We specifically focus on non-synonymous SNPs (nsSNPs), i.e. SNPs located in coding regions and resulting in amino acid variation in protein products of genes. It was shown in several recent studies that impact of amino acid allelic variants on protein structure/function can be reliably predicted via analysis of multiple sequence alignments and protein 3D-structures. As we demonstrated in an earlier work, these predictions correlate with the effect of natural selection seen as an excess of rare alleles. Therefore, predictions at the molecular level reveal SNPs affecting actual phenotypes.

PolyPhen (=Polymorphism Phenotyping) is an automatic tool for prediction of possible impact of an amino acid substitution on the structure and function of a human protein. This prediction is based on straightforward empirical rules which are applied to the sequence, phylogenetic and structural information characterizing the substitution.

For a given amino acid substitution in a human protein, PolyPhen performs several steps:

References

• DISULFID, THIOLEST, THIOETH bond or
• BINDING, ACT_SITE, LIPID, METAL, SITE, MOD_RES, CARBOHYD, SE_CYS site

PolyPhen also checks if the substitution site is located in the region annotated as

• TRANSMEM, SIGNAL, PROPEP

For a substitution in an annotated or predicted transmembrane region, PolyPhen uses the PHAT transmembrane specific matrix score to evaluate possible functional effect of a nsSNP in the transmembrane region.

SWALL

SwissProt Help

TMHMM

Coils

SignalP

PHAT

After the BLAST search, the set of hits is filtered to retain hits that have
(a) sequence identity to the input sequence in the range 30- 94%, inclusively, and
(b) lehgth of alignment with the query sequence not smaller than 50.
Sequence identity is defined as the number of matches divided by the complete alignment length.

The resulting multiple alignment is used by the new version of the PSIC software (Position-Specific Independent Counts) to calculate the so-called profile matrix. Elements of the matrix (profile scores) are logarithmic ratios of the likelihood of given amino acid occurring at a particular position to the likelihood of this amino acid occurring at any position (background frequency).

PolyPhen computes the absolute value of the difference between profile scores of both allelic variants in the polymoprphic position. Big values of this difference may indicate that the studied substitution is rarely or never observed in the protein family. PolyPhen also shows the number of aligned sequences at the query position. This number may be used to assess the reliability of profile score calculations.

BLAST

nrdb

PSIC paper

PSIC server

(a) sequence identity threshold is set to 50%, since this value guarantees the conservation of basic structural characteristics
(b) minimal hit length is set to 100
(c) maximal number of gaps is set to 20

By default, a hit is rejected if its amino acid at the corresponding position differs from the amino acid in the input sequence. The position of the substitution is then mapped onto the corresponding positions in all retained hits. Hits are sorted according to the sequence identity or E-value of the sequence alignment with the input protein.

PDB

PQS

• Secondary structure (according to the DSSP nomenclature)
• Solvent accessible surface area (absolute value in Ų)
• Phi-psi dihedral angles

DSSP

• Normed accessible surface area: the absolute value divided by the maximal area defined as a 99%-quantile of surface area distribution for this particular amino acid type in PDB
• Change in accessible surface propensity resulting from the substitution. Accessible surface propensities (knowledge-based hydrophobic "potentials") are logarithmic ratios of the likelihood of given amino acid occuring at a site with a particular accessibility to the likelihood of this amino acid occuring at any site (background frequency)
• Change in residue side chain volume measured in ų. Side chain volumes are here
• Region of the phi-psi map (Ramachandran map) derived from the residue dihedral angles. Ramachandran map is here
• Normalised B-factor (temperature factor) for the residue. B-factor, or temperature factor, is used in crystallographic studies of macromolecules to characterise the "mobility" of an atom. It is believed [Chasman D, Adams RM (2001)] that the values of B-factor of a residue may be correlated with its tolerance to amino acid substitutions.

By default, contacts are calculated for all found hits with known structure. This is essential for the cases when several PDB(PQS) entries correspond to one protein, but carry different information about complexes with other macromolecules and ligands (for example, see Fig.2 in [Sunyaev et al 2001]).

PolyPhen checks three types of contacts for a variable amino acid residue:

PDB HETATM

PQS

• probably damaging, i.e., it is with high confidence supposed to affect protein function or structure
• possibly damaging, i.e., it is supposed to affect protein function or structure
• benign, most likely lacking any phenotypic effect
• unknown, when in some rare cases, the lack of data do not allow PolyPhen to make a prediction

Prediction basis and Substitution effect are described below.

(1) FT, sequence annotation (or prediction) being a fragment of SWALL feature table (FT) describing the substitution position,
(2) alignment, PSIC profile scores derived from multiple alignment,
(3) structure, structural information, obtained if a search against structural database was successful.

The presence of all three data sources indicates the highest reliability of a prediction. However, as a rough estimate one can expect that approximately only ~10% of all sequences have homologous proteins with known structure.

• sequence annotation
• sequence prediction
• multiple alignment
• structure

   1. structural
      1.1. buried site
         1.1.1. hydrophobicity disruption
         1.1.2. overpacking
         1.1.3. cavity creation
      1.2. bond formation
         1.2.1. covalent bond
             1.2.1.1. disulphide
             1.2.1.2. thioesther
             1.2.1.3. thioether
         1.2.2. non-covalent
             1.2.2.1. hydrogen
             1.2.2.2. salt bridge (electrostatic)

   2. functional
      2.1. indirect
      2.2. functional site
         2.2.1. signal peptide
         2.2.2. transmembrane
         2.2.3. ligand binding
         2.2.4. protein interaction

The purpose of introduction of this classification is, on the one hand, to suggest an explanation of a damaging effect without strict reference to the prediction rule, which relies on technical details. On the other hand, this classification leaves room for further improvement of a method.

Protein identifier (ACC or ID) from the SWALL database which is case-insensitive, e.g., pexa_human, XYZ_HUMAN, P12345, p12345, aah01234, etc. PolyPhen maps this value to primary accession number and works with it.

Amino acid sequence in FASTA format which should obey the "classical" FASTA format, e.g., provide sequence identifier

User is supposed to complete only one of the fields above.

Position is checked not to exceed the protein length

Substitution is given by two amino acid variants; the first one is checked to correspond to the actual protein sequence, whereas the second is checked to differ from the first one.

Description is an optional short string (up to 60 characters) providing descriptive name and/or comment for your query. It will be displayed in the query management page to facilitate identifying particular query instances which may be useful when you submit a large number of them.

SWALL

FASTA format

Sort hits by (Identity/E-value)
Hits are sorted according to the sequence identity or E- value (default) of the sequence alignment with the input protein.

Map to mismatch (No/Yes)
By default, a hit is rejected if its amino acid at the corresponding position differs from the amino acid in the input sequence. Mapping to mismatching amino acid residue should be used with caution only when a protein with known structure and matching amino acid can not be found.

Calculate structural parameters (For first hit only/For all hits)
In some cases a user may want to check the conservation of structural parameters of a residue in all hits. By default, parameters are calculated for the first hit only, since they are expected to be very close in all homologous structures.

Calculate contacts (For first hit only/For all hits)
Contrary to the structural parameters, contacts are by default calculated for all found hits with known structure. This is essential for the cases when several PDB(PQS) entries correspond to one protein, but carry different information about complexes with other macromolecules and ligands (for example, see Fig.2 in [Sunyaev et al 2001])

Minimal alignment length (integer number, default: 100)
PolyPhen will filter out hits with structure whose alignment length with the query sequence is smaller than the given value.

Minimal identity in alignment (floating point value, not exceeding 1, default: 0.5)
Hits with structure whose sequence identity to the query sequence is smaller than the given threshold are filtered out

Maximal gap length in alignment (integer number, default: 20)
PolyPhen will filter out hits with structure whose alignment with the query sequence contains gaps with total length greater than this value

Threshold for contacts (floating point value, default: 6.0Å)
PolyPhen will report residue contacts below this threshold

PDB

PQS

SRS

SNP data collection page

HGVBase

Flanking genomic sequences of SNPs from HGVbase with length 25 bp each have been translated in all six possible frames and searched for exact match in the hs_swall protein set which contains all human proteins from the SWALL database.

Protein sequences and genomic fragments have been pre-processed by SEG, XNU, RepeatMasker, and DUST programs which are used to filter out areas of low compositional complexity, regions containing internal repeats of short periodicity, and known known human genomic repeat sequences. ALU subfamily proteins were also excluded from the set. We did not consider SNP entries which have N's in their flanking sequences, since this makes translation ambiguous.

We required that at least one translated flanking sequence should have an exact match with a database protein sequence. In case this match has been detected, we further required that the second flanking sequence has either exact match with the protein sequence or matches the protein sequence in all positions until the end of the protein or conventional exon/intron border is observed. The mapping quality is given for each SNP:

LR, both flanks completely match the protein sequence;
L?, exact match of left flank and partial match of the right one;
?R, vice versa

Filtering sequences

Detailed prediction statistics can be found in the SNP data collection page .

* asterisk matching everything,
? question mark matching exactly one symbol,
[] brackets to set a character class

For example, search for "transcription factor [12] " in the description field gives 12 hits in transcription factors of type 1 and 2. Note that unless set otherwise, search engine tries to match the search text to any part of the searched field, not to the whole field. Other search parameters are self-explanatory.