Splice Site Consensus Sequence Research Articles

P1065: CONGENITAL ERYTHROCYTOSIS DUE TO HETEROZYGOUS VARIANTS IN THE BISPHOSPHOGLYCERATE MUTASE GENE

Background: Congenital erythrocytosis is an inherited condition characterized by increased red blood cell mass, reflected by persistently elevated hemoglobin (Hb) and hematocrit levels. Erythrocytosis can be primary due to an intrinsic defect of erythropoiesis, or secondary, including germline variants in genes involved in the oxygen-sensing pathway and Hb variants with altered oxygen affinity. A very rare cause involves variants in bisphosphoglyerate mutase (BPGM), which regulates the red blood cell concentration of 2,3-bisphosphoglycerate (2,3-BPG), an important modifier of Hb-oxygen affinity. Aims: We report three European families with erythrocytosis and partial BPGM deficiency due to heterozygosity for variants in the BPGM gene, including one novel variant. Methods: The study was performed on adults of three families with JAK2-negative erythrocytosis. DNA analysis concerned sequencing of relevant coding exons, including flanking splice-site consensus sequences, of the eight major genes involved in secondary congenital erythrocytosis (EPOR, HBB, HBA1, HBA2, VHL, EPAS1, ELGN1, and BPGM). Identified variants were evaluated using Ensembl, gnomAD, PolyPhen-2, and SIFT. BPGM enzyme activity was determined as described by Beutler. Quantitative analysis of 2,3-BPG was performed using liquid chromatography tandem mass spectrometry. Hb-oxygen affinity, reflected by p50 levels (the oxygen pressure when Hb is 50% saturated with oxygen), was analyzed using the Hemox Analyzer (TCS Scientific). Results: In the first family (Table 1), a novel missense variant (c.535C>T p.(Arg179Cys) was identified in BPGM. Both the 65-year-old mother with complaints of headaches, and her 39-year-old son with a medical history of retinal vein occlusion were heterozygous for this variant. Allele frequency in gnomAD was 3.98-5. The predicted effect ranged from either deleterious (SIFT) to benign (HumVar model of Polyphen-2). Subsequent functional analysis showed low-normal BPGM activity, whereas 2,3-BPG and p50 levels were decreased (Table 1). In the second and third families, three male individuals were heterozygous for the c.269G>A p.(Arg90His) missense variant in BPGM. This variant was only once previously reported, and has an allele frequency of 1.06-5. All affected subjects had mild erythrocytosis without any additional clinical features, and showed decreased BPGM activity, decreased 2,3-BPG levels and decreased p50 (Table 1). Image:Summary/Conclusion: We describe five individuals from three families in which heterozygosity for missense variants in BPGM is associated with congenital erythrocytosis. Our findings further strengthen the hypothesis of an autosomal dominant inheritance pattern of BPGM variants. Our data also demonstrate the added value of functional analyses to validate gene panel and in silico analysis results for their clinical relevance. Future cases may help to interpret the impact of BPGM variants on hematological and clinical phenotype.

New insights into minor splicing-a transcriptomic analysis of cells derived from TALS patients.

Minor intron splicing plays a central role in human embryonic development and survival. Indeed, biallelic mutations in RNU4ATAC, transcribed into the minor spliceosomal U4atac snRNA, are responsible for three rare autosomal recessive multimalformation disorders named Taybi–Linder (TALS/MOPD1), Roifman (RFMN), and Lowry–Wood (LWS) syndromes, which associate numerous overlapping signs of varying severity. Although RNA-seq experiments have been conducted on a few RFMN patient cells, none have been performed in TALS, and more generally no in-depth transcriptomic analysis of the ∼700 human genes containing a minor (U12-type) intron had been published as yet. We thus sequenced RNA from cells derived from five skin, three amniotic fluid, and one blood biosamples obtained from seven unrelated TALS cases and from age- and sex-matched controls. This allowed us to describe for the first time the mRNA expression and splicing profile of genes containing U12-type introns, in the context of a functional minor spliceosome. Concerning RNU4ATAC-mutated patients, we show that as expected, they display distinct U12-type intron splicing profiles compared to controls, but that rather unexpectedly mRNA expression levels are mostly unchanged. Furthermore, although U12-type intron missplicing concerns most of the expressed U12 genes, the level of U12-type intron retention is surprisingly low in fibroblasts and amniocytes, and much more pronounced in blood cells. Interestingly, we found several occurrences of introns that can be spliced using either U2, U12, or a combination of both types of splice site consensus sequences, with a shift towards splicing using preferentially U2 sites in TALS patients’ cells compared to controls.

COSSMO: predicting competitive alternative splice site selection using deep learning

MotivationAlternative splice site selection is inherently competitive and the probability of a given splice site to be used also depends on the strength of neighboring sites. Here, we present a new model named the competitive splice site model (COSSMO), which explicitly accounts for these competitive effects and predicts the percent selected index (PSI) distribution over any number of putative splice sites. We model an alternative splicing event as the choice of a 3′ acceptor site conditional on a fixed upstream 5′ donor site or the choice of a 5′ donor site conditional on a fixed 3′ acceptor site. We build four different architectures that use convolutional layers, communication layers, long short-term memory and residual networks, respectively, to learn relevant motifs from sequence alone. We also construct a new dataset from genome annotations and RNA-Seq read data that we use to train our model.ResultsCOSSMO is able to predict the most frequently used splice site with an accuracy of 70% on unseen test data, and achieve an R2 of 0.6 in modeling the PSI distribution. We visualize the motifs that COSSMO learns from sequence and show that COSSMO recognizes the consensus splice site sequences and many known splicing factors with high specificity.Availability and implementationModel predictions, our training dataset, and code are available from http://cossmo.genes.toronto.edu.Supplementary information Supplementary data are available at Bioinformatics online.

Novel diagnostic tool for prediction of variant spliceogenicity derived from a set of 395 combined in silico/in vitro studies: an international collaborative effort.

Variant interpretation is the key issue in molecular diagnosis. Spliceogenic variants exemplify this issue as each nucleotide variant can be deleterious via disruption or creation of splice site consensus sequences. Consequently, reliable in silico prediction of variant spliceogenicity would be a major improvement. Thanks to an international effort, a set of 395 variants studied at the mRNA level and occurring in 5′ and 3′ consensus regions (defined as the 11 and 14 bases surrounding the exon/intron junction, respectively) was collected for 11 different genes, including BRCA1, BRCA2, CFTR and RHD, and used to train and validate a new prediction protocol named Splicing Prediction in Consensus Elements (SPiCE). SPiCE combines in silico predictions from SpliceSiteFinder-like and MaxEntScan and uses logistic regression to define optimal decision thresholds. It revealed an unprecedented sensitivity and specificity of 99.5 and 95.2%, respectively, and the impact on splicing was correctly predicted for 98.8% of variants. We therefore propose SPiCE as the new tool for predicting variant spliceogenicity. It could be easily implemented in any diagnostic laboratory as a routine decision making tool to help geneticists to face the deluge of variants in the next-generation sequencing era. SPiCE is accessible at (https://sourceforge.net/projects/spicev2-1/).

Characterization of a rare variant (c.2635-2A>G) of the MSH2 gene in a family with Lynch syndrome.

Lynch syndrome is caused by germline mutations in one of the mismatch repair genes ( MLH1, MSH2, MSH6, and PMS2) or in the EPCAM gene. Lynch syndrome is defined on the basis of clinical, pathological, and genetic findings. Accordingly, the identification of predisposing genes allows for accurate risk assessment and tailored screening protocols. Here, we report a family case with three family members manifesting the Lynch syndrome phenotype, all of which harbor the rare variant c.2635-2A>G affecting the splice site consensus sequence of intron 15 of the MSH2 gene. This mutation was previously described only in one family with Lynch syndrome, in which mismatch repair protein expression in tumor tissues was not assessed. In this study, we report for the first time the molecular characterization of the MSH2 c.2635-2A>G variant through in silico prediction analysis, microsatellite instability, and mismatch repair protein expression experiments on tumor tissues of Lynch syndrome patients. The potential effect of the splice site variant was revealed by three splicing prediction bioinformatics tools, which suggested the generation of a new cryptic splicing site. The potential pathogenic role of this variant was also revealed by the presence of microsatellite instability and the absence of MSH2/MSH6 heterodimer protein expression in the tumor cells of cancer tissues of the affected family members. We provide compelling evidence in favor of the pathogenic role of the MSH2 variant c.2635-2A>G, which could induce an alteration of the canonical splice site and consequently an aberrant form of the protein product (MSH2).

Search for Hidden Germline Mutations in Bilateral Retinoblastoma

Retinoblastoma (RB) is a cancer in the retina that develops in children usually under the age of five and can cause blindness, loss of the affected eye, or death by metastasis, if not diagnosed and treated early. It arises as a result of bi‐allelic inactivation of the RB1 gene in a retinal cell that prevents the production of the pRB protein, causing uncontrollable cell proliferation in the retina. The Genetic Diagnostic Laboratory in the Perelman School of Medicine at the University of Pennsylvania has used Sanger sequencing to identify disease‐causing mutations in the RB1 gene in thousands of individuals diagnosed with bilateral retinoblastoma. Current testing focuses on coding regions and splice site consensus sequences, and various mutations have been found in 97% of individuals tested. This study aimed to identify pathogenic exonic or deep intronic mutations in the germline of six individuals which belong to the 3% of mutation‐negative cases. For three of these individuals, RNA isolated from the lymphocytes of the whole blood was converted into cDNA, RB1 transcript amplified with gene specific primers and sequenced by Sanger sequencing. The cDNA of RB1 had indicated one sample had a heterozygous deletion of exon 10, but the cause of the exonic deletion was unknown. To investigate this, several primer pairs were designed to amplify and sequence regions flanking the deleted region in the genomic DNA, and an inversion between exons 10 and 11 was suspected. For the other two patients, who are sisters, an exonic deletion of exon 2 was found but proven to be benign. For the other three individuals, previous Sanger sequencing of exons had not indicated the presence of any pathogenic mutations. An AmpliSeq panel of three hundred and four primer pairs was designed to amplify and sequence both coding and non‐coding regions spanning the RB1 gene. Amplicons were prepared using genomic DNA isolated from these three individuals and next generation sequencing on the Ion Torrent PGM platform was performed. Upon analysis, three pathogenic mutations present at low allele frequencies were identified in each individual. These were low frequency mosaic mutations that may not be detected through Sanger sequencing. Therefore, the AmpliSeq design panel can provide information about intronic mutations and low frequency mosaic mutations, and it can serve as an assay complimentary to Sanger sequencing when investigating missing mutations in RB1 gene.Support or Funding InformationJennifer Richards‐Yutz, Tappan Ganguly, Eric Toorens, Jennifer Rosado, Richard Grant, Kimberly Moran, Arupa Ganguly. Genetic Diagnostic Laboratory at the University of Pennsylvania, Philadelphia, PAThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

OR24 Alternative splicing of HLA-C adds another layer of polymorphism to the hyperpolymorphic HLA genes

Aim Based on DNA sequencing the Human Leukocyte Antigen (HLA) gene system is the most polymorphic one amongst the human genome. For many genes it is known that isoforms can be present due to alternative splicing. In the IPD-IMGT/HLA database, a number of structural splice variants for HLA have been described and annotated, based on DNA polymorphism in the splice site consensus sequence. But data on mRNA transcript sequences are still lacking. The lack of RNA/cDNA SBT analysis thus underestimates the actual number of HLA splice variants. In the present study, we performed cDNA sequencing to study alternative splicing of HLA-C. Methods RNA was isolated from fresh peripheral blood mononuclear cells of healthy individuals and from B cell lines using the Qiagen RNeasy kit, followed by cDNA synthesis with the Superscript III first-strand synthesis system from Thermo Fisher Scientific. Sanger Sequencing was performed after HLA-C group-specific amplification of cDNA. Twelve of the fourteen different HLA-C allele groups were studied, C * 17 and C * 18 were not available. Results Sequencing of HLA-C revealed the presence of different mRNA splice variants. One of the most intriguing splice variants involved exon 5 skipping. This mRNA variant is co-expressed together with the mature spliced transcript. The presence of this variant is depending on the allele group, implicating a role for polymorphism in this alternative splicing process. Furthermore, since exon 5 encodes the transmembrane region, the translation of this mRNA variant might result in soluble HLA-C molecules. Conclusions Some HLA-C allele groups show alternative splicing resulting in exon 5 skipping. The functional implication of this exon 5 skipping on the HLA-C expression and the influence on T and NK cell recognition is further investigated.

A novel splicing mutation in the albumin gene (c.270+1G>T) causes analbuminaemia in a German infant.

Congenital analbuminaemia is a rare autosomal recessive disorder manifested by the presence of a very low amount of circulating serum albumin. The clinical diagnosis may be challenging because of the absence of unambiguous symptoms and because hypoalbuminemia may have many causes different from a genetic lack of the protein. We describe the clinical and molecular characterization of a new case of congenital analbuminaemia in an infant of apparently non-consanguineous parents from Treves, Germany. For molecular diagnosis, we used our strategy, based on the screening of the albumin gene by single-strand conformation polymorphism, heteroduplex analysis and direct DNA sequencing, which revealed that the proband is homozygous and both parents are heterozygous, for a novel G > T transversion at nucleotide c.270+ 1, the first base of intron 3. The mutation inactivates the strongly conserved GT dinucleotide at the 5' splice site consensus sequence of this intron. In conclusion, we report the clinical findings and the molecular defect of this case, which contributes to a better understanding of the biological mechanism of congenital analbuminaemia.

Dinoflagellate Gene Structure and Intron Splice Sites in a Genomic Tandem Array.

Dinoflagellates are one of the last major lineages of eukaryotes for which little is known about genome structure and organization. We report here the sequence and gene structure of a clone isolated from a cosmid library which, to our knowledge, represents the largest contiguously sequenced, dinoflagellate genomic, tandem gene array. These data, combined with information from a large transcriptomic library, allowed a high level of confidence of every base pair call. This degree of confidence is not possible with PCR‐based contigs. The sequence contains an intron‐rich set of five highly expressed gene repeats arranged in tandem. One of the tandem repeat gene members contains an intron 26,372 bp long. This study characterizes a splice site consensus sequence for dinoflagellate introns. Two to nine base pairs around the 3′ splice site are repeated by an identical two to nine base pairs around the 5′ splice site. The 5′ and 3′ splice sites are in the same locations within each repeat so that the repeat is found only once in the mature mRNA. This identically repeated intron boundary sequence might be useful in gene modeling and annotation of genomes.

The low information content of Neurospora splicing signals: implications for RNA splicing and intron origin.

When we expressed a small (0.9 kb) nonprotein-coding transcript derived from the mitochondrial VS plasmid in the nucleus of Neurospora we found that it was efficiently spliced at one or more of eight 5′ splice sites and ten 3′ splice sites, which are present apparently by chance in the sequence. Further experimental and bioinformatic analyses of other mitochondrial plasmids, random sequences, and natural nuclear genes in Neurospora and other fungi indicate that fungal spliceosomes recognize a wide range of 5′ splice site and branchpoint sequences and predict introns to be present at high frequency in random sequence. In contrast, analysis of intronless fungal nuclear genes indicates that branchpoint, 5′ splice site and 3′ splice site consensus sequences are underrepresented compared with random sequences. This underrepresentation of splicing signals is sufficient to deplete the nuclear genome of splice sites at locations that do not comprise biologically relevant introns. Thus, the splicing machinery can recognize a wide range of splicing signal sequences, but splicing still occurs with great accuracy, not because the splicing machinery distinguishes correct from incorrect introns, but because incorrect introns are substantially depleted from the genome.

Splicing: still so much to learn.

Splicing: still so much to learn.

Compound heterozygous mutations in RIPPLY2 associated with vertebral segmentation defects.

Segmentation defects of the vertebrae (SDV) are caused by aberrant somite formation during embryogenesis and result in irregular formation of the vertebrae and ribs. The Notch signal transduction pathway plays a critical role in somite formation and patterning in model vertebrates. In humans, mutations in several genes involved in the Notch pathway are associated with SDV, with both autosomal recessive (MESP2, DLL3, LFNG, HES7) and autosomal dominant (TBX6) inheritance. However, many individuals with SDV do not carry mutations in these genes. Using whole-exome capture and massive parallel sequencing, we identified compound heterozygous mutations in RIPPLY2 in two brothers with multiple regional SDV, with appropriate familial segregation. One novel mutation (c.A238T:p.Arg80*) introduces a premature stop codon. In transiently transfected C2C12 mouse myoblasts, the RIPPLY2 mutant protein demonstrated impaired transcriptional repression activity compared with wild-type RIPPLY2 despite similar levels of expression. The other mutation (c.240-4T>G), with minor allele frequency <0.002, lies in the highly conserved splice site consensus sequence 5' to the terminal exon. Ripply2 has a well-established role in somitogenesis and vertebral column formation, interacting at both gene and protein levels with SDV-associated Mesp2 and Tbx6. We conclude that compound heterozygous mutations in RIPPLY2 are associated with SDV, a new gene for this condition.

T.P.30: Recurrent episodes of myoglobinuria, mental retardation and epilepsy but no haemolysis in two brothers with phosphoglycerate kinase deficiency

We report two brothers, aged 14 and 16, with a mild intellectual deficiency. Both brothers presented recurrent episodes of generalized weakness following effort since the age of 4. At the age of 8, after spending twenty minutes playing in a swimming pool, the youngest brother presented an episode of weakness, together with a painful swelling of the left thigh and myoglobinuria (max CK 540,000 UI/l). He was hospitalized in an intensive care unit and treated by intravenous fluids and loop diuretics. 4 months later, a muscle biopsy disclosed abnormal vacuolisation with slightly increased muscle lipid content. An extensive workup excluded a mitochondrial disease, a defect of fatty acid oxidation and Mac Ardle disease. At the age of 13, the youngest brother presented a refractory status epilepticus, complicated by rhabdomyolysis and acute renal failure (max CK 710,000 UI/l). Physical examination of both brothers, between acute episodes, was completely normal and CK level was approximately 500 UI/l ( N PGK1 gene sequencing revealed a splice site consensus sequence change that co-segregates with decreased PGK activity. Its putative functional consequences are currently under investigation. Conclusion: Phosphoglycerate kinase (PGK) deficiency, a rare defect in glycolysis pathway, has its place in the differential diagnosis of metabolic myopathies. The association of exercise intolerance and recurrent episodes of myoglobinuria with haemolytic anaemia, intellectual deficiency or epilepsy is highly suggestive of the disease.

Congenital analbuminemia caused by a novel aberrant splicing in the albumin gene

Introduction:Congenital analbuminemia is a rare autosomal recessive disorder manifested by the presence of a very low amount of circulating serum albumin. It is an allelic heterogeneous defect, caused by variety of mutations within the albumin gene in homozygous or compound heterozygous state. Herein we report the clinical and molecular characterization of a new case of congenital analbuminemia diagnosed in a female newborn of consanguineous (first degree cousins) parents from Ankara, Turkey, who presented with a low albumin concentration (< 8 g/L) and severe clinical symptoms.Materials and methods:The albumin gene of the index case was screened by single-strand conformation polymorphism, heteroduplex analysis, and direct DNA sequencing. The effect of the splicing mutation was evaluated by examining the cDNA obtained by reverse transcriptase - polymerase chain reaction (RT-PCR) from the albumin mRNA extracted from proband’s leukocytes.Results:DNA sequencing revealed that the proband is homozygous, and both parents are heterozygous, for a novel G>A transition at position c.1652+1, the first base of intron 12, which inactivates the strongly conserved GT dinucleotide at the 5′ splice site consensus sequence of this intron. The splicing defect results in the complete skipping of the preceding exon (exon 12) and in a frame-shift within exon 13 with a premature stop codon after the translation of three mutant amino acid residues.Conclusions:Our results confirm the clinical diagnosis of congenital analbuminemia in the proband and the inheritance of the trait and contribute to shed light on the molecular genetics of analbuminemia.

Spliceosome-mediated decay (SMD) regulates expression of nonintronic genes in budding yeast

We uncovered a novel role for the spliceosome in regulating mRNA expression levels that involves splicing coupled to RNA decay, which we refer to as spliceosome-mediated decay (SMD). Our transcriptome-wide studies identified numerous transcripts that are not known to have introns but are spliced by the spliceosome at canonical splice sites in Saccharomyces cerevisiae. Products of SMD are primarily degraded by the nuclear RNA surveillance machinery. We demonstrate that SMD can significantly down-regulate mRNA levels; splicing at canonical splice sites in the bromodomain factor 2 (BDF2) transcript reduced transcript levels roughly threefold by generating unstable products that are rapidly degraded by the nuclear surveillance machinery. Regulation of BDF2 mRNA levels by SMD requires Bdf1, a functionally redundant Bdf2 paralog that plays a role in recruiting the spliceosome to the BDF2 mRNA. Interestingly, mutating BDF2 5' splice site and branch point consensus sequences partially suppresses the bdf1Δ temperature-sensitive phenotype, suggesting that maintaining proper levels of Bdf2 via SMD is biologically important. We propose that the spliceosome can also repress protein-coding gene expression by promoting nuclear turnover of spliced RNA products and provide an insight for coordinated regulation of Bdf1 and Bdf2 levels in the cell.

Molecular basis of acid ceramidase deficiency in a neonatal form of Farber disease: Identification of the first large deletion in ASAH1 gene

Farber disease, also known as Farber's lipogranulomatosis, is a clinically heterogeneous autosomal recessive disease caused by mutations in the ASAH1 gene. This gene codes for acid ceramidase, a lysosomal heterodimeric enzyme that hydrolyzes ceramide into sphingosine and fatty acid. To date, less than 25 distinct mutations have been identified in Farber patients, but no large deletions have yet been reported. In this work, cultured fibroblasts from a Farber patient with the rare neonatal form of Farber disease were studied to elucidate the molecular basis of this extremely severe phenotype. Direct sequencing of ASAH1 genomic DNA revealed the causative heterozygous mutation in the donor splice site consensus sequence of intron 11, g.24491A>G (c.917+4A>G), that resulted in the absence of detectable mRNA. Subsequent analysis of ASAH1 mRNA showed total skipping of exons 3 to 5. Long-range PCR and sequencing led to the identification of a gross deletion of ASAH1 gene, g.8728_18197del (c.126-3941_382+1358del) predicting the synthesis of a truncated polypeptide, p.Tyr42_Leu127delinsArgfs*10. Accordingly, no molecular forms corresponding to precursor or proteolytically processed mature protein were observed. These findings indicate that any functionally active acid ceramidase is absent in patient cells, underscoring the severity of the clinical phenotype. Molecular findings in the non-consanguineous parents confirmed the compound heterozygous ASAH1 genotype identified in this Farber case. This work unravels for the first time the mutations underlying the neonatal form of Farber disease and represents the first report of a large deletion identified in the ASAH1 gene. Screening for gross deletions in other patients in whom the mutation present in the second allele had not yet been identified is required to elucidate further its overall contribution for the molecular pathogenesis of this devastating disease.

A Mutant mRNA Expression in an Endomyocardial Biopsy Sample Obtained from a Patient with a Cardiac Variant of Fabry Disease Caused by a Novel Acceptor Splice Site Mutation in the Invariant AG of Intron 5 of the α-Galactosidase A Gene

We herein describe the case of a 58-year-old man who presented with dilated-phase hypertrophic cardiomyopathy (HCM) and required an implantable cardioverter defibrillator implant. Subsequently, the patient was diagnosed with Fabry disease (FD), which was suspected based on the results of an endomyocardial biopsy and diagnosed following demonstration of deficient α-galactosidase A (GLA) activity. Molecular studies showed a novel point mutation in the 3' splice site consensus sequence of intron 5 in the gene encoding GLA that created a new splicing site, resulting in the expression of mutant mRNA. FD should be considered a cause of HCM in patients with severe tachyarrhythmia without other remarkable manifestations of FD.

Clinical utility gene card for: Rothmund–Thomson syndrome

1.5 Mutational spectrum Biallelic mutations in the RECQL4 gene are associated with Rothmund–Thomson (RTS) and two additional recessive disorders: RAPADILINO (RAdial hypoplasia, PAtellae hypoplasia and cleft or arched PAlate, DIarrhea and DIslocated joints, LIttle size and LImb malformation, slender NOse and NOrmal intelligence) and Baller– Gerold syndrome (BGS). More than 60 disease-causing mutations have been reported, of which at least 40 have been detected in RTS patients.1,2 The types of observed mutations are as follows, in order of decreasing prevalence: nonsense or frameshift mutations; splicing alterations, including substitutions at canonical splice junctions or at splice-site consensus sequences and subtle intronic deletions that reduce intron size below the threshold (o80 bp) required for correct splicing;3,4 and missense mutations. There are a few recurrent mutations, among which the most common, exon 9 c.1573delT (p.Cys525AlafsX33), has been detected in patients with all three RECQL4-associated diseases. This truncating mutation accounts for approximately one-third of RTS mutations and has only been found in compound heterozygous patients from multiple ethnic backgrounds. A deletion of the entire gene has never been identified.

Analysis of F8 mRNA in haemophilia A patients with silent mutations or presumptive splice site mutations

Mutation screenings in haemophilia A (HA) patients identified a great variety of mutations in the factor VIII gene (F8): intron 22 or intron 1 inversions, missense mutations, nonsense mutations, small or large deletions, insertions, duplications and splice site mutations. Mutations which do not result in amino acid substitutions (silent mutations) and intronic variants located outside the splice site consensus sequences cannot be easily classified as causative for HA. In these cases, special prediction software algorithms are applied to estimate their impact on splicing. Here, we present mRNA analysis of novel F8 mutations with possible impact on splicing in four HA patients with silent mutations and seven patients with intronic variants close to or within splice site consensus sequences. Seven of eleven mutations examined in vitro could be shown to have an effect on F8 mRNA splicing and the results were compared to in silico predictions. In addition, to validate the splice site prediction software Alamut v2.0 (Interactive Biosoftware), we compared published F8 mRNA analyses with the results of the in silico prediction. In general, the results of the splice site prediction tools of Alamut were in good accordance with the experimental F8 mRNA analyses, but a fundamental discrepancy between in silico and in vitro analyses was obtained in some cases. In conclusion, this study shows that the functional classification of potential splicing mutations should not only rely on prediction software, but be rather based on mRNA analysis experiments.

Aberrant transcript produced by a splice donor site deletion in the TECTA gene is associated with autosomal dominant deafness in a Brazilian family

We ascertained a Brazilian family with nine individuals affected by autosomal dominant nonsyndromic sensorineural hearing loss. The bilateral hearing loss affected mainly mid-high frequencies, was apparently stable with an early onset. Microsatellites close to the DFNA8/DFNA12 locus, which harbors the TECTA gene, showed significant multipoint lod scores (3.2) close to marker D11S4107. Sequencing of the exons and exon–intron boundaries of the TECTA gene in one affected subject revealed the deletion c.5383+5delGTGA in the 5′ end of intron 16, that includes the last two bases of the donor splice site consensus sequence. This mutation segregates with deafness within the family. To date, 33 different TECTA mutations associated with autossomal dominant hearing loss have been described. Among them is the mutation reported herein, first described by Hildebrand et al. (2011) in a UK family. The audioprofiles from the UK and Brazilian families were similar. In order to investigate the transcripts produced by the mutated allele, we performed cDNA analysis of a lymphoblastoid cell line from an affected heterozygote with the c.5383+5delGTGA and a noncarrier from the same family. The analysis allowed us to identify an aberrant transcript with skipping of exon 16, without affecting the reading frame. One of the dominant TECTA mutations already described, a synonymous substitution in exon 16 (c.5331G<A), was also shown to affect splicing, resulting in an aberrant transcript lacking exon 16. Despite the difference in the DNA level, both the synonymous substitution in exon 16 (c.5331G<A) and the mutation described herein affect splicing of exon 16, leading to its skipping. At the protein level they would have the same effect, an in-frame deletion of 37 amino-acids (p.S1758Y/G1759_N1795del) probably leading to an impaired function of the ZP domain. Thus, like the TECTA missense mutations associated with dominant hearing loss, the c.5383+5delGTGA mutation does not have an inactivating effect on the protein.