Sequence-based mutation analysis BCKDHA
Subset of mutations
Reference amino acid | Mutated amino acid | Secondary Structure | |||||
---|---|---|---|---|---|---|---|
Position | Residue | Properties | Structure | Residue | Properties | Structure | |
29 | G | tiny, small | E | charged, polar | C | ||
125 | Q | acidic, polar | E | charged, polar | C | ||
166 | Y | hydrophobic, aromatic, polar | N | acidic, polar, small | H | ||
249 | G | tiny, small | S | polar, small, tiny, hydroxylic | H | ||
264 | C | sulphur containing, hydrophobic, tiny, small, polar | W | hydrophobic, aromatic, polar | E | ||
265 | R | charged, positive (basic), polar | W | hydrophobic, aromatic, polar | E | ||
326 | I | aliphatic, hydrophobic | T | hydroxylic, hydrophobic, small, polar | E | ||
361 | I | aliphatic, hydrophobic | V | aliphatic, hydrophobic, small | H | ||
409 | F | aromatic, hydrophobic | C | sulphur containing, hydrophobic, tiny, small, polar | C | ||
438 | Y | hydrophobic, aromatic, polar | N | acidic, polar, small | C |
Position | AA1/AA2 | BLOSUM62 | PAM1 | PAM250 | |||
---|---|---|---|---|---|---|---|
score | worst | score | worst | score | worst | ||
29 | G/E | -2 | -4 (I, L) | 7 | 0 (I, W, Y) | 9 | 2 (W) |
125 | Q/E | 2 | -3 (C, F, I) | 27 | 0 (F, W, Y) | 7 | 1 (C, F, W) |
166 | Y/N | -2 | -3 (D, G, P) | 3 | 0 (R, D, Q, G, K, M, P) | 2 | 1 (A, R, D, Q, E, G, K, P) |
249 | G/S | 0 | -4 (I, L) | 21 | 0 (I, W, Y) | 11 | 2 (W) |
264 | C/W | -2 | -4 (E) | 0 | 0 (N, D, Q, E, G, L, K, M, F, W) | 1 | 1 (R, N, D, Q, E, L, K, M, F, W) |
265 | R/W | -3 | -3 (W, V, F, I, C) | 8 | 0 (D, E, G, Y) | 7 | 1 (F) |
326 | I/T | -1 | -4 (G) | 7 | 0 (G, A, P, W) | 4 | 1 (W) |
361 | I/V | 3 | -4 (G) | 33 | 0 (G, H, P, W) | 9 | 1 (W) |
409 | F/C | -2 | -4 (P) | 0 | 0 (D, C, Q, E, K, P, V) | 1 | 1 (R, D, C, Q, E, G, K, P) |
438 | Y/N | -2 | -3 (D, G, P) | 3 | 0 (R, D, Q, G, K, M, P) | 2 | 1 (A, R, D, Q, E, G, K, P) |
Annotation: H = helix, E = beta-sheet, C = coil
PSSM
In order to get a human-readable PSSM-File PsiBlast was run using the following command: blastpgp -i sequence.fasta -j 5 -d /data/blast/nr/nr -C profile.ckp -u 1 -J T
Multiple Sequence Alignment
To find the homologue sequences to BCKDHA we used BLAST. It found 250 homologous sequences, 25 of them are mammals.
ID | Accession | Entry name |
---|---|---|
sp | P11178 | ODBA_BOVIN |
sp | P12694 | ODBA_HUMAN |
sp | Q8HXY4 | ODBA_MACFA |
sp | P50136 | ODBA_MOUSE |
sp | A5A6H9 | ODBA_PANTR |
sp | P11960 | ODBA_RAT |
tr | Q6ZSA3 | Q6ZSA3_HUMAN |
tr | E7ESE6 | E7ESE6_HUMAN |
tr | B2R8A9 | B2R8A9_HUMAN |
tr | Q658P7 | Q658P7_HUMAN |
tr | E7EW46 | E7EW46_HUMAN |
tr | B4DP47 | B4DP47_HUMAN |
tr | Q59EI3 | Q59EI3_HUMAN |
tr | F1N5F2 | F1N5F2_BOVIN |
tr | B1PK12 | B1PK12_PIG |
tr | E2RPW4 | E2RPW4_CANFA |
tr | B2LSM3 | B2LSM3_SHEEP |
tr | F1RHA0 | F1RHA0_PIG |
tr | F1PI86 | F1PI86_CANFA |
tr | D2HMT3 | D2HMT3_AILME |
tr | Q2TBT9 | Q2TBT9_BOVIN |
tr | Q3U3J1 | Q3U3J1_MOUSE |
tr | Q99L69 | Q99L69_MOUSE |
tr | Q5EB89 | Q5EB89_RAT |
tr | B1WBN3 | B1WBN3_RAT |
With this 25 results we made a multiple alignment by using CLUSTALW. The alignment with all mammalian homologous was quite bad because of the sequences "Q6ZSA3" and "E2RPW4". These two sequences are much longer than the other one. So we removed those sequences and realigned the other sequences.
SNAP
To run SNAP we used the command:
snapfun -i BCKDHA.fasta -m mutations.txt -o SNAP.out
nsSNP | Prediction | Reliability Index | Expected Accuracy |
---|---|---|---|
G29E | Neutral | 0 | 53% |
Q125E | Non-neutral | 1 | 63% |
Y166N | Non-neutral | 2 | 70% |
G249S | Neutral | 1 | 60% |
C264W | Non-neutral | 4 | 82% |
R265W | Non-neutral | 4 | 82% |
I326T | Non-neutral | 3 | 78% |
I361V | Neutral | 4 | 85% |
F409C | Non-neutral | 4 | 82% |
Y438N | Non-neutral | 4 | 82% |
A second SNAP run was performed where all ten chosen mutation positions were mutated by all possible substitutions.
SIFT
SIFT was run with default parameters (Selected databse to search: UniRef90 2011 Apr, Median conservation of sequences: 3.00, Remove sequences more then 90 percent identical to query.)
Polyphen2
Position | AA1/AA2 | HumDiv | HumVar | ||||||
---|---|---|---|---|---|---|---|---|---|
prediction | Score | Sensitivity | Specificity | prediction | Score | Sensitivity | Specificity | ||
29 | G/E | benign | 0.025 | 0.96 | 0.80 | benign | 0.018 | 0.96 | 0.52 |
125 | Q/E | possibly damaging | 0.759 | 0.85 | 0.93 | benign | 0.285 | 0.87 | 0.75 |
166 | Y/N | probably damaging | 0.997 | 0.40 | 0.98 | probably damaging | 0.964 | 0.59 | 0.93 |
249 | G/S | benign | 0.145 | 0.93 | 0.86 | benign | 0.292 | 0.86 | 0.75 |
264 | C/W | probably damaging | 1.000 | 0.00 | 1.00 | probably damaging | 1.000 | 0.00 | 1.00 |
265 | R/W | probably damaging | 1.000 | 0.00 | 1.00 | probably damaging | 1.000 | 0.00 | 1.00 |
326 | I/T | probably damaging | 0.997 | 0.40 | 0.98 | probably damaging | 0.998 | 0.16 | 0.99 |
361 | I/V | benign | 0.039 | 0.95 | 0.82 | benign | 0.178 | 0.89 | 0.70 |
409 | F/C | probably damaging | 0.998 | 0.27 | 0.99 | probably damaging | 0.939 | 0.64 | 0.92 |
438 | Y/N | probably damaging | 1.000 | 0.00 | 1.00 | probably damaging | 0.987 | 0.49 | 0.96 |
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