Cryptic Exon Research Articles

BG05 Artificial intelligence prediction of splice site mutation consequences in junctional epidermolysis bullosa: signposts to severity

Abstract Over one-fifth of disease-causing mutations in junctional epidermolysis bullosa (JEB) are splice site variants. These affect pre-mRNA splicing through the activation of cryptic splice sites and/or exon skipping, resulting in altered mRNA and translated proteins. Prediction of the precise location of activated cryptic splice sites is vital, as these can potentially alter the reading frame of the resulting transcript. Out-of-frame transcripts contain premature termination complexes (PTCs), which generally result in nonfunctional protein and often correlate with severe JEB in conjunction with other PTC-introducing mutations. SpliceAI is a 32-layer convolutional neural network that examines 10 000 nucleotides of flanking sequence around a variant of interest to predict its effects on RNA splicing. It was used to predict splice site mutation consequences in five patients with JEB with LAMB3 mutations. Genotypes, predicted transcripts and JEB subtypes are summarized in Table 1. SpliceAI predicted PTCs in cases 1 and 2, where transcripts were either out of frame or novel PTCs were introduced following aberrant splicing. An in-frame transcript with no PTC was predicted for case 3, which may explain this individual’s mild phenotype. Reverse transcriptase polymerase chain reaction data were reviewed for cases 4 and 5, and two predicted transcripts were confirmed. Two additional in-frame transcripts not predicted by SpliceAI were found to be produced in low levels. It is likely that these in-frame transcripts generated partially functional protein, alleviating JEB severity to a degree. This study highlights the importance of examining RNA transcripts for accurate genotype–phenotype correlation. In silico tools such as SpliceAI show potential in elucidating mechanisms of severity. They may facilitate selection of cases for further laboratory investigation and can guide which regions of the gene or transcript to sequence for confirmatory analyses. Where RNA analyses were available, SpliceAI correctly predicted two of the resultant transcripts. Further validation of this tool is required with additional cases in this rare disease.Table 1Genotypes, predicted transcripts and JEB subtypes found with SpliceAICaseZygosityMutation (LAMB3)Exon skipping predictionCryptic splice site activation predictionPredicted outcome commentsJEB subtype1Homozygousc.2701+1G>AOOF exon 18 skipping20 nt excluded from exon 18Both outcomes OOFSevere2Heterozygousc.565-2A>G, c.2914C>T (p.R972X)OOF exon 7 skipping117 nt included from intron 6 (including TGA PTC at c.565-60)In-frame cryptic activation with novel PTC introducedSevereOOF exon skipping3Heterozygousc.1705C>T (p.R569X), c.943+2T>COOF exon 9 skipping27 nt included from intron 9In-frame cryptic activationIntermediateOOF exon skipping4Homozygousc.298+5G>COOF exon 4 skipping64 nt included from intron 4Both outcomes OOFIntermediate5Heterozygousc.3119G>A (p.W1040X), c.629-12T>APredicted to be unlikely10 nt included from intron 7OOF cryptic activationIntermediatent, nucleotides; OOF, out of frame.

All reported non-canonical splice site variants in GLA cause aberrant splicing

BackgroundFabry disease is an X-linked lysosomal storage disorder caused by insufficient α-galactosidase A (GLA) activity resulting from variants in the GLA gene, which leads to glycosphingolipid accumulation and life-threatening, multi-organ complications. Approximately 50 variants have been reported that cause splicing abnormalities in GLA. Most were found within canonical splice sites, which are highly conserved GT and AG splice acceptor and donor dinucleotides, whereas one-third were located outside canonical splice sites, making it difficult to interpret their pathogenicity. In this study, we aimed to investigate the genetic pathogenicity of variants located in non-canonical splice sites within the GLA gene.Methods13 variants, including four deep intronic variants, were selected from the Human Gene Variant Database Professional. We performed an in vitro splicing assay to identify splicing abnormalities in the variants.ResultsAll candidate non-canonical splice site variants in GLA caused aberrant splicing. Additionally, all but one variant was protein-truncating. The four deep intronic variants generated abnormal transcripts, including a cryptic exon, as well as normal transcripts, with the proportion of each differing in a cell-specific manner.ConclusionsValidation of splicing effects using an in vitro splicing assay is useful for confirming pathogenicity and determining associations with clinical phenotypes.

Characterization of the genomic and transcriptional structure of chicken NRG4 gene.

Neuregulin 4 (NRG4) is an important adipocytokine, which plays crucial roles in maintaining energy balance, regulating glucose and lipid metabolism, and preventing non-alcoholic fatty liver disease in mammals. At present, the genomic organization, transcript and protein isoforms of human NRG4 gene have been fully explored. Previous studies in our laboratory have shown that the NRG4 gene is expressed in chicken adipose tissue, but the chicken NRG4 (cNRG4) genomic structure, transcript and protein isoforms are still unknown. To this end, in this study, the genomic and transcriptional structure of the cNRG4 gene were systematically investigated using rapid amplification of cDNA ends (RACE) and reverse transcription-polymerase chain reaction (RT-PCR). The results showed that the coding region (CDS) of the cNRG4 gene was small, but it had a very complex transcriptional structure characterized by multiple transcription start sites, alternative splicing, intron retention, cryptic exons, and alternative polyadenylation, thus leading to production of four 5?UTR isoforms (cNRG4 A, cNRG4 B, cNRG4 C, and cNRG4 D) and six 3?UTR isoforms (cNRG4 a, cNRG4 b, cNRG4 c, cNRG4 d, cNRG4 e, and cNRG4 f) of the cNRG4 gene. The cNRG4 gene spanned 21,969 bp of genomic DNA (Chr.10:3,490,314~3,512,282) and consisted of 11 exons and 10 introns. Compared with the cNRG4 gene mRNA sequence (NM_001030544.4), two novel exons and one cryptic exon of the cNRG4 gene were identified in this study. Bioinformatics analysis, RT-PCR, cloning and sequencing analysis showed that the cNRG4 gene could encode three protein isoforms (cNRG4-1, cNRG4-2 and cNRG4-3). This study lays a foundation for further research on the function and regulation of the cNRG4 gene.

Deep intronic mutation in CRTAP results in unstable isoforms of the protein to induce type I collagen aggregation in a lethal type of osteogenesis imperfecta type VII

Genetic mutations are involved in Mendelian disorders. Unbuffered intronic mutations in gene variants can generate aberrant splice sites in mutant transcripts, resulting in mutant isoforms of proteins with modulated expression, stability, and function in diseased cells. Here, we identify a deep intronic variant, c.794_1403A>G, in CRTAP by genome sequencing of a male fetus with osteogenesis imperfecta (OI) type VII. The mutation introduces cryptic splice sites in intron-3 of CRTAP, resulting in two mature mutant transcripts with cryptic exons. While transcript-1 translates to a truncated isoform (277 amino acids) with thirteen C-terminal non-wild-type amino acids, transcript-2 translates to a wild-type protein sequence, except that this isoform contains an in-frame fusion of non-wild-type twenty-five amino acids in a tetratricopeptide repeat sequence. Both mutant isoforms of CRTAP are unstable due to the presence of a unique ‘GWxxI’ degron, which finally leads to loss of proline hydroxylation and aggregation of type I collagen. Although type I collagen aggregates undergo autophagy, the overall proteotoxicity resulted in death of the proband cells by senescence. In summary, we present a genetic disease pathomechanism by linking a novel deep intronic mutation in CRTAP to unstable mutant isoforms of the protein in lethal OI type VII.

Multi-omics identifies large mitoribosomal subunit instability caused by pathogenic MRPL39 variants as a cause of pediatric onset mitochondrial disease.

MRPL39 encodes one of 52 proteins comprising the large subunit of the mitochondrial ribosome (mitoribosome). In conjunction with 30 proteins in the small subunit, the mitoribosome synthesizes the 13 subunits of the mitochondrial oxidative phosphorylation (OXPHOS) system encoded by mitochondrial Deoxyribonucleic acid (DNA). We used multi-omics and gene matching to identify three unrelated individuals with biallelic variants in MRPL39 presenting with multisystem diseases with severity ranging from lethal, infantile-onset (Leigh syndrome spectrum) to milder with survival into adulthood. Clinical exome sequencing of known disease genes failed to diagnose these patients; however quantitative proteomics identified a specific decrease in the abundance of large but not small mitoribosomal subunits in fibroblasts from the two patients with severe phenotype. Re-analysis of exome sequencing led to the identification of candidate single heterozygous variants in mitoribosomal genes MRPL39 (both patients) and MRPL15. Genome sequencing identified a shared deep intronic MRPL39 variant predicted to generate a cryptic exon, with transcriptomics and targeted studies providing further functional evidence for causation. The patient with the milder disease was homozygous for a missense variant identified through trio exome sequencing. Our study highlights the utility of quantitative proteomics in detecting protein signatures and in characterizing gene-disease associations in exome-unsolved patients. We describe Relative Complex Abundance analysis of proteomics data, a sensitive method that can identify defects in OXPHOS disorders to a similar or greater sensitivity to the traditional enzymology. Relative Complex Abundance has potential utility for functional validation or prioritization in many hundreds of inherited rare diseases where protein complex assembly is disrupted.

Cytoplasmic aggregation of mutant FUS causes multistep RNA splicing perturbations in the course of motor neuron pathology.

Dysfunction of the RNA-binding protein (RBP) FUS implicated in RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. Mutations affecting FUS nuclear localization can drive RNA splicing defects and stimulate the formation of non-amyloid inclusions in affected neurons. However, the mechanism by which FUS mutations contribute to the development of ALS remains uncertain. Here we describe a pattern of RNA splicing changes in the dynamics of the continuous proteinopathy induced by mislocalized FUS. We show that the decrease in intron retention of FUS-associated transcripts represents the hallmark of the pathogenesis of ALS and is the earliest molecular event in the course of progression of the disease. As FUS aggregation increases, the pattern of RNA splicing changes, becoming more complex, including a decrease in the inclusion of neuron-specific microexons and induction of cryptic exon splicing due to the sequestration of additional RBPs into FUS aggregates. Crucially, the identified features of the pathological splicing pattern are also observed in ALS patients in both sporadic and familial cases. Our data provide evidence that both a loss of nuclear FUS function due to mislocalization and the subsequent cytoplasmic aggregation of mutant protein lead to the disruption of RNA splicing in a multistep fashion during FUS aggregation.

Biofluid Biomarkers in the Prognosis of Amyotrophic Lateral Sclerosis: Recent Developments and Therapeutic Applications.

Amyotrophic lateral sclerosis is a neurodegenerative disease characterized by the degeneration of motor neurons for which effective therapies are lacking. One of the most explored areas of research in ALS is the discovery and validation of biomarkers that can be applied to clinical practice and incorporated into the development of innovative therapies. The study of biomarkers requires an adequate theoretical and operational framework, highlighting the "fit-for-purpose" concept and distinguishing different types of biomarkers based on common terminology. In this review, we aim to discuss the current status of fluid-based prognostic and predictive biomarkers in ALS, with particular emphasis on those that are the most promising ones for clinical trial design and routine clinical practice. Neurofilaments in cerebrospinal fluid and blood are the main prognostic and pharmacodynamic biomarkers. Furthermore, several candidates exist covering various pathological aspects of the disease, such as immune, metabolic and muscle damage markers. Urine has been studied less often and should be explored for its possible advantages. New advances in the knowledge of cryptic exons introduce the possibility of discovering new biomarkers. Collaborative efforts, prospective studies and standardized procedures are needed to validate candidate biomarkers. A combined biomarkers panel can provide a more detailed disease status.

Abstract 6542: Detection of androgen receptor splice variants from clinical sequencing

Abstract Androgen receptor (AR) splice variants (AR-Vs) have been widely studied on its important role in prostate cancer (PC) progression. In particular, AR-Vs lacking ligand binding domains, such as AR splice variant 7 (AR-v7) that links exon 3 and cryptic exon 3 or AR variant that losses exons 5 to 7 (ARv567es), have been demonstrated as one of the resistant mechanisms to androgen deprivation therapy in PC. Despite its clinical importance, the current algorithms to detect fusions such as TopHat-Fusion have limitation to detect intra-chromosomal rearrangements including AR-Vs. In this respect, we propose a novel approach to identify AR-Vs from targeted RNA sequencing (RNA-seq) dataset. In short, our two-step approach first gathers soft-clipped or divided reads that are adjacent to the splicing sites of AR-Vs and then yields the number of splitting reads that support the existence of AR-Vs. Next, the algorithm selects the paired-end reads whose one side and the other side are mapped to the preceding and following exons, respectively. The final number of spanning reads are calculated following to the removal of low-quality reads. We validated the proposed approach using two large-scale, independent RNA-seq datasets of PC samples: 34 samples from Seoul National University Hospital (SNUH) and 558 samples from The Cancer Genome Atlas (TCGA) dataset. Overall, our approach successfully identified samples with putative AR-Vs, including AR-v7 and ARv567es. In addition, our approach successfully detected AR-v7 with reliable number of supporting reads, from the RNA-seq dataset of AR-v7-expressing cell line. Finally, statistical analyses of 34 SNUH PC patients suggested potential detection threshold in clinical sequencing settings (at least 10 split reads and 1% proportion). In the SNUH dataset, The AR-v7 positive group, which included 6 patients (17.1%) with values above the threshold, had higher AR-v7 expression levels than the negative group (p=4.8E-04). The similar pattern was also found in the analysis of TCGA dataset. Owing to its flexibility, we also confirmed that the developed approach can be used to detection of other AR-Vs, including AR-v3 and ARv567es. We expect that the further in-depth analyses including larger samples and clinical outcomes can discover clinical applicability of AR-Vs. Citation Format: Suhyun Hwangbo, Sungyoung Lee, Sheehyun Kim, Hongseok Yun. Detection of androgen receptor splice variants from clinical sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6542.

LKB1 isoform expression modulates T cell plasticity downstream of PKCθ and IL-6

Following activation, CD4 T cells undergo metabolic and transcriptional changes as they respond to external cues and differentiate into T helper (Th) cells. T cells exhibit plasticity between Th phenotypes in highly inflammatory environments, such as colitis, in which high levels of IL-6 promote plasticity between regulatory T (Treg) cells and Th17 cells. Protein Kinase C theta (PKCθ) is a T cell-specific serine/threonine kinase that promotes Th17 differentiation while negatively regulating Treg differentiation. Liver kinase B1 (LKB1), also a serine/threonine kinase and encoded by Stk11, is necessary for Treg survival and function. Stk11 can be alternatively spliced to produce a short variant (Stk11S) by transcribing a cryptic exon. However, the contribution of Stk11 splice variants to Th cell differentiation has not been previously explored. Here we show that in Th17 cells, the heterogeneous ribonucleoprotein, hnRNPLL, mediates Stk11 splicing into its short splice variant, and that Stk11S expression is diminished when Hnrnpll is depleted using siRNA knock-down approaches. We further show that PKCθ regulates hnRNPLL and, thus, Stk11S expression in Th17 cells. We provide additional evidence that exposing induced (i)Tregs to IL-6 culminates in Stk11 splicing downstream of PKCθAltogether our data reveal a yet undescribed outside-in signaling pathway initiated by IL-6, that acts through PKCθ and hnRNPLL to regulate Stk11 splice variants and facilitate Th17 cell differentiation. Furthermore, we show for the first time, that this pathway can also be initiated in developing iTregs exposed to IL-6, providing mechanistic insight into iTreg phenotypic stability and iTreg to Th17 cell plasticity.

TDP-43 and other hnRNPs regulate cryptic exon inclusion of a key ALS/FTD risk gene, UNC13A.

A major function of TAR DNA-binding protein-43 (TDP-43) is to repress the inclusion of cryptic exons during RNA splicing. One of these cryptic exons is in UNC13A, a genetic risk factor for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The accumulation of cryptic UNC13A in disease is heightened by the presence of a risk haplotype located within the cryptic exon itself. Here, we revealed that TDP-43 extreme N-terminus is important to repress UNC13A cryptic exon inclusion. Further, we found hnRNP L, hnRNP A1, and hnRNP A2B1 bind UNC13A RNA and repress cryptic exon inclusion, independently of TDP-43. Finally, higher levels of hnRNP L protein associate with lower burden of UNC13A cryptic RNA in ALS/FTD brains. Our findings suggest that while TDP-43 is the main repressor of UNC13A cryptic exon inclusion, other hnRNPs contribute to its regulation and may potentially function as disease modifiers.

A deep intronic TCTN2 variant activating a cryptic exon predicted by SpliceRover in a patient with Joubert syndrome.

The recent introduction of genome sequencing in genetic analysis has led to the identification of pathogenic variants located in deep introns. Recently, several new tools have emerged to predict the impact of variants on splicing. Here, we present a Japanese boy of Joubert syndrome with biallelic TCTN2 variants. Exome sequencing identified only a heterozygous maternal nonsense TCTN2 variant (NM_024809.5:c.916C >T, p.(Gln306Ter)). Subsequent genome sequencing identified a deep intronic variant (c.1033+423G>A) inherited from his father. The machine learning algorithms SpliceAI, Squirls, and Pangolin were unable to predict alterations in splicing by the c.1033+423G>A variant. SpliceRover, a tool for splice site prediction using FASTA sequence, was able to detect a cryptic exon which was 85-bp away from the variant and within the inverted Alu sequence while SpliceRover scores for these splice sites showed slight increase (donor) or decrease (acceptor) between the reference and mutant sequences. RNA sequencing and RT-PCR using urinary cells confirmed inclusion of the cryptic exon. The patient showed major symptoms of TCTN2-related disorders such as developmental delay, dysmorphic facial features and polydactyly. He also showed uncommon features such as retinal dystrophy, exotropia, abnormal pattern of respiration, and periventricular heterotopia, confirming these as one of features of TCTN2-related disorders. Our study highlights usefulness of genome sequencing and RNA sequencing using urinary cells for molecular diagnosis of genetic disorders and suggests that database of cryptic splice sites predicted in introns by SpliceRover using the reference sequences can be helpful in extracting candidate variants from large numbers of intronic variants in genome sequencing.

STK36 splice site variant in an Australian Shepherd dog with primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) represents a group of diseases characterized by impaired movement of cilia and subsequent health problems in diverse organ systems, notably the respiratory tract. Almost 50 candidate genes for PCD are known in humans. In this study, we investigated an Australian Shepherd dog with a history of recurrent respiratory infections and nasal discharge. A transmission electron microscopy investigation led to the diagnosis of PCD with central pair defect, in which the normal 9:2 arrangement of respiratory cilia was altered and reduced to a 9:0 arrangement. Whole genome sequencing data from the affected dog was obtained and searched for variants in PCD candidate genes that were not present in 918 control genomes from different breeds. This revealed a homozygous single base pair exchange at a splice site of STK36, XM_038585732.1:c.2868-1G>A. The mutant allele was absent from 281 additionally genotyped Australian Shepherd dogs. RT-PCR confirmed aberrant splicing in the affected dog with the skipping of exon 20 and the insertion of a cryptic exon, which is predicted to lead to a premature stop codon and truncation of 36% of the STK36 wild-type open reading frame, XP_038441660.1:(p.Met957Profs*11). STK36 variants were previously reported to cause PCD in humans and mice. The knowledge from other species together with the absence of the mutant allele in more than 1000 control dogs suggests STK36:c.2868-1G>A as the most likely candidate variant for PCD in the investigated case.

Neurodegeneration: 2023 update.

This paper reviews ten highly impactful studies published in the previous year selected by the author from the neurodegenerative neuropathology literature. As in previous years, the focus is to highlight human tissue-based experimentation most relevant to neuropathologists. A concerted effort was made to balance the selected studies across disease categories, approaches, and methodologies to capture the breadth of the research landscape. Studies include an integrated proteomic and transcriptomic study of Alzheimer disease (AD) and new consensus diagnostic neuropathological criteria for progressive supranuclear palsy. A number of studies looking at TAR DNA-binding protein 43 (TDP-43) are highlighted. One examined interaction between AD and limbic age-related TDP-43 encephalopathy (LATE) and yet another demonstrated how TDP-43 represses cryptic exon inclusion in UNC13A, suggesting a novel pathogenic mechanism. Most surprisingly, three cryogenic electron microscopy (cryo-EM) studies showed that TMEM106B filaments form the core of TDP-43-positive inclusions. Cryo-EM revealed a prion protein amyloid structure from aggregates in Gerstmann-Sträussler-Scheinker disease. There was an elegant functional genomic study cataloging microglial gene expression in the human brain. A study shed light on how APOE influences chronic traumatic encephalopathy. A pathoanatomical study tested the dual hit hypothesis of Lewy body progression throughout the nervous system. And finally, deep learning continues to show its promise with application of a weakly supervised multiple instance learning paradigm to assess aging post-mortem brains.

Germline variants associated with toxicity to immune checkpoint blockade.

Immune checkpoint inhibitors (ICIs) have yielded remarkable responses but often lead to immune-related adverse events (irAEs). Although germline causes for irAEs have been hypothesized, no individual variant associated with developing irAEs has been identified. We carried out a genome-wide association study of 1,751 patients on ICIs across 12 cancer types. We investigated two irAE phenotypes: (1) high-grade (3-5) and (2) all-grade events. We identified 3 genome-wide significant associations (P < 5 × 10-8) in the discovery cohort associated with all-grade irAEs: rs16906115 near IL7 (combined P = 3.6 × 10-11; hazard ratio (HR) = 2.1); rs75824728 near IL22RA1 (combined P = 3.5 × 10-8; HR = 1.8); and rs113861051 on 4p15 (combined P = 1.2 × 10-8, HR = 2.0); rs16906115 was replicated in 3 independent studies. The association near IL7 colocalized with the gain of a new cryptic exon for IL7, a critical regulator of lymphocyte homeostasis. Patients carrying the IL7 germline variant exhibited significantly increased lymphocyte stability after ICI initiation, which was itself predictive of downstream irAEs and improved survival.

Targeting a Putative Intronic Splicing Silencer Salvages Expression from the Recurrent SBDS C.258+2T>C Mutant Allele in Shwachman-Diamond Syndrome Patient Cells and Mouse Model

Shwachman-Diamond syndrome (SDS) is an autosomal recessive inherited bone marrow failure syndrome and leukemia predisposition disorder due to biallelic mutations of SBDS, whose gene product positively regulates 80S ribosome assembly. In addition to predisposition to pancytopenia, myelodysplasia and acute myeloid leukemia, patients frequently experience exocrine pancreatic insufficiency, skeletal dysplasia, cardiac abnormalities, and immune dysfunction. Faithful animal models are lacking to study hematopoiesis and clonal evolution in SDS. Almost all patients carry at least one copy of the SBDS c.258+2T>C mutation that disrupts the intron 2 5' splice site (SS). We hypothesize that this recurrent mutant allele allows for sufficient residual SBDS expression to promote SBDS cellular function and permit organismal development, and thus residual expression could be potentiated through novel therapeutic approaches. Here we present RNA-seq analysis that shows residual expression of full-length SBDS originating from the non-canonical SS of the SBDS c.258+2T>C allele. Most transcripts were the product of aberrant splicing from a cryptic exon 2 SS and exon 2 skipping, both of which result in nonsense transcripts. Prior animal models of SDS have relied on complete or tissue-specific Sbds knock-out and thus fail to replicate the splicing defect and residual SBDS expression. To develop a mouse model to test novel sequence-specific therapeutic strategies, we generated, by CRISPR/Cas9-mediated HDR, mice with a humanized intron 2 5' SS containing the c.258+2T>C mutation at the endogenous mouse Sbds gene (c.199+2T>C). Homozygous Sbds SS mutant knock-in mouse embryos were found to be viable until at least mid-gestation (E14.5) though live births were not observed. In contrast, constitutive Sbds knock-out mice are reported to succumb to early embryonic lethality by E8.51. Using isolated MEFs, we demonstrate that the humanized mouse Sbds mutant allele recapitulates low-level residual expression, aberrant splicing from a cryptic exon 2 SS, and exon 2 skipping, although exon skipping appears to be greater and residual expression lower as compared to the human mutant allele. Consistent with the characteristic SDS molecular defect, homozygous Sbds c.199+2T>C MEFs show impaired 80S ribosome assembly, accumulation of EIF6 on the 60S ribosomal subunit, and induction of a p53-dependent stress response. To explore strategies to further increase residual wild type SBDS transcript expression from the SBDS c.258+2T>C allele, we designed anti-sense oligonucleotides (ASOs) targeting potential splice regulatory sequences. We evaluated 38 antisense oligos, identifying several that block a putative intronic splicing silencer (ISS) that suppresses normal splicing from the non-canonical c.258+2T>C SS, with the most potent ASO resulting in 4.0-fold increase in SBDS expression (p<0.01) in fibroblasts from 4 different SDS patients. Disruption of sequences within this putative ISS by Cas12a nuclease-mediated indels result in 1.4 and 2.4 fold increased SBDS/Sbds expression from SDS patient fibroblasts and homozygous MEFs respectively (p<0.05). Together these studies present a novel mouse model for SDS recapitulating the recurrent SS mutation with residual intact splicing and indicate that salvaging residual SBDS expression by modulating aberrant splicing is a promising strategy for SDS therapeutics.

Non-Canonical Exon Usage in Pediatric Leukemia: Implications for Chemoresistance

B-lymphoblastic leukemia (B-ALL) is a heterogeneous, chromosome translocation-driven disease where the prevalence of somatic mutations and copy number variations is relatively low. Previous B-ALL whole exome sequencing efforts by other groups have focused upon mutations acquired under therapeutic pressure, but they have not identified universal resistance gene(s). Instead, relapse-specific mutations occurred in multiple genetic loci often involved in resistance to either glucocorticoids or purine analogs (e.g., 6-mercaptopurine, or 6-MP). The most prevalent target, NT5C2, encodes the enzyme 5'-nucleotidase/cytosolic II involved in 6-MP catabolism, but these gain-of-function mutations were found in a minority of relapsed/refractory (r/r) B-ALL samples (~25%). This lack of concordance between the genotype and the phenotype suggested a possibility that instead of mutations, NT5C2 is predominantly affected by post-transcriptional events, such as aberrant mRNA splicing (AS). To address this possibility, we have analyzed several richly annotated RNA-Seq datasets, including NCI TARGET, which presently includes several hundred baseline childhood B-ALL samples as well as 48 paired diagnostic and relapse samples. In baseline B-ALL samples, we indeed discovered an abnormally spliced NT5C2 mRNA isoform containing the cryptic in-frame exon 4a (NT5ex4a). Of note, NT5ex4a levels were further increased in relapses compared to diagnostic samples, consistent with its putative role in chemoresistance. Furthermore, NT5ex4a mapped to full-length protein-coding transcripts and resulted in inclusion of 8 extra amino acids near the ATP-binding effector site 2. Using bacterially produced protein, we demonstrated that at low concentrations of ATP NT5ex4a exhibited elevated enzymatic activity compared to the canonical isoform. Consistent with this biochemical finding, in reconstituted NT5C2low B-ALL cells NT5ex4a conferred the same level of resistance to 6-MP as the variant with the R238W hotspot mutation (2-log difference in IC50). Conversely, NT5ex4a-KO CRISPR/Cas9 engineered cells reduced cell survival in the presence of 6-MP. The role of NT5ex4a in chemoresistance in vivo was further confirmed in B-ALL cells expressing this alternative isoform. Therefore, inclusion of NT5C2 exon 4a phenocopies relapse-specific mutations and could serve as both a valuable predictive biomarker in B-ALL and potentially chronic myelogenous (CML) and acute myeloid leukemia (AML). Additionally, at least in vitro, expression of this non-canonical isoform conferred collateral sensitivity to the purine biosynthesis inhibitor Mizoribine, suggesting the existence of a therapeutic window to treat leukemias with dysregulated splicing.

14. Providing more answers for patients with supplemental RNA analysis

<h3>Introduction</h3> To help resolve the clinical significance of variants predicted to alter splicing, and identify splice-altering variants in regions outside of the reportable range of our routine hereditary cancer DNA sequencing tests, we initiated an IRB-approved RNA-sequencing research study at Invitae. <h3>Methods</h3> We developed an RNA-sequencing assay run on whole blood for 63 transcripts from the Invitae Multi-Cancer panel and an algorithm for detecting alterations in splicing caused by nearby DNA variants. Our algorithm was designed to detect statistically significant alterations in splicing of a specific transcript relative to a panel of controls. Samples with previously determined positive (n=52) or negative RNA effects (n=70) were used for validation. RNA analysis was also prospectively performed for >13000 patients. <h3>Results</h3> Accuracy was 99.7%, with 97.8% sensitivity and 99.7% specificity for the 122 validation samples. Reproducibility was 100%. 723/13175 (5.5%) were found to have a significant splicing-alteration associated with a DNA variant. Thirty-four patients (0.26%) were found to have a cryptic exon (32) or an intron inclusion event (2) associated with a DNA variant beyond our standard reportable range (+/- 20 bp around each exon). RNA analysis led to a more definitive classification in 4.9% of individuals (Likely Pathogenic/Pathogenic=0.4%; Likely Benign/Benign=4.5%). <h3>Conclusions</h3> Invitae's RNA sequencing assay is improving the accuracy of variant classification within the reportable range of our tests, and enabling new variant discovery in regions outside our reportable range. These results support the integration of RNA analysis into the genetic testing workflow.

Morphologic and Molecular Patterns of Polymyositis With Mitochondrial Pathology and Inclusion Body Myositis.

To characterize morphological and molecular underpinnings of polymyositis with mitochondrial pathology (PM-Mito) in comparison with sporadic inclusion body myositis (IBM) and to define common and distinct pathophysiologic features with a focus on interferon (IFN)-associated inflammation and T-cell response. In this cross-sectional study, skeletal muscle biopsy samples and clinical and laboratory data from patients with PM-Mito and IBM were analyzed at Charité university hospital in Berlin, Germany. All available PM-Mito biopsy samples, an equal number of randomly selected IBM biopsy samples, and randomly selected nondiseased controls (NDCs) were included in the study. Biopsy samples were studied by histopathology, immunohistochemistry, and quantitative PCR (qPCR) and compared with biopsies derived from NDCs. Primary outcomes included cell counts for immunohistochemistry and gene expression (fold-change values compared with those in NDCs) for qPCR. Twenty-five skeletal muscle biopsy samples of patients with PM-Mito and IBM were included in the study and compared with 5 biopsy samples from NDCs. PM-Mito and IBM qualitatively harbored a strikingly similar molecular signature and shared important histopathologic features. Expression of IFN-induced guanylate-binding protein (GBP)6 and T-cell function-related KLRG1 distinguished IBM from PM-Mito biopsies with IBM patients showing significantly higher expression of GBP6 and KLRG1. Cryptic exon expression was detected in both patient groups with IBM patients showing higher expression levels. Skeletal muscle biopsies from IBM patients showed significantly more GBP6+ cells and KLRG1+ lymphocytes in comparison with biopsies from patients with PM-Mito. CD45+, CD68+, CD57+, PD1+, and CD8+ cytotoxic T cells were also significantly more abundant in patients with IBM. Clinically, patients with PM-Mito presented with a spectrum of muscle-related symptoms including myalgia, proximal paraparesis, proximal tetraparesis, and incomplete IBM-like patterns. Thirteen of 14 (93%) patients with PM-Mito for whom clinical follow-up was available later developed clinically defined IBM. Notably, 2 follow-up biopsies obtained 5 and 7 years after the first ones were available in this cohort, both showing histopathologic progress to net IBM including GBP6 and KLRG1 upregulation. Our combined data suggest that specific IFN-mediated inflammation plays a key role in both IBM and PM-Mito. GBP6 was identified as a new molecule of type II IFN-induced inflammation distinguishing IBM from PM-Mito. Skeletal muscles from both groups harbor dysfunctional T cells of similar type, albeit in different quantity. T-cell senescence exemplified by KLRG1 positivity does not play a significant role in PM-Mito. Based on these findings, we propose to include PM-Mito in the spectrum of IBM (IBM-spectrum disease [IBM-SD]) as a possible early form of this disease. The establishment of IBM-SD as a larger entity could potentially have a significant effect on the design of trials and therapeutic interventions.

P08 - Identification of functional single nucleotide polymorphisms in cryptic exon 3 of the androgen receptor gene

P08 - Identification of functional single nucleotide polymorphisms in cryptic exon 3 of the androgen receptor gene

NOS1AP is a novel molecular target and critical factor in TDP-43 pathology.

Many lines of evidence have highlighted the role played by heterogeneous nuclear ribonucleoproteins in amyotrophic lateral sclerosis. In this study, we have aimed to identify transcripts co-regulated by TAR DNA-binding protein 43 kDa and highly conserved heterogeneous nuclear ribonucleoproteins which have been previously shown to regulate TAR DNA-binding protein 43 kDa toxicity (deleted in azoospermia-associated protein 1, heterogeneous nuclear ribonucleoprotein -Q, -D, -K and -U). Using the transcriptome analyses, we have uncovered that Nitric Oxide Synthase 1 Adaptor Protein mRNA is a direct TAR DNA-binding protein 43 kDa target, and in flies, its modulation alone can rescue TAR DNA-binding protein 43 kDa pathology. In primary mouse cortical neurons, we show that TAR DNA-binding protein 43 kDa mediated downregulation of Nitric Oxide Synthase 1 Adaptor Protein expression strongly affects the NMDA-receptor signalling pathway. In human patients, the downregulation of Nitric Oxide Synthase 1 Adaptor Protein mRNA strongly correlates with TAR DNA-binding protein 43 kDa proteinopathy as measured by cryptic Stathmin-2 and Unc-13 homolog A cryptic exon inclusion. Overall, our results demonstrate that Nitric Oxide Synthase 1 Adaptor Protein may represent a novel disease-relevant gene, potentially suitable for the development of new therapeutic strategies.