Splice Site Of Intron Research Articles

The Prevalence of Rare Variants Potentially Important for the Response to Medicines Used for CVD Treatment in the Lithuanian Population

Cardiovascular disease (CVD) is one of the leading causes of death among Europeans and around the world. Major factors that are considered relevant for the efficacy or adverse drug reactions (ADRs) caused by drugs used for CVD treatment have been identified through GWAS studies mostly using low density common variant genotyping arrays. In this study whole exome sequencing (WES) was carried out to identify genomic variants relevant for CVD treatment. The study group included 98 (49 males and 49 females) self-reported healthy unrelated individuals from the Lithuanian population with at least three generations of Lithuanian ethnicity and residency in the same ethnolinguistic region. After performing WES genetic loci of 55 genes that were reported as relevant to the efficacy or ADRs caused by drugs used for CVD treatment were analyzed using LifeScopeTM and ANNOVAR software. The analysis of small indels in the selected regions revealed 13 exonic frameshift indels and a single intronic splice site variant that may affect the efficacy or cause ADRs in patients treated for CVD. Over 400 potentially relevant SNVs in different frequencies were detected in the genes reported to be important for CVD treatment. The strength of evidence analysis identified 14 SNVs most likely to be relevant for CVD treatment. The frequency distribution of several variant alleles that have been shown to be highly important for CVD treatment in the CYP2D6 (rs16947) and ADRB1 (rs1801253) as well as several variants in the CYP2C9 and NAT2 genes were determined to be significantly (p<0.05) different from other Caucasian populations showing the potential benefit of pharmacogenomic testing prior to treatment in the Lithuanian population. The research was carried out under the LITGEN project. LITGEN project (VP1-3.1-SMM-07-K-01-013) was funded by the European Social Fund under the Global Grant measure.

A novel BRD4-NUT fusion in an undifferentiated sinonasal tumor highlights alternative splicing as a contributing oncogenic factor in NUT midline carcinoma.

NUT midline carcinoma (NMC) is a fatal cancer that arises in various tissues along the upper midline of the body. The defining molecular feature of NMC is a chromosomal translocation that joins (in the majority of cases) the nuclear testis gene NUT (NUTM1) to the bromodomain protein family member 4 (BRD4) and thereby creating a fusion oncogene that disrupts cellular differentiation and drives the disease. In this study, we report the case of an adolescent NMC patient presenting with severe facial pain, proptosis and visual impairment due to a mass arising from the ethmoid sinus that invaded the right orbit and frontal lobe. Treatment involved radical resection, including exenteration of the affected eye with the view to consolidate treatment with radiation therapy; however, the patient experienced rapid tumor progression and passed away 79 days post resection. Molecular analysis of the tumor tissue identified a novel in-frame BRD4-NUT transcript, with BRD4 exon 15 fused to the last 124 nucleotides of NUT exon 2 (BRD4-NUT ex15:ex2Δnt1–585). The partial deletion of NUT exon 2 was attributed to a mid-exonic genomic breakpoint and the subsequent activation of a cryptic splice site further downstream within the exon. Inhibition of the canonical 3′ acceptor splice site of NUT intron 1 in cell lines expressing the most common NMC fusion transcripts (PER-403, BRD4-NUT ex11:ex2; PER-624, BRD4-NUT ex15:ex2) induced alternative splicing from the same cryptic splice site as identified in the patient. Detection of low levels of an in-frame BRD4-NUT ex11:ex2Δnt1–585 transcript in PER-403 confirmed endogenous splicing from this alternative exon 2 splice site. Although further studies are necessary to assess the clinical relevance of the increasing number of variant fusions described in NMC, the findings presented in this case identify alternative splicing as a mechanism that contributes to this pathogenic complexity.

Late-onset spastic paraplegia: Aberrant SPG11 transcripts generated by a novel splice site donor mutation

We identified a novel homozygous mutation in the splice site donor (SSD) of intron 30 (c.5866+1G>A) in consanguineous Japanese SPG11 siblings showing late-onset spastic paraplegia using the whole-exome sequencing. Phenotypic variability was observed, including age-at-onset, dysarthria and pes cavus. Coding DNA sequencing revealed that the mutation affected the recognition of the constitutive SSD of intron 30, splicing upstream onto a nearby cryptic SSD in exon 30. The use of constitutive splice sites of intron 29 was confirmed by sequencing. The mutant transcripts are mostly subject to degradation by the nonsense-mediated mRNA decay system. SPG11 transcripts, escaping from the nonsense-mediated mRNA decay pathway, would generate a truncated protein (p.Tyr1900Phefs5X) containing the first 1899 amino acids and followed by 4 aberrant amino acids. This study showed a successful clinical application of whole-exome sequencing in spastic paraplegia and demonstrated a further evidence of allelic heterogeneity in SPG11. The confirmation of aberrant transcript by splice site mutation is a prerequisite for a more precise molecular diagnosis.

Unmasking a novel disease gene NEO1 associated with autism spectrum disorders by a hemizygous deletion on chromosome 15 and a functional polymorphism

BackgroundAutism spectrum disorders (ASD) are neurodevelopmental disorders with a high degree of heritability, but the genetic basis is exceedingly heterogeneous. Microdeletions of chromosome 15q24 have been demonstrated to be recurrent genomic alterations in ASD patients. Of interest, neuronal migration genes are of particular relevance to the pathogenesis of ASD. NEO1 is located in 15q24 and encodes for neogenin, a membrane receptor involved in cortical interneuron migration and axon guidance. We postulated that the ASD patient has one copy of the NEO1 gene deleted and the other copy disrupted by intragenic mutation. ResultsWe identify genetic changes in both alleles of NEO1 in two individuals from a cohort of 66 Han Chinese patients with ASD. In one patient, we detected a hemizygous 1.97-Mb deletion at 15q23q24.1 encompassing the NEO1 gene, a missense variant in NEO1, c.3388C>T (p.Arg1130Cys), and a duplication, c.2204-14_2204-2dup, in the acceptor splice site of intron 14 of NEO1. Furthermore, we identified a second patient was a compound heterozygote for NEO1. A novel missense variant in NEO1, c.302G>A (p.Arg101His), in addition to c.3388C>T and c.2204-14_2204-2dup was detected in the second patient. The c.3388C>T is a single nucleotide polymorphism with allele frequency of 0.045 in Han Chinese individuals. In silico and functional analyses indicated that p.Arg1130Cys, located at the nuclear localization signal (NLS) domain of neogenin led to defective nuclear translocation of neogenin. ConclusionsThe hemizygous 15q deletion unmasks the recessive functional polymorphism in NEO1 which plays a pivotal role in cortical interneuron development. Our study provides the first evidence linking NEO1 with ASD in humans.

Compound heterozygous DUOX2 gene mutations (c.2335-1G>C/c.3264_3267delCAGC) associated with congenital hypothyroidism. Characterization of complex cryptic splice sites by minigene analysis

Iodide Organification defects (IOD) represent 10% of cases of congenital hypothyroidism (CH) being the main genes affected that of TPO (thyroid peroxidase) and DUOX2 (dual oxidasa 2). From a patient with clinical and biochemical criteria suggestive with CH associated with IOD, TPO and DUOX2 genes were analyzed by means of PCR-Single Strand Conformation Polymorphism analysis and sequencing. A novel heterozygous compound to the mutations c.2335-1G>C (paternal mutation, intron 17) and c.3264_3267delCAGC (maternal mutation, exon 24) was identified in the DUOX2 gene. Ex-vivo splicing assays and subsequent RT-PCR and sequencing analyses were performed on mRNA isolated from the HeLa cells transfected with wild-type and mutant pSPL3 expression vectors. The wild-type and c.2335-1G>C mutant alleles result in the complete inclusion or exclusion of exon 18, or in the activation of an exonic cryptic 5′ ss with the consequent deletion of 169 bp at the end of this exon. However, we observed only a band of the expected size in normal thyroid tissue by RT-PCR. Additionally, the c.2335-1G>C mutation activates an unusual cryptic donor splice site in intron 17, located at position −14 of the authentic intron 17/exon 18 junction site, with an insertion of the last 14 nucleotides of the intron 17 in mutant transcripts with complete and partial inclusion of exon 18. The theoretical consequences of splice site mutation, predicted with the bioinformatics NNSplice, Fsplice, SPL, SPLM and MaxEntScan programs were investigated and evaluated in relation with the experimental evidence. These analyses confirm that c.2335-1G>C mutant allele would result in the abolition of the authentic splice acceptor site. The results suggest the coexistence in our patient of four putative truncated proteins of 786, 805, 806 and 1105 amino acids, with conservation of peroxidase-like domain and loss of gp91phox/NOX2-like domain. In conclusion a novel heterozygous compound was identified being responsible of IOD. Cryptic splicing sites have been characterized in DUOX2 gene for the first time. The use of molecular biology techniques is a valuable tool for understanding the molecular pathophysiology of this type of thyroid defects.

Molecular, biochemical, and structural analysis of a novel mutation in patients with methylmalonyl-CoA mutase deficiency

BackgroundMethylmalonic aciduria (MMA) is an inborn error of metabolism resulting from genetic defects in methylmalonyl-CoA mutase (MCM). This enzyme is encoded by the MUT gene and is required for the degradation of odd-chain fatty acids, the amino acids valine, isoleucine, methionine, and threonine, and cholesterol. MethodThree unrelated affected patients with isolated MMA and their parents were studied. The MUT gene was analyzed by PCR and sequencing of its entire coding region and the highly conserved exon-intron splice junctions. The homology modeling of the novel mutation found in the MUT gene was performed using the online Swiss-Prot server for automated modeling and then analyzed with special bioinformatics software to better study the structural effects caused by the mutation. ResultWe found one homozygous nucleotide change in intron 12 of the MUT gene (c.2125-3 C>G). The variant is located near the highly conserved acceptor splice site of intron 12. A region at the C-terminus of the protein from ASP709 to GLN748 has been deleted by the alteration of c.2125-3 C>G in intron 12 of the MUT gene. Further studies of the novel mutation in the MUT gene by means of homology modeling revealed abnormalities in the protein's structure, which causes the protein to act malfunctioning and also the mRNA expression analysis of MUT gene confirmed these results. ConclusionWe report this novel mutation, including its clinical and biochemical features and genetic defects, in the MUT gene of three patients affected with isolated MMA. Structural analyses of the mutated protein identified changes in the energy and stereochemical features of the protein that unfortunately altered the protein's functionalities. Therefore, we demonstrate that a novel splice site mutation in intron 12 of the MUT gene is a potential highly pathogenic allele via inhibition of alternative splicing.

Identification of two novel mutations in CDHR1 in consanguineous Spanish families with autosomal recessive retinal dystrophy.

Inherited retinal dystrophies present extensive phenotypic and genetic heterogeneity, posing a challenge for patients’ molecular and clinical diagnoses. In this study, we wanted to clinically characterize and investigate the molecular etiology of an atypical form of autosomal recessive retinal dystrophy in two consanguineous Spanish families. Affected members of the respective families exhibited an array of clinical features including reduced visual acuity, photophobia, defective color vision, reduced or absent ERG responses, macular atrophy and pigmentary deposits in the peripheral retina. Genetic investigation included autozygosity mapping coupled with exome sequencing in the first family, whereas autozygome-guided candidate gene screening was performed by means of Sanger DNA sequencing in the second family. Our approach revealed nucleotide changes in CDHR1; a homozygous missense variant (c.1720C > G, p.P574A) and a homozygous single base transition (c.1485 + 2T > C) affecting the canonical 5’ splice site of intron 13, respectively. Both changes co-segregated with the disease and were absent among cohorts of unrelated control individuals. To date, only five mutations in CDHR1 have been identified, all resulting in premature stop codons leading to mRNA nonsense mediated decay. Our work reports two previously unidentified homozygous mutations in CDHR1 further expanding the mutational spectrum of this gene.

A nucleotide substitution at the 5′ splice site of intron 1 of rice HEADING DATE 1 (HD1) gene homolog in foxtail millet, broadly found in landraces from Europe and Asia

We investigated genetic variation of a rice HEADING DATE 1(HD1) homolog in foxtail millet. First, we searched for a rice HD1 homolog in a foxtail millet genome sequence and designed primers to amplify the entire coding sequence of the gene. We compared full HD1 gene sequences of 11 accessions (including Yugu 1, a Chinese cultivar used for genome sequencing) from various regions in Europe and Asia, found a nucleotide substitution at a putative splice site of intron 1, and designated the accessions with the nucleotide substitution as carrying a splicing variant. We verified by RT-PCR that this single nucleotide substitution causes aberrant splicing of intron 1. We investigated the geographical distribution of the splicing variant in 480 accessions of foxtail millet from various regions of Europe and Asia and part of Africa by dCAPS and found that the splicing variant is broadly distributed in Europe and Asia. Differences of heading times between accessions with wild type allele of the HD1 gene and those with the splicing variant allele were unclear. We also investigated variation in 13 accessions of ssp. viridis, the wild ancestor, and the results suggested that the wild type is predominant in the wild ancestor.

Nucleotide sequence composition adjacent to intronic splice sites improves splicing efficiency via its effect on pre-mRNA local folding in fungi.

RNA splicing is the central process of intron removal in eukaryotes known to regulate various cellular functions such as growth, development, and response to external signals. The canonical sequences indicating the splicing sites needed for intronic boundary recognition are well known. However, the roles and evolution of the local folding of intronic and exonic sequence features adjacent to splice sites has yet to be thoroughly studied. Here, focusing on four fungi (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus nidulans, and Candida albicans), we performed for the first time a comprehensive high-resolution study aimed at characterizing the encoding of intronic splicing efficiency in pre-mRNA transcripts and its effect on intron evolution. Our analysis supports the conjecture that pre-mRNA local folding strength at intronic boundaries is under selective pressure, as it significantly affects splicing efficiency. Specifically, we show that in the immediate region of 12–30 nucleotides (nt) surrounding the intronic donor site there is a preference for weak pre-mRNA folding; similarly, in the region of 15–33 nt surrounding the acceptor and branch sites there is a preference for weak pre-mRNA folding. We also show that in most cases there is a preference for strong pre-mRNA folding further away from intronic splice sites. In addition, we demonstrate that these signals are not associated with gene-specific functions, and they correlate with splicing efficiency measurements (r = 0.77, P = 2.98 × 10−21) and with expression levels of the corresponding genes (P = 1.24 × 10−19). We suggest that pre-mRNA folding strength in the above-mentioned regions has a direct effect on splicing efficiency by improving the recognition of intronic boundaries. These new discoveries are contributory steps toward a broader understanding of splicing regulation and intronic/transcript evolution.

Analysis of USH2A gene mutation in a Chinese family affected with Usher syndrome

To investigate the disease-causing mutation in a Chinese family affected with Usher syndrome type II. All of the 11 members from the family underwent comprehensive ophthalmologic examination and hearing test, and their genomic DNA were isolated from venous leukocytes. PCR and direct sequencing of USH2A gene were performed for the proband. Wild type and mutant type minigene vectors containing exon 42, intron 42 and exon 43 of the USH2A gene were constructed and transfected into Hela cells by lipofectamine reagent. Reverse transcription (RT)-PCR was carried out to verify the splicing of the minigenes. Pedigree analysis and clinical diagnosis indicated that the patients have suffered from autosomal recessive Usher syndrome type II. DNA sequencing has detected a homozygous c.8559-2A>G mutation of the USH2A gene in the proband, which has co-segregated with the disease in the family. The mutation has affected a conserved splice site in intron 42, which has led to inactivation of the splice site. Minigene experiment has confirmed the retaining of intron 42 in mature mRNA. The c.8559-2A>G mutation in the USH2A gene probably underlies the Usher syndrome type II in this family. The splice site mutation has resulted in abnormal splicing of USH2A pre-mRNA.

The expanding large-spored Metschnikowia clade: Metschnikowia matae sp. nov., a yeast species with two varieties from the Brazilian Atlantic Forest.

Fifty-two yeast isolates from flowers and associated nitidulid beetles of the Brazilian Atlantic Forest (Mata Atlântica) region were found to represent a new species in the large-spored Metschnikowia clade. The species is heterothallic, haploid, and allogamous, and produces asci with two aciculate ascospores that can reach 80 μm in length, as is typical in the clade. Analysis of sequences of the ribosomal RNA gene cluster indicates that the new species is closely related to Metschnikowia lochheadii, which ranges across Central America to northern Brazil, occurs as an adventive species in Hawaii, but is rarely found in central Brazil. The species is not readily distinguishable from relatives based on morphology or growth responses, but is well delineated from M. lochheadii on reproductive isolation. Based on an intron splice site PCR screen, we selected 26 isolates for further study. The sequence of the region that includes the complete internal transcribed spacer/5.8S rRNA gene segment as well as the D1/D2 domains of the large subunit rRNA gene contained three polymorphic segments and 14 haplotypes were identified. Of these, a single divergent isolate from the southernmost of four sampled localities exhibited diminished mating success when crossed with others. We describe two varieties, Metschnikowia matae var. matae sp. nov. var. nov. (type UFMG-CM-Y395(T), CBS 13986(T), NRRL Y-63736(T); allotype UFMG-CM-Y391(A), CBS 13987(A), NRRL Y-63735(A)) and Metschnikowia matae var. maris sp. nov. var. nov. (type UFMG-CM-Y397(T), CBS 13985(T), NRRL Y-63737(T)). We also report on the discovery of the h (+) mating type of Candida ipomoeae and transfer of the species to Metschnikowia ipomoeae comb. nov. (allotype UWOPS 12-660.1(A), CBS 13988(A), NRRL Y-63738(A)).

Phenylalanine hydroxylase deficiency in south Italy: Genotype–phenotype correlations, identification of a novel mutant PAH allele and prediction of BH4 responsiveness

We investigated the mutation spectrum of the phenylalanine hydroxylase gene (PAH) in a cohort of patients from 33 Italian PKU families. Mutational screening of the known coding region, including conventional intron splice sites, was performed by direct sequencing of the patients' genomic DNA.Thirty-three different disease causing mutations were identified in our patient group, including 19 missense, 6 splicing, 3 nonsense, 5 deletions, with a detection rate of 100%. The most prevalent mutation was the IVS10−11G>A, accounting for 12.1% of PKU alleles studied. Other frequent mutations were: p.R261Q (9.1%), p.P281L (7.6%), and p.R408W (6.1%). We also identified one novel missense mutation, p.H290Q. A spectrum of 31 different genotypes was observed and a genotype based predictions of BH4-responsiveness were assessed. Among all genotypes, 13 were predicted to be BH4-responsive represented by thirteen PKU families. In addition, genotype–phenotype correlations were performed. This study reveals the importance of a full genotyping of PKU patients and the prediction of BH4-responsiveness, not only because of the definitive diagnosis and prediction of the optimal diet, but also to point out those patients that could benefit from new therapeutic approach. They may potentially benefit from BH4 therapy which, combined with a less strict diet, or eventually in special cases as monotherapy, may contribute to reduce nutritional deficiencies and minimize neurological and psychological dysfunctions.

A new low linolenic acid allele of GmFAD3A gene in soybean PE1690

Relative fatty acid content of soybean oil is about 12 % palmitic acid, 4 % stearic acid, 23 % oleic acid, 54 % linoleic acid, and 8 % linolenic acid. To improve oxidative stability and quality of oil, breeding programs have mainly focused on reducing saturated fatty acids, increasing oleic acid, and reducing linolenic acid in soybean oil. The objective of this study was to characterize the low linolenic acid trait in seed oil of PE1690, which was derived from an ethyl methanesulfonated-mediated mutagenesis. PE1690 DNA was used to examine the genomic sequence of a candidate gene, and we found a point mutation which substituted guanine to adenine in the second intron splice site of the GmFAD3A gene. Mutation of PE1690 at the first base of the second intron caused missplicing which led to the inclusion of 334 bp of the second intron into the coding sequence. The amino acid sequence of the mutant was analyzed based on cDNA sequence of GmFAD3A in PE1690. The position 128 amino acid, which codes for tryptophan (TGG) in PE1690, was changed to a premature stop codon (TGA). Therefore, the single base mutation in the PE1690 GmFAD3A gene renders the desaturase enzyme nonfunctional. Based on a genetic marker that we developed, the association of the PE1690 derived GmFAD3A allele with linolenic acid level was tested in 89 F2 progeny of a cross between Pungsannamul (wild type) × PE1690. The results showed that the mutation of GmFAD3A in PE1690 was associated with reduced linolenic acid content in the soybean seed oil. A newly developed low linolenic acid soybean PE1690 provides a good genetic resource to improve the quality of soybean oil.

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.

Interordinal gene capture, the phylogenetic position of Steller’s sea cow based on molecular and morphological data, and the macroevolutionary history of Sirenia

The recently extinct (ca. 1768) Steller’s sea cow (Hydrodamalis gigas) was a large, edentulous North Pacific sirenian. The phylogenetic affinities of this taxon to other members of this clade, living and extinct, are uncertain based on previous morphological and molecular studies. We employed hybridization capture methods and second generation sequencing technology to obtain >30kb of exon sequences from 26 nuclear genes for both H. gigas and Dugong dugon. We also obtained complete coding sequences for the tooth-related enamelin (ENAM) gene. Hybridization probes designed using dugong and manatee sequences were both highly effective in retrieving sequences from H. gigas (mean=98.8% coverage), as were more divergent probes for regions of ENAM (99.0% coverage) that were designed exclusively from a proboscidean (African elephant) and a hyracoid (Cape hyrax). New sequences were combined with available sequences for representatives of all other afrotherian orders. We also expanded a previously published morphological matrix for living and fossil Sirenia by adding both new taxa and nine new postcranial characters. Maximum likelihood and parsimony analyses of the molecular data provide robust support for an association of H. gigas and D. dugon to the exclusion of living trichechids (manatees). Parsimony analyses of the morphological data also support the inclusion of H. gigas in Dugongidae with D. dugon and fossil dugongids. Timetree analyses based on calibration density approaches with hard- and soft-bounded constraints suggest that H. gigas and D. dugon diverged in the Oligocene and that crown sirenians last shared a common ancestor in the Eocene. The coding sequence for the ENAM gene in H. gigas does not contain frameshift mutations or stop codons, but there is a transversion mutation (AG to CG) in the acceptor splice site of intron 2. This disruption in the edentulous Steller’s sea cow is consistent with previous studies that have documented inactivating mutations in tooth-specific loci of a variety of edentulous and enamelless vertebrates including birds, turtles, aardvarks, pangolins, xenarthrans, and baleen whales. Further, branch-site dN/dS analyses provide evidence for positive selection in ENAM on the stem dugongid branch where extensive tooth reduction occurred, followed by neutral evolution on the Hydrodamalis branch. Finally, we present a synthetic evolutionary tree for living and fossil sirenians showing several key innovations in the history of this clade including character state changes that parallel those that occurred in the evolutionary history of cetaceans.

Two novel splicing mutations in the SLC45A2 gene cause Oculocutaneous Albinism Type IV by unmasking cryptic splice sites

Oculocutaneous albinism (OCA) is characterized by hypopigmentation of the skin, hair and eye, and by ophthalmologic abnormalities caused by a deficiency in melanin biosynthesis. OCA type IV (OCA4) is one of the four commonly recognized forms of albinism, and is determined by mutation in the SLC45A2 gene. Here, we investigated the genetic basis of OCA4 in an Italian child. The mutational screening of the SLC45A2 gene identified two novel potentially pathogenic splicing mutations: a synonymous transition (c.888G>A) involving the last nucleotide of exon 3 and a single-nucleotide insertion (c.1156+2dupT) within the consensus sequence of the donor splice site of intron 5. As computer-assisted analysis for mutant splice-site prediction was not conclusive, we investigated the effects on pre-mRNA splicing of these two variants by using an in vitro minigene approach. Production of mutant transcripts in HeLa cells demonstrated that both mutations cause the almost complete abolishment of the physiologic donor splice site, with the concomitant unmasking of cryptic donor splice sites. To our knowledge, this work represents the first in-depth molecular characterization of splicing defects in a OCA4 patient.

The dynamic loss and gain of introns during the evolution of the Brassicaceae

Sequence comparison allows the detailed analysis of evolution at the nucleotide and amino acid levels, but much less information is known about the structural evolution of genes, i.e. how the number, length and distribution of introns change over time. We constructed a parsimonious model for the evolutionary rate of intron loss (IL) and intron gain (IG) within the Brassicaceae and found that IL/IG has been highly dynamic, with substantial differences between and even within lineages. The divergence of the Brassicaceae lineages I and II marked a dramatic change in the IL rate, with the common ancestor of lineage I losing introns three times more rapidly than the common ancestor of lineage II. Our data also indicate a subsequent declining trend in the rate of IL, although in Arabidopsis thaliana introns continue to be lost at approximately the ancestral rate. Variations in the rate of IL/IG within lineage II have been even more remarkable. Brassica rapa appears to have lost introns approximately 15 times more rapidly than the common ancestor of B.rapa and Schenkiella parvula, and approximately 25 times more rapidly than its sister species Eutrema salsugineum. Microhomology was detected at the splice sites of several dynamic introns suggesting that the non-homologous end-joining and double-strand break repair is a common pathway underlying IL/IG in these species. We also detected molecular signatures typical of mRNA-mediated IL, but only in B.rapa.

440. Optimization of RNA Trans-Splicing for the β-Globin Gene; Detecting Trans-Splicing Events Targeting Endogenous β-Globin Pre-mRNA in Human Erythroid Cells

Sickle cell disease is caused by a point mutation in exon 1 of the β-globin gene (3 exons and 2 introns). RNA splicing between two distinct pieces of pre-mRNA, known as trans-splicing, represents a potential therapeutic strategy allowing replacement of the mutated exon 1 with a normal exon. Induction of exogenous mRNA splicing using spliceosome-mediated trans-splicing requires an RNA trans-splicing molecule (RTM) which imitates endogenous cis-splicing elements. However, clinical application of trans-splicing for globin disorders will require high efficiency, thus we sought to optimize the efficiency of trans-splicing targeting the β-globin gene to replace the exon 1 among human erythroid cells.To optimize the binding domain targeting the β-globin gene, the randomized binding domains of 20-600b were generated by sonicating the β-globin gene, and these were inserted into RTM-expressing plasmids downstream of the 5’ half of GFP and a 5’ splice site. In addition, we designed a target plasmid which encodes the β-globin gene and an artificial 3’ splice site connected to 3’ site of the other half of GFP. In transfection of both RTM and target plasmids, we selected 6 candidates from several thousand RTMs, which produced the brightest GFP-positive cells (maximal trans-splicing), and interestingly, all 6 binding domains targeted the β-globin intron 2.To evaluate efficiency of trans-splicing for human endogenous β-globin RNA, we constructed lentiviral vectors encoding RTMs which contain the γ-globin cDNA connected to a 5’ splice site and 3 candidates (1E1; 0.6kb, 2D10; 0.7kb, and 13-1; 0.8kb) of β-globin binding domains (selected from the 6 candidates). Human CD34+ cells were transduced with these RTM-encoding lentiviral vectors, and these cells were differentiated to erythroid cells in vitro. Trans-splicing was detected by RT-PCR and sequencing in RTM 2D10 and 13-1 but not in RTM 1E1, suggesting that longer binding domains improve trans-splicing efficiency. To elongate the binding domain in RTM 13-1, we designed RTM BGin2 (containing 5’ splice site of β-globin intron 2; 0.9kb) and BGex1-in2 (containing whole β-globin sequence except exon 3; 1.3kb). In both RTM BGin2 and BGex1-in2, ~4-fold higher trans-splicing was detected by RT-qPCR than RTM 13-1, which resulted in ~0.07% trans-splicing efficiency as compared to endogenous human β-globin RNA, implying that the 5’ splice site of intron 2 should be included as a binding domain for efficient trans-splicing.In summary, we developed a screening system to select more efficient binding domains for trans-splicing. For the first time, we obtained detectable levels of spliceosome-mediated trans-splicing (~0.07%) for endogenous β-globin RNA in human erythroid cells which were transduced with RTM-expressing lentiviral vectors. Further optimization is required to develop trans-splicing based gene therapy.

57. Seamless Targeted Correction of CYBB Exon 5 Mutations Restores Granulocyte Function in X-Linked Chronic Granulomatous Disease iPSCs

X-linked chronic granulomatous disease (X-CGD) is an immune deficiency resulting from lack of production of microbicidal reactive oxygen species (ROS) by phagocytic cells. Mutations causing X-CGD can occur throughout the >30-kb CYBB gene encoding gp91phox, with the majority of patients exhibiting a single causative mutation within one of the 13 exons or adjoining intronic splice sites, resulting in a loss of gp91phox protein expression. We previously demonstrated targeted “safe-harbor” gene therapy of X-CGD in iPSCs through insertion of a codon-optimized CYBB minigene into the AAVS1 locus, resulting in constitutive gp91phox expression from a CAG promoter. Another approach targeting insertion of the codon-optimized minigene or normal CYBB cDNA to the start site of endogenous CYBB resulted in little or no gp91phox expression or ROS activity in iPSC-derived granulocytes, suggesting that regulatory elements downstream of the CYBB promoter may be required for efficient expression at this locus. In order to maintain normal regulation of gp91phox expression with minimal alterations in corrected cells, we now demonstrate a strategy for seamless targeted repair of CYBB exon 5 mutations using an exon 5-specific TALEN pair or CRISPR, along with a donor plasmid containing the corrected 146-bp exon 5 interrupted by a piggyBac transposon cassette containing a PGK promoter and puroDtk gene for positive/negative selection, and flanked by ~900-bp upstream and downstream intron sequences for homologous recombination. iPSCs from X-CGD patients with exon 5 458T>G mutation or 461A deletion were nucleofected with donor and TALEN or CRISPR expression plasmids. Both TALENs and CRISPR enabled efficient targeted insertion in 82-98% of puromycin-resistant clones, 25-65% of which contained off-target inserts, for an overall efficiency of 29-75% of selected clones containing only the targeted insert. Targeted iPSC clones lacking off-target inserts were nucleofected with “excision-only” piggyBac transposase expression plasmid and selected with ganciclovir for removal of the PGK-puroDtk cassette, leaving only a silent codon change to accommodate the TTAA sequence left behind after piggyBac excision, without additional alterations detected beyond correction of the CYBB mutation. Corrected iPSCs maintained pluripotency and upon in vitro granulocyte differentiation exhibited restoration of gp91phox expression and ROS production comparable to normal blood neutrophils (3100% of normal gp91phox expression and 85-99% of normal ROS activity by mean fluorescence intensity in DHR assay). Our findings demonstrate efficient and seamless targeted repair of exon 5 mutations in X-CGD iPSCs with an exon replacement strategy, resulting in normal regulation of gp91phox expression and functional correction of the disease defect in iPSC-derived granulocytes. The development of similar approaches for the other exons of CYBB will enable seamless repair of the majority of causative mutations found in X-CGD patients.

1 GENOMICS AND EPIGENOMICS

s of the 13th International Symposium on Myelodysplastic Syndromes (MDS) Washington, DC, USA, April 29–May 2, 2015 Invited Speaker Presentations 1 GENOMICS AND EPIGENOMICS M.E. Figueroa1 1Pathology, University of Michigan, Ann Arbor, USA Epigenetic deregulation is a hallmark of MDS and the MDS/ MPN. Moreover, the DNA methyltransferase inhibitors (DMTi) Azacytidine and Decitabine (DAC) represent the only effective therapies for these disorders. However, little is understood about the precise nature and extent of epigenetic deregulation in the different forms of MDS or about how DMTis work in MDS and MDS/MPN. We have studied DNA methylation, gene expression and mutational profiles in patients with MDS and MDS/MPN. We first compared de novo and therapy-related MDS (t-MDS) and determined that these two forms of MDS are profoundly divergent, with marked hypermethylation in de novo MDS while t-MDS had widespread hypomethylation which affected the WNT and cadherin signaling pathways. Next we analyzed mechanisms of response to DMTi in a cohort of 40 CMML patients all of whom had been uniformly treated with DAC 20 mg/m2/day x 5 days. A comparison of profiles at diagnosis revealed no significant correlation between the presence of specific mutations and the likelihood of response to DAC. However, both DNA methylation and gene expression differences were identified that distinguished DAC-sensitive from DAC-resistant patients. Using a machine learning approach these DNA methylation differences could be harnessed into building an epigenetic classifier predictive of response, which when applied to an independent cohort of 28 patients was capable of predicting response to DAC with 87% accuracy. Finally, gene expression analysis demonstrated that DACresistant patients show high levels of CXCL4 and CXCL7. Treatment of normal CD34+ cells as well as primary CMML mononuclear cells with CXCL4 and CXCL7 completely abrogated the effects of DAC in vitro, indicating that upregulation of these chemokines may play a role in inducing primary resistance to DAC in CMML. 2 MOLECULAR DIAGNOSTICS R. Bejar1 1Hematology/Oncology, University of California San Diego/Moores Cancer Center, San Diego, USA Establishing a diagnosis of MDS can be challenging as it requires quantification of morphologic bone marrow features like dysplasia and blast proportion. Many patients with unexplained cytopenias fail to meet diagnostic criteria for MDS and are left without a clear diagnosis. The discovery of recurrent somatic mutations in patients with MDS has led to the speculation that these genetic lesions could have diagnostic utility in patients with idiopathic cytopenias of undetermined significance (ICUS) as they do with in myeloproliferative neoplasms and AML subtypes. However, using somatic mutations to establish a diagnosis of MDS is complicated by several issues: (1) no single gene is mutated in the majority of cases, (2) no genetic lesion is highly specific for MDS, (3) clonal hematopoiesis is strikingly common in older individuals, most of whom do not have clinical evidence of a hematologic disorder, (4) we lack data on outcomes for ICUS patients with clonal cytopenias (CCUS). Given these caveats, can genetic sequencing inform the diagnosis of MDS? Genetically, patients with CCUS more closely resemble patients with MDS than age matched controls with somatic mutations (i.e., clonal hematopoiesis of indeterminate potential or CHIP). Rates of clonality are higher in ICUS patients, particularly if they have at least equivocal evidence of dysplasia. Somatic variant allele fractions are also higher in CCUS. Genetic testing may identify alternative diagnoses in cytopenic patients. Finally, somatic mutations may identify diagnostic subgroups with comparable clinical features or risk. While we do not have the data needed to include somatic mutations in the diagnostic definition of MDS, it would be very useful to formally define CCUS so that these patients can be recognized and followed to determine their eventual outcomes. 3 MODELING SPLICING FACTOR MUTATIONS IN MDS M. Walter1 1Department of Medicine, Washington University, St. Louis, USA Somatic mutations in spliceosome genes occur in up to ~50% of MDS patients. Mutations in SF3B1, SRSF2, U2AF1, and ZRSR2 are the most frequently mutated spliceosome genes in MDS. These genes encode factors that are involved in recognition of the 3’-intronic splice site and are mutually exclusive of one another in patient samples, implying that they may contribute similarly to MDS pathogenesis or, alternatively, may not be tolerated by a cell when they co-occur. It is unclear how these mutations contribute to disease. We examined the in vivo hematopoietic consequences of mutant U2AF1 expression using a doxycyclineinducible transgenic mouse model. Mice expressing mutant U2AF1(S34F) display altered hematopoiesis, including peripheral blood leukopenia, an increase in hematopoietic progenitor cells, bone marrow lineage perturbations, and a competitive disadvantage of stem cells compared to wild-type U2AF1 0145-2126 – see front matter © 2015 Elsevier Ltd. All rights reserved.