Table 3. Description of DPYD variations along with in silico / in vitro functionality.
| SNP or INDEL position (or rs if any) | Nucleotide change and nomenclature alias (if any) |
Location |
AA change | In silico pathogenicity prediction (impact on splicing signal) |
In vitro functionality [15] / [16] |
wt/wt |
Case number var/wt |
var/var |
MAF (%) | Significant association with deficient phenotype* | Significant association with increased toxicity** |
|---|---|---|---|---|---|---|---|---|---|---|---|
| rs145438244 | c.-672T>C | 5-UTR | 241 | 1 | 0 | 0.2 | nt | nt | |||
| rs61787828 | c.-477T>G | 5-UTR | 214 | 27 | 1 | 6.0 | NS | NS | |||
| 98386496 | c.-18G>A | 5-UTR | 241 | 1 | 0 | 0.2 | na, nt | nt | |||
| 98348989 | c.40-69_40-59del | Intron 1 | (No impact on splicing) | 242 | 1 | 0 | 0.2 | nt | nt | ||
| 98348894 | c.76G>A | Exon 2 | A26T | Pathogenic (ESE site broken) |
- / - | 241 | 1 | 0 | 0.2 | nt | nt |
| rs1801265 | c.85T>C (*9A) | Exon 2 | C29R | Benign (ESE site broken, new ESS) |
- / Slightly deficient | 161 | 74 | 7 | 18.2 | NS | No*** |
| rs371587702 | c.194C>T | Exon 3 | T65M | Pathogenic (ESE site broken) |
Benign / - | 241 | 1 | 0 |
0.2 |
nt |
nt |
| 98205969 | c.300C>A | Exon 4 | F100L | Pathogenic (ESE site broken) |
F100[FS] very deficient / - | 241 | 1 | 0 |
0.2 |
na, nt |
nt |
| rs56276561 | c.483+18G>A # | Intron 5 | (No impact on splicing) | 238 | 4 | 0 | 0.8 | NS | NS | ||
| 98186503 | c.483+563T>C | Intron 5 | (No impact on splicing) | 241 | 1 | 0 |
0.2 |
nt |
nt | ||
| 98186337 | c.483+729G>A | Intron 5 | (No impact on splicing) | 241 | 1 | 0 |
0.2 |
nt |
nt | ||
| rs56066952 | c.483+834A>G | Intron 5 | (No impact on splicing) | 240 | 2 | 0 | 0.4 | nt | nt | ||
| rs55684412 | c.483+837A>G | Intron 5 | (No impact on splicing) | 195 | 44 | 3 | 10.3 | NS | NS | ||
| 98185786 | c.483+1280A>G | Intron 5 | (No impact on splicing) | 242 | 1 | 0 | 0.2 | nt | nt | ||
| rs61786599 | c.483+1342T>A | Intron 5 | (No impact on splicing) | 202 | 40 | 1 | 8.6 | NS | NS | ||
| rs61786598 | c.483+1344T>A | Intron 5 | (No impact on splicing) | 192 | 48 | 3 | 11.1 | NS | NS | ||
| 98185721 | c.483+1345_483+1354del | Intron 5 | (No impact on splicing) | 242 | 1 | 0 | 0.2 | nt | nt | ||
| rs199919864 | c.483+1346A>T | Intron 5 | (No impact on splicing) | 242 | 1 | 0 | 0.2 | na, nt | nt | ||
| 98185720 | c.483+1345_483+1346dup | Intron 5 | (No impact on splicing) | 241 | 2 | 0 | 0.4 | nt | nt | ||
| 98185711 | c.483+1354_483+1355insAA | Intron 5 | (No impact on splicing) | 242 | 1 | 0 | 0.2 | nt | nt | ||
| 98185705 | c.483+1360_483+1361dup | Intron 5 | (No impact on splicing) | 233 | 10 | 0 | 2.1 | NS | NS | ||
| rs75848562 | c.483+1366A>G | Intron 5 | (No impact on splicing) | 232 | 11 | 0 | 2.3 | NS | NS | ||
| rs142148197 | c.483+1689G>A | Intron 5 | (No impact on splicing) | 242 | 1 | 0 | 0.2 | nt | nt | ||
| rs2297595 | c.496A>G | Exon 6 | M166V | Benign (New ESS site and cryptic acceptor splice site) |
Proficient / Slightly deficient | 198 | 42 | 3 | 9.9 | NS | NS |
| 98060744 | c.851-22T>C | Intron 8 | (No impact on splicing) | 241 | 1 | 0 | 0.2 | nt | nt | ||
| rs183385770 | c.1025A>G | Exon 10 | D342G | Pathogenic (ESE site broken, new ESS site) |
D342N very deficient / - | 241 | 1 | 0 | 0.2 |
nt |
nt |
| 98058849 | c.1053T>C | Exon 10 | A351A | Benign (No impact on splicing) |
241 | 1 | 0 | 0.2 | nt |
nt | |
| 98058804 | c.1098C>T | Exon 10 | G366G | Probably pathogenic (New ESS site) |
241 | 1 | 0 | 0.2 | na, nt | nt | |
| rs56293913 | c.1129-15T>C | Intron 10 | (No impact on splicing) | 191 | 46 | 5 | 11.6 | NS | NS | ||
| rs61622928 | c.1218G>A | Exon 11 | M406I | Benign (No impact on splicing) |
Benign / Benign | 242 | 1 | 0 | 0.2 | nt | nt |
| rs56038477 | c.1236G>A # | Exon 11 | E412E | Benign (No impact on splicing) |
239 | 4 | 0 | 0.8 | NS | NS | |
| rs72549304 | c.1475C>T | Exon 12 | S492L | Pathogenic (New ESS site) |
Very deficient / - | 241 | 1 | 0 | 0.2 | na, nt | nt |
| rs199469537 | c.1524 +16C>A | Intron 12 | (No impact on splicing) | - | 241 | 1 | 0 | 0.2 | na, nt | nt | |
| rs1801158 | c.1601G>A (*4) | Exon 13 | S534N | Probably benign (New ESS site) |
- / Slightly deficient | 234 | 8 | 0 | 1.7 | NS | NS |
| rs1801159 | c.1627A>G | Exon 13 | I543V | Benign (No impact on splicing) |
- / Benign | 161 | 73 | 8 | 18.4 | NS | NS |
| rs55886062 | c.1679T>G (*13) | Exon 13 | I560S | Pathogenic (No impact on splicing) |
Very deficient / - | 242 | 1 | 0 | 0.2 | na, nt | nt |
| 97981200 | c.1740+82del | Intron 13 | (No impact on splicing) | 242 | 1 | 0 | 0.2 | nt | nt | ||
| rs59086055 | c.1774C>T | Exon 14 | R592W | Pathogenic (New ESS site) |
Very deficient / - | 241 | 1 | 0 | 0.2 | nt | nt |
| rs17376848 | c.1896T>C | Exon 14 | F632F | Benign (New ESS site) |
227 | 15 | 1 | 3.5 | NS | NS | |
| rs3918290 | c.1905+1G>A (*2A) | Splice intron 14 | Pathogenic (Alteration of the donor site) |
Very deficient / | 240 | 3 | 0 | 0.6 | Yes | Yes | |
| rs369990607 | c.1905+17A>G | Intron 14 | (No impact on splicing) | 242 | 1 | 0 | 0.2 | nt | nt | ||
| rs12078940 | c.1906-24G>A | Intron 14 | (No impact on splicing) | 241 | 1 | 0 | 0.2 | nt | nt | ||
| 97771825 | c.2087G>A | Exon 17 | R696H | Probably pathogenic (No impact on splicing) |
- / - | 241 | 1 | 0 | 0.2 | nt | nt |
| rs55846082 | c.2179+28C>T | Intron 17 | (No impact on splicing) | 241 | 1 | 0 | 0.2 | nt | nt | ||
| rs138637410 | c.2179+29G>A | Intron17 | (No impact on splicing) | 241 | 1 | 0 | 0.2 | na, nt | nt | ||
| rs1801160 | c.2197G>A (*6) | Exon 18 | V732I | Benign (No impact on splicing) |
V732G benign / V732I Slightly deficient |
226 | 16 | 0 | 3.3 | NS | NS |
| rs67376798 | c.2846A>T | Exon 22 | D949V | Probably pathogenic (ESE site broken, new ESS site) |
Moderately deficient / Moderately deficient | 240 | 3 | 0 | 0.6 | Yes | Yes |
| rs56160474 | c.*274T>C | 3-UTR | 158 | 75 | 9 | 19.2 | NS | NS | |||
| rs188501488 | c.*432T>A | 3-UTR | 240 | 2 | 0 | 0.4 | nt | nt | |||
| rs291592 | c.*768G>A | 3-UTR | 85 | 122 | 35 | 39.7 | NS | NS | |||
| rs291593 | c.*780C>T | 3-UTR | 151 | 78 | 13 | 21.5 | NS | NS | |||
| rs17470762 | c.*900T>C | 3-UTR | 222 | 19 | 1 | 4.3 | NS | NS | |||
| rs41285690 | c.*1062A>G | 3-UTR | 238 | 4 | 0 | 0.8 | NS | NS | |||
| 97543343 | c.*1189G>A | 3-UTR | 241 | 1 | 0 | 0.2 | nt | nt |
SNP and INDEL positions are given relative to genome build 37 HG19 (reference = nucleotide A of the translation initiation codon ATG).
In silico pathogenicity of coding variants was predicted using UMD-Predictor system [27]. The potential impact of exonic and intronic variations on splicing signal types was predicted using Human Splicing Finder system [28].
In vitro functionality derived from two published in vitro functional studies [15,16] reporting DPD enzyme activity of missense DPYD variants transgenically expressed in mammalian cells. In both studies, deficiency and proficiency were based on statistical comparison relative to wild-type DPD activity (100% activity). p value considered statistically significant was 0.05 in the study by Offer [15] and 0.001 in the study by van Kuilenburg [16]. Statistically significant DPD deficiency was classified as “Very deficient” for DPD activity ≤25% wild-type DPYD, “Moderately deficient” for DPD activity within 25–60% that of wild-type, and “Slighly deficient” for DPD activity >60% that of wild-type. Otherwise, variant functionality was considered benign (not statistically significant) or proficient when significantly greater than that of wild-type.–means that the variant was not tested in vitro.
MAF means minor allelic frequency, expressed as a percentage.
* Impact of each DPYD variation on phenotype (UH2/U or U) was tested by means of non-parametric Mann-Whitney test for variants present in at least 3 patients (see Statistics section and Fig 2 for details).
** Impact of each DPYD variation on digestive/hemato/neurotoxicity (grade 3-4-5 or grade 4–5) was tested by means of Fisher Exact test for variants present in at least 3 patients (see Statistics section).
*** Patients bearing variant allele C29R significantly experienced less toxicity than wt patients (p = 0.041).
# Variant linked to haplotype B3 comprising synonymous variant E412E and three intronic variants c.483+18G>A, c.680+139G>A and c.959-51T>C.
ESE means exonic splicing enhancer; ESS means exonic splicing silencer.
nt means not tested due to scarcity of variant carriers (less than 3 patients bearing at least one variant allele).
na means that DPD phenotype was not available (lack of validated UH2/U or U plasma concentration).
NS means not significant (p≥0.05).
Yes means that a significant relationship (p<0.05) was observed (see details in the Results section).