DISORDER DETECTIVES
Examining human chromosome disorders
DISORDER DETECTIVES: Examining human chromosome disorders
Published by HudsonAlpha Institute for Biotechnology, 2010
Educational Outreach
HudsonAlpha Institute for Biotechnology
601 Genome Way
Huntsville, Alabama 35806
http://education.hudsonalpha.org
Permission is hereby granted to teachers to photocopy any pages or figures in this laboratory kit for classroom use.
Teachers may also make transparencies of pages in the Teacher’s guide. Requests or reprinting for photocopying for
distribution outside of the typical classroom setting should be made to HudsonAlpha Educational Outreach via email
at edoutreach@hudsonalpha.org.
This kit is intended for educational purposes only. It is not to be used for research or diagnostic purposes.
Image Credits: human chromosome spread on Chromoscan board and human karyotype and ideogram in instructor
manual: University of Washington, Department of Pathology
http://www.pathology.washington.edu/. Human chromosome packaging in instructor manual modified from
National Institute of General Medical Sciences http://images.nigms.nih.gov/. Human chromosome ideograms on
Chromoscan board redrawn from Mittleman F (ed) 1995) ISCN 1995: An International System for Human Cytogenetic
Nomenclature. Karger, Basel, 114 pp.
Funding for the development of this module and its associated activities has been provided in part by the
Educational Outreach Program at the HudsonAlpha Institute for Biotechnology, located in Huntsville, Alabama.
The Institute, a unique partnership between scientific researchers and biotechnology companies, has a strong
commitment to educating today’s youth about opportunities in biotechnology. For more information about the
ongoing work at HudsonAlpha, please visit www.hudsonalpha.org.
A previous version of this product was funded in part by a grant awarded under the Workforce Innovation in
Regional Economic Development (WIRED) Initiative as implemented by the U.S. Department of Labor’s Employment
& Training Administration. The information contained in this product was created by a grantee organization and
does not necessarily reflect the official position of the U.S. Department of Labor. All references to non-governmental
companies or organizations, their services, products or resources are offered for informational purposes and should
not be construed as an endorsement by the Department of Labor.
This product is copyrighted by the institution that created it and is intended for individual organizational, noncommercial use only.
FOREWORD
In the broadest sense, biotechnology is the use of biological processes, organisms or systems to develop products
aimed to improve some aspect of life. Biotechnology at its roots is a very old science, stretching back 7,000 years
to the creation of bread, cheese, wine, and vinegar (which all depend on harnessing and modifying some biological
process). The field has expanded dramatically over the last quarter century, powered by our understanding of DNA,
the recipe card inside the nucleus of our cells. This recipe card provides the instructions to make proteins and all the
structures of the cell.
The DNA is packaged into individual units known as chromosomes. Humans typically have 46 chromosomes that
can be uniquely identified through a staining process known as banding. The collection of identified chromosomes
is known as a karyotype. Scientists can study the karyotype to identify alterations in chromosome number and
structure that may result in a broad range of clinical outcomes ranging from asymptomatic chromosomal variation
to Down syndrome or certain types of cancers.
This classroom activity offers students the chance to take on the role of a cytogeneticist, reviewing a patient history
and assembling that patient’s karyotype. Once assembled, the karyotype is analyzed for numerical or structural
anomalies that may link to the clinical symptoms.
Additional student content including web-based exercises and activities are included in an online supplement that
accompanies this activity - www.hudsonalpha.org/education/kits/disorder-detectives. Career profiles of individuals
who work in fields related to this activity, such as genetic counselors and laboratory technicians, are also included at
this site.
ACKNOWLEDGEMENTS
This activity was developed by the HudsonAlpha Institute for Biotechnology through the combined work of Dr. Bob
Zahorchak, Jennifer Carden, Madelene Loftin, Dr. Adam Hott and Dr. Neil Lamb. The Chromoscan board and layout
for the instructor manual were designed by J.D. Frey. Kelly East developed the online supplement that accompanies
this lab. Additional assistance in preparation and modification was provided by Michelle Morris, April Reis, Kelly Hill
and Dedra, Griffin, Rebecca and Lisa Herod.
Thanks also to Steve Ricks, Robin Nelson, Martha Anne Allison and the outstanding Science in Motion Biology
Specialists at the Alabama Math, Science, and Technology Initiative (part of the Alabama State Department of
Education) for their assistance in piloting the initial version of the activity.
CONTENTS
I.
Foreword
4
II.
Acknowledgements
4
III. Disorder Detectives: examining human chromosome disorders 1. Overview
7
2. Learning Objectives
7
3. Suggested Unit Correlative
7
4. Materials
8
IV. Timeframe of Activity
10
V.
1. Day One: (Can be completed in 20-25 minutes)
10
2. Day Two: Lab exercise (Can be completed in 45-60 minutes)
10
3. Optional extension of the activity (Day Three or at home)
10
Instructor Introduction and Background
VI. Instructor Protocol
6
7
11
16
A. Prior to Day One
16
B. Day One – Description of Karyotyping
16
C. Day Two – Assembling the Karyotype
16
VII. Instructor’s Key to Patient Karyotypes
18
VIII. Student Handout
24
1. Overview
24
2. Background
24
3. Protocol for the Karyotyping Activity:
26
4. Kit contents
9
IX. General Information for the instructor Regarding Each
21
X.
21
Chromosomal Disorder
DISORDER DETECTIVES: EXAMINING HUMAN CHROMOSOME
DISORDERS Overview
This activity will provide students with a hands-on experience studying human chromosomes and connecting
various karyotypes with chromosomal disorders. Students will take on the role of a cytogeneticist working in a
hospital or clinic. Students will be given a case study and a set of patient chromosome decals and asked to arrange
the chromosomes on a prepared board into a completed karyotype. Students will then analyze the karyotype and
diagnose their patient. Many types of chromosomal anomalies are presented, though normal karyotypes are also
represented.
Learning Objectives
The Student will:
• describe the anatomy of a human chromosome.
• describe how a karyotype is created and how it is used to diagnosis chromosomal disorders.
• construct a simulated karyotype.
• diagnosis patient disorders based on karyotype analysis.
• relate chromosomal abnormalities to clinical symptoms.
• identify types of chromosomal changes resulting from nondisjunction, deletion, inversion and translocation.
Suggested Unit Correlative
It is recommended that this activity be completed after a study of meiosis, as many chromosomal disorders occur
during oogenesis and spermatogenesis. Basic chromosome structure (centromere, telomere, p and q arm), the
process of chromatin packing, and the various categories of numerical and structural chromosome disorders should
also be addressed ahead of time.
Materials
The Disorder Detectives kit contains enough material for use by a class of 30 students, working in pairs. The kit
includes the following components:
• 15 “Chromoscan” boards with various case studies and space for karyotype assembly
• 15 sets of chromosome decals (reusable vinyl stickers), color coded by case
• Instructor manual, including student handout pages
Teachers will need to provide copies of student background information and the “Cytogeneticist Report for
G-Banded Karyotype” to complete the patient karyotype information and diagnosis. These pages are in the
instructor manual and permission is granted to teachers to photocopy student pages for classroom use.
Note: The first time the Disorder Detectives kit is used, the chromosome decals
will need to be separated from each other and placed on the “cryostorage”
section found on the right side of the Chromoscan boards. Two complete sets
of chromosome decals are provided: one set is to be used with the gameboards
while the other set serves as a “backup” to replace missing or damaged decals.
Separate the sets at the perforation in the middle of the decal page. Set aside the
chromosomes labeled spare set. The chromosome decals are color-coded and
correspond to a specific case study on the Chromoscan board – match the color of
the chromosome decals to the color that surrounds the case study and cryostorage
section of the board. After a student assembles the karyotype and completes the
exercise, the chromosomes need to be returned and stored in random order to the
“cryostorage” section of the board.
The chromosomes can be cleaned by rinsing carefully in warm water to remove
lint, dust and debris. Pat gently with a paper towel or soft dry cloth. DO NOT use
harsh cleaners or chemicals on the chromosomes.
Find more information regarding additional kits, upgrades, resources and
replacement pieces — hudsonalpha.org/education/kits
8
Kit contents
1
detach spare chromosome
set and store in box
Manual
15 chromosome decal sets
15 spare decal sets
2
Unfold Chromoscan boards
15 Chromoscan boards
Classroom kit box
4
5
3
Match decal sets to
Chromoscan board
Students will match chromosome decals to images
on the Chromoscan board following instructions in
the student handout
After a student assembles the
karyotype and completes the exercise,
the chromosomes need to be returned
and stored in random order to the
“cryostorage” section of the board
Chromoscan boards and decals are now
ready for the next class or to be folded
and stored in the box
THE SCIENCE OF PROGRESS
9
TIMEFRAME OF ACTIVITY
Day One: (Can be completed in 20-25 minutes)
Discuss with students how and why a karyotype is created. Note that karyotyping is the standard method to identify
chromosome number and structure. However, small deletions and insertions are below the resolution of a karyotype
and cannot be detected by this method. These abnormalities require other methods of detection such as FISH or
array CHG. In addition, single nucleotide changes require a detection method such as direct DNA sequencing or
microarray analysis.
Day Two: Lab exercise (Can be completed in 45-60 minutes)
• Students will receive case study and assemble karyotype.
• Students will use the “Cytogeneticist Report for G-Banded Karyotype” to describe the karyotype and create a
diagnosis for the patient, including patients with chromosomally normal karyotypes.
• The various results will be discussed with students, exploring options where additional testing may
be necessary.
Optional extension of the activity (Day Three or at home)
Students will use available resources, such as textbooks or online references, to identify additional clinical features
associated with the chromosomal diagnosis as well as patient and caregiver resources such as support groups or
nearby specialists.
If the classroom is scheduled on a block format (90 minute segments), both the overview of karyotyping and the lab
activity can be performed in a single day.
10
INSTRUCTOR INTRODUCTION AND BACKGROUND
Note to instructor: The student guide also addresses these topics, but in much less
detail. Additional content is provided here to support the instructor in discussing the
concepts of karyotyping and cytogenetics with the class.
Additional student content including web-based exercises and activities can be
found at the online supplement designed to accompany this exercise —
http://www.hudsonalpha.org/education/kits/disorder-detectives
What is a chromosome?
The DNA of all living organisms is organized into discrete packets called chromosomes. Many bacteria have a single
circular chromosome. Most multicellular organisms have several large linear chromosomes, contained in the nucleus
of each cell. Most human cells contain 46 chromosomes, grouped into two sets of 23, a maternal set contributed by
the mother’s egg and a paternal set provided from the father’s sperm. (Note that the body cells are diploid, while
the gametes (egg and sperm) are haploid). The maternal and paternal chromosomes of a pair are called homologous
chromosomes, or homologs. Within each set of chromosomes there is one sex chromosome and 22 other
chromosomes, called autosomes. There are two types of sex chromosomes, classified as “X” and “Y”. Typically,
(although not always as we shall see), males have both an “X” and “Y” sex chromosome, while females have two
“X” sex chromosomes.
Number of Chromosomes Across Various Organisms
Organism
Diploid Chromosome Number
D. melanogaster (fruitfly)
8
C. elegans (roundworm)
12
Z. maize (corn)
20
F. catus (domestic cat)
38
M. musculus (mouse)
40
H. sapiens (human)
46
O. aries (sheep)
54
B. tarus (cow)
60
C. familiaris (dog)
78
What is the structure of a chromosome?
The human genome consists of 3 billion base pairs of DNA. This is roughly equivalent to the amount of information
contained in a stack of telephone books the height of the Washington monument. If the chromosomes from a single
cell were unwound and placed end to end, there would be nearly 6 feet of DNA. For this information to fit into the
nucleus, it must be substantially condensed. The DNA wraps around scaffolding proteins known as histones, much
like string might be wrapped around a set of beads. The wrapped histones are then coiled into larger groups called
nucleosomes, which stack together to form chromatin strands. Most of the time, the chromosomes are present as
long tangled chromatin strands and individual chromosomes cannot be distinguished from one another.
Before a cell undergoes mitosis or meiosis, its DNA is replicated. The two copies of each chromosome, called sister
chromatids, are temporarily held together at a specific location on the chromosome called the centromere. The
DNA strands undergo even more condensation, packing into a compact, easily movable form – the familiar shape
we typically associate with a chromosome. As the cell moves out of prophase and into metaphase, individual
chromosomes can be easily identified.
THE SCIENCE OF PROGRESS
11
How are chromosomes classified?
The chromosomes can be uniquely identified based on several features. They vary in size and the autosomes are
numbered roughly from largest to smallest. The largest chromosome, chromosome #1, contains over 3,ooo genes
and is approximately 240 million base pairs long. Chromosome 21 is the smallest, containing only a few hundred
genes distributed across approximately 46 million basepairs. Note that early cytogeneticists misclassified the
relative size of chromosomes 21 and 22: Chromosome 22 is actually the larger of the two. With regards to the sex
chromosomes, the X chromosome has around 1400 genes and approximately 150 million base pairs while the Y
chromosome has over 200 genes and about 50 million base pairs.
The relative location of the centromere along the chromosome is another distinguishing feature. The centromere
divides the chromosome into two regions, also called arms. Metacentric chromosomes have arms of roughly equal
lengths. The arms of submetacentric chromosomes are slightly unequal; the shorter arm is called the p arm (from
petit, the French term for small) and the longer arm is the q arm (so called because q follows p in the alphabet).
Acrocentric chromosomes have a centromere that is even closer to one end of the chromosome (known as the
telomere).
While relative size and centromere position are helpful in identifying specific chromosomes, several appear identical
by these criteria alone. Chromosome identification was greatly revolutionized by the discovery that certain dyes
would produce stained bands on the chromosome. These reproducible patterns of dark and light bands uniquely
identify each chromosome. Consequently, chromosome banding has become a standard tool for cytogenetic
analysis. Various dyes produce different banding patterns – for the purposes of this activity we will focus on the
pattern produced by the Giemsa stain, known as G-banding. It is important to note that each band DOES NOT
indicate the location of a single gene – for some chromosomal regions, hundreds of genes may be present in a band
while other regions may have relatively few genes per band.
What is a karyotype?
A karyotype is an organized profile of an individual’s chromosomes. Generally the chromosomes have been
stained, identified and organized in a specific order. This allows a scientist called a cytogeneticist to examine the
chromosomes and quickly identify alterations that may result in a genetic disorder.
A word about chromosome and karyotype nomenclature: There is a very precise
system for describing parts of each chromosome. The p and q arm have been
described above. Each arm is subdivided into regions, numbered consecutively
from the centromere. Regions are then divided into light and dark bands, numbered
within the region. Large bands are further subdivided into smaller sub-bands as
necessary, allowing for more precise localization. Sub-band numbers are given
after band numbers, separated by a decimal point. For example, 15q21.3 refers
to the long arm of chromosome 15, region 2, band 1, sub-band 3. The correct
pronunciation of this location would be “15q two one point three”.
When summarizing the karyotype of an individual, the total number of
chromosomes are written first, followed by the sex chromosomes present. A
typical male has a 46, XY karyotype and 46, XX describes a typical female. If a
chromosome abnormality is identified, it is written last. Extra chromosomes are
recorded with a + symbol, a translocation is noted by a t, a deletion by del, and an
inversion with inv. For example, 47 XY +21 describes a male with an extra copy
of chromosome 21 (commonly known as Down syndrome), while 46, XX del 5p
describes a female missing the p arm of chromosome 5.
12
Note that it is not necessary for students to master the complexities of
chromosome nomenclature for this activity. Simply describing the disorders using
words or phrases is fine.
How is a G-banded karyotype created?
As described above, chromosomes are only visible when fully condensed in actively dividing cells. A karyotype can
be obtained from blood, skin or other samples, but few if any cells will be in the process of dividing. Therefore,
the sample is grown in the laboratory for 2-3 days using a chemical that stimulates the cells to undergo division. A
second chemical is then added to the cells which halts the cell cycle in metaphase, providing many cells with fully
condensed chromosomes. The cells are then swollen and dropped onto a glass slide to break open the nucleus.
The chromosomes are stained with Geimsa stain and observed under a microscope. A digital image is taken.
Computer programs help the laboratory scientist identify each chromosome and arrange them in a predetermined
order, usually from largest to smallest with the sex chromosomes placed at the end. The cytogeneticist counts the
chromosomes, identifies each one and confirms that its structure appears normal. Ten cells are typically analyzed for
each sample.
Why do some karyotypes show chromosomes that look like
an “X”, but the chromosomes from other karyotypes look like
an “I”?
Because karyotypes are prepared from cells that have replicated their
DNA in preparation to divide, each chromosome has two identical
sister chromatids joined at the centromere. Early karyotyping protocols
produced stained chromosomes where the sister chromatids were
separated, resulting in the “X” shape. More recent laboratory
techniques result in chromosomes where the sister chromatids are
tightly pressed together, appearing more like an “I”.
For the Disorder Detectives activity, students will be working with
drawings of G-banded chromosomes obtained from the recent
preparation methods. Geneticists use these types of diagrams, called
ideograms, as standard representations for chromosomes. This makes
it easier for students to compare the banding patterns and identify
chromosomal abnormalities.
x
- +
CryoS
CryoStorage.stor
C
ryoStorage.stor
Chromosome Preparation Protocol
1
2
3
4
5
Add cell sample to culture media
Induce cell growth and division;
culture up to two weeks
Arrest cells in metaphase
Swell and drop cells onto
microscope slide. Cells burst
spreading out chromosomes
Stain with Giemsa dye and
observe under microscope
A generalized flowchart of the G-banding
protocol is included on right side of the
student’s Chromoscan board.
When are Chromosome Disorders Most Commonly Observed?
Chromosome abnormalities have been identified in a number of key clinical situations:
• Chromosome abnormalities have been found in over half of embryos that spontaneously abort during the first
trimester.
• Approximately 1 of every 200 newborns has clinical symptoms caused by a chromosome anomaly.
• When a couple is dealing with infertility or recurrent miscarriages, chromosome disorders such as
translocations and inversions may be present in the man or woman. These do not cause any symptoms in the
adult, but can lead to abnormal eggs or sperm.
• Cells often acquire chromosome abnormalities as they progress from normal to cancerous. Many times there
are specific outcomes that correspond with a particular cancer or clinical outcome, such as how fast the cancer
metastasizes or how well it responds to a particular treatment.
What types of samples are often obtained for karyotyping?
Cells may be obtained from various sources for karyotype analysis, including:
• Blood – Blood samples are the most common sample obtained for karyotyping. Chromosomes are examined
from lymphocytes (white blood cells) because in mammals the erythrocytes (red blood cells) expel their
nucleus to provide more space for hemoglobin.
• Skin or other tissues – Cells may be obtained from the inside of the cheek by gently swabbing with a soft
brush. In other instances, larger samples may be required, e.g., if the chromosome abnormality is present in a
cancerous tumor, a biopsy of the region will be required.
THE SCIENCE OF PROGRESS
13
• Chorionic villi - The chorionic villi are wispy projections that make up most of the placenta. These structures
were formed from the embryo very early in development. Chorionic Villus Sampling (CVS) involves removing
some of the chorionic villi from the placenta. This is an early prenatal test that can be conducted at 10 - 13
weeks’ gestation, but carries a 1-2% risk of miscarriage. CVS may be performed by inserting a catheter through
the cervix or across the abdomen until it reaches the placenta and gently suctioning a tissue sample. The results
may be available in as few as 7 days due to the presence of rapidly dividing chorionic villi cells. A more lengthy
analysis may also be performed by culturing slower growing villi cells.
• Amniotic fluid – Amniotic fluid surrounds the fetus and contains fetal cells that have been shed during
development. The process of withdrawing this fluid using a hollow needle is called amniocentesis. It is
conducted between 14 and 20 weeks’ gestation and has a 1% or less risk of miscarriage. Doctors use an
ultrasound device to locate the fetus and identify a safe entry point for the needle. The fetal cells present in the
amniotic fluid are slow growing and may require two weeks to provide enough dividing cells for karyotyping.
Because of this, it can be nearly three weeks after the amniocentesis before final results are reported.
What types of chromosome anomalies are identified by G-banded karyotyping?
Numerical Changes:
• Atypical sets of chromosomes such as triploidy (69 chromosomes – three complete sets)
• Altered numbers for one particular chromosome- Monosomy (one chromosome too few) and Trisomy (one
chromosome too many)
Numerical changes can result from atypical cell division in either mitosis or meiosis. Instead of dividing the
chromosomes evenly, the chromosomes segregate improperly. If the error occurs in meiosis and the affected
egg or sperm participates in fertilization, all the cells of the developing embryo will have an abnormal number of
chromosomes. If the error occurs during mitosis, only the daughter cells that develop from the abnormal cell will be
affected and the remainder of the cells will have the typical chromosome number.
Structural Changes:
• Translocations – A translocation occurs when two chromosomes swap segments. If two acrocentric
chromosomes are joined at their centromeres (deleting their p arm fragments, which generally only contain
repeating DNA sequences), the resulting chromosome is known as a Robertsonian translocation.
• Deletions - Deletions involve the loss of a segment of the chromosome. Deletions may occur at the ends of a
chromosome or involve the loss of material in the middle of a chromosome arm.
• Inversions - Inversions occur when part of a chromosome is placed in the wrong orientation compared to the
rest of the chromosome. The inverted region may involve the centromere or may only be concentrated on a
single chromosome arm.
• Duplications - Duplications occur when a segment of the chromosome is replicated and inserted next to the
original copy.
Like numerical changes, structural changes can also occur in meiosis or mitosis. Structural abnormalities are often
due to chromosome breaks that are improperly repaired. In some cases, the structural change is unnoticed in
the individual. This is often the case in translocations and inversions where no genetic material is lost or gained.
However, when these chromosomes attempt to initiate meiosis during egg or sperm formation, it becomes difficult
to correctly match the homologous pairs. Often egg and sperm are produced with extra or missing chromosome
fragments, leading to infertility, repeated miscarriages or offspring with serious clinical symptoms.
Note: Sample karyotypes of many of these anomalies can be viewed at
Cytogenetics Gallery by the Department of Pathology, University of Washington,
Seattle — http://www.pathology.washington.edu/galleries/Cytogallery/
14
What types of anomalies will not be identified by G-banded karyotyping?
Translocations, deletions, inversions, insertions or duplications that are under 1 million base pairs in size are below
the resolution of standard karyotyping. Individuals with these types of disorders will have an apparently typical
G-banded karyotype. (An example of this is seen in the lab activity. Case K describes an individual with 22q11.2 deletion
syndrome, a microdeletion on chromosome 22 that is frequently too small to be observed by standard G-banding.) A
set of more recent techniques, collectively titled molecular cytogenetics, has been developed that can identify many
of these smaller changes. FISH (Fluorescent In Situ Hybridization) can detect changes as small as ten or twenty
thousand nucleotides in size. FISH can also be used on dividing or nondividing cells, reducing the laboratory time
required by karyotyping. An even more advanced technique, called aCGH (array comparative genome hybridization)
can detect abnormalities only a few thousand nucleotides in size and can scan across the entire genome in a single
sweep.
Because FISH and aCGH can detect small changes, they are often used as a follow-up method when a standard
G-banded karyotype appears normal, but clinical symptoms strongly suggest a chromosome disorder. However, if
the symptoms are caused by very small genetic mutations, i.e. a single nucleotide change or a deletion/insertion of
100 basepairs or less, more direct molecular techniques such as DNA sequencing must be performed for detection
and diagnosis.
What benefit does karyotyping provide?
Prenatally, the results of a karyotype can provide answers or a diagnosis. Parents may utilize this information to
identify a care team to be present at the birth or to make an informed reproductive decision. In postnatal cases, the
results can be used to diagnose complicated syndromes.
In both prenatal and postnatal cases, the results can be used to identify additional risks or symptoms that require
surveillance. Implications for future children as well as risks that may be present among other family members can
also be revealed. While many chromosome disorders do not run in families, translocations, duplications, deletions
and inversions may be passed from generation to generation. Genetic counselors can help parents understand test
results and provide information as parents or patients reach decisions regarding next steps.
Often a pregnant woman is offered a blood test to help identify certain chromosome disorders – is this
a karyotype?
In the US, as in many other countries, pregnant women are offered a screening test for a number of disorders,
including neural tube defects and certain chromosomal trisomies. Although the word ‘screening’ is often used
loosely as a synonym for ‘testing’, the two are not identical and the maternal blood test is not used to create a
karyotype for the fetus. The screening tests measure specific proteins found in the blood of pregnant women
to identify who should be offered more extensive (and often more invasive and expensive) testing, such as
amniocentesis to obtain fetal cells for karyotyping. In the lab activity, cases B,C,G and J address women who have
undergone these serum screening tests. The screening test is not 100% sensitive and often women who are carrying
a normal fetus have an abnormal screening test and must deal with the stress of deciding whether or not to
undergo amniocentesis. At the same time, there are a small number of cases where truly abnormal pregnancies go
undetected by the maternal blood screen.
While the risk of having a child with a chromosome abnormality rises sharply for older woman, screening on the
basis of maternal age alone is not very sensitive. Because there are so many more pregnancies among younger
women, the majority of babies with chromosome anomalies are in fact born to younger women. To improve the
sensitivity of the screening process, several proteins in the mother’s blood are measured. The overall distribution
of these proteins is predictive (but not diagnostic) for a number of disorders, including trisomy 13, 18, 21 and neural
tube defects, especially when coupled with a fetal ultrasound. Previously these proteins were measured during
the second trimester of a woman’s pregnancy, although new screening techniques are being developed that offer
results much earlier. Women with a positive screening test are then offered the option of having more invasive
diagnostic testing.
THE SCIENCE OF PROGRESS
15
INSTRUCTOR PROTOCOL
Prior to Day One
Students should have a basic understanding of chromosomes and chromosome abnormalities before beginning this
activity. This understanding should include:
•
•
•
•
•
the process of meiosis, including oogenesis and spermatogenesis.
differences between meiosis and mitosis.
basic chromosome structure (centromere, telomere, p and q arm).
the process of chromatin packing.
the various categories of numerical and structural chromosome disorders, including monosomy, aneuploidy,
translocation, deletion and inversion.
Make copies of the student background information, protocol and “Cytogenetics Report for G-Banded Karyotype”
for each student in the class. The student information is found following the instructor materials in this manual.
Permission is granted to teachers to reprint or photocopy in classroom quantities the pages or sheets needed for
the students.
Day One – Description of Karyotyping
Using the background information found in this manual, discuss with students why a physician would want to
examine a patient’s chromosomes, including the role of chromosomal abnormalities in miscarriages, birth defects
and cancer.
Introduce the concept of a karyotype and familiarize students with the basic protocol used to obtain a standard
G-banded chromosome preparation. Remind students that the only time chromosomes are visible under a light
microscope is during cell division, so samples to be karyotyped must be living cells that can be induced to divide.
Review the various types of cell sources that may be used to obtain a karyotype. This may involve explaining the
processes of CVS and amniocentesis if these topics have not previously been discussed.
Note the types of chromosome anomalies that can be identified by G-banding and discuss those abnormalities that
standard karyotyping will not identify. Note that molecular techniques such as FISH, aCHG and DNA sequencing can
detect these changes.
Explore the concept of maternal screening, if not previously discussed. Several of the case studies involve pregnant
women and maternal serum screening. Make sure students understand the difference between a screening
procedure and a diagnostic test.
Day Two – Assembling the Karyotype
Provide each student with a copy of the “Cytogenetics Report for G-Banded Karyotype”. Although students will
work in pairs, each student will complete a copy of this report.
Distribute one of the 15 Chromoscan boards containing a case study and set of patient chromosome decals to each
pair. Each case study has been assigned a letter A-O and a unique color. The color of the patient chromosomes
should match the color that surrounds the case study and cryostorage regions of the Chromoscan board. Have
students confirm the color match.
Have each student begin by selecting a single chromosome decal from the cryostorage area of the board and sketch
that chromosome on their Cytogenetics Report. Students need to note the centromere, telomere, and p and q arms
on the diagram and classify the chromosome based on centromere position.
16
Instruct students to write a brief explanation on the Cytogenetics Report describing how a karyotype is prepared.
Have students read the case study found on the left side of the Chromoscan board. Based on that information,
students will need to begin completing the initial patient information on the Cytogenetics Report.
To make the process of karyotype assembly less complex, one of each of the homologous chromosomes is already
illustrated on the Chromoscan board. Have the student pairs identify the other homolog from the chromosome
decals and place it on the board in the proper position.
Once the karyotype is completed, instruct the student pair to analyze it for chromosomal anomalies, paying
particular attention to chromosome number and large-scale structure.
Have students develop a diagnosis by referring to the “Example of Findings Commonly Identified by Karyotyping”
located at the end of their information packet.
Student pairs will complete the Cytogenetics Report on their patient, using information found in the case study and
the results of the karyotype analysis.
Two post-activity questions are included at the bottom of the Cytogenetics Report. Tell student pairs to discuss
these questions and formulate their answers.
At the end of the activity, students should return the chromosome decals to the “cryostorage” region of the board
in random order, preparing the board for the next group’s use.
Have students check carefully around desks and lab tables to make sure all chromosome decals have been collected
and returned to the board.
As an optional follow-on activity, the students may use available print and online
resources to learn more about their diagnosis. The Cytogenetics Report includes
a section where notes for the patient’s caregiver may be included. While such
information would not typically be included on a true karyotype lab report, it would
be communicated to the family during a subsequent visit with the physician or
genetic counselor. Students may identify support groups or physicians in their
region who work with these types of diagnoses. Students who have patients with
normal karyotypes can be assigned one of the other variants for this activity.
THE SCIENCE OF PROGRESS
17
INSTRUCTOR’S KEY TO PATIENT KARYOTYPES
Note to instructor: The case studies marked with a * will require more intense analysis of the chromosomes in
order to properly diagnose the results. These cases may be more appropriate for advanced students or may require
additional assistance from the instructor or other classmates.
Case Case Color Reason for Referral
Source of Cells
A
newborn with several clinical findings, including low
birthweight, misshapen head and clenched fist
pregnancy with advanced maternal age; positive maternal
serum screen
pregnancy with advanced maternal age; maternal serum screen
did not lower risks
newborn with unusual clinical findings, including cat-like cry
blood
newborn with several serious birth defects who died at one
week of age
newborn with low birthweight
blood sample taken before
child died
blood
B
C*
D
E
F
G
amniocytes
blood
H
pregnancy with advanced maternal age; maternal serum screen amniocytes
did not lower risks
adult male with infertility issues
blood
I*
adult female experiencing weight loss, exhaustion
J
pregnancy with advanced maternal age; maternal serum screen amniocytes
did not lower risks
20 week fetus with heart abnormality and cleft palate identified amniocytes
by ultrasound
K*
L
M
N*
O*
18
amniocytes
blood
pregnancy with advanced maternal age, mother wants answers chorionic villi
in first trimester (before maternal serum screening is offered)
newborn with atypical features including low set ears and
blood
webbing at neck and between fingers
20 week fetus with very small gestational size from ultrasound amniocytes
pregnancy with advanced maternal age, mother wants answers chorionic villi
in first trimester (before maternal serum screening is offered)
Total Chromosome #, Sex
Chrosomes Present & Gender
Karyotype Finding
Diagnosis
47 XX
(female)
trisomy 18
Edward Syndrome
47 XX
(female)
trisomy 21
Down Syndrome
46 XY
(male)
Down Syndrome
46 XX
(female)
robertsonian translocation
with chr. 14/21
5p deletion
47 XY
(male)
trisomy 13
Patau Syndrome
46 XY
(male)
no anomalies identified
none
46 XX
(female)
no anomalies identified
none
47 XXY
(male)
XXY
Klinefelter Syndrome
46 XX
(female)
Chronic Myelogenous Leukemia
47 XXX (female)
reciprocal translocation
with chr. 9/22
XXX
46 XY
(male)
no anomalies identified
47 XYY
(male)
XYY
22q11.2 microdeletion syndrome – a microdeletion
of 22q that often is so small that it is only identified
by molecular techniques like FISH or aCGH
XYY male
45 X
(female)
monosomy X
Turner Syndrome
46 XY
(male)
46 XX
(female)
inversion on chr. 3 involving
the p arm, centromere and
q arm
inversion on chr. 9
none – this is an identified variant in the human
population that does not appear to be associated
with clinical symptoms
none – this is an identified variant in the human
population that does not appear to be associated
with clinical symptoms
Cri du chat
Trisomy X female
THE SCIENCE OF PROGRESS
19
GENERAL INFORMATION FOR THE INSTRUCTOR REGARDING EACH
CHROMOSOMAL DISORDER
Chromosome
Diagnosis
Incidence and Clinical Findings
Finding & Case ID
trisomy 18
Edward Syndrome occurs in 1 per 3,000 livebirths
Case A
trisomy 21
Clinical features present include heart and kidney defects, clenched hands,
a small head and jaw, fluid-filled cysts in the brain and anomalies in the
digestive system.
Down Syndrome
Case B and C
5p deletion
Clinical features include congenital heart defects, digestive blockages,
childhood leukemia, respiratory and hearing problems and developmental
and intellectual delay (although there is a wide range of delay present,
from mild to severe).
Cri du Chat
Life expectancy for a child born with Down Syndrome is 60 years.
occurs in 1 per 37,000 livebirths
Patau Syndrome
Clinical features present at birth often include low birth weight, poor
muscle tone, small head and a high-pitched cat-like cry. As they grow,
many individuals have trouble with language, as well as feeding difficulties,
delays in walking and significant intellectual delays.
occurs in 1 per 16,000 livebirths
Case D
trisomy 13
Case E
XXY
Case H
50% of babies carried to term are stillborn, less than 10% survive their first
year; a small number survive into adulthood, although with significant
developmental and intellectual delays
occurs in approximately 1 per 800 livebirths (most commonly occurring
chromosomal condition)
Clinical features include heart defects, brain or spinal cord abnormalities,
small or poorly developed eyes, extra fingers or toes, cleft lip and or
palate, weak muscle tone, and severe intellectual delay.
Klinefelter
Syndrome
More than 80% of children with trisomy 13 die in the first month and only
5-10% survive their first year.
occurs in 1 in 500 to 1 in 1,000 newborn males (does not occur in females)
Klinefelter syndrome affects different stages of physical, language and
social development. The most common symptom is infertility. Individuals
often produce a lower level of testosterone, leading to reduced secondary
sex characteristics and delayed puberty. Mild learning and language
problems have also been linked to Kleinfelter syndrome.
Many people with Klinefelter syndrome have few noticeable symptoms
and often the condition goes undiagnosed until adulthood.
20
Available Treatments
Associated Support Groups
Surgery can correct abnormalities in the heart or digestive tract. Trisomy 18 Foundation
www.trisomy18.org
While the disability is significant, those children who survive
infancy can advance to some degree (interact with their family,
smile and acquire skills such as rolling over or even self-feeding)
Many of the clinical features, such as heart and digestive
anomalies, leukemia and hearing problems, can be treated –
most individuals live relatively healthy lives.
Early intervention (physical therapy, speech and language
therapy) can help enhance the development of children with
Down Syndrome.
National Down Syndrome Society
www.ndss.org
National Association for Down Syndrome
www.nads.org
No specific treatment is available for this syndrome. Half
Five P Minus Society
of children with Cri du Chat learn sufficient verbal skills to
www.fivepminus.org
communicate. The cat-like cry becomes less apparent over time.
Surgery can correct abnormalities in the heart or digestive tract, SOFT – support organization for trisomy 18, 13
but no specific treatment is available for this syndrome.
and related disorders
www.trisomy.org
While the disability is significant, those children who survive
infancy can advance to some degree (interact with their family,
smile and acquire skills such as rolling over or even self-feeding)
Treatments may include physical, speech and occupational
therapy, as well as testosterone replacement therapy.
KS&A
www.genetic.org
American Association for Klinefelter Syndrome
Information and Support
www.aaksis.org
Klinefelter Syndrome Support Group
klinefeltersyndrome.org
THE SCIENCE OF PROGRESS
21
9/22 reciprocal
translocation
Case I
trisomy X
Chronic
Myelogenous
Leukemia (CML)
Triple X syndrome
22q11.2 deletion
syndrome
Females with Triple X syndrome may be taller than average, but there are
generally no unusual physical features. There may be an increased risk of
learning disabilities or delayed speech and language development. Some
females have no symptoms at all.
occurs in an estimated 1 in 4,000 livebirths, although the incidence may be
higher, as mild cases are often not detected
47, XYY male
Clinical features may include heart defects, cleft palate, kidney
malfunction and differences in the formation of the spine. Developmental
delay and non-verbal learning disabilities may also be present.
occurs in 1 per 10,000 newborn boys (does not occur in females)
Turner Syndrome
There are generally no unusual physical features associated with 47, XYY,
although males may be taller than average. There is an increased risk of
learning disabilities and delayed development of speech and language
skills.
affects about 1 in 2,500 liveborn girls (does not occur in males)
Case K
XYY
Case L
monosomy X
Case M
22
CML is one of four main types of leukemia, a cancer of the bone marrow
and blood. In CML, early myeloid cells (precursors to white blood cells,
red blood cells and platelet-making cells) fail to mature normally and
build up, crowding out normal bone marrow cells. Most cases occur in
adults. Individuals with CML often tire easily, have shortness of breath, an
enlarged spleen and experience weight loss.
CML patients have a chromosomal translocation that begins in a single
cell between chromosomes 9 and 22. Part of chromosome 9 becomes
connected to chromosome 22 (and part of chromosome 22 breaks off
and attaches to chromosome 9). The chromosome 22 that has a piece of
chromosome 9 is also known as the Philadelphia Chromosome. The break
on chromosome 9 involves a gene called Abl. The break on chromosome
22 occurs at a gene called Bcr. The Philadelphia Chromosome joins the
upstream portion of Bcr with the downstream part of Abl. This new hybrid
Bcr-Abl gene combination is responsible for the CML cancer.
occurs in 1 per 1,000 newborn girls (does not occur in males)
Case J
22q11.2
microdeletion
In the United States, just over 5,000 new cases of CML are diagnosed each
year.
Females with Turner syndrome are usually infertile and do not enter
puberty. Affected girls are usually short, may have low-set ears and
extra folds of skin connecting the tops of the shoulders to the sides of
the neck. They may also have heart or kidney disorders. Women with
Turner syndrome have normal intelligence, although learning disabilities
associated with spatial concepts may be present. Some females have very
mild symptoms, while others have major impacts from the disorder.
Three new drugs have been approved to treat CML since 2001
– Gleevec, Sprycel and Tasinga. All three work by targeting the
Bcr-Abl gene and shutting off the protein the gene produces.
In certain cases, a bone marrow transplant may be necessary.
About 70% of patients with a transplant are cured of their CML.
There are serious risks associated with this process however
and the treatment is most successful in younger patients.
The Leukemia & Lymphoma Society
www.leukemia-lymphoma.org
American Cancer Society
www.cancer.org
Treatment, if needed, is based upon symptoms – speech
therapy, learning strategies etc.
KS&A
www.genetic.org
Treatment is based on the type of symptoms that are
present. For example, heart defects are treated surgically.
Early intervention and speech therapies are started to treat
developmental delays.
The International 22q11.2 Deletion Syndrome
Foundation
www.22q.org
Treatment, if needed, is based upon symptoms – speech
therapy, learning strategies etc.
KS&A
www.genetic.org
Growth hormone often helps girls reach heights close to
average. Sex hormone replacement therapy will induce the
features of puberty, maintain secondary sexual development
and protect their bones from osteoporosis.
Turner Syndrome Society of the United States
www.turnersyndrome.org
THE SCIENCE OF PROGRESS
23
DISORDER DETECTIVES: EXAMINING HUMAN CHROMOSOME
DISORDERS
STUDENT HANDOUT
Overview
You and a partner will take on the role of a cytogeneticist working in a hospital. A case study will be given to you
for review, along with a set of patient chromosomes. You and your partner will arrange the chromosomes on a
prepared board into a completed karyotype. You will analyze the karyotype and diagnose your patient. Your patient
may have one of the many types of recognized chromosomal abnormalities, though normal karyotypes are also
represented. Be careful and use your observation skills as things are not always as simple as they seem.
Background
In order to think like a cytogeneticist, here are a few things you will need to know
What is a chromosome?
The DNA of all living organisms is organized into discrete packets called chromosomes. Most human cells contain 46
chromosomes, grouped into two sets of 23, one set contributed by the mother’s egg and the other set contributed
from the father’s sperm. The maternal and paternal chromosomes of a pair are called homologous chromosomes,
or homologs. Within each set of chromosomes there is one sex chromosome and 22 other chromosomes, called
autosomes. There are two types of sex chromosomes, classified as “X” and “Y”. Typically males have both an “X”
and “Y” sex chromosome, while females have two “X” sex chromosomes.
What is the structure of a chromosome?
Most of the time, the chromosomes are present as long tangled chromatin strands composed of DNA tightly
wrapped around histone proteins and further condensed and stacked. At this stage, individual chromosomes cannot
be distinguished from one another.
During cell division, the DNA is replicated and even further condensed. The two copies of each chromosome, called
sister chromatids, are temporarily held together at a specific location on the chromosome called the centromere. At
this point, individual chromosomes can be identified.
How are chromosomes classified?
Chromosomes vary in size and shape. Centromere location is also a feature utilized to distinguish one chromosome
from another. Metacentric chromosomes have arms of roughly equal lengths. The arms of submetacentric
chromosomes are slightly unequal; the shorter arm is called the p arm and the longer arm is the q arm. Acrocentric
chromosomes have a centromere that is even closer to the end of the chromosome (which is called the telomere).
When stained with Geimsa stain, different chromosomes have different banding patterns. These patterns of dark
and light bands uniquely identify each chromosome. Each band DOES NOT indicate the location of a single gene – for
some chromosomal regions, hundreds of genes may be present in a band while other regions may have relatively
few genes per band.
What is a karyotype?
A karyotype is an organized profile of an individual’s chromosomes. Generally the chromosomes have been
stained, identified and organized in a specific order. This allows a scientist called a cytogeneticist to examine the
chromosomes and quickly identify alterations that may result in a genetic disorder. Chromosomes are typically
prepared for karyotyping with the sister chromatids so closely aligned that they appear as a single structure (in other
words they do not look like an “X”, but instead appear as an “I”.
What types of samples are often obtained for karyotyping?
Cells may be obtained from various sources for karyotype analysis, including:
• Blood
• Skin or other tissues
24
• Chorionic villi - The chorionic villi are part of the placenta. Chorionic Villus Sampling (CVS) involves removing
some of the chorionic villi so the cells can be analyzed. This test can be conducted at 10 - 13 weeks’ gestation,
but carries a 1-2% risk of miscarriage.
• Amniotic fluid – Amniotic fluid surrounds the fetus and contains fetal cells that have been shed. The process of
withdrawing this fluid using a hollow needle is called amniocentesis. It is conducted between 14 and 20 weeks’
gestation and has a 1% or less risk of miscarriage.
Examples of Findings Commonly Identified by Karyotyping
Common Karyotype Findings
Associated Clinical Symptom
9/22 translocation
Chronic Myelogenous Leukemia
5p deletion
Cri du Chat
22q11.2 deletion
22q11.2 Deletion syndrome
Trisomy 21
Down Syndrome
Trisomy 18
Edward Syndrome
XXY
Klinefelter Syndrome
46 chromosomes (XX)
Typical Female
46 chromosomes (XY)
Typical Male
3p25q21 inversion (1 normal chr 3, 1 inverted chr 3) No clinical symptoms present
9p11q12 inversion (1 normal chr 9, 1 inverted chr 9) No clinical symptoms present
Trisomy 13
Patau Syndrome
14/21 translocation (1 normal chr 14, 1 chr14/21
Robertsonian Translocation – Down Syndrome
translocation, 2 normal chr 21)
Monosomy X
Turner Syndrome
Trisomy X
XYY
XXX
XYY
What benefits are provided by karyotyping?
Prenatally, the results of a karyotype can provide answers or a diagnosis. Parents may utilize this information to
identify a care team to be present at the birth or to make an informed reproductive decision. In postnatal cases, the
results can be used to diagnose complicated syndromes.
Often a pregnant woman is offered a blood test to help identify certain chromosome disorders – is this
a karyotype?
Pregnant women are offered a screening test for a number of disorders, including neural tube defects and certain
chromosomal disorders. Although the word ‘screening’ is often used loosely as a synonym for ‘testing’, the two are
not identical and the maternal blood test is not used to create a karyotype for the fetus.
The screening tests measure specific proteins found in the blood of pregnant women to identify who should be
offered more extensive (and often more invasive and expensive) testing, such as amniocentesis to obtain fetal cells
for karyotyping. The screening test is not 100% sensitive and often women who are carrying a normal fetus have an
abnormal screening test and must deal with the stress of deciding whether or not to undergo amniocentesis. At
the same time, there are a small number of cases where truly abnormal pregnancies go undetected by the maternal
blood screen.
THE SCIENCE OF PROGRESS
25
Protocol for the Karyotyping Activity:
Make sure you receive a copy of the “Cytogenetics Report for G-Banded Karyotype” from your teacher.
You will also receive a Chromoscan board containing a case study and set of patient chromosomes (reusable decals).
Each case study has a Case ID (letters A-O) and a unique color. The color of the patient chromosomes matches
the color that is printed around the case study section of the Chromoscan board. Confirm that the colors of the
chromosomes and the board match.
Select a chromosome decal from the cryostorage area of the board and sketch it on your Cytogenetics Report,
noting the centromere, telomere, and p and q arms. Note the centromere position as well.
Read the case study found on the left side of the board.
On the Cytogenetics Report, record patient information such as name, case ID, reason for referral, patient age and
source of cells
To make the process of karyotype assembly less complex, one of each of the homologous chromosomes is already
illustrated on the board. Identify the other homolog and place it on the board in the proper position.
Once the karyotype is completed, analyze it for chromosomal anomalies, paying particular attention to chromosome
number and structure.
Record chromosome number, gender and chromosomal findings on the Cytogenetics Report.
Determine the suggested diagnosis by looking at the “Examples of Findings Commonly Identified by Karyotyping”
located in your student handout.
Complete the Cytogenetics Report on your patient to include patient diagnosis.
Briefly explain on the Cytogenetics Report how a karyotype is prepared. A summary of the technique can be found
on the right hand side of the board where the chromosomes are stored.
Discuss the questions found at the bottom of the Cytogenetics Report and write out your answers.
At the end of the activity, return the chromosome decals to the “cryostorage”
region of the Chromoscan board in random order, to prepare the board for the next
group’s use. Check carefully around your desks and lab tables to make sure all the
chromosomes have been collected and returned to the board.
homologous chromosome set
case study
protocol
cryostorage
example chromosome preparation
26
Cytogenetics Report for G-Banded Karyotype
Select a chromosome from the cryostorage area. Sketch the chromosome labeling the p arm, q arm, centromere
and telomere.
Centromere type:
__________metacentric __________submetacentric __________acrocentric
Patient Name
Case Study ID
Age
Why is the patient being referred for karyotyping?
Source of Cells for Karyotyping
_____ Blood
_____ Amniocytes
_____ Chorionic Vili
_____ Other (specify) ___________
Total Number of Chromosomes Observed
Gender:
Chromosomal Findings
_____ no observable chromosomal abnormalities
_____ monosomy (chromosome # ______)
_____ trisomy (chromosome # ______)
_____ deletion (chromosome # ______, arm ________)
_____ insertion (chromosome # ______, arm _________)
_____ translocation (chromosome #s ________ and _______)
_____ inversion (chromsome # ______, arm(s) _____________)
Patient Diagnosis
(Optional) On a separate sheet of paper attach notes for patient’s caregiver with additional implications associated
with the diagnosis including life expectancy, complications, available treatments and support group information.
Briefly explain how a karyotype is prepared.
There are only three autosomal trisomic conditions (Patau, Edward, and Down Syndrome) where a fetus will
survive to birth. Why do you think this is so?
Why are microdeletions and microinsertions difficult to diagnosis using karyotyping?
28
INSTRUCTOR NOTES
THE SCIENCE OF PROGRESS