Deciphering the impact of coding and non-coding SCN1A gene variants on RNA splicing.

Variants that disrupt normal pre-mRNA splicing are increasingly being recognized as a major cause of monogenic disorders. The SCN1A gene, a key epilepsy gene that is linked to various epilepsy phenotypes, is no exception. Approximately 10% of all reported variants in the SCN1A gene are designated as splicing variants, with many located outside of the canonical donor and acceptor splice sites, and most have not been functionally investigated. However, given its restricted expression pattern, functional analysis of splicing variants in the SCN1A gene could not be routinely performed. In this study, we conducted a comprehensive analysis of all reported SCN1A variants and their potential to impact SCN1A splicing and conclude that splicing variants are substantially misannotated and under-represented. We created a splicing reporter system consisting of 18 splicing vectors covering all 26 protein-coding exons with different genomic contexts and several promoters of varying strengths in order to reproduce the wild-type splicing pattern of the SCN1A gene, revealing cis-regulatory elements essential for proper recognition of SCN1A exons. Functional analysis of 95 SCN1A variants was carried out, including all 68 intronic variants reported in the literature, located outside of the splice sites canonical dinucleotides; 21 exonic variants of different classes (synonymous, missense, nonsense and in-frame deletion) and six variants observed in patients with epilepsy. Interestingly, almost 20% of tested intronic variants had no influence on SCN1A splicing, despite being reported as causative in the literature. Moreover, we confirmed that the majority of predicted exonic variants affect splicing unravelling their true molecular mechanism. We used functional data to perform genotype-phenotype correlation, revealing distinct distribution patterns for missense and splice-affecting 'missense' variants and observed no difference in the phenotype severity of variants leading to in-frame and out-of-frame isoforms, indicating that the Nav1.1 protein is highly intolerant to structural variations. Our work demonstrates the importance of functional analysis in proper variant annotation and provides a tool for high-throughput delineation of splice-affecting variants in SCN1A in a whole-gene manner.

Homozygous deep intronic variant in SNX14 cause autosomal recessive Spinocerebellar ataxia 20: a case report.

Autosomal recessive spinocerebellar ataxia type 20, SCAR20 (MIM: 616354) is a rare syndromic form of hereditary ataxias. It characterized by the presence of progressive ataxia, intellectual developmental disorder, autism and dysmorphic features. The disease caused by biallelic variants in SNX14 gene that lead to loss of protein function. Typically, these variants result in the formation of a premature stop codon, a shift in the reading frame or a variant in canonical splicing sites, as well as gross rearrangements. Here we present the first case of a deep intronic variant c.462-589A>G in SNX14 identified in two sisters with SCAR20 from a consanguineous family. This variant resulted in the inclusion of a pseudo-exon 82 nucleotides long and the formation of a premature stop codon, leading to the production of a truncated protein (NP_722523.1:p.Asp155Valfs*8). Following an extensive diagnostic search, the diagnosis was confirmed using trio whole genome sequencing. This case contributes to expanding the spectrum of potential genetic variants associated with SCAR20.

Evaluation of Pathogenicity and Causativity of Variants in the MPZ and SH3TC2 Genes in a Family Case of Hereditary Peripheral Neuropathy.

The implementation of NGS methods into clinical practice allowed researchers effectively to establish the molecular cause of a disorder in cases of a genetically heterogeneous pathology. In cases of several potentially causative variants, we need additional analysis that can help in choosing a proper causative variant. In the current study, we described a family case of hereditary motor and sensory neuropathy (HMSN) type 1 (Charcot-Marie-Tooth disease). DNA analysis revealed two variants in the SH3TC2 gene (c.279G>A and c.1177+5G>A), as well as a previously described variant c.449-9C>T in the MPZ gene, in a heterozygous state. This family segregation study was incomplete because of the proband's father's unavailability. To evaluate the variants' pathogenicity, minigene splicing assay was carried out. This study showed no effect of the MPZ variant on splicing, but the c.1177+5G>A variant in the SH3TC2 gene leads to the retention of 122 nucleotides from intron 10 in the RNA sequence, causing a frameshift and an occurrence of a premature stop codon (NP_078853.2:p.Ala393GlyfsTer2).

Clinical and Functional Characteristics of the E92K CFTR Gene Variant in the Russian and Turkish Population of People with Cystic Fibrosis.

The pathogenic variant E92K (c.274G > A) of the CFTR gene is rare in America and Europe, but it is common for people with cystic fibrosis from Russia and Turkey. We studied the effect of the E92K genetic variant on the CFTR function. The function of the CFTR channel was studied using the intestinal current measurements (ICM) method. The effects of CFTR modulators on the restoration of the CFTR function were studied in the model of intestinal organoids. To assess the effect of E92K on pre-mRNA splicing, the RT-PCR products obtained from patients' intestinal organoid cultures were analyzed. Patients with the genetic variant E92K are characterized by an older age of diagnosis compared to homozygotes F508del and a high frequency of pancreatic sufficiency. The results of the sweat test and the ICM method showed partial preservation of the function of the CFTR channel. Functional analysis of CFTR gene expression revealed a weak effect of the E92K variant on mRNA-CFTR splicing. Lumacaftor (VX-809) has been shown to restore CFTR function in an intestinal organoid model, which allows us to consider the E92K variant as a promising target for therapy with CFTR correctors.

Annotation of uORFs in the OMIM genes allows to reveal pathogenic variants in 5'UTRs.

An increasing number of studies emphasize the role of non-coding variants in the development of hereditary diseases. However, the interpretation of such variants in clinical genetic testing still remains a critical challenge due to poor knowledge of their pathogenicity mechanisms. It was previously shown that variants in 5'-untranslated regions (5'UTRs) can lead to hereditary diseases due to disruption of upstream open reading frames (uORFs). Here, we performed a manual annotation of upstream translation initiation sites (TISs) in human disease-associated genes from the OMIM database and revealed ∼4.7 thousand of TISs related to uORFs. We compared our TISs with the previous studies and provided a list of 'high confidence' uORFs. Using a luciferase assay, we experimentally validated the translation of uORFs in the ETFDH, PAX9, MAST1, HTT, TTN,GLI2 and COL2A1 genes, as well as existence of N-terminal CDS extension in the ZIC2 gene. Besides, we created a tool to annotate the effects of genetic variants located in uORFs. We revealed the variants from the HGMD and ClinVar databases that disrupt uORFs and thereby could lead to Mendelian disorders. We also showed that the distribution of uORFs-affecting variants differs between pathogenic and population variants. Finally, drawing on manually curated data, we developed a machine-learning algorithm that allows us to predict the TISs in other human genes.

Splice-site variant in the RPS7 5'-UTR leads to a decrease in the mRNA level and development of Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by erythroid aplasia. Pathogenic variants in ribosomal protein (RP) genes, GATA1, TSR2, and EPO, are considered to be the etiology of DBA. Variants in 5'-untranslated regions (UTRs) of these genes are poorly studied and can complicate the variant interpretation. We investigated the functional consequences NM_001011.4:c.-19 + 1G > T variant in the donor splice-site of the RPS7 5'-UTR. This variant was found in a family where two sons with DBA were carriers. Father, who also had this variant, developed myelodysplastic syndrome, which caused his death. Search for candidate causal variants and copy number variations in DBA-associated genes left RPS7 variant as the best candidate. Trio whole exome sequencing analysis revealed no pathogenic variants in other genes. Functional analysis using luciferase expression system revealed that this variant leads to disruption of splicing. Also, a decrease in the levels of mRNA and protein expression was detected. In conclusion, the established consequences of 5'-UTR splice-site variant c.-19 + 1G > T in the RPS7 gene provide evidence that it is likely pathogenic.

Processed pseudogene insertion in GLB1 causes Morquio B disease by altering intronic splicing regulatory landscape.

Morquio B disease (MBD) is an ultra-rare lysosomal storage disease, which represents the relatively mild form of GLB1-associated disorders. In this article, we present the unique case of "pure" MBD associated with an insertion of the mobile genetic element from the class of retrotransposons. Using whole-genome sequencing (WGS), we identified an integration of the processed pseudogene NPM1 deep in the intron 5 of GLB1. The patient's mRNA analysis and the detailed functional analysis revealed the underlying molecular genetic mechanism of pathogenesis, which is an alteration of the GLB1 normal splicing. By co-expression of minigenes and antisense splice-modulating oligonucleotides (ASMOs), we demonstrated that pseudogene-derived splicing regulatory motifs contributed to an activation of the cryptic exon located 36 bp upstream of the integration site. Blocking the cryptic exon with ASMOs incorporated in the modified U7 small nuclear RNA (modU7snRNA) almost completely restored the wild-type splicing in the model cell line, that could be further extended toward the personalized genetic therapy. To our knowledge, this is the second reported case of the processed pseudogene insertion for monogenic disorders. Our data emphasizes the unique role of WGS in identification of such rare and probably underrepresented in literature types of disease-associated genetic variants.

A Founder Mutation in the POMC 5'-UTR Causes Proopiomelanocortin Deficiency Through Splicing-Mediated Decrease of mRNA.

CONTEXT: The syndrome of adrenal insufficiency, obesity, and red hair is a rare autosomal recessive disorder. The majority of disease-causing variants associated with the syndrome are located in the coding region of the POMC gene. OBJECTIVE: This work describes 7 unrelated patients who shared a novel homozygous mutation in the 5'-untranslated region (UTR) of the POMC gene and functionally characterize this novel variant. METHODS: Whole-exome sequencing (WES) with autozygosity mapping, Sanger sequencing, model expression system studies, and RNA sequencing were used for identification of the disease-causing variant and its subsequent functional characterization. Seven unrelated patients of the Perm Tatar ethnic group presented with hypoglycemia and excessive weight gain, low plasma adrenocorticotropin, and cortisol. Five of 7 children had red hair; 6 of 7 patients also showed signs of bronchial obstruction. RESULTS: WES showed shared autozygosity regions overlapping the POMC gene. Sanger sequencing of the POMC 5'-UTR detected a homozygous variant chr2:25391366C > T (hg19) at the splice donor site of intron 1. As demonstrated by the model expression system, the variant led to a significant decrease in the POMC messenger RNA level. Analyses of the patients' haplotypes were suggestive of the founder effect. We estimate that the mutation must have occurred at least 4.27 generations ago (95% CI, 0.86-7.67). CONCLUSION: This report presents a new molecular mechanism of POMC deficiency and contributes to the information on phenotypic variability in patients with this disorder.

Additive effect of frequent polymorphism and rare synonymous variant alters splicing in twin patients with Niemann-Pick disease type C.

Niemann-Pick disease type C (NP-C) (OMIM#257220) is a rare lysosomal storage disorder caused by pathogenic variants in either the NPC1 or NPC2 genes. It manifests with a wide spectrum of clinical symptoms and variable age of onset. We studied the impact of the frequent polymorphic variant c.2793 C > T (p.Asn931 = ), located in the donor splice site (SS) of NPC1 exon 18 on the penetrance of the rare synonymous variant c.2727 C > T (p.Cys909 = ), identified in two 55 y.o. twins with an adult onset form of NP-C. The patients' diagnosis was supported by biochemical analysis and positive filipin test. Analysis of the patients' cDNA showed that the c.2727 C > T variant leads to cryptic donor SS activation and frameshift deletion in the NPC1 exon 18. However, the minigene assay demonstrated that this exon shortening takes place only in the presence of the frequent polymorphic variant c.2793 C > T. Results of the transcript specific qPCR showed that only the presence in the NPC1 exon 18 of both variants leads to significant decrease of wild type (WT) transcript isoform.

Complex Transposon Insertion as a Novel Cause of Pompe Disease.

Pompe disease (OMIM#232300) is an autosomal recessive lysosomal storage disorder caused by mutations in the GAA gene. According to public mutation databases, more than 679 pathogenic variants have been described in GAA, none of which are associated with mobile genetic elements. In this article, we report a novel molecular genetic cause of Pompe disease, which could be hardly detected using routine molecular genetic analysis. Whole genome sequencing followed by comprehensive functional analysis allowed us to discover and characterize a complex mobile genetic element insertion deep in the intron 15 of the GAA gene in a patient with infantile onset Pompe disease.

Investigation of LINC00493/SMIM26 Gene Suggests Its Dual Functioning at mRNA and Protein Level.

The amount of human long noncoding RNA (lncRNA) genes is comparable to protein-coding; however, only a small number of lncRNAs are functionally annotated. Previously, it was shown that lncRNAs can participate in many key cellular processes, including regulation of gene expression at transcriptional and post-transcriptional levels. The lncRNA genes can contain small open reading frames (sORFs), and recent studies demonstrated that some of the resulting short proteins could play an important biological role. In the present study, we investigate the widely expressed lncRNA LINC00493. We determine the structure of the LINC00493 transcript, its cell localization and influence on cell physiology. Our data demonstrate that LINC00493 has an influence on cell viability in a cell-type-specific manner. Furthermore, it was recently shown that LINC00493 has a sORF that is translated into small protein SMIM26. The results of our knockdown and overexpression experiments suggest that both LINC00493/SMIM26 transcript and protein affect cell viability, but in the opposite manner.

Health Characteristics of Patients with Cystic Fibrosis whose Genotype Includes a Variant of the Nucleotide Sequence c.3140-16T>A and Functional Analysis of this Variant.

Cystic fibrosis (CF) is the most common monogenic autosomal recessive disease, associated with pathogenic variants in the CFTR gene. The splicing variant c.3140-16T>A (3272-16T>A) has been described previously and, according to the Russian CF Patients Registry, occurs with a frequency of 0.34%. The phenotypic features of CF patients with the c.3140-16T>A variant were compared with those of patients with the genotype F508del/F508del. Patients with the allele c.3140-16T>A had higher average age and age at diagnosis, and the allele was present in a greater proportion of adults. Patients carrying the c.3140-16T>A allele were characterised by better physical development indicators, both in adults and in children, had preserved pancreatic function, as well as the absence of a number of complications, and required pancreatic enzyme replacement therapy less often than patients with the F508del/F508del genotype. Sweat test values also were lower in patients with the c.3140-16T>A genotype. According to the results of clinical and laboratory studies, the phenotype of patients with the genetic variant c.3140-16T>A can be considered "mild". Functional CFTR protein activity in the presence of c.3140-16T>A was evaluated using intestinal current measurements (ICM) and the forskolin-induced swelling assay on organoids obtained from patients' rectal biopsies. c.3140-16T>A had high residual CFTR channel activity and was amenable to effective pharmacological correction with thea VX-770 potentiator. To evaluate the effect of the variant on CFTR pre-mRNA splicing we performed a minigene assay, as well as RT-PCR analysis of RNA isolated from the nasal epithelium and rectal biopsy of patients. We showed that the c.3140-16T>A variant creates a novel acceptor AG dinucleotide within CFTR intron 19, resulting in a 14-nucleotide extension of exon 20. This frameshift produces a premature termination codon and triggers mRNA degradation by the nonsense-mediated decay (NMD) mechanism. Moreover, we observed that the c.3140-16T>A allele could produce a residual amount of normally spliced transcript, thus explaining the patient's mild phenotype.

Upstream ORF frameshift variants in the PAX6 5'UTR cause congenital aniridia.

Congenital aniridia (AN) is a severe autosomal dominant panocular disorder associated with pathogenic variants in the PAX6 gene. Previously, we performed a molecular genetic study of a large cohort of Russian patients with AN and revealed four noncoding nucleotide variants in the PAX6 5'UTR. 14 additional PAX6-5'UTR variants were also reported in the literature, but the mechanism of their pathogenicity remained unclear. In the present study, we experimentally analyze five patient-derived PAX6 5'UTR-variants: four variants that we identified in Russian patients (c.-128-2delA, c.-125dupG, c.-122dupG, c.-118_-117del) and one previously reported (c.-52+5G>C). We show that the variants lead to a decrease in the protein translation efficiency, while mRNA expression level is not significantly reduced. Two of these variants also affect splicing. Furthermore, we predict and experimentally validate the presence of an evolutionarily conserved small uORF in the PAX6 5'UTR. All studied variants lead to the frameshift of the uORF, resulting in its extension. This extended out-of-frame uORF overlaps with the downstream CDS and thereby reduces its translation efficiency. We conclude that the uORF frameshift may be the main mechanism of pathogenicity for at least 15 out of 18 known PAX6 5'UTR variants. Moreover, we predict additional uORFs in the PAX6 5'UTR.

Functional Analysis of the PCCA and PCCB Gene Variants Predicted to Affect Splicing.

It is estimated that up to one-third of all variants causing inherited diseases affect splicing; however, their deleterious effects and roles in disease pathogenesis are often not fully characterized. Given their prevalence and the development of various antisense-based splice-modulating approaches, pathogenic splicing variants have become an important object of genomic medicine. To improve the accuracy of variant interpretation in public mutation repositories, we applied the minigene splicing assay to study the effects of 24 variants that were predicted to affect normal splicing in the genes associated with propionic acidemia (PA)-PCCA and PCCB. As a result, 13 variants (including one missense and two synonymous variants) demonstrated a significant alteration of splicing with the predicted deleterious effect at the protein level and were characterized as spliceogenic loss-of-function variants. The analysis of the available data for the studied variants and application of the American College of Medical Genetics and the Association for Molecular Pathology (ACMG/AMP) guidelines allowed us to precisely classify five of the variants and change the pathogenic status of nine. Using the example of the PA genes, we demonstrated the utility of the minigene splicing assay in the fast and effective assessment of the spliceogenic effect for identified variants and highlight the necessity of their standardized classification.

Mutation in PHACTR1 associated with multifocal epilepsy with infantile spasms and hypsarrhythmia.

A young boy with multifocal epilepsy with infantile spasms and hypsarrhythmia with minimal organic lesions of brain structures underwent DNA diagnosis using whole-exome sequencing. A heterozygous amino-acid substitution p.L519R in a PHACTR1 gene was identified. PHACTR1 belongs to a protein family of G-actin binding protein phosphatase 1 (PP1) cofactors and was not previously associated with a human disease. The missense single nucleotide variant in the proband was shown to occur de novo in the paternal allele. The mutation was shown in vitro to reduce the affinity of PHACTR1 for G-actin, and to increase its propensity to form complexes with the catalytic subunit of PP1. These properties are associated with altered subcellular localization of PHACTR1 and increased ability to induce cytoskeletal rearrangements. Although the molecular role of the PHACTR1 in neuronal excitability and differentiation remains to be defined, PHACTR1 has been previously shown to be involved in Slack channelopathy pathogenesis, consistent with our findings. We conclude that this activating mutation in PHACTR1 causes a severe type of sporadic multifocal epilepsy in the patient.

Recessive myotonia congenita caused by a homozygous splice site variant in CLCN1 gene: a case report.

BACKGROUND: Myotonia congenita is a rare neuromuscular disease, which is characterized by a delay in muscle relaxation after evoked or voluntary contraction. Myotonia congenita can be inherited in a dominant (Thomsen disease) and recessive form (Becker disease) and both are caused by pathogenic variants in the CLCN1 gene. Noncanonical splice site variants are often classified as variants of uncertain significance, due to insufficient accuracy of splice-predicting tools. Functional analysis using minigene plasmids is widely used in such cases. Moreover, functional analysis is very useful in investigation of the disease pathogenesis, which is necessary for development of future therapeutic approaches. To our knowledge only one noncanonical splice site variant in the CLCN1 gene was functionally characterized to date. We further contribute to this field by evaluation the molecular mechanism of splicing alteration caused by the c.1582 + 5G > A in a homozygous state. CASE PRESENTATION: We report a clinical case of an affected 6-y.o boy with athletic appearance due to muscle hypertrophy, calf muscle stiffness, cramping and various myotonic signs in a consanguineous family with no history of neuromuscular disorders. The neurological examination showed percussion-activated myotonia in the hands and legs. Plasma creatine kinase enzyme and transaminases levels were normal. Electromyography at the time of examination shows myotonic runs in the upper and lower extremities. CONCLUSIONS: Functional analysis of the variant in a minigene system showed alteration of splicing leading to loss of function, thereby confirming that the variant is pathogenic.

Various haploinsufficiency mechanisms in Pitt-Hopkins syndrome.

Pitt-Hopkins syndrome is a rare neurodevelopment disorder caused by haploinsufficiency of the transcription factor 4 (TCF4). The main clinical symptoms of Pitt-Hopkins syndrome are severe development delay, intellectual disability, characteristic facial phenotype, and breathing abnormalities, including episodic hyperventilation. Different pathogenic variants can lead to Pitt-Hopkins syndrome. The most common are large deletions at 18q21 encompassing the TCF4 gene and frameshifting/nonsense single nucleotide variants. However, variants in noncoding regions can also lead to Pitt-Hopkins syndrome by disrupting the normal pre-mRNA splicing machinery. Here we describe three patients with Pitt-Hopkins syndrome caused by a large deletion in chromosome 18, a nonsense variant, and a novel variant located in intron 11 of TCF4 c.922+5G > A. Using RT-PCR analysis and minigene splicing assay we showed that this intronic variant leads to exon 11 skipping resulting in a formation of a premature stop codon. To our knowledge, this is the first functional annotation of a splicing variant in Pitt-Hopkins syndrome.

Analysis of candidate genes expected to be essential for melanoma surviving.

INTRODUCTION: Cancers may be treated by selective targeting of the genes vital for their survival. A number of attempts have led to discovery of several genes essential for surviving of tumor cells of different types. In this work, we tried to analyze genes that were previously predicted to be essential for melanoma surviving. Here we present the results of transient siRNA-mediated knockdown of the four of such genes, namely, UNC45A, STK11IP, RHPN2 and ZNFX1, in melanoma cell line A375, then assayed the cells for their viability, proliferation and ability to migrate in vitro. In our study, the knockdown of the genes predicted as essential for melanoma survival does not lead to statistically significant changes in cell viability. On the other hand, for each of the studied genes, mobility assays showed that the knockdown of each of the target genes accelerates the speed of cells migrating. Possible explanation for such counterintuitive results may include insufficiency of the predicting computational models or the necessity of a multiplex knockdown of the genes. AIMS: To examine the hypothesis of essentiality of hypomutated genes for melanoma surviving we have performed knockdown of several genes in melanoma cell line and analyzed cell viability and their ability to migrate. METHODS: Knockdown was performed by siRNAs transfected by Metafectene PRO. The levels of mRNAs before and after knockdown were evaluated by RT-qPCR analysis. Cell viability and proliferation were assessed by MTT assay. Cell migration was assessed by wound healing assay. RESULTS: The knockdown of the genes predicted as essential for melanoma survival does not lead to statistically significant changes in cell viability. On the other hand, for each of the studied genes, mobility assays showed that the knockdown of each of the target genes accelerates the speed of cells migrating. CONCLUSION: Our results do not confirm initial hypothesis that the genes predicted essential for melanoma survival as a matter of fact support the survival of melanoma cells.

CTNS mRNA molecular analysis revealed a novel mutation in a child with infantile nephropathic cystinosis: a case report.

BACKGROUND: Cystinosis is an autosomal recessive lysosomal storage disorder characterized by accumulation of cystine in lysosomes throughout the body. Cystinosis is caused by mutations in the CTNS gene that encodes the lysosomal cystine carrier protein cystinosin. CTNS mutations result in either complete absence or reduced cystine transporting function of the protein. The diagnosis of nephropathic cystinosis is generally based on measuring leukocyte cystine level, demonstration of corneal cystine crystals by the slit lamp examination and confirmed by genetic analysis of the CTNS gene. CASE PRESENTATION: A boy born to consanguineous Caucasian parents had the characteristic clinical features of the infantile nephropathic cystinosis including renal Fanconi syndrome (polydipsia/polyuria, metabolic acidosis, hypokalemia, hypophosphatemia, low molecular weight proteinuria, glycosuria, cystine crystals in the cornea) and elevated WBC cystine levels. Initially we performed RFLP analysis of the common in the Northern European population 57-kb deletion of proband's DNA, then a direct Sanger sequencing which revealed no mutations in the coding part of the CTNS gene. To confirm the diagnosis we performed RT-PCR analysis of total RNA obtained from patient-derived fibroblasts in combination with cDNA sequencing. This revealed the skipping of exon 4 and exon 5 in the CTNS in our patient. Therefore, we detected a novel 9-kb homozygous deletion in the CTNS gene at genomic DNA level, spanning region from intron 3 to intron 5. In order to identify the inheritance pattern of the deletion we analyzed DNA of proband's mother and father. Both parents were found to be heterozygous carriers of the CTNS mutation. CONCLUSIONS: Analysis of CTNS gene transcript allowed to identify a large homozygous deletion in the patient with infantile nephropathic cystinosis. Mutational detection at RNA level may be an efficient tool to establish the genetic defect in some cystinosis patients.