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28
Table 1. List of genes analyzed by target sequencing in this study
No.
Gene
Location
10
11
12
13
14
15
16
RPL11
RPL5
RPS27
RPS7
RPL35A
RPS14
RPS10
RPS24
RPS26
RPS17
RPS17L
RPL26
RPL27
TP53
RPS19
GATA1
Chromosome 1
Chromosome 1
Chromosome 1
Chromosome 2
Chromosome 3
Chromosome 5
Chromosome 6
Chromosome 10
Chromosome 12
Chromosome 15
Chromosome 15
Chromosome 17
Chromosome 17
Chromosome 17
Chromosome 19
Chromosome X
29
Table 2. List of variants cloned into the minigene construct
Variant
Location
mRNA
of
variant
In vitro (minigene assay)
In silico (MaxEnt score)
Genetic region
Original
Variant
Novel
Splice site
score
splice
Result
cloned
score
Reference
site
score
No.
c.72-92A>G
Intron 2
N/A
Introns 1–3
Normal transcript
10.14
10.14
[19]
No.
c.356+18G>
Intron 4
N/A
Intron 3–exon 6
Normal transcript
8.84
8.84
[20]
No.
c.411+1G>A
Intron 5
Exon 5 skipping
Intron 3–exon 6
Normal transcript
11.08
2.90
[40]
c.411+6G>C
Intron 5
N/A
Intron 3–exon 6
Normal transcript
11.08
9.88
[20]
c.412-3C>G
Intron 5
Normal transcript
Intron 3–exon 6
Exon 6 skipping or
13.52
1.50
No.
No.
and insertion of
insertion of AG
AG
between exons 5
between
exons 5 and 6
and 6
N/A: not available
30
8.93
Our case
Figure 1. Schema for the hybrid minigene
31
Figure 2. Transcript analysis in our patient and his family
32
Figure 3. In vitro splicing analysis for c.72-92A>G, c.356+18G>C, and c.411+6G>C
variants
33
Figure legends
Figure 1. Schema for the hybrid minigene
The H492 vector has two cassette exons, A and B, between which is a multicloning site.
The H492 vector also has cytomegalovirus (CMV) enhancer-promotor and bovine growth
hormone (BGH) gene polyadenylation site.
Figure 2. Transcript analysis in our patient and his family
(A) The RPS19 genomic DNA sequences of the patient and his parents. A heterozygous
single-base substitution of C to G was detected in the patient. (B) Electrophoretic gel of
the RT-PCR products obtained from the control and the patient. A single band was
observed for each PCR product. The products were almost identical sizes. (C) PCR
products were subcloned and sequenced. Wild-type and an otherwise wild-type RPS19
sequence with two bases inserted between exons 5 and 6 were identified. (D) RT-PCR
amplified products of minigene construct transcripts. As shown in the gel images, a 295bp smaller band and 2-bp larger band are produced by the minigene construct with the
c.412-3C>G variant in both in HEK 293T and Hela cells. Transcripts from the minigene
construct with the c.412-3C>G variant show skipping of exon 6 and that the larger
transcript has two bases inserted between exons 5 and 6. (E) Quantitative RT-PCR using
mRNA from the patient and a normal control was performed to compare RPS19 and
34
RPL5 expression. Relative quantification (RQ) of each gene was calculated, which is a
fold change compared with the calibrator, HPRT-1. RPS19 expression level in the patient,
(RQ = 0.581) was approximately half that measured in the control (RQ = 1.000). The
RPL5 (RQ = 0.961) expression levels in the patient were almost equal to those in the
control (RQ = 1.000).
Figure 3. In vitro splicing analysis for c.72-92A>G, c.356+18G>C, and c.411+6G>C
variants
RT-PCR amplified products of minigene construct transcripts. (a, b, and c) Gel images
and sequences of transcripts from the minigenes with c.72-92A>G (a), c.356+18G>C (b),
and c.411+6G>C (c). Electrophoresis results reveal a band the same size as that produced
by the wild-type construct. Transcript analysis shows that each transcript is the same as
that of the wild-type construct. (d) As shown in the gel images, the transcript from the
wild-type construct was larger than that from the minigene with the c.411+1G>A variant
in both HEK 293T and Hela cells. The transcripts from the c.411+1G>A variant skipped
exon 5.
35
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