Bioinformatic Predictions of Splicing Alterations

Supplementary Materials

Methods

Bioinformatic predictions of splicing alterations

Four different splice site prediction algorithms were used to evaluate the effect of VUS on the canonical acceptor and donor splice sites: SpliceSiteFinder-like (SSF, see Splice Site Prediction byNeural Network (NNS, MaxEntScan( andHuman Splicing Finder (HSF, programs were interrogated simultaneously using the integrated software Alamut v.2.0 (Interactive Biosoftware;

VUS were also analyzed for possible splicing enhancer (ESE) alterations by using ESEfinder 3.0 ( and RESCUE-ESE ( addition, we used the in silico tool EX-SKIP ( to compare the ESE/ESS profile of the wild-type and the mutant versions of the entire BRCA2 exon 7. This program calculates, for each segment, the total number of ESSs and ESEs and their ratio by using RESCUE-ESEs, FAS-ESSs, PESEs/PESSs, neighborhood inferenceand EIE/IIEs.6,8-11

Bioinformatic predictions of protein alterations

Three different prediction algorithms were used to evaluate the effect of VUS on protein function: Align GVGD (Grantham Variation, Grantham Deviation, PolyPhen-2 (Polymorphism Phenotyping v2, and SIFT (Sorting Intolerant From Tolerant,

References

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Table S1 Bioinformatic predictions of5’ or 3’ splice site alterations for the selected BRCA2VUS

BRCA2 Exon 7 Variants(1) / In silico prediction relative to 3' ss / 5' ss(2)
Position
of interest / SSF
[0-100]* / MaxEntScan
[0-16]* / NNS
[0-1]* / HSF
[0-100]*
c.517 G>T / c.517 (natural 3' ss) / 94.42 > 88.18
(-6.6%) / 10.00 > 9.10
(-9.1%) / 0.98 > 0.93
(-4.9%) / 96.60 > 92.30
(-4.4%)
c.520C>T / no change / no change / no change / no change
c.522T>G / no change / no change / no change / no change
c.572 A>G / c.571 (new 5' ss) / 0 > 81.32 / 0 > 8.14 / 0 > 0.99 / 0 > 86.44
c.587G>A / no change / no change / no change / no change
c.617C>G / no change / no change / no change / no change
c.625C>T / no change / no change / no change / no change
c.631G>A / c.631 (natural 5' ss) / 78.15 > 66.01
(-15.5%) / 6.84 > 0 / NP / 83.37 > 72.79
(-12.7%)
c.561 (cryptic 5' ss) / no change
(70.35) / no change
(1.48) / NP / no change
(76.57)

(1) Variants that alter splice site usage, as determined experimentally in the minigene assay, are indicated in bold; (2) The score values obtained with the four in silico tools(SSF, MaxEntScan, NNS and HSF)are shown for the wild type (wt) and variant splice sites (wt > variant) and the score changes relative to the wt are expressed in percentage and indicated between brackets. The potential creation of a splice site, as well as the score of cryptic splice site, are also indicated.* Score range of each bioinformatics prediction algorithm. NP, not predicted.

Table S2 Bioinformatic predictions of splicing regulatory elements modifications for the selected BRCA2 VUS, using ESEfinder and RESCUE-ESE

BRCA2
Exon 7
Variants(1) / ESEfinder / RESCUE-ESE
Motif / Sequence(2)
(wt/variant) / Score (wt/variant) / Sequence(2)
(wt/variant) / Match(3)
(wt/variant)
c.517G>T / SF2/ASF / cccag(G/T)G / 3.53/b.t.(4) / g(G/T)GTCG / 0/1
SF2/ASF
(IgM-BRCA1) / cccag(G/T)G / 4.09/2.54
SRp40 / tcccag(G/T) / 3.93/b.t.
SRp55 / ag(G/T)GTC / b.t./2.71
c.520C>T / no change / no change
c.522T>G / SF2/ASF / CG(T/G)CAGA / b.t./2.38 / no change
SF2/ASF
(IgM-BRCA1) / CG(T/G)CAGA / b.t./3.00
SC35 / GGTCG(T/G)CA / b.t./2.98
c.572A>G / no change / G(A/G)TATG / 1/0
c.587G>A / no change / GTCAA(G/A) / 1/0
c.617C>G / SC35 / TT(C/G)TACTG / 3.04/b.t. / no change
SC35 / (C/G)TACTGTG / b.t./3.11
SRp40 / (C/G)TACTGT / 3.03/b.t.
c.625C>T / SC35 / TG(C/T)TCATA / 2.44/b.t. / no change
SRp55 / (C/T)TCATA / b.t./2.86
c.631G>A / SF2/ASF / CATA(G/A)gt / 3.32/b.t. / no change
SF2/ASF
(IgM-BRCA1) / CATA(G/A)gt / 2.90/b.t.
SRp40 / TCATA(G/A)g / 3.95/3.38
SRp55 / TA(G/A)gta / b.t./3.02

(1) Variants that induce exon skipping by altering potential splicing regulatory elements, as determined experimentally in the minigene assay, are indicated in bold; (2) Putativesplicing regulatory sequences. Exonic and intronic nucleotides are indicated in upper and lower case, respectively; (3) Match, presence (1) or absence (0) of putative ESEs; (4) b.t., below threshold. We used as threshold values the ones set up by default by the developers of the ESEfinder algorithm: 1.956 for SF2/ASF, 1.867 for SF2/ASF(IgM-BRCA1), 2.383 for SC35, 2.67 for SRp40 and 2.676 for SRp55.

Table S3 Bioinformatic predictions of ESS/ESE ratio of wild-type and variantBRCA2 exon 7, calculated using EX-SKIP

BRCA2
Exon 7
Wt and variants(1) / ESS
(total) / ESE
(total) / ESS/ESE
(ratio) / Fold change in ESS/ESE ratio of variantsrelative to wt
wt / 62 / 89 / 0.70 / n.a.(2)
c.517G>T / 63 / 89 / 0.71 / 1.01
c.520C>T / 67 / 87 / 0.77 / 1.1
c.522T>G / 63 / 87 / 0.72 / 1.03
c.572A>G / 64 / 86 / 0.74 / 1.06
c.587G>A / 59 / 85 / 0.69 / 0.99
c.617C>G / 64 / 86 / 0.74 / 1.06
c.625C>T / 71 / 86 / 0.83 / 1.19
c.631G>A / 62 / 89 / 0.70 / 1

(1)Variants that induce exon skipping by altering potential splicing regulatory elements, as determined experimentally in the minigene assay, are indicated in bold; (2) n.a., not applicable

Table S4 Bioinformatic predictions of protein function alterations for the selected BRCA2 VUS, using Align GVGD, PolyPhen-2 and SIFT

BRCA2 / Align GVGD
(Class(2)) / PolyPhen-2 / SIFT
Nucleotide variant(1) / Protein variant
c.517G>T / p.Gly173Cys / C0 / Probably damaging
(score: 0.998) / Tolerated
(score: 0.12)
c.520C>T / p.Arg174Cys / C0 / Possibly damaging
(score: 0.802) / Deleterious
(score: 0.00)
c.522T>G / p.Arg174Arg / n.a.(2) / n.a. / n.a.
c.572A>G / p.Asp191Gly / C15 / Probably damaging
(score: 1.00) / Deleterious
(score: 0.01)
c.587G>A / p.Ser196Asn / C45 / Probably damaging
(score: 1.00) / Deleterious
(score: 0.00)
c.617C>G / p.Ser206Cys / C15 / Probably damaging
(score: 0.999) / Tolerated
(score: 0.11)
c.625C>T / p.Leu209Phe / C0 / Probably damaging
(score: 0.999) / Deleterious
(score: 0.04)
c.631G>A / p.Val211Ile / C0 / Possibly damaging
(score: 0.734) / Tolerated
(score: 0.37)

(1)Variants inducing splicing alterations are indicated in bold; (2)Classes given by Align GVGD ordered from most likely to interfere with protein function (C65) to least likely (C0); (2)n.a., not applicable

Figure S1 Comparison of the predicted effect on BRCA2 exon 7 splicing with the experimental data obtained by using the functional splicing minigene assay, for the c.517G>T (A), c.572A>G (B) and c.631G>A (C). The data obtained by using the functional splicing minigene assay are shown in the left panel (Wt, wild-type; VUS, variant; V, vector). The position of the variants is marked with a vertical arrow. The in silico predictions are depicted in the right panelby using the Alamut software. A schematic representation of the transcripts is also shown with black boxes representing the minigene exons.

Figure S2Effects of c.520C>T, c.587G>A and c.617C>G variants on BRCA2 exon 7splicing, by using the minigene assay: comparison of the results in HeLa, HBL-100 and MCF-7 cell lines

Minigene constructs, carrying either the wild-type (Wt) or the mutant sequences of BRCA2 exon 7, were transiently expressed in HeLa, HBL-100 or MCF-7 cell lines. The splicing patterns were monitored by RT-PCR analysis and ethidium bromide agarose gel electrophoresis. Transcripts corresponding to the inclusion (+Exon 7) or the skipping (ΔExon 7) of the exon are indicated.

Figure S3 Effects of c.520C>T and c.617C>G variants in patient RNA analyzed by RT-PCR targeting BRCA2 exons 3 to 8. The splicing patterns were monitored by RT-PCR analysis, using a forward primer located in BRCA2 exon 3 and a reverse primer located in BRCA2 exon 8, from peripheral blood RNA of control individuals (Control 1 and Control 2) and from two different patients, carrying BRCA2 c.520C>T (upper panel) or from two different blood sampling from the same patient carrying c.617C>G (lower panel). After ethidium bromide agarose gel electrophoresis, the different RT-PCR products were sequenced. The identity of the transcripts is indicated on the right.