Finding disease genes

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Finding disease genes

• Inherited diseases are caused by defects in DNA
  (genes) which are passed from generation to
  generation

How do you find the gene and the defect within it?
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Finding disease genes

• What kind of DNA defect are we looking for?
• Changes in DNA which cause changes in the protein
  product

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Finding disease genes

• Point mutations - single nucleotide changes
• Missense mutations
• Nonsense mutations
• Splice-site mutations
• Insertions/deletions
• Small number of nucleotides involved
• Large-scale changes

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Finding disease genes

• Why do small changes to a protein cause disease?
• Loss of function
• Gain of function
• Novel function

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Finding disease genes

• Where do we start to look for the gene? Narrow it
  down
• Autosome (chromosome 1-22)?
• X chromosome?
• Mitochondria?

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Modes of Inheritance
Autosomal Dominant
-Single mutant allele causes disease
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-Half of offspring are affected
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-Males and females equally at risk
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Modes of Inheritance
Autosomal Recessive
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-Two mutant alleles required to produce disease
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-25 of offspring affected
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Modes of Inheritance
X-linked
-Only males affected
X X
X Y
-No male-to-male transmission
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Modes of Inheritance
Mitochondrial
-All of offspring of females are affected
-No offspring of males are affected
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Finding disease genes-Methods

• Positional Cloning
• Start with a family or families with the same
  inherited disease
• Determine chromosomal location using linkage
• Test candidate genes in the region for mutations

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Finding disease genes-Methods

• Candidate Gene Cloning/Functional Cloning
• Identify a gene with an important function
• Ask What disease might be caused by defects in
  this gene?
• Test patients with disease for mutations in this
  gene

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Finding disease genes-Examples

• Retinitis Pigmentosa (RP)
• Phenotype
• Degeneration of rod cells in the retina
• Night blindness, loss of peripheral vision,
  eventual blindness
• Genetic heterogeneity
• Dominant, recessive, X-linked forms - many genes
  still unidentified

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Finding disease genes-Examples

• Our labs goal was to find the RP1 gene by
  positional cloning
• Resources The RP01 family
• Multigeneration autosomal dominant family
• Extensive pedigree
• What is the gene causing disease in this family?

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Finding disease genes-Examples

• Preparation for positional cloning
• Family ascertainment in RP01
• Blood collection
• Clinical evaluation
• Linkage mapping - narrowing of region

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Finding disease genes-Examples

• Linkage mapping

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Finding disease genes-Examples

• Linkage mapping - RP1
• Markers tested on every chromosome
• Location narrowed down to chromosome 8
• 5 years spent trying to narrow the region further
• The smaller the region, the fewer genes you need
  to test
• Kept looking for good candidate genes in the
  region
• Retina expressed/retina specific

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Finding disease genes-Examples

• Linkage mapping - RP1
• Found a single retinal expressed gene in the
  region
• Tested the gene for mutations - found a nonsense
  mutation causing truncation of 2/3 of the protein
• All affected family members have this mutation,
  normal people do not
• Called the new gene RP1 and have been trying to
  figure out what it does ever since

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RP1 in photoreceptors

• RP1 localizes to the connecting cilium between
  the outer and inner segments
• RP1 interacts with microtubules

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Finding disease genes-Examples

• Functional cloning - Rhodopsin
• Transmembrane protein, large of the total rod
  cell protein, absorbs photon of light and changes
  shape to start phototransduction cascade gt rod
  cells cant function without it

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Finding disease genes-Examples

• Do mutations in rhodopsin cause RP?
• Ascertain a large number of patients with RP and
  screen the rhodopsin gene for mutations
• Result Rhodopsin mutations are found in about
  25 of patients with autosomal dominant RP

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Conclusions

• Gene identification is important - but its only
  the beginning