The prevalent c.903+469T>C mutation in MTRR causes the cblE type of homocystinuria by strengthening an SRSF1 binding site in an ESE leading to activation of a pseudoexon. We hypothesized that other splicing regulatory elements (SREs) are also critical for MTRR pseudoexon inclusion. We demonstrate that the MTRR pseudoexon is on the verge of being recognized and is therefore vulnerable to several point mutations that disrupt a fine-tuned balance between the different SREs. Normally, pseudoexon inclusion is suppressed by a hnRNP A1 binding exonic splicing silencer (ESS). When the c.903+469T>C mutation is present two ESEs abrogate the activity of the ESS and promote pseudoexon inclusion. Blocking the 3'splice site or the ESEs by SSOs is effective in restoring normal splicing of minigenes and endogenous MTRR transcripts in patient cells. By employing an SSO complementary to both ESEs, we were able to rescue MTRR enzymatic activity in patient cells to approximately 50% of that in controls. We show that several point mutations, individually, can activate a pseudoexon, illustrating that this mechanism can occur more frequently than previously expected. Moreover, we demonstrate that SSO blocking of critical ESEs is a promising strategy to treat the increasing number of activated pseudoexons.

• MeSH
• buněčné linie MeSH
• exony * MeSH
• ferredoxin-NADP-reduktasa genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• homocystinurie enzymologie   genetika   MeSH
• kultivované buňky MeSH
• lidé MeSH
• megaloblastová anemie enzymologie   genetika   MeSH
• místa sestřihu RNA MeSH
• mutace * MeSH
• oligonukleotidy * MeSH
• regulační sekvence ribonukleových kyselin * MeSH
• sestřih RNA * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Retinitis pigmentosa (RP) is a hereditary disease affecting tens of thousands of people world-wide. Here we analyzed the effect of an amino acid substitution in the RNA helicase DHX38 (Prp16) causing RP. DHX38 has been proposed as the helicase important for the 2nd step of splicing. We showed that DHX38 associates with key splicing factors involved in both splicing steps but did not find any evidence that the RP mutations changes DHX38 interaction profile with the spliceosome. We further downregulated DHX38 and monitored changes in splicing. We observed only minor perturbations of general splicing but detected modulation of ~70 alternative splicing events. Next, we probed DHX38 function in splicing of retina specific genes and found that FSCN2 splicing is dependent on DHX38. In addition, RHO splicing was inhibited specifically by expression of DHX38 RP variant. Finally, we showed that overexpression of DHX38 promotes usage of canonical as well as cryptic 5' splice sites in HBB splicing reporter. Together, our data show that DHX38 is a splicing factor that promotes splicing of cryptic splice sites and regulate alternative splicing. We further provide evidence that the RP-linked substitution G332D modulates DHX38 splicing activity.

• MeSH
• DEAD-box RNA-helikasy * genetika   MeSH
• lidé MeSH
• místa sestřihu RNA MeSH
• mutace MeSH
• retinopathia pigmentosa * genetika   MeSH
• sestřih RNA MeSH
• sestřihové faktory * genetika   MeSH
• spliceozomy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Mutations affecting splicing underlie the development of many human genetic diseases, but rather rarely through mechanisms of pseudoexon activation. Here, we describe a novel c.1092T>A mutation in the iduronate-2-sulfatase (IDS) gene detected in a patient with significantly decreased IDS activity and a clinical diagnosis of mild mucopolysaccharidosis II form. The mutation created an exonic de novo acceptor splice site and resulted in a complex splicing pattern with multiple pseudoexon activation in the patient's fibroblasts. Using an extensive series of minigene splicing experiments, we showed that the competition itself between the de novo and authentic splice site led to the bypass of the authentic one. This event then resulted in activation of several cryptic acceptor and donor sites in the upstream intron. As this was an unexpected and previously unreported mechanism of aberrant pseudoexon inclusion, we systematically analysed and disproved that the patient's mutation induced any relevant change in surrounding splicing regulatory elements. Interestingly, all pseudoexons included in the mature transcripts overlapped with the IDS alternative terminal exon 7b suggesting that this sequence represents a key element in the IDS pre-mRNA architecture. These findings extend the spectrum of mechanisms enabling pseudoexon activation and underscore the complexity of mutation-induced splicing aberrations. KEY MESSAGE: Novel exonic IDS gene mutation leads to a complex splicing pattern. Mutation activates multiple pseudoexons through a previously unreported mechanism. Multiple cryptic splice site (ss) activation results from a bypass of authentic ss. Authentic ss bypass is due to a competition between de novo and authentic ss.

• MeSH
• bodová mutace MeSH
• exony MeSH
• glykoproteiny genetika   MeSH
• introny MeSH
• lidé MeSH
• messenger RNA genetika   MeSH
• místa sestřihu RNA MeSH
• mladiství MeSH
• mukopolysacharidóza II genetika   MeSH
• mutace MeSH
• sestřih RNA MeSH
• Check Tag
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH

Mutations in SNRP200 gene cause autosomal-dominant retinal disorder retinitis pigmentosa (RP). The protein product of SNRNP200 is BRR2, a DExD/H box RNA helicase crucial for pre-mRNA splicing. In this study, we prepared p.S1087L and p.R1090L mutations of human BRR2 using bacterial artificial chromosome recombineering and stably expressed them in human cell culture. Mutations in BRR2 did not compromise snRNP assembly and both mutants were incorporated into the spliceosome just as the wild-type (wt) protein. Surprisingly, cells expressing RP mutants exhibited increased splicing efficiency of the LDHA gene. Next, we found that depletion of endogenous BRR2 enhanced usage of a β-globin cryptic splice site while splicing at the correct splice site was inhibited. Proper splicing of optimal and cryptic splice sites was restored in cells expressing BRR2-wt but not in cells expressing RP mutants. Taken together, our data suggest that BRR2 is an important factor in 5'-splice-site recognition and that the RP-linked mutations c.3260C>T (p.S1087L) and c.3269G>T (p.R1090L) affect this BRR2 function.

• MeSH
• alternativní sestřih MeSH
• beta-globiny genetika   metabolismus   MeSH
• HeLa buňky MeSH
• klonování DNA MeSH
• lidé MeSH
• místa sestřihu RNA genetika   MeSH
• mutace * MeSH
• prekurzory RNA genetika   metabolismus   MeSH
• reportérové geny MeSH
• retinopathia pigmentosa genetika   MeSH
• ribonukleoproteiny malé jaderné genetika   MeSH
• RNA-helikasy genetika   MeSH
• spliceozomy MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Biallelic pathogenic GJB2 gene mutations cause pre-lingual genetic hearing loss in up to 50% of individuals with bilateral sensorineural hearing loss worldwide. Sequencing of the entire GJB2 gene-coding region in Czech patients with pre-lingual bilateral hearing loss revealed that 10.3% of Czech patients carry only one monoallelic pathogenic mutation in the coding region of the GJB2 gene, which is significantly more than the population frequency of 3.4%. The 309-kb GJB6 deletion, frequent in Spain and France, is very rare in the Czech population. In order to evaluate the impact of the IVS1 + 1 G to A splice site mutation in the non-coding part of the GJB2 gene among Czech patients, we tested all available patients with pre-lingual hearing loss with only one monoallelic mutation in the coding part of GJB2. By sequencing of the exon 1 region of the GJB2 gene and HphI restriction analysis in 20 Czech patients we identified nine patients carrying IVS1 + 1 G to A. Testing for this mutation explained deafness in 45% of Czech GJB2 monoallelic patients. This mutation represents now 4% of GJB2 pathogenic mutations in Czech patients and is the third most common GJB2 mutation found in our cohort of 242 unrelated Czech patients with prelingual hearing loss. A similar frequency may also be expected in other Central European or Slavic populations.

• MeSH
• alely MeSH
• financování organizované MeSH
• genetické testování MeSH
• kohortové studie MeSH
• konexiny genetika   MeSH
• lidé MeSH
• místa sestřihu RNA genetika   MeSH
• mutace MeSH
• mutační analýza DNA MeSH
• oboustranná nedoslýchavost diagnóza   epidemiologie   genetika   MeSH
• restrikční mapování MeSH
• Check Tag
• lidé MeSH
• Geografické názvy
• Česká republika MeSH

About 4% of all BRCA1 and BRCA2 alterations reported to the Breast Information Core database are splice site variants. Only a limited number of them have been studied at the RNA level. By BRCA1 and BRCA2 mutation analysis of breast/ovarian cancer families, we identified two novel and eight previously reported potential splice site mutations, never characterized at the cDNA level before. RT-PCR was performed to determine whether these variants disrupted correct splicing. To ensure efficient detection of transcripts containing premature termination codons, a nonsense-mediated mRNA decay inhibitor was added to the lymphoblastoid cell lines of the patients before RNA extraction. We found that BRCA1 IVS3+3A>C, 4304G>A (in the last codon of exon 12), and IVS19+2delT and BRCA2 IVS6+1G>A, IVS23-2A>G, and IVS24+1G>A lead to aberrant transcripts in lymphocytes. Therefore, they were considered to be true pathogenic mutations, predisposing carriers to cancers of the hereditary breast/ovarian cancer syndrome. BRCA2 IVS24-16T>C is a frequent polymorphism in linkage disequilibrium, with a polymorphic stop codon in exon 27, K3326X. BRCA1 IVS2-14C>T and BRCA2 IVS9-5insT and IVS25+9A>C represent rare variants, not disrupting normal splicing in blood lymphocytes. However, some of the alterations may act differently, qualitatively and/or quantitatively, in breast or ovarian tissues. The data provided in this paper allowed more accurate risk estimation of patients and relatives carrying the mutations described herein and have facilitated genetic counseling. Furthermore, our study is important for a better understanding of splicing mechanisms and revealed new patterns of alternative splicing in BRCA1 and BRCA2. Copyright 2003 Wiley-Liss, Inc.

• MeSH
• čtecí rámce genetika   MeSH
• dospělí MeSH
• genetická predispozice k nemoci * genetika   MeSH
• geny BRCA1 * MeSH
• geny BRCA2 * MeSH
• lidé středního věku MeSH
• lidé MeSH
• místa sestřihu RNA * genetika   MeSH
• mutace * MeSH
• nádory prsu u mužů genetika   patologie   MeSH
• nádory prsu genetika   patologie   MeSH
• nádory vaječníků genetika   patologie   MeSH
• polymorfismus genetický * genetika   MeSH
• posunová mutace genetika   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• transformované buněčné linie MeSH
• věk při počátku nemoci MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• práce podpořená grantem MeSH

The DNA damage checkpoint kinase, CHK2, promotes growth arrest or apoptosis through phosphorylating targets such as Cdc25A, Cdc25C, BRCA1, and p53. Both germline and somatic loss-of-function CHEK2 mutations occur in human tumours, the former linked to the Li-Fraumeni syndrome, and the latter found in diverse types of sporadic malignancies. Here we examined the status of CHK2 by genetic and immunohistochemical analyses in 53 breast carcinomas previously characterized for TP53 status. We identified two CHEK2 mutants, 470T>C (Ile157Thr), and a novel mutation, 1368insA leading to a premature stop codon in exon 13. The truncated protein encoded by CHEK2 carrying the 1368insA was stable yet mislocalized to the cytoplasm in tumour sections and when ectopically expressed in cultured cells. Unexpectedly, we found CHEK2 to be subject to extensive alternative splicing, with some 90 splice variants detected in our tumour series. While all cancers expressed normal-length CHEK2 mRNA together with the spliced transcripts, we demonstrate and/or predict some of these splice variants to lack CHK2 function and/or localize aberrantly. We conclude that cytoplasmic sequestration may represent a novel mechanism to disable CHK2, and propose to further explore the significance of the complex splicing patterns of this tumour suppressor gene in oncogenesis.

• MeSH
• alternativní sestřih * MeSH
• checkpoint kinasa 2 MeSH
• imunohistochemie MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• mutace * MeSH
• mutační analýza DNA MeSH
• nádory prsu * genetika   metabolismus   MeSH
• polymerázová řetězová reakce MeSH
• protein-serin-threoninkinasy * genetika   metabolismus   MeSH
• sekvence nukleotidů MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH

PURPOSE: To confirm and define a molecular basis for a case of mucolipidosis type IV (ML IV) with an extremely atypical phenotype pattern. DESIGN: Observational case report of a patient with ML IV with disease progression restricted to ocular symptoms. METHODS: Complete ophthalmologic and neurologic examination. Ultrastructural examination of white blood cells, skin, conjunctiva, and corneal epithelium. The MCOLN1 gene was sequenced from cDNA and the proportion of splicing variants were assessed by quantitative allele-specific polymerase chain reaction. RESULTS: Absence of any neurological abnormalities. Retinal pathologic features were the main cause of visual disability: low visual acuity and cloudy corneas since 2 years of age, progressive decrease in visual acuity since the age of 9 years. Ultrastructural examination showed storage lysosomes filled with either concentric membranes or lucent precipitate in corneal and conjunctive epithelia and in vascular endothelium. Cultured fibroblasts were free of any autofluorescence. Sequencing of the MCOLN1 gene identified compound heterozygosity for D362Y and A-->T transition leading to the creation of a novel donor splicing site and a 4-bp deletion from exon 13 at the mRNA level. Both normal and pathologic splice forms were detected in skin fibroblasts and leukocytes, with the normal form being more abundant. CONCLUSIONS: The case of this patient with ML IV is unique and is characterized by a curious lack of generalized symptoms. In this patient, the disorder was limited to the eyes and appeared without the usual psychomotor deterioration. The resulting phenotype is the mildest seen to date.

• MeSH
• alternativní sestřih genetika   MeSH
• degenerace retiny genetika   patologie   MeSH
• dítě MeSH
• elektroretinografie MeSH
• epitelové buňky ultrastruktura   MeSH
• fenotyp MeSH
• fibroblasty ultrastruktura   MeSH
• financování organizované MeSH
• kationtové kanály TRPM genetika   MeSH
• kůže ultrastruktura   MeSH
• leukocyty ultrastruktura   MeSH
• lidé MeSH
• lyzozomy genetika   ultrastruktura   MeSH
• messenger RNA genetika   MeSH
• mukolipidózy genetika   patologie   MeSH
• mutace MeSH
• mutační analýza DNA MeSH
• nemoci rohovky genetika   patologie   MeSH
• nemoci spojivky genetika   patologie   MeSH
• polymerázová řetězová reakce MeSH
• rohovkový epitel ultrastruktura   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• kazuistiky MeSH

Deep intronic mutations are often ignored as possible causes of human diseases. A deep intronic mutation in the MTRR gene, c.903+469T>C, is the most frequent mutation causing the cblE type of homocystinuria. It is well known to be associated with pre-mRNA mis-splicing, resulting in pseudoexon inclusion; however, the pathological mechanism remains unknown. We used minigenes to demonstrate that this mutation is the direct cause of MTRR pseudoexon inclusion, and that the pseudoexon is normally not recognized due to a suboptimal 5' splice site. Within the pseudoexon we identified an exonic splicing enhancer (ESE), which is activated by the mutation. Cotransfection and siRNA experiments showed that pseudoexon inclusion depends on the cellular amounts of SF2/ASF and in vitro RNA-binding assays showed dramatically increased SF2/ASF binding to the mutant MTRR ESE. The mutant MTRR ESE sequence is identical to an ESE of the alternatively spliced MST1R proto-oncogene, which suggests that this ESE could be frequently involved in splicing regulation. Our study conclusively demonstrates that an intronic single nucleotide change is sufficient to cause pseudoexon activation via creation of a functional ESE, which binds a specific splicing factor. We suggest that this mechanism may cause genetic disease much more frequently than previously reported.

• MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• exony genetika   MeSH
• ferredoxin-NADP-reduktasa genetika   MeSH
• homocystinurie klasifikace   enzymologie   genetika   MeSH
• introny genetika   MeSH
• jaderné proteiny metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• místa sestřihu RNA genetika   MeSH
• molekulární sekvence - údaje MeSH
• mutace genetika   MeSH
• mutantní proteiny genetika   MeSH
• proteiny vázající RNA metabolismus   MeSH
• sekvence nukleotidů MeSH
• sestřih RNA genetika   MeSH
• vazba proteinů MeSH
• vitamin B 12 metabolismus   MeSH
• výpočetní biologie MeSH
• zesilovače transkripce genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In the inbred SHR/OlaIpcv rat colony, we identified males with small testicles and inability to reproduce. By selectively breeding their parents, we revealed the infertility to segregate as an autosomal recessive Mendelian character. No other phenotype was observed in males, and females were completely normal. By linkage using a backcross with Brown Norway strain, we mapped the locus to a 1.2Mbp segment on chromosome 7, harboring 35 genes. Sequencing of candidate genes revealed a G to A substitution in a canonical 'AG' splice site of intron 37 in Sbf1 (SET binding factor 1, alias myotubularin-related protein 5). This leads to either skipping exon 38 or shifting splicing one base downstream, invariantly resulting in frameshift, premature stop codon and truncation of the protein. Western blotting using two anti-Sbf1 antibodies revealed absence of the full-length protein in the mutant testis. Testicles of the mutant males were significantly smaller compared with SHR from 4weeks, peaked at 84% wild-type weight at 6weeks and declined afterward to 28%, reflecting massive germ cell loss. Histological examination revealed lower germ cell number; latest observed germ cell stage were round spermatids, resulting in the absence of sperm in the epididymis (azoospermia). SBF1 is a member of a phosphatase family lacking the catalytical activity. It probably modulates the activity of a phosphoinositol phosphatase MTMR2. Human homozygotes or compound heterozygotes for missense SBF1 mutations exhibit Charcot-Marie-Tooth disease (manifested mainly as progressive neuropathy), while a single mouse knockout reported in the literature identified male infertility as the only phenotype manifestation.

• MeSH
• alternativní sestřih genetika   MeSH
• intracelulární signální peptidy a proteiny genetika   MeSH
• krysa rodu rattus MeSH
• mutace genetika   MeSH
• mužská infertilita etiologie   metabolismus   patologie   MeSH
• potkani inbrední SHR MeSH
• regulace genové exprese * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH