Minigene Constructs Research Articles

Evaluation of Suspected Autosomal Alport Syndrome Synonymous Variants.

Alport syndrome is an inherited disorder characterized by progressive renal disease, variable sensorineural hearing loss, and ocular abnormalities. Although many pathogenic variants in COL4A3 and COL4A4 have been identified in patients with autosomal Alport syndrome, synonymous mutations in these genes have rarely been identified. We conducted in silico splicing analysis using Human Splicing Finder (HSF) and Alamut to predict splicing domain strength and disruption of the sites. Furthermore, we performed in vitro splicing assays using minigene constructs and mRNA analysis of patient samples to determine the pathogenicity of four synonymous variants detected in four patients with suspected autosomal dominant Alport syndrome (COL4A3 [c.693G>A (p.Val231=)] and COL4A4 [c.1353C>T (p.Gly451=), c.735G>A (p.Pro245=), and c.870G>A (p.Lys290=)]). Both in vivo and in vitro splicing assays showed exon skipping in two out of the four synonymous variants identified (c.735G>A and c.870G>A in COL4A4). Prediction analysis of wild-type and mutated COL4A4 sequences using HSF and Alamut suggested these two variants may lead to the loss of binding sites for several splicing factors, e.g., in acceptor sites and exonic splicing enhancers. The other two variants did not induce aberrant splicing. This study highlights the pitfalls of classifying the functional consequences of variants by a simple approach. Certain synonymous variants, although they do not alter the amino acid sequence of the encoded protein, can dramatically affect pre-mRNA splicing, as shown in two of our patients. Our findings indicate that transcript analysis should be carried out to evaluate synonymous variants detected in patients with autosomal dominant Alport syndrome.

Non-canonical splice junction processing increases the diversity of RBFOX2 splicing isoforms.

The underlying mechanisms of splicing regulation through non-canonical splice junction processing remain largely unknown. Here, we identified two RBFOX2 splicing isoforms by alternative 3' splice site selection of exon 9; the non-canonical splice junction processed RBFOX2 transcript (RBFOX2-N.C.) was expressed by the selection of the 3' splice GG acceptor sequence. The cytoplasmic localization of RBFOX2-C., a canonical splice junction-processed RBFOX2 transcript, was different from that of RBFOX2-N.C., which showed nuclear localization. In addition, we confirmed that RBFOX2-C. showed a significantly stronger localization into stress granules than RBFOX2-N.C. upon sodium arsenite treatment. Next, we investigated the importance of non-canonical 3' splice GG sequence selection of specific cis-regulatory elements using minigene constructs of the RBFOX2 gene. We found that the non-canonical 3' splice GG sequence and suboptimal branch point site adjacent region were critical for RBFOX2-N.C. expression through a non-canonical 3' splice selection. Our results suggest a regulatory mechanism for the non-canonical 3' splice selection in the RBFOX2 gene, providing a basis for studies related to the regulation of alternative pre-mRNA splicing through non-canonical splice junction processing.

Massively parallel reporter assays discover de novo exonic splicing mutants in paralogs of Autism genes.

To determine the contribution of defective splicing in Autism Spectrum Disorders (ASD), the most common neurodevelopmental disorder, a high throughput Massively Parallel Splicing Assay (MaPSY) was employed and identified 42 exonic splicing mutants out of 725 coding de novo variants discovered in the sequencing of ASD families. A redesign of the minigene constructs in MaPSY revealed that upstream exons with strong 5’ splice sites increase the magnitude of skipping phenotypes observed in downstream exons. Select hits were validated by RT-PCR and amplicon sequencing in patient cell lines. Exonic splicing mutants were enriched in probands relative to unaffected siblings -especially synonymous variants (7.5% vs 3.5%, respectively). Of the 26 genes disrupted by exonic splicing mutations, 6 were in known ASD genes and 3 were in paralogs of known ASD genes. Of particular interest was a synonymous variant in TNRC6C - an ASD gene paralog with interactions with other ASD genes. Clinical records of 3 ASD patients with TNRC6C variant revealed respiratory issues consistent with phenotypes observed in TNRC6 depleted mice. Overall, this study highlights the need for splicing analysis in determining variant pathogenicity, especially as it relates to ASD.

Splicing Outcomes of 5' Splice Site GT>GC Variants That Generate Wild-Type Transcripts Differ Significantly Between Full-Length and Minigene Splicing Assays.

Combining data derived from a meta-analysis of human disease-associated 5′ splice site GT>GC (i.e., +2T>C) variants and a cell culture-based full-length gene splicing assay (FLGSA) of forward engineered +2T>C substitutions, we recently estimated that ∼15–18% of +2T>C variants can generate up to 84% wild-type transcripts relative to their wild-type counterparts. Herein, we analyzed the splicing outcomes of 20 +2T>C variants that generate some wild-type transcripts in two minigene assays. We found a high discordance rate in terms of the generation of wild-type transcripts, not only between FLGSA and the minigene assays but also between the different minigene assays. In the pET01 context, all 20 wild-type minigene constructs generated the expected wild-type transcripts; of the 20 corresponding variant minigene constructs, 14 (70%) generated wild-type transcripts. In the pSPL3 context, only 18 of the 20 wild-type minigene constructs generated the expected wild-type transcripts whereas 8 of the 18 (44%) corresponding variant minigene constructs generated wild-type transcripts. Thus, in the context of a particular type of variant, we raise awareness of the limitations of minigene splicing assays and emphasize the importance of sequence context in regulating splicing. Whether or not our findings apply to other types of splice-altering variant remains to be investigated.

A homozygous duplication of the FGG exon 8-intron 8 junction causes congenital afibrinogenemia. Lessons learned from the study of a large consanguineous Turkish family.

Congenital afibrinogenemia is the most severe congenital fibrinogen disorder, characterized by undetectable fibrinogen in circulation. Causative mutations can be divided into two main classes: null mutations with no protein production at all and missense mutations producing abnormal protein chains that are retained inside the cell. The vast majority of cases are due to single base pair mutations or small insertions or deletions in the coding regions or intron-exon junctions of FGB, FGA and FGG. Only a few large rearrangements have been described, all deletions involving FGA. Here we report the characterization of a 403 bp duplication of the FGG exon 8-intron 8 junction accounting for congenital afibrinogenemia in a large consanguineous family from Turkey. This mutation, which had escaped detection by Sanger sequencing of short polymerase chain reaction (PCR) amplicons of coding sequences and splice sites, was identified by studying multiple alignments of reads obtained from whole exome sequencing of a heterozygous individual followed by PCR amplification and sequencing of a larger portion of FGG. Because the mutation duplicates the donor splice site of intron 8, we predicted that the impact of the mutation would be on FGG transcript splicing. Analysis of mRNA produced by cells transiently transfected with normal or mutant minigene constructs showed that the duplication causes production of several aberrant FGG transcripts generating premature truncating codons.

A Missense Mutation rs781536408 (c.2395G>A) of TYK2 Affects Splicing and Causes Skipping of Exon18 in vivo.

TYK2 variants can impact disease onset or progression. In our previous study, we identified abnormal splicing that happened near rs781536408 in the TYK2 gene. The purpose of this research was to examine the effect of the mutation on alternative splicing in vivo and in vitro. Whole exome sequencing was performed to identify the mutations followed by bidirectional Sanger sequencing. Then the minigene analysis was carried out based on HeLa and HEK293T cell lines. The results showed that rs781536408 (c.2395G>A, p.G799R) was homozygous in the patient, but heterozygous in parents. PCR amplification confirmed the abnormal splicing in the somatic cells of the patients, but not in the parents. Sanger sequencing results showed that there was a skipping of exon18 near the mutation. For minigene analysis, there was no difference between the wild-type and the mutant type in the two minigene construction strategies, indicating that mutation c.2395G>A had no effect on splicing in vitro. Combining the results of in vivo, we speculated that the effect of the mutation on splicing was not absolute, but rather in degree.

A drug-repositioning screen using splicing-sensitive fluorescent reporters identifies novel modulators of VEGF-A splicing with anti-angiogenic properties

Alternative splicing of the vascular endothelial growth factor A (VEGF-A) terminal exon generates two protein families with differing functions. Pro-angiogenic VEGF-Axxxa isoforms are produced via selection of the proximal 3′ splice site of the terminal exon. Use of an alternative distal splice site generates the anti-angiogenic VEGF-Axxxb proteins. A bichromatic splicing-sensitive reporter was designed to mimic VEGF-A alternative splicing and was used as a molecular tool to further investigate this alternative splicing event. Part of VEGF-A’s terminal exon and preceding intron were inserted into a minigene construct followed by the coding sequences for two fluorescent proteins. A different fluorescent protein is expressed depending on which 3′ splice site of the exon is used during splicing (dsRED denotes VEGF-Axxxa and EGFP denotes VEGF-Axxxb). The fluorescent output can be used to follow splicing decisions in vitro and in vivo. Following successful reporter validation in different cell lines and altering splicing using known modulators, a screen was performed using the LOPAC library of small molecules. Alterations to reporter splicing were measured using a fluorescent plate reader to detect dsRED and EGFP expression. Compounds of interest were further validated using flow cytometry and assessed for effects on endogenous VEGF-A alternative splicing at the mRNA and protein level. Ex vivo and in vitro angiogenesis assays were used to demonstrate the anti-angiogenic effect of the compounds. Furthermore, anti-angiogenic activity was investigated in a Matrigel in vivo model. To conclude, we have identified a set of compounds that have anti-angiogenic activity through modulation of VEGF-A terminal exon splicing.

Investigating pY240‐PTEN:Ki‐67 Interaction and its Role in DNA Damage Response

Glioblastoma (GBM) is the most aggressive and common malignant primary brain tumor. Standard of care for GBM patients involves chemotherapy and ionizing radiation which induce DNA damage to kill tumor cells; however resistance to these treatment modalities commonly develop through various mechanisms. One novel mechanism of radioresistance identified in the Furnari lab is through enhanced homologous recombination DNA damage response mediated by nuclear-localized tyrosine-phosphorylated PTEN (pY240-PTEN) recruited to chromatin through interaction with the PP1 Binding Domain (PP1BD) of Ki-67. The objective of this study is to characterize the regulation and molecular interactions involved in pY240-PTEN and Ki-67 binding. Biotin-tagged synthetic peptides based on the Ki-67 PP1BD are being used in streptavidin pull down experiments to interrogate the critical amino acids required for pY240-PTEN:Ki-67 interaction. Orthogonally, a minigene expressing the Ki-67 PP1BD and Leucine-Rich Repeat domain (required for tethering to chromatin) fused to a C-terminal mNeonGreen is employed in glioma stem-like cell lines to interrogate the impact of specific amino acid mutations on PTEN:Ki-67 interaction and subsequent disruption of pY240-PTEN mediated DNA damage response. Since phosphorylation of the PP1BD has been shown to affect binding of Ki-67 to other phosphatases, namely PP1γ, the peptides and minigene construct will additionally be used to investigate how phosphorylation of the PP1BD can affect PTEN:Ki-67 interactions. Specifically, phosphorylation of the Ki-67 PP1BD by Aurora B Kinase and Cyclin-Dependent Kinase 1 will be investigated through phosphorylated synthetic peptides and minigene constructs harboring mutations of candidate residues to non-phosphorylatable alanine, or phosphomimetic glutamic acid or aspartic acid. The insight into this interaction will be leveraged in future studies to evaluate competitor peptides based on Ki-67 for their ability to disrupt this binding and sensitize cells to radiation, as well as to guide CRISPR/Cas9 Ki-67 knockin mutations to determine the effects of Ki-67 mutation in vivo.

A cell culture model/mimic to investigate the biochemical consequences of cdkal1 mutations that result in unprocessed, non‐functional insulin and Type 2 Diabetes Mellitus

The primary objective of the project is to develop a cell culture model that mimics the pancreatic beta islets to study the biochemical consequences of single nucleotide polymorphisms (SNPs) found in the intron-5 of cdkal1 gene which codes for a tRNA modification enzyme. SNPs in cdkal1 gene has been pointed out for its role in the development of Type 2 Diabetes Mellitus (T2DM) from past few years by human Genome Wide Association Studies. Still we lack a model system to investigate the consequences of cdkal1 mutations that result in unprocessed, non-functional insulin leading to the development of T2DM. Hypothesis Incorrectly processed cdkal1 mRNA results in untranslated or incorrectly translated Cdkal1 protein. Cdkal1 protein is a thiomethyl transferase that adds an ms2 moiety to the already modified N6-threonyl carbamoyl adenosine (t6A37) in the 37 position of tRNA. Improperly processed Cdkal1 protein will not be able to modify tRNALys3. Proinsulin will not be correctly translated and processed resulting in little or no insulin secreted from the β- cells in pancreatic islets. To uncover the pathogenesis of the human cdkal1-SNP associated T2DM, the current project will produce a cell culture model of the cdkal1 gene SNP-associated human T2DM. With the model, we will be able to discover the biochemical properties of the insulin, if translated at all. In a cdkal1-knockout (KO) cells, we will rescue the insulin production with WT human cdkal1, and then discover the effects of intron-5 SNPs of the cdkal1 gene. SNPs could affect mRNA processing. Methods and preliminary results We have successfully knocked down the cdkal1 gene in a murine β-cell line (NIT1) inducible for insulin. Our recent knockdown studies in NIT1 cell lines showed that under KCl insulin induction, unprocessed insulin mRNA increases by ~40% relative to mature insulin mRNA (Fig. B) and cdkal1 mRNA is reduced by ~40%. Subsequent reduction in tRNA ms2 modification and insulin production was also observed upon cdkal1 knockdown. As 100% gene knockout is required to study the effects of SNPs, we now have a murine β-cell line (MIN-cdkal1-null); the parent of that cell line (MIN-cdkal1-het) produces insulin assessed with a glucose- stimulated insulin release (GSIR) assay. The MIN-cdkal1-null having a cdkal1 knockout will be an advantageous tool to evaluate the state of insulin production when Cdkal1 is not modifying tRNALys3. A variant of mini-gene reporter constructs carrying potential SNPs have been designed and will be expressed and analyzed in knockout cell lines to evaluate cdkal1 mRNA processing and splicing events. We are positioned to answer important questions that connect Cdkal1 protein biochemistry to T2DM like, are the human intronic SNPs of the cdkal1 gene cause incorrect processing of cdkal1 mRNA? What is the status of the insulin produced, if at all translated - is proinsulin unprocessed, aggregated or degraded and not secreted? In the long-term, the results of this study will stimulate new insights into the human health implications of tRNA modification enzymes and the role(s) of modifications not just in the pathogenesis of cdkal1 SNP-associated T2DM, but also in a variety of human diseases.

Congenital ataxia due to novel variant in ATP8A2.

Congenital ataxias are a heterogeneous group of disorders characterized by congenital or early-onset ataxia. Here, we describe two siblings with congenital ataxia, who acquired independent gait by age 4 years. After 16 years of follow-up they presented near normal cognition, cerebellar ataxia, mild pyramidal signs, and dystonia. On exome sequencing, a novel homozygous variant (c.1580-18C > G - intron 17) in ATP8A2 was identified. A new acceptor splice site was predicted by bioinformatics tools, and functionally characterized through a minigene assay. Minigene constructs were generated by PCR-amplification of genomic sequences surrounding the variant of interest and cloning into the pCMVdi vector. Altered splicing was evaluated by expressing these constructs in HEK293T cells. The construct with the c.1580-18C > G homozygous variant produced an aberrant transcript, leading to retention of 17 bp of intron 17, by the use of an alternative acceptor splice site, resulting in a premature stop codon by insertion of four amino acids. These results allowed us to establish this as a disease-causing variant and expand ATP8A2-related disorders to include less severe forms of congenital ataxia.

Targeting novel human transient receptor potential ankyrin 1 splice variation with splice-switching antisense oligonucleotides.

Activation of transient receptor potential ankyrin 1 (TRPA1) channels by both environmental irritants and endogenous inflammatory mediators leads to excitation of the nerve endings, resulting in acute sensation of pain, itch, or chronic neurogenic inflammation. As such, TRPA1 channels are actively pursued as therapeutic targets for various pathological nociception and pain disorders. We uncovered that exon 27 of human TRPA1 (hTRPA1) could be alternatively spliced into hTRPA1_27A and hTRPA1_27B splice variants. The resulting channel variants displayed reduced expression, weakened affinity to interact with WT, and suffered from complete loss of function because of disruption of the C-terminal coiled-coil domain. Using a human minigene construct, we revealed that binding of splicing factor serine/arginine-rich splicing factor 1 (SRSF1) to the exonic splicing enhancer was critical for the inclusion of intact exon 27. Knockdown of SRSF1, mutation within exonic splicing enhancer, or masking SRSF1 binding with antisense oligonucleotides promoted alternative splicing within exon 27. Finally, antisense oligonucleotides-induced alternative splicing produced transcript and protein variants that could be functionally determined as diminished endogenous TRPA1 activity in human Schwann cell-line SNF96.2 and hiPSCs-derived sensory neurons. The outcome of the work could potentially offer a novel therapeutic strategy for treating pain by targeting alternative splicing of hTRPA1.

Generation of neoantigen-specific T cells for adoptive cell transfer for treating head and neck squamous cell carcinoma

ABSTRACT Adoptive cell therapy using TCR-engineered T cells (TCR-T cells) represents a promising strategy for treating relapsed and metastatic cancers. We previously established methods to identify neoantigen-specific TCRs based on patients’ PBMCs. However, in clinical practice isolation of PBMCs from advanced-stage cancer patients proves to be difficult. In this study, we substituted blood-derived T cells for tumor-infiltrating lymphocytes (TILs) and used an HLA-matched cell line of antigen-presenting cells (APCs) to replace autologous dendritic cells. Somatic mutations were determined in head and neck squamous cell carcinoma resected from two patients. HLA-A*02:01-restricted neoantigen libraries were constructed and transferred into HLA-matched APCs for stimulation of patient TILs. TCRs were isolated from reactive TIL cultures and functionality was tested using TCR- T cells in vitro and in vivo. To exemplify the screening approach, we identified the targeted neoantigen leading to recognition of the minigene construct that stimulated the strongest TIL response. Neoantigen peptides were used to load MHC-tetramers for T cell isolation and a TCR was identified targeting the KIAA1429D1358E mutation. TCR-T cells were activated, exhibited cytotoxicity, and secreted cytokines in a dose-dependent manner, and only when stimulated with the mutant peptide. Furthermore, comparable to a neoantigen-specific TCR that was isolated from the patient’s PBMCs, KIAA1429D1358E-specific TCR T cells destroyed human tumors in mice. The established protocol provides the required flexibility to methods striving to identify neoantigen-specific TCRs. By using an MHC-matched APC cell line and neoantigen-encoding minigene libraries, autologous TILs can be stimulated and screened when patient PBMCs and/or tumor material are not available anymore. Abbreviations: Head and neck squamous cell carcinoma (HNSCC); adoptive T cell therapy (ACT); T cell receptor (TCR); tumor-infiltrating lymphocytes (TIL); cytotoxic T lymphocyte (CTL); peripheral blood mononuclear cell (PBMC); dendritic cell (DC); antigen-presenting cells (APC)

Reversion mutation of cDNA CA8-204 minigene construct produces a truncated functional peptide that regulates calcium release in vitro and produces profound analgesia in vivo.

Intracellular calcium is critical in orchestrating neuronal excitability and analgesia. Carbonic anhydrase-8 (CA8) regulates intracellular calcium signaling through allosteric inhibition of neuronal inositol trisphosphate receptor 1 (ITPR1) to produce profound analgesia. Recently, we reported the "G" allele at rs6471859 represents cis-eQTL regulating alternative splicing of a 1697bp transcript (CA8-204G) with a retained intron, alternative polyadenylation site and a new stop codon producing a functional 26kDa peptide with an extended exon 3. In this study we show the reversion mutation (G to C) at rs6471859 within the CA8-204G expression vector also produced a stable 1697bp transcript (CA8-204C) coding for a smaller peptide (~ 22kDa) containing only the first three CA8 exons. Surprisingly, this peptide inhibited ITPR1 (pITPR1) activation, ITPR1-mediated calcium release in vitro; and produced profound analgesia in vivo. This is the first report showing CA8-204C codes for a functional peptide sufficient to regulate calcium signaling and produce profound analgesia.

Phenotype Driven Analysis of Whole Genome Sequencing Identifies Deep Intronic Variants that Cause Retinal Dystrophies by Aberrant Exonization.

PurposeTo demonstrate the effectiveness of combining retinal phenotyping and focused variant filtering from genome sequencing (GS) in identifying deep intronic disease causing variants in inherited retinal dystrophies.MethodsAffected members from three pedigrees with classical enhanced S-cone syndrome (ESCS; Pedigree 1), congenital stationary night blindness (CSNB; Pedigree 2), and achromatopsia (ACHM; Pedigree 3), respectively, underwent detailed ophthalmologic evaluation, optical coherence tomography, and electroretinography. The probands underwent panel-based genetic testing followed by GS analysis. Minigene constructs (NR2E3, GPR179 and CNGB3) and patient-derived cDNA experiments (NR2E3 and GPR179) were performed to assess the functional effect of the deep intronic variants.ResultsThe electrophysiological findings confirmed the clinical diagnosis of ESCS, CSNB, and ACHM in the respective pedigrees. Panel-based testing revealed heterozygous pathogenic variants in NR2E3 (NM_014249.3; c.119-2A>C; Pedigree 1) and CNGB3 (NM_019098.4; c.1148delC/p.Thr383Ilefs*13; Pedigree 3). The GS revealed heterozygous deep intronic variants in Pedigrees 1 (NR2E3; c.1100+1124G>A) and 3 (CNGB3; c.852+4751A>T), and a homozygous GPR179 variant in Pedigree 2 (NM_001004334.3; c.903+343G>A). The identified variants segregated with the phenotype in all pedigrees. All deep intronic variants were predicted to generate a splice acceptor gain causing aberrant exonization in NR2E3 [89 base pairs (bp)], GPR179 (197 bp), and CNGB3 (73 bp); splicing defects were validated through patient-derived cDNA experiments and/or minigene constructs and rescued by antisense oligonucleotide treatment.ConclusionsDeep intronic mutations contribute to missing heritability in retinal dystrophies. Combining results from phenotype-directed gene panel testing, GS, and in silico splice prediction tools can help identify these difficult-to-detect pathogenic deep intronic variants.

Preparation of a Functional Rat LDL Receptor Minigene

The majority of the low-density lipoprotein (LDL) receptors present in the body are expressed in the liver. Therefore, plasma LDL levels significantly correlate with changes in the activity of the hepatic LDL receptor. Based on this, there is a need to understand the regulatory mechanisms that control the hepatic expression of the low-density lipoprotein (LDL) receptor. Herein, we have prepared a functional rat LDL receptor minigene construct that can produce mRNA after splicing. Sequence analysis suggests that this construct has the potential to code for a truncated version of LDL receptor protein. This minigene could be used as a research tool to identify small molecules, natural products, and regulators of the LDL receptor gene that could be developed into LDL receptor-specific activators for therapeutic use.

The Liberfarb syndrome, a multisystem disorder affecting eye, ear, bone, and brain development, is caused by a founder pathogenic variant in the PISD gene

PurposeWe observed four individuals in two unrelated but consanguineous families from Portugal and Brazil affected by early-onset retinal degeneration, sensorineural hearing loss, microcephaly, intellectual disability, and skeletal dysplasia with scoliosis and short stature. The phenotype precisely matched that of an individual of Azorean descent published in 1986 by Liberfarb and coworkers. MethodsPatients underwent specialized clinical examinations (including ophthalmological, audiological, orthopedic, radiological, and developmental assessment). Exome and targeted sequencing was performed on selected individuals. Minigene constructs were assessed by quantitative polymerase chain reaction (qPCR) and Sanger sequencing. ResultsAffected individuals shared a 3.36-Mb region of autozygosity on chromosome 22q12.2, including a 10-bp deletion(NM_014338.3:c.904-12_904-3delCTATCACCAC), immediately upstream of the last exon of the PISD (phosphatidylserine decarboxylase) gene. Sequencing of PISD from paraffin-embedded tissue from the 1986 case revealed the identical homozygous variant. In HEK293T cells, this variant led to aberrant splicing of PISD transcripts. ConclusionWe have identified the genetic etiology of the Liberfarb syndrome, affecting brain, eye, ear, bone, and connective tissue. Our work documents the migration of a rare Portuguese founder variant to two continents and highlights the link between phospholipid metabolism and bone formation, sensory defects, and cerebral development, while raising the possibility of therapeutic phospholipid replacement.

Two autopsy cases of sudden unexpected death from Dravet syndrome with novel de novo SCN1A variants

AimDravet syndrome (DS) is characterized by high epilepsy-related premature mortality with a markedly young age at death, however, autopsy report of sudden unexpected death with DS has been fewer than expected. MethodsWe report two autopsy cases with sudden unexpected death from DS. Case 1 was a 13-year-old male who drowned in a bathtub, and Case 2 was a 3-year-old female who died while sleeping. In Case 1, the blood concentration of the anticonvulsant, valproic acid, was below the recommended therapeutic range. Neuropathological investigation and genetic analysis of 402 cardiovascular disease-related and 146 epilepsy-related genes by next generation sequencing were applied. ResultsNo significant neuronal loss with gliosis was observed in the brain of either patient. Although possible mild malformations of cortical development were found in both, the degree thereof was similar to that of age-matched controls. Genetic analysis identified a novel variant in SCN1A intron 23 (c.4477-3T > C) in Case 1 that falls outside of the minor splicing consensus sequence. In vitro splicing functional assays with minigene constructs revealed that this intronic variant leads to a 2-bp insertion immediately before exon 24 that results in protein truncation. Similarly, a novel de novo missense mutation of unknown significance, SCN1A_Arg187Pro, was identified in Case 2. In both cases, we also identified cardiomyopathy-related variants classified as likely pathogenic; however, the effect of these variants at death was minimal because there was an absence of pathological change indicating inherited cardiomyopathy. ConclusionThe present cases emphasize the need for multifaceted examination of DS cases so as to obtain a definitive autopsy diagnosis and to explore the mechanism of sudden unexpected death.

P.106Mutation-specific therapy for X-linked myotubular myopathy

X-linked myotubular myopathy (XLMTM) is a severe congenital myopathy due to mutations in MTM1 encoding a 3-phosphoinositides phosphatase myotubularin. XLMTM patients display severe generalized hypotonia at birth accompanied by respiratory insufficiency and pathologically show small-size fibers with peripheral halo, centrally located nuclei, disorganized perinuclear organelles in the muscle. Among 78 patients with pathologically diagnosed as XLMTM in our cohort, causative mutations were not identified in 20 cases by targeted re-sequencing panel for congenital myopathy, MTM1 Sanger sequencing nor Whole-exome sequencing. In order to validate deep intronic mutations and aberrant splicing events, we performed RNA-seq analysis on 14 cases who had unreported variants or no-mutations in exonic regions of MTM1. Sashimi plot revealed retentions of intronic sequence in patient1 (between exon 5 and 6) and patient 2 (between exon 11 and 12), respectively. In patient 3, exon 4 skipping was observed due to a synonymous mutation in exon 4. These aberrant splicing were predicted to cause frameshift and subsequently create premature stop codon in these patients. The in vitro splicing experiments with artificial hybrid minigene constructs confirmed those mutations exclusively produced abnormal splicing products. Lastly, Myoblasts from patient 2 showed perinuclear distribution of mitochondria in addition to the abnormal splicing, and treatment with antisense morpholino oligonucleotides rescued those phenotypes. In conclusion, RNA-seq analysis can uncover deep intronic mutations and synonymous mutations which lead abnormal splicing events as the cause of muscle diseases such as XLMTM. We propose that antisense therapy is potentially practical for diseases arising from this type of mutation.

Abstract 467: Identification of Novel Pathogenic Mutations in Non-Canonical RNA Splice Sites in Congenital Heart Disease

Rationale: Congenital heart disease (CHD) occurs in about 1 in 100 live births, yet known genetic causes explain less than 20% of CHD cases. While variants that cause frameshift, nonsense, start/stop site gain or loss, and canonical splice site alterations are readily categorized as being pathogenic or loss-of-function (LOF), interpreting the clinical significance of variants without obvious functional consequences remains challenging. Here, we aim to improve classification of variants of unknown significance (VUS) in non-canonical RNA splice sites that may be pathogenic for CHD. Methods: We tested candidate LOF de novo (DNV) and rare (p < 2E-5) inherited variants from whole exome sequencing of 4474 CHD probands and their parents in the NHLBI Pediatric Cardiac Genetics Consortium. Briefly, variants underwent computational selection to prioritize VUS in splice regions that are likely to alter splicing (“high-likelihood VUS”). These variants then underwent in vitro analysis including Minigene construction, transfection, RNA isolation, and sequencing to confirm splicing outcomes. Results: Preliminary results limited to DNV variants showed that 163 of 2678 (6.08%) were high-likelihood VUS. Subsequent analysis in vitro assay of high-likelihood VUS yielded 53 as splice-altering (p < 0.05) and thus LOF. Combined with previously identified 366 DNV LOF variants, the addition of these splice-altering variants represents a 15.3% increase in total LOF DNV variant calls. This includes new pathogenic mutations in known CHD genes such as KMT2D . In one case, a CHD proband with features of Kabuki Syndrome without a definitive diagnosis was found to have a splice-altering variant in KMT2D . We have extended this assay to 34518 rare, inherited variants in the same cohort, of which 868 (2.54%) are in genes previously associated with CHD. Conclusion: Consideration of non-canonical RNA splice sites in this assay increased the yield of LOF mutations from traditional sequencing methods by 15.3% in the CHD cohort. Further analysis of splice-altering variants in both known and unknown pathogenic genes will improve diagnostic classification of VUS and gene-based diagnosis of CHD.

Functional Characterization of Argininosuccinate Lyase Gene Variants by Mini-Gene Splicing Assay.

ObjectiveArgininosuccinate lyase (ASL) gene mutations account for argininosuccinic aciduria (ASA). This study aimed to design a minigene construct of ASL gene in order to investigate the impact of variants on splicing.MethodsThe peripheral blood samples were collected from the family members, and genomic DNA was extracted for gene diagnosis using the total exon sequencing method. The novel mutation gene was cloned into pEGFP-C1 vector, and the pathogenicity of the mutation was examined in cultured cells in vitro.ResultsThe clinical diagnosis of the proband as ASA was clear. Two pathogenic mutations, c.281G>T (p.Arg94Leu) and c.208-15 T>A were detected in the ASL gene, and the two mutations had not been reported. The minigene expression in vitro confirmed that c.208-15 T>A could cause aberrant splicing, resulting in the retention of 13 bp in intron 2 to exon 3.ConclusionTwo new pathogenic mutations of ASL gene, c.208-15 T>A and c.281G>T, were found in an ASA family, which enriches the mutational profile of the ASL gene and provides a basis for genetic diagnosis of ASA. Minigenes are optimal approaches to determine whether the intron mutation can cause aberrant splicing.