Activation Of Cryptic Splice Sites Research Articles

Characterization of NEB pathogenic variants in patients reveals novel nemaline myopathy disease mechanisms and omecamtiv mecarbil force effects.

Nebulin, a critical protein of the skeletal muscle thin filament, plays important roles in physiological processes such as regulating thin filament length (TFL), cross-bridge cycling, and myofibril alignment. Pathogenic variants in the nebulin gene (NEB) cause NEB-based nemaline myopathy (NEM2), a genetically heterogeneous disorder characterized by hypotonia and muscle weakness, currently lacking curative therapies. In this study, we examined a cohort of ten NEM2 patients, each with unique pathogenic variants, aiming to understand their impact on mRNA, protein, and functional levels. Results show that pathogenic truncation variants affect NEB mRNA stability and lead to nonsense-mediated decay of the mutated transcript. Moreover, a high incidence of cryptic splice site activation was found in patients with pathogenic splicing variants that are expected to disrupt the actin-binding sites of nebulin. Determination of protein levels revealed patients with either relatively normal or markedly reduced nebulin. We observed a positive relation between the reduction in nebulin and a reduction in TFL, or reduction in tension (both maximal and submaximal tension). Interestingly, our study revealed a pathogenic duplication variant in nebulin that resulted in a four-copy gain in the triplicate region of NEB and a much larger nebulin protein and longer TFL. Additionally, we investigated the effect of Omecamtiv mecarbil (OM), a small-molecule activator of cardiac myosin, on force production of type 1 muscle fibers of NEM2 patients. OM treatment substantially increased submaximal tension across all NEM2 patients ranging from 87 to 318%, with the largest effects in patients with the lowest level of nebulin. In summary, this study indicates that post-transcriptional or post-translational mechanisms regulate nebulin expression. Moreover, we propose that the pathomechanism of NEM2 involves not only shortened but also elongated thin filaments, along with the disruption of actin-binding sites resulting from pathogenic splicing variants. Significantly, our findings highlight the potential of OM treatment to improve skeletal muscle function in NEM2 patients, especially those with large reductions in nebulin levels.

Activation of Cryptic Donor Splice Sites within the UDP-Glucuronosyltransferase (UGT)1A First-Exon Region Generates Variant Transcripts That Encode UGT1A Proteins with Truncated Aglycone-Binding Domains.

The human UDP-glucuronosyltransferases (UGTs) have crucial roles in metabolizing and clearing numerous small lipophilic compounds. The UGT1A locus generates nine UGT1A mRNAs, 65 spliced transcripts, and 34 circular RNAs. In this study, our analysis of published UGT-RNA capture sequencing (CaptureSeq) datasets identified novel splice junctions that predict 24 variant UGT1A transcripts derived from ligation of exon 2 to unique sequences within the UGT1A first-exon region using cryptic donor splice sites. Of these variants, seven (1A1_n1, 1A3_n3, 1A4_n4, 1A5_n1, 1A8_n2, 1A9_n2, 1A10_n7) are predicted to encode UGT1A proteins with truncated aglycone-binding domains. We assessed their expression profiles and deregulation in cancer using four RNA sequencing (RNA-Seq) datasets of paired normal and cancerous drug-metabolizing tissues from large patient cohorts. Variants were generally coexpressed with their canonical counterparts with a higher relative abundance in tumor than in normal tissues. Variants showed tissue-specific expression with high interindividual variability but overall low abundance. However, 1A8_n2 showed high abundance in normal and cancerous colorectal tissues, with levels that approached or surpassed canonical 1A8 mRNA levels in many samples. We cloned 1A8_n2 and showed expression of the predicted protein (1A8_i3) in human embryonic kidney (HEK)293T cells. Glucuronidation assays with 4-methylumbelliferone (4MU) showed that 1A8_i3 had no activity and was unable to inhibit the activity of 1A8_i1 protein. In summary, the activation of cryptic donor splice sites within the UGT1A first-exon region expands the UGT1A transcriptome and proteome. The 1A8_n2 cryptic donor splice site is highly active in colorectal tissues, representing an important cis-regulatory element that negatively regulates the function of the UGT1A8 gene through pre-mRNA splicing. SIGNIFICANT STATEMENT: The UGT1A locus generates nine canonical mRNAs, 65 alternately spliced transcripts, and 34 different circular RNAs. The present study reports a series of novel UDP-glucuronosyltransferase (UGT)1A variants resulting from use of cryptic donor splice sites in both normal and cancerous tissues, several of which are predicted to encode variant UGT1A proteins with truncated aglycone-binding domains. Of these, 1A8_n2 shows exceptionally high abundance in colorectal tissues, highlighting its potential role in the first-pass metabolism in gut through the glucuronidation pathway.

Genotype–Phenotype Correlation in Junctional Epidermolysis Bullosa: Signposts to Severity

Junctional epidermolysis bullosa (JEB) is a rare autosomal recessive genodermatosis with a broad spectrum of phenotypes. Current genotype-phenotype paradigms are insufficient to accurately predict JEB subtype and characteristics from genotype, particularly for splice site mutations, which account for over a fifth of disease-causing mutations in JEB. This study evaluated genetic and clinical findings from a JEB cohort, investigating genotype-phenotype correlations through bioinformatic analyses and comparison with previously reported mutations.Eighteen unique mutations in LAMB3, LAMA3, LAMC2 or COL17A1 were identified from seventeen individuals. Seven had severe JEB, nine intermediate JEB and one laryngo-onycho-cutaneous syndrome. Seven mutations were previously unreported. Deep phenotyping was completed for all intermediate JEB cases and demonstrated substantial variation between individuals. Splice site mutations underwent analysis with SpliceAI, a state-of-the-art artificial intelligence tool, in order to predict resultant transcripts. Predicted functional effects included exon skipping and cryptic splice site activation, which provided potential explanations for disease severity and in most cases correlated with lamimin-332 immunofluorescence. RT-PCR was performed for one case to investigate resultant transcripts produced from the splice site mutation.This study expands the JEB genomic and phenotypic landscape. AI tools show potential for predicting functional effects of splice site mutations and may identify candidates for confirmatory laboratory investigation. Investigation of RNA transcripts will help to further elucidate genotype-phenotype correlations for novel mutations.

Comprehensive genetic analysis reveals the mutational landscape of ABCA4-associated retinal dystrophy in a Chinese cohort

PurposeTo depict the variant profiles of the ABCA4 gene in a large Chinese cohort of patients with ABCA4-associated retinal dystrophy (ABCA4-RD). MethodsWe recruited 290 unrelated Chinese patients with ABCA4-RD and did ABCA4 mutational screening by a combination of Sanger sequencing, targeted exome sequencing, entire ABCA4 locus sequencing, and whole genome sequencing (WGS). The pathogenicity of variants was assessed using in silico tools or in vitro splicing assays following the American College of Medical Genetics and Genomics guidelines. ResultsTwo hundred sixty-eight distinct pathogenic variants were identified, and 57 were novel. In 580 alleles, 22 noncoding region variants outside canonical splice sites and 4 structural variations were found in 44 alleles accounting for 7.6% of all alleles. Bioinformatics analysis showed the complex mechanism of aberrant splicing productsnatural splice site disruption, branch point destruction, and cryptic splice site activation. Correspondingly, minigene assays validated the various abnormal splicing products, including exon skipping, exon elongation, partial exon deletion, and pseudoexon insertion. WGS identified the first inversion variation in ABCA4. ConclusionsThis study systematically depicted the variant profiles of ABCA4 and revealed the missing alleles of patients with ABCA4-RD in a large Chinese cohort. Our findings demonstrated the complexity of molecular diagnosis of Mendelian diseases and the efficiency of WGS for detecting structural variants.

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.

SF3B1 facilitates HIF1-signaling and promotes malignancy in pancreatic cancer.

Mutations in the splicing factor SF3B1 are frequently occurring in various cancers and drive tumor progression through the activation of cryptic splice sites in multiple genes. Recent studies also demonstrate a positive correlation between the expression levels of wild-type SF3B1 and tumor malignancy. Here, we demonstrate that SF3B1 is a hypoxia-inducible factor (HIF)-1 target gene that positively regulates HIF1 pathway activity. By physically interacting with HIF1α, SF3B1 facilitates binding of the HIF1 complex to hypoxia response elements (HREs) to activate target gene expression. To further validate the relevance of this mechanism for tumor progression, we show that a reduction in SF3B1 levels via monoallelic deletion of Sf3b1 impedes tumor formation and progression via impaired HIF signaling in a mouse model for pancreatic cancer. Our work uncovers an essential role of SF3B1 in HIF1 signaling, thereby providing a potential explanation for the link between high SF3B1 expression and aggressiveness of solid tumors.

Characterisation of a novel OPA1 splice variant resulting in cryptic splice site activation and mitochondrial dysfunction

Autosomal dominant optic atrophy (DOA) is an inherited optic neuropathy that results in progressive, bilateral visual acuity loss and field defects. OPA1 is the causative gene in around 60% of cases of DOA. The majority of patients have a pure ocular phenotype, but 20% have extra-ocular features (DOA +). We report on a patient with DOA + manifesting as bilateral optic atrophy, spastic paraparesis, urinary incontinence and white matter changes in the central nervous system associated with a novel heterozygous splice variant NM_015560.2(OPA1):c.2356-1 G > T. Further characterisation, which was performed using fibroblasts obtained from a skin biopsy, demonstrated that this variant altered mRNA splicing of the OPA1 transcript, specifically a 21 base pair deletion at the start of exon 24, NM_015560.2(OPA1):p.Cys786_Lys792del. The majority of variant transcripts were shown to escape nonsense-mediated decay and modelling of the predicted protein structure suggests that the in-frame 7 amino acid deletion may affect OPA1 oligomerisation. Fibroblasts carrying the c.2356-1 G > T variant demonstrated impaired mitochondrial bioenergetics, membrane potential, increased cell death, and disrupted and fragmented mitochondrial networks in comparison to WT cells. This study suggests that the c.2356-1 G > T OPA1 splice site variant leads to a cryptic splice site activation and may manifest in a dominant-negative manner, which could account for the patient’s severe syndromic phenotype.

Epimutations in both the TESK2 and MMACHC promoters in the Epi-cblC inherited disorder of intracellular metabolism of vitamin B12

Backgroundepi-cblC is a recently discovered inherited disorder of intracellular vitamin B12 metabolism associating hematological, neurological, and cardiometabolic outcomes. It is produced by an epimutation at the promoter common to CCDC163P and MMACHC, which results from an aberrant antisense transcription due to splicing mutations in the antisense PRDX1 gene neighboring MMACHC. We studied whether the aberrant transcription produced a second epimutation by encompassing the CpG island of the TESK2 gene neighboring CCDC163P.MethodsWe unraveled the methylome architecture of the CCDC163P–MMACHC CpG island (CpG:33) and the TESK2 CpG island (CpG:51) of 17 epi-cblC cases. We performed an integrative analysis of the DNA methylome profiling, transcriptome reconstruction of RNA-sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-Seq) of histone H3, and transcription expression of MMACHC and TESK2.ResultsThe PRDX1 splice mutations and activation of numerous cryptic splice sites produced antisense readthrough transcripts encompassing the bidirectional MMACHC/CCDC163P promoter and the TESK2 promoter, resulting in the silencing of both the MMACHC and TESK2 genes through the deposition of SETD2-dependent H3K36me3 marks and the generation of epimutations in the CpG islands of the two promoters.ConclusionsThe antisense readthrough transcription of the mutated PRDX1 produces an epigenetic silencing of MMACHC and TESK2. We propose using the term 'epi-digenism' to define this epigenetic disorder that affects two genes. Epi-cblC is an entity that differs from cblC. Indeed, the PRDX1 and TESK2 altered expressions are observed in epi-cblC but not in cblC, suggesting further evaluating the potential consequences on cancer risk and spermatogenesis.

A Severe Dementia Syndrome Caused by Intron Retention and Cryptic Splice Site Activation in STUB1 and Exacerbated by TBP Repeat Expansions.

Heterozygous pathogenic variants in the STIP1 homologous and U-box containing protein 1 (STUB1) gene have been identified as causes of autosomal dominant inherited spinocerebellar ataxia type 48 (SCA48). SCA48 is characterized by an ataxic movement disorder that is often, but not always, accompanied by a cognitive affective syndrome. We report a severe early onset dementia syndrome that mimics frontotemporal dementia and is caused by the intronic splice donor variant c.524+1G>A in STUB1. Impaired splicing was demonstrated by RNA analysis and in minigene assays of mutated and wild-type constructs of STUB1. The most striking consequence of this splicing impairment was retention of intron 3 in STUB1, which led to an in-frame insertion of 63 amino acids (aa) (p.Arg175_Glu176ins63) into the highly conserved coiled-coil domain of its encoded protein, C-terminus of HSP70-interacting protein (CHIP). To a lesser extent, activation of two cryptic splice sites in intron 3 was observed. The almost exclusively used one, c.524+86, was not predicted by in silico programs. Variant c.524+86 caused a frameshift (p.Arg175fs*93) that resulted in a truncated protein and presumably impairs the C-terminal U-box of CHIP, which normally functions as an E3 ubiquitin ligase. The cryptic splice site c.524+99 was rarely used and led to an in-frame insertion of 33 aa (p.Arg175_Glu176ins33) that resulted in disruption of the coiled-coil domain, as has been previously postulated for complete intron 3 retention. We additionally detected repeat expansions in the range of reduced penetrance in the TATA box-binding protein (TBP) gene by excluding other genes associated with dementia syndromes. The repeat expansion was heterozygous in one patient but compound heterozygous in the more severely affected patient. Therefore, we concluded that the observed severe dementia syndrome has a digenic background, making STUB1 and TBP important candidate genes responsible for early onset dementia syndromes.

How to Design U1 snRNA Molecules for Splicing Rescue.

Mutations affecting constitutive splice donor sites (5'ss) are among the most frequent genetic defects that disrupt the normal splicing process. Pre-mRNA splicing requires the correct identification of a number of cis-acting elements in an ordered fashion. By disrupting the complementarity of the 5'ss with the endogenous small nuclear RNA U1 (U1 snRNA), the key component of the spliceosomal U1 ribonucleoprotein, 5'ss mutations may result in exon skipping, intron retention or activation of cryptic splice sites. Engineered modification of the U1 snRNA seemed to be a logical method to overcome the effect of those mutations. In fact, over the last years, a number of in vitro studies on the use of those modified U1 snRNAs to correct a variety of splicing defects have demonstrated the feasibility of this approach. Furthermore, recent reports on its applicability in vivo are adding up to the principle that engineered modification of U1 snRNAs represents a valuable approach and prompting further studies to demonstrate the clinical translatability of this strategy.Here, we outline the design and generation of U1 snRNAs with different degrees of complementarity to mutated 5'ss. Using the HGSNAT gene as an example, we describe the methods for a proper evaluation of their efficacy in vitro, taking advantage of our experience to share a number of tips on how to design U1 snRNA molecules for splicing rescue.

Unraveling the molecular basis underlying nine putative splice site variants of von Willebrand factor

Approximately 10% of von Willebrand factor (VWF) gene variants are suspected to disrupt messenger RNA (mRNA) processing, the number of which might be underestimated due to the lack of transcript assays. In the present study, we provided a detailed strategy to evaluate the effects of nine putative splice site variants (PSSVs) of VWF on mRNA processing as well as protein properties and establish their genotype-phenotype relationships. Eight of nine PSSVs affected VWF splicing: c.322A>T, c.1534-13_1551delinsCA, and c.8116-2del caused exon skipping; c.221-2A>C, c.323+1G>T, and c.2547-13T>A resulted in the activation of cryptic splice sites; c.2684A>G led to exon skipping and activation of a cryptic splice site; c.2968-14A>G created a new splice site. The remaining c.5171-9del was likely benign. The efficiency of nonsense-mediated mRNA decay (NMD) was much higher in platelets compared to leukocytes, impairing the identification of aberrant transcripts in 4 of 8 PSSVs. The nonsense variant c.322A>T partially impaired mRNA processing, leaking a small amount of correct transcripts with c.322T (p.Arg108*), while the missense variant c.2684A>G totally disrupted normal splicing of VWF, rather than produced mutant protein with the substitution of Gln895Arg. The results of this study would certainly add novel insights into the molecular events behind von Willebranddisease.

DMD – CLINICAL CARE: EP.98 Clinical and molecular characterisation of unusual DMD mutations

The dystrophinopathies encompass a spectrum of clinical phenotypes associated with mutations in the DMD gene. The clinical impact of most mutations can be predicted by the reading frame rule, but uncommon mutations continue to shed light on pathophysiological mechanisms of the disease and inform therapeutic development. We report here clinical, histological, and molecular findings in 5 such patients, including muscle histology, quantitative immunofluorescence (IF) analysis, western blot (WB), and RNA-Seq. Expression of dystrophin of increased molecular weight (536 kDa) was detected in an entirely asymptomatic 16 y.o. boy who was incidentally found to carry an in-frame duplication of exons 10-27. Serum CK, muscle histology, and dystrophin expression by IF were essentially normal. Such complete absence of clinical or laboratory pathology is highly unusual in comparison to other in-frame duplications. An out-of-frame deletion of exon 2 was detected during evaluation for global developmental delay in a 4 y.o. with no observable muscle weakness; consistent with utilization of the downstream (exon 5) internal ribosome entry site (IRES), dystrophin expression was only mildly reduced. In contrast, a boy with exon 3 nonsense mutation (c.103C>T) had a severe phenotype and absent dystrophin expression, indicating lack of IRES-induced translation. An intermediate phenotype was observed in a boy with an exon 3 splice donor site mutation (c.186+1G>A), who showed reduced but not absent dystrophin expression. A final patient had a canonical splice donor site mutation in the last nucleotide of exon 45 (c.6614G>T) that did not result in cryptic splice site activation or exon 45 exclusion, as is typical for splice site mutations. WB showed abundant expression of a 256 kDa dystrophin, undetectable with C-terminal antibodies. RNA-Seq revealed instead the formation of a novel terminal exon, encompassing exon 45 and a portion of intron 45 comprising a pseudo-3’UTR region, a previously unreported molecular mechanism.

Mutation spectrum of the OPA1 gene in a large cohort of patients with suspected dominant optic atrophy: Identification and classification of 48 novel variants

Autosomal dominant optic atrophy is one of the most common inherited optic neuropathies. This disease is genetically heterogeneous, but most cases are due to pathogenic variants in the OPA1 gene: depending on the population studied, 32–90% of cases harbor pathogenic variants in this gene. The aim of this study was to provide a comprehensive overview of the entire spectrum of likely pathogenic variants in the OPA1 gene in a large cohort of patients. Over a period of 20 years, 755 unrelated probands with a diagnosis of bilateral optic atrophy were referred to our laboratory for molecular genetic investigation. Genetic testing of the OPA1 gene was initially performed by a combined analysis using either single-strand conformation polymorphism or denaturing high performance liquid chromatography followed by Sanger sequencing to validate aberrant bands or melting profiles. The presence of copy number variations was assessed using multiplex ligation-dependent probe amplification. Since 2012, genetic testing was based on next-generation sequencing platforms. Genetic screening of the OPA1 gene revealed putatively pathogenic variants in 278 unrelated probands which represent 36.8% of the entire cohort. A total of 156 unique variants were identified, 78% of which can be considered null alleles. Variant c.2708_2711del/p.(V903Gfs*3) was found to constitute 14% of all disease-causing alleles. Special emphasis was placed on the validation of splice variants either by analyzing cDNA derived from patients´ blood samples or by heterologous splice assays using minigenes. Splicing analysis revealed different aberrant splicing events, including exon skipping, activation of exonic or intronic cryptic splice sites, and the inclusion of pseudoexons. Forty-eight variants that we identified were novel. Nine of them were classified as pathogenic, 34 as likely pathogenic and five as variant of uncertain significance. Our study adds a significant number of novel variants to the mutation spectrum of the OPA1 gene and will thereby facilitate genetic diagnostics of patients with suspected dominant optic atrophy.

Activation of cryptic donor splice sites by non-coding and coding PAX6 variants contributes to congenital aniridia

BackgroundThe paired-domain transcription factor paired box gene 6 (PAX6) causes a wide spectrum of ocular developmental anomalies, including congenital aniridia, Peters anomaly and microphthalmia. Here, we aimed to functionally assess...

Characterization of C9orf72 haplotypes to evaluate the effects of normal and pathological variations on its expression and splicing.

Expansion of the hexanucleotide repeat (HR) in the first intron of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in Caucasians. All C9orf72-ALS/FTD patients share a common risk (R) haplotype. To study C9orf72 expression and splicing from the mutant R allele compared to the complementary normal allele in ALS/FTD patients, we initially created a detailed molecular map of the single nucleotide polymorphism (SNP) signature and the HR length of the various C9orf72 haplotypes in Caucasians. We leveraged this map to determine the allelic origin of transcripts per patient, and decipher the effects of pathological and normal HR lengths on C9orf72 expression and splicing. In C9orf72 ALS patients' cells, the HR expanded allele, compared to non-R allele, was associated with decreased levels of a downstream initiated transcript variant and increased levels of transcripts initiated upstream of the HR. HR expanded R alleles correlated with high levels of unspliced intron 1 and activation of cryptic donor splice sites along intron 1. Retention of intron 1 was associated with sequential intron 2 retention. The SNP signature of C9orf72 haplotypes described here enables allele-specific analysis of transcriptional products and may pave the way to allele-specific therapeutic strategies.

<p>A Novel Splice-Site Mutation in the <em>ELN</em> Gene Suggests an Alternative Mechanism for Vascular Elastinopathies</p>

The ELN gene encodes elastin, a fundamental protein of the extracellular matrix that confers elasticity to different tissues including blood vessels. The formation of elastin fibers is a complex process involving monomer coacervation and subsequent crosslinking. Mutations in exons 1–29 of the ELN gene have been linked to supravalvular aortic stenosis (SVAS) whereas mutations in exons 30–33 are associated with autosomal dominant cutis laxa (ADCL). This striking segregation has led to the hypothesis that distinct molecular mechanisms underlie both diseases. SVAS is believed to arise through haploinsufficiency while ADCL is hypothesized to be caused by a dominant negative effect. Here, we describe a patient with SVAS harboring a novel splice-site mutation in the last exon of ELN. The location of this mutation is not consistent with current knowledge of SVAS, since all mutations reported in the C-terminus have been found in ADCL patients, and a thorough evaluation did not reveal significant skin involvement in this case. RT-PCR analysis of skin tissue showed that C-terminal mutations in the region can lead to the production of aberrant transcripts through intron retention and activation of cryptic splice sites and suggest that disruption of the very last exon can lead to functional haploinsufficiency potentially related to SVAS.

EFTUD2 missense variants disrupt protein function and splicing in mandibulofacial dysostosis Guion-Almeida type.

Pathogenic variants in the core spliceosome U5 small nuclear ribonucleoprotein gene EFTUD2/SNU114 cause the craniofacial disorder mandibulofacial dysostosis Guion-Almeida type (MFDGA). MFDGA-associated variants in EFTUD2 comprise large deletions encompassing EFTUD2, intragenic deletions and single nucleotide truncating or missense variants. These variants are predicted to result in haploinsufficiency by loss-of-function of the variant allele. While the contribution of deletions within EFTUD2 to allele loss-of-function are self-evident, the mechanisms by which missense variants are disease-causing have not been characterized functionally. Combining bioinformatics software prediction, yeast functional growth assays, and a minigene (MG) splicing assay, we have characterized how MFDGA missense variants result in EFTUD2 loss-of-function. Only four of 19 assessed missense variants cause EFTUD2 loss-of-function through altered protein function when modeled in yeast. Of the remaining 15 missense variants, five altered the normal splicing pattern of EFTUD2 pre-messenger RNA predominantly through exon skipping or cryptic splice site activation, leading to the introduction of a premature termination codon. Comparison of bioinformatic predictors for each missense variant revealed a disparity amongst different software packages and, in many cases, an inability to correctly predict changes in splicing subsequently determined by MG interrogation. This study highlights the need for laboratory-based validation of bioinformatic predictions for EFTUD2 missense variants.

Two novel ECHS1 variants, affecting splicing and reducing enzyme activity, is associated with mitochondrial encephalopathy in infant: a case report

BackgroundShort-chain enoyl-CoA hydratase (ECHS1) is a multifunctional mitochondrial matrix enzyme involved in the second step of mitochondrial fatty acid β-oxidation. Mitochondrial diseases resulting from ECHS1 mutations are often characterised by encephalopathy, deafness, epilepsy, optic atrophy, cardiomyopathy, dystonia, and lactic acidosis. In this study, we report two novel heterogeneous variants, c.414 + 5G > A (in intron 3) and c.310C > G (in CDS), of ECHS1 in an infant with mitochondrial encephalopathy.Case presentationThe two novel variants, c.414 + 5G > A (Chr10:135183403) in intron 3 and c.310C > G (Chr10:135183512) in CDS, were identified by next generation sequencing (NGS). A minigene assay was used to analyse the function of the c.414 + 5G > A variant. ECHS1 enzyme activity was measured by spectrophotometry in the patient-derived myoblasts. The 2-year old patient presented with mitochondrial encephalopathy since birth. Clinical features were encephalopathy, epilepsy, and hindered psychomotor and language development. Serum lactate and blood ammonia levels were elevated, and brain magnetic resonance imaging showed abnormal signals in the bilateral frontal, parietal, and occipital cortices and brainstem and basal ganglia. We found two novel heterogeneous variants in ECHS1 in this patient. Minigene assay revealed the c.414 + 5G > A variant as the cause of intronic cryptic splice site activation and 39 bp deletion in mature mRNA. In silico analysis predicted that c.310C > G might change glutamine (Q) to glutamic acid (E) in the 104th amino acid sequence (p.Q104E). To investigate the impact of these two variants on protein function, we constructed a 3D model of human ECHS1 and showed that the variants might alter the highly conserved region in close proximity to the active site, which might hinder, or even halt, enzymatic activity. The experimental assay showed that ECHS1 enzyme activity in the patient-derived myoblasts decreased compared to that in control.ConclusionsOur findings are the first to report a mitochondrial encephalopathy infant carrying two novel ECHS1 variants, c.414 + 5G > A and c.310C > G, which might be deleterious variants, function as pathogenicity markers for mitochondrial encephalopathy, and facilitate disease diagnosis.

In vitro functional characterization of splicing variants of the APOB gene found in familial hypobetalipoproteinemia

Familial hypobetalipoproteinemia type 1 (FHBL-1) is a codominant disorder characterized by greatly reduced plasma levels of total cholesterol, low-density lipoprotein cholesterol, and apolipoprotein B. Rare exonic pathogenic variants of APOB gene (nonsense variants, minute deletions/insertions and nonsynonymous variants) have been frequently reported in subjects with FHBL-1. Also, rare intronic variants of APOB located at intron/exon junctions and assumed to affect splicing have been reported. However, the pathogenicity of most of these intronic variants remains to be established. The objective of this study was the invitro functional characterization of six splicing variants of APOB gene identified in seven putative FHBL-1 heterozygotes. ApoB minigenes harboring each variant were expressed in COS-1cells and their transcripts were sequenced. Four novel variants (c.237+1G>A, c.818+5G>A, c.3000-1G>T, and c.3842+1G>A), predicted in silico to obliterate splice site activity, were found to generate abnormal transcripts. The abnormal transcripts were generated by the activation of cryptic splice sites or exon skipping. All these transcripts harbored a premature termination codon and were predicted to encode truncated apoBs devoid of function. The predicted translation products were: i) p.(Lys41Serfs*2) and p.(Val80Ilefs*10) for c.237+1G>A; ii) p.(Asn274*) for c.818+5G>A; iii) p.(Leu1001Alafs*10) for c.3000-1G>T, and iv) p.(Ser1281Argfs*2) for c.3842+1G>A. Two previously annotated rare variants (c.905-15C>G and c.1618-4G>A) with uncertain effect in silico were found to generate only wild-type transcripts. These invitro minigene expression studies support the assignment of pathogenicity to four novel splice site variants of APOB gene found in FHBL-1.

Activation of cryptic splice sites in three patients with chronic granulomatous disease

BackgroundChronic granulomatous disease (CGD) is a primary immune deficiency caused by mutations in the genes encoding the structural components of the phagocyte NADPH oxidase. As a result, the patients cannot generate sufficient amounts of reactive oxygen species required for killing pathogenic microorganisms.MethodsWe analyzed NADPH oxidase activity and component expression in neutrophils, performed genomic DNA and cDNA analysis, and used mRNA splicing prediction tools to evaluate the impact of mutations.ResultsIn two patients with CGD, we had previously found mutations that cause aberrant pre‐mRNA splicing. In one patient an exonic mutation in a cryptic donor splice site caused the deletion of the 3' part of exon 6 from the mRNA of CYBB. This patient suffers from X‐linked CGD. The second patient, with autosomal CGD, has a mutation in the donor splice site of intron 1 of CYBA that activates a cryptic donor splice site downstream in intron 1, causing the insertion of intronic sequences in the mRNA. The third patient, recently analyzed, also with autosomal CGD, has a mutation in intron 4 of CYBA, 15 bp from the acceptor splice site. This mutation weakens a branch site and activates a cryptic acceptor splice site, causing the insertion of 14 intronic nucleotides into the mRNA.ConclusionWe found three different mutations, one exonic, one in a donor splice site and one intronic, that all caused missplicing of pre‐mRNA. We analyzed these mutations with four different splice prediction programs and found that predictions of splice site strength, splice enhancer and splice silencer protein binding and branch site strength are all essential for correct prediction of pre‐mRNA splicing.