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 - A single nucleotide point mutation in a codon, such that the resulting amino acid changes
• Nonsense - A single nucleotide point mutation in a codon, such that the resulting codon represents a polymerase stop signal
• Splicing - A mutation, which influences the splicing of the gene
• Regulatory - A mutation, which influences the regulation of the gene
• Small/Gross deletions - A mutation, which deletes some/more nucleotides in the gene
• Small/Gross insertions - A mustation, which inserts some/more nucleotides in the gene
• Small indels - A deletion followed by an insertion after the nucleotides affected
• Gross duplications - A mutation, which results in the copy of a piece of the DNA
• Complex rearrangements - A mutation, which results in a changed order of the sequence parts of a gene

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

• Repeat variations - A mutation, which affects a repeated sequence in the gene

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

dbSNP

Clarifications on insertions and deletions

The impact of insertions and deletions in coding regions on chromosomes can be differently. Fatal for a protein are insertions or deletions which introduce a frame shift. This happens when the length of the insertion or deletion is not divisible by 3 without producing a rest. Or in other words: Len mod 3 != 0 . Where Len is the length of the insertion or deletion.

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.

Processing Data

A self written Perl script is helping us to parse the important information out of the FlatFile we downloaded in the previous step. In the current version the following information is parsed out of the FlatFile: identifier, triplet reference/mutated, allele reference/mutated , frame of the mutation, residue reference/mutated and residue position.

All annotations we retrieved are annotated for the mRNA sequence with the GenBank accession number NM_000277.1 which contains the coding sequences of our PAH protein with the accession number NP_000268.1 which is exactly the same protein and therefore has also the same sequence as the UniProt entry for PAH with the accession number P00439. Thus, no mapping to other residue coordinates was required to map these mutations to our mutation map.

With our perl script at hand (the code is accessible here: dbSNP Silent Mutations Parser) and the results in FlatFile format, we are only missing the CDS sequence of NM_000277.1 in order to get the used triplet for each residue. This CDS sequence can be found in the CCDS database of NCBI with the accession number CCDS9092.1.

Now, with all data at hand we can run our perl script in different ways to retrieve different outputs for different purposes. Currently the following three outputs with the following commands can be generated:

• Generates a WikiTable of found silent mutations: ./parse.pl wiki snp_result.txt ccds_pah.fasta
• Generates CSVs which can be used by our mapping tool to generate the mutation map: ./parse.pl map snp_result.txt ccds_pah.fasta
• Generates a human readable one liner for each silent mutation: ./parse.pl list snp_result.txt ccds_pah.fasta

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	66	GAA	GAG	A	G	3	E	E
rs75065106	258	CTG	TTG	C	T	1	L	L
rs62651567	323	ACA	ACG	A	G	3	T	T
rs62508648	367	CTG	CTA	G	A	3	L	L
rs61747292	321	CTC	CTT	C	T	3	L	L
rs59326968	426	AAT	AAC	T	C	3	N	N
rs17852374	36	TCA	TCG	A	G	3	S	S
rs1801152	414	TAC	TAT	C	T	3	Y	Y
rs1801151	400	AGG	CGG	A	C	1	R	R
rs1801150	399	GTA	GTT	A	T	3	V	V
rs1801147	203	TGC	TGT	C	T	3	C	C
rs1801146	137	AGC	AGT	C	T	3	S	S
rs1801145	10	GGC	GGG	C	G	3	G	G
rs1126758	232	CAG	CAG	A	G	3	Q	Q
rs1042503	245	GTG	GTA	G	A	3	V	V
rs772897	385	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".

Mapping

position	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20
sequence	M	S	T	A	V	L	E	N	P	G	L	G	R	K	L	S	D	F	G	Q
annotations	CM920539,missense,M1I,ATGt-ATA									rs1801145,silent,G10G,GGC-GGG						CM010947,missense,S16P,cTCT-CCT				CM971121,nonsense,Q20Term,aCAG-TAG
annotations	CM890092,missense,M1V,cATG-GTG
position	21	22	23	24	25	26	27	28	29	30	31	32	33	34	35	36	37	38	39	40
sequence	E	T	S	Y	I	E	D	N	C	N	Q	N	G	A	I	S	L	I	F	S
annotations		HM972014,missense,T22K,ACA-AAA														rs17852374,silent,S36S,TCA-TCG			CM910280,missense,F39L,TTCt-TTG	CM961063,missense,S40L,TCA-TTA
position	41	42	43	44	45	46	47	48	49	50	51	52	53	54	55	56	57	58	59	60
sequence	L	K	E	E	V	G	A	L	A	K	V	L	R	L	F	E	E	N	D	V
annotations	CM000543,missense,L41P,CTC-CCC					CM930535,missense,G46S,tGGT-AGT	CM993577,missense,A47E,GCA-GAA	CM910281,missense,L48S,TTG-TCG				CM981426,missense,L52S,TTG-TCG	CM981427,missense,R53H,CGC-CAC	CM068057,missense,L54S,TTA-TCA	CM971122,missense,F55L,TTTg-TTG	CM920540,missense,E56D,GAGg-GAT			CM992368,missense,D59Y,tGAT-TAT
annotations													CM010948,missense,R53C,gCGC-TGC
position	61	62	63	64	65	66	67	68	69	70	71	72	73	74	75	76	77	78	79	80
sequence	N	L	T	H	I	E	S	R	P	S	R	L	K	K	D	E	Y	E	F	F
annotations	CM024619,missense,N61D,aAAC-GAC				CM981428,missense,I65N,ATT-AAT	rs117308669,silent,E66E,GAA-GAG	CM950882,missense,S67P,aTCT-CCT	CM920542,missense,R68S,AGAc-AGT	CM045074,missense,P69S,aCCT-TCT	CM981429,missense,S70P,tTCT-CCT						CM010950,missense,E76A,GAG-GCG	CM981430,nonsense,Y77Term,TATg-TAG	CM973035,missense,E78K,tGAA-AAA
annotations	CM992369,missense,N61K,AACc-AAG				CM920541,missense,I65T,ATT-ACT											CM992370,missense,E76G,GAG-GGG
annotations					CM024620,missense,I65S,ATT-AGT											CM071057,nonsense,E76Term,tGAG-TAG
annotations					CM010949,missense,I65V,cATT-GTT
position	81	82	83	84	85	86	87	88	89	90	91	92	93	94	95	96	97	98	99	100
sequence	T	H	L	D	K	R	S	L	P	A	L	T	N	I	I	K	I	L	R	H
annotations	CM950883,missense,T81P,cACC-CCC			CM961066,missense,D84Y,gGAT-TAT			CM961067,missense,S87R,tAGC-CGC		CM971123,missense,P89S,gCCT-TCT					CM981431,missense,I94S,ATC-AGC				CM930537,missense,L98S,TTG-TCG		CM992944,missense,H100R,CAT-CGT
position	101	102	103	104	105	106	107	108	109	110	111	112	113	114	115	116	117	118	119	120
sequence	D	I	G	A	T	V	H	E	L	S	R	D	K	K	K	D	T	V	P	W
annotations		CM993578,missense,I102T,ATT-ACT	CM045080,missense,G103S,tGGT-AGT	CM920543,missense,A104D,GCC-GAC						CM010951,missense,S110L,TCA-TTA	CM920544,nonsense,R111Term,aCGA-TGA								CM043044,missense,P119S,gCCC-TCC	CM961068,nonsense,W120Term,TGG-TAG
position	121	122	123	124	125	126	127	128	129	130	131	132	133	134	135	136	137	138	139	140
sequence	F	P	R	T	I	Q	E	L	D	R	F	A	N	Q	I	L	S	Y	G	A
annotations		CM971124,missense,P122Q,CCA-CAA		CM930538,missense,T124I,ACC-ATC					CM971125,missense,D129G,GAC-GGC			CM981432,missense,A132V,GCC-GTC		CM071056,nonsense,Q134Term,tCAG-TAG			rs1801146,silent,S137S,AGC-AGT
annotations									CM010952,missense,D129Y,gGAC-TAC
position	141	142	143	144	145	146	147	148	149	150	151	152	153	154	155	156	157	158	159	160
sequence	E	L	D	A	D	H	P	G	F	K	D	P	V	Y	R	A	R	R	K	Q
annotations			CM961069,missense,D143G,GAT-GGT		CM961070,missense,D145V,GAC-GTC	CM983349,missense,H146Y,cCAC-TAC	CM971126,missense,P147S,cCCT-TCT	CM950884,missense,G148S,tGGT-AGT			CM010953,missense,D151H,aGAT-CAT			CM010954,missense,Y154N,gTAC-AAC	CM981433,missense,R155H,CGT-CAT		CM056667,missense,R157K,AGA-AAA	CM930539,missense,R158W,aCGG-TGG		CM010955,missense,Q160P,CAG-CCG
annotations											CM971127,missense,D151G,GAT-GGT			CM056672,missense,Y154H,gTAC-CAC	CM000544,missense,R155P,CGT-CCT		CM056668,missense,R157I,AGA-ATA	CM890093,missense,R158Q,CGG-CAG
annotations																		CM015340,missense,R158P,CGG-CCG
position	161	162	163	164	165	166	167	168	169	170	171	172	173	174	175	176	177	178	179	180
sequence	F	A	D	I	A	Y	N	Y	R	H	G	Q	P	I	P	R	V	E	Y	M
annotations	CM920545,missense,F161S,TTT-TCT			CM941128,missense,I164T,ATT-ACT	CM971128,missense,A165T,tGCC-ACC	CM990993,nonsense,Y166Term,TACa-TAA	CM011945,missense,N167S,AAC-AGC	CM992946,missense,Y168H,cTAC-CAC	CM992371,missense,R169H,CGC-CAC	CM010957,missense,H170R,CAT-CGT	CM941129,missense,G171A,GGG-GCG	CM961071,nonsense,Q172Term,gCAG-TAG	CM920546,missense,P173T,gCCC-ACC	CM930540,missense,I174T,ATC-ACC	CM961072,missense,P175A,cCCT-GCT	CM992372,missense,R176Q,CGA-CAA	CM993954,missense,V177M,aGTG-ATG	CM941131,missense,E178G,GAA-GGA	CM015341,missense,Y179H,aTAC-CAC
annotations				CM992945,missense,I164V,cATT-GTT	CM010956,missense,A165P,tGCC-CCC	CM994293,nonsense,Y166Term,TACa-TAG	CM981434,missense,N167I,AAC-ATC			CM011946,missense,H170D,cCAT-GAT	CM010958,missense,G171R,tGGG-AGG			CM990994,missense,I174V,cATC-GTC		CM010959,missense,R176P,CGA-CCA	CM961073,missense,V177L,aGTG-CTG
position	181	182	183	184	185	186	187	188	189	190	191	192	193	194	195	196	197	198	199	200
sequence	E	E	E	K	K	T	W	G	T	V	F	K	T	L	K	S	L	Y	K	T
annotations		CM950885,missense,E182G,GAA-GGA	CM000545,missense,E183Q,aGAA-CAA				CM950886,missense,W187R,aTGG-CGG			CM981436,missense,V190A,GTG-GCG				CM950887,missense,L194P,CTG-CCG
annotations							CM981435,missense,W187C,TGGg-TGC
position	201	202	203	204	205	206	207	208	209	210	211	212	213	214	215	216	217	218	219	220
sequence	H	A	C	Y	E	Y	N	H	I	F	P	L	L	E	K	Y	C	G	F	H
annotations	CM010960,missense,H201R,CAT-CGT		CM981438,missense,C203Y,TGC-TAC		CM003162,missense,E205A,GAG-GCG	CM992373,missense,Y206D,gTAC-GAC	CM971129,missense,N207S,AAT-AGT					CM983350,missense,L212P,CTT-CCT	CM961074,missense,L213P,CTT-CCT			CM010961,nonsense,Y216Term,TACt-TAG	CM034743,missense,C217R,cTGT-CGT	CM930545,missense,G218V,GGC-GTC
annotations	CM981437,missense,H201Y,cCAT-TAT		rs1801147,silent,C203C,TGC-TGT		CM068056,missense,E205K,tGAG-AAG	CM043311,missense,Y206C,TAC-TGC	CM981440,missense,N207D,cAAT-GAT										CM961075,missense,C217G,cTGT-GGT
annotations						CM056674,nonsense,Y206Term,TACa-TAA											CM015342,missense,C217Y,TGT-TAT
annotations						CM981439,nonsense,Y206Term,TACa-TAG
position	221	222	223	224	225	226	227	228	229	230	231	232	233	234	235	236	237	238	239	240
sequence	E	D	N	I	P	Q	L	E	D	V	S	Q	F	L	Q	T	C	T	G	F
annotations	CM910282,missense,E221G,GAA-GGA	CM971130,missense,D222G,GAT-GGT		CM961076,missense,I224T,ATT-ACT	CM990995,missense,P225R,CCC-CGC	CM004891,missense,Q226H,CAGc-CAC				CM081727,missense,V230A,GTT-GCT	CM000546,missense,S231F,TCT-TTT	CM990996,nonsense,Q232Term,tCAA-TAA	CM010963,missense,F233L,TTCc-TTA		CM043312,missense,Q235P,CAG-CCG			CM920548,missense,T238P,cACT-CCT	CM034744,missense,G239D,GGT-GAT	CM003163,missense,F240S,TTC-TCC
annotations		CM010962,missense,D222V,GAT-GTT		CM920547,missense,I224M,ATTc-ATG	CM971131,missense,P225T,tCCC-ACC	CM024631,nonsense,Q226Term,cCAG-TAG				CM994779,missense,V230G,GTT-GGT	CM950888,missense,S231P,tTCT-CCT	rs1126758,silent,Q232Q,CAG-CAG							CM971132,missense,G239A,GGT-GCT	CM973036,missense,F240V,tTTC-GTC
annotations					CM994634,missense,P225A,tCCC-GCC														CM990997,missense,G239V,GGT-GTT
annotations																			CM941132,missense,G239S,tGGT-AGT
position	241	242	243	244	245	246	247	248	249	250	251	252	253	254	255	256	257	258	259	260
sequence	R	L	R	P	V	A	G	L	L	S	S	R	D	F	L	G	G	L	A	F
annotations	CM930548,missense,R241C,cCGC-TGC	CM920549,missense,L242F,cCTC-TTC	CM900176,nonsense,R243Term,cCGA-TGA	CM920550,missense,P244L,CCT-CTT	CM930549,missense,V245E,GTG-GAG	CM010964,missense,A246D,GCT-GAT	CM043314,missense,G247D,GGC-GAC	CM961078,missense,L248P,CTG-CCG	CM973037,missense,L249H,CTT-CAT			CM941134,missense,R252Q,CGG-CAG		CM973038,missense,F254I,tTTC-ATC	CM920553,missense,L255V,cTTG-GTG		CM010966,missense,G257D,GGC-GAC	rs75065106,silent,L258L,CTG-TTG	CM910286,missense,A259V,GCC-GTC
annotations	CM950889,missense,R241L,CGC-CTC		CM910283,missense,R243Q,CGA-CAA		CM961077,missense,V245L,tGTG-CTG	CM981441,missense,A246V,GCT-GTT	CM920551,missense,G247V,GGC-GTC	CM010965,missense,L248R,CTG-CGG	CM030919,missense,L249P,CTT-CCT			CM920552,missense,R252G,tCGG-GGG			CM910285,missense,L255S,TTG-TCG		CM973039,missense,G257V,GGC-GTC		CM962433,missense,A259T,gGCC-ACC
annotations			CM993955,missense,R243L,CGA-CTA		rs1042503,silent,V245V,GTG-GTA		CM043315,missense,G247S,tGGC-AGC		CM950890,missense,L249F,gCTT-TTT			CM910284,missense,R252W,tCGG-TGG					CM010968,missense,G257S,tGGC-AGC
annotations							CM043313,missense,G247R,tGGC-CGC										CM010967,missense,G257C,tGGC-TGC
position	261	262	263	264	265	266	267	268	269	270	271	272	273	274	275	276	277	278	279	280
sequence	R	V	F	H	C	T	Q	Y	I	R	H	G	S	K	P	M	Y	T	P	E
annotations	CM910288,nonsense,R261Term,cCGA-TGA		CM930550,missense,F263L,TTCc-TTG	CM010969,missense,H264L,CAC-CTC	CM983351,missense,C265G,cTGC-GGC		CM056666,missense,Q267E,aCAG-GAG	CM010970,missense,Y268H,gTAC-CAC	CM981443,missense,I269N,ATC-AAC	CM950892,missense,R270K,AGA-AAA	CM010971,missense,H271Y,aCAT-TAT	CM900177,nonsense,G272Term,tGGA-TGA	HM972015,missense,S273P,aTCC-CCC	CM003327,missense,K274E,cAAG-GAG	CM992374,missense,P275R,CCC-CGC	CM034746,missense,M276K,ATG-AAG	CM910291,missense,Y277D,gTAT-GAT	CM045073,missense,T278S,ACC-AGC		CM890094,missense,E280K,cGAA-AAA
annotations	CM910287,missense,R261Q,CGA-CAA				CM981442,missense,C265Y,TGC-TAC		CM056670,missense,Q267H,CAGt-CAC		CM981444,missense,I269L,cATC-CTC	CM930551,missense,R270S,AGAc-AGT			CM910289,missense,S273F,TCC-TTC		CM024621,missense,P275L,CCC-CTC	CM034745,missense,M276R,ATG-AGG	CM931193,missense,Y277C,TAT-TGT	CM981445,missense,T278I,ACC-ATC		CM030920,missense,E280A,GAA-GCA
annotations	CM950891,missense,R261P,CGA-CCA															CM910290,missense,M276I,ATGt-ATT		CM931194,missense,T278A,tACC-GCC		CM045310,missense,E280G,GAA-GGA
annotations																CM930552,missense,M276V,cATG-GTG
position	281	282	283	284	285	286	287	288	289	290	291	292	293	294	295	296	297	298	299	300
sequence	P	D	I	C	H	E	L	L	G	H	V	P	L	F	S	D	R	S	F	A
annotations	CM043045,missense,P281A,aCCT-GCT	CM043316,missense,D282G,GAC-GGC	CM981446,missense,I283N,ATC-AAC		CM981447,missense,H285Y,cCAT-TAT	CM040236,missense,E286K,tGAG-AAG		CM010972,missense,L288F,TTGg-TTC		CM055475,missense,H290R,CAT-CGT			CM045076,missense,L293M,cTTG-ATG		CM990998,nonsense,S295Term,TCA-TGA		CM971133,missense,R297H,CGC-CAC		CM920554,missense,F299C,TTT-TGT	CM950893,missense,A300V,GCC-GTC
annotations	CM992948,missense,P281S,aCCT-TCT	CM930554,missense,D282N,tGAC-AAC	CM930555,missense,I283F,cATC-TTC														CM961079,missense,R297C,tCGC-TGC
annotations	CM910292,missense,P281L,CCT-CTT
position	301	302	303	304	305	306	307	308	309	310	311	312	313	314	315	316	317	318	319	320
sequence	Q	F	S	Q	E	I	G	L	A	S	L	G	A	P	D	E	Y	I	E	K
annotations			CM981448,missense,S303P,tTCC-CCC	CM010974,missense,Q304R,CAG-CGG		CM920556,missense,I306V,aATT-GTT		CM010975,missense,L308V,cCTT-GTT	CM941135,missense,A309D,GCC-GAC	CM024622,missense,S310Y,TCT-TAT	CM880057,missense,L311P,CTG-CCG	CM040237,missense,G312V,GGT-GTT	CM992375,missense,A313V,GCA-GTA	CM040238,missense,P314T,aCCT-ACT	CM993956,missense,D315Y,tGAT-TAT		CM024624,missense,Y317H,aTAC-CAC	CM003164,missense,I318T,ATT-ACT		CM961081,missense,K320N,AAGc-AAC
annotations			CM010973,missense,S303A,tTCC-GCC							CM010976,missense,S310F,TCT-TTT			CM014737,missense,A313T,tGCA-ACA	CM024623,missense,P314S,aCCT-TCT
annotations														CM961080,missense,P314H,CCT-CAT
position	321	322	323	324	325	326	327	328	329	330	331	332	333	334	335	336	337	338	339	340
sequence	L	A	T	I	Y	W	F	T	V	E	F	G	L	C	K	Q	G	D	S	I
annotations	rs61747292,silent,L321L,CTC-CTT	CM920558,missense,A322T,cGCC-ACC	rs62651567,silent,T323T,ACA-ACG	CM041815,missense,I324V,aATT-GTT	CM000547,missense,Y325C,TAC-TGC	CM920559,nonsense,W326Term,TGG-TAG	CM990999,missense,F327L,TTTa-TTG	CM010977,missense,T328A,tACT-GCT		CM000548,missense,E330D,GAGt-GAC	CM941136,missense,F331L,gTTT-CTT	CM993957,missense,G332E,GGG-GAG	CM950894,missense,L333F,gCTC-TTC	CM010978,missense,C334S,TGC-TCC		CM930557,nonsense,Q336Term,aCAA-TAA	CM010979,missense,G337V,GGA-GTA	CM941137,missense,D338Y,aGAC-TAC		CM034747,missense,I340T,ATA-ACA
annotations		CM920557,missense,A322G,GCC-GGC			CM981449,nonsense,Y325Term,TACt-TAG						CM961082,missense,F331C,TTT-TGT	CM045077,missense,G332V,GGG-GTG				CM043317,missense,Q336R,CAA-CGA
position	341	342	343	344	345	346	347	348	349	350	351	352	353	354	355	356	357	358	359	360
sequence	K	A	Y	G	A	G	L	L	S	S	F	G	E	L	Q	Y	C	L	S	E
annotations	CM981450,nonsense,K341Term,aAAG-TAG	CM930558,missense,A342T,gGCA-ACA	CM068055,missense,Y343D,aTAT-GAT	CM000550,missense,G344V,GGT-GTT	CM920560,missense,A345T,tGCT-ACT	CM056675,missense,G346R,tGGG-AGG	CM961083,missense,L347F,gCTC-TTC	CM920561,missense,L348V,cCTG-GTG	CM910293,missense,S349P,gTCA-CCA	CM981453,missense,S350T,aTCC-ACC		CM981454,missense,G352R,tGGT-CGT			CM991001,nonsense,Q355Term,aCAG-TAG	CM962547,missense,Y356H,gTAC-CAC	CM003165,missense,C357G,cTGC-GGC		CM930561,nonsense,S359Term,TCA-TGA
annotations	CM010981,missense,K341T,AAG-ACG	CM971134,missense,A342P,gGCA-CCA	CM010982,missense,Y343F,TAT-TTT	CM981451,missense,G344S,tGGT-AGT	CM010983,missense,A345S,tGCT-TCT	CM991000,missense,G346R,tGGG-CGG			CM056673,missense,S349A,gTCA-GCA	CM015343,missense,S350Y,TCC-TAC		CM993958,missense,G352C,tGGT-TGT				CM941138,nonsense,Y356Term,TACt-TAA
annotations	CM010980,missense,K341R,AAG-AGG			CM000549,missense,G344R,tGGT-CGT					CM043318,nonsense,S349Term,TCA-TAA							CM930560,nonsense,Y356Term,TACt-TAG
annotations									CM981452,missense,S349L,TCA-TTA
position	361	362	363	364	365	366	367	368	369	370	371	372	373	374	375	376	377	378	379	380
sequence	K	P	K	L	L	P	L	E	L	E	K	T	A	I	Q	N	Y	T	V	T
annotations		CM971135,missense,P362T,gCCA-ACA	CM068054,missense,K363N,AAGc-AAT				CM993959,missense,L367P,CTG-CCG		CM081726,missense,L369V,gCTG-GTG		CM950895,missense,K371R,AAG-AGG	CM961084,missense,T372S,gACA-TCA	CM994329,missense,A373T,aGCC-ACC				CM010984,missense,Y377C,TAC-TGC
annotations							CM056671,missense,L367L,CTGg-CTA
annotations							rs62508648,silent,L367L,CTG-CTA
position	381	382	383	384	385	386	387	388	389	390	391	392	393	394	395	396	397	398	399	400
sequence	E	F	Q	P	L	Y	Y	V	A	E	S	F	N	D	A	K	E	K	V	R
annotations					rs772897,silent,L385L,CTG-CTC	CM950896,missense,Y386C,TAT-TGT	CM061883,nonsense,Y387Term,TACg-TAA	CM930564,missense,V388M,cGTG-ATG	CM993579,missense,A389E,GCA-GAA		CM045075,missense,S391I,AGT-ATT			CM961085,missense,D394H,tGAT-CAT	CM043319,missense,A395D,GCC-GAC				rs1801150,silent,V399V,GTA-GTT	CM056669,missense,R400K,AGG-AAG
annotations							CM950897,missense,Y387H,tTAC-CAC	CM981455,missense,V388L,cGTG-CTG							CM981456,missense,A395G,GCC-GGC					CM034748,missense,R400T,AGG-ACG
annotations															CM930566,missense,A395P,tGCC-CCC					rs1801151,silent,R400R,AGG-CGG
position	401	402	403	404	405	406	407	408	409	410	411	412	413	414	415	416	417	418	419	420
sequence	N	F	A	A	T	I	P	R	P	F	S	V	R	Y	D	P	Y	T	Q	R
annotations		CM030921,missense,F402L,cTTT-CTT				CM992376,missense,I406T,ATA-ACA	CM971136,missense,P407S,aCCT-TCT	CM920562,missense,R408Q,CGG-CAG		CM000551,missense,F410S,TTC-TCC	CM003166,nonsense,S411Term,TCA-TGA		CM920563,missense,R413P,CGC-CCC	CM910294,missense,Y414C,TAC-TGC			CM973397,missense,Y417N,aTAC-AAC	CM043046,missense,T418N,ACC-AAC
annotations							CM993960,missense,P407L,CCT-CTT	CM870016,missense,R408W,tCGG-TGG		CM068058,missense,F410C,TTC-TGC			CM981457,missense,R413S,tCGC-AGC	rs1801152,silent,Y414Y,TAC-TAT			CM024632,missense,Y417H,aTAC-CAC	CM920565,missense,T418P,cACC-CCC
annotations													CM010985,missense,R413C,tCGC-TGC
position	421	422	423	424	425	426	427	428	429	430	431	432	433	434	435	436	437	438	439	440
sequence	I	E	V	L	D	N	T	Q	Q	L	K	I	L	A	D	S	I	N	S	E
annotations		CM993580,missense,E422K,tGAG-AAG		CM993961,missense,L424S,TTG-TCG		rs59326968,silent,N426N,AAT-AAC				CM010986,missense,L430P,CTT-CCT				CM043320,missense,A434D,GCT-GAT
position	441	442	443	444	445	446	447	448	449	450	451	452
sequence	I	G	I	L	C	S	A	L	Q	K	I	K
annotations							CM961086,missense,A447D,GCC-GAC
annotations							CM045079,missense,A447P,tGCC-CCC

Discussion

dbSNP

At first, we were quite surprised that we found only 16 silent mutation for the PAH gene. Normally, silent mutations are expected to appear more frequent in coding regions than missense/nonsense mutations because they are not subject to positive selection pressure. However, we assume that the few known silent mutations in PAH is probably a result of lack of data.

In a next step we analyzed the frequencies of the different possible allele mutations with the following results:

Allele Mutation	Absolute Frequency	Relative Frequency
A->T	1	0.0625
A->C	1	0.0625
A->G	4	0.25
T->A	0	0
T->C	1	0.0625
T->G	0	0
C->A	0	0
C->T	5	0.3125
C->G	1	0.0625
G->A	2	0.125
G->T	0	0
G->C	1	0.0625
Purine->Purine	6	0.375
Purine->Pyrimidine	3	0.1875
Pyrimidine->Pyrimidine	6	0.375
Pyrimidine->Purine	1	0.0625

We observed that C -> T and A -> G are the most frequent mutations with a relative frequency of 0.3125 and 0.25 respectively. The first most frequent mutation (C -> T) is a mutation from a pyrimidine base (C) to another pyrimidine base (T) and the second most frequent mutation is a mutation from a purine base (A) to another purine base (G). Also, in general purine -> purine and pyrimidine -> pyrimidine mutations are the most frequent ones with a relative frequency of 0.375 and 0.375 respectively.

This observation somehow reflects our expectations. Since it is more likely that a base gets wrongly incorporated during replication with a base of the same type. The reason for this is that the DNA-polymerase is differentiating the different nucleobase by shape. Adenine and guanine have almost the same shape since they have two rings. The same applies fro cytosine and thymine which also hardly differ regarding to their shape (both have only one ring). Hence, it is more likely for a DNA-polymerase to mistake a purine base with another purine base and to mistake a pyrimidine base with another pyrimidine base than to mistake a purine base with a pyrimidine and vice versa.