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At least 253 records · Page 14Linked to original sources
PubMed1999

Reported in vivo splice-site mutations in the factor IX gene: severity of splicing defects and a hypothesis for predicting deleterious splice donor mutations.

Small consensus sequences have been defined for RNA splicing, but questions about splicing in humans remain unanswered. Analysis of germline mutations in the factor IX gene offers a highly advantageous system for studying the mutational process in humans. In a sample of 860 families with hemophilia B, 9% of independent mutations are likely to disrupt splicing as their primary mode of action. This includes 26 splicing mutations reported herein. When combined with the factor IX splice mutations reported by others, at least 104 independent mutations have been observed, 80 of which are single base substitutions within the splice donor and splice acceptor consensus sequences. After analysis of these mutations, the following inferences emerge: (1) the susceptibility of a splice donor sequence to deleterious mutation depends on the degree of similarity with the donor consensus sequence, suggesting a simple "5-6 hypothesis" for predicting deleterious vs. neutral mutations; (2) the great majority of mutations that disrupt the splice donor or splice acceptor sequences result in at least a 100-fold decrement in factor IX coagulant activity, indicating that the mutations at these sites generally function as an on/off switch; (3) mutations that create cryptic splice junctions or that shorten but do not interrupt the polypyrimidine tract in the splice acceptor sequence can reduce splicing by a variable amount; and (4) there are thousands of potential donor-acceptor consensus sequence combinations in the 38-kb factor IX gene region apparently not reduced by evolutionary selective pressure, presenting an apparent paradox; i.e., mutations in the donor and acceptor consensus sequences at intron/exon splice junctions can dramatically alter normal splicing, yet, appropriately spaced, good matches to the consensus sequences do not predispose to significant amounts of alternative splicing.

Computer Simulation↗
PubMed1993

Gaucher disease as a paradigm of current issues regarding single gene mutations of humans.

Gaucher disease is a glycolytic storage disease caused by a deficiency in activity of the catabolic enzyme glucocerebrosidase. Over 35 different mutations have been documented, including missense and nonsense point mutations, splicing mutations, deletions and insertions, a fusion gene, and examples of gene conversion. Gaucher disease is most common in the Ashkenazi Jewish population, in which just five of the mutations in this population account for 98% of the disease-producing alleles. Each of these mutations is found in the context of a single haplotype, a finding consistent with a single origin of each mutation. Although it is clear that these mutations provide a selective advantage in the Jewish population and thus constitute a balanced polymorphism, the nature of the advantage is unknown. Gaucher disease can be treated symptomatically, by administration of the missing enzyme, by allogeneic bone marrow transplantation, and potentially by gene transfer into hematopoietic stem cells. Increasing understanding of this disease has, as in other genetic disorders, created a host of social and ethical dilemmas regarding matters such as the cost of treatment for rare diseases and the advantages and disadvantages of population-targeted genetic screening.

Amino Acid Sequence↗
PubMed1994

[Molecular genetics of inherited metabolic diseases--its application to the investigation of pathogenesis and the diagnosis of Fabry disease].

Fabry disease is an X-linked inborn error of glycosphingolipid catabolism resulting from the deficient activity of the lysosomal hydrolase, alpha-galactosidase. Patients with classic Fabry disease of early onset show diverse clinical manifestations caused by generalized vasculopathy. Recent clinical and enzymatic examinations have revealed another form of this disease; progressive cardiomyopathy of late onset without other systemic signs or symptoms. Efforts were directed to identify the specific mutations in the alpha-galactosidase gene and to clarify the phenotype/genotype correlations. A variety of mutations, including deletions, nonsense mutations, splicing mutations and amino acid substitutions caused the classic form of Fabry manifestations, which resulted in the complete deficiency of alpha-galactosidase activity. Single base substitutions were detected in the upstream region of alpha-galactosidase gene exon 6, which resulted in residual enzyme activity. The degree of the expressed residual enzyme activity might determine the clinical phenotypes. Heterozygotes were successfully identified by gene analysis and immunofluorescence imaging diagnosis using confocal laser scanning microscopy.

DNA↗
PubMed2003

A specific GFP expression assay, penetrance estimate, and histological assessment for a putative splice site mutation in BRCA1.

Genetic testing for cancer predisposing mutations in BRCA1 and BRCA2 has been of benefit to many individuals from breast and ovarian cancer-prone kindreds. However, a function has not been assigned to many of the domains that make up these complex proteins and hence, the significance of many sequence variants, including missense mutations, splice-site mutations, and in-frame deletions/insertions, remains unclear. We identified a putative splice site mutation (IVS6-2delA) in BRCA1 in a family attending a Familial Cancer Centre that had a significant history of both breast and ovarian cancer. This sequence variant was not novel but the exact effect on mRNA splicing and hence the biological impact of this sequence variation was unclear and therefore the finding was unable to be used in genetic counseling of the family. Via the construction of novel GFP-based expression fusion constructs, we demonstrated that this sequence variation prevented normal splicing of the BRCA1 transcript. By combining these data with an assessment of the histopathological features of the breast carcinomas in this family and mutation penetrance estimate we were able to conclude that this BRCA1 variant conveyed an increased risk of breast cancer.

Adult↗
PubMed2002

Identification of 14 novel CTNS mutations and characterization of seven splice site mutations associated with cystinosis.

Cystinosis is an autosomal recessive disorder characterized by intra-lysosomal accumulation of cystine. Three disease forms exist, infantile, juvenile, and ocular nonnephropathic cystinosis, delineated on the basis of severity of symptoms and age of onset. Mutations in the causative gene, CTNS, which encodes cystinosin, the seven transmembrane domain lysosomal cystine transporter, have been identified in all forms confirming their allelic status. By screening for mutations in the CTNS exons and promotor region, we report 14 novel mutations associated with cystinosis: 11 underlying infantile cystinosis, two juvenile cystinosis, and one associated with an atypical form of the disease. These mutations, all situated in the exons or immediately flanking intronic sequences, comprise in-frame insertions and deletions, as well as missense, nonsense, and putative splice-site mutations. Furthermore, we confirmed the putative splice-site mutations we have reported to date (five novel and two previously reported) by isolation of RNA from the affected carriers and characterization of the resultant transcripts using RT-PCR. Since the cloning of CTNS, we have screened for mutations in 108 affected individuals, which has resulted in a high mutation detection rate of 95.8%. Interestingly, the few undetectable mono- or bi-allelic mutations segregated mostly in the noninfantile forms, suggesting that these individuals carry mutations either in the introns or in unidentified regulatory sequences.

Adolescent↗
PubMed2001

Novel compound heterozygous mutations for lipoprotein lipase deficiency. A G-to-T transversion at the first position of exon 5 causing G154V missense mutation and a 5' splice site mutation of intron 8.

We systematically investigated the molecular defects causing a primary LPL deficiency in a Japanese male infant (patient DI) with fasting hyperchylomicronemia (type I hyperlipoproteinemia) and in his parents. Patient DI had neither LPL activity nor immunoreactive LPL mass in the pre- and post-heparin plasma. The patient was a compound heterozygote for novel mutations consisting of a G-to-T transversion at the first nucleotide of exon 5 [+1 position of 3' acceptor splice site (3'-ass) of intron 4] and a T-to-C transition in the invariant GT at position +2 of the 5' donor splice site (5'-dss) of intron 8 (Int8/5'-dss/t(+2)c). The G-to-T transversion, although affecting the 11 nucleotide of the 3'-consensus acceptor splice site, resulted in a substitution of Gly(154) to Val (G154V; GG(716)C(-->)GTC). The mutant G154V LPL expressed in COS-1 cells was catalytically inactive and hardly released from the cells by heparin. The Int8/5'-dss/t(+2)c mutation inactivated the authentic 5' splice site of intron 8 and led to the utilization of a cryptic 5'-dss in exon 8 as an alternative splice site 133 basepairs upstream from the authentic splice site, thereby causing joining of a part of exon 8 to exon 9 with skipping of a 134-bp fragment of exon 8 and intron 8. These additional mutations in the consensus sequences of the 3' and 5' splice sites might be useful for better understanding the factors that are involved in splice site selection in vivo.

Adult↗
PubMed2002

A new type of mutation causes a splicing defect in ATM.

Disease-causing splicing mutations described in the literature primarily produce changes in splice sites and, to a lesser extent, variations in exon-regulatory sequences such as the enhancer elements. The gene ATM is mutated in individuals with ataxia-telangiectasia; we have identified the aberrant inclusion of a cryptic exon of 65 bp in one affected individual with a deletion of four nucleotides (GTAA) in intron 20. The deletion is located 12 bp downstream and 53 bp upstream from the 5' and 3' ends of the cryptic exon, respectively. Through analysis of the splicing defect using a hybrid minigene system, we identified a new intron-splicing processing element (ISPE) complementary to U1 snRNA, the RNA component of the U1 small nuclear ribonucleoprotein (snRNP). This element mediates accurate intron processing and interacts specifically with U1 snRNP particles. The 4-nt deletion completely abolished this interaction, causing activation of the cryptic exon. On the basis of this analysis, we describe a new type of U1 snRNP binding site in an intron that is essential for accurate intron removal. Deletion of this sequence is directly involved in the splicing processing defect.

Adult↗
PubMed1992

Genetic basis of galactosemia.

Classic galactosemia is an inborn error of galactose metabolism and results from deficiency of the ubiquitously expressed enzyme galactose-1-phosphate uridyltransferase (GALT). Nine missense mutations, three splicing mutations, three GALT protein polymorphisms, and one silent nucleotide substitution have been identified to date. Most of the disease-causing mutations are rare among patients. The most common mutation, Q188R, has a frequency of only one-fourth in the patient population examined. Three classes of disease-causing mutations have been reported: CRM+ missense mutations (the most common class), CRM- missense mutations, and splicing mutations. Thus, galactosemia is heterogeneous at the molecular level, which is noteworthy in light of the well-documented clinical variability observed in this disorder. It has also been shown that eight of nine galactosemia missense mutations occur in evolutionarily well-conserved domains, suggesting that they affect functionally and/or structurally important residues. In contrast, all protein polymorphisms alter variable amino acids which presumably are not important for the enzyme's function.

Amino Acid Sequence↗
PubMed1994

Gene alterations and clinical characteristics of xeroderma pigmentosum group A patients in Japan.

BACKGROUND AND DESIGN: The responsible gene for xeroderma pigmentosum group A was recently identified. This study was performed to detect the gene alteration of xeroderma pigmentosum group A complementing gene in 29 xeroderma pigmentosum group A patients in Japan and to analyze whether genetic alterations in the xeroderma pigmentosum group A complementing gene are related to the clinical features. RESULTS: Of 29 patients, 25 (86%) had a mutation at the splicing junction of intron 3 and exon 4 in the homozygous state that was detected by the polymerase chain reaction and the AlwN I-restriction fragment length polymorphism. Patients having a splicing mutation at intron 3 in the homozygous state develop severe skin manifestations from early infancy and severe progressive neurologic abnormalities, including sensorineural hearing impairment, brain atrophy, mental retardation, areflexia, and ataxia. Four patients were heterozygous for splicing mutation in intron 3. Among them, at least three patients showed milder skin symptoms and milder neurologic abnormalities than patients with the homozygous splicing mutation. Two patients (single-ovum twins) revealed a compound heterozygote of splicing mutation of intron 3 and a nonsense mutation of exon 6. One patient had the splicing mutation and another mutation at the last codon of exon 5, a type of mutation that has never been reported. CONCLUSION: These data indicate that different genetic alterations in the xeroderma pigmentosum group A complementing gene may induce different clinical features.

Adolescent↗
PubMed1987

A trans-acting suppressor restores splicing of a yeast intron with a branch point mutation.

Splicing of introns from Saccharomyces cerevisiae pre-mRNA requires the conserved sequence TACTAAC; the 3'-most A residue is utilized as the site of branch formation. We showed previously that the transcript from an actin-HIS4 gene fusion containing the mutation TACTAAC to TACTACC (designated C259) is spliced inefficiently, thereby preventing growth on the histidine precursor histidinol. By selecting for growth on histidinol, we have identified a mutant in which the splicing of the C259 transcript is increased fourfold; splicing of other mutated introns is not significantly improved. The mutant locus encodes a trans-acting suppressor. A single mutation, rna16-1, is sufficient for suppression; however, suppression is maximized in heterozygous diploids containing both rna16-1 and the wild-type allele RNA16. In addition, wild-type pre-mRNAs (and lariat intermediates) accumulate in rna16-1 cells. We propose that the RNA16 locus encodes a component of the splicing machinery.

Genes, Fungal↗
PubMed1992

Three nonsense mutations responsible for group A xeroderma pigmentosum.

The molecular basis of xeroderma pigmentosum (XP) group A was studied and 3 nonsense mutations of the XP-A complementing gene (XPAC) were identified. One was a nucleotide transition altering the Arg-228 codon (CGA) to a nonsense codon (TGA). This transition creates a new cleavage site for the restriction endonuclease HphI. Of 21 unrelated Japanese XP-A patients examined, 1 (XP39OS) was a homozygote for this mutation and 3 were compound heterozygotes for this mutation and for the splicing mutation of intron 3 reported previously which is the most common mutation in Japanese patients and creates a new cleavage site for the restriction endonuclease AlwNI. The second mutation was a nucleotide transition altering the Arg-207 codon (CGA) to a nonsense codon (TGA). A Palestinian patient (XP12RO) who had severe symptoms of XP was homozygous for this mutation. The third mutation was a nucleotide transversion altering the Tyr-116 codon (TAT) to a nonsense codon (TAA). This transversion creates a new cleavage site for the restriction endonuclease MseI. Of the Japanese patients, 2 with severe clinical symptoms had this mutant allele. One was a compound heterozygote for this mutation and for the splicing mutation, and the other was heterozygous for this mutation and homozygous for the splicing mutation. Although most XP-A patients such as XP12RO have severe skin symptoms and neurological abnormalities of the de Sanctis-Cacchione syndrome, patient XP39OS was an atypical XP-A patient who had mild skin symptoms and minimal neurological abnormalities. Our results suggest that the clinical heterogeneity in XP-A is due to different mutations in the XPAC gene. Moreover, our data indicate that almost all Japanese cases of XP-A are caused by one or more of the 3 mutations, i.e., the splicing mutation of intron 3 and the 2 nonsense mutations of codons 116 and 228. Therefore, by restriction fragment length polymorphism analysis of PCR-amplified DNA sequences using the 3 restriction enzymes described above, rapid and reliable diagnosis of XP-A can be achieved in almost all Japanese subjects including prenatal cases and carriers.

Base Sequence↗
PubMed1993

Four novel cystic fibrosis mutations in splice junction sequences affecting the CFTR nucleotide binding folds.

Single cases of the four novel splice site mutations 1525-1 G-->A (intron 9), 3601-2 A-->G (intron 18), 3850-3 T-->G (intron 19), and 4374 + 1 G-->T (intron 23) were detected in the CFTR gene of cystic fibrosis patients of Indo-Iranian, Turkish, Polish, and German descent. The nucleotide substitutions at the +1, -1, and -2 positions all destroy splice sites and lead to severe disease alleles associated with features typical of gastrointestinal and pulmonary cystic fibrosis disease. The 3850-3 T-to-G change was discovered in a very mildly affected 33-year-old delta F508 compound heterozygote, suggesting that the T-to-G transversion at the less conserved -3 position of the acceptor splice site may retain some wildtype function.

Adolescent↗
PubMed1996

Splice site mutations in a xeroderma pigmentosum group A patient with delayed onset of neurological disease.

XP12BE is a commonly studied XP-A cell line that exhibits slightly increased resistance to UV compared with the majority of XP-A cell lines. The elevated UV survival is common to a subset of XP-A cell lines and correlates with delayed onset of the neurological disease in patients. We identified the XPA mutations in XP12BE by single strand conformation polymorphism (SSCP) analyses and nucleotide sequencing. XP12BE is a compound heterozygote and both mutations affect mRNA splicing. One mutation is a G to C transversion within the splice donor site of intron 4 that is common to several cell lines from XP-A patients with delayed onset of neurological disease. The other mutation is a G to T transversion at the same position as a G to C transversion in the splice acceptor site of intron 3 that is common in Japanese XP-A patients. We also demonstrated the persistence of the XP12BE mutations in cell line 2-O-A2 which has been shown to express XPA protein. These results suggest that the intron 4 splice donor mutation likely produces some, at least partially functional, XPA protein that accounts for the increased UV survival of XP-A cell lines derived from patients with delayed onset of neurological disease.

Alleles↗
PubMed2002

Outcome of donor splice site mutations accounting for congenital afibrinogenemia reflects order of intron removal in the fibrinogen alpha gene (FGA).

Congenital afibrinogenemia (Mendelian Inheritance in Man #202400) is a rare, autosomal recessive disorder characterized by the complete absence of circulating fibrinogen. Our recent studies on the molecular basis of the disease showed that the most common genetic defect is a donor splice mutation in fibrinogen alpha gene (FGA) intron 4, IVS4+1G>T. Two other FGA donor splice mutations, in intron 1 (IVS1+3A>G) and intron 3 (IVS3+1_+4delGTAA), were identified in afibrinogenemia patients. Because it was impossible to directly study the effect of these mutations on mRNA splicing in patient hepatocytes, we used a transfected cell approach, which previously allowed us to show that the common IVS4 mutation causes afibrinogenemia due to the activation of multiple cryptic donor splice sites. In this study, analysis of the IVS3delGTAA mutation showed exon 3 skipping in 99% of transcripts and exons 2 and 3 skipping in 1% of transcripts. The different outcomes of these donor splice mutations appear to follow the model proposed in a study of fibrillar collagen genes, where donor splice mutations occurring in a rapidly spliced intron with respect to upstream introns lead in most cases to exon skipping, while mutations in later-spliced introns lead to intron inclusion or cryptic splice-site utilization. Indeed, we found that in FGA intron 3 was preferentially spliced first, followed by intron 2, intron 4, and intron 1.

Afibrinogenemia↗
PubMed2004

Characterization of two novel BRCA1 germ-line mutations involving splice donor sites.

Deleterious BRCA1 mutations have significant clinical implications for the patients that carry them. Point mutations in critical functional domains and frameshift mutations that lead to early termination of protein translation are associated with a 60-80% risk of breast cancer and a 20-40% risk of ovarian cancer. In contrast, the significance of mutations located in intronic regions of BRCA1, even in the setting of a family history of breast and ovarian cancer, is not always clear. Some of these mutations occur in splice donor/acceptor consensus sites. These mutations can affect heteronuclear RNA (hnRNA) processing, leading to the loss of functional BRCA1 protein and thus may be disease-associated. However, it is important to verify the effect of these mutations, because splicing alterations cannot be predicted from genomic sequence alone. We report here the characterization of two novel BRCA1 mutations identified in families seen in our cancer risk evaluation clinic that alter splice donor sites of BRCA1. We show that both mutations alter transcript splicing and result in truncated BRCA1. IVS17 + 1G --> T leads to inclusion of part of intron 17 after the coding sequence of exon 17, resulting in early termination of BRCA1 protein following codon 1692. 252del5insT abolishes the splice donor site in exon 3, leading to the skipping of exon 5 and BRCA1 protein truncation following codon 45. Thus, both mutations result in loss of BRCA1 function, and carriers of these mutations should be counseled in the same manner as carriers of other truncating BRCA1 mutations.

BRCA1 Protein↗
PubMed1994

A 5' splice site mutation affecting the pre-mRNA splicing of two upstream exons in the collagen COL1A1 gene. Exon 8 skipping and altered definition of exon 7 generates truncated pro alpha 1(I) chains with a non-collagenous insertion destabilizing the triple helix.

A heterozygous de novo G to A point mutation in intron 8 at the +5 position of the splice donor site of the gene for the pro alpha 1(I) chain of type I procollagen, COL1A1, was defined in a patient with type IV osteogenesis imperfecta. The splice donor site mutation resulted not only in the skipping of the upstream exon 8 but also unexpectedly had the secondary effect of activating a cryptic splice site in the next upstream intron, intron 7, leading to re-definition of the 3' limit of exon 7. These pre-mRNA splicing aberrations cause the deletion of exon 8 sequences from the mature mRNA and the inclusion of 96 bp of intron 7 sequence. Since the mis-splicing of the mutant allele product resulted in the maintenance of the correct codon reading frame, the resultant pro alpha 1(I) chain contained a short non-collagenous 32-amino-acid sequence insertion within the repetitive Gly-Xaa-Yaa collagen sequence motif. At the protein level, the mutant alpha 1(I) chain was revealed by digestion with pepsin, which cleaved the mutant procollagen within the protease-sensitive non-collagenous insertion, producing a truncated alpha 1(I). This protease sensitivity demonstrated the structural distortion to the helical structure caused by the insertion. In long-term culture with ascorbic acid, which stimulates the formation of a mature crosslinked collagen matrix, and in tissues, there was no evidence of the mutant chain, suggesting that during matrix formation the mutant chain was unable to stably incorporated into the matrix and was degraded proteolytically.

Amino Acid Sequence↗
PubMed2005

[Novel splice-site mutation in the pre-mRNA splicing gene PRPF31 in a Chinese family with autosomal dominant retinitis pigmentosa].

OBJECTIVE: To identify mutations in a four-generation Chinese family with retinitis pigmentosa and to investigate its clinical phenotype. METHODS: Ophthalmic and electrophysiological examinations of patients with RP were performed. A genome-wide scan and linkage analysis were conducted in this family. Direct genomic sequencing was used to evaluate the candidate gene. A restriction assay was used to confirm the mutation status in this family, and to exclude it as a polymorphism in a reference population. RESULTS: The mutation gene was mapped close to RP11 in chromosomal region 19q13.4 by linkage analysis. A novel single heterozygous base substitution (G > C) was detected at the beginning of intron 8 in the PRPF31 gene whereby the consensus GT dinucleotide of the intron 8 splice donor site was changed to CT. The mutation was co-segregated completely with the disease phenotype, but was absent in the unaffected relatives and in 100 reference subjects, thus supporting its pathogenic nature in this family. The clinical findings of RP patients were consistent with a relatively severe and diffuse type of RP. CONCLUSION: A novel splice site mutation (IVS8 + 1G > C) in the PRPF31 gene caused retinitis pigmentosa in the four-generation Chinese RP family studied.

Asian People↗