Biology Van
Revised 2/06
A Study of Alcaptonuria
Safety notes:
1. Human urine is not used for this lab; therefore, no special safety precautions are
necessary.
2. The alcaptonuria test solution is poisonous and will permanently stain clothing and
paper products. Be careful not to spill it!
Vocabulary1:
Gene- The unit of transmission of hereditary characteristics in an organism. A gene
is usually a segment of DNA occupying a specific place on a particular chromosome.
Each gene influences the inheritance and development of some characteristic.
Recessive Gene- A subordinate gene
Dominant Gene- A prevailing gene
Locus- The position in a chromosome of a particular gene or allele.
Allele-2 One of 2 or more alternate forms of a gene occupying the same locus in a
particular chromosome.
Consanguinity3- descended from the same ancestor.
Enzyme- A complex protein substance produced in living cells that causes or
accelerates chemical reactions in other substances in an organism without
being permanently changed itself.
Genotype- The genetic makeup of an organism as distinguished from its physical
appearance or phenotype.
Oxidation- the loss of electrons. A reaction which removes electrons from the
compound.
Phenotype- The external appearance of an organism resulting from the interaction of
its genotype and its environment.
Punnett Square4- A checkerboard representation of predictable genotypes of children
based on the genotypes of the parents. The parent genotypes are on the outside of the
square, while possible child genotypes are found in each individual square. The
punnett square assumes that there is an equal probability that the parent will pass on
either of its two gene forms (alleles) to each child. In following square, A codes for
the “normal” dominant allele, while a codes for the recessive allele. The disease is
found when 2 recessive alleles are together (aa).
A
1
a  Parent
A
AA
Aa  Child
a
Aa
aa
Genotype
¼ AA
½ Aa
¼ aa
Phenotype
¾ normal
¼ diseased
Definitions, unless otherwise noted, are from The American Heritage Dictionary of Science.
Definition is from Glossary of Genetics- Classical & Molecular.
3
Definition is from Merriam-Webster Dictionary.
4
Definition is from World of Genetics and Genetics.
2
Biology Van
Revised 2/06
Introduction:
Genotypes are passed on from one generation to the next through the inheritance of
chromosomes, which possess thousands of genes. The inheritance of the disease being
studied here can be explained by using the principles discovered by Gregor Mendel in his pea
plant 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,
students will also be able to predict the possible inheritance of the next generation of the
families involved.
In this laboratory, a biochemical test on “urine” samples will be performed 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. Students will apply the principles of Mendelian genetics in a realistic experimental
situation.
2. Students will learn about a biochemical pathways and how errors in it can cause disease.
3. Students will become familiar with genetic relationships of families through pedigree
analysis.
4. Students will 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.
Biology Van
Revised 2/06
Alcaptonuria Pathway:
Biochemical Pathway
One enzyme that is needed for the breakdown of
tyrosine is homgentisic 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 homogentistic acid is almost
undetectable in body fluids. The blocked step,
caused by the deficient enzyme, is shown with an X
in the pathway on the left.
The gene location for this mutated gene is on
chromosome 3 (3q21-q23). Alcaptonuria appears in
both sexes with almost 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. If an
individual is heterozygous, they are referred to as a
“carrier” because of their potential to pass on the
recessive gene to their children.
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, or placed in a solution such
as sodium hydroxide (NaOH), stains become darker.
Later in life, ochronosis (o-kron- sis) may occur. Ochronosis is when connective tissues
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 (called ochronotic arthropathy), 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.
Alcaptonuria is rare worldwide; however, disease incidence is higher in some parts of the
world such as Northern Ireland, and at other locations where consanguinity 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.
Pedigrees:
Figure 2 shows the pedigrees of four families that can be analyzed. Individuals with
alcaptonuria are darkened in after being confirmed by lab tests. In family I, the father must
Biology Van
Revised 2/06
be Aa in order for his son, to have alcaptonuria (genotype aa). The daughter must also be Aa,
inheriting the A allele from her father and the a allele from her mother. In family II, only the
daughter has alcaptonuria, therefore, she must have inherited one allele from her father and
her mother, both carriers. In family III, we can determine the genotype of all individuals
except the two paternal grandparents. Knowing that the affected individuals are homozygous
recessive and normal individuals are either heterozygous or homozygous dominant, we can
do pedigree analysis and say with certainty that all normal individuals are heterozygous
including one of the paternal grandparents. What we can’t prove without further testing is:
Which paternal grandparent is heterozygous? What is the genotype of the other paternal
grandparent? Is it AA or Aa? In family IV, the girl in the third generation is marked “?”. She
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 (and a Punnett square) to say that she has a
50% chance of being alcaptonuric and 50% chance of being heterozygous.
Materials:
urine samples in labeled cuvettes
alcaptonuria test solution
ruler
gloves
Procedure:
1. Obtain the cuvette rack containing the urine samples of family members.
2. Mark the pedigree (Figure 3) with the following labels, which correspond to the labels on
the urine samples.
PGF - paternal grandfather
PGM - paternal grandmother
MGF - maternal grandfather
MGM - maternal grandmother
M - mother
F - father
MA - maternal aunt
PA - paternal aunt
MU - maternal uncle
PU - paternal uncle
B - brother
S - sister
3. Test the urine of each person by adding 2 drops of the alcaptonuria test solution into each
sample.
4. Agitate the tubes and look for positive tests (formation of dark precipitate).
5. Determine which family members have the disease and mark them on the pedigree
(Figure 3) by coloring in their symbol.
6. Create Punnett squares to determine the probabilities of each generation’s genotypes.
7. Mark the genotype in the two lines below each person’s symbol on Figure 3.
8. Dispose of the urine properly (it may be flushed down the sink) and thoroughly rinse
out all tubes with water only. Tube racks with tubes in them may then be placed upside
down on a paper towel to dry.
Biology Van
Revised 2/06
Figure 2
= male
= marriage
= generations
= female
= affected individual
= carrier
Family I
Family II
Aa
aa
aa
Aa
a
Aa
A?
aa
A
a
AA
Aa
Aa
aa
a
A
Aa Aa
a
aa
A
aa
Aa
a
Family III
A
Aa
Aa
aa
A?
Aa
A?
a
A AA
Aa
a
Aa
aa
A
a
a
Aa
aa
a
Aa
aa
Aa
aa
Family IV
aa
aa
Aa
Aa
Aa
Aa
aa
Aa
?
? = ½ Aa;
½ aa
Biology Van
Revised 2/06
References
Barnhart RK. 1986. The American heritage dictionary of science. Boston: Houghton Mifflin.
p 173, 197, 248, 250, 463, 488, 549.
Lerner KL, Lerner BW, editors. 2002. World of genetics. Volume 2. New York: Gale Group.
p 577-579.
O’Brien WM, La Du BN, Bunim JJ. 1963. Biochemical, pathologic, and clinical aspects of
alcaptonuria, ochronosis and ochronotic arthropathy. Am J of Med 34: 813-838.
ScienceDirect. <www.sciencedirect.com>. Accessed 2004 Jun 8.
Phomphutkul C, Introne WJ, Perry MB, Bernardini and others. Natural history of
alcaptonuria. The New Eng J of Med 347: 2111-2122. ProQuest. <http://proquest.umi.com>.
Accessed 2004 Jun 8.
Rieger R, Michaelis A, Green MM. 1991. Glossary of genetics; classical and molecular. 5th
ed. New York: Springer-Verlag. p 16-17.
Robinson R, editor. 2003. Genetics. Volume 3. New York: Mcamillian Refernce USA.
p 194.
Stenn FF, Milgram JW, Lee SL, Weigand RJ, Veis A. 1977. Biochemical identification of
homogentisic acid pigment in an ochronotic Egyptian mummy. Sci 197: 566-568. JSTOR.
<www.jstor.org>. Accessed 2004 Jun 8.
Biology Van
Revised 2/06
Figure 3
I.
1__
2__
3__
4__
II.
1__
2__
3__
4__
1__
2__
III.
5__
6__
Biology Van
Revised 2/06
Name_________________________
Student Evaluation
A Study of Alcaptonuria
Data: Circle the people who had a positive test result.
PGF
PGM
MGF
MGM
M
F
MA
PA
MU
PU
B
S
Analysis/Conclusions:
1. Is alcaptonuria caused by a gene mutation or a chromosome mutation? Explain!
2. Is alcaptonuria a sex-linked trait? Why or why not?
3. Can you determine the homozygous A from the heterozygous genotype by looking at the
phenotype of the individual? If yes, how? If no, why not?
4. Why are recessive trait diseases not common in human populations?
5. Explain how a genetic counselor would use information from a pedigree obtained from a
biochemical test, to predict patterns of inheritance in members of the next generation.
6. Could a genetic disease “suddenly” show up in a family? If so, how would you explain the
inheritance of the disease state to an affected child’s parents?
Biology Van
Revised 2/06
Teacher Notes:
A Study of Alcaptonuria
Approximate time needed to complete lab: 40 minute period
Attached are three possible keys to the lab. However there are various possibilities for the
genotypes of each individual so you may design your own pedigree as long as it follows
reasonable probabilities.
Also, attached is a list of references with more in depth information about Alcaptonuria for
your use.
Answers to Evaluation questions:
1. Alcaptonuria is caused by a gene mutation. The mutated gene changes the function of
one enzyme.
2. Alcaptonuria is not a sex-linked disease. It is found equally in males and females. Also
the mutation is found on chromosome 3, not an X or Y chromosome.
3. No, the phenotype is the same for both AA and Aa (it shows the dominant allele).
4. Recessive trait diseases are not common because in order for them to occur, both parents
must have at least one recessive allele. Even when both parents are carriers, there is only
a 1/4 chance that their child will have the disease. If one parent has the disease and the
other is a carrier, there is still only a ½ chance that their child will also have the disease.
5. A genetic counselor would use a pedigree to make a Punnett square (or use probabilities)
to predict patterns of inheritance in members of the next generation.
6. Since the disease is a recessive disease it may not have shown in either family before.
Carriers of the disease will not show the phenotype. When a child receives both recessive
alleles from their parents, the phenotype will “show up”. However the genes will not
suddenly change in a family (except in a rare mutational change.) The explanation to the
parents should explain carriers, recessive alleles, and pedigree analysis.
Biology Van
Revised 2/06
State Standards
3.1.7. E- Identify change as a variable in describing natural and physical systems.
 Describe fundamental science and technology concepts that could solve practical
problems.
 Explain how ratio is used to describe change.
3.3.10. C- Describe how genetic information is inherited and expressed.
 Compare and contrast the function of mitosis and meiosis.
 Describe mutations’ effects on a trait’s expression.
 Distinguish different reproductive patterns in living things
(e.g., budding, spores, fission).
 Compare random and selective breeding practices and their results (e.g., antibiotic
resistant bacteria).
 Explain the relationship among DNA, genes and chromosomes.
 Explain different types of inheritance (e.g., multiple allele, sex-influenced traits).
 Describe the role of DNA in protein synthesis as it relates to gene expression.
3.2.12. C- Apply the elements of scientific inquiry to solve multi-step problems.
 Generate questions about objects, organisms and/or events that can be answered
through scientific investigations.
 Evaluate the appropriateness of questions.
 Design an investigation with adequate control and limited variables to investigate a
question.
 Organize experimental information using analytic and descriptive techniques.
 Evaluate the significance of experimental information in answering the question.
 Project additional questions from a research study that could be studied.
3.3.12. B- Analyze the chemical and structural basis of living organisms.
 Identify and describe factors affecting metabolic function (e.g., temperature, acidity,
hormones).
 Evaluate metabolic activities using experimental knowledge of enzymes.
 Evaluate relationships between structure and functions of different anatomical parts
given their structure.
 Describe potential impact of genome research on the biochemistry and physiology of
life.
3.3.12. C- Explain gene inheritance and expression at the molecular level.
 Analyze gene expression at the molecular level.
 Describe the roles of nucleic acids in cellular reproduction and protein synthesis.
 Describe genetic engineering techniques, applications and impacts.
 Explain birth defects from the standpoint of embryological development and/or changes
in genetic makeup.
Biology Van
Revised 2/06
Key 1
MGM
MGF
PGM
2 A? (most likely AA)
3 Aa
PGF
I.
1 aa
MA
MU
M
4 Aa
F
PU
PA
II.
1 Aa
2 Aa
3 Aa
4 aa
5 A?
6 A?
(most likely Aa)
S
B
III.
1 Aa
2 aa
Key 2
MGM
MGF
PGM
2 aa
3 Aa
PGF
I.
1 Aa
MA
MU
M
4 Aa
F
PU
PA
II.
1 Aa
2 aa
3 aa
4 Aa
5 A?
(most likely Aa)
S
B
III.
1 aa
2 Aa
6 aa
Biology Van
Revised 2/06
Key 3
MGM
MGF
PGM
PGF
I.
1 Aa
MA
2 Aa
MU
3 Aa
M
4 Aa
F
PU
PA
II.
1 aa
2 A?
3 Aa
4 Aa
5 aa
(most likely Aa)
6 A?
(most likely Aa)
S
B
III.
1 Aa
2 aa