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PubMed1994

Analysis of mutations and alternative splicing patterns in the CFTR gene using mRNA derived from nasal epithelial cells.

Ten to fifteen percent of CF chromosomes carry mutations which are not detected by routine screening of the CFTR gene for known mutations. Many techniques have been used to screen the CFTR gene for these remaining mutations. Most of the methods use genomic DNA, and since the CFTR gene contains 27 exons, are necessarily labour intensive. We have screened the entire coding region of CFTR, by chemical cleavage of 7 overlapping segments of amplified cDNA. Using this method we have identified 4 sequence changes which had not been detected by screening genomic DNA, and successfully detected 10 out of 13 known mutations. In addition, we have identified 8 alternatively spliced forms of CFTR mRNA, 4 of which have not been described previously. These include transcripts lacking a) exon 3, b) exons 2 + 3, c) exons 9 + 12, and d) the final 357 bp of exon 15 as a result of use of the cryptic splice donor site CA2863/GTTCGT).

Base Sequence↗
PubMed1998

Two aberrant splicings caused by mutations in the insulin receptor gene in cultured lymphocytes from a patient with Rabson-Mendenhall's syndrome.

Rabson-Mendenhall's syndrome is one of the most severe forms of insulin resistance syndrome. We analyzed an English patient described elsewhere and found novel mutations in both alleles of the insulin receptor gene. One is a substitution of G for A at the 3' splice acceptor site of intron 4, and the other is an eight-base pair deletion in exon 12. Both decrease mRNA expression in a cis-dominant manner, and are predicted to produce severely truncated proteins. Surprisingly, nearly normal insulin receptor levels were expressed in the patient's lymphocytes, although the level of expression assessed by immunoblot was approximately 10% of the control cells. Insulin binding affinity was markedly reduced, but insulin-dependent tyrosine kinase activity was present. Analyzing the insulin receptor mRNA of the patient's lymphocytes by reverse transcription PCR, we discovered aberrant splicing caused by activation of a cryptic splice site in exon 5, resulting in a four-amino acid deletion and one amino acid substitution, but restoring an open reading frame. Skipped exon 5, another aberrant splicing, was found in both the patient and the mother who had the heterozygotic mutation, whereas activation of the cryptic splice site occurred almost exclusively in the patient. Transfectional analysis in COS cells revealed that the mutant receptor produced by cryptic site activation has the same characteristics as those expressed in patient's lymphocytes. We speculate that this mutant receptor may be involved in the relatively long survival of the patient by rescuing otherwise more severe phenotypes resulting from the complete lack of functional insulin receptors.

Alleles↗
PubMed1998

Characterization of exon skipping mutants of the COP1 gene from Arabidopsis.

The removal of introns from pre-mRNA requires accurate recognition and selection of the intron splice sites. Mutations which alter splice site selection and which lead to skipping of specific exons are indicative of intron/exon recognition mechanisms involving an exon definition process. In this paper, three independent mutants to the COP1 gene in Arabidopsis which show exon skipping were identified and the mutations which alter the normal splicing pattern were characterized. The mutation in cop1-1 was a G-->A change 4 nt upstream from the 3' splice site of intron 5, while the mutation in cop1-2 was a G-->A at the first nucleotide of intron 6, abolishing the conserved G within the 5' splice site consensus. The effect of these mutations was skipping of exon 6. The mutation in cop1-8 was G-->A in the final nucleotide of intron 10 abolishing the conserved G within the 3' splice site consensus and leading to skipping of exon 11. The splicing patterns surrounding exons 6 and 11 of COP1 in these three mutant lines of Arabidopsis provide evidence for exon definition mechanisms operating in plant splicing.

Arabidopsis↗
PubMed2001

A novel splice acceptor site mutation of protein S gene in affected individuals with type I protein S deficiency: allelic exclusion of the mutant gene.

Sequencing studies of the protein S gene (PROS1) in a Japanese patient suffering from recurrent thrombosis revealed the following. The proband and his first daughter, but not the second daughter, were having the type I protein S (PS) deficiency due to a novel point mutation from A to G at the intronic acceptor splice site in intron 13 of the PROS1. In the affected daughter, exclusion of the aberrant allele was assessed by the BstX1 dimorphism of PROS1 at Pro626 (CCG/CCA). The reduced PS activities in the proband and his first daughter were apparently due to defective production of mRNA from the mutant allele.

Alleles↗
PubMed2006

Alpha-thalassemia phenotype induced by the new IVS-II-2 (T --> A) splice donor site mutation on the alpha2-globin gene.

We present a family of North European extraction referred for a refractory non iron depleted microcytic anemia. The proband, a 36 year-old male, presented with chronic borderline anemia and microcytic hypochromic parameters. No abnormal hemoglobin (Hb) fractions were observed on high performance liquid chromatography (HPLC) or on alkaline electrophoresis. Gap-polymerase chain reaction (gap-PCR) excluded the seven common alpha-thalassemia (thal) deletion defects. However, the beta/alpha-globin chain synthesis ratio measured in vitro was unbalanced, indicating a reduced expression of the alpha-globin genes. Direct sequencing of the alpha-globin genes revealed heterozygosity for a T --> A transversion at the IVS-II-2 position of the alpha2 gene. This is the first IVS-II splice donor site mutation described on the alpha2-globin gene.

Adult↗
PubMed2003

Heterogous dystrophin mRNA produced by a novel splice acceptor site mutation in intermediate dystrophinopathy.

The molecular background of an intermediate type of dystrophinopathy [Duchenne and Becker muscular dystrophy (DMD/BMD)] remains to be clarified, and out-of -frame and in-frame mutations of the dystrophin gene are shown to be causes of DMD and BMD, respectively. In a boy with this disease, dystrophin mRNA extracted from lymphocytes and muscle were analyzed both qualitatively and quantitatively using reverse transcription PCR. Three different dystrophin mRNA were found to be produced via the use of three cryptic splice acceptor sites resulting from a novel point mutation of 2831-2A>G at the conserved splice acceptor site of intron 20. One of three mRNA showed an insertion of six nucleotides of intron 20 between exons 20 and 21 (dys+6) that encoded two novel amino acids in the rod domain of dystrophin. Two other mRNA species showed an insertion of seven nucleotides of intron 20 between exons 20 and 21 (dys+7) or a seven-nucleotide deletion in exon 21 (dys-7). Quantitative analysis of each dystrophin mRNA expressed in the boy's skeletal muscle disclosed that around 95% and 5% of dystrophin mRNAs were dys-7 and dys+6, respectively, whereas these two mRNA were almost equally expressed in lymphocytes. It is suggested that production of a small fraction of in-frame mRNA in muscle explains the molecular background of the intermediate type of dystrophinopathy in the index case. This finding underlines the potential of genetic therapeutic strategies aimed to modify mRNA in DMD to generate a much milder disease.

Adolescent↗
PubMed1996

[Screening for mutations in the PAH gene in Chinese: a new splice and a novel polymorphic mutation].

OBJECTIVE: to screening for novel mutations in the phenylalanine hydroxylase (PAH) genes in Chines as to investigate the molecular basis of phenylketonuria and to raise its diagnosis rate. METHODS: We applied the polymorase chain reaction (PCR) on amplified DNA fragment harboring exon 4 of the PAH gene. For novel mutations detected, the amplified fragments were sequenced by using ds-DNA cycle-sequencing method. RESULTS: Seven out of 24 cases of PKU had different kinds of band-shifting, which are subjected to two patterns of polymorphism. The first one, only in one case, has one mutation band migrating before the normal bands, while, the second one, which was common among the cases, had both the mutation bands running ahead of the normal bands. By sequence analysis, these polymorphic patterns corresponded to two noval mutations. One was a GT-->AT substitution at the 5' splice donor site of intron 4, and the other is an A to C transversion at position-11 of intron 3. CONCLUSION: The two mutations are both first reported. The 5' donor splicing mutation may result in skipping of the preceeding exon during RNA processing. The A to C transversion, which accounted for about 20% of the Chinese PKU alleles is a polymorphism changes.

DNA Mutational Analysis↗
PubMed2000

The allele-specific suppressor sup-39 alters use of cryptic splice sites in Caenorhabditis elegans.

Mutations in the Caenorhabditis elegans sup-39 gene cause allele-specific suppression of the uncoordination defect of unc-73(e936). e936 is a point mutation that changes the canonical G at the 5' end of intron 16 to a U. This mutation activates three splice donors, two of which define introns beginning with the canonical GU. Use of these two cryptic splice sites causes loss of reading frame; interestingly these messages are not substrates for nonsense-mediated decay. The third splice donor, used in 10% of steady-state e936 messages, is the mutated splice donor at the wild-type position, which defines an intron beginning with UU. In the presence of a sup-39 mutation, these same three splice donors are used, but the ratio of messages produced by splicing at these sites changes. The percentage of unc-73(e936) messages containing the wild-type splice junction is increased to 33% with a corresponding increase in the level of UNC-73 protein. This sup-39-induced change was also observed when the e936 mutant intron region was inserted into a heterologous splicing reporter construct transfected into worms. Experiments with splicing reporter constructs showed that the degree of 5' splice site match to the splicing consensus sequence can strongly influence cryptic splice site choice. We propose that mutant SUP-39 is a new type of informational suppressor that alters the use of weak splice donors.

Alleles↗
PubMed2000

A novel splice-acceptor site mutation (IVS13-2A>T) of polycystic kidney disease 1 (PKD1) gene resulting in an RNA processing defect with a 74-nucleotide deletion in exon 14 of the mRNA transcript.

Autosomal dominant polycystic kidney disease (ADPKD) occurs mainly from mutations of polycystic kidney disease 1 (PKD1) gene. A novel mutation of the PKD1 gene due to a nucleotide substitution in splice-acceptor site of IVS13 (AG->TG) was identified by analyses of PKD1-cDNA and genomic DNA. The IVS13-2A>T substitution resulted in an inactivation of this splice site and utilization of cryptic splice acceptor site in exon 14, causing a 74-nucleotide deletion of this exon in the PKD1-mRNA transcript. The abnormal transcript was present ectopically in the patients' lymphocytes. The partial deletion of PKD1-mRNA leads to frameshift translation and introduces a termination signal at codon 1075. The truncated protein with about one quarter of the full-length polycystin-1 is most likely inactive. Thus, the effect of this mutation would be "loss-of-function" type. Allele specific amplification (ASA) was developed to detect the mutation in DNA samples of other family members. The mutation was present in 11 affected but absent in 13 unaffected family members, corresponding to the results of linkage analysis. In addition, it was not observed in DNA samples of 57 unrelated healthy individuals. Hum Mutat 15:115, 2000.

Amino Acid Sequence↗
PubMed1999

The identical 5' splice-site acceptor mutation in five attenuated APC families from Newfoundland demonstrates a founder effect.

Inherited mutations of the APC gene predispose carriers to multiple adenomatous polyps of the colon and rectum and to colorectal cancer. Mutations located at the extreme 5' end of the APC gene, however, are associated with a less severe disease known as attenuated adenomatous polyposis coli (AAPC). Many individuals with AAPC develop relatively few colorectal polyps but are still at high risk for colorectal cancer. We report here the identification of a 5' APC germline mutation in five separately ascertained AAPC families from Newfoundland, Canada. This disease-causing mutation is a single basepair change (G to A) in the splice-acceptor region of APC intron 3 that creates a mutant RNA without exon 4 of APC. The observation of the same APC mutation in five families from the same geographic area demonstrates a founder effect. Furthermore, the identification of this germline mutation strengthens the correlation between the 5' location of an APC disease-causing mutation and the attenuated polyposis phenotype.

Adenomatous Polyposis Coli↗
PubMed1990

Genetic defect in muscle phosphofructokinase deficiency. Abnormal splicing of the muscle phosphofructokinase gene due to a point mutation at the 5'-splice site.

The genetic defect in muscle phosphofructokinase deficiency (type VII glycogenosis, Tarui disease) was investigated. Six cDNAs for muscle phosphofructokinase, including a full-length clone, were isolated from a non-amplified library of muscle from a patient. By sequence analysis of these clones, a 75-base in-frame deletion was identified. The rest of the sequence was identical to that of the normal cDNA, except for a silent base transition at position 516 (ACT (Thr) to ACC (Thr]. The deletion was located in the 3'-terminal region of exon 13 (numbered with reference to the rabbit muscle phosphofructokinase gene (Lee, C.-P., Kao, M.-C., French, B.A., Putney, S.D., and Chang, S.H. (1987) J. Biol. Chem. 262, 4195-4199]. Genomic DNA of the patient was amplified by polymerase chain reaction. Sequence analysis of the amplified DNA revealed a point mutation from G to T at the 5'-end of intron 13. This mutation changed the normal 5'-splice site of CAG:GTATGG to CAG:TTATGG. A cryptic splice site of ACT:GTGAGG located 75 bases upstream from the normal splice site was recognized and spliced in the patient.

Base Sequence↗
PubMed1999

The same mutation affecting the splicing of WT1 gene is present on Frasier syndrome patients with or without Wilms' tumor.

Denys-Drash and Frasier syndromes are rare human disorders that associate nephropathy with gonadal and genital abnormalities. In DDS there is a predisposition to Wilms' tumor. Heterozygous point mutations in the Wilms' tumor, type1 gene (WT1), particularly those altering the zinc finger (ZF) encoding exons, have been reported in most DDS patients, while mutations in intron 9 of the same gene cause FS. This paper describes two cases of DDS, one FS and one patient with Wilm's tumor and intersex genitalia, in which mutations were searched by sequencing the exons 8 and 9 of WT1 gene. Patient 1 carried a missense point mutation in exon 8 (ZF2), converting a CGA-Arg codon to a TGA-stop codon. Patient 2 presented a single nucleotide deletion within exon 9 (ZF3) introducing a premature chain termination at codon 398. Patients 3 and 4 had a C-->T transition at position +4 of the second alternative splice donor site of exon 9 (this mutation was detected in peripheral blood and in tumor derived DNA of patient 3). However, patient 3 had previously developed a Wilms' tumor. This is the first case of Wilms' tumor development in a phenotypically and genetically confirmed case of FS.

Adolescent↗
PubMed1998

Splicing error due to a splice acceptor site mutation in the ALD gene identified in a Japanese childhood cerebral adrenoleukodystrophy case.

X-linked adrenoleukodystrophy (ALD) is characterised by progressive multifocal demyelination of central nervous system and adrenocortical insufficiency. This disorder has been also associated with mutations in the ALD gene, encoding an ATP-binding transporter which is located in the peroxisomal membrane. Defect of the gene may lead to impaired peroxisomal beta-oxidation and increase of serum and tissue very long chain fatty acids (VLCFAs). Here we report the results of analysis of the ALD gene in a Japanese patient with childhood cerebral ALD. In our patient, by sequencing of the ALD cDNA, an 8 bp insertion (ACCCCCAG) was detected at the start of exon 9, which corresponded to nucleotides from position -1 to -8 of the 3' splice acceptor site of intron 8. Sequencing of genomic DNA showed a single nucleotide substitution at position-10 of the 3' splice acceptor site of intron 8 replacing a G with an A which activated a cryptic splice acceptor site. It is concluded that a splicing error was induced by a point mutation that created a novel splice acceptor site. This insertion created a nonsense codon downstream, producing a truncated ALD protein. His mother and younger sister with increased VLCFA ratios were heterozygotes of normal and mutant ALD genes.

ATP-Binding Cassette Transporters↗
PubMed2004

A frameshift mutation and alternate splicing in human brain generate a functional form of the pseudogene cytochrome P4502D7 that demethylates codeine to morphine.

A frameshift mutation 138delT generates an open reading frame in the pseudogene, cytochrome P4502D7 (CYP2D7), and an alternate spliced functional transcript of CYP2D7 containing partial inclusion of intron 6 was identified in human brain but not in liver or kidney from the same individual. mRNA and protein of the brain variant CYP2D7 were detected in 6 of 12 human autopsy brains. Genotyping revealed the presence of the frameshift mutation 138delT only in those human subjects who expressed the brain variant CYP2D7. Genomic DNA analysis in normal volunteers revealed the presence of functional CYP2D7 in 4 of 8 individuals. In liver, the major organ involved in drug metabolism, a minor metabolic pathway mediated by CYP2D6 metabolizes codeine (pro-drug) to morphine (active drug), whereas norcodeine is the major metabolite. In contrast, when expressed in Neuro2a cells, brain variant CYP2D7 metabolized codeine to morphine with greater efficiency compared with the corresponding activity in cells expressing CYP2D6. Morphine binds to micro-opioid receptors in certain regions of the central nervous system, such as periaqueductal gray, and produces pain relief. The brain variant CYP2D7 and micro-opioid receptor colocalize in neurons of the periaqueductal gray area in human brain, indicating that metabolism of codeine to morphine could occur at the site of opioid action. Histio-specific isoforms of P450 generated by alternate splicing, which mediate selective metabolism of pro-drugs within tissues, particularly the brain, to generate active drugs may play an important role in drug action and provide newer insights into the genetics of metabolism.

Alternative Splicing↗
PubMed1989

Genetic analysis of the role of human U1 snRNA in mRNA splicing: I. Effect of mutations in the highly conserved stem-loop I of U1.

The 5' splice site mutation known as hr440 can be suppressed efficiently in vivo by a compensatory base change in U1 small nuclear RNA (snRNA). We have now begun a second-site reversion analysis of this suppressor U1-4u snRNA (which has a C----U change at position 4) to identify U1 nucleotides that are essential for mRNA splicing. Point mutations in U1-4u that disrupt the structure of stem-loop I or alter phylogenetically conserved nucleotides within the loop cause loss of suppression. The level of suppressor activity observed for most mutants correlated with the abundance of the corresponding suppressor RNA, suggesting that mutations in stem-loop I cause loss of suppression by destabilizing U1 snRNA or the U1 snRNP (small nuclear ribonucleoprotein particle). We favor the interpretation that incompletely or improperly assembled U1 snRNPs are unstable, because two severe point mutations in stem-loop I were found to decrease the binding of U1 snRNP-specific proteins in vitro. In a separate set of experiments, we found that increasing the distance between stem-loop I and the 5' end of U1 snRNA also inhibited suppression but did not affect assembly or stability of the U1 snRNP. This suggests that the relationship between the 5' splice site and the body of the U1 snRNP is important for mRNA splicing.

Gene Expression Regulation↗
PubMed1996

Mutation detection in exons 1-14 of the adenomatous polyposis coli gene: identification of an alternatively spliced transcript.

Mutations in the APC gene are responsible for the dominantly inherited colon cancer syndrome, familial adenomatous polyposis (FAP). We have designed PCR primers which allow amplification by RT-PCR of exons 1-14 of the APC gene in six overlapping segments. The amplicons have been screened for the presence of mutations in patients affected with FAP using heteroduplex analysis. One patient has been identified with an alternatively spliced transcript involving exon 14 and a single base insertion mutation within the same exon.

Alternative Splicing↗
PubMed2005

Disease-associated mutations and alternative splicing alter the enzymatic and motile activity of nonmuscle myosins II-B and II-C.

Human families with single amino acid mutations in nonmuscle myosin heavy chain (NMHC) II-A (MYH9) and II-C (MYH14) have been described as have mice generated with a point mutation in NMHC II-B (MYH10). These mutations (R702C and N93K in human NMHC II-A, R709C in murine NMHC II-B, and R726S in human NMHC II-C) result in phenotypes affecting kidneys, platelets, and leukocytes (II-A), heart and brain (II-B), and the inner ear (II-C). To better understand the mechanisms underlying these defects, we characterized the in vitro activity of mutated and wild-type baculovirus-expressed heavy meromyosin (HMM) II-B and II-C. We also expressed two alternatively spliced isoforms of NMHC II-C which differ by inclusion/exclusion of eight amino acids in loop 1, with and without mutations. Comparison of the actin-activated MgATPase activity and in vitro motility shows that mutation of residues Asn-97 and Arg-709 in HMM II-B and the homologous residue Arg-722 (Arg-730 in the alternatively spliced isoform) in HMM II-C decreases both parameters but affects in vitro motility more severely. Analysis of the transient kinetics of the HMM II-B R709C mutant shows an extremely tight affinity of HMM for ADP and a very slow release of ADP from acto-HMM. Although mutations generally decreased HMM activity, the R730S mutation in HMM II-C, unlike the R730C mutation, had no effect on actin-activated MgATPase activity but decreased the rate of in vitro motility by 75% compared with wild type. Insertion of eight amino acids into the HMM II-C heavy chain increases both actin-activated MgATPase activity and in vitro motility.

Actins↗