6 months and 35 years, 13 were still living and 2 had died after surgery.

Conclusions

Xp11.2 translocation RCC is the predominant form of pediatric RCC, associated with advanced stage at presentation. Nephrectomy is the usual treatment for RCC but NSS is an option for patients with tumors measuring < 7 cm. Patients with N + M0 maintained a favorable prognosis following surgery alone.     

A novel Czech kindred with familial medullary thyroid carcinoma and Hirschsprung's disease

Purpose

The RET proto-oncogene is involved in neural crest disorders. Activating germline mutations in the RET proto-oncogene cause the development of familial medullary thyroid carcinoma (FMTC) or medullary thyroid carcinoma (MTC) as a part of multiple endocrine neoplasia type 2 (MEN2) syndrome. Inactivating germline mutations in the RET proto-oncogene are detected in Hirschsprung's disease (HSCR). Only in a very small number of families are these 2 diseases expressed together.

Methods

This study presents a novel Czech kindred with FMTC-HSCR phenotype. Two family members (mother and daughter) were tested for RET germline mutations in exons 10, 11, 13, 14, 15, and 16.

Results

Direct fluorescent sequencing of genomic DNA revealed a heterozygous mutation in the RET proto-oncogene in exon 10 at codon C609Y in both persons tested. This family was reclassified, thanks to genetic screening from the apparently sporadic MTC-HSCR to FMTC-HSCR.

Conclusion

The germline mutation was detected because of the systematic genetic screening of the RET proto-oncogene, which is useful for genetic counseling of potential risk of HSCR and MTC in other family members. This family could be added to the small worldwide cohort of families with MEN2A/FMTC-HSCR.     

Association analysis of the PHOX2B gene with Hirschsprung disease in the Han Chinese population of Southeastern China

Background

Hirschsprung disease (HSCR, OMIM 142623) is a complex congenital disorder characterized by intestinal obstructions caused by the absence of the intestinal ganglion cells of the nerve plexuses in variable lengths of the digestive tract. The PHOX2B gene is involved in neurogenesis and disruption of Phox2b in mice results in a HSCR-like phenotype. The first association study of the PHOX2B gene with HSCR derived from Chinese population in Hong Kong; here, we address the question of whether PHOX2B acts as a predisposing factor in HSCR pathogenesis in Chinese population in mainland.

Methods

To investigate the contribution of PHOX2B to the HSCR phenotype, polymerase chain reaction amplification and direct sequencing were used to screen PHOX2B coding regions and intron/exon boundaries for mutations and polymorphisms in 102 patients with HSCR and 96 ethnically matched controls, in Han Chinese populations of Southeastern China.

Results

In this study, we genotyped 4 single nucleotide polymorphisms (SNPs) (including 1 novel SNP) located within the PHOX2B gene. Statistically significant differences were found for c.701 A > G and IVS2 + 100 A > G, and the log-additive model was accepted as the best inheritance model (odds ratio [OR], 1.79; 95% confidence interval [CI], 1.11-2.87) for IVS2 + 100 A > G. We also showed that the haplotype-A G A N composed of 4 SNPs exhibited significant association with the disease (P = .03); this haplotype was more frequently observed in cases than in controls (OR, 2.31; 95% CI, 1.11-4.82).

Conclusions

Our study provided further evidence that the PHOX2B gene is involved in the susceptibility to HSCR in the Han Chinese population. Our findings are in accordance with the involvement of PHOX2B in the signaling pathways governing the development of enteric neurons.     

Mutation analysis of the motor neuron and pancreas homeobox 1 (MNX1, former HLXB9) gene in Swedish patients with Currarino syndrome

Background

Currarino syndrome (CS) is a triad consisting of partial sacral agenesis, presacral mass, and anorectal malformations, typically anal stenosis but the phenotype varies. The main cause of this monogenic disorder is mutations in the motor neuron and pancreas homeobox 1 gene. We describe the clinical and genetic findings in 4 unrelated Swedish cases with CS and their relatives.

Methods

We performed mutation analysis of the motor neuron and pancreas homeobox 1 gene in 4 cases with CS by DNA sequence analysis as well as multiplex ligation-dependent probe amplification. In addition, array comparative genome hybridization was performed in 2 cases. Including relatives, totally, 14 individuals were analyzed.

Results

We found 2 previously described mutations, 1 de novo nonsense mutation (p.Gln212X) and 1 maternally inherited frameshift mutation (p.Pro18ProfsX38). In the family with the frameshift mutation, we also detected the same maternally inherited mutation in 3 of the proband's 4 brothers, who displayed varying symptoms. All mutation carriers had presacral tumors, although 2 were asymptomatic.

Conclusion

Our findings emphasize the need for genetic counseling and mutation analysis in patients with CS to detect tumors early. It shows the importance of evaluation of the sacrum and the presacral region in patients with anal stenosis with or without funnel anus. Family members of index cases should be considered for evaluation even if they are asymptomatic.     

Motility of the anorectum after “Rectoplasty with posterior triangular colonic flap” in Hirschsprung’s disease

Clinical assessment of fecal continence and anorectal manometry were carried out in 22 patients with Hirschsprung’s disease who had undergone “Rectoplasty with posterior triangular colonic flap”. Satisfactory postoperative continence was achieved in 18 out of these 22 patients. The result of anorectal manometry disclosed that normal rectal compliance (reservoir function of the rectum), together with normal resting pressure of the anorectum and rectoanal reflex, is indispensable for an adequate postoperative continence.     

Familial associations in medullary thyroid carcinoma with Hirschsprung disease: the role of the RET-C620 “Janus” genetic variation

Introduction

Hirschsprung disease (HSCR) is associated with the later development of multiple endocrine neoplasia (MEN2), because RET gene variations are associated with both conditions. Specifically, HSCR-MEN2 cosegregation mostly relates to the cysteine-rich area at the RET-620 (the “Janus gene”).

Aim

The aim of this study was to explore the clinical and genetic associations of HSCR-MEN2 in a cohort of HSCR patients.

Methods

RET gene variation was evaluated by heteroduplex single-strand conformational polymorphism analysis and validated with automated sequencing techniques in HSCR patients (including 18 kindreds). Those with RET C620 variations were subjected to familial evaluation for coexisting HSCR-MEN2.

Results

A cohort of 118 patients with HSCR (n = 89) or medullary thyroid carcinoma (n = 29) were studied, including 3 families where a RET-620 point mutation was identified. No C618, C609, or C611 variations were detected. In 1 remarkable 6-generational family (family 3), HSCR in early generations seemed to be later replaced by MEN2A. In the other 2 families with total colonic aganglionosis, a relative with a medullary thyroid carcinoma was identified.

Conclusion

Gene mutation in the RET-620 position carries significant risk and may be part of a targeted investigation of high-risk areas in HSCR. We propose an alternative hypothesis of endoplasmic reticulum control to explain the changing phenotypic expression.     

Novel mutations of RET gene in Korean patients with sporadic Hirschsprung's disease

Background/Purpose

Hirschsprung's disease (HSCR) is a congenital abnormality that can cause an intestinal obstruction. Although HSCR demonstrates a sex-modified polygenic inheritance with contributions from multiple genes, mutations in the RET gene are believed to be the major sign of susceptibility in the development of disease. The allele frequency of polymorphisms was mostly tested in the American and European population, but the data of an ethnically diverse nonwhite population are unclear.

Methods

All 21 exons and intron/exon boundaries of the RET gene in 18 Korean patients with sporadic HSCR and 84 normal individuals were screened using polymerase chain reaction amplification and direct sequencing.

Results

A total of 11 different nucleotide substitutions were identified. Of these, 2 were new missense mutations (C558Y, cysteine-rich domain; R844W, tyrosine kinase domain) and 9 previously described variants. This study also analyzed the haplotypes for the association between the variants identified with HSCR, but the estimated RET haplotypes did not show any disease risk.

Conclusions

This study identified additional mutations of RET gene, which represents the first comprehensive genetic dissection of sporadic HSCR disease in Koreans.     

A novel mutation of TGF beta1 in a Chinese family with Camurati-Engelmann disease

Camurati–Engelmann disease (CED) [OMIM 131300] is a rare autosomal dominant disorder characterized by bone pain and osteosclerosis affecting the diaphysis of long bones. It has been previously reported that CED is caused by mutations of the transforming growth factor beta 1 (TGFβ1) gene on chromosome 19q13.1–q13.3. Until now, seven mutations (LLL12-13ins, Y81H, R156C, R218C, R218H, H222D, C225R) in Australian, French, Belgian, Japanese, and European families have been reported and these data showed that there was no correlation between the nature of the mutations and the variability of the clinical manifestations. In this study, we found a Chinese family with CED and observed some intra-familial clinical variability and symptoms that became more severe with the age. A new TGFβ1 mutation (E169K) in exon 2 was identified in the Chinese family using polymerase chain reaction, direct sequencing analysis of PCR products and single-strand conformation polymorphism analysis. This mutation has not been previously reported in other countries in the world.     

An association study of HFE gene mutation with idiopathic male infertility in the Chinese Han population

Mutations in the haemochromatosis gene (HFE) influence iron status in the general population of Northern Europe, and excess iron is associated with the impairment of spermatogenesis. The aim of this study is to investigate the association between three mutations (C282Y, H63D and S65C) in the HFE gene with idiopathic male infertility in the Chinese Han population. Two groups of Chinese men were recruited: 444 infertile men (including 169 with idiopathic azoospermia) and 423 controls with proven fertility. The HFE gene was detected using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The experimental results demonstrated that no C282Y or S65C mutations were detected. Idiopathic male infertility was not significantly associated with heterozygous H63D mutation (odds ratio=0.801, 95% confidence interval=0.452–1.421, χ²=0.577, P=0.448). The H63D mutation frequency did not correlate significantly with the serum luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone (T) levels in infertile men (P=0.896, P=0.404 and P=0.05, respectively). Our data suggest that the HFE H63D mutation is not associated with idiopathic male reproductive dysfunction.     

Fabry病家系的α-半乳糖苷酶A基因突变研究

目的 通过检测3个Fabry病家系基因突变类型明确基因诊断,并进行家系成员的基因型检测.方法 通过PCR和直接测序的方法,对3个Fabry家系的先证者及部分家系成员外周血DNA进行α-半乳糖苷酶A编码GLA基因7个外显子及其相邻内含子的DNA序列检测.结果 （1）先证者1的GLA基因7号外显子内1142位点发生碱基缺失（1142delG）,1142位碱基G的缺失导致蛋白质翻译在390位氨基酸提前终止,该突变国内外均未见报道;（2）先证者2的GLA基因6号外显子内902位点存在1个错义突变,碱基G被A取代,导致其编码的第301位氨基酸由精氨酸变为谷氨酰胺（902G＞A,R301Q）;（3）先证者3的GLA基因3号外显子内484位点存在1个错义突变,碱基T被C取代,导致其编码的第142位氨基酸由半胱氨酸变为精氨酸（484T＞C,C142R）.在3个家系的部分成员中进行基因检测,检出GLA突变基因携带者共6例,其中男性半合子1例,女性杂合子5例,突变类型均与相应先证者符合.100条正常X染色体对照中均未发现上述位点异常.结论 本研究在3个Fabry病家系中检出3种GLA基因突变,其中1142delG为新发现的突变,并在3个家系的部分家系成员中检出男性半合子1例,女性杂合子5例.     

Multiple variants in UGT1A1 gene are factors to develop indirect hyper-bilirubinemia

Most Taiwanese patients with hyper-bilirubinemia have genetic abnormalities in the uridine diphosphoglucuronate-glucuronosyltransferase 1A1 (UGT1A1) gene beyond the variants in the TATA box upstream of UGT1A1 associated with Gilbert’s syndrome. To investigate the role of UGT1A1 in the pathogenesis of indirect hyper-bilirubinemia, we prospectively studied 97 consecutive patients with indirect hyper-bilirubinemia for genotypes of promoter [(TA)₆TAA6, (TA)₇TAA7] and coding region [nucleotide (nt)-211, nt-686, nt-1,091 and nt-1,456] of UGT1A1. Thirty-six of the patients (45.6%) were found to have Gilbert’s syndrome with 7/7 genotype; among them, 14 also carried variants at nt-686. Forty-two patients (43.3%) had the 6/7 genotype; among them, 36 patients were found to have one or more variants in the coding region. Patients with higher serum total bilirubin are associated with higher likelihood of carrying Gilbert’s syndrome genotype: 60.0% (P=0.007) patients with serum total bilirubin level ≥2.5 mg/dL carried the Gilbert’s syndrome genotype, while only 23.9% of patients with serum total bilirubin level <2.5 mg/dL carry the same genotype (P=0.0006). Forty-one of the 61 non-Gilbert’s patients had one homogenous variants or two or more heterozygous variants in UGT1A1. Further studies are necessary to confirm the role of one homo-zygous variant or two or more hetero-zygous variants in UGT1A1 gene as factors for indirect hyper-bilirubinemia.     

A novel BMPR2 gene mutation associated with exercise-induced pulmonary hypertension in septal defects

Abstract

Objective. Our study aimed to investigate the relationship between exercise-induced pulmonary arterial hypertension and genetic changes related to the transforming growth factor-β (TGF-β) signalling pathway in patients with cardiac septal defects. Design. In a population-based group of 44 patients (age 13–25 years) with either isolated ventricular septal defect (n=27) or isolated atrial septal defect (n=17), right ventricular systolic pressure response to submaximal exercise was studied by echocardiography and classified as normal (≤45 mmHg), borderline (45–50 mmHg) or abnormal (>50 mmHg). Three genes related to TGF-β, bone morphogenetic protein receptor type 2 (BMPR2), activin receptor-like kinase 1 (ALK1) and endoglin (ENG), were analyzed by DNA sequencing (only BMPR2) and multiplex ligand-dependent probe amplification (BMPR2, ALK1 and ENG). Results. Pressure response was borderline in five and abnormal in nine patients. Five patients showed mutations in exon 12 of the bone morphogenetic protein receptor type 2 gene. The previously described polymorphism S775N (c. 2324, G > A) was found in three patients with normal pressure response. The mutation Y589C (c. 1766, A > G), which has not been described previously, was found in two of 14 patients with borderline/abnormal pressure response. Conclusion. Genetic changes in the BMPR2 gene may be overrepresented in patients with cardiac septal defects and exercise-induced pulmonary hypertension.