The document discusses DNA sequence variation, focusing on different types of polymorphisms and mutations, including their definitions, classifications, and implications for diseases. It outlines the importance of distinguishing between disease-causing mutations and neutral variations, and emphasizes understanding polymorphisms such as SNPs and CNVs. The text also provides insight into the methodologies used for detecting mutations and the relevance of genetic variations in the context of human health.

1. Author(s): David Ginsburg, 2009
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3. DNA Sequence Variation
M1 Patients and Populations
David Ginsburg, MD
August 13, 2008
Fall 2008

4. Learning Objectives
• Understand the meaning of DNA sequence and
amino acid polymorphisms.
• Recognize the different types of DNA sequence
polymorphisms:
– RFLP, VNTR, 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

5. Chromosomes, DNA, and Genes
Proteins
Jfdwolff (wikimedia)
Wikimedia Commons

6. TRANSCRIPTION
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 5.1

7. 1983 2001
Huntington Draft human
Disease genome
gene 1989 sequence
1956 1981mapped Positional cloning
Glu 6 Val in Transgenic mice without deletion
sickle hemoglobin (CF)
1944 1970 1975 1995
1985 1st complete
DNA is the 1953 First restriction Southern PCR
genetic Double enzyme blotting bacterial
material helix genome
sequence
1945 1950 1955 1960 1965 1970 1975 1980 1985 1990 1995 2000
1972 1986 1990 1996
1949 1966 Recombinant Complete yeast
Positional First NIH-
Abnl Hemoglobin Completion of the genome
plasmids cloning (CGD, approved
in sickle cell genetic code sequence
muscular gene
anemia dystrophy, therapy
retinoblastoma experiment
Since 2001: 1987
Knockout
Complete human genome complete; human mice
hapmap;
Other genomes: mouse, rat, dog, chimp, chicken, Gelehrter, Collins and Ginsburg: Principles of Medical
Genetics 2E; Figure 5.4
zebrafish…18 vertebrates, 8 invertebrates,
>500 microbial genomes.

8. DNA Samples and PCR
DNA for analysis
cheek swab
Blood sample urine sample
Forensic specimens
Source: Dennis Myts (wikimedia)
Repeat
DNA cycles
primers
Target
sequence Amplified
product
D. Ginsburg

9. SINGLE-STRANDED
DNA PROBES
FOR GENE A
C
D
B MIXTURE OF
+ SINGLE-STRANDED
DNA MOLECULES
E A
F
C
C
D
B B D
A A
E
E
F F
ONLY A FORMS A STABLE A, C, E ALL FORM
DOUBLE-STRANDED COMPLEXES STABLE COMPLEXES
STRINGENT HYBRIDIZATION REDUCED-STRINGENCY HYBRIDIZATION
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E;
Figure 5.8

10. Hybridization array: gene chip
DNA chip v6.0:
~1 million SNPs
RNA expression:
All ~ 20,000 genes
CGH:
Survey whole genome
Ricardipus (wikipedia)
for large deletions,
insertions,
rearrangements

11. Image of DNA
sequencing
process removed

12. DNA Sequencing
Source Undetermined

13. New Sequencing Technologies
Source Undetermined

14. Learning Objectives
• Understand the meaning of DNA sequence and
amino acid polymorphisms.
• Recognize the different types of DNA sequence
polymorphisms:
– RFLP, VNTR, 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

15. 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·phism
Pronunciation: "päl-i-'mor-"fiz-&m
Function: 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

16. Mutations
A mutation is a change in the normal base pair sequence
Dennis Myts (wikimedia)
• Can be:
– a single base pair substitution
– a deletion or insertions of 1 or more base pairs (indel)
– a larger deletion/insertion or rearrangement

17. Polymorphisms and Mutations
• Genetic polymorphism:
– Common variation in the population:
• Phenotype (eye color, height, etc)
• genotype (DNA sequence polymorphism)
– Minor allele frequency > 1%
• DNA (and amino acid) sequence variation:
– Most common allele < 0.99 = polymorphism
(minor allele > 1%)
– Variant alleles < 0.01 = rare variant
• Mutation-- any change in DNA sequence
– Silent vs. amino acid substitution vs. other
– neutral vs. disease-causing
• Common but incorrect usage:
– mutation vs. polymorphism
• balanced polymorphism= disease + polymorphism

18. All DNA sequence variation arises via
mutation of an ancestral sequence
< 1% > 1%
Rare variant or
Normal private polymorphism
polymorphism
Example: Factor V Leiden
Disease Disease mutation (thrombosis)
5% allele frequency
Common but incorrect usage:
a disease-causing mutation OR a polymorphism

19. Learning Objectives
• Understand the meaning of DNA sequence and
amino acid polymorphisms.
• Recognize the different types of DNA sequence
polymorphisms:
– RFLP, VNTR, 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

20. 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
• Insertions or deletions
– INDEL – small (few nucleotides) insertion or deletion
• Rearrangement (inversion, duplication, complex
rearrangement)
• CNV: Copy Number Variation

21. VNTR
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 5.19

22. STR
Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 5.22

23. SNP
Allele 1 A U G A A G U U U G G C G C A U U G C A A
Allele 2 A U G A A G U U U G G U G C A U U G C A A
Adapted from ASCO teaching slides
• Most are silent
• Intragenic
• Promoters and other regulatory sequences
• Introns
• Exons
– 5 and 3 untranslated regions
– Coding sequence (~1-2% of genome)

24. Learning Objectives
• Understand the meaning of DNA sequence and
amino acid polymorphisms.
• Recognize the different types of DNA sequence
polymorphisms:
– RFLP, VNTR, 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

25. Silent Sequence Variants
(Synonymous SNP)
mRNA
Normal A U G A A G U U U GGC GC A U UG C A A
Protein Met Lys Phe Gly Ala Leu Gln
Sequence mRNA
A U G A A G U U U GGU GC A U UG C A A
variant
Protein Met Lys Phe Gly Ala Leu Gln
Adapted from ASCO teaching slides
Changes that do not alter the encoded amino acid

26. Missense Mutations
mRNA
Normal A U G A A G U U U GGC GC A U UG C A A
Protein Met Lys Phe Gly Ala Leu Gln
mRNA
A U G A A G U U U AGC GC A U UG C A A
Missense Protein Met Lys Phe Ser Ala Leu Gln
Adapted from ASCO teaching slides
Missense: changes to a codon for another amino acid
(can be harmful mutation or neutral variant)
(nonsynonymous SNP)

27. Nonsense Mutations
mRNA
Normal A U G A A G U U U GGC GC A U UG C A A
Protein Met Lys Phe Gly Ala Leu Gln
mRNA
A U G U A G U U U GGC GC A U UG C A A
Nonsense Protein Met
STOP
Adapted from ASCO teaching slides
Nonsense: change from an amino acid codon to a
stop codon, producing a shortened protein
(nonsynonymous SNP)

28. Frameshift Mutations
mRNA
Normal A U G A A G U U U GGC GC A U UG C A A
Protein Met Lys Phe Gly Ala Leu Gln
mRNA
Frameshift A U G A A G U U G GC G C A U UGC A A
Protein Met Lys Leu Ala
STOP
Adapted from ASCO teaching slides
Frameshift: insertion or deletion of base pairs, producing
a stop codon downstream and (usually) shortened protein

29. Splice-site Mutations
Exon 1 Intron Exon 2 Intron Exon 3
Exon 2
Exon 1 Exon 3
Altered mRNA
Adapted from ASCO teaching slides
Splice-site mutation: a change that results in altered RNA sequence

30. Other Types of Mutations
• Mutations in regulatory regions of the gene
• Large deletions or insertions
• Chromosomal translocations or inversions

31. 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.

32. Learning Objectives
• Understand the meaning of DNA sequence and
amino acid polymorphisms.
• Recognize the different types of DNA sequence
polymorphisms:
– RFLP, VNTR, 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

33. 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 genome

34. 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 X
• Rare disease-causing mutation vs. private
polymorphism (rare variant)

35. Learning Objectives
• Understand the meaning of DNA sequence and
amino acid polymorphisms.
• Recognize the different types of DNA sequence
polymorphisms:
– RFLP, VNTR, 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

36. Additional Source Information
for more information see: http://open.umich.edu/wiki/CitationPolicy
Slide 5: Jfdwolff (wikimedia) http://creativecommons.org/licenses/by-sa/3.0/;
http://commons.wikimedia.org/wiki/File:Chromosome_zh-tw.svg
Slide 6: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 5.1
Slide 7: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 5.4
Slide 8: D. Ginsburg
Slide 9: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 5.8
Slide 10: CC: BY-SA: Ricardipus (wikipedia) http://creativecommons.org/licenses/by-sa/2.0/deed.en
Slide 11: Image of DNA sequencing process removed
Slide 12: Undetermined
Slide 13: Undertirmined
Slide 16: Dennis Myts (wikimedia)
Slide 21: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 5.19
Slide 22: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 5.22
Slide 23: Adapted from ASCO teaching slides
Slide 25: Adapted from ASCO teaching slides
Slide 26: Adapted from ASCO teaching slides
Slide 27: Adapted from ASCO teaching slides
Slide 28: Adapted from ASCO teaching slides
Slide 29: Adapted from ASCO teaching slides
Slide 31: Nature 444:444, Nov 2006.