Forward vs Reverse
        Genetics

"Classical" Mendelian Genetics versus "Reverse" Molecular Genetics:
Genetics and molecular biology of Alkaptonuria, an inborn error of metabolism


    In the first classical genetics approach to a human trait, the physician Archibald Garrod in 1902 observed "Black Urine Disease" (Alkaptonuria, AKU) in his patients (Step 1). The urine and diapers of infants with Alkaptonuria darken upon exposure to air, and adults show darkening of the cartilage in the ears and nose. Chemical analysis identified a high level of a substance called alkapton in their urine (Step 2). From the pattern of inheritance (pedigree) observed in families under his care (two unaffected parents, and unaffected and affected children in an approximate 3:1 ratio) (Step 3), Garrod deduced that the condition was inherited as a recessive trait, following the reasoning of Gregor Mendel whose work in 1867 had recently been rediscovered, and was widely discussed. Following Mendelian Rules, the birth of an alkaptonuric child to two unaffected parents suggests that she had two recessive alleles (aa) at some Gene for the trait. The parents must both then be Aa, and do not show the condition because A is dominant to a. The other, unaffected children are either AA or Aa (shown as A-). Garrod further suggested that the condition was due to the absence of an enzyme to metabolize (break down) alkapton. Subsequent biochemical analysis showed that alkapton (now called Homogentisic Acid) is metabolized by Homogentisic Acid Oxidase (HGO) to Maleylacetoacetic Acid (Step 4). Thus, genetic analysis of a cross in a pedigree allows inference of the existence and recessive nature of a gene for the trait Alkaptonuria. The nature of the gene is unknown.

   
Molecular Genetics in the 21st century  proceeds from knowledge of the 3,200 Mbp human genome, completed in 2003 (Step 1). Based on knowledge of the amino acid sequences of HGO in other organisms, bioinformatic analysis of all 23 pairs of chromosomes mapped the gene for Homogentisic Acid Oxidase (HGO) to Band 2 on the long (q) arm of Chromosome 3 (3q2) (Step 2). Detailed sequence analysis of this region identified an HGO gene locus with 14 expressed exons and 13 intervening introns (Step 3). DNA sequence analysis of multiple individuals shows a larger number of Single Nucleotide Polymorphism (SNP) variants in particular exons. Two of these SNPs are predicted to cause amino acid substitutions in the protein products of Exons 10 & 12.  These are respectively a change of Pro to Ser at residue 230 (P230S), and substitution of Glu for Val at residue 300 (V300G) (Step 4). A child with Alkaptonuria is born to unaffected parents: DNA sequencing shows that the parents have the two different allelic variants (Step 5), each in combination with an alternative "+" allele. DNA sequence analysis shows that the unaffected siblings have the three possible combinations of the "+", P230S, and V300G alleles. The affected child has inherited both the P230S and V300G alleles, both of which are non-functional. HOMEWORK: what SNPs are responsible for the two amino acid substitutions?

    Garrod's analysis in the early 20th century is an example of  Classical or Mendelian Genetics: given observable phenotypic variation, he inferred the genotypic nature of inheritance from an analysis of pedigrees. This differs slightly from what Mendel did, which was to arrange controlled crosses and measured the observed outcomes. The molecular analysis in the early 21st century is often called "Reverse Genetics", because detailed knowledge of the molecular genotype predicts how it produces a disease phenotype arises in certain pedigrees. Also, because the Central Dogma states "DNA makes RNA makes Protein", moving from an enzyme defect to the underlying DNA variant implies a "reversal" of molecular logic.

    For the advanced student: Mendel's experiments carefully isolated and bring together exactly two allelic variants of each gene considered, A and a. Garrod's interpretation was that any individual with Alkaptonuria combined two copies of the same "a", such that the individual was an aa homozygote. This assumption implies that there are only two alleles, the "normal" A allele carried by most people, and the "disease" a allele found in affected persons. This was for long time the standard interpretation: by extension most individuals were homozygous for a standard "wild type" allele, whereas a minority were homozygous for a "disease" allele. The molecular analysis shows instead the combination of two different alleles
that produce the same phenotype: the individual is a compound heterozygote. True homozygosity would require that the parents have inherited the identical allele from a more or less distant ancestor. One result of the Human Genome Project is to demonstrate extensive heterogeneity in the alleles associated with any particular genetic condition, such that compound heterozygosity is far more frequent than previously expected.

     Step 3 of the analysis presented above (observation of a 3:1 ratio of unaffected to affected children) is an exaggeration. Garrod's actual data departed from 3:1 due to a phenomenon called ascertainment bias. Investigate and explain.





Archibald Garrod  (ca. 1908)

Figure  © 2016 by Steven M Carr, after  ©2002 by Griffiths et al.; All text material ©2024 by Steven M Carr