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).

Functional investigation of SCN1A deep-intronic variants activating poison exons inclusion.

Dravet syndrome is a devastating epileptic syndrome characterized by intractable epilepsy with an early age of onset, regression of developmental milestones, ataxia, and motor deficits. Loss-of-function pathogenic variants in the SCN1A gene are found in the majority of patients with Dravet syndrome; however, a significant number of patients remain undiagnosed even after comprehensive genetic testing. Previously, it was shown that intronic elements in the SCN1A gene called poison exons can incorporate into SCN1A mRNA, leading to haploinsufficiency and potentially causing Dravet syndrome. Here, we developed a splicing reporter assay for all described poison exons of the SCN1A gene and validated it using previously reported and artificially introduced variants. Overall, we tested 18 deep-intronic single nucleotide variants and one complex allele in the SCN1A gene. Our approach is capable of evaluating the effect of both variants affecting cis-regulatory sequences and splice-site variants, with the potential to functionally annotate every possible variant within these elements. Moreover, using antisense-modified uridine-rich U7 small nuclear RNAs, we were able to block poison exon incorporation in mutant constructs, an approach that could be used as a promising therapeutic intervention in Dravet syndrome patients with deep-intronic variants.

Clinical and Genetic Characteristics of Calvarial Doughnut Lesions with Bone Fragility in Three Families with a Reccurent SGMS2 Gene Variant.

Calvarial doughnut lesions (CDL) with bone fragility with or without spondylometaphyseal dysplasia (MIM: #126550) is a rare autosomal dominant skeletal disorder characterized by low bone mineral density, spinal and peripheral fractures, and specific sclerotic lesions of the cranial bones. In the current classification of skeletal disorders, the disease is included in the group of bone fragility disorders along with osteogenesis imperfecta. The disease is caused by pathogenic variants in the SGMS2 gene, the protein product of which is sphingomyelin synthase 2, which primarily contributes to sphingomyelin (SM) synthesis-the main lipid component of the plasma membrane essential for bone mineralization. To date, 15 patients from eight families with CDL with bone fragility have been described in the literature, and a recurrent variant c.148C>T (p.Arg50Ter) in the SGMS2 gene has been identified, which was found in patients from six families. We diagnosed the disease in 11 more patients from three unrelated families, caused by the same heterozygous nonsense variant c.148C>T (p.Arg50Ter) in the SGMS2 gene. Our results show wide interfamilial and intrafamilial phenotypic variability in patients with a detected recurrent variant in the SGMS2 gene, the presence of which must be taken into consideration in the diagnosis of the disease. The primary analysis of this variant will contribute to optimal molecular genetic diagnostics, which can reduce diagnostic costs and time.

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.

Variable clinical presentation of split hand/foot malformation syndrome in a family with microduplication of 10q24.32: a case report.

SHFM (Split Hand/Foot Malformation) is a heterogeneous group of disorders characterized by the presence of clefts in the hands and feet, along with syndactyly of the digits. In this article, we describe a family in which two members exhibit characteristic developmental abnormalities associated with SHFM, presenting with variable clinical features. Using whole-genome sequencing, we identified a microduplication of a chromosomal segment on locus 10q24.32, specifically spanning positions 102934495 to 103496555, encompassing genes BTRC, POLL, FBXW4 and LBX1 in the proband. Genomic duplications, including these genes, were previously described in patients diagnosed with the third type of SHFM. We validated the presence of this structural rearrangement in 7 family members, including the proband and the proband's father. Remarkably, further investigation demonstrated that the detected duplication exhibits a mosaic state in the phenotypically normal paternal grandmother of the proband, thereby providing a plausible explanation for the absence of a pathological phenotype in her.

Hyperammonemia in Russia Due to Carbonic Anhydrase VA Deficiency Caused by Homozygous Mutation p.Lys185Lys (c.555G>A) of the CA5A Gene

Hyperammonemia due to carbonic anhydrase VA deficiency (OMIM# 615751) is a rare, life-threatening hereditary disease caused by biallelic mutations in the CA5A gene, presenting as encephalopathic hyperammonemia of unexplained origin during the neonatal period and infancy. Here, we present a detailed description of a 5-year-old patient with the homozygous mutation p.Lys185Lys (c.555G>A) in the CA5A gene. This variant was previously described by van Karnebeek et al. in 2014 in a boy of Russian origin. We found a high frequency of carriers of this mutation in Russia; 1:213, which is 7 times higher than the expected frequency calculated based on data on Western European populations. Thus, targeted testing for the mutation p.Lys185Lys (c.555G>A) in the CA5A gene should be useful for early detection by selective screening in neonatal intensive care units.

Case report: Unusual episodic myopathy in a patient with novel homozygous deletion of first coding exon of MICU1 gene.

We present a patient with unusual episodes of muscular weakness due to homozygous deletion of exon 2 in the MICU1 gene. Forty-three patients from 33 families were previously described with homozygous and compound heterozygous, predominantly loss of function (LoF) variants in the MICU1 gene that lead to autosomal recessive myopathy with extrapyramidal signs. Most described patients developed muscle weakness and elevated CK levels, and half of the patients had progressive extrapyramidal signs and learning disabilities. Our patient had a few severe acute episodes of muscle weakness with minimal myopathy features between episodes and a strongly elevated Creatinine Kinase (CK). Whole exome sequencing (WES) was performed and the homozygous deletion of exon 2 was suspected. To validate the diagnosis, we performed an RNA analysis of all family members. To investigate the possible impact of this deletion on the phenotype, we predicted a new Kozak sequence in exon 4 that could lead to the formation of a truncated MICU1 protein that could partly interact with MCU protein in a mitochondrial Ca2+ complex. We suspect that this unusual phenotype of the proband with MICU1-related myopathy could be explained by the presence of the truncated but partly functional protein. This work helps to define the clinical polymorphism of MICU1 deficiency better.

Meier-Gorlin Syndrome: Clinical Misdiagnosis, Genetic Testing and Functional Analysis of ORC6 Mutations and the Development of a Prenatal Test.

Meier−Gorlin syndrome (MGS) is a rare genetic developmental disorder that causes primordial proportional dwarfism, microtia, the absence of or hypoplastic patellae and other skeletal anomalies. Skeletal symptoms overlapping with other syndromes make MGS difficult to diagnose clinically. We describe a 3-year-old boy with short stature, recurrent respiratory infections, short-rib dysplasia, tower head and facial dysmorphisms who was admitted to the Tomsk Genetic Clinic to verify a clinical diagnosis of Jeune syndrome. Clinical exome sequencing revealed two variants (compound heterozygosity) in the ORC6 gene: c.2T>C(p.Met1Thr) and c.449+5G>A. In silico analysis showed the pathogenicity of these two mutations and predicted a decrease in donor splicing site strength for c.449+5G>A. An in vitro minigene assay indicated that variant c.449+5G>A causes complete skipping of exon 4 in the ORC6 gene. The parents requested urgent prenatal testing for MGS for the next pregnancy, but it ended in a miscarriage. Our results may help prevent MGS misdiagnosis in the future. We also performed in silico and functional analyses of ORC6 mutations and developed a restriction fragment length polymorphism and haplotype-based short-tandem-repeat assay for prenatal genetic testing for MGS. These findings should elucidate MGS etiology and improve the quality of genetic counselling for affected families.

Congenital myopathy as a new phenotype caused by two undescribed variants in ASCC1 gene.

We present a patient with congenital myopathy and an inborn epiphysiolysis of the ulna. Whole-exome sequencing analysis revealed two novel mutations in Activation Signal Cointegrator Complex 1 (ASCC1) gene in a compound heterozygous state-a splicing variant c.395-2A>G and a deletion of the first two coding exons. Homozygous and compound heterozygous LoF variants in ASCC1 gene lead to a severe phenotype of spinal muscular atrophy with congenital bone fractures 2 (SMABF2). All patients described to date presented with a severe muscular hypotony, inborn fractures, and passed away shortly after birth while our proband had moderate hypotony, no fractures, but epiphysiolysis and he was 3.5 years old at the time of examination. To explain the phenotype of our patient, we performed an RNA analysis of all family members. We discovered that the c.395-2A>G variant results in two aberrant mRNA isoforms. We also validated the deletion of two exons in ASCC1 gene that lead to the increased expression of this truncated transcript by 1.8 times. To investigate the possible impact of this deletion on the phenotype we predicted a new Kozak sequence in exon 4 that could lead to the formation of a truncated protein with shortened KH domain and a full RNA ligase-like domain. We suggest that this unexpectedly different phenotype of the proband with ASCC1-related disorder could be explained by the presence of the truncated protein with an increased expression.

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.

Case Report: Phenotype-Driven Diagnosis of Atypical Dravet-Like Syndrome Caused by a Novel Splicing Variant in the SCN2A Gene.

Febrile-associated epileptic encephalopathy is a large genetically heterogeneous group that is associated with pathogenic variants in SCN1A, PCDH19, SCN2A, SCN8A, and other genes. The disease onset ranges from neonatal or early-onset epileptic encephalopathy to late-onset epilepsy after 18 months. Some etiology-specific epileptic encephalopathies have target therapy which can serve as a clue for the correct genetic diagnosis. We present genetic, clinical, electroencephalographic, and behavioral features of a 4-year-old girl with epileptic encephalopathy related to a de novo intronic variant in the SCN2A gene. Initial NGS analysis revealed a frameshift variant in the KDM6A gene and a previously reported missense variant in SCN1A. Due to lack of typical clinical signs of Kabuki syndrome, we performed X-chromosome inactivation that revealed nearly complete skewed inactivation. Segregation analysis showed that the SCN1A variant was inherited from a healthy father. The proband had resistance to multiple antiseizure medications but responded well to sodium channel inhibitor Carbamazepine. Reanalysis of NGS data by a neurogeneticist revealed a previously uncharacterized heterozygous variant c.1035-7A>G in the SCN2A gene. Minigene assay showed that the c.1035-7A>G variant activates a cryptic intronic acceptor site which leads to 6-nucleotide extension of exon 9 (NP_066287.2:p.(Gly345_Gln346insTyrSer). SCN2A encephalopathy is a recognizable severe phenotype. Its electro-clinical and treatment response features can serve as a hallmark. In such a patient, reanalysis of genetic data is strongly recommended in case of negative or conflicting results of DNA analysis.

mRNA analysis revealed a novel pathogenic EIF2S3 variant causing MEHMO syndrome.

EIF2S3 pathogenic variants have been shown to cause MEHMO syndrome - a rare X-linked intellectual disability syndrome. In most cases, DNA diagnostics of MEHMO syndrome is performed using exome sequencing. We describe two cousins with profound intellectual disability, severe microcephaly, microgenitalism, hypoglycemia, epileptic seizures, and hypertrichosis, whose clinical symptoms allowed us to suspect MEHMO syndrome. To confirm this diagnosis, we designed an mRNA analysis for the EIF2S3 gene. It is a cost-effective method to detect coding sequence variants in multi-exonic genes, as well as splicing defects and allelic imbalance. Our mRNA sequence analysis revealed a novel EIF2S3 variant c.820C>G in both cousins. We also found the same variant in female family members in the heterozygous state. To investigate the pathogenicity of the c.820C>G variant, we performed expression analysis, which showed that the DDIT3 transcript level was significantly increased in the patient relative to the controls. We, thus, demonstrate that mRNA analysis is an efficient tool for performing genetic testing in patients with distinct phenotypic features.

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.

Case Report: Functional Investigation of an Undescribed Missense Variant Affecting Splicing in a Patient With Dravet Syndrome.

Pathogenic variants in the SCN1A gene are associated with a spectrum of epileptic disorders ranging in severity from familial febrile seizures to Dravet syndrome. Large proportions of reported pathogenic variants in SCN1A are annotated as missense variants and are often classified as variants of uncertain significance when no functional data are available. Although loss-of-function variants are associated with a more severe phenotype in SCN1A, the molecular mechanism of single nucleotide variants is often not clear, and genotype-phenotype correlations in SCN1A-related epilepsy remain uncertain. Coding variants can affect splicing by creating novel cryptic splicing sites in exons or by disrupting exonic cis-regulation elements crucial for proper pre-mRNA splicing. Here, we report a novel case of Dravet syndrome caused by an undescribed missense variant, c.4852G>A (p.(Gly1618Ser)). By midigene splicing assay, we demonstrated that the identified variant is in fact splice-affecting. To our knowledge, this is the first report on the functional investigation of a missense variant affecting splicing in Dravet syndrome.