Startle disease in an Italian family by mutation (K276E): The alpha-subunit of the inhibiting glycine receptor.
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Chromosomes, Human, Pair 5↗
Biomedical subjects
M Seri
Publications and source records attributed to M Seri.
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).
DNA Mutational Analysis↗
A Cys634Gly substitution of the RET proto-oncogene in a family with recurrence of multiple endocrine neoplasia type 2A and cutaneous lichen amyloidosis.
Germ-line mutations of the RET proto-oncogene, involving five cysteine residues at codons 609, 611, 618, 620 and 634, are associated with two variants of the inherited cancer syndrome multiple endocrine neoplasia type 2: type 2A and familial medullary thyroid carcinoma. The association of multiple endocrine neoplasia type 2A with the dermatological disorder cutaneous lichen amyloidosis has already been reported, and mutations in the Cys634 have been identified in different families. We describe here an additional pedigree in which multiple endocrine neoplasia type 2A and cutaneous lichen amyloidosis cosegregate. A Cys634Gly was identified by direct sequencing of the RET proto-oncogene exon 11 in the affected individuals. The mutation creates a new HaeIII site, and restriction analysis performed on all family members rules out the presence of the altered allele in two children and consequently the risk of developing thyroid tumors. These results emphasize the role of molecular analysis of the RET proto-oncogene in diagnosing presymptomatically those individuals at risk of inheriting the disease allele.
Adolescent↗
Molecular cloning and mapping of a human cDNA (PA2G4) that encodes a protein highly homologous to the mouse cell cycle protein p38-2G4.
We have identified a novel human gene with strong homology to the mouse Pa2g4 cell cycle gene. This novel gene (called PA2G4) belongs to a gene family with members in several chromosome regions: 3q24-q25, 6q22, 9q21, 12q13, 18q12, 20p12 and Xq25. A composite cDNA of 1697 nucleotides was isolated. The sequence of this cDNA predicts a protein of 394 amino acids. The deduced amino acid sequence of this human protein shows very strong homology to the mouse protein p38-2G4. The cDNA analyzed probably corresponds to a functional copy found at 12q13.
Animals↗
Assignment of mouse Gfra1, the homologue of a new human HSCR candidate gene, to the telomeric region of mouse chromosome 19.
Glial cell line-derived neurotrophic factor (Gdnf) and its alpha receptor (Gfra1) interact with the Ret receptor triggering its tyrosine kinase activity. As Gdnf and Ret have been linked to the development of Hirschsprung disease (HSCR), it seems likely that Gfra1 could also be a susceptibility gene for HSCR. A further HSCR candidate gene is represented by the human homologue of the Dom (Dominant megacolon) mouse mutation, mapped to Chr 15, for which the gene has not yet been identified. In order to test if Gfra1 could be the Dom gene or if it represents a new possible HSCR locus we have undertaken the mapping of the mouse Gfra1. Using specific PCR primers on a somatic cell hybrid mapping panel and fluorescence in situ hybridization with an expressed sequence tag (EST) cDNA clone corresponding to the mouse Gfra1, we mapped the gene to mouse chromosome 19D2-D3, a region with known homology with human chromosome 10q24-->q26.
Animals↗
Inheritance of Niikawa-Kuroki (Kabuki makeup) syndrome.
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Abnormalities, Multiple↗
Chromosomal localization of the genes (CLNS1A and CLNS1B) coding for the swelling-dependent chloride channel ICln.
ICln is a cloned chloride channel paramount for regulatory volume decrease. Two different loci that carry the coding region for ICln were identified in the human genome. By PCR strategies an intronless copy of the gene was located on chromosome 6 at position 6p12.1-6q13 (CLNS1B). By fluorescence in situ hybridization a copy carrying introns with a putative length of 19 kb was located at chromosome 11 on position 11q13.5-q14.1 (CLNS1A). The characterization and chromosomal localization of the ICln gene offer the opportunity to study the regulatory sites of this gene in greater detail and could be helpful in establishing linkages between ICln and potential human diseases.
Chloride Channels↗
Time-dependent current decline in cyclic GMP-gated bovine channels caused by point mutations in the pore region expressed in Xenopus oocytes.
1. Amino acids with a charged or a polar residue in the putative pore region, between lysine 346 and glutamate 372 of the alpha-subunit of the cGMP-gated channel from bovine rods were mutated to a different amino acid. The mRNA encoding for the wild-type, i.e. the alpha-subunit, or mutant channels was injected in Xenopus laevis oocytes. 2. When glutamate 363 was mutated to asparagine, serine or alanine, the current activated by a steady cGMP concentration declined in mutant channels. No current decline was observed when glutamate 363 was mutated to aspartate, glutamine or glycine, when theronine 359, 360 and 364 were mutated to alanine or when other charged residues in the pore region were neutralized. 3. The amount of current decline and its time course were significantly voltage dependent. In mutant E363A the current decline developed within about 1.5 s at -100 mV, but in about 6 s at +100 mV. In the same mutant, the current declined to about 55% of its initial level at +100 mV and to about 10% at -100 mV. 4. The current decline in mutants E363A, E363S and E363N was only moderately dependent on the cGMP concentration (from 10 to 1000 microM) and was not caused by a reduced affinity of the mutant channels for cGMP. Analysis of current fluctuations at a single-channel level indicated that current decline was primarily caused by a decrease of the open probability. 5. The wild-type channel was not permeable to dimethylammonium. When glutamate 363 was replaced by a smaller residue such as serine, mutant channels became permeable to dimethylammonium. 6. The current decline observed in mutant channels is reminiscent of desensitization of ligand-gated channels and of inactivation of voltage-gated channels. These results suggest also that gating and permeation through the cGMP-gated channel from bovine rods are intrinsically coupled and that glutamate 363 is part of the molecular structure controlling both the gating and the narrowest region of the pore.
Amino Acid Sequence↗
Strategies for the Identification of Intron-Exon Boundaries and Point Mutations: The Example of the RET Proto-Oncogene
For any given gene, the identification of exon boundaries and the corresponding flanking intron sequences is the essential prerequisite for asking interesting questions related to its structural organization, spanning from the study of its evolution to the molecular mechanisms underlying possible alternative splicing. In addition, this type of knowledge allows us to carry out mutation screenings for whole exons when the gene in question is known or suspected to be responsible for a human disease phenotype. Several different strategies already used to establish the genomic organization of genes are presented in detail, along with appropriate examples, and they are compared to each other with respect to their requirements, efficiency, and applicability. Particular emphasis is given to the detailed description of the strategy used to study the intron-exon junctions and to establish the intron flanking sequences of the RET proto-oncogene, whose mutations cause Hirschsprung disease (or congenital megacolon) as well as different types of thyroid cancer.
Journal Article↗
Prevalence and parental origin of de novo RET mutations in Hirschsprung's disease.
In contrast with the reported almost exclusive paternal origin of de novo mutations in MEN 2A, FMTC and MEN 2B, de novo mutations in Hirschsprung patients arise both on paternal and maternal chromosomes. This distinctive feature of RET mutations associated with Hirschsprung's disease and of the RET mutations associated with thyroid cancer indicates a basic biological difference between the mutational events leading to the different phenotypes.
DNA Mutational Analysis↗
RET mutations in exons 13 and 14 of FMTC patients.
RET is a receptor tyrosine kinase gene which is responsible for three different inherited cancer syndromes namely multiple endocrine neoplasia type 2A (MEN 2A), type 2B (MEN 2B) and familial medullary thyroid carcinoma (FMTC) as well as for Hirschsprung disease (HSCR), a congenital disorder affecting the intestinal motility. Germ-line mutations in the RET exons 10 and 11 were demonstrated in the majority of the MEN 2A and FMTC patients. On the other hand, one codon of RET exon 16 is preferentially changed in MEN 2B patients. Recently, a germ-line mutation in the exon 13 was described in one FMTC family as well as in four sporadic MTCs. In the present study, we observed the same exon 13 mutation in two FMTC families. In addition, we identified a previously unreported substitution of RET exon 14 in two unrelated FMTC families. Both mutations segregate with the disease in these four FMTC families and involve the tyrosine kinase domain of RET. Haplotype analysis using polymorphic markers tightly linked to the RET gene indicates that in each pedigree the mutation arose as an independent event.
Adult↗
DNA polymorphisms and conditions for SSCP analysis of the 20 exons of the ret proto-oncogene.
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Base Sequence↗
A rare G6490-->A substitution at the last nucleotide of exon 10 of the glucocerebrosidase gene in two unrelated Italian Gaucher patients.
Mutation screening of the glucocerebrosidase gene by SSCP analysis revealed an abnormal pattern of exon 10 in two unrelated Italian Gaucher patients. Direct sequencing of the mutated samples identified a G6490-->A transition. The same mutation has been described before in a Japanese patient with Gaucher disease type III. The clinical phenotype of our patients was type I in one whose second allele carried the N370S mutation and type II in the other one with a L444P mutation. In this latter the G6490-->A substitution cancels a normal Msp I site, while on the opposite chromosome the T6433-->C mutation (L444P) introduces a new Msp I site. Thus, digestion with Msp I of the amplified exon 10 is a useful method for identifying the two mutations simultaneously.
Adenine↗
Lung tissue resistance and hysteretic moduli of lung parenchyma.
Lung tissue resistance (Rti) represents a large and labile component of total pulmonary resistance, but the mechanism is unknown. One hypothesis that has received some support in the literature is that on exposure to contractile agonists airway smooth muscle shortens and then, by the agency of elastic interdependence, induces distortion in surrounding parenchyma. Parenchymal distortion induced in the vicinity of a constricted airway is a pure shear deformation, but currently there are no data available for shear hysteresivity. Guided by a microstructural model, we have assigned stiffness and hysteresivity to microstructural elements and then computed how those properties are expressed at the macroscale in bulk hysteresivities for both shear and volumetric expansion. Hysteresivity for volumetric expansion is shown to be a stiffness-weighted average of hysteresivities of all microstructural components. But as the hysteresivity of microstructural elements increases, that for shear deformation increases to some degree but eventually attains a plateau. Blunted hysteretic response in shear seems to be an intrinsic property of pressure-supported structures, like the lung, that require an inflating pressure to ensure mechanical stability. The analysis indicates that that part of Rti attributable to parenchymal distortion can be at most a small fraction of that attributable to volumetric expansion. These results are purely theoretical in nature, and this suggests that caution is necessary in their interpretation. However, the mechanical basis of the results is sufficiently general to conclude that the hypothesis that parenchymal distortion secondary to bronchoconstriction can account for Rti and its changes seems to be implausible.
Airway Resistance↗
Point mutations affecting the tyrosine kinase domain of the RET proto-oncogene in Hirschsprung's disease.
Hirschsprung's disease is a genetic disorder of neural crest development affecting 1 in 5,000 births. It is characterized by the absence of intramural ganglion cells in the hindgut, which often results in partial to complete intestinal obstruction during the first years of life. An autosomal dominant gene causing this disease was recently mapped to chromosome 10q11.2 (refs 1,2), using an interstitial deletion of this region isolated in a cell hybrid. It was subsequently localized to a 250-kilobase interval which contains the RET proto-oncogene. Using flanking intronic sequences as primers to amplify 12 of the 20 exons of RET from genomic DNA of 27 Hirschsprung's disease patients, we have now identified four mutations (one frameshift and three missense) that totally disrupt or partially change the structure of the tyrosine kinase domain of the RET protein (Ret). Mutations in the extracellular cysteine-rich domain of Ret have been identified previously in patients with multiple endocrine neoplasia type 2A, and a targeted mutation in the tyrosine kinase domain of the same gene produces intestinal aganglionosis and kidney agenesis in homozygous transgenic mice. Our results support the hypothesis that RET, in addition to its potential role in tumorigenesis, plays a critical role in the embryogenesis of the mammalian enteric nervous system.
Base Sequence↗
Variability of clinical phenotype in a large Alport family with Gly 1143 Ser change of collagen alpha 5(IV)-chain.
In a large Italian family with adult-onset Alport syndrome, molecular analysis of the COL4A5 gene, which encodes the alpha 5(IV)-chain of glomerular basement membrane collagen, revealed a GGC-->AGC change in exon 38, resulting in substitution of a serine for a glycine in position 1143 of the polypeptide chain, between interruptions 19 and 20 of the triple helical domain. The mutation leads to loss of a restriction site for the enzyme Msp I, and could thus be easily recognized in several female and male relatives. Among relatives of both sexes who carried the same mutation, the clinical phenotype of Alport syndrome was variable as for the onset of renal failure and the presence of associated ear and eye abnormalities.
Adolescent↗
Heterogeneity and low detection rate of RET mutations in Hirschsprung disease.
Mutations in some exons of the RET proto-oncogene were recently observed in Hirschsprung patients. Using DNA polymorphisms and single-strand conformation polymorphism analysis for the whole coding sequence of the RET proto-oncogene, 82 unrelated Hirschsprung patients were screened systematically. A total of 4 complete deletions of RET and 12 point mutations were identified, each present in no more than one patient and distributed along the whole gene. De novo mutations could be documented in 4 patients. Southern blot and fluorescence in situ hybridization analysis carried out in a restricted number of patients did not reveal any deletion of RET. The low efficiency in detecting mutations of RET in Hirschsprung patients (20%) may originate mainly from genetic heterogeneity.
Blotting, Southern↗
De-novo COL4A5 gene mutations in Alport's syndrome.
Before the advent of direct molecular gene analysis the diagnosis of Alport syndrome was operationally based on three of the four classical clinical criteria. Recently, mutations have been identified in the COL4A5 gene, which is involved in X-linked Alport syndrome. Here we describe two de-novo mutations in two unrelated children, a male and a female, both with early onset of the nephropathy, but with only one of the diagnostic criteria, i.e. electron-microscopy alterations. Because of the significant estimated proportion of de-novo mutations this diagnosis should be considered in children with early signs of nephropathy, even without a suggestive family history or clinical picture (ocular or audiologic abnormalities). In the future the diagnosis of Alport syndrome will probably be made on the basis of both clinical findings and molecular analysis. Now Alport syndrome is clearly underdiagnosed.
Adolescent↗