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Transcript of Author(s): David Ginsburg, M.D., 2012 License: Unless otherwise noted, this material is made...
Author(s): David Ginsburg, M.D., 2012
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DNA Sequence Variation
David Ginsburg, MD
M1 Patients and Populations
Fall 2012
Relationships with IndustryUMMS faculty often interact with pharmaceutical, device, and biotechnology companies to improve patient care, and develop new therapies. UMMS faculty disclose these relationships in order to promote an ethical & transparent culture in research, clinical care, and teaching.•I am a member of the Board of Directors for Shire plc.•I am a member of the Scientific Advisory Boards for Portola Pharmaceuticals and Catalyst Biosciences.•I benefit from license/patent royalty payments to Boston Children’s Hospital (VWF) and the University of Michigan (ADAMTS13).
Disclosure required by the UMMS Policy on Faculty Disclosure of Industry Relationships to Students and Trainees .
• Understand the meaning of DNA sequence and amino acid polymorphisms.
• Recognize the different types of DNA sequence polymorphisms:– STR, SNP, CNV
• Know the different classes of DNA mutation:– Point mutations (silent, missense, nonsense, frameshift,
splicing, regulatory) insertion/deletions, rearrangements
• Understand how to distinguish a disease-causing mutation from a neutral DNA sequence variation
Learning Objectives
Chromosomes, DNA, and Genes
CellCellNucleusNucleus
ChromosomesChromosomes
Gene
ProteinProtein
Adapted from Adapted from ASCO teaching slidesASCO teaching slides
TRANSCRIPTION
Other specificpromoter elements
e.g. CACCC in -globin“CAP SITE”
Transcriptionstart site
“CCAAT” Box
“TATA” Box
ATGInitiation Codon
GT GTAG AG
GU GUAG AG
TissueSpecificelementsEnhancer
GENE
TAA, TGA, or TAGstop codon
AATAAAPolyadenylationsignalSite for addition of (A)n
mRNA PECURSOR
MATURE mRNA
5’ untranslated
region
3’ untranslated
region
INTRON 1 INTRON 2
3’AAAAACAP
5’
AAAAACAP
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 5.1
1944DNA is the
genetic material
1949Abnl Hemoglobin
in sickle cell anemia
1953Double helix
1956Glu 6 Val in sickle hemoglobin
1966Completion of the
genetic code
1970First restriction
enzyme
1972Recombinant
plasmids
1975Southern blotting
1981Transgenic mice
1983Huntington
Disease gene
mapped
1985PCR
1986Positional
cloning (CGD, muscular
dystrophy, retinoblastoma
1945 1950 1955 1960 1965 1970 1975 1980 1985 1990 1995 2000
1987Knockout
mice
1989Positional cloning without deletion (CF)
1990First NIH-approved gene therapy experiment
1996Complete yeast genome sequence
19951st complete bacterialgenome sequence
2001Draft human
genome sequence
Since 2001:Complete human genome (~100 individual genomes, 1000 genomes in progress) Growing index of human variation: human hapmap, dbSNP ; 1000 genomesComplete genomes of >6500 other species
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 5.4
DNA Samples and PCR
Blood sampleBlood sample
DNA for analysisDNA for analysischeek swabcheek swaburine sampleurine sampleForensic specimensForensic specimens
DNADNARepeat Repeat cyclescycles
Amplified Amplified productproduct
Target Target sequencesequence
primersprimers
Hybridization array: gene chipHybridization array: gene chip
DNA chip v6.0:
~1 million SNPs
RNA expression:
All ~ 20,000 genes
CGH:
Survey whole genome for large deletions, insertions, rearrangements
Ricardipus (wikipedia)
Image of DNA sequencing
process removed
DNA Sequencing
New Sequencing Technologies
• Understand the meaning of DNA sequence and amino acid polymorphisms.
• Recognize the different types of DNA sequence polymorphisms:– STR, SNP, CNV
• Know the different classes of DNA mutation:– Point mutations (silent, missense, nonsense, frameshift,
splicing, regulatory) insertion/deletions, rearrangements
• Understand how to distinguish a disease-causing mutation from a neutral DNA sequence variation
Learning Objectives
DNA Sequence Variation
• DNA Sequence Variation:– Human to human: ~0.1% (1:1000 bp)
• Human genome = 3X109 bp X 0.1% =~3X106 DNA common variants
– Human to chimp: ~1-2%– More common in “junk” DNA: introns, intergenic regions
• poly·mor·phismPronunciation: "päl-i-'mor-"fiz-&mFunction: noun: the quality or state of existing in or assuming different forms: as a (1) : existence of a species in several forms independent of the variations of sex (2) : existence of a gene in several allelic forms (3) : existence of a molecule (as an enzyme) in several forms in a single species
MutationsA mutation is a change in the “normal” base pair sequence
• Can be:– a single base pair substitution– a deletion or insertions of 1 or more base pairs (indel)– a larger deletion/insertion or rearrangement
Adapted from Adapted from ASCO teaching slidesASCO teaching slides
Polymorphisms and Mutations• Genetic polymorphism:
– Common variation in the population:• Phenotype (eye color, height, etc)• genotype (DNA sequence polymorphism)
– Frequency of minor allele(s) > 1%
• DNA (and amino acid) sequence variation:– Most common allele < 0.99 = polymorphism
(minor allele(s) > 1%)– Variant alleles < 0.01 = rare variant
• Mutation-- any change in DNA sequence– Silent vs. amino acid substitution vs. other– neutral vs. disease-causing– 1X10-8/bp/generation (~70 new mutations/individual)
• balanced polymorphism= disease + polymorphism
• Common but incorrect usage: – “mutation vs. polymorphism”
Common but incorrect usage:
“a disease-causing mutation” OR “a polymorphism”
All DNA sequence variation arises via mutation of an ancestral sequence
“Normal”
“Disease”
< 1% > 1%
Rare variant or “private”
polymorphismpolymorphism
Disease mutationExample: Factor V Leiden
(thrombosis) 5% allele frequency
• Understand the meaning of DNA sequence and amino acid polymorphisms.
• Recognize the different types of DNA sequence polymorphisms:– STR, SNP, CNV
• Know the different classes of DNA mutation:– Point mutations (silent, missense, nonsense, frameshift,
splicing, regulatory) insertion/deletions, rearrangements
• Understand how to distinguish a disease-causing mutation from a neutral DNA sequence variation
Learning Objectives
Types of DNA Sequence Variation• RFLP: Restriction Fragment Length Polymorphism• VNTR: Variable Number of Tandem Repeats
– or minisatellite– ~10-100 bp core unit
• SSR : Simple Sequence Repeat – or STR (simple tandem repeat) – or microsatellite– ~1-5 bp core unit
• SNP: Single Nucleotide Polymorphism– Commonly used to also include rare variants (SNVs)
• Insertions or deletions– INDEL – small (few nucleotides) insertion or deletion
• Rearrangement (inversion, duplication, complex rearrangement)– CNV: Copy Number Variation
STR
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 5.22
SNP
• Most are “silent”• Intragenic• Promoters and other regulatory sequences• Introns• Exons
– 5’ and 3’ untranslated regions– Coding sequence (~1-2% of genome)
Allele 1Allele 1 A U G A A G U U U G G C G C A U U G A AC
Allele 2Allele 2 A U G A A G U U U G G U G C A U U G A AC
Human Chromosome 4
2010
• 23,653,737 total human entries in dbSNP
http://www.ncbi.nlm.nih.gov/projects/SNP/
• Chromosome 4– 4,311,728 SNPs
• ~1M SNP chip commercially available
1981 1991 1994 1996
3 markers 53 markers 393 markers 791 markers
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 10.3
Copy Number Variation (CNV)• Kb to Mb in size (average ~250 Kb)• >>2000 known, affect ~12% of human genome• ? ~100 / person• ? Role in human disease/normal traits
Nature 444:444, Nov 2006.
• Understand the meaning of DNA sequence and amino acid polymorphisms.
• Recognize the different types of DNA sequence polymorphisms:– STR, SNP, CNV
• Know the different classes of DNA mutation:– Point mutations (silent, missense, nonsense, frameshift,
splicing, regulatory) insertion/deletions, rearrangements
• Understand how to distinguish a disease-causing mutation from a neutral DNA sequence variation
Learning Objectives
Silent Sequence Change(Synonymous SNP)
NormalNormalmRNAmRNA
ProteinProtein
A U G
Met
A A G
Lys
U U U
Phe
G G C
Gly
G C A
Ala
U U G
Leu
A A
Gln
C
Sequence Sequence variantvariant
mRNAmRNA
ProteinProteinA U G
Met
A A G
Lys
U U U
Phe
G G U
Gly
G C A
Ala
U U G
Leu
A A
Gln
C
G
Changes that do not alter the encoded amino acidChanges that do not alter the encoded amino acid
Adapted from Adapted from ASCO teaching slidesASCO teaching slides
MissenseMissense
Missense: changes to a codon for another amino acid Missense: changes to a codon for another amino acid
(can be harmful mutation or neutral variant(can be harmful mutation or neutral variant))
mRNAmRNA
ProteinProtein
NormalNormalmRNAmRNA
ProteinProtein
A U G
Met
A A G
Lys
U U U
Phe
G G C
Gly
G C A
Ala
U U G
Leu
A U G
Met
A A G
Lys
U U U
Phe
A G C
Ser
G C A
Ala
U U G
Leu
A A
Gln
C
A A
Gln
C
Adapted from Adapted from ASCO teaching slidesASCO teaching slides
Missense Mutation(Nonynonymous SNP)
Nonsense: change from an amino acid codon to a Nonsense: change from an amino acid codon to a stop codon, producing a shortened proteinstop codon, producing a shortened protein
NonsenseNonsense
mRNAmRNA
ProteinProtein
NormalNormalmRNAmRNA
ProteinProtein
A U G
Met
A A G
Lys
U U U
Phe
G G C
Gly
G C A
Ala
U U G
Leu
A U G
Met
U A G U U U G G C G C A U U G
A A
Gln
C
A AC
Adapted from Adapted from ASCO teaching slidesASCO teaching slides
Nonsense Mutation(Nonynonymous SNP)
FrameshiftFrameshift U G C A AA U G
Met
A A G
Lys
G C G
Ala
C A UU U
U
G
Leu
Frameshift: insertion or deletion of base pairs, producing Frameshift: insertion or deletion of base pairs, producing a stop codon downstream and (usually) shortened proteina stop codon downstream and (usually) shortened protein
mRNAmRNA
ProteinProtein
NormalNormalmRNAmRNA
ProteinProtein
A U G
Met
A A G
Lys
U U U
Phe
G G C
Gly
G C A
Ala
U U G
Leu
A A
Gln
C
Adapted from Adapted from ASCO teaching slidesASCO teaching slides
Frameshift Mutations
Exon 1Exon 1 IntronIntron Exon 2Exon 2 IntronIntron Exon 3Exon 3
Exon 1Exon 1 Exon 3Exon 3Altered mRNAAltered mRNA
Splice-site mutation: a change that results in altered RNA sequenceSplice-site mutation: a change that results in altered RNA sequence
Exon 2Exon 2
Adapted from Adapted from ASCO teaching slidesASCO teaching slides
Splice-site Mutations
Other Types of Mutations
• Mutations in regulatory regions of the geneMutations in regulatory regions of the gene
• Large deletions or insertionsLarge deletions or insertions
• Chromosomal translocations or inversionsChromosomal translocations or inversions
Potentially pathogenic CNV detected in ~10-20% of unexplained intellectual disability
J. Med. Genet. 2004, 41:241.
• Understand the meaning of DNA sequence and amino acid polymorphisms.
• Recognize the different types of DNA sequence polymorphisms:– STR, SNP, CNV
• Know the different classes of DNA mutation:– Point mutations (silent, missense, nonsense, frameshift,
splicing, regulatory) insertion/deletions, rearrangements
• Understand how to distinguish a disease-causing mutation from a neutral DNA sequence variation
Learning Objectives
Tests to Detect Mutations
• Many methods/technologies
• Rapidly changing
• DNA sequencing– Most direct and informative– The gold standard– Targeted region (known mutation)– “Whole” gene (unknown mutation)– … Whole exome / whole genome
How do we distinguish a disease causing mutation from a silent sequence variation?
• Obvious disruption of gene– large deletion or rearrangement– frameshift– nonsense mutation
• Functional analysis of gene product– expression of recombinant protein– transgenic mice
• New mutation by phenotype and genotype
• Computer predictions• Disease-specific mutation databases
– Same/similar mutation in other patients, not in controls
• Rare disease-causing mutation vs. private “polymorphism” (rare variant)
X
• Understand the meaning of DNA sequence and amino acid polymorphisms.
• Recognize the different types of DNA sequence polymorphisms:– STR, SNP, CNV
• Know the different classes of DNA mutation:– Point mutations (silent, missense, nonsense, frameshift,
splicing, regulatory) insertion/deletions, rearrangements
• Understand how to distinguish a disease-causing mutation from a neutral DNA sequence variation
Learning Objectives
Slide 10: CC: BY-SA: Ricardipus (wikipedia) http://creativecommons.org/licenses/by-sa/2.0/deed.en
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