Splicing Mutation In Intron Research Articles

Identification and characterization of a novel intronic splicing mutation in CSF1R-related leukoencephalopathy.

Colony stimulating factor 1 receptor (CSF1R)-related leukoencephalopathy is a rapidly progressing neurodegenerative disease caused by CSF1R gene mutations. This study aimed to identify and investigate the effect of a novel intronic mutation (c.1754-3C>G) of CSF1R on splicing. A novel intronic mutation was identified using whole-exome sequencing. To investigate the impact of this mutation, we employed various bioinformatics tools to analyze the transcription of the CSF1R gene and the three-dimensional structure of its encoded protein. Furthermore, reverse transcription polymerase chain reaction (RT-PCR) was performed to validate the findings. A novel mutation (c.1754-3C>G) in CSF1R was identified, which results in exon 13 skipping due to the disruption of the 3' splice site consensus sequence NYAG/G. This exon skipping event was further validated in the peripheral blood of the mutation carrier through RT-PCR and Sanger sequencing. Protein structure prediction indicated a disruption in the tyrosine kinase domain, with the truncated protein showing significant structural alterations. Our findings underscore the importance of intronic mis-splicing mutations in the diagnosis and management of CSF1R-related leukoencephalopathy.

Molecular and functional correction of a deep intronic splicing mutation in CFTR by CRISPR-Cas9 gene editing

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CFTR gene. The 10th most common mutation, c.3178-2477C>T (3849+10kb C>T), involves a cryptic, intronic splice site. This mutation was corrected in CF primary cells homozygous for this mutation by delivering pairs of guide RNAs (gRNAs) with Cas9 protein in ribonucleoprotein (RNP) complexes that introduce double-strand breaks to flanking sites to excise the 3849+10kb C>T mutation, followed by DNA repair by the non-homologous end-joining pathway, which functions in all cells of the airway epithelium. RNP complexes were delivered to CF basal epithelial cell by a non-viral, receptor-targeted nanocomplex comprising a formulation of targeting peptides and lipids. Canonical CFTR mRNA splicing was, thus, restored leading to the restoration of CFTR protein expression with concomitant restoration of electrophysiological function in airway epithelial air-liquid interface cultures. Off-target editing was not detected by Sanger sequencing of in silico-selected genomic sites with the highest sequence similarities to the gRNAs, although more sensitive unbiased whole genome sequencing methods would be required for possible translational developments. This approach could potentially be used to correct aberrant splicing signals in several other CF mutations and other genetic disorders where deep-intronic mutations are pathogenic.

Novel c.2654+1G>A mutation in the CSF1R gene in a family with CSF1R‐microglial encephalopathy

AbstractBackgroundThe colony‐stimulating factor‐1 receptor (CSF1R) is a transmembrane tyrosine kinase receptor that is mainly expressed in the brain‐resident microglia. Thus, microglial dysfunction due to CSF1R mutation may play a primary and pivotal role in the pathogenesis of CSF1R‐microglial encephalopathy. Neither the functional consequences of all known CSF1R mutations nor the full genetic spectrum of mutations causing CSF1R‐microglial encephalopathy have been fully elucidated. We aimed to describe a novel heterozygous splicing variant of the CSF1R gene that caused CSF1R‐microglial encephalopathy in a Han Chinese family.MethodThe demographic data, detailed medical history, and clinical manifestations were collected from a family with CSF1R‐microglial encephalopathy. Some family members also underwent neuropsychological evaluation, structural and functional magnetic resonance imaging (MRI), and whole‐exome sequence analyses.ResultFour generations of this family, including 27 members, presented with a medical history of a dominant hereditary pattern (Figure 1). Seven of these 27 individuals had died, while five presented with similar cognitive impairments clinically, including the proband, and his sister, father, uncle, and grandmother during their life. Brain MRI of the proband (Figure 2) and his sister (Figure 3) depicted symmetric confluent and diffuse deep white matter changes, atrophy of the frontoparietal lobes, and thinning of the corpus callosum. The brother of the proband remained asymptomatic, and the structural brain MRI only revealed minimal white matter changes; however, pseudo‐continuous arterial spin labeling (pCASL) demonstrated marked reduction in the cerebral blood flow (CBF) in the bilateral deep white matter and corpus callosum (Figure 4). Whole‐exome sequencing was performed for seven family members, which identified a novel splice‐site heterozygous mutation (c.2319+1C>A) in intron 20 of the CSF1R gene in four members (Figure 5).ConclusionWe identified a novel splicing mutation (c.2319+1C>A) in intron 20 of CSF1R as the cause of CSF1R‐microglial encephalopathy in a Han Chinese family, which broadens the genetic spectrum of CSF1R‐microglial encephalopathy. We opine that clinically feasible pCASL techniques may be more sensitive to the alterations in CBF of associated with microglial density and cellular changes. This study highlights the potential value of pCASL techniques to improve the clinical diagnostic accuracy of CSF1R‐microglial encephalopathy.

Mechanism of Early Flowering in a Landrace Naked Barley eam8.l Mutant

The EARLY MATURITY 8 (EAM8) gene of barley is homologous to the EARLY FLOWERING 3 (ELF3) gene in Arabidopsis, as loss-of-function mutations in this circadian clock gene promote rapid flowering. A previous study demonstrated that the early flowering phenotype of a hulless barley, Lalu, was due to allele eam8.l carrying an alternative splicing mutation in intron 3 that led to intron retention. In the present study, we verified that eam8.l encoded a truncated protein. Although EAM8 was expressed at a higher level in Lalu than in other barley lines with a longer growth period, it did not negatively regulate flowering time. This result further proved that the eam8.l protein was nonfunctional in regulating flowering in barley. The early flowering phenotype of Lalu plants was strongly dependent on the biosynthesis of gibberellin (GA). The eam8.l mutation stopped the suppression of GA biosynthesis, and Lalu accumulated excessive GA, especially in leaves. This was achieved through the upregulated expression of genes in the GA pathway, including GA20ox2, LFY1, SOC1, PAP2, and FPF3. The mutation of the EAM8 gene also abolished the inhibition of FLOWERING LOCUS T-like (FT1) gene expression at night. During the night, expression levels of the FT1 gene were higher than those during the day in Lalu. However, the GA-dependent pathway and FT1 gene mechanism are two independent pathways that promote flowering in Lalu. Alleles of EAM8, therefore, demonstrated an important breeding value in barley, which is probably effective in many other day-length-sensitive crop plants as well; thus, they could be used to tune adaptation in different geographic regions and climatic conditions, a critical issue in times of global warming.

Integrative assessment of CIP2A overexpression and mutational effects in human malignancies identifies possible deleterious variants

KIAA1524 is the gene encoding the human cancerous inhibitor of PP2A (CIP2A) protein which is regarded as a novel target for cancer therapy. It is overexpressed in 65%–90% of tissues in almost all studied human cancers. CIP2A expression correlates with cancer progression, disease aggressivity in lung cancer besides poor survival and resistance to chemotherapy in breast cancer. Herein, a pan-cancer analysis of public gene expression datasets was conducted showing significant upregulation of CIP2A in cancerous and metastatic tissues. CIP2A overexpression also correlated with poor survival of cancer patients. To determine the non-coding variants associated with CIP2A overexpression, 5′UTR and 3′UTR variants were annotated and scored using RegulomeDB and Enformer deep learning model. The 5′UTR variants rs1239349555, rs1576326380, and rs1231839144 were predicted to be potential regulators of CIP2A overexpression scoring best on RegulomeDB annotations with a high “2a” rank of supporting experimental data. These variants also scored the highest on Enformer predictions.Analysis of the 3′UTR variants of CIP2A predicted rs56255137 and rs58758610 to alter binding sites of hsa-miR-500a-5 and (hsa-miR-3671, hsa-miR-5692a) respectively. Both variants were also found in linkage disequilibrium with rs11709183 and rs147863209 respectively at r2 ≥ 0.8. The aforementioned variants were found to be eQTL hits significantly associated with CIP2A overexpression. Further, analysis of rs11709183 and rs147863209 revealed a high “2b” rank on RegulomeDB annotations indicating a probable effect on DNAse transcription factors binding. The MuTarget analysis indicated that somatic mutations in TP53 are significantly associated with upregulated CIP2A in human cancers. Analysis of missense SNPs on CIP2A solved structure predicted seven deleterious effects. Four of these variants were also predicted as structurally and functionally destabilizing to CIP2A including; rs375108755, rs147942716, rs368722879, and rs367941403. Variant rs1193091427 was predicted as a potential intronic splicing mutation that might be responsible for the novel CIP2A variant (NOCIVA) in multiple myeloma. Finally, Enrichment of the Wnt/β-catenin pathway within the CIP2A regulatory gene network suggested potential of therapeutic combinations between FTY720 with Wnt/β-catenin, Plk1 and/or HDAC inhibitors to downregulate CIP2A which has been shown to be essential for the survival of different cancer cell lines.

592: Molecular and functional correction of a deep intronic splicing mutation in CFTR by CRISPR/Cas9 gene editing

592: Molecular and functional correction of a deep intronic splicing mutation in CFTR by CRISPR/Cas9 gene editing

Novel Compound Missense and Intronic Splicing Mutation in ALDH18A1 Causes Autosomal Recessive Spastic Paraplegia.

Background: Hereditary spastic paraplegia (HSP) caused by mutations in ALDH18A1 have been reported as spastic paraplegia 9 (SPG9), with autosomal dominant and autosomal recessive transmission (SPG9A and SPG9B). SPG9 is rare and has shown phenotypic and genotypic heterogeneity in previous reports.Methods: This study screened ALDH18A1 mutations in autosomal recessive HSP patients using combined whole exome sequencing and RNA splicing analysis. We conducted in silico investigations, co-segregation analysis, and ELISA-based analysis of P5CS (Δ1-pyrroline-5-carboxylate synthetase; encoded by ALDH18A1) concentration to validate the pathogenicity of the detected ALDH18A1 variants. All previously reported bi-allelic ALDH18A1 mutations and cases were reviewed to summarize the genetic and clinical features of ALDH18A1-related HSP.Results: A novel missense mutation c.880T>C, p.S294P and an intronic splicing mutation c.-28-13A>G were both detected in ALDH18A1 in an autosomal recessive family presenting with a complicated form HSP. ELISA assays revealed significantly decreased P5CS concentration in the proband's plasma compared with that in the healthy controls. Moreover, review of previously reported recessive cases showed that SPG9B patients in our cohort presented with milder symptoms, i.e., later age at onset and without cognitive impairment.Conclusion: The present study expands the genetic and clinical spectrum of SPG9B caused by ALDH18A1 mutation. Our work defines new genetic variants to facilitate future diagnoses, in addition to demonstrating the highly informative value of splicing mutation prediction in the characterization of disease-related intronic variants.

Optimization and Validation of Multimodular, Long-Range PCR–Based Next-Generation Sequencing Assays for Comprehensive Detection of Mutation in Tuberous Sclerosis Complex

The genetic diagnosis of tuberous sclerosis complex is difficult because of its broad spectrum of mutations. In addition to point mutations in coding regions, intragenic or chromosomal-level large deletions, deep intronic splicing mutations, and mosaic mutations represent a significant proportion of the mutations. In this study, multimodular, long-range PCR-based next-generation sequencing assays were optimized and validated using >100 samples with known TSC1 and TSC2 variants. Multiplex, long-range PCR covering the entire genomic region of both genes detected all 138 known variants; however, it also yielded false-positive results. Intragenic large deletions were detected with accurate breakpoint sequences. Chromosomal-level deletions were estimated by discordant allele segregation in the family and confirmed by DNA microarray. Deep intronic mutations were verified using a combination of long-range DNA PCR and full-length mRNA sequencing. DNA samples were mixed to simulate mosaic mutations, and most variants were detected but could not be distinguished from equivalently detected false-positive results. Repeated false-positive results were classified, and the strategy of selecting the common variants detected in the duplicate analysis and eliminating known false-positive results improved the sensitivity (85.2%) and positive predictive value (96.6%) of a 10% mosaic simulation. Long-range PCRbased next-generation sequencing is a highly versatile genetic test; however, confirmation tests remain necessary for clinical use because false-positive results cannot be completely eliminated from single experiments.

CONGENITAL MYOPATHIES 1 – NEMALINE: P.26 A new intronic mutation in nebulin myopathy

The aim of this study was to demonstrate functional and clinical presentation of a new homozygous intronic mutation in nebulin. Nebulin encodes for a giant protein component of the cytoskeletal matrix that coexists with the thick and thin filaments within the sarcomeres of skeletal muscle. Of the 183 exons in the nebulin gene, at least 43 are alternatively spliced, although exons 143 and 144 are not found in the same transcript. We report the case of a 6 yo boy, born from consanguineous parents with typical phenotypic presentation for nemaline myopathy, proven on muscle biopsy. Muscle biopsy showed a significant amount of nemaline bodies. Muscle MRI was strongly suggestive of nebulin related myopathy but gene panel for congenital myopathies was negative. Exome sequencing was performed and did not reveal specific findings. Approach with SNP to show loss of heterozygosity demonstrates that nebulin gene was of interest. Deep sequencing of nebulin revealed an intronic homozygous splicing mutation between exon 144 and exon 145. Cloning of the biopsy PCR fragment revealed that a large proportion of the isoform with exon 144 contained an 117 additional base pairs probably due to the creation of a new splicing site as predicted by in silico analysis. Exon 143 and 144 cannot coexist in the same isoform but in our patient functional studies demonstrated an altered exon 144/143 isoform ratio. Nebulin protein sequence encoded by exon 143 is highly conserved across species, suggesting that it may have a special function, such as species interaction with another protein. For example, the binding site for KLHL40 is within S21, but its precise location is not known. Therefore this intronic mutation alters nebulin protein by direct functional alteration of the transcript and by destabilizing other transcripts, particularly exon 143, whose interaction in KLHL40 binding site could explain the high amount of nemaline inclusion bodies in the muscle biopsy as seen in KLHL40 related nemaline myopathy. Some unresolved cases of nemaline myopathy could be explained by intronic mutations in nebulin. New diagnostic approaches could help to explain these cases.

Endoplasmic Reticulum Stress Activation in Alport Syndrome Varies Between Genotype and Cell Type.

Alport syndrome is a hereditary progressive chronic kidney disease caused by mutations in type IV collagen genes COL4A3/4/5. X-linked Alport syndrome (XLAS) is caused by mutations in the COL4A5 gene and is the most common form of Alport syndrome. A strong correlation between the type of COL4A5 mutation and the age developing end-stage renal disease in male patients has been found. Mutation to the α (IV) chain causes retention of the protein to the endoplasmic reticulum lumen, which causes endoplasmic reticulum stress (ERS) and subsequent exertion of deleterious intracellular effects through the activation of ERS. The exact time point that mutant type IV collagen α chain exerts its deleterious effects remains elusive. In this study, we explored the relationship between the COL4A5 genotype and cell type in ERS activation. We obtained skin fibroblasts from Alport syndrome patients with different COL4A5 mutation categories [i.e., a missense mutation (c.4298G > T, p.Gly1433Val) in exon 47, a splicing mutation (c.1949–1G > A) in intron 25 and an insertion (c.573_c.574insG, p. Pro193Alafs*23) in exon 10], and then reprogrammed these fibroblasts into induced pluripotent stem cells (iPSCs). Interestingly, no significant dysregulation of ERS pathway markers was observed for the three COL4A5 mutant iPSCs; however, significant activation of ERS in COL4A5 mutant fibroblasts was observed. In addition, we found that the activation levels of some ERS markers in fibroblasts varied among the three COL4A5 mutation types. Mutant COL4A5 proteins were demonstrated to have different effects on cells at different stages of ontogenesis, providing a theoretical basis for choosing the timing of intervention. The observed differences in activation of ERS by the COL4A5 mutant fibroblasts may contribute to the intracellular molecular mechanisms that describe the correlation between genotype and clinical features in XLAS.

Antisense-Mediated Skipping of Dysferlin Exons in Control and Dysferlinopathy Patient-Derived Cells.

Dysferlinopathies encompass a spectrum of progressive muscular dystrophies caused by the lack of dysferlin due to missense mutations in the dysferlin gene or mutations causing premature truncation of protein translation. Dysferlin is a modular protein, and dysferlins lacking one or more repetitive domains have been shown to retain functionality. As such, antisense-mediated exon skipping has been proposed as a therapy for dysferlinopathy. By skipping the mutated exon, the reading frame would be maintained, while the mutation would be bypassed, thus allowing production of an internally deleted, but partially functional, dysferlin. We previously showed that dysferlin exon skipping is feasible in control cell lines. We here evaluated exon skipping and dysferlin protein restoration in patient-derived cells requiring the skipping of exon 9, 29, 30, or 34. Exon 30 skipping was possible at high efficiency, but did not result in increased dysferlin. We discovered that the alleged exon 30 mutation was in fact a polymorphism and identified a splicing mutation in intron 28 as the disease-causing mutation. While exon skipping was feasible for each of the other cell lines, no increases in dysferlin protein could be detected by western blotting.

ATP6V1B1 recurrent mutations in Algerian deaf patients associated with renal tubular acidosis

Hereditary distal renal tubular acidosis (dRTA) is a rare disorder characterized by metabolic acidosis due to impaired renal acid excretion. To date, three genes (ATP6V1B1, ATP6V0A4 and SLC4A1) have been reported to be responsible for this genetic disorder. Notably, mutations of ATP6V1B1 gene, which encode B1-subunit of H + -ATPase pump cause distal renal tubular acidosis often, associated with sensorineural hearing loss (SNHL). Furthermore, enlarged vestibular aqueduct (EVA) was also described in some patients with ATP6V1B1 mutations. Four Algerian unrelated patients presented with dRTA and SNHL were recruited. The ATP6V1B1 gene was preferentially analyzed in all these patients by Sanger sequencing.We identified two previously reported variants in ATP6V1B1 gene: a frameshift mutation (c.1155dupC: p.(Ile386Hisfs*56) in exon 12 and a splicing mutation in intron 2 (c.175-1G > C: p?). Both mutations were homozygous in affected members. Interestingly, one patient with p.(Ile386Hisfs*56) mutation presented profound SNHL and bilateral enlarged vestibular aqueduct (EVA).Our study indicates the importance contribution of ATP6V1B1 gene mutations to the pathogenesis of the dRTA in the Algerian population and will contribute to introducing principles to predict the characteristics of the dRTA in patients. Thus, screening for this gene could allow rapid patient management and provide adequate genetic counseling.

A deep intronic splice mutation of STAT3 underlies hyper IgE syndrome by negative dominance

Heterozygous in-frame mutations in coding regions of human STAT3 underlie the only known autosomal dominant form of hyper IgE syndrome (AD HIES). About 5% of familial cases remain unexplained. The mutant proteins are loss-of-function and dominant-negative when tested following overproduction in recipient cells. However, the production of mutant proteins has not been detected and quantified in the cells of heterozygous patients. We report a deep intronic heterozygous STAT3 mutation, c.1282-89C>T, in 7 relatives with AD HIES. This mutation creates a new exon in the STAT3 complementary DNA, which, when overexpressed, generates a mutant STAT3 protein (D427ins17) that is loss-of-function and dominant-negative in terms of tyrosine phosphorylation, DNA binding, and transcriptional activity. In immortalized B cells from these patients, the D427ins17 protein was 2 kDa larger and 4-fold less abundant than wild-type STAT3, on mass spectrometry. The patients' primary B and T lymphocytes responded poorly to STAT3-dependent cytokines. These findings are reminiscent of the impaired responses of leukocytes from other patients with AD HIES due to typical STAT3 coding mutations, providing further evidence for the dominance of the mutant intronic allele. These findings highlight the importance of sequencing STAT3 introns in patients with HIES without candidate variants in coding regions and essential splice sites. They also show that AD HIES-causing STAT3 mutant alleles can be dominant-negative even if the encoded protein is produced in significantly smaller amounts than wild-type STAT3.

Mutations in ANAPC1, Encoding a Scaffold Subunit of the Anaphase-Promoting Complex, Cause Rothmund-Thomson Syndrome Type 1

Rothmund-Thomson syndrome (RTS) is an autosomal-recessive disorder characterized by poikiloderma, sparse hair, short stature, and skeletal anomalies. Type 2 RTS, which is defined by the presence of bi-allelic mutations in RECQL4, is characterized by increased cancer susceptibility and skeletal anomalies, whereas the genetic basis of RTS type 1, which is associated with juvenile cataracts, is unknown. We studied ten individuals, from seven families, who had RTS type 1 and identified a deep intronic splicing mutation of the ANAPC1 gene, a component of the anaphase-promoting complex/cyclosome (APC/C), in all affected individuals, either in the homozygous state or in trans with another mutation. Fibroblast studies showed that the intronic mutation causes the activation of a 95bp pseudoexon, leading to mRNAs with premature termination codons and nonsense-mediated decay, decreased ANAPC1 protein levels, and prolongation of interphase. Interestingly, mice that were heterozygous for a knockout mutation have an increased incidence of cataracts. Our results demonstrate that deficiency in the APC/C is a cause of RTS type 1 and suggest a possible link between the APC/C and RECQL4 helicase because both proteins are involved in DNA repair and replication.

Genotype-phenotype correlation study and mutational and hormonal analysis in a Chinese cohort with 21-hydroxylase deficiency.

BackgroundSteroid 21‐hydroxylase deficiency (21OHD) is the most common enzymatic defect, but the genotype–phenotype associations have not been well established in Chinese patients. Here, a Chinese 21OHD cohort was enrolled to investigate the clinical, biochemical, and genetic characteristics of this disorder.MethodsMutation analysis of CYP21A2 gene, 21‐hydroxylase activity assays and in silico predictions of protein structure were performed. Genotype–phenotype associations were analyzed in both the cohort and 487 Chinese CAH patients ever reported.ResultsAmong the total cohort (72 patients), 47 patients (65.3%) were diagnosed as salt‐wasting (SW) phenotype, 11 (15.3%) were simple virilizing (SV) type, and 14 (19.4%) were nonclassic (NC) type. The value of FSH and LH for prediction of the SW phenotype was up to 0.862 and 0.669, respectively. Overall, the detection rate of CYP21A2 mutation was 97.9%, which revealed 25 mutations and 36 genotypes. Four novel mutations (p.L199X, p.E321del, p.H393Q, and p.L459‐P464del) were detected and induced a significantly reduced 21‐hydroxylase activity. Generally, disease severity can be predicted with the genotypes. The most common genotypes in Chinese population were I2G/I2G (12.5%), I2G/Large lesion (12.1%), I173N/I2G (10.3%), and I173N/Large lesion (9.2%). The SW form of CAH is prominent in deletion or intronic splice mutations, namely I2G/I2G (18.6%), I2G/Large lesion (17.2%) and Large lesion/Large lesion (8.6%).ConclusionFour novel mutations were identified and a high consistency of genotype–phenotype association was found in SW CAH. Moreover, FSH and LH levels were proved to be a promising marker for predicting the severity of the disease.

Mutations in GPAA1, Encoding a GPI Transamidase Complex Protein, Cause Developmental Delay, Epilepsy, Cerebellar Atrophy, and Osteopenia

Approximately one in every 200 mammalian proteins is anchored to the cell membrane through a glycosylphosphatidylinositol (GPI) anchor. These proteins play important roles notably in neurological development and function. To date, more than 20 genes have been implicated in the biogenesis of GPI-anchored proteins. GPAA1 (glycosylphosphatidylinositol anchor attachment 1) is an essential component of the transamidase complex along with PIGK, PIGS, PIGT, and PIGU (phosphatidylinositol-glycan biosynthesis classes K, S, T, and U, respectively). This complex orchestrates the attachment of the GPI anchor to the C terminus of precursor proteins in the endoplasmic reticulum. Here, we report bi-allelic mutations in GPAA1 in ten individuals from five families. Using whole-exome sequencing, we identified two frameshift mutations (c.981_993del [p.Gln327Hisfs∗102] and c.920delG [p.Gly307Alafs∗11]), one intronic splicing mutation (c.1164+5C>T), and six missense mutations (c.152C>T [p.Ser51Leu], c.160_161delinsAA [p.Ala54Asn], c.527G>C [p.Trp176Ser], c.869T>C [p.Leu290Pro], c.872T>C [p.Leu291Pro], and c.1165G>C [p.Ala389Pro]). Most individuals presented with global developmental delay, hypotonia, early-onset seizures, cerebellar atrophy, and osteopenia. The splicing mutation was found to decrease GPAA1 mRNA. Moreover, flow-cytometry analysis of five available individual samples showed that several GPI-anchored proteins had decreased cell-surface abundance in leukocytes (FLAER, CD16, and CD59) or fibroblasts (CD73 and CD109). Transduction of fibroblasts with a lentivirus encoding the wild-type protein partially rescued the deficiency of GPI-anchored proteins. These findings highlight the role of the transamidase complex in the development and function of the cerebellum and the skeletal system.

Splicing regulation and dysregulation of cholinergic genes expressed at the neuromuscular junction.

We humans have evolved by acquiring diversity of alternative RNA metabolisms including alternative means of splicing and transcribing non-coding genes, and not by acquiring new coding genes. Tissue-specific and developmental stage-specific alternative RNA splicing is achieved by tightly regulated spatiotemporal regulation of expressions and activations of RNA-binding proteins that recognize their cognate splicing cis-elements on nascent RNA transcripts. Genes expressed at the neuromuscular junction are also alternatively spliced. In addition, germline mutations provoke aberrant splicing by compromising binding of RNA-binding proteins, and cause congenital myasthenic syndromes (CMS). We present physiological splicing mechanisms of genes for agrin (AGRN), acetylcholinesterase (ACHE), MuSK (MUSK), acetylcholine receptor (AChR) α1 subunit (CHRNA1), and collagen Q (COLQ) in human, and their aberration in diseases. Splicing isoforms of AChET , AChEH , and AChER are generated by hnRNP H/F. Skipping of MUSK exon 10 makes a Wnt-insensitive MuSK isoform, which is unique to human. Skipping of exon 10 is achieved by coordinated binding of hnRNP C, YB-1, and hnRNP L to exon 10. Exon P3A of CHRNA1 is alternatively included to generate a non-functional AChR α1 subunit in human. Molecular dissection of splicing mutations in patients with CMS reveals that exon P3A is alternatively skipped by hnRNP H, polypyrimidine tract-binding protein 1, and hnRNP L. Similarly, analysis of an exonic mutation in COLQ exon 16 in a CMS patient discloses that constitutive splicing of exon 16 requires binding of serine arginine-rich splicing factor 1. Intronic and exonic splicing mutations in CMS enable us to dissect molecular mechanisms underlying alternative and constitutive splicing of genes expressed at the neuromuscular junction. This is an article for thespecial issue XVth International Symposium on Cholinergic Mechanisms.

CRISPR/Cas9-Mediated Genome Editing as a Therapeutic Approach for Leber Congenital Amaurosis 10

As the most common subtype of Leber congenital amaurosis (LCA), LCA10 is a severe retinal dystrophy caused by mutations in the CEP290 gene. The most frequent mutation found in patients with LCA10 is a deep intronic mutation in CEP290 that generates a cryptic splice donor site. The large size of the CEP290 gene prevents its use in adeno-associated virus (AAV)-mediated gene augmentation therapy. Here, we show that targeted genomic deletion using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system represents a promising therapeutic approach for the treatment of patients with LCA10 bearing the CEP290 splice mutation. We generated a cellular model of LCA10 by introducing the CEP290 splice mutation into 293FT cells and we showed that guide RNA pairs coupled with SpCas9 were highly efficient at removing the intronic splice mutation and restoring the expression of wild-type CEP290. In addition, we demonstrated that a dual AAV system could effectively delete an intronic fragment of the Cep290 gene in the mouse retina. To minimize the immune response to prolonged expression of SpCas9, we developed a self-limiting CRISPR/Cas9 system that minimizes the duration of SpCas9 expression. These results support further studies to determine the therapeutic potential of CRISPR/Cas9-based strategies for the treatment of patients with LCA10.

Hybridization Capture-Based Next-Generation Sequencing to Evaluate Coding Sequence and Deep Intronic Mutations in the NF1 Gene

Neurofibromatosis 1 (NF1) is one of the most common genetic disorders and is caused by mutations in the NF1 gene. NF1 gene mutational analysis presents a considerable challenge because of its large size, existence of highly homologous pseudogenes located throughout the human genome, absence of mutational hotspots, and diversity of mutations types, including deep intronic splicing mutations. We aimed to evaluate the use of hybridization capture-based next-generation sequencing to screen coding and noncoding NF1 regions. Hybridization capture-based next-generation sequencing, with genomic DNA as starting material, was used to sequence the whole NF1 gene (exons and introns) from 11 unrelated individuals and 1 relative, who all had NF1. All of them met the NF1 clinical diagnostic criteria. We showed a mutation detection rate of 91% (10 out of 11). We identified eight recurrent and two novel mutations, which were all confirmed by Sanger methodology. In the Sanger sequencing confirmation, we also included another three relatives with NF1. Splicing alterations accounted for 50% of the mutations. One of them was caused by a deep intronic mutation (c.1260 + 1604A > G). Frameshift truncation and missense mutations corresponded to 30% and 20% of the pathogenic variants, respectively. In conclusion, we show the use of a simple and fast approach to screen, at once, the entire NF1 gene (exons and introns) for different types of pathogenic variations, including the deep intronic splicing mutations.

New intronic splicing mutation in the LMNA gene causing progressive cardiac conduction defects and variable myopathy

BackgroundMost of mutations in the LMNA gene are unique and have been found in only a few unrelated families. The clinical interpretation of new genetic variants, especially beyond the coding area and canonical splice sites, is proving to be difficult and requires advanced investigation. MethodsThis study included patients with progressive cardiac conduction defects with neuromuscular involvement. The clinical evaluation included medical history and 24-h Holter monitoring. The genetic evaluation included mutation screening in the LMNA gene by the Sanger sequence. Sanger sequencing was followed by RT-PCR of the target fragment of cDNA. In silico modeling was performed with CCBulder and Modeller software. ResultsThe diagnosis of limb-girdle muscular dystrophy type 1B (LGMD1B) was established. The new intronic variant c.513+45T>G was found in the LMNA gene in the proband and affected daughter. The insertion of 45bp was confirmed in the proband's cDNA. The structural and possible functional effects of the aberrant protein were predicted. ConclusionsVariant c.513+45T>G in the LMNA gene likely translates into the longer lamin A/C proteins with additional 15 amino acids. This variant is thought to be pathogenic. Intronic variants in the LMNA gene located beside canonic splice sites may be responsible for some genotype-negative cases with clinical phenotype of laminopathies.