Task 8 (MSUD)

Results

Lab journal

Visualization of mutations

• Original residues and mutations in BCKDHA (1U5B chain A)
• 

  Original residues of BCKDHA are marked in red.

• 

  Mutant residues are marked in orange.

SIFT

The following table shows the prediction of mutation effects by SIFT. 6 out of 10 mutations are predicted to affect protein function.

mutation	prediction	score	median sequence conservation	sequences represented at position
P38H	tolerated	0.16	3.16	17
M82L	tolerated	0.56	3.03	43
T151M	tolerated	0.05	3.03	46
A222T	affect protein function	0.04	3.03	46
C264W	affect protein function	0.00	3.03	46
R265W	affect protein function	0.00	3.03	46
R314Q	affect protein function	0.01	3.03	46
R346H	affect protein function	0.00	3.03	46
I361V	tolerated	0.12	3.03	46
Y413H	affect protein function	0.00	3.03	46

Polyphen2

The prediction resuls of Polyphen2 are shown in the following table. 7 out of 10 mutations are predicted to be probably damaging (at least for the trained set HumDiv). For the mutation T151M, this does not agree with the results of SIFT. But for this mutations the Polyphen scores are also the lowest of all mutations that are predicted to be damaging. So Polyphen sees an uncertainty for this mutation too, if it is damaging.

	HumDiv				HumVar
mutation	prediction	score	sensitivity	specificity	prediction	score	sensitivity	specificity
P38H	benign	0.118	0.93	0.96	benign	0.042	0.93	0.62
M82L	benign	0.002	0.99	0.30	benign	0.006	0.97	0.45
T151M	probably damaging	0.963	0.78	0.95	possibly damaging	0.622	0.80	0.85
A222T	probably damaging	1.000	0.00	1.00	probably damaging	0.943	0.65	0.91
C264W	probably damaging	0.989	0.72	0.97	possibly damaging	0.887	0.71	0.89
R265W	probably damaging	1.000	0.00	1.00	probably damaging	1.000	0.00	1.00
R314Q	probably damaging	1.000	0.00	1.00	probably damaging	0.998	0.18	0.98
R346H	probably damaging	1.000	0.00	1.00	probably damaging	0.993	0.47	0.96
I361V	benign	0.386	0.90	0.89	possibly damaging	0.610	0.80	0.83
Y413H	probably damaging	1.000	0.00	1.0	probably damaging	0.999	0.09	0.99

MutationTaster

Except the mutation P38H, all the other 9 selected mutations are predicted by MutationTaster to be disease causing.

mutation	prediction	mutation score	prediction probability
P38H	polymorphism	2.1	0.99
M82L	disease causing	0.41	0.99
T151M	disease causing	2.21	0.93
A222T	disease causing	1.58	0.99
C264W	disease causing	5.86	0.99
R265W	disease causing	2.75	0.99
R314Q	disease causing	1.17	0.99
I361V	disease causing	0.79	0.99
R346H	disease causing	0.79	0.99
Y413H	disease causing	2.26	0.99

SNAP

Mutation	Pathogenic	RI	Probability
P38H	Neutral	4	72%
M82L	Neutral	5	78%
T151M	Non-neutral	7	85%
A222T	Non-neutral	6	80%
C264W	Non-neutral	8	91%
R265W	Non-neutral	7	85%
R314Q	Non-neutral	6	80%
R346H	Non-neutral	4	71%
I361V	Neutral	6	82%
Y413H	Non-neutral	8	91%

Summary

Following are the selected mutations from HGMD and dbSNP. Changes in physicochemical properties, secondary structures, amino acid substitution scores, conservation and prediction results of different programs are also shown in the table:

mutation	physicochemical changes	secondary structure	BLOSUM62 score	PAM250 score	PSSM conservation WT	PSSM conservation mutant	MSA conservation WT	MSA conservation mutant	SIFT	Polyphen2	MutationTaster	SNAP	DB
P38H	small/fixed conformation/apolar to large/aromatic/polar	-	-2	0	0.31	0	0.29	0	tolerated	benign	polymorphism	neutral	dbSNP
M82L	sulfur-containing to aliphatic/branched	E	2	4	0.12	0.59	0.36	0.01	tolerated	benign	disease causing	neutral	dbSNP
T151M	small/polar to apolar/sulfur-containing	-	-1	-1	0.08	0	0.8	0	tolerated	probably/possibly damaging	disease causing	non-neutral	HGMD
A222T	apolar to polar	H	0	1	0.64	0.04	0.38	0	affect protein function	probably damaging	disease causing	non-neutral	dbSNP
C264W	small/sulfur-containing to large/aromatic	E	-2	-8	0.52	0	0.91	0	affect protein function	probably/possibly damaging	disease causing	non-neutral	HGMD
R265W	positively-charged to aromatic/larger	E	-3	2	0.12	0	0.36	0.38	affect protein function	probably damaging	disease causing	non-neutral	HGMD
R314Q	guanidin/large/positively-charged to amin	H	1	1	0.23	0	0.35	0	affect protein function	probably damaging	disease causing	non-neutral	dbSNP
R346H	hydrophilic to hydrophobic/aromatic	-	0	2	0.92	0	0.90	0	affect protein function	probably damaging	disease causing	non-neutral	HGMD
I361V	smaller	H	3	4	0.64	0.10	0.89	0.01	tolerated	benign/possibly damaging	disease causing	neutral	dbSNP
Y413H	uncharged to positively-charged	-	2	0	0.74	0.01	0.90	0	affect protein function	probably damaging	disease causing	non-neutral	HGMD

Discussion

In six mutations (A222T, C264W, R265W, R314Q, R346H and Y413H) the prediction programs agree, that they could affect the protein function or be disease causing, respectively. Among these, there are the lowest BLOSUM62 and PAM250 scores but also higher scores. If the results of evolutionary conservation analysis by using a PSSM or a MSA are taken together, these mutations show a much higher conservation for the WT amino acid than for the mutant. They also show obvious changes in their physicochemical properties like change in charge, hydrophobicity or size. It is interesting that two of them (A222T and R314Q) are not included in HGMD, so it might be that they are disease causing but it is not known yet, or they are tolerated although there are these hints that they might change the protein function. The fact that the two mutations also lie in secondary structure elements argues for the first.

Only for the mutation P38H the programs agree that it has no effect. This is surprising because it has bad scores in the scoring matrices and also shows no conservation in the mutant amino acid. But because it lies near the end of the protein, the residue seems to have no crucial function, why it can be exchanged without effect. This view is also consistant with the mutation not beeing listed in HGMD.

For the remaining three muations (M82L, T151M and I361V), the programs come to different results, if it changes the protein function or not. For M82L and I361V it is unlikely, that they cause the disease, because they do not present high changes in the properties of the amino acids. Also the BLOSUM and PAM scores are comparatively high and the mutant amino acid is often observed in the conservation analysis - at least for the PSSM, which was derived from all homologs, but also for the MSA with mammalian homologs the mutant was still observed. But M82L and I361V also lie in secondary structure elements, so we cannot say definitely, that they are not disease causing. For T151M, the change of polarity, low BLOSUM and PAM scores and no conservation for the mutant amino acid argues for this mutation being disease causing - it actually is included in HGMD.

So in summary, most of the mutations that are known to cause MSUD and are consequently listed in HGMD could be identified with the predictions. For some mutations (e. g. A222T and R314Q) there are hints that they might affect the protein function, but at the moment it has not been published that they are disease causing. There is always an uncertainty, because if it has not been shown that a mutation is disease causing, does not mean that it is not disease causing. But the analyses have pointed out, that a mutation that introduces great changes in the physicochemical properties, has a high conservation for the WT and no conservation for the mutant amino acid and also lies in secondary structure elements, will likely change the function of the protein and therefore might lead to a disease.