Exonic Splicing Enhancer Site Research Articles

Investigating the influence of the SIRT6 gene and alternative splicing on canine longevity: an in-depth bioinformatics analysis and experimental confirmation via NGS-based targeted sequencing.

Sirtuin 6 (SIRT6) has many functions, but its most notable contribution lies in the intricate regulation of cell senescence and lifespan. The effect of the SIRT6 gene on body size and longevity in dogs has not been extensively studied, particularly with regard to alternative splicing mechanisms. To address this gap, the present study used a comprehensive approach that integrated bioinformatics analysis, DNA sequence analysis, and next-generation sequencing-based targeted sequencing analyses. Our results show that, according to the reference genomes of different dog breeds, the canine SIRT6 gene exhibits different variants according to the dog breed. Except for the exonic variant g.55,146,051C > T (rs851065050) detected in the Boxer breed, all variants obtained from other genomes were determined to be intronic variants. The g.56,075,604 G > T (rs3343377774) intronic variant previously detected in the Labrador Retriever breed was only detected in the small breed group in our study. As a result of in silico analysis, the g.56,075,604 G > T variant has an exonic splicing enhancer (ESE) site; this variant has created the motif of the binding site for the splicing factor ESE_SRp55. The g.55.146,051C > T variant was associated with a change in the ratio of exonic splicing silencer/ESE binding motifs. This change indicates an increased probability of exon skipping for the mutant allele. Thus, as with the intronic variant g.56,075,604 G > T, the mis-splicing induced by the exonic variant g.55,146,051C > T could potentially be associated with an altered distribution of regulatory splicing factors of the canine SIRT6 gene.

Identification of a Novel Splice Site Mutation in RUNX2 Gene in a Family with Rare Autosomal Dominant Cleidocranial Dysplasia.

Background:Pathogenic variants of RUNX2, a gene that encodes an osteoblast-specific transcription factor, have been shown as the cause of CCD, which is a rare hereditary skeletal and dental disorder with dominant mode of inheritance and a broad range of clinical variability. Due to the relative lack of clinical complications resulting in CCD, the medical diagnosis of this disorder is challenging, which leaves it underdiagnosed. Methods:In this study, nine healthy and affected members of an Iranian family were investigated. PCR and sequencing of all exons and exon-intron boundaries of RUNX2 (NM_001024630) gene was performed on proband. Co-segregation analysis was conducted in the other family members for the identified variant. Additionally, a cohort of 100 Iranian ethnicity-matched healthy controls was screened by ARMS-PCR method. Results:The novel splice site variant (c.860-2A>G), which was identified in the intron 6 of RUNX2 gene, co-segregated with the disease in the family, and it was absent in healthy controls. Pathogenicity of this variant was determined by several software, including HSF, which predicts the formation or disruption of splice donor sites, splice acceptor sites, exonic splicing silencer sites, and exonic splicing enhancer sites. In silico analysis predicted this novel variant to be disease causing. Conclusion:The identified variant is predicted to have an effect on splicing, which leads to exon skipping and producing a truncated protein via introducing a premature stop codon.

A single-nucleotide polymorphism induced alternative splicing in Tacr3 involves in hypoxic-ischemic brain damage

Single-nucleotide polymorphism (SNP) and Alternative splicing (AS) were found to be implicated in certain diseases, nevertheless, the contributions of mRNA SNPs and AS to pathogenesis in developing rat brains with hypoxic-ischemic encephalopathy (HIE) remained largely vague. Additionally, the disease associated with Tacr3 was normosmic congenital hypogonadotropic hypogonadism, while the relationship between HIE and Tacr3 remained largely elusive. The current study was designed to investigate the differentially expressed mRNAs and related SNPs as well as AS in neonatal rats subjected to HIE to identify if the exhibition of AS was associated with SNPs under pathological condition. Firstly, we used postnatal day 7 Sprague-Dawley rats to construct neonatal HIE model, and analyzed the expression profiles of SNP mRNA in hypoxic-ischemic (HI) and sham brains by using RNA sequencing. Then four genes, including Mdfic, Lpp, Bag3 and Tacr3, connecting with HIE and exhibiting SNPs and AS were identified by bioinformatics analysis. Moreover, combined with exonic splicing enhancer (ESE) and alternative splice site predictor (ASSP) analysis, we found that Tacr3 is associated specifically with HIE through 258547789 G > A SNP in inside the Alt First Exon and 258548573 G > A SNP in outside the Alt First Exon. Taken together, our study provides new evidence to understand the role of Tacr3 in HIE and it is possibly a potential target for the treatment of HIE in future clinic trial.

A Genetic Variant of ASCT2 Hampers In Vitro RNA Splicing and Correlates with Human Longevity.

Given the role of amino acid regulation for physiological and pathological cell proliferation, we investigated whether the variability of solute carrier family 1, member 5 (SLC1A5, namely ASCT2), encoding for ASCT2 protein, a major glutamine transporter, is related to longevity. A total of 607 differently aged unrelated individuals, 351 very old subjects (≥85 years, range 85-106 years, mean age 93.82 ± 4.44 years) and 256 younger controls (<85 years, range 64-84 years, mean age 73.60 ± 5.70 years) were analyzed. Single-nucleotide polymorphisms (SNPs) were selected by a tagging SNP approach prioritizing those most likely to be of functional relevance. Genotyping was carried out using iPLEX Gold Genotyping Assay (Sequenom MassARRAY), and a logistic regression analysis was used to examine the associations between genotypes and the longevity phenotype. Age-associated variants were predicted to be involved in the alteration of an exonic splicing enhancer (ESE) site by Human Splicing Finder (HSF) v.3. Minigene constructs containing the alleles associated with longevity allowed the assessment of the functional effects of the polymorphisms. Two polymorphisms were found associated with human longevity (rs3027958 and rs1644343). Indeed, the major alleles of both SNPs positively influence the probability to become long-lived. In vitro assays suggested that the minor allele of rs3027958 alters an ESE site, thus inducing intron retention. We provide evidence about the functional role of intronic variants of the ASCT2 gene in hampering the splicing process, and the effect of these variants on survival, confirming the crucial role played by this amino acid transporter in modulating longevity.

Mutation in an exonic splicing enhancer site causing chronic granulomatous disease

In a male patient suffering from X-linked chronic granulomatous disease (CGD) we found a c.389G>T mutation in exon 5 of the CYBB gene. We have analyzed why 95% of the transcripts of this gene lacked exon 5, leading to a frameshift and premature termination codon. The mutation was located in a region comprising three putative exonic splicing enhancer binding sites, for SRSF1, SRFS2 and SRFS6, according to the ESEfinder Tool (http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi). With the Analyser Splice Tool we calculated the probability of skipping of exon 5 in CYBB mRNA, and by means of Sroogle the number of putative binding motifs for splicing enhancer and splicing silencer proteins (http://astlab.tau.ac.il/index.php). These analyses clarify why this exon was skipped in the majority of the mRNA. The normally spliced transcript contains an amino acid change p.Arg130Leu. This poorly expressed transcript gives rise to a protein with low expression but presumably normal activity, leading to a respiratory burst activity in the patient's neutrophils of about 15% of normal.

Abstract 1396: Role of epigenetic reader and alternative mRNA splicing variant MBD2 variant 2 in triple-negative breast cancer

Abstract Background: Among all breast cancer patients, those diagnosed with the triple-negative breast cancer (TNBC) subtype have the worst prognosis, in part attributable to the fact that TNBC lacks targets for effective molecularly-targeted therapies. The concept that EGFR inhibitor drugs could be used as a targeted treatment against TNBC has been put forth because roughly 50% of TNBC express high levels of EGFR. However clinical trials targeting EGFR did not significantly improve patient outcomes. Our recent work offers a potential explanation as to why EGFR inhibitors failed and supports an innovative therapeutic approach combining an EGFR tyrosine kinase inhibitor with the novel antioxidant biologic, CAT-SKL. Our data indicate that inhibition of cancer stem cell-like cells depends on antioxidant-induced downregulation of an alternative mRNA splice variant of the methyl-CpG binding domain 2 gene, MBD2_v2. Objective and Methods: The purpose of the present study was to evaluate the importance of MBD2_v2 in TNBC and better understand how CAT-SKL regulates its expression. Using TNBC cell lines we investigated the effects of MBD2_v2 expression on tumor initiation capacity. Furthermore we studied MBD2_v2 mRNA levels in an in vivo tumor xenograph mouse model of TNBC and in human TNBC samples using an existing database. Then by RNA-seq analysis we assessed how CAT-SKL regulates MBD2_v2 expression in TNBC cells through differential gene expression. Results: The results support that MBD2_v2 expression is regulated by redox signaling in TNBC cells, which is tightly linked to TNBC cell tumor initiation capacity. Furthermore, MBD2_v2 was upregulated in tumors harvested from overweight mice, which also displayed increased tumor take rate, suggesting that a pro-inflammatory tumor microenvironment could play a role in promoting MBD2_v2 expression. In addition, RNA-seq analysis identified an overrepresentation of mRNA splicing factors with expression downregulated by CAT-SKL treatment of TNBC cells. From this gene set two splicing co-factors are highlighted: SRRM1 and CPSF3. These co-factors are critical for splicing factor function, 3’ end formation and polyadenylation; and both have been linked via protein-protein interaction to a relevant exonic splicing enhancer site in exon 3 of the MBD2 _v2 transcript. Finally, according to the Kaplan Meier Plotter database high MBD2_v2 levels in BC were associated with shorter relapse free survival. Conclusion: Our investigation sheds light on the relevance of MBD2_v2 expression in TNBC, and how a select antioxidant regulates its expression. The application of which holds promise of a novel, targeted therapeutic modality for TNBC, the subtype with the worst prognosis and highest need for treatment options. Citation Format: Emily A. Girsch, Bin Bao, Cristina Mitrea, Gregory Dyson, Julie Boerner, Lisa Polin, Stan R. Terlecky, Aliccia Bollig-Fischer. Role of epigenetic reader and alternative mRNA splicing variant MBD2 variant 2 in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1396. doi:10.1158/1538-7445.AM2017-1396

Novel multiple endocrine neoplasia type 1 variations in patients with sporadic primary hyperparathyroidism.

Background and Objectives:Primary hyperparathyroidism (PHPT) can occur either as a sporadic case or in association with syndromes such as multiple endocrine neoplasia. Multiple endocrine neoplasia type 1 (MEN1) is a rare autosomal-dominant disease resulting from mutations in MEN1 gene encoding a 621 amino acid long tumor suppressor protein “menin.” We report here the results of MEN1 screening in 31 patients diagnosed with sporadic PHPT.Materials and Methods:Diagnosis of sporadic PHPT was made when blood urea and serum creatinine were normal, serum parathyroid hormone was high, and parathyroid enlargement could be localized on ultrasound and/or parathyroid scan. A total of 31 patients and 50 healthy volunteers were recruited for molecular analysis after taking informed consent.Results:Major symptoms at presentation were bone pain, fatigue, muscle weakness, and renal stones. Molecular genetic analysis revealed the presence of two novel intronic variations, c. 913-79T>A and c. 784-129T>A which by human splicing finder are predicted to cause potential alteration of splicing by either activating an intronic cryptic acceptor site or converting a conserved exonic splicing silencer sequence to an exonic splicing enhancer site. Apart from these, two reported polymorphisms rs144677807 and rs669976 were seen only in patients and none of the controls. Other reported polymorphisms rs2071313 and rs654440 were identified both in controls and patients.Conclusions:This is the first study of MEN1 gene screening in sporadic PHPT in India reporting on the clinical and genetic findings, wherein two novel intronic variations c. 913-79T>A and c. 784-129T>A were identified showing their possible role in disease causation.

Risk given by AGT polymorphisms in inducing susceptibility to essential hypertension among isolated populations from a remote region of China: A case-control study among the isolated populations.

Hypertension is a serious risk factor affecting up to 30% of the world's population with a heritability of more than 30-50%. The aim of this study was to investigate the contribution of the polymorphisms localized in the angiotensinogen (AGT) gene, a main component of the renin-angiotensin-aldosterone system, in inducing the susceptibility to essential hypertension (EH) among isolated populations (Yi and Hani minorities) with low prevalence rate from the remote region of Yunnan in China. A case-control association study was performed, and all subjects were genotyped for the seven single nucleotide polymorphisms localized in the AGT region by polymerase chain reaction-restriction fragment length polymorphism analysis. Three polymorphisms, i.e. rs5046, rs5049, and rs2478544, were significantly associated with EH among the Hani minority. The associations, found in the Yi minority, did not reach a conclusive level of statistical significance. The polymorphisms of rs2478544 and rs5046 caused the transformations of exonic splicing enhancer sites and transcription factor binding sites, respectively, in the bioinformatic analyses. The haplotype-rs5046T, rs5049A, rs11568020G, rs3789679C, rs2478544C was susceptible for EH among the Hani minority. Our findings suggested that the AGT polymorphisms have played a vital role in determining an individual's susceptibility to EH among the isolated population, which would be helpful for EH management in the remote mountainous region of Yunnan in China.

A Korean case of neurofibromatosis type 1 with an exonic splicing enhancer site mutation

【Neurofibromatosis type 1 (NF1) is an autosomal dominant disease characterized by neurological, cutaneous, and ophthalmological manifestations. A 33-year-old woman with typical symptoms of NF1 visited Ajou University Hospital. Screening of the whole-messenger RNA region of NF1 at the complementary DNA level by polymerase chain reaction-direct sequencing confirmed the presence of an NF1 mutation at the genomic level. The mutation analysis revealed an in-frame skipping of exon 46 (c.6757_6858del) caused by a point mutation (c. 6792C>A) in exon 46. In this report, we have described the first Korean case of a proband with NF1 that carries an allele with an exon 46 deletion caused by an exonic splicing enhancer site mutation, leading to the skipping of the whole of exon 46 (c.6757_6858del).】

Genetic variants affecting alternative splicing of human cholesteryl ester transfer protein

Cholesteryl ester transfer protein (CETP) plays an important role in reverse cholesterol transport, with decreased CETP activity increasing HDL levels. Formation of an alternative splice form lacking exon 9 (Δ9-CETP) has been associated with two single nucleotide polymorphisms (SNPs) in high linkage disequilibrium with each other, namely rs9930761 T>C located in intron 8 in a putative splicing branch site and rs5883 C>T in a possible exonic splicing enhancer (ESE) site in exon 9. To assess the relative effect of rs9930761 and rs5883 on splicing, mini-gene constructs spanning CETP exons 8 to 10, carrying all four possible allele combinations, were transfected into HEK293 and HepG2 cells. The minor T allele of rs5883 enhanced splicing significantly in both cell lines whereas the minor C allele of rs9930761 did not. In combination, the two alleles did not yield greater splicing than the rs5883 T allele alone in HepG2 cells. These results indicate that the genetic effect on CETP splicing is largely attributable to rs5883. We also confirm that Δ9-CETP protein is expressed in the liver but fails to circulate in the blood.

A compensatory effect upon splicing results in normal function of the CYP2A6*14 allele

A synonymous variant in the first exon of CYP2A6, rs1137115 (51G>A), defines the common reference allele CYP2A6*1A, and is associated with lower mRNA expression and slower in-vivo nicotine metabolism. Another common allele, CYP2A6*14, differs from CYP2A6*1A by a single variant, rs28399435 (86G>A, S29N). However, CYP2A6*14 shows in-vivo activity comparable with that of full-function alleles, and significantly higher than CYP2A6*1A. rs1137115A is predicted to create an exonic splicing suppressor site overlapping an exonic splicing enhancer (ESE) site in the first exon of CYP2A6, whereas rs28399435A is predicted to strengthen another adjacent ESE, potentially compensating for rs1137115A. Using an allelic expression assay to assess cDNAs produced from rs1137115 heterozygous liver biopsy samples, lower expression of the CYP2A6*1A allele is confirmed while CYP2A6*14 expression is found to be indistinguishable from that of rs1137115G alleles. Quantitative PCR assays to determine the relative abundance of spliced and unspliced or partially spliced CYP2A6 mRNAs in liver biopsy samples show that *1A/*1A homozygotes have a significantly lower ratio, due to both a reduction in spliced forms and an increase in unspliced or partially spliced CYP2A6. These results show the importance of common genetic variants that effect exonic splicing suppressor and ESEs to explain human variation regarding clinically-relevant phenotypes.

MYH7-Mutation Associated Allelic Imbalance in Familial Hypertrophic Cardiomyopathy: Molecular Mechanisms and Correlation with Disease Prognosis

Familial Hypertrophic Cardiomyopathy (FHC) is an autosomal dominant disease of the heart. The severity of the disease ranges from mild cases to sudden cardiac death or progression to heart failure. FHC is mostly caused by mutations in genes encoding for sarcomeric proteins, 30-40% of the patients are affected by missense mutations in one allele of the β-myosin heavy chain (MYH7).To gain further insights into the mechanisms of FHC-progression in heterozygous patients we performed a comparative expression analysis of the wildtype and the mutated MYH7 allele. We have analyzed samples from Musculus soleus and myocardium of genotyped and clinically well-characterized FHC-patients with different MYH7-mutations. We demonstrated an unequal allelic mRNA expression for each mutation analysed. The ratios of the mutated mRNA ranged from 29% to 66% in a mutation-specific manner. They were comparable in myocardium and soleus muscle and, importantly, were essentially the same at the protein level. Intriguingly, we observed a correlation between life expectancy and fraction of mutated mRNA or protein. Thus, the allelic imbalance may provide a novel factor underlying the progression of FHC.Our results suggest that the allelic imbalance is induced by differential regulation of the mutated MYH7 mRNA-expression. Thus we aimed to identify molecular mechanisms that may account for the mutation-related different mRNA levels. Bioinformatical analysis revealed that mutation V606M disrupts an exonic splicing enhancer site. Additionally, for the mutation R723G severe changes in the mRNA secondary structure were predicted. Alternative splicing variants of the V606-allele and a structure-related increased stability of the R723G-allele thus may provide potential factors inducing altered levels of mutated MYH7-mRNA.

The dual effect of the lupus-associated polymorphism rs10516487 on BANK1 gene expression and protein localization

Numerous loci have been found genetically associated with complex diseases, but only in a few cases has the functional variant and the molecular mechanism behind it been identified. Recently, the association of the BANK1 gene with systemic lupus erythematosus (SLE) was described. Here, we investigated the role of the associated polymorphisms on gene function and found that SNP rs17266594 located in the branch point consensus sequence has negligible effect on splicing or gene expression. The non-synonymous SNP rs10516487 located in exon 2 influenced splicing efficiency by creating an exonic splicing enhancer site for the SRp40 factor. Further, this same SNP generates protein isoforms with differential and measurable self-association properties. The full-length protein isoform containing the R61 variant forms larger protein scaffold complexes in the cell cytoplasm compared with the protective BANK1-61H variant. We also observed that, contrary to the full-length isoforms, the short Δ2 isoform of BANK1 displays a homogeneous cytoplasmic distribution, underscoring the potential role of the exon 2-coded protein domain in the scaffolding function of BANK1. We provide evidence that the non-synonymous SNP rs10516487 (G>A; R61H) shows a dual nature by first, influencing mRNA splicing and consequently the quantity of protein, and, second, by producing a risk variant-containing protein isoform with increased potential for multimerization.

Alternative splicing of CD200 is regulated by an exonic splicing enhancer and SF2/ASF

CD200, a type I membrane glycoprotein, plays an important role in prevention of inflammatory disorders, graft rejection, autoimmune diseases and spontaneous fetal loss. It also regulates tumor immunity. A truncated CD200 (CD200tr) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200. Thus, it is important to explore the mechanism(s) controlling alternative splicing of CD200. In this study, we identified an exonic splicing enhancer (ESE) located in exon 2, which is a putative binding site for a splicing regulatory protein SF2/ASF. Deletion or mutation of the ESE site decreased expression of the full-length CD200. Direct binding of SF2/ASF to the ESE site was confirmed by RNA electrophoretic mobility shift assay (EMSA). Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200. In vivo studies showed that viral infection reversed the alternative splicing pattern of CD200 with increased expression of SF2/ASF and the full-length CD200. Taken together, our data suggest for the first time that SF2/ASF regulates the function of CD200 by controlling CD200 alternative splicing, through direct binding to an ESE located in exon 2 of CD200.

Characterization of the c.190T&gt;C missense mutation in BRCA1 codon 64 (Cys64Arg)

In the Milan area (Northern Italy), we identified a family characterized by a high prevalence of ovarian and breast cancer cases (5 out of 6 subjects, over 3 generations), and a predominant prevalence of ovarian lesions (4 out of 5 patients). Analysis of BRCA1 and BRCA2 genes allowed the identification of the missense c.190T>C mutation in codon 64 (Cys64Arg) of BRCA1. The aims of the present investigation were to characterize the functional implications of the c.190T>C mutation at the molecular level, and to search whether additional polymorphisms might be linked to the peculiar phenotypic features observed in the Italian pedigree. Molecular modelling studies suggested that substitution of the cysteine 64 with an arginine likely disrupts the architecture of the BRCA1 RING finger domain, responsible for the interaction with BARD1, essential for the tumor-suppressor activity of the BRCA1-BARD1 complex. By splicing site information analysis, exonic splicing enhancer site characterization, and analysis of transcript fragment length and sequence, we showed that the c.190T>C mutation was able to modulate the splicing of exon 5 in a fashion opposite to the c.190T>G transversion, responsible for the functionally-related Cys64Gly amino acid substitution. Genotyping of BRCA1 and BRCA2 in the Italian family revealed the presence of two significant polymorphisms: the cancer-associated c.2612C>T SNP in BRCA1, and the c.-26G>A SNP in the BRCA2 gene, acting as an ovarian cancer risk modifier in carriers of deleterious BRCA1 mutations. Analysis of these SNPs in a genotypically-unrelated Polish family, characterized by prevalent breast neoplasms in carriers of the c.190T>C mutation, revealed a genetic profile consistent with the hypothetic role of both polymorphisms.

Guidelines for Antisense Oligonucleotide Design and Insight Into Splice-modulating Mechanisms

Antisense oligonucleotides (AONs) can interfere with mRNA processing through RNase H-mediated degradation, translational arrest, or modulation of splicing. The antisense approach relies on AONs to efficiently bind to target sequences and depends on AON length, sequence content, secondary structure, thermodynamic properties, and target accessibility. We here performed a retrospective analysis of a series of 156 AONs (104 effective, 52 ineffective) previously designed and evaluated for splice modulation of the dystrophin transcript. This showed that the guanine-cytosine content and the binding energies of AON-target and AON-AON complexes were significantly higher for effective AONs. Effective AONs were also located significantly closer to the acceptor splice site (SS). All analyzed AONs are exon-internal and may act through steric hindrance of Ser-Arg-rich (SR) proteins to exonic splicing enhancer (ESE) sites. Indeed, effective AONs were significantly enriched for ESEs predicted by ESE software programs, except for predicted binding sites of SR protein Tra2beta, which were significantly enriched in ineffective AONs. These findings compile guidelines for development of AONs and provide more insight into the mechanism of antisense-mediated exon skipping. On the basis of only four parameters, we could correctly classify 79% of all AONs as effective or ineffective, suggesting these parameters can be used to more optimally design splice-modulating AONs.

K45R variant of squalene synthase increases total cholesterol levels in two study samples from a French Canadian population

Squalene synthase is an important component of the cholesterol biosynthetic pathway, and inhibitors of this enzyme have been shown to lower plasma cholesterol levels. Previously, we sequenced the squalene synthase gene, FDFT1 (farnesyl-diphosphate farnesyltransferase), and identified several SNPs, including a nonsynonymous variant, rs11549147:A>G (K45R). To examine the possible association of K45R with plasma lipid traits, we tested 887 individuals from 149 families from the founder population of Saguenay-Lac St. Jean (SLSJ), Quebec. K45R was associated with increased total cholesterol (TC) (P=0.035) and non-high-density lipoprotein cholesterol (non-HDL-C) (P=0.01). These results were replicated in an independent sample of unrelated individuals (P=0.0008 for TC, P=0.004 for non-HDL-C). This SNP also influenced low-density lipoprotein cholesterol (P=0.042) and HDL-C (P=0.025) in the family-based sample, and triglycerides (TG) (P=0.007) in the unrelated subjects. The lysine (K) in codon 45 is conserved across 11 mammals and lies in a potential exonic splicing enhancer (ESE) site. These results suggest that this coding variant in the squalene synthase gene influences plasma cholesterol levels, possibly by affecting the intracellular production of cholesterol.

PTEN c.511C&gt;T Nonsense Mutation in a BRRS Family Disrupts a Potential Exonic Splicing Enhancer and Causes Exon Skipping

Bannayan-Riley-Ruvalcaba syndrome (BRRS) is an autosomal dominant disorder characterized by macrocephaly, intestinal hamartomatous polyps, lipomas and pigmented macules of the glans penis. We identified a Thai family affected with BRRS. In addition to typical manifestations of BRRS, the proband has a large hepatic AVM which is rarely found in BRRS. The molecular analysis revealed affected members were heterozygous for an exon skipping-associated nonsense mutation c.511C>T in the PTEN gene. The mutation was previously assumed to be deleterious by causing a change to a termination codon, Q171X. We, herein, found that another pathogenic effect was splicing related by disrupting a potential exonic splicing enhancer (ESE) and causing an entire exon 6 skipping. The results prompted us to investigate other reported missense/nonsense mutations in the PTEN gene. We found that they do not colocalize with ESE sites, suggesting that most of their pathogenic effects are not through ESE disruption.

Therapeutic Modulation of DMD Splicing by Blocking Exonic Splicing Enhancer Sites with Antisense Oligonucleotides

Antisense oligonucleotides (AONs) can be used to correct the disrupted reading frame of Duchenne muscular dystophy patients (DMD). We have a collection of 121 AONs, of which 79 are effective in inducing the specific skipping of 38 out of the 79 different DMD exons. All AONs are located within exons and were hypothesized to act by steric hindrance of serine-arginine rich (SR) protein binding to exonic splicing enhancer (ESE) sites. Indeed, retrospective in silico analysis of effective versus ineffective AONs revealed that the efficacy of AONs is correlated to the presence of putative ESE sites (as predicted by the ESEfinder and RESCUE-ESE software). ESE predicting software programs are thus valuable tools for the optimization of exon-internal antisense target sequences.

Germline Missense Mutations Affecting KRAS Isoform B Are Associated with a Severe Noonan Syndrome Phenotype

Noonan syndrome (NS) is a developmental disorder characterized by short stature, facial dysmorphia, congenital heart disease, and multiple skeletal and hematologic defects. NS is an autosomal dominant trait and is genetically heterogeneous. Gain of function of SHP-2, a protein tyrosine phosphatase that positively modulates RAS signaling, is observed in nearly 50% of affected individuals. Here, we report the identification of heterozygous KRAS gene mutations in two subjects exhibiting a severe NS phenotype with features overlapping those of cardiofaciocutaneous and Costello syndromes. Both mutations were de novo and affected exon 6, which encodes the C-terminal portion of KRAS isoform B but does not contribute to KRAS isoform A. Structural analysis indicated that both substitutions (Val152Gly and Asp153Val) perturb the conformation of the guanine ring-binding pocket of the protein, predicting an increase in the guanine diphosphate/guanine triphosphate (GTP) dissociation rate that would favor GTP binding to the KRASB isoform and bypass the requirement for a guanine nucleotide exchange factor.