Functional Splicing Assay Research Articles

Evaluation in Monogenic Diabetes of the Impact of GCK, HNF1A, and HNF4A Variants on Splicing through the Combined Use of In Silico Tools and Minigene Assays

Variants in GCK, HNF1A, and HNF4A genes are the three main causes of monogenic diabetes. Determining the molecular etiology is essential for patients with monogenic diabetes to benefit from the most appropriate treatment. The increasing number of variants of unknown significance (VUS) is a major issue in genetic diagnosis, and assessing the impact of variants on RNA splicing is challenging, particularly for genes expressed in tissues not easily accessible as in monogenic diabetes. The in vitro functional splicing assay based on a minigene construct is an appropriate approach. Here, we performed in silico analysis using SpliceAI and SPiP and prioritized 36 spliceogenic variants in GCK, HNF1A, and HNF4A. Predictions were secondarily compared with Pangolin and AbSplice-DNA bioinformatics tools which include tissue-specific annotations. We assessed the effect of selected variants on RNA splicing using minigene assays. These assays validated splicing defects for 33 out of 36 spliceogenic variants consisting of exon skipping (15%), exonic deletions (18%), intronic retentions (24%), and complex splicing patterns (42%). This provided additional evidence to reclassify 23 out of 31 (74%) VUS including missense, synonymous, and intronic noncanonical splice site variants as likely pathogenic variants. Comparison of in silico analysis with minigene results showed the robustness of bioinformatics tools to prioritize spliceogenic variants, but revealed inconsistencies in the location of cryptic splice sites underlying the importance of confirming predicted splicing alterations with functional splicing assays. Our study underlines the feasibility and the benefits of implementing minigene-splicing assays in the genetic testing of monogenic diabetes after a prior in-depth in silico analysis.

SEPT–GD: A decision tree to prioritise potential RNA splice variants in cardiomyopathy genes for functional splicing assays in diagnostics

BackgroundSplice prediction algorithms currently used in routine DNA diagnostics have limited sensitivity and specificity, therefore many potential splice variants are classified as variants of uncertain significance (VUSs). However, functional assessment of VUSs to test splicing is labour-intensive and time-consuming. We developed a decision tree to prioritise potential splice variants for functional studies and functionally verified the outcome of the decision tree. Materials and methodsWe built the decision tree, SEPT–GD, by setting thresholds for the splice prediction programs implemented in Alamut. A set of 343 variants with known effects on splicing was used as control for sensitivity and specificity. We tested SEPT–GD using variants from a Dutch cardiomyopathy cohort of 2002 patients that were previously classified as VUS and predicted to have a splice effect according to diagnostic rules. We then selected 12 VUSs ranked by SEPT–GD to functionally verify the predicted effect on splicing using a minigene assay: 10 variants predicted to have a strong effect and 2 with a weak effect. RT-PCR was performed for nine variants. Variant classification was re-evaluated based on the functional test outcome. ResultsCompared to similar individually tested algorithms, SEPT–GD shows higher sensitivity (91 %) and comparable specificity (88 %) for both consensus (dinucleotides at the start and end of the intron, GT at the 5′ end and AG at the 3′ end) and non-consensus splice-site variants (excluding middle of exon variants). Using clinical diagnostic criteria, 1295 unique variants in our cardiomyopathy cohort had originally been classified as VUSs, with 57 predicted by Alamut to have an effect on splicing. Using SEPT–GD, we prioritised 31 variants in 40 patients. In the minigene assay, all 12 variants showed results concordant with SEPT-GD predictions. RT-PCR confirmed the minigene results for two variants, TMEM43 c.1000 + 5G > T and TTN c.25922–6 T > G. Based on all outcomes, the SGCD c.4-1G > A and CSRP3 c.282-5_285del variants were reclassified as likely pathogenic. ConclusionSEPT–GD outperforms the tools commonly used for RNA splicing prediction and improves prioritisation of variants in cardiomyopathy genes for functional splicing analysis in a diagnostic setting.

Activation of cryptic donor splice sites by non-coding and coding PAX6 variants contributes to congenital aniridia

BackgroundThe paired-domain transcription factor paired box gene 6 (PAX6) causes a wide spectrum of ocular developmental anomalies, including congenital aniridia, Peters anomaly and microphthalmia. Here, we aimed to functionally assess...

Last Nucleotide Substitutions of COL4A5 Exons Cause Aberrant Splicing

IntroductionCOL4A5 is a causative gene of X-linked Alport syndrome (XLAS). Male patients with XLAS with nonsense variants have the most severe phenotypes of early onset end-stage kidney disease (ESKD); those with splicing variants have middle phenotypes and those with missense variants have the mildest phenotypes. Therefore, genotyping for male patients with XLAS can be used to predict kidney prognosis. Single-base substitutions at the last nucleotide position in each exon are known to affect splicing patterns and could be splicing variants. Nevertheless, in XLAS, these variants are generally considered to be missense variants, without conducting a transcript analysis, which underestimates some patients as having mild phenotypes. This study aimed to investigate whether single-base substitutions at the last nucleotide position of COL4A5 exons cause aberrant splicing.MethodsIn total, 20 variants were found in the Human Gene Mutation Database (n = 14) and our cohort (n = 6). We performed functional splicing assays using a hybrid minigene analysis and in vivo transcript analyses of patients’ samples when available. Then, we investigated genotype–phenotype correlations for patients with splicing variants detected in this study by comparing data from our previous studies.ResultsAmong the 20 variants, 17 (85%) caused aberrant splicing. Male patients with splicing variants had more severe phenotypes when compared with those with missense variants. Findings from the in vivo analyses for 3 variants were identical to those from the minigene assay.ConclusionOur study revealed that most single-base substitutions at the last nucleotide position of COL4A5 exons result in splicing variants, rather than missense variants, thereby leading to more severe phenotypes.

Usefulness of functional splicing analysis to confirm precise disease pathogenesis in Diamond-Blackfan anemia caused by intronic variants in RPS19

Diamond-Blackfan anemia (DBA) is mainly caused by pathogenic variants in ribosomal proteins and 22 responsible genes have been identified to date. The most common causative gene of DBA is RPS19 [NM_001022.4]. Nearly 180 RPS19 variants have been reported, including three deep intronic variants outside the splicing consensus sequence (c.72-92A > G, c.356 + 18G > C, and c.411 + 6G > C). We also identified one case with a c.412-3C > G intronic variant. Without conducting transcript analysis, the pathogenicity of these variants is unknown. However, it is difficult to assess transcripts because of their fragility. In such cases, in vitro functional splicing assays can be used to assess pathogenicity. Here, we report functional splicing analysis results of four RPS19 deep intronic variants identified in our case and in previously reported cases. One splicing consensus variant (c.411 + 1G > A) was also examined as a positive control. Aberrant splicing with a 2-bp insertion between exons 5 and 6 was identified in the patient samples and minigene assay results also identified exon 6 skipping in our case. The exon 6 skipping transcript was confirmed by further evaluation using quantitative RT-PCR. Additionally, minigene assay analysis of three reported deep intronic variants revealed that none of them showed aberrant splicing and that these variants were not considered to be pathogenic. In conclusion, the minigene assay is a useful method for functional splicing analysis of inherited disease.

Functional analysis of suspected splicing variants in CLCN5 gene in Dent disease 1

BackgroundIn recent years, the elucidation of splicing abnormalities as a cause of hereditary diseases has progressed. However, there are no comprehensive reports of suspected splicing variants in the CLCN5 gene in Dent disease cases. We reproduced gene mutations by mutagenesis, inserted the mutated genes into minigene vectors, and investigated the pathogenicity and onset mechanisms of these variants.MethodsWe conducted functional splicing assays using a hybrid minigene for six suspected splicing variants (c.105G>A, c.105+5G>C, c.106−17T>G, c.393+4A>G, c.517−8A>G, c.517−3C>A) in CLCN5. We extracted information on these variants from the Human Gene Mutation Database. We reproduced minigene vectors with the insertion of relevant exons with suspected splicing variants. We then transfected these minigene vectors into cultured cells and extracted and analyzed the mRNA. In addition, we conducted in silico analysis to confirm our minigene assay results.ResultsWe successfully determined that five of these six variants are pathogenic via the production of splicing abnormalities. One showed only normal transcript production and was thus suspected of not being pathogenic (c.106−17T>G).ConclusionWe found that five CLCN5 variants disrupted the original splice site, resulting in aberrant splicing. It is sometimes difficult to obtain mRNA from patient samples because of the fragility of mRNA or its low expression level in peripheral leukocytes. Our in vitro system can be used as an alternative to in vivo assays to determine the pathogenicity of suspected splicing variants.

Determination of the pathogenicity of known COL4A5 intronic variants by in vitro splicing assay

X-linked Alport syndrome (XLAS) is a congenital renal disease caused by mutations in COL4A5. In XLAS cases suspected of being caused by aberrant splicing, transcript analysis needs to be conducted to determine splicing patterns and assess the pathogenicity. However, such analysis is not always available. We conducted a functional splicing assay using a hybrid minigene for seven COL4A5 intronic mutations: one was identified by us and six were found in the Human Gene Mutation Database. The minigene assay revealed exon skipping in four variants, exon skipping and a 10-bp insertion in one variant, and no change in one variant, which appeared not to be pathogenic. For one variant, our assay did not work. The results of all three cases for which transcript data were available were consistent with our assay results. Our findings may help to increase the accuracy of genetic test results and clarify the mechanisms causing aberrant splicing.

406 An in vitro splicing assay reveals the pathogenicity of intronic variants in ABCC6, the gene at fault in pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, is caused by inactivating mutations in the ABCC6 gene predominantly expressed in hepatocytes. Different types of mutations have been identified in ABCC6 including canonical splice site mutations affecting pre-mRNA splicing. Splicing variants in ABCC6 are often overlooked due to the unavailability of liver biopsies from patients with such variants to determine their consequences on splicing by transcriptional analysis. Thus, the interpretation of intronic variants purely relies on computational algorithms to predict their effects on splicing. Very often the prediction programs do not provide a consensus as to the effect of variants for disrupting normal splicing. Our recent ABCC6 mutation analysis of over 400 patients with PXE shows that this continues to be a problem: among 7 intronic variants in ABCC6 with conflicting results by current splice site prediction algorithms, 3 variants were found to affect splicing. These three intronic variants were analyzed in an in vitro functional splicing assay using expression of minigene constructs in transfected HepG2 cells. Total RNA was extracted and RT-PCR was performed to analyze the splice pattern followed by Sanger sequencing. Two of the variants, c.3883-46A>G and c.2070+5G>A, caused skipping of the adjacent exon, predicted to cause out-of-frame translation and premature stop of translation. One variant, c.3883-6G>A, created a new splice acceptor site resulting in a 4-bp retention from the intronic region and causing frame-shift of translation. The results provided evidence that intronic variants away from the canonical splice sites disrupted splicing, clearly demonstrating the presence of mutations beyond the canonical splice sites.

Intronic variants of SLC26A4 gene enhance splicing efficiency in hybrid minigene assay

The SLC26A4 genomic sequence screening in autoimmune thyroid diseases (AITD) revealed different variants types with possible pathogenic effects. Although intronic variants may have more detrimental effects than those coding, they are poorly explored. Thus, in a first assessment, our bioinformatics analysis of intronic variants predicted a pathogenic effect of c.1002-9A>C, c.1545-5T>G and c.1544+9C>T variants. Validating these variants pathogenicity may provide new clues on the AITD physiopathology. Variants were explored in a general population by PCR-RFLP. These variants effects on the mRNA processing was assessed using functional splicing assay based in DNA hybrid minigene in HeLa cell lines. The constructs splicing efficiency was investigated by real time PCR. Our results revealed that c.1002-9A>C is a rare allele (minor frequency allele (MFA)=0.007) whereas c.1545-5T>G and c.1544+9C>T are low frequency variants. The RT-PCR analysis showed that these variants did not affect the mRNA processing. However, quantifying the transcripts generated from minigene constructs proved an mRNA splicing enhancement. Our study suggests a pathogenic effect of three intronic variants on the mRNA splicing efficiency using a DNA Hybrid minigene. By quantifying these transcripts, we unveil the limit of standard RT-PCR in analyzing a splicing minigene assay.

Genetic screening of the FLCN gene identify six novel variants and a Danish founder mutation

Pathogenic germline mutations in the folliculin (FLCN) tumor suppressor gene predispose to Birt-Hogg-Dubé (BHD) syndrome, a rare disease characterized by the development of cutaneous hamartomas (fibrofolliculomas), multiple lung cysts, spontaneous pneumothoraces and renal cell cancer. In this study, we report the identification of 13 variants and three polymorphisms in the FLCN gene in 143 Danish patients or families with suspected BHD syndrome. Functional mini-gene splicing analysis revealed that two intronic variants (c.1062+2T>G and c.1177-5_1177-3del) introduced splicing aberrations. Eleven families exhibited the c.1062+2T>G mutation. Combined single nucleotide polymorphism array-haplotype analysis showed that these families share a 3-Mb genomic fragment containing the FLCN gene, revealing that the c.1062+2T>G mutation is a Danish founder mutation. On the basis of in silico prediction and functional splicing assays, we classify the 16 identified variants in the FLCN gene as follows: nine as pathogenic, one as likely pathogenic, three as likely benign and three as polymorphisms. In conclusion, the study describes the FLCN mutation spectrum in Danish BHD patients, and contributes to a better understanding of BHD syndrome and management of BHD patients.

Functional splicing assay of DSPP mutations in hereditary dentin defects

Dentin sialophosphoprotein (DSPP) gene mutations have been identified in isolated hereditary dentin defects; however, the genotype-phenotype correlations are poorly understood. We performed in vitro splicing assays to test the hypothesis that DSPP mutations in splice junctions as well as proposed missense/nonsense mutations experimentally result in aberrant pre-mRNA splicing. The genomic fragment of the human DSPP gene was cloned into the pSPL3 splicing vector, and previously reported as well as informative de novo mutations were then introduced by PCR mutagenesis. The COS-7 cells were transfected with each plasmid vector, and total RNA was isolated. RT-PCR result was analyzed, and the band intensity of the product was calibrated using ImageJ. The predictions by others of exon 3 skipping in specific DSPP mutations have been validated and a cryptic splicing donor site has been identified. However, the degree of mutational effect on pre-mRNA splicing varied considerably depending on the changed nucleotide. The predictions of exon 3 skipping in specific DSPP mutations have been validated, and a cryptic splicing donor site has been identified. Our data may provide insight into the contribution of DSPP mutations in the pathogenesis and genotype-phenotype correlations of hereditary dentin defects.

Contribution of bioinformatics predictions and functional splicing assays to the interpretation of unclassified variants of the BRCA genes

A large fraction of sequence variants of unknown significance (VUS) of the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 may induce splicing defects. We analyzed 53 VUSs of BRCA1 or BRCA2, detected in consecutive molecular screenings, by using five splicing prediction programs, and we classified them into two groups according to the strength of the predictions. In parallel, we tested them by using functional splicing assays. A total of 10 VUSs were predicted by two or more programs to induce a significant reduction of splice site strength or activation of cryptic splice sites or generation of new splice sites. Minigene-based splicing assays confirmed four of these predictions. Five additional VUSs, all at internal exon positions, were not predicted to induce alterations of splice sites, but revealed variable levels of exon skipping, most likely induced by the modification of exonic splicing regulatory elements. We provide new data in favor of the pathogenic nature of the variants BRCA1 c.212+3A>G and BRCA1 c.5194-12G>A, which induced aberrant out-of-frame mRNA forms. Moreover, the novel variant BRCA2 c.7977-7C>G induced in frame inclusion of 6 nt from the 3' end of intron 17. The novel variants BRCA2 c.520C>T and BRCA2 c.7992T>A induced incomplete skipping of exons 7 and 18, respectively. This work highlights the contribution of splicing minigene assays to the assessment of pathogenicity, not only when patient RNA is not available, but also as a tool to improve the accuracy of bioinformatics predictions.

Functional splicing assay supporting that c.70 + 5G > A mutation in the MPV17 gene is disease causing

Mitochondrial DNA depletion syndrome (MDS) is a group of disorders characterized by a quantitative reduction of the mitochondrial DNA copy number and inherited as autosomal recessive traits. Patients affected by this group of diseases present with a wide variety of symptoms depending on the altered gene. MPV17 is one of the genes causing combined encephalopathy and liver failure and at present there is no treatment for this devastating disease. The gene codes for an inner mitochondrial membrane protein, but its function is still unknown, and therefore, the only way to offer prenatal diagnosis relies on DNA studies. Consequently, mutations have to be well characterized. We previously described a patient homozygous for a novel intronic mutation in the MPV17 gene (c.70 + 5G > A). Here we report the use of a functional splicing assay based on the use of minigenes to support that c.70 + 5G > A mutation is disease causing. We carried out three prenatal diagnoses on three consecutive pregnancies of the previously described family. After two affected fetuses, a healthy baby was born homozygous for the wild-type allele.

Highlights of This Issue

Aberrant expression of inducible nitric oxide synthase (iNOS) in the tumor microenvironment has been linked to progression, metastasis, and poor prognosis of human cancers, including cutaneous melanoma. Here, Sikora and colleagues tested the efficacy of iNOS-directed targeted therapy for the treatment of established human melanoma xenografts in SCID/NOD mice. Oral treatment with the iNOS-selective small molecule antagonist N6-(1-Iminoethyl)-L-lysinedihydrochloride (L-nil) inhibited melanoma growth, decreased formation of intratumoral microvessels, and enhanced the efficacy of cisplatin chemotherapy. These data support the hypothesis that iNOS expression supports tumor growth in vivo and suggest targeted iNOS inhibition is a promising therapeutic approach.Although there are hundreds of drugs currently in development for cancer, a number of constraints limit the ability of researchers to effectively evaluate all of these agents in Phase I trials. In this issue of CCR Focus, guest editors Patricia LoRusso and Lesley Seymour explore some of the barriers related to Phase I clinical trials, and discuss strategies for managing and overcoming these barriers. Some of the topics addressed include different approaches to Phase I trial design, the incorporation of biomarkers into early trials, good practices for data collection and reporting, and requirements for preclinical testing prior to conducting a first-in-human trial.The CpG island methylator phenotype (CIMP) represents a subgroup of colorectal cancers characterized by frequent promoter hypermethylation. Here, Dahlin and colleagues investigated the impact of this phenotype on patient prognosis in two large patient groups. Compared to patients with CIMP-negative tumors, CIMP-low tumor patients had poorer survival, regardless of the status of microsatellite instability screening. CIMP-high tumor patients, on the other hand, had poorer survival only in the context of microsatellite stable colorectal cancer. These findings contribute to an increased understanding of molecular subtypes and prognostic factors in colorectal cancer.A large proportion of BRCA gene variants have unknown clinical significance. To investigate the effect of BRCA DNA variants on splicing efficiency, Sanz and colleagues developed a strategy combining electronic prediction and RT-PCR–based functional splicing assays of lymphocyte mRNA or hybrid minigenes in HeLa and MCF10A cells. Anomalous RNA processing was observed in 28 out of the 57 assayed variants, and variants affected splice sites and splicing enhancers and silencers. Aberrant splicing may therefore represent a relevant pathogenic mechanism in hereditary breast/ovarian cancer, and further study of these splicing defects may increase the proportion of families that benefit from genetic counselling.

Functional Complementation in Yeast Allows Molecular Characterization of Missense Argininosuccinate Lyase Mutations

Deficiency of argininosuccinate lyase (ASL) causes argininosuccinic aciduria, an urea cycle defect that may present with a severe neonatal onset form or with a late onset phenotype. To date phenotype-genotype correlations are still not clear because biochemical assays of ASL activity correlate poorly with clinical severity in patients. We employed a yeast-based functional complementation assay to assess the pathogenicity of 12 missense ASL mutations, to establish genotype-phenotype correlations, and to screen for intragenic complementation. Rather than determining ASL enzyme activity directly, we have measured the growth rate in arginine-free medium of a yeast ASL(null) strain transformed with individual mutant ASL alleles. Individual haploid strains were also mated to obtain diploid, "compound heterozygous" yeast. We show that the late onset phenotypes arise in patients because they harbor individual alleles retaining high residual enzymatic activity or because of intragenic complementation among different mutated alleles. In these cases complementation occurs because in the hybrid tetrameric enzyme at least one active site without mutations can be formed or because the differently mutated alleles can stabilize each other, resulting in partial recovery of enzymatic activity. Functional complementation in yeast is simple and reproducible and allows the analysis of large numbers of mutant alleles. Moreover, it can be easily adapted for the analysis of mutations in other genes involved in urea cycle disorders.

Determination of splice-site mutations in Lynch syndrome (hereditary non-polyposis colorectal cancer) patients using functional splicing assay

Lynch syndrome (hereditary non-polyposis colorectal cancer) is an inherited disease caused by germ-line mutation in mismatch repair genes such as MLH1, MSH2, and MSH6. The mutations include missense and nonsense mutations, small insertions and deletions, and gross genetic alterations including large deletions and duplications. In addition to these genetic changes, mutations in introns are also involved in the pathogenesis. However, it is sometimes difficult to interpret correctly the pathogenicity of variants in exons as well as introns. To evaluate the effect of splice-site mutations in two Lynch syndrome patients, we carried out a functional splicing assay using minigenes. Consequently, this assay showed that the mutation of c.1731+5G>A in MLH1 led to exon15 skipping, and that the mutation of c.211+1G>C in MSH2 created an activated cryptic splice-site 17-nucleotides upstream in exon1. These aberrant splicing patterns were not observed when wild type sequence was used for the assay. We also obtained concordant results by RT-PCR experiments with transcripts from the patients. Furthermore, additional functional splicing assays using two different intronic mutations described in earlier studies revealed splicing alterations that were in complete agreement with the reports. Therefore, functional splicing assay is helpful for evaluating the effects of genetic variants on splicing.

In vivo and in vitro splicing assay of SLC12A1 in an antenatal salt-losing tubulopathy patient with an intronic mutation

Type I Bartter syndrome (BS), an inherited salt-losing tubulopathy, is caused by mutations of the SLC12A1 gene. While several intronic nucleotide changes in this gene have been detected, transcriptional analysis had not been conducted because mRNA analysis is possible only when renal biopsy specimens can be obtained or occasionally when mRNA is expressed in the leukocytes. This report concerns a type I BS patient due to compound heterozygosity for the SLC12A1 gene. Genomic DNA sequencing disclosed the presence of two novel heterozygous mutations of c.724 + 4A > G in intron 5 and c.2095delG in intron 16, but it remains to be determined whether the former would be likely to influence the transcription. In this report, we conducted both in vivo assay of RT-PCR analysis using RNA extracted from the proband's urinary sediments and in vitro functional splicing study by minigene construction, and obtained evidence that this intronic mutation leads to complete exon 5 skipping. To the best of our knowledge, this is the first study to use non-invasive methods for both an in vivo assay and an in vitro functional splicing assay of inherited kidney disease. These analytical assays could be adapted for all inherited kidney diseases.

The effect of disease-associated HRPT2 mutations on splicing

Mutations in the tumour suppressor HRPT2 occur in patients with parathyroid carcinoma, kidney tumours and Hyperparathyroidism-Jaw Tumour syndrome. Disruption of exonic splicing through mutation of donor/acceptor splice sites or exonic splice enhancer (ESE) sites leads to loss of function of a number of major tumour suppressors including BRCA1, APC and MLH1. Given that the effect of HRPT2 mutations on splicing has not been widely studied, we used an in vitro splicing assay to determine whether 17 HRPT2 mutations located in hot-spot and other exons predicted to disrupt ESE consensus sites led to aberrant splicing. Using two independent web-based prediction programs, the majority of these mutations were predicted to disrupt ESE consensus sites; however, aberrant splicing of HRPT2 transcripts was not observed. Canonical donor or acceptor splice site mutations were also investigated using this splicing assay and transcripts assessed from tumour tissue. Splice site mutations were shown to lead to either exon skipping or retention of intronic sequences through the use of cryptic splice sites comprised of non-classical splicing signals. Aberrant splicing caused by disruption of ESE sites does not appear to have a major role in HRPT2-associated disease; however, premature truncation of parafibromin as the result of canonical donor or acceptor splice site mutations is associated with pathogenicity. Functional splicing assays must be undertaken in order to confirm web-based software predictions of the modification of putative ESE sites by disease-associated mutations.

Analysis of the involvement of the NR2E3 gene in autosomal recessive retinal dystrophies

The nuclear receptor protein NR2E3 is postulated to play an important role in rod and cone photoreceptor development. NR2E3 gene mutational analyses were carried out in 103 unrelated subjects with different retinal diseases. A total of 14 different sequence variants were identified, including 3 mutations, 6 rare sequence variants and five polymorphisms. One of three mutations is novel (a frameshift mutation: c.1034_1038del5bp). Five of the six rare sequence variants and one of the polymorphisms identified are novel. Splice prediction programs and functional splicing assays were performed to study three of these variants. The c.119-2 A>C mutant allele construction produces, in addition to the normal one, an abnormal transcript of 180 bp resulting from an aberrant splicing with skipping of exon 2 and the generation of a premature stop codon in exon 3. These experimental data confirm the splice predictions made by the computer programs. The obtained results reinforce the idea that NR2E3 gene is involved in several retinal diseases without a clear genotype-phenotype correlation.

Modulation of Fox-Regulated Alternative Splicing Events during Erythropoiesis.

A switch in the alternative splicing pattern in protein 4.1R pre-mRNA, involving activation of exon 16 splicing, occurs in late erythropoiesis. Because the inclusion of exon 16 leads to synthesis of protein isoforms with high affinity for spectrin and actin, and mechanical strengthening of the membrane, this switch is critical for normal erythyroid differentiation. Previous studies have shown that Fox-2 protein is a splicing regulator that binds to intronic enhancer sequences downstream of exon 16, and that this binding stimulates the inclusion of exon 16. Fox binding sites are also located downstream of other tissue-specific alternative exons, some of which exhibit variable splicing efficiencies, leading us to investigate whether splicing switch strength is influenced by variations in the sequence of the Fox binding-site motif (UGCAUG) and by the number of these protein binding sites. A series of minigenes was created in which protein 4.1R E16 splicing efficiency was measured in transfection studies. Mutation of the first U residue of the Fox binding site resulted in a significantly weaker, but still Fox dependent activation of splicing, whereas mutation of the terminal G residue dramatically reduced enhancer activity. Insertion of two wild-type UGCAUG elements enhanced splicing substantially in a Fox protein concentration-dependent manner, and four elements gave even stronger inclusion. To test whether the reduced splicing efficiency observed for minigenes containing mutated enhancer elements was due to a reduction in Fox binding, surface plasmon resonance (SPR) was employed. Our binding studies indicate that full-length mouse Fox-2a bound to the UGCAUG sequence with high affinity (KD ∼100nM). Furthermore, in agreement with our functional splicing assays, Fox binding affinity for the hexameric sequence was reduced with mutations in position one, and almost eliminated with mutations in position six. There was an excellent correlation between binding affinity of Fox protein for the enhancer motif, and strength of enhancer activity measured in functional splicing assays. Additional studies demonstrated that Fox enhancer activity can be reduced by the presence of a closely linked binding site for hnRNP A1, a known splicing inhibitor. Taken together, these studies demonstrate that the composition and number of binding sites for Fox can significantly affect the efficiency of splicing for E16. It is likely that through these combinatorial mechanisms, Fox splicing factors are able to modulate the efficiency with which tissue specific exons in the erythroid alternative splicing program can be spliced into mature mRNA.