RNA Splicing Defects in Hypertrophic Cardiomyopathy: Implications for Diagnosis and Therapy

Figures

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  Figure 1. Precursor messenger RNA (pre-mRNA) splicing. (A) Exons are represented by boxes and introns by lines. The most conserved nucleotides at the 5’ splice site (5’ss), branch point (BP), polypyrimidine tract (PY), and 3’ splice site (3’ss) are indicated. (B) The two transesterification reactions that result in the excision of introns from pre-mRNA are represented.

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  Figure 2. Spliceosome assembly for pre-mRNA splicing. (A) Spliceosome assembly is initiated through binding of the U1 snRNP to the 5’ splice site (ss), SF1 to the branch point (BP), U2AF65 to the polypyrimidine tract (PY), and U2AF35 to the 3’ splice site. (B) Next, the U2 snRNP displaces SF1 and binds the BP. (C) Recruitment of the U4/U6.U5 tri-snRNP. (D) Conformational rearrangements involving the release of the U1 and U4 snRNPs lead to catalytic activation. (E) Ligated exons are released from the spliceosome, and the remaining snRNPs persist, associated with the intron lariat. (F) Subsequently, snRNPs dissociate from the excised intron.

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  Figure 3. Splicing regulatory sequence elements. Regulatory elements are located in exons (exonic splicing enhancer (ESE) or exonic splicing silencer (ESS)) and in introns (intronic splicing enhancer (ISE) or intronic splicing silencer (ISS)). Enhancers (green) bind SR proteins and promote spliceosome assembly at nearby splice sites. Silencers (red) bind members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family and inhibit spliceosome assembly.

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  Figure 4. Splicing mutations. (A) Schematics illustrating the consequences of cis-acting mutations on pre-mRNA splicing. (B) Representation of a frameshift deletion introduced by the activation of a cryptic splice site within an exon and degradation of the resulting mRNA by nonsense-mediated decay (NMD).

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  Figure 5. Hypertrophic cardiomyopathy (HCM) mutations. The bar graphs depict the number and type of HCM-associated variants in the indicated genes. All variants reported as pathogenic or likely pathogenic in the National Center for Biotechnology Information’s ClinVar database (http://www.ncbi.nlm.nih.gov/clinvar/) were considered.

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  Figure 6. HCM splice site mutations reported in the ClinVar database. Position of splice site mutations reported in the ClinVar database for the eight HCM-associated genes shown in Figure 5. The number of variants per position is indicated. The 5′ splice site (ss) is also known as the donor (D) splice site, whereas the 3’ site is also known as the acceptor (A) splice site. Position D + 1 corresponds to the first intronic nucleotide downstream of the depicted exon, D + 2 to the second, and so forth. Position A-1 corresponds to the last intronic nucleotide upstream of the depicted exon, A-2 to the previous one, and so forth.

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  Figure 7. Recently reclassified MYBPC3 splicing mutations. Exons are represented by boxes and introns by lines. Exonic nucleotides are indicated by capital letters and intronic nucleotides by small-case letters. (A) Variant c.654+5G>C created a cryptic donor splice site within exon 5, leading to a truncated exon with an in-frame deletion of 48 pb [63]. (B) Variant c.1624+4A>T caused the skipping of exon 17, leading to a frameshift [63]. (C) Deep intronic variant c.1090+453C>T created a new splice donor sequence, which led to the inclusion of a 77-bp pseudo-exon in the mRNA, causing a frameshift and the introduction of a premature termination codon (PTC) [62]. (D) Deep intronic variant c.1091-575A>C created a new splice acceptor sequence, which led to the inclusion of an 85-bp pseudo-exon in the mRNA, causing a frameshift and the introduction of a PTC [62]. (E) Synonymous variant c.2274C>T (p.Gly758Gly) caused the truncation of exon 23 by 36 nucleotides, leading to an in-frame deletion of 12 amino acids [62].