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Higuchi T, Yoshizawa K, Hatata T, Yoshizawa K, Takamizawa S, Kobayashi J, Kubota N, Hidaka E. Novel Causative RET Mutation in a Japanese Family with Hirschsprung's Disease: Case Report and Factors Impacting Disease Severity. J Pediatr Genet 2020; 11:240-244. [DOI: 10.1055/s-0040-1718385] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2020] [Accepted: 08/29/2020] [Indexed: 10/23/2022]
Abstract
Abstract
RET gene variances confer susceptibility to Hirschsprung's disease (HSCR) with pathogenetic mutations being identified in half of familial cases. This investigation of familial HSCR was aimed to clarify the relationship between genetic mutations and clinical phenotype using next-generation sequencing. A novel c2313C > G(D771E) RET mutation was identified in all three affected family members. The mutation involved the kinase domain, which is believe to impair RET activity and intestinal function. A second RET mutation, c1465G > A(D489N), was found only in the extensive aganglionosis case. We conclude that the novel c2313C > A(D771E) mutation in RET may be pathogenic for HSCR, while the c1465C > G(D489N) mutation may be related to phenotype severity.
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Affiliation(s)
- Tsukasa Higuchi
- Department of General Pediatrics, Nagano Children's Hospital, Azumino, Japan
- Life Science Research Center, Nagano Children's Hospital, Azumino, Japan
| | - Kazuki Yoshizawa
- Department of Pediatric Surgery, Nagano Children's Hospital, Azumino, Japan
| | - Tomoko Hatata
- Department of Pediatric Surgery, Nagano Children's Hospital, Azumino, Japan
| | - Katsumi Yoshizawa
- Department of Pediatric Surgery, Nagano Children's Hospital, Azumino, Japan
| | - Shigeru Takamizawa
- Department of Pediatric Surgery, Nagano Children's Hospital, Azumino, Japan
| | - Jun Kobayashi
- Life Science Research Center, Nagano Children's Hospital, Azumino, Japan
- Department of Clinical Laboratory, Nagano Children's Hospital, Azumino, Japan
| | - Noriko Kubota
- Life Science Research Center, Nagano Children's Hospital, Azumino, Japan
- Department of Clinical Laboratory, Nagano Children's Hospital, Azumino, Japan
| | - Eiko Hidaka
- Life Science Research Center, Nagano Children's Hospital, Azumino, Japan
- Department of Clinical Laboratory, Nagano Children's Hospital, Azumino, Japan
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Exome Sequencing Identifies RET Associated Hirschsprung Disease in a Fetus with Echogenic Bowel. JOURNAL OF FETAL MEDICINE 2019. [DOI: 10.1007/s40556-019-00212-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
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Virtanen VB, Salo PP, Cao J, Löf-Granström A, Milani L, Metspalu A, Rintala RJ, Saarenpää-Heikkilä O, Paunio T, Wester T, Nordenskjöld A, Perola M, Pakarinen MP. Noncoding RET variants explain the strong association with Hirschsprung disease in patients without rare coding sequence variant. Eur J Med Genet 2019; 62:229-234. [DOI: 10.1016/j.ejmg.2018.07.019] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2018] [Revised: 06/06/2018] [Accepted: 07/17/2018] [Indexed: 02/04/2023]
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Moore SW. Advances in understanding the association between Down syndrome and Hirschsprung disease (DS-HSCR). Pediatr Surg Int 2018; 34:1127-1137. [PMID: 30218169 DOI: 10.1007/s00383-018-4344-z] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 09/10/2018] [Indexed: 02/07/2023]
Abstract
The clinical association between Trisomy 21 (Down syndrome) and aganglionosis (Hirschsprung disease; DS-HSCR) is well-established, being of the order of 5% and remains the most common congenital association with Hirschsprung disease. However, little consensus exists as to the possible etiologic and genetic factors influencing this association. Recent research has identified a number of levels at which development of the enteric nervous system is potentially affected in Trisomy 21. These include a decreased central pool of available neuroblasts for migration into the enteric nervous system, abnormal neuroblast type, poor synaptic nerve function and early germline gene-related influences on the migrating neuroblasts due to genetic mutations of a number of important developmental genes, and possible somatic mutations resulting from alterations in the local tissue microenvironment. In this paper, we review available evidence for this association. In addition, we provide evidence of both germline and somatic gene mutations suggesting causation. Although the picture is complex, recent associations between specific RET proto-oncogene variations have been shown to be significant in Down syndrome patients with Hirschsprung disease, as they probably interfere with vital RET functions in the development of the autonomic and enteric nervous systems, increasing the risk of disturbed normal function. In addition, we explore potential role of other facilitatory influence of other susceptibility genes as well as potential other chromosome 21 gene actions and the microenvironment on the Down syndrome gastro-intestinal tract. The various ways in which trisomy of chromosome influences the enteric nervous system are becoming clearer. The sum of these effects influences the outcome of surgery in Down syndrome patients with Hirschsprung Disease.
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Affiliation(s)
- S W Moore
- Division of Paediatric Surgery, Faculty of Medicine and Health Sciences, University of Stellenbosch, PO Box 241, Cape Town, South Africa.
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Abstract
PURPOSE During the past two decades several genes have been identified that control morphogenesis and differentiation of the enteric neuron system (ENS). These genes, when mutated or deleted, interfere with ENS development. RET gene is the major gene causing Hirschsprung's disease (HD). Mutations in RET gene are responsible for 50% of familial HD cases and 15-20% of sporadic cases. The aim of this meta-analysis was to determine the incidence of RET gene mutations in patients with HD and to correlate RET mutations with the extent of aganglionosis. METHODS A systematic literature-based search for relevant cohorts was performed using the terms "Hirschsprung's disease AND RET Proto-oncogene", "Hirschsprung's disease AND genetic polymorphism" and "RET Gene". The relevant cohorts of HD were systematically searched for reported mutations in the RET gene (RET+). Data on mutation site, phenotype, and familial or sporadic cases were extracted. Combined odds ratio (OR) with 95% CI was calculated to estimate the strength of the different associations. RESULTS In total, 23 studies concerning RET with 1270 individuals affected with HD were included in this study. 228 (18%) of these HDs were RET+. Of these 228, 96 (42%) presented as rectosigmoid, 81 (36%) long segment, 18 (8%) as TCA, 16 (7%) as total intestinal aganglionosis and 17 (7%) individuals were RET+ but no extent of aganglionosis was not reported. In the rectosigmoid group, no significant association between phenotype and RET mutation could be shown (P = 0.006), whereas a clear association could be shown between long-segment disease, total colonic- and total intestinal aganglionosis and RET mutations (P = 0.0002). Mutations most often occurred in Exon 13 (24) and showed significant association with rectosigmoid disease (P = 0.004). No significance could be shown between RET+ and sporadic cases (P = 0.53), albeit a trend towards RET+ and Familial cases could be observed (P = 0.38). CONCLUSIONS The association with the RET gene and HD is well recognized. This study showed a clear association between RET+ mutations and the long-segment, total colonic- and total intestinal aganglionosis. Exon 13 appears to be a mutational "hot spot" in rectosigmoid disease.
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Moore SW, Zaahl M. Clinical and genetic correlations of familial Hirschsprung's disease. J Pediatr Surg 2015; 50:285-8. [PMID: 25638620 DOI: 10.1016/j.jpedsurg.2014.11.016] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/25/2014] [Accepted: 11/02/2014] [Indexed: 02/04/2023]
Abstract
BACKGROUND The risk of familial transmission in Hirschsprung's disease (HSCR) currently lacks correlation between the clinical phenotype and the underlying genetic factors. The aim of this study was to clinically evaluate familial HSCR transmission and to correlate with the genetic background. METHODS Clinical and gene analysis of familial HSCR patients were explored. DNA from 45 patients (35 kindreds) was screened for genetic variations of the RET, and EDNRB genes were screened for genetic variation by semi-automated bi-directional sequencing analysis and matched to controls. MAIN RESULTS Male:female ratio (3:1) had a female proband in 4 families. Aganglionosis was significantly more frequent with total colonic aganglionosis (TCA) in 40% familial cases (viz: 17/43 (43%) vs. 19/342 non-familial patients (5.6%) (p<0.01)). Transmission of S-HSCR was observed in 13 (31%), which was associated with EDNRB variation. RET gene promoter variation correlated with extended aganglionosis in 6/35 kindreds (17%). In 3 kindreds, both significant EDNRB and RET mutations were identified and where present were associated with increased penetrance in succeeding generations. An increased penetrance with succeeding generations occurred in 6 (14%). In a further 3 generation family, extensive variations in exon 6, 13, and 18 affected 3 males with progressive penetration and aganglionic length, including total intestinal aganglionosis in the further offspring. RET and MEN association was noted in 5 kindreds (14.3%) related to RET variations at Cysteine sites. CONCLUSIONS Cumulative effects of the RET and EDNRB genes contribute to long-segment and total colonic aganglionosis.
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Affiliation(s)
- Sam W Moore
- Division of Paediatric Surgery, University of Stellenbosch, Tygerberg, Western Cape, South Africa.
| | - Monique Zaahl
- Division of Paediatric Surgery, University of Stellenbosch, Tygerberg, Western Cape, South Africa
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Ngo DN, So MT, Gui H, Tran AQ, Bui DH, Cherny S, Tam PKH, Nguyen TL, Garcia-Barcelo MM. Screening of the RET gene of Vietnamese Hirschsprung patients identifies 2 novel missense mutations. J Pediatr Surg 2012; 47:1859-64. [PMID: 23084198 DOI: 10.1016/j.jpedsurg.2012.05.020] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/06/2012] [Revised: 05/14/2012] [Accepted: 05/15/2012] [Indexed: 12/28/2022]
Abstract
BACKGROUND/PURPOSE Hirschsprung disease (HSCR; megacolon, congenital aganglionosis) is a congenital disorder characterized by the absence of ganglion cells along variable segments of the gut. Both rare (RV) and common variants of the RET gene are associated with HSCR. The aim of this study is to assess, for the first time, the variation in the RET gene of Vietnamese HSCR patients. METHODS We used Sanger sequencing to screen the coding sequence of the RET gene of 97 Vietnamese HSCR patients of Southern Chinese ancestry. The healthy population consisted of 250 Southern Chinese individuals with no diagnosis of HSCR. RESULTS We detected 8 heterozygous RVs distributed among 13 patients (13.40%) and that were not present in healthy individuals. Among those variants, there were 2 novel and deleterious (R133C [c.397 C>T]; R144C [c.430 C>T]) missense amino acid substitutions, 2 novel silent variants (P667P [c.2001 A>T]; Y809Y [c.2427 C>T]), and 4 previously described missense substitutions (R114H [c.341 G>A]; V292M [c.874 G>A]; G533S [c.1597 G>A]; R982C [c.2944 C>T]). As expected, the common RET coding sequence variants rs1800858 (A45A [c.135 G>A]) and rs1800861 (L769L [c.2307 T>G]) were highly associated with the disease. CONCLUSIONS The identification of novel deleterious variants together with the fact RET RVs are virtually unique to HSCR patients indicates that the RET gene is a target for mutations among Hirschsprung patients of any population.
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Affiliation(s)
- Diem-Ngoc Ngo
- Department of Human Genetics, National Hospital of Pediatrics, Hanoi, Vietnam
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Hyndman BD, Gujral TS, Krieger JR, Cockburn JG, Mulligan LM. Multiple functional effects of RET kinase domain sequence variants in Hirschsprung disease. Hum Mutat 2012; 34:132-42. [PMID: 22837065 DOI: 10.1002/humu.22170] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2011] [Accepted: 07/16/2012] [Indexed: 01/08/2023]
Abstract
The REarranged during Transfection (RET) gene encodes a receptor tyrosine kinase required for maturation of the enteric nervous system. RET sequence variants occur in the congenital abnormality Hirschsprung disease (HSCR), characterized by absence of ganglia in the intestinal tract. Although HSCR-RET variants are predicted to inactivate RET, the molecular mechanisms of these events are not well characterized. Using structure-based models of RET, we predicted the molecular consequences of 23 HSCR-associated missense variants and how they lead to receptor dysfunction. We validated our predictions in biochemical and cell-based assays to explore mutational effects on RET protein functions. We found a minority of HSCR-RET variants abrogated RET kinase function, while the remaining mutants were phosphorylated and transduced intracellular signals. HSCR-RET sequence variants also impacted on maturation, stability, and degradation of RET proteins. We showed that each variant conferred a unique combination of effects that together impaired RET protein activity. However, all tested variants impaired RET-mediated cellular functions, including cell transformation and migration. Our data indicate that the molecular mechanisms of impaired RET function in HSCR are highly variable. Although a subset of variants cause loss of RET kinase activity and downstream signaling, enzymatic inactivation is not the sole mechanism at play in HSCR.
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Affiliation(s)
- Brandy D Hyndman
- Division of Cancer Biology and Genetics, Cancer Research Institute, and Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON, Canada
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Abstract
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.
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Gartside MG, Chen H, Ibrahimi OA, Byron SA, Curtis AV, Wellens CL, Bengston A, Yudt LM, Eliseenkova AV, Ma J, Curtin JA, Hyder P, Harper UL, Riedesel E, Mann GJ, Trent JM, Bastian BC, Meltzer PS, Mohammadi M, Pollock PM. Loss-of-function fibroblast growth factor receptor-2 mutations in melanoma. Mol Cancer Res 2009; 7:41-54. [PMID: 19147536 DOI: 10.1158/1541-7786.mcr-08-0021] [Citation(s) in RCA: 100] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
We report that 10% of melanoma tumors and cell lines harbor mutations in the fibroblast growth factor receptor 2 (FGFR2) gene. These novel mutations include three truncating mutations and 20 missense mutations occurring at evolutionary conserved residues in FGFR2 as well as among all four FGFRs. The mutation spectrum is characteristic of those induced by UV radiation. Mapping of these mutations onto the known crystal structures of FGFR2 followed by in vitro and in vivo studies show that these mutations result in receptor loss of function through several distinct mechanisms, including loss of ligand binding affinity, impaired receptor dimerization, destabilization of the extracellular domains, and reduced kinase activity. To our knowledge, this is the first demonstration of loss-of-function mutations in a class IV receptor tyrosine kinase in cancer. Taken into account with our recent discovery of activating FGFR2 mutations in endometrial cancer, we suggest that FGFR2 may join the list of genes that play context-dependent opposing roles in cancer.
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Affiliation(s)
- Michael G Gartside
- Division of Cancer and Cell Biology, Translational Genomics Research Institute, Phoenix, AZ 85004, USA
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Abstract
Down syndrome (DS) is the most common chromosomal abnormality occurring in humans. Up to 77% of DS children have associated gastrointestinal (GI) abnormalities, which may be structural or functional in nature. Functional disturbances may, in turn, affect the outcome of corrective surgical procedures, prompting to caution. It is becoming clear that the processes affecting the enteric nervous system (ENS) in DS not only affect the micro-anatomy but also nerve function, and there is some histological evidence of ENS variations in both human and DS animal models. This suggests that developmental disorders of the ENS are probably fundamental to the functional GI disturbances encountered in patients with DS. The anomalous brain development, function and resulting intellectual impairment associated with DS appears to result from the genetic imbalance created by the trisomy of chromosome 21. The possible links between the brain, GI and ENS involvement are not as yet entirely clear. Neurotropic factors affecting brain development during embryogenesis are probably interlinked with ENS development, but the precise mechanism of how this occurs has yet to be established. This study explores what is known about the ENS dysfunction in DS and reviews the possible importance of chromosome 21 located and other genes in its etiology. Functional motor disturbances of the esophagus and colon are not uncommon and may be congenital or acquired in nature. The most prominent of these include esophageal dysmotility syndromes (e.g. achalasia, gastroesophageal reflux, dysphagia) as well as a higher incidence of chronic constipation and Hirschsprung's disease (HSCR) (2-15%) occurring in association with DS. Chromosome 21 itself is thought to be the site of a modifier gene for HSCR. Recently identified candidate genetic mechanisms provide unique insights into the genetic background of the neurological and cognitive disorders associated with DS. Although the role of the triplicated chromosome 21 and genetic dosage remain important, the additional role of other chromosome 21 genes in the etiology of ENS developmental anomalies remains undetermined and requires ongoing research.
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Affiliation(s)
- S W Moore
- Division of Paediatric Surgery, Department of Surgical Sciences, Faculty of Health Sciences, University of Stellenbosch, P.O. Box 19063, Tygerberg, 7505, South Africa.
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Abstract
BACKGROUND The cause of Hirschsprung's disease (HD) remains unclear, but currently there are two theories: the mutation of the RET gene and the change of enteric microenvironment. This study was undertaken to elucidate the cause of HD by assessing the expression of laminin (LN), laminin gene, and the RET gene in the aganglionic segment, transitional zone and normal segment of the colon in patients with HD. METHODS Specimens of the aganglionic segment, transitional zone, and normal segment of the colon from 27 cases of HD were stained immunohistologically by a PV 9000 polymer detection system. Photos were taken by the RS image system, and the staining area of each image was calculated by a JD 801 image analysis system. The qualitative expressions of the laminin gene and RET gene of these three segments in the 27 cases were detected by reverse transcription-polymerase chain reaction (RT-PCR), and the difference of the expressions was shown by the alpha 9900 image analysis system. The quantitative expressions of the laminin gene and RET gene in the three segments were detected by real-time quantitative PCR, and the difference of the expression was shown by SDS software. RESULTS The laminin and laminin gene were expressed in all the three segments. The expression was higher in the aganglionic segment than in the dilated segment, and the expression decreased stepwisely from the aganglionic segment to the normal segment, while the expression of the RET gene was opposite, showing an increased segmenting from the aganglionic segment to the normal segment. The correlation between the expressions of the two genes was negatively correlated. CONCLUSIONS The highly increased expression of LN in the aganglionic segment may cause early differentiation, early maturation and premature ecesis of enteric nervous cells. The change of the microenvironment of colon wall may be the cause of HD. The negative correlation between the expression of the two genes may be closely related to the occurrence of HD.
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Moore SW, Zaahl MG. A review of genetic mutation in familial Hirschsprung's disease in South Africa: towards genetic counseling. J Pediatr Surg 2008; 43:325-9. [PMID: 18280283 DOI: 10.1016/j.jpedsurg.2007.10.021] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/06/2007] [Accepted: 10/09/2007] [Indexed: 10/22/2022]
Abstract
BACKGROUND Hirschsprung's disease (HSCR) represents a complex disorder of signaling molecules, resulting from the effects of at least 9 known susceptibility genes. Affected families carry 200 times higher risk, but genetic counseling via pedigree analysis is difficult and the significance of genetic variations is unclear. This study evaluated a set of patients affected by HSCR with familial recurrence to evaluate factors of greatest value in genetic counseling. PATIENTS AND METHODS One hundred twenty patients with HSCR (including 18 kindreds) were screened for genetic variations of the 2 major susceptibility genes (RET and endothelin B receptor [EDNRB]) and compared with 60 control samples (20 per ethnic group). Familial recurrence patterns were studied for patient sex, pattern of recurrence, presence of associated syndromic features, and genetic features of major susceptibility genes. Polymerase chain reaction and HEX-SSCP analysis were performed on DBA extracted from blood/microdissected tissue samples. SSCP variants were validated and automated sequencing techniques performed on polymerase chain reaction products showing conformational variants in acrylamide gel. RESULTS Familial cases had a male-female ratio of 1.5:1, male-to-male transmission (n = 10; 2 father to son), female-to-male transmissions (n = 4; 3 female carriers, female-to-female (n = 4; 2 mother to daughter), and 1 paternal RET deletion-female with very long segment aganglionosis. Increasing gene penetrance occurred in 3 pedigrees. An increased incidence of long segment HSCR was noted in families with recurrence and appeared important. No consistent mendelian trends or specific genetic sites were observed, but 3 suggested autosomal dominant and recessive in a further 3. Identified genetic variations included deletions, frame shifts, and missense mutations, as well as a number of significant single nucleotide polymorphism variations. Transmitted RET mutations occurred in 5 (30%) of 16 kindreds. Splice RET mutation plus variants of exon 17 (973L) affected 2 children with identical total colonic aganglionosis. In a 3-generation family, variations in RET exons 6, 13, and 18 (928) affected 3 male children with increasing penetration to recur as total intestinal aganglionosis in a grandchild. CONCLUSIONS Mendelian transmission appears mediated by the RET proto-oncogene. EDNRB mutations suggest haplotypic gene-gene interaction. Genetic counseling remains a challenge in HSCR because of its multfactorial etiology.
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Affiliation(s)
- Samuel W Moore
- Division of Pediatric Surgery, University of Stellenbosch, Tygerberg, South Africa.
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Guan T, Li JC, Li MJ, Tou JF. Polymerase chain reaction-single strand conformational polymorphism analysis of rearranged during transfection proto-oncogene in Chinese familial hirschsprung’s disease. World J Gastroenterol 2005; 11:275-9. [PMID: 15633231 PMCID: PMC4205417 DOI: 10.3748/wjg.v11.i2.275] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the relationship between mutations of rearranged during transfection (RET) proto-oncogene and Chinese patients with Hirschsprung’s disease (HD), and to elucidate the genetic mechanism of familial HD patient at the molecular level.
METHODS: Genomic DNA was extracted from venous blood of probands and their relatives in two genealogies. Polymerase chain reaction (PCR) products, which were amplified using specific primers (RET, exons 11, 13, 15 and 17), were electrophoresed to analyze the single-strand conformational polymorphism (SSCP) patterns. The positive amplified products were sequenced. Forty-eight sporadic HD patients and 30 normal children were screened for mutations of RET proto-oncogene simultaneously.
RESULTS: Three cases with HD in one family were found to have a G heterozygous insertion at nucleotide 18974 in exon 13 of RET cDNA (18974insG), which resulted in a frameshift mutation. In another family, a heterozygosity for T to G transition at nucleotide 18888 in the same exon which resulted in a synonymous mutation of Leu at codon 745 was detected in the proband and his father. Eight RET mutations were confirmed in 48 sporadic HD patients.
CONCLUSION: Mutations of RET proto-oncogene may play an important role in the pathogenesis of Chinese patients with HD. Detection of mutated RET proto-oncogene carriers may be used for genetic counseling of potential risk for HD in the affected families.
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Affiliation(s)
- Tao Guan
- Department of Lymphology, Institute of Cell Biology, Zhejiang University Medical College, Hangzhou 310031, Zhejiang Province, China
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Affiliation(s)
- Paul K H Tam
- Department of Surgery and Genome Research Centre, The University of Hong Kong, Queen Mary Hospital K15, Pokfulam, Hong Kong, P.R. China.
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Abstract
AIM: To investigate the mutation of EDNRB gene and EDN-3 gene in sporadic Hirschsprung’s disease (HD) in Chinese population.
METHODS: Genomic DNA was extracted from bowel tissues of 34 unrelated HD patients which were removed by surgery. Exon 3, 4, 6 of EDNRB gene and Exon 1, 2 of EDN-3 gene were amplified by polymerase chain reaction (PCR) and analyzed by single strand conformation polymorphism (SSCP).
RESULTS: EDNRB mutations were detected in 2 of the 13 short-segment HD. One mutant was in the exon 3, the other was in the exon 6. EDN-3 mutation was detected in one of the 13 short-segment HD and in the exon 2. Both EDNRB and EDN-3 mutations were detected in one short-segment HD. No mutations were detected in the ordinary or long-segment HD.
CONCLUSION: The mutations of EDNRB gene and EDN-3 gene are found in the short-segment HD of sporadic Hirschsprung’s disease in Chinese population, which suggests that the EDNRB gene and EDN-3 gene play important roles in the pathogenesis of HD.
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Affiliation(s)
- Xiang-Long Duan
- Department of General Surgery, Second Hospital of Xi'an Jiaotong University, Xi'an 710004, Shaanxi Province, China.
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Solari V, Ennis S, Yoneda A, Wong L, Messineo A, Höllwarth ME, Green A, Puri P. Mutation analysis of the RET gene in total intestinal aganglionosis by wave DNA fragment analysis system. J Pediatr Surg 2003; 38:497-501. [PMID: 12632375 DOI: 10.1053/jpsu.2003.50087] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
BACKGROUND/PURPOSE Total intestinal aganglionosis (TIA) extending from the duodenum to the rectum is the most rare form of Hirschprung's disease (HSCR) and usually is fatal. RET is the major gene associated with HSCR, and germline mutations of this gene account for up 50% of familial and up to 15 to 20% of sporadic cases in HSCR. The aim of this study was to investigate DNA variants in the RET gene in TIA patients using the WAVE DNA Fragment Analysis System. METHODS Genomic DNA was extracted from whole blood samples from 6 patients with TIA. Polymerase chain reaction (PCR) amplification of the 21 exons of RET was performed using published oligonucleotide primers. Heteroduplexes were followed by the WAVE DNA Fragment Analysis System with the DNASep cartridge. RESULTS WAVE system technology detected 16 variants in the RET gene in the 6 patients with TIA. Three patients had a significant mutation in exon 8, 11, and 15, respectively. Thirteen RET polymorphic variants also were detected in the 6 patients, with L746L variant in exon 13 occurring in 4 patients. CONCLUSIONS WAVE system technology is an efficient method for the detection of DNA sequence variants. Our findings suggest that not only RET mutations but also RET polymorphic variants may contribute to the occurrence of TIA.
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Affiliation(s)
- V Solari
- Children's Research Centre, Our Lady's Hospital for Sick Children, University College Dublin, Crumlin, Ireland
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Zaahl MG, du Plessis L, Warnich L, Kotze MJ, Moore SW. Significance of novel endothelin-B receptor gene polymorphisms in Hirschsprung's disease: predominance of a novel variant (561C/T) in patients with co-existing Down's syndrome. Mol Cell Probes 2003; 17:49-54. [PMID: 12628594 DOI: 10.1016/s0890-8508(03)00003-3] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
Several genes have been implicated in the pathogenesis of Hirschsprung's disease (HSCR). In a previous study performed, five novel (V202M, E480K, IVS10-2A/G, D771N, IVS19-9C/T) mutations and one previously described mutation (P937L) have been identified in the RET proto-oncogene in 20% of the study population. To further investigate the involvement of other genes, mutation analysis of the endothelin-B receptor (EDNRB) gene was performed in 52 unrelated sporadic HSCR patients, including 38 non-syndromic and 14 patients with HSCR and Down's syndrome. Six novel (178G/A, 552C/T, 561C/T, 702C/T, IVS3-6C/T and IVS4 + 3A/G) sequence variants and one previously described (831G/A) polymorphism were identified. Statistically significant differences were achieved for six (178G/A, 552C/T, 561C/T, 702C/T, IVS3-6C/T and 831G/A) of these variants. The T-allele of the 561C/T polymorphism was over represented in the HSCR/Down's syndrome patient group (36% representing 5 of 14) compared to normal controls (6% representing 5 of 84) (p < 0.002, chi(2) with Yates correction = 12.14), suggesting that the 561C/T variant is associated with a low penetrance effect in patients with this complex phenotype. Detection of the 178G/A polymorphism in only non-syndromic HSCR patients, provide further support for an important role of specific sequence variants in the EDNRB gene in the HSCR/Down's syndrome phenotype.
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Affiliation(s)
- M G Zaahl
- Division of Human Genetics, Faculty of Health Sciences, University of Stellenbosch, Tygerberg, South Africa.
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Abstract
AIM: To investigate the pathogenic mechanism of Hirschsprung’s disease (HD) at the molecular level and to elucidate the relationship between RET oncogene and Chinese patients with HD.
METHODS: Exon 13 of RET oncogene from 20 unrelated HD patients was analyzed with polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). The positive amplifying products were then sequenced. According to the results of SSCP and DNA sequence, SSCP was done as well for the samples from the family other members of some cases with mutated RET gene.
RESULTS: SSCP analysis indicated that mobility abnormality existed in 4 unrelated HD patients. Direct DNA sequence analysis identified a missense mutation, T to G at the nucleotide 18888 and a frameshift mutation at the nucleotide 18926 insG. In a HD family, the sicked child and his father were the same heterozygous missense mutation (T to G at nucleotide 18888).
CONCLUSION: Among Chinese HD patients, RET gene mutations may exist in considerable proportion with different patterns. These new discoveries indicate that RET mutations may play an important role in the pathogenesis of unrelated HD in the Chinese population. PCR-SSCP combined with DNA sequence can be used as a tool in the genetic diagnosis of HD.
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Affiliation(s)
- Ji-Cheng Li
- Department of Lymphology, Department of Histology and Embryology, Zhejiang University Medical College, Hangzhou 310031, Zhejiang Province, China.
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