Biomedical impact of splicing mutations revealed through exome sequencing

Splicing is a cellular mechanism, which dictates eukaryotic gene expression by removing the noncoding introns and ligating the coding exons in the form of a messenger RNA molecule. Alternative splicing (AS) adds a major level of complexity to this mechanism and thus to the regulation of gene expression. This widespread cellular phenomenon generates multiple messenger RNA isoforms from a single gene, by utilizing alternative splice sites and promoting different exon-intron inclusions and exclusions. AS greatly increases the coding potential of eukaryotic genomes and hence contributes to the diversity of eukaryotic proteomes. Mutations that lead to disruptions of either constitutive splicing or AS cause several diseases, among which are myotonic dystrophy and cystic fibrosis. Aberrant splicing is also well established in cancer states. Identification of rare novel mutations associated with splice-site recognition, and splicing regulation in general, could provide further insight into genetic mechanisms of rare diseases. Here, disease relevance of aberrant splicing is reviewed, and the new methodological approach of starting from disease phenotype, employing exome sequencing and identifying rare mutations affecting splicing regulation is described. Exome sequencing has emerged as a reliable method for finding sequence variations associated with various disease states. To date, genetic studies using exome sequencing to find disease-causing mutations have focused on the discovery of nonsynonymous single nucleotide polymorphisms that alter amino acids or introduce early stop codons, or on the use of exome sequencing as a means to genotype known single nucleotide polymorphisms. The involvement of splicing mutations in inherited diseases has received little attention and thus likely occurs more frequently than currently estimated. Studies of exome sequencing followed by molecular and bioinformatic analyses have great potential to reveal the high impact of splicing mutations underlying human disease.     

Listening to silence and understanding nonsense: exonic mutations that affect splicing

Point mutations in the coding regions of genes are commonly assumed to exert their effects by altering single amino acids in the encoded proteins. However, there is increasing evidence that many human disease genes harbour exonic mutations that affect pre-mRNA splicing. Nonsense, missense and even translationally silent mutations can inactivate genes by inducing the splicing machinery to skip the mutant exons. Similarly, coding-region single-nucleotide polymorphisms might cause phenotypic variability by influencing splicing accuracy or efficiency. As the splicing mechanisms that depend on exonic signals are elucidated, new therapeutic approaches to treating certain genetic diseases can begin to be explored.     

Contribution of rare and common variants determine complex diseases—Hirschsprung disease as a model

Finding genes for complex diseases has been the goal of many genetic studies. Most of these studies have been successful by searching for genes and mutations in rare familial cases, by screening candidate genes and by performing genome wide association studies. However, only a small fraction of the total genetic risk for these complex genetic diseases can be explained by the identified mutations and associated genetic loci. In this review we focus on Hirschsprung disease (HSCR) as an example of a complex genetic disorder. We describe the genes identified in this congenital malformation and postulate that both common ‘low penetrant’ variants in combination with rare or private ‘high penetrant’ variants determine the risk on HSCR, and likely, on other complex diseases. We also discuss how new technological advances can be used to gain further insights in the genetic background of complex diseases. Finally, we outline a few steps to develop functional assays in order to determine the involvement of these variants in disease development.     

影响RNA剪接的基因变异

发现和正确解读疾病相关突变是遗传病分子诊断和临床指导的关键。尽管二代测序技术的应用显著改善了突变检测效率,但解读突变的生物学效应仍然存在挑战。目前对基因检测结果的解读更多地关注突变对蛋白质结构和功能的影响,而忽视了基因变异对RNA剪接的影响。越来越多的证据显示引起RNA剪接异常的基因变异在疾病发生中发挥重要作用。本文对影响RNA剪接的主要突变类型和确认方法进行介绍,以期为准确判断突变的遗传效应提供参考。     

Genetic suppression of intronic +1G mutations by compensatory U1 snRNA changes in Caenorhabditis elegans

Mutations to the canonical +1G of introns, which are commonly found in many human inherited disease alleles, invariably result in aberrant splicing. Here we report genetic findings in C. elegans that aberrant splicing due to +1G mutations can be suppressed by U1 snRNA mutations. An intronic +1G-to-U mutation, e936, in the C. elegans unc-73 gene causes aberrant splicing and loss of gene function. We previously showed that mutation of the sup-39 gene promotes splicing at the mutant splice donor in e936 mutants. We demonstrate here that sup-39 is a U1 snRNA gene; suppressor mutations in sup-39 are compensatory substitutions in the 5' end, which enhance recognition of the mutant splice donor. sup-6(st19) is an allele-specific suppressor of unc-13(e309), which contains an intronic +1G-to-A transition. The e309 mutation activates a cryptic splice site, and sup-6(st19) restores splicing to the mutant splice donor. sup-6 also encodes a U1 snRNA and the mutant contains a compensatory substitution at its 5' end. This is the first demonstration that U1 snRNAs can act to suppress the effects of mutations to the invariant +1G of introns. These findings are suggestive of a potential treatment of certain alleles of inherited human genetic diseases.     

Codon bias and the folding dynamics of the cystic fibrosis transmembrane conductance regulator

Synonymous or silent mutations are often overlooked in genetic analyses for disease-causing mutations unless they are directly associated with potential splicing defects. More recent studies, however, indicate that some synonymous single polynucleotide polymorphisms (sSNPs) are associated with changes in protein expression, and in some cases, protein folding and function. The impact of codon usage and mRNA structural changes on protein translation rates and how they can affect protein structure and function is just beginning to be appreciated. Examples are given here that demonstrate how synonymous mutations alter the translational kinetics and protein folding and/or function. The mechanism for how this occurs is based on a model in which codon usage modulates the translational rate by introducing pauses caused by nonoptimal or rare codons or by introducing changes in the mRNA structure, and this in turn influences co-translational folding. Two examples of this include the multidrug resistance protein (p-glycoprotein) and the cystic fibrosis transmembrane conductance regulator gene (CFTR). CFTR is also used here as a model to illustrate how synonymous mutations can be examined using in silico predictive methods to identify which sSNPs have the potential to change protein structure. The methodology described here can be used to help identify “non-silent” synonymous mutations in other genes.     

Exonic Splicing Mutations Are More Prevalent than Currently Estimated and Can Be Predicted by Using In Silico Tools

The identification of a causal mutation is essential for molecular diagnosis and clinical management of many genetic disorders. However, even if next-generation exome sequencing has greatly improved the detection of nucleotide changes, the biological interpretation of most exonic variants remains challenging. Moreover, particular attention is typically given to protein-coding changes often neglecting the potential impact of exonic variants on RNA splicing. Here, we used the exon 10 of MLH1, a gene implicated in hereditary cancer, as a model system to assess the prevalence of RNA splicing mutations among all single-nucleotide variants identified in a given exon. We performed comprehensive minigene assays and analyzed patient’s RNA when available. Our study revealed a staggering number of splicing mutations in MLH1 exon 10 (77% of the 22 analyzed variants), including mutations directly affecting splice sites and, particularly, mutations altering potential splicing regulatory elements (ESRs). We then used this thoroughly characterized dataset, together with experimental data derived from previous studies on BRCA1, BRCA2, CFTR and NF1, to evaluate the predictive power of 3 in silico approaches recently described as promising tools for pinpointing ESR-mutations. Our results indicate that ΔtESRseq and ΔHZ_EI-based approaches not only discriminate which variants affect splicing, but also predict the direction and severity of the induced splicing defects. In contrast, the ΔΨ-based approach did not show a compelling predictive power. Our data indicates that exonic splicing mutations are more prevalent than currently appreciated and that they can now be predicted by using bioinformatics methods. These findings have implications for all genetically-caused diseases.     

De novo mutations in human genetic disease

New mutations have long been known to cause genetic disease, but their true contribution to the disease burden can only now be determined using family-based whole-genome or whole-exome sequencing approaches. In this Review we discuss recent findings suggesting that de novo mutations play a prominent part in rare and common forms of neurodevelopmental diseases, including intellectual disability, autism and schizophrenia. De novo mutations provide a mechanism by which early-onset reproductively lethal diseases remain frequent in the population. These mutations, although individually rare, may capture a significant part of the heritability for complex genetic diseases that is not detectable by genome-wide association studies.     

RNA splicing: disease and therapy

The majority of human genes that encode proteins undergo alternative pre-mRNA splicing and mutations that affect splicing are more prevalent than previously thought. The mechanism of pre-mRNA splicing is highly complex, requiring multiple interactions between pre-mRNA, small nuclear ribonucleoproteins and splicing factor proteins. Regulation of this process is even more complicated, relying on loosely defined cis-acting regulatory sequence elements, trans-acting protein factors and cellular responses to varying environmental conditions. Many different human diseases can be caused by errors in RNA splicing or its regulation. Targeting aberrant RNA provides an opportunity to correct faulty splicing and potentially treat numerous genetic disorders. Antisense oligonucleotide therapies show particular promise in this area and, if coupled with improved delivery strategies, could open the door to a multitude of novel personalized therapies.     

Assessing the functional characteristics of synonymous and nonsynonymous mutation candidates by use of large DNA constructs

As we identify more and more genetic changes, either through mutation studies or population screens, we need powerful tools to study their potential molecular effects. With these tools, we can begin to understand the contributions of genetic variations to the wide range of human phenotypes. We used our catalogue of molecular changes in patients with carbamyl phosphate synthetase I (CPSI) deficiency to develop such a system for use in eukaryotic cells. We developed the tools and methods for rapidly modifying bacterial artificial chromosomes (BACs) for eukaryotic episomal replication, marker expression, and selection and then applied this protocol to a BAC containing the entire CPSI gene. Although this CPSI BAC construct was suitable for studying nonsynonymous mutations, potential splicing defects, and promoter variations, our focus was on studying potential splicing and RNA-processing defects to validate this system. In this article, we describe the construction of this system and subsequently examine the mechanism of four putative splicing mutations in patients deficient in CPSI. Using this model, we also demonstrate the reversible role of nonsense-mediated decay in all four mutations, using small interfering RNA knockdown of hUPF2. Furthermore, we were able to locate cryptic splicing sites for the two intronic mutations. This BAC-based system permits expression studies in the absence of patient RNA or tissues with relevant gene expression and provides experimental flexibility not available in genomic DNA or plasmid constructs. Our splicing and RNA degradation data demonstrate the advantages of using whole-gene constructs to study the effects of sequence variation on gene expression and function.     

RNA splicing promotes translation and RNA surveillance

Aberrant mRNAs harboring premature termination codons (PTCs or nonsense codons) are degraded by the nonsense-mediated mRNA decay (NMD) pathway. mRNAs transcribed from genes that naturally acquire PTCs during lymphocyte development are strongly downregulated by PTCs. Here we show that a signal essential for this robust mRNA downregulatory response is efficient RNA splicing. Strong mRNA downregulation can be conferred on a poor NMD substrate by either strengthening its splicing signals or removing its weak introns. Efficient splicing also strongly promotes translation, providing a molecular explanation for enhanced NMD and suggesting that efficient splicing may have evolved to enhance both protein production and RNA surveillance. Our results suggest simple approaches for increasing protein expression from expression vectors and treating human genetic diseases caused by nonsense and frameshift mutations.     

ESEfinder: A web resource to identify exonic splicing enhancers

Point mutations frequently cause genetic diseases by disrupting the correct pattern of pre-mRNA splicing. The effect of a point mutation within a coding sequence is traditionally attributed to the deduced change in the corresponding amino acid. However, some point mutations can have much more severe effects on the structure of the encoded protein, for example when they inactivate an exonic splicing enhancer (ESE), thereby resulting in exon skipping. ESEs also appear to be especially important in exons that normally undergo alternative splicing. Different classes of ESE consensus motifs have been described, but they are not always easily identified. ESEfinder (http://exon.cshl.edu/ESE/) is a web-based resource that facilitates rapid analysis of exon sequences to identify putative ESEs responsive to the human SR proteins SF2/ASF, SC35, SRp40 and SRp55, and to predict whether exonic mutations disrupt such elements.     

Interpretation, Stratification and Evidence for Sequence Variants Affecting mRNA Splicing in Complete Human Genome Sequences

Information theory-based methods have been shown to be sensitive and specific for predicting and quantifying the effects of non-coding mutations in Mendelian diseases. We present the Shannon pipeline software for genome-scale mutation analysis and provide evidence that the software predicts variants affecting mRNA splicing. Individual information contents (in bits) of reference and variant splice sites are compared and significant differences are annotated and prioritized. The software has been implemented for CLC-Bio Genomics platform. Annotation indicates the context of novel mutations as well as common and rare SNPs with splicing effects. Potential natural and cryptic mRNA splicing variants are identified, and null mutations are distinguished from leaky mutations. Mutations and rare SNPs were predicted in genomes of three cancer cell lines (U2OS, U251 and A431), which were supported by expression analyses. After filtering, tractable numbers of potentially deleterious variants are predicted by the software, suitable for further laboratory investigation. In these cell lines, novel functional variants comprised 6-17 inactivating mutations, 1-5 leaky mutations and 6-13 cryptic splicing mutations. Predicted effects were validated by RNA-seq analysis of the three aforementioned cancer cell lines, and expression microarray analysis of SNPs in HapMap cell lines.     

Splicing in disease: disruption of the splicing code and the decoding machinery

Human genes contain a dense array of diverse cis-acting elements that make up a code required for the expression of correctly spliced mRNAs. Alternative splicing generates a highly dynamic human proteome through networks of coordinated splicing events. Cis- and trans-acting mutations that disrupt the splicing code or the machinery required for splicing and its regulation have roles in various diseases, and recent studies have provided new insights into the mechanisms by which these effects occur. An unexpectedly large fraction of exonic mutations exhibit a primary pathogenic effect on splicing. Furthermore, normal genetic variation significantly contributes to disease severity and susceptibility by affecting splicing efficiency.     

Most rare missense alleles are deleterious in humans: implications for complex disease and association studies

The accumulation of mildly deleterious missense mutations in individual human genomes has been proposed to be a genetic basis for complex diseases. The plausibility of this hypothesis depends on quantitative estimates of the prevalence of mildly deleterious de novo mutations and polymorphic variants in humans and on the intensity of selective pressure against them. We combined analysis of mutations causing human Mendelian diseases, of human-chimpanzee divergence, and of systematic data on human genetic variation and found that ~20% of new missense mutations in humans result in a loss of function, whereas ~27% are effectively neutral. Thus, the remaining 53% of new missense mutations have mildly deleterious effects. These mutations give rise to many low-frequency deleterious allelic variants in the human population, as is evident from a new data set of 37 genes sequenced in >1,500 individual human chromosomes. Surprisingly, up to 70% of low-frequency missense alleles are mildly deleterious and are associated with a heterozygous fitness loss in the range 0.001-0.003. Thus, the low allele frequency of an amino acid variant can, by itself, serve as a predictor of its functional significance. Several recent studies have reported a significant excess of rare missense variants in candidate genes or pathways in individuals with extreme values of quantitative phenotypes. These studies would be unlikely to yield results if most rare variants were neutral or if rare variants were not a significant contributor to the genetic component of phenotypic inheritance. Our results provide a justification for these types of candidate-gene (pathway) association studies and imply that mutation-selection balance may be a feasible evolutionary mechanism underlying some common diseases.     

RNA repair for haemophilia A

The mainstay of gene transfer studies is the use of wild-type cDNAs to effect phenotypic correction of diseases. However, this strategy is not feasible for genetic diseases caused either by mutations of large genes or by dominant-negative mutations, or where the regulation of the gene is critical. In this review, we will discuss a novel RNA reprogramming strategy - spliceosome-mediated RNA trans-splicing - where the pre-messenger RNA is modified by the splicing of two independent RNA species. The use of trans-splicing to effect phenotypic change in the hereditary bleeding disorder haemophilia A will be discussed.     

Increased Probability of Co-Occurrence of Two Rare Diseases in Consanguineous Families and Resolution of a Complex Phenotype by Next Generation Sequencing

Massively parallel sequencing of whole genomes and exomes has facilitated a direct assessment of causative genetic variation, now enabling the identification of genetic factors involved in rare diseases (RD) with Mendelian inheritance patterns on an almost routine basis. Here, we describe the illustrative case of a single consanguineous family where this strategy suffered from the difficulty to distinguish between two etiologically distinct disorders, namely the co-occurrence of hereditary hypophosphatemic rickets (HRR) and congenital myopathies (CM), by their phenotypic manifestation alone. We used parametric linkage analysis, homozygosity mapping and whole exome-sequencing to identify mutations underlying HRR and CM. We also present an approximate approach for assessing the probability of co-occurrence of two unlinked recessive RD in a single family as a function of the degree of consanguinity and the frequency of the disease-causing alleles. Linkage analysis and homozygosity mapping yielded elusive results when assuming a single RD, but whole-exome sequencing helped to identify two mutations in two genes, namely SLC34A3 and SEPN1, that segregated independently in this family and that have previously been linked to two etiologically different diseases. We assess the increase in chance co-occurrence of rare diseases due to consanguinity, i.e. under circumstances that generally favor linkage mapping of recessive disease, and show that this probability can increase by several orders of magnitudes. We conclude that such potential co-occurrence represents an underestimated risk when analyzing rare or undefined diseases in consanguineous families and should be given more consideration in the clinical and genetic evaluation.     

Genetic therapies for RNA mis-splicing diseases

RNA mis-splicing diseases account for up to 15% of all inherited diseases, ranging from neurological to myogenic and metabolic disorders. With greatly increased genomic sequencing being performed for individual patients, the number of known mutations affecting splicing has risen to 50-60% of all disease-causing mutations. During the past 10years, genetic therapy directed toward correction of RNA mis-splicing in disease has progressed from theoretical work in cultured cells to promising clinical trials. In this review, we discuss the use of antisense oligonucleotides to modify splicing as well as the principles and latest work in bifunctional RNA, trans-splicing and modification of U1 and U7 snRNA to target splice sites. The success of clinical trials for modifying splicing to treat Duchenne muscular dystrophy opens the door for the use of splicing modification for most of the mis-splicing diseases.