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Results for “Splicing mutations”

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PubMed2001

Reduced expression of the epithelial adhesion ligand laminin 5 in the skin causes intradermal tissue separation.

Laminin 5, the major keratinocyte adhesion ligand, is found in the lamina lucida subregion of the epidermal basement membrane of the skin, where it colocalizes with the anchoring filaments. Mutations in the genes encoding laminin 5 cause junctional epidermolysis bullosa, an inherited skin blistering disease characterized by abnormal hemidesmosomes and cleavage of the lamina lucida leading to epidermal detachment. In this work we describe the genetic basis of a new subtype of lethal inherited epidermolysis bullosa associated with reduced skin reactivity to laminin 5, presence of mature hemidesmosomes, and intradermal cleavage of the skin. The epidermolysis bullosa patients were heterozygous for a nonsense mutation (Q896X) and a splice site mutation (764-10T-->G) in the gene (LAMC2) for the gamma2 chain of laminin 5. The nonsense mutation causes accelerated decay of the corresponding mRNA, while the splice site mutation results in maturation of a cryptic wild-type gamma2 mRNA leading to reduced expression of wild-type laminin 5. In vitro studies using the probands' keratinocytes showed that secretion of reduced amounts of functional laminin 5 in the patient, although permitting formation of hemidesmosomes, fail to restore efficient cell adhesion. Our results provide the first evidence that laminin 5 contributes to the firm adhesion of the epithelial basement membrane to the underlying stroma. They also show that a low expression level of laminin 5 induces assembly of mature hemidesmosomes in vivo but fails to assure a stable cohesion of the dermal-epidermal junction.

3T3 Cells↗

PubMed2001

Splice donor site mutation in the lysosomal neuraminidase gene causing exon skipping and complete loss of enzyme activity in a sialidosis patient.

Sialidosis is a lysosomal storage disease caused by the deficiency of alpha-N-acetylneuraminidase (NEU1; sialidase), the key enzyme for the intralysosomal catabolism of sialylated glycoconjugates. We have identified a homozygous transversion in the last intron (IVSE +1 G>C) in neu1 of a sialidosis patient. Sequencing of the truncated cDNA revealed an alternatively spliced neu1 transcript which lacks the complete sequence of exon 5. Skipping of exon 5 leads to a frameshift and results in a premature termination codon. This is the first description of an intronic point mutation causing a complete deficiency of the lysosomal neuraminidase activity.

Amino Acid Sequence↗

PubMed2000

Novel point mutation in the splice donor site of exon-intron junction 6 of the androgen receptor gene in a patient with partial androgen insensitivity syndrome.

Androgen receptor (AR) gene mutations have been shown to cause androgen insensitivity syndrome with altered sexual differentiation in XY individuals, ranging from a partial insensitivity with male phenotype and azoospermia to a complete insensitivity with female phenotype and the absence of pubic and axillary sexual hair after puberty. In this study we present an 11-yr-old XY girl, with clinical manifestations peculiar for impaired androgen biological action, including female phenotype, blind-ending vagina, small degree of posterior labial fusion, and absence of uterus, fallopian tubes, and ovaries. At the time of the diagnosis the patient had a FSH/LH ratio according to the puberal stage, undetectable 17beta-estradiol, and high levels of testosterone (80.1 ng/mL). After bilateral gonadectomy, performed at the age of 11 yr, histological examination showed small embryonic seminiferous tubules containing prevalently Sertoli cells and occasional spermatogonia together with abundant fibrous tissue. Molecular study of the patient showed a guanine to thymine transversion in position +5 of the donor splice site in the junction between exon 6 and intron 6 of the AR gene. The result of RT-PCR amplification of the AR messenger ribonucleic acid from cultured genital skin fibroblasts of the patient suggests that splicing is defective, and intron 6 is retained in most of the receptor messenger ribonucleic acid molecules. We show by immunoblotting that most of the expressed protein lacks part of the C-terminal hormone-binding domain, and a small amount of normal receptor is observed. This is probably responsible for the reduced binding capacity in genital skin fibroblasts of the patient. The molecular basis of the alteration in this case is a novel, uncommon mutation, leading to a phenotype indicative of a partial androgen insensitivity syndrome, Quigley's grade 5.

Androgen-Insensitivity Syndrome↗

PubMed2002

Evaluation of candidate genes MAP2K4, MADH4, ACVR1B, and BRCA2 in familial pancreatic cancer: deleterious BRCA2 mutations in 17%.

It is estimated that familial aggregation and genetic susceptibility play a role in as many as 10% of pancreatic ductal adenocarcinomas. To investigate the role of germ-line mutations in the etiology of pancreatic cancer, we have analyzed samples from patients with pancreatic cancer enrolled in the NFPTR for mutations in four tumor suppressor candidate genes: (a) MAP2K4; (b) MADH4; (c) ACVR1B; and (d) BRCA2 by direct sequencing of constitutional DNA. These genes are mutated in clinically sporadic pancreatic cancer, but germ-line mutations are either not reported or anecdotal in familial pancreatic cancer. Pancreatic cancer patient samples were selected from kindreds in which three or more family members were affected with pancreatic cancer, at least two of which were first-degree relatives. No mutations were identified in mitogen-activated protein kinase kinase 4 (0 of 22), MADH4 (0 of 22), or ACVR1B (0 of 29), making it unlikely that germ-line mutations in these genes account for a significant number of inherited pancreatic cancers. BRCA2 gene sequencing identified five mutations (5 of 29, 17.2%) that are believed to be deleterious and one point mutation (M192T) unreported previously. Three patients harbored the common 6174delT frameshift mutation, one had the splice site mutation IVS 16-2A > G, and one had the splice site mutation IVS 15-1G > A. Two of the five BRCA2 mutation carriers reported a family history of breast cancer, and none reported a family history of ovarian cancer. These findings confirm the increased risk of pancreatic cancer in individuals with BRCA2 mutations and identify germ-line BRCA2 mutations as the most common inherited genetic alteration yet identified in familial pancreatic cancer.

Activin Receptors, Type I↗

PubMed2005

Mutation of acceptor splice site of the SEDL gene in X-linked spondyloepiphyseal dysplasia tarda causes the activation of cryptic splice site.

OBJECTIVE: To further investigate the genetic basis of hereditary X-linked spondyloepiphyseal dysplasia tarda (SEDL) and provide useful information for the prevention and treatment of the disease. METHODS: RT-PCR and cDNA sequencing were used to test mRNA expression of SEDL gene in a patient with 13 bp deletion of SEDL gene involving the acceptor splice site of intron 5. RESULTS: Of two different sizes of mRNA products identified in the patient, the 393 bp product was created due to the activation of cryptic splice site within exon 6; the 433 bp product was completely consistent with the part of genomic sequence on chromosome 8. CONCLUSION: The intragenic deletion that occurred in the acceptor splice site of the 3'region of intron 5 and the 5' coding region of exon 6 results in the activation of a cryptic splice site within exon 6, which causes 47 bp deletion of the resulting mRNA followed by a frameshift that would add two missense amino acids and then be followed by a termination codon (D109-S123del; S124fsX126). In addition, the mutation may activate the transcription of pseudogene SEDLP2 on chromosome 8 to partly complement the function of SEDL protein.

Adolescent↗

PubMed1997

Aberrant splicing but not mutations of TSG101 in human breast cancer.

The 11p15 gene TSG101 was recently reported to undergo frequent large intragenic deletions in human breast cancer. Here we show that that is generally not the case, but the gene shows aberrant splicing, based on the following observations: identical products were observed in matching normal and fetal tissues; deleted cDNA sequence revealed canonical splicing donor and acceptor site sequences; and genomic Southern blots showed no intragenic deletions in all 72 tumors studied. Nevertheless, relaxation of RNA splicing fidelity may be an oncodevelopmental marker in cancer and may play a general role in other genes and tumors.

Base Sequence↗

PubMed1999

Structure of tau exon 10 splicing regulatory element RNA and destabilization by mutations of frontotemporal dementia and parkinsonism linked to chromosome 17.

Coding region and intronic mutations in the tau gene cause frontotemporal dementia and parkinsonism linked to chromosome 17. Intronic mutations and some missense mutations increase splicing in of exon 10, leading to an increased ratio of four-repeat to three-repeat tau isoforms. Secondary structure predictions have led to the proposal that intronic mutations and one missense mutation destabilize a putative RNA stem-loop structure located close to the splice-donor site of the intron after exon 10. We have determined the three-dimensional structure of this tau exon 10 splicing regulatory element RNA by NMR spectroscopy. We show that it forms a stable, folded stem-loop structure whose thermodynamic stability is reduced by frontotemporal dementia and parkinsonism linked to chromosome 17 mutations and increased by compensatory mutations. By exon trapping, the reduction in thermodynamic stability is correlated with increased splicing in of exon 10.

Base Sequence↗

PubMed2000

Mutation analysis of the GLUT2 gene in patients with Fanconi-Bickel syndrome.

Fanconi-Bickel syndrome (FBS) is an autosomal recessive disorder manifesting hepatorenal glycogen accumulation, Fanconi nephropathy, and impaired utilization of glucose and galactose. Several mutations in a gene encoding a glucose transporter, GLUT2, have recently been reported in patients with FBS. We performed molecular analysis on three Japanese patients and found four novel mutations: a splice-site mutation (IVS2-2A>G), a nonsense mutation (Q287X), and two missense mutations (L389P and V423E). Heterozygotes of L389P or V423E mutation from the patients' families showed renal glucosuria. These data suggested that GLUT2 gene defects may be a cause of renal glucosuria.

Adolescent↗

PubMed2002

tau Exon 10 expression involves a bipartite intron 10 regulatory sequence and weak 5' and 3' splice sites.

tau mutations that deregulate alternative exon 10 (E10) splicing cause frontotemporal dementia with parkinsonism chromosome 17-type by several mechanisms. Previously we showed that E10 splicing involved exon splicing enhancer sequences at the 5' and 3' ends of E10, an exon splicing silencer, a weak 5' splice site, and an intron splicing silencer (ISS) within intron 10 (I10). Here, we identify additional regulatory sequences in I10 using both non-neuronal and neuronal cells. The ISS sequence extends from I10 nucleotides 11-18, which is sufficient to inhibit use of a weakened 5' splice site of a heterologous exon. Furthermore, ISS function is location-independent but requires proximity to a weak 5' splice site. Thus, the ISS functions as a linear sequence. A new cis-acting element, the intron splicing modulator (ISM), was identified immediately downstream of the ISS at I10 positions 19-26. The ISM and ISS form a bipartite regulatory element, within which the ISM functions when the ISS is present, mitigating E10 repression by the ISS. Additionally, the 3' splice site of E10 is weak and requires exon splicing enhancer elements for efficient E10 inclusion. Thus far, tau FTDP-17 splicing mutations affect six predicted cis-regulatory sequences.

Alternative Splicing↗

PubMed1997

Both the polypyrimidine tract and the 3' splice site function prior to the first step of splicing in fission yeast.

While it is known that several trans -acting splicing factors are highly conserved between Schizosaccharomyces pombe and mammals, the roles of cis -acting signals have received comparatively little attention. In Saccharomyces cerevisiae, sequences downstream from the branch point are not required prior to the first transesterification reaction, whereas in mammals the polypyrimidine tract and, in some introns, the 3' AG dinucleotide are critical for initial recognition of an intron. We have investigated the contribution of these two sequence elements to splicing in S.pombe. To determine the stage at which the polypyrimidine tract functions, we analyzed the second intron of the cdc2 gene (cdc 2-Int2), in which pyrimidines span the entire interval between the branch point and 3' splice site. Our data indicate that substitution of a polypurine tract results in accumulation of linear pre-mRNA, while expanding the polypyrimidine tract enhances splicing efficiency, as in mammals. To examine the role of the AG dinucleotide in cdc 2-Int2 splicing, we mutated the 3' splice junction in both the wild-type and pyrimidine tract variant RNAs. These changes block the first transesterification reaction, as in a subset of mammalian introns. However, in contrast to the situation in mammals, we were unable to rescue the first step of splicing in a 3' splice site mutant by expanding the polypyrimidine tract. Mutating the terminal G in the third intron of the nda 3 gene (nda 3-Int3) also blocks the first transesterification reaction, suggesting that early recognition of the 3' splice site is a general property of fission yeast introns. Counter to earlier work with an artificial intron, it is not possible to restore the first step of splicing in cdc 2-Int2 and nda 3-Int3 3' splice site mutants by introducing compensatory changes in U1 snRNA. These results highlight the diversity and probable redundancy of mechanisms for identifying the 3' ends of introns.

Alleles↗

PubMed1993

SPL1-1, a Saccharomyces cerevisiae mutation affecting tRNA splicing.

A genetic approach was used to isolate and characterize Saccharomyces cerevisiae genes affecting tRNA processing. Three mutants were isolated which were able to process and utilize splicing-deficient transcripts from inactivated Schizosaccharomyces pombe suppressor tRNA genes. Extragenic recovery of suppressibility was verified by the suppression of nonsense mutations in LEU2, HIS4, and ADE1. One mutant, SPL1-1, was chosen for detailed analysis on the basis of its increased synthesis of mature suppressor tRNA over wild-type cell levels as determined by Northern (RNA) analysis. This mutant exhibited strong suppression exclusively with the defective tRNA gene used in the mutant selection. Genetic analysis revealed that a single, dominant, haplo-lethal mutation was responsible for the suppression phenotype. The mutation mapped on chromosome III to an essential 1.5-kb open reading frame (L. S. Symington and T. D. Petes, Mol. Cell. Biol. 8:595-604, 1988), recently named NFS1 (S. G. Oliver et al., Nature [London] 357:38-46, 1992), located adjacent (centromere proximal) to LEU2.

Amino Acid Sequence↗

PubMed2001

Mutation spectrum and splicing variants in the OPA1 gene.

Optic atrophy type 1 (OPA1, MIM 165500) is a dominantly inherited optic neuropathy that features low visual acuity leading in many cases to legal blindness. We have recently shown, with others, that mutations in the OPA1 gene encoding a dynamin-related mitochondrial protein, underlie the dominant form of optic atrophy. Here we report that OPA1 has eight mRNA isoforms as a result of the alternative splicing of exon 4 and two novel exons named 4b and 5b. In addition, we screened a cohort of 19 unrelated patients with dominant optic atrophy by direct sequencing of the 30 OPA1 exons (including exons 4b and 5b) and found mutations in 17 (89%) of them of which 8 were novel. A majority of these mutations were truncative (65%) and located in exons 8 to 28, but a number of them were amino acid changes predominantly found in the GTPase domain (exons 8 to 15). We hypothesize that at least two modifications of OPA1 may lead to dominant optic atrophy, that is alteration in GTPase activity and loss of the last seven C-terminal amino acids that putatively interact with other proteins.

Alternative Splicing↗

PubMed2001

Mutations of MLC1 (KIAA0027), encoding a putative membrane protein, cause megalencephalic leukoencephalopathy with subcortical cysts.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is an autosomal recessive disorder characterized by macrocephaly, deterioration of motor functions with ataxia, and spasticity, eventuating in mental decline. The brain appears swollen on magnetic resonance imaging, with diffuse white-matter abnormalities and the invariable presence of subcortical cysts. MLC was recently localized on chromosome 22q(tel). We have narrowed down the critical region by linkage analysis of 11 informative families with MLC to a region of approximately 250 kb, containing four known genes. One family with two patients who were siblings did not display linkage between the MLC phenotype and any of the analyzed microsatellite markers on chromosome 22q(tel), suggesting genetic heterogeneity and the existence of at least a second MLC locus. The maximum two-point LOD score for the 11 families was 6.6 at recombination fraction .02. Twelve different mutations in seven informative and six uninformative families were found in one of the candidate genes, KIAA0027, which we renamed "MLC1." The gene encodes a putative membrane protein with eight predicted transmembrane domains. The patients of one family were compound heterozygotes for mutations that both introduced stop codons. The mutations further included frameshifts, splice-acceptor mutations, a putative splice-donor mutation, and amino acid substitutions of residues in predicted transmembrane domains. These data provide strong evidence that mutations of MLC1 cause the disease.

Alleles↗

PubMed2001

ATP7A gene mutations in 16 patients with Menkes disease and a patient with occipital horn syndrome.

Genomic DNA of 17 unrelated Japanese males with Menkes disease and 2 Japanese males with occipital horn syndrome were studied for mutations in the ATP7A gene. Using SSCP analysis and direct sequencing of the exons and the 5'-upstream region of the gene amplified by PCR, we identified 16 mutations in 16 of 17 males with Menkes disease, including 4 deletions, 2 insertions, 6 nonsense mutations, 2 missense mutations, and 2 splice-site mutations. All these mutations were those that affect the function of the gene. Of the two males with occipital horn syndrome, one had a splice-site mutation in intron 6 that led to normal-size and smaller-size transcripts. The amount of the normal-size transcripts in his cultured skin fibroblasts was 19% of the normal level. His serum copper and ceruloplasmin levels were normal, whereas his cultured skin fibroblasts contained increased levels of copper. These findings indicate that his mild clinical manifestations were due to the presence of normal-size and presumably functional transcripts of the gene. DNA sequencing analysis of the exons and 5'-upstream region of the ATP7A gene in 20 normal individuals and the 19 affected males identified 25 polymorphisms.

Adenosine Triphosphatases↗

PubMedDate not supplied

NPC1 gene mutations in Japanese patients with Niemann-Pick disease type C.

Complementary and genomic DNAs isolated from the fibroblasts of 10 Japanese (7 late infantile, 2 juvenile, and 1 adult form of the disease) and one Caucasian patient with Niemann-Pick disease type C were analyzed for mutations in the NPC1 gene. Fourteen novel mutations were found including small deletions and point mutations. A one-base deletion and a point mutation caused splicing errors. The mutations were not clustered in any particular region of the gene and were found both in and out of the transmembrane domains. Three patients were homozygous, five were compound heterozygous, and the remaining three were suspected of being compound hetrozygous with an unknown error in one of their NPC1 alleles. Of the 14 mutations, the G1553A substitution that caused a splicing error of exon 9 appeared to be relatively common in Japanese patients, because two patients were homozygous and one patient was compound heterozygous for this mutation.

Adolescent↗

PubMed1999

Lysosomal acid lipase mutations that determine phenotype in Wolman and cholesterol ester storage disease.

Mechanisms producing the divergent phenotypes, Wolman disease (WD) and cholesterol ester storage disease (CESD), associated with the genetic deficiency of human lysosomal acid lipase/cholesterol ester hydrolase (hLAL) function were investigated with the determination of HLAL activity levels, mRNA and protein expression, and defects in structural gene sequences in cells from three WD and five CESD patients. Measured with natural substrates, HLAL activities were all below 2% of normal, regardless of phenotype. Immunoblotting showed a lack of detectable hLAL protein in all mutant fibroblasts. Four CESD, but no WD genomes contained at least one allele with a specific exon 8 splice junction mutation, c.894 G>A, that encodes a shortened form of hLAL mRNA. Other CESD mutations were identical in type to the WD defects: nucleotide deletions (positions 397, 684, 980), insertions (594), or substitutions (193, 347) that result in premature terminations precluding any function. The only exception was a substitution at nucleotide 866 in the CESD case without an exon 8 splicing mutation; expression of the predicted S289C change in a transfection assay produced a low, but clearly measurable, level of acid esterase activity. Although it is not easily demonstrated in conventional assays, CESD is distinct from WD in that at least one mutant allele has the potential to produce enough residual enzymatic function to ameliorate the phenotype; in the majority of CESD cases this may come from a single, easily detected, splicing mutation in one allele.

Adult↗

PubMed2000

A novel HLA-B39 allele (HLA-B3916) due to a rare mutation causing cryptic splice site activation.

A novel HLA-B*39 variant, found in an African patient with sickle cell anemia undergoing bone marrow transplantation is described. Initially suspected by inconsistent serological typing (B-blank, Bw6), then recognized by PCR-SSP, and finally characterized by nucleotide sequencing, this novel allele is designated HLA-B*3916. It differs from HLA-B*3910 by a point mutation (G to C) at position 17 of exon 3 causing glutamine to histidine change at codon 96 of alpha(2) domain, a conserved position among HLA class I alleles. cDNA sequence analysis further revealed the presence of both normally and abnormally spliced mRNA species in established cell lines. The abnormal species correspond to partial truncation of exon 3 presumably due to the nucleotide change in exon 3, which constitutes a new consensus acceptor splice site within this exon. We postulate that the observed blank is essentially the consequence of qualitative change in a critical region of this novel antigen as abnormal mRNA species are relatively less abundant than normal species. Because the residue 96 of the HLA class I heavy chain is directly involved in interaction with alpha(2)m, another interesting possibility is that an aminoacid change in this position would perturb such interaction and consequently could affect the serological specificity of B*3916, or its expression or both.

Amino Acid Sequence↗

PubMed2003

Characterization of 17 novel endoglin mutations associated with hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia type 1 (HHT1) is a vascular dysplasia caused by mutations in the endoglin (ENG) gene and associated with epistaxis, telangiectases, and a high incidence of pulmonary arteriovenous malformations. To efficiently detect deletions and insertions, we optimized a quantitative multiplex polymerase chain reaction (QMPCR) analysis. We report 17 novel mutations, of which six were detected by QMPCR. Three deletions occurring in intronic sequences were associated with a single copy of exons 9a-14, exon 5, and exons 7-8, respectively. A transient 70kDa monomeric mutant protein resulted from the in-frame deletion of exons 7 and 8 but no mutant protein was present in the other cases. Deletion (in exon 10) or insertion (in exon 7) of two nucleotides, as well as a 1-bp deletion in the small exon 9a were found by QMPCR. Sequencing was required to detect single nucleotide deletions/insertions in exons 2, 5, 6, and 8. No mutant proteins were associated with these frame shift mutations. Two novel splice site mutations resulted in skipping of exons 2 and 4, respectively, while a previously reported intron 3 splice mutant was observed as a de novo mutation. We also report five novel nonsense and missense mutations, including one de novo. Review of the 80 HHT1 families reported to date indicates that 10% would not be resolved by sequencing and that an additional 25% could be revealed by QMPCR performed prior to sequencing. Thus the use of QMPCR accelerates genetic screening for HHT1 and resolves mutations affecting whole exons.

Adolescent↗