AKU: From classical genetics to gene cloning

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AKU From classical genetics to gene cloning

• Classical genetics
• Garrod - inborn errors of metabolism (1908)
• alkaptonuria accumulation of homogentisic acid
  in urine
• congenital inborn defect
• rare but increased frequency with consanguinity
• autosomal recessive

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AKU From classical genetics to gene cloning

• Biochemical genetics
• Enzymatic deficiency of AKU demonstrated in 1958
• Homogentisate 1,2 dioxygenase (HGO)
• Mapping
• Trait mapped by homozygosity mapping and by
  comparative mapping (mouse gene on chr 16,
  sharing conserved synteny with human 3q)

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AKU From classical genetics to gene cloning

• Functional cloning - getting there
• fungal hmgA was starting point to search human
  EST databse
• retrieved sequence and clone
• sequenced clone - encoded peptide with 52
  identify with fungal hmgA
• bacterially expressed protein had HGO activity
• expressed in human liver and kidney

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AKU From classical genetics to gene cloning

• Functional cloning - proving its there
• retrieved genomic clone
• HGO mapped by somatic cell hybrid and FISH
  analysis - HGO maps to AKU!
• mutations found in affected patients
• mutant protein lacks HGO activity