Sequence-based mutation analysis (Phenylketonuria)

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Summary

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Mutation dataset

Lab journal

SNPs
AA - three letter AA - one letter Nucleotides
Ala259Val A259V C776T
Arg123Ile R123I G368T
Gln20Leu Q20L A59T
Gln172His Q172H G516T
Gly103Ser G103S G307A
Ile421Thr I421T T1262C
Lys341Thr K341T A1022C
Phe392Ser F392S T1175C
Pro416Gln P416Q C1247A
Thr266Ala T266A A796C

Analyze SNPs

The complete results of the prediction tools can be found in the Lab journal.
Looking at the substitution matrices you can see that the substitutions analyzed in this task never have the worst values.
As 2pah begins at position 118 for amino acids 20 and 103 the substitutions cannot be shown with pymol. A comination of the other eight mutations can be seen in <xr id="all_mutations"/>. None of those amino acids participates in forming the binding site, so it is not disturbed by any of the substitutions that are examined here.

<figure id ="all_mutations">

All eight substitutions shown in the catalytic domain of 2pah (green), where the original amino acids are shown in yellow and the mutated one in purple.

</figure>

Ala-259-Val

<figure id="A259V">

Mutation of alanine (yellow) to valine (purple) at position 259 of 2pah (green).

</figure>

Properties alanine is small, non-polar, neutral and hydrophobic
valine is aliphatic, small, non-polar, neutral and hydrophobic
Structure LOOP
Substitution Matrices middle ranged value → neutral substitution
PSSM alanine seems to be highly conserved with a value of 70. Valine has a value of 1.
SIFT TOLERATED with a score of 0.16
PolyPhen2 probably damaging with a score of 1.000
SNAP non-neutral, RI = 2
MutationTaster disease causing
⇒ prediction: non-neutral

For the substitution of alanine to valine the properties do not change very much as the only difference is that valine is aliphatic. This can also be seen in the substitution matrices where such an exchange has a middle ranged value and therefore indicates a neutral substitution. Additionally SIFT predicts this mutation to be tolerable, but the score is not very high. When looking at the PSSM matrix that was created with psiblast a high conservation of alanine on position 259 can be seen, where valine only has a low tolerable value here. Furthermore the other three prediction tools all predict a non-neutral effect of this substitution where most notably PolyPhen2 has a very high score. SNAP only has a reliability index of two, but an accuracy of 70%. Looking at the structure itself (<xr id="A259V"/>) you can see that the substitution is located in a LOOP, however it is directly beneath a HELIX or the last amino acid of it, which is predicted to get lost by MutationTaster if the amino acid is changed. In this case, to decide if the substitution is neutral or not is really hard as the different features suggest both. However, we predict this mutation to be non-neutral.

Arg-123-Ile

<figure id="R123I">

Mutation of arginine (yellow) to isoleucine (purple) at position 123 of 2pah (green).

</figure>

Properties arginine is positively charged, polar and hydrophilic
isoleucine is aliphatic, neutral, non-polar and hydrophobic
Substitution Matrices low value → bad substitution
Structure LOOP
PSSM arginine seems to be highly conserved with a value of 49. Isoleucine has a value of 7.
SIFT AFFECT PROTEIN FUNCTION with a score of 0.00
PolyPhen2 possibly damaging with a score of 0.807 / 0.582
SNAP neutral, RI = 0
MutationTaster disease causing
⇒ prediction: non-neutral

Here a lot of the different features indicate a non-neutral and probably damaging substitution, which begins with the complete different properties of the two amino acids. Furthermore the substitution matrices show low values and arginine is high conserved on position 123. Beside of SNAP all prediction tools forecast a bad substitution, however PolyPhen2 only indicates a possibly damaging mutation. Nevertheless SNAP itself has the lowest reliability index of 0. This substitution lies in a LOOP and a similar conformation of the two amino acids in the molecule can be seen in <xr id="R123I"/>. Altogether we predict that the substitution of arginine to isoleucine at position 123 of the PAH protein is non-neutral and might affect function.

Gln-20-Leu

Properties glutamine is neutral, polar and hydrophilic
leucine is aliphatic, neutral, non-polar and hydrophilic
Substitution Matrices low value → bad substitution
Structure LOOP
PSSM seems to be not conserved as the value for aspartic acid (17) is higher than for glutamine (14).

leucine has a value of 6.

SIFT TOLERATED with a score of 0.90
PolyPhen2 benign with a score of 0.000
SNAP neutral, RI = 0
MutationTaster disease causing
⇒ prediction: neutral

The exchange of glutamine to leucine at position 20 is predicted to be neutral, as there is much indication for this by most features. Only the substitution matrices indicate a bad substitution. Glutamine seems to be not conserved at this position, but flexible as there is a higher value for aspartic acid in PSSM. SIFT and PolyPhen2 both get high scores for being a neutral substitution, where SNAP has a very low reliability index. Only MutationTaster predicts it to be disease causing. Again the substitution can be found in a LOOP region.

Gln-172-His

<figure id="Q172H">

Mutation of glutamine (yellow) to histidine (purple) at position 172 of 2pah (green).

</figure>

Properties glutamine is neutral, polar and hydrophilic
histidine is positively charged, polar and hydrophobic
Substitution Matrices middle ranged → neutral substitution
Structure LOOP
PSSM ...
...
SIFT AFFECT PROTEIN FUNCTION with a score of 0.03.
PolyPhen2 possibly damaging with a score of 0.705 / benign with a score of 0.170
SNAP neutral, RI = 4
MutationTaster disease causing
⇒ prediction: neutral

Gly-103-Ser

Properties glycine is small, neutral, non-polar and hydrophilic
serine is small, neutral, polar and hydrophilic
Substitution Matrices middle ranged value → neutral substitution
Structure HELIX
PSSM glycine seems to be completely unconserved as every amino acid has the same value 5.
SIFT TOLERATED with a score of 0.05
PolyPhen2 benign with a score of 0.003 / 0.006
SNAP neutral, RI = 6
MutationTaster disease causing
⇒ prediction: neutral

Ile-421-Thr

<figure id="I421T">

Mutation of isoleucine (yellow) to threonine (purple) at position 421 of 2pah (green).

</figure>

Properties isoleucine is aliphatic, neutral, non-polar and hydrophobic
threonine is small, neutral, polar and hydrophobic
Substitution Matrices low value for Blosum62, middle ranged value for PAM1/250 → bad or neutral substitution
Structure STRAND
PSSM isoleucine with a value of 32 seems to be not conserved as valine has a value of 41.

threonine has a value of 0.

SIFT AFFECT PROTEIN FUNCTION with a score of 0.00.
PolyPhen2 possibly damaging with a score of 0.667 / probably damaging with a score of 0.913
SNAP non-neutral, RI = 2
MutationTaster disease causing
⇒ prediction: non-neutral

Lys-341-Thr

<figure id="K341T">

Mutation of lysine (yellow) to threonine (purple) at position 341 of 2pah (green).

</figure>

Properties lysine is positively charged, polar and hydrophobic
threonine is small, neutral, polar and hydrophobic
Substitution Matrices low value for Blosum62, middle ranged value for PAM1/250 → bad or neutral substitution
Structure STRAND
PSSM lysine seems to be conserved with a value of 39.

threonine has a value of 1.

SIFT AFFECT PROTEIN FUNCTION with a score of 0.00.
PolyPhen2 probably damaging with a score of 1.000 / 0.996
SNAP non-neutral, RI = 3
MutationTaster disease causing
⇒ prediction: non-neutral

Phe-392-Ser

<figure id="F392S">

Mutation of phenylalanine (yellow) to serine (purple) at position 392 of 2pah (green).

</figure>

Properties phenylalanine is aromatic, neutral, non-polar and hydrophobic
serine is small, neutral, polar and hydrophilic
Substitution Matrices low value → bad substitution
Structure HELIX
PSSM phenylalanine seems to be highly conserved with a value of 49. Serine has a value of 0.
SIFT AFFECT PROTEIN FUNCTION with a score of 0.00.
PolyPhen2 probably damaging with a score of 1.000
SNAP non-neutral, RI = 1
MutationTaster disease causing
⇒ prediction: non-neutral

Pro-416-Gln

<figure id="P416Q">

Mutation of proline (yellow) to glutamine (purple) at position 416 of 2pah (green).

</figure>

Properties proline is small, neutral, non-polar and hydrophilic
glutamine is neutral, polar and hydrophilic
Substitution Matrices low value → bad substitution
Structure LOOP
PSSM proline seems to be highly conserved with a value of 66. Glutamine has a value of 0.
SIFT AFFECT PROTEIN FUNCTION with a score of 0.00.
PolyPhen2 probably damaging with a score of 0.996 / 0.985
SNAP non-neutral, RI = 1
MutationTaster disease causing
⇒ prediction: non-neutral

Thr-266-Ala

<figure id="T266A">

Mutation of threonine (yellow) to alanine (purple) at position 266 of 2pah (green).

</figure>

Properties threonine is small, neutral, polar and hydrophobic
alanine is small, non-polar, neutral and hydrophobic
Substitution Matrices middle ranged value &rarr neutral substitution
Structure HELIX
PSSM threonine seems to be conserved with a value of 43. Alanine has a value of 29.
SIFT AFFECT PROTEIN FUNCTION with a score of 0.00.
PolyPhen2 probably damaging with a score of 1.000
SNAP neutral, RI = 2
MutationTaster disease causing
⇒ prediction: non-neutral

Mammalian Homologous Sequences

We used a normal BLAST search on the mammalian database of Uniprot and filter the results per hand for double entries. Altogether we found 22 homologues sequences (<xr id="IDs"/>). <figtable id="IDs">

UniProt-IDs homologue to P00439
H2Q6R0 G3S964 G1R3M2 G7PJC2 F7HMW9 F7I717 F7BKF9
F6XY00 E2R366 G1T8B6 H2NIF5 M3YKN3 M9P0Q7 H0WTI6
M3W9R1 G3TIW0 G1LIM6 Q2KIH7 G1P4I7 P16331 M9P0Y5
UniProt IDs of the 22 mammalian homologues sequences of the PAH protein. The only ID that has a sequence that is not 100% identical to P00439 is marked in green.

</figtable>

For the creation of the multiple alignment we used clustalw. Nevertheless, only F6XY00 has a slightly different sequence. Like E2R366 F6XY00 is a homologue sequence of the human PAH in canis familiaris. On the positions of our substitutions the sequences are conserved.

Prediction

SNP-Prediction
SNP Prediction Validation
Ala259Val neutral/non-neutral??? HGMD
Arg123Ile non-neutral ? dbSNP
Gln20Leu neutral wrong HGMD
Gln172His neutral ? dbSNP
Gly103Ser neutral wrong HGMD
Ile421Thr non-neutral ? dbSNP
Lys341Thr non-neutral correct HGMD
Phe392Ser non-neutral ? dnSNP
Pro416Gln non-neutral correct HGMD
Thr266Ala non-neutral correct dbSNP

References

<references/>

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