Human SNPs resulting in premature stop codons and protein truncation

SNPs

SNPs annotated 'premature termination codon SNPs' were retrieved from the dbSNP database build 120 (http://www.ncbi.nlm.nih.gov/SNP/) [19]. We have annotated such SNPs as X-SNPs throughout this paper. There was a total of 977 X-SNPs in the dbSNP database; however, only 119 of them were presented with minor allele frequency information. Among these SNPs, only the ones that were found in at least two chromosomes with a sample size of ≥ 20 chromosomes were further analysed (herein annotated as validated X-SNPs). The X-SNPs that are located on the transcripts annotated as 'predictions', 'pseudogenes', 'similar to' or 'open reading frames' were excluded from this study. In total, 28 X-SNPs were in agreement with all of the above requirements.

BLAST analyses

To map the SNP sequences on transcripts, SNP-flanking sequences of X-SNPs were blasted against the transcripts in GenBank (http://www.ncbi.nih.gov/Genbank/)[20] using the BLAST against gene transcripts tool (http://lpgws.nci.nih.gov:80/perl/blast2/) [21], as explained by Savas et al [22] One mismatch in the SNP-flanking sequence/transcript alignment was allowed. The SNP-flanking sequences were also blasted against the human genome using the NCBI BLAST tool (http://www.ncbi.nlm.nih.gov/BLAST/)[23] to ensure that the SNP sequences are not derived from multiple genomic regions [24], as explained in a further paper by Savas et al. [25]

Alternatively spliced transcript variants (ASTVs)

Information relating to ASTVs was retrieved from the Ref Seq resource of NCBI (http://www.ncbi.nlm.nih.gov/RefSeq/) [26].

Candidate transcripts for nonsense-mediated mRNA decay

Blasting the transcript sequence against the human genome identified the genomic structures of transcripts. The subsequent manual analysis of the exon - intron boundaries identified X-SNPs that can lead to nonsense-mediated mRNA decay (NMD): the transcripts with an SNP introducing a PTC located ≥ 50 nucleotides upstream of an exon - intron junction are considered candidates to undergo NMD [13, 27–29]. Blasting the transcript sequence against the human genome identified the genomic structures of transcripts. The subsequent manual analysis of the exon - intron boundaries identified X-SNPs that can lead to nonsense-mediated mRNA decay (NMD): the transcripts with an SNP introducing a PTC located ≥ 50 nucleotides upstream of an exon - intron junction are considered candidates to undergo NMD [13, 27–29].

Possible biological consequences of X-SNPs

We then carried out a theoretical evaluation of the possible biological consequences of the identified X-SNPs at the mRNA and protein levels. For example, NMD is a surveillance system that specifically eliminates transcripts that contain PTCs as a result of mutations in DNA or errors in RNA processing [15]. NMD usually requires a downstream intron and at least 50 - 55 nucleotides before the downstream exon - intron junction in order for a PTC to be recognised [27, 28]. Based on the 50 - 55 nucleotide rule, we analysed the locations of the X-SNPs with respect to the exon - intron boundaries and predicted that eight (28.6 per cent) X-SNPs (AGT-Q53X, APOC4-W47X, EPHX1-W97X, MS4A12-Q71X, POLE2- K443X, SERPINB11-E90X, SMUG1-Q3X and ZNF34- Q56X) may potentially cause mRNA degradation via NMD. Thus, at least these eight X-SNPs are likely to result in loss of gene function. Exceptions to this rule have also been reported [17], however, which suggests that the proportion of PTC-containing mRNAs undergoing mRNA degradation may, in fact, be larger. The reported allele frequencies of these X-SNPs ranged from rare (AGT-Q53X 0 -5 per cent; APOC4-W47X 0 - 5 per cent; EPHX1-W97X 0 - 2.2 per cent; POLE2-K443X 0 - 1.2 per cent; SMUG1-Q3X 1.2 - 1.4 per cent) to common (MS4A12-Q71X 41.5 - 45.8 per cent; SERPINB11-E90X 22.9 - 52.1 per cent; ZNF34-Q56X 27 per cent) (Table 1). Individuals with a homozygous state for two of these X-SNPs, namely MS4A12-Q71X and SERPINB11-E90X, were reported in dbSNP submissions, suggesting that, in such individuals, the levels of these truncated protein products are likely to be reduced by the NMD mechanism.

If no NMD occurs and the protein products are translated, then the PTCs lead to protein truncation at the C-terminus - the consequences of which vary depending on the degree of the truncation. For example, 17 (60.7 per cent) X-SNPs led to moderate to severe truncation at the C-terminus of the proteins (deletion of > 50 per cent of the amino acid sequence), which is likely radically to alter protein structure and function (Table 1, Figure 1). As an extreme example, SMUG1-Q3X, when translated, would yield only a two-amino acid peptide, which would presumably be non-functional (loss of function). Also, PTCs can destabilise the protein products by altering the protein-folding state or kinetics[14, 31] and may cause proteolysis. In addition, they may act as dominant-negative mutations[15] or cause exon skipping and alter the open reading frame [32]. Alternatively, mRNA molecules bearing a PTC closer to the 5' end can be still translated if an in-frame translationable AUG start codon is present downstream of the PTC [33–35]. Such N-terminal truncated proteins can be fully or partially functional. For example, in the case of SMUG1-Q3X, there is an inframe AUG located at the 18th codon of the SMUG1 gene, which can be experimentally evaluated to determine if an N-terminal truncated SMUG1 protein is produced and functional. To summarise, the stability, structure and function of the protein products or transcripts may be affected by the X-SNPs described in this study, and experimental approaches are needed to evaluate their true biological effects.

Possible evolutionary explanations of common X-SNPs

The small number of validated X-SNPs identified suggests infrequent occurrence of the PTC-introducing variations in the human genome and thus agrees with the presence of selection against them [18]. By contrast with rare PTC-introducing mutations observed in human diseases, however, X-SNPs analysed in this study represented commonly occurring variations in humans: 22 X-SNPs (78.6 per cent) were found with minor allele frequencies of ≥ 5 per cent in at least one sample panel analysed (common X-SNPs) compared with only six rare X-SNPs (with < 5 per cent minor allele frequencies).

How can we explain the abundance of such common (and perhaps deleterious) X-SNPs in the human population? Possible scenarios are summarised in Figure 2. For example, one explanation could be that the truncated protein product may still be functional in the presence of the X-SNP. For instance, LPL-S474X was located only one amino acid prior to the natural termination codon; thus, it may not really alter the protein properties and thus may not be deleterious to cell function at all. Alternatively, the protein may not be essential for the fitness of human beings; in this case, the evolutionary pressure is relieved, which can lead to toleration of an increase in allele frequency of premature stop codons in human populations.

Another possibility is that X-SNPs may be capable of affecting protein function/the organism per se, but other factors might modify their effects. Here, we will assume that these PTCs represent both the strongly deleterious mutations that are a result of selection and quickly removed from the populations, as well as the slightly deleterious mutations that are subject to both selection and drift [36]. For example, these X-SNPs may be hot-spot mutations, where the new mutations introduce (slightly) deleterious alleles and thus increase the allele frequency, despite the selection. In order to assess whether some of these X-SNPs might in fact represent the hot-spot mutations, we analysed the immediate flanking sequences of each X-SNP. As a result, we found that 25 per cent (7/28) of X-SNPs (all common) had occurred at CpG dinucleotides (Table 1). These data suggest that these X-SNPs might have arisen from spontaneous deamination of methylcytosine leading to a thymine, and thus may represent hot-spot mutations [37].

Additionally, diploidy was suggested to relieve the tension of purifying selection and increase the tolerance for PTCs [38], which predicts a recessive effect or loss of function. All but one gene (MAGEE2) in Table 1 were located in autosomal chromosomes, which may also help to explain the frequency of the naturally occurring PTC polymorphisms in humans. Moreover, it is also likely that, even though (slightly) deleterious in a homozygous state, some X-SNPs can confer selective advantage to heterozygotes [39]. Alternatively, epistatic interactions of additional mutations, either on the same or different genes, may compensate for the (slightly) deleterious effects of the X-SNP [16, 40]. Furthermore, X-SNPs may be beneficial at present conditions, which may favour the positive selection of the X-SNPs and increase their allele frequencies. Moreover, if a PTC is located at the 5' end of a gene and there is a nearby in-frame initiation codon after that PTC, then the protein translation can re-initiate and a peptide with aminotruncation may be produced [33–35]. Depending on the nature and extent of the truncation, the truncated peptide can fully or partially function and thus can, completely or to some extent, rescue the phenotype. There is a need for further studies to elucidate the molecular basis of the discrepancy and the determination of the biological differences between human disease-related mutations and naturally occurring stop codon-creating polymorphisms.

Frequency spectrum of X-SNPs in different human populations

Comparison of the population(s) and the minor allele frequencies of X-SNP entries in the dbSNP database[19] presented great variability across different human populations, at least in some cases (Table 1). For example, HSP4-R246X, IL17RB-Q484X, LPL-S474X, MS4A12-Q71X, OVCH2-W556X, SERPINB11-E90X, TAAR9-Q61X and TRPM1-E1305X were detected in samples from African, Asian and Caucasian backgrounds. This might mean that either these X-SNPs have been inherited from a common ancestor or they represent hot-spot mutations (HSP4-R246X and IL17RB Q484X occurred at CpG dinucleotides and thus might in fact be hot-spot mutations; see Table 1). By contrast, CYPC19-W212X (African and Asian), EPHX1-W97X (Asian and Hispanic) and OAS2-W720X (African and European) were detected in some populations but not in others. In addition, there were three X-SNPs that were found exclusively in one population: FUT2-R297X and LCE5A-R79X in Asian and LIG4-W46X in African samples. Either different selection in different populations or the occurrence of founder effect/genetic drift may explain the population spectrum of these SNPs [16, 41].