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PubMed1987

Adenovirus proteins from both E1B reading frames are required for transformation of rodent cells by viral infection and DNA transfection.

To determine the requirements for the individual Ad2 E1B proteins during the transformation of rodent cells, viral mutants were constructed with genetic lesions disrupting the coding sequence of either the 175 amino acid residue (175R) or the 495 amino acid residue (495R) E1B proteins. Point mutations generating stop codons very early in the coding sequences were constructed to prevent the expression of amino-terminal protein fragments which might have biological activity. Mutant virus pm1722 contains a point mutation that terminates translation of the 175R protein after three amino acids. It was completely defective for transformation of CREF cells in virion- and DNA-mediated assays. In HeLa cells, pm1722 replicated as well as wild-type virus but produced an extreme cytopathic effect and fragmentation of host-cell DNA. Nonetheless, we provide evidence that the observed transformation defect is not due to the death of transformed cells. The mutant virus dl1520, a double mutant unable to synthesize the 495R protein, was also extremely defective for the transformation of CREF cells in virion- and viral DNA-mediated assays. This result is in contrast to studies with other Ad5 mutants with lesions in the equivalent protein. Possible explanations for this difference are discussed. Replication of dl1520 in HeLa cells was significantly reduced compared to wild-type. Studies with a third mutant virus, pm2022, which contains a stop codon after the second codon of the 495R protein, suggest that very low levels of 495R protein activity are sufficient for a productive infection and significant transforming activity.

Adenovirus Early Proteins↗
PubMed1994

Mutation specific PCR and direct solid phase sequencing assay for the detection of hepatitis B virus pre-C/C mutants in anti-HBe-positive, chronic hepatitis B.

Sequence analysis of the HBV DNA from patients with anti-HBe+, chronic hepatitis B revealed that the lack of HBeAg is mostly due to a single G-->A transition at nucleotide position 1896, resulting in a translational stop codon. A point mutation-specific polymerase chain reaction (msPCR) for the detection of this genetic variant was established. Two serologically defined groups of patients with symptomatic chronic hepatitis B (HBeAg+ n = 14, anti-HBe+ n = 11) were included in this study. Viral DNA from 43 sera (26 eAg+/17 anti-HBe+) was amplified twice, using two different sets of PCR primers. Each set contained the same -strand primer, but the +strand primers differed at their 3'-end, thus being complementary only to the wild-type or to the mutant DNA. Unspecific amplification was ruled out by choosing high annealing temperatures (66 degrees C) and cloned HBV-DNA as specificity controls. Furthermore, the results were compared to our findings obtained with direct solid-phase sequencing of the amplified viral DNA. Using msPCR, we found the mutation in four of 26 of the eAg+ sera and in 17 of 17 of our anti-HBe+ samples. Mixed virus populations were identified in 13 of 21 cases. Compared to the sequencing results, msPCR is more sensitive and less time consuming for the detection of the stop codon and thus is suitable as a rapid and specific screening method.

Base Sequence↗
PubMed1999

A phagemid vector using the E. coli phage shock promoter facilitates phage display of toxic proteins.

Phage display is a powerful tool with which to adapt the specificity of protease inhibitors. To this end, a library of variants of the potato protease inhibitor PI2 was introduced in a canonical phagemid vector. Although PI2 is a natural trypsin inhibitor, we were unable to select trypsin-binding variants from the library. Instead, only mutants carrying deletions or amber stop codons were found. Bacteria carrying these mutations had a much faster growth rate than those carrying the wt PI2-encoding gene, even when the promoter was repressed. To overcome these problems, two new phagemid vectors for g3-mediated phage display were constructed. The first vector has a lower plasmid copy number, as compared to the canonical vector. Bacteria harboring this new vector are much less affected by the presence of the PI2-g3 fusion gene, which appears from a markedly reduced growth retardation. A second vector was equipped with the promoter of the Escherichia coli psp operon, instead of the lac promoter, to control the PI2-g3 gene fusion expression. The psp promoter is induced upon helper phage infection. A phagemid vector with this promoter controlling a PI2-g3 gene fusion did not affect the viability of the host. Furthermore, both new vectors were shown to produce phage particles that display the inhibitor protein and were therefore considered suitable for phage display. The inhibitor library was introduced in both new vectors. Trypsin-binding phages with inhibitory sequences were selected, instead of sequences with stop codons or deletions. This demonstrates the usefulness of these new vectors for phage display of proteins that affect the viability of E. coli.

Amino Acid Sequence↗
PubMed1996

A new alternatively spliced exon between v9 and v10 provides a molecular basis for synthesis of soluble CD44.

The numerous isoforms of murine CD44 contain a common peptide region that is encoded by exons 1-5, 16-18, and 20 and variant regions derived from exons 6-15, usually referred to as v1-v10. We have obtained evidence for expression of an additional exon between v9 (or exon 14) and the exon previously termed v10 (or exon 15). Thus, we now number the variant exons as follows: v1-v9 (exons 6-14), v10 (exon 15), and v11 (exon 16); the remaining 3'-exons become exons 17-21 (newly numbered exons are underlined). The new exon, now termed exon v10, contains 93 base pairs and can be internally spliced; the 5'-region is termed v10a, and the 3'-region, v10b. Stop codons are positioned in v10a such that translated protein would be truncated prior to the transmembrane domain and secreted as a soluble protein. We have also found that the previously described v9 exon (now termed v9a), which is 90 base pairs in length, is actually the 5'-region of a longer exon of 142 base pairs (the 3'-region is termed v9b) and thus arises by internal splicing of the longer exon. Using reverse transcription-polymerase chain reaction, four different cDNAs for CD44 isoforms that use different combinations of the new exonic sequences have been found. The mRNAs containing the new exonic sequences are restricted in their expression; to date, we have demonstrated their presence in murine G8 myoblasts in culture and in embryonic muscle and cartilage tissues in vivo. Of these new isoforms, the predominant, full-length amplified product is encoded by exons 1-5, exon 13 (v8), the 5'-part of exon 14 (v9a), exon 15 (v10), exon 16 (v11), exons 17-19, and exon 21. When COS-7 cells were transfected with v10-containing cDNA constructs, the cells secreted low molecular weight soluble CD44 into their medium. Thus, the stop codons within the new exon v10 provide a molecular basis for de novo synthesis of soluble CD44 isoforms.

Alternative Splicing↗
PubMed1998

Identification of recurrent and novel mutations in the LDL receptor gene in Spanish patients with familial hypercholesterolemia. Mutations in brief no. 135. Online.

We used the single strand conformation polymorphism (SSCP) method to investigate 13 apparently unrelated Spanish patients with familial hypercholesterolemia (FH) for mutations in the promoter region and the 18 exons and their flanking intron sequences of the low density lipoprotein (LDL) receptor gene. We found 16 aberrant SSCP patterns, and the underlying mutations were characterized by DNA sequencing. Five novel missense mutations, Q71E, C74G, C95R, C281Y and D679E, and one nonsense mutation, Q133X, were identified. We also found six missense mutations, S156L, D200Y, D200G, E256K, T413K and C646Y, and one stop codon mutation, W(-18)X, that were previously described in patients from other populations. A new frameshift mutation, 2085del19, was found in one patient. We also identified three splicing mutations; two of them are novel mutations, 1706-10G->A and 2390-1G->A, and the other one has been reported recently, 313+1G->C. Four patients were found to carry two different mutations in the same allele: Q71E and 313+1G->C; C95R and D679E; W(-18)X and E256K, and C281Y and 1706-10G->A. Our results demonstrate that there is a broad spectrum of mutations in the LDL receptor gene in the Spanish population.

DNA Mutational Analysis↗
PubMed2002

Non-stop decay--a new mRNA surveillance pathway.

Gene expression is an inherently complex process and errors often occur during the transcription and processing of mRNAs. Several surveillance mechanisms have evolved to check the fidelity at each step of mRNA manufacture. Two recent reports describe the identification of a novel pathway in eukaryotes that recognizes and degrades mRNAs that lack a stop codon. The non-stop decay mechanism releases ribosomes stalled at the 3' end of a mRNA and stimulates the exosome to rapidly degrade the transcript.

3' Untranslated Regions↗
PubMed1997

Decreased Stability of Transforming Growth Factor beta Type II Receptor mRNA in RER+ Human Colon Carcinoma Cells

Transforming growth factor beta (TGF-beta) is a potent inhibitor of cell growth and tumor progression. Previous work has shown that loss of functional TGF-beta type II receptor (RII) due to a frameshift mutation in the 5' half of the RII gene leads to TGF-beta resistance in a highly progressed, RER+ human colon carcinoma cell line designated HCT116. Expression of this mutated RII gene was highly repressed in RER+ cell lines such as HCT116 and RKO, as analyzed by RNase protection assays. Nuclear run-on and RII promoter-reporter (CAT) assays showed that the transcriptional levels of the RII gene in these RER+ cells were not reduced, compared to RII-expressing cells. However, the half-lives of the RII mRNA, as analyzed by RNase protection assays following actinomycin D treatment, were significantly decreased. This suggested that the decreased expression of the RII gene mutant was due to decreased mRNA stability. Furthermore, RII mRNA from HCT116 transfected with wild-type RII had a longer half-life than the endogenous mutated RII mRNA. A dominant negative RII mutant, which encodes a similarly truncated RII protein as HCT116 but lacks the extensive 3' untranslated region of RII mRNA, gave the same half-life as endogenous wild-type RII mRNA. We conclude that the frameshift mutation which results in a premature stop codon in the 5' half of the mRNA transcript accounts for the reduced RII mRNA levels in RER+ cells.

Journal Article↗
PubMed1998

Amelioration of polyuria in nephrogenic diabetes insipidus due to aquaporin-2 deficiency.

OBJECTIVE: We have recently reported a large cluster of patients with nephrogenic diabetes insipidus (NDI) due to an autosomal recessive aquaporin-2 (AQP-2) early-stop codon. This paper describes the clinical manifestations and evaluation of therapeutic approaches to this new entity. PATIENTS AND DESIGN: Nine patients with an AQP-2 mutation were studied. Urine osmolality was measured in five patients before and at 3 x 30 min intervals after desmopressin given in increasing doses of 5-100 micrograms. Urinary prostaglandins PGE2 and 6-keto PGF1 alpha, were extracted from 24-h urine samples and estimated by radioimmunoassays. Eight NDI patients were given a combination of a low-sodium diet and hydrochlorothiazide. Four to 11 weeks later, ibuprofen was added, and the patients were retested within the following 4-9 weeks. RESULTS: Urine osmolality remained unchanged after supra-pharmacological doses of desmopressin, at 60-70 mOsm/kg. Urinary PGE2 in control subjects was 0.74 +/- 0.1 microgram/g creatinine (mean +/- SD) compared to 5.0 +/- 2.6 micrograms/g creatinine in AQP-2 deficient patients (P < 0.05). Urinary 6-keto PGF1 alpha, was 0.20 +/- 0.03 microgram/g creatinine in controls and 0.75 +/- 0.31 microgram/g creatinine in AQP-2 deficiency (P < 0.05). Urinary volumes decreased by a mean 31% on a low-salt diet and hydrochlorothiazide, and by a mean of 38% on the combination therapy. Plasma osmolality decreased by a mean 15 mOsm/kg on the low-salt diet and hydrochlorothiazide, and by 22 mOsm/kg on the combination therapy. Urinary osmolality increased from a mean 80 mOsm/kg to 96 mOsm/kg on the low-salt diet and hydrochlorothiazide, and to 146 mOsm/kg on the combination therapy. CONCLUSION: AQP-2 deficiency in these patients with an early-stop codon is associated with complete unresponsiveness of the collecting duct to vasopressin, implying an indispensable role for AQP-2 in vasopressin antidiuresis. Urinary PGE2 and 6-keto PGF1 alpha are elevated, the former being extremely high, apparently due to the extreme vasopressin unresponsiveness. Combination therapy with a combination of a low-salt diet, thiazide and non-steroidal anti-inflammatory drug is partially effective.

6-Ketoprostaglandin F1 alpha↗
PubMed2004

[Analysis, identification and correction of some errors of model refseqs appeared in NCBI Human Gene Database by in silico cloning and experimental verification of novel human genes].

We found that human genome coding regions annotated by computers have different kinds of many errors in public domain through homologous BLAST of our cloned genes in non-redundant (nr) database, including insertions, deletions or mutations of one base pair or a segment in sequences at the cDNA level, or different permutation and combination of these errors. Basically, we use the three means for validating and identifying some errors of the model genes appeared in NCBI GENOME ANNOTATION PROJECT REFSEQS: (I) Evaluating the support degree of human EST clustering and draft human genome BLAST. (2) Preparation of chromosomal mapping of our verified genes and analysis of genomic organization of the genes. All of the exon/intron boundaries should be consistent with the GT/AG rule, and consensuses surrounding the splice boundaries should be found as well. (3) Experimental verification by RT-PCR of the in silico cloning genes and further by cDNA sequencing. And then we use the three means as reference: (1) Web searching or in silico cloning of the genes of different species, especially mouse and rat homologous genes, and thus judging the gene existence by ontology. (2) By using the released genes in public domain as standard, which should be highly homologous to our verified genes, especially the released human genes appeared in NCBI GENOME ANNOTATION PROJECT REFSEQS, we try to clone each a highly homologous complete gene similar to the released genes in public domain according to the strategy we developed in this paper. If we can not get it, our verified gene may be correct and the released gene in public domain may be wrong. (3) To find more evidence, we verified our cloned genes by RT-PCR or hybrid technique. Here we list some errors we found from NCBI GENOME ANNOTATION PROJECT REFSEQs: (1) Insert a base in the ORF by mistake which causes the frame shift of the coding amino acid. In detail, abase in the ORF of a gene is a redundant insertion, which causes a reading frame shift in the translation of an alternative protein, such as LOC124919 is wrong form of C17 orf32 (with mouse and rat orthologs determined by us). (2) Put together by mistake (with force). This is a wrong assembly of non-relating cDNA segment, such as LOC147007 is wrong form of C17orf32. (3) Mistakenly insert a base or one section of cDNA in the ORF which causes it ending beforehand, only coding cDNA sequence of N-terminal amino acids, incomplete. For example, LOC123722 is wrong form of SPRYD1, and even the human hypothetical gene LOC126250 or PDCD5 is wrong form of our PDCD5 (TFAR19). (4) Incomplete, only coding cDNA sequence of C-terminal amino acids. For example, human LOC149076 and mouse LOC230761 are wrong form of our verified human ZNF362 and mouse Zfp362, respectively. (5) Incomplete, only coding one section of coding protein cDNA sequence of correct gene ORF, lacking N-terminal and C-terminal amino acids sequence, and at the same time, mistakenly anticipates the first non-initiation codon amino acid of the incomplete protein amino acid as the initiation codon, e.g. anticipating L as M. For example, LOC200084 is wrong form of ZNF362. (6) Mistakenly insert a base or one section of cDNA in the ORF, wrongly causing unwanted termination codon before the insertion, so the coding protein lacks the first part of the amino acids. For example, the GenBank Acc. No. AL096883 ( LOCUS No. HS323M22B) is wrong form of an experimentally verified human NM_012263 with mouse ortholog of BC010510 determined. (7) It may regard the polluted genomic sequence as complete gene cDNA sequence and anticipate the so-called single exon gene, even the real one, only a small ORF in the very long single exon mRNA, while there really exists termination code in the same phase of the upper part of the ORF initiation code, no other characters accord with the gene's condition. For example, LOC91126 is wrong form of ZNF362. (8) The anticipated genes only have ORF which has no EST proofs on both terminal sides. Depending on this ORF, a complete gene cDNA with double support of EST and human genome (there are termination codes at the same phase of the upper part of ORF) which indicates the anticipated ORF reference sequence may be incorrect. For example, LOC164395 may be wrong form of novel human gene bankit4590055. (9) A similar but smaller protein-coding gene is anticipated in the range of the human genome sequence that has the support of EST experimental proof, so other new anticipated gene may be incorrect. For example, LOC167563 may be wrong form of CMYA5. However,these errors can be corrected or avoided by using our strategy. Here we give one example in detail: Comparision of the sequence SPRYD1 with human hypothetical gene LOC123722. The TAA bases in the position of 478-480 in LOC123722 cDNA is redundant, which causes a reading frame shift in the translation of an alternative protein. The redundancy of GTAAA of LOC123722 is not supported by our experimental clone,and is almost fully rejected by human EST alignment, and is shown as the next intron sequence by genomic GT/AG organization analysis. The verification of cDNA or genomic DNA sequence of SPRYD1 implies that LOC123722 has a wrong stop codon within its ORF because of the prediction program, thus being not complete cds. To sum up, by combining bioinformatics analyses with experimental verification, we have found that there are many errors of at least nine kinds appeared in NCBI GENOME ANNOTATION PROJECT REFSEQs through BLAST of our cloned genes in non-redundant database, and our strategy is helpful in correcting them, such as LOC14907, LOC200084 and LOC91126 (all of them should be ZNF362, but are three different kinds of wrong forms of ZNF362), three model reference sequences predicted from NCBI contig NT_004511 by automated computational analysis using gene prediction method, or such as LOC124919 and LOC147007 (both should be C17orf32, but are two different kinds of wrong forms of C17orf32), two model reference sequences predicted from NCBI contig NT_010808 by automated computational analysis using gene prediction method. Therefore, the correct identification and annotation of novel human genes may be still a heavy task, which can be finished within a long period of time. So human genome coding regions annotated by computer should be used with caution. The articles published in the past did not clearly point out the existence of mistakes in the NCBI human gene mode reference sequence. At the Seventh International Human Genome Conference held in April 2002, we first published the researching result on this aspect in the communication form of Posterly insert a base or one section of cDNA in the ORF, wrongly causing unwanted termination codon before the insertion, so the coding protein lacks the first part of the amino acids. For example, the GenBank Acc. No. AL096883 ( LOCUS No. HS323M22B) is wrong form of an experimentally verified human NM_012263 with mouse ortholog of BC010510 determined. (7) It may regard the polluted genomic sequence as complete gene cDNA sequence and anticipate the so-called single exon gene, even the real one, only a small ORF in the very long single exon mRNA, while there really exists termination code in the same phase of the upper part of the ORF initiation code, no other characters accord with the gene's condition. For example, LOC91126 is wrong form of ZNF362. (8) The anticipated genes only have ORF which has no EST proofs on both terminal sides. Depending on this ORF, a complete gene cDNA with double support of EST and human genome (there are termination codes at the same phase of the upper part of ORF) which indicates the anticipated ORF reference sequence may be incorrect. For example, LOC164395 may be wrong form of novel human gene bankit4590055. (9) A similar but smaller protein-coding gene is anticipated in the range of the human genome sequence that has the support of EST experimental proof, so other new anticipated gene may be incorrect. For example, LOC167563 may be wrong form of CMYA5. However, these errors can be corrected or avoided by using our strategy. Here we give one example in detail: Comparision of the sequence SPRYD1 with human hypothetical gene LOC123722. The TAA bases in the position of 478-480 in LOC123722 cDNA is redundant, which causes a reading frame shift in the translation of an alternative protein. The redundancy of GTAAA of LOC123722 is not supported by our experimental clone, and is almost fully rejected by human EST alignment, and is shown as the next intron sequence by genomic GT/AG organization analysis. The verification of cDNA or genomic DNA sequence of SPRYD1 implies that LOC123722 has a wrong stop codon within its ORF because of the prediction program, thus being not complete cds. To sum up, by combining bioinformatics analyses with experimental verification, we have found that there are many errors of at least nine kinds appeared in NCBI GENOME ANNOTATION PROJECT REFSEQs through BLAST of our cloned genes in non-redundant database, and our strategy is helpful in correcting them, such as LOC14907, LOC200084 and LOC91126 (all of them should be ZNF362, but are three different kinds of wrong forms of ZNF362), three model reference sequences predicted from NCBI contig NT_004511 by automated computational analysis using gene prediction method, or such as LOC124919 and LOC147007 (both should be C17orf32, but are two different kinds of wrong forms of C17orf32), two model reference sequences predicted from NCBI contig NT_010808 by automated computational analysis using gene prediction method. Therefore, the correct identification and annotation of novel human genes may be still a heavy task, which can be finished within a long period of time. So human genome coding regions annotated by computer should be used with caution. (ABSTRACT TRUNCATED)

Amino Acid Sequence↗
PubMed1989

Apolipoprotein B48 RNA editing in chimeric apolipoprotein EB mRNA.

Apolipoprotein (apo) B occurs in two forms, apoB100 (512 kDa) and apoB48 (240 kDa); both are derived from the same gene. A novel mechanism involving editing of the apoB mRNA causes the formation of apoB48; the first base of codon 2153 is changed from cytosine to uracil, converting a glutamine codon to a premature stop codon. To identify the apoB mRNA sequence elements recognized by the apoB mRNA editing mechanism, two apoB cDNA fragments (354 and 63 base pairs) with codon 2153 near their centers were inserted into a high expression vector of another secreted apolipoprotein, apoE. The resulting vectors, pHEB-354 and -63, were transfected into Chinese hamster ovary cells, HepG2 cells, and apoB48-producing CaCo-2 cells. The secreted chimeric apolipoproteins (apoEB354 and apoEB63) were analyzed for premature truncation, and the mRNA was analyzed for the presence of an edited base. The pHEB-354 construct produced a truncated protein only in CaCo-2 cells, whereas pHEB-63 produced no truncated protein in any of the three cell types. The mRNA was converted to cDNA and amplified by the polymerase chain reaction technique. Differential hybridization of the polymerase chain reaction products with CAA (Gln) and TAA (Stop) specific probes detected an edited base only in cDNA from CaCo-2 cells transfected with pHEB-354, in agreement with the protein analysis. We conclude that the nucleotide sequence of the apoB cDNA insert in pHEB-354 contains sufficient information to be edited in CaCo-2 cells. In these cells, a cryptic polyadenylation site was activated in the edited pHEB-354 mRNA. As a result, CaCo-2 cells transfected with pHEB-354 produced a short, edited pHEB-354 mRNA and a long, unedited pHEB-354 mRNA. Chinese hamster ovary cells transfected with pHEB-354 or CaCo-2 cells transfected with pHEB-63 produced only a full length transcript. Amplification of the pHEB-354 cDNA using 3'-primers upstream and downstream of the poly(A) addition site and hybridization with the TAA probe confirmed these results. This unusual mRNA editing apparently occurs before polyadenylation, probably in the nucleus.

Adenocarcinoma↗
PubMed1996

Complete nucleotide sequences of hepatitis B virus genomes associated with epidemic fulminant hepatitis.

Pre-core/core mutants are frequently observed in patients with fulminant hepatitis. To investigate the extent of molecular characteristics of hepatitis B virus (HBV) genomes implicated in the development of fulminant hepatitis, full-length HBV genomes were sequenced directly from sera of two patients with epidemic fatal fulminant hepatitis, after amplification by the polymerase chain reaction. These two genomes, of 3215 nucleotides, were 99.6% identical, indicating that a common source of HBV potentially caused fulminant hepatitis. Thirty unique nucleotide mutations were commonly found in the two entire HBV genomes. Three were located in the stem-loop structure, changing this element to a more stable structure. Twenty-five unique amino acid substitutions were found in each open reading frame, except for the X and pre-surface 2 genes. One was located in the pre-surface 1 gene; two were in the surface gene; three were in the pre-core gene, including codons 28 (tryptophan to stop codon) and 29 (glycine to aspartic acid); eight were in the core gene; and 11 were in the polymerase gene. The pre-core mutations at codons 28 and 29 were common to the two HBV strains reported previously in patients with epidemic fulminant hepatitis. Thus, HBV genomes associated with epidemic fatal fulminant hepatitis have numerous unique mutations, located mainly in the polymerase gene, as well as the pre-core/core gene, including mutations in the stem-loop structure of the pregenome encapsidation signal sequence. These mutations may be associated with the development of fulminant hepatitis.

Aged↗
PubMed1996

Alterations of retinoblastoma, p53, p16(CDKN2), and p15 genes in human astrocytomas.

BACKGROUND: Alterations of the suppressor genes, such as the retinoblastoma (RB), p53, p16(CDKN2), and p15 genes, have been reported in human gliomas. These genes have been suggested as the cell cycle regulatory genes at the G1-S checkpoint. METHODS: Alterations of the RB, p53, p16(CDKN2), and p15 genes in human astrocytomas were screened by single strand conformation polymorphism analysis of polymerase chain reaction products (PCR-SSCP analysis) and then confirmed by dideoxy sequencing. In addition, the expression of RB and p16 protein was examined by Western blot analysis. RESULTS: Aberrations of the RB gene were found in 3 of 23 surgical astrocytoma specimens (13%). Mutations were found at codon 754 in exon 22 (Val-->Gly), codon 519 in exon 17 (Thr-->Pro), and one base deletion at codon 903 resulting in stop codon at codon 905 in exon 26. These mutational locations were all near the regions associated with the functional domains of the RB gene. Aberrations of the p53 gene were found in 4 cases (17.4%). These mutations were found at codons 146 (Trp-->Gly) and 165 (Gln-->His) in exon 5, codon 73 (Val-->Glu) in exon 4, and codon 313 (Ser-->Asn) in exon 9. In addition, alterations of the p16(CDKN2) gene were found, with 5 cases (21.7%) having homozygous deletions, and 2 cases (8.7%) harboring point mutations. No p15 gene alteration was detected. The expression of p16 protein was undetectable in 10 cases (43.5%) by Western blot analysis, demonstrating an inverse correlation with the expression of RB protein. CONCLUSIONS: A few cases had overlapping alterations, and the incidence of one or more RB, p53, or p16(CDKN2) changes appeared to be relatively high in human astrocytomas. These results suggest that cell cycle regulatory gene alterations may play an important role in the development of gliomas.

Astrocytoma↗
PubMed2000

Germline mutations of the dpc4 gene in Korean juvenile polyposis patients.

Juvenile polyposis is an uncommon condition characterized by the development of multiple (usually more than 5) juvenile polyps in the gastrointestinal tract, especially in the colon. This disease usually occurs during childhood, and is inherited in an autosomal dominant fashion. It has been suggested that the dpc4 (deleted in pancreatic carcinoma, locus 4) gene, which is located on chromosome 18q21.1, might cause juvenile polyposis. The dpc4 (smad4) gene is a candidate tumor-suppressor gene and may play a role in the TGF-beta-signaling pathway. To confirm the idea that alterations of the dpc4 gene may result in juvenile polyposis, we screened 5 Korean juvenile-polyposis patients by PCR-SSCP (single-strand conformation polymorphism) analysis and bi-directional sequencing. There were germline mutations of the dpc4 gene in 3 out of the 5 patients: 2 had a genetic alteration in exon 9 and the third had a mutation in exon 8. These germline mutations occurred in the C-terminus of the dpc4 gene, similar to most published mutations. One patient exhibited a non-sense mutation (codon 388), which changed a glutamine codon (CAG) to a stop codon (TAG). The second patient harbored a mis-sense mutation (codon 390), causing a non-conservative amino-acid change . The third patient had a mis-sense mutation in exon 8 (codon 361), which altered an arginine codon (CGC) into a histidine codon (CAC).

Adenomatous Polyposis Coli↗
PubMed2000

Prenatal diagnosis of a novel COL1A1 mutation in osteogenesis imperfecta type I carried through full term pregnancy.

Prenatal diagnosis was performed in a family where the father has osteogenesis imperfecta (OI) type I, with a novel mutation in the COL1A1 gene: a C to T change at position c3076 (c.3076C-->T) leading to a change of arginine at codon 848 to a stop codon (R848X). Prenatal diagnosis by chorionic villous sampling (CVS) was performed during the fourth pregnancy, and revealed that the fetus is a carrier of the same COL1A1 mutation. The possibility of phenotypic variability was discussed with the parents. They elected to carry the pregnancy to term, and a male child with mild OI was born. This is the first reported case where OI was diagnosed prenatally, and the parents opted to carry the pregnancy to term. It illustrates the potential use of DNA-based analysis for early prenatal diagnosis of OI, and the complexities of genetic counselling.

Adult↗
PubMed1993

Linkage disequilibrium between TAP2 variants and HLA class II alleles; no primary association between TAP2 variants and insulin-dependent diabetes mellitus.

The TAP1 and TAP2 genes, located in the HLA class II region, encode subunits of a peptide transporter. Both genes display limited genetic variability; four different nucleotide substitutions have been found in the TAP2 gene. Here studies on linkage disequilibrium between TAP2 variants and HLA class II alleles are reported, in an attempt to evaluate whether TAP2 variants are associated with insulin-dependent diabetes mellitus (IDDM). As reported previously, a significant decrease of homozygosity for TAP2 alleles encoding alanine at residue 665 (665 Ala) and glutamine at 687 (687 Gln) paralleled by an increase in homozygosity for TAP2 alleles encoding threonine at residue 665 (665 Thr) and a stop codon at 687 (687 Stop), was found in both Finnish and Norwegian IDDM patients compared to random controls. However, a strong linkage disequilibrium between these TAP2 polymorphisms and given HLA-DR and -DQ genes was observed among healthy controls. The frequent 665 Thr and 687 Stop variants were in linkage disequilibrium both with the DR4-DQ8 and the DR3-DQ2 haplotypes, haplotypes which are strongly associated with IDDM. In contrast, the DR1-DQ5 and DR13-DQ6 (e.g. DQB1*0603) haplotypes, which are decreased among IDDM patients, were associated with the 665 Ala and 687 Gln variants. Thus, when DR- and DQ-matched patients and controls were compared, associations of the investigated TAP2 variants and IDDM were no longer detectable. These data, therefore, indicate that the associations previously found between certain TAP2 variants and IDDM are secondary to a primary association between this disease and particular DQ alpha beta heterodimers.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Hepatitis B virus carriers without precore mutations in hepatitis B e antigen-negative stage show more severe liver damage.

Hepatitis B e antigen (HBeAg) is considered to be a major target for the immune response in chronic hepatitis B. The G-->A mutation at nucleotide 1896 may mediate viral escape by creating a TAG stop codon in the precore region, thus preventing HBeAg production. This mutation frequently evolves during HBe seroconversion if thymine, but rarely if cytosine, is present in position 1858. Applying a combination of polymerase chain reaction (PCR) and restriction enzyme action, we have studied the relation of the TAG mutation and the nucleotide (nt) 1858 variants to liver damage assessed by histology activity index (HAI) scoring in 175 chronic hepatitis B virus (HBV) carriers. A TAG mutation was found in 68 of 71 (96%) of HBeAg-negative carriers infected with a T-1858 strain, but not in any of 33 carriers infected exclusively with a C-1858 strain. Four patients showed a mutation of the precore start codon, and 2 had a TAA stop mutation at codon 2. HBeAg-positive infection with a mixture of wild-type and TAG mutant virus indicated active liver damage, because 8 of 9 (89%) of such patients had a Knodell HAI greater than or equal to 8. In HBeAg-negative stage, both inflammation and fibrosis were more pronounced in carriers infected with wild-type HBV compared with precore mutants. C-1858 strains were associated with more inflammation and fibrosis compared with T-1858 strains. C-1858 strains were found in 71% of northern European, 17% of southern European, 31% of African, 2% of Middle Eastern, and 10% of Far Eastern carriers. Analysis of these variants and mutants may prove useful for clinical evaluation and choice of therapy, and may be facilitated by the methods described.

Adolescent↗
PubMed1998

Homology-based gene prediction using neural nets.

We have developed and implemented a method for computational gene identification called GIN (gene identification using neural nets and homology information) that has been particularly designed to avoid false positive predictions. It thus predicts 55% of all genes tested correctly, has a specificity of 99%, but also has an overall accuracy of 92% on a benchmark set of 570 vertebrate genes constructed by Burset and Guigo. The method combines homology searches in protein and expressed sequence tag databases with several neural networks designed to recognize start codons, Poly(A) signals, stop codons, and splice sites. Predicted exons are assembled into genes using a homology-based scoring function. GIN is able to recognize multiple genes within genomic DNA as demonstrated by the identification of a globin gene (gamma-globin-1(G)) that has not been annotated as a coding region in the widely used the test set of Burset and Guigo. Furthermore, GIN identifies more than 107 other protein hits in noncoding regions and classifies them into possible pseudogenes or splice variants.

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