Identification of Genes for Human Disease

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Identification of Genes for Human Disease
Reverse Genetics
Reverse Genetics
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Modes of Inheritance

• Autosomal Dominant
• Autosomal Recessive
• X-Linked
• Multifactorial

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Useful numbers

• The haploid human genome has
• 3 X 109 base pairs
• This contains approximately
• 50,000 - 100,000 genes

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The Human Genome

• Single Copy DNA (60)
• Mainly Euchromatic regions
• Genes, Pseudogenes
• Repetitive DNA (40)
• Mainly Heterochromatic regions
• Tandem repeats
• Microsatellite repeats
• Minisatellite repeats
• Interspersed repeats
• Alphoid repeats

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How to find your gene Monogenic Disorders -

• Identify large families with the disease
• Clinical Assessment.
• Identify a chromosomal locus
• Linkage.
• Refine the candidate interval
• Run more markers, construct a contig.
• Identify Candidate Genes
• Databases, exon trapping, direct selection.
• Mutation Analysis
• Sequencing, southern blotting, SSCP, ....

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Mapping Approach
Slow

• Whole genome scan
• Concentrate on gene rich areas
• Informative chromosomal rearrangement
• Deletion
• Translocation
• Candidate gene approach

Fast
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DNA markers

• Restriction Fragment Length Polymorphisms
• VNTRs - minisattelites
• Single Nucleotide Polymorphisms
• Microsattelite Repeats
• They are not usually genes themselves, just flags
  on the gene map

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Marker Location / Map Construction

• Linkage - CEPH families
• Need crossovers to map
• FISH
• Somatic Cell Hybrids
• eg Chromosome specific somatic cell hybrids
• Radiation Reduced Somatic Cell Hybrids
• Can only relate locus to other known loci

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Gene Map
WWW.gdb.org
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Using Polymorphic Markers
(CA)5
(CA)7
Chromosome 9
PCR
Electrophoresis
7 repeats
5 repeats
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Linkage

• When particular alleles at two different loci are
  inherited together more often than you would
  expect by chance, they are in
• LINKAGE DISEQUILIBRIUM
• This implies they map close together on the same
  chromosome.

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LOD score

• The statistical measure of the evidence for
  linkage
• Log10

Likelihood of data given ?
Likelihood of data given ?0.5
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• Z gt 3.0 Favours linkage at plt0.05
• Z lt -2.0 Linkage excluded
• Can link marker to marker
• or Marker to phenotype

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A
C
D
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C
D
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Disease vs. D9S63 4.52 3.03 1.05
0.70 0.21 0.00
Disease vs. D9S61 - 1.03 1.52
1.01 0.21 0.00
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When Linkage Doesnt Work

• Is the mode of inheritance correct
• Is the clinical assessment correct
• Is there non-paternity
• Is there locus heterogeneity
• Is the family big enough

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Candidate Interval
FLT4 FGFR4 Unknown
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Positional CloningTypes of Clones

• A means of isolating and replicating a stretch of
  DNA
• Somatic Cell Hybrids Entire Chromosomes
• YACs 1 Megabase
• BACs, PACs 100 Kilobases
• Cosmids 50 Kilobases
• Phage Clones 20 Kilobases
• Plasmids 10 Kilobases

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Positional Cloning

• Libraries
• A collection of recombinant DNA clones
• Genomic
• cDNA
• Contig
• Overlapping genomic clones across the candidate
  region

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YAC contig
D9S315
D9S60
D9S63
D9S61
ENG
AK1
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Identifying Genes
Old

• Zoo Blotting
• Identification of CpG islands
• Screen a cDNA library
• Computer databases of known Genes
• Computer databases of ESTs
• cDNA selection
• Exon Trapping
• Sequence database searching (BLAST)
• Large scale computer analysis of sequence (eg.
  GRAIL)

New
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Use of Computers (1)Identifying Genes in a
candidate interval

• Whole Genome Sequencing
• Searching for known ESTs
• Searching for homology to known proteins
• Human or other species
• Identification of ORFs
• Identification of Exons
• Identification of promotors
• Contig Construction
• Gene maps/databases

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Use of Computers (2)Aids in analysis of genes

• Intron / Exon boundary identification
• From whole genome sequence
• Boundaries in homologous genes
• Identification of functional protein domains

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Use of Computers (3)Aids in mutation analysis

• Automated sequencing
• Identification of conserved amino acids
• across species
• between homologous proteins
• Construction of phenotype/genotype databases

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Mutation AnalysisScreening for Unknown Mutations
Large

• Chromosome analysis
• Pulsed Field Gel Electrophoresis
• Southern Blotting
• PCR amplification - point mutation detection
• Sequencing
• Heteroduplex detection
• SSCP
• DGGE
• DPLC

Small
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A Mutation Can Cause Disease by

• Abolishing protein production
• Creating non-functioning protein
• Creating a poorly functioning protein
• Allowing accumulation of protein in cell
• Abnormal activation of protein
• Causing gain of function of protein

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Regulation of Protein Quantity

• Rate of Transcription
• Stability of mRNA Transcript
• Stability of Protein

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Types of mutation in DNA sequences

• The cat sat on the mat Wild type
• The cat stop
• The car sat on the mat Missense
• The cat spa to nth ema t Insertion
• The cas ato nt hem at Deletion (out of
  frame)
• The cat on the mat Deletion (in frame)
• The cat cat sat on the mat Triplet expansion
  
• The tas tac on the mat Inversion

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Heteroduplex Analysis
Mutant Type
PCR
Wild Type
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Heteroduplex Analysis
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Mutations in the ALK1 Gene
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SSCP Analysis
Mutant Type
PCR
Denature
Wild Type
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A Point Mutation in the Endoglin Gene On
Chromosome 9
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• EGLN_MOUSE ENDOGLIN PRECURSOR
• Query 1 RPKTGSQDQEVHRTVFMRLNIISPDLSGCTSKGL 34
• RP T SQ EVTV MRLNISPDLSG KGL
• Sbjct 554 RPSTLSQ--EVYKTVSMRLNIVSPDLSG---KGL 582
• EGLN_PIG ENDOGLIN PRECURSOR
• Query 1 RPKTGSQDQEVHRTVFMRLNIISPDLSGCTSKGL 34
• RPT S D VHT RLNISP G KGL
• Sbjct 554 RPRTWSLD--VHKTASTRLNIVSP---GLPDKGL 582

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Sequencing ACGT tracking
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Mutation AnalysisScreening for Known Mutations

• Allele Specific Oligonucleotide Hybridisation
• Altered Restriction Endonuclease Site
• ARMS
• RGPCR

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Restriction Endonucleases
BamHI
5---GGATCC---3 3---CCTAGG---5
5--GGATC C--3 3--C CTAGG--5
BamHI
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Detection of Mutation that Creates Restriction
Site
280bp
280bp
HhaI
160bp
160bp
120bp
Mutation Creates HhaI site
120bp
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Restriction Digest with HhaI
Wt/Wt Wt/M uncut Ladder
Normal PCR product
Digested Mutant Product
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Is a Mutation Pathogenic

• Does it have a significant effect on protein
• Stop/frameshift
• Change a conserved amino acid
• Is it present in all affected family members
• Is it ever observed in the absence of disease
• What happens in the mouse model

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Mapping Disease Genes - When you dont know the
mode of inheritance

• Model Free Linkage Analysis
• Non-parametric Linkage Analysis
• Association Studies
• APM method
• Sib pair analysis
• Association Studies with Internal Controls (ASIC)
• Haplotype Relative Risk (HRR)
• Transmission Disequilibrium Test (TDT)

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Mapping Disease Genes Multifactorial
Inheritance
Genetic Factors
Environmental Factors
Phenotype
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Mapping Disease Genes Multifactorial Inheritance

• Establish Heritability of Trait
• Identify a Sufficient Cohort of Patients
• Methods of Analysis
• Association Studies
• Model Free Linkage Methods
• Non Parametric Linkage Methods
• Identify Candidate Genes
• How to Narrow the Candidate Interval ?

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Once You Have Your Gene

• Use in clinical diagnosis
• Pre-Symptomatic testing
• Does mutation type affect prognosis
• Does mutation type affect response to treatment
• What does the gene do - what does this tell us
• Does the gene suggest novel treatment strategies
• Does the gene apply to other human diseases

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• Sahoo T. Johnson EW. Thomas JW. Kuehl PM. Jones
  TL. Dokken CG. Touchman JW. Gallione CJ. Lee-Lin
  SQ. Kosofsky B. Kurth JH. Louis DN. Mettler G.
  Morrison L. Gil-Nagel A. Rich SS. Zabramski JM.
  Boguski MS. Green ED. Marchuk DA. Mutations in
  the gene encoding KRIT1, a Krev-1/rap1a binding
  protein, cause cerebral cavernous malformations
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  1999
• Laberge-le Couteulx S. Jung HH. Labauge P.
  Houtteville JP. Lescoat C. Cecillon M. Marechal
  E. Joutel A. Bach JF. Tournier-Lasserve E.
  Truncating mutations in CCM1, encoding KRIT1,
  cause hereditary cavernous angiomas. Nature
  Genetics. 23(2)189-93, 1999
• Type 1 neurofibromatosis gene identification of
  a large transcript disrupted in three NF1
  patients
• Science. 249(4965)181-6, 1990 Jul 13.
• A major segment of the neurofibromatosis type 1
  gene cDNA sequence, genomic structure, and point
  mutations.
• Cell. 62(1)193-201, 1990 Jul 13.

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Bedtime Reading

• Human Molecular Genetics 2
• Strachan and Reid
• www.hgmp.mrc.ac.uk
• www.ncbi.nlm.nih.org
• www.gdb.org