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ISSN: 2155-9880
Journal of Clinical & Experimental Cardiology
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Splice Site Mutation-Induced Alteration of Selective Regional Activity Correlates with the Role of a Gene in Cardiomyopathy

Jia Fei and Shi-You Chen*
Department of Physiology and Pharmacology, University of Georgia, USA
Corresponding Author : Shi-You Chen, Ph.D.
Department of Physiology and Pharmacology
The University of Georgia Athens
GA30602, USA
Tel: 706-542-8284
E-mail: [email protected]
Received: December 05, 2012; Accepted: January 03, 2013; Published: January 05, 2013
Citation: Fei J, Chen SY (2013) Splice Site Mutation-Induced Alteration of Selective Regional Activity Correlates with the Role of a Gene in Cardiomyopathy. J Clin Exp Cardiolog S12:004. doi:10.4172/2155-9880.S12-004
Copyright: © 2013 Fei J, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Splice site mutations of a number of genes are found to induce cardiomyopathy. The mutation alters the thermodynamic activity of nucleotide fragment around the splice sites, which causes exon skipping, leading to dysfunction of certain genes. In the present study, we analyzed the selective and regional characteristic of splice site mutations for genes contributed to the development of cardiomyopathy. 93 splice site mutations in total 16genes involved in cardiomyopathy were analyzed for the information content (Ri). Exon skipping (Ri value<0) appeared to correlate with hypertrophic cardiomyopathy and arrhythmogenic right ventricular dysplasia, the two cardiomyopathies with the highest mortality. We also found that most of the exon skipping was due to the donor splice site mutations instead of the acceptor site mutations at the splice junction. Our studies revealed that the regional preference of mutations at the donor sites is an important factor in determining whether or not the gene plays a role in the development of cardiomyopathies

Cardiomyopathy; Splice site; Exon skipping; Bioinformatics
Cardiomyopathy represents a cluster of severe heart muscle diseases or syndromes leading to heart failure including hypertrophic cardiomyopathy (HCM), catecholaminergic polymorphic ventricular tachycardia (CPVT), dilated cardiomyopathy (DCM), arrhythmogenic right ventricular dysplasia (ARVD), and restrictive cardiomyopathy (RCM) [1]. Patients with cardiomyopathies have a high risk of arrhythmia or sudden cardiac death [2]. Gene mutations have been identified in both inheritable cardiomyopathies [3] and acquired cardiomyopathies [4].
Gene mutations such as deletion, insertion, or fragment shifting normally do not alter RNA splice. However, nucleotide substitution at the junction of primary or natural splicing sites (NSS) can alter property of the splicing sites [5]. Moreover, most substitutions induce exon skipping or interruption of cryptic splicing sites (CSS) or alternative splicing sites (ALSS), resulting in genetic disorders [6-8]. Exon skipping is considered as the major consequence of abnormal splice. In addition, a number of CSSs are found near or around NSS with splice potentials. Activation or abnormal splice of these CSSs frequently induce genetic diseases [6]. Moreover, abnormal splice of intervening sequence (IVS) or intron has also been shown to alter functions of eukaryotic genes [9]. Therefore, it is very important to predict and analyze the impact of splicing site mutations on genetic disorder. In this report, an informative evaluation using Informationcontent Model has been conducted to evaluate effects of nucleotide substitutions on cardiomyopathies. Information theory-based model explains nucleotide variations in splice sites and has been frequently used to predict activities of normal or mutant splice sites in order to identify CSS [5]. Individual information reflects thermodynamic entropy and free energy of binding [10]. The information content (Ri, in bits) of a gene sequence describes the degree of the sequence contributing to the conservation of the entire gene [10]. Thus, the total effects of nucleotide alterations can evaluated based on cumulative Ri at every position in the splice-site [5]. By using the Information-content Model, we established the effect of regional activity alteration of splice sites in 16 genes on the development of cardiomyopathies, which predict the mechanism of a gene in inducing cardiomyopathy.
Selection and phenotypes of cardiomyopathies
Four main types of cardiomyopathies, i.e., HCM, CPVT, DCM and ARVD, were selected for this study. RCM was not included because RCM was not found to be a consequence of splice site mutation. Cardiac muscle myosin heavy chain 7-β (MYH7) mutation induces left ventricular non-compaction cardiomyopathy, which is classified as a HCM [11]. HCM phenotypes include increased left ventricular wall thickness, asymmetric septal hypertrophy and/or abnormal ECG [12,13]. CPVT is characterized by bidirectional ventricular tachycardia (VT) [14]. DCM is defined as decreased ejection fraction (<50%), left ventricular shortening fraction (<28%), and/or increased left ventricular internal diastolic diameter (>2.7 cm/m2 body surface area) [15,16]. ARVD displays fibro-fatty penetration into right ventricular wall (RV) and RV wall thinning due to loss of cardiomyocyte [17].
Selection of candidate genes with mutation in cardiomyopathy
The human gene mutation database namely Biobase Database ( database) [18] was used to select genes which mutations are involved in cardiomyopathies. A total of 16genes were found to be involved in cardiomyopathies due to splicing site mutations, and 93 splice site mutations were analyzed for their Ri values (Table 1).
Informatics analysis of splicing site mutations
DNA sequences with 150 nucleotides upstream and downstream of normal or mutated splicing sites were obtained from Genbank. The Ri of each sequence was calculated using Delila system for Human Splice Junction Analysis ( [5,10,19-21]. The user license was approved by National Institutes of Health.
The minimal information content (Ri-min) of a normal splice sites was calculated based on the boundary of nonfunctional site and normal transcript production. Comparison of multiple splice sites from a number of genes has established that an average value of 2.4 represents the Ri-min for the normal splice sites [5].
Ri for both donor and acceptor sites in normal (Ri(n)) or mutated sequences (Ri(m)) was also calculated using Delila system. The correlation of Ri value with splice site mutation was determined by the following criteria: Ri(m) ≤ 0 bit predicts that a junction mutation induces an exon skipping. Ri(m)<2.4 but >0 bits expects that a mutation completely inhibits or abolishes an normal splice site and contributes to an severe phenotype; Ri(m)>2.4 bits with a lower Ri(n) of adjacent normal site forecasts that a mutation reduces the activity of an natural splice site resulting in a mild phenotype [5]; Ri(m)>Ri(n) predicts that a mutation activates CSS. This newly-generated CSS would have its own Ri. Ri(m)=Ri(n) suggests that a mutation does not affect normal splice sites [22].
Statistical analysis
Ri values were analyzed and showed a non-normal distribution. Ri was presented as mean (average) ± standard error of the mean (SEM). The significant difference between groups was analyzed using Mann Whitney test. A two-tailed value of p<0.05 indicates statistical significance [5].
Gene mutation generated different impacts on splice site
To study the effect of gene mutation on the property of splice sites of genes involved in cardiomyopathy, we analyzed Ri of 93 splice site mutations found in 16 genes as aforementioned. We found that a great number of splice site mutations caused exon skipping (Table 2, Mutation allele #1, 10, 13, 14, 15, 18, 21, 22, 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 41, 42, 43, 45, 47, 48, 49, 50, 51, 53, 56 and Table 3, # 6, 10, 11, 12, 16, 17, 21, 26, 27, 32). A less number of splice site mutations led to complete abolishment of natural splice sites (Table 2, Mutation allele #2, 5, 9, 11, 12, 20, 38, 39, 44, 46 and Table 3, #37). A number of mutations inhibited the activities of NSS and induced moderate cardiomyopathy phenotype (Table 2, Mutation allele #3, 4, 6, 7, 8, 16, 17, 19, 27, 37, 40, 54 and Table 3, # 2, 3, 4, 5, 7, 8, 13, 14, 20, 24, 25, 28, 30, 31, 33, 34, 35, 36, 37). In addition, several mutations activated CSS (Table 3, Mutation allele #2, 5, 9, 14, 15, 22, 25, 35). Only a few mutations had no effect on NSS (Table 2, Mutation allele #54 and Table 3, #1, 18, 19, 23, 29, 36).
Exon skipping was caused by donor site mutation
To determine what splice site mutation causes exon skipping, we quantified Ri value in both donor and acceptor sites of the genes with exon skipping, and found that Ri values at mutated donor sites were significantly decreased to -5.19 ± 0.79 as compared with their corresponding natural Ri value (7.17 ± 0.43 bits) (P<0.0001). In contrast, Ri values at mutated acceptor sites were reduced to -3.31 ± 1.09 as compared with their corresponding natural acceptor sites (4.77 ± 1.09) (P<0.0001). Since the difference of Ri value at the donor sites was much greater than the acceptor sites (14.3 ± 2.15 vs. 8.08 ± 0.22) (P<0.05), the donor site mutation, rather than the acceptor site mutation, was likely to be the major factor in inducing exon skipping.
Abolishment of normal splice sites was predicted by the similar reduction of Ri in both mutated donor and acceptor sites
A number of genes (Table 2, Mutation allele #2, 5, 9, 11, 12, 20, 38, 39, 44, 46 and Table 3, #37) involved in severe cardiomyopathies exhibited splice site mutations different from these found in Exon skipping. Analysis of these genes revealed that Ri values at the donor sites was dramatically reduced as compared to the natural splice sites (0.82 ± 0.21 vs. 8.31 ± 0.64) (P<0.0001). Ri values at the acceptor sites were also reduced as compared to the natural sequences (1.70 ± 0.75 vs. 10.0 ± 1.11) (P<0.0001). However, unlike Exon skipping, Ri value reductions at donor and acceptor sites were not significantly different (7.18 ± 1.32 vs. 8.30 ± 0.38, P>0.05). Based on the established requirement for Ri value (2.4) of a normal splice site, it was predicted that no normal splicing occurred in these genes.
Leaky splice was induced by either the donor or acceptor site mutations
Leaky splice was caused by an incomplete inhibition of natural splice due to splice site mutations. These mutations had a reduced Ri values that were greater than Ri-min (2.4 bits) and were observed to induce mild cardiomyopathy phenotypes. Compared to NSS, these mutations significantly decreased the Ri in mutated donor sites (5.04 ± 0.51 vs. 9.05 ± 0.40) (p<0.0001, n=12). The mutations may also significantly reduced Ri value of mutated acceptor sites (5.43 ± 0.52 vs. 10.73 ± 0.76) (p<0.0001). There were no significant difference in Ri values between donor and acceptor sites (4.01 ± 0.59 vs. 4.88 ± 0.88), similar to splice elimination. Therefore, leaky splice was not caused by regional mutations.
The nearby CSS detection and classification
CSS activated by splice site mutations in or around splice sites occurred as a result of new formation of secondary cryptic splice sites with an abnormal Ri value. CSS may be categoRized as following:
1. CSS with a high Ri value: Mutations induced an increased CSS Ri value accompanied by a decreased or unaltered Ri value of NSS. In cardiomyopathy related genes, two CSSs were found at donor site mutations (Table 2, # 22, 23) and four at acceptor site mutations (Table 3, #2, 3, 22, 28). This type of CSS was shown in Figure 1.
2. CSS with a low Ri value: The newly-created CSS was characterized by a Ri value lower than that of NSS after mutations (Table 3, # 5, 7, 30, 34, 35, 36). Desmoplakin (DSP) gene exhibited this type of CSS, as shown in Figure 2.
3. CSS with residual splice: This type of CSS occurred when splice site mutations had no effect on Ri values (Table 3, # 2, 8, 9, 35, 37). This phenomenon is likely to result in the competition between NSS and CSS.
Splice site mutation regional effect on cardiomyopathy phenotype
ARVD, DCM and HCM were selected to analyze if the regional mutation of a gene was a determining factor for its involvement in the development of a specific type of cardiomyopathy. As shown in table 4, ARVD was associated with exon skipping due to donor site mutation while ARVD was associated with leaky splice due to acceptor site mutation of certain genes. Both exon skipping and leaky splice showed equal contributions to DCM due to donor site mutation of different genes. The activation of CSS or leaky splice was involved in DCM as a result of acceptor site mutation. Exon skipping was the major cause of donor site mutation-induced HCM. Interestingly, acceptor site mutation-induced HCM may be due to exon-skipping, leaky splice, CSS, or splice elimination.
It is evident that gene mutations result in cardiomyopathies. Splice site mutations play a critical role in the onset of cardiomyopathies. Nucleotide substitutions significantly induce exon skipping, splice abolishment and leaky splice. Although mutations contribute to the significant change of Ri value in both donor and acceptor sites, the significant effect is only evident on exon skipping between donor and acceptor sites, suggesting that donor sites are more susceptible to mutation than acceptor sites in the development of cardiomyopathy.
The relationship analyses indicate that mutation-induced Ri value alteration contributes to the selective splice site abnormalities. These abnormalities selectively occur at either donor or acceptor sites depending on the types of cardiomyopathies. Such regional abnormalities appear to determine particular genes that may be involved in certain cardiomyopathies. For example, MYBPC3 mutations induce hypertrophic cardiomyopathy. Most of the splice site mutations are found in donor sites. Similar phenomena are observed in Laminin α1 gene and Plakophilin 2, in which donor site mutations cause DCM and ARVD, respectively. Moreover, donor site mutationinduced exon skipping with large decline of Rivalue (Ri< 0) is closely associated with the onset of ARVD and HCM. Comparing to the other types of cardiomyopathies, ARVD and HCM are recognized as the most threatening disorders due to sudden death. Therefore, donor site mutation may be useful for predicting the potential role of a particular gene in inducing severe cardiomyopathies. The smaller Ri alterations induced by the relatively mild splice site mutations randomly occur in most of less severe cardiomyopathies. Exon skipping, splice abolishment, and leaky splice due to acceptor site mutations are found in all types of cardiomyopathies, suggesting that mutation in acceptor sites has less selective effects.
CSS obtains its own Ri value although it is lower compared to NSS. Most of CSS occur in acceptor sites because acceptor sites appear to be used for defining and recognizing exons in vertebrate species [23]. It has been reported that certain nucleotide compositions such as alternative GC-AG introns exhibit weak donor sites but enhanced spliceosome intron-recognizing ability in acceptor sites in human [24]. Most of the newly-created CSS have relative low Ri value compared to the mutant sites, suggesting that CSS activation may not be the major cause for cardiomyopathy. The function of CSS in cardiomyopathies requires further investigations. Individual information analysis simulates thermodynamic entropy of nucleotides, and thus reflects the importance of the regional splice site mutation in the selection of genes that may be involved in the onset of cardiomyopathies. This finding may be useful for developing a novel method to screen candidate genes potentially implicated in these diseases.
This work was supported by grants from National Institutes of Health (HL093429 and HL107526 to Dr. Chen).

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