Paola Griseri

Double Heterozygosity for a RET Substitution Interfering with Splicing and an EDNRB Missense Mutation in Hirschsprung Disease

The financial support of Telethon–Italy (grant E791) is gratefully acknowledged. This work was also funded by the Italian Telethon Foundation, the Italian Ministry of Health, and the European Community (contract MH4-CT97-2107).

Single nucleotide polymorphic alleles in the 5′ region of the RET proto-oncogene define a risk haplotype in Hirschsprung’s disease

Hirschsprung’s disease is a congenital disorder characterised by intestinal obstruction caused by the absence of parasympathetic intrinsic ganglion cells along variable lengths of the colon.1 The high proportion of sporadic cases (80–90%), the variable expressivity, the incomplete sex dependent penetrance, and the involvement of several genes, most of which are yet to be identified, show a complex pattern of inheritance for this disorder.2–4 The RET proto-oncogene is the major gene involved in Hirschsprung’s disease, accounting for a high proportion of both familial (about 50%) and sporadic cases (10–15%). Five to ten per cent of patients show alterations in other genes such as the glial cell line derived neurotrophic factor ( GDNF ), neurturin ( NTN ), endothelin 3 ( EDN3 ), endothelin B receptor ( EDNRB ), endothelin converting enzyme 1 ( ECE1 ), transcriptional factor SOX10 , and Smad interacting protein 1 ( SIP1 ).1,5 The small number of affected patients with known mutations confirms the involvement of modifier genes or additional genetic risk factors, some of which are already mapped,3,4,6 in the aetiology of the disease. According to what was expected for a complex inheritance pattern, several common polymorphisms of the RET proto-oncogene have been associated with a variable risk of developing Hirschsprung’s disease. Moreover, specific RET haplotypes have been found to have either protective or predisposing effects, or to modulate the severity of the resulting phenotype.6–12 In particular, specific haplotypes comprising the rarer allele of a single nucleotide polymorphism (SNP) of exon 2 (A45A) have been strongly associated with Hirschsprung’s disease, whereas the haplotype including the rarer allele of exon 14 SNP (S836S) has shown a low penetrant protective effect against the disease.11,12 Recently, Borrego et al have extended the genetic analysis of the SNP2 associated predisposing haplotype for Hirschsprung’s disease, hypothesising the existence of a …

Letters to the EditorDouble Heterozygosity for a RET Substitution Interfering with Splicing and an EDNRB Missense Mutation in Hirschsprung Disease

The financial support of Telethon–Italy (grant E791) is gratefully acknowledged. This work was also funded by the Italian Telethon Foundation, the Italian Ministry of Health, and the European Community (contract MH4-CT97-2107).

A Rare Haplotype of the RET Proto-Oncogene Is a Risk-Modifying Allele in Hirschsprung Disease

Hirschsprung disease (HSCR) is a common genetic disorder characterized by intestinal obstruction secondary to enteric aganglionosis. HSCR demonstrates a complex pattern of inheritance, with the RET proto-oncogene acting as a major gene and with several additional susceptibility loci related to the Ret-signaling pathway or to other developmental programs of neural crest cells. To test how the HSCR phenotype may be affected by the presence of genetic variants, we investigated the role of a single-nucleotide polymorphism (SNP), 2508C-->T (S836S), in exon 14 of the RET gene, characterized by low frequency among patients with HSCR and overrepresentation in individuals affected by sporadic medullary thyroid carcinoma. Typing of several different markers across the RET gene demonstrated that a whole conserved haplotype displayed anomalous distribution and nonrandom segregation in families with HSCR. We provide genetic evidence about a protective role of this low-penetrant haplotype in the pathogenesis of HSCR and demonstrate a possible functional effect linked to RET messenger RNA expression.

Molecular mechanisms of RET‐induced Hirschsprung pathogenesis

The RET proto‐oncogene is the major gene involved in the pathogenesis of Hirschsprung (HSCR), a complex genetic disease characterized by lack of ganglia along variable lengths of the gut. Here we present a survey of the different molecular mechanisms through which RET mutations lead to the disease development. Among these, loss of function, gain of function, apoptosis, aberrant splicing and decreased gene expression are exemplified and considered with respect to their pathogenetic impact. In particular, RET transcription regulation represents a new insight into the outline of HSCR susceptibility, and having reached important progress in the last few years, deserves to be reviewed. Notably, gene expression impairment seems to be at the basis of the association of HSCR disease with several RET polymorphisms, allowing us to define a predisposing haplotype spanning from the promoter to exon 2. Putative functional variants, in the promoter and in intron 1, and proposed as low penetrant predisposing alleles, are presented and discussed. Finally, based on the RET mutation effects thus summarized, we attempt to derive conclusions which may be useful for HSCR risk prediction and genetic counselling.

Haplotypes of the Human RET Proto‐oncogene Associated with Hirschsprung Disease in the Italian Population Derive from a Single Ancestral Combination of Alleles

The RET proto‐oncogene is the major gene involved in the complex genetics of Hirschsprung disease (HSCR), or aganglionic megacolon, showing causative loss‐of‐function mutations in 15–30% of the sporadic cases. Several RET polymorphisms and haplotypes have been described in association with the disease, suggesting a role for this gene in HSCR predisposition, also in the absence of mutations in the coding region. Finally, the presence of a functional variant in intron 1 has repeatedly been proposed to explain such findings. Here we report a case‐control study conducted on 97 Italian HSCR sporadic patients and 85 population matched controls, using 13 RET polymorphisms distributed throughout the gene, from the basal promoter to the 3′UTR. Linkage disequilibrium and haplotype analyses have shown increased recombination between the 5′ and 3′ portions of the gene and an over‐representation, in the cases studied, of two haplotypes sharing a common allelic combination that extends from the promoter up to intron 5. We propose that these two disease‐associated haplotypes derive from a single founding locus, extending up to intron 19 and successively rearranged in correspondence with a high recombination rate region located between the proximal and distal portions of the gene. Our results suggests the possibility that a common HSCR predisposing variant, in linkage disequilibrium with such haplotypes, is located further downstream than the previously suggested interval encompassing intron 1.

Induction of RET Dependent and Independent Pro-Inflammatory Programs in Human Peripheral Blood Mononuclear Cells from Hirschsprung Patients

Hirschsprung disease (HSCR) is a rare congenital anomaly characterized by the absence of enteric ganglia in the distal intestinal tract. While classified as a multigenic disorder, the altered function of the RET tyrosine kinase receptor is responsible for the majority of the pathogenesis of HSCR. Recent evidence demonstrate a strong association between RET and the homeostasis of immune system. Here, we utilize a unique cohort of fifty HSCR patients to fully characterize the expression of RET receptor on both innate (monocytes and Natural Killer lymphocytes) and adaptive (B and T lymphocytes) human peripheral blood mononuclear cells (PBMCs) and to explore the role of RET signaling in the immune system. We show that the increased expression of RET receptor on immune cell subsets from HSCR individuals correlates with the presence of loss-of-function RET mutations. Moreover, we demonstrate that the engagement of RET on PBMCs induces the modulation of several inflammatory genes. In particular, RET stimulation with glial-cell line derived neurotrophic factor family (GDNF) and glycosyl-phosphatidylinositol membrane anchored co-receptor α1 (GFRα1) trigger the up-modulation of genes encoding either for chemokines (CCL20, CCL2, CCL3, CCL4, CCL7, CXCL1) and cytokines (IL-1β, IL-6 and IL-8) and the down-regulation of chemokine/cytokine receptors (CCR2 and IL8-Rα). Although at different levels, the modulation of these “RET-dependent genes” occurs in both healthy donors and HSCR patients. We also describe another set of genes that, independently from RET stimulation, are differently regulated in healthy donors versus HSCR patients. Among these “RET-independent genes”, there are CSF-1R, IL1-R1, IL1-R2 and TGFβ-1, whose levels of transcripts were lower in HSCR patients compared to healthy donors, thus suggesting aberrancies of inflammatory responses at mucosal level. Overall our results demonstrate that immune system actively participates in the physiopathology of HSCR disease by modulating inflammatory programs that are either dependent or independent from RET signaling.

The involvement of the RET variant G691S in medullary thyroid carcinoma enlightened by a meta‐analysis study

Medullary thyroid carcinoma (MTC) is a rare tumor, partially explained by mutations in the rearranged during transfection (RET) proto‐oncogene. The nonsynonymous RET polymorphism G691S has been reported as associated with MTC, but findings are discordant. We sought to clarify the role of G691S in MTCs through in silico analysis, genetic association in our patients and a meta‐analysis with extensive literature revision. Ninety‐three Italian patients were compared to 85 healthy individuals. Results were included in a meta‐analysis together with 11 case‐control association studies identified through PubMed, EMBASE and Web of Science, with a combined sample of 968 cases and 2,115 controls. No association of G691S with MTC was found in our sample; however, we observed an excess of homozygotes for the variant, significantly higher among females. The overall allelic association in the meta‐analysis was significant under the fixed‐effect model (odds ratio [OR] = 1.22 [95% confidence intervals: 1.06–1.39], p = 0.0049), but borderline under the random effect model (OR = 1.21 [0.99–1.46], p = 0.0575), with a moderate/high heterogeneity (I2 = 44.6%, p = 0.047). Under the recessive model of transmission, applied to the eight studies with available genotype frequencies, results were significant under both effect models (OR = 2.016 and OR = 2.022, p = 0.0004). No heterogeneity was anymore detectable. In silico analyses on G691S confirmed a change of the phosphorylation pattern that might account for the enhanced signaling transduction previously reported for G691S in several cancers, thus also explaining its overrepresentation in MTCs. The G691S variant allele does increase the risk for MTC, with a recessive mechanism of action, apparently more evident among females.

Complex pathogenesis of Hirschsprung's disease in a patient with hydrocephalus, vesico-ureteral reflux and a balanced translocation t(3;17)(p12;q11)

Hirschsprungs disease (HSCR), a congenital complex disorder of intestinal innervation, is often associated with other inherited syndromes. Identifying genes involved in syndromic HSCR cases will not only help understanding the specific underlying diseases, but it will also give an insight into the development of the most frequent isolated HSCR. The association between hydrocephalus and HSCR is not surprising as a large number of patients have been reported to show the same clinical association, most of them showing mutations in the L1CAM gene, encoding a neural adhesion molecule often involved in isolated X-linked hydrocephalus. L1 defects are believed to be necessary but not sufficient for the occurrence of the intestinal phenotype in syndromic cases. In this paper, we have carried out the molecular characterization of a patient affected with Hirschsprungs disease and X-linked hydrocephalus, with a de novo reciprocal balanced translocation t(3;17)(p12;q21). In particular, we have taken advantage of this chromosomal defect to gain access to the predisposing background possibly leading to Hirschsprungs disease. Detailed analysis of the RET and L1CAM genes, and molecular characterization of MYO18A and TIAF1, the genes involved in the balanced translocation, allowed us to identify, besides the L1 mutation c.2265delC, different additional factors related to RET-dependent and -independent pathways which may have contributed to the genesis of enteric phenotype in the present patient.

Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes.

BackgroundHirschsprung disease (HSCR), which is congenital obstruction of the bowel, results from a failure of enteric nervous system (ENS) progenitors to migrate, proliferate, differentiate, or survive within the distal intestine. Previous studies that have searched for genes underlying HSCR have focused on ENS-related pathways and genes not fitting the current knowledge have thus often been ignored. We identify and validate novel HSCR genes using whole exome sequencing (WES), burden tests, in silico prediction, unbiased in vivo analyses of the mutated genes in zebrafish, and expression analyses in zebrafish, mouse, and human.ResultsWe performed de novo mutation (DNM) screening on 24 HSCR trios. We identify 28 DNMs in 21 different genes. Eight of the DNMs we identified occur in RET, the main HSCR gene, and the remaining 20 DNMs reside in genes not reported in the ENS. Knockdown of all 12 genes with missense or loss-of-function DNMs showed that the orthologs of four genes (DENND3, NCLN, NUP98, and TBATA) are indispensable for ENS development in zebrafish, and these results were confirmed by CRISPR knockout. These genes are also expressed in human and mouse gut and/or ENS progenitors. Importantly, the encoded proteins are linked to neuronal processes shared by the central nervous system and the ENS.ConclusionsOur data open new fields of investigation into HSCR pathology and provide novel insights into the development of the ENS. Moreover, the study demonstrates that functional analyses of genes carrying DNMs are warranted to delineate the full genetic architecture of rare complex diseases.