SNP

molecular function, evolution and disease

Md Imtiyaz Hassan, Ph.D
Effect on mo|ecu|ar funct|on
Phenotype
Natura| se|ect|on
Hed|ca| Cenet|cs
8tructura| |o|ogy
|ochem|stry
Evo|ut|onary Cenet|cs
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Predicting the effect of mutations in proteins
hy is this useful?
Understanding variation in molecular
function and structure
Evolutionary genetics: comparison of
polymorphism and divergence rates between different
functional categories is a robust way to detect
selection
inkage analysis
Rare
lassical association studies
Control Disease
Common
"uant|tat|ve tra|t
endelists Biometricians
Forces to maintain
variation:
Selection
utation
ommon disease / ommon variant
Trade off (antagonistic pleiotropy)
Balancing selection
Recent positive selection
Reverse in direction of selection
Examples
POE Alzheimer`s disease
% Hypertension
P3 Hypertension
PA1 Type 2 diabetes
ndividual human genome is a target for
deleterious mutations !
~40 oI human Mendelian diseases are
due to hypermutable sites
Frequency oI deleterious variants is
directly proportional to mutation rate
(638
ultiple mostly rare variants
any deleterious alleles in mutation-selection
balance
Examples
Plasma level of HD-
Plasma level of D-
olorectal adenomas
Harmful mutations
Function: damaging
EvoIution: deIeterious
Phenotype: detrimentaI
dvantageous
pseudogenization (Zhang et
al. 2006)
Gain of function disease
mutations
Sickle Cell nemia

N E V T T A R G F S - P K D V V R
R E S A T T V T G F S - P A D V F V Q
G G S R S V A S G T - F S G Y D Q V
T P G T T T V S G F S - S S Y D G V
G Q K A K R P E - - - - K G H P V V F Y
G Q E A T E P - - - - S G H S A V F Y
G Q Q V T S F P - - - - S G H S Y Y
R K D V S T V V G F N - P G D S V E T
G Q K T K Q Q N - - - - F N H D T Y Y
R D K A T F T F V V G S D - K D A H T E
S K S A T T R V S N V N A D G E V S
G A R T S N T F S D - - - S A S Q Y F Y
G A S Q R K Y S Y - - - S A T P Y F Y
N G A P K T V V D E S E K N V N V T N
E A T V T T V V S N - - A P Y G V N V S T

Profile



Ia -1.2 1.1 -0.6 -0.8 0.3 ... ...
rg 0.6 -0.3 -0.3 -0.5 0.6 ... ...
sn -1.1 -0.5 -0.5 -0.7 0.4 ... ...
sp -0.9 -0.3 -0.3 -0.5 0.6 ... ...
Cys 0.4 -0.5 0.6 0.8 -0.3 ... ...
GIn ... ... ... ... ... ... ...
... ... ... ... ... ... ... ...

protein
muItipIe aIignment
profiIe
PolyPhen
Prediction rate of damaging
substitutions
possibly probably
Disease mutations
Divergence
82 57
9 3
Polymorphism
27 15
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10 of PolyPhen false-positives are due to
compensatory substitutions
Neutral mutation model
uman ACCTTGAAAT
Chimpanzee ACCTTAAAAT
Baboon ACCTTAAAAT
!rob(TC->TGC) !rob(TGC->TC)
!rob(XY
1
Z->XY
2
Z) 64x3 matrix
Strongly detrimental mutations
Effectively neutral mutations
ildly deleterious mutations
ildly deleterious mutations
54 genes, 757 individuals
inflammatory response
236 genes, 46-47 individuals
DNA repair and
cell cycle pathways
518 genes, 90-95 individuals
Fitness and selection coefficient
iId type New mutation

1
= 4

2
= 3
Fitness
1
N
1
N
2
= 1 - s
SeIection coefficient
lassical association studies
Control Disease
Common
Cenet|c po|ymorph|sm
0enefic PoIymorphism: A difference in DMA sequence omong
individuoIs, groups, or popuIofions.
0enefic Mufofion: A chonge in fhe nucIeofide sequence of o
DMA moIecuIe.
0enefic mufofions ore o kind of genefic poIymorphism.
SingIe nucIeotide
PoIymorphism
{point mutution}
Repeut heterogeneity
Senetic Vuriution
8NP
8|ng|e Nuc|eot|de Po|ymorph|sms
A SingIe MucIeofide PoIymorphism is o source vorionce in o genome.
A SMP ("snip") is o singIe bose mufofion in DMA.
SMPs ore fhe mosf simpIe form ond mosf common source of
genefic poIymorphism in fhe humon genome (907 of oII humon DMA poIymorphisms).
There ore fwo fypes of nucIeofide bose subsfifufions resuIfing in SMPs:
-%runsition: subsfifufion befween purines (A, 0) or befween
pyrimidines (C, T). Consfifufe fwo fhirds of oII SMPs.
-%runsversion: subsfifufion befween o purine ond o pyrimidine.
8NP
nsteud of using restriction enzymes these ure found by direct sequencing
%hey ure etremeIy usefuI for mupping
Murkers
CIussicuI MendeIiun 100
RFLPs 7000
SNPs 1,410

-----------------------CGGCT
-----------------------TGGCT
SNPs occur every 300-1000 bp uIong the 3 biIIion Iong humun genome
Muny SNPs huve no effect on ceII function
Human Genome and SNPs
W uman genome is (mostly sequenced, attention turning to
the evaluation oI variation
W Alterations in DNA involving a single base pair are called
single nucleotide polymorphisms, or SNPs
W Map oI ~1.4 million SNPs (Feb 2001
W t is estimated that ~60,000 SNPs occur within exons
Goals of SNP nitiatives
W mmediate goals:
Detection/identiIication oI all SNPs estimated to be
present in the human genome
nterest also in other organisms, e.g. potatoes(!
Establishment oI SNP Database(s
SNPs
umons ore geneficoIIy 99 per cenf idenficoI: if is fhe
finy percenfoge fhof is differenf
Much of our genefic voriofion is coused by singIe-nucIeofide
differences in our DMA : fhese ore coIIed singIe nucIeofide
poIymorphisms, or SMPs.
As o resuIf, eoch of us hos o unique genofype fhof fypicoIIy differs in obouf fhree miIIion nucIeofides
from every ofher person.
SMPs occur obouf once every 300-I000 bose poirs in fhe genome, ond fhe frequency of o porficuIor
poIymorphism fends fo remoin sfobIe in fhe popuIofion.
8ecouse onIy obouf 3 fo b percenf of o persons DMA sequence codes for fhe producfion of profeins,
mosf SMPs ore found oufside of "coding sequences".
onger term goals: Areas oI SNP
Application
W ene discovery and mapping
W Association-based candidate polymorphism testing
W Diagnostics/risk proIiling
W #esponse prediction
W omogeneity testing/study design
W ene Iunction identiIication etc.
Polymorphism
W Technical deIinition: most common variant (allele occurs
with less than 99 Irequency in the population
W Also used as a general term Ior variation
W Many types oI DNA polymorphisms, including #FPs,
VNT#s, micro-satellites
W ighly polymorphic` many variants
SNPs in Genetic Analysis
W Abundance lots
W Position throughout genome
W aplotype patterns groups oI SNPs may provide
exploitable diversity
W #apid and eIIicient to genotype
W ncreased stability over other types oI mutation
W #ecombination patterns e.g. hot spots`
od|ng Reg|on 8NPs
Occos|ono||y, o SNF moy octuo||y couse o d|seose.
SNFs w|th|n o cod|ng sequence ore o| port|cu|or |nterest to
reseorchers becouse they ore more ||ke|y to o|ter the b|o|og|co|
|unct|on o| o prote|n.
W%ypes of cod|ng reg|on 8NPs
8ynonymous: the subst|tut|on causes no am|no ac|d change to the prote|n |t produces. %h|s |s a|so ca||ed a
s||ent mutat|on.
Non-8ynonymous: the subst|tut|on resu|ts |n an a|terat|on of the encoded am|no ac|d. A m|ssense mutat|on
changes the prote|n by caus|ng a change of codon. A nonsense mutat|on resu|ts |n a m|sp|aced term|nat|on.
0ne ha|f of a|| cod|ng sequence 8NPs resu|t |n non-synonymous codon changes.
ntergen|c 8NPs
% kesearchers have found that most SNs are not respons|b|e for a d|sease state because they are |ntergen|c
SNs
% Instead they serve as b|o|og|ca| markers for p|npo|nt|ng a d|sease on the human genome map because
they are usua||y |ocated near a gene found to be assoc|ated w|th a certa|n d|sease
% Sc|ent|sts have |ong known that d|seases caused by s|ng|e genes and |nher|ted accord|ng to the |aws of
Mende| are actua||y rare
% Most common d|seases ||ke d|abetes are caused by mu|t|p|e genes I|nd|ng a|| of these genes |s a d|ff|cu|t
task
% kecent|y there has been focus on the |dea that a|| of the genes |nvo|ved can be traced by us|ng SNs
% 8y compar|ng the SN patterns |n affected and nonaffected |nd|v|dua|spat|ents w|th d|abetes and
hea|thy contro|s for examp|esc|ent|sts can cata|og the spec|f|c DNA var|at|ons that under||e suscept|b|||ty
for d|abetes
PoIymorphic Sifes PeveoIed in Sequencing PoIymorphic Sifes PeveoIed in Sequencing
edium- and Low-throughput SNP Genotyping
. S! Discovery and validation.
. Data base mining, "resequencing on microarrays, de novo
sequencing of EST libraries.
B. Genotyping of pooled samples for determining heterozygosity.
. How many S!s are to be typed in how many samples?
. What degree of multiplexing is possible for the before-typing !CR
reactions?
B. What degree of multiplexing is possible for the genotyping
reactions?
. What is the appropriate platform given the size of the project, the
budget and the degree of automation desired?
uIy Z003 NC buiId 34
Ped ~ of Ieosf I SMP per I00 kb
8Iock ~ 0ops in genome coveroge
9Z% of genome within 100kb of u SNP
3% of genome within 0 kb of u SNP
0% of genome within 1 kb of u SNP
Z% of genome within kb of u SNP
Mopping I00I Coveroge: IIo,Z04 SMPs
Chemistry/DemuItipIeing/Detection Options in SNP Senotyping
AIIeIe-Specific
ybridi;ofion
AIIeIe-Specific
Exfend + Ligofe
AIIeIe-Specific
PCP
Sequenom iPIex
TM
Moss Spec.
"DAS,
AmpIicon T
m
FIuor Pes Energy
Tronsfer-FPET
Luminex I00 FIow
Cyfomefry
SingIe MucIeofide
Primer Exfension
OIigonucIeofide
Ligofion Assoy
CopiIIory
EIecfrophoresis
omogeneous
Semi-omogen.
FIuorescence
SoIid phose
microorroy
SoIid phose
microspheres
Moss
Specfromefry
A8I SMPIex
TM
A8I SMoPShof
TM
FIuorescence
PoIori;ofion
Microorroy
Minisequencing
Perkin-EImer
FP-TDI
A8I Toqmon
TM
b'-MucIeose
IIIumino
8eodArroy
TM
En;yme Chemisfry DemuIfipIexing Defecfion Mefhod PIofform/Compony

T
T
C
C
ddC-biot or dd-biot

T

T

Single Base !rimer Extension,
"Minisequencing
llele-specific
!rimer Extension
llele-specific !rimer
Extension and Ligation
llele-specific
Hybridization
T

T

LSO
!robes
SBE !rimer

Short GC
T

G
C
Long GC
!CR only: T
m
-shift
!rimers
Enzymatic Options in SNP Genotyping
dd-biot, dT!, dTT!, dGT!
SNP Senotyping on euds/Microurruys
8e|ect|on of 8NPs
0es|gn of PR and "%ag"
8E|A8PE pr|mers
Preparat|on of beads w|th "Ant|-
%ag" pr|mers
Hu|t|p|ex PR
yc||c 8E|A8PE w|th b|ot(f|uor.}-
ddN%P|dN%P
apture of products on
beads
8|gna| measurement |n f|ow
cytometer|scanner
Pastinen, et al., en. #es. 7,
606, 1997
SingIe 8ose Exfension (S8E) of Torgefs on Microorroys SingIe 8ose Exfension (S8E) of Torgefs on Microorroys
S8E (Minisequencing) of Torgef DMA wifh S8E (Minisequencing) of Torgef DMA wifh
0Ioss 0Ioss--immobiIi;ed primers immobiIi;ed primers
llele llele- -Specific Extension & dentification in CE: Specific Extension & dentification in CE:
"Minisequencing (B Sa!Shot "Minisequencing (B Sa!Shot
TM TM
) )
dR6G
dR110
Degree of Multiplexing Depends on Resolution in CE Degree of Multiplexing Depends on Resolution in CE
AB SNaPshot

on 3130xl
0en. Pes. 9: 49Z, I999
Fluorescence Polarization
0en. Pes. 9: 49Z, I999
S8E (Minisequencing) wifh Defecfion by FIuorescence PoIori;ofion S8E (Minisequencing) wifh Defecfion by FIuorescence PoIori;ofion
!CR mplification
Single Base Extension
S! Treatment
MLD-TOF Mass Spec
Spot on 384-place Chips
Genotyping by SBE and Mass Spectrometry Genotyping by SBE and Mass Spectrometry
AIIeIe AIIeIe--specific Primer Exfension (ASPE) wifh Choin specific Primer Exfension (ASPE) wifh Choin
Terminofion Terminofion
&se of AIIeIe &se of AIIeIe- -specific Probes in Senotyping by MeIting Curve specific Probes in Senotyping by MeIting Curve
AnuIysis: "DASH" AnuIysis: "DASH"
ne base
mismatch
Matched
eterozygote
Nuture iotech, 17: 7 1999
ntercalating dye
ong, ef oI., 8iofechniques 39: 88b, Z00b
Use of Modified T Use of Modified T
mm
--shiffing Primers in 0enofyping shiffing Primers in 0enofyping
8eod Arroys: DMA immobiIi;ed on siIico or poIysfyrene beods, rondom orroy requires
decoding sfeps.
I) Lynx (www.Iynxgen.com). In rows. Limifed fo co. Z0 boses/reod.
Z) IIIumino 8eodChip (www.iIIumino.com). In efched microweIIs.
3) Luminex coded microspheres (Iuminexcorp.com). Meosuremenfs by fIow
cyfomefry.
4) 4b4 LifeSciences (www.4b4.com). CIonoI ompIificofion ond sequencing on Z8 p
beods. Minimum I00 boses/reod.
eud %echnoIogies for SNP Senotyping/Sene eud %echnoIogies for SNP Senotyping/Sene
Epression und MussiveIy PuruIIeI Sequencing Epression und MussiveIy PuruIIeI Sequencing
(nof currenfIy supporfed in CIF) (nof currenfIy supporfed in CIF)
Lynx/SoIexo 8eod Arroys for 0ene Expression ond MPSS Lynx/SoIexo 8eod Arroys for 0ene Expression ond MPSS
CIones on 8eods
8renner ef oI., PMAS 97: Ioob, Z000, ond
Mofure 8iofech. I8: o30, Z000
Seporofe Iooded from unIooded beods
(FACS), Iigofe fo onfi-fog.
I.8 x I0
Ib
unique Togs
fog
CompefifiveIy hybridi;e beods wifh
IobeIed Iibrories, fhen sorf by
FACS, OP,
Sequence signofures wifh fype IIs
res. en;. & IobeIed, encoded
odopfors.
Expression profiIing wifh IIIumino 8eodChips in MicroweIIs Expression profiIing wifh IIIumino 8eodChips in MicroweIIs
0en. Pes. I4: 870 & Z347, Z004
TofoI sefup cosfs, sofeIIife
fociIify $o000.
umonPef-8: Z4k probes,
$I00/sompIe, $b0 IobeIing.
Pondom Iooding of beods in
efched 3 pm microweIIs
Decoding by SequenfioI
hybridi;ofion: II0IZZ0Z. 3
8
~ oboI
codes. (4
8
~ ob,b3o)
b'
3'
IIIumino AIIeIe Specific IIIumino AIIeIe Specific
Primer Exfension (ASPE) ond Primer Exfension (ASPE) ond
Ligofion Ligofion
ASOs ond LSOs
Cy3 ond Cyb-IobeIed
universoI primers
Luminex coded microspheres ond muIfipIexed ossoys Luminex coded microspheres ond muIfipIexed ossoys
0reen Ioser: Up fo I00 differenf fronscripfs con be monifored simuIfoneousIy in high-fhroughpuf
by fIow cyfomefry, e.g., wifh "PP genes in Arobidopsis, 0en. Pes. II: I888, Z00I ond ZI7 miPMAs
in humon concers, Mofure 43b: 834, Z00b.
Ped Ioser: Coding is in rofio of red ond oronge fIuorescence inside microsphere.
SMP 0enofyping Cosfs by PIofform SMP 0enofyping Cosfs by PIofform
PIofform #SMPs/
sompIe
# sompIes $OIigo
Sef/$SMP
$Mix/SMP $ per SMP Min $
IIIumino (UCLA) Ib3o 488 0.09 o9,89Z
A8 SMPIex (A8I
3730)
48 b000
b00
7Z/0.0I44
7Z/0.0I44
0.04
0.Z0
0.078
0.ZI4
I4,840
A8 SMoPshof
(A8I 3I00)
b0 b00 b0/0.I0 0.47o 0.b7o I4,400
A8 Toqmon (A8I
7700)
I 7b0 3I0/0.4I3 0.7b I.ZI 9I0
AIIeIe-specific
PCP
b0 b000
b00
I7.o0/0.003b
I7.o0/0.03b
0.4ZZ 0.43
S.-H. Lcc ct a!., Thcov. App!. Gcnct. IIo:I, ioo