Splice Donor Research Articles

Full genome sequence analysis of a novel adenovirus from a captive polar bear (Ursus maritimus)

The presence of a novel adenovirus (AdV) was detected by PCR and sequencing, in the internal organs of a captive polar bear that had died in the Budapest zoo. The virus content of the samples proved to be high enough to allow for conventional Sanger sequencing on PCR-amplified genomic fragments. With this approach, the sequence of the entire genome of the putative polar bear adenovirus 1 (PBAdV-1) was obtained. Although the genome was found to be short, consisting of 27,952 base pairs merely, with a relatively balanced G + C content of 46.3 %, its organisation corresponded largely to that of a typical mastadenovirus. Every genus-common gene could be identified except that of protein IX. The short E3 region of the PBAdV-1 consisted of two novel, supposedly type-specific ORFs only, whereas no homologue of any of the E3 genes, usually conserved in mastadenoviruses, such as for example that of the 12.5 K protein, were present. In the E4 region, only the highly conserved gene of the 34 K protein was found besides two novel ORFs showing no homology to any known E4 ORFs. In silico sequence analysis revealed putative splicing donor and acceptor sites in the genes of the E1A, IVa2, DNA-dependent DNA polymerase, pTP, 33 K proteins, and also of U exon protein, all being characteristic for mastadenoviruses. Phylogenetic calculations, based on various proteins, further supported that the newly-detected PBAdV is the representative of a new species within the genus Mastadenovirus, and may represent the evolutionary lineage of adenoviruses that coevolved with carnivorans.

Characterization of splice-altering mutations in inherited predisposition to cancer

Mutations responsible for inherited disease may act by disrupting normal transcriptional splicing. Such mutations can be difficult to detect, and their effects difficult to characterize, because many lie deep within exons or introns where they may alter splice enhancers or silencers or introduce new splice acceptors or donors. Multiple mutation-specific and genome-wide approaches have been developed to evaluate these classes of mutations. We introduce a complementary experimental approach, cBROCA, which yields qualitative and quantitative assessments of the effects of genomic mutations on transcriptional splicing of tumor suppressor genes. cBROCA analysis is undertaken by deriving complementary DNA (cDNA) from puromycin-treated patient lymphoblasts, hybridizing the cDNA to the BROCA panel of tumor suppressor genes, and then multiplex sequencing to very high coverage. At each splice junction suggested by split sequencing reads, read depths of test and control samples are compared. Significant Z scores indicate altered transcripts, over and above naturally occurring minor transcripts, and comparisons of read depths indicate relative abundances of mutant and normal transcripts. BROCA analysis of genomic DNA suggested 120 rare mutations from 150 families with cancers of the breast, ovary, uterus, or colon, in >600 informative genotyped relatives. cBROCA analysis of their transcripts revealed a wide variety of consequences of abnormal splicing in tumor suppressor genes, including whole or partial exon skipping, exonification of intronic sequence, loss or gain of exonic and intronic splicing enhancers and silencers, complete intron retention, hypomorphic alleles, and combinations of these alterations. Combined with pedigree analysis, cBROCA sequencing contributes to understanding the clinical consequences of rare inherited mutations.

Charcot-Marie-Tooth type 4B2 demyelinating neuropathy in miniature Schnauzer dogs caused by a novel splicing SBF2 (MTMR13) genetic variant: a new spontaneous clinical model.

BackgroundCharcot-Marie-Tooth (CMT) disease is the most common neuromuscular disorder in humans affecting 40 out of 100,000 individuals. In 2008, we described the clinical, electrophysiological and pathological findings of a demyelinating motor and sensory neuropathy in Miniature Schnauzer dogs, with a suspected autosomal recessive mode of inheritance based on pedigree analysis. The discovery of additional cases has followed this work and led to a genome-wide association mapping approach to search for the underlying genetic cause of the disease.MethodsFor genome wide association screening, genomic DNA samples from affected and unaffected dogs were genotyped using the Illumina CanineHD SNP genotyping array. SBF2 and its variant were sequenced using primers and PCRs. RNA was extracted from muscle of an unaffected and an affected dog and RT-PCR performed. Immunohistochemistry for myelin basic protein was performed on peripheral nerve section specimens.ResultsThe genome-wide association study gave an indicative signal on canine chromosome 21. Although the signal was not of genome-wide significance due to the small number of cases, the SBF2 (also known as MTMR13) gene within the region of shared case homozygosity was a strong positional candidate, as 22 genetic variants in the gene have been associated with demyelinating forms of Charcot-Marie-Tooth disease in humans. Sequencing of SBF2 in cases revealed a splice donor site genetic variant, resulting in cryptic splicing and predicted early termination of the protein based on RNA sequencing results.ConclusionsThis study reports the first genetic variant in Miniature Schnauzer dogs responsible for the occurrence of a demyelinating peripheral neuropathy with abnormally folded myelin. This discovery establishes a genotype/phenotype correlation in affected Miniature Schnauzers that can be used for the diagnosis of these dogs. It further supports the dog as a natural model of a human disease; in this instance, Charcot-Marie-Tooth disease. It opens avenues to search the biological mechanisms responsible for the disease and to test new therapies in a non-rodent large animal model. In particular, recent gene editing methods that led to the restoration of dystrophin expression in a canine model of muscular dystrophy could be applied to other canine models such as this before translation to humans.

Protein trap: a new Swiss army knife for geneticists?

The protein trap is a powerful tool for genetic and biochemical studies of gene function in the animal kingdom. Although the original protein trap was developed for flies, it can be easily adapted to other multicellular organisms, both known models and ones with an unsequenced genome. The protein trap has been successfully applied to the fruit fly, crustaceans Parhyale hawaiensis, zebrafish, andinsect and animal cell cultures. This approach is based on the integration into genes of an artificial exon that carries DNA encoding a fluorescent marker, standardized immunoepitopes, an integrase docking site, and splice acceptor and donor sites. The protein trap for cell cultures additionally contains an antibiotic resistance gene, which facilitates the selection of trapped clones. Resulting chimeric tagged mRNAs can be interfered by dsRNA against GFP (iGFPi-in vivo GFP interference), or the chimeric proteins can be efficiently knocked down by deGradFP technology. Both RNA and protein knockdowns produce a strong loss of function phenotype in tagged cells. The fluorescent and protein affinity tags can be used for tagged protein localisation within the cell and for identifying their binding partners in their native complexes. Insertion into protein trap integrase docking sites allows the replacement of trap contents by any new constructs, including other markers, cell toxins, stop-codons, and binary expression systems such as GAL4/UAS, LexA/LexAop and QF/QUAS, that reliably reflect endogenous gene expression. A distinctive feature of the protein trap approach is that all manipulations with a gene or its product occur only in the endogenous locus, which cannot be achieved by any other method.

Short communication: A splice site mutation in CENPU is associated with recessive embryonic lethality in Holstein cattle

A genome scan for homozygous haplotype deficiency coupled with whole-genome sequence data analysis is a very effective method to identify embryonic lethal mutations in cattle. Among other factors, the power of the approach depends on the availability of a greater amount of genotyping and sequencing data. In the present study, we analyzed the largest known panel of Illumina BovineSNP50 (Illumina Inc., San Diego, CA) genotypes, comprising 401,896 Holstein animals, and we report the mapping of a new embryonic lethal haplotype on chromosome 27, called HH7. We fine mapped the locus in a 2.0-Mb interval using an identical-by-descent approach and analyzed genome sequence data from 4 carrier and 143 noncarrier Holstein bulls to identify the causative mutation. We detected a strong candidate variant in the gene encoding centromere protein U (CENPU), a centromere component essential for proper chromosome segregation during mitosis. The mutant allele is a deletion of 4 nucleotides located at position +3 to +6 bp after the splicing donor site of exon 11. Cross-species nucleotide alignment revealed that the nucleotide at position +3 is entirely conserved among vertebrates, suggesting that it plays an important role in the regulation of CENPU splicing. For verification, we genotyped the candidate variant in 232,775 Holstein individuals and did not observe any homozygotes, whereas 16 were expected (Poisson P-value = 1.1 × 10-7; allele frequency = 0.8%). In addition, genotyping of 250,602 animals from 19 additional breeds revealed that the mutant allele is restricted to animals of Holstein descent. Finally, we estimated the effect of the candidate variant on 2 fertility traits in at-risk mating (i.e., between carrier bulls and daughters of carrier bulls) versus non-risk mating. In agreement with a recessive lethal inheritance pattern, we observed a marked reduction in both conception rate and 56-d nonreturn rate in heifers and cows. The effect on 56-d nonreturn rate suggests that a substantial proportion of homozygous mutants die before 35 d after insemination, which is consistent with the early embryonic death previously reported in CENPU-/- mouse embryos. In conclusion, we demonstrate that with more than 400,000 genotypes, we can map very rare recessive lethal mutations segregating at a frequency below 1% in the population. We recommend performing new analyses regularly as data are accumulating.

Compound heterozygous LPIN2 pathogenic variants in a patient with Majeed syndrome with recurrent fever and severe neutropenia: case report

BackgroundMajeed syndrome is a rare, autosomal recessive autoinflammatory disorder first described in 1989. The syndrome starts during infancy with recurrent relapses of osteomyelitis typically associated with fever, congenital dyserythropoietic anemia (CDA), and often neutrophilic dermatosis. Mutations in the LPIN2 gene located on the short arm of chromosome 18 have been identified as being responsible for Majeed syndrome.Case presentationWe report an 8-month-old boy, who presented with recurrent fever, mild to moderate anemia, and severe neutropenia. Erythrocyte sedimentation rate and C-reactive protein were elevated. Molecular testing identified a paternal splicing donor site variant c.2327 + 1G > C and a maternal frameshift variant c.1691_1694delGAGA (Arg564Lysfs*3) in LPIN2.ConclusionsOnly a few cases with LPIN2 mutation have been reported, mainly in the Middle East with homozygous variants. Our patient exhibited a mild clinical phenotype and severe neutropenia, different from previous reports.

Altered Genomic and Epigenetic Profiling of Myeloma Bone Marrow Stromal Cells Identifies Targets for Current and Future Immunotherapeutic Approaches

Background: Novel drug discoveries have shifted the treatment paradigms of most hematological malignancies including multiple myeloma (MM), but minimal residual disease and drug resistance underlie relapses in MM. Although many genetic and epigenetic alterations regulate MM progression, MM cells are not autonomous. Dynamic interactions between MM cells and cells of the bone marrow (BM) microenvironment have been reported by our group and others. MM plasma cells (PCs) depend on interactions with bone marrow stromal cells (BMSCs) for their survival and growth, but little is known about the specific genetic events taking place in the MM BM microenvironment. Methods: Here we report a detailed analysis of the genetic and epigenetic events that are characteristic of MM BMSC as compared to HD-BMSC interacting with BM PCs. To evaluate genetic and epigenetic landscapes, RNA was extracted from bulk sorted populations of 16 MM-BMSC, 3 HD-BMSC, and 10 autologous MM cells. We prepared libraries for 32 samples using the NEBNext Ultra II Stranded Poly A kit, and then sequenced on the NextSeq 500, PE150. Sequencing data were analyzed using a custom computational and statistical pipeline at the Department of Biostatistics, School of Public Health and Partek software. Results: Unsupervised clustering showed that MM-BMSC samples clustered as a distinct and completely separate cluster from HD-BMSC and autologous MM cells. Gene level analyses of these three groups identified 990 genes differentially expressed (upregulated or downregulated, P< 0.005). Sequential filtration analyses of the differentially expressed genes in MM-BMSC identified significant deregulation of : transcripts in the Jak-STAT signaling pathway (JAK3, PIM1, IL6, CSF2R, AKT1/2, BCL2L1, CDKN1A and range of IL transcripts); genes encoding extracellular matrix interacting proteins (CD36, CD49, LAMA3, CD44, CD47); and various plasma membrane proteins that define different subpopulations of hematopoietic cells. These genes were deregulated in >24% of MM-BMSC samples analyzed as compared to HD-BMSC samples. These transcripts were downregulated in autologous tumor cells. Next, we interrogated the epigenomic landscape and identified the splicing signature of MM-BMSC as compared to HD-BMSC, and autologous MM cells. Comparison of the splicing patterns (exon skipping, intron retention, novel splice acceptor and/or donor activation) of these three distinct groups showed that a total of 2,100 genes were differentially expressed and 566 were alternatively spliced among the three groups (P < 0.01). These analyses identified a limited number of the transcripts with ~3% significantly spliced in MM-BMSC compared to HD-BMSC. However, comparing MM-BMSC splicing events to MM cells splicing events, we identified >30% of genes which were alternatively spliced in MM cells but not in MM-BMSC. Further, gene enrichment and pathway analyses identified a selective set of transcripts that were alternatively and differentially spliced in MM-BMSC including genes involved in MAPK and Ras signaling pathways, homologous recombination, mismatch repair, and adherens junction. Conclusions: Taken together, our studies identified marked differences between important stromal elements in MM- and HD-BM. We identified genes that were specifically upregulated/suppressed in MM-BMSC compared to MM-cells and HD-BMSC. Within MM BMSCs, we identified several splicing events on genes of signaling pathways implicated in development and progression of MM. Furthermore, altered splicing events identified on these transcripts represent potential new immunotherapeutic targets. Disclosures Chu: Gilead: Honoraria; Celgene: Honoraria; Teva: Consultancy; Amgen Inc.: Honoraria; AstraZeneca: Honoraria. Anderson:C4 Therapeutics: Other: Scientific founder ; Gilead Sciences: Other: Advisory Board; OncoPep: Other: Scientific founder ; Sanofi-Aventis: Other: Advisory Board; Janssen: Other: Advisory Board.

Splicing mutation in LAMP2 gene leading to exon skipping and cardiomyopathy development

Mutations in LAMP2 (Xq24) encoding a lysosomal-associated membrane protein 2 crucial to the maintenance and adhesion of the lysosome cause a Danon disease. It is an X-linked glycogen storage disease that includes cardiomyopathy (hypertrophic or dilated), skeletal myopathy and mental retardation. Here, we report a woman, carrying the mutation c.864 + 1G > A (IVS6 + 1G > A, rs727503119) in heterozygosity and affected with late-onset dilated cardiomyopathy. We demonstrated the effects of the mutation on the splicing LAMP2 in cardiac muscle. The variant c.864 + 1G > A is located in the canonical splice donor site in intron 6. Functional analysis reveals that mutation c.864 + 1G > A leads to the skipping of exon 6 in cardiac muscle. The protein loses 41 amino acids in the second luminal domain of LAMP2 without frameshift and premature termination of protein synthesis. Using qRT-PCR we revealed a significantly low level of wild-type LAMP2 mRNA in patient that can be explained by X-inactivation.

RNA Splicing Defects in Cancer-Linked Genes Indicate Mutation or Focal Gene Deletion and Are Associated with TKI Resistance in CML

Background Mutated cancer genes in patients (pts) with TKI failure and blast crisis (BC) CML have recently been described. RUNX1 mutations, namely single nucleotide variants (SNVs) and indels, were the most frequently detected besides BCR-ABL1 [reviewed in Branford, Kim Leuk 2019]. They were found in ~18% of pts, although splice variants were rarely described. RNA splicing events were associated with focal deletion of IKZF1 and RUNX1 in TKI resistant pts that were identified by copy number analysis and RNAseq [Branford Blood 2018]. Novel splicing associated with mutation of cancer genes is an unexplored area of study in resistance. RNA sequencing can assess the functional effect of splice site variants, which lead to splicing errors due to the use of alternative or cryptic splice sites and cause alterations to protein function. Aim We determined whether novel splicing can identify cancer genes with potential altered function. Methods RNAseq analysis was performed for 48 pts at diagnosis and 33 at BC using a protocol that preserved intron-retaining precursor RNA. Coverage of intron-exon borders was sufficient to detect intronic splice region variants. The STAR aligner was used to bioinformatically collate unannotated RNA splice junctions. 54 cancer genes were assessed and aberrant splice events were filtered based on the number of samples in which a splice junction occurred. Manual inspection of the splice junctions was performed using the Integrative Genomics Viewer. This approach identified previously verified aberrant splicing associated with IKZF1 and RUNX1 deletions. Results Ten previously undetected novel splice junctions were revealed in 9/33 pts (27%) in BC within key tumor suppressor genes CDKN2A/B (5), RB1 (1), ATM (1), and RUNX1 (3). The aberrant splicing pattern of CDKN2A and RB1 (Fig A/B) in 6 pts suggested large deletions, as previously described in our cohort with IKZF1 and RUNX1 deletions. Breakpoints associated with deletions ranging from 53 to 181 Kb were detected in the 5 pts with CDKN2A aberrant splicing. Similarly, a 90 Kb deletion of exons 18-27 of the RB1 gene led to the aberrant splicing. The pts transformed to lymphoid BC (median 5 months). 4 of these 6 pts were tested at diagnosis and the deletions were not detected, indicating they were gained at resistance. The aberrant splicing patterns of ATM and RUNX1 did not predict large deletions. These were related to somatic SNVs at canonical splice sites in ATM and in 2 of the pts with RUNX1 aberrant splicing. A splice acceptor site SNV in ATM resulted in skipping of exon 61 (Fig C) and protein truncation. This novel SNV has not been reported in any population or somatic variant database. Two pts in myeloid BC at 28 and 48 months after diagnosis had an identical somatic RUNX1 mutation at the canonical splice donor site of exon 5. The SNV was not detectable prior to imatinib treatment in both pts. The splice site SNV led to activation of a cryptic splice site within exon 5 in both pts (Fig D), which predicted premature termination. While this mutation is novel, an adjacent intronic SNV occurs in familial platelet disorder, leading to activation of the same cryptic splice site. The atypical RUNX1 splicing of the 3rd patient was associated with retention of 55 bp of intron 6 as a cryptic exon (Fig E), leading to protein truncation. A deep intronic SNV identified at lymphoid BC at 6 months of imatinib was detected near the cryptic exon by RNAseq and verified as somatic by DNA Sanger sequencing. This was predicted to activate cryptic RNA splicing elements and lead to intron sequence retention in a RUNX1 transcript. We sequenced the diagnosis sample using an RNA-based gene panel method under development that provides enhanced sensitivity of variant detection. The same pattern of atypical splicing was observed and the intronic SNV was present at low level. The RUNX1 mutation at diagnosis may have contributed to early BC. To our knowledge this is the first report of a RUNX1 truncating variant in CML involving a cryptic exon. Conclusion Enhanced bioinformatic analysis of RNAseq data has revealed a high proportion of pts with truncating mutations in cancer genes indicated by novel RNA splicing (27% pts in BC). Using RNA-based sequencing allows an evaluation of the potential functional effect of variants that are not apparent by DNA-based mutation analysis. We suggest that future studies include RNA sequencing to detect a broader spectrum of mutations associated with TKI resistance. Disclosures Shanmuganathan: Gilead: Other: Travel Support; Janssen: Other: Travel Support; Amgen: Other: Travel Support; Bristol-Myers Squibb: Honoraria, Other: Travel Support; Novartis: Honoraria, Other: Travel Support. Yeung:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria; Amgen: Honoraria. Scott:Celgene: Honoraria. Hughes:Novartis, Bristol-Myers Squibb, Celgene: Research Funding; Novartis, Bristol-Myers Squibb: Consultancy, Other: Travel. Branford:Cepheid: Consultancy, Honoraria; Qiagen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau.

Long-read nanopore sequencing resolves a TMEM231 gene conversion event causing Meckel-Gruber syndrome.

The diagnostic deployment of massively parallel short-read next-generation sequencing (NGS) has greatly improved genetic test availability, speed, and diagnostic yield, particularly for rare inherited disorders. Nonetheless, diagnostic approaches based on short-read sequencing have a poor ability to accurately detect gene conversion events. We report on the genetic analysis of a family in which 3 fetuses had clinical features consistent with the autosomal recessive disorder Meckel-Gruber syndrome (MKS). Targeted NGS of 29 known MKS-associated genes revealed a heterozygous TMEM231 splice donor variant c.929+1A>G. Comparative read-depth analysis, performed to identify a second pathogenic allele, revealed an apparent heterozygous deletion of TMEM231 exon 4. To verify this result we performed single-molecule long-read sequencing of a long-range polymerase chain reaction product spanning this locus. We identified four missense variants that were absent from the short-read dataset due to the preferential mapping of variant-containing reads to a downstream TMEM231 pseudogene. Consistent with the parental segregation analysis, we demonstrate that the single-molecule long reads could be used to show that the variants are arranged in trans. Our experience shows that robust validation of apparent dosage variants remains essential to avoid the pitfalls of short-read sequencing and that new third-generation long-read sequencing technologies can already aid routine clinical care.

Determination of Pathogenicity of Breast Cancer 1 Gene Variants using the American College of Medical Genetics and Genomics and the Association for Molecular Pathology Guidelines.

ObjectivesMolecular diagnostic laboratories screen for mutations in disease-causing genes in order to confirm a clinical diagnosis. The classification of DNA variants as ‘pathogenic’ or ‘likely pathogenic’ mutations creates a workflow bottleneck, which becomes increasingly challenging as greater number of genes are screened. The classification challenge is also acute if there are conflicting reports regarding pathogenicity and differing classification criteria between laboratories. This study aimed to compare two procedures for the classification of variants in the breast cancer (BRCA)1 gene.MethodsThis bioinformatic study was conducted at LabPLUS, Auckland, New Zealand, from February to June 2017. DNA was extracted from peripheral blood samples of 30 patients and gene library construction was carried out using a commercially available targeted panel for the BRCA1 and BRCA2 genes. The genes were subsequently sequenced and the sequence data analysed. The guidelines published by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) provides a comprehensive framework for the interpretation of variants in genes that are associated with Mendelian disorders. The use of these guidelines were compared to the variant classifications that were achieved by reference to those reported in the BRCA Exchange database.ResultsThe results showed concordance between the two classification protocols for a panel of 30 BRCA1 gene variants, although the transparency in following the ACMG/AMP guidelines provides a diagnostic laboratory with a generalisable approach that allows laboratory-directed revisions to be undertaken in light of new information.ConclusionThe ACMG/AMP-based guidelines were applied to a cohort of patients with BRCA1 gene variants. The use of these guidelines provides a system which creates consistency in variant interpretation and supports subsequent clinical management.

Recurrent NUS1 canonical splice donor site mutation in two unrelated individuals with epilepsy, myoclonus, ataxia and scoliosis - a case report

BackgroundWe encountered two unrelated individuals suffering from neurological disorders, including epilepsy and scoliosis.Case presentationWhole-exome sequencing identified the same recurrent, de novo, pathogenic variant in NUS1 [NM_138459.4:c.691 + 1C > A] in both individuals. This variant is located in the conserved cis-prenyltransferase domain of the nuclear undecaprenyl pyrophosphate synthase 1 gene (NUS1), which encodes the Nogo-B receptor, an essential catalyst for protein glycosylation. This variant was confirmed to create a new splice donor site, resulting in aberrant RNA splicing resulting in a 91-bp deletion in exon 3 in both individuals. The mutant mRNA was partially degraded by nonsense mediated mRNA decay. To date, only four de novo variants and one homozygous variant have been reported in NUS1, which cause developmental and epileptic encephalopathy, early onset Parkinson’s disease, and a congenital disorder of glycosylation. Seven patients, including our two patients, have presented with epileptic seizures and intellectual disabilities.ConclusionsOur study strongly supports the finding that this recurrent, de novo, variant in NUS1 causes developmental and epileptic encephalopathy with involuntary movement, ataxia and scoliosis.

RARE LOSS-OF-FUNCTION VARIANT ANALYSIS IN LONE ATRIAL FIBRILLATION

Atrial fibrillation (AF) is a complex heritable disorder that substantially increases the risk for stroke, sudden death and all-cause mortality. Lone AF is a subset defined by early diagnosis (less than 60 years) and with no evidence of cardiopulmonary disease. Genome-wide association studies have associated loci near genes for structural integrity and function of muscles with AF susceptibility. In addition, some studies have identified rare loss-of-function (LoF) variants in dilated cardiomyopathy (DCM) genes in lone AF. The increased burden of these variants in lone AF patients may indicate a shared predisposition between AF and DCM. We performed a retrospective study on 252 adult patients and a control group of 503 healthy European subjects. Exome sequencing and microarray assays were performed at the McGill Genome Center while exome data from the controls was retrieved from 1KG Consortium. Principal component analysis was performed to define a group with common ancestry using SVS software. Variants within candidate DCM genes were filtered by minor allele frequency of ≤0.1%, a Combined Annotation Dependent Depletion (CADD) Phred score of >20 and a LoF variant type (frameshift, splice acceptor, splice donor and stop gain). These factors were retrieved from public annotation databases and applied using VarSeq software for variant analysis. All variants were confirmed with Sanger sequencing. Cascade screening was attempted on all cases positive for variants. The AF cohort, an ethnically homogenous composite of 181 lone AF cases, had 6 heterozygous LoF variants, including 4 novel variants, in 6 individuals, while the controls had 4 in 503 individuals. Half of variants in the cases were in the TTN gene (3/6). The prevalence of rare LoF variants in the lone AF cohort was 3.3% while in the control cohort 0.79%, the odds ratio of rare LoF variants found in lone AF patients compared to controls is 4.28 (95%CI: 1.19-15.33, P-value: 2.5E-02). Cascade screening in one family demonstrated co-segregation of one of the rare TTN variants and AF. Patients with lone AF carry a significant burden of heterozygous LoF variants in DCM genes. Our results are in agreement with a previous study identifying TTN variants in lone AF patients. Further, we identified novel variants in different DCM genes and confirmed the co-segregation of a TTN variant and AF in one family. Our findings may indicate a common genetic predisposition between lone AF and DCM, which may help elucidate novel pathophysiologic mechanisms, and improve diagnosis and treatment.

P.140RNA-Seq shows an absence of off-target splicing effects in AAV9-U7snRNA mediated skipping of DMD exon 2

Duplications of the DMD exon 2 account for 10% of all duplication mutations causing Duchenne muscular dystrophin. In the Dup2 mouse model, skipping of a duplicated exon can restore wild-type dystrophin, as we have shown by use of a viral-based approach using AAV9 vector containing four copies of a non-coding U7snRNA with additional sequences targeting the DMD exon 2 splice acceptor and donor sites (the scAAV9.U7.ACCA vector). Following systemic delivery of clinically relevant doses to Dup2 mice, we assessed whether this vector resulted in off-target exon skipping in other genes by comparing vector- and untreated (n=3 per group) at day 90 post-injection. Skeletal muscle, liver, and heart were used for RNA-Seq analysis using Illumina technology, and sequences were analyzed for differential gene expression and for detection of local splicing variation (LSV) using MAJIQ software. Splice junction quantification in skeletal and cardiac muscles showed robust skipping of DMD exon 2, confirming the general validity of quantifying treatment-induced alternative splicing from the mapped RNA-seq reads. Only 11 viable candidate off-target LSVs, mapping to 6 genes, were identified, and all were known alternative splicing events annotated in the Vertebrate Alternative Splicing and Transcription Database (VastDB), a large resource of alternative splicing events detected by RNA-seq from a diverse set of vertebrate cells and tissues. Only 1 splice event showed alterations in both skeletal and cardiac tissue, and binding energy prediction suggests that the alterations are indirect effect of treatment on alternative splicing instead of a direct off-target effect due to modified U7 snRNA–target pre-mRNA binding. Differential gene expression showed reversal of known dystrophic gene expression signatures, including the downregulation of transcripts consistently identified as observed as upregulated in skeletal muscle from dystrophic mice and humans. These results suggest that specific transcriptional responses are related to restoration of dystrophin levels due to exon 2 skipping in skeletal muscle and heart, rather than changes in mRNA levels due to off-target U7.ACCA snRNA interactions directly with differentially expressed transcripts.

P.160The spectrum of disease-causing and normal variation in the nebulin gene

The nebulin gene (NEB) with 183 exons is one of the largest genes in human. More than 240 different disease-causing variants in NEB have been identified in some 220 families. Around 50% of nemaline myopathies (NM) are caused by mutations in NEB. Mutations in NEB also cause core-rod myopathy, distal forms of NM, distal myopathy without nemaline rods, and multiple pterygium syndrome. The majority (95%) of the disease-causing variants in NEB are recessive point mutations, mainly nonsense and splice site mutations, and small indels, causing frameshifts. The remaining 5% of mutations are large copy number variations (CNVs). In addition to the recurrent CNVs in the triplicate region and the founder mutation with world-wide occurrence, deletion of exon 55, 11 different large pathogenic duplications and deletions have been identified in NEB. Until recently, all identified mutations in NEB have been recessive. Now it is known that large in-frame deletions in NEB, leading to the expression of substantially smaller proteins, can have a dominant-negative effect, and cause dominant distal forms of NM. The genome aggregation database (gnomAD) contains exome and genome data from 141,456 unrelated individuals, excluding patients with severe pediatric disorders. The total number of different NEB variants listed in gnomAD is 10,634. Most of the variants (96%) are very rare, only present in a few heterozygotes. Intronic variants comprise 43%, missense variants 40%, synonymous variants 14%, loss-of-function variants 3%, and UTR variants 0.2% of the total variation. It is noteworthy that 12.5% of the intronic variants are in the splice regions (excluding splice donor and acceptor sites), and may affect splicing. Rare variants are the most common variant type in NEB, complicating the interpretation of pathogenicity. Therefore, the pathogenicity of especially missense and splice site variants in NEB requires verification by functional analyses at the protein and mRNA level.

Novel synonymous and missense variants in FGFR1 causing Hartsfield syndrome

Hartsfield syndrome is a rare clinical entity characterized by holoprosencephaly and ectrodactyly with the variable feature of cleft lip/palate. In addition to these symptoms patients with Hartsfield syndrome can show developmental delay of variable severity, isolated hypogonadotropic hypogonadism, central diabetes insipidus, vertebral anomalies, eye anomalies, and cardiac malformations. Pathogenic variants in FGFR1 have been described to cause phenotypically different FGFR1-related disorders such as Hartsfield syndrome, hypogonadotropic hypogonadism with or without anosmia, Jackson-Weiss syndrome, osteoglophonic dysplasia, Pfeiffer syndrome, and trigonocephaly Type 1. Here, we report three patients with Hartsfield syndrome from two unrelated families. Exome sequencing revealed two siblings harboring a novel de novo heterozygous synonymous variant c.1029G>A, p.Ala343Ala causing a cryptic splice donor site in exon 8 of FGFR1 likely due to gonadal mosaicism in one parent. The third case was a sporadic patient with a novel de novo heterozygous missense variant c.1868A>G, p.(Asp623Gly).

A deep intronic mutation of c.1166-285 T > G in SLC46A1 is shared by four unrelated Japanese patients with hereditary folate malabsorption (HFM)

Hereditary folate malabsorption (HFM) is an autosomal recessive disease caused by mutations in SLC46A1 encoding the proton-coupled folate transporter (PCFT). HFM patients present with various clinical features including megaloblastic anemia, thrombocytopenia, combined immunodeficiency and neurodevelopmental disorders. In this study, we report the same deep intronic mutation of c.1166-285 T > G shared by four unrelated Japanese patients with HFM. This mutation was shown to generate a cryptic splice donor site for a 168-bp insertion of intron 3 sequences, leading to premature termination in the middle of this insertion. This mutation could be a founder mutation in the Japanese population, but also could be a hot-spot and could be present in undiagnosed HFM patients worldwide because of the difficulty to detect this mutation.

300 Assessment of Non-Canonical Splicing Variants in ABCC6 and Comparison with Bioinformatics Predictions

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, is caused by inactivating mutations in the ABCC6 gene predominantly expressed in hepatocytes. Different types of mutations have been identified in ABCC6 including canonical splice site mutations affecting pre-mRNA splicing. Non-canonical splicing variants near or within exon-intron boundary in ABCC6 are under-recognized due to the lack of liver biopsies from patients carrying such variants, and consequently, inability to determine their consequences on splicing by transcriptional analysis. The interpretation of such variants has primarily relied on the prediction by in silico algorithms; often such programs do not provide consensus predictions. Among seven non-canonical splicing variants identified in ABCC6 in patients with PXE, five variants were found to affect splicing in in vitro minigene splicing assays in transfected HepG2 cells. Four of the variants, c.1868-5T>G, c.2070+5G>A, c.3735G>A (p.E1245=), and c.3883-46A>G, caused skipping of the respective adjacent exon. One variant, c.3883-6G>A, created a new splicing donor site resulting in a 4-bp retention from the intronic region. The results provide experimental evidence that these non-canonical variants disrupt splicing, suggesting the presence of pathogenic mutations beyond the canonical splice sites, including silent variants in the coding region.

Combining Engineered U1 snRNA and Antisense Oligonucleotides to Improve the Treatment of a BBS1 Splice Site Mutation

Manipulation of pre-mRNA processing is a promising approach toward overcoming disease-causing mutations and treating human diseases. We show that a combined treatment applying two splice-manipulating technologies improves therapeutic efficacies to correct mutation-induced splice defects. Previously, we identified a family affected by retinitis pigmentosa caused by the homozygous BBS1 splice donor site mutation c.479G > A. The mutation leads to both exon 5 skipping and intron 5 retention. We developed a therapeutic approach applying lentivirus-mediated gene delivery of engineered U1 small nuclear RNA (U1), which resulted in increased levels of correctly spliced BBS1. Herein, we show that the therapeutic effect of the engineered U1 efficiently reverted exon skipping but failed to reduce the intron retention. To complement the engineered U1 treatment, we identified four different antisense oligonucleotides (AONs) that block intron 5 retention in BBS1 transcripts. A treatment using engineered U1 in combination with AONs showed the highest therapeutic efficacy and increased the amount of correctly spliced BBS1 transcripts. We did not detect elevated levels of apoptotic cell death in AON-treated cell lines. In conclusion, engineered U1 or AONs provide efficient therapies with complementary effects and can be combined to increase efficacy of therapeutic approaches to correct splice defects.

Mutation Spectrum of Cancer-Associated Genes in Patients With Early Onset of Colorectal Cancer.

Background: Colorectal cancer (CRC) incidence is rising worldwide, as well as in the Republic of Kazakhstan, while its occurrence is also increasing in the younger population. Hereditary forms associated with the development of colon and rectal cancer and early-onset CRC have never been studied in the population of Kazakhstan. The aim of this research was to investigate the spectrum of CRC-related gene mutations to determine which mutations cause early onset of CRC in the Kazakhstan population.Methods: The study included 125 unrelated patients from Kazakhstan (range 17–50 years in age) with early onset CRC. Genomic DNA was obtained from peripheral blood of the patients. Next-generation sequencing was performed using the TruSightCancer Kit on the MiSeq platform. The Studio Variant was used to annotate and interpret genetic variants.Results: Bioinformatics analysis of Next-generation sequencing data revealed 11,152 variants from 85 genes, of them, 3,790 missense, 6,254 synonymous variants, 44 3′UTR variants, 10 frameshift variants, five stop-gain variants, four in-frame deletions, two splice donors, one splice acceptor variant, and 1,042 intron or non-coding variants. APC, BRCA2/1, ALK, BRIP1, EGFR, FANCA, FANCD2, FANCI, HNF1A, MEN1, NSD1, PMS2, RECQL4, RET, SLX4, WRN, and XPC genes mutated most often. According to the ACMG guidelines and LOVD/ClinVar databases, 24 variants were pathogenic (10 frameshifts, five missenses, five stop-gain, one in-frame deletion, and three splice-site mutations), and 289 were VUS with population frequency <1%, 131 of them were attributed as deleterious. In the study, 50% of all pathogenic mutations found in Kazakhstani patients with early CRC onset were identified in the subgroups with a family history of CRC and primary multiple tumors. In APC, pathogenic mutations were most often (21%).Conclusion: Pathogenic and likely pathogenic mutations were found in 20 (16%) out of 125 patients. Eight novel pathogenic mutations detected in FANCI, APC, BMPR1, ATM, and DICER1 genes have not been reported in previous literature. Given the high frequency and wide spectrum of mutations, NGS analysis must be carried out in families with a history of CRC/CRC-related cancers with the purpose to identify cause-effective mutations, clarify the clinical diagnosis, and prevent the development of the disease in other family members.