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Results for “Splicing mutations”

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PubMed2003

Variations of the human glucocorticoid receptor gene (NR3C1): pathological and in vitro mutations and polymorphisms.

Glucocorticoid (GC) resistance can occur in a number of diseases. It can be either generalized (i.e., familial glucocorticoid resistance) or localized (i.e., asthma). In many cases, a reason for this resistance to steroids lies with mutations or polymorphisms present in the glucocorticoid receptor gene (GR/NR3C1) that belongs to a large family of nuclear receptors. A number of GC-resistant cell lines have been isolated in vitro, some of which arose or may have arisen in vivo. These and the mutations defined in them are included in this review as well as mutations engineered in plasmids and expressed in vitro. It also lists polymorphisms and the individual studies where association-related studies have been performed. NR3C1 is located on chromosome 5q31 and contains 10 exons that code for a 777 amino acid protein. There are two naturally occurring isoforms of the NR3C1, GRalpha (functional) and GRbeta (no hormone-binding ability). A total of 15 missense, three nonsense, three frameshift, one splice site, and two alternative spliced mutations have been reported in the NR3C1 gene associated with glucocorticoid resistance as well as 16 polymorphisms. Mutation and polymorphism data for NR3C1 will soon be found on the newly created locus-specific database.

Animals↗

PubMed2003

Neurofibromatosis 1: a novel NF1 mutation in an 11-year-old girl with a giant cell granuloma.

We report an 11-year-old girl who presented with a painless unilateral enlargement of the nasal bridge. Because of multiple café-au-lait spots and a positive family history, neurofibromatosis 1was diagnosed. On a computed tomographic scan, a unilocular radiolucency measuring 1.2 x 2 cm was seen in the anterior wall of the maxillary sinus, which was surgically removed. Histology revealed a central giant cell granuloma. Hyperparathyroidism, which can present with an osseous tumor and similar histology, was excluded. Molecular analysis uncovered a novel splice mutation (A4268G) in this neurofibromatosis 1 family, affecting our patient as well as her mother and brother. This article focuses on the variability of the neurofibromatosis 1 phenotype in this family and the possible relationship between central giant cell granuloma and neurofibromatosis 1.

Child↗

PubMedDate not supplied

Tetrahydrobiopterin responsiveness: results of the BH4 loading test in 31 Spanish PKU patients and correlation with their genotype.

Tetrahydrobiopterin (BH4) responsiveness in patients with mutations in the phenylalanine hydroxylase (PAH) gene is a recently recognized subtype of hyperphenylalaninemia characterized by a positive BH4 loading test. According to recent estimates, this phenotype may be quite common, suggesting that a large group of individuals may benefit from BH4 substitution, eliminating the need of life-long dietary restrictions. This underscores the importance of identifying BH4-responsive patients in each population, establishing the association with specific PAH mutations. In this work, we describe the results of a pilot study performed with 31 Spanish PAH-deficient patients subjected to a BH4 loading test. Overall, 11/31 (37%) showed a positive response with a 30% decrease in blood Phe levels 8 h after the BH4 challenge, and three additional patients, considered slow responders, showed this decrease only after 12-16 h. We report for the first time a patient homozygous for a splicing mutation with a slow response, suggesting an effect of BH4 supplementation on PAH gene expression. Most of the responsive patients belong to the mild hyperphenylalaninemia (MHP) or mild phenylketonuria phenotypic groups. In MHP patients we report for the first time the results of parallel single Phe doses confirming the utility of these analyses for a better evaluation of the response. Genotype analysis confirms the involvement in the response of specific mutations (D415N, S87R, R176L, E390G, and A309V) present in hemizygous patients, and provide relevant information for the discussion of the potential mechanisms underlying BH4 responsiveness.

Adolescent↗

PubMed1995

A splice donor site mutation results in the insertion of five extra amino acids into P53 from SEWA mouse sarcoma cells.

The status of the p53 gene in SEWA-AS12-ADH (S-ADH) cells, a subline of the mouse sarcoma cell line SEWA, was examined. Immunoprecipitation with wild-type (wt) or mutant P53-specific monoclonal antibodies (mAb) showed that both wt and mutant P53 were produced. Sequence analysis of the p53 cDNA and genomic DNA revealed a single nucleotide (nt) substitution at a splice donor site at the beginning of intron 7. As a result of this mutation, an alternative splice site 15 nt further 3' in intron 7 is used. The P53 protein translated from this aberrantly spliced mRNA carries an Arg258-->Ser substitution, followed by an insertion of 5 extra amino acids. This is the first example of a splice-site mutation in the mouse p53 gene.

Alternative Splicing↗

PubMed1993

Complementation cloning of an MHC class II transactivator mutated in hereditary MHC class II deficiency (or bare lymphocyte syndrome).

Hereditary major histocompatibility complex (MHC) class II deficiency (or bare lymphocyte syndrome) is a form of severe primary immunodeficiency with a total lack of MHC class II expression. It is due to a defect in the regulation of MHC class II genes. A novel gene was isolated by complementation cloning, using an MHC class II-negative mutant cell line. This gene (CIITA) functions as a transactivator of MHC class II gene expression and restores expression of all MHC class II isotypes in mutant cells. In addition, CIITA fully corrects the MHC class II regulatory defect of cells from patients with bare lymphocyte syndrome. In this disease we have identified a splicing mutation that results in a 24 amino acid deletion in CIITA, resulting in loss of function of the transactivator. Hence, the CIITA gene is essential for MHC class II gene expression and has been shown to be responsible for hereditary MHC class II deficiency.

Amino Acid Sequence↗

PubMed2006

An inherited mutation leading to production of only the short isoform of GATA-1 is associated with impaired erythropoiesis.

Acquired somatic mutations in exon 2 of the hematopoietic transcription factor GATA-1 have been found in individuals with Down syndrome with both transient myeloproliferative disorder and acute megakaryoblastic leukemia. These mutations prevent the synthesis of the full-length protein but allow the synthesis of its short isoform, GATA-1s. Experiments in mice suggest that GATA-1s supports normal adult megakaryopoiesis, platelet formation and erythropoiesis. Here we report a mutation, 332G --> C, in exon 2 of GATA1, leading to the synthesis of only the short isoform in seven affected males from two generations of a family. Hematological profiles of affected males demonstrate macrocytic anemia, normal platelet counts and neutropenia in most cases. Altogether, data suggest that GATA-1s alone, produced in low or normal levels, is not sufficient to support normal erythropoiesis. Moreover, this is the first study to indicate that a germline splicing mutation does not lead to leukemia in the absence of other cooperating events, such as Down syndrome.

Adolescent↗

PubMed2002

Germline mutation of ARF in a melanoma kindred.

Familial melanoma predisposition is associated with germline mutations at the CDKN2A/ARF locus in up to 40% of families. The exact role of the two proteins encoded by this complex locus in this predisposition is unclear. Most mutations affect either CDKN2A only or products of both genes. Recently a deletion affecting ARF-specific exon 1beta was reported in a family with melanoma and neural tumours. However, the possibility of this deletion also altering the CDKN2A transcript could not be excluded. More convincingly, a 16 base pair insertion in exon 1beta has been reported in an individual with multiple melanomas suggesting a direct role for ARF in melanoma predisposition. We report here a splice mutation in exon 1beta in a family with melanoma that results in ARF haploinsufficiency. The mutation was observed in a mother and daughter with melanoma. A sibling of the mother with breast cancer also had this mutation. Analysis of the melanoma from one individual revealed a 62 bp deletion in exon 3 of the wildtype allele and loss of the mutant allele; these somatic changes would affect both CDKN2A and ARF. These somatic events suggest that concomitant inactivation of both ARF and CDKN2A may be necessary for melanoma development and that mutations in ARF and CDKN2A possibly confer different levels of susceptibility to melanoma, with the former associated with lesser predisposition. In this situation, the events follow a 'three-hit' model as observed in tumours from FAP patients with an attenuated phenotype. Overall, the data suggest a direct role for ARF haploinsufficiency in melanoma predisposition and co-operation between ARF and CDKN2A in tumour formation, consistent with recent observations in Cdkn2a-specific knockout mice.

Adult↗

PubMed1996

Mutant isoforms of the anti-Müllerian hormone type II receptor are not expressed at the cell membrane.

Anti-Müllerian hormone, a member of the transforming growth factor beta superfamily, produces early regression of Müllerian ducts in the male fetus through binding to a serine/threonine kinase receptor, homologous to type II receptors of the transforming growth factor beta (TGF-beta) family. A splice mutation of this receptor, described in a patient with abnormal retention of Müllerian derivatives, generates two mutant isoforms, one lacking the second exon and the other bearing an insertion of 12 bases between exons 2 and 3. Using hemagglutinin-tagged recombinant receptors, we have visualized wild type and mutant receptors in COS cells by Western blotting and immunoprecipitation. The 82-kDa, endoglycosidase H-insensitive, mature form of the wild type receptor is reduced to 68 kDa by N-glycosidase F treatment. Mutant receptor isoforms, 73 and 63 kDa for the long and short form, respectively, are sensitive to endoglycosidase H, suggesting that they are retained in the endoplasmic reticulum. Indeed, only the wild type receptor was expressed on the cell surface and bound iodinated anti-Müllerian hormone. These results provide a biological explanation for the failure of the mutant receptor to induce Müllerian regression.

Alternative Splicing↗

PubMedDate not supplied

[Mutational analysis of the PKD1 and PKD2 (type 1 and 2 dominant autosomal polycystic kidney) genes].

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease. It is caused by mutations in at least two different genes: PKD1 and PKD2. The study of mutations in these genes is very difficult nowadays. In this study we have analyzed the non reiterated region of the PKD1 gene and all the exons and intron exon boundaries of the PKD2 gene. The technique used to study these genes have been single strand conformation analysis and heteroduplex. We have found 25 differences within the DNA sequence of the PKD1 gene with respect to the published sequence. Seven of these changes correspond to nonsense, missense, frameshifting and splicing mutations. The rest of changes correspond to polymorphisms or rare DNA variants. In the PKD2 gene we have identified 8 new mutations and one polymorphism. Six of these mutations are frameshifting, one is missense and the other one is a large deletion of the PKD2 gene. The rate of mutation detection within the PKD1 gene has been 4% and the rate for PKD2 has been 100%. We have not observed any correlation between genotype and phenotype either in the PKD1 nor in the PKD2 gene. The mutation analysis of ADPKD genes is very difficult, specially for the PKD1 gene. The rate of mutation detection is higher in the PKD2 gene but the global efficacy of the technique is very low as PKD2 represents only 15% of ADPKD patients. Nowadays linkage analysis is still the most useful technique for the molecular diagnosis of ADPKD patients.

DNA Mutational Analysis↗

PubMed2000

A novel St(a) glycophorin produced via gene conversion of pseudoexon III from glycophorin E to glycophorin A gene.

Stone (St(a)) is a variant antigen carried on human erythrocyte MNSs glycophorins (GPSt(a)) that are genetically associated with splicing mutations in GPA genes or with hybrid formation between GPA and GPB genes. Here we identify the first and rare gene conversion event in which GPE, the third member of the family, recombined with GPA, giving rise to a GPA-E-A hybrid gene encoding the St(a) antigen. Western blot detected expression in the proband of both GPA and GPSt(a) on the plasma membrane. Southern blot showed a new restriction fragment from the GPSt(a) gene, indicating an altered exon III-intron 3 junction. Sequencing of RT-PCR products identified one full-length and two shortened glycophorin cDNAs. The shortened forms were derived from GPSt(a) lacking one (exon III) and two exons (exon III and IV), respectively. To define the molecular basis for exon skipping, the genomic region spanning exon III of the GPSt(a) gene was amplified and sequenced. This revealed transfer from GPE to GPA of a DNA segment containing the pseudoexon III and its silent donor splice site. Thus, the inactivation of GPA exon III by conversion of a silent GPE donor splice site portrays a new molecular mechanism for St(a) antigen expression in human erythrocytes.

Alleles↗

PubMed1992

The second human beta B2-crystallin gene is a pseudogene.

Comparison of the partial sequences of the human beta B2-1- and beta B2-2-crystallin genes with orthologous rat or calf sequences shows that the fourth exon sequence of the human beta B2-2 gene contains a one triplet deletion and a mutated splice acceptor site. No transcripts from the beta B2-2-crystallin gene could be detected in the human lens. These data suggest that the human beta B2-2-crystallin gene is a pseudogene.

Base Sequence↗

PubMed2002

A hMLH1 genomic mutation and associated novel mRNA defects in a hereditary non-polyposis colorectal cancer family.

Hereditary non-polyposis colorectal cancer (HNPCC), or Lynch syndrome I, is responsible for as high as 10% of all colorectal cancers (CRCs) newly diagnosed in any given year. This disorder has an autosomal dominant inheritance pattern and is almost fully penetrant (>85%). It occurs when there is a mutation in any one of six mismatch repair genes: hMLH1, hMSH2, hPMS1, hPMS2, hMSH3 and hMSH6. Mutations in these genes allow mistakes in tumor suppressor genes and oncogenes to accumulate which eventually leads to cancer. The founder of an HNPCC family in the Creighton University Hereditary Cancer Institute database was known to produce truncated hMLH1 protein, a product of one of the aforementioned mismatch repair genes. Lymphoblasts were isolated from ten members of this HNPCC family (six affected and four unaffected) and two persons from outside this family (both unaffected controls). RNA and DNA were purified from these lymphoblasts which had been transformed by the Epstein-Barr virus (EBV). The hypothesis was that a mutation in the hMLH1 gene perpetuated defects in its mRNA and functional protein. hMLH1 RNA transcripts were detected in reverse transcriptase polymerase chain reactions (RT-PCR) whereby total poly A(+) RNA was converted to a complementary DNA (cDNA), amplified using hMLH1 specific primers, purified and cycle sequenced. Likewise, DNA was employed as template for PCR amplification of hMLH1 exons; PCR products were then directly cycle sequenced. Affected family members were found to produce hMLH1 mRNA lacking exons 6 and 7 (and wild-type mRNA). A splicing mutation at 546--2 (two bases 5' to exon 7) was located in the genomic DNA samples from the six family members with the HNPCC phenotype. This mutation caused deletion of exon 7 from the mRNA. None of the four unaffected family members or the two unaffected persons outside of this family had the above defects in their hMLH1 mRNA and DNA.

Adaptor Proteins, Signal Transducing↗

PubMed1997

E2F-dependent mitogenic stimulation of the splicing of transcripts from an S phase-regulated gene.

There is one class of genes whose expression increases at the G1/S transition of the cell cycle. One of these genes codes for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2), an enzyme that controls glycolysis. The cell-cycle regulation of the PFK-2 gene depends on a binding site for the transcription factor E2F located at the 5'end of the first exon and involves not only a transcriptional, but also a post-transcriptional, mechanism. We have investigated this mechanism by studying in Rat-1 fibroblasts mature and immature mRNAs from the endogenous PFK-2 gene and from stably expressed transgenes containing PFK-2 gene regions. An increase in precursor mRNA half-life and processing took place at the G1/S transition. Transgenes with a mutated E2F binding site or with mutated splice sites lost the regulation by serum, indicating that both an intact E2F binding site and an efficient splicing reaction are necessary for proper mitogenic stimulation. In quiescent cells, the transgene lacking the E2F binding site was more efficiently spliced than the wild-type construct. These results indicate that, in the wild-type gene, precursor mRNA splicing is blocked in G0and that this block requires the E2F binding site. The data provide evidence for a coupling between stimulation of promoter activity and increased mRNA splicing in the mitogenic regulation of S phase-regulated genes.

Animals↗

PubMed1994

Development of RNA-SSCP protocols for the identification and screening of CFTR mutations: identification of two new mutations.

A strategy is described that allows a rapid and accurate identification and screening of cystic fibrosis gene mutations. It consists of setting up and developing RNA single strand conformation polymorphism (rSSCP) protocols, a technique based on the large repertoire of secondary structure of single-stranded RNA. By incorporating the T7 phage promoter sequence into PCR primers, it is possible to carry out rSSCP and compare it to standard single-strand conformation polymorphism (SSCP). Several parallel tests indicate that rSSCP detects a higher fraction of single base changes, and is less time consuming than SSCP since it requires only one fairly short electrophoretic run. Using this technique we were able to identify two new splicing mutations in introns 5 (711 + 5G-->A) and 10 (1717-8G-->A) of the CFTR gene.

Cystic Fibrosis↗

PubMed1993

Molecular heterogeneity in osteogenesis imperfecta type I.

Osteogenesis imperfecta (OI) type I is characterized by bone fragility without significant deformity, osteopenia, normal stature, blue sclerae, and autosomal dominant inheritance. Dermal fibroblasts from most affected individuals produce about half the expected amount of type I collagen, suggesting that the OI type I phenotype results from a variety of mutations which alter the apparent expression of either COL1A1 or COL1A2, the genes encoding the chains of type I collagen. Short-pulse labeling of dermal fibroblasts with [3H]proline from affected individuals in 19 families indicates that most have alterations in the expected 2:1 synthetic ratio of pro alpha 1(I): pro alpha 2(I), with most having decreased production of pro alpha 1(I). Ratios of COL1A1:COL1A2 mRNA from these individuals, using slot-blot hybridization, indicate that they fall into different groups, but that most have decreased COL1A1 mRNA levels, compared with controls. These data suggest that most of our OI I families have COL1A1 mutations. Copy number and size of the COL1A1 gene by restriction endonuclease analysis of genomic DNA from affected individuals are normal in the families examined. We have identified one 3 generation family in which all affected members have one normal COL1A1 allele and another with a 5 base-pair deletion near the 3' end of the gene. The deletion creates a shift in the translational reading-frame and predicts the synthesis of an elongated pro alpha 1(I) chain. In a second family, a father and a son have a single exon deletion that results from a splicing mutation. Chemical cleavage analysis of amplified cDNA from affected individuals in different regions of the COL1A1 gene, including the promoter, suggests that several individuals have point mutations within the coding region of the gene, while one individual may have a small deletion within the alpha 1(I) carboxyl-terminal propeptide region. Our data provide evidence for significant molecular heterogeneity within the OI type I phenotype and indicate that a variety of mutations can result in decreased synthesis of type I collagen.

Cells, Cultured↗

PubMed1999

Loss-of-function mutations of SURF-1 are specifically associated with Leigh syndrome with cytochrome c oxidase deficiency.

Mutations of SURF-1, a gene located on chromosome 9q34, have recently been identified in patients affected by Leigh syndrome (LS), associated with deficiency of cytochrome c oxidase (COX), the terminal component of the mitochondrial respiratory chain. To investigate to what extent SURF-1 is responsible for human disorders because of COX deficiency, we undertook sequence analysis of the SURF-1 gene in 46 unrelated patients. We analyzed 24 COX-defective patients classified as having typical Leigh syndrome (LS(COX)), 6 patients classified as Leigh-like (LL(COX)) cases, and 16 patients classified as non-LS(COX) cases. Frameshift, stop, and splice mutations of SURF-1 were detected in 18 of 24 (75%) of the LS(COX) cases. No mutations were found in the LL(COX) and non-LS(COX) group of patients. Rescue of the COX phenotype was observed in transfected cells from patients harboring SURF-1 mutations, but not in transfected cell lines from 2 patients in whom no mutations were detected by sequence analysis. Loss of function of SURF-1 protein is specifically associated with LS(COX), although a proportion of LS(COX) cases must be the result of abnormalities in genes other than SURF-1. SURF-1 is the first nuclear gene to be consistently mutated in a major category of respiratory chain defects. DNA analysis can now be used to accurately diagnose LS(COX), a common subtype of Leigh syndrome.

Child, Preschool↗

PubMed2005

Association of two mutations in the CHEK2 gene with breast cancer.

The 1100delC mutation of the cell cycle checkpoint kinase 2 (CHEK2) gene confers an increased risk for breast cancer, but the clinical impact of other CHEK2 gene variants remains unclear. We determined the frequency of two functionally relevant CHEK2 gene mutations, I157T and IVS2+1G > A, in two large series of breast cancer cases and controls from two independent populations. Our first series consisted of a hospital-based cohort of 996 German breast cancer cases and 486 population controls, and the second series consisted of 424 breast cancer patients and 307 population controls from the Republic of Belarus. The missense substitution I157T was identified in 22/996 cases (2.2%) vs. 3/486 controls (0.6%; OR = 3.6, 95% CI 1.1-12.2, p = 0.044) in the German population and in 24/424 cases (5.7%) vs. 4/307 controls (1.3%; OR = 4.5, 95% CI 1.6-13.2, p = 0.005) in the Byelorussian cohorts. The splicing mutation IVS2+1G > A was infrequent in both populations, being observed in 3/996 German and 4/424 Byelorussian patients (0.3% and 0.9%, respectively) and in 1/486 German controls (0.2%; adjusted OR = 4.0, 95% CI 0.5-30.8, p = 0.273). Heterozygous CHEK2 mutation carriers tended to be diagnosed at an earlier age in both populations, but these differences did not reach statistical significance. Family history of breast cancer did not differ between carriers and noncarriers. Our data indicate that the I157T allele, and possibly the IVS2+1G > A allele, of the CHEK2 gene contribute to inherited breast cancer susceptibility.

Adult↗

PubMed1997

Skipping of exon 14 and possible instability of both the mRNA and the resultant truncated protein underlie a common cholesteryl ester transfer protein deficiency in Japan.

Among the Japanese population, a G-to-A mutation at the beginning of intron 14 of the human cholesteryl ester transfer protein (CETP) gene is a frequent cause of CETP deficiency characterized by markedly increased HDL cholesterol. The resulting abnormalities responsible for null CETP deficiency were studied in detail. The CETP mRNA transcripts amplified by polymerase chain reaction from the monocyte-derived macrophages of two homozygous patients were both found to be normal except for the whole deletion of exon 14. The deletion causes a shift of reading frame and introduces a premature termination codon downstream. Examination of the macrophage RNA from heterozygotes suggested the increased instability of the abnormal mRNA in the cytoplasm, because the amount of the aberrant transcript was nearly one third that of a normal transcript in the cytoplasm, while they were equal in the nucleus. Although this indicated the synthesis of a mutant CETP that lacks about 15% at its carboxy terminus, immunoblot analysis demonstrated that the abnormal CETP was virtually absent in both the media and cell lysates of transfected COS-1 cells, which massively expressed the mutant CETP mRNA. These results elucidate the primary abnormality due to the common CETP splicing mutation to be the exon skipping of mRNA, which decreases the level of mRNA and produces a truncated protein that should be rapidly degraded intracellularly.

Alternative Splicing↗