"Forward" 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 crosses in a pedigree allows inference of the
existence and recessive nature of a gene for the trait
Alkaptonuria. The physical nature of the gene was at that
time entirely unknown.
Molecular Genetics in the 21st century proceeds from knowledge of the 3,200 Mbp and 20,050 protein-coding genes in the 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 a standard, functional allele, designated "+", that has both the P230 and V300 forms of the gene. 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 measure proportions in the observed outcomes. This is how the science of Genetics was understood for more than 50 years, which only in the molecular era was sometimes described as "Forward Genetics". Molecular Biology in the late 20th and early 21st centuries is sometimes called "Reverse Genetics", because detailed knowledge of the molecular genotype predicts how it produces a disease phenotype that arises in certain pedigrees. Confusion arises with the formulation of the Central Dogma as "DNA makes RNA makes Protein", which refers to the forward transfer of information. The logic of the inference is from detection of an enzyme defect to the underlying DNA variant, and thence to phenotype, which implies a "reversal" of molecular logic.
HOMEWORK: 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 ascertainment bias. Investigate and explain.
Molecular Genetics in the 21st century proceeds from knowledge of the 3,200 Mbp and 20,050 protein-coding genes in the 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 a standard, functional allele, designated "+", that has both the P230 and V300 forms of the gene. 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 measure proportions in the observed outcomes. This is how the science of Genetics was understood for more than 50 years, which only in the molecular era was sometimes described as "Forward Genetics". Molecular Biology in the late 20th and early 21st centuries is sometimes called "Reverse Genetics", because detailed knowledge of the molecular genotype predicts how it produces a disease phenotype that arises in certain pedigrees. Confusion arises with the formulation of the Central Dogma as "DNA makes RNA makes Protein", which refers to the forward transfer of information. The logic of the inference is from detection of an enzyme defect to the underlying DNA variant, and thence to phenotype, which implies a "reversal" of molecular logic.
HOMEWORK: 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 ascertainment bias. Investigate and explain.
Archibald Garrod (ca. 1908)