In-frame Skipping Of Exon Research Articles

Analysis of the functional consequences of targeted exon deletion in COL7A1 reveals prospects for dystrophic epidermolysis bullosa therapy.

Genetically evoked deficiency of collagen VII causes dystrophic epidermolysis bullosa (DEB)-a debilitating disease characterized by chronic skin fragility and progressive fibrosis. Removal of exons carrying frame-disrupting mutations can reinstate protein expression in genetic diseases. The therapeutic potential of this approach is critically dependent on gene, protein, and disease intrinsic factors. Naturally occurring exon skipping in COL7A1, translating collagen VII, suggests that skipping of exons containing disease-causing mutations may be feasible for the treatment of DEB. However, despite a primarily in-frame arrangement of exons in the COL7A1 gene, no general conclusion of the aptitude of exon skipping for DEB can be drawn, since regulation of collagen VII functionality is complex involving folding, intra- and intermolecular interactions. To directly address this, we deleted two conceptually important exons located at both ends of COL7A1, exon 13, containing recurrent mutations, and exon 105, predicted to impact folding. The resulting recombinantly expressed proteins showed conserved functionality in biochemical and in vitro assays. Injected into DEB mice, the proteins promoted skin stability. By demonstrating functionality of internally deleted collagen VII variants, our study provides support of targeted exon deletion or skipping as a potential therapy to treat a large number of individuals with DEB.

ACOL7A1variant leading to in-frame skipping of exon 15 attenuates disease severity in recessive dystrophic epidermolysis bullosa

A<i>COL7A1</i>variant leading to in-frame skipping of exon 15 attenuates disease severity in recessive dystrophic epidermolysis bullosa

Haploinsufficiency of the NF-κB1 Subunit p50 in Common Variable Immunodeficiency

Common variable immunodeficiency (CVID), characterized by recurrent infections, is the most prevalent symptomatic antibody deficiency. In ∼90% of CVID-affected individuals, no genetic cause of the disease has been identified. In a Dutch-Australian CVID-affected family, we identified a NFKB1 heterozygous splice-donor-site mutation (c.730+4A>G), causing in-frame skipping of exon 8. NFKB1 encodes the transcription-factor precursor p105, which is processed to p50 (canonical NF-κB pathway). The altered protein bearing an internal deletion (p.Asp191_Lys244delinsGlu; p105ΔEx8) is degraded, but is not processed to p50ΔEx8. Altered NF-κB1 proteins were also undetectable in a German CVID-affected family with a heterozygous in-frame exon 9 skipping mutation (c.835+2T>G) and in a CVID-affected family from New Zealand with a heterozygous frameshift mutation (c.465dupA) in exon 7. Given that residual p105 and p50—translated from the non-mutated alleles—were normal, and altered p50 proteins were absent, we conclude that the CVID phenotype in these families is caused by NF-κB1 p50 haploinsufficiency.

A TALEN-Exon Skipping Design for a Bethlem Myopathy Model in Zebrafish.

Presently, human collagen VI-related diseases such as Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) remain incurable, emphasizing the need to unravel their etiology and improve their treatments. In UCMD, symptom onset occurs early, and both diseases aggravate with ageing. In zebrafish fry, morpholinos reproduced early UCMD and BM symptoms but did not allow to study the late phenotype. Here, we produced the first zebrafish line with the human mutation frequently found in collagen VI-related disorders such as UCMD and BM. We used a transcription activator-like effector nuclease (TALEN) to design the col6a1ama605003-line with a mutation within an essential splice donor site, in intron 14 of the col6a1 gene, which provoke an in-frame skipping of exon 14 in the processed mRNA. This mutation at a splice donor site is the first example of a template-independent modification of splicing induced in zebrafish using a targetable nuclease. This technique is readily expandable to other organisms and can be instrumental in other disease studies. Histological and ultrastructural analyzes of homozygous and heterozygous mutant fry and 3 months post-fertilization (mpf) fish revealed co-dominantly inherited abnormal myofibers with disorganized myofibrils, enlarged sarcoplasmic reticulum, altered mitochondria and misaligned sarcomeres. Locomotion analyzes showed hypoxia-response behavior in 9 mpf col6a1 mutant unseen in 3 mpf fish. These symptoms worsened with ageing as described in patients with collagen VI deficiency. Thus, the col6a1ama605003-line is the first adult zebrafish model of collagen VI-related diseases; it will be instrumental both for basic research and drug discovery assays focusing on this type of disorders.

Mutations of IFT81, encoding an IFT-B core protein, as a rare cause of a ciliopathy

Results Homozygosity for IFT81 mutations were identified in two consanguineous sporadic cases. The first individual harbored a splice site change predicted to result in an inframe exon skipping; the second carried a 4 bp deletion resulting in a loss-of-stop with extension of the deduced protein by 10 amino acids. The spectrum of IFT81-related disease expression included nephronophthisis, retinal dystrophy, cerebellar atrophy, and polydactyly. Fibroblasts from one affected individual showed no difference to control cells with regard to IFT81 localization or binding to IFT25, but a statistically significant decrease in ciliated cell abundance was noted. GLI2 expression and ciliary localization were impaired suggesting altered sonic hedgehog signaling.

FUBP1: a new protagonist in splicing regulation of the DMD gene.

We investigated the molecular mechanisms for in-frame skipping of DMD exon 39 caused by the nonsense c.5480T>A mutation in a patient with Becker muscular dystrophy. RNase-assisted pull down assay coupled with mass spectrometry revealed that the mutant RNA probe specifically recruits hnRNPA1, hnRNPA2/B1 and DAZAP1. Functional studies in a human myoblast cell line transfected with DMD minigenes confirmed the splicing inhibitory activity of hnRNPA1 and hnRNPA2/B1, and showed that DAZAP1, also known to activate splicing, acts negatively in the context of the mutated exon 39. Furthermore, we uncovered that recognition of endogenous DMD exon 39 in muscle cells is promoted by FUSE binding protein 1 (FUBP1), a multifunctional DNA- and RNA-binding protein whose role in splicing is largely unknown. By serial deletion and mutagenesis studies in minigenes, we delineated a functional intronic splicing enhancer (ISE) in intron 38. FUBP1 recruitment to the RNA sequence containing the ISE was established by RNA pull down and RNA EMSA, and further confirmed by RNA-ChIP on endogenous DMD pre-mRNA. This study provides new insights about the splicing regulation of DMD exon 39, highlighting the emerging role of FUBP1 in splicing and describing the first ISE for constitutive exon inclusion in the mature DMD transcript.

Mutation‐dependent effects on mRNA and protein expressions in cultured keratinocytes of Hailey–Hailey disease

Hailey-Hailey disease (HHD) is a dominantly inherited skin disease caused by mutations in ATP2C1 gene, which encodes secretory pathway Ca(2+) /Mn(2+) -ATPase protein 1. The precise mechanism remains unclear. In this study, to understand molecular basis of HHD, we examined expression of mRNA and protein in cultured keratinocytes derived from three HHD patients with different mutations. We showed that reduced expression of mRNA and protein in patient with p.Gln504X, but not in patients with p.Pro307His and c.1308+1G>A. RT-PCR analysis for patient with c.1308+1G>A revealed in-frame exon skipping. Reduction of mRNA and protein in p.Gln504X was considered to be caused by nonsense-mediated mRNA decay. p.Pro307His located adjacent to Ca(2+) -binding residue may induced conformational change, which leads to defective Ca(2+) transport. In-frame shorter transcript caused by c.1308+1G>A may have slightly reduced activity, which accounted for mild phenotype of the patient. These results clarified the pathogenic effects of different causative mutations in development of skin lesions.

A Korean case of neurofibromatosis type 1 with an exonic splicing enhancer site mutation

【Neurofibromatosis type 1 (NF1) is an autosomal dominant disease characterized by neurological, cutaneous, and ophthalmological manifestations. A 33-year-old woman with typical symptoms of NF1 visited Ajou University Hospital. Screening of the whole-messenger RNA region of NF1 at the complementary DNA level by polymerase chain reaction-direct sequencing confirmed the presence of an NF1 mutation at the genomic level. The mutation analysis revealed an in-frame skipping of exon 46 (c.6757_6858del) caused by a point mutation (c. 6792C>A) in exon 46. In this report, we have described the first Korean case of a proband with NF1 that carries an allele with an exon 46 deletion caused by an exonic splicing enhancer site mutation, leading to the skipping of the whole of exon 46 (c.6757_6858del).】

Novel physiological RECQL4 alternative transcript disclosed by molecular characterisation of Rothmund–Thomson Syndrome sibs with mild phenotype

Rothmund-Thomson syndrome is a rare genodermatosis caused by biallelic mutations of the RECQL4 gene and is characterised by poikiloderma, sparse hair, eyelashes and/or eyebrows, small stature, skeletal and dental abnormalities and cancer predisposition. Mutations predicted to result in the loss of RECQL4 protein have been associated with osteosarcoma risk, but mutation(s)-phenotype correlations are better addressed by combined DNA and RNA analyses. We describe two siblings with a mild phenotype, mainly restricted to the skin, who carry the unreported paternal c.2272C>T alteration in exon 14 and the previously reported maternal exon 15 c.2492_2493delAT, both predicted to result in premature termination codons (p.(Arg758*), p.(His831Argfs*52)). However real-time and transcript analysis showed, in the carrier father and affected daughter, increased levels of a novel RECQL4 physiological alternative transcript with partial in-frame skipping of exon 14, generated by increased usage of a weak cryptic splice site. This alternative transcript is expressed in all controls and tested tissues, its upregulation is specific to the paternal c.2272C>T mutation and depends on the abrogation of the binding motifs for SF2 and SRp55 serine/arginine-rich proteins with bypass of the mutation site located in the skipped exon 14 portion. Moreover, in the proband the increased levels of the alternative transcript, likely encoding a protein isoform with residual activity, may compensate for the dearth of the canonical transcript with the c.2492_2493delAT, accounting for the mild clinical phenotype of the siblings. Our results emphasise the value of RNA analysis to better predict the effects of RECQL4 mutations on the clinical phenotype.

In frame exon skipping in UBE3B is associated with developmental disorders and increased mortality in cattle.

BackgroundInherited developmental diseases can cause severe animal welfare and economic problems in dairy cattle. The use of a small number of bulls for artificial insemination (AI) carries a risk that recessive defects rapidly enrich in the population. In recent years, an increasing number of Finnish Ayrshire calves have been identified with signs of ptosis, intellectual disability, retarded growth and mortality, which constitute an inherited disorder classified as PIRM syndrome.ResultsWe established a cohort of nine PIRM-affected calves and 38 unaffected half-siblings and performed a genome-wide association study (GWAS) to map the disease to a 700-kb region on bovine chromosome 17 (p = 1.55 × 10-9). Whole genome re-sequencing of an unaffected carrier, its affected progeny and 43 other unaffected animals from another breed identified a G > A substitution mutation at the last nucleotide of exon 23 in the ubiquitin protein ligase E3B encoding gene (UBE3B). UBE3B transcript analysis revealed in-frame exon skipping in the affected animals resulting in an altered protein lacking 40 amino acids, of which 20 are located in the conserved HECT-domain, the catalytic site of the UBE3B protein. Mutation screening in 129 Ayrshire AI bulls currently used in Finland indicated a high carrier frequency (17.1%). We also found that PIRM syndrome might be connected to the recently identified AH1 haplotype, which has a frequency of 26.1% in the United States Ayrshire population.ConclusionWe describe PIRM syndrome in cattle, which is associated with the mutated UBE3B gene. The bovine phenotype resembles human Kaufman oculocerebrofacial syndrome, which is also caused by mutations in UBE3B. PIRM syndrome might be connected with the recently identified AH1 haplotype, which is associated with reduced fertility in the US Ayrshire population. This study enables the development of a genetic test to efficiently reduce the high frequency of mutant UBE3B in Ayrshires, significantly improving animal health and reducing economic loss.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-890) contains supplementary material, which is available to authorized users.

Abstract P4-04-09: Extensive novel hybrid isoforms revealed by RNA sequencing of 120 primary breast cancer samples

Abstract Introduction Recent studies of next generation sequencing (NGS) have highlighted the extensive transcriptional heterogeneity of cancer cells. Alternative splicing is one of the evolutionary process by which cells and tissues achieve their specificity within central dogma. Also it is highly assumed to contribute to oncogenesis and thought to be a critical mechanism how cancer cells gain resistance to therapeutic agents and adapt to various circumstances. Relevance of differential splicing in breast cancer biology is mostly unknown. We performed whole transcriptome sequencing (RNA-Seq) to reveal novel splicing alterations among 120 primary breast cancer samples. Materials and Method Total RNA was prepared using the Illumina TrueSeq™ RNA sample Preparation Kit and TrueSeq mRNA library was constructed. Clustering and sequencing was done using Illumina HiSeq 2000. RNA-Seq reads were aligned to human reference genome(hg19) using TopHat software and expression was measured using cufflinks software. We used tissues extracted from previously collected 120 fresh-frozen primary breast cancer samples obtained after surgical resection whose clinicopathological data are available. Patients undergone neoadjuvant systemic therapies or stage lV disease at diagnosis were excluded. Thirty-six(30%) cases occurred distant metastasis during follow up. Hormone receptor(HR) was positive in 61(50.8%) samples, 20(16.7%) had HER2 oncogene overexpression and 36(30%) were triple negative breast cancer. Results and discussion Total 11345 novel isoforms were detected among 120 tumors. Isoforms of pseudo-genes and exon skipping of the ‘non-coding exon’ were excluded. Splice variants detected in normal reference were sorted out as well. 4045 were in-frame exon skipping and 4960 were off-frame exon skipping which may lead to protein truncation. 5036 were private exon skipping and 3969 isoforms were detected recurrently in more than 2 samples. To minimize false positivity we confined ‘exon skipping’ analysis to those with the expression level (Fragments per kilo-base of exon per million fragments mapped, FPKM) of the skipped exon below 0.1 compared to the adjacent exons. Mean number of exon skipping events per sample was 196.8 (range 75-299, SD 35.9). There were no differences in numbers of exon skipping event among breast cancer subtypes nor distant metastasis. We have identified novel exon skipping in ESR1, CHEK2, EIF3E, FGFR, MAP2K, PIK3R2, TERT, VAV3 genes which is strongly suspected to be novel driver isoforms and is under validation process. Conclusion We performed whole-transcriptome sequencing with a large set of primary breast cancer samples and revealed extensive transcriptional heterogeneity by isoform profiling. As distinguishing the natural transcriptomic dynamics from oncogenic ‘driver’ isoform is a major challenge, validation and functional studies are ongoing. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-04-09.

In Vitro and Ex Vivo Investigation of Aberrant and Alternative Splicing of Von Willebrand Factor.

AbstractAbstract 2179Von Willebrand disease (VWD) is the most commonly inherited bleeding disorder with a symptomatic prevalence of 1:1000. Type 1 VWD comprises ∼80% of VWD cases and is defined by mild to moderate deficiencies of the large multimeric glycoprotein von Willebrand factor (VWF). Sequence variation in consensus splice sites is a common culprit in the induction of aberrant splicing, and occurs in approximately 10% of type 1 VWD cases. Investigation of the VWF mRNA is required in order to determine whether these sequence variants are causative mutations and the manner in which they affect splicing. In this study, aberrant VWF splicing from a consensus splice site mutation was investigated using mRNA from patient platelets and patient-derived blood outgrowth endothelial cells (BOEC). Both aberrant and alternative VWF splice forms were identified and their effects were further characterized through expression studies in HEK293T cells as well as further investigation of the patient BOEC.Patient T152 presented with a type 1 VWD phenotype: VWF:Ag 0.49IU/mL, VWF:RCo 0.42IU/mL, FVIII:C 1.16IU/mL and an abnormal bleeding score of 8. Direct sequencing of the patient‘s VWF gene revealed c.5842+1G>C in intron 34. This variation was thought to cause the type 1 VWD phenotype through aberrant splicing. Reverse transcription of the VWF mRNA detected different splice variants within the platelet and BOEC RNA. Wildtype VWF mRNA as well as transcripts encoding in-frame double exon skipping of exons 33 and 34 were found in both RNA sources. An additional VWF transcript which skipped exon 33 and introduced a premature termination codon in exon 34 was present in the BOEC RNA. Sequencing of this transcript determined it was produced from the patient’s normal allele and may be a product of alternative splicing. qRT-PCR quantification determined the patient VWF mRNA to be 51±5% wildtype, 28±4% exon33–34 skipped and 21±9% exon 33 skipped transcripts. The skipping of exon 33 was confirmed as an alternative transcript comprising 12±8% of VWF mRNA in 4 normal individuals.VWF expression vectors were created through site-directed mutagenesis for all three of these transcripts. Calcium-phosphate mediated transfections of these mutant vectors into HEK293T cells with varying wildtype:mutant ratios (100:0, 90:10, 75:25, 50:50, 0:100) for both mutant transcripts, as well as in the biological ratio (51:28:21) determined as above from the patient qRT-PCR. The alternative exon 33 skipping transcript does not decrease VWF secretion compared to wildtype (p>0.5). The presence of even 10% of the double exon skipping mutant transcript decreased secretion of VWF to 62± 1%(p<0.05) of wildtype, demonstrating a dominant negative effect. Similar to the plasma VWF:Ag level of 0.49 IU/mL, VWF secretion decreased to 46± 8% (p<0.01) when transfected at biological mRNA ratios. When stimulated with PMA, the transfections mimicking the patient biological VWF mRNA ratio increased VWF secretion 22±10% which approximates the patient’s clinical DDAVP response. A similar degree of increase was seen in PMA stimulated wildtype transfections. Multimer analysis of concentrated transfection media showed that the in-frame exon 33–34 is normally multimerized, which is consistent with the patient plasma multimers.BOEC isolated from the patient enabled analysis of these VWF mutants ex vivo. Patient BOEC expressed 48±10% (p<0.01) less VWF relative to BOEC from normal individuals. Upon stimulus by thrombin and PMA for 24 hours, normal BOEC showed a 28±13% and 93±70% increase in VWF secretion respectively. Similar increases in secretion (35±11% and 120±32% respectively) were seen with T152’s stimulated BOEC. Immunofluorescent staining of these BOEC showed abnormal morphology of Weibel-Palade bodies (WPBs). WPBs are characteristically cigar shaped; however, the patient’s WPBs appear shortened and rounded relative to their normal counterparts.We therefore conclude that the consensus splice site mutation c.5842+1G>C causes aberrant double exon skipping of exons 33 and 34 in this individual and is responsible for type 1 VWD through a dominant negative decrease in secretion. Additionally, the presence of VWF lacking exons 33 and 34 also affects the formation of WPBs. The alternative transcript which skips exon 33 was found to have no effect on VWF secretion and may be degraded by nonsense mediated decay. Disclosures:James:CSL-Behring, Baxter, Bayer: Honoraria, Research Funding.

P03 Reducing toxic N-terminal huntingtin fragments in HD using exon skipping

BackgroundSeveral studies have implicated the importance of proteolytic cleavage of mutant huntingtin in HD pathogenesis (Thornberry et al 1997). Huntingtin fragments within the striatum of human HD brains clearly differ...

RAPADILINO RECQL4 mutant protein lacks helicase and ATPase activity

The RecQ family of helicases has been shown to play an important role in maintaining genomic stability. In humans, this family has five members and mutations in three of these helicases, BLM, WRN and RECQL4, are associated with disease. Alterations in RECQL4 are associated with three diseases, Rothmund–Thomson syndrome, Baller–Gerold syndrome, and RAPADILINO syndrome. One of the more common mutations found in RECQL4 is the RAPADILINO mutation, c.1390+2delT which is a splice-site mutation leading to an in-frame skipping of exon 7 resulting in 44 amino acids being deleted from the protein (p.Ala420–Ala463del). In order to characterize the RAPADILINO RECQL4 mutant protein, it was expressed in bacteria and purified using an established protocol. Strand annealing, helicase, and ATPase assays were conducted to characterize the protein's activities relative to WT RECQL4. Here we show that strand annealing activity in the absence of ATP is unchanged from that of WT RECQL4. However, the RAPADILINO protein variant lacks helicase and ssDNA-stimulated ATPase activity. These observations help explain the underlying molecular etiology of the disease and our findings provide insight into the genotype and phenotype association among RECQL4 syndromes.

Novel peripheral myelin protein 22 (PMP22) micromutations associated with variable phenotypes in Greek patients with Charcot-Marie-Tooth disease

ARTICLE Sir, We read with interest the paper by Taioli et al . (2011) reporting eight different micromutations of PMP22 in patients with inherited demyelinating neuropathies. Small frameshift insertions/deletions (indels), nonsense nucleotide substitutions and splice-site mutations led to phenotypically and pathologically variable neuropathies. Patients with mutations causing a premature or delayed stop codon had a phenotype within the hereditary neuropathy with liability to pressure palsies (HNPP) spectrum. One patient with a splice-site mutation causing an in-frame skipping of exon 4 had a CMT1 phenotype. As the authors state, micromutations of PMP22 are considered very rare causes of Charcot–Marie–Tooth (CMT) in most populations and this certainly includes our experience of PMP22 analysis in cases with CMT from the UK. We presently report three novel PMP22 micromutations identified in a Greek cohort of patients with CMT that were associated with variable clinical phenotypes, ranging from severe CMT1 of the Dejerine–Sottas disease spectrum to HNPP. These novel cases, taken together with the findings of Taioli et al . (2011) illustrate that, despite some exceptions, in-frame indels in PMP22 usually lead to CMT1 of varying severity, whereas frameshift changes more often cause HNPP. DNA was extracted from peripheral blood following standard procedures. Patients were previously found negative for the common duplication/deletion in chromosome 17 and for mutations in GJB1 and MPZ . The four coding exons (exons 2–5) of PMP22 with adjacent intronic sequences were amplified by polymerase chain reaction and sequenced on an Applied Biosystems 3730XL genetic analyser. Fragment analysis was performed with GeneMapper® software after running fragments on the 3730XL with a LIZ500 standard. RNA was obtained from peripheral blood using the Qiagen/PreAnalytix™ blood RNA system. Complementary DNA was synthesized using the Applied Biosystems high capacity complementary DNA reverse transcription kit. A 391 bp fragment of complementary DNA spanning the regions of interest …

Identification of a novel mutation in the exon 2 splice donor site of the POU1F1/PIT‐1 gene in Japanese identical twins with mild combined pituitary hormone deficiency

To date, approximately 35 different POU1F1 mutations have been described in patients with familial and sporadic combined pituitary hormone deficiency (CPHD) from different ethnic backgrounds. The majority are missense mutations clustered within the conserved POU-specific and POU-homeo domains, encoded by exons 4 and 6, respectively. This study aimed to identify the molecular basis and clinical characteristics of a Japanese CPHD family with a novel POU1F1 mutation. The POU1F1 gene was sequenced in identical twin brothers with mild CPHD. The mutation identified was also evaluated in family members as well as 188 Japanese controls and then examined in functional studies. A novel heterozygous splice site mutation (Ex2 + 1G>T; c.214 + 1G>T) was detected. This mutation was also present in their undiagnosed mother, but not in any of the controls. In vitro splicing studies suggested this mutation to result in an in-frame skipping of exon 2, thus producing an internally deleted protein lacking most of the R2 transactivation subdomain (TAD-R2). Heterologous expression studies of the mutated POU1F1 protein showed only modest reductions in its transactivation activities in HEK293T cells, while acting as a dominant-negative inhibitor of the endogenous activities of POU1F1 in pituitary GH3 cells. This is the first report of a mutation at the exon 2 donor splice site of POU1F1, affecting TAD-R2. The addition of this mutation to the growing list of pathological POU1F1 mutations may provide deeper insights into clinical heterogeneity in the expressions of individual mutations and a better understanding of the structure-function relationships of POU1F1.

Gene Therapeutic Approach Using Mutation-adapted U1 snRNA to Correct a RPGR Splice Defect in Patient-derived Cells

Retinitis pigmentosa (RP) is a disease that primarily affects the peripheral retina and ultimately causes visual impairment. X-chromosomal forms of RP are frequently caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. We show that the novel splice donor site (SDS) mutation c.1245+3A>T in intron 10 of RPGR cosegregates with RP in a five-generation Caucasian family. The mutation causes in-frame skipping of exon 10 from RPGR transcripts in patient-derived primary fibroblasts. To correct the splice defect, we developed a gene therapeutic approach using mutation-adapted U1 small nuclear RNA (U1). U1 is required for SDS recognition of pre-mRNAs and initiates the splice process. The mutation described herein interferes with the recognition of the SDS by U1. To overcome the deleterious effects of the mutation, we generated four U1 isoforms with increasing complementarity to the SDS. Lentiviral particles were used to transduce patient-derived fibroblasts with these U1 variants. Full complementarity of U1 corrects the splice defect partially and increases recognition of the mutant SDS. The therapeutic effect is U1-concentration dependent as we show for endogenously expressed RPGR transcripts in patient-derived cells. U1-based gene therapeutic approaches constitute promising technologies to treat SDS mutations in inherited diseases including X-linked RP.

Early onset collagen VI myopathies: Genetic and clinical correlations

Mutations in the genes encoding the extracellular matrix protein collagen VI (ColVI) cause a spectrum of disorders with variable inheritance including Ullrich congenital muscular dystrophy, Bethlem myopathy, and intermediate phenotypes. We extensively characterized, at the clinical, cellular, and molecular levels, 49 patients with onset in the first 2 years of life to investigate genotype-phenotype correlations. Patients were classified into 3 groups: early-severe (18%), moderate-progressive (53%), and mild (29%). ColVI secretion was analyzed in patient-derived skin fibroblasts. Chain-specific transcript levels were quantified by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and mutation identification was performed by sequencing of complementary DNA. ColVI secretion was altered in all fibroblast cultures studied. We identified 56 mutations, mostly novel and private. Dominant de novo mutations were detected in 61% of the cases. Importantly, mutations causing premature termination codons (PTCs) or in-frame insertions strikingly destabilized the corresponding transcripts. Homozygous PTC-causing mutations in the triple helix domains led to the most severe phenotypes (ambulation never achieved), whereas dominant de novo in-frame exon skipping and glycine missense mutations were identified in patients of the moderate-progressive group (loss of ambulation). This work emphasizes that the diagnosis of early onset ColVI myopathies is arduous and time-consuming, and demonstrates that quantitative RT-PCR is a helpful tool for the identification of some mutation-bearing genes. Moreover, the clinical classification proposed allowed genotype-phenotype relationships to be explored, and may be useful in the design of future clinical trials.

Membrane Trafficking of Large Conductance Calcium-activated Potassium Channels Is Regulated by Alternative Splicing of a Transplantable, Acidic Trafficking Motif in the RCK1-RCK2 Linker

Trafficking of the pore-forming α-subunits of large conductance calcium- and voltage-activated potassium (BK) channels to the cell surface represents an important regulatory step in controlling BK channel function. Here, we identify multiple trafficking signals within the intracellular RCK1-RCK2 linker of the cytosolic C terminus of the channel that are required for efficient cell surface expression of the channel. In particular, an acidic cluster-like motif was essential for channel exit from the endoplasmic reticulum and subsequent cell surface expression. This motif could be transplanted onto a heterologous nonchannel protein to enhance cell surface expression by accelerating endoplasmic reticulum export. Importantly, we identified a human alternatively spliced BK channel variant, hSloΔ579–664, in which these trafficking signals are excluded because of in-frame exon skipping. The hSloΔ579–664 variant is expressed in multiple human tissues and cannot form functional channels at the cell surface even though it retains the putative RCK domains and downstream trafficking signals. Functionally, the hSloΔ579–664 variant acts as a dominant negative subunit to suppress cell surface expression of BK channels. Thus alternative splicing of the intracellular RCK1-RCK2 linker plays a critical role in determining cell surface expression of BK channels by controlling the inclusion/exclusion of multiple trafficking motifs.

Hypodysfibrinogenaemia due to production of mutant fibrinogen alpha-chains lacking fibrinopeptide A and polymerisation knob ‘A’

Inherited disorders of fibrinogen are rare and affect either the quantity (hypofibrinogenaemia and afibrinogenaemia) or the quality of the circulating fibrinogen (dysfibrinogenaemia) or both (hypodysfibrinogenaemia). Extensive allelic heterogeneity has been found for all these disorders: in congenital afibrinogenaemia for example more than 40 mutations, the majority in FGA , have been identified in homozygosity or in compound heterozygosity. Numerous mutations have also been identified in patients with hypofibrinogenaemia, many of these patients are in fact heterozygous carriers of afibrinogenaemia mutations. Despite the number of genetic analyses performed, the study of additional patients still allows the identification of novel mutations. Here we describe the characterization of a novel FGA intron 2 donor splice-site mutation (Fibrinogen Montpellier II) identified in three siblings with hypodysfibrinogenaemia. Functional analysis of RNA produced by the mutant minigene in COS-7 cells revealed that the mutation led to the in-frame skipping of exon 2. Western blot analysis of COS-7 cells expressing an exon 2 deleted FGA cDNA revealed that an alpha-chain lacking exon 2, which codes in particular for fibrinopeptide A and polymerisation knob 'A', has the potential to be assembled into a hexamer and secreted. Analysis of precipitated fibrinogen from patient plasma showed that the defect leads to the presence in the circulation of alpha-chains lacking knob 'A' which is essential for the early stages of fibrin polymerisation. Fibrin made from purified patient fibrinogen clotted with thrombin displayed thinner fibers with frequent ends and large pores.