Genetics and Molecular Biology Tutorial II Computational Perspective

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Genetics and Molecular Biology Tutorial II --
Computational Perspective

• The goal is to introduce some topics to
  individuals with a minimal background in
  genetics/biology, and yet try to provide some
  examples of topics to maintain the interest of
  individuals with extensive biological/genetics
  backgrounds.

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Outline

• Gene structure
• genomic structure vs mRNA structure
• coding and noncoding exons
• introns
• primary transcript processing
• aside -- nonsense mediated mRNA degradation
• alternative splicing and differential
  polyadenylation
• evolutionary conservation of coding and noncoding
  sequences

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Outline

• Genomic structure
• repetitive sequences
• LINES and SINES
• example -- Y chromosome palindromes
• C value paradox
• genomes of model organisms
• example
• yeast genome and gene-chip
• single/double knockouts
• cross-species sequence similarities for putative
  function identification
• example -- chaperonine

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Fundamental Genetics and Probability Concepts

• meiosis and sampling
• patterns of inheritance
• monogenic and complex inheritance
• phenocopy
• reduced penetrance
• DNA variation
• polymorphisms, SNPs, and mutations
• positional cloning

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Gene Structure
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Transcript Processing

• DNA -gt pre-mRNA -gt mRNA -gt protein

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Nonsense mediated mRNA degradation

• unknown mechanism
• more rapidly degrades mRNA containing
• Lykke-Andersen, mRNA quality control Marking
  the message for life or death. Current Biology,
  11, 2001.

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Nonsense Mediated mRNA Degradation
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Genome Structure -- repeat classes
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C-Value ParadoxHartl, Molecular melodies in
high and low C, Nat. Rev. Genetics, Nov 20001

• refers to the massive, counterintuitive and
  seemingly arbitrary differences in genome size
  observed in eukaryotic organisms
• Drosophila melanogaster 180 Mb
• Podisma pedestris 18,000 Mb
• difference is difficult to explain in view of
  apparently similar levels of evolutionary,
  developmental, and behavioral complexity

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Alternative Splicing

• Every conceivable pattern of alternative
  splicing is found in nature. Exons have multiple
  5 or 3 splice sites alternatively used (a, b).
  Single cassette exons can reside between 2
  constitutive exons such that alternative exon is
  either included or skipped ( c ). Multiple
  cassette exons can reside between 2 constitutive
  exons such that the splicing machinery must
  choose between them (d). Finally, introns can be
  retained in the mRNA and become translated.
• Graveley, Alternative splicing increasing
  diversity in the proteomic world. Trends in
  Genetics, Feb., 2001.

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Classic View of Gene No Longer Valid -- Strachan
pg 185
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Alternative Splicing Example -- Graveley 2001
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Alternative PolyAdenylation

• common in human RNA (Edwards-Gilbert 1997)
• in many genes, 2 or more poly-A signals in 3 UTR
• alternative transcripts can show tissue
  specificity
• alternative poly-A signals may be brought into
  play following alternative splicing

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Edwards-Gilbert. Nucleic Acids Res, 13, 1997
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• Evolution of the mitochondrial genome and origin
  of eukaryotic cells

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Evolutionary Conservation of Coding and Noncoding
Sequences

• Sequencing of H. sapiens and model organisms is
  basis for comparative genomics
• Generally, functional solutions (encoded as
  genes) across organisms allows us to compare gene
  sequences and infer function
• protein functional/structural region domains
• Intergenic regions are generally not conserved
  (always exceptions)

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Example - MKKS (UniGene Clusters)

• human rat 87.4
• human mouse 84.9
• human cow 87.1
• mouse rat 97.8
• rat cow 91.0
• mouse cow 85.1
• frog rat 62.5

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Example - MKKS
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Computational Approach to Using Conserved Regions

• Problem -- want to screen genes for mutations
• Conventional approach -- screen all exons of a
  single gene
• Alternative -- identify domains with in multiple
  genes, and screen domains first, to optimize
  screening time and resources

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Cross-Species Similarities

• yeast
• gene chip for hybridization/expression
• complete genome (first eukaryote)
• singe knockouts and double knockouts

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Fundamental Genetics

• meiosis
• Hs are diploid
• meiosis produces haploid gametes
• mechanism for transmission of genetic material to
  offspring
• recombination by cross-over (Holliday structure)
  or by independent segregation of homologous pairs

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Fundamental Genetics (Background for Linkage
Analysis)

• Rule of Segregation
• offspring receive ONE allele (genetic material)
  from the pair of alleles possessed by BOTH
  parents
• Rule of Independent Assortment
• alleles of one gene can segregate independently
  of alleles of other genes
• (Linkage Analysis relies on the violation of
  Independent Assortment Rule)

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Genetic Marker Prelude to LA

• A genetic marker allows for the observation of
  the genetic state at a particular genomic
  location (locus).
• A genotype is the measured state of a genetic
  marker.
• May never be feasible to sequence cases directly.
• An informative marker is often heterozygous,
  or polymorphic and enables the observation of
  the inheritance of genetic material.

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Monogenic and Polygenic Diseases

• monogenic (Mendelian) -- one gene
• simple (dominant and recessive) Mendelian
  inheritance
• direct correspondence between one gene mutation
  and one disorder
• majority of disease genes found are monogenic
• polygenic -- (complex) multiple genes
• heterogeneity and epistasis
• combinatorics
• no longer have direct correspondence between one
  gene and disorder
• majority of disorders are probably polygenic
• complexity of organisms and observed pathways

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...Mongenic and Polygenic Diseases

• phenocopy
• reduced penetrance
• Example -- sickle cell anemia
• classic recessive disorder
• defect in red blood cells (hemoglobin)
• but infant hemoglobin gene can leak
• wide range of phenotypes

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Examples
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Examples
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Example
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BBS4 Pedigree
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Hardy-Weinberg Equilibrium

• Rule that relates allelic and genotypic
  frequencies in a population of diploid, sexually
  reproducing individuals if that population has
  random mating, large size, no mutation or
  migration, and no selection
• Assumptions
• allelic frequencies will not change in a
  population from one generation to the next
• genotypic frequencies are determined in a
  predictable way by allelic frequencies
• the equilibrium is neutral -- if perturbed, it
  will reestablish within one generation of random
  mating at the new allelic frequency

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H-W

• f(AA) p2
• f(Aa) 2pq
• f(aa) q2
• (pq)2
• (p2 q2 r2 2pq 2pr 2qr) (pqr)2

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Dominant and Recessive Penetrance
Modeledpenetrance P(pt gt)

• DD Dd dd
• 1 1 0
• DD Dd dd
• 0.9 0.9 0.0

• DD Dd dd
• 0 0 1
• DD Dd dd
• 0 0 0.8

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D-R Heterogeneous, DD Epistatic

• AA Aa aa
• BB 1 1 0
• Bb 1 1 0
• bb 1 1 1
• reduced penetrance
• 3,9,27,81,243 3n

• AA Aa aa
• BB 1 1 0
• Bb 1 1 0
• bb 0 0 0

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Dom-Rec Heterozygous
Screen genes A, B?, b
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Uninformative Marker
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Informative Marker
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• Given the following observations family
  structure, affection status, genotypes, and
  disease allele frequencies. Assuming a model for
  the disease, can we calculate the probability
  that these observations fit an assumed model???

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Linkage
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Linkage Analysis

• Goal find a marker linked to a disease gene.
• LOD score log of likelihood ratio
• LR?data k Pdata ?
• theta estimate of genetic distance
  (recombination fraction) between marker and
  disease
• proportion of recombinant gametes/total gametes

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Linkage Analysis

• Linkage analysis calculates the likelihood that
  the inheritance pattern of the phenotype
  (disease) is supported by the observed
  inheritance patterns (genotypes) in a pedigree.
• few monogenic models, easy to test
• more difficult to find models explaining
  inheritance in polygenic models
• parameter maximization

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Linkage Analysis Programs

• FASTLINK - 2 point
• O(n2), where n number of markers
• GeneHunter - multipoint, 2 point
• O(n2), where n number of people

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Allele Sharing

• tries to show that affected family members
  inherit the same chromosomal regions more often
  than expected by chance

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Allele Sharing Example
Needs at least sibs.
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Association Studies

• Allelic association studies provide the most
  powerful method for locating genes of small
  effect contributing to complex diseases and
  traits. Daniels, Am J Hum Genet 621189-1197,
  1998.
• Linkage analysis
• genome wide screen, 400 markers 10 cM (10 MB),
  association needs 4000 polymorphic markers
• generally need nuclear family or larger
• Association finds linkage disequilibruim

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Association Studies

• Association is simply a statistical statement
  about the co-occurrence of alleles or phenotypes.
  Allele A is associated with disease D if people
  who have D also have A more (or maybe less) often
  than would be predicted from the individual
  frequencies of D and A in the population. Pg.
  286 Human Molecular Genetics 2, Tom Strachan

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Examples

• HLA-DR4 (antigen marker)
• 36 in UK
• 78 with rheumatoid arthritis
• CF( RFLP markers XV2.c (X1,X2), KM19(K1,K2))
• Marker Alleles CF(case) Normal(control)
• X1, K1 3 49
• X1, K2 147 19
• X2, K1 8 70
• X2, K2 8 25
• CF associated with X1, K2 in 89 (Strachan)

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Linkage Disequilibrium

• linkage equilibrium (aka Hardy-Weinberg) is true
  if
• P(gt1,gt1gt2,gt2) P(gt1,gt1)P(gt2,gt2)
  where P(haplotype)
• case vs controls
• TDT (heterozygous marker transmitted), HRR
  (untransmitted alleles as control)
• allelic associations (outbred populations)
  maintained at only lt 1cM

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Equilibrium
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SNPs

• Single-Nucleotide Polymorphisms
• 1 every 1000 bp (estimated)
• 2,972,052 SNPs submitted to dbSNP
• dbSNP summary link
• 50 of all SNPs are in question
• 10 of UTRs have SNPs
• 100,000 - 500,000 SNPs needed
• Why dont we do this?

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Homozygosity Mapping
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Positional Cloning
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Disease Gene Identification

• SSCP -- single strand conformational polymorphism
• PCR -- polymerase chain reaction
• primers amplify template sequence
• direct sequencing
• BBS2 (Bardet-Biedl Syndrome)

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BBS2 genetic mapping
C16
1 2 3 4 5 6 7 8 9 10 11 12
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BBS2 genetic mapping
unaffected
affected
C16
1 2 3 4 5 6 7 8 9 10 11 12
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BBS4 Gene (Direct Sequencing)
(Hs.26471)
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BBS4 Deletion (by PCR)
exons 3 4
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BBS4 Mutations (direct sequencing)
(R295P)
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Summary

• Disease Gene Identification
• challenges
• interval localization
• genotyping and genetic markers, linkage analysis,
  allele sharing, association studies (SNiPs),
  homozygosity mapping
• disease gene identification techniques
• Take home
• A complex disorder (with interacting genes) has
  yet to be characterized

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Demo -- installing a database

• A database organizes data
• Most common
• relational database (oracle, sybase)
• perceived as a collection of tables,
• where table is an unordered collection of rows
• each row has a fixed number of fields, and each
  field can store a predefined type of data value
  (date, integer, string, etc.)
• simplest
• flat file

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Databases

• NCBI
• BLAST
• Amazon
• Yahoo
• Several of our own
• genotypes
• rat ESTs
• eye clones from differential display
• micro-array data

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