Exon structure and flanking intronic sequences of the human RET proto-oncogene

Using a PCR strategy based on an initial set of 15 couples of primers designed from the known cDNA sequence, we identified 18 introns in the human RET proto-oncogene and sequenced the corresponding 5' and 3' exon-intron junctions. This approach was successful in locating all the introns contained in fragments short enough to be amplified by PCR. Thus 19 exons were identified which, together with the previously reported exon subjected to alternative splicing, brings the total number of RET exons to 20. This information is relevant for the screening of recently reported missense mutations of RET which cause Multiple Endocrine Neoplasia 2A (MEN2A) and for the search of additional point mutations of the same gene which might cause two other neural crest disorders, MEN2B and Hirschsprung disease, mapping in the same region as MEN2A.     

Identification of novel mutations in the human EXT1 tumor suppressor gene

Hereditary multiple exostoses (EXT) is a genetically heterogeneous bone disorder caused by genes segregating on human chromosomes 8, 11, and 19 and designated EXT1, EXT2 and EXT3, respectively. Recently, the EXT1 gene has been isolated and partially characterized and appears to encode a tumor suppressor gene. We have identified six mutations in the human EXT1 gene from six unrelated multiple exostoses families segregating for the EXT gene on chromosome 8. One of the mutations we detected is the same 1-bp deletion in exon 6 that was previously reported in two independent EXT families. The other five mutations, in exons 1, 6, 9, and the splice junction at the 3' end of exon 2, are novel. In each case, the mutation is likely to result in a truncated or nonfunctional EXT1 protein. These results corroborate and extend the previous report of mutations in this gene in two EXT families, and provide additional support for the EXT1 gene as the cause of hereditary multiple exostoses in families showing linkage to chromosome 8.     

Biallelic mutations of the Tsc2 gene in chemically induced rat renal cell carcinoma

A number of cancer genes have been identified by the study of hereditary human cancers and shown to be involved in sporadic genesis of the same tumors. We have identified a germline mutation in the rat homologue of the human tuberous sclerosis (TSC2) predisposing gene in the Eker rat model. In this study, we searched for mutations of the Tsc2 gene in chemically induced non-Eker rat renal cell carcinomas (RCs). N-ethyl-N-hydroxyethylnitrosamine (EHEN)- and diethylnitrosamine (DEN)-induced non-Eker rat primary RCs were subjected to polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis using specific primers covering all exons of the Tsc2 gene (41 coding exons and 1 non-coding exon). We simultaneously searched for mutations in the Vhl gene, a rat homologue of von Hippel-Lindau disease (VHL) gene, as well as the Tsc2 gene. Mutations in the Vhl gene were not detected in any rat RCs (0/8). In contrast, Tsc2 gene mutations were detected at a high frequency in EHEN-induced RCs (2/3) and DEN-induced RCs (3/5) (total 5/8) (p < 0.05). By a direct cloning approach utilizing PCR analysis in 2 applicable cases, we could demonstrate the presence of intragenic somatic mutations in both alleles of the Tsc2 gene. Our results suggest that Tsc2 gene inactivation plays an important role in EHEN- and DEN-induced RCs as well as in Eker rat RCs.     

Mutation analysis of coding sequences of the entire transforming growth factor beta type II receptor gene in sporadic human colon cancer using genomic DNA and intron primers

Mutations in the transforming growth factor beta type II receptor (TGFbeta RII) gene have been detected in several human cancers exhibiting microsatellite instability. To extend analyses of this gene, we previously investigated the exon-intron organization of the TGFbeta RII gene and defined seven exons and flanking intron sequences. In this study, we further determined the nucleotide sequences surrounding these seven exons and designed eight sets of intron-based primers to examine the entire coding region of the TGFbeta RII gene. Using these primers, we screened genomic DNA sequences from 30 sporadic colorectal cancers for mutations of the TGFbeta RII gene. TGFbeta RII mutations were detected in two of 30 tumors and both displayed microsatellite instability. One had a deletion in a polyadenine tract in exon 3 and the other had a point mutation in the kinase domain located in exon 7. There were no mutations in exons 1, 2, 4, 5 and 6. These results further implicate the polyadenine tract and kinase domain as mutational hotspots in the TGFbeta RII gene in cells with genomic instability and suggest that TGFbeta RII gene mutations occur rarely in cells lacking genomic instability.     

Effect of YM435, a dopamine DA1 receptor agonist, in a canine model of ischemic acute renal failure

In mammals, one of the Mad homologues, Smad2, was reported to be a mediator of TGF-beta signaling, and was found mutated in some cases of colon and lung cancers. To extend the analysis of this gene, we previously investigated the genomic organization of the human Smad2 gene and defined the structure of 12 exons and flanking introns. In this study, we designed 11 sets of intron-based primers to examine the entire coding region of the Smad2 gene. By the PCR-SSCP method using these primers, we screened genomic DNA sequences of colorectal cancers for mutations of the Smad2 gene. Though there was no mutation within all exons of the Smad2 gene, two of 60 sporadic colorectal cancers displayed deletions in the polypyrimidine tract preceding exon 4. Deletions of this region were also detected in colon cancer cell lines, and were clustered within cells exhibiting microsatellite instability. Deletions in the polypyrimidine tract had various effects on pre-mRNA splicing, but had no effect on the splicing of the Smad2 gene in these cases. However, our data support the idea that the polypyrimidine tract in the splicing acceptor site is a target of mutations in mismatch repair-deficient tumors.     

Self-organization of binocular disparity tuning by reciprocal corticogeniculate interactions

Nramp2 is a gene encoding a transmembrane protein that is important in metal transport, in particular iron. Mutations in nramp2 have been shown to be associated with microcytic anemia in mk/mk mice and defective iron transport in Belgrade rats. Nramp2 contains a classical iron responsive element in the 3' untranslated region that confers iron dependent mRNA stabilization. In this report, we describe a splice variant form of human nramp2 that has the carboxyl terminal 18 amino acids substituted with 25 novel amino acids and has a new 3' untranslated region lacking a classical iron-responsive element. This splice form of nramp2, nramp2 non-IRE, was found to be derived from splicing of an additional exon into the terminal coding exon. The nramp2 gene is comprised of 17 exons and spans more than 36 kb. It contains an additional 5' exon and intron (exon and intron 1) and an additional 3' exon (exon 17) and intron (intron 16) as compared to nramp1, a homologous gene. The additional exons and introns account for much of the difference in length between nramp2 (> 36 kb) and nramp1 (12 kb). The exon-intron borders of nramp2 exons 3-15 are homologous to nramp1 exons 2-14. The nramp2 5' regulatory region contains two CCAAT boxes but lacks a TATA box. The 5' regulatory region of nramp2 also contains five potential metal response elements (MRE's) that are similar to the MRE's found in the metallothionein-IIA gene, three potential SP1 binding sites and a single gamma-interferon regulatory element. Five single nucleotide mutations or polymorphisms were identified within the nramp2 gene. One of these, 1303C-->A, occurs in the coding region of nramp2 and results in an amino acid change from leucine to isolecine. A polymorphism, 1254T/C, also occurs in the coding region of nramp2 but does not cause an amino acid change. The other 3 polymorphisms are within introns (IVS2 + 11A/G, IVS4 + 44C/A, and IVS6 + 538G/Gdel). In addition, a polymorphic microsatellite TATATCTATATATC (TA)6-7 (CA)10-11 CCCCCTATA (TATC)3 (TCTG)5 TCCG (TCTA)6 was identified in intron 3. Analysis of cDNA derived by direct amplification of reversed transcribed RNA or cDNA clones isolated from a library provide evidence of skipping of exons 10 and 12 of nramp2. Deletion of either of these exons would result in a sequence that remains in frame yet would generate a protein that would lack transmembrane spanning region 7 or 8 respectively. The deletion of a single transmembrane domain would have severe topological consequences. The coding region of the nramp2 gene of hemochromatosis patients with or without mutations in the hemochromatosis gene, HFE, were examined and found to be normal. One hemochromatosis patient, with a normal HFE genotype, was heterozygous for the 1303C-->A mutation. Furthermore, in an examination of hemochromatosis patients with mutant HFE and normal HFE genes, we did not observe a linkage disequilibrium of either group with a particular nramp2 haplotype. These data suggest that mutations in nramp2 are not commonly associated with hemochromatosis.     

Somatic mutations in the kinase domain of the Met/hepatocyte growth factor receptor gene in childhood hepatocellular carcinomas

The MET protooncogene encodes a transmembrane tyrosine kinase identified as the receptor of a polypeptide known as hepatocyte growth factor/scatter factor. We performed PCR-based single-strand conformational polymorphism and sequencing analysis of the tyrosine kinase domain of the MET gene (exon 15-19) in 75 primary liver cancers. Three missense mutations were detected exclusively in 10 childhood hepatocellular carcinomas (HCCs), while no mutations were detected in 16 adult HCCs, 21 cholangiocarcinomas, or 28 hepatoblastomas. The extremely short incubation period from hepatitis B virus infection to the genesis of childhood HCC as compared with the adult HCC suggests that there may be an additional mechanism that accelerates the carcinogenesis of childhood HCC. Our results indicate that mutations of the tyrosine kinase domain of the MET gene may be involved in the acceleration of the carcinogenesis in childhood HCC.     

Heterogeneous mutations in the gene encoding the alpha-subunit of the stimulatory G protein of adenylyl cyclase in Albright hereditary osteodystrophy

Albright hereditary osteodystrophy (AHO) is an inherited disorder associated with deficient activity of the alpha-subunit of the guanine nucleotide-binding regulatory protein (Gs alpha) that couples receptors to adenylyl cyclase. To identify mutations that lead to Gs alpha deficiency, we isolated genomic DNA from patients with AHO and used the polymerase chain reaction to amplify exons of the Gs alpha genes. DNA was amplified using intron-specific oligonucleotide primers flanking exons of the Gs alpha gene. To optimize our ability to detect mutations, one oligonucleotide from each primer pair was synthesized with a 5' GC-clamp. Amplified Gs alpha gene fragments were analyzed by denaturing gradient gel electrophoresis in order to detect mutations that alter the melting point of the double-stranded DNA fragment. Using this technique, we have identified and characterized three mutations and one neutral polymorphism. The polymorphism, located in exon 5, consisted of a T-->C substitution that conserves the isoleucine residue at codon 131 (ATT-->ATC). Two mutations were missense mutations, which in one family consisted of a nucleotide substitution (T-->C) in exon 4 that results in replacement of Leu by Pro at codon 99 of the Gs alpha molecule. Affected subjects in a second family had a single base (C-->T) mutation in exon 6 that resulted in replacement of Arg by Cys at codon 165. A 4-base pair deletion (GTGG) in exon 8 at position +214 was identified in one Gs alpha allele from each affected subject in the third family. This mutation causes a frameshift after the codon for Gln213 that results in a premature stop codon 81 base pair after the deletion. Immunoblot analysis of plasma membranes prepared from cultured fibroblasts or erythrocytes indicated that levels of immunoactive Gs alpha protein were decreased in all affected subjects. We conclude that heterogeneous mutations in the gene encoding Gs alpha, including deletions and single amino acid substitutions, are responsible for Gs alpha deficiency in AHO.     

Fine structure of the human ceruloplasmin gene

We characterized the genomic region corresponding to the human ceruloplasmin cDNA previously reported. Using PCR-direct sequencing methods, we determined precise intron/exon boundaries and intron-exon composition of the gene in the region. The gene region spanned about 50 kb and was composed of 19 exons and 18 introns. The lengths of exons and introns range from 107 to over 267 bp and from 0.44 to 10.0 kb, respectively. The translation initiation codon and the termination codon were located in exons 1 and 19, respectively. The nucleotide sequences of the introns were also determined in the region around the intron/exon boundaries for 24-220 bp. All the sequences around the intron/exon boundaries were consistent with the 5' and 3' consensus sequences for splice junctions of transcribed genes. Putative lariat sequences were identified between -17 and -42 nucleotides from the 3' splice junction for all 18 introns.     

Characterization of the human dihydropyrimidine dehydrogenase gene

Dihydropyrimidine dehydrogenase (DPD) catabolizes endogenous pyrimidines and pyrimidine-based antimetabolite drugs. A deficiency in human DPD is associated with congenital thymine-uraciluria in pediatric patients and severe 5-fluorouracil toxicity in cancer patients. The dihydropyrimidine dehydrogenase gene (DPYD) was isolated, and its physical map and exon-intron organization were determined by analysis of P1, PAC, BAC, and YAC clones. The DPYD gene was found to contain 23 exons ranging in size from 69 bp (exon 15) to 961 bp (exon 23). A physical map derived from a YAC clone indicated that DPYD is at least 950 kb in length with 3 kb of coding sequence and an average intron size of about 43 kb. The previously reported 5' donor splice site mutation present in pediatric thymine-uraciluria and cancer patients can now be assigned to exon 14. All 23 exons were sequenced from a series of human DNA samples, and three point mutations were identified in three racial groups as G1601A (exon 13, Ser534Asn), A1627G (exon 13, Ile543Val), and G2194A (exon 18, Val732Ile). These studies, which have established that the DPYD gene is unusually large, lay a framework for uncovering new mutations that are responsible for thymine-uraciluria and toxicity to fluoropyrimidine drugs.     

Fibronectin synthesis by human tubular epithelial cells in culture: effects of PDGF and TGF-beta on synthesis and splicing

Mutations of p53 tumor suppressor gene are the most common genetic alterations in a variety of human carcinomas. The sites of p53 mutations, however, vary in different cancers. The present study was designed to characterize p53 mutations in 40 primary human renal cancer specimens using hot-start-PCR-single-strand conformation polymorphism (SSCP) analysis, sequencing of PCR product and immunohistochemistry. DNA extracted from microdissected paraffin-embedded sections was amplified by hot-start-PCR using oligonucleotide primers specific for exons 4-9 of p53. The mutations were analyzed by PCR-SSCP technique and the generated fragments were denatured and analyzed by 6% polyacrylamide gel electrophoresis. The samples showing a band shift were denatured and sequenced using the Sequenase Version 2.0 DNA Sequencing Kit (US Biochemical, Cleveland, Ohio). Genomic DNA from control samples containing wild-type p53 alleles was sequenced in parallel for confirming mutations in samples that were positive for p53 in the PCR-SSCP analysis. The results of these experiments demonstrate that: (1) there were mutations in p53 exon 5 and 8 in 35% (14 out of 40 samples) of human renal cancer tissues as revealed by PCR-SSCP analysis; (2) DNA sequencing of samples showing frame-shift have hot spot of p53 mutation on exon 8 at codon 244 (GGC-->TGC) and exon 5 at codon 132 [AAG (Lys)-->AGG (Arg)]. This mutation in p53 exon 5 at codon 132 is novel and has not yet been reported; (3) immunohistochemical staining of p53 in renal cancer tissue using mouse anti-human p53 monoclonal antibody, clone PAb 1801, correlated with the p53 mutation assessed by PCR-SSCP. No correlation was found between p53 mutations and tumor stage and grade of renal cancer.     

The usefulness of single-strand DNA conformation polymorphism analysis to detect mutations in protein C deficiency

The standard approach for the molecular genetic analysis of protein C deficiency, polymerase chain reaction (PCR) amplification followed by direct sequencing, although very accurate, is time-consuming. The aim of this study is to investigate the usefulness of a simplified, time-saving screening method for the detection of protein C mutations consisting of the combination of multiplex PCR amplifications using the same primers that were designed for sequencing, followed by single-strand DNA conformation polymorphism (SSCP) electrophoresis analysis performed with one set of conditions. The study was designed in two phases. First, we tested six known point mutations located in different exons of the protein C gene by SSCP. Second, we prospectively studied nine patients with protein C deficiency type I using SSCP as the first screening technique. All the exons were amplified with a common PCR protocol, either as single fragments or as multiplex combinations of several of them. In the retrospective study, three out of the six point mutations were visible as a band shift: 40 T-->G (exon 2), 1432 C-->T (exon 3) and 7253 C-->T (exon 8). In the prospective analysis SSCP detected three different mutations. These mutations were: 6128 T-->C (exon 7), 6216 C-->T (exon 7) and in two probands 8631 C-->T (exon 9). In the five remaining patients we identified only two different mutations by direct sequencing: 6246 G-->A (exon 7) in two patients and 8589 G-->A (exon 9) in four patients. In summary, the results from both studies show that only 60% of all mutations can be detected using this simplified method. It also suggests that a multiple set of conditions, smaller PCR fragments, or both, should be used in order to achieve a sensitivity comparable to sequencing.     

Rapid sequencing of the p53 gene with a new automated DNA sequencer

p53 is the most commonly mutated gene in human cancers. Approximately 90% of the p53 gene mutations are localized between domains encoding exons 5 to 8. Sequencing methods currently available are tedious and time-consuming and are not suitable for routine laboratory testing. In an effort to identify a simple and rapid sequencing method, we analyzed 16 preselected breast tumors and 18 preselected ovarian tumors, using a newly developed automated DNA sequencer. p53 gene mutations had been previously identified in these tumors, using a conventional automated sequencing procedure. Exons 5 to 8 were amplified by PCR, and the PCR products were subsequently subjected to cycle sequencing with the Sanger chain termination method, using Cy5.5-labeled primers. The sequencing mixture was then resolved on a newly developed automated DNA sequencer that can sequence approximately 300 bases of DNA in 30 min. Of these 16 breast tumors, two had mutations in exon 5, four in exon 6, three in exon 7, and three in exon 8. Of the 18 ovarian tumors, two had mutations in exon 5, five in exon 6, two in exon 7, and three in exon 8. In all cases, we identified the same mutations by both the new and the conventional sequencing procedures. Most mutations affected an arginine codon. These data demonstrate that the new method has the capability to provide accurate sequencing information in a fraction of the time and labor in comparison with current automated sequencing techniques. When such procedures are used, DNA sequencing may become a routine tool for identifying clinically important mutations for diagnosis and prognosis of patients with genetic, malignant, infectious, and other diseases.