Figure 1.
Analysis of the two E+1 mutations of the BTK gene, O13 (c.1482G>T) and HK08 (c.1883G>A).
(A) Schematic sequences of mutated acceptor splice sites. Introns are shown in lower-case, and exons are shown in capital letters. Mutated nucleotides are bold and underlined. The PPS are singly underlined and other polypyrimidine stretches are dashed underlined. (B) RT-PCR of minigenes transfected into HeLa cells. cDNA bands originating from O13 mutated minigene are numbered as follows: 1) cryptic 3′ss utilization 81 nt upstream of the authentic splice site (the aberrant exon starts at c.1350-81G), 2) normally spliced RNA, 3) skipping of mutated exon. (C) RT-PCR from RNA extracted from patients’ blood. P = patient’s sample, HC = healthy control sample. The O13 cDNA bands are numbered as in (B).
Figure 2.
The sequences are ordered according to the length of their longest PPS. Exons whose splicing was shown to depend on intact E+1 position are underlined. The PPS are singly underlined and other polypyrimidine stretches are dashed underlined. Sites of mutations are showed in bold. Seq. = sequence. (A) Sequences of the test set. (B) Sequences of the borderline set.
Figure 3.
Results of the splicing minigene analyses.
RT-PCR analysis of the literature-derived E+1 variations. The splicing affecting sequences are underlined. (A) The test set sequences. cDNA bands originating from BTK exon 10 mutated minigene are numbered as follows: 1) cryptic 3′ss utilization 31 nt upstream of the authentic splice site (the aberrant exon starts at c.840-31G), 2) normally spliced RNA. (B) The borderline set sequences.
Figure 4.
Selected results of in silico predictions and other parameters of AG-dependent and AG-independent splice sites.
The boxplots show the values, median and interquartile range of the values predicted for the test set sequences. The values of scores are shown in the original units of the tools, differences are counted as ratios of the absolute score (or percentile) difference to the wild type score (or percentile). Other values are simply numbers of nucleotides. Asterisks indicate statistically significant differences between the two sets of values (*at p<0.05; **at p<0.01; ***at p<0.001). Abbreviations: Ab = group of sequence variants that lead to aberrant splicing; diff. = difference; Non = group of sequence variants that do not disrupt the process of splicing; nt = nucleotide(s); perc. = percentile; Py in 25 nt = number of pyrimidines in 25 nucleotides upstream of acceptor splice site.
Table 1.
Predicted value ranges in the test set of G+1 mutations obtained from the instruments evaluating the overall strength of the 3′splice site.
Table 2.
Comparison of value ranges describing particular intronic parameters in the test set of G+1 mutations.
Table 3.
Proposed cut-off values for the in silico tools that discriminate AG-dependent 3′ss from AG-independent 3′ss.
Table 4.
Results of combined predictions in discrimination of G+1-dependent 3′ss from G+1-independent 3′ss.