Low frequency of RET mutations in Hirschsprung disease in Sweden

Hirschsprung disease is a congenital malformation, where absence of intramural ganglia in the hindgut results in a defect in the coordination of peristaltic movement. This leads to ileus in the newborn or, more often, constipation in children and adults. The disease affects one in 5000 live births. Siblings of affected cases are at an increased risk (4%) of developing the disease. Among cases, males are affected more often than females.
The first major susceptibility gene for Hirschsprung disease is the RET proto-oncogene on 10q11.2. Germline RET mutations in Hirschsprung disease are mainly inactivating, and have been reported to account for up to 20 and 50% of sporadic and familial cases, respectively. We have screened Swedish population-based samples from 62 sporadic cases and seven familial cases of Hirschsprung disease with single strand conformation polymorphism (SSCP), and found five mutations.     

Congenital central hypoventilation syndrome: Mutation analysis of the receptor tyrosine kinase RET

Congenital central hypoventilation syndrome (CCHS) usually occurs as an isolated phenotype. However, 16% of the index cases are also affected with Hirschsprung disease (HSCR). Complex segregation analysis suggests that CCHS is familial and has the same inheritance pattern with or without HSCR. We postulate that alteration of normal function of the receptor tyrosine kinase, RET, may contribute to CCHS based on RET's expression pattern and the identification of RET mutations in HSCR patients. To further explore the nature of the inheritance of CCHS, we have undertaken two main routes of investigation: cytogenetic analysis and mutation detection. Cytogenetic analysis of metaphase chromosomes showed normal karyotypes in 13 of the 14 evaluated index cases; one index case carried a familial pericentric inversion on chromosome 2. Mutation analysis showed no sequence changes unique to index cases, as compared to control individuals, and as studied by single strand conformational polymorphism (SSCP) analysis of the coding region of RET. We conclude that point mutations in the RET coding region cannot account for a substantial fraction of CCHS in this patient population, and that other candidate genes involved in neural crest cell differentiation and development must be considered. © 1996 Wiley-Liss, Inc.     

Frequency of RET mutations in long- and short-segment Hirschsprung disease

Hirschsprung disease, or congenital aganglionic megacolon, is a genetic disorder of neural crest development affecting 1:5,000 newborns. Mutations in the RET proto-oncogene, repeatedly identified in the heterozygous state in both long- and short-segment Hirschsprung patients, lead to loss of both transforming and differentiating capacities of the activated RET through a dominant negative effect when expressed in appropriate cellular systems. The approach of single-strand conformational polymorphism analysis established for all the 20 exons of the RET proto-oncogene, and previously used to screen for point mutations in Hirschsprung patients allowed us to identify seven additional mutations among 39 sporadic and familial cases of Hirschsprung disease (detection rate 18%). This relatively low efficiency in detecting mutations of RET in Hirschsprung patients cannot be accounted by the hypothesis of genetic heterogeneity, which is not supported by the results of linkage analysis in the pedigrees analyzed so far. Almost 74% of the point mutations in our series, as well as in other patient series, were identified among long segment patients, who represented only 25% of our patient population. The finding of a C620R substitution in a patient affected with total colonic aganglionosis confirms the involvement of this mutation in the pathogenesis of different phenotypes (i.e., medullary thyroid carcinoma and Hirschsprung). Finally the R313Q mutation identified for the first time in homozygosity in a child born of consanguineous parents is associated with the most severe Hirschsprung phenotype (total colonic aganglionosis with small bowel involvement). Hum Mutat 9:243–249, 1997. © 1997 Wiley-Liss, Inc.     

Low RET mutation frequency and polymorphism analysis of the RET and EDNRB genes in patients with Hirschsprung disease in Taiwan

Hirschsprung disease (HSCR), or congenital intestinal aganglionosis, is a relatively common disorder characterized by the absence of ganglion cells in the nerve plexuses of the lower digestive tract, resulting in intestinal obstruction in neonates. Mutations in genes of the RET receptor tyrosine kinase and endothelin receptor B (EDNRB) signaling pathways have been shown to be associated in HSCR patients. In this study, we collected genomic DNA samples from 55 HSCR patients in central Taiwan and analyzed the coding regions of the RET and EDNRB genes by PCR amplification and DNA sequencing. In the 55 patients, an A to G transition was detected in two (identical twin brothers). The mutation was at the end of RET exon 19 at codon 1062 (Y1062C), a reported critical site for the signaling pathways. Single nucleotide polymorphisms (SNP) in exons 2, 7, 11, 13, and 15 of RET and exon 4 of EDNRB in the HSCR patients or controls were detected. The differences between patients and controls in allele distribution of the five RET polymorphic sites were statistically significant. The most frequent genotype encompassing exons 2 and 13 SNPs (the polymorphic sites with the highest percentage of heterozygotes) was AA/GG in patients, which was different from the AG/GT in the normal controls. Transmission disequilibrium was observed in exons 2, 7, and 13, indicating nonrandom association of the susceptibility alleles with the disease in the patients. This study represents the first comprehensive genetic analysis of HSCR disease in Taiwan.     

RET基因调控区遗传变异与先天性巨结肠患病风险研究

目的先天性巨结肠（HSCR）是一种复杂的先天性疾病,RET是其主要的易感性基因。本研究对RET非编码区单核甘酸多态性（SNP）-5G〉A（rs10900296）,-1A〉C（rs10900297）和intron1 C〉T（rs2435357）进行分型分析,评估RET基因调控区SNPs及单倍型与先天性巨结肠之间的相关性。方法选取115名病例组病人和139名对照组正常人群,应用聚合酶链反应（PCR）技术和直接测序的方法进行基因分型。回归模型中使用OR值和95%置信区间（CI）作为基因型危险性的评价指标。结果 -5G〉A,-1A〉C,intron1C〉T各基因型频率在病例和对照人群的分布具有显著差异。-5 AA（OR=6.26,95%CI=3.62-10.83）,-1 CC（OR=7.54,95%CI=2.06-27.66）和intron1 TT（OR=19.22,95% CI=7.54-48.99）基因型均能显著增加HSCR发病的风险。单倍型A-C-T（OR=6.28,95% CI=3.77-10.46）和双体型A-C-T/A-C-T（OR=13.62,95% CI=3.48-53.30）分析同样表明与HSCR发病风险存在较强的相关性,并呈现出一定的累积效应。结论 RET基因调控区的基因多态性可能与HSCR的发病易感性有关,支持RET通路的常见变异在HSCR的发展过程中起着重要的作用的假设。     

Clinical spectrum of MEN2A in a large family caused by the infrequent RET mutation Cys609Phe

Mutations in RET proto‐oncogene cause multiple endocrine neoplasia 2A (MEN2A). Mutations in codons 609 and 611 are not frequent. We identified two MEN2A families with the Cys609Phe RET mutation, which turned out to be the same family. This mutation has been described a couple of times with no clinical details. We have characterized the clinical phenotype of this large kindred. A 54‐year‐old woman, with a medullary thyroid carcinoma (MTC), and a 33‐year‐old woman, who was operated on for an adrenal pheochromocytoma, were the index cases. 35 relatives were studied. Sixteen turned out to be carriers and 12 of them have been operated on. This family showed eight patients with C‐cell hyperplasia, six patients affected by MTC and two showing pheochromocytoma. A papillary thyroid carcinoma was also found, together with the MTC, in one of the carriers. The phenotype in this large kindred is clearly of MEN2A. In carriers presenting the Cys609Phe mutation, the timing of the presentation of the syndrome is highly unpredictable. Therefore, a strict follow up of MTC must be carried out because of risk, and pheochromocytoma should not be ignored. These results reinforce the scarce data observed on this particular mutation.     

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.     

Analysis of RET, ZEB2, EDN3 and GDNF Genomic Rearrangements in 80 Patients with Hirschsprung Disease (Using multiplex ligation-dependent probe amplification)

Hirschsprung disease (HSCR) is transmitted in a complex pattern of inheritance and is mostly associated with variants in the RET proto-oncogene. However, RET mutations are only identified in 15–20% of sporadic HSCR cases and solely in 50% of the familial cases. Since genomic rearrangements in particularly sensitive areas of the RET proto-oncogene and/or associated genes may account for the HSCR phenotype in patients without other detectable RET variants, the aim of the present study was to identify rearrangements in the coding sequence of RET as well as in three HSCR-associated genes ( ZEB2 , EDN3 and GDNF ) in HSCR patients by using Multiplex Ligation-dependent Probe Amplification (MLPA). We have screened 80 HSCR patients for genomic rearrangements in RET, ZEB2, EDN3 and GDNF and did not identify any deletion or amplification in these four genes in all patients. We conclude that genomic rearrangements in RET are rare and were not responsible for the HSCR phenotype in individuals without identifiable germline RET variants in our group of patients, yet this possibility cannot be excluded altogether because the confidence to identify variation in at least two percent of the individuals was only 95%.     

A Novel Point Variant in NTRK3, R645C, Suggests a Role of this Gene in the Pathogenesis of Hirschsprung Disease

Hirschsprung disease (HSCR) is a developmental disorder characterized by the absence of ganglion cells in the myenteric and submucosal plexuses due to a defect in the migration process of neural crest neuroblasts. Manifestation of the disease has been linked to the dysfunction of two principal signalling pathways involved in the enteric nervous system (ENS) formation: the RET-GDNF and the EDN3-EDNRB receptor systems. However, the NTF3/NTRK3 signalling pathway plays an essential role in the development of the ENS suggesting a potential role for those genes in the pathogenesis of HSCR. We have sought to evaluate the candidature of the NTRK3 gene, which encodes the TrkC receptor, as a susceptibility gene for Hirschsprung disease. Using dHPLC technology we have screened the NTRK3 coding region in 143 Spanish HSCR patients. A total of four previously described polymorphisms and 12 novel sequence variants were detected. Of note, the novel R645C mutation was detected in 2 affected siblings of a HSCR family also carrying a RET splicing mutation. Using bioinformatics tools we observed that the presence of an additional cysteine residue might implicate structural alterations in the mutated protein. We propose haploinsufficiency as the most probable mechanism for the NTRK3 R645C mutation. NTRK3 and RET mutations in this family only appear together in the HSCR patients, suggesting that they per se are necessary but not sufficient to produce the phenotype. In addition, it is quite probable that the contribution of other still unidentified modifier genes, may be responsible for the different phenotypes (length of aganglionosis) in the two affected members.     

Differential Dimerization of Variants Linked to Enhanced S‐Cone Sensitivity Syndrome (ESCS) Located in the NR2E3 Ligand‐Binding Domain

NR2E3 encodes the photoreceptor‐specific nuclear hormone receptor that acts as a repressor of cone‐specific gene expression in rod photoreceptors, and as an activator of several rod‐specific genes. Recessive variants located in the ligand‐binding domain (LBD) of NR2E3 cause enhanced short wavelength sensitive‐ (S‐) cone syndrome (ESCS), a retinal degeneration characterized by an excess of S‐cones and non‐functional rods. We analyzed the dimerization properties of NR2E3 and the effect of disease‐causing LBD missense variants by bioluminescence resonance energy transfer (BRET²) protein interaction assays. Homodimerization was not affected in presence of p.A256V, p.R039G, p.R311Q, and p.R334G variants, but abolished in presence of p.L263P, p.L336P, p.L353V, p.R385P, and p.M407K variants. Homology modeling predicted structural changes induced by NR2E3 LBD variants. NR2E3 LBD variants did not affect interaction with CRX, but with NRL and rev‐erbα/NR1D1. CRX and NRL heterodimerized more efficiently together, than did either with NR2E3. NR2E3 did not heterodimerize with TLX/NR2E1 and RXRα/NR2C1. The identification of a new compound heterozygous patient with detectable rod function, who expressed solely the p.A256V variant protein, suggests a correlation between LBD variants able to form functional NR2E3 dimers and atypical mild forms of ESCS with residual rod function.     

Spectrum of ATP7B mutations and genotype–phenotype correlation in large‐scale Chinese patients with Wilson Disease

Wilson disease (WD), an inherited disorder associated with ATP7B gene, has a wide spectrum of genotypes and phenotypes. In this study, we developed a rapid multiplex PCR‐MassArray method for detecting 110 mutant alleles of interest, and used it to examine genomic DNA from 1222 patients and 110 healthy controls. In patients not found to have any mutation in the 110 selected alleles, PCR‐Sanger sequencing was used to examine the ATP7B gene. We identified 88 mutations, including 9 novel mutations. Our analyses revealed p.Arg778Leu, p.Arg919Gly and p.Thr935Met showed some correlations to phenotype. The p.Arg778Leu was related to younger onset age and lower levels of ceruloplasmin (Cp) and serum copper, while p.Arg919Gly and p.Thr935Met both indicated higher Cp levels. Besides, the p.Arg919Gly was related to neurological subtype, and p.Thr935Met showed significant difference in the percentage of combined neurological and visceral subtype. Moreover, for ATP7B mutations, the more severe impact on ATP7B protein was, the younger onset age and lower Cp level presented. The feasibility of presymptomatic DNA diagnosis and predicting clinical manifestation or severity of WD would be facilitated with identified mutations and genotype–phenotype correlation precisely revealed in the study.     

Mendelian Disorders of Cornification Caused by Defects in Intracellular Calcium Pumps: Mutation Update and Database for Variants in ATP2A2 and ATP2C1 Associated with Darier Disease and Hailey–Hailey Disease

The two disorders of cornification associated with mutations in genes coding for intracellular calcium pumps are Darier disease (DD) and Hailey–Hailey disease (HHD). DD is caused by mutations in the ATP2A2 gene, whereas the ATP2C1 gene is associated with HHD. Both are inherited as autosomal‐dominant traits. DD is mainly defined by warty papules in seborrheic and flexural areas, whereas the major symptoms of HHD are vesicles and erosions in flexural skin. Both phenotypes are highly variable. In 12%–40% of DD patients and 12%–55% of HHD patients, no mutations in ATP2A2 or ATP2C1 are found. We provide a comprehensive review of clinical variability in DD and HHD and a review of all reported mutations in ATP2A2 and ATP2C1. Having the entire spectrum of ATP2A2 and ATP2C1 variants allows us to address the question of a genotype–phenotype correlation, which has not been settled unequivocally in DD and HHD. We created a database for all mutations in ATP2A2 and ATP2C1 using the Leiden Open Variation Database (LOVD v3.0), for variants reported in the literature and future inclusions. This data may be of use as a reference tool in further research on treatment of DD and HHD.     

Characterization of All Possible Single‐Nucleotide Change Caused Amino Acid Substitutions in the Kinase Domain of Bruton Tyrosine Kinase

Knowledge about features distinguishing deleterious and neutral variations is crucial for interpretation of novel variants. Bruton tyrosine kinase (BTK) contains the highest number of unique disease‐causing variations among the human protein kinases, still it is just 10% of all the possible single‐nucleotide substitution‐caused amino acid variations (SNAVs). In the BTK kinase domain (BTK‐KD) can appear altogether 1,495 SNAVs. We investigated them all with bioinformatic and protein structure analysis methods. Most disease‐causing variations affect conserved and buried residues disturbing protein stability. Minority of exposed residues is conserved, but strongly tied to pathogenicity. Sixty‐seven percent of variations are predicted to be harmful. In 39% of the residues, all the variants are likely harmful, whereas in 10% of sites, all the substitutions are tolerated. Results indicate the importance of the entire kinase domain, involvement in numerous interactions, and intricate functional regulation by conformational change. These results can be extended to other protein kinases and organisms.     

Bardet‐Biedl syndrome in Denmark—report of 13 novel sequence variations in six genes

Bardet‐Biedl syndrome (BBS) is an autosomal recessive disease characterized by retinal dystrophy, polydactyly, obesity, learning disabilities, renal involvement, and male hypogenitalism. BBS is genetically heterogeneous with mutations of 14 genes, accounting for approximately 70% of cases. Triallelic inheritance has been suggested in about 5% of cases. Forty‐nine unrelated BBS patients were screened for mutations by DHPLC analysis in BBS1, BBS2, BBS4, BBS6/MKKS, BBS10, and BBS12. The selected genes either account for more than 5% of the mutational load or are commonly reported in triallelic inheritance. Eight patients with only one or no BBS mutation were further investigated by single nucleotide polymorphism (SNP) analysis. In total, mutations were detected in 44 patients. Twenty percent had two mutations in BBS1, 18% in BBS2, 4% in BBS9, 43% in BBS10, and 2% in BBS12. Five patients were heterozygous for a sequence variation in BBS6/MKKS. We found eight patients with three sequence variations in two genes, which could be explained by triallelic inheritance, by the prevalence of heterozygous carriers or the third sequence variations representing rare polymorphisms. All changes found in a second BBS gene were amino acid substitutions. Genotype–phenotype correlations suggest a milder phenotype for BBS1 compared to BBS2 and BBS10, which we ascribe to the hypomorphic p.Met390Arg‐mutation. Hum Mutat 31:429–436, 2010. © 2010 Wiley‐Liss, Inc.     

Neuronal Phenotype and Tyrosine Kinase Receptor Expression in Cocultures of Dorsal Root Ganglion and Skeletal Muscle Cells

The neuropeptide‐immunoreactive (IR) and neurofilament‐IR neurons are two major phenotypical classes in dorsal root ganglion (DRG). Tyrosine kinase receptor (Trk)A, TrkB, and TrkC are three members of the Trk family which may be relevant to neuronal phenotypes. Whether target skeletal muscle cells generate their expression remains unclear. Neurons containing substance P (SP), calcitonin gene‐related peptide (CGRP), neurofilament 200 (NF‐200), TrkA, TrkB, and TrkC were quantified using immunohistochemistry in rat DRG neuronal cultures and cocultures of DRG neurons and skeletal muscle cells. The percentage of NF‐200 and TrkC‐expressing neurons in cocultures of DRG neurons and skeletal muscle cells was significantly higher, 26.86% ± 3.17% (NF‐200) and 27.74% ± 3.63% (TrkC) compared with 20.92% ± 1.98% (NF‐200) and 16.70% ± 3.68% (TrkC) in DRG cultures; whereas the percentage of SP, CGRP, TrkA, and TrkB‐expressing neurons was not changed significantly by the addition of target skeletal muscle cells. Thus, target skeletal muscle cells may influence neurofilament‐phenotype and TrkC receptor but not neuropeptide‐phenotype and TrkA and TrkB receptors. Anat Rec, 2009. © 2008 Wiley‐Liss, Inc.