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PubMed2025

Utility of genome sequencing and group-enrichment to support splice variant interpretation in Marfan syndrome.

PURPOSE: To quantify the impact of noncanonical FBN1 splice site variants in undiagnosed Marfan syndrome (MFS), a connective tissue disorder associated with skeletal abnormalities and familial thoracic aortic aneurysm disease (FTAAD). METHODS: A systematic analysis of ultrarare FBN1 variants was performed using genome sequencing data from the 100,000 Genomes Project. Variants were annotated with SpliceAI and the significance of enrichment among individuals with FTAAD was assessed using Fisher's exact test. Experimental validation used RNA sequencing, reverse transcriptase polymerase chain reaction, minigene constructs, and replication analysis was with data from UK Biobank. RESULTS: Using aggregate data for 78,195 individuals, we identified 13,864 singleton single-nucleotide variants in FBN1 of which 21 were predicted to affect splicing (SpliceAI > 0.5). Incidence of candidate splice variants in individuals recruited with FTAAD (9/703) was significantly elevated compared with that seen in non-FTAAD participants (12/77,492; odds ratio = 84, P = 9.7 × 10-14). Additional analysis uncovered a further 14 families harboring 11 different FBN1 splice variants. A total of 20 candidate splice variants in 23 families were identified, of which 70% lay beyond the ±8 splice regions. RNA testing confirmed the predicted splice aberration in 16 of 20 and for 9 of 20, pseudoexonization was the likely splicing anomaly. CONCLUSION: Our findings indicate that noncanonical splice variants may account for approximately 3% of families with undiagnosed FTAAD, highlighting the importance of incorporating analysis of introns and confirmatory RNA testing into genetic testing for Marfan syndrome.

Humans↗
PubMed2001

Molecular defects underlying the Kell null phenotype.

Expression of the Kell blood group system is dependent on two proteins, Kell and XK, that are linked by a single disulfide bond. Kell, a type II membrane glycoprotein, is a zinc endopeptidase, while XK, which has 10 transmembrane domains, is a putative membrane transporter. A rare phenotype termed Kell null (Ko) is characterized by the absence of Kell protein and Kell antigens from the red cell membrane and diminished amounts of XK protein. We determined the molecular basis of eight unrelated persons with Ko phenotypes by sequencing the coding and the intron-exon splice regions of KEL and, in some cases, analysis of mRNA transcripts and expression of mutants on the cell surface of transfected cells. Six subjects were homozygous: four with premature stop codons, one with a 5' splice site mutation, G to A, in intron 3, and one with an amino acid substitution (S676N) in exon 18. Two Ko persons with premature stop codons had identical mutations in exon 4 (R128Stop), another had a different mutation in exon 4 (C83Stop), and the fourth had a stop codon in exon 9 (Q348Stop). Two Ko persons were heterozygous for two mutations. One had a 5' splice site mutation (G to A) in intron 3 of one allele that caused aberrant splicing and exon skipping, and the other allele had an amino acid substitution in exon 10 (S363N). The other heterozygote had the same amino acid substitution in exon 10 (S363N) in one allele and a premature stop codon in exon 6 (R192Stop) in the other allele. The S363N and S676N mutants, expressed in 293T cells, were retained in a pre-Golgi compartment and were not transported to the cell surface, indicating that these mutations inhibit trafficking. We conclude that several different molecular defects cause the Kell null phenotype.

Adult↗
PubMed1998

Human G(olf) gene polymorphisms and vulnerability to bipolar disorder.

Two intronic polymorphisms of the human alpha subunit of the olfactory G-protein (G(olf)) are described. They were detected with single-stranded conformational polymorphism (SSCP) methods and confirmed by sequencing both strands. These single base pair (bp) substitutions occur in introns 3 (an A/G at 35 bp 3' from the exon 3/intron 3 5' splice site) and 10 (an T/G at 7 bp 5' from the 3' splice site). Both polymorphisms are relatively common, with minor allele frequencies of 31% (intron 3) and 16% (intron 10). The intron 3 variant shows no linkage disequilibrium with an intron 5 (CA)n microsatellite located approximately 50 kb 3' from the intron 3 variant, among a small group of German individuals with schizophrenia. The intron 3 variant is interesting because it may create an 'in-frame' cryptic splice site which, if activated, would add 12 residues to exon 3. The intron 10 variant is interesting because a purine is substituted for a pyrimidine in the 'polypyrimidine' tract of the 3' splice site, a single base substitution of the type which has been associated with aberrant splicing in the androgen receptor gene.

Alleles↗
PubMed2004

RNA modulation, repair and remodeling by splice switching oligonucleotides.

Targeting splicing by antisense oligonucleotides allows RNA modifications that are not possible with RNA interference or other antisense techniques that destine the RNA for destruction. By changing the ratio of naturally occurring splice variants the expression of mRNA is modulated. By preventing the use of an aberrant splice site created by a mutation and enforcing re-selection of correct splice sites the RNA is repaired. Antisense induced skipping of the exon that carries a nonsense mutation remodels the mRNA and restores the reading frame of the defective protein. All of the above approaches have clinical applications. Modulation of splice variants is particularly important since close to 60% of all genes code for alternatively spliced pre-mRNA.

Alternative Splicing↗
PubMed1997

Frame shift mutation, exon skipping, and a two-codon deletion caused by splice site mutations account for pyruvate kinase deficiency.

Three novel splice site mutations and two novel missense mutations were identified by molecular analysis of pyruvate kinase (PK) deficiency associated with hereditary nonspherocytic hemolytic anemia. A Nepalese PK variant, PK Kowloon, was found to have a homozygous transversion at the 5'-splice site of the seventh intervening sequence (IVS) of the L-type PK gene (Ivs7[+1]gt --> tt). Using a reverse transcription polymerase chain reaction (RT-PCR) assay, we showed that the R-type PK mRNA in the proband's reticulocytes included the seventh IVS between the seventh and eighth exon, introducing a stop codon 3 nucleotides downstream of the mutated site. Consequently, the translational product may lack 44% of the R-PK polypeptide. A transition at the last nucleotide of exon 9 (1269GCG --> GCA) was found in a Japanese PK variant, PK 'Kamata.' The mutation did not alter the amino acid sequence, but caused skipping of the ninth exonic sequence in the R-PK transcripts. As a result, the affected R-type PK lost 51 amino acid residues (373Met-423Ala del). A transversion at the splice acceptor site of the third IVS (Ivs 3[-2]ag --> tg) was identified in PK 'Aomori.' The mutation resulted in aberrant splicing at a cryptic splice site within exon 4, causing deletion of two codons in the aberrant R-PK transcript (95 Gly-96 Pro --> del). Both PK 'Kamata' and PK 'Aomori' had a missense mutation on the other allele, 1044AAG --> AAT (348Lys --> Asn) and 1075CGC --> TGC (359Arg --> Cys), respectively. Although both 348Lys and 359Arg were located in the sixth loop of A domain (beta/alpha)8 barrel, which has been shown to contain the substrate and cation binding sites, the degree of anemia was much more severe in PK 'Kamata' than PK 'Aomori,' possibly because the 51 amino acid deletion of PK 'Kamata' but the 2 amino-acid deletion of PK 'Aomori' may abolish PK catalytic activity.

Anemia, Hemolytic, Congenital Nonspherocytic↗
PubMed1992

Mutations which alter splicing in the human hypoxanthine-guanine phosphoribosyltransferase gene.

A large proportion of mutations at the human hprt locus result in aberrant splicing of the hprt mRNA. We have been able to relate the mutation to the splicing abnormality in 30 of these mutants. Mutations at the splice acceptor sites of introns 4, 6 and 7 result in splicing out of the whole of the downstream exons, whereas in introns 1, 7 or 8 a cryptic site in the downstream exon can be used. Mutations in the donor site of introns 1 and 5 result in the utilisation of cryptic sites further downstream, whereas in the other introns, the upstream exons are spliced out. Our most unexpected findings were mutations in the middle of exons 3 and 8 which resulted in splicing out of these exons in part of the mRNA populations. Our results have enabled us to assess current models of mRNA splicing. They emphasize the importance of the polypyrimidine tract in splice acceptor sites, they support the role of the exon as the unit of assembly for splicing, and they are consistent with a model proposing a stem-loop structure for exon 8 in the hprt mRNA.

Base Sequence↗
PubMed1984

One base substitution in IVS-2 causes a beta +-thalassemia phenotype in a Chinese patient.

Two beta-globin genes derived from a Chinese patient with a beta +-thalassemia phenotype have been cloned and sequenced. A four-nucleotide deletion in codon 41 and 42 was found in one of the clones and a C to T substitution at position 654 of IVS-2 was detected in the other clone. The former change, which causes premature termination at codon 59, was found previously in another Chinese patient with homozygous beta 0-thalassemia. The latter change, which has not been reported so far, creates a sequence much like the consensus sequence of the 5' splice site. These results suggest that the new base change found in IVS-2 causes aberrant splicing of the beta-globin mRNA precursor. Thus, this patient is doubly heterozygous for beta 0- and beta +-thalassemia.

Base Sequence↗
PubMed2024

Dysfunction of ATP7B Splicing Variant Caused by Enhanced Interaction With COMMD1 in Wilson Disease.

BACKGROUND & AIMS: The association between Wilson disease and various ATP7B mutations is well-established; however, the molecular mechanism underlying the functional consequence of these mutations, particularly the splicing mutations, remains unclear. This study focused on the ATP7B c.1543+1G>C variant, to reveal a universal pathogenic mechanism of the ATP7B mutants with altered N-terminus. METHODS: The splicing assay and RNA pull-down were performed to explore the mechanism of the aberrant splicing. The ATP7B knockout HuH-7 cell line and Atp7b-/- mice were created, and the functional consequence of the mutant ATP7B were evaluated in vitro and in vivo. RESULTS: The c.1543+1G>C mutation resulted in the skipping of ATP7B exon 3, and the mutant ATP7B showed a loss of trans-Golgi network localization and was degraded via the ubiquitin-proteasome pathway, facilitated by enhanced interactions with COMMD1. Elevated intercellular copper concentration and reduced survival rate were observed in HuH-7 cells expressing mutant ATP7B. Restoration of wild-type ATP7B in Atp7b-/- mice resulted in a substantial improvement in phenotype, whereas mice treated with mutant ATP7B did not demonstrate equivalent benefits. CONCLUSIONS: Our research investigated the pathogenicity and mechanism of ATP7B c.1543+1G>C variant, with particular focus on its enhanced interaction with COMMD1 as a potential universal mechanism contributing to the dysfunction of various ATP7B variants. These findings provide a foundation for the development of innovative therapeutic strategies that target abnormal splicing events in a range of hereditary diseases, including Wilson disease.

Copper-Transporting ATPases↗
PubMed2000

A novel mutation in the pendrin gene associated with Pendred's syndrome.

OBJECTIVE: Pendred's syndrome is an autosomal recessive disorder characterized by goitre, sensorineural deafness and iodide organification defect. It is one of the most frequent causes of congenital deafness, accounting for about 10% of hereditary hearing loss. It is caused by mutations in the pendrin (PDS) gene, a 21 exon gene located on chromosome 7. The aim of this study was to examine an Italian family affected with Pendred's syndrome at the molecular level. PATIENTS: Thirteen subjects belonging to a family from Southern Italy were evaluated for the clinical and genetic features of Pendred's syndrome. MEASUREMENTS: Exons 2-21 of the PDS gene were amplified from peripheral leucocytes by the polymerase chain reaction; mutation analysis was performed by single strand conformation polymorphism, direct sequencing and restriction analysis. RESULTS: The index patient had the classical triad of the syndrome and harboured two mutations in the PDS gene in the form of compound heterozygosity. He was found to be heterozygous for a cytosine to adenosine mutation at nucleotide 1523 in exon 13 and for a IVS 1001 + 1G --> A mutation. The former is a novel mutation which results in a change of 508 threonine to asparagine in the putative eleventh transmembrane domain. The latter mutation in the donor splice site has already been described in other patients and is thought to lead to aberrant splicing and premature protein truncation. Three subjects who were heterozygous for one mutation had normal phenotypes. Two subjects had sensorineural deafness and were heterozygous for a single mutation. Goitre was found only in patients with Pendred's syndrome and was absent in all other individuals, albeit residing in an iodine-deficient area. CONCLUSIONS: We have identified a novel mutation in the pendrin gene causing Pendred's syndrome, and confirm that molecular analysis is a useful tool for a definitive diagnosis. This is particularly relevant in cases such as in the subjects of our family in which the clinical features might be misleading and other genetics factors might be responsible for deafness.

Adult↗
PubMed1996

Differential expression of dystrophin isoforms in strains of mdx mice with different mutations.

Mutations in the dystrophin gene are responsible for Duchenne and Becker muscular dystrophy (DMD/BMD). Studies of dystrophin expression and function have benefited from use of the mdx mouse, an animal model for DMD/BMD. Here we characterized mutations in three additional strains of mdx mice, the mdx2cv, mdx4cv and mdx5cv alleles. The mutation in the mdx2cv mouse was found to be a single base change in the splice acceptor sequence of dystrophin intron 42. This mutation leads to a complex pattern of aberrant splicing that generates multiple transcripts, none of which preserve the normal open reading frame. In the mdx5cv allele, the dystrophin mRNA contains a 53 bp deletion of sequences from exon 10. Analysis of the genomic DNA uncovered a single A to T transversion in exon 10. Although this base change does not alter the encoded amino acid, a new splice donor was created (GTGAG) that generates a frameshifting deletion in the processed mRNA. In the mdx4cv allele, direct sequencing revealed a C to T transition in exon 53, creating an ochre codon (CAA to TAA). The differential location of these mutations relative to the seven known dystrophin promoters results in a series of mdx mouse mutants that differ in their repertoire of isoform expression, such that these mice should be useful for studies of dystrophin expression and function. The mdx4cv and mdx5cv strains may be of additional use in gene transfer studies due to their low frequency of mutation reversion.

Alleles↗
PubMed2006

A novel splicing mutation in KCNQ2 in a multigenerational family with BFNC followed for 25 years.

PURPOSE: A large multigenerational family with benign familial neonatal convulsions (BFNC) was revisited to identify the disease-causing mutation and to assess long-term outcome. METHODS: We supplemented the original data with recent clinical and neurophysiologic data on patients and first-degree relatives, including information on seizure recurrence. We conducted linkage analysis at the EBN1 and EBN2 loci, followed by mutation analysis of KCNQ2. We evaluated the qualitative effect of the KCNQ2 mutation at the messenger RNA (mRNA) level by using reverse-transcribed total RNA isolated from leukocytes. RESULTS: Thirteen relatives had a history of neonatal convulsions, 11 of whom showed remission within 2 months. One patient showed an atypical course of neonatal convulsions, developing photosensitive myoclonic epilepsy at age 13 years. We found suggestive linkage of the BFNC phenotype to the 20q13-EBN1 locus (lod score, 2.03) and an intronic mutation IVS14-6 C>A in KCNQ2 segregating with the trait in all affected members, but absent in 100 unrelated control subjects. This mutation creates a new, preferentially used, splice site. Alternative splicing adds 4 nt containing a premature stop codon to the transcript, resulting in a truncated protein after position R588. CONCLUSIONS: We detected and characterized a novel splicing mutation in the brain-specific KCNQ2 gene by using easily accessible blood leukocytes. Aberrant splicing cosegregates with BFNC but not with photosensitivity.

Adolescent↗
PubMed1996

Molecular heterogeneity of late-onset forms of globoid-cell leukodystrophy.

Globoid-cell leukodystrophy (GLD) is an autosomal recessive inherited disorder caused by the deficiency of galactocerebrosidase, the lysosomal enzyme responsible for the degradation of the myelin glycolipid galactocerebroside. Although the most common form of the disease is the classical infantile form (Krabbe disease), later-onset forms also have been described. We have analyzed the galactocerebrosidase gene in 17 patients (nine families) with late-onset GLD and in 1 patient with classical Krabbe disease. Half of the patients were heterozygous for the large gene deletion associated with the 502C-->T polymorphism, the most common mutation in infantile patients. Several novel mutations that result in deficient galactocerebrosidase activity were also identified in these patients. They include the missense mutations R63H, G95S, M101L, G268S, Y298C, and I234T; the nonsense mutation S7X; a one-base deletion (805delG); a mutation that interferes with the splicing of intron 1; and a 34-nt insertion in the RNA, caused by the aberrant splicing of intron 6. All of these genetic defects are clustered in the first 10 exons of the galactocerebrosidase gene and therefore affect the 50-kD subunit of the mature enzyme. Studies on the distribution and enzymatic activity of the polymorphic alleles 1637T/C (I546/T546) provided support for previous data that had indicated the existence of two galactocerebrosidase forms with different catalytic activities in the general population. Our data also indicate that the mutations occur preferentially in the "low activity" 1637C allele.

Adolescent↗
PubMed1991

Control of adenovirus alternative RNA splicing: effect of viral DNA replication on RNA splice site choice.

The primary transcripts of most adenovirus transcription units are processed into multiple, alternatively spliced mRNAs. The relative concentrations of such differentially processed mRNAs changes during the infectious cycle. The factors that control this temporal shift in mRNA abundance have not yet been characterized. In the experiments presented here we have examined mRNA synthesis from three viral transcription units: two early regions E1a and E1b, and late region L1. We show that viral DNA replication plays a key role in the control of cytoplasmic mRNA expression from these regions. In the absence of efficient late protein synthesis, viral DNA replication was sufficient to induce a substantial fraction of the E1a, E1b and L1 transcripts to shift from the early to the late pattern of mRNA structure. The shift was not complete under the conditions used, suggesting that viral proteins, although not essential for the process, play an important regulatory role. The requirement for late viral protein synthesis differed between the three transcription units examined. This dependence was most pronounced for correct L1 mRNA production. Viral DNA replication was sufficient to trigger a significant shift in L1 alternative 3' splice site selection. However, in the absence of late translation the L1 pre-mRNA was aberrantly spliced.

Adenovirus Early Proteins↗
PubMed2004

Analysis of the LAMB3 gene in a junctional epidermolysis bullosa patient reveals exonic splicing and allele-specific nonsense-mediated mRNA decay.

How splicing, the process of intron removal in pre-messenger RNA (mRNA), is carried out with such fidelity in human cells is still not understood, although some general rules are being proposed mainly by in vitro experiments. These rules are currently being redefined by analysis of splicing mechanisms in patients presenting splicing defects. We analysed material of a patient suffering from junctional epidermolysis bullosa, a heritable blistering skin disease. Absence of laminin-5 protein together with hypoplastic hemidesmosomes at the dermo-epidermal junction in the patient's skin was shown by immunohistochemical analysis and immunoelectron microscopy. Subsequent DNA analysis revealed heterozygosity for the mutations R635X and 3009C-->T in the LAMB3 gene. The latter did not alter codon translation, but introduced an exonic splice site in exon 20. Interestingly, this exonic splice site, which presented a splice score of only 68.6, was preferentially used by the spliceosome over the wild-type splice site at the exon 20-intron 20 border, which showed a splice score of 92.2. LAMB3 mRNA was still detectable in RT-PCR analysis although the aberrantly spliced mRNA leads to a stop codon in exon 21, 5' of the commonly assumed 3' border for nonsense-mediated mRNA decay. These results describe an exception to the proposed rules of pre-mRNA splicing and RNA degradation.

Adult↗
PubMed2001

JC virus T' proteins encoded by alternatively spliced early mRNAs enhance T antigen-mediated viral DNA replication in human cells.

Alternative splicing of the JC Virus (JCV) precursor early mRNA yields five transcripts that encode proteins that regulate the life cycle of this human polyomavirus. Large T protein (TAg) mediates viral DNA replication and oncogenic activities, and small t protein influences these functions under certain conditions. Recently, three new early proteins, T'(135), T'(136), and T'(165), were discovered that contain sequences overlapping amino-terminal TAg functional domains. Initial studies with the T' proteins suggested they contribute to viral DNA replication and transformation. Mutation of a donor splice site utilized by all three T' mRNAs creates a mutant that exhibits a 10-fold decrease in viral DNA replication compared to wild type JCV. To assess the influence that individual T' proteins have on the replication process, a set of T' acceptor site mutants was created in which the unique second acceptor splice site of each T' mRNA was altered to eliminate production of one, two or all three T' mRNAs. The patterns of early mRNA and protein expression in these seven mutants were examined, and it was found that mutation of the T'(135) acceptor site resulted in the utilization of cryptic splice sites and the generation of new T' species. Additional mutations were made to prevent these aberrant splicing reactions prior to measuring DNA replication potential of the mutants. DpnI assays revealed that each T' protein contributes to TAg-mediated DNA replication activity. The three single mutants that express two T' proteins and the double mutant that only produces T'(136), exhibited levels of replication equivalent to that of wild type virus, whereas the two double mutants that fail to express T'(136) replicated about twofold less efficiently than wild-type JCV. Replication activity of the triple acceptor site mutant, like that of the T' donor site mutant from an earlier study, was impaired significantly.

Alternative Splicing↗
PubMed2000

Pathology of early-onset Alzheimer's disease cases bearing the Thr113-114ins presenilin-1 mutation.

Most cases of familial presenile Alzheimer's disease are caused by mutations in the presenilin-1 (PSEN-1) gene, most of these mutations being missense mutations. A mutation in the splice donor site of intron 4 of PSEN-1 has been described recently which results in aberrant splicing of PSEN-1 mRNA, causing insertion of an additional amino acid, Thr113-114ins, into the protein. We studied the neuropathology of four cases bearing this mutation in an attempt to clarify the pathology of this hereditary form of Alzheimer's disease and to determine whether it differs from other familial forms of the disease. The disease presented as a progressive cognitive decline, myoclonus and seizures developing later in the disease, a feature common to PSEN-1-linked Alzheimer's disease. The course of the disease was relatively rapid, death occurring approximately 6 years after onset. Pathology in the intron 4 cases demonstrated a severe Alzheimer's disease pathology with abundant deposition of ss-amyloid (Ass) 1-42 senile plaques and the formation of neurofibrillary tangles. Amyloid angiopathy was present in these cases and was readily demonstrated by Ass 1-40 staining, particularly in the cerebellum. Cases with the intron 4 mutation appear clinically and pathologically similar to other cases of early-onset Alzheimer's disease bearing PSEN-1 mutations.

Adult↗
PubMed2001

A deep intronic mutation in CDKN2A is associated with disease in a subset of melanoma pedigrees.

Germline mutations of CDKN2A at 9p21 have been shown to predispose to disease in melanoma pedigrees worldwide. However, there remains a significant proportion of melanoma pedigrees with evidence of linkage to 9p21 in which mutations in CDKN2A have not been detected. Investigation of other potential tumour suppressor genes at 9p21 and the promotor of CDKN2A has been unable to explain genetic predisposition to melanoma in these pedigrees. Here we describe a mutation, IVS2-105 A/G, deep in intron 2 of CDKN2A, detected in six English melanoma pedigrees. The mutation creates a false GT splice donor site 105 bases 5' of exon 3 and has been demonstrated to result in aberrant splicing of the mRNA. This is the most common mutation identified in English families to date. The presence of this deep intronic mutation in a relatively large number of kindreds, indicates that it may account for a significant proportion of 9p21-linked melanoma pedigrees with no detectable mutations in the coding region of CDKN2A. In addition, the identification of one deep intronic mutation in CDKN2A indicates the possibility of the existence of other similar splicing mutations located elsewhere in the CDKN2A introns.

Alleles↗
PubMed2026

An antisense antidote to oncogenic poison exons.

Splicing factors are frequently mutated in myeloid cancers, causing splicing aberrations that derail the expression of tumor suppressor genes. In SRSF2 mutated cancers, a key oncogenic splicing event is the inclusion of a "poison" exon that introduces an early stop codon in EZH2 mRNA, causing its destabilization. In this issue of Genes & Development, Islam et al. (doi:10.1101/gad.353628.126) define how mutant SRSF2 binding to the poison exon mediates its inclusion and identify an antisense oligonucleotide that represses the exon to restore EZH2 function and rescues hematopoietic defects. Thus, targeting of poison exons, many of which show protumorigenic and antitumorigenic properties, is a promising new avenue to treat cancer.

Oligonucleotides, Antisense↗