"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 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 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.
For
the advanced student: Mendel's
experiments carefully isolated and brought together
exactly two allelic variants of each gene, A
& 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
tacitly assumes that there are only two
alleles, the "normal" A allele
carried by most people, and the "disease" a
allele found in affected persons. This became the
standard interpretation: most individuals were
homozygous AA for a standard "wild
type" allele A, whereas a
minority were homozygous aa for a "disease"
allele a. Early molecular analysis began to
show instead that genetic defects might occur at
several places in the DNA of any gene. The
combination of two different alleles a'a
could produce a compound
heterozygote, with the genetic
phenotype of an aa homozygote.
True homozygosity would
require that an individual inherited an allele identical
by descent (autozygous)
from a more or less distant ancestor. One result of
the Human Genome Project
is to demonstrate extensive heterogeneity among the
alleles associated with any particular genetic
condition, such that compound heterozygosity
is far more frequent than
previously expected.
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)
Figure © 2016 by Steven M Carr,
after ©2002 by Griffiths et al.; All text
material ©2025 by Steven M Carr