Fabry:Sequence-based mutation analysis

Fabry Disease » Sequence-based mutation analysis

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The following analyses were performed on the basis of the α-Galactosidase A sequence. Please consult the journal for the commands used to generate the results.

Dataset preparation

Q279E
N215S
I289V
S65T
R356W
V316I
P323T
P40S
R118H
A143T

Amino acid properties

<figtable id="tab:aaProp"> Physicochemical properties of the chosen SNPs and changes of properties between wildtype (wt) and mutant (mt). Used abbreveations in this table:
AA: Amino Acid, Pol: Side-chain polarity, Charge: Side-chain charge at pH 7.4, HI: Hydropathy index, RM: Residue Mass, iP: isoelectric point

SNP	wt AA	wt Pol	wt Charge	wt HI	wt RM	wt iP	mt AA	mt Pol	mt Charge	mt HI	mt RM	mt iP	change in Pol	change in Charge	change in HI	change in RM	change in iP
Q279E	Q	polar	neutral	−3.5	128.131	5.65	E	polar	negative	−3.5	129.116	3.15	none	neutral to negative	0	0.985	-2.5
N215S	N	polar	neutral	−3.5	114.104	5.41	S	polar	neutral	−0.8	87.078	5.68	none	none	−2.7	-27.026	0.27
I289V	I	nonpolar	neutral	4.5	113.160	6.05	V	nonpolar	neutral	4.2	99.133	6.00	none	none	-0.3	-14.027	-0.045
S65T	S	polar	neutral	−0.8	87.078	5.68	T	polar	neutral	−0.7	101.105	5.60	none	none	0.1	14.027	-0.08
R356W	R	polar	positive	−4.5	156.188	10.76	W	nonpolar	neutral	−0.9	186.213	5.89	polar to nonpolar	positive to neutral	3.6	30.025	-4.87
V316I	V	nonpolar	neutral	4.2	99.133	6.00	I	nonpolar	neutral	4.5	113.160	6.05	none	none	0.3	14.027	0.05
P323T	P	nonpolar	neutral	−1.6	97.117	6.30	T	polar	neutral	−0.7	101.105	5.60	nonpolar to polar	none	0.9	3.988	-0.7
P40S	P	nonpolar	neutral	−1.6	97.117	6.30	S	polar	neutral	−0.8	87.078	5.68	nonpolar to polar	none	0.8	-10.039	-0.62
R118H	R	polar	positive	−4.5	156.188	10.76	H	polar	pos(10%), neutr(90%)	−3.2	137.142	7.60	none	positive to pos(10%), neutr(90%)	1,3	-19.046	-3.16
A143T	A	nonpolar	neutral	1.8	71.079	6.01	T	polar	neutral	−0.7	101.105	5.60	nonpolar to polar	none	−2.5	30.026	-0.41

</figtable>

Simple structural analysis

• Now take into consideration where in the protein the mutation occurs and document: Create a picture with PyMOL showing the original and mutated residue in the protein. Use PyMOL for this. More thorough structural analyses will be introduced in the next task.

Location

<figtable id="tab:Location"> Physicochemical properties of the chosen SNPs and changes of properties between wildtype (wt) and mutant (mt)

SNP	SecStruc Psipred	SecStruc Psipred long	SecStruc Reprof	SecStruc Reprof long	SecStruc DSSP	SecStruc DSSP long
Q279E	H	CCCCCCCHHHHHHHHHHHHHH	H	EECCCCCCHHHHHHHHHHHHH	H	CCCCC--HHHHHHHHHHHHHC
N215S	H	CCCCCCCCCCHHHHHCCCCCC	H	CECCCCCCCCHHHHHHHHHHH	H	HHHCCCC---HHHHCCC-CEE
I289V	H	HHHHHHHHHHHCCCEEEECCC	H	HHHHHHHHHHHHHCHHCCCCC	C	HHHHHHHHHHCC--EEE-C-C
S65T	H	CCCCCCCCCCHHHHHHHHHHH	H	CCCCCCCHHHHHHHHHHHHHH	H	CCC-CCCC-CHHHHHHHHHHH
R356W	C	CCEEEEEEECCCCCCEEEEEE	C	HHHHHHHHHHCCCCCCCCHHH	-	CEEEEEEEE---CCC-EEEEE
V316I	H	HHHCCCCHHHHHHCCCCCCCC	E	HHHHCCCCCEEEECCCCCCCC	H	HHHHHH-HHHHHHHC-CC---
P323T	C	HHHHHHCCCCCCCCCEEEEEC	C	CCEEEECCCCCCCCCCEECCC	C	HHHHHHHC-CC----EEEE-C
P40S	C	CCCCCCCCCCCCCCCCCCCCC	C	HHCCCCCCCCCCCHHHHHHEE	-	---CC--CC--EEEECHHHHC
R118H	H	CCCCCCCCHHHHHHHHHHCCC	H	CCCCCCHHHHHHHHHHHCCCC	H	-CCC-CCHHHHHHHHHHHCC-
A143T	C	EECCCCCCCCCCCCCCCCHHH	C	EEECCCCCCCCCCCCCCCCCC	C	EEECCCE-CCCCE--CCCHHH

</figtable>

Substitution matrices

<figtable id="tab:Subsmatr"> Substitution values for all SNPs, both substitution matrices

SNP	Value BLOSUM62	Value PAM250
Q279E	2	2
N215S	1	1
I289V	3	4
S65T	1	1
R356W	-3	2
V316I	3	4
P323T	-1	0
P40S	-1	1
R118H	0	2
A143T	0	1

</figtable>

PSSM

• Getting a bit closer to evolution you will have to create a PSSM (position specific scoring matrix) for your protein sequence using PSI-BLAST (5 iterations). How conserved are the WT residues in your mutant positions? How is the frequency of occurrence (conservation) for the mutant residue type? Anything interesting?

Multiple sequence alignment

• And another step close to evolution: Identify all mammalian homologous sequences. Create a multiple sequence alignment for them with a method of your choice. Using this you can now calculate conservation for WT and mutant residues again. Compare this to the matrix- and PSSM-derived results.

Scoring methods

SIFT

<figtable id="tab:Sift"> Sift Scores

SNP	Prediction	Sift Score	Sequences represented at this position
P40S	AFFECT PROTEIN FUNCTION	0.00	41
S65T	AFFECT PROTEIN FUNCTION	0.01	45
R118H	be TOLERATED	0.06	48
A143T	AFFECT PROTEIN FUNCTION	0.01	48
N215S	AFFECT PROTEIN FUNCTION	0.01	48
Q279E	AFFECT PROTEIN FUNCTION	0.00	48
I289V	AFFECT PROTEIN FUNCTION	0.05	48
V316I	be TOLERATED	0.75	48
P323T	AFFECT PROTEIN FUNCTION	0.01	48
R356W	AFFECT PROTEIN FUNCTION	0.01	47

</figtable>

Median sequence conservation: 2.99

SIFT

Polyphen2

<figtable id="tab:Polyphen"> Polyphen Scores

SNP	rs ID	Sec Struc	Prediction	pph2 Class	pph2 Prob	pph2 FPR	pph2 TPR	pph2 FDR
Q279E	rs28935485	H	probably damaging	deleterious	0.983	0.0387	0.745	0.0657
N215S	rs28935197	.	benign	neutral	0.048	0.167	0.941	0.194
I289V	?	H	probably damaging	deleterious	0.975	0.0436	0.762	0.072
S65T	?	.	probably damaging	deleterious	0.995	0.0277	0.681	0.0521
R356W	?	.	probably damaging	deleterious	1	0.00026	0.00018	0.0109
V316I	?	H	benign	neutral	0.308	0.113	0.904	0.144
P323T	?	T	possibly damaging	deleterious	0.612	0.091	0.872	0.124
P40S	?	.	probably damaging	deleterious	1	0.00026	0.00018	0.0109
R118H	?	H	benign	neutral	0.015	0.209	0.956	0.229
A143T	?	T	probably damaging	deleterious	1	0.00026	0.00018	0.0109

</figtable>

Polyphen2

SNAP

• • SNAP is installed on the VirtualBox and should be used command-line only. -- As blast is the bottleneck of SNAP, and you are doing that anyway, we might as well look at all possible substitutions in the position of our mutations. This way we can learn much more about the nature of the given mutation: Is our mutation problematic because we introduce an unwanted effect, or because the WT residue is essential and by mutating we remove that?

Results and Conclusion

• Compare ALL results and create an overview table.
• Try to come up with a consensus between all the findings requested above.
• Check whether you are right in the HGMD – were you able to predict a change?