Lab Journal - Task 8 (PAH)

Mutation dataset

For the generation of the mutation dataset the following five SNPs from the HGMD database were used (25th June 2013): <figtable id="mutds_hgmd">

Missense mutations (SNPs) from HGMD
Accession Number	Codon change	Sequence position	Amino acid change	Codon number	Disease	Reference
CM000542	CAG⇒CTG	59	Gln(Q)-Leu(L)	20	Hyperphenylalaninaemia	Hennermann (2000) Hum Mutat 15, 254
CM045080	GGT⇒AGT	307	Gly(G)-Ser(S)	103	Phenylketonuria	Lee (2004) J Hum Genet 49, 617
CM910286	GCC⇒GTC	776	Ala(A)-Val(V)	259	Phenylketonuria	Labrune (1991) Am J Hum Genet 48, 1115
CM010981	AAG⇒ACG	1022	Lys(K)-Thr(T)	341	Phenylketonuria	Tyfield (1997) Am J Hum Genet 60, 388
CM090791	CCA⇒CAA	1247	Pro(P)-Gln(Q)	416	Hyperphenylalaninaemia	Dobrowolski (2009) J Inherit Metab Dis 32, 10

Detailed information about the five SNPs taken of HGMD. The first column represents the SNP ID and the second the change of the nucleotide which leads to a codon change. This change happens on the position found in column three, whereas four and five tell about the amino acids that are exchanged and which position this is in the amino acid sequence. The last column shows with wich disease the SNP is associated.

</figtable>

Furthermore, the following five mutations from dbSNP were added (25th June 2013): <figtable id="mutds_dbSNP">

Missense mutations (SNPs) from dbSNP
Reference SNP	Codon change	Sequence position	Amino acid change	Codon number	Disease
rs199475681	AGA⇒ATA	368	Arg(R)-Ile(I)	123	?
rs192592111	CAG⇒CAT	516	Gln(Q)-His(H)	172	?
rs62508752	ACA⇒CCA	796	Thr(T)-Ala(A)	266	Phenylketonuria
rs199475695	TTT⇒TCT	1175	Phe(F)-Ser(S)	392	?
rs199475696	ATT⇒ACT	1262	Ile(I)-Thr(T)	421	?

Detailed information about the five SNPs taken of dbSNP. The first column represents the SNP ID and the second the change of the nucleotide which leads to a codon change. This change happens on the position found in column three, whereas four and five tell about the amino acids that are exchanged and which position this is in the amino acid sequence. The last column shows with which disease the SNP is associated, if annotated. Those indicate with '?' are not associated with any disease yet.

</figtable>

Analyze SNPs

PSSM

We created the PSSM matrix using the standard parameter of PsiBlast and five iterations. In the right matrix you can see, that for some of the ten residues the amino acid is not conserved as they have higher values for other amino acids. SNPs are marked in red.

Last position-specific scoring matrix computed, weighted observed percentages rounded down,
information per position, and relative weight of gapless real matches to pseudocounts
         A  R  N  D  C  Q  E  G  H  I  L  K  M  F  P  S  T  W  Y  V    A  R  N  D  C  Q  E  G  H  I  L  K  M  F  P  S  T  W  Y  V
 20 Q   -2  1  2  3 -4  2  2 -1  2 -3 -1  2 -2 -3 -3 -1 -1 -4 -3 -1    0  7 13 17  0 14 11  3  5  0  6 16  0  0  0  0  2  0  0  6 0.32 inf
103 G    0  0  0  0  2  0  0 -1  1  0 -1  0  1  0  0  0  0 -2  1  0    5  5  5  5  5  5  5  5  5  5  5  5  5  5  5  5  5  0  5  5 0.03 inf
123 R   -3  6 -1 -3 -3  0 -2 -3  0  0 -1  3 -1 -4 -3 -1  1 -4 -3 -3    1 49  1  0  1  3  0  1  2  7  4 16  1  0  0  2 10  0  0  0 0.72 inf
172 Q   -1  0  0  3 -4  5  2 -1  0 -3 -3  1 -1 -4 -3 -1  0 -4 -3 -1    5  4  2 16  0 29 11  5  1  1  2  9  1  0  0  3  6  0  0  4 0.43 inf
259 A    6 -2 -2 -3 -3 -1 -2 -1 -3 -2 -2 -2 -1 -4 -3  1 -2 -4 -2 -2   70  2  1  0  0  3  1  3  0  2  4  1  1  0  0  9  1  0  1  1 0.93 inf
266 T    3 -1 -1 -3 -3 -3 -3  0 -3 -3 -3  0 -3 -4 -3  0  5 -4  0 -2   29  3  1  0  0  0  0  6  0  0  1  6  0  0  0  5 43  0  3  1 0.70 inf
341 K   -2  4 -1 -2 -1  1  1 -3 -1 -3 -1  4 -1  0 -2 -1 -1 -1 -2 -2    0 28  0  0  2  2 10  0  0  0  8 39  1  7  0  1  1  1  0  1 0.44 inf 
392 F   -4 -5 -5 -5  0 -5 -5 -3 -3  3  2 -5  1  6 -5 -4 -3 -2  2  1    0  0  0  0  2  0  0  2  0 16 17  0  2 49  0  0  0  0  6  7 0.98 inf
416 P    0 -4  1 -3 -5 -3 -2 -3 -2 -2 -5 -2 -4 -1  7 -1 -3 -6 -4 -4    9  0  9  0  0  0  2  1  1  3  0  2  0  4 66  4  0  0  0  0 1.60 inf
421 I   -1 -4 -4 -4 -3 -1 -4 -1 -4  4  1 -3  0 -2 -4 -3 -2 -4 -3  4    5  0  0  0  0  4  0  6  0 32  9  0  1  0  0  0  0  0  0 41 0.62 inf

SIFT

• A259V

Substitution at pos 259 from A to V is predicted to AFFECT PROTEIN FUNCTION with a score of 0.00.
   Median sequence conservation: 3.01
   Sequences represented at this position:77

• R123I

Substitution at pos 123 from R to I is predicted to be TOLERATED with a score of 0.05.
   Median sequence conservation: 3.01
   Sequences represented at this position:76

• Q20L

Substitution at pos 20 from Q to L is predicted to be TOLERATED with a score of 0.16.
   Median sequence conservation: 3.20
   Sequences represented at this position:42

• Q172H

Substitution at pos 172 from Q to H is predicted to AFFECT PROTEIN FUNCTION with a score of 0.03.
   Median sequence conservation: 3.01
   Sequences represented at this position:77

• G103S

Substitution at pos 103 from G to S is predicted to be TOLERATED with a score of 0.90.
   Median sequence conservation: 3.02
   Sequences represented at this position:72

• I421T

Substitution at pos 421 from I to T is predicted to AFFECT PROTEIN FUNCTION with a score of 0.00.
   Median sequence conservation: 3.00
   Sequences represented at this position:74

• K341T

Substitution at pos 341 from K to T is predicted to AFFECT PROTEIN FUNCTION with a score of 0.00.
   Median sequence conservation: 3.01
   Sequences represented at this position:77

• F392S

Substitution at pos 392 from F to S is predicted to AFFECT PROTEIN FUNCTION with a score of 0.00.
   Median sequence conservation: 3.01
   Sequences represented at this position:77

• P416Q

Substitution at pos 416 from P to Q is predicted to AFFECT PROTEIN FUNCTION with a score of 0.00.
   Median sequence conservation: 3.00
   Sequences represented at this position:74

• T266A

Substitution at pos 266 from T to A is predicted to AFFECT PROTEIN FUNCTION with a score of 0.00.
   Median sequence conservation: 3.01
   Sequences represented at this position:77

PolyPhen2

• A259V

HumDiv
This mutation is predicted to be probably damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00)
HumVar
This mutation is predicted to be probably damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00)

• R123I

HumDiv
This mutation is predicted to be possibly damaging with a score of 0.807 (sensitivity: 0.84; specificity: 0.93)
HumVar
This mutation is predicted to be possibly damaging with a score of 0.582 (sensitivity: 0.81; specificity: 0.83)

• Q20L

HumDiv
This mutation is predicted to be benign with a score of 0.000 (sensitivity: 1.00; specificity: 0.00)
HumVar
This mutation is predicted to be benign with a score of 0.000 (sensitivity: 1.00; specificity: 0.00)

• Q172H

HumDiv
This mutation is predicted to be possibly damaging with a score of 0.705 (sensitivity: 0.86; specificity: 0.92)
HumVar
This mutation is predicted to be benign with a score of 0.170 (sensitivity: 0.89; specificity: 0.72)

• G103S

HumDiv
This mutation is predicted to be benign with a score of 0.003 (sensitivity: 0.98; specificity: 0.44)
HumVar
This mutation is predicted to be benign with a score of 0.006 (sensitivity: 0.97; specificity: 0.45)

• I421T

HumDiv
This mutation is predicted to be possibly damaging with a score of 0.667 (sensitivity: 0.86; specificity: 0.91)
HumVar
This mutation is predicted to be probably damaging with a score of 0.913 (sensitivity: 0.69; specificity: 0.90)

• K341T

HumDiv
This mutation is predicted to be probably damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00)
HumVar
This mutation is predicted to be probably damaging with a score of 0.996 (sensitivity: 0.36; specificity: 0.97)

• F392S

HumDiv
This mutation is predicted to be probably damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00)
HumVar
This mutation is predicted to be probably damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00)

• P416Q

HumDiv
This mutation is predicted to be probably damaging with a score of 0.996 (sensitivity: 0.55; specificity: 0.98)
HumVar
This mutation is predicted to be probably damaging with a score of 0.985 (sensitivity: 0.55; specificity: 0.94)

• T266A

HumDiv
This mutation is predicted to be probably damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00)
HumVar
This mutation is predicted to be probably damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00)

SNAP

A259V          Non-neutral          Reliability Index: 2          Expected Accuracy: 70%
R123I              Neutral          Reliability Index: 0          Expected Accuracy: 53%
Q20L               Neutral          Reliability Index: 0          Expected Accuracy: 53%
Q172H              Neutral          Reliability Index: 4          Expected Accuracy: 85%
G103S              Neutral          Reliability Index: 6          Expected Accuracy: 92%
I421T          Non-neutral          Reliability Index: 2          Expected Accuracy: 70%
K341T          Non-neutral          Reliability Index: 3          Expected Accuracy: 78%
F392S          Non-neutral          Reliability Index: 1          Expected Accuracy: 63%
P416Q          Non-neutral          Reliability Index: 1          Expected Accuracy: 63%
T266A              Neutral          Reliability Index: 2          Expected Accuracy: 69%

MutationTaster

Gene: ENSG00000171759
Transcript: ENST00000553106
For affected protein features only those are reported that are annotated as "lost" and not as "might get lost".

• A259V - C776T

Prediction: disease causing, Model: simple_aae, prob: ~1 (classification due to ClinVar)
Summary: 
•	amino acid sequence changed
•	known disease mutation at this position (HGMD CM910286)
•	known disease mutation: rs118203921 (pathogenic)
•	protein features (might be) affected (HELIX: 251-259, lost)
splice sites: Acc marginally increased

• R123I - G368T

Prediction: disease causing, Model: simple_aae, prob: ~1
Summary
•	amino acid sequence changed
•	listed as SNP
•	protein features (might be) affected
•	splice site changes (Acc increased)

• Q20L - A59T

Prediction: disease causing, Model: simple_aae, prob: ~0.95
Summary
•	amino acid sequence changed
•	known disease mutation at this position (HGMD CM000542)
•	protein features (might be) affected
•	splice site changes (Donor lost, acc marginally increased)

• Q172H - G516T

Prediction: disease causing, Model: simple_aae, prob: ~1
Summary 	
•	amino acid sequence changed
•	protein features (might be) affected 
•	splice site changes (Acc marginally increased, donor lost, acc gained)

• G103S - G307A

Prediction: disease causing, Model: simple_aae, prob: ~1
Summary
•	amino acid sequence changed
•	known disease mutation at this position (HGMD CM045080)
•	protein features (might be) affected (Domain ACT: lost)
•	splice site changes (Donor gained)

• I421T - T1262C

Prediction: disease causing, Model: simple_aae, prob: ~1
Summary
•	amino acid sequence changed
•	listed as SNP
•	protein features (might be) affected (STRAND: 420-424, lost)

• K341T - A1022C

Prediction: disease causing, Model: simple_aae, prob: ~1
Summary
•	amino acid sequence changed
•	known disease mutation at this position (HGMD CM010980)
•	known disease mutation at this position (HGMD CM010981)
•	protein features (might be) affected (STRAND: 339-342, lost)

• F392S - T1175C

Prediction: disease causing, Model: simple_aae, prob: ~1
Summary
•	amino acid sequence changed
•	listed as SNP
•	protein features (might be) affected (HELIX: 392-403, lost)
•	splice site changes (Donor increased)

• P416Q - C1247A

Prediction: disease causing, Model: simple_aae, prob: ~1
Summary
•	amino acid sequence changed
•	known disease mutation at this position (HGMD CM090791)
•	protein features (might be) affected (TURN: 416-419, lost)
•	splice site changes (Acc marginally increased, donor increased, donor gained)

• T266A - A796C

Prediction: disease causing, Model: simple_aae, prob: ~1
Summary
•	amino acid sequence changed
•	listed as SNP
•	protein features (might be) affected 
•	splice site changes (Acc increased, acc gained, donor increased)