Acceptor Site Of Exon Research Articles

A New Genetic Immunodeficiency Syndrome Caused By Autosomal Dominant Mutations in LCP1 Identified By Mosaic Tetraploidy and Severe Neutropenia in Unrelated Pedigrees

The spectrum of genetic abnormalities causing immune deficiencies is still far from complete. We identified independent kindreds in the Netherlands (NL) and the USA in which affected members presented with severe chronic neutropenia (SCN), but lacked the thus far known mutations causing SCN. Bone marrow morphology of the patients showed large hyper-segmented and binucleate neutrophils. In addition to neutropenia, lymphopenia and abnormalities in B cell development were seen, indicating that the hematopoietic defect was not restricted to the neutrophil lineage. An additional remarkable and thus far little-known feature was the presence of cells in the bone marrow with a tetraploid karyotype (Hochberg et al., Pediatr Blood Cancer 2008). Pedigree analysis of the NL and USA families revealed a hereditary pattern consistent with an autosomal dominant inheritance (Figure 1). Comparative whole exome sequencing of both pedigrees identified LCP1, encoding the actin-bundling protein L-plastin, as the single candidate gene, completely segregating affected from unaffected individuals. The LCP1 mutations in the pedigrees are not identical, but give rise to the same altered transcript. The LCPNLmutation is a single nucleotide substitution that disrupts the splice acceptor site at the 5' side of exon 8. In contrast, the mutation LCP1USA causes a deletion of 25 nucleotides at the boundary of intron 7 and exon 8. Theoretically, the LCP1USAmutation could cause a frameshift deletion in exon 8 resulting in a premature stop codon in exon 8. However, because this mutation also disrupts the 5' splice acceptor site of exon 8, it could also lead to alternative RNA splicing, using an alternate cryptic splice acceptor site in exon 8. Sanger sequencing on cDNA derived from mRNA from NL-I unveiled that the splice-site mutation indeed results in an alternative splicing variant with an in-frame loss of 24 nucleotides at the start of exon 8. Next generation RNA sequencing on bone marrow cells from NL-I and USA-III (Figure 1) revealed that the identical alternative splice variant is expressed from LCP1USA and LCP1NL. In both pedigrees, there is approximately a fifty percent drop in reading depth from the start of exon 8 up until the next available splice acceptor site, consistent with a heterozygous 24-nucleotide deletion from the start of exon 8. The resulting in-frame deletion of 8 amino acids affects a highly conserved region between actin binding domain (ABD) 1 and ABD2, thereby predicted to affect the horseshoe-like 3D structure of L-plastin. LCP1USA and LCP1NL will henceforth collectively be termed LCP1ex8mut. Confocal microscopy analysis of HeLa cells expressing LCP1ex8mut showed that mutant L-plastin colocalized with F-actin in excessively formed filopodia, which was not seen in cells expressing wild-type LCP1 (LCP1wt). In addition, binucleate cells were present in cells expressing LCP1ex8mut, but not LCP1wt. A number of cells in G2M phase showed accumulation of F-actin and L-plastin on the contractile ring, a characteristic seen only in cells expressing LCP1ex8mut. This finding suggests that mutant L-plastin interferes with successful completion of cell division. Accumulation of mutated LCP1 on the contractile ring thus could explain the inability of neutrophil progenitors to properly go through cytokinesis, hence giving rise to neutropenia and tetraploidy. Finally, because a recently developed mouse model revealed that phosphorylation of serine 5 (Ser5) of L-plastin is essential for its function in host-pathogen interactions and immune defense in vivo (Anaya et al., J Immunol 2021), we mutated Ser5 to alanine (Ser5Ala) in LCP1ex8mut. This did not affect the function of LCP1ex8mut, suggesting that the mutation confers activation of L-plastin independent of upstream serine/threonine kinase pathways. Altogether, these findings establish that the mutations confer a gain of function rather than haploinsufficiency. In conclusion, by comparing independent pedigrees, we have identified a new immune deficiency syndrome caused by autosomal dominant activating mutations in LCP1 leading to defective neutrophil production and B cell development, which at least in part results from disturbed cytokinesis causing tetraploidy. Figure1. Pedigree information of the NL and USA family. Solid black squares or circles indicate neutropenic males or females, respectively. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Expression and characterisation of Fmr1 splice variants during folliculogenesis in the rat.

The FMR1 gene consists of 17 exons and codes for the FMRP protein. FMR1 is involved in four genetic disorders depending on the CGG repeats length in its 5'UTR: the full mutation is responsible for the Fragile X syndrome while the premutation is associated with the Fragile X-associated Tremor/Ataxia Syndrome, Fragile X-associated Primary Ovarian Insufficiency (FXPOI) and Fragile X-associated neuropsychiatric disorders. FMR1 presents multiple isoforms resulting from skipping of exons 12 and 14 and the use of alternative splice sites in exons 15 and 17. To investigate the expression of Fmr1 splicing variants during folliculogenesis in the rat. We used preantral, early antral and preovulatory follicles to isolate RNA and characterise, by fluorescent PCR followed by sequencing, all the isoforms present in the different follicular stages. We identified two isoforms resulting from splicing of exon 12, six isoforms resulting from splicing of exon 14 and 15 and one isoform for exon 17. The expression levels of the isoforms vary within each follicular stage but not between different stages of folliculogenesis. Importantly, we identify for the first time in rat, an isoform that contains exon 12 and two isoforms, one that includes and one that excludes exon 14 and use the third acceptor site in exon 15. Characterisation of the different FMR1 variants expressed during folliculogenesis will help to understand the potential distinct cellular roles of each of them and the possible implication in the development of FXPOI.

A Novel Mutation Causing GATA2 Haploinsufficiency Activates a Cryptic Splice Site in Exon 3

A 48-year old woman presented with clinical symptoms consistent with GATA2 haploinsufficiency: recurrent human papilloma virus associated cervical intraepithelial neoplasia, extensive deep vein thrombosis of the lower extremity, a history of hepatitis C virus infection and recurrent pneumonias complicated by cryptogenic organizing pneumonia. Infectious agents isolated from the lungs included mycoplasma pneumonia, serratia marcescens, mycobacterium intracellulare and aspergillus nidulans . Laboratory findings revealed monocytopenia, B, NK and CD4 T cell lymphopenia. However, analysis of the bone marrow ruled out myelodysplastic syndrome and showed a normal karyotype. Sanger sequencing from genomic DNA isolated from peripheral blood did not reveal any published missense, nonsense or complex mutation associated with GATA2 haploinsufficiency, but showed a novel, synonymous mutation in exon 3 (c.351C>G, p.T117T). In order to elucidate the pathomechanism of the mutation, we performed an in depth in silico evaluation of the mutated sequence and analysed cDNA from the patient bone marrow and fibroblasts for aberrant splicing patterns by GATA2 specific RT-PCR and Sanger sequencing.In silico studies of the mutation showed that the mutated cytosine at position 351 is conserved across most species (www.ensembl.org) and predicted aberrations of splicing, especially the activation of a crypticsplice site in exon 3 (www.fruitfly.org). GATA2 specific RT-PCR showed comparable results for the patient and controls and did not show any aberrant bands at visual inspection. Sanger sequencing of these RT-PCR amplified GATA2 specific transcripts from patient bone marrow and blood identified a clear “C” call for the wild type allele and a very weak “G” call for the mutated allele. In order to test if the mutated allele was expressed at minor frequency we cloned the transcripts into a topoisomerase I-activated pCR™4-TOPO® TA Vector. Sequencing of 60 clones showed an overrepresentation of the wild type allele (42/60 clones, 70%) in comparison to the mutated allele (8/70 clones, 13%). Further clones (10/60, 17%) showed an aberrantly spliced cDNA using the predicted cryptic splice donor site at cDNA position 351 and a cryptic splice acceptor site in exon 3 (cDNA position 487). The aberrant transcript showed a deletion of 136bp in exon 3 and introduction of a premature stop codon after 517bp. This presumably leads to a truncated GATA2 protein disrupting the DNA binding domains. The data support that GATA2 haploinsufficiency in our patient is caused by decreased expression of the mutated allele and abrogation of functional protein expression from the aberrantly spliced mRNA.In summary, we report a novel mutation type leading to GATA2 haploinsufficiency. The described, synonymous mutation activates a cryptic splice donor site in GATA2 exon 3. This mutation type and mutations affecting splicing in general have so far not been described in GATA2 haploinsufficiency. Our finding underscores the need to thoroughly investigate also rare “silent” mutations in genetic counselling and diagnosis. DisclosuresNo relevant conflicts of interest to declare.

Insulin-like androgenic gland hormone from the shrimp Fenneropenaeus merguiensis: Expression, gene organization and transcript variants.

Male sex differentiation in the crustacean is best known to be controlled by the insulin-like androgenic gland hormone (IAG). In this report, the cDNA and gene of the shrimp Fenneropenaeus merguiensis FmIAG were studied and characterized. FmIAG gene shares a high sequence identity in the coding region as well as the promoter region with that of F. chinensis. FmIAG gene is most likely consists of 5 exons and 4 introns. The cDNA reported here is the most abundant transcript that retained cryptic intron 4. The use of different splicing acceptor sites in exon 2 can produce a long-form FmIAG transcript variant with 6 additional amino acids inserted. Splicing of cryptic intron 4 would produce a transcript variant with a different C-terminal end. Therefore 4 different FmIAG transcripts can be produced from the FmIAG gene. During the molt cycle, the expression level of FmIAG was low in the early intermolt, increase steadily towards the late premolt and decreased rapidly in the early postmolt. In addition to the androgenic gland, FmIAG is also expressed in the hepatopancreas and ovary of adult females. Unilateral eyestalk ablation caused a significant increase in FmIAG transcript suggesting that the eyestalk consists of inhibiting factor(s) that suppressesFmIAGexpression. To explore the function of FmIAG in males, injection of FmIAG dsRNA knock-down the expression of FmIAG and up-regulated the expression of the vitellogenin gene in the testis and hepatopancreas. Interestingly a CHH-like gene identified in the androgenic gland was down-regulated. CHH-like gene knock-down resulted in altered expression of FmIAG in males suggesting that the CHH-like may be involved in FmIAG's regulation. RT-PCR with specific primers to the different transcript variant were used to determine if there is an association of different sizes of male and the type of IAG transcript. Results indicated that a high percentage of the large male shrimp expressed the long-form of FmIAG. The results suggested that FmIAG may be useful as a size marker for male shrimp aquaculture. In summary, the results of this study have expanded our knowledge of shrimp insulin-like androgenic gland hormone in male sex development and its potential role as a marker gene for growth regulation in shrimp.

Restoration of Dystrophin Protein Expression by Exon Skipping Utilizing CRISPR-Cas9 in Myoblasts Derived from DMD Patient iPS Cells.

Duchenne muscular dystrophy (DMD) is a congenital X-linked disease caused by mutations in the gene encoding the dystrophin protein, which is required for myofiber integrity. Exon skipping therapy is an emerging strategy for restoring the open reading frame of the dystrophin gene to produce functional protein in DMD patients by skipping single or multiple exons. Although antisense oligonucleotides are able to target pre-mRNA for exon skipping, their half-lives are short and any therapeutic benefit is transient. In contrast, genome editing by DNA nucleases, such as the CRISPR-Cas9 system, could offer permanent correction by targeting genomic DNA. Our laboratory previously reported that disrupting the splicing acceptor site in exon 45 by plasmid delivery of the CRISPR-Cas9 system in iPS cells, derived from a DMD patient lacking exon 44, successfully restored dystrophin protein expression in differentiated myoblasts. Herein, we describe an optimized methodology to prepare myoblasts differentiated from iPS cells by mRNA transfection of the CRISPR-Cas9 system to skip exon 45 in myoblasts, and evaluate the restored dystrophin by RT-PCR and Western blotting.

Developmental regulation of SMN expression: pathophysiological implications and perspectives for therapy development in spinal muscular atrophy.

Spinal muscular atrophy (SMA), the predominant form of motoneuron disease in children and young adults is caused by loss of function of the SMN protein. On the basis of a disrupted splice acceptor site in exon 7, transcripts from a second SMN gene in humans called SMN2 cannot give rise to SMN protein at sufficient levels for maintaining function of motoneurons and motor circuits. First clinical trials with Spinraza/Nusinersen, a drug that counteracts disrupted splicing of SMN2 transcripts, have shown that elevating SMN levels can successfully interfere with motoneuron dysfunction. This review summarizes current knowledge about the pathophysiological alterations in Smn-deficient motoneurons, which lead to defective neuromuscular transmission and altered spinal circuit formation. Both pathological mechanisms are important targets for therapeutic intervention. However, the developmental time window when therapeutic interventions ideally should start is not known. Endogenous SMN expression both from SMN1 and SMN2 genes is high at early developmental stages and declines progressively in humans and mice. Thus, therapeutic SMN upregulation should start just before SMN declines below a critical threshold, and before irreversible defects occur at neuromuscular junctions and in spinal circuits. Previous results indicate that loss of Smn function leads to synaptic dysfunction during a stage of neuromuscular development when synaptic strength determines which synapses are maintained or not. This time window appears as an important target for therapy, which possibly could be supported by additional strategies that strengthen synaptic transmission.

Abstract 892: Functional characterization of EIF1AX mutations in thyroid cancer predicts for gain of function by increasing translational rate with concomitant derepression of upstream inputs from mTOR

Abstract EIF1AX (eukaryotic translation initiation factor 1A) is a component of the translation pre-initiation complex (PIC). Recurrent EIF1AX mutations, first reported in uveal melanomas, are found in ∼1% of papillary thyroid cancers in a mutually exclusive manner with other oncogenic driver events (BRAF, RAS, and oncogenic fusions). By contrast, they are enriched in advanced thyroid cancers (9% of anaplastic and 11% poorly-differentiated thyroid cancers), and are strongly associated with RAS mutations (p<0.0001). EIF1AX mutations cluster in the N-terminal (NTT) or C-terminal tails (CTT). EIF1AX NTT missense mutations in thyroid cancer occur within the first 15 amino acids, whereas the CTT mutation disrupts a splice acceptor site at exon 6 (A113splice). A113splice is the most prevalent defect and is private to this disease, and results in two differentially spliced mRNAs: (1) Cryptic splice variant: by use of a cryptic acceptor site in exon 6 that leads to a 132 AA protein that excludes 12 AA; (2) Truncated splice variant: by retaining intron 5, leading to a 115 AA truncated protein. EIF1AX mutants retain the ability to recruit the ternary complex, as shown by co-IP with EIF2 after ectopic expression of NTT or A113splice EIF1AX mutants in HEK293T cells, or after CRISPR-mediated knock-in of A113splice into Cal62 RAS-mutant thyroid cancer cells. The EIF1AX mutants had greater affinity to EIF5 compared to WT, consistent with a more stable PIC. As translation initiation is a rate-limiting step, the altered affinity of EIF1AX mutants to PIC components could impact the rate of protein synthesis. We tested this by L-azidohomoalanine labeling, which showed contrasting roles of the two A113-generated splice variants expressed at endogenous levels: i.e. the cryptic splice variant increased protein synthetic rate, whereas the truncated splice variant strongly inhibited protein translation. Despite inhibiting translation, the truncated splice variant showed a paradoxical increase in 4EBP1 phosphorylation. Upon A113splice knock-in, where both variants are expressed, translation is increased, which we hypothesize results from the combined effects of 4EBP1 phosphorylation, caused by relief of negative feedback events upstream in the pathway, with increased PIC assembly caused by the cryptic splice variant. We are currently determining whether the altered rate of protein synthesis is global or selective. Of note, cells expressing the cryptic, but not the truncated splice form, showed a transforming phenotype as assessed by soft agar colony formation. As EIF1AX mutations co-occur with RAS mutations in advanced thyroid cancers, it is likely that RAS-induced PI3K-AKT/mTOR signaling may provide a further cooperative benefit and play a key role in disease progression, and in generating specific tumor cell dependencies. Citation Format: Gnana P. Krishnamoorthy, Inigo Landa, Jeffrey A. Knauf, James Nagarajah, Gunnar Rätsch, Hans-Guido Wendel, James A. Fagin. Functional characterization of EIF1AX mutations in thyroid cancer predicts for gain of function by increasing translational rate with concomitant derepression of upstream inputs from mTOR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 892.

Fryns Syndrome Associated with Recessive Mutations in PIGN in two Separate Families.

Fryns syndrome is an autosomal recessive condition characterized by congenital diaphragmatic hernia (CDH), dysmorphic facial features, distal digital hypoplasia, and other associated malformations, and is the most common syndromic form of CDH. No gene has been associated with this condition. Whole-exome sequence data from two siblings and three unrelated individuals with Fryns syndrome were filtered for rare, good quality, coding mutations fitting a recessive inheritance model. Compound heterozygous mutations in PIGN were identified in the siblings, with appropriate parental segregation: a novel STOP mutation (c.1966C>T: p.Glu656X) and a rare (minor allele frequency <0.001) donor splice site mutation (c.1674+1G>C) causing skipping of exon 18 and utilization of a cryptic acceptor site in exon 19. A further novel homozygous STOP mutation in PIGN (c.694A>T: p.Lys232X) was detected in one unrelated case. All three variants affected highly conserved bases. The two remaining cases were negative for PIGN mutations. Mutations in PIGN have been reported in cases with multiple congenital anomalies, including one case with syndromic CDH. Fryns syndrome can be caused by recessive mutations in PIGN. Whether PIGN affects other syndromic and non-syndromic forms of CDH warrants investigation.

A novel FOXP1-PDGFRA fusion gene in myeloproliferative neoplasm with eosinophilia

We identified a novel fusion gene, FOXP1-PDGFRA, in a patient with myeloproliferative neoplasm (MPN) with eosinophilia, harboring the chromosome abnormality t(3;4)(p13;q12). The patient responded well to imatinib and has remained in molecular remission for 3 years. This is the seventh fusion gene involving PDGFRA in MPN with eosinophilia. PDGFRA was truncated in its autoinhibitory domain, as in other PDGFRA-related MPNs, and was fused to FOXP1 at its functional forkhead domain. Comparing genomic DNA with mRNA sequences provides the possibility that the splicing process near the breakpoint junction in the FOXP1-PDGFRA fusion gene may use the normal splice donor site for intron 23a of FOXP1 and the cryptic splice acceptor site in exon 12 of PDGFRA. This is the first report to describe the FOXP1-PDGFRA fusion gene in MPN.

A canonical splice site mutation in GIPC3 causes sensorineural hearing loss in a large Pakistani family.

With homozygosity mapping we have identified two large homozygous regions on chromosome 3q13.11-q13.31 and chromosome 19p13.3-q31.32 in a large Pakistani family suffering from autosomal recessive nonsyndromic hearing impairment (arNSHI). The region on chromosome 19 overlaps with the previously described deafness loci DFNB15, DFNB72 and DFNB95. Mutations in GIPC3 have been shown to underlie the nonsyndromic hearing impairment linked to these loci. Sequence analysis of all exons and exon-intron boundaries of GIPC3 revealed a homozygous canonical splice site mutation, c.226-1G>T, in GIPC3. This is the first mutation described in GIPC3 that affects splicing. The c.226-1G>T mutation is located in the acceptor splice site of intron 1 and is predicted to affect the normal splicing of exon 2. With a minigene assay it was shown to result in the use of an alternative acceptor site in exon 2, resulting in a frameshift and a premature stop codon. This study expands the mutational spectrum of GIPC3 in arNSHI.

T.P.33 A cutting-edge approach to Spinal Muscular Atrophy treatment using antisense oligonucleotides

Abstract Spinal Muscular Atrophy (SMA), a severe neuromuscular disorder characterised by the loss of motor neurons, is typically caused by deletion of the Survival Motor Neuron (SMN) 1 gene. The loss of the SMN1 gene product is incompatible with life, however in humans the presence of one or more copies of the near-identical SMN2 modifies disease severity. A single C>T base change in exon 7 of SMN2 results in abnormal splicing with the majority of transcripts missing exon 7 that encodes a crucial oligomerisation domain, and therefore results in a non-functional protein. The C>T variation simultaneously abrogates an exonic splicing enhancer, and creates a silencing element that leads to the exclusion of this exon from the mature gene transcript. Hence, redirecting splicing of SMN2 pre-mRNA has become a potential therapeutic target with the aim of re-enforcing exon 7 recognition and inclusion to increase levels of the full-length transcript and functional protein. Antisense oligomers (AOs) can interfere with splice site recognition by the spliceosome, and depending upon design can promote exon skipping or exon inclusion in the mature transcript. In treating SMA, efforts have focused on either enhancing exon 7 recognition, or masking splice silencing motifs located in introns 6 or 7. We present a third strategy where targeting the acceptor site of exon 8 with AOs induced exon and intron 7 inclusion in the transcript. The retention of intron 7 occurs after the normal stop codon and results in a longer than normal 3′ untranslated region. Western blot analysis demonstrated increased SMN expression in various SMA patient fibroblasts transfected with these AOs. “GEMs” are intranuclear aggregates of SMN and are barely quantifiable in fibroblasts from SMA patients. Immunofluorescent GEM staining showed substantial increases in abundance of GEMs in the AO treated SMA fibroblasts, indicating this strategy has considerable therapeutic potential.

Abstract 2170: The SWI/SNF nucleosome-remodeling gene PBRM1 - Another tumor suppressor gene in renal cell carcinomas

Abstract The polybromodomain containing gene PBRM1 was recently discovered to harbor 41% truncating somatic mutations in clear cell renal cell carinomas (ccRCC). Next to the VHL tumor suppressor gene this gene is thought to be the second major cancer gene in ccRCCs. PBRM1 (also called BAF180) belongs to the SWI/SNF-B or PBAF complex and provides functional specificity to this class of chromatin remodelers by binding to acetylated histones via its six bromodomains, two BAH domains, and one HMG domain (the latter binding to nucleosomal DNA). PBRM1 is located on chromosome 3p21 in close vicinity to VHL and SETD2 genes arguing for a putative gatekeeper function in cases where VHL loss alone is insufficient for ccRCCs tumorigenesis. PBRM1 codes for at least three major transcripts of about 7500nt in length whose expression was measured by Q-PCR in 49 cases of paired normal and tumor tissue samples of RCC patients who underwent radical tumor nephrectomy at our clinic between 06/2009 and 10/2010. 20/49 cases showed a marked upregulation of PBRM1 expression whereas in 29/49 cases PBRM1 was clearly downregulated. To assess the contribution of somatic mutations for PBRM1 downregulation we resequenced 29 PBRM1 exons of 48 tumor tissues. Specific truncating mutations were detected in 6 (15.4%) of 39 ccRCCs. These mutations include 5 deletions in exons 1, 10, 16, 23, and 24, as well as one insertion in exon 16. All these heterozygous mutations lead to a decrease of PBRM1 mRNA expression, that was verified by Q-PCR measurements. In addition, 6 coding SNPs (cSNPs) were revealed as well as 5 SNPs in intronic regions, one of the latter is in close vicinity to the splice acceptor site of exon 9. Concurrent expression of three major transcripts (var. 1,2, and 4) is seen in all 46 matched normal and tumor samples analyzed so far. In 22 (47.8%) samples we detected a dominance of var.1 (NM_018165) in normal tissue when compared to the corresponding tumor sample, that preferentially expresses var.4 (NM_181042). So far our data set of 15.4% truncating mutations in 39 cases of ccRCC could not match the exceptional high number of 41% reported in the initial report by the Sanger Institute. Truncating mutations of PBRM1 have been described in breast cancers but were absent in lung carcinomas, despite the fact that LOH at 3p21 is frequently seen in these cancers. Understanding the contribution of PBRM1 expression changes, be it on the genomic level (mutation) and/or mRNA (differential splicing), to clinical disease progression and outcome in kidney cancer needs further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2170. doi:1538-7445.AM2012-2170

The expression analysis of mouse interleukin-6 splice variants argued against their biological relevance

Alternative splicing generates several interleukin-6 (IL-6) isoforms; for them an antagonistic activity to the wild-type IL-6 has been proposed. In this study we quantified the relative abundance of IL-6 mRNA isoforms in a panel of mouse tissues and in C2C12 cells during myoblast differentiation or after treatment with the Ca(2+) ionophore A23187, the AMP-mimetic AICAR and TNF-α. The two mouse IL-6 isoforms identified, IL-6δ5 (deletion of the first 58 bp of exon 5) and IL-6δ3 (lacking exon 3), were not conserved in rat and human, did not exhibit tissue specific regulation, were expressed at low levels and their abundance closely correlated to that of full-length IL-6. Species-specific features of the IL-6 sequence, such as the presence of competitive 3' acceptor site in exon 5 and insertion of retrotransposable elements in intron 3, could explain the production of IL-6δ5 and IL-6δ3. Our results argued against biological significance for mouse IL-6 isoforms.

The Arabic allele: A single base pair substitution activates a 10-base downstream cryptic splice acceptor site in exon 12 of LDLR and severely decreases LDLR expression in two unrelated Arab families with familial hypercholesterolemia

Familial hypercholesterolemia (FH) is a monogenic autosomal dominant disorder caused by defects in LDLR. Few reports describe FH mutations among Arabs. We describe a mutation in LDLR of two unrelated Arab families. We investigated 19 patients using DNA sequencing, RFLP, and real-time (RT) PCR. DNA sequencing showed a base pair substitution (c.1706-2 A>T) in the splice acceptor site of LDLR intron 11. Our results were confirmed by RFLP on 2% agarose gel. In silico analysis predicted a new cryptic splice site downstream of the original position generating a 10-base deletion from the beginning of exon 12; (c.1706-1715del.ATCTCCTCAG). cDNA sequencing of exon 12 confirmed the computational analysis. The deletion was visualized on 4% agarose gel. The deletion generates a frameshift and a premature termination codon (c.1991-1993; p.(Asp569Valfs*93). RT-PCR revealed that LDLR mRNA is 9.3% ± 6.5 and 17.9% ± 8.0 for FH homozygote and heterozygote individuals respectively, compared to a healthy family control. We predict a class II LDLR mutation that leads to a truncated receptor missing exons 14–18. We called this mutation “the Arabic allele”. We expect a significant contribution of this mutation to the prevalence of FH among Arabs. Also, we propose that the severe down regulation of LDLR mRNA expression is due to nonsense-mediated-decay

Decreased Expression in Nuclear Factor-κB Essential Modulator Due to a Novel Splice-Site Mutation Causes X-linked Ectodermal Dysplasia with Immunodeficiency

X-linked ectodermal dysplasia with immunodeficiency (XL-ED-ID) is caused by hypomorphic mutations in NEMO, which encodes nuclear factor-kappaB (NF-κB) essential modulator. We identified a novel mutation, 769-1 G>C, at the splicing acceptor site of exon 7 in NEMO in a Japanese patient with XL-ED-ID. Although various abnormally spliced NEMO messenger RNAs (mRNAs) were observed, a small amount of wild-type (WT) mRNA was also identified. Decreased NEMO protein expression was detected in various lineages of leukocytes. Although one abnormally spliced NEMO protein showed residual NF-κB transcription activity, it did not seem to exert a dominant-negative effect against WT-NEMO activity. CD4(+) T cell proliferation was impaired in response to measles and mumps, but not rubella. These results were consistent with the clinical and laboratory findings of the patient, suggesting the functional importance of NEMO against specific viral infections. The 769-1 G>C mutation is responsible for decreased WT-NEMO protein expression, resulting in the development of XL-ED-ID.

Abstract LB-97: Identification of a novel splice variant of Hyaluronan Synthase-2 (HAS2) in ovarian cancer stem cells

Abstract We have identified a novel cellular structure consisting of a chain of 4–72 cells with a pericellular sugar coat (glycocalyx) in metastatic human ovarian cancer (OvCa) ascites samples. We have termed these “catenae” (Latin for “chains”). Catenae develop clonally and are exclusively composed of cancer stem cells (CSC) since a single catena cell forms intraperitoneal tumor (Abs #1376, AACR 2011). The glycocalyx is necessary for the maintenance of cancer stem cell state of catena and confers resistance to small molecule, antibody and immune cell-mediated killing. Microarray gene expression analysis identified hyaluronan synthase-2 (HAS2) as the most upregulated gene in catena compared to differentiated OvCa monolayer cells. HAS2 enzyme catalyzes the production of hyaluronan, which is an essential component of the catena glycocalyx. 454 deep sequencing analysis of the HAS2 gene in catenae showed that the read coverage was specific to the exon 1 and exon 4. Limited coverage was detected for exon 2 and exon 3, which suggested the possibility of an alternative splice variant. RT-PCR analysis of HAS2 identified a band of 1115 bp as a new splice variant of the HAS2 gene lacking exon 2, which we have termed the “Greenwich variant”. The Greenwich variant is in the same sequence frame as the wild-type HAS2 and represents a novel form of HAS2 protein starting from amino acid 215 to the C-terminus. The variant still has half the cytoplasmic enzymatic domain and it retains three extracellular domains. The Greenwich variant was expressed in catena cells with high CSC (50–100%) frequencies but not in differentiated OvCa monolayer cells with low CSC (1–5%) frequencies. The variant was present in 14 out of 16 (88%) ascites samples from patients with advanced stage metastatic serous ovarian adenocarcinoma and in 15 out of 25 (60%) metastatic serosal cancer samples of other histologies including breast, lung, gastric, colon and pancreatic cancer. Deep sequencing analysis identified 2 intronic point mutations at the splicing acceptor site of exon 4 of the HAS2 gene in catena cells. 5 out of 220 OvCa patients in The Cancer Genome Atlas database had HAS2 mutations. 4 out of 5 of these mutations were located at the exon 2-exon 3 junction. These mutations could result in alternative splicing of HAS2 and generation of the Greenwich variant. We also analyzed the expression of miRNAs in catena cells by Agilent arrays. hsa-miR-23b was the most downregulated miRNA in catena compared to differentiated OvCa cells. hsa-miR-23b is a negative regulator of HAS2 gene, and our results suggest that downregulation of hsa-miR-23b is associated with upregulation of wild-type HAS2 and the Greenwich variant. The Greenwich variant is a novel CSC-specific marker and its presence could be linked to catena formation, increase in cancer stem cell frequency, drug resistance, metastasis to serosal cavities (ascites, pleural and pericardial effusions) and poor prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-97. doi:10.1158/1538-7445.AM2011-LB-97

Splice Variants of the Dual Specificity Tyrosine Phosphorylation-regulated Kinase 4 (DYRK4) Differ in Their Subcellular Localization and Catalytic Activity

Dual specificity tyrosine phosphorylation-regulated kinases, DYRKs, are a family of conserved protein kinases that play key roles in the regulation of cell differentiation, proliferation, and survival. Of the five mammalian DYRKs, DYRK4 is the least studied family member. Here, we show that several splice variants of DYRK4 are expressed in tissue-specific patterns and that these variants have distinct functional capacities. One of these variants contains a nuclear localization signal in its extended N terminus that mediates its interaction with importin α3 and α5 and that is capable of targeting a heterologous protein to the nucleus. Consequently, the nucleocytoplasmic mobility of this variant differs from that of a shorter isoform in live cell imaging experiments. Other splicing events affect the catalytic domain, including a three-amino acid deletion within subdomain XI that markedly reduces the enzymatic activity of DYRK4. We also show that autophosphorylation of a tyrosine residue within the activation loop is necessary for full DYRK4 kinase activity, a defining feature of the DYRK family. Finally, by comparing the phosphorylation of an array of 720 peptides, we show that DYRK1A, DYRK2, and DYRK4 differ in their target recognition sequence and that preference for an arginine residue at position P -3 is a feature of DYRK1A but not of DYRK2 and DYRK4. Therefore, we highlight the use of subcellular localization as an important regulatory mechanism for DYRK proteins, and we propose that substrate specificity could be a source of functional diversity among DYRKs.

Functional Complementation ofGlra1spd-ot, a Glycine Receptor Subunit Mutant, by Independently Expressed C-Terminal Domains

The oscillator mouse (Glra1(spd-ot)) carries a 9 bp microdeletion plus a 2 bp microinsertion in the glycine receptor alpha1 subunit gene, resulting in the absence of functional alpha1 polypeptides from the CNS and lethality 3 weeks after birth. Depending on differential use of two splice acceptor sites in exon 9 of the Glra1 gene, the mutant allele encodes either a truncated alpha1 subunit (spd(ot)-trc) or a polypeptide with a C-terminal missense sequence (spd(ot)-elg). During recombinant expression, both splice variants fail to form ion channels. In complementation studies, a tail construct, encoding the deleted C-terminal sequence, was coexpressed with both mutants. Coexpression with spd(ot)-trc produced glycine-gated ion channels. Rescue efficiency was increased by inclusion of the wild-type motif RRKRRH. In cultured spinal cord neurons from oscillator homozygotes, viral infection with recombinant C-terminal tail constructs resulted in appearance of endogenous alpha1 antigen. The functional rescue of alpha1 mutants by the C-terminal tail polypeptides argues for a modular subunit architecture of members of the Cys-loop receptor family.

Silent exonic mutations in the low‐density lipoprotein receptor gene that cause familial hypercholesterolemia by affecting mRNA splicing

In a large group of patients with the clinical phenotype of familial hypercholesterolemia, such as elevated low-density lipoprotein (LDL) cholesterol and premature atherosclerosis, but without functional mutations in the genes coding for the LDL receptor and apolipoprotein B, we examined the effect of 128 seemingly neutral exonic and intronic DNA variants, discovered by routine sequencing of these genes. Two variants, G186G and R385R, were found to be associated with altered splicing. The nucleotide change leading to G186G resulted in the generation of new 3'-splice donor site in exon 4 and R385R was associated with a new 5'-splice acceptor site in exon 9 of the LDL receptor gene. Splicing of these alternate splice sites leads to an in-frame 75-base pair deletion in a stable mRNA of exon 4 in case of G186G and R385R resulted in a 31-base pair frame-shift deletion in exon 9 and non-sense-mediated mRNA decay.

Identification of cis-acting elements involved in Acetylcholinesterase RNA alternative splicing

The 3′ end of Acetylcholinesterase (AChE) pre-mRNA is processed by a complex mechanism of alternative splicing. Three different transcripts are generated and called R, H and T according respectively to the intron (intron 4′) or exons (5 or 6) retained in the mature RNA. The relative expression of the specific transcripts depends on cell type, developmental stage or pathophysiological conditions. The aim of our study was to identify sequences involved in AChE pre-mRNA splicing choices. For this purpose, we constructed a minigene in which the constitutive exons were fused and followed by the entire alternative domain without 3′ UTR. We transfected the wild-type or minigene mutated in the alternative domain in muscle or COS-7 cells and identified the splicing products by RPA, RT-PCR and sedimentation coefficients of the enzymatic molecular forms. We find that the alternative splicing domain contains most of the necessary signals to control splicing choices in skeletal muscle cells with the coding sequences of the domain having little effect on the splicing outcome. A branch point at an unusual location 278 nt from the 3′ acceptor site of exon 6 is characterized. We further identify several regulatory sequences in the non-coding sequence of exon 5 that regulate the splicing pattern. Sequences that control the splice to exon 5 and those which influence intron 4′ retention or splicing to exon 6 appear to be distinct.