APPLIED CYTOGENETICS
GENETICS 202
Jon Bernstein
Department of Pediatrics
September 29th, 2015
Learning Goals – Applied Cytogenetics


Learn normal chromosome structure and nomenclature
Gain familiarity with the results of errors in Meiosis and Mitosis, how
they are diagnosed and how the behave in families
◦ Numerical chromosome abnormalities
◦ Isochromosomes
◦ Ring chromosomes
◦ Translocations
 Reciprocal and Robertsonian translocations
◦ Inversions
◦ Chromosomal deletions and duplications
 Microdeletions and microduplications
(See short videos on coursework for additional detail)
Scales of Genetic Variation
ACTGATCCG
ACTGATAACCCG
ACTGCG
Structural Variant (SV)
ACTGATCCG
ACTGGTCCG
Small insertion or deletion (indel) Single nucleotide variant (SNV)
Nature Reviews Genetics 12, 363-376 (May 2011)
Learning Goals – Applied Cytogenetics

Understand the methods, strengths and limitations of
commonly used cytogenetic tests
◦ Karyotype
◦ FISH (Fluorescence in situ hybridization)
◦ Array CGH (comparative genomic hybridization)
◦ (See short videos on coursework for additional
detail)
Applied Cytogenetics

Outline for lecture
◦ The karyotype, normal chromosome structure
◦ Gallery of cytogenetic tests and anomalies
◦ Clinical cases demonstrating
 Microscopic chromosome abnormalities
 Seen on karyotype, Down syndrome, Turner syndrome, Inversion
 Submicroscopic chromosome abnormalities
 Seen by FISH or CGH, 22q11 deletion syndrome
Visualizing chromosomes and their types
#1
Basepair #1
#9
#14
Basepair #247 Million
Metacentric
Cells to Tissues, Stanford Cytogenetics Lab
Submetacentric
Acrocentric
Karyotype Resolution
High Resolution Karyotype, 550
bands or greater
Standard Resolution
Karyotype, 400 bands
Gallery of cytogenetic tests
wikipedia.org
Karyotype
DNA Microarray / Array Comparative
Genomic Hybridization
FISH – Fluorescence in situ hybridization
Test characteristics
Resolution (size of variants detected)
Ability to assess copy number (quantity of DNA)
Ability to assess location (position of DNA)
Gallery of anomalies
Trisomy
Interpreting cytogenetic test results

What am I seeing?
◦ Is there a gain or loss of genetic material?

Who has the change -- is it new or inherited?
 If new, when and where could the change have
occurred?
 If inherited, did it malsegregate during meiosis?
Interpreting cytogenetic test results

What does this result mean?
◦ Does this test result support a specific diagnosis?
◦ Can the genetic change be passed on by the patient?
◦ Are the patient’s parents or other relatives at risk of
having a child with a similar genetic change?
Clinical Case #1


You have just
examined an infant
girl with low muscle
tone and subtly
distinctive facial
features.
A chromosome
analysis is ordered
and the following
karyotype is seen:
New Clinical Genetics 2e
Andrew Read and Dian Donnai
ISBN: 9781904842804
© Scion Publishing Ltd, 2011
Diagnostic Karyotype
New Clinical Genetics 2e
Andrew Read and Dian Donnai
ISBN: 9781904842804
© Scion Publishing Ltd, 2011
Down Syndrome


Occurs in 1/600 - 1/1000 births
Due to extra #21 chromosome q arm
◦ ~95% of cases with trisomy 21, 47,XX +21 or 47,XY+21
◦ Due to non–disjunction, typically in maternal meiosis I
 Diagnosis can be confirmed by chromosome
analysis
Down Syndrome – Health Risks
Newborn Period
Childhood
Adulthood
Congenital heart disease Intellectual disability
Dementia
Duodenal atresia
Hypothyroidism
Obesity
Celiac disease
Male infertility
Dry skin
Recurrence risk counseling for Down syndrome
 For trisomy 21 the estimated recurrence risk is
the greater of 1% or the maternal age related
risk.
 A minority of cases of Down syndrome are
due to a Robertsonian translocation and have
a higher risk of familial recurrence.
Robertsonian Translocation

Translocation involving acrocentric
chromosomes
◦ 13, 14, 15, 21, 22
Acrocentric
Breaks are at/near the centromere
 A centric fusion of q arms
 Acrocentric short arms contain redundant
material

Robertsonian Translocation
New Clinical Genetics 2e
Andrew Read and Dian Donnai
ISBN: 9781904842804
© Scion Publishing Ltd, 2011
45, XY, der(14;21)(q10;q10)
Robertsonian Translocation
Recurrence risk counseling for Down syndrome

In translocation Down syndrome where the
mother carries a Robertsonian translocation the
risk of recurrence is estimated to be 10%.
◦ What if the translocation is a new occurrence?

When a parent carries a 21;21 isochromosome
the risk of recurrence is estimated to be 100%
Isochromosmes


Metacentric chromosome
consisting of genetically
identical arms with all loci
equidistant from the
centromere
http://ghr.nlm.nih.gov/handbook/illustrations/isochromosomes
Believed to result from
transverse rather than
longitudinal division of the
centromere in meiosis or
mitosis
Clinical Case #2
Your are evaluating a
young girl in clinic with
short stature. As an
infant she had “puffy
feet.”
 A chromosome analysis
is ordered and the
following karyotype is
seen:

Stanford Medical Genetics
http://learn.genetics.utah.edu/content/disorders/whataregd/turner/
Diagnostic karyotype
Turner Syndrome, 45,X Karyotype

Birth prevalence 1:2500-1:5000
◦ Represents 1-2% of all conceptuses with Turner
syndrome; >90% result in pregnancy loss
◦ Reason for decreased viability incompletely understood

Paternal X much more likely to be missing
◦ No maternal age effect
◦ Low recurrence risk
Turner syndrome
•Growth and
development
•Short stature
•Normal intelligence
•Difficulties with
spatial orientation
and math
• Cardiac anomalies
– Coarctation of the aorta
– Bicuspid aortic valve
• Renal Anomalies
– Horseshoe kidney
• Other
– Congenital lymphedema
– Osteoporosis
Other forms of Turner syndrome - mosaicism

Mosaicism: The presence of two or more
genetically different cell lines in an individual.
◦ Degree of mosaicism can vary from tissue to tissue
◦ The level of mosaicism in one tissue is not predictive of
the level in others

Mosaic Turner syndrome
◦ 45,X and another cell line - often 46,XX
Other forms of Turner syndrome – ring
chromosome
Formed by a double break, with the
“broken ends” reuniting into a ring
configuration
Usually associated with some deletion of
the chromosomal ends
Clinical Case #3

A 32 year old man is a carrier of a reciprocal
translocation. What implications may this have for his
offspring?
Balanced Reciprocal Translocation
New Clinical Genetics 2e
Andrew Read and Dian Donnai
ISBN: 9781904842804
© Scion Publishing Ltd, 2011
Segregation of a reciprocal translocation
Reciprocal translocations

Carriers of balanced reciprocal translocations
typically have no clinical manifestations
◦ No net gain or loss of genetic information

Reciprocal translocation carriers, like
Robertsonian translocation carriers, are at risk
to have offspring with unbalanced
chromosome complements
Clinical Case #4

A newborn boy has a cleft palate and a short
jaw.

Amniocentesis karyotype (400 band
resolution) was reported as normal.
Array CGH
New Clinical Genetics 2e
Andrew Read and Dian Donnai
ISBN: 9781904842804
© Scion Publishing Ltd, 2011
Array CGH

An array CGH study is
performed and shows
an 8 Mb duplication
on 18p and a 7.5 Mb
deletion on 18q.
Synthesis question

Why might the amniocentesis karyotype have been
reported as normal?
◦ An error?
◦ Differences in the pre and postnatal testing methods?
A deletion and a duplication - two
independent events?

Possibly.
A deletion and a duplication - two independent
events?
 Or, the result of meiotic segregation of a
pericentric inversion.
◦ A chromosomal segment spanning the centromere is
reversed in orientation
◦ Typically, no or minimal genetic material lost or
gained in carrier
Chromosome Inversions
Segregation of pericentric inversions
Stoler et al., N Engl J Med 2004;351:2319-26.
Modern Genetic Analysis.
Griffiths AJF, Gelbart WM, Miller JH, et al.
New York: W. H. Freeman; 1999.
Copyright © 1999, W. H. Freeman and Company.
NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.
http://www.ncbi.nlm.nih.gov/books/NBK21367/
Fluorescence in situ Hybridization
Interphase
Stanford Cytogenetics Lab – Athena Cherry
http://www.genome.gov/glossary/?id=65
Metaphase
wikipedia.org
FISH study for case 4
Red=18p probe
Green = 18q probe
Normal control
Patient
Patient’s parent
Chromosome Inversions

Pericentric inversion carriers can produce
gametes with duplication or deletion of
chromosome ends
◦ Depending on the inversion, approximately 10% risk
to have a child with an unbalanced karyotype

Low risk for paracentric inversion carrier to have
child with abnormal karytoype
◦ Recombinant chromosomes are acentric /dicentric and not
viable
Case #4 summary – Increased resolution ->able to
detect smaller changes

Normal amniocentesis karyotype
◦ Resolution only 400 bands

8p duplication and 8q deletion detected by aCGH
and confirmed with position information by FISH
◦ Resolution is submicroscopic, smaller than visible bands
◦ These two events are the result of a parental pericentric
inversion (balanced)
Clinical Case #5

Pre-school aged girl
with a history of
difficulty with
articulation.

Array CGH shows an
interstitial deletion at
chromosome 22q11.2
New Clinical Genetics 2e
Andrew Read and Dian Donnai
ISBN: 9781904842804
© Scion Publishing Ltd, 2011
22q11.2 deletion syndrome
Contiguous gene deletion syndrome resulting
from interstitial microdeletion at 22q11.2
 Clinical features

◦
◦
◦
◦
Cardiac Anomalies
Immune Dysfunction
Craniofacial Anomalies
Developmental Difficulties
Low Copy Repeats on 22q

Deletions and duplications at 22q11 due to low copy repeats.
Genome Res. 2007. 17: 451-460.
Synthesis question

What are the chances that the parent of a child
with a mosaic 22q11 deletion syndrome would
carry the deletion?
Mosaic
microdeletion
Array CGH - Variants of Unknown Significance
Is it inherited or de novo (new)
 Has it been seen before in other unrelated individuals
◦ The medical literature
◦ Online databases

 Decipher http://decipher.sanger.ac.uk/
Lecture Summary

Chromosome abnormalities observed under the light
microscope include:
◦
◦
◦
◦
◦
◦

Numerical abnormalities
Robertsonian translocations
Isochromosomes
Ring chromosomes
Inversions
Reciprocal translocations
The recurrence risk for a chromosome anomaly depends on
the type of the anomaly and its origin
Summary

There is a wide variety of submicroscopic
variation in the human genome
◦ Microdeletions
◦ Microduplications
Copy number variants/Structural variants
 Pathogenic microduplications and microduplications can
result in a variety of syndromes
Review Question

An increased risk of recurrence in Down syndrome is
potentially associated with the presence of:
◦ A) Mosaicism
◦ B) A Robertsonian translocation
◦ C) A ring chromosome
◦ D) A metacentric chromosome
Review Question

Which type of translocation is more likely to result in a
phenotype due to disruption of genetic material at a
breakpoint?
◦ A) Robertsonian translocation
◦ B) Reciprocal translocation
Review Question

You have performed array CGH on a patient with
multiple congenital anomalies. Which cytogenetic
abnormalities would be detectable by CGH?
◦ A) Robertsonian translocation
◦ B) Inversion
◦ C) Balanced reciprocal translocation
◦ D) Ring chromosome
Case for small group session

Chief complaint (CC)
◦ You are asked to evaluate a 5 year old girl with a
history of developmental delay currently hospitalized
for poorly controlled seizures.
History of present illness (HPI)
In addition to developmental delay and
seizures, on exam the patient also has fleshy
hands and thin toenails. She has a history of
multiple urinary tract infections.
The patient was born at term (40 weeks). Her
mother's pregnancy was notable for febrile
illness in the second trimester.
Family History (FH)
The patient has an older brother and a younger
sister who are developing typically.
Her parents have also had a pregnancy which
ended in a miscarriage.
The patient has a paternal cousin through her
father’s sister with mild intellectual disability.