[1] F. Peyvandi, I. Garagiola, G. Young, The past and future of haemophilia: diagnosis, treatments,
and its complications, Lancet 388(10040) (2016) 187-97.
[2] S. Yoshitake, B.G. Schach, D.C. Foster, E.W. Davie, K. Kurachi, Nucleotide sequence of the gene
for human factor IX (antihemophilic factor B), Biochemistry 24(14) (1985) 3736-50.
[3]
CDC
Hemophilia
Mutation
Project
(CHAMP
CHBMP).
https://www.cdc.gov/ncbddd/hemophilia/champs.html.
[4] EAHD Factor IX Gene (F9) Variant Database. https://f9-db.eahad.org/index.php.
[5] R. Vaz-Drago, N. Custodio, M. Carmo-Fonseca, Deep intronic mutations and human disease,
Hum Genet 136(9) (2017) 1093-1111.
[6] R.D. Bagnall, N.H. Waseem, P.M. Green, B. Colvin, C. Lee, F. Giannelli, Creation of a novel
donor splice site in intron 1 of the factor VIII gene leads to activation of a 191 bp cryptic exon in two
haemophilia A patients, Br J Haematol 107(4) (1999) 766-71.
[7] G. Castaman, S.H. Giacomelli, M.E. Mancuso, G. D'Andrea, R. Santacroce, S. Sanna, E.
Santagostino, P.M. Mannucci, A. Goodeve, F. Rodeghiero, Deep intronic variations may cause mild
hemophilia A, J Thromb Haemost 9(8) (2011) 1541-8.
[8] H. Inaba, T. Koyama, K. Shinozawa, K. Amano, K. Fukutake, Identification and characterization
of an adenine to guanine transition within intron 10 of the factor VIII gene as a causative mutation in
a patient with mild haemophilia A, Haemophilia 19(1) (2013) 100-5.
25
Koya ODAIRA
[9] B. Pezeshkpoor, N. Zimmer, N. Marquardt, I. Nanda, T. Haaf, U. Budde, J. Oldenburg, O. ElMaarri, Deep intronic 'mutations' cause hemophilia A: application of next generation sequencing in
patients without detectable mutation in F8 cDNA, J Thromb Haemost 11(9) (2013) 1679-87.
[10] C.Y. Chang, C.L. Perng, S.N. Cheng, S.H. Hu, T.Y. Wu, S.Y. Lin, Y.C. Chen, Deep intronic
variant c.5999-277G>A of F8 gene may be a hot spot mutation for mild hemophilia A patients without
mutation in exonic DNA, Eur J Haematol 103(1) (2019) 47-55.
[11] J. Feng, Q. Liu, J. Drost, S.S. Sommer, Deep intronic mutations are rarely a cause of hemophilia
B, Hum Mutat 14(3) (1999) 267-8.
[12] K. Shinozawa, K. Yada, T. Kojima, K. Nogami, M. Taki, K. Fukutake, A. Yoshioka, A. Shirahata,
M. Shima, J.H.I.S. study group on, Spectrum of F8 Genotype and Genetic Impact on Inhibitor
Development in Patients with Hemophilia A from Multicenter Cohort Studies (J-HIS) in Japan,
Thromb Haemost
(2020).
[13] T. Kojima, M. Tanimoto, T. Kamiya, Y. Obata, T. Takahashi, R. Ohno, K. Kurachi, H. Saito,
Possible absence of common polymorphisms in coagulation factor IX gene in Japanese subjects, Blood
69(1) (1987) 349-52.
[14] Human Genome Variation Society. http://www.hgvs.org/mutnomen/.
[15] Splicing site Site prediction Prediction. https://www.fruitfly.org/seq_tools/splice.html.
[16] Netgene2. http://www.cbs.dtu.dk/services/NetGene2.
26
Koya ODAIRA
[17] K. Odaira, S. Tamura, N. Suzuki, M. Kakihara, Y. Hattori, M. Tokoro, S. Suzuki, A. Takagi, A.
Katsumi, F. Hayakawa, S. Okamoto, A. Suzuki, T. Kanematsu, T. Matsushita, T. Kojima, Apparent
synonymous mutation F9 c.87A>G causes secretion failure by in-frame mutation with aberrant
splicing, Thromb Res 179 (2019) 95-103.
[18]
Acetone
precipitation
of
proteins,
Tech
Tip
#49.
https://tools.thermofisher.com/content/sfs/brochures/TR0049-Acetoneprecipitation.pdf?CID=Social_LAB.
[19] A. Ferlini, N. Galie, L. Merlini, C. Sewry, A. Branzi, F. Muntoni, A novel Alu-like element
rearranged in the dystrophin gene causes a splicing mutation in a family with X-linked dilated
cardiomyopathy, Am J Hum Genet 63(2) (1998) 436-46.
[20] H.R. Madden, S. Fletcher, M.R. Davis, S.D. Wilton, Characterization of a complex Duchenne
muscular dystrophy-causing dystrophin gene inversion and restoration of the reading frame by induced
exon skipping, Hum Mutat 30(1) (2009) 22-8.
[21] B. Baskin, W.T. Gibson, P.N. Ray, Duchenne muscular dystrophy caused by a complex
rearrangement between intron 43 of the DMD gene and chromosome 4, Neuromuscul Disord 21(3)
(2011) 178-82.
[22] M.M. Khelifi, A. Ishmukhametova, P. Khau Van Kien, D. Thorel, D. Mechin, S. Perelman, J.
Pouget, M. Claustres, S. Tuffery-Giraud, Pure intronic rearrangements leading to aberrant pseudoexon
inclusion in dystrophinopathy: a new class of mutations?, Hum Mutat 32(4) (2011) 467-75.
27
Koya ODAIRA
[23] M.G. Reese, F.H. Eeckman, D. Kulp, D. Haussler, Improved splice site detection in Genie, J
Comput Biol 4(3) (1997) 311-23.
[24] S. Brunak, J. Engelbrecht, S. Knudsen, Prediction of human mRNA donor and acceptor sites
from the DNA sequence, J Mol Biol 220(1) (1991) 49-65.
[25] D. Scalet, I. Maestri, A. Branchini, F. Bernardi, M. Pinotti, D. Balestra, Disease-causing variants
of the conserved +2T of 5' splice sites can be rescued by engineered U1snRNAs, Hum Mutat 40(1)
(2019) 48-52.
[26] A.R. Kornblihtt, Promoter usage and alternative splicing, Curr Opin Cell Biol 17(3) (2005) 2628.
28
Koya ODAIRA
Figure Legends
Fig 1. Identification of F9 intronic structural variation.
A) F9 exon copy number analysis by MLPA. Data were normalized using pooled gDNA of healthy
male subjects. B) Genomic long‐range PCR with primer set, F9_Ex1‐3_PCR_Fw vs. Rv. N: Pooled
normal male gDNA, P: patient gDNA. C) Densitometry analysis of genomic long‐range PCR amplicon.
To examine the size alteration of PCR amplicon derived from the patient’s gDNA, gel mobility of each
amplicon was measured by densitometry analysis of the image presented in panel B. D) Breakpoint
junction DNA sequencing. The patient carried a 28‐bp deletion (from NC_000023.11: g.139,534,834
to g.139,534,861) and a 476‐bp insertion (NC_000012.12: g.54,285,853_54,286,328inv) into F9
intron 1. The 476‐bp insertion was an identical sequence of a part of inverted HNRNPA1 exon 12.
29
Koya ODAIRA
Fig 2. Cell‐based transcript analysis of the F9 intronic structural variation.
A) Diagram of the predicted anomalous splicing pattern in variant F9 intron 1. This diagram was
designed based on the splice‐site prediction in silico using Splice Site Prediction and NetGene2 (Table
1). B) Design of variant F9 exon‐trap vector, pET01 F9 int 1 and 2 variant. The PCR‐amplified DNA
alignment of variant F9 was inserted into a previously constructed pET01 F9 int 1 and 2 WT (15). C)
Result of RT‐PCR in COS‐7 cells transfected with pET01 F9 int 1 and 2 WT or pET01 F9 int 1 and 2
variant. Abnormal amplicons of 553 bp (V1 transcript) and 729 bp (V2 transcript) were detected in
cells transfected with the variant, and 448‐bp amplicon (N1 transcript) was significantly reduced. D)
Direct sequencing of RT‐PCR amplicon. The N1 transcript (448‐bp amplicon) was a normal‐type
(protein‐coding) transcript composed of F9 exons 1, 2, and 3. The V1 (553 bp) and V2 (729 bp)
transcripts were aberrant transcripts containing pseudoexon(s). DNA sequences of the boundary region
between exons or pseudoexons were displayed. The asterisk indicates a PTC within pseudoexon 2.
pEx indicates a pseudoexon. E) Quantification of N1 transcript (normal F9 mRNA) in COS-7 cells
transfected with the variant exon-trap vector. The transcription diagram shows the design of the N1
transcript-specific primer set. The amount of N1 transcript was normalized with respect to the GAPDH
expression level. n = 5 (independently transfected samples).
30
Koya ODAIRA
Fig 1. Identification of F9 intronic structural variation.
31
Koya ODAIRA
Fig 2. Cell‐based transcript analysis of the F9 intronic structural variation.
32
Koya ODAIRA
Table 1. In silico splicing simulation of F9 structural variation
Splice Site Prediction
NetGene2
pseudo -Int 1
pseudo-Int 2
pseudo -Int 3
Donor Site Score
0.83
0.96
0.60
Acceptor Site Score
n.d
0.98
0.94
Donor Site Score
0.7
0.39
0.60
Acceptor Site Score
0.16
0.56
n.d
Score ranged from 0.4 to 1.0 for Splice Site Prediction and from 0.0 to 1.0 for NetGene2. Higher
scores indicate more potential splice sites.
n.d: not detected.
33
Koya ODAIRA
Supplementary data
Table of contents
1. Supplementary Fig. 1. Restriction enzyme fragment analysis.
2. Supplementary Fig. 2. Genomic DNA sequence of breakpoint junction in the patient F9.
3. Supplementary Fig. 3. Result of cell-based transcript analysis using HEK293 cells.
4. Supplementary Fig. 4. FIX protein expression analysis using splicing-competent vector with F9
intron 1 variation.
5. Supplementary table. Oligonucleotide primers.
34
Koya ODAIRA
Supplementary Figure Legends
Supplementary Fig. 1. Restriction enzyme fragment analysis.
A) Long‐range genomic PCR amplicons ranging from F9 5′‐untranslated region to intron 3 were
digested with PstI. Upper panel indicates an image of agarose gel electrophoresis analysis. Abnormal
fragments (magenta arrowhead) were detected in the PCR amplicons derived from the patient’s gDNA.
In the control gDNA pooled with healthy male subjects, the PstI-digensted fragments were detected as
1857 bp, 2280 bp, and 3012 bp. In the gDNA of the patient, the PstI-digensted fragments were detected
as 1857 bp, 2280 bp, and approximately 3500 bp. Lower panel is an illustration to explain the result
of PstI-digensted fragments analysis. (B) Long‐range genomic PCR amplicons ranging from F9 5′‐
untranslated region to intron 3 were digested with HpaII. Upper panel indicates an image of agarose
gel electrophoresis analysis. Abnormal fragments (magenta arrowhead) were detected in the PCR
amplicons derived from the patient’s gDNA. In the control gDNA pooled with healthy male subjects,
the HpaII -digensted fragments were detected as 1479 bp, 2326 bp, and 3344 bp. In the gDNA of the
patient, the HpaII -digensted fragments were detected as approximately 2100 bp, 2326 bp, and 3344
bp. Lower panel is an illustration to explain the result of HpaII -digensted fragments analysis. These
observations indicated that an approximately 500 bp DNA alignment was inserted into the central part
of the F9 intron 1. N: Control gDNA pooled with healthy male subjects. P: patient’s gDNA.
35
Koya ODAIRA
Supplementary Fig. 2. Genomic DNA sequence of breakpoint junction in the patient F9.
The sequences displayed are the DNA alignments of the patient’s gDNA, the reference F9 intron 1
(NC_000023.11:g.139534803_139534882), and the inserted sequence of HNRNPA1 on chromosome
12 (NC_000012.12:g.54285853_54286328inv). Microhomology sequences are indicated by
underlined characters.
36
Koya ODAIRA
Supplementary Fig. 3. Result of cell-based transcript analysis using HEK293 cells.
A) Result of RT-PCR in HEK293 cells transfected with pET01 F9 int 1 and 2 WT or pET01 F9 int 1
and 2 variant. Abnormal amplicons were detected in cells transfected with the variant, and 448 bp
amplicon (N1 transcript) was significantly reduced. B) Quantification of N1 transcript (normal F9
mRNA) in HEK293 cells transfected with the variant exon-trap vector. The amount of N1 transcript
was normalized with respect to the GAPDH expression level. n = 5 (independently transfected
samples).
37
Koya ODAIRA
Supplementary Fig. 4. FIX protein expression analysis using splicing-competent vector with F9
intron 1 variation.
A) Design of a splicing-competent FIX expression vector. The PCR-amplified DNA alignment of
normal or variant F9 intron 1 was inserted into a pcDNA3.1-based FIX expression vector that we
previously constructed [17]. B) Result of RT-PCR in HEK293 cells transfected with FIXwt int1(+)
(WT) or FIXvariant int1(+) (Variant). Normal amplicon is indicated as N1 transcript. C)
Quantification of N1 transcript in HEK293 cells transfected with FIXvariant int1(+). The amount of
N1 transcript was normalized with respect to the GAPDH expression level. n = 4 (independently
transfected samples). D) Western blot analysis of rFIX produced by HEK293 cells transfected with
pcDNA3.1 (Mock), FIXwt int1(+) or FIXvariant int1(+). Here, 10 μg protein in cell lysate and 5 μg
protein in culture media precipitation were loaded. a-tubulin was used as a loading control. E)
Quantification of secreted rFIX from HEK293 cells transfected with FIXwt int1(+) or FIXvariant
int1(+). n=4 (independently transfected samples). F) The specific coagulant activity of rFIXvariant.
The specific activity was calculated as the ratio between the coagulant activity and the secreted protein
level. The dotted line indicates the specific activity of rFIX-WT.
38
Koya ODAIRA
Supplementary Fig. 1. Restriction enzyme fragment analysis.
39
Koya ODAIRA
Supplementary Fig. 2. Genomic DNA sequence of breakpoint junction in the patient F9.
40
Koya ODAIRA
Supplementary Fig. 3. Result of cell-based transcript analysis using HEK293 cells.
41
Koya ODAIRA
Supplementary Fig. 4. FIX protein expression analysis using splicing-competent vector with F9
intron 1 variation.
42
Koya ODAIRA
Supplementary table. Oligonucleotide primers.
Name of oligonucleotide
Sequence (5'-3')
Application
F9_ex1_Fw
AATCAGACTAACTGGACCAC
F9 exon sequence
F9_ex1_Rv
TATCTAAAAGGCAAGCATAC
F9 exon sequence
F9_ex2.3_Fw
ATGATGTTTTCTTTTTTGCT
F9 exon sequence
F9_ex2.3_Rv
GGTTGGACTGATCTTTCTG
F9 exon sequence
F9_ex4_Fw
TTCTAAGCAGTTTACGTGCC
F9 exon sequence
F9_ex4_Rv
GTAGCTTCTTGAACTCATATCC
F9 exon sequence
F9_ex5_Fw
CCCCCAATGTATATTTGACC
F9 exon sequence
F9_ex5_Rv
CCGTCCTTATACTAGAAGCC
F9 exon sequence
F9_ex6_Fw
AATACTGATGGGCCTGCT
F9 exon sequence
F9_ex6_Rv
AACTTGCCTAAATACTTCTCAC
F9 exon sequence
F9_ex7_Fw
CCAATATTTTGCCTATTCCT
F9 exon sequence
F9_ex7_Rv
CTTCTGGTATGGAAATGGCT
F9 exon sequence
F9_ex8-1_Fw
TGTGTATGTGAAATACTGTTTG
F9 exon sequence
F9_ex8-1_Rv
TTATAGATGGTGAACTTTGTAGA
F9 exon sequence
F9_ex8-2_Fw
TTGGATCTGGCTATGTAAGT
F9 exon sequence
F9_ex8-2_Rv
AGTTAGTGAGAGGCCCTG
F9 exon sequence
F9_Ex1-3_PCR_Fw
AATCAGACTAACTGGACCAC
Variant specific PCR in Fig. 1B
F9_Ex1-3_PCR_Rv
GGTTGGACTGATCTTTCTG
Variant specific PCR in Fig. 1B
Int1_fusion_Fw
ATAGTAAGCCATTTTTATATCGGAG
Construction of exon-trap vector in Fig. 2B
Int1_fusion_Rv
TTCACTCGTTTGCAATGCT
Construction of exon-trap vector in Fig. 2B
pET01_Int1wt_inversePCR_Fw
pET01_Int1wt_inversePCR_Rv
ATTGCAAACGAGTGAAGGAAATTGAGA
Construction of exon-trap vector in Fig. 2B
AATATGG
AAAATGGCTTACTATTTGCATATAACCT
Construction of exon-trap vector in Fig. 2B
AAACACATCCTC
FIX_int1_infusion_Fw
GTGCTGAATGTACAGGTTTGTTTCCTT Construction of splicing-competent FIX
TTTTAAAATACATTG
expression vector in supplementary Fig.4A
FIX_int1_infusion_Rv
CATGATCAAGAAAAACTGAAATGTAAA Construction of splicing-competent FIX
AGAATAATTCTTTAGTTT
expression vector in supplementary Fig.4A
FIX_ex1_inv_Rv
CTGTACATTCAGCACTGAGTAGATATC Construction of splicing-competent FIX
CTAAAAG
expression vector in supplementary Fig.4A
FIX_ex2_inv_Fw
TTTTTCTTGATCATGAAAACGCCAACAA Construction of splicing-competent FIX
AA
expression vector in supplementary Fig.4A
ET-PCR-primer2
GATCGATCTGCTTCCTGGCCC
RT-PCR in Fig. 2C, supplementary Fig.3A
ET-PCR-primer3
CTGCCGGGCCACCTCCAGTGCC
RT-PCR in Fig. 2C, supplementary Fig.3A
pcDNA3.1_Fw
AGTGCTTACTGGCTTATCGAAAT
RT-PCR in supplementary Fig. 4B
GAPDH_qRT_Fw
GGCTGCTTTTAACTCTGGTA
RT-qPCR in Fig. 2E, supplementary Fig. 3B,
supplementary Fig. 4C
GAPDH_qRT_Rv
CATGGGTGGAATCATATTGG
RT-qPCR in Fig. 2E, supplementary Fig. 3B,
supplementary Fig. 4C
F9_WT_RT-qPCR_Fw
TACTCAGTGCTGAATGTACAGTTTT
RT-qPCR in Fig. 2E
F9_WT_RT-qPCR_Fw2
CTCAGTGCTGAATGTACAGTTTTTC
RT-qPCR in supplementary Fig. 3B,
supplementary Fig. 4C
F9_WT_RT-qPCR_Rv
TGAACAAACTCTTCCAATTTACCTG
RT-PCR in supplementary Fig. 4B, RT-qPCR
in Fig. 2E, supplementary Fig. 3B,
supplementary Fig. 4C
43
...
論文の公開元へ
