Sequence-based mutation analysis Gaucher Disease

The aim of this task was to carry out a thorough analysis of ten mutations and to classify them as disease-causing and non-disease causing. The mutations have been selected by another group from our [Researching SNPs Gaucher Disease|set of collected mutations] such that their impact had been unknown for us prior to this task. Technical details are reported in our protocol.

Mutations

<figtable id="tab:mutations">

Nr	Position	From	To
1	99	H	R
2	211	V	I
3	150	E	K
4	236	L	P
5	248	W	R
6	509	L	P
7	351	W	C
8	423	A	D
9	482	D	N
10	83	R	S

Randomly selected mutations from HGMD and dbSNP which were used for the sequence-based mutation analysis. </figtable>

Physicochemical properties

<figtable id="tab:props">

Nr	Wildtype					Mutant					Severe change
	AA	Charge	Polarity	Size	Aromatic	AA	Charge	Polarity	Size	Aromatic
1	H	negative	polar	large	no	R	negative	polar	large	no	no
2	V	neutral	unpolar	medium	no	I	neutral	unpolar	medium	no	no
3	E	positive	polar	large	no	K	negative	polar	large	no	yes
4	L	neutral	unpolar	medium	no	P	neutral	unpolar	medium	no	no
5	W	neutral	unpolar	large	yes	R	negative	polar	large	no	yes
6	L	neutral	unpolar	medium	no	P	neutral	unpolar	medium	no	no
7	W	neutral	unpolar	large	yes	C	neutral	polar	small	no	yes
8	A	neutral	unpolar	small	no	D	positive	polar	medium	no	yes
9	D	positive	polar	medium	no	N	neutral	polar	medium	no	no
10	R	negative	polar	large	no	S	neutral	polar	small	no	no

Physicochemical properiets of the wildtype and mutatant amino acid which were used to classify the mutation as severe or non-severe. </figtable>

Structural analysis

<figtable id="tab:structure">

Nr	Mutation	Accessibility	2nd structure	Domain region	Mutation
1	H99R	exposed	C	no
2	V211I	exposed	C	no
3	E150K	exposed	C	no
4	L236P	exposed	C	no
5	W248R	buried	H	yes
6	L509P	exposed	S	yes
7	W351C	exposed	S	yes
8	A423D	buried	C	yes
9	D482N	exposed	C	yes
10	R83S	exposed	C	yes

Location of mutations in 2nt0_A. Blue: wildtype; Red: mutant. </figtable>

<figure id="fig:structure_all">

2nt0_A along with the selected wildtype residues from <xr id="tab:mutations"/> in blue.

</figure>

Substitution scores

BLOSUM62 scores

The scores were taken from the BLOSUM62 matrix.

<figtable id="tab:subst_blosum">

Nr	Mutation	Score mutation	Score min	Score max
1	H99R	0	-3	8
2	V211I	3	-3	4
3	E150K	1	-4	5
4	L236P	-3	-4	4
5	W248R	-3	-4	11
6	L509P	-3	-4	4
7	W351C	-2	-4	11
8	A423D	-2	-3	4
9	D482N	1	-4	6
10	R83S	-1	-3	5

BLOSUM62 scores of the selected mutations </figtable>

PSSM of all hits

PSSM.

<figtable id="tab:subst_pssm_all">

Nr	Mutation	Score mutation	Score min	Score max	Conservation
1	H99R	0	-4	2
2	V211I	4	-4	4
3	E150K	0	-4	5
4	L236P	0	-3	1
5	W248R	-2	-3	4
6	L509P	-6	-6	4
7	W351C	-3	-6	9
8	A423D	-3	-3	3
9	D482N	4	-4	4
10	R83S	0	-3	2

Position specific substitution scores derived from all significant hits after 5 rounds PSI-BLAST. </figtable>

Scoring Mutants

SIFT

The predicted results from SIFT Blink is shown here:

Substitution at pos 83 from R to S is predicted to be TOLERATED with a score of 0.17.
   Median sequence conservation: 2.12
   Sequences represented at this position:77

Substitution at pos 99 from H to R is predicted to be TOLERATED with a score of 0.64.
   Median sequence conservation: 2.14
   Sequences represented at this position:80

Substitution at pos 150 from E to K is predicted to be TOLERATED with a score of 0.76.
   Median sequence conservation: 2.10
   Sequences represented at this position:86

Substitution at pos 211 from V to I is predicted to be TOLERATED with a score of 0.56.
   Median sequence conservation: 2.09
   Sequences represented at this position:86

Substitution at pos 236 from L to P is predicted to AFFECT PROTEIN FUNCTION with a score of 0.02.
   Median sequence conservation: 2.09
   Sequences represented at this position:86

Substitution at pos 248 from W to R is predicted to AFFECT PROTEIN FUNCTION with a score of 0.00.
   Median sequence conservation: 2.09
   Sequences represented at this position:86

Substitution at pos 351 from W to C is predicted to AFFECT PROTEIN FUNCTION with a score of 0.00.
   Median sequence conservation: 2.10
   Sequences represented at this position:87

Substitution at pos 423 from A to D is predicted to AFFECT PROTEIN FUNCTION with a score of 0.01.
   Median sequence conservation: 2.10
   Sequences represented at this position:85

Substitution at pos 482 from D to N is predicted to be TOLERATED with a score of 0.69.
   Median sequence conservation: 2.18
   Sequences represented at this position:66

Substitution at pos 509 from L to P is predicted to AFFECT PROTEIN FUNCTION with a score of 0.00.
   Median sequence conservation: 2.10
   Sequences represented at this position:79