Task 7: Research SNPs

Lab journal Task 7

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HGMD (The Human Gene Mutation Database)

The search results for the HFE gene contain the different types of mutations that are specified in <xr id="hgmd"/>:

<figtable id="hgmd">

Table 1: Table of the different mutation types that were found for HFE in the HGMD.

mutation type	definition	number
missense, nonsense	mutation that leads to a change of amino acid or a stop codon	28
splicing	mutation that affects mRNA splicing	3
regulatory	substitiution causing abnormal regulation	1
small deletion	micro deletion (<= 20 bp)	4
small insertion	micro insertions (<= 20 bp)	1
small indel	micro indels (<= 20 bp)	0
gross deletion	delition > 20 bp	2
gross insertions/duplications	insertion > 20bp	0
complex rearrangments	rearrangements of stretches of the DNA sequence	1
repeat variations	differences in repeat length	0

</figtable>

In total, we found 40 mutation in the public version of the database and 49 in the non-public version.

<figtable id="hgmd missense">

Table 2: 28 missense and nonsense mutations for HFE from the HGMD. All of them are disease causing.

accession number	codon change	aa change	codon number
CM032270	AGGc-AGC	Arg-Ser	6
CM091838	TTG-TGG	Leu-Trp	46
CM994469	cGTG-ATG	Val-Met	53
CM994470	cGTG-ATG	Val-Met	59
HM971246	CATg-CAC	His-His	63
CM960827	tCAT-GAT	His-Asp	63
CM990718	gAGT-TGT	Ser-Cys	65
CM033969	tCGC-TGC	Arg-Cys	66
CM020721	cCGA-TGA	Arg-Term	71
CM990719	aGGG-CGG	Gly-Arg	93
CM990720	ATT-ACT	Ile-Thr	105
CM990721	CAAg-CAC	Gln-His	127
CM091839	aGAC-AAC	Asp-Asn	129
CM091840	TACg-TAG	Tyr-Term	138
CM004810	gGAG-CAG	Glu-Gln	168
CM004106	gGAG-TAG	Glu-Term	168
CM004107	TGG-TAG	Trp-Term	169
CM015326	GCC-GTC	Ala-Val	176
CM081301	CTG-CCG	Leu-Pro	183
CM034097	CGG-CAG	Arg-Gln	224
CM101181	cCAG-TAG	Gln-Term	233
CM024530	tGTA-TTA	Val-Leu	272
CM994771	aGAG-AAG	Glu-Lys	277
CM960828	TGC-TAC	Cys-Tyr	282
CM004391	TGC-TCC	Cys-Ser	282
CM032271	CAG-CCG	Gln-Pro	283
HM030028	GTG-GCG	Val-Ala	295
CM990722	AGG-ATG	Arg-Met	330

</figtable>

The 28 missense and nonsense mutations for HFE are listed in <xr id="hgmd missense"/> together with the amino acid (aa) change and the codon number. They are all connected with the hemochromatosis phenotype.

dbSNP

dbSNP was searched for non-synonymous and silent (synonymous) mutations of the HFE gene. Silent mutations are mutations in the nucleotide sequence that do not lead to a change in the amino acid sequence of the protein.

<figtable id="dbSNP all">

Table 3: All 49 mutations in the HFE gene from dbSNP.

cluster ID	Function	codon number	codon pos	nucleotide change	aa change
rs149342416	missense	6	3	G -> C	Arg -> Ser
rs114758821	synonymous	7	3	G -> A	Pro -> Pro
rs368895240	synonymous	10	3	C -> T	Leu -> Leu
rs201657128	missense	14	1	C -> G	Leu -> Val
rs143662783	missense	17	2	C -> T	Thr -> Ile
rs148161858	missense	23	2	G -> A	Arg -> His
rs2242956	missense	35	2	T -> C	Met -> Thr
rs377254261	missense	37	2	C -> T	Ala -> Val
rs147297176	synonymous	58	3	C -> T	Phe -> Phe
rs147426902	synonymous	63	3	T -> C	His -> His
rs139523708	missense	67	2	G -> A	Arg -> His
rs62625342	synonymous	76	3	C -> T	Ser -> Ser
rs376650371	missense	97	3	G -> A	Met -> Ile
rs199988202	missense	106	2	T -> C	Met -> Thr
rs200706856	missense	129	1	G -> A	Asp -> Asn
rs201885016	missense	130	2	A -> G	Asn -> Ser
rs369790080	synonymous	132	3	C -> T	Thr -> Thr
rs372789940	missense	141	1	G -> A	Asp -> Asn
rs199879669	missense	157	1	G -> C	Ala -> Pro
rs145475682	missense	162	1	G -> T	Ala -> Ser
rs148480830	synonymous	162	3	C -> G	Ala -> Ala
rs144170531	missense	166	1	A -> G	Lys -> Glu
rs146519482	missense	168	1	G -> C	Glu -> Gln
rs199916850	missense	183	2	T -> C	Leu -> Pro
rs140957442	nonsense	192	1	C -> T	Gln -> [Te
rs4986950	missense	217	2	C -> T	Thr -> Ile
rs144797937	missense	224	1	C -> T	Arg -> Trp
rs62625346	missense	224	2	G -> A	Arg -> Gln
rs140515012	missense	245	1	C -> G	Pro -> Ala
rs150402693	missense	251	3	C -> A	Phe -> Leu
rs182920795	synonymous	253	3	T -> A	Pro -> Pro
rs202068193	missense	256	1	G -> A	Val -> Ile
rs143846467	missense	259	2	A -> G	Asn -> Ser
rs140080192	missense	277	1	G -> A	Glu -> Lys
rs369354634	synonymous	281	3	G -> A	Thr -> Thr
rs201310322	synonymous	292	3	C -> T	Pro -> Pro
rs143175221	missense	295	2	T -> C	Val -> Ala
rs114038675	synonymous	298	3	G -> A	Glu -> Glu
rs372856303	synonymous	301	3	G -> A	Pro -> Pro
rs147519426	missense	315	2	T -> G	Val -> Gly
rs148632352	synonymous	315	3	T -> C	Val -> Val
rs371192232	synonymous	317	3	C -> T	Val -> Val
rs141229562	missense	318	1	G -> A	Val -> Ile
rs150716212	missense	322	2	T -> C	Ile -> Thr
rs138993448	missense	327	2	T -> C	Ile -> Thr
rs368122334	missense	340	2	G -> C	Gly -> Ala
rs35201683	synonymous	342	3	C -> T	Tyr -> Tyr
rs370285936	missense	343	2	T -> A	Val -> Asp
rs146508927	missense	347	2	G -> A	Arg -> His

</figtable>

In total, we found 49 SNPs in the transcript variant 1 of the HFE gene. They are listed in <xr id="dbSNP all"/>. The column "Function" states if the mutation is synonymous or non-synonymous.

SNPdbe

35 mutations that are associated with the human HFE protein were found in SNPdbe.

<figtable id="exp evidence">

Table 4: Types of experimental evidence and their occurence among the 35 mutations associated with HFE.

exp. evidence	count
1000Genome,freq,cluster	6
by cluster	6
by cluster,freq	2
Not validated	13
by freq	8

</figtable>

Not all mutations have experimental evidence, over one third is not validated, see <xr id="exp evidence"/>.

<figtable id="snpdbe">

Table 5: Mutations in the HFE protein from SNPdbe.

dbSNP	Mutation	Disease association	Experimental evidence
rs1799945	H63D	In hereditary haemochromatosis (HH) (PMD)	1000Genome,freq,cluster
rs1800562	C282Y	hemochromatosis (SwissVar)	1000Genome,freq,cluster
rs1800730	S65C	hemochromatosis (SwissVar). In hereditary hemochromatosis patient who had resulted positive to screening for iron overload (PMD)	1000Genome,freq,cluster
rs2242956	M35T	N/A	by cluster
rs4986950	T217I	N/A	by cluster,freq
rs28934595	Q127H	hemochromatosis (SwissVar). In variegate porphyria (VP) (PMD)	by cluster
rs28934596	I105T	hemochromatosis (SwissVar). In hemochromatosis (PMD)	by cluster
rs28934597	G93R	hemochromatosis (SwissVar). In hemochromatosis (PMD)	by cluster
rs28934889	V53M	N/A	1000Genome,freq,cluster
rs62625346	R224Q	N/A	by cluster,freq
rs28934890	V59M	N/A	Not validated
rs111033558	R330M	hemochromatosis (SwissVar). In hereditary haemochromatosis (HH) (PMD)	by cluster
rs111033563	Q283P	hemochromatosis (SwissVar)	by cluster
rs149342416	R6S	hemochromatosis (SwissVar)	by freq
rs140080192	E277K	N/A	1000Genome,freq,cluster
rs143175221	V295A	hemochromatosis (SwissVar)	by freq
rs148161858	R23H	N/A	1000Genome,freq,cluster
N/A	M106T	N/A	Not validated
rs146519482	E168Q	N/A	Not validated
N/A	L183P	N/A	Not validated
rs138176635	E252G	N/A	Not validated
rs138993448	I327T	N/A	Not validated
rs139523708	R67H	N/A	Not validated
rs140515012	P245A	N/A	Not validated
rs141229562	V318I	N/A	Not validated
rs143662783	T17I	N/A	by freq
rs143846467	N259S	N/A	Not validated
rs144170531	K166E	N/A	by freq
rs144797937	R224W	N/A	by freq
rs145475682	A162S	N/A	by freq
rs146508927	R347H	N/A	by freq
rs147519426	V315G	N/A	by freq
rs149662565	P160T	N/A	Not validated
rs150402693	F251L	N/A	Not validated
rs150716212	I322T	N/A	Not validated

</figtable>

A list of the 35 mutations and their experimental evidence can be found in <xr id="snpdbe"/>.

OMIM

OMIM (Online Mendelian Inheritance in Man) also contains information about HFE, but only a small amount of all known mutations can be found.

<figtable id="omim">

Table 6: Mutations from OMIM that are associated with the HFE protein .

dbSNP accession	Phenotype	Mutation
rs1799945	HEMOCHROMATOSIS	HIS63ASP
rs1800730	HEMOCHROMATOSIS	SER65CYS
rs28934889	HFE POLYMORPHISM	VAL53MET
rs28934595	HEMOCHROMATOSIS	GLN127HIS
rs111033558	HEMOCHROMATOSIS	ARG330MET
rs28934596	HEMOCHROMATOSIS	ILE105THR
rs28934597	HEMOCHROMATOSIS	GLY93ARG
rs111033563	HEMOCHROMATOSIS	GLN283PRO

</figtable>

A complete list of the mutations can be found in <xr id="omim"/>. Two of the mutations are poylmorphisms of the HFE protein and 8 of them cause the disease hemochromatosis.

Databases comparison

<figtable id="comparison">

Table 6: Comparison of HGMD, dnSNP, SNPdbe and OMIM with respect to the date of the last update, the information contained in the databases and the number of entries.

database	last update	version	what information	where from	# entries homo sapiens	# HFE mutations
HGMD	spring 2013	public 2013.1 (mainly 3 year old data)	Collection of published gene lesions in the human genome that cause inherited diseases.	Only from publications. Journals are searched manually and by computational means each week.	99869	28 missense/nonsense
dbSNP	26.06.2012	Build 137	Short nucleotide sequence variations in different organisms (common and rare)	Submissions from laboratories but also private research companies.	192,678,553	10 synonymous, 41 non-synonymous, 10 disease causing SNPs and 162 SNPs in the UTR
SNPdbe	05.03.2012	-	Annotations for single amino acid substitutions (SAASs), e.g. functional effect (experimental, predicted), associated disease, evol. conservation,...	Based on entries from SwissProt, dbSNP, 1000 Genomes, PMD	967879	10 disease associated, 25 other
OMIM	daily	-	Compendium of human genes, genetic phenotypes and diseases. 3,035 genes with phenotype-causing mutations known.	Information from publications and databases is reviewed and summed up in texts by scientists.	21,934	2 8 disease causing mutations, 2 other

</figtable>

<xr id="comparison"/> contains a comparison of the four databases used in this task. It clearly shows that OMIM is most up to date and also the private version of HGMD is from 2013, but dbSNP, SNPdbe and execially the public HGMD have not been updated for over a year. dbSNP is very large and is the only database that also contains synonymous SNPs.

Mutation map

In total, we could collect a set of 72 point mutations from the different databases, containing missense, nonsense mutations and also synonymous SNPs. The complete list can be viewed in the Lab journal Task 7.

<figure id="mutation map">

Figure 1: Mutation map for the HFE protein. Disease causing mutations are marked in red and non disease causing mutations with blue. The protein sequence with the MHC antigen-recognition domain (green) and the Immunoglobulin domain (blue) are shown below. dc: disease causing

</figure>

<xr id="mutation map"/> shows the mutation map for HFE. Disease causing mutations (red) and non disease causing mutations (blue) are nearly equally distributed over the protein sequence. Nevertherless, there are some region where only dc mutation are located. Those regions are probably especially relevant for the protein function and can not be mutated without a dicrease in function.

<figtable id="mutations struc">

Figure 2: Visualisation of the location of the different mutations in the pdb structure for 1A6Z chain A (HFE). The structure is shown in three different orientations. The protein domains are coloured accoording to <xr id="mutation map"/> with the MHC antigen-recognition domain in green and the Immunoglobulin domain in blue. Disease causing mutations are marked in red and non disease causing mutations in grey.

</figtable>

In addition to the mutation map, <xr id="mutations struc"/> shows a 3D visualisation of the location of the mutated amin acids in the protein structure (1A6Z,A). Most disease causing mutations are located in secondary structure regions, but there are also some mutations that are located in loops. Therefore, it is difficult to tell which regions of the structure are important for the function when only looking at the the location of the mutations.

References

Stenson et al (2009), The Human Gene Mutation Database (HGMD®): 2008 Update. Genome Med 1(1):13
http://www.ncbi.nlm.nih.gov/books/NBK3848/
ftp://ftp.ncbi.nih.gov/pub/factsheets/Factsheet_SNP.pdf
https://www.rostlab.org/services/snpdbe/