Genetic and Molecular Epidemiology

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Genetic and Molecular Epidemiology
Lecture III Molecular and Genetic Measures Jan
20, 2009 Joe Wiemels AC-34 (Parnassus) 514-0577 j
oe.wiemels_at_ucsf.edu
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Lecture
Review of the structure of genes genetic
variation and mutation PCR (polymerase chain
reaction) and DNA amplification
methods Detection of mutations and
polymorphisms low and high throughput
techniques Other genetic markers
microsatellites Microarray techniques SNPs,
gene expression, etc.
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Gene structure
Each cell has two chromosomes, therefore there
are two physical copies of each gene. The
position of a gene is called a locus, and the
exact form of the gene is called an allele. Each
gene can exist in the form of two alleles
1378 genes on chromosome 7, 159,000,000 base
pairs.
TAS2R38 gene (PTC TASTE RECEPTOR)
Chromosome 7
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Anatomy of a Gene
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Expression blots and arrays Exon splice
variants microRNA
Detection with antibodies, mass spec, proteomics
Genotyping Mutations Chromosomes
DNA RNA PROTEIN
Translation
Transcription
Occurs in the nucleus
Occurs in the cytoplasm
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Mutation
DNA variation
A gene variant limited to less than 1 of the
population, or in one family line only, or even
in one cell (when talking about cancer)
Single Nucleotide Polymorphism (SNP) A single
position in the human genome where more than one
type of nucleotide (a variant) is prevalent in
the population (at least 1 or 5 prevalence
dependent on your purposes). These average 1 in
1000 base pairs or 3 million common SNPs per
genome.
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Where are mutations/polymorphisms likely to have
an effect on gene function and disease?

• Promoter region affect expression levels
• Coding region (exons) affect protein structure
• Exon splice sites affect protein structure
• Mutations in genes that control the expression of
  other genes can have profound effects on
  expression.

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Gene promoters are 500-2000 bp upstream from
the coding region.
Promoter of the human CDKN1B gene
Transcription factors are proteins which bind
within promoters and control gene expression.
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Mutations in Exons can result in altered protein
structure
Example of a missense base pair change
M P N S G N V
ATGTTTAATAGCGGTAATGTT ATGTTTAATAGCAGTAATGTT
M P N S S N V
This is a G-gtA SNP which results in a missense
mutation
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Classes of SNPs in coding sequences
(exons) Synonymous (silent) substitutions Do
not cause amino acid change (but still may be
functional) Nonsense mutation cause the
formation of a stop codon (ie., TAA or TGA, and a
truncated protein (completely disables a
protein) Nonsynonymous (missense) mutations
cause change of amino acid, but may be
conservative (like for like amino acid) or
nonconservative (dissimilar amino acid)
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Examples of diseases caused by.
Missense mutation/polymorphism cause variable or
loss of function sickle cell anemia
(hemoglobin) HBB - GAG-GTG in 20th
codon thrombosis, early miscarriage, heart
disease, colon cancer MTHFR (30 carrier
frequency) ALS SOD1, breast cancer -
BRCA1/2 Nonsense mutation/polymorphism cause
complete loss Duchene Muscular
dystrophy dystrophin, DMD Cystic Fibrosis
CFTR
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Examples of diseases caused by.
Synonymous mutation/polymorphism affects
expression or protein folding infantile spinal
muscular atrophy UBE1 (reduction in
expression) hearing loss TECTA (exonic splice
enhancer) Promoter/enhancer mutation/polymorphism
affects expression Drug and caffeine
metabolism CYP3A4 thymidine and folate
metabolism TK activation of compounds in bone
marrow/granulocytes MPO
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A disease has a genetic component, what do you do
now?

No idea of the gene whole genome scan of genetic
markers SNPs or microsatellites Fair idea of
the gene candidate gene SNPs at medium
throughput You know what the gene is, but no
idea of the genetic alteration DNA sequencing.
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Genetic Markers
Polymorphic variation scattered around genome
used to help identify disease genes. Most
genetic variation is non-functional, but may be
physically linked to a functional genetic
element. Genetic markers may segregate with a
disease
Microsatellites (a million per genome) Single
Nucleotide Polymorphisms (3 million in
genome)
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A common genetic marker
Microsatellite (aka STS, sequence tagged site)
highly polymorphic DNA sequence feature (not
functionally polymorphic). A simple repeat
sequence that invites slippage-mispair during
replication, and hence many polymorphic
variations in size in the population.
DNA sequence, showing alternating ACACACAC
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Microsatellites are diagnosed by size
Every individual has 2 alleles
Individual people

nearly everyone is heterozygous
Size of DNA fragment
3 separate microsatellite polymorphisms analyzed
in multiplex
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Single Nucleotide Polymorphism
For usefulness as a genetic marker, it should be
common (gt5 allele frequency) Only two variants,
so much less information per test than a
microsatellite Whole genome disease scan
requires far more tests than microsatellite, but
each test is far less expensive
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How do we test for genetic variants?Many
Genetic Analyses begin with PCR
Polymerase Chain Reaction (PCR) specific
amplification of a single gene sequence 2
synthetic oligonucleotides can find their
complementary DNA sequences among 3 billion
nucleotide sequence. Able to faithfully amplify
a specific sequence 1030 times.
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Testing for functional SNsP (background)
Alleles different forms of a gene at the same
locus.
TAS2R38 3 polymorphisms C145G (G variant at
position 145) P49A C785T (T variant at
position 785) V262A G886A (A variant at
position 886) V296I wild type (C, C, and G at
each position, respectively)
WT
8 potential haplotype alleles based on 3 SNPs
P49A
V262A
V296I
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Genotyping in MGE TICR Individuals

1. Get genomic DNA from subject (buccal cell
   demonstration in class)
2. Isolate DNA on Autogen 3000
3. Lyses cells with detergent and digests protein
   with Proteinase K
4. Removes protein with Phenol
5. Concentrates DNA using ethanol precipitation,
   rehydrates DNA in buffered water.

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Genotyping in MGE - TICR Individuals (continued)
Purified genomic DNA will be amplified in the
region of the polymorphisms, then a readout
performed
PCR amplification is a standard method, but there
are many methods to read the polymorphism
Cellular DNA is 3 X 109 base pairs, a gamish of
sequence but only a few copies of the gene of
interest
Two PCR primers (oligonucleotides) will be able
to make billions of copies of one small segment,
crowding out the rest of the genomic DNA
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PCR design for TAS2R38 polymorphism
These probes are used to diagnose the SNP.
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PCR protocol 10 ng of DNA mixed with 10 pmoles
each PCR primer 1 pmoles each probe 2.5 umoles
each dNTP Reaction buffer (salts including
MgCl2) Taq polymerase (thermostable DNA
polymerase) The temperature of the mixture is
cycled 35 times 65 degrees 30 seconds 72
degrees 30 seconds 94 degrees 15 seconds
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Detection of PCR products using Electrophoresis
gel.
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individuals
PCR product

PCR products for a SNP are all the same size
this gel is not diagnostic for the SNPs
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Taqman allelic discrimination genotyping (for
taste receptor TASR32)
There are four oligonucleotides in the reaction
mix -- two PCR primers and two probes each
labelled different color and each matching
different SNP allele.
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Taqman Genotyping - Real-time PCR
hets
homozygotes
homozygotes
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DNA sequencing the method to obtain the genotype
of a new mutation (for example, in a cancer
family)
Prior to sequencing, one first amplifies a
sequence by PCR or cloning in a bacterial vector.
Then, using ONE primer, adds fluorescent labeled
dideoxy chain terminators and DNA polymerase.
ddNTPs will cap the sequence.
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DNA sequencing
The products of the sequencing reaction are
separated on a gel mixture that can separate
fragments by one base pair.
Larger fragments
Smaller fragments
Useful when you suspect a gene, but dont know
the variant. This one is BRAF gene in leukemia
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Many genotyping platforms available today
Taqman genotyping Low throughput Fluorescence
Polarization (Pui Kwok) Low Luminex medium
Massive parallel genotyping High throughput,
useful for whole genome scans Affymetrix Illumi
na Ultradeep or next generation sequencing
Illumina (Solexa), Applied Biosystems, 454 (Roche)
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Illumina GoldenGate technologyfor 384-6000 SNPs
at a time (medium, not whole genome)
96-well plate, each with bead array
45,000 beads
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Illumina Infinium assay up to 1 million SNPs
(for whole genome study)
Bead array on slide
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(No Transcript)
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Microarray basics

• Some Applications for Microarray
• SNP genotyping (eg Affymetrix, Illumina)
• Gene expression patterns - comparing one tissue
  to another (Affymetrix, Superarray, etc)
• Gene deletion or amplification arrayCGH (for
  cancer applications, Albertson and Pinkel, UCSF)
• microRNA (UCSF Gladstone, Ambion)
• Pathogen identification (DeRisi, UCSF)

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Types of Microarrays
Spotted (early technology) cDNA (for expression,
100s - 1000s bases) oligonucleotide (less than
100 bp) BAC clone (100-200,000 bases, for
array-based comparative genomic
hybridization) Chemically synthesized
oligonucleotides (Affymetrix, NimbleGen,
Agilent) expression gene resequencing SNP
genotyping array-based CGH
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Spotted microarray for gene expression (oligos or
cloned genes)
The microrarray may have immobilized
oligonucleotides (eg., virochip, UCSF) or cloned
genes
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Affymetrix arrays have 25 bp oligonucleotides,
very short, but massive parallel probes for
redundancy. One color array.
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The virochip (UCSF) is a spotted microarray.
Hybridization of a clinical RNA (cDNA) sample can
identify specific viral expression
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Gene Expression of Breast Cancer predicts
disease-free outsome (Nature 2002 Friend et al)
Figure 2 Supervised classification on prognosis
signatures. a, Use of prognostic reporter genes
to identify optimally two types of disease
outcome from 78 sporadic breast tumours into a
poor prognosis and good prognosis group (for
patient data see Supplementary Information Table
S1). b, Expression data matrix of 70 prognostic
marker genes from tumours of 78 breast cancer
patients (left panel). Each row represents a
tumour and each column a gene, whose name is
labelled between b and c. Genes are ordered
according to their correlation coefficient with
the two prognostic groups. Tumours are ordered by
the correlation to the average profile of the
good prognosis group (middle panel). Solid line,
prognostic classifier with optimal accuracy
dashed line, with optimized sensitivity. Above
the dashed line patients have a good prognosis
signature, below the dashed line the prognosis
signature is poor. The metastasis status for each
patient is shown in the right panel white
indicates patients who developed distant
metastases within 5 years after the primary
diagnosis black indicates patients who continued
to be disease-free for at least 5 years. c, Same
as for b, but the expression data matrix is for
tumours of 19 additional breast cancer patients
using the same 70 optimal prognostic marker
genes. Thresholds in the classifier (solid and
dashed line) are the same as b. (See Fig. 1 for
colour scheme.)
NOW A CLINICAL ASSAY!! ONCOTYPE
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ONCOTYPE routine at Kaiser