Background
Methods
Patients and samples
Primer design and PCR of promoter regions
Amplicon next-generation sequencing analysis
Linkage disequilibrium analysis
Calculation of the BCR-ABL1 halving time
Genotyping of SNPs in the SLC22A4 and SLC22A5 promoters
Genotyping of SNP rs1050152
Cell lines
Expression analysis of SLC22A4 and SLC22A5 in cell lines
Statistical analysis
Results
In silico analysis of the SLC and ABC genes
Identification of SNPs in the promoters of SLC and ABC genes associated with patient response
Linkage disequilibrium analysis
a. | |||||||
Gene Functional consequence | SNP | Position | Regulome DB score | eQTL (Monocytes) | Histone modification Chromatin state Blood cells | Protein binding K562 | |
SLC22A4
Intron 3 | rs156322 A/G | chr5:131653924 | 1f |
SLC22A4
SLC22A5
|
Quiescent/Low
T, B and NK cells from PB
Strong transcription
Primary neutrophils, monocytes, MNC from PB
Weak transcription
Primary HSC
Genic enhancers
K562 | NA | |
SLC22A4
Intron 3 | rs272842 C/T | chr5:131656516 | 1f |
SLC22A4
SLC22A5
|
Quiescent/Low
T, B and NK cells from PB
Strong transcription
Primary neutrophils and monocytes from PB
Weak transcription
Primary HSC, MNC from PB, K562 | NA | |
SLC22A4
Intron 5 | rs272889 C/T | chr5:131665377 | 1f |
SLC22A4
SLC22A5
|
Quiescent/Low
Primary T, B and NK cells from PB
Strong transcription
MNC from PB
Weak transcription
T-helper naïve from PB, K562, primary HSC | NA | |
SLC22A4
Intron 8 | rs272872 C/T | chr5:131675863 | 1f |
SLC22A4
SLC22A5
|
Quiescent/Low
primary T, B and NK cells from PB
Strong transcription
MNC from PB
Weak transcription
T-helper naïve from PB, K562, primary HSC | NA | |
SLC22A5
Intron 1 | rs2631363 C/T | chr5:131707094 | 2b | NA | NA | RBBP5, POLR3A | |
SLC22A5
Intron 1 | rs2631362 C/T | chr5:131707292 | 1f |
SLC22A4
SLC22A5
|
Enhancers
MNC and T cells from PB
Flanking active TSS
K562
Weak transcription
MNC from PB, primary HSC | RBBP5 | |
SLC22A5
Intron 1 | rs274570 A/G | chr5:131713225 | 1f |
SLC22A4
SLC22A5
|
Quiescent/Low
Primary T cells from PB
Weak transcription
primary HSC, MNC from PB, K562 | NA | |
SLC22A5
Intron 2 | rs274567 A/G | chr5:131714408 | 1f |
SLC22A4
SLC22A5
|
Quiescent/Low
T regulatory and helper cells, primary neutrophils from PB
Strong transcription
K562, primary T cells from PB, primary HSC G-CSF mobilized female and male
Weak transcription
primary HSC, primary T, B and NK cells from PB | NA | |
SLC22A5
Intron 2 | rs183898 C/G | chr5:131716901 | 3a | NA |
Quiescent/Low
Primary neutrophils from PB, T regulatory and helper cells from PB
Weak Transcription
B cells, MNC form PB, K562, primary HSC short term culture | NA | |
SLC22A5
Intron 3 | rs274559 C/T | chr5:131720069 | 1f |
SLC22A4
SLC22A5
|
Quiescent/Low
primary neutrophils from PB, T regulatory and helper cells from PB
Strong Transcription
K562
Weak Transcription
primary HSC, primary NK cells from PB, T and B cells from PB, MNC from PB | NA | |
b. | |||||||
Gene Functional consequence | SNP | Position | Regulome DB score | Histone modification Chromatin state Blood cells | Protein binding K562 | Motif (cell type) | Position-Weight Matrix for TF binding Ref. sequence of SNP |
SLC22A4
Promoter | rs460089 C/G | chr5:131629771 | 2a | NA | POLR2A, PHF8, HMGN3, UBTF, CTCF, ATF3, USF1, MAX, MYC, CBX3, UBTF, ZBTB7A, CCNT2, E2F6, IRF1, BCLAF1, ETS1 | MYC:MAX (K562) | |
SLC22A4
Intron 3 | rs270606 C/T | chr5:131650866 | 3a | NA | HDAC8 | STAT5A (NA) | |
Downstream from SLC22A4
(LOC553103 intron 3) | rs272868 C/G | chr5:131680750 | 3a |
Quiescent/Low
Primary T and B cells from PB
Strong transcription MNC from PB
Weak transcription
T-helper naïve from PB, K562, primary HSC | STAT1, STAT2, CEBPB | MEOX2 (NA) | |
SLC22A5
NC transcript variant, UTR variant 5 prime | rs13180043 C/T | chr5:131705586 | 2b | NA | POLR2A, PHF8, TAF1, ETS1, IRF1, MYC, UBTF, RBBP5, SAP30, MAX, E2F6, NRF1, HMGN3, KDM5B, E2F1 | HIC1 (K562) | |
SLC22A5
NC transcript variant, UTR variant 5 primes | rs13180295 G/A | chr5:131705587 | 2b | NA | POLR2A, PHF8, TAF1, ETS1, IRF1, MYC, UBTF, RBBP5, SAP30, MAX, E2F6, NRF1, HMGN3, KDM5B, E2F1 | HIC1 (K562) |
The rs460089-GC is associated with a shorter BCR-ABL1 halving time and better outcome
a. | ||||||||||||
Response to imatinib at 3 months | rs460089 (G/C) | rs2631365 (T/C) | rs13180043 (C/T) | |||||||||
GG | GC | CC |
P value Fept
| TT | TC | CC |
P value Fept
| CC | CT | TT |
P value Fept
| |
EMR (n = 86) | 42 | 33 | 11 | 0.470 | 31 | 45 | 10 | 0.671 | 77 | 9 | 0 | 0.220 |
non-EMR (n = 31) | 19 | 8 | 4 | 12 | 14 | 5 | 25 | 6 | 0 | |||
b. | ||||||||||||
rs460089 (G/C)a
| rs2631365 (T/C)a
| rs13180043 (C/T) a
| ||||||||||
GG | GC | median/P valueMMT
| TT | TC | median/P valueMMT
| CC | CT | median/P value
MMT
| ||||
HT6 median of days (number of cases) | 33.10 (61) | 21.25 (43) | 27.00 p <0.00001 | 32.29 (44) | 25.41 (54) | 27.04
p = 0.223 | 26.46 (100) | 28.34 (17) | 27.00
p = 0.177 | |||
c. | ||||||||||||
rs2631365-TC | rs13180043-CC | |||||||||||
Optimal resp. | Non-optimal resp. |
P value Fept
| Optimal resp. | Non-optimal resp. |
P value Fept
| |||||||
rs460089-GG | 6 | 15 | 0.00006 | 26 | 33 | 0.0023 | ||||||
rs460089-GC | 27 | 8 | 30 | 8 | ||||||||
rs460089-CC | 4 | 0 | 9 | 6 | ||||||||
d. | ||||||||||||
rs460089-GG_ rs2631365-TC (number) | rs460089-GC_ rs2631365_TC (number) | median value/P value MMT
| rs460089-GG_ rs13180043-CC (number) | rs460089-GC_ rs13180043-CC (number) | median value/P value MMT
| |||||||
HT6 median of days (number of cases) | 36.34 (18) | 21.48 (34) | 25.41/0.004 | 33.150 (50) | 21.251 (37) | 27.100/0.002 | ||||||
e. | ||||||||||||
Haplotype rs460089_rs270606_rs156322_rs272872_rs272868_rs2631362_rs274570_rs183898 | European population | |||||||||||
G_G_T_G_G_A_C_C | 0.68 | |||||||||||
C_A_C_A_C_G_T_G | 0.32 | |||||||||||
G_A_C_A_C_G_T_G | 0.003 | |||||||||||
C_G_T_G_G_A_C_C | 0.001 | |||||||||||
Haplotype rs2631365_rs272842_rs272889_rs2631363_rs274567_rs274559 | ||||||||||||
T_G_G_A_C_A | 0.58 | |||||||||||
C_A_A_G_T_G | 0.42 | |||||||||||
C_A_G_A_C_G | 0.004 | |||||||||||
C_A_A_A_C_A | 0.001 | |||||||||||
C_G_G_A_C_A | 0.001 |
Identification of a cluster of genotypes associated with response and event-free survival
Multivariate analysis determined rs460089 predictive of outcome
No MMR N (%) | MMR N (%) | OR | 95% CI OR |
P value | Number | Events N | HR | CI (95% HR) |
P value | |
---|---|---|---|---|---|---|---|---|---|---|
Univariate | ||||||||||
Imatinib dose | ||||||||||
Imatinib 400/day | 33 (73) | 75 (89) | 0 | 0 | 0 | 108 | 37 | 0 | 0 | 0 |
Imatinib 200–300 mg/day | 12 (27) | 9 (11) | 2.86 | (1.16–7.89) | 0.0251 | 21 | 11 | 1.64 | (0.84–3.23) | 0.1482 |
Risk score | ||||||||||
Eutos Low | 31 (72) | 74 (88) | 0 | 0 | 0 | 105 | 34 | 0 | 0 | 0 |
Eutos High | 12 (27) | 10 (12) | 2.86 | (1.12–7.32) | 0.0452 | 22 | 12 | 1.53 | (1.10–2.13) | 0.0121 |
Sokal Low | 13 (31) | 42 (50) | 0 | 0 | 0 | 55 | 17 | 0 | 0 | 0 |
Sokal Intermediate | 11 (26) | 30 (36) | 1.18 | (0.47–3.00) | 0.8130 | 41 | 9 | 0.79 | (0.46–2.13) | 0.5772 |
Sokal High | 18 (43) | 12 (14) | 4.85 | (1.86–12.65) | 0.0019 | 30 | 19 | 1.69 | (1.22–2.36) | 0.0016 |
Hasford Low | 11 (26) | 30 (36) | 0 | 0 | 0 | 41 | 11 | 0 | 0 | 0 |
Hasford Intermediate | 18 (43) | 47 (56) | 1.04 | (0.43–2.52) | 1.0000 | 65 | 21 | 1.13 | (0.55–2.35) | 0.7344 |
Hasford High | 13 (31) | 7 (8) | 5.06 | (1.60–15.99) | 0.0058 | 20 | 13 | 1.71 | (1.14–2.55) | 0.0095 |
SNPs | ||||||||||
rs460089 | ||||||||||
GG | 35 (78) | 35 (42) | 0 | 0 | 0 | 70 | 37 | 0 | 0 | 0 |
CC | 4 (9) | 11 (13) | 0.36 | (0.10–1.25) | 0.1530 | 15 | 2 | 0.43 | (0.21–0.87) | 0.0197 |
GC | 6 (13) | 38 (45) | 0.15 | (0.06–0.43) | <0.0001 | 44 | 9 | 0.28 | (0.13–0.58) | 0.0006 |
rs13180043 | ||||||||||
CC | 35 (78) | 77 (92) | 0 | 0 | 0 | 112 | 41 | 0 | 0 | 0 |
CT | 10 (2) | 7 (8) | 3.14 | (1.11–8.94) | 0.0321 | 17 | 10 | 1.91 | (0.95–3.81) | 0.0677 |
rs2631365 | ||||||||||
TT | 19 (42) | 31 (37) | 0 | 0 | 0 | 50 | 20 | 0 | 0 | 0 |
CC | 6 (14) | 13 (15) | 0.75 | 0.24–2.32 | 0.7808 | 19 | 4 | 0.61 | (0.35–1.05) | 0.0767 |
TC | 20 (44) | 40 (48) | 0.82 | 0.73–1.79 | 0.6903 | 60 | 24 | 1.00 | (0.55–1.82) | 0.9900 |
Multivariate | ||||||||||
Imatinib dose | ||||||||||
Imatinib 400/day | 33 (73) | 75 (89) | 0 | 0 | 0 | 108 | 37 | 0 | 0 | 0 |
Imatinib 200–300 mg/day | 12 (27) | 9 (11) | 4.17 | (1.42–12.18) | 0.0191 | 21 | 11 | 1.98 | (1.01–3.90) | 0.0481 |
SNP | ||||||||||
rs460089 | ||||||||||
GG | 35 (78) | 35 (42) | 0 | 0 | 0 | 70 | 37 | 0 | 0 | 0 |
CC | 4 (9) | 11 (13) | 0.53 | (0.27–1.02) | 0.0740 | 15 | 2 | 0.40 | (0.20–0.83) | 0.0141 |
GC | 6 (13) | 38 (45) | 0.28 | (0.06–0.42) | 0.0003 | 44 | 9 | 0.28 | (0.14–0.59) | 0.0008 |