"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