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Chromosomal basis of heredity

Lecture one
6
th
February 2012
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Why an interest in Clinical Genetics?

An understanding of genetics is becoming
increasingly important for health care practitioners
Rare familiar traits (e.g. cystic fibrosis,
haemophilia, phenylketonuria, etc.) have been
recognised for some time
Now thought that most major diseases (e.g.
cancer, heart disease, diabetes, psychiatric
disorders, etc.) have a genetic component
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Introduction to


We are going to explore various aspects of
clinical genetics, including:
chromosomal disorders
single gene disorders
multifactorial disorders
mitochondrial disorders
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Introduction to


We are going to examine genetic variation at:
an individual level, and
a population level
We are also going to study a range of more
specialist topics including:
developmental genetics
immunogenetics
the genetics of cancer

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Review of chromosomal basis
of heredity
Gregor Mendel:
is universally acknowledged as the founder of
genetics
formulated the laws of inheritance in the 1860s
but knew nothing about meiosis
Link between Mendels theory and the observed
behaviour of chromosomes at meiosis was not made
until the beginning of the 20
th
century
It then became clear that Mendels determinants (now
known as genes) were physical units on chromosomes
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Structure of eukaryotic chromosomes
The nucleus contains linear chromosomes composed of
double stranded DNA coiled around histone proteins
The packaging of this complex, called chromatin, varies:
During interphase (G1, S & G2), DNA is relatively
loosely associated with the histones, the chromosomes
are very long and thin, and hence can only be observed
under an electron microscope
At the start of nuclear division (mitosis or meiosis),
DNA becomes very tightly coiled around the histones (a
process known as condensation), hence the
chromosomes become much shorter and thicker, and
can now be seen under a light microscope
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Cell Cycle
copyright cmassengale
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Karyotype
A karyotype is defined as either the chromosomal
constitution of an individual or the representation (as a
photograph or diagram) of that chromosomal constitution
The term ideogram is also used for diagrams

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Human karyotype analysis
Conventional karyotyping utilises mitotic metaphase
chromosomes
Samples of lymphocyte precursor cells (from bone
marrow) or chorionic villi cells (obtained via chorion
biopsy at 9-14 weeks gestation) usually contain sufficient
dividing cells for direct examination
Most other tissue samples (e.g. T lymphocytes from
venous blood, skin fibroblasts, foetal cells shed into the
amniotic fluid and obtained via amniocentesis at 16-18
weeks gestation, etc.) require culturing in vitro, a process
that can take up to three weeks
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Human karyotype analysis
Dividing cells are arrested at metaphase using a spindle
inhibitor such as colchicine
Cells are then spread on a glass slide in the presence of a
hypotonic saline solution that causes the nuclei to rupture
and the chromosomes to separate
Chromosomes were observed under the light microscope
as early as the mid 19
th
century
Human cytogenetics dates from 1956 when Tjio and
Levan established that the normal human chromosome
number in somatic cells was 46 with:
22 pairs of autosomes, and
1 pair of sex chromosomes (XY in males and XX in
females)

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Human karyotype analysis
Traditionally, chromosomes were cut out from a photograph
and glued on to card
This stage is now largely undertaken electronically utilising
digital images
Whichever method is used, the 22 pairs of autosomes are
arranged according to:
length, from longest (chromosome 1) to shortest
(chromosome 22), and
position of centromere
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Human karyotype analysis
Chromosome arms are respectively designated p (for short)
and q (for long):





By convention, p arms are shown uppermost in karyotypes
The p and q arms of metacentric chromosomes have been
designated by international agreement
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Human karyotype analysis
Human chromosomes were originally divided into seven
groups: A (1-3), B (4-5), C (6-12 plus X), D (13-15), E (16-18),
F (19-20) and G (21-22 plus Y):
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Human karyotype analysis
Staining procedures are normally used to visualise
chromosomes
As these techniques improved, initially individual
chromosomes could be identified, and then smaller and
smaller chromosomal sections could be recognised
The most common procedure, G-banding, uses the protease
trypsin to denature chromosomal proteins followed by
treatment with Giemsa
As Geimsa binds differentially, characteristic patterns of dark
and light bands result
The darker stained G-bands are rich in adenine and thymine
while the lighter stained G-bands are rich in guanine and
cytosine
The latter have been found to contain more genes
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Ideogram of human
G-banded chromosomes

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G-banded human chromosomes
Such banding has greatly facilitated the identification of
individual chromosomes and their subdivision into a series of
regions
In current nomenclature:
each chromosome arm is divided into regions numbered
outwards from the centromere (p1, p2, etc.)
then each region is subdivided into bands also
numbered outwards from the centromere (p11, p12, etc.)
Thus on the short arm of chromosome 2, 2p12 lies
centromeric to 2p13 but telomeric to 2p11
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Regions and bands on chromosome 2
(at low resolution)

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Regions and bands on chromosome 7
(at a range of resolutions)
At higher resolutions,
further subdivisions
are sometimes used
(e.g. 7q31.32 refers
to chromosome 7,
long arm, region 3,
band 1, sub-band 3,
sub-sub-band 2, etc.)

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Other banding techniques
Other less commonly used techniques include:
Q (quinacrine) banding
C (centromeric) banding
NOR (nucleolar organiser region) staining which visualises
the satellite stalks present on the p arms of all acrocentric
chromosomes except Y

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Fluorescence in situ hybridisation (FISH)
Is a technique where one or more fluorescently labelled
DNA probes are applied directly to the chromosomes
Unlike conventional karyotyping, interphase chromosomes
can be analysed and hence a rapid diagnosis of a suspected
genetic abnormality can be obtained
Individual genes can be localised and abnormalities
beyond the resolution of the light microscope can be seen
Applications of FISH include the detection of:
submicroscopic deletions
translocations (such as the Philadelphia chromosome
and translocation of the SRY locus from Yp11 often to X)
trisomy (using centromeric probes)

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Multicolour (M) - FISH
(also known as spectral karyotyping)
The so-called chromosome painting technique uses the
same fluorochrome to label a range of sequence probes for
an individual chromosome
Then, if each of the 24 different chromosomes (1-22, X
and Y) are labelled with a different fluorochrome, the whole
karyotype can be observed:

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Multicolour (M) - FISH
(also known as spectral karyotyping)
The main application is the visualisation of chromosomal
rearrangements that occur during the development of some
types of cancer





Note that M-FISH does not as yet offer the degree of
resolution provided by conventional chromosome analysis
Hence FISH and M-FISH are currently used to supplement
conventional analysis rather than to replace it

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Chromosomal mutation
Two broad categories of mutation occur:
point mutation where only a single base or a few
bases are altered
chromosomal mutation where an entire chromosome
or a large region of a chromosome is altered
Chromosomal mutations can be detected by karyotype
analysis
Chromosomal mutations are of two main types:
changes in chromosome number
chromosome rearrangements

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Changes in chromosome number
May arise from:
euploidy, the loss or gain of whole sets of
chromosomes so euploid cells contain exact
multiples of the haploid number (n)
aneuploidy, the loss or gain of individual
chromosomes so aneuploid cells have
incomplete sets of chromosomes

Reminder: n = 23 in humans
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Euploidy: monoploids (n)
The term monoploid is used for euploid mutants with only
one set of chromosomes





Monoploids arise in diploid species via parthenogenesis, viz.
the development of an egg without fertilisation
Monoploid animal embryos (including humans) do not
develop far


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Euploidy: polyploids (>2n)
Polyploids have more than two sets of chromosomes
For example:
triploids (3n) have three sets of chromosomes
tetraploids (4n) have four sets of chromosomes

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Euploidy: polyploids (>2n)
Mature polyploid animals are rare
Approximately1% of human conceptions are triploid but
most die before birth
Those that are born alive (1 in 10,000 live births) do not
survive long

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Euploidy: polyploids (>2n)
Polyploidy can occur spontaneously in a number of ways:
An egg may be fertilised by more than one sperm, a
condition known as dispermy. This is the most common
cause of triploidy
Failure of reduction division at meiosis will led to diploid
gametes. Fertilisation of such a diploid gamete with a
normal haploid gamete will then result in triploidy
In females, one of the polar bodies (usually non viable
products of meiosis) may fuse with the ovum to produce a
diploid gamete. Fertilisation with a normal haploid sperm
will then result in triploidy

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Aneuploidy
Aneuploidy arises as a consequence of non-disjunction of
chromosomes (i.e. failure to separate properly) at mitosis or
meiosis, respectively resulting in mosaic formation or
abnormal gametes
Aneuploids fall into a number of groups, for example:
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Human aneuploidy
All types of autosomal monosomy are lethal
Approximately 4% of human conceptions are
trisomic
While the gain of an autosome is not usually
compatible with survival beyond early pregnancy,
the following are observed in live births:
trisomy 13 (Patau syndrome)
trisomy 18 (Edwards syndrome)
trisomy 21 (Down syndrome)

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Down syndrome (trisomy 21)

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Down syndrome (trisomy 21)

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Down syndrome (trisomy 21)

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Human aneuploidy
The presence of an extra sex chromosome does not
apparently compromised embryonic survival and the
following sex chromosome trisomics are observed in live
births:
47,XXY (Klinefelter syndrome)
47,XYY (XYY syndrome or double Y syndrome)
47,XXX (triple X syndrome or trisomy X)
Loss of an X chromosome generating 45,Y is lethal
Loss of either an X or a Y chromosome generating 45,X
(Turner syndrome) is observed in live births but most such
embryos are lost during pregnancy
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Klinefelter syndrome (trisomy XXY)

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Klinefelter syndrome (trisomy XXY)

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Turner syndrome (monosomy X0)

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Turner syndrome (monosomy X0)

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Summary of human aneuploidy
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Chromosome rearrangements
Several different classes of structural rearrangement are
recognised:
Deletion


Duplication


Inversion

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Examples of human chromosome
rearrangements
Deletion:
Cri du chat syndrome results from a deletion involving
the terminal region of chromosome 5p
Both Angelman and Prader-Willi syndromes result from
a microdeletion involving chromosome 15q
Duplication
A small proportion of Down syndrome cases result from
the duplication of the q arm of chromosome 21. Such
patients have 46 chromosomes, including one normal 21
and one "isochromosome" (i.e. a chromosome with the
extra copy of the q arm replacing the p arm)

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Chromosome rearrangements
Structural rearrangement also include several types of
translocation:
Reciprocal translocation


Non reciprocal translocation


Robertsonian translocation





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Human chromosome translocations
The carriers of either reciprocal translocations or
Robertsonian translocations are at risk of having children with
unbalanced chromosome complements:
Familiar Down syndrome can be caused by a Robertsonian
translocation between chromosomes 14 and 21
The risk of a carrier producing a child with Down syndrome
varies with:
the chromosome pairing arrangements during meiosis
which parent is the carrier - 10-15% risk for female
carriers and 1-2% risk for male carriers


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Other outcomes of human chromosome
rearrangements
Some 20% of cancers are thought to result from inversions,
translocations and transpositions as such chromosomal
rearrangements may change changing the location of growth
regulating genes
For example, the so-called Philadelphia translocation
between chromosomes 9 and 22 is characteristic of chronic
myeloid leukaemia

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Clinical cytogenetics
Cytogenetic investigations are undertaken in the following
circumstances:
suspected chromosomal abnormality
infertility or multiple miscarriages
stillbirth or neonatal death
multiple congenital anomalies and/or developmental
retardation
undiagnosed mental retardation
disorders of sexual function
certain malignancies
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Clinical cytogenetics
Total chromosome count is normally determined in 10-15
cells (increased to a minimum of 30 cells if mosaicism is
suspected)
A detailed G-banding pattern analysis is undertaken in 3-5
cells
The formal report utilises standard abbreviations that can be
interpreted by health care practitioners
Most cytogenetic laboratories also provide written guidance
where findings are complex
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Abbreviations used in cytogenetic reports
p short arm
q long arm
cen centromere
tel telomere
del deletion
dup duplication
+ gain of
chromosome
- loss of
chromosome
i isochromosome
ins insertion
inv inversion
r ring
t translocation
rcp reciprocal
translocation
rob Robertsonian
translocation
mos mosaicism


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Examples of karyotype reports
46,XX Normal female
46,XY Normal male
45,X Female with Turner syndrome
47,XY,+21 Male with Down syndrome
47,XXY Male with Klinefelter syndrome
69,XXX Triploid female
46,XX,del(5p) Female with cri-du-chat
syndrome

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Examples of karyotype reports (cont)
45,XX,rob(14;21)(q10;q10)
Female with a balanced 14;21 Robertsonian
translocation (by convention the positions of
the centromeres are designated as q10;q10)

46,XX,rob(14;21)(q10;q10),+21
Female with an unbalanced 14;21
Robertsonian translocation resulting in Down
syndrome


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Karyotypes observed with
Down syndrome
Chromosome abnormality Frequency
Trisomy 21

Maternal origin
Paternal origin
95%

90%
5%
14/21 translocation up to 4%
Isochromosome 21 up to 1%
Mosaic trisomy 21 < 1%
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Fate of one
million implanted
human zygotes