Splicing Analysis of MYO5B Noncanonical Variants in Patients with Low Gamma-Glutamyltransferase Cholestasis
Table 2
In silico and functional analysis for MYO5B intronic and synonymous variants.
Variants
Location
Human splicing finder
Splice AI (score)
varSEAK (class)
MutationTaster
MaxEntScan
Minigene splicing assay
Blood RNA analysis
Allele frequency in gnomAD (hom/het/total)†
Original classification
Reclassification
c.1669-35A>C
Intron 13
Alteration of the WT branch point may affect splicing
Acceptor loss (0.14)
No splicing effect (1)
No abrogation of potential splice sites
6.33⇒6.33
Inserting 96 nt of intron 13/normal splicing (~17%)
NA
—
VUS (PM2_P+PP3+PP4)
Likely pathogenic (PM2_P+PP4+PS3)
c.455+8T>C
Intron 4
No significant impact on splicing signals
No splicing effect (0)
No splicing effect (1)
Donor increased
9.22⇒9.22
No splicing effect
NA
0/71/280946
VUS (PM2_P+PM3_P+PP3+PP4)
Likely benign (PM3_P+PP4++BS3)
c.2415-6C>G
Intron 19
Alteration of the WT acceptor site, most probably affecting splicing
No splicing effect (0)
Likely no splicing effect (2)
Acceptor gained
10.21⇒6.81 (-33.3%)
No splicing effect
NA
0/169/279700
VUS (PM2_P+PP3)
Likely benign (PP3+BS3)
c.613-11G>A
Intron 5
Alteration of the WT acceptor site, most probably affecting splicing
Acceptor loss (0.84) Acceptor gain (0.99)
Use of de novo splice site 9 nt upstream of 3 splice site and loss of function for authentic splice site (5)
Donor increased Donor gained
9.06⇒3.19 (-64.8%)
Inserting 9 nt of intron 5
Inserting 9 nt of intron 5
—
VUS (PM2_P+PP3+PP4)
Likely pathogenic (PM2_P+PP3+PP4+PS3)
c.4221G>A/p.(=)
Exon 31
Alteration of the WT donor site, most probably affecting splicing
Donor loss (0.32)
Loss of function for authentic splice site, exon skipping (5)
Alteration within used splice site, likely to disturb normal splicing
6.29⇒1.46 (-76.8%)
Exon 31 skipping/normal splicing (~30%)
NA
—
VUS (PM2_P+PM3+PP3+PP4)
Likely pathogenic (PM2_P+PM3+PP3+PP4+PS3)
c.4852+11A>G
Intron 36
No significant impact on splicing signals
No splicing effect (0)
No splicing effect (1)
No abrogation of potential splice sites
8.89⇒8.89
No splicing effect
NA
—
VUS (PM2_P+PM3_P+PP4)
Likely benign (PM2_P+PM3_P+PP4+BS3)
c.2090+3A>T
Intron 17
Alteration of the WT donor site, most probably affecting splicing
Donor loss (0.85)
Loss of function for authentic splice site, strong decrease of score for authentic splice site, exon skipping (5)
Donor lost
8.63⇒0.49 (-94.3%)
Inserting 185 nt of intron 17
NA
—
VUS (PM2_P+PM3_P+PP3+PP4)
Likely pathogenic (PM2_P+PM3_P+PP3+PP4+PS3)
c.1322+5G>A
Intron 10
Alteration of the WT donor site, most probably affecting splicing
Donor loss (0.28)
Loss of function for authentic splice site, strong decrease of score for authentic splice site, exon skipping (5)
No abrogation of potential splice sites
6.03⇒-0.42 (-107%)
134 nt deletion of exon10/normal splicing (~20%)
NA
—
VUS (PM2_P+PM3+PP3+PP4)
Likely pathogenic (PM2_P+PM3+PP3+PP4+PS3)
c.2414+5G>T
Intron 19
Alteration of the WT donor site, most probably affecting splicing
Donor loss (0.67) Donor gain (0.42)
Strong decrease of score for authentic splice site, use of a cryptic site 27 nt upstream of 5 splice site (5)
No abrogation of potential splice sites
9.60⇒4.28 (-55.4%)
71 nt and 27 nt deletions of exon 19, respectively
NA
—
VUS (PM2_P+PM3+PP3+PP4)
Likely pathogenic (PM2_P+PM3+PP3+PP4+PS3)
c.2349A>G/p.(=)
Exon 19
Activation of a cryptic donor site, potential alteration of splicing
Donor loss (0.09) Donor gain (0.25)
No splicing effect (1)
No abrogation of potential splice sites
9.60⇒9.60
71 nt deletion of exon 19/normal splicing (~35%)
NA
0/1/249462
VUS (PM2_P+PM3+PP3+PP4)
Likely pathogenic (PM2_P+PM3+PP3+PP4+PS3)
c.3045+3A>T
Intron 22
Alteration of the WT donor site, most probably affecting splicing
Donor loss (0.52)
Loss of function for authentic splice site, strong decrease of score for authentic splice site, exon skipping (5)
Donor lost
9.55⇒5.42 (-43.2%)
125 nt and 4 nt deletion of exon 22, respectively/normal splicing (~10%)
NA
—
VUS (PM2_P+PM3_P+PP3+PP4)
Likely pathogenic (PM2_P+PM3_P+PP3+PP4+PS3)
Abbreviations: NA, not applicable; —, not reported; nt, nucleotides; VUS, Variant of uncertain significance; PM2_P, PM2_Supporting; PM3_P, PM3_Supporting. PM2: absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project; PM3: for recessive disorders, detected in trans with a pathogenic or likely pathogenic variant; PP3: multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.); PP4: patient’s phenotype or family history is highly specific for a disease with a single genetic etiology; PS3: well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product; BS3: well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing. †The number of homozygotes/heterozygotes/total in population. Variants in boldface indicate novel.
