Potential Splice Sites Research Articles

A novel cancer risk prediction score for the natural course of FA patients with biallelic BRCA2/FANCD1 mutations.

Biallelic germline mutations in BRCA2 occur in the Fanconi anemia (FA)-D1 subtype of the rare pediatric disorder, FA, characterized clinically by severe congenital abnormalities and a very high propensity to develop malignancies early in life. Clinical and genetic data from 96 FA-D1 patients with biallelic BRCA2 mutations were collected and used to develop a new cancer risk prediction score system based on the specific mutations in BRCA2. This score takes into account the location of frameshift/stop and missense mutations relative to exon 11 of BRCA2, which encodes the major sites for interaction with the RAD51 recombinase, and uses the MaxEnt and HBond splicing scores to analyze potential splice site perturbations. Among 75 FA-D1 patients with ascertained BRCA2 mutations, 66 patients developed 102 malignancies, ranging from one to three independent tumors per individual. The median age at the clinical presentation of peripheral embryonal tumors was 1.0, at the onset of hematologic malignancies 1.8 and at the manifestation of CNS tumors 2.7years, respectively. Patients who received treatment lived longer than those without. Using our novel scoring system, we could distinguish three distinct cancer risk groups among FA-D1 patients: in the first, patients developed their initial malignancy at a median age of 1.3years (n = 36, 95% CI = 0.9-1.8), in the second group at 2.3years (n = 17, 95% CI = 1.4-4.4) and in the third group at 23.0years (n = 22, 95% CI = 4.3-n/a). Therefore, this scoring system allows, for the first time, to predict the cancer manifestation of FA-D1 patients simply based on the type and position of the mutations in BRCA2.

Unpredicted Aberrant Splicing Products Identified in Postmortem Sudden Cardiac Death Samples.

Molecular screening for pathogenic mutations in sudden cardiac death (SCD)-related genes is common practice for SCD cases. However, test results may lead to uncertainty because of the identification of variants of unknown significance (VUS) occurring in up to 70% of total identified variants due to a lack of experimental studies. Genetic variants affecting potential splice site variants are among the most difficult to interpret. The aim of this study was to examine rare intronic variants identified in the exonic flanking sequence to meet two main objectives: first, to validate that canonical intronic variants produce aberrant splicing; second, to determine whether rare intronic variants predicted as VUS may affect the splicing product. To achieve these objectives, 28 heart samples of cases of SCD carrying rare intronic variants were studied. Samples were analyzed using 85 SCD genes in custom panel sequencing. Our results showed that rare intronic variants affecting the most canonical splice sites displayed in 100% of cases that they would affect the splicing product, possibly causing aberrant isoforms. However, 25% of these cases (1/4) showed normal splicing, contradicting the in silico results. On the contrary, in silico results predicted an effect in 0% of cases, and experimental results showed >20% (3/14) unpredicted aberrant splicing. Thus, deep intron variants are likely predicted to not have an effect, which, based on our results, might be an underestimation of their effect and, therefore, of their pathogenicity classification and family members’ follow-up.

RNA Analysis and Clinical Characterization of a Novel Splice Variant in the NSD1 Gene Causing Familial Sotos Syndrome.

BackgroundSotos syndrome is an autosomal dominant disorder characterized by overgrowth, macrocephaly, distinctive facial features and learning disabilities. Haploinsufficiency of the nuclear receptor SET domain-containing protein 1 (NSD1) gene located on chromosome 5q35 is the major cause of the syndrome. This syndrome shares characteristics with other overgrowth syndromes, which can complicate the differential diagnosis.MethodsGenomic DNA was extracted from peripheral blood samples of members of the same family and targeted exome analysis was performed. In silico study of the variant found by next-generation sequencing was used to predict disruption/creation of splice sites and the identification of potential cryptic splice sites. RNA was extracted from peripheral blood samples of patients and functional analyses were performed to confirm the pathogenicity.ResultsWe found a novel c.6463 + 5G>A heterozygous NSD1 gene pathogenic variant in a son and his father. Molecular analyses revealed that part of the intron 22 of NSD1 is retained due to the destruction of the splicing donor site, causing the appearance of a premature stop codon in the NSD1 protein.ConclusionsOur findings underline the importance of performing RNA functional assays in order to determine the clinical significance of intronic variants, and contribute to the genetic counseling and clinical management of patients and their relatives. Our work also highlights the relevance of using in silico prediction tools to detect a potential alteration in the splicing process.

Functional assays of non-canonical splice-site variants in inherited retinal dystrophies genes

Inherited retinal dystrophies are a group of disorders characterized by the progressive degeneration of photoreceptors leading to loss of the visual function and eventually to legal blindness. Although next generation sequencing (NGS) has revolutionized the molecular diagnosis of these diseases, the pathogenicity of some mutations casts doubts. After the screening of 208 patients with a panel of 117 genes, we obtained 383 variants that were analysed in silico with bioinformatic prediction programs. Based on the results of these tools, we selected 15 variants for their functional assessment. Therefore, we carried out minigene assays to unveil whether they could affect the splicing of the corresponding gene. As a whole, seven variants were found to induce aberrant splicing in the following genes: BEST1, CACNA2D4, PRCD, RIMS1, FSCN2, MERTK and MAK. This study shows the efficacy of a workflow, based on the association of the Minimum Allele Frequency, family co-segregation, in silico predictions and in vitro assays to determine the effect of potential splice site variants identified by DNA-based NGS. These findings improve the molecular diagnosis of inherited retinal dystrophies and will allow some patients to benefit from the upcoming gene-based therapeutic strategies.

Differences in SCN1A intronic variants result in diverse aberrant splicing patterns and are related to the phenotypes of epilepsy with febrile seizures

ObjectiveIntronic variants of the SCN1A gene are detected in patients with epilepsy with febrile seizures (EFS), which includes a series of phenotypes with different severities. However, the pathogenicity of intronic variants and their genotype-phenotype correlation remain under characterized. The purpose of this study was to determine the changes in mRNA splicing caused by SCN1A intronic variants associated with EFS and their association with phenotypes. MethodsFive SCN1A intronic variants detected in patients with focal epilepsy with antecedent febrile seizures plus (FEFS+) and Dravet syndrome (DS) were molecularly cloned. Through an in vitro minigene splicing assay, their influence on mRNA splicing was qualitatively and quantitatively compared and analyzed using reverse-transcription polymerase chain reaction (RT-PCR) and fluorescence quantitative PCR (Q-PCR). ResultsThe severe phenotype of DS-associated variants c.602 + 1G > A and c.4853-1G > C, which occurred in canonical splice sites of introns, caused exon skipping and little retention of full-length mRNA, while the milder phenotype of FEFS+-associated variants c.473 + 5G > A, c.473 + 5G > C and c.4853-25T > A, which occurred in potential splice sites or in deep intronic regions, presented partial exon skipping or intronic insertion and significantly higher retention of full-length mRNA at different levels. Full-length mRNA retention was negatively correlated with the location of intronic variants and phenotype severity. ConclusionThe different aberrant splicing patterns resulting from SCN1A intronic variants with different positions represent a potential molecular mechanism for phenotypic differences in EFS. This research provides valuable clues for functional studies on the pathogenicity of intronic variants and for the evaluation of genotype-phenotype correlations.

Characterization of Rheumatoid Arthritis Risk-Associated SNPs and Identification of Novel Therapeutic Sites Using an In-Silico Approach.

Single-nucleotide polymorphisms (SNPs) are reported to be associated with many diseases, including autoimmune diseases. In rheumatoid arthritis (RA), about 152 SNPs are reported to account for ~15% of its heritability. These SNPs may result in the alteration of gene expression and may also affect the stability of mRNA, resulting in diseased protein. Therefore, in order to predict the underlying mechanism of these SNPs and identify novel therapeutic sites for the treatment of RA, several bioinformatics tools were used. The damaging effect of 23 non-synonymous SNPs on proteins using different tools suggested four SNPs, including rs2476601 in PTPN22, rs5029941 and rs2230926 in TNFAIP3, and rs34536443 in TYK2, to be the most damaging. In total, 42 of 76 RA-associated intronic SNPs were predicted to create or abolish potential splice sites. Moreover, the analysis of 11 RA-associated UTR SNPs indicated that only one SNP, rs1128334, located in 3'UTR of ETS1, caused functional pattern changes in BRD-BOX. For the identification of novel therapeutics sites to treat RA, extensive gene-gene interaction network interactive pathways were established, with the identification of 13 potential target sites for the development of RA drugs, including three novel target genes. The anticipated effect of these findings on RA pathogenesis may be further validated in both in vivo and in vitro studies.

Bovine TMEM95 gene: Polymorphisms detecting in five Chinese indigenous cattle breeds and their association with growth traits

Background: Transmembrane protein 95 ( TMEM95 ) plays a role in male fertility. Previous studies showed that genes with a significant impact on reproductive traits can also affect the growth traits of livestock. Thus, we speculated that the genetic variation of TMEM95 gene may have effects on growth traits of cattle. Results: Two SNPs were genotyped. The rs136174626 and rs41904693 were in the intron 4 and 3′-untranslated region, respectively. The linkage disequilibrium analysis illustrated that these two loci were not linked. The rs136174626 was associated with six growth traits of Nanyang cattle, four traits of Luxi cattle, and three traits of Ji’an cattle. For rs41904693 locus, the GG individuals had greater body height and abdominal girth in Ji’ an cattle than TT and TG individuals. In Jinnan cattle, GG and TT individuals had greater body height, height at hip cross, body length, and heart girth than TG individuals. The potential splice site prediction results suggest that the rs136174626 may influence the splicing efficiency of TMEM95, and the miRNA binding site prediction results showed that the rs41904693 may influence the expression of TMEM95 by affecting the binding efficiency of Bta-miR-1584 and TMEM95 3′-UTR. Conclusions: The findings of the study suggested that the two SNPs in TMEM95 could be a reliable basis for molecular breeding in cattle. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Times New Roman; mso-bidi-theme-font:minor-bidi;}

Modifying splice site usage with ModCon: Maintaining the genetic code while changing the underlying mRNP code

Codon degeneracy of amino acid sequences permits an additional “mRNP code” layer underlying the genetic code that is related to RNA processing. In pre-mRNA splicing, splice site usage is determined by both intrinsic strength and sequence context providing RNA binding sites for splicing regulatory proteins. In this study, we systematically examined modification of splicing regulatory properties in the neighborhood of a GT site, i.e. potential splice site, without altering the encoded amino acids.We quantified the splicing regulatory properties of the neighborhood around a potential splice site by its Splice Site HEXplorer Weight (SSHW) based on the HEXplorer score algorithm. To systematically modify GT site neighborhoods, either minimizing or maximizing their SSHW, we designed the novel stochastic optimization algorithm ModCon that applies a genetic algorithm with stochastic crossover, insertion and random mutation elements supplemented by a heuristic sliding window approach.To assess the achievable range in SSHW in human splice donors without altering the encoded amino acids, we applied ModCon to a set of 1000 randomly selected Ensembl annotated human splice donor sites, achieving substantial and accurate changes in SSHW. Using ModCon optimization, we successfully switched splice donor usage in a splice site competition reporter containing coding sequences from FANCA, FANCB or BRCA2, while retaining their amino acid coding information.The ModCon algorithm and its R package implementation can assist in reporter design by either introducing novel splice sites, silencing accidental, undesired splice sites, and by generally modifying the entire mRNP code while maintaining the genetic code.

Discovery of driver non-coding splice-site-creating mutations in cancer

Non-coding mutations can create splice sites, however the true extent of how such somatic non-coding mutations affect RNA splicing are largely unexplored. Here we use the MiSplice pipeline to analyze 783 cancer cases with WGS data and 9494 cases with WES data, discovering 562 non-coding mutations that lead to splicing alterations. Notably, most of these mutations create new exons. Introns associated with new exon creation are significantly larger than the genome-wide average intron size. We find that some mutation-induced splicing alterations are located in genes important in tumorigenesis (ATRX, BCOR, CDKN2B, MAP3K1, MAP3K4, MDM2, SMAD4, STK11, TP53 etc.), often leading to truncated proteins and affecting gene expression. The pattern emerging from these exon-creating mutations suggests that splice sites created by non-coding mutations interact with pre-existing potential splice sites that originally lacked a suitable splicing pair to induce new exon formation. Our study suggests the importance of investigating biological and clinical consequences of noncoding splice-inducing mutations that were previously neglected by conventional annotation pipelines. MiSplice will be useful for automatically annotating the splicing impact of coding and non-coding mutations in future large-scale analyses.

Implications of SARS-CoV-2 Mutations for Genomic RNA Structure and Host microRNA Targeting.

The SARS-CoV-2 virus is a recently-emerged zoonotic pathogen already well adapted to transmission and replication in humans. Although the mutation rate is limited, recently introduced mutations in SARS-CoV-2 have the potential to alter viral fitness. In addition to amino acid changes, mutations could affect RNA secondary structure critical to viral life cycle, or interfere with sequences targeted by host miRNAs. We have analysed subsets of genomes from SARS-CoV-2 isolates from around the globe and show that several mutations introduce changes in Watson–Crick pairing, with resultant changes in predicted secondary structure. Filtering to targets matching miRNAs expressed in SARS-CoV-2-permissive host cells, we identified ten separate target sequences in the SARS-CoV-2 genome; three of these targets have been lost through conserved mutations. A genomic site targeted by the highly abundant miR-197-5p, overexpressed in patients with cardiovascular disease, is lost by a conserved mutation. Our results are compatible with a model that SARS-CoV-2 replication within the human host is constrained by host miRNA defences. The impact of these and further mutations on secondary structures, miRNA targets or potential splice sites offers a new context in which to view future SARS-CoV-2 evolution, and a potential platform for engineering conditional attenuation to vaccine development, as well as providing a better understanding of viral tropism and pathogenesis.

Novel deep intronic mutation in the coagulation factor XIII a chain gene leading to unexpected RNA splicing in a patient with factor XIII deficiency

BackgroundCoagulation factor XIII (FXIII) plays an essential role in maintaining hemostasis by crosslinking fibrin. Deficiency in FXIII affects clot stability and increases the risk of severe bleeding. Congenital FXIII deficiency is a rare disease. Recently, we identified a Chinese family with FXIII deficiency and investigated the pathogenesis of congenital FXIII deficiency, contributing non-coding pathogenic variants.MethodsWe performed common tests, coding sequencing by targeted next-generation sequencing (NGS), whole-genome sequencing and splice-sites prediction algorithms. The pathogenesis was investigated via minigene and nonsense-mediated mRNA decay (NMD) by experiments in vitro.ResultsThe proband is homozygote for a novel deep intronic c.799-12G > A mutation in the F13A1 gene. Through direct sequencing of the minigenes mRNA, we found 10 bases of intron 6 insert in the mRNA of mutant minigenes mRNA. The relative expression of EGFP-F13A1 was higher by suppression of NMD in vitro. Furthermore, we found the proband with enhanced thrombin generation (TG).ConclusionWe reported a novel deep intronic c.799-12G > A mutation of F13A1 which produced a new acceptor site and frame shifting during translation introducing a premature termination codon. Our results support the premature termination codon triggered NMD. We need to pay attention to the position of potential alterable splicing sites while counselling and genetic test. The finding of enhanced TG indicated that we should be aware of the risk of thrombosis in patients with FXIII deficiency during replacement therapy.

Abstract GMM-025: ERBB FAMILY GENOMIC ALTERATIONS IN GYNECOLOGICAL MALIGNANCIES

Abstract Activation of HER2 or EGFR signaling is a common, targetable oncogenic mechanism in solid tumors such as breast, gastroesophageal, lung and colorectal carcinomas. In addition, ERBB3 activation has been shown to confer sensitivity to HER2-targeted therapies, and ERBB4 mutation is reported a mechanism of resistance. From a database of 14,692 comprehensive genomic profiles for both common and rare gynecological malignancies, we extracted 9,121 ovarian tumor profiles and evaluated the prevalence of genomic alterations (GA) in the ERBB family (EGFR, ERBB2, ERBB3, ERBB4), including base substitutions, small indels, amplification (AMP), and rearrangements (RE). At least 50ng of DNA was extracted from FFPE specimens and sequenced to high, uniform median coverage (>500X) by hybridization capture of exons from up to 406 cancer-related genes and select introns from up to 31 genes commonly rearranged in cancer. For some samples, RNA sequencing of 265 genes was also performed. Tumor mutational burden (TMB, mutations/Mb [mut/Mb]) was determined on ~1.1 Mbp of sequenced DNA, and microsatellite instability (MSI) and overall genomic loss of heterozygosity was evaluated for subsets of the samples. Of 9,121 tumors, most were serous carcinomas (n = 6,847). A total of 615 (6.7%) tumors had ≥1 oncogenic GA in an ERBB gene, with 40 (0.4%) having ≥2 ERBB GA. ERBB+ were most often mucinous (15.9%) or clear cell (13.9%) carcinomas. Serous carcinomas were ERBB+ in 4.3-7.1% of samples. Patient age was comparable in both the ERBB+ and ERBB- tumors (median 61 y vs. 61 y, range 19-94 y vs. 2-95 y). ERBB2 (HER2) AMP was the most common ERBB GA; 2.7% of serous carcinomas had amplified ERBB2. GA distribution by ERBB gene was as follows: ERBB2 4.5% (n=394; of those AMP were 78.4%, n=309), ERBB3 0.8% (n=87; AMP 50.6%, n=44), EGFR 0.9% (n=90; AMP 45.6%, n=41), and ERBB4 0.6% (n=66; AMP 65.1%, n=43). Known or potential oncogenic rearrangements, fusions, and splice site GA were observed in both ERBB2 (n=20) and EGFR (n=10), including a kinase domain duplication in EGFR and exon 16 deletions in ERBB2. Although ERBB2 AMP is concordant with HER2+ by IHC or FISH, 70/615 (11.4%) HER2-driven samples harbored targetable, oncogenic GA that would not be detected by these methods, including the well-characterized A775_G776insYVMA (n=13), S310Y/F (n=10), and V842I (n=8). In ERBB+ tumors, the most commonly co-occurring GA affected TP53 (76.6%), PIK3CA (19.5%), MYC (15.7%), TERC (14.6%), ARID1A (18.3%), and CCNE1 (20.5%). Pathways likely to confer some level of resistance to ERBB-targeted therapies were often mutated: PI3K-AKT-MTOR 35.0% (n=215), RAS-RAF-MEK 18.2% (n=112), and FGFR 5.5% (n=34). ERBB+ tumors had slightly higher TMB scores: median 3.5 mut/Mb vs 2.6 mut/Mb, and average 6.7 vs 3.7 mut/Mb. Of 502 samples analyzed for microsatellite instability, 12 were MSI high. In conclusion, nearly 7% of ovarian tumors harbor potentially targetable GA in an ERBB family gene, including HER2 and EGFR. Of these, 49.6% of samples would not be positive by HER2 IHC or FISH. Further exploration into the efficacy of HER2- or EGFR-targeted therapies, and the effects of co-altered pathways, in ovarian carcinomas may be promising. Citation Format: Laurie M Gay, Jo-Anne Vergilio, J Keith Killian, Shakti Ramkissoon, Eric Severson, Sugganth Daniel, Amanda Hemmerich, Jeffrey S Ross, Julia A Elvin. ERBB FAMILY GENOMIC ALTERATIONS IN GYNECOLOGICAL MALIGNANCIES [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-025.

246-LB: Rare Noncoding Intronic HNF1A Sequence Variants Cause Aberrant mRNA Processing in MODY

The transcription factor Hepatocyte nuclear factor-1α (HNF1A) is important for normal pancreas development and function. Some HNF1A genetic variants are associated with type 2 diabetes, others can cause Maturity-Onset Diabetes of the Young (MODY3). Since variations in the HNF1A gene give different clinical consequences, novel sequence variants require thorough clinical and functional analysis. Here we investigate, in vitro, the consequence of rare non-coding intronic sequence variants of HNF1A from the Norwegian MODY registry. To evaluate the effect of four potential splice site mutations, fragments of intron/exon/intron genomic DNA from patient blood was amplified and cloned between two exons in a pCAS2 splicing minigene vector. Total RNA was isolated from HeLa and HepG2 cells transfected with minigene plasmids (wt or mutant) and analyzed by RT-PCR followed by agarose gels and sanger sequencing. All selected variants showed alternative mRNA splicing compared to wt by the minigene assay. The major splicing event of mutant c.526+1G>A showed the activation of a cryptic donor site, causing loss of the last 31 nucleotides of exon 2. In mutant c.714-1G>A, the major splicing event showed loss of 38 nucleotides in exon 4, due to activation of a novel cryptic acceptor site. In the event of c.956-2A>G, sequencing showed a complete skipping of the subsequent exon 5. The c.1502-6G>A variant causes activation of a cryptic acceptor site in intron 7 which leads to a four-nucleotide retention of the intronic sequence. The consequences of all variants were additionally evaluated using in silico splice site predictions by the integrated software tool Alamut v.2.6. Predictions of cryptic sites and the in vitro nucleotide changes observed were fully in agreement. These four intronic HNF1A variants likely cause MODY3 due to abnormally spliced mRNAs. By combining in silico predictions and the pCAS2 minigene expression system, we here provide further evidence of the variants role as pathogenic MODY3 mutations. Disclosure M.H. Solheim: None. M. Nasrollahzadeh Khakiani: None. J. Molnes: None. I. Aukrust: None. P. Njølstad: None. S. Johansson: None.

Blepharospasm: A genetic screening study in 132 patients

IntroductionBlepharospasm is a common type of focal dystonia that involves involuntary eyelid spasms and eye closure. In familial cases, an autosomal dominant pattern of inheritance is noted with reduced penetrance. Few genes have been associated with the disease including GNAL and CIZ1. A whole exome sequencing study published lately suggested TOR2A and REEP4 as potential candidate genes. MethodsSanger sequencing of GNAL, CIZ1, TOR2A and REEP4 exons including exon-intron boundaries in 132 patients diagnosed primarily with blepharospasm and/or Meige's syndrome. ResultsAll variants detected in GNAL, CIZ1 and TOR2A seem to be benign. Sequencing of REEP4 revealed the presence of two nonsynonymous SNVs, one potential splice site variant and one indel all predicted to be damaging by in silico algorithms. ConclusionSequencing REEP4 in larger blepharospasm cohorts and functional studies will need to be performed to further elucidate the association between REEP4 and the disease.

Characterization of the IVS-II-821 (A>C) (HBB: c.316-30A>C) Mutation in a β-Thalassemia Phenotype in Iran

β-Thalassemia (β-thal) is the most frequently observed hereditary blood disorder that results from genetic defects causing deficient synthesis of hemoglobin (Hb) polypeptide chains. Detecting thalassemia mutations are necessary for prenatal diagnosis (PND) programs leading a better quality of life for the patients, as well as a reduction in the cost of their medical care. There are more than 900 different genomic mutations of the β-globin gene described in the human hemoglobin variant (HbVar) database. In this study, we identified a mid-intronic mutation at IVS-II-821 (A>C) (HBB: c.316-30A>C) position in the HBB gene of an Iranian proband and two of her siblings that was associated with β-thal clinical features. Direct DNA sequence analysis was performed by mutation scanning of the β-globin gene. Based on the observed β-thal phenotype and bioinformatics analysis results, we concluded that this β-globin gene mutation was associated with a mild phenotype of β-thal through activating potential splice sites by creating exonic splicing enhancers (ESEs), exon-identity element (EIE) and exonic splicing regulatory sequences (ESRs) sites.

Unraveling the effect of silent, intronic and missense mutations on VWF splicing: contribution of next generation sequencing in the study of mRNA.

Large studies in von Willebrand disease patients, including Spanish and Portuguese registries, led to the identification of >250 different mutations. It is a challenge to determine the pathogenic effect of potential splice site mutations on VWF mRNA. This study aimed to elucidate the true effects of 18 mutations on VWF mRNA processing, investigate the contribution of next-generation sequencing to in vivo mRNA study in von Willebrand disease, and compare the findings with in silico prediction. RNA extracted from patient platelets and leukocytes was amplified by RT-PCR and sequenced using Sanger and next generation sequencing techniques. Eight mutations affected VWF splicing: c.1533+1G>A, c.5664+2T>C and c.546G>A (p.=) prompted exon skipping; c.3223-7_3236dup and c.7082-2A>G resulted in activation of cryptic sites; c.3379+1G>A and c.7437G>A) demonstrated both molecular pathogenic mechanisms simultaneously; and the p.Cys370Tyr missense mutation generated two aberrant transcripts. Of note, the complete effect of three mutations was provided by next generation sequencing alone because of low expression of the aberrant transcripts. In the remaining 10 mutations, no effect was elucidated in the experiments. However, the differential findings obtained in platelets and leukocytes provided substantial evidence that four of these would have an effect on VWF levels. In this first report using next generation sequencing technology to unravel the effects of VWF mutations on splicing, the technique yielded valuable information. Our data bring to light the importance of studying the effect of synonymous and missense mutations on VWF splicing to improve the current knowledge of the molecular mechanisms behind von Willebrand disease. clinicaltrials.gov identifier:02869074.

Clinical and genetic analysis of two unrelated patients with Angelman syndrome and novel UBE3A mutations

To explore the genetic cause for two familial Angelman syndrome cases and correlation between the clinical phenotypes and their genetic basis. Karyotyping analysis and microarray assay were carried out to exclude chromosome anomalies and uniparental disomy. The UBE3A gene was analyzed for potential point mutations, deletions, insertions and splice site mutations. Reverse transcription PCR was used to evaluate splicing mutation of the RNA transcripts. DNA sequencing showed the proband of family 1 has carried a novel maternal UBE3A splice acceptor site mutation, resulting in a guanine-to-cytosine transversion (IVS15-1G>C). Reverse transcription PCR revealed the proband and his mother both carried heterozygous mutant transcripts with loss of 54 and 59 nucleotides in exon 16, respectively. The proband displayed severe mental retardation, ataxia, seizures and inappropriate laughter. The siblings of family 2 has carried a novel maternal missense mutation in exon 16 of the UBE3A gene (c.2540C>T). She also presented with mental retardation, absent speech, mild ataxia and inappropriate laughter. The novel IVS15-1G>C and c.2540 C>T mutations of the UBE3A gene probably underlie the AS in the two families. Compared with small-scale mutations, larger fragments mutations can produce more severe phenotypes.

X-linked Alport syndrome associated with a synonymous p.Gly292Gly mutation alters the splicing donor site of the type IV collagen alpha chain 5 gene.

X-linked Alport syndrome (XLAS) is a progressive hereditary nephropathy caused by mutations in the type IV collagen alpha chain 5 gene (COL4A5). Although many COL4A5 mutations have previously been identified, pathogenic synonymous mutations have not yet been described. A family with XLAS underwent mutational analyses of COL4A5 by PCR and direct sequencing, as well as transcript analysis of potential splice site mutations. In silico analysis was also conducted to predict the disruption of splicing factor binding sites. Immunohistochemistry (IHC) of kidney biopsies was used to detect α2 and α5 chain expression. We identified a hemizygous point mutation, c.876A>T, in exon 15 of COL4A5 in the proband and his brother, which is predicted to result in a synonymous amino acid change, p.(Gly292Gly). Transcript analysis showed that this mutation potentially altered splicing because it disrupted the splicing factor binding site. The kidney biopsy of the proband showed lamellation of the glomerular basement membrane (GBM), while IHC revealed negative α5(IV) staining in the GBM and Bowman's capsule, which is typical of XLAS. This is the first report of a synonymous COL4A5 substitution being responsible for XLAS. Our findings suggest that transcript analysis should be conducted for the future correct assessment of silent mutations.

Molecular characterization of ten F8 splicing mutations in RNA isolated from patient's leucocytes: assessment of in silico prediction tools accuracy.

Although 8% of reported FVIII gene (F8) mutations responsible for haemophilia A (HA) affect mRNA processing, very few have been fully characterized at the mRNA level and/or systematically predicted their biological consequences by in silico analysis. This study is aimed to elucidate the effect of potential splice site mutations (PSSM) on the F8 mRNA processing, investigate its correlation with disease severity, and assess their concordance with in silico predictions. We studied the F8 mRNA from 10 HA patient's leucocytes with PSSM by RT-PCR and compared the experimental results with those predicted in silico. The mRNA analysis could explain all the phenotypes observed and demonstrated exon skipping in six cases (c.222G>A, c.601+1delG, c.602-11T>G, c.671-3C>G, c.6115+9C>G and c.6116-1G>A) and activation of cryptic splicing sites, both donor (c.1009+1G>A and c.1009+3A>C) and acceptor sites (c.266-3delC and c.5587-1G>A). In contrast, the in silico analysis was able to predict the score variation of most of the affected splice site, but the precise mechanism could only be correctly determined in two of the 10 mutations analysed. In addition, we have detected aberrant F8 transcripts, even in healthy controls, so this must be taken into account as they could mask the actual contribution of some PSSM. We conclude that F8 mRNA analysis using leucocytes still constitutes an excellent approach to investigate the transcriptional effects of the PSSM in HA, whereas prediction in silico is not always reliable for diagnostic decision-making.

In silico prediction of splice-altering single nucleotide variants in the human genome.

In silico tools have been developed to predict variants that may have an impact on pre-mRNA splicing. The major limitation of the application of these tools to basic research and clinical practice is the difficulty in interpreting the output. Most tools only predict potential splice sites given a DNA sequence without measuring splicing signal changes caused by a variant. Another limitation is the lack of large-scale evaluation studies of these tools. We compared eight in silico tools on 2959 single nucleotide variants within splicing consensus regions (scSNVs) using receiver operating characteristic analysis. The Position Weight Matrix model and MaxEntScan outperformed other methods. Two ensemble learning methods, adaptive boosting and random forests, were used to construct models that take advantage of individual methods. Both models further improved prediction, with outputs of directly interpretable prediction scores. We applied our ensemble scores to scSNVs from the Catalogue of Somatic Mutations in Cancer database. Analysis showed that predicted splice-altering scSNVs are enriched in recurrent scSNVs and known cancer genes. We pre-computed our ensemble scores for all potential scSNVs across the human genome, providing a whole genome level resource for identifying splice-altering scSNVs discovered from large-scale sequencing studies.