Sequence-based mutation analysis of ARSA

Intro

We randomly picked 10 missense mutations from dbSNP and HGMD. The mutations are listed below, together with a pymol mutagenesis image and a description of the properties of the mutations.

mutation	position	reference	mutation	both
Asp-Asn	29
Description of Asp-Asn Paste description of mutation here
Pro - Ala	136
Description of Pro-Ala Paste description of mutation here
Gln-His	153
Description of Gln-His Paste description of mutation here
Trp-Cys	193
Description of Trp-Cys Paste description of mutation here
Thr-Met	274
Description of Thr-Met Paste description of mutation here
Phe -Val	356
Description of Phe-Val Paste description of mutation here
Thr-Ile	409
Description of Thr-Ile Paste description of mutation here
Asn-Ser	440
Description of Asn-Ser Paste description of mutation here
Cys-Gly	489
Description of Cys-Gly Paste description of mutation here
Arg-His	496
Description of Arg-His Paste description of mutation here

SNAP

We ran snap using the following command:

snapfun -i ARSA.fasta -m mutants.txt -o snap.out

output:

nsSNP	Prediction	Reliability Index	Expected Accuracy
-----	------------	-------------------	-------------------
D29N	Non-neutral		7			96%
Q153H	 Neutral 		0			53%
T274M	Non-neutral		6			93%
T409I	Non-neutral		1			63%
C489G	Non-neutral		5			87%
W193C	Non-neutral		3			78%
F356V	 Neutral 		1			60%
N440S	Non-neutral		2			70%
R496H	 Neutral 		1			60%
P136A	Non-neutral		4			82%

In order to analyze all possible combinations of amino acid substitutions from the above mutated positions, we used the Generate Mutants tool on http://rostlab.org/services/snap/submit to create all possible exchanges from the following pattern: referenceAminoAcidPosition* . Then we again executed snap:

snapfun -i ARSA.fasta -m all_mutants.txt -o snap_all.out

Next, we wrote a perl script to parse and summarize the SNAP output in the following table, which shows which amino acid substitutions are Non-neutral or Neutral:

{!border="1" style="text-align:center; border-spacing:0;" ! ref\mutation || A || R || N || D || C || Q || E || G || H || I || L || K || M || F || P || S || T || W || Y || V |- | D29 || Non-neutral || Non-neutral || Non-neutral || || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral |- | Q153 || Non-neutral || Non-neutral || Neutral || Non-neutral || Non-neutral || || Non-neutral || Non-neutral || Neutral || Non-neutral || Non-neutral || Non-neutral || Neutral || Non-neutral || Neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral |- | T274 || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || || Non-neutral || Non-neutral || Non-neutral |- | T409 || Neutral || Non-neutral || Neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || || Non-neutral || Non-neutral || Non-neutral |- | C489 || Non-neutral || Non-neutral || Non-neutral || Non-neutral || || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral |- | W193 || Non-neutral || Non-neutral || Neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || || Non-neutral || Non-neutral |- | F356 || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Neutral || Non-neutral || Non-neutral || Neutral || Neutral || Neutral || Non-neutral || Neutral || || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Neutral || Neutral |- | N440 || Non-neutral || Non-neutral || || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral |- | R496 || Non-neutral || || Non-neutral || Non-neutral || Non-neutral || Neutral || Non-neutral || Non-neutral || Neutral || Non-neutral || Non-neutral || Neutral || Non-neutral || Neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Neutral || Non-neutral |- | P136 || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral || || Non-neutral || Non-neutral || Non-neutral || Non-neutral || Non-neutral |- |}

Multiple sequence alignments

First, we downloaded the HSSP file for ARSA to get all proteins, which are homologuous to it. Then we downloaded all mammalian protein sequences from Uniprot. This was achieved by searching for the term taxonomy:40674, which codes for all mammalian protein sequences. We saved all sequences in one multiple fasta file. Then we extracted all homologuous mammalian proteins to human ARSA by mapping the ids from the HSSP file to sequence ids in the multi fasta file. This yielded 75 homologuous mammalian sequences to human ARSA.
Next, we calculated multiple sequence alignments of these proteins (including ARSA) with Muscle. The Jalview images of the alignments are shown below.

Multiple sequence alignments of all 75 homologuous sequences using muscle

pos	conservation - reference	conservation - mutant
29	0.86	0
153	0.14	0
274	0.87	0
409	0.35	0.16
489	0.80	0.05
193	0.13	0
356	0.15	0
440	0.15	0
496	0.14	0.01
136	0.93	0

PSI-BLAST

blastpgp -i ARSA.fasta -d /data/blast/nr/nr -e 10E-6 -j 5 -Q psiblast.mat -o psiblast_eval10E_6.it.5.new.txt

Last position-specific scoring matrix computed, weighted observed percentages rounded down, information per position, and relative weight of gapless real matches to pseudocounts
          A  R  N  D  C  Q  E  G  H  I  L  K  M  F  P  S  T  W  Y  V   A   R   N   D   C   Q   E   G   H   I   L   K   M   F   P   S   T   W   Y   V
  29 D   -5 -5 -2  8 -7 -3 -1 -4 -4 -6 -7 -4 -6 -7 -5 -3 -4 -7 -6 -6    0   0   0 100   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  2.49 1.56
 153 Q    3  2 -1  4 -4 -1 -1 -2  0 -2 -3 -3  4 -2 -3 -1 -2 -3 -2 -2   26  10   3  23   0   3   3   3   2   2   1   1  13   2   1   3   2   0   1   2  0.53 1.48
 274 T   -3 -4 -3 -4 -2 -4 -4 -5 -5 -4 -4 -4 -3 -5 -4  1  8 -6 -5 -3    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   7  92   0   0   0  1.94 1.62
 409 T   -1  0  0 -1 -2 -1 -1  0 -1 -1 -1  0 -1 -1  3  0  1  6  0 -1    5   5   5   4   1   3   4   8   1   3   6   5   1   2  13   6   8  11   3   4  0.26 0.95
 489 C    2 -1  1 -4  8 -4 -4 -2 -1 -1 -2 -3 -1 -4 -4  0  0  5 -1 -3   15   4   8   0  36   0   0   2   1   3   3   1   1   0   0   6   5   9   2   0  0.99 1.22
 440 N   -5 -3  6  5 -6 -2 -1 -4 -3 -6 -6 -3 -6 -6  2 -2 -3 -6 -6 -5    0   1  46  36   0   1   2   0   0   0   0   1   0   0  10   1   1   0   0   0  1.48 1.67
 356 F   -3 -1 -5 -5 -3  0 -1 -6  1  3  0 -1  0  2 -6 -3 -2 -3  5  3    1   4   0   0   1   5   4   0   3  18   8   5   2   8   0   1   2   0  20  20  0.59 1.62
 193 W   -2  4  2  3 -5  0  0 -2  0 -3 -4  1 -3 -1 -2 -1 -2  1  1 -3    3  25  11  16   0   4   5   3   2   2   1   7   0   2   2   4   2   2   5   2  0.46 1.45
 136 P   -3 -5 -5 -5 -6 -4 -4 -5 -5 -6 -6 -4 -6 -7  9 -4 -4 -7 -6 -5    1   0   0   0   0   0   0   0   0   0   0   0   0   0  98   0   0   0   0   0  3.03 1.61
 496 R   -3  1  0 -3 -4  1  1 -1  1 -3  1  1 -2  2  4  0 -3 -1 -1 -3    1   7   4   1   0   5  10   4   3   1  16   9   0   9  20   8   1   1   1   1  0.34 0.96