Giovanna Patrone

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 …

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.

A 5′-CG-3′-rich region in the promoter of the transcriptionally frequently silenced RET protooncogene lacks methylated cytidine residues

In a large proportion of familial and sporadic cases of Hirschsprung disease (HSCR) mutations in the RET (rearranged during transfection) protooncogene have been described. We have investigated the structure of the RET gene promoter and have analysed a region of approximately 1000 nucleotides in its promoter and 5′-upstream segments for the occurrence of 5-methyldeoxy-cytidine (5-mC) residues by using the bisulfite protocol of the genomic sequencing method. With an estimated sensitivity of about 93% of this technique, not a single 5-mC residue could be detected in the control region of a gene that seems to be silenced or exhibit low activity in many adult tissues. In these experiments, the DNAs of peripheral white blood cells (PWBC) from four healthy individuals, from seven patients with familial HSCR, as well as DNAs from different human tissues and from a human embryonic kidney (HEK) cell line have been included. In a DNA segment starting 790 nucleotides upstream of the transcriptional start site of the RET gene, a few 5-mC residues have been identified. This region possibly constitutes the transition site from an unmethylated promoter to a more extensively methylated region in the human genome. The data presented are remarkable in that a highly 5′-CG-3′-enriched segment of the human genome with 49 5′-CG-3′ dinucleotide pairs in 400 bp within the putative promoter region is completely devoid of 5-mC residues, although this control region is not actively transcribed in most adult human tissues. By hybridization of a PCR-amplified RET protooncogene cDNA probe harboring exons 9–15 to a membrane (Clontech) containing poly-A selected RNAs from 50 different human tissues, weak RET protooncogene expression in many of the neural cell derived tissues has been detected. RNAs extracted from many other human tissues do not share sequence homologies to this 32P-labeled probe. Mechanisms other than DNA methylation obviously play the crucial role in the inactivation of the RET gene promoter in these tissues. We have also demonstrated by the in vitro premethylation of a RET promoter-chloramphenicol acetyltransferase (CAT) gene construct and transient transfection experiments into neuroblastoma cells that the transcriptional activity of the RET promoter is decreased by HpaII (5′-CCGG-3′) methylation and abolished by SssI (5′-CG-3′) methylation. Hence, the RET promoter region is sensitive to this regulatory signal. However in vivo, DNA methylation of the promoter region seems not to be the predominant regulatory mechanism for the RET protooncogene. Possibly, in adults the RET gene can be occasionally activated.

Sequence and characterisation of the RET proto-oncogene 5' flanking region: Analysis of retinoic acid responsiveness at the transcriptional level

The RET proto‐oncogene encodes a receptor tyrosine kinase expressed during neural crest development. RET expression is enhanced in vitro by several differentiating agents, including retinoic acid (RA), which up‐regulates RET expression in neuroblastoma cell lines. In the present work we sequenced and analysed a 5 kbp genomic fragment 5′ to RET. Three deletion fragments of this region were tested for their RA inducibility in transient transfection assays and failed to support the hypothesis of a direct transcriptional activation. Finally, our functional analysis of a candidate RA response element present in the RET promoter provides new hints for the understanding of the interaction between nuclear receptors and their specific recognition sites.

Rescue of human RET gene expression by sodium butyrate: a novel powerful tool for molecular studies in Hirschsprung disease

Background: The RET gene encodes a tyrosine kinase receptor involved in different human neurocristopathies, such as specific neuroendocrine tumours and Hirschsprung disease (HSCR). Gene expression is developmentally regulated and the RET transcript is undetectable in most adult cells, including lymphocytes. The impossibility of performing functional studies on RET mRNA has to date limited the detection and characterisation of an indefinite proportion of gene anomalies that cannot be identified by conventional DNA genomic screening in HSCR cases. Aims: Development of a protocol suitable to activate RET expression in RET negative cell lines and therefore to investigate directly RET mRNA, extending the conventional gene mutation analysis to detection of splicing anomalies and impaired expression of the RET gene. Methods: The effect of sodium butyrate (NaB), a histone deacetylase inhibitor, on rescuing RET expression was tested by one round of reverse transcription- polymerase chain reaction from total RNA of treated lymphoblasts from both HSCR patients and control individuals. Results: Analysis of RET expression was possible by NaB treatment of RET negative cells, such as lymphoblasts. This treatment allowed us to detect impaired RET expression as well as a splicing defect in two HSCR patients previously believed to be devoid of any gene abnormality. Conclusions: The full application of the proposed protocol in most of the unexplained HSCR cases will allow us to establish the precise role of RET not only in causing but also in predisposing to HSCR pathogenesis.

Cell-line specific transcription rates of the RET gene and functional domains in its minimal promoter

To gain insight into the RET gene transcriptional regulation we analysed its mRNA expression, transcription rate, and promoter activity in different cell lines, showing that RET transcription is highly cell-line specific. By using a panel of promoter deletion constructs in transient transfection assays we identified the −147/+53 fragment as the main functional element. GMSA experiments indicated binding by Sp1 and a CACCC binding protein to multiple sites within the promoter. Upon deletion analysis Sp1 appeared to be the main positive regulator of promoter activity. The −147/+53 sequence did not reproduce the cell-line specific activity of the endogenous gene in vitro, raising the question of how the regulated pattern of RET transcription is achieved at the molecular level. To address this topic, both the search for far-sited tissue-specific enhancers/silencers and the study of chromatin structure within the RET locus should be envisaged.

Erratum: Cell-line specific chromatin acetylation at the Sox10-Pax3 enhancer site modulates the RET proto-oncogene expression (FEBS 26252) (FEBS Letters (2002) 523 (123-127) PII: S0014579302029575)

aLaboratory of Molecular Genetics, G. Gaslini Institute, Largo G. Gaslini 5, 16148 Genova, Italy bLaboratory of Pathology, Center of Biotechnology, University of Urbino, 61029 Urbino (PU), Italy cDepartment of Internal Medicine, Angiology and Hepatology, University of Bologna, 40100 Bologna, Italy dDepartment of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy eDepartment of Oncology, Biology and Genetics, University of Genova, 16100 Genova, Italy

Erratum to: Cell-line specific chromatin acetylation at the Sox10-Pax3 enhancer site modulates theRETproto-oncogene expression (FEBS 26252): [FEBS Letters 523 (2002) 123-127]

aLaboratory of Molecular Genetics, G. Gaslini Institute, Largo G. Gaslini 5, 16148 Genova, Italy bLaboratory of Pathology, Center of Biotechnology, University of Urbino, 61029 Urbino (PU), Italy cDepartment of Internal Medicine, Angiology and Hepatology, University of Bologna, 40100 Bologna, Italy dDepartment of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy eDepartment of Oncology, Biology and Genetics, University of Genova, 16100 Genova, Italy