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 beta strand 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 that are basic. Histidine forms a ring structure and is aromatic, 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 almost the same and the mutation may be tolerated. Only the different structure of Arginine may influence the function.

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, aliphatic, polar and neutral and 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

Substitution Matrices

We used the following matrices:

• BLOSUM62
• PAM1
• PAM250

Nr.	Substitution	BLOSUM62	PAM1	PAM250
1	Gly - Ser	0 (worst: -4)	16 (worst: 0)	9 (worst: 1)
2	Asp - Asn	1 (worst: -4)	36 (worst: 0)	7 (worst: 1)
3	His - Arg	0 (worst: -3)	10 (worst: 0)	6 (worst: 1)
4	Arg - Gln	1 (worst: -3)	9 (worst: 0)	5 (worst: 1)
5	Pro - Leu	-3 (worst: -4)	3 (worst: 0)	5 (worst: 1)
6	Ser - Asn	1 (worst: -3)	20 (worst: 0)	5 (worst: 1)
7	Asn - Ser	1 (worst: -4)	34 (worst: 0)	8 (worst: 1)
8	His - Arg	0 (worst: -3)	10 (worst: 0)	6 (worst: 1)
9	Met - Val	1 (worst: -3)	17 (worst: 0)	4 (worst: 1)
10	Leu - Pro	-3 (worst: -4)	2 (worst: 0)	3 (worst: 1)

Multiple Alignment

Figure 3: Multiple alignment of glucecerebrosidase and the found mammal sequences

Usage

• protein blast with the option mammals

We found the following sequences:

Accession	Description
NP_000148.2	glucosylceramidase isoform 1 precursor [Homo sapiens]
AAA35880.1	glucocerebrosidase [Homo sapiens] linked to AAA35880.1Genome view with mapviewer linked to AAA35880.1
BAH13365.1	unnamed protein product [Homo sapiens]
EAW53100.1	glucosidase, beta; acid (includes glucosylceramidase), isoform CRA_a [Homo sapiens] ked to EAW53100.1
NP_001008997.1	glucosylceramidase precursor [Pan troglodytes] NP_001008997.1Gene info linked to NP_001008997.1Genome view with mapviewer linked to NP_001008997.1
XP_003259439.1	PREDICTED: glucosylceramidase-like isoform 1 [Nomascus leucogenys]
XP_003259440.1	PREDICTED: glucosylceramidase-like isoform 2 [Nomascus leucogenys]
NP_001127488.1	glucosylceramidase precursor [Pongo abelii] NP_001127488.1Genome view with mapviewer linked to NP_001127488.1
NP_001128784.1	DKFZP469B0323 protein [Pongo abelii]
AAA35877.1	lysosomal glucocerebrosidase precursor [Homo sapiens]
AAA35873.1	glucocerebrosidase precursor (5' end put.); putative [Homo sapiens]
BAH13232.1	unnamed protein product [Homo sapiens]
XP_002760131.1	PREDICTED: glucosylceramidase isoform 2 [Callithrix jacchus]
XP_002760130.1	PREDICTED: glucosylceramidase isoform 1 [Callithrix jacchus]
2WKL_A	Chain A, Velaglucerase Alfa
3KE0_A	Chain A, Crystal Structure Of N370s Glucocerebrosidase At Acidic Ph
2V3D_A	Chain A, Acid-Beta-Glucosidase With N-Butyl-DeoxynojirimycinubChem BioAssay Info linked to 2V3D_A
1Y7V_A	Chain A, X-Ray Structure Of Human Acid-Beta-Glucosidase Covalently Bound To Conduritol B Epoxide
XP_001498700.1	PREDICTED: similar to putative lysosomal glucocerebrosidase [Equus caballus]d to XP_001498700.1
XP_855035.1	PREDICTED: similar to glucocerebrosidase precursor [Canis familiaris]
XP_002715375.1	PREDICTED: glucocerebrosidase-like [Oryctolagus cuniculus]
NP_001005730.1	glucosylceramidase precursor [Sus scrofa]
XP_002928339.1	PREDICTED: glucosylceramidase-like [Ailuropoda melanoleuca]
NP_001039886.1	glucosylceramidase precursor [Bos taurus] d to NP_001039886.1Gene info linked to NP_001039886.1Genome view with mapviewer linked to NP_001039886.1
DAA31806.1	glucosylceramidase precursor [Bos taurus]
BAH13605.1	unnamed protein product [Homo sapiens]
NP_001165283.1	glucosylceramidase isoform 3 precursor [Homo sapiens]
BAH12898.1	unnamed protein product [Homo sapiens]
EFB14309.1	hypothetical protein PANDA_018263 [Ailuropoda melanoleuca]
NP_001121111.1	glucosidase, beta, acid [Rattus norvegicus]
EDL15229.1	glucosidase, beta, acid, isoform CRA_a [Mus musculus]
NP_032120.1	glucosylceramidase isoform 1 [Mus musculus]
XP_002760132.1	PREDICTED: glucosylceramidase isoform 3 [Callithrix jacchus]
NP_001165282.1	glucosylceramidase isoform 2 [Homo sapiens]
BAH13357.1	unnamed protein product [Homo sapiens]
XP_003259441.1	XP_003259442.1| PREDICTED: glucosylceramidase-like isoform 4 [Nomascus leucogenys]
XP_001374060.1	PREDICTED: glucosylceramidase-like [Monodelphis domestica]
BAH13467.1	unnamed protein product [Homo sapiens]
XP_002760133.1	PREDICTED: glucosylceramidase isoform 4 [Callithrix jacchus]
BAG58248.1	unnamed protein product [Homo sapiens]
BAG60020.1	unnamed protein product [Homo sapiens]
CAI95088.1	glucosidase, beta; acid, pseudogene [Homo sapiens]
BAA02546.1	glucocerebrosidase [Homo sapiens]
BAG52175.1	unnamed protein product [Homo sapiens]
BAH13574.1	unnamed protein product [Homo sapiens]
XP_002810119.1	PREDICTED: glucosylceramidase-like [Pongo abelii]
AAA35876.1	D-glucosyl-N-acylsphingosine glucohydrolase [Homo sapiens]
CAE06503.1	putative lysosomal glucocerebrosidase precursor [Sus scrofa]
AEB01891.1	beta-glucosidase [Platanista minor]
ABM68996.1	glucocerebrosidase [Sotalia fluviatilis]
ABX59623.1	glucocerebrosidase [Lagenorhynchus australis]
ABX59621.1	glucocerebrosidase [Lagenodelphis hosei]
ABX59628.1	glucocerebrosidase [Peponocephala electra]
AAF63861.1	beta-acid glucosidase [Equus caballus]
ACY77522.1	glucosidase beta acid [Balaenoptera bonaerensis]
ACY77443.1	glucosidase beta acid [Eubalaena australis]
ACY77479.1	glucosidase beta acid [Balaenoptera bonaerensis]
ACY77364.1	glucosidase beta acid [Megaptera novaeangliae]
ACY77382.1	glucosidase beta acid [Megaptera novaeangliae]
ABX59629.1	glucocerebrosidase [Peponocephala electra]
ABX59635.1	glucocerebrosidase [Phocoenoides dalli]
AEB01886.1	beta-glucosidase [Feresa attenuata]
AEB01881.1	beta-glucosidase [Lissodelphis borealis]
AEB01885.1	beta-glucosidase [Lagenorhynchus acutus]
AEB01888.1	beta-glucosidase [Neophocaena phocaenoides]
AAA35874.1	glucocerebrosidase precursor [Homo sapiens] AAA35874.1
ABX59637.1	glucocerebrosidase [Phocoena phocoena]
ABX59639.1	glucocerebrosidase [Inia geoffrensis boliviensis]
AEB01890.1	beta-glucosidase [Ziphius cavirostris]
ABX59620.1	glucocerebrosidase [Tursiops truncatus]
ABX59634.1	glucocerebrosidase [Phocoenoides dalli]
ACY77528.1	glucosidase beta acid [Cephalorhynchus hectori]
AEB01889.1	beta-glucosidase [Monodon monoceros]
ACY77530.1	glucosidase beta acid [Globicephala macrorhynchus]
AAA35875.1	glucocerebrosidase (alt.) precursor [Homo sapiens]75.1
ACY77529.1	glucosidase beta acid [Cephalorhynchus hectori]
NP_001071509.1	phospholipase D3 [Bos taurus] e info linked to NP_001071509.1Gene info linked to NP_001071509.1Genome view with mapviewer linked to NP_001071509.1
BAE88528.1	unnamed protein product [Macaca fascicularis]
Q4R583.1	unnamed protein product [Macaca fascicularis]
XP_001094274.1	PREDICTED: phospholipase D3 isoform 4 [Macaca mulatta]94274.1Gene info linked to XP_001094274.1Genome view with mapviewer linked to XP_001094274.1
XP_002762177.1	PREDICTED: phospholipase D3-like [Callithrix jacchus]
AAB16799.1	HU-K4 [Homo sapiens]
AAH00553.2	PLD3 protein [Homo sapiens]
XP_001498901.1	PREDICTED: phospholipase D family, member 3 isoform 1 [Equus caballus] linked to XP_001498901.1
XP_866921.1	PREDICTED: similar to phospholipase D3 isoform 2 isoform 3 [Canis familiaris]
CAH93251.1	hypothetical protein [Pongo abelii]
NP_001026866.1	phospholipase D3 [Homo sapiens]fo linked to NP_001026866.1Genome view with mapviewer linked to NP_001026866.1
NP_001126871.1	phospholipase D3 [Pongo abelii]
XP_866933.1	PREDICTED: similar to phospholipase D3 (predicted) isoform 4 [Canis familiaris]
XP_853110.1	PREDICTED: similar to phospholipase D3 (predicted) isoform 2 [Canis familiaris]
XP_866956.1	PREDICTED: similar to phospholipase D3 (predicted) isoform 6 [Canis familiaris]
NP_001012167.1	phospholipase D3 [Rattus norvegicus]

Mutation Nr.	Position	Amino acid change	Conservation all (Jalview)	Conservation best 25 (Jalview)
1	49	Gly - Ser	0.0	11.0
2	63	Asp - Asn	4.0	11.0
3	99	His - Arg	3.0	8.0
4	159	Arg - Gln	0.0	11.0
5	221	Pro - Leu	0.0	11.0
6	310	Ser - Asn	0.0	11.0
7	409	Asn - Ser	0.0	11.0
8	350	His - Arg	0.0	9.0
9	455	Met - Val	0.0	11.0
10	509	Leu - Pro	0.0	9.0

Secondary Structure

The secondary structure is assigned as in Task 3 - Sequence-based predictions.

Mutation Nr.	Position	Amino acid change	Secondary structure (Uniprot)	PSIPRED	Jpred3	DSSP
1	49	Gly - Ser	Beta sheet	Coil	-	Turn
2	63	Asp - Asn	-	Coil	-	-
3	99	His - Arg	-	Coil	-	-
4	159	Arg - Gln	Beta sheet	Beta sheet	Beta sheet	Bend
5	221	Pro - Leu	-	Coil	-	-
6	310	Ser - Asn	-	Coil	-	Turn
7	409	Asn - Ser	Helix	Helix	Helix	Helix
8	350	His - Arg	Beta sheet	Beta sheet	-	Bend
9	455	Met - Val	Helix	Coil/Helix	Helix	Helix
10	509	Leu - Pro	Beta sheet	Beta sheet	Beta sheet	Bend

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

Discussion

Mutation 1: Gly - Ser (Pos. 49/10)

BLOSUM62	PAM1	PAM250	Conservation (all/best 25)	Secondary structure (Uniprot)	Psipred	Jpred3	DSSP
0 (worst: -4)	16 (worst: 0)	9 (worst: 1)	0.0/11.0	Beta sheet	Coil	-	Turn

SNAP	SIFT	Polyphen2
Prediction: Neutral Reliability Index: 3 Expected Accuracy: 78%	Prediction: tolerated Score: 0.51 Sequence Conservation: 3.05	Prediction: probably damaging Score: 0.997 Sensitivity: 0.40 Specificity: 0.98

Mutation number 1 is the change from the aliphatic, unpolar and neutral amino acid Glycine to the aliphatic, polar and neutral amino acid Serine. The BLOSUM62, PAM1 und PAM250 indicate that this substitution is very common as its values are very high. Especially the PAM matrices with a score of 16 and 9 have high values.
Anyway the conservation is very high. The reason why it has a conservation of 0 for all found proteins is, because the alignment of the proteins has gaps there. But if you align only the best 25 found homologues there is total conservation and no other protein shows an amino acid exchange to Serine.
In Uniprot this position is assigned as part of a beta sheet, whereas the different tools predicted it as part of a coil or turn. As it is at the exterior of the protein as you can see at the visualization of figure 2, it should not affect the function of the protein that much.
SNAP and SIFT classify the mutation as neutral with an accuracy of 78% and tolerated with a score of 0.51 whereas Polyphen2 predicts it as probably damaging with a score of 0.997. As the mutation is listed in HGMD it is associated with Gaucher Disease and should therefore be damaging and affect the protein's function.
It may be not classified as damaging because the change from Glycine to Serine is common and the position of the mutation does not indicate a big influence.

Mutation 2: Asp - Asn (Pos. 63/24)

BLOSUM62	PAM1	PAM250	Conservation (all/best 25)	Secondary structure (Uniprot)	Psipred	Jpred3	DSSP
1 (worst: -4)	36 (worst: 0)	7 (worst: 1)	4.0/11.0	-	Coil	-	-

SNAP	SIFT	Polyphen2
Prediction: Non-neutral Reliability Index: 5 Expected Accuracy: 87%	Prediction: tolerated Score: 0.06 Sequence Conservation: 3.05	Prediction: probably damaging Score: 1.000 Sensitivity: 0.00 Specificity: 1.00

Mutation number 2 is the exchange from the aliphatic, polar and acidic Aspartic acid to the aliphatic, polar and neutral amino acid Aparagine. Therefore it loses its acidic chracter and therefore may change the structure of the protein.
The BLOSUM62, PAM1 and PAM250 show that the substitution has relatively high values, so it is very common. But if you look at the alignment the conservation is also very high. There is no protein found with a substitution to Asparagine.
If you look at the structure you can see that it is part of a coil at the exterior of the protein. This is also the result of our secondary structure predictions. So the mutation may not influence the function of the protein that much, because it is not in the functional part of the protein.
SNAP predicts the mutation as non-neutral with an accuracy of 87%. Polyphen2 also assigns the mutation as probably damaging with a score of even 1.0. Only SIFT predicts it as tolerated but only with a score of 0.06. So the mutation may be damaging.
As the mutation is listed in HGMD it affects the function of the protein. It is associated with Gaucher disease 1 and was published in 2005. <ref>Identification and functional characterization of five novel mutant alleles in 58 Italian patients with Gaucher disease type 1. Miocić S, Filocamo M, Dominissini S, Montalvo AL, Vlahovicek K, Deganuto M, Mazzotti R, Cariati R, Bembi B, Pittis MG. Hum Mutat. 2005</ref>

Mutation 3: His - Arg (Pos. 99/60)

BLOSUM62	PAM1	PAM250	Conservation (all/best 25)	Secondary structure (Uniprot)	Psipred	Jpred3	DSSP
0 (worst: -3)	10 (worst: 0)	6 (worst: 1)	3.0/8.0	-	Coil	-	-

SNAP	SIFT	Polyphen2
Prediction: Neutral Reliability Index: 5 Expected Accuracy: 89%	Prediction: tolerated Score: 0.62 Sequence Conservation: 3.04	Prediction: benign Score: 0.000 Sensitivity: 1.00 Specificity: 1.00

Mutation number 3 is the change from the aromatic, polar and slightly basic Histidine to the aliphatic, polar and very basic Arginine. As they are both basic and have the same charge the exchange may not influence the function of the protein that much.
The values in the different substitution matrices are again relatively high. So the change from Histidine to Arginine is common. The interesting part here is the alignment. Histidine is not highly conserved. If you look at that position there are also many sequences with an Arginine. So there are proteins of other mammals which were mutated during evolution in that way and this exchange seems not to influence the function of the protein.
Histidine is part of a coil in the secondary structure and is situated in the exterior of the protein. So it is not at the functional part of the protein and the influence may be low.
SNAP, SIFT and Polyphen2 predict the mutation as neutral, tolerated and benign. Although the scores are not that high it is exactly what we expected. On the one hand the mutation is not listed in HGMD and therefore not associated with Gaucher Disease and on the other hand the conservation and the alignment show that the mutation should be tolerated.

Mutation 4: Arg - Gln (Pos. 159/120)

BLOSUM62	PAM1	PAM250	Conservation (all/best 25)	Secondary structure (Uniprot)	Psipred	Jpred3	DSSP
1 (worst: -3)	9 (worst: 0)	5 (worst: 1)	0.0/11.0	Beta sheet	Beta sheet	Beta sheet	Bend

SNAP	SIFT	Polyphen2
Prediction: Non-neutral Reliability Index: 7 Expected Accuracy: 96%	Prediction: affect protein function Score: 0.03 Sequence Conservation: 3.01	Prediction: probably damaging Score: 1.000 Sensitivity: 0.00 Specificity: 1.00

Mutation number 4 is the change from the aliphatic, polar and very basic Arginine to the aliphatic, polar and neutral Glutamine. Concerning the substitution matrices the exchange is not rare. The values are relatively high, so it is common.
The alignment shows a very high conservation for the best 25 hits. All but two have also an Arginine at that position, the others show a substitution with Tryptophan but no Glutamine. The low conservation for all sequences is again because of gaps in the alignment.
This Arginine is part of a beta sheet and plays an important role in forming hydrogen bonds with the active site. So a mutation should affect the function of the protein. As the amino acid change is from basic to neutral it loses chemical properties that are necessary for the protein's function.
SNAP, SIFT and Polyphen2 predict the mutation as damaging. The accuracy of SNAP with 96% is very high and also the prediction of Polyphen2 has a score of 1.00. In HGMD it is associated with Gaucher Disease 1 which was already published in 1988. <ref>Gaucher disease type 1: cloning and characterization of a cDNA encoding acid beta-glucosidase from an Ashkenazi Jewish patient. Graves PN, Grabowski GA, Eisner R, Palese P, Smith FI. DNA. 1988 Oct;7(8):521-8.</ref>

Mutation 5: Pro - Leu (Pos. 221/182)

BLOSUM62	PAM1	PAM250	Conservation (all/best 25)	Secondary structure (Uniprot)	Psipred	Jpred3	DSSP

SNAP	SIFT	Polyphen2
Prediction: Non-neutral Reliability Index: 5 Expected Accuracy: 87%	Prediction: affect protein function Score: 0.00 Sequence Conservation: 3.01	Prediction: probably damaging Score: 1.000 Sensitivity: 0.00 Specificity: 1.00

Mutation number 5 is the change from the heterocyclic, unpolar and neutral Proline to aliphatic, unpolar and neutral Leucine. As Proline forms a ring structure the mutation may change the structure of the protein. Apart from that the polarity remains the same and so the chemical properties.
The substitution is not as common as the mutations mentioned before. BLOSUM62 has with -3 a very low value and also the PAM matrices show with the values 3 and 5 that the substitution is rare. So the mutation may not be tolerated. If you look at the alignment you can also see a high conservation. The found sequences share the Proline at this position.
The Proline is situated in the interior of the protein at a coil. As the interior is the functional part of the protein the mutation may not be tolerated. But it is not part of the active site.
SNAP, SIFT and Polyphen2 predict the mutation as non-neutral, "affect protein function" and probably damaging and that is what we expected. In HGMD it is also associated with Gaucher Disease 2. The mutation was published 2004. <ref>Functional analysis of 13 GBA mutant alleles identified in Gaucher disease patients: Pathogenic changes and "modifier" polymorphisms. Montfort M, Chabás A, Vilageliu L, Grinberg D. Hum Mutat. 2004 Jun;23(6):567-75.</ref>

Mutation 6: Ser - Asn (Pos. 310/271)

BLOSUM62	PAM1	PAM250	Conservation (all/best 25)	Secondary structure (Uniprot)	Psipred	Jpred3	DSSP
1 (worst: -3)	20 (worst: 0)	5 (worst: 1)	0.0/11.0	-	Coil	-	Turn

SNAP	SIFT	Polyphen2
Prediction: Neutral Reliability Index: 0 Expected Accuracy: 53%	Prediction: tolerated Score: 0.54 Sequence Conservation: 3.01	Prediction: benign Score: 0.100 Sensitivity: 0.94 Specificity: 0.85

Mutation number 6 is the change from the aliphatic, polar and neutral Serine to the aliphatic, polar and neutral Asparagine. They are both zwitterions and share chemical properties. The substitution matrices show relatively high values, so the substitution is common.
The conservation is very high. If you look at the best 25 hits they all have Serine at this position. If you look at more there are also some sequences with a Glycine at this position. But there is no change to Asparagine.
This position is part of a coil in the exterior of the protein and not near the active site. Therefore the mutation might not influence the function of the protein that much.
SNAP predicts the mutation as neutral with an expected accuracy of 53%. SIFT also predicts the mutation as tolerated with a score of 0.54 as well as Polyphen2 which predicts it as benign. But the mutation is associated with Gaucher Disease as published 1997.<ref>Identification and expression of acid beta-glucosidase mutations causing severe type 1 and neurologic type 2 Gaucher disease in non-Jewish patients. Grace ME, Desnick RJ, Pastores GM. J Clin Invest. 1997 May 15;99(10):2530-7</ref> So the prediction is wrong. The mutation is not tolerated and changes the function of the protein. Although the scores are not that high it was not predicted as damaging.

Mutation 7: Asn - Ser (Pos. 409/370)

BLOSUM62	PAM1	PAM250	Conservation (all/best 25)	Secondary structure (Uniprot)	Psipred	Jpred3	DSSP
1 (worst: -4)	34 (worst: 0)	8 (worst: 1)	0.0/11.0	Helix	Helix	Helix	Helix

SNAP	SIFT	Polyphen2
Prediction: Non-neutral Reliability Index: 1 Expected Accuracy: 63%	Prediction: affect protein function Score: 0.05 Sequence Conservation: 3.02	Prediction: possibly damaging Score: 0.573 Sensitivity: 0.88 Specificity: 0.91

Mutation number 7 is the substitution of the aliphatic, polar and neutral Asparagine to the also aliphatic, polar and neutral Serine. This is the most common mutation found in gaucher disease type 1 patients.
Concerning the substitution matrices the mutation is very common. PAM1 has with 34 a very high value as well as PAM250 with 8. Only BLOSUM62 does not show that tendency because it has only a value of 1.
The conservation in the alignment is very high. All sequences show an Asparagine at that position. So there are no mutations to Serine which would be accepted.
The mutation is situated at a helix in the interior of the protein. So it may affect its function and therefore it may not be tolerated.
SNAP predicts the mutation as non-neutral, SIFT as affecting the protein function and Polyphen2 as possibly damaging. It is interesting, that Polyphen2 predicts it as only possibly damaging and not probably damaging. We know that the mutation is associated with Gaucher Disease 1, which was first published in 1988<ref>Genetic heterogeneity in type 1 Gaucher disease: multiple genotypes in Ashkenazic and non-Ashkenazic individuals. Tsuji S, Martin BM, Barranger JA, Stubblefield BK, LaMarca ME, Ginns EI. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2349-52.</ref> and is supported by a lot of other sources given in HGMD.

Mutation 8: His - Arg (Pos. 350/311)

BLOSUM62	PAM1	PAM250	Conservation (all/best 25)	Secondary structure (Uniprot)	Psipred	Jpred3	DSSP
0 (worst: -3)	10 (worst: 0)	6 (worst: 1)	0.0/9.0	Beta sheet	Beta sheet	-	Bend

SNAP	SIFT	Polyphen2
Prediction: Non-neutral Reliability Index: 8 Expected Accuracy: 96%	Prediction: affect protein function Score: 0.00 Sequence Conservation: 3.11	Prediction: probably damaging Score: 1.0 Sensitivity: 0.00 Specificity: 1.00

Mutation number 8 is the substitution of the aromatic, polar and slightly basic Histidine to the aliphatic, polar and strong basic Arginine. It is situated in the interior of the protein and part of a beta sheet. So the mutation may cause and change in the protein's function.
Concerning BLOSUM62 the amino acid replacement is rare because it has only a value of 0. The values for PAM1 and PAM250 are higher with 10 and 6 which would indicate a common substitution. The conservation is relatively high, there are only two sequences with a substitution to Glutamic acid but no change to Arginine. Therefore the mutation may not be accepted.
SNAP predicts the mutation as non-neutral, SIFT as "affect protein function" and Polyphen2 as probably damaging. And the mutation is associated to Gaucher Disease 2 as published in 1999<ref>Is the perinatal lethal form of Gaucher disease more common than classic type 2 Gaucher disease? Stone DL, van Diggelen OP, de Klerk JB, Gaillard JL, Niermeijer MF, Willemsen R, Tayebi N, Sidransky E. Eur J Hum Genet. 1999 May-Jun;7(4):505-9.</ref> So the prediction is right.

Mutation 9: Met - Val (Pos. 455/416)

BLOSUM62	PAM1	PAM250	Conservation (all/best 25)	Secondary structure (Uniprot)	Psipred	Jpred3	DSSP
1 (worst: -3)	17 (worst: 0)	4 (worst: 1)	0.0/11.0	Helix	Coil/Helix	Helix	Helix

SNAP	SIFT	Polyphen2
Prediction: Non-neutral Reliability Index: 3 Expected Accuracy: 78%	Prediction: tolerated Score: 0.12 Sequence Conservation: 3.01	Prediction: probably damaging Score: 0.999 Sensitivity: 0.14 Specificity: 0.99

Mutation number 9 is the substitution of the aliphatic, unpolar and neutral Methionine to the aliphatic, unpolar and neutral Valine. The interesting thing is that Methionine contains a sulfur. Therefore it has other chemical properties than Valine. As it is also situated at a helix at an important part of the protein this may affect the protein's function.
The values in the substitution matrices of BLOSUM62 and PAM250 are relatively low so the substitution is rare. The conservation in the alignment is also very high and there is no other amino acid at this position.
SNAP and Polyphen2 predict the mutation as non-neutral and probably damaging. Only SIFT predicts it as tolerated. In HGMD it is associated with Gaucher Disease 2 which was published in 2005<ref>Novel mutations in type 2 Gaucher disease in Chinese and their functional characterization by heterologous expression. Tang NL, Zhang W, Grabowski GA, To KF, Choy FY, Ma SL, Shi HP. Hum Mutat. 2005 Jul;26(1):59-60.</ref> So the prediction of SIFT is wrong, SNAP and Polyphen2 predict what we expected.

Mutation 10: Leu - Pro (Pos. 509/470)

BLOSUM62	PAM1	PAM250	Conservation (all/best 25)	Secondary structure (Uniprot)	Psipred	Jpred3	DSSP

SNAP	SIFT	Polyphen2
Prediction: Non-neutral Reliability Index: 1 Expected Accuracy: 63%	Prediction: affect protein function Score: 0.01 Sequence Conservation: 3.09	Prediction: probably damaging Score: 0.978 Sensitivity: 0.75 Specificity: 0.96

Mutation number 10 is the change from the aliphatic, unpolar and neutral Leucine to the heterocyclic, unpolar and neutral Proline. Proline forms a ring structure so the change might also influence the protein folding. It is part of a beta sheet but at the exterior of the protein. So the change may not affect the function of the protein.
The values in the substitution matrices are very low, in BLOSUM62 as well as in the PAM matrices. So it is not a common substitution. The conservation is relatively high, although there are some substitutions to Valine and Phenylalanine.
SNAP, SIFT and Polyphen2 predict the mutation as non-neutral and probably damaging. But we do not know if it has such an influence on the protein's function. The mutation is only found in dbSNP and not in HGMD.