A Study of Alcaptonuria
Teacher Notes
Introduction:
Genetic features are passed on from one generation to the next through the inheritance of chromosomes,
which possess thousands of bits of genetic information called genes. Genes are actually portions of the DNA
molecules - the part of chromosomes that stores the genetic codes. The inheritance of the disease being studied
here can be explained by using the principles discovered by Gregor Mendel in his garden pea experiments.
Laws of probability and pedigree analysis will be used to study the inheritance of a recessive gene that leads to
a defective protein (in this case an enzyme).
This exercise is a simulation, which traces the inheritance of the biochemical disorder Alcaptonuria through
2 to 3 generations in families, permitting the genotypes of past and present family members to be determined.
By using probabilities and/or Punnett squares, you will also be able to predict the possible inheritance of the
next generation of the families involved.
Alcaptonuria is classified as a mild genetic disease because it does not cause fetal damage and does not lead
to mental or physical retardation. It is often described as a biochemical disorder because the mutant gene
involved leads to the formation of a defective enzyme. The enzyme is part of a biochemical pathway, which
causes the breakdown of amino acids such as phenylalanine and tyrosine to the waste products water and
carbon dioxide.
Biochemical Pathway:
Tyrosine  P-hydroxyphenylpyruvic acid  Homogentisic acid  X 
 Maleylacetoacetic acid  additional steps  water and carbon dioxide
The enzyme, which is changed by the mutant gene, is called homogentisic acid oxidase. When this enzyme is
altered, the Homogentisic acid is broken down slowly or inefficiently and is found in high concentration in
body fluids such as blood and urine. In a normal individual homogentisic acid is almost undetectable in body
fluids. The blocked step is shown with an X in the pathway above.
The gene location for this mutated gene is on chromosome 3. Alcaptonuria appears in both sexes with about
equal frequency. The wild type (normal) allele is dominant, A, and the alcaptonuria allele is recessive, a. Only
individuals who are homozygous recessive, aa, have alcaptonuria. Individuals who are homozygous dominant,
AA, or heterozygous, Aa, are both normal and cannot be distinguished from one another.
Symptoms and the diagnosis of alcaptonuria are due to the fact that homogentisic acid turns black when
oxidized. Diapers of affected infants stain black as the homogentisic acid oxidizes in air. Oxidation increases
at higher pH so if diapers are washed in alkaline soaps, stains become darker. Later in life connective tissues
often become gray or black as oxidized products of homogentisic acid are deposited. There may also be dark
spots in the cartilage of the ear and on the sclera of the eye. In middle age an arthritic condition, especially in
the lumbar region of the spine, hips, and knees, may develop. Degeneration of the spinal disks may also occur.
Blood and urine samples of diseased individuals show highly elevated levels of homogentisic acid. None of
the symptoms is life threatening and alcaptonurics appear to have normal lives.
Alcaptonuria is rare worldwide; however, disease incidence is higher in some parts of the world such as
Northern Ireland, and at other locations where inbreeding still occurs. Statistics for this disease are difficult to
gather because affected individuals may be unaware that they are affected. Since their urine is acidic, affected
individuals would probably not observe darkened urine since homogentisic acid oxidizes slowly at low pH.
Figure 1 shows the pedigrees of four families that can be analyzed. Individuals with alcaptonuria are darkened
in after being confirmed by lab tests. Work through one of two of the pedigrees as a large group. Then ask lab
groups to discuss the others. Go over them as a large group before proceeding to the actual lab.
 In family I, individual 1 must be Aa in order for his son, individual 2, to have alcaptonuria (genotype aa).
The daughter (#4) must also be Aa, inheriting the A allele from her father and the a allele from her mother.
 In family II, only the daughter (#4) has alcaptonuria. She must have inherited one allele from her father and
one from her mother, both normal people. Therefore her parents both have to be heterozygous (Aa) and are
called carriers.
 In family III, we can determine the genotype of all individuals except the two paternal grandparents.
Knowing that the affected individuals are recessive and normal individuals are dominant, we can do
pedigree analysis and say with certainty that all normal individuals are heterozygous including one of the
paternal grandparents. What you can’t know for certain is:
1. Which paternal grandparent is heterozygous?
2. The genotype of the other paternal grandparent (is it AA or Aa?).
 In family IV, the girl in the third generation who is marked “?” has not had a urine test done so her
phenotype is unknown. Pedigree analysis proves that her mother has the genotype Aa and the fact that her
father is an alcaptonuric means he has the genotype aa. Genetic counselors use these facts to say that she
has a 50% chance of being alcaptonuric and 50% chance of being heterozygous.
In this laboratory, students will perform a biochemical test on “urine” samples to determine the presence or
absence of homogentisic acid. Results of this test will then be used to determine the genotypes of the family
members involved in a pedigree provided.
Objectives:
1.
2.
3.
4.
To apply the principles of Mendelian genetics in a realistic experimental situation.
To learn about chemical pathways and how errors in them can cause disease.
To become familiar with genetic relationships of families through pedigree analysis.
To learn how genetic counselors integrate information they have obtained in order to establish genotypes
of related individuals and to predict patterns of inheritance in members of the next generation.
Safety Notes:
1. Safety glasses must be worn at all times during this lab.
2. The alcaptonuria test solution is poisonous and will permanently stain clothing and paper products. Be
careful not to spill it!
Materials:





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Water and yellow food coloring to make “urine”
Starch to add to the affected test tubes
urine samples in marked test tubes (in plastic rack)
small bottles of Alcaptonuria test solution (iodine)
paper
ruler
safety glasses
Procedure:
1.
2.
3.
4.
5.
6.
7.
8.
Review Mendelian genetics, pedigree analysis, and biochemical pathways and discuss how diseases such
as this one can be studied by combining these areas of analysis.
Obtain the test tube rack containing the urine samples of family members. These samples have already
been placed into the tubes.
Mark the pedigree (Figure 2) with the following labels, which correspond to the labels on the urine
samples.
Test the urine of each person by pipeting 2 drops of the Alcaptonuria test solution into each
sample.
Agitate tubes and look for positive tests (formation of dark color).
Determine which family members have the disease and mark them on the pedigree (Figure 2)
by coloring in their symbol.
With the genetic background you have for this disease, mark the genotype in the two lines
below each person’s symbol on Figure 2. Write a short paragraph below Figure 2 describing
what you have done.
Dispose of the urine properly and clean all glassware.
Figure 1
Family I
Family II
Aa
aa
aa
Aa
Aa
A?
For this couple, stress that we
know that at least one of them
must be Aa. The other could be
AA or Aa. There is no way to
know which one is for sure is Aa
with the information given.
Family III
Aa
Aa or
AA
Aa
Aa
Aa
aa
Aa or
AA
aa
Family IV
aa
aa
Aa
Aa
Aa
Aa
aa
Aa
Aa
Aa
aa
Be sure to discuss why
we can know that some
of the unaffected
individuals are
heterozygous and why
we can’t know—at
least at this time— the
genotype of the son in
Family II. Stress that if
he happens to have a
child with alcaptonuria,
then we would know his
genotype. Otherwise, we
just know that he is A
something.
Teacher Key: Add the starch solution to the test tubes for the shaded individuals
I.
1 MGM
2 MGF
3 PGM
4 PGF
aa
Aa
Aa
Aa
II.
5 MA
6 MU
7M
8F
9 PU
Aa
aa
Aa
Aa
A?
11 S
12 B
aa
10 PA
aa
III.
A?
Analysis/Conclusions:
1.
2.
3.
4.
5.
Is alcaptonuria caused by a gene mutation or a chromosome mutation? Explain!
Alcaptonuria is caused by a gene mutation. The mutated gene changes the function
of one enzyme.
Can you determine the AA genotype from the Aa genotype by using a biochemical t t
test?No, the genotypes AA and Aa are expressed the same.
No, the genotypes AA and Aa are expressed the same.
Explain how a genetic counselor would use information from a biochemical test to
predict patterns of inheritance in members of the next generation.
A genetic counselor would use the results of the biochemical test to assign genotypes
to the individuals in the family. This information would be used to predict the
genotypes of any future offspring.
You are a genetic counselor. A baby has been born with the disease alcaptonuria.
How would you explain the inheritance of the disease to the child’s parents?
Since the disease is a recessive disease it may not have shown in either family before.
The student may also have an explanation of a recessive disease.
Can you know the sister’s genotype for certain? Why or why not?
We know that she doesn’t have alcaptonuria, but since her parents are both
heterozygous, she could be AA or Aa.