UNIT 7: KARYOTYPING

detects this compliments or measures the

number.
TRANS OUTLINE
● comes from the Greek word “Karyon” which
means nucleus.
I. DEFINITION OF TERMS
A. KARYOTYPE
B. INFORMATION ON THE KARYOTYPE
C. KARYOLOGY
D. IDIOGRAM OR KARYOGRAM
E. ASYMMETRIC KARYOTYPE
F. SYMMETRIC KARYOTYPE
II. TYPES OF KARYOTYPE
A. BRIEF IDEOGRAMS OF CHROMOSOME
III. KARYOTYPING PROCEDURE
A. REAGENTS AND MATERIALS NEEDED
B. PROCEDURE: 5 MAJOR STEPS
◦ Short Term Lymphocyte Culture
◦ Harvesting of Lymphocytes
◦ Fixing the Cells
◦ Making the Chromosome Slides
◦ Slide Analysis
IV. CHROMOSOME BANDING
A. HISTORY
B. WHY STUDY BANDING PATTERN?
V. CLASSIFICATION OF BANDING TECHNIQUES
INFORMATION ON THE KARYOTYPE
A. Q BANDING TECHNIQUES
◦ Advantages
● Size of chromosome
◦ Disadvantages
● Position of centromere
B. G BANDING TECHNIQUES
● Presence of secondary constrictions
◦ Advantages
● Size of satellites
◦ Disadvantages
C. N BANDING TECHNIQUES
KARYOLOGY
◦ Advantages
D. C BANDING TECHNIQUES
● the study of whole sets of chromosomes.
◦ Advantages
VI. REPRESENTATION OF A CHROMOSOME
IDIOGRAM OR KARYOGRAM
BAND
VII. ISCN
● the standard format of representing
chromosomes as a diagram when the haploid
DEFINITION OF TERMS set of chromosomes of an organism are
ordered in a series of decreasing size.
KARYOTYPE ○ standard diagrammatic representation
of your karyotype.
● is the number and appearance of ● easily tells you the position of chromosomes.
chromosomes in the nucleus of a eukaryotic
cell.
● used for the complete set of chromosomes in a
species or individual organism for a test that
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 (a) A symmetric and (b) an asymmetric karyotype
ASYMMETRIC KARYOTYPE (redrawn from Stebbins, Chromosomal Evolution in Higher
Plants).
● show larger differences between smaller and
larger chromosomes in a set. Have more
Species showing a greater asymmetry are more
acrocentric chromosomes and relatively
advanced. How?
advanced features.
● organisms are more evolved if they have an
DEGREE OF ASYMMETRY
asymmetric karyotype.
● Proportion of metacentric, acrocentric
SYMMETRIC KARYOTYPE
chromosomes in a set.
● Ratio between size of largest and smallest
● show lesser difference between smaller and
chromosomes in a set.
larger chromosomes in a set. Have more
metaphase chromosomes and no advanced
INTERPRETATION
features.
● more symmetrical, more metacentric
● Higher the proportion of acrocentric
centromeres, means less advanced features.
chromosomes, Greater the value of size ratio,
more asymmetrical is a karyotype.
TYPES OF KARYOTYPE
● The more asymmetric, the more advanced
features are there in the organism.
In 1931 G.A. Levitzky, a Russian scientist, suggested
that in flowering plants there is a predominant trend
towards karyotype asymmetry. This trend has been
carefully studied in the genus Crepis of the family
compositae.

● Not flowering plant = symmetric karyotype

● Flowering plant = asymmetric karyotype

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 IDEOGRAMS OF CHROMOSOME thus providing a genome-wide snapshot of an
individual’s chromosomes.
○ If there are aberrations, they can be
seen in your karyotype.
● Karyotypes are prepared using standardized
staining procedures that reveal characteristic
structural features for each chromosome.
○ Stain imparts color. By staining, you
can look at the bands and therefore,
will help us identify what chromosome
type is present.
● Karyotyping analysis can reveal subtle
structural changes such as chromosomal
translocations, deletions, duplications, or
inversions. Moreover, it can reveal changes in
chromosome number associated with
aneuploid conditions, such as Trisomy 21
(Down syndrome).
● Any gross genetic changes can be detected by
karyotyping.
○ Will involve several megabases or more
of your DNA.

● If there is a more submetacentric chromosome,

then the gender is Female.
● If the number of acrocentric chromosomes is more
than 5, then the gender is Male. Euchromatin and Heterochromatin.
● Heterochromatin:
KARYOTYPING PROCEDURE ○ dark staining and tightly packed.
■ transcription factors cannot
● Karyotyping is the process of pairing and readily access the DNA
ordering all the chromosomes of an organism, sequences.

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 ■ plays a lesser role in
of nucleosomes. Transcription factors can bind the
transcription and translation. DNA and genes are expressed.
○ mostly contains DNA repeating
sequences but is not translated into
codon/protein.
● Euchromatin:
○ light staining and loosely coiled.
○ less compact and evenly spaced.
■ allows the DNA segment in the
histones available for
transcription and translation.
○ contain protein-encoding genes.
○ plays a bigger role in translation to a
protein than the former.

REAGENTS AND MATERIALS NEEDED

MATERIALS REAGENTS EQUIPMENT

Sterile 5 mL Glacial acetic

Centrifuge
syringe acid

21-gauge Incubator at
Methanol
syringe needle 37°C CO2

Conical tubes KCl (hypotonic

Refrigerator
(15 mL) solution)

Green-top
Vacutube
RPMI Growth Inverted
(contains
Medium Microscope
heparin as
anticoagulant)

Fetal Bovine
Glass slides
Serum

Phytohemagglu
Pasteur Pipette
tinin

Colcemid (or
The level of compaction is driven by the methylation of colchicine which
DNA. Pipettor and
is used to arrest
Pipette tips
mitosis at
(UP) Methylation of DNA and histones causes metaphase)
nucleosomes to pack tightly together. Transcription
factors cannot bind the DNA, and genes are not Serological
expressed. Giemsa Dye
pipettes
(DOWN) Histone acetylation results in loose packing Trypsin
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 PROCEDURE: 5 MAJOR STEPS Similar to RPMI, it will also provide
nutrients to allow the cells to
live/survive.
○ Antibiotics - to prevent microbial
contamination which can invalidate
the test. If it is contaminated,
microorganisms will grow instead of
cells.
○ Phytohemagglutinin (PHA) - a
mitogen. It is the reagent that induces
mitotic activity.
■ Phyto - means plant. Reagent
derived from plants typically
Phaseolus or winged beans.
● The cultured blood cells will be grown at 37 °C
incubator for 3 days.
○ The temperature mimics body
temperature. We are trying to trick cells
to think that it is still inside the body
Overview of the procedure. so that it will grow.
1. Collect blood from the patient.
2. Blood needs to be cultured. Add a few drops of ● Note that the cells must be in logarithmic
blood to the culture medium with phase because splitting of a cell line 2 days
phytohemagglutinin which will stimulate before harvesting, and changing the medium 1
mitosis, allowing cells to grow and multiply. day before harvesting, stimulates cell
3. To support the culture of blood, incubate at 37 °C proliferation significantly.
(body temperature) for 2 to 3 days. ○ Logarithmic phase - means that cells
4. Once the cells are actively dividing, add
are actively dividing. There are more
colchicine for about 2 hours. It will stop mitosis
in the metaphase. cells that are alive and actively
5. Transfer cells into a tube. Fix, then put on a slide. dividing as compared to cells that are
6. Allow cells to swell, chromosomes will enlarge to dying.
visualize it easier. ○ Change culture medium in 2nd day
7. Drop cells into a slide then examine under a ○ After 3 days, lymphocytes can now be
microscope.
collected.
8. Process chromosomes that you will see in a
karyotype.
2. HARVESTING OF LYMPHOCYTES

1. SHORT TERM LYMPHOCYTE CULTURE ● Addition of pre-warmed colcemid (also known

as colchicine), the reagent that arrests the cell
● Culture blood to target lymphocytes. cycle at metaphase stage, into the culture and
● The collected blood will be grown in vitro by incubates for 15 mins.
adding cell culture growth medium (RPMI), ○ Colchicine - medication used for gout
fetal bovine serum, antibiotics, and patients (high uric acid; gouty
phytohemagglutinin (PHA). arthritis). It can reduce levels of uric
○ culture growth medium - typically acid in the blood. But in karyotyping, it
RPMI which will allow cells to grow. It is used to stop mitosis in the
will provide nutrients to allow the cells metaphase stage by disrupting cell
to live/survive. spindle which is necessary for cell
○ Fetal bovine serum - a supplement division.
that will support the growth of blood.

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 ○ Pre-warmed means it is incubated at and adding thymidine for 5.5 h before colcemid
37 °C to avoid thermal shock. treatment (Hirai et al., 1994).
● Optimal exposure time to colcemid requires a
balance between proliferative activity index of 3. FIXING THE CELLS
cells and concentration of colcemid
○ If the cells have a high proliferative ● The cell suspension in the hypotonic state will
index, they can divide actively and be centrifuged for 1200 RPM for 5 mins.
rapidly, they need a shorter length of ● The cell pellet will be treated with fixative
exposure to a high concentration of solution (absolute methanol:glacial acetic
colcemid. acid; 3:1) or Carnoy’s fixative and will be
○ However, if the cells are slow growing centrifuged at 1200 RPM for 5 mins
or have a low proliferative index, they ● The process will be repeated 3x and the final
will require longer exposure to addition of fixative solution will require
colcemid or incubate it overnight with incubation at 4 °C for 10 mins.
a lower concentration. ● Fixation refers to the process of preserving
○ Prolonged exposure or high cells to prevent cellular degradation.
concentration of colcemid, increases
proportion of chromosomes at late 4. MAKING THE CHROMOSOME SLIDES
metaphase resulting in shortening of
chromosomes. However, Short ● 5 or 6 cold slides will be layered next to each
exposure at a high concentration other in a paper towel.
reduces the total yield of metaphases. ● 2 or 3 drops of the samples will be dropped
Therefore, you need to strike a balance onto each slide and dry them spontaneously
between proliferative index of cells and ● The slide will be stained by GTG-banding
concentration of colcemid. (G-bands by Trypsin using Giemsa), the most
● Centrifuge the tube at 1000 RPM for 10 mins common method of staining chromosomes for
and the cell pellet was resuspended in warm differentiation which uses trypsin that digests
hypotonic solution (can be KCl or sodium the chromosomes at regions rich in basic
citrate) and the solution was mixed. amino acids (Arg and Lys).
○ Centrifuge after exposure to colcemid
for about 1 to 2 hours. 5. SLIDE ANALYSIS
○ KCl or sodium citrate is often added to
make it hypotonic. (Cells will swell to ● Slides that will be chosen for analysis and
make the visualization of visualization must be:
chromosomes easier) ○ Properly trypsinized chromosomes
● Incubate at room temperature for 15 mins. ○ Clearly defined metaphase spreading
● Optional: additional modifications that allow for ● Requires a microscope with automated
enrichment of long (prometaphase) computer software program primarily,
chromosomes by using Actinomycin D or Cytovision TM by Applied Imaging Inc. which
ethidium bromide (added before harvesting), follows the International System of Human
or bromodeoxyuridine (BrdU), added before Cytogenetic Nomenclature (ISCN) that
colcemid treatment. Some of these reagents arranges chromosomes according to size and
are carcinogenic. banding patterns.
● Cell synchronization can significantly increase
the total yield of metaphase chromosomes. CHROMOSOME BANDING
Cells are arrested at S phase by adding an
excess amount of BrdU overnight (16 h). After ● A Band is a part of a chromosome which is
this, the block is released by washing the cells clearly distinguishable from its adjacent
segments by appearing darker or lighter with
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 various banding methods (Paris conference, ● GC and AT rich regions.
1971) ● Constitutive Heterochromatin Region.

Always metaphase chromosomes whose size has

condensed and whose diameter is increased are used for
chromosome banding studies after fixing the stage.

HISTORY

● In 1958, Caspersson et al., published there first

paper describing the use of quinacrine
mustard to stain chromosome there by
ushered in a new era of chromosome banding Overview of Banding Techniques and when it was
● The Paris report (1971) was the first attempt to developed.
provide nomenclature for chromosome
banding in any species and thus its
recommendations have been adopted to
nonhuman species as well.

WHY STUDY BANDING PATTERN?

● This allows you to see smaller pieces of the

chromosome, so that you could identify
smaller structural chromosome abnormalities
not visible on a routine analysis.

The differences between the banding techniques.

● Q-banding (Quinacrine banding), most common,
which uses a fluorescent microscope – useful for
chromosomes and bands.
● G-banding is one of the most common since it's
the least expensive and use a normal or
CLASSIFICATION OF BANDING TECHNIQUES brightfield microscope
● R-banding (Reverse banding), same as
Based on G-banding but stained differently, and still uses a

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 brightfield microscope. regions.
● C-banding (Centromeric banding), uses a ● Light regions are Guanine-Cytosine rich regions.
brightfield microscope.
● N-bandding (NOR banding), uses a brightfield
microscope

TYPE OF BANDING STAINING SUMMARY

Giemsa stain
G-Banding
AT-rich regions stain darker
than GC-rich regions

Quinacrine fluorescent dye

stains AT-rich regions
- Essentially same with
Q-Banding
G-banding but Q-banding
uses fluorescent dyes
instead

Banding pattern is opposite

G-banding Slide 33. Example of R-banding.
R-Banding
- GC-rich regions is darker ● Opposite of G-banding
than AT-rich regions ● Darker bands are G-C rich regions.
● Light bands are A-T rich regions.
Stains heterochromatic
regions close to the
centromeres
C-Banding
Usually stains the entire long
arm of the Y chromosome

Slide 34. Example of Q-banding.

● Similar to G-banding but uses fluorescent dye
● Darker portions/bands are Adenine-Thymine rich
regions.
● Light regions/bands are Guanine-Cytosine rich
regions.
Slide 32. Example of G-banding.
● Uses Giemsa
● Darker portions are Adenine-Thymine rich

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 Slide 35. Example of C-banding Slide 37. Example shows a normal male.
● Uses denaturation with barium hydroxide ● If TWO COPIES of X are present = FEMALE
followed by Giemsa ● If only ONE COPY of X = XO Syndrome (one of the
● Dark bands only represent heterochromatin two X chromosomes is completely or partially
near the centromere! absent)

Q BANDING TECHNIQUES

Slide 36. Example shows a normal male.

● 1-23 with correct pairings and size; no inclusions
or inversions Slide 38. Q Banding Techniques
● X and Y present = MALE ● Stained with Quinacrine Mustard
● Subjected to UV light to see banding pattern
● Dark staining = Region Rich in AT bases
○ AT region quenches dye & fluorescence,
situated in heterochromatin region
● Light staining = Region rich in GC bases
○ GC region quenches dye but do not
fluorescence, situated in euchromatin
region
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ADVANTAGES: ADVANTAGES:
● Simple and Versatile. ● Used in identification of bands rich in Sulfur
● Used where G band is not accepted. content.
● Used in study of chromosome ● Used in the identification of chromosomal
heteromorphism. abnormalities
● Gene Mapping.
DISADVANTAGES:
● Tendency to fade during examination. DISADVANTAGES:
○ Photo-degradation ● Not used in plants.
■ Fluorescence is lost
○ Chromophore - absorb light of a N BANDING TECHNIQUES
particular wavelength due to a
chemical bond formed between dye
and light.
■ Can be non-specific
○ UV light breaks the chemical bond.

G BANDING TECHNIQUES

Slide 42. N Banding Techniques

● NOR banding techniques
● Air dried chromosome is treated with 5%
Trichloroacetic acid at 95ᵒC for 30 min. Next,
treated with 0.1N HCl (hydrochloric acid ) at 60ᵒC
for 30 min. Then, banding pattern in structural
non-histone proteins linked to NOR region can be
seen.
Slide 40. G Banding Techniques
● Most commonly used
ADVANTAGES:
● Chromosome is digested using weak trypsin /
urea/ protease to denature protein. Once ● Used in the identification of Nucleolar
digested/ protein is denatured, it is treated with organizer regions.
Giemsa, to see banding patterns. ● Superior banding pattern for plants.
○ For Giemsa, MethyleneAzure +,
Methylene Violet +, or Methylene Blue + DISADVANTAGES:
can be used ● Chemicals used (Trichloroacetic acid, HCl) can
○ Interaction of the DNA with thiazine &
generate fumes.
eosin components of stain brightens
sulfur rich regions

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 C BANDING TECHNIQUES REPRESENTATION OF A CHROMOSOME BAND

● Each human chromosome has a short arm

("p" for "petit") and long arm ("q" for
"queue"), separated by a centromere.
● Each chromosome arm is divided into regions,
or cytogenetic bands, that can be seen using a
microscope and special stains.
● The cytogenetic bands are labeled p1, p2, q1, q2,
etc., counting from the centromere out toward
the telomeres.
● At higher resolutions, sub-bands can be seen
within the bands. The sub-bands are also
numbered from the centromere out toward the
telomere

Example 1: The cytogenetic map location of a gene is

7q31.2.
● This indicates that the gene is on chromosome
7, q arm, band 3, sub-band 1, and sub-sub-band
2.
Slide 40. C Banding Techniques ● The ends of the chromosomes are labeled ptel
● Chromosome is treated with alkali solution, to and qtel.
denature DNA. Then, wash with Sodium citrate at
60ᵒC for 30 min (Repetitive DNA renature but
Example 2: The notation 7qtel refers to the end of the
unique DNA do not renature). Then, stained with
Giemsa to see banding pattern at long arm of chromosome 7.
heterochromatin region.
○ Only heterochromatin is only stained by ISCN
Giemsa since Sodium citrate will
renature the repetitive DNA ● International System for Human Cytogenetic
■ Heterochromatin is made up of
Nomenclature
repeating sequences of DNA
■ Euchromatin is made up of ● Each area of chromosome given number
protein and codeine sequences. ● Lowest number of closest (proximal) to
(unique sequences) centromere
● Do not play a role in ● Highest number at tips (distal) to centromere
translation of protein,
and cannot be
accessed by
transcription factors =
sodium citrate will NOT
RENATURE

ADVANTAGES:
● Identification of chromosomes particularly in
insects and plants.
● Identification of bivalents at diakinesis using
both centromere positions.
● Paternity testing.
● Gene mapping.

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 ● Nomenclature: p (petit) arm = short arm; q = long ● In terms of biological sex, the person is a female
arm. Band numbering proceed outwards from the because it lacks a Y chromosome.
centromere e.g., bands p11 (one-one NOT eleven) or ● There is a problem in the chromosome 21
p23 (p two-three NOT twenty-three) (trisomy 21)
● 47 chromosomes

● The patient is a female

● The patient has trisomy 18 that results in a total
of 47 chromosomes

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 ● The patient is a female ● The patient has only one X chromosome which
● The patient has trisomy 13 that results in a total would indicate a turner syndrome
of 47 chromosomes ● Only 45 chromosomes are present

● There is a portion of chromosome 15 that has ● The female patient has trisomy of X chromosome
been deleted which would result into 47 chromosomes
● This is a patient with Prader Willi syndrome
where there is an absence or non expression of a
group of genes in chromosome number 15

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 ● The patient has tetrasomy of X chromosome ● The patient has Jacob syndrome (XYY sex
which would result into 48 chromosomes in total chromosomes)

● The patient has klinefelter syndrome (XXY sex

chromosomes)

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 UNIT 8: FISH
● used to check the cause of trisomies,
microdeletion syndromes, etc.
TRANS OUTLINE

I. FLUORESCENCE IN-SITU HYBRIDIZATION

(FISH)
A. SPECIMEN TYPES FOR FISH
II. FISH PROBES
A. TYPES OF FISH PROBES
o Locus Specific Probe
o Alphoid or Centromeric Repeat
o Subtelomere Probe
o Whole Chromosome Probes
o Pre-Natal Fish Probes
What is FISH?
III. STEPS FOR FISH
● Fluorescence In-Situ Hybridization. Detects and
IV. CELL SCORING IN FISH localizes specific DNA sequences using
V. APPLICATION OF FISH fluorescently labeled complementary DNA
VI. SPECIALIZED AND EVOLVING probes.
TECHNOLOGIES ● Probe: short sequence of DNA complementary to
A. COMPARATIVE GENOMIC HYBRIDIZATION target we want to detect
(CGH) ON METAPHASE CELLS ● Denature: to separate DNA strands and allow
B. MULTIPLEX FISH (M-FISH) probe access to target DNA.
C. MULTICOLOR BANDING (mBAND) ● Hybridize: together to bind probe to target DNA.
● Analyze: probe signals using a fluorescent
ANALYSIS
microscope.
D. FIBER FISH
E. PRIMED IN SITU LABELING (PRINS)
F. REVERSE FISH SPECIMEN TYPES FOR FISH
VII. COMPARATIVE GENOMIC HYBRIDIZATION

FLUORESCENCE IN-SITU HYBRIDIZATION (FISH) Metaphase FISH Interphase FISH

Gold Standard and May also be done on
● A cytogenetic technique that uses fluorescent routinely done. uncultured specimens.
probes that bind specifically to a part of Done on cultured cells. Advantageous in the rapid
chromosomes complementary to its sequence. screening of many nuclei
● useful in detecting and mapping the presence for prenatal diagnosis and
or absence of particular DNA sequences within newborn studies.
chromosomes. Allows direct Also beneficial in the
● FISH is applied to provide specific localization visualization of study of samples with a
of genes on chromosomes. chromosomes and low mitotic index such as
● rapid diagnosis of trisomies and exact position of most solid tumors.
microdeletions is acquired using specific signals.
probes.

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1 of 13
 Useful in the detection Major disadvantage is the
of structural changes in inability to detect
the genome. unknown structural
chromosomal changes.
Samples: Samples:
1. Amniocytes 1. Amniocytes - for
2. Chorionic ploidy analysis
villous cells during prenatal
3. Lymphocytes studies.
4. Cells from bone 2. Peripheral Blood
marrow Smears - for
aspirates or ploidy analysis in
solid tumors. newborns.
5. Fibroblasts. 3. Bone Marrow
Aspirate Smear or
Direct Harvest -
translocation or
copy number
analysis in cancer
studies.

FISH PROBES

● Complementary sequences of target nucleic

acids (DNA, RNA, or Nucleic Acid analogs)
tagged or labeled with fluorophores.
● Designed to hybridized with the
complementary sequence
● Size ranges from 20 to 1000 base pairs.
● Direct Labelling TYPES OF FISH PROBES
○ fluorophores are directly attached to
the probe. LOCUS SPECIFIC PROBE
○ less sensitive.
○ most common and one step only ● binds to a particular region of a chromosome.
○ ex: FITC, rhodamine, and cyanines. ● used when only a small portion of a gene is
● Indirect Labelling isolated and wants to determine on which
○ chemical conjugation of the nucleic chromosome the gene is located, or how many
acid with a nonfluorescent molecule copies of a gene exist within a particular
that can bind fluorescent material genome.
after hybridization. ● Locus = defined by DNA sequence
○ ex: Biotin and Diogoxigenin.
SINGLE COLOR FISH DOUBLE COLOR FISH
PROBE PROBE

Designed to cover a gene Designed to cover any 2

of interest. genes for the detection
of the aberrations.

Allows simultaneous

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 detection of numerical
abnormalities of 2 to 3
regions in one FISH
assay.

ALPHOID OR CENTROMERIC REPEAT

● Generated from repetitive sequences found in

the middle of each chromosome.
● Used to determine whether an individual has
the correct number of chromosomes or if there
is aneuploidy in the patient’s genome.

WHOLE CHROMOSOME PROBES

● Collection of smaller probes that bind to the

whole length of the chromosome.
● Useful in the examination of chromosomal
aberrations.
○ Find missing chromosomes

SUBTELOMERE PROBE

● Specific to the subtelomere region of the

chromosome.
● Useful in the detection of subtelomere
deletions and rearrangements.

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 PRE-NATAL FISH PROBES
CELL SCORING IN FISH
● Comprise of different combinations of
fluorophore-labeled probes specific for Single color FISH counting guidelines:
chromosomes 13, 18, 21, X, and/or Y.
PICTURES RULES

Don’t count, skip over.

This could be 2 nuclei
with 1 signal to each
other or one twisted
nucleus.

Count as 2 signals. One

is very compact, the
other is diffuse.

STEPS FOR FISH

Don’t count; skip over.
Steps: Observer cannot
1. Probe and target DNAs are denatured using determine which
high-temperature incubation in a nucleus contains the
formamide/salt solution. signals.
2. Probe sequences hybridize to the
complementary target sequences, and
nonspecific binding is eliminated via stringent Count as 2 signals. 1
washing. signal in a split.
3. The probe hybridization is detected with
fluorescence microscopy.

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 Count as 3 signals. Count as 1 red signal
and 1 green signal. The
red signal is diffuse.

Count as 3 signals. 1 is
split. APPLICATION OF FISH
1. Detection and characterization of
chromosome.

Count as 4 signals.

Two color (e.g. X and Y) counting guidelines:

PICTURES RULES
Slide 22. Example of FISH to a single copy target using a
Don’t count. Nuclei are
cosmid (SNRPN) to the Prader-Willi “critical region”
overlapping and all areas
localized to 15q11-13.
of both nuclei are not
visible.

Count as 1 red signal

and 1 green signal. The
red signal is diffuse.

Don’t count. Nuclei are

too close together to
determine boundaries.
Slide 22. Partial metaphase spread from a patient with a
duplication involving chromosome 11.

2. Detection and analysis of prenatal

chromosomal abnormalities.

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 Slide 23. Prenatal ploidy assessment utilizing Abbott Slide 25. ERBB2 (HER2) analysis for carcinoma of the
Molecular AneuVysion analysis of uncultured amniotic breast.
fluid cells using unique copy probes for the long arms of
chromosomes 13, 18, 21, X, and Y.
SPECIALIZED AND EVOLVING TECHNOLOGIES
3. Study of chromosomal abnormalities
associated with cancer. COMPARATIVE GENOMIC HYBRIDIZATION (CGH) ON
METAPHASE CELLS

● A technique that uses DNA from the cells of

interest, rather than using a standard
karyotype, for chromosomal analysis.
● This can be very useful, especially in some
cancers when only DNA is available rather than
any growing cells.
● This technology has been used successfully for
clinical analysis, particularly with cases that
have a low (or no) mitotic index.
● It is not useful for detecting balanced
rearrangements.

Slide 24. FISH panels for B cell disorders. Results from a

peripheral blood sample from a patient with CLL,
hybridized with the Abbott Molecular CLL probe panel with
addition of an MYB probe.

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 ● Whole chromosome probe
● Ratio-labeled probes are used to create a
distinct computer-generated false color for
each chromosome.
● Useful for complex rearrangements, such as
those seen in neoplastic disorders and solid
tumors.

Slide 27. The utility of metaphase CGH is illustrated by

the CGH profiles of a case with an insertion of unknown
material into the short arm of chromosome 4. The
chromosomal profiles reveal a gain of 15q (highlighted in
orange).

Slide 31. M-FISH of the pre-B ALL derived cell line RS4; 11
showing (A) blended colours generated by merging the
separated fluorochrome images, (B) pseudocolours
generated using the colour scheme in Fig. 1, and (C) colour
karyotype showing the t(4;11)(q21;a23), i(7q) and trisomy 8.
● each chromosome has individual color

MULTICOLOR BANDING (mBAND) ANALYSIS

● Uses chromosome-specific mixtures of partial

chromosome paints that are labeled with
various fluorochromes.
● colors different bands
● A computed program analyzes metaphase
chromosome data and produces a
pseudocolored, banded karyotype with an
estimated resolution of 550 bands, regardless
of chromosome length.
MULTIPLEX FISH (M-FISH) ● Advantageous for the determination of
breakpoints and the analysis of
● A technique that allows the investigator to intrachromosomal rearrangements and can be
view a karyotype so that each chromosome is particularly useful in preparations with shorter
“painted” with a different color. chromosomes.

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 Slide 35. Fiber-FISH analyzing the association of CL14
and CL34 repeats with telomeric DNA. (a) Five
representative fiber-FISH signals derived from probes
pAtT4 (red) and CL14 (green). (b) Five representative
fiber-FISH signals derived from probes pAtT4 (red) and
CL34 (green). Only the most proximal part of each
CL14/CL34 signal was included in each image. Note the
different sizes of the telomeric DNA and gap in the
junctions, indicating these signals may be derived from
different chromosomal ends. (c) Fiber-FISH analysis of
telomere (yellow), CL34 (red), and CL14 (green). The CL34
signals within the 4 images show significantly different
Slide 33. Multicolor banding. (a) Region-specific probes sizes. Only the most proximal part of each CL14 signal was
labeled with different partial chromosome patins (PCP) included in the image.
and computer false color (MetaSystems’ mBAND)
produces a definable number of colored bands per PRIMED IN SITU LABELING (PRINS)
chromosome, regardless of chromosome length. (b) This
example shows an abnormal X chromosome (right ● Essentially PCR on a slide.
homolog of each pair). ● Amplifying sequence is exponential
● Not homologous pair ● Primers of interest are hybridized on a slide
and then subjected to cycles of denaturation,
reannealing, and elongation that are used to
FIBER FISH
incorporate labeled nucleotides. The labels are
then detected fluorescently, or labeled
● A technique that is almost entirely used for
nucleotides are incorporated during the
research.
reaction.
● It allows the chromosomes to be stretched out
● Can differentiate hybridization with the alpha
and elongated.
satellite sequences for chromosomes 13 and
○ Stretched out = All sequences are exposed!
21, something that cannot be done with
● The probes are applied and can be physically
traditional FISH.
ordered on the fibers.
● This provides a much higher spatial resolution
and allows for correct orientation and
placement of probes and for precise mapping
of probes.

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 Slide 37. Metaphase chromosomes are subjected to Slide 39. Reverse FISH of a patient with an abnormal
PRINS with alpha satellite oligonucleotides specific for chromosome 8 (a) G-banding suggested a duplication of
chromosomes X, 11, and 17. Bright yellow fluorescein bands 8p23.1 p23.3. Two pairs of chromosomes 8 are
staining is seen at the centromeres of these shown; arrows indicate the additional band. This band
chromosomes. was microdissected, and the DNA was amplified, labeled,
and used as a FISH probe (b, c) Hybridization to normal
chromosomes.
REVERSE FISH

(b) The same metaphase is imaged with reverse DAPI to

● Used to identify material of unknown origin.
approximate G-banding patterns and identify the 2
● This unidentified material, such as marker
chromosomes 8, and with (c) typical DAPI staining. Arrows
chromosome or duplication, is flow sorted or
indicate both chromosomes 8 (d) Hybridization back to a
microdissected off a slide after G-banding. The
metaphase from the patient, demonstrating that 1
DNA from this material is extracted,
chromosomes 8 contains a duplication (arrow). The
PCR-amplified and labeled with a
reverse FISH results confirm the initial interpretation.
fluorochrome. This is then used as a probe and
hybridized to normal or patient metaphase
chromosomes to identify the origin of the *Under CGH but walang specified na topic or
unknown material. subtopic
● Copy-number variations (CNVs) are
alterations of the DNA of a genome that results
in the cell having an abnormal number of
copies of one or more sections of the DNA.
● Large regions of the genome have been deleted
(fewer than the normal number) or duplicated
(more than the normal number) on certain
chromosomes.
● Amplifications and deletions can contribute to
tumorigenesis.
● Amplification is the most common change
seen in malignancies.
● Detection and mapping provides an approach
to associate an aberration with a disease
phenotype and localizing critical genes -
Biomarkers.

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 ● Prognosis and therapeutics. ● CGH is based onco-hybridization of 2
● Resistance and susceptibility to disease. differentially labeled genomic DNAs (eg. tumor
○ Ex: HIV and SLE and normal) to human metaphase
● Mental retardation, developmental delay and chromosome spreads.
seizure disorders. ● It is based on the co-hybridization of
● Dysmorphic features and multiple congenital differentially labelled test and reference DNA
anomalies. onto metaphase spreads, which usually have
● Schizophrenia and autism spectrum disorder. been prepared from peripheral blood
lymphocytes of a healthy donor.
COMPARATIVE GENOMIC HYBRIDIZATION ● The signal intensity ratios of the 2 labels along
the chromosomes then reflect DNA copy
● Comparative Genomic Hybridization (CGH) or number changes in the test genome relative to
Chromosomal Microarray Analysis (CMA) is a the reference genome.
molecular-cytogenetic method for the analysis ● Resolution is limited to about 3 - 10 Mb.
of copy number changes (gains/losses) in the
DNA content of a given subject’s DNA and often
in tumor cells.
● First described in 1993 by Kallioniemi et. al.
● DNA from subject tissue and from normal
control tissue (reference) are each labeled with
different tags.
● Hybridized to metaphase chromosomes or, for
array- or matrix- CGH.
● Regional differences in the fluorescence ratio
of gains/losses vs. control DNA can be detected
and used for identifying abnormal regions in
the genome.
● CGH, a special FISH technique (dual probes), is
applied for detecting all genomic imbalances.
● The basics of technique is comparison of total
genomic DNA of the given sample DNA (e.g. Slide 14.
tumor DNA) with total genomic DNA of normal
cells.
● Typically, an identical amount of both tumor
and normal DNAs is labeled with 2 different
fluorescent dyes; the mixture is added and
hybridized to a normal lymphocyte metaphase
slide.
● A fluorescent microscope equipped with a CCD
camera and an image analysis systems are
used for evaluation.
● CGH is used to determine copy number
alterations of genome in cancer and those
cells whose karyotype is hard or impossible to Slide 15. After extraction of test DNA (i.e. from a tumor
prepare or analyze. sample) and normal DNA (i.e. from peripheral blood), the
● CGH will detect only unbalanced chromosomal samples are differentially labeled with discernable
changes. Structural chromosome aberrations fluorochromes (i.e. tumor DNA with FITC [green] and
such as balanced reciprocal translocations or control DNA with RITC [red]).
inversions can not be detected, as they do not
change the copy number.

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 ● The genomes are combined with an excess of ● A gain of chromosomal region in the test
cot-1 DNA and hybridized to metaphase sample would result in an increased intensity
chromosomes. of green fluorescence.
● Background hybridization due to repetitive ● A loss within a chromosomal region in the
DNA sequences is a common problem in tumor would be indicated by a shift towards
assays. red intensities.
● Cot-1 DNA blocking reagent blocks repetitive ● CGH analysis software measures fluorescence
DNA sequences and prevents nonspecific intensity values along the length of the
hybridization. chromosomes and translates the ratios into
chromosome profiles.
● The ratio of green to red fluorescence values is
used to quantitate genetic imbalances in
tumor samples.

● Images of metaphase spreads are then

acquired (charged coupled device) CCD camera
and fluorochrome-specific optical filter sets to
capture the FITC and TRITC fluorescence.
● Differences in fluorescence intensity values
between tumor and control DNA represent
gains and losses of specific chromosomes or
chromosomal regions.

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 ARRAY CGH: THE COMPLETE PROCESS

Step 1 - 3: Patient and control DNA are labeled with

fluorescent dyes and applied to the microarray.

Step 4: Patient and control DNA compete to attach, or

hybridize, to the microarray.

Step 5: The microarray scanner measures the

fluorescent signals.
Step 6: Compute software analyzes the data and
generates a plot.

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2H-MT | A.Y. 2022-2023 Page 13 of 13
 UNIT 9: PEDIGREE ANALYSIS AND GENETIC COUNSELING

GENETIC COUNSELING
TRANS OUTLINE
● In 1947, “Genetic counseling” (new field) was
coined by Sheldon Reed, a geneticist who
I. GENETIC COUNSELING advises physicians on how to explain heredity
A. REASONS FOR PATIENT TO SEE GENETIC to patients with single-gene diseases (cystic
COUNSELOR fibrosis, huntington, etc).
B. GENETIC COUNSELING INVOLVES THE ● In 1971, the first batch of trained genetic
FOLLOWING counselors were trained; nowadays, they
o Genetic Counseling should have a Master’s degree.
II. GENETIC TESTING TECHNIQUES ● Help patients and their families:
III. PEDIGREE ANALYSIS ○ Understand the inheritance pattern of
a specific medical condition.
A. PEDIGREE CHART
○ Interpret genetic testing results, and
o Analyzing Pedigrees
○ Evaluate the risk.
o Pedigree Chart Symbols
○ Example:
IV. MODES OF INHERITANCE AND THEIR ■ Your child has chromosomal
PROPERTIES aberration, e.g. Down Syndrome.
A. AUTOSOMAL DOMINANT Consult a genetic counselor if you
B. AUTOSOMAL RECESSIVE want to know the probability that
C. X-LINKED DOMINANT your next child will also have it.
D. X-LINKED RECESSIVE ■ Pregnant women can get tested to
E. MITOCHONDRIAL know if the baby has
V. CONDENSED PEDIGREE CLUES, NOT RULES chromosomal aberration. Genetic
counselor can then help the
A. ALGORITHM
mother to decide if she will
B. EXAMPLES
continue or terminate the
VI. PEDIGREE ANALYSIS IN REAL LIFE pregnancy.
VII. PEDIGREE SHOWS TRAIT AND DISEASE ● Lead the patient to take genetic testing if
INHERITANCE necessary.
A. AUTOSOMAL RECESSIVE TRAIT ● Have medical, scientific, and communication
o Phenylketonuria (Pku) skills because genetic counseling addresses
B. AUTOSOMAL DOMINANT TRAIT medical, psychological, sociological, and
o Huntington’s Disease ethical issues.
C. X-LINKED RECESSIVE PEDIGREE ● Work directly with patients and be part of a
o X-Linked Recessive Diseases healthcare team that works in a medical
center.
D. X-LINKED DOMINANT PEDIGREE
o X-Linked Dominant Diseases
REASONS FOR PATIENT TO SEE GENETIC COUNSELOR
E. Y-LINKED INHERITANCE
o Y-Linked Genes 1. Family history of cancer.
F. MITOCHONDRIAL 2. Family history of multifactorial disease.
a. Example: sickle cell anemia
3. Family history of abnormal chromosomes.
4. Elevated risk of single-gene disease.

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 ○ Needle is inserted into the abdomen of
GENETIC COUNSELING INVOLVES THE FOLLOWING the mother guided by an ultrasound.
○ Another option: Chorionic villus
1. Family history assessment. sampling (CVS)
a. Investigation of family tree starts with the ■ Collects mother’s placental
proband tissue through the vaginal
2. Pedigree construction and identifying family canal by inserting a speculum,
member/s who is/are at risk. retract and open the vaginal
a. Similar to creating a family tree, except it canal, then swab a piece of
traces which members of each generation placental tissue.
has the disorder ■ More accurate than
b. Basis of what genetic test will be used amniocentesis but has a
3. Genetic Testing (includes Karyotyping) and higher probability of
discussion of results and treatments. miscarriage

● Proband - one who availed the services of a genetic ● Pedigree Analysis

counselor to address the concern about their ○ Genetic analysis method to determine
probability of having a genetic disease. disease family history.
○ Across generations
GENETIC COUNSELING ○ To determine pattern of trait if it is:
■ Autosomal, X-linked,
mitochondrial, or Y-linked

PEDIGREE ANALYSIS

● Genetic counselors deduce dominance and

distinguish autosomal from X-linked
inheritance.
● He traces the history of some variant
phenotype back through the history of the
family and draws up a family tree (pedigree)
using the standard symbols.
● The clues in the pedigree have to be interpreted
differently depending on whether one of the
contrasting phenotypes is a rare disorder or
whether both phenotypes of a pair are common
GENETIC TESTING TECHNIQUES morphs of a polymorphism.

● Karyotype PEDIGREE CHART

○ Tests for abnormalities of
chromosomes. ● “Family tree”
○ That traces transmittance of genetic
● Amniocentesis trait
○ To test baby prenatally ● Drawn with standard genetic symbols, showing
○ Collection of amniotic fluid. inheritance patterns for specific phenotypic
○ Less accurate karyotype but is also characters.
less risky
■ FISH, CGH, PCR can also be
performed to the amniotic fluid

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 ANALYZING PEDIGREES CAN REVEAL
We will see if the trait is:
1. Whether a trait is dominant or recessive. ● Dominant or recessive
a. Dominant - only 1 allele needs to be ● Genotype
inherited ● Probabilities or Phenotypes
b. Recessive - 2 alleles, cannot be
expressed unless it is homozygous
2. The type of chromosome, autosomal or sex, a ● Squares = males; Circles = females.
trait is linked to. ○ With number inside - shorthand; if 2 =
a. Female two child, and so on
i. X-linked Dominant - 1 copy of X ● Shaded squares or circles signify the
ii. X-Linked Recessive - 2 copy of X presence of a trait of interest.
b. Male ● Rows are generations labeled with Roman
i. Trait is expressed even if it is numerals.
x-linked Recessive since there is ○ The oldest generation comprises the
no additional X top row, with each subsequent
3. Genotypes of family members. generation on separate rows.
4. Probabilities of phenotypes in future
○ Within each generation or row, family
generations. For families with a history of
members may be labeled numerically
autosomal or sex-linked diseases, this
from left to right and referred to by
information can be crucial to family planning.
their generation and position.
● Horizontal line connecting 2 parents is called a
PEDIGREE CHART SYMBOLS
marriage line, although marriage is not
necessarily involved.
● A vertical line of descent extending downward
from a marriage line connects to a horizontal
sibling line. Individuals connected to the line
of descent via the sibling line are offspring.
● Individuals that are not directly connected to
the sibling line entered the family via marriage
lines, and are not biological offspring of the
preceding generation.
● Arrow towards shaded shape, where the
investigation/pedigree started - Proband
● Slash - deceased
● Two symbol connected by an arch - fraternal
twin
○ Additional line between two shapes -
identical
● Diamond - unknown sex
● Cross inside diamond (or square if male,
circle if female) - born dead after 9 months
pregnancy
● Single bar between Square and circle -
marriage or mating, not necessarily married
Slide 16. Symbols used in human pedigree analysis. ● Double bar - marriage between relatives
(After W. F. Bodmer and L. L. Cavalli-Sforza, Genetics, ○ PH law - 3rd degree: first cousin
Evolution, and Man. Copyright © 1976 by W. H. Freeman ○ Higher chance of manifestation of
and Company.) recessive trait and congenital defects

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 ○ Genes are very close, hence, there is no
variation or biodiversity
○ Discouraged since it weakens genetic pool
● Broken line - extramarital, not married

Slide 20. Pedigree Symbols

Example:

Slide 18. Pedigree

Proband: III - 3rd generation

● sex - female
● First child
Slide 19. Pedigree Chart
II - 2nd generation
● Parent who has the trait: Mother
○ Deceased
● Parents siblings - 3
○ 1st child - Male
○ 2nd child (Mother) - only the mother
has the trait
○ 3rd - Male

I - 1st generation
● Grandparent who has the trait: Grandfather
○ deceased

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 MODES OF INHERITANCE AND THEIR PROPERTIES ○ Color Blindness

● Autosomal: chromosome 1 to 22 MITOCHONDRIAL

● X-linked: X chromosome
● Only females can pass on mitochondrial
AUTOSOMAL DOMINANT conditions to their children (maternal
inheritance).
● One mutated allele causes the disease. ● Both males and females can be affected.
● Each affected person usually has one affected ○ But can only be transmitted in the female’s
parent. next generation and not in male’s
● Appears in every generation of an affected ○ Apo sa babaeng anak
family (Vertical) ● Can appear in every generation of a family.
● Examples: ● Examples:
○ Marfan Syndrome ○ LHON: Leber’s Hereditary Optic
○ Achondroplasia Neuropathy
○ Huntington disease
○ Myotonic dystrophy

AUTOSOMAL RECESSIVE CONDENSED PEDIGREE CLUES, NOT RULES

● 2 mutated alleles needed to cause the disease.

INHERITANCE
● Parents are usually unaffected heterozygotes. CLUES
PATTERNS
● Not typically seen in every generation
(Horizontal). ● Approximately half of
● Examples: everybody.
Autosomal
○ Beta thalassemia ● Males and Females are
Dominant
○ Cystic Fibrosis affected.
● All generations
○ Homocystinuria
● Rare
X-LINKED DOMINANT ● Skips generations.
Autosomal
● Males and Females are
Recessive
● Females are more frequently affected than affected
males. ● Consanguinity (▢ = ◯)
● No male-to-male transmission.
● Some females can have
● Examples:
it.
○ Rett Syndrome X-linked ● All generations (no
○ Hypophosphatemia Dominant skipped generations)
(Sex-lined ● Males get it from
X-LINKED RECESSIVE dominant) affected mothers and
give it to their
● Males are more frequently affected than daughters.
females.
● Rare
● Both parents of an affected daughter must be ● Males predominantly
carriers. X-linked have it.
● Fathers cannot pass X-linked traits to their Recessive ● Generally skips
sons. (Sex-linked generations.
● Examples: recessive) ● Males generally get it
○ Hemophilia from unaffected
mothers.
○ Duchenne Muscular Dystrophy

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 ● All males, all the time,
all generations (must be
Y-linked
direct descendents of
the family).

● Every child of an
Mitochondrial
affected mother is
or Maternal
affected.

ALGORITHM
**Memorize to interpret pedigree

● Does offspring with disease have a parent

with disease? (Y/N)

○ If YES
■ Dominant (does not skip generations)

● Is there male to male transmission

of disease? (Y/N)
○ If YES EXAMPLES
■ Autosomal dominant
○ If NO
Example 1
■ Do daughters of affected
males have the disease?
(Y/N)
● If YES
○ X-linked dominant
● If NO
○ mitochondrial

○ If NO
■ Recessive (can skip generations)
● Predominantly males with disease?
(Y/N)
○ If YES
■ X-linked recessive
○ If NO
■ Autosomal recessive
Proband: offspring #1 in the 4th generation
If it doesn’t fit the questions: ● Proband: Female
● Check if it is only being transmitted to males ○ 3 siblings
○ If YES, it is Y-linked ■ Only 2 has the trait: 1st and
● Go back to the clues 3rd
● Parents:
○ Consanguineous between 1st
cousins (in the father side)
● Generations

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 ○ 4 generations Example 3
○ 1st to 3rd do not have the trait

QUESTIONS:
● Does offspring with disease have a parent with
disease?
○ NO
■ Recessive (can skip generations)
● Predominantly males with disease?
○ NO
■ Autosomal recessive

What is the pattern of inheritance?

● Autosomal recessive

Example 2 Proband: offspring #6 in the 3rd generation

● Proband: Male

QUESTIONS:
● Does offspring with disease have a parent with
disease?
○ YES
■ Dominant (does not skip generations)
● Is there male to male transmission of
disease? (1st gen to 1st offspring in
the 2nd gen)
○ YES
■ Autosomal dominant

What is the pattern of inheritance?

● Autosomal dominant
Proband: offspring #4 in the 4th generation
● Proband: Male
● Generations Example 4
○ 4 generations
○ Affected: II, III, and 4

QUESTIONS:
● Does offspring with disease have a parent with
disease?
○ NO
■ Recessive (can skip generations)
● Predominantly males with disease?
○ YES
■ X-linked recessive

What is the pattern of inheritance?

● X-linked recessive

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 Proband: offspring #1 in the 3rd generation Example 6
● Proband: Male
QUESTIONS:
● Does offspring with disease have a parent with
disease?
○ YES
■ Dominant (does not skip generations)
● Is there male to male transmission of
disease? (1st gen to 1st offspring in
the 2nd gen)
○ YES
■ Autosomal dominant

What is the pattern of inheritance?

● Autosomal dominant

Example 5 Proband: offspring #5 in the 3rd generation

● Proband: Male

QUESTIONS:
● Does offspring with disease have a parent with
disease?
○ NO
■ Recessive (can skip generations)
● Predominantly males with disease?
○ YES
■ X-linked recessive

What is the pattern of inheritance?

● X-linked recessive

Example 7
Proband: offspring in the 4th generation
● Proband: Female

QUESTIONS:
● Does offspring with disease have a parent with
disease?
○ NO
■ Recessive (can skip generations)
● Predominantly males with
disease?
○ If NO
■ Autosomal recessive
** check clues: consanguinity

What is the pattern of inheritance?

● Autosomal recessive Proband: offspring #2 in the 3rd generation
● Proband: Female

2H-MT | A.Y. 2022-2023 Page 8 of 13

 ● Unknown since it is not
QUESTIONS: shown in the pedigree
● Does offspring with disease have a parent with
disease? What is the pattern of inheritance?
○ YES ● X-linked dominant
■ Dominant (does not skip generations) ○ Closest
● Is there male to male transmission of ○ Favors the gender
disease? (1st gen to 1st offspring in ■ More female is affected
the 2nd gen)
○ NO
Example 9
■ Do daughters of affected
males have the disease?
● YES
○ X-linked dominant

What is the pattern of inheritance?

● X-linked dominant

Example 8

Proband: offspring #1 in the 3rd generation

● Proband: Female

QUESTIONS:
● Does offspring with disease have a parent with
disease?
○ YES
■ Dominant (does not skip generations)
● Is there male to male transmission of
disease? (1st gen to 1st offspring in
Proband: offspring #3 in the 3rd generation
the 2nd gen)
● Proband: Female
○ NO
■ Mitochondrial
QUESTIONS:
● Does offspring with disease have a parent with
What is the pattern of inheritance?
disease?
● Mitochondrial dominant
○ YES
○ Pag nanay ang may trait, lahat ng anak
■ Dominant (does not skip generations)
niya ay may trait
● Is there male to male transmission of
○ Pero anak na babae lang ang pwede mag
disease?
pamana ng trait
○ NO
■ Do daughters of affected
males have the disease?

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 Example 10 PHENYLKETONURIA (PKU)

● A human metabolic disease caused by a

mutation in a gene encoding a
phenylalanine-processing enzyme which leads
to mental retardation if not treated; inherited
as an autosomal recessive phenotype.

Proband: offspring #2 in the 4th generation

● Proband: Male

What is the pattern of inheritance?

● Y-linked dominant
○ Lahat ng males may trait

PEDIGREE ANALYSIS IN REAL LIFE

AUTOSOMAL DOMINANT TRAIT
● Keep in mind that pedigrees don’t always give
the pattern of inheritance. Many factors (like ● These are requiring only one copy of the
the environment or multiple genes) might be determining allele to influence phenotype
involved! ○ Will always manifest
● The algorithm will work in most cases but if it does ● Freckles and polydactylism (extra fingers or
not, look at the given clues. toes) are examples.
● Autosomal dominant diseases, such as
PEDIGREE SHOWS TRAIT AND DISEASE INHERITANCE Huntington’s disease, afflict ~50% of offspring
with one affected parent.
AUTOSOMAL RECESSIVE TRAIT ● Many of these diseases do not cause
symptoms until later in life and or after
● Traits caused by genes on autosomes and reproductive age.
requiring two allele copies to influence a ● Children can inherit these diseases from
phenotype are autosomal recessive. unknowingly affected parents, highlighting the
○ Example: Mother and Father are both importance of analyzing family history.
heterozygous, if they both passed on
the trait, you will have the trait. HUNTINGTON’S DISEASE
● Several disorders are autosomal recessive,
including cystic fibrosis, Tay-Sachs disease, ● Very common example
and maple syrup urine disease. ● Majority of the offspring will possess the trait since
● Most people with these diseases have it is always present
heterozygous parents who do not have the ● An autosomal dominant genetic disorder that
condition but carry a causal allele. affects the central nervous system of human
beings.

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 ● This disease typically shows up when a person
reaches their mid 30's or 40's, with no earlier
signs or symptoms.
● The nerve cells of an affected individual
quickly degenerate in certain parts of the brain,
which leads to symptoms that include
dementia, loss of memory, and severely
decreased mental capacity, muscle rigidity,
and loss of bodily function and muscle
coordination.
● Huntington's usually shortens an affected
persons lifespan, with the average person
dying 15-20 years after onset of the disease,
making the life expectancy around 50-60 years
of age.
● Genders affected
○ males must receive defective gene
from carrier mother
■ carrier mother's sons have 50%
of having disease
○ affected males give copy to all of their
daughters
● Generations affected
○ skips generations
■ male-to-male transmission not
allowed
■ diseases passes through
carrier daughters
● Pathology
○ defects in enzymatic genes
■ similar to AR diseases
X-LINKED RECESSIVE PEDIGREE ● Presentation timing
○ usually after puberty
● Trait is rare in pedigree. ● Other notes
● Trait skips generations. ○ only one defective copy necessary for
● Affected fathers DO NOT pass to their sons. disease in males
● Males are more often affected than females . ■ because males are hemizygous
○ Favors the males because of the genotype for X chromosome
● Usually manifests after puberty. ○ two defective copies necessary for
● Kapag si tatay ang may trait, napapamana lang ang disease in females
trait sa anak na babae pero hindi sa anak na lalaki. ■ can be affected with just one
Lahat ng anak na babae ay magiging carrier. defective copy if normal X
chromosome is inactivated to
Barr body
● called manifesting
heterozygotes
○ phenotype
usually milder
than affected
males

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 X-LINKED DOMINANT PEDIGREE
X-LINKED RECESSIVE DISEASES

● Red-green color blindness

● Hemophilia
○ Hemophilia in European royalty

● Trait is common in pedigree

● Affected fathers pass to ALL of their daughters,
but not their sons
○ Because females only have to have 1
copy
● Does not skip generations
● Kapag si tatay ang may trait, napapamana lang ang
■ Disastrous for most royal trait sa anak na babae pero hindi sa anak na lalaki.
families since males carry the Lahat ng anak na babae ay magmamanifest ng trait.
crown
■ Consanguineous/in-breeding ● Genders affected
marriage since they want to ○ male and female at equal frequency
retain the royal blood ● Generations affected
○ does not skip generations
● X-linked ichthyosis ■ only possibility is reduced
penetrance
○ females of affected fathers are always
affected
■ male-to-male transmission not
seen
○ male or females of affected mothers
can be affected
● Pathology
○ defects in structural genes
● Presentation timing
○ usually after puberty

X-LINKED DOMINANT DISEASES

● X-linked dominant diseases are extremely

○ “Kaliskis ng isda” unusual
● Often, they are lethal (before birth) in males
and only seen in females

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 ● Examples: ● BPY2 (basic protein on the Y chromosome),
○ incontinentia pigmenti (skin lesions) ● AZF1 (azoospermia factor 1),
○ X-linked rickets (bones ● DAZ (deleted in azoospermia),
soften/deform) ● RBM1 (RNA binding motif protein, Y
■ Bow leg - legs bend like a bow chromosome, family 1, member A1),
and arrow ● RBM2 (RNA binding motif protein 2) and
● UTY (ubiquitously transcribed TPR gene on Y
chromosome).

MITOCHONDRIAL

● Mitochondrial DNA comes only from the mother

○ Napapamana ng nanay sa lahat ng anak
pero anak na babae lang ang makakapag
pamana sa further generation, it will stop
with the males
● Genders affected
○ male and females at equal frequency
● Generations affected
○ does not skip generations
○ only transmitted from affected female
■ gives to all offspring
Y-LINKED INHERITANCE
■ due to the fact that the sperm
do not contribute mitochondria
● Inheritance of genes on the Y chromosome
to the zygote
● Since only males normally have a Y
● Pathology
chromosome, Y-linked genes can only be
○ defects in electron transport/oxidative
transmitted from father to son
phosphorylation process
● Y-linked inheritance is also called holandric
■ presents as
inheritance
neuropathies/myopathies
● neurons and muscle
Y-LINKED GENES
cells require high
amounts of energy and
A number of genes were are known to be Y-linked
depend on
including:
mitochondria
● ASMTY (which stands for acetylserotonin
● Presentation timing
methyltransferase),
○ usually after puberty
● TSPY (testis-specific protein),
● Other notes
● IL3RAY (interleukin-3 receptor),
○ variable expression due to
● SRY (sex-determining region),
heteroplasmy
● TDF (testis determining factor),
■ a small percentage of
● ZFY (zinc finger protein), PRKY (protein kinase,
mitochondria within a cell are
Y-linked),
affected leading to variable
● AMGL (amelogenin),
severity
● CSF2RY (granulocyte-macrophage
colony-stimulating factor receptor, alpha
subunit on the Y chromosome),
● ANT3Y (adenine nucleotide translocator-3 on
the Y),
● AZF2 (azoospermia factor 2),

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