Methods of Human genetics

Karyotype Analysis of Human

Chromosomes
(Cytogenetic study)

• Karyotype preparation and analysis

• Cells (from blood, amniotic fluid, etc)
are grown in vitro (in a cell culture
dish) to increase their number
• Cell division is then arrested
in metaphase with colchicine
(prevents mitotic spindle from
forming)
• Cells are centrifuged and lysed to
release chromosomes
• Chromosomes are stained,
photographed, and grouped by size and
banding patterns
 CHROMOSOMAL
MUTATIONS
• Alterations in
chromosome number: Alterations in
chromosome
• (Polyploidy
structure;
• Aneuploidy)
Deletion
Duplication
Inversion
Translocation
 Alterations in
chromosome number
Nondisjunction occurs when either
homologues fail to separate during
anaphase I of meiosis, or sister
chromatids fail to separate during
anaphase II. The result is that one gamete
has 2 copies of one chromosome and the
other has no copy of that chromosome.
(The other chromosomes are distributed
normally.)
• If either of these gametes unites with
another during fertilization, the result
is aneuploidy (abnormal chromosome
number)
• A trisomic cell has one extra chromosome
(2n +1) = example: trisomy 21. (Polyploidy
refers to the condition of having three
homologous chromosomes rather then two)

• A monosomic cell has one missing

chromosome (2n - 1) = usually lethal except
for one known in humans: Turner's syndrome
(monosomy XO).
• The frequency of nondisjunction is quite
high in humans, but the results are
usually so devastating to the growing
zygote that miscarriage occurs very
early in the pregnancy.

• If the individual survives, he or she

usually has a set of symptoms - a
syndrome - caused by the abnormal dose
of each gene product from that
chromosome.
• 1. Human disorders due to
chromosome alterations in autosomes
(Chromosomes 1-22). There only 3
trisomies that result in a baby that can
survive for a time after birth; the
others are too devastating and the baby
usually dies in utero.
• A. Down syndrome (trisomy 21): The
result of an extra copy of chromosome
21. People with Down syndrome are 47,
21+. Down syndrome affects 1:700
children and alters the child's
phenotype either moderately or
severely:
• characteristic facial
features, short stature;
heart defects
• susceptibility to respiratory
disease, shorter lifespan
• prone to developing early
Alzheimer's and leukemia
• often sexually
underdeveloped and sterile,
usually some degree of
mental retardation.
• Down Syndrome is correlated
with age of mother but can
also be the result of
nondisjunction of the
father's chromosome 21.
•  
• Karyotype of a boy
with Down
Syndrome:
• Patau syndrome (
trisomy 13): serious
eye, brain,
circulatory defects
as well as cleft
palate. 1:5000 live
births. Children
rarely live more than
a few months
• Edward's syndrome
(trisomy 18):
almost every organ
system affected
1:10,000 live births.
Children with full
Trisomy 18 generally
do not live more than
a few months.
• Nondisjunction of
the sex
chromosomes (X or
Y chromosome):
Can be fatal, but
many people have
these karyotypes
and are just fine!
• Klinefelter syndrome
: 47, XXY males.
Male sex organs;
unusually small
testes, sterile.
Breast enlargement
and other feminine
body
characteristics.
Normal intelligence.
• 47, XYY males: Individuals
are somewhat taller than
average and often have
below normal intelligence.
At one time (~1970s), it
was thought that these men
were likely to be criminally
aggressive, but this
hypothesis has been
disproven over time.
• 3. Trisomy
X: 47, XXX
females. 1:1000 live
births - healthy and
fertile - usually
cannot be
distinguished from
normal female
except by karyotype
• Monosomy X (Turner's
syndrome): 1:5000 live
births; the only viable
monosomy in humans -
women with Turner's have
only 45 chromosomes!!! XO
individuals are genetically
female, however, they do
not mature sexually during
puberty and are sterile.
Short stature and normal
intelligence. (98% of these
fetuses die before birth)
 Alterations in
chromosome structure:
• Sometimes, chromosomes break,
leading to 4 types of changes in
chromosome structure:
• Deletion: a portion of one chromosome
is lost during cell division. That
chromosome is now missing certain
genes. When this chromosome is
passed on to offspring the result is
usually lethal due to missing genes.
• Example - Cri du chat (cry of the cat
): A specific deletion of a small portion
of chromosome 5; these children have
severe mental retardation, a small head
with unusual facial features, and a cry
that sounds like a distressed cat.
• Duplication: if the fragment joins the
homologous chromosome, then that
region is repeated
• Translocation: a fragment of a chromosome is moved
("trans-located") from one chromosome to another -
joins a non-homologous chromosome. The balance of
genes is still normal (nothing has been gained or lost)
but can alter phenotype as it places genes in a new
environment. Can also cause difficulties in egg or
sperm development and normal development of a
zygote. Acute Myelogenous Leukemia is caused by
this translocation:
 Prenatal detection of
genetic conditions
• Some conditions can be detected before
birth (in utero). From conception until
about the eighth week of pregnancy, a
pregnant woman is carrying an embryo;
• from the eighth week until birth the term
fetus is used rather than embryo.
• In the technique of amniocentesis a small amount of amniotic fluid is
withdrawn from the sac in which fetus is developing in the mother’s uterus.
• The fluid itself is analyzed for the
presence or absence of sertain enzymes
that might indicate a genetic defect in
fetus. Also amniotic fluid usually contains
cells that have been shed from the
surface of the fetus. Growing the cells in
the laboratory can reveal additional
information.
• Instead of amniocentesis, chorionic
villus sampling is sometimes used for
prenatal detection.
• This technique is a type of a biopsy of
placenta.
• Because the part of placenta biopsied is
tissue derived from the fetus, not from
the mother, the cells sampled by this
technique are fetal cells
• Chorionic villus sampling can be performed
earlier in pregnancy that amniocentesis can.
Certain low, but nonzero risks are
associated with amniocentesis or chorionic
villus sampling, including mechanical injury
to the growing fetus and a risk that the
pregnancy will be prematurely terminated.
Because of this risks and other reasons,
these tests are not performed routinely on
every expected mother
• After fetal cells have been obtained by
either amniocentesis or chorionic villus
sampling,chromosomes from these cells
are analysed for evidence of Down,
Klinefelter or Turner syndromes. Also
if there is a reason, other tests are
done.
• Biochemical reactions products can be
detected in the fluid.
• More than 350 genetic diseases are
detectable through prenatal tests,
including Tay-Sachs disease, cystic
fibrosis and phenylketonuria.
 Testing newborns and
adults
Other tests are done on newborns or
adults. Tests are simple and
inexpensive.
For example, many hospitals routinely
screen newborns all infants at birth
for phenylketonuria.
 Twin studies
Twin studies, although limited by
complicating factors, provide the best
source for separating genetic
contributions to the trait being studied
from environmental influences
   There are two different kinds of twins;
identical and fraternal.  1.) Identical twins
come from one egg which is fertilized by one
sperm.  That single fertilized egg, or zygote,
then splits into two separate identical
embryos which continue to grow. 

A
2.) Fraternal twins come from
two separate eggs that are
ovulated at the same time. 
In this case the two eggs
are fertilized by two
different sperm.  Fraternal
twins are just like an
ordinary brother and
sister, except that they
are born at the same time. 
They have a different
genetic makeup and tend to
be very independent of
each other.
• Twin studies are conducted all over the world,
and help to distinguish between what is
influenced by genetics and what is influenced
by environment.  This means that they can
determine whether a disorder is something
you are born with, or whether it is something
that is developed based on the surroundings.
• Because identical
twins have the same
genetic makeup and
come from the same
mother, it can be
assumed that any
differences between
the twins is caused by
environmental
factors. 
• Fraternal twins have
the same pre-birth
environment as well,
but have different
DNA so they tend to
have many
differences between
them.
Concordance rates in MZ and DZ
Trait or Concordance Concordance Heritability
disease rate in MZ rate in DZ
Blood 0,55 0,25 0,62
pressure
Body fat 0,73 0,22 >1,0
IQ 0,76 0,51 0,50
Measles 0,95 0,87 0,16
Spina bifida 0,72 0,33 0,78

Cleft 0,38 0,08 0,60

lip/palate
• Concordance rates and correlation coefficients for
a number of traits are given . The concordance
rates for contagious diseases like measles are quite
similar in MZ and DZ twins. This is expected, since
a contagious disease is unlikely to be influenced
markedly by genes. On the other hand, the
concordance rates are quite dissimilar for
schizophrenia and bipolar affective disorder,
indicating a sizable genetic component for these
diseases. The MZ correlation for der-matoglyphics
(fingerprints), which is determined al­most entirely
by genes, is close to 1.0.
• HC = (CMZ - CDZ)/(1 - CDZ).
 Human Allelic Disorders
(Recessive)
• The first Mendelian trait in humans
was described in 1905 (brachydactly)
by Dr. Farabee (no relation to your
author). Now more than 3500 human
genetic traits are known.
• Albinism, the lack of pigmentation in skin, hair, and eyes, is
also a Mendelian human trait. Homozygous recessive (aa)
individuals make no pigments, and so have face, hair, and
eyes that are white to yellow. For heterozygous parents
with normal pigmentation (Aa), two different types of
gametes may be produced: A or a. From such a cross 1/4 of
the children could be albinos. The brown pigment melanin
cannot be made by albinos. Several mutations may cause
albinism: 1) the lack of one or another enzyme along the
melanin-producing pathway; or 2) the inability of the
enzyme to enter the pigm
• Phenylketonuria (PKU) is recessively inherited disorder
whose sufferers lack the ability to synthesize an enzyme to
convert the amino acid phenylalanine into tyrosine
Individuals homozygous recessive for this allele have a
buildup of phenylalanine and abnormal breakdown products in
the urine and blood. The breakdown products can be harmful
to developing nervous systems and lead to mental
retardation. 1 in 15,000 infants suffers from this problem.
PKU homozygotes are now routinely tested for in most
states. If you look closely at a product containing Nutra-
sweet artificial sweetener, you will see a warning to PKU
sufferers since phenylalanine is one of the amino acids in the
sweetener. PKU sufferers are placed on a diet low in
phenylalanine, enough for metabolic needs but not enough to
cause the buildup of harmful intermediates.
• Tay-Sachs Disease is an autosomal recessive resulting in
degeneration of the nervous system. Symptoms manifest after
birth. Children homozygous recessive for this allele rarely survive
past five years of age. Sufferers lack the ability to make the
enzyme N-acetyl-hexosaminidase, which breaks down the GM2
ganglioside lipid. This lipid accumulates in lysosomes in brain cells,
eventually killing the brain cells. Although rare in the general
population (1 in 300,000 births), it was (until recently) higher (1 in
3600 births) among Jews of eastern central European descent.
One in 28 American Jews is thought to be a carrier, since 90% of
the American Jewish population emigrated from those areas in
Europe. Most Tay-Sachs babies born in the US are born to non-
Jewish parents, who did not undergo testing programs that most
US Jewish prospective parents had.
• Sickle-cell anemia is an autosomal recessive we
have discussed in other sections. Nine-percent of
US blacks are heterozygous, while 0.2% are
homozygous recessive. The recessive allele causes
a single amino acid substitution in the beta chains
of hemoglobin. When oxygen concentration is low,
sickling of cells occurs. Heterozygotes make
enough "good beta-chain hemoglobin" that they do
not suffer as long as oxygen concentrations
remain high, such as at sea-level.
 Human Allelic Disorders
(Dominant)

• Autosomal dominants are rare,

although they are (by definition)
more commonly expressed.
• Achondroplastic dwarfism occurs,
even though sufferers have reduced
fertility.
• Huntington's disease (also referred to as Woody
Guthrie's disease, after the folk singer who died
in the 1960s) is an autosomal dominant resulting in
progressive destruction of brain cells. If a parent
has the disease, 50% of the children will have it
(unless that parent was homozygous dominant, in
which case all children would have the disease).
The disease usually does not manifest until after
age 30, although some instances of early onset
phenomenon are reported among individuals in
their twenties.
• Polydactly is the presence of a sixth digit.
In modern times the extra finger has been
cut off at birth and individuals do not know
they carry this trait. One of the wives of
Henry VIII had an extra finger. In certain
southern families the trait is also more
common. The extra digit is rarely
functional and definitely causes problems
buying gloves, let alone fitting them on
during a murder trial.
• Lethal Alleles
• G. Mendel’s First Law, which predicts a 3:1 ratio among the F2 generation in a
• monohybrid cross, makes the implicit assumption that all offspring of the
• cross are equally viable and this live long enough to be counted.
• H. Many alleles, however, are absolutely necessary or at least extremely vital for
• survival or health of an embryo during early development. Usually, if a gene
• is that important, the body makes more than enough gene product to
• compensate for the loss of one copy of the gene. Therefore, mutations in such
• genes are usually recessive and are called recessive lethal alleles because
• they cause the death of the embryo only in the homozygous state. Often, the
• development of such embryos is arrested very early and the embryo is either
• shed by the mother as a spontaneous miscarriage or is “reabsorbed” by the
• mother’s body.
• I. Each one of us is a carrier for a few lethal alleles. Fortunately, it is unlikely
• that we will mate with someone who is a carrier for a lethal or sublethal
• allele
• at the same gene locus. If we do, an average of ј of our children will either be
• miscarried or severely malformed.
• J. The inheritance of a lethal allele is difficult to follow in a family because only
• normal embryos will survive to term. Whether the miscarriage was caused by
• homozygosity for a lethal allele or for some other reason may never be
• known. However, the fertility of the couple is reduced by 25%.
• Environmental Influence
• A. Some traits that are under the control of a single gene are also strongly
• influenced by the environment. This can also affect the expected Mendelian
• ratios in a monohybrid cross
• B. One example is temperaturesensitive
• alleles. The proteins coded by these
• alleles only operate at low temperatures, not at higher ones
• C. Himalayan rabbits and Siamese cats have a temperature sensitive allele in a
• gene that codes for an enzyme that makes dark pigment. The enzyme is only
• active in parts of the body that are relatively cool (ears, nose, extremities),
• while the core body areas are too warm for the protein to fold properly
• D. Most cats and rabbits that are homozygous for this allele therefore have
• pigmented fur on their extremities (nose, ears, paws)
• E. However, if these cats or rabbits are raised in a tropical climate, they often do
• not express the pigment at all and are white all over
• VI. Hormonal Influence
• H. Some alleles are dominant in one sex and recessive in the other due to the
• presence or absence of sex hormones
• I. The classic human example of a hormonally influenced allele is the allele that
• causes malepattern
• baldness
• i. Earlyonset
• baldness (usually before age 25)
• ii. Hair is progressively lost in the pattern show below
• iii. The allele (B) is autosomal and is dominant over the normal
• allele (b = no baldness) in men
• iv. However, the allele is recessive (b) in women, which is why
• women rarely suffer from pattern baldness. Moreover, women
• who are homozygous for the pattern baldness allele (bb) have a
• much later onset of phenotype than males and they tend to lose
• hair in a more random pattern
• v. Male production of DHT appears to be responsible for the
• dominance of the allele in men. Men who do not produce
• testosterone (due to abnormalities of the genitalia or accidental
• loss of the testicles) do not get pattern bald, even if they carry a
• copy of the baldness allele
• Homozygous