Merce Garcia-Barcelo

Genetic basis of Hirschsprung’s disease

Hirschsprung’s disease (HSCR) is a developmental disorder characterized by the absence of ganglion cells in the lower digestive tract. Aganglionosis is attributed to a disorder of the enteric nervous system (ENS) whereby ganglion cells fail to innervate the lower gastrointestinal tract during embryonic development. HSCR is a complex disease that results from the interaction of several genes and manifests with low, sex-dependent penetrance and variability in the length of the aganglionic segment. The genetic complexity observed in HSCR can be conceptually understood in light of the molecular and cellular events that take place during the ENS development. DNA alterations in any of the genes involved in the ENS development may interfere with the colonization process, and represent a primary etiology for HSCR. This review will focus on the genes known to be involved in HSCR pathology, how they interact, and on how technology advances are being employed to uncover the pathological processes underlying this disease.

Hirschsprung's disease

Hirschsprungs disease (HSCR) is characterized by absence of the enteric nervous system in a variable portion of the distal gut. Affected infants usually present in the days after birth with bowel obstruction. Despite surgical advances, long-term outcomes remain variable. In the last 2 decades, great advances have been made in understanding the genes and molecular biological mechanisms that underlie the disease. In addition, our understanding of normal enteric nervous system development and how motility develops in the developing fetus and infant has also increased. This review aims to draw these strands together to explain the developmental and biological basis of HSCR, and how this knowledge may be used in the future to aid children with HSCR.

Chinese patients with sporadic Hirschsprung’s disease are predominantly represented by a single RET haplotype

Hirschsprung’s disease is a developmental disorder characterised by the absence of ganglion cells in the nerve plexuses of the lower digestive tract. The Hirschsprung phenotype is variable and can be classified into two groups: SSA, or short segment aganglionosis, which includes patients with aganglionosis as far as the rectosigmoid junction; and LSA, or long segment aganglionosis, which includes patients with aganglionosis beyond the rectosigmoid junction. The condition presents in the neonatal period as a failure to pass meconium, chronic severe constipation, colonic distension, secondary electrolyte disturbances, and sometimes enterocolitis and bowel perforation. The estimated population incidence is 1/5000 live births, although this is a representative value. The highest incidence is in Asian populations (2.8 per 10 000 life births) and the lowest in Hispanics (1 per 10 000 life births).1 The male to female (M:F) ratio is approximately 4:1 for SSA patients and approximately 1:1 for LSA patients.1 Approximately 20% of cases are familial. The recurrence risks for siblings of SSA variant probands ranges between 1.5% and 3.3%, while the risks for those of LSA variant probands varies from 2.9% to 17.6%.1 Hirschsprung’s disease is often associated with chromosomal abnormalities, with other neurodevelopmental disorders such as Waardenburg syndrome type 4, and with a variety of additional isolated anomalies and syndromes.1,2 The disease has a complex genetic aetiology, and many studies indicate the receptor tyrosine kinase gene ( RET ) as the major susceptibility gene for Hirschsprung’s disease.1–14 RET mutations are also associated with multiple endocrine neoplasia type 2 (MEN2),15 and medullary thyroid carcinoma.16 Papillary thyroid carcinoma is associated with RET somatic rearrangements.17 Mutations in the RET gene account for up to 50% of the familial cases and anywhere between 7% and 35% of the sporadic cases, and they lack genotype–phenotype correlation.5– …

Implications of Endocrine Gland–Derived Vascular Endothelial Growth Factor/Prokineticin-1 Signaling in Human Neuroblastoma Progression

Purpose: Neuroblastoma is a common pediatric tumor that is derived from improperly differentiated neural crest cells (NCC). We recently revealed that endocrine gland–derived vascular endothelial growth factor/prokineticin-1 (EG-VEGF/Prok-1) is a key factor mediating the growth and differentiation of enteric NCCs during development. In this report, we further elucidate its role in neuroblastoma progression. Experimental Design: We studied the expression and copy number of EG-VEGF/Prok-1 receptors (PK-R1 and PK-R2) in 26 neuroblastoma tumors by real-time reverse transcription-PCR and immunohistochemical analysis. Implication of EG-VEGF/Prok-1 signaling in neuroblastoma progression was further shown in a neuroblastoma cell line (SK-N-SH). Results: We found that all neuroblastoma samples from stages II to IV expressed both PK-R1 and PK-R2. Kruskall-Wallis signed rank tests revealed that the expression level of PK-R1 transcript is associated with the stages and metastasis of the neuroblastoma (P < 0.05), and PK-R2 is persistently higher in advanced-stage neuroblastoma samples. About 38% of the neuroblastoma tumors (10:26) possessed MYCN amplification, whereas no PK-R1 and PK-R2 amplifications were detected, suggesting that the overexpression of the receptors was not due to gene amplification. Subsequent functional studies showed that EG-VEGF/Prok-1 activates the Akt pathway to induce the proliferation of neuroblastoma cells. Targeted down-regulation studies revealed that EG-VEGF/Prok-1–mediated proliferation requires the presence of these two receptors, and that PK-R2 is essential for inhibiting apoptosis. In vitro migration and invasion assays also indicated that EG-VEGF/Prok-1 significantly enhances the cell migration/invasion of SK-N-SH. Conclusions: Our study has shown for the first time that aberrant EG-VEGF/Prok-1 signaling favors neuroblastoma progression and could be a potential target for future neuroblastoma treatment.

Liver Intestine-Cadherin (CDH17) Haplotype Is Associated with Increased Risk of Hepatocellular Carcinoma

Purpose: Hepatocellular carcinoma (HCC), the most common form of liver cancer, is a leading cause of cancer death worldwide. We previously showed that aberrant mRNA splicing of the liver intestine-cadherin gene CDH17 in liver tissues was triggered by the specific constellation of two CDH17 single nucleotide polymorphisms (651T and IVS6+35G). CDH17 aberrant splicing was highly associated with tumor dissemination and shorter survival of HCC patients. Consequently, it is highly relevant to assess whether the presence of these single nucleotide polymorphisms in the general population represents a risk to the development of HCC. Experimental Design: We conducted a case-control study including 164 HCC and 99 cirrhosis patients and 293 healthy controls. Genotyping was done by PCR and direct sequencing. Odds ratio (OR) and χ2 analysis were used to analyze genotypes and haplotypes. Results: Genotypes 651TT [OR, 2.62; 95% confidence interval (95% CI), 1.34-5.03] and IVS6+35 GG (OR, 1.95; 95% CI, 1.04-3.62) were highly associated with HCC disease. The 651T (C>T) and IVS6+35G (A>G) alleles were also overrepresented in HCC patients and, in particular, the T-G haplotype was the most prevalent in HCC patients when compared with healthy controls (OR, 1.57; 95% CI, 1.167-2.109; P = 0.004), which was in agreement with the aberrant splicing observed in tumor tissues. There was no significant difference in genotype and allele frequencies between cirrhosis patients and controls. Conclusion: The functional T-G haplotype of CDH17 (651 C>T and IVS6+35A>G) is a genetic susceptibility factor for the development of HCC in a Chinese population.

Correlation between genetic variations in Hox clusters and Hirschsprung's disease.

Interactions between migrating neural crest cells and the environment of the gut are crucial for the development of the enteric nervous system (ENS). A key signalling mediator is the RET‐receptor‐tyrosine‐kinase which, when defective, causes Hirschprungs disease (HSCR, colon aganglionosis). RET mutations alone cannot account for the variable HSCR phenotype, invoking interactions with as yet unknown, and probably inter‐related, loci involved in ENS development. Homeobox (HOX) genes have a major role in gut development as depicted by the enteric Hox code. We investigated whether DNA alterations in HOX genes, either alone or in combination with RET, are implicated in HSCR. Genotyping effort was minimized by applying the HapMap data on Han Chinese from Beijing (CHB). 194 HSCR patients and 168 controls were genotyped using Sequenom technology for 72 tag, single nucleotide polymorphisms (SNPs) distributed along the HOX clusters. The HapMap frequencies were compared to those in our population and standard statistics were used for frequency comparisons. The multifactor‐dimensionality‐reduction method was used for multilocus analysis, in which RET promoter SNP genotypes were included. Genetic interactions were found between two HOX loci (5′‐HOXA13 and 3′UTR‐HOXB7) and the RET loci tested. Minor allele frequencies (MAF) of the SNPs tested in our sample were not significantly different from those reported by HapMap when the sample sizes of the populations compared were considered. This is the first evaluation of the HOX genes in HSCR and the first application of HapMap data in a Chinese population. The interacting HOX loci may affect the penetrance of the RET risk allele. HapMap data for the CHB population correlated well with the general Chinese population.

Is there a role for the IHH gene in Hirschsprung's disease?

Abstract  Hirschsprung disease (HSCR) is characterized by the absence of ganglion cells along a variable length of the intestine. HSCR has a complex genetic aetiology with 50% of the patients unexplained by mutations in the major HSCR genes. The Ihh gene is involved in the development of the enteric nervous system (ENS) and Ihh mutant mice present with a phenotype reminiscent of HSCR. The requirement of Ihh signalling for the proper development of the ENS, together with the evidence presented by the Ihh murine model, prompted us to investigate the involvement of the human IHH gene in HSCR. Sequence analysis revealed seven single nucleotide polymorphisms, six of which were new. Allele and haplotype frequencies were compared between cases and controls, and, among the cases, between genders, between different phenotypes, and between patients with different mutation status in the main HSCR genes. Despite the involvement of IHH in the development of the ENS, IHH is not a major gene in HSCR. Nevertheless, as the manifestation of the HSCR phenotype is genetic background dependent, polymorphic loci should be tested simultaneously to characterize gene–gene interaction. The involvement of IHH in other intestinal anomalies should be investigated.

Allele-specific expression at the RET locus in blood and gut tissue of individuals carrying risk alleles for Hirschsprung disease.

RET common variants are associated with Hirschsprung disease (HSCR; colon aganglionosis), a congenital defect of the enteric nervous system. We analyzed a well‐known HSCR‐associated RET haplotype that encompasses linked alleles in coding and noncoding/regulatory sequences. This risk haplotype correlates with reduced level of RET expression when compared with the wild‐type counterpart. As allele‐specific expression (ASE) contributes to phenotypic variability in health and disease, we investigated whether RET ASE could contribute to the overall reduction of RET mRNA detected in carriers. We tested heterozygous neuroblastoma cell lines, ganglionic gut tissues (18 HSCR and 14 non‐HSCR individuals) and peripheral blood mononuclear cells (PBMCs; 16 HSCR and 14 non‐HSCR individuals). Analysis of the data generated by SNaPshot and Pyrosequencing revealed that the RET risk haplotype is significantly more expressed in gut than in PBMCs (P = 0.0045). No ASE difference was detected between patients and controls, irrespective of the sample type. Comparison of total RET expression levels between gut samples with and without ASE, correlated reduced RET expression with preferential transcription from the RET risk haplotype. Nonrandom RET ASE occurs in ganglionic gut regardless of the disease status. RET ASE should not be excluded as a disease mechanism acting during development.

Male and female differential reproductive rate could explain parental transmission asymmetry of mutation origin in Hirschsprung disease

Hirschsprung disease (HSCR, aganglionic megacolon) is a complex and heterogeneous disease with an incidence of 1 in 5000 live births. Despite the multifactorial determination of HSCR in the vast majority of cases, there is a monogenic subgroup for which private rare RET coding sequence mutations with high penetrance are found (45% of HSCR familial cases). An asymmetrical parental origin is observed for RET coding sequence mutations with a higher maternal inheritance. A parent-of-origin effect is usually assumed. Here we show that a differential reproductive rate for males and females also leads to an asymmetrical parental origin, which was never considered as a possible explanation till now. In the case of HSCR, we show a positive association between penetrance of the mutation and parental transmission asymmetry: no parental transmission asymmetry is observed in sporadic RET CDS mutation carrier cases for which penetrance of the mutation is low, whereas a parental transmission asymmetry is observed in affected sib-pairs for which penetrance of the mutation is higher. This allows us to conclude that the explanation for this parental asymmetry is that more severe mutations have resulted in a differential reproductive rate between male and female carriers.