Sequence based mutation analysis of GBA

TODO: Change images for mutation 8

Subset of SNPs

Figure 1: Selected mutations highlighted on structure of 10GS. The different colors indicate, in which database the mutation is listed: dbSNP (blue), HGMD (red), both (green) CHANGE (Mut. 8)

The ten SNPs shown in the table below and highlighted in Figure 1 were chosen for the analysis in this task. It was tried to include SNPs all over the protein, in order to investigate the influence of mutations in several parts of the protein. The mutated residues forming hydrogenbonds with the active site of glucocerebrosidase are included, as these should result in either a mal- or nonfunctioning protein [TODO: Insert mutation at position 311 (His)]. It was not easy to find missense mutations only listed in dbSNP, as most of them were listed in HGMD, too.

Nr.	SNP ID/Accession Number	Database	Position including SP	Position without SP	Amino Acid Change	Codon Change	Remarks
1	CM081634	HGMD	49	10	Gly - Ser	cGGC-AGC
2	rs74953658, CM050263	dbSNP, HGMD	63	24	Asp - Asn	tGAC-AAC
3	rs1141820	dbSNP	99	60	His - Arg	CAC - CGC	suspected, status not validated
4	CM880035	HGMD	159	120	Arg - Gln	CGG-CAG	synonymos mutation at this position listed in dbSNP; forming hydrogen bond with active site
5	rs80205046, CM041347	dbSNP, HGMD	221	182	Pro - Leu	CCC - CTC
6	rs74731340, CM970620	dbSNP, HGMD	310	271	Ser - Asn	AGT - AAT
7	CM880036	HGMD	409	370	Asn - Ser	AAC-AGC	most common mutation found in gaucher disease type 1 patients
8	CM993703	HGMD	350	311	His - Arg	CAT-CGT	severe form of gaucher disease 2; forming hydrogen bond with active site
9	rs80020805, CM052245	dbSNP, HGMD	455	416	Met - Val	cATG-GTG
10	rs113825752	dbSNP	509	470	Leu - Pro	CTT - CCT

Physicochemical Properties and Changes

Figure 2: Comparison of the wildtype (red) and mutated (blue) amino acids mapped to the structure of 1OGS. CHANGE (Mut. 8)

Mutation 1

The wildtype amino acid, Glycine is nonpolar, whereas the mutated amino acid Serin is polar. This different polarity, could be an indication, that the mutation is damaging. Looking at the structure, one can see, that this residue (pos. 10 in mature protein) is situated in a loop at the exterior of the protein. Therefore a substitution should not affect the function of the protein that much.

Mutation 2

In this mutation the Aspartic acid as an acidic amino acid is replaced by its derivative Asparagine. So it loses its acidic character. Therefore it could have an effect on the folding of the protein. If you look at the structure you see, that it is not in the interior of the protein, so the mutation should also not affect the function.

Mutation 3

Histidine is mutated to Arginine, which are both amino acids with a positively charged functional group. Histidine forms a ring structure, whereas Arginine forms a straight chain. The residue is also situated at the exterior of the protein, so its influence is not so strong. As they have the same charge the propertiers are the same and the mutation may be tolerated.

Mutation 4

In this mutation the positively charged Arginine is replaced by the charged amino acid Glutamine. Glutamine is a zwitterion and can be positively charged as well as negatively. It is situated in the interior of the protein, so it could also affect the function of the protein.

Mutation 5

Proline is the amino acid, which forms a ring structure. Therefore it has a great influence on the folding of the protein. Both, Proline and Leucine are zwitterions. So the chemical properties are the same. As it is also in the interior of the protein it could influence the function of the protein.

Mutation 6

Serine is a hydroxylic amino acid which is mutated in the acidic amino acid Asparagine. They are both zwitterions, therefore they share chemical propertiers. The structure is also very similar. As it is positioned at the exterior the influence of the mutation may be not that strong.

Mutation 7

Asparagine is an acidic amino acid whereas Serine is hydroxylic. This mutation is exactly the other way round than mutation six. But this amino acid is situated in the interior of the protein as part of a helix. So the mutation could affect the function of the protein.

Mutation 8

Histidine and Arginine are both basic amino acids. Histidine builds an imidazole ring, which is protonated. The residue is in the interior of the protein and involved in the proteins function. So the mutation may not be tolerated.

Mutation 9

Methionine is a sulfur containing amino acid which is mutated to Valine. Their chemical properties differ so the mutation might affect the folding or the function of the protein. If you look at the structure you see that it is placed at a helix.

Mutation 10

Leucine is mutated to Proline, which is the amino acid that forms a ring structure. Therefore the structure of the protein might change. If you look at the structure you see that it is part of a beta sheet, which might be disordered by the mutation and so affect the protein's function.

Mutation Analysis

PSSM

Usage

• command line: blastpgp -i gbaseq.fasta -j 5 -d /data/blast/nr/nr -C profile.ckp

SNAP

Usage

• command line: snapfun -i gbaseq.fasta -m mutations.txt -o snapfun_out.out

Mutation Nr.	AA change	Prediction	Reliability Index	Expected Accuracy
1	G49S	Neutral	3	78%
2	D63N	Non-neutral	5	87%
3	H99R	Neutral	5	89%
4	R159Q	Non-neutral	7	96%
5	P221L	Non-neutral	5	87%
6	S310N	Neutral	0	53%
7	N409S	Non-neutral	1	63%
8	H350R	Non-neutral	8	96%
9	M455V	Non-neutral	3	78%
10	L509P	Non-neutral	1	63%

SIFT

Usage

• Webserver: http://sift.jcvi.org/www/SIFT_seq_submit2.html
• Input: sequence in fasta format, substitutions of interest (Example for mutation 1: G49S)

Results

Mutation Nr.	Prediction	Score	Sequence Conservation
1	tolerated	0.51	3.05
2	tolerated	0.06	3.05
3	tolerated	0.62	3.04
4	affect protein function	0.03	3.01
5	affect protein function	0.00	3.01
6	tolerated	0.54	3.01
7	affect protein function	0.05	3.02
8	affect protein function	0.00	3.11
9	tolerated	0.12	3.01
10	affect protein function	0.01	3.09

Coming soon: Multiple alignment of sequence with its homologs + Conditional Probability Matrix

Polyphen2

Usage

• Webserver: http://genetics.bwh.harvard.edu/pph2/
• Input: sequence in fasta format, position and substitution of mutation

Results

The results of PolyPhen-2 (HumDiv) are listed in the table below. Two mutations (Numbers 3 and 6) have been predicted to be harmless. Interestingly, the most common mutation found in gaucher disease patients is only classified as possibly damaging.

Mutation Nr.	Prediction	Score	Sensitivity	Specificity
1	probably damaging	0.997	0.40	0.98
2	probably damaging	1.000	0.00	1.00
3	benign	0.000	1.00	0.00
4	probably damaging	1.000	0.00	1.00
5	probably damaging	1.000	0.00	1.00
6	benign	0.100	0.94	0.85
7	possibly damaging	0.573	0.88	0.91
8	probably damaging	1.0	0.00	1.00
9	probably damaging	0.999	0.14	0.99
10	probably damaging	0.978	0.75	0.96