Sequence-Based Mutation Analysis Hemochromatosis

Hemochromatosis>>Task 6: Sequence-based mutation analysis

Short task description

Detailed description: Sequence-based mutation analysis

Protocol

A protocol with a description of the data acquisition and other scripts used for this task is available here.

TODO: Fill in references!!

SNPs

From MSUD: M35T V53M G93R Q127H A162S L183P T217I R224W E277K C282S

Amino acid features

As a first estimation of the SNPs' effects we looked at the physicochemical properties of the wildtype and mutated amino acids (see <xr id="aa_features"/>). We collected the hydropathy index, the polarity, the isoelectric point (charge), and the van der Waals volume of the corresponding amino acids.

The most striking differences can be spotted in the following mutations:

• G93R which goes from neutral hydrophobicity to highly hydrophilic, from uncharged to positive, and triples in size.
• L183P has a change of 5.4 in hydrophobicity (from hydrophobic to neutral).
• T217I, the opposite of L183P, which goes from neutral (-0.7) to hydrophobic (4.5).
• R224W becomes neutral (former hydrophilic) and also loses its positive charge.
• E277K changes from acidic (negative charge) to basic (positive charge).

Based on this information alone, these SNPs could be considered disease causing.

<figtable id="aa_features">

Table 1: Comparison of the physicochemical properties between the wildtype (wt) and mutant (mt) amino acids. From left to right: the hydropathy index, the polarity, the isoelectric point (pI), and the van der Waals volume.

Mutation	Hydrophobicity (wt)	Hydrophobicity (mt)	Polarity (wt)	Polarity (mt)	pI (wt)	pI (mt)	v.d.W. volume (wt)	v.d.W. volume (mt)
M35T	1.9	-0.7	nonpolar	polar	5.74	5.60	124	93
V53M	4.2	1.9	nonpolar	nonpolar	6.00	5.74	105	124
G93R	-0.4	-4.5	nonpolar	polar	6.06	10.76	48	148
Q127H	-3.5	-3.2	polar	polar	5.65	7.60	114	118
A162S	1.8	-0.8	nonpolar	polar	6.01	5.68	67	73
L183P	3.8	-1.6	nonpolar	nonpolar	6.01	6.30	124	90
T217I	-0.7	4.5	polar	nonpolar	5.60	6.05	93	124
R224W	-4.5	-0.9	polar	nonpolar	10.76	5.89	148	163
E277K	-3.5	-3.9	polar	polar	3.15	9.60	109	135
C282S	2.5	-0.8	polar	polar	5.05	5.68	86	73

</figtable>

Evolutionary analysis

The next step was to look at the evolutionary constraints for each mutation. Starting with simple statistics such as BLOSUM62, PAM1, and PAM250, and then moving to more sophisticated methods such as PSI-BLAST's PSSM and multiple sequence alignments.

BLOSUM62/PAM1/PAM250

When looking at all three matrices (cf. <xr id="bpp_matrices"/>) G93R, L183P, and R224W stand out as the most unlikely mutations of all 10 SNPs. M35T and T217I, while still quite rare, don't show such a strong signal. V53M and C282S are special cases. While BLOSUM62 ranks V53M as a more or less common mutation, PAM1/250 classify it as very rare. For C282S its the other way around.

<figtable id="bpp_matrices">

Table 2: Summary of the BLOSUM62, PAM1, and PAM250 scores for the different mutations. For a better readability the values of the PAM1 and PAM250 matrices are multiplied by 10000.

Mutation	BLOSUM62	PAM1	PAM250
M35T	-1	6	500
V53M	1	4	200
G93R	-2	0	200
Q127H	0	20	700
A162S	1	28	900
L183P	-3	2	300
T217I	-1	7	400
R224W	-3	2	200
E277K	1	7	800
C282S	-1	11	700

</figtable>

PSSM

We calculated the PSSM-Matrix of our sequence with 5 iterations against the big-database TODO:REF.

The important values can be found in <xr id="pssm_matrix_important_positions"/>

<figtable id="pssm_matrix_important_positions">

Table 3: The mutation depending position scores extracted from the PSSM Matrix.

Mutation	wt		mt
	PSSM-value	frequency	PSSM-value	frequency
M35T	3	16%	5	78%
V53M	5	99%	1	1%
G93R	3	29%	-2	1%
Q127H	2	16%	-2	0%
A162S	5	100%	1	0%
L183P	4	95%	-3	0%
T217I	2	16%	-2	0%
R224W	6	100%	-3	0%
E277K	6	100%	0	0%
C282S	10	100%	-1	0%

</figtable>

These values lead to our following conclusions:

M35T would not be predicted as a disease causing mutation as the mutant is occuring frequently.

V53M would most probably be predicted as a disease causing mutation, because the mutant type occurs more often than expected. Another evidence is the high wildtype conservation at this position. The only thing that does not fit the prediction is, that the mutation is seen more often than expected, which could be a sign of a non-disease-causing mutation.

G93R is hard to predict based on the given numbers, as the wildtype is not very conserved with 29%, but because the mutation gets a value of -2 (meaning the occurrence of this mutation is fewer than expected) this position is more likely to be disease causing. In total, 7 different amino acids were observed at this position (A, R, D, E, G, T, V).

Q127H, predicted by only the conservation of wild type and mutation would be quite difficult. The conclusion would be that (like for G93R) the position is disease causing because the mutant type occurrence is lower than expected. Another fact supporting this prediction is, that only 3 different amino acids are observed at position 127 (Q, E, G), which might be an indicator of the importance of this position for the protein.

A162S would be predicted as a disease causing mutation, based on the 100% conservation of the wild type.

L183P would be predicted as a disease causing mutation, based on the wildtype conservation (95%) and the low frequency of occurence (lower than expected) of the mutated position).

T217I is another difficult prediction case when only looking at wildtype and mutation type conservation. Probably it would be predicted as disease causing because of the lower-than-expected frequency of the mutant type. Another supporting fact for this prediction is, that only two amino acids were observed at that position, meaning the sequence is fairly conserved.

R224W would be predicted as a disease causing mutation because of the high wildtype conservation (100%), supported by the lower than expected frequency of the mutant type.

E277K would be predicted as a disease causing mutation because of the high wildtype conservation (100%).

C282S would be predicted as a disease causing mutation because of the high wildtype conservation (100%), supported by the lower than expected frequency of the mutant type.

MSA conservation

The following <xr id="msa_table"/> has been retrieved through the MSAs of the HFE-sequence and homologs (found via PSIBlast). The MSAs can be seen here (Muscle MSA) and here (ClustalW MSA).

<figtable id="msa_table">

Table 4: Table showing the conservation values, consensus and consensus percentage of different MSAs. The used MSAs were from Muscle and ClustalW and inherited the HFE sequence as well as found homologs (found via PSIBlast).

Mutation	Muscle			Clustal
	conservation score	consensus	consensus percentage	conservation score	consensus	consensus percentage
M35T	10	T	98%	10	T	98%
V53M	10	V	99%	10	V	99%
G93R	5	A	58%	5	A	58%
Q127H	10	V	99%	10	V	99%
A162S	11	A	100%	11	A	100%
L183P	9	L	92%	9	L	92%
T217I	10	S	99%	10	S	99%
R224W	11	R	100%	11	R	100%
E277K	10	E	99%	10	E	99%
C282S	11	C	100%	11	C	100%

</figtable>

The predictions based on these informations would be:

M35T: not disease causing (as the consensus in 98% of the positions T occurs)

V53M: disease causing

Q127H: disease causing

A162S: disease causing

L183P: disease causing

T217I: disease causing

R224W: disease causing

E277K: disease causing

C282S: disease causing

M35T

• Secondary structure assignment: sheet

SEQ (mt):     LRSHSLHYLFTGASEQDLGLS
DSSP (wt):     CCEEEEEEEEEEECCCCCCE
PsiPred (wt): CCCCCCCEEEEEEECCCCCCC
PsiPred (mt): CCCCCCCEEEEEEECCCCCCC

<figtable id="M35T_pymol">

Table TODO: ...

	Wildtype.	Mutant.

</figtable>

M35T is located in the "sheet complex" of the MHC I domain. According to PyMol it forms two hydrogen bonds with the neighboring sheet (cf. <xr id="M35T_pymol"/>) which should stabilize the formation. These bonds are still present in the mutated protein. The right figure also shows that this substitution causes almost no clashes (shown as green blocks). This suggests that M35T is a not a disease causing mutation.

V53M

• Secondary structure assignment: sheet

SEQ (mt):     GLSLFEALGYMDDQLFVFYDH
DSSP (wt):    CCECCEEEEEECCEEEEEEEC
PsiPred (wt): CCCEEEEEEEECCEEEEEEEC
PsiPred (mt): CCCEEEEEEEECCEEEEEEEC

<figtable id="V53M_pymol">

Table TODO: ...

	Wildtype.	Mutant.

</figtable>

V53M seems to also have a stabilizing function (cf. hydrogen bonds shown in <xr id="V53M_pymol"/>) in the MHC I domain's sheet complex. Although this stabilization is retained by the mutant, the size of the methionine causes several clashes with one of the helices in the same domain. This means that the whole domain needs to undergo some structural corrections to fit in the mutation which might cause a decrease or loss in function.

G93R

• Secondary structure assignment: helix

SEQ (mt):     MWLQLSQSLKRWDHMFTVDFW
DSSP (wt):    HHHHHHHHHHHHHHHHHHHHH
PsiPred (wt): HHHHHHHHHHHHHHHHHHHHH
PsiPred (mt): HHHHHHHHHHHHHHHHHHHHH

<figtable id="G93R_pymol">

Table TODO: ...

	Wildtype.	Mutant.

</figtable>

G93R is within the first big helix of HFE's MHC I domain. As a helical residue the hydrogen bonds are very important for stability. As shown in <xr id="G93R_pymol"/> the new residue not only retains these bonds, it also forms additional ones. There are also no clashes with other residues which is very surprising when considering the triplication in size (cf. <xr id="aa_features"/>). On the other hand it must also be considered that this residue is located on the outside of the protein. The new bulk might as well prevent proper complex formation with other proteins or the new hydrogen bonds could make the protein too stiff.

Q127H

• Secondary structure assignment: coil

SEQ (mt):     TLQVILGCEMHEDNSTEGYWK
DSSP (wt):    EEEEEEEEEECCCCCEEEEEE
PsiPred (wt): EEEEECCCCCCCCCCCCCEEE
PsiPred (mt): CEEEECCCEECCCCCCCCCCE

<figtable id="Q127H_pymol">

Table TODO: ...

	Wildtype.	Mutant.

</figtable>

Q127H is again within the sheet complex of the MHC I domain. It's just behind a sheet and seems to help in forming and stabilizing the loop to the next (neighboring) sheet (see <xr id="Q127H_pymol"/>). The substitution of the glutamine with a histidine causes the loss of one of the hydrogen bonds as well as some clashes with other residues. As the loss of the stabilizing bond might have severe effects on HFE's tertiary structure this mutation could be considered disease causing.

A162S

• Secondary structure assignment: helix

SEQ (mt):     TLDWRAAEPRSWPTKLEWERH
DSSP (wt):    HCEEEECCHHHHHHHHHHHCC
PsiPred (wt): CCCEECCCCCHHHHHHHHHHH
PsiPred (mt): CCCEECCCCCHHHHHHHHHHH

<figtable id="A162S_pymol">

Table TODO: ...

	Wildtype.	Mutant.

</figtable>

A162S is at the beginning of a helix (within MHC I domain). The mutation causes some clashes as well as the formation of several new hydrogen bonds (cf. <xr id="A162S_pymol"/>). Unlike the previous mutations this one is buried within the protein which means that the increase in size does not directly affect the protein surface and therefore is unlikely to directly affect complex formation (although the additional stability through the new hydrogen bonds might). Overall this mutation seems unlikely to be disease causing.

L183P

• Secondary structure assignment: helix (trusting DSSP)

SEQ (mt):     KIRARQNRAYPERDCPAQLQQ
DSSP (wt):    CHHHHHHHHHHHHHHHHHHHH
PsiPred (wt): HHHHHHHHCCCCCCHHHHHHH
PsiPred (mt): HHHHHHHHCCCCCCHHHHHHH

<figtable id="L183P_pymol">

Table TODO: ...

	Wildtype.	Mutant.

</figtable>

T217I

• Secondary structure assignment: coil

SEQ (mt):     PPLVKVTHHVISSVTTLRCRA
DSSP (wt):    CCEEEEEEEECCCCEEEEEEE
PsiPred (wt): CCCEEEECCCCCCCCEEEEEE
PsiPred (mt): CCCEEEECCCCCCCCEEEEEE

<figtable id="T217I_pymol">

Table TODO: ...

	Wildtype.	Mutant.

</figtable>

R224W

• Secondary structure assignment: sheet

SEQ (mt):     HHVTSSVTTLWCRALNYYPQN
DSSP (wt):    EEECCCCEEEEEEEEEEECCC
PsiPred (wt): CCCCCCCCEEEEEECCCCCCC
PsiPred (mt): CCCCCCCCEEEEEECCCCCCC

<figtable id="R224W_pymol">

Table TODO: ...

	Wildtype.	Mutant.

</figtable>

E277K

• Secondary structure assignment: helix (trusting DSSP)

SEQ (mt):     WITLAVPPGEKQRYTCQVEHP
DSSP (wt):    EEEEEECCCHHHHEEEEEECC
PsiPred (wt): EEEEEECCCCCCCEEEEEECC
PsiPred (mt): EEEEEECCCCCCCEEEEEECC

<figtable id="E277K_pymol">

Table TODO: ...

	Wildtype.	Mutant.

</figtable>

C282S

• Secondary structure assignment: sheet

SEQ (mt):     VPPGEEQRYTSQVEHPGLDQP
DSSP (wt):    ECCCHHHHEEEEEECCCCCCC
PsiPred (wt): ECCCCCCCEEEEEECCCCCCC
PsiPred (mt): ECCCCCCCCEEEEECCCCCCC

<figtable id="C282S_pymol">

Table TODO: ...

	Wildtype.	Mutant.

</figtable>

Predictions

SIFT

<figtable id="sift_results">

Table 5: The predictions and scores of each mutation (calculated by SIFT).

Mutation	Score	Prediction
M35T	1.00	TOLERATED
V53M	0.00	AFFECT PROTEIN FUNCTION
G93R	0.26	TOLERATED
Q127H	0.01	AFFECT PROTEIN FUNCTION
A162S	0.02	AFFECT PROTEIN FUNCTION
L183P	0.00	AFFECT PROTEIN FUNCTION
T217I	0.92	TOLERATED
R224W	0.00	AFFECT PROTEIN FUNCTION
E277K	0.04	AFFECT PROTEIN FUNCTION
C282S	0.00	AFFECT PROTEIN FUNCTION

</figtable>

SNAP

<figtable id="snap_results">

Table 6: The predictions and scores of each mutation (calculated by SNAP).

Mutation	Prediction	Reliability	Expected Accuracy
M35T	Neutral	1	53%
V53M	Neutral	6	49%
G93R	Neutral	0	51%
Q127H	Neutral	7	85%
A162S	Neutral	8	91%
L183P	Non-neutral	1	60%
T217I	Non-neutral	1	60%
R224W	Non-neutral	6	77%
E277K	Neutral	1	53%
C282S	Non-neutral	2	63%

</figtable>

PolyPhen2

<figtable id="polyphen2_results">

Table 7: The prediction of each mutation (calculated by PolyPhen2).

Mutation	Prediction
M35T	possibly damaging
V53M	possibly damaging
G93R	possibly damaging
Q127H	benign
A162S	possibly damaging
L183P	possibly damaging
T217I	benign
R224W	possibly damaging
E277K	possibly damaging
C282S	possibly damaging

</figtable>

References

<references/>