Task 5: Mapping point mutations

Task description

A detailed task description can be found here: Mapping point mutations

SNP databases

HGMD

• HGMD
• Searched for PAH
• 429 Missense/Nonsense mutations known by HGMD Professional

There are several mutation types known for PAH:

• Missense/nonsense
• Splicing
• Regulatory
• Small deletions
• Small insertions
• Small indels
• Gross deletions
• Gross insertions/duplications
• Complex rearrangements

One additional category of mutation is known, but is not recorded for PAH

• Repeat variations

Reference Sequence

The reference sequence is given by the accession number NM_000277.1, whose entry contains the following amino acid sequence:

MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEEN DVNLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDI GATVHELSRDKKKDTVPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQ FADIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCG FHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPM YTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLC KQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESF NDAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQK IK

SNPdb

Methodology

Retrieving Data

Figure 1: Shows the query and result for our silent mutation search

We searched in dbSNP for silent mutations in coding regions. This means we only considered those SNPs which alter the triplet but not the amino acid.

To do so we used the Entrez interface of NCBI which is accessible under this URL:

•  ://www.ncbi.nlm.nih.gov/sites/entrez?Db=snp

The advantage of this Entrez interface is that we can construct arbitrary complex queries to restrict our result set.

We constructed the following query to search for SNPs which are considered silent in the coding regions of the human PAH gene (see figure 1):

• "synonymous-codon"[Function_Class] AND PAH[GENE] AND "human"[ORGN] AND "snp"[SNP_CLASS]

Results of this query can be accessed directly via the following URL:

• http://www.ncbi.nlm.nih.gov/sites/entrez?Db=snp&Cmd=DetailsSearch&Term=%22synonymous-codon%22%5BFunction_Class%5D+AND+PAH%5BGENE%5D+AND+%22human%22%5BORGN%5D+AND+%22snp%22%5BSNP_CLASS%5D

We decided to download the results as FlatFile. This seemed to be the most simple format to process and contains almost all information we need.

Results

We could find the following silent mutations in dbSNP:

Identifier	AA-Position	Reference Triplet	Mutated Triplet	Reference Allele	Mutated Allele	Frame	Reference Residue	Mutated Residue
rs117308669	65	GAA	GAG	A	G	3	E	E
rs75065106	257	CTG	TTG	C	T	1	L	L
rs62651567	322	ACA	ACG	A	G	3	T	T
rs62508648	366	CTG	CTA	G	A	3	L	L
rs61747292	320	CTC	CTT	C	T	3	L	L
rs59326968	425	AAT	AAC	T	C	3	N	N
rs17852374	35	TCA	TCG	A	G	3	S	S
rs1801152	413	TAC	TAT	C	T	3	Y	Y
rs1801151	399	AGG	CGG	A	C	1	R	R
rs1801150	398	GTA	GTT	A	T	3	V	V
rs1801147	202	TGC	TGT	C	T	3	C	C
rs1801146	136	AGC	AGT	C	T	3	S	S
rs1801145	9	GGC	GGG	C	G	3	G	G
rs1126758	231	CAG	CAG	A	G	3	Q	Q
rs1042503	244	GTG	GTA	G	A	3	V	V
rs772897	384	CTG	CTC	G	C	3	L	L

Comparing the annotation of HGMD and SNPdb

Alignment of the reference sequences

We decided to use the sequence of PAH of Uniprot (see UniProt).

• MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDV
NLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPW
FPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYM
EEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGF
RLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFA
QFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSE
KPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQR
IEVLDNTQQLKILADSINSEIGILCSALQKIK

Alignment with the reference sequence used in HGMD

The resulting alignment shows a 100% identity without any gaps. Therefore it is a "self-alignment".