GTS368 Semester Test 2

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THE HUMAN KARYOTYPE

• The human genome consists of 25 different DNA molecules partitioned between two
genomes: one in the nucleus (nuclear genome) and another in the mitochondria
(mitochondrial genome, mtDNA).
• In the nucleus there are either 23 or 24 different linear DNA molecules: 23 in females (since
they have two X chromosomes in addition to their autosomes) and 24 in males (since they
have one X and one Y chromosome in addition to their autosomes).
• These DNA molecules are immensely long ranging in size from 48 Mb to 249 Mb.
• In mitochondria, there is just one type of DNA molecule: a circular DNA molecule that is
comparatively tiny and is only about 16.6 kb in length.
• There are many copies of the mtDNA in a cell, but only two copies of nuclear DNA
molecules present in diploid cells.
THE HUMAN GENOME PROJECT
• Chromosomes can be stained with a certain dye such as Giemsa to reveal the alternating
pattern of dark and light bands for each chromosome.
• The alternating pattern of bands reflects different staining intensities, that in turn reflects
differences in chromatin organisation along chromosomes as a result of differences in base
composition, as well as differences in gene and exon density.
• The resolution of the original physical map is low, and even a high-resolution chromosome map
the average size of a band is several mega bases of DNA.
• Obtaining the complete DNA sequence of the human nuclear genome was a long drawn out
process that was driven by an international collaborative effort between many research teams,
known as the human genome project.
• After it was realised that many regions of the DNA have highly variable DNA sequences it was
possible to define multiple polymorphic DNA markers and map them to individual chromosomes.
• Panels of mapped polymorphic markers were then used to build a genetic map by looking at
their segregation in defined reference families.
• The genetic map was then used as a scaffold to map short DNA sequences to chromosomal
regions which were in turn used to fish out clones with much longer DNA sequences that could
be allocated to defined positions on each chromosome.
• Ultimately, a map for each chromosome was constructed based on many DNA clones with long
inserts that could be ordered as a series of clones with partly overlapping DNA sequences.
• Finally, DNAs from selected clones were sequences and used to build chromosome-wide DNA
sequences.
• Not all regions of DNA were sequenced; a low priority was given to the DNA of heterochromatin
where the chromatin is highly condensed throughout the cell cycle and believed to be
transcriptionally inactive.
• Heterochromatin was believed to have very few genes and the underlying DNA is technically
difficult to sequence as it largely consists of highly repetitive noncoding tandem repeats.
• The focus was on sequencing the euchromatin component of our genome which was known to
be transcriptionally active and suspected to contain virtually all of our genes.
CHROMOSOMAL ABNORMALITIES
• Many large scale changes to our DNA sequences that cause diseases are more readily
studied at the level of chromosomes, as are changes in chromosome copy number resulting
from errors in chromosome segregation.
• In standard cytogenic karyotyping (profiling of chromosomes observable under a light
microscope based on size, shape, and number of chromosomes), suitable metaphase or
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prometaphase chromosome preparations are chemically stained to reveal a pattern of

alternating light and dark bands under light microscopy, which are examined to identify
chromosome abnormalities.
• Karyotyping is important for diagnostic purposes and the karyotype may be visualised under
microscope by staining chromosomes.
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• Chromosome abnormalities can be classified as constitutional where it is present in all
nucleated cells of the body and so must have been present very early in development, or
somatic/acquired where it is present only in certain cells or tissues of a person who is
therefore a genetic mosaic (possessing two populations of cells with altered chromosome
or DNA content deriving from the same zygote).
• Constitutional abnormalities arise as a result of an abnormal sperm or egg, through
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abnormal fertilisation, or through an abnormal event in the very early embryo.
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• Chromosomal abnormalities (whether constitutional or somatic) can be subdivided into to
categories: structural abnormalities (which arise through chromosome breakage events that
are not repaired) and numerical abnormalities (changes in chromosome number that often
arise through errors in chromosome segregation).
CHROMOSOME PREPARATION AND CHROMOSOME BANDING METHODS
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To study chromosomes under the light microscope, the chromosomes must be suitably
condensed and so metaphase or prometaphase chromosome preparations are required.
It I • A peripheral blood sample is taken and separated white blood cells are stimulated to divide
using a mitogen (agent that stimulates mitosis) such as phytohemagglutinin.
I 2 • The white blood cells are grown in a rich culture medium containing a spindle disrupting
agent such as colcemid to maximise the number of metaphase cells (cells that enter
ios metaphase but cannot progress through the rest of M phase).
• Prometaphase preparations can also be obtained which have slightly less-condensed
E sw chromosomes, making analysis easier.
fg.fiii • Chromosome banding involves treating chromosome preparations with denaturing agents.
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Alternatively, they are digested with enzymes and then exposed to a dye that can bind to
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to • Some dyes preferentially bind to AT-rich sequences, others bind to GC-rich sequences. The
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dyes show differential binding to different regions across a chromosome that will reflect the
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g relative frequencies of AT and GC base pairs.
• The most commonly used method in human chromosome banding is G-banding where the
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chromosomes are treated with trypsin and stained with Giemsa which preferentially binds to
AT-rich regions producing alternating dark bands (Giemsa positive AT-rich) and light bands
(Giemsa negative GC-rich).
• Genes are preferentially associated with GC-rich regions (because of CG islands in
promoters) and so dark bands indicate gene poor regions while dark bands indicate gene
rich regions.
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• A G-banded karyotype has several advantages: it allows for whole genome analysis and
gives an overall impression of the genome.
• No prior knowledge of the DNA sequence or molecular genetics is required and so it can be
used blindly.
• This karyotype can detect balanced chromosome changes as well as polyploidies and
mosaics.

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• However, the G-banded karyotype is low resolution (1-10 Mb) and requires culturing to
obtain metaphase chromosomes which may take a lot of time. The process is also very
labour intensive and specialised as very little automation is possible.
• The final drawback is that it is time consuming with a time delay to results (3-14 days) which
may be frustrating in times where results are needed urgently, e.g., with pregnant mothers.
CHROMOSOME BANDING NOMENCLATURE
• Human chromosome nomenclature is decided periodically by the International Standing

Committee on Human Cytogenic Nomenclature (ISCN).
• It assigns numbers 1-22 to autosomes based on perceived size, and uses the symbols p
• I
and q to denote the short and long arms of a chromosome.
Depending on the position of the centromere, chromosomes are described as
metacentric (centromere at or close to the middle of the chromosome), submetacentric
(centromere some distance from the middle and from telomeres), or acrocentric
(centromere close to the telomere).
• Each chromosome arm is subdivided into a number of regions, according to consistent
and distinct morphological features (depending on the size of the chromosome arm,
there may beMMM from one to three regions).
• Each region is in turn divided into bands, then into sub-bands and sub-sub-bands
according to the banding resolution.
• The numbering of regions, bands, sub-bands, and sub-sub-bands is done according to
the relative proximity to the centromere, where the region closest to the centromere is
region 1 and that closest to the telomere is region.LAST
• Nomenclature of 4q21 indicates a band on the long arm of chromosome 4 in region 2
which is closest to the centromere (band 1). Mk
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In chromosome nomenclature the words proximal and distal are used to indicateEthe
relative position on a chromosome with respect to the centromere.
• Thus, proximal Xq means the segment of the long arm of the X that is closest to the
centromere. Similarly, distal 3p means the portion of the short arm of chromosome 3
that is most distant from the centromere (= closest to the telomere). triplicate
• Triploidy is a form of polyploidy where all chromosomes (autosomes and sex
69 X
chromosomes) are present in triplicates as opposed to duplicates in a normal diploid,
hence the nomenclature would be 69,XXX or 69,XXY or 69,XYY.
MONE TRIP
• Trisomy is also a numerical abnormality where only one chromosome (autosome) has
an extra copy present, e.g., Down syndrome where an extra copy of chromosome 21 is
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present (47,XX,+21).
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• Monosomy refers to when a loss of a chromosome has occurred, e.g., 45,X (sex 1
chromosome lost) or 45,XY, -14.
• Mosaicism is a phenomenon where some cells in the body have normal numbers of
chromosomes (46,XX) while others have some form of polyploidy (47,XXX) NORM
SOME
STRUCTURAL CHROMOSOMAL ABNORMALITIES BUT
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• Abnormal chromosome breaks caused by double stranded DNA breaks occur as a result of
unrepaired damage to DNA or through faults in the recombination process.
• Chromosome breaks that occur during the G2 phase (after DNA has replicated) are really
chromatid breaks because they affect only one of the two sister chromatids.

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• Those that occur during the G1 phase that are not repaired by S phase where DNA
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replication occurs become chromosome breaks where both sister chromatids are affected.
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• A cell with highly damaging chromosome breaks may often be removed by triggering cell
death mechanisms; if it survives with unrepaired breaks, chromosomes with structural
abnormalities can result.
• Errors in recombination that produce structural chromosome abnormalities can occur at
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meiosis.
• Paired homologs are normally subjected to recombination mechanisms that ensure the Bmi
breakage and re-joining of non-sister chromatids, but if recombination occurs between
mispaired homologs, the resulting products may have structural abnormalities.
• Intrachromatid recombination can also be a source of structural abnormalities.
• A form of somatic recombination also occurs naturally in B and T cells in which the cellular
DNA undergoes programmed rearrangements to make antibodies and T cell receptors.
• Abnormalities in these recombination processes can also cause structural chromosomal
abnormalities that may be associated with cancer. Structural chromosome abnormalities are
often the result of incorrect joining together of two broken chromosome ends.
FLUORESCENCE IN SITU HYBRIDIZATION (FISH)
• The essence of chromosome FISH is to fix chromosome preparations on microscopic

slides, treat the slides so as to denature DNA, and hybridize fluorescently labelled probes of
interest to the denatured DNA.
• The locations of the fluorescent signals are recorded against a background stain that binds
to all DNA sequences.
• Chromosome FISH is often used to confirm regions of chromosome duplication or deletion
that have been suggested by other screening methods and can also be used to screen for
the amplification of specific oncogenes that are associated with particular types of cancer,
such as the amplification of the MYCN gene in neuroblastoma.
• Another major application is in detecting translocations, most notably acquired
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translocations that are common in cancer.
Recurrent translocations are associated with certain types of cancer; often, the translocation
DNA
involves breakages in specific genes, producing hybrid genes that are inappropriately
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• FISH combines karyotyping and hybridization techniques, and uses DNA probes labelled
with fluorescent dyes to detect specific chromosomes or chromosome regions using
fluorescence microscopy.
• In the FISH technique, interphase/metaphase chromosomes are fixed onto slides and
treated to denature DNA to produce single strands.
• The single stranded DNA probe is then prepared as a cloned piece of the genome that is
complementary to the sequence of interest on the DNA.
• The probe is labelled with a fluorescent dye and denatured before use in hybridization of
the probe to the DNA of interest.
• We then localize the fluorescent signals against a background stain that binds to all DNA
sequences.
• Different probes can be used in FISH. Gene/locus specific probes are for detecting the
presence/absence/location of a particular gene. NORMAL
• Repetitive DNA probes are used to detect centromere repeats (alpha-satellites) or
telomere repeats so as to detect the number of copies of a particular chromosome.
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• Whole chromosome probes are used for chromosome painting which is useful to detect
chromosome rearrangements. These are pools of fragments that covers entire
chromosomes/chromosome arms.
• Different fluorochromes are available and thus we can detect multiple probes
simultaneously.
• Interphase FISH is used for diagnostic purposes where aneuploid prenatal screening is
done on interphase cells from amnionic fluid.
• FISH has a higher resolution depending on application for which it used as well as probe,
and also has the advantage that we can use both interphase and metaphase cells.
• It is a quick technique to detect abnormal chromosome numbers and many fluorochromes
are available such that we can detect many probes simultaneously, which is advantageous.
• However, the disadvantages are that we need prior information such as the sequence of the
area of interest to make the probe.
• It is also not a general screening tool.
• Spectral karyotyping can evaluate a complete karyotype in a single experiment. In this
experiment, we have 24 different chromosome painting probes (one for each chromosome).
• Individual chromosomes are obtained by flow cytometry and each chromosome is labelled
with specific combinations of fluorescent dyes to give unique fluorescent signals which are
then analysed by a computer and assigned a specific colour in order to generate
photographs.
• This can be used to detect chromosome rearrangements in cancer cells as well as detect
differences in ploidy.
• This method can evaluate the complete karyotype in a single experiment (advantageous).
MONOGENIC INHERITANCE
• Genes are functional units of DNA that make some product needed by cells, either the
polypeptide chain of a protein or a functional noncoding RNA.
• Single gene disorders are diseases in which the genetic contribution is determined primarily
by one gene locus. Although individually rare, single-gene disorders are important
contributors to disease.
• Knowledge of single-gene disorders also provides a framework for understanding the more
complex genetic susceptibility to common disease
• The term phenotype may be used broadly to describe the observable characteristics of a
person, an organ, or a cell, but geneticists also use the word phenotype in a narrower sense
to describe only those specific manifestations that arise in response to the differential
expression of just one or a small number of genes. These manifestations may be harmful,
and we can talk of a disease phenotype.
• When the observable manifestations are not disease-associated we normally refer to a
character or trait, for example blue eyes or blood group O. We can measure and record
aspects of the phenotype, such as anatomical and morphological features, behaviour, or
cognitive functions.
• Genetic variation (changes in the base sequence of our DNA) is the primary influence on
the phenotype but it is not the only determinant of the phenotype: environmental factors
make a contribution, too, as do epigenetic effects.
• An individual gene or DNA sequence in our nuclear DNA has a unique chromosomal
location that defines its position, its locus.

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• An allele means an individual copy of a gene or other DNA sequence that is carried at a
locus on a single chromosome. Because we are diploid, we normally have two alleles at any
one chromosomal locus, one inherited on a chromosome passed down from mother (the
maternal allele) and one inherited from father (the paternal allele).
• The term genotype describes the combination of alleles that a person possesses at a single
locus (or at a number of loci).
• If both alleles are the same at an individual locus, a person is said to be homozygous at
that locus and may be referred to as a homozygote. If the alleles are different, even by a
single nucleotide, the person is said to be heterozygous at that locus, a heterozygote.
• Although we are essentially diploid, men have two types of sex chromosomes, X and Y,
which are very different in both structure and gene content. As a result, most DNA
Hemi sequences on X do not have a direct equivalent (allele) on Y, and vice versa.
r • Men are therefore hemizygous for such loci (because they normally only have one allele).
Women normally have two alleles at each locus on the X chromosome.
• For some genetic characters in humans, a particular genotype at a single locus is the
primary determinant, being both necessary and sufficient for the character to be expressed
under normal circumstances.
• Such characters are often said to be Mendelian, but that implies that a chromosomal locus
is involved; a more accurate term is monogenic (which takes in to account both
•
chromosomal loci and loci on mitochondrial DNA).
WPTby PRIMARILY ONEGENE
Although collectively important, individual single-gene disorders are rare, and common
genetic disorders depend on multiple genetic loci.
• When a human monogenic disorder (or trait) is determined by a nuclear gene, the disorder
(or trait) is said to be dominant if it is manifested in the heterozygote (who carries a normal
allele and a mutant allele), or recessive if it is not.
• Dominant & recessive refer to phenotypic manifestation, not necessarily a single gene. A
disorder inherited as dominant, may involve several contiguous genes (transmitted as a
single locus).
• Sometimes two different phenotypes that result from mutations at a single gene locus can
be simultaneously displayed by the heterozygote and are said to be co-dominant. For
example, the AB blood group is the result of co-dominant expression of the A and B blood
group phenotypes that are determined by different alleles at the ABO blood group locus.
PEDIGREE ANALYSIS
• A pedigree is a graphical representation of a family tree that uses the standard symbols
depicted below:

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• Generations are often labelled with Roman numerals that increase from top to bottom of the
page (toward the youngest generation). Individuals within each generation are given Arabic
numerals that increase from left to right.
• An extended family covering many generations may be described as a kindred. A family
member through whom the family is first ascertained (brought to the attention of health care
professionals) is known as the proband (also called propositus—feminine proposita) and
may be marked with an arrow.
• The term sib (sibling) is used to indicate a brother or sister, and a series of brothers and
sisters is known as a sibship.
• According to the number of steps in the pedigree that links two family members, they may
be classified relatives of the first degree (parent and child; sibs); second degree
(grandparent and grandchild; uncle/aunt and nephew/niece; half-sibs); third degree (first
cousins), and so on.
• Couples who have one or more recent ancestors in common are said to be
consanguineous.
• Pedigrees can often rule out, but not necessarily prove a certain mode of inheritance.
• To construct a pedigree, we obtain info about as many possible members of a family
(minimum 3-4 generations) and then go back chronologically from the proband to the
ancestors.
• We place the eldest generation at the top of the pedigree chart followed by successive
generations using Roman numerals, with each generation on a separate horizontal line.
• We then assign genotypes using the standard symbols and analyse the pedigree for a
specific pattern.
• When analysing a pedigree, some aspects to consider are whether or not the trait skips a
generation, the sex ratio of males:females showing the phenotype, the transmission (male-
male, female-male, etc.) and also if consanguinity is involved.
THE BASICS OF MENDELIAN AND MITOCHONDRIAL DNA INHERITANCE PATTERNS
• Mendelian characters are determined by chromosomal loci, either on an autosome (human
chromosomes 1 to 22) or on a sex chromosome (X or Y).
•

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• Females are diploid for all loci (they have 23 pairs of homologous chromosomes). Males are
different. Like females they have two copies of each autosomal locus and of
pseudoautosomal sequences at the tips of the sex chromosomes.
• However, they are hemizygous for the great majority of loci on the X and the Y (males have
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only one copy of the great majority of loci that are located on the X and the Y but outside
the pseudoautosomal regions). As a result of the above, there are five basic Mendelian
inheritance patterns.
• Autosomal disorders often affect males and females at equal frequencies while X-linked
disorders may appear at different frequencies depending on whether they are dominant or
recessive.
• Females may be homozygous or heterozygous for X-linked disorders depending on X-
chromosome inactivation.
• Recessive disorders are mostly due to loss-of-function mutations of a protein. In these
disorders, one functional allele expresses sufficient (50%) protein for normal physiological
function.
• Dominant disorders manifest despite protein being expressed from a normal allele. In pure
ayyy dominance disorders, the disorder is equally severe in homozygous and heterozygous
individuals while in incomplete dominance disorders the disorder is more severe in
kids • homozygotes.
EESSIVE Mode of inheritance is seldom unambiguous and cannot be completely certain by
RopNot inspecting a single pedigree due to a limited number of children, and the proportion of
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affected individuals not being reliable for recessive conditions, carrier parents may by good
REFÉI.eefortune not have an affected child.
• Only when cloned copy of gene is available, can inheritance be defined with certainty.
AUTOSOMAL DOMINANT INHERITANCE
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• A dominantly inherited disorder is one that is manifested in heterozygotes: affected
persons usually carry one mutant allele and one normal allele at the disease locus.
• In autosomal dominant inheritance, the disease locus is present on an autosome (any
chromosome other than the X or the Y), and so an affected person can be of either sex.
• When an affected person has children with an unaffected person, each child would
normally have a 50% chance of developing the disease (the affected parent can transmit
either the mutant allele or the normal allele).
• Affected persons often have an affected parent. Because the disorders are rare, affected
individuals are almost always heterozygotes. Very occasionally, however, affected
homozygotes are born to parents who are both affected heterozygotes.
• According to the effect of the mutation on the gene product, the affected homozygotes
may show the same phenotype as the affected heterozygote. More commonly, affected
homozygotes have a more severe phenotype than affected heterozygotes.
• The disease phenotype occurs in every generation.
• Sometimes a child is born with severe dominant disease but the family has no prior
history. This can be explained by the disease being due to a new mutation where the
mutant allele arose in either maternal or paternal gamete and the parents are
phenotypically normal.
• The recurrence risk for offspring is low and maintenance of the defective allele in the
population depends on the fitness of the heterozygote.
• If the heterozygote cannot reproduce the disorder can only manifest if the affected
person received a defective allele from the gamete of the normal parent.
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AUTOSOMAL RECESSIVE INHERITANCE
• A person affected by an autosomal recessive disorder can be of either sex and is usually
born to unaffected parents who are heterozygotes (the parents would be described as
asymptomatic carriers because they carry one mutant allele without being affected).
• Affected individuals carry two mutant alleles at the disease locus, one inherited from
each parent. Assuming that both parents of an affected child are phenotypically normal
carriers, the chance that each future child born to these parents is also affected is
normally 25%.
• When an autosomal recessive disorder is quite frequent, carriers will be common. In that
case an affected child may often be born to two parents who carry different mutant
alleles, and the affected individual with two different mutant alleles would be described
as a compound heterozygote.
• A feature of many recessive disorders, especially rare conditions, is that affected
individuals often have two identical mutant alleles because the parents are close
relatives; such couples are said to be consanguineous.
• For rare disorders when there is doubt concerning the mode of inheritance, known
Is parental consanguinity will strongly indicate autosomal recessive inheritance in a

pedigree in which affected individuals have unaffected parents.
SEX CHROMOSOMES
• The X and Y chromosomes are morphologically and functionally different and are known as
a heteromorphic pair.
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• The X chromosome is quite large, approx. 155Mb in size, and is extremely gene rich with
many important genes.
• The majority of the genes are not involved in determining gender but rather encode
housekeeping genes and specialised functions.
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• Females have 2 X chromosomes while males have one, however the quantity of protein
from X-linked genes is equivalent in females and males because of X-inactivation.
• X-linked genes do not act alone but interact with proteins encoded by autosomal genes,
and the ratio of A:X gene dosage is important to have a normal phenotype.
• In contrast, the Y chromosome is small (59 Mb) and gene poor, with a large part of it
consisting of repetitive, non-coding DNA and being heterochromatic and not exhibiting
expression of genes in those regions.
• Many Y-linked genes also show testis-specific expression that is important for normal
spermatogenesis. For example, deletions of the AZF a, b, and c causes azoospermia.
• Despite the differences in the two sex chromosomes, they contain regions of homology
known as pseudo-autosomal regions where they have some homologous gene pairs.
• These PARs are required for X-Y pairing in male meiosis and are important for the correct
segregation of chromosomes. The two sex chromosomes can only exchange sequences
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here during crossing over. MEIOSIS POINT OFXCHANGE
•
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There is a major PAR region on the tips of the p arms of the X and Y chromosomes known
as PAR1 which is a site of obligatory cross over and occurs approximately 5 kb from the
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SRY gene.
• The minor PAR2 is on the tips of the q arms of the two sex chromosomes and cross over in
this region is not necessary or sufficient for meiosis.
• As a result of recombination in male meiosis, the individual X–Y gene pairs in the
pseudoautosomal regions are effectively alleles.
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• An individual allele in these regions can move locations between the X and Y chromosomes
and so is neither X-linked nor Y-linked; instead, the pattern of inheritance resembles
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autosomal inheritance
X-CHROMOSOME INACTIVATION Lot
•
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Having different numbers of chromosomes usually has severe, often lethal
consequences—the loss of just one of our 46 chromosomes is lethal except for 45,X
(Turner syndrome), and having an extra chromosome is usually lethal or results in a
developmental syndrome such as trisomy 21 (Down syndrome).
• This happens because of problems with gene dosage: for some of our genes, the
amount of gene product made must be tightly controlled (having one or three copies of
these genes can be harmful because too little or too much product is made).
• The sex difference regarding the Y chromosome is minimized by the conspicuous lack
of genes on the Y. Most of the very few genes on the Y chromosome have male-specific
functions, or they have an equivalent gene copy on the X (these X–Y gene pairs are
largely concentrated at the tips of the sex chromosomes in the pseudoautosomal
regions).
• To compensate for having different numbers of X chromosomes in males and females, a
special mechanism is needed to silence genes on one of the two X chromosomes in
each female cell (X-inactivation), that is to say, men are constitutionally hemizygous for
most genes on the X chromosome, and women are functionally hemizygous for the
same genes.
OR • The X-inactivation mechanism is initiated after a cellular mechanism counts the number
of X chromosomes in each cell of the early embryo. If the number of X chromosomes is
two (or more), all except one of the multiple X chromosomes is inactivated.
• Each such X chromosome is induced to form a highly condensed chromosome that is
OR mostly transcriptionally inactive, known as a Barr body.
• In humans the initial decision to inactivate one of the two X chromosomes is randomly
made in the preimplantation embryo, beginning at around the eight-cell stage; some
cells inactivate the paternal X and the remainder inactivate the maternal X.
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• Once a cell has chosen which X to inactivate in the early embryo, that pattern of X-
inactivation is continued in all descendant cells.
• Thus, a female who is heterozygous at a disease locus will be a genetic mosaic,
containing cell clones in which the normal allele is expressed and clones in which the
mutant allele is expressed.
• X-inactivation is stable through mitosis but not across the generations. A woman’s
Y't maternal X can equally well have been the active or inactive one in her mother, and has
the same chance as her paternal X of being inactivated in her own cells.
MECHANISM OF X-CHROMOSOME INACTIVATION
•
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Inactivation of a human X chromosome is initiated at an X-inactivation centre (XIC) at Xq13.
The XIC is a minimal region on the C-chromosome that is both necessary and required to
trigger inactivation.
• It then propagates along the whole length of the chromosome in what may be an extreme
example of the tendency of heterochromatin to spread.
• The transient pairing of the two XIC sequences is probably the mechanism by which the X
chromosomes are counted. Within this region, the XIST gene encodes a 17 kb spliced and
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poucomb tTE
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polyadenylated ncRNA, an X-inactivation-specific transcript that is expressed only from the
inactive X chromosome.
• XIST is centrally involved in spreading heterochromatinization outward from the XIC: both
XIST RNA and the Polycomb proteins that it recruits seem to spread along the inactive X to
initiate gene silencing along the length of the chromosome.
• As a result, the inactive X carries modifications typical of heterochromatin (H3K9me2,
H3K9me3, H3K27me3, unmethylated H3K4, deacetylated H4, and frequent replacement of
histone H2A by the macro-H2A histone variant).
•
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In differentiated cells that have already undergone X-inactivation, loss of XIST does not
cause reactivation. That is, XIST is needed to establish X-inactivation but not to maintain it.
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X-LINKED RECESSIVE INHERITANCE
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In X-linked recessive disorders, affected individuals are mostly male, and affected males
are usually born to unaffected parents.
• The mother of an affected male is quite often a carrier (and clearly so if she has affected
male relatives).
• A distinguishing feature is that there is no male-to-male transmission because males
pass a Y chromosome to sons. However, a pedigree may appear to show male-to-male
transmission when an affected man (with a condition such as haemophilia, for example)
and a carrier woman produce an affected son.
• The same parents could each potentially transmit a mutant X to produce an affected
daughter. In X-linked recessive disorders, female carriers with a single mutant allele can
occasionally be quite severely affected and are known as manifesting heterozygotes.
• Because of X-inactivation, female carriers of an X-linked mutation are mosaics: some of
their cells have the normal X chromosome inactivated and other cells have the mutant X
inactivated, as seen most readily in skin disorders.
• Manifesting heterozygotes can occur by chance: most cells of a tissue that is critically
important in disease development have an inactivated X carrying the normal allele.
• Manifesting heterozygotes can occasionally occur because of non-random X-inactivation
that can happen when there is some advantage in inactivating the normal X
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chromosome instead of the mutant X chromosome.
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For example, an X-linked disorder may manifest in a woman who has an X-autosome
translocation in which the breakpoint on the X is the cause of the disorder.
• If the X-autosome translocation chromosome were to be inactivated, neighbouring
autosomal genes would also be silenced, causing gene dosage problems and so the
normal X is preferentially inactivated.
• Skewing of X-inactivation can often work in the other direction: some female carriers are
asymptomatic because of non-random inactivation of the mutant X chromosome.
• A few genes on the X have active counterparts on the Y, notably in the terminal
pseudoautosomal regions.
• X-inactivation is therefore not a blanket inactivation of the entire chromosome, because
no dosage compensation is needed for genes on the X that have functional equivalents
on the Y.
•
BEEF fromdespite
However, unlike in mouse, in which only a small number of genes escape X-inactivation
and are not coated by Xist RNA, about 15% of genes on the human X somehow escape
inactivation.

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iIA
E
SKEWED X-CHROMOSOME INACTIVATION CAUSES DISEASE
• Translocations between X-chromosome and autosomes can cause disease if they disrupt
an important gene on the X-chromosome.
• In cells where the new variant X chromosome (der(X)) is inactivated, there is cell death due
to inactivation of the autosomal region.
• Only cells where the normal X chromosome is inactivated will survive, however, since there
is a breakpoint in the important gene on the X chromosome, no protein is expressed for the
disrupted gene.
• If the break has disrupted an important gene on the X chromosomes, females will have no
active copy of that gene and express an X-linked disease.
X-LINKED DOMINANT INHERITANCE
• Affected individuals with an X-linked dominant disorder can be of either sex, and usually
at least one parent is affected. However, there are significantly more affected females
than affected males, and affected females typically have milder (but more variable)
expression than affected males.
•
9 or
The excess of affected females arises because there is no male-to-male transmission of
the disorder.
•
0 to 8 Y xlinkeddoesn'tmatter
All children born to an affected mother (and an unaffected father) have a 50% chance of
being affected, but an affected father with a single X chromosome will consistently have
unaffected sons (they do not inherit his X chromosome), but his daughters will always be
at risk because they will always inherit his affected X.
• The milder phenotype seen in affected females is a result of X-inactivation—the mutant
allele is located on an inactivated X in a proportion of their cells.
• For certain X-linked dominant disorders, virtually all affected individuals are female: the
phenotype is so severe in males that they die in the prenatal period, but the milder
phenotype of affected females allows them to survive and reproduce.
Y-LINKED INHERITANCE
00 ON it
• The Y-specific region of the Y chromosome is a non-recombining male-specific region.
• In Y-linked inheritance, males only should be affected and there should be exclusive
male-to-male transmission. However, because of the lack of genes, Y-linked disorders
are rare.
• The SRY gene is required for the development of maleness and is passed from father to
son on the Y chromosome.
MITOCHONDRIAL INHERITANCE
REACTIVE 8 8
• The mitochondrial genome is a small (16.5 kb) circular genome that has 37 genes.
É
species • It is much more prone to mutation than nuclear DNA, partly because of its proximity to
reactive oxygen species since the mitochondrion is a major source of reactive oxygen
species in the cell and as a result, mutations in mitochondrial DNA (mtDNA) are a
significant cause of human genetic disease.
In
abig
•
•
Tissues that have a high energy requirement— such as muscle and brain—are primarily
affected in mtDNA disorders.
Individuals with a mitochondrial DNA disorder can be of either sex, but affected males
do not transmit the condition to any of their children because the sperm does contribute
mtDNA to the zygote, but the paternal mtDNA is destroyed in the very early embryo
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(after being tagged by ubiquitin), and a father’s mtDNA sequence variants are not
observed in his children.
• Inheritance occurs exclusively through the mother (matrilineal inheritance). An
additional, common feature of mitochondrial DNA disorders is that the phenotype is
highly variable within families.
HETEROPLASMY
• Each cell contains multiple mitochondria, and there are often several hundred to
thousands of mtDNA copies per cell.
• In some affected persons every mtDNA molecule carries the causative mutation
(homoplasmy), but affected individuals frequently have cells with a mixed population of
normal and mutant mtDNAs (heteroplasmy).
• The clinical features depend mostly on the proportion of mutant to normal mtDNA
molecules in the cells of tissues with high energy requirements.
• Although a human egg cell is haploid for nuclear DNA, it contains more than 100,000
mtDNA molecules. A heteroplasmic mother can give rise to children who differ widely
from her and from each other in the ratio of mutant to normal mtDNA molecules in their
tissues (variable heteroplasmy).
• As a result, there can be very significant clinical variability between affected members of
the same family.
r Variability
some mm mm
THOUSANDS
f NB
inPeexpression
All W MM
homoplasmy

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ORIGINS OF HUMAN GENETIC VARIABILITY

• Genetic variation describes naturally occurring genetic differences among individuals of the
same species. Underlying genetic variation are changes in DNA sequences.
• Mutation describes both a process that produces altered DNA sequences (either a change
in the base sequence or in the number of copies of a specific DNA sequence) and the
outcome of that change (the altered DNA sequence).
• Inherited genetic variation occurs as a result of germline cells and exists between and
within individuals while post zygotic or somatic genetic variation occurs throughout life,
and mostly occurs randomly with minimal consequence.
• Individuals differ from each other mostly because our DNA sequences differ, but genetic
variation is not the only explanation for differences in phenotype (our observable
characteristics).
• A fertilized egg cell can split in two in early development and give rise to genetically
identical twins (monozygotic twins) that nevertheless grow up to be different: although
hugely important, genetic variation is not the only influence on phenotype.
• During development, additional effects on the phenotype occur by a combination of
stochastic (random) factors, differential gene– environment interactions and epigenetic
variation that is not attributable to changes in base sequence.
• The major sources of variation are mutations and chromosome recombination.
• Allele frequencies can be affected by a variety of factors including mating patterns
(inbreeding/outbreeding), genetic drift, distribution (preserves genetic variation) and
migration.
• Changes in relative allele frequencies at the population level can influence genetic variation
over successive generations. Fs
GENETIC VARIATION BECAUSE OF MUTATIONS s A 3
• Natural errors in various processes that affect chromosome and DNA function—
chromosome segregation, recombination, and DNA replication—are important contributors
to genetic variation.
• Endogenous errors in the above processes may often not have harmful consequences, but
some of them make important contributions to disease.
• Each time the DNA of a human diploid cell replicates, 6 billion nucleotides need to be
inserted in the correct order to make new DNA molecules.
• Not surprisingly, DNA polymerases very occasionally insert the wrong nucleotide, resulting
in mispaired bases (a base mismatch).
• In the great majority of cases, the errors are quickly corrected by the DNA polymerase itself.
The major DNA polymerases engaged in replicating our DNA have an intrinsic 3ʹ→5ʹ
exonuclease activity with a proofreading function. If, by error, the wrong base is inserted,
the 3ʹ→5ʹ exonuclease is activated and degrades the newly synthesized DNA strand from its
3ʹ end, removing the wrongly inserted nucleotide and a short stretch before it, then the DNA
polymerase resumes synthesis again.
• If mispaired bases are not eliminated by the DNA polymerase, a DNA mismatch repair
system is activated.
• Another type of DNA replication error commonly occurs within regions of DNA where there
are short tandem oligonucleotide repeats.

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• If, for example, the DNA polymerase encounters a 30-nucleotide sequence with 15
sequential repeats of AT dinucleotide there will be an increased chance that during DNA
replication a mistake is made in aligning the growing DNA strand with its template strand.
• A frequent result is that the template strand and newly synthesized strand pair up out of
register by one (or sometimes more) repeat units, causing replication slippage. Errors like
this are also often repaired successfully by the DNA mismatch repair system.
• Although the vast majority of DNA changes caused by DNA replication errors are identified
and corrected, some persist. That happens because although we have many very effective
DNA repair pathways, DNA repair is also not 100% effective: unrepaired changes in DNA
sequence are an important source of mutations.
• Chemical damage to DNA can involve the cleavage of covalent bonds in the sugar–
phosphate backbone of DNA, causing single-strand or double-strand breaks.
• Alternatively, bases are deleted (by cleavage of the N-glycosidic bond connecting a base to
a sugar), or they are chemically modified in some way.
• Many base modifications involve replacing certain groups on bases or adding a chemical
group: a methyl or larger alkyl group, or some other large chemical. Sometimes base
modification involves the formation of covalent bonds between two bases (crosslinking); the
bases may be on the same strand or on complementary DNA strands.
• Chemically modified bases may block DNA or RNA polymerases, and cause base
mispairing; if not repaired, they may induce mutations.
• Most of the chemical damage to our DNA arises spontaneously and is unavoidable. Three
major types of chemical change occur:
1) Hydrolytic damage. Hydrolysis is inevitable in the aqueous environment of cells. It can
disrupt bonds that hold bases to sugars, cleaving the base from the sugar to produce an
abasic site—loss of purine bases (depurination) is particularly common. Hydrolysis also
strips amino groups from some bases (deamination), leaving a carbonyl (C=O) group.
Cytosines are often deaminated to give uracil, which base pairs with adenine. Adenine is
occasionally deaminated to produce hypoxanthine, which effectively behaves like
guanine by base pairing with cytosine.
2) Oxidative damage. Normal cellular metabolism generates some strongly electrophilic
(and therefore highly reactive) molecules or ions. The most significant are reactive
oxygen species (ROS) formed by the incomplete one-electron reduction of oxygen,
including superoxide anions (O2–), hydrogen peroxide (H2O2), and hydroxyl radicals
(OH•). ROS are generated in different cellular locations and have important roles in
certain intercellular and intracellular signalling pathways, but they mostly originate in
mitochondria (where electrons can prematurely reduce oxygen). Endogenous ROS
attack covalent bonds in sugars, causing DNA strands to break. They also attack DNA
bases, especially purines and many derivatives are produced from each base. Some of
the base derivatives are highly mutagenic, such as 7,8-dihydro-8-oxoguanine which
base pairs with adenine; others are not mutagenic but nevertheless block DNA and RNA
polymerases.
3) Aberrant DNA methylation. Many cytosines in our DNA are methylated by
methyltransferases. Cells also use S-adenosylmethionine (SAM) as a methyl donor in a
non-enzymatic reaction to methylate different types of molecules, but sometimes SAM
can inappropriately methylate DNA to produce harmful bases. Some of these bases
distort the double helix, disrupting crucial DNA–protein interactions.

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• A minority of the chemical damage to our DNA is caused by external agents that can induce
mutation (mutagens), including radiation and harmful chemicals in the environment. Ionizing
radiation (such as X-rays and gamma rays, and so on) interacts with cellular molecules to
generate ROS that break chemical bonds in the sugar–phosphate backbone, breaking DNA
strands. Non-ionizing ultraviolet radiation causes covalent bonding between adjacent
pyrimidines on a DNA strand (pyrimidine dimers).
• As a person ages, some sequences of their DNA remain the same however, other
experience mutation and DNA damage and so it is unlikely to find a perfect copy of the
entire original genome they had as a new born.
• Cells with a high turnover rate such as the intestinal epithelium and skin cells usually have
high mutation rates as compared to other cells since they are constantly exposed to
mutagens on alcohol, food, and sun rays.
THE SCALEOF HUMAN GENETIC VARIATION

• Human genetic variation occurs by changes to our base sequence that can be classified
into two categories.
• First, some changes to our DNA sequence do not affect the DNA content (that is, the
number of nucleotides is unchanged).
• Quite often a single nucleotide is substituted for a different nucleotide. More rarely, multiple
nucleotides at a time may be sent to a new location without net loss or gain of DNA content:
these balanced translocations and inversions that result in chromosome breakage without
net loss or gain of DNA may sometimes have no effect on the phenotype or be harmful.
• A second class of DNA change causes a net loss or gain of DNA sequence: there is a
change in copy number of a DNA sequence that can be large or small.
• The largest changes in copy number result from abnormal chromosome segregation,
producing fewer or more chromosomes than normal, and therefore a change in the copy
number of whole nuclear DNA molecules.
• They are almost always harmful: many result in spontaneous abortion and some give rise to
developmental syndromes.
• Sometimes the change in copy number can mean the deletion or insertion of a single
nucleotide. In between are copy number changes that range from altered numbers of
specific short oligonucleotide sequences to megabase lengths of DNA.
• The most common DNA changes are on a small scale and involve only a single nucleotide
or a very small number of nucleotides, called point mutations.
• They often have no obvious effect on the phenotype; in that case they would be considered
to be neutral mutations. That happens because 90% or so of our DNA is poorly conserved
and of questionable value and seems to tolerate small changes in DNA sequence without
obvious effect.
NUCLEOTIDE DIVERSITY
• This is a concept of molecular genetics which is used to measure the degree of genetic
variation or mutation within a population.
• Mutations are random and can occur anywhere in the genome however there is
considerable variation in the amount of mutation observed in different regions of the
genome.

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• The protein coding fraction of the genome has the smallest number of variants per kilobase
(kb) because these changes would compromise protein structure and function of important
proteins for development and function of the organism.
• Intergenic regions and repeat fractions of the genome experience almost double the
number of mutations of protein coding regions.
• These differences arise because different regions of DNA evolve at different rates. The
protein coding regions evolve at a relatively slow rate while in intergenic regions and repeat
fractions of the genome mostly neutral mutations occur almost always and at a much higher
rate.
• Mutations are defined as permanent and transmissible changes in DNA sequences resulting
from failure of DNA repair.
• These often occur during replication where DNA undergoes frequent chemical changes that
usually undergo repair but some go unrepaired and result in mutation.
• Mutations are classified based on where they occur (somatic/germline) as well as the
length of the nucleotide sequence they affect (gene level/chromosomal).
• Gene level mutations often occur as point mutations which can be silent (do not alter the
encoded amino acid), missense (change the codon to one that codes for a different amino
acid, which can be harmful or neutral), and nonsense (changes a codon encoding an amino
acid to one encoding a stop codon which will result in production of a truncated/shortened
protein).
• Frameshift mutations also occur by insertion or deletion of bases which changes the
reading frame of the nucleotide sequence. This may produce a stop codon downstream
which will produce a truncated protein.
• Chromosomal mutations alter longer stretched of DNA ranging from multiple genes to entire
chromosomes.
• Missense mutations can also be classified as being conservative (replacing it with a similar
one) or nonconservative (replacing it with a completely different one).
• A nucleotide substitution that replaces one amino acid by another of the same chemical
class is a conservative substitution and often has minimal consequences for how the protein
functions.
POLYMORPHISM
• For any locus, if more than one DNA variant is common in the population (above a
frequency of 0.01), the DNA variation is described as a polymorphism.
• Alleles with frequencies of less than 1% are by convention called “rare variants”.
• The most common type of genetic variation in the human genome is due to single
nucleotide substitutions, where for example, one variant may have a G at a defined position
and another variant a C.
• This type of variation produces single nucleotide variants (SNVs). If two or more alternative
DNA variants of this type exceed a frequency of 0.01 in the population, the DNA variation is
commonly described as single nucleotide polymorphism or SNP.
• The pattern of SNV in the human genome is non-random for two reasons: first, there is a
mechanistic reason: different DNA regions and different DNA sequences can undergo
different mutation rates. For example, mitochondrial DNA has a much higher mutation rate
than nuclear DNA.
• A second reason for non-random variation comes from our evolutionary ancestry. In
general, the nucleotides found at SNP sites are not particularly susceptible to mutation (the

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germ-line single nucleotide mutation rate is about 1.1 × 10–8 per generation, roughly 1
nucleotide per 100 Mb, and SNPs are stable over evolutionary time).
• Instead, the alternative nucleotides at SNP sites mark alternative ancestral chromosome
segments that are common in the present-day population.
• Using SNPs to define ancestral chromosome segments is important in mapping genetic
determinants of disease.
• SNPs are found in coding and most commonly non-coding regions and occur with very high
frequency (about 1 in 1000 bases to 1 in 100-300 bases).
• The abundance of SNPs and the ease with which they can be measured make these
genetic variations significant as SNPs close to a gene act as a marker to that gene.
• Some point mutations create DNA variants that differ by the presence or absence of a
single nucleotide, or by a relatively small number (2-100) of nucleotides at a specific
position. This is an example of insertion/deletion variation or polymorphism (indel).
• These are often simple polymorphisms that have only two alleles, so the indel is either
present or absent.
• A subset of SNPs or indels leads to the gain or loss of a restriction site, in which case
cutting the DNA with the relevant restriction nuclease can generate restriction fragment
length polymorphism.
• Repetitive DNA accounts for a large fraction of the human genome. Tandem copies of quite
short DNA repeats (1 bp to fewer than 200 bp) are common, and those with multiple
tandem repeats are especially prone to DNA variation. A continuous sequence of multiple
tandem repeats is known as an array.
• The repeated sequences are classified as belonging to three classes, according to the total
length of the array and genomic location: •
1) satellite DNA (array length: often from 20 kb to many hundreds of kilobases; located at
centromeres and some other heterochromatic regions)
2) minisatellite DNA (array length: 100 bp to 20 kb; found primarily at telomeres and sub
telomeric locations)
3) microsatellite DNA (array length: fewer than 100 bp long; widely distributed in
euchromatin).
• The instability of tandemly repeated DNA sequences results in DNA variants that differ in
the numbers of tandem repeats (variable number of tandem repeats polymorphism, or
VNTR polymorphism).
• Because microsatellite DNA arrays (usually called microsatellites) are frequently
distributed within human euchromatin (roughly once every 30 kb) and are often highly
polymorphic, they have been widely used in genetic mapping.
• Microsatellites have very short repeats (one to four base pairs long), so microsatellite
polymorphisms are sometimes also known as short tandem repeat polymorphisms
(STRPs).
• Unlike SNPs (which almost always have just two alleles), microsatellite polymorphisms
usually have multiple alleles.
• The variation in copy number arises as a result of replication slippage: during DNA
replication, the nascent (newly synthesized) DNA strand slips relative to the template strand
so that the two strands are slightly out of alignment.
• Individual microsatellite polymorphisms can be typed by first using PCR to amplify a short
sequence containing the array, and then separating the PCR products according to size by
polyacrylamide gel electrophoresis.
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• They have been used extensively in both family studies and DNA profiling (to establish
identity for legal and forensic purposes). However, unlike SNP assays, it is not easy to
automate assays of micro-satellite polymorphisms.
HOW GENETIC VARIATION RESULTS IN DISEASE

• Pathogenic mutations do not occur haphazardly in our DNA. For example, single nucleotide
substitutions, the most common type of pathogenic mutation, are not random: certain types
of DNA sequence are more vulnerable to point mutation (mutation hotspots).
• Two mutational hotspots in our genome are CpG nucleotides/CG-rich regions due to the
spontaneous hydrolysis of cytosine, as well as microsatellites due to the way they are prone
to DNA slippage and indel formation.
• The genetic variation that causes disease may do so in two broad ways. First, it may cause
a change in the sequence of the gene product. The result may be a loss of function: the
mutant gene product may simply be incapable of carrying out its normal task (or may have
a significantly reduced ability to work normally; a hypomorph).
• Alternatively, it may acquire an altered function (or occasionally a new function; a
neomorph) that is harmful in some way (causing cells to die or to behave inappropriately).
• Loss of function and gain of function quite often involve a change in protein structure.
• A second broad way in which mutations cause disease is by changing the amount of gene
product made.
• Some small-scale pathogenic mutations in protein-coding genes result in unstable mRNAs
and failure to make the normal gene product. Other mutations can result in altered gene
copy number or adversely affect regulatory sequences controlling gene expression so that
too little or too much product is made.
• That is a problem for a subset of our genes whose expression must be closely controlled,
keeping quite tight limits on the amount of gene product made.
• Although most pathogenic mutations affect individual genes, some mutations (and
chromosome abnormalities) can simultaneously affect multiple genes.
• Large-scale deletions and duplications, for example, result in a simultaneous change in the
copy number of multiple genes, with adverse effects. Additionally, a mutation in a regulatory
gene can indirectly have consequences for how the many different target genes controlled
by that gene are expressed.
NONSENSE-MEDIATED DECAY
• Various RNA surveillance mechanisms monitor RNA integrity, checking for splicing
accidents and occasional errors in base incorporation during transcription.
• These errors frequently give rise to in-frame premature termination codons (PTCs) that
could be dangerous—the aberrant transcripts could give rise to truncated proteins that
might have the potential to interfere with the function of normal proteins.
• To protect cells, an mRNA surveillance mechanism known as nonsense-mediated decay
(NMD) degrades most mRNA transcripts that have an in-frame PTC.
• The primary NMD pathway is dependent on RNA splicing (single exon genes escape NMD
because they do not undergo RNA splicing).
• Multi-subunit protein complexes, called exon-junction complexes, are deposited shortly
before the 3ʹ end of each transcribed exon during RNA splicing and remain bound at
positions close to the exon–exon boundaries in mature RNA.
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• The first ribosome to bind and move along the mRNA displaces each of these complexes in
turn before disengaging from the mRNA at the natural stop codon.
• If there is an in-frame PTC, however, the ribosome detaches from the mRNA at an early
stage; some exon junction complexes remain bound to the RNA, which usually signals
mRNA destruction.
• However, in-frame PTCs within or just before the last exon often escape NMD and are
translated to give truncated proteins. Nonsense mutations, frameshifting insertions and
deletions, and certain splicing mutations (such as those that result in retention of an intron)
ETYIyi.gg
can activate nonsense-mediated decay. am
POINT MUTATIONS WITHIN SPLICE SITES
• Pathogenic mutations can also occur in additional cis-acting splice regulatory elements,
including splice enhancer and splice silencer sequences in exons and introns.
• Mutations like this may be less readily identified as pathogenic mutations, and can explain
why some synonymous substitutions are pathogenic.
• By chance, sometimes a sequence may be almost identical to a genuine splice donor or
splice acceptor site (a latent or cryptic splice site) and changing a single nucleotide can
cause it to become a novel splice site.
• Activation of cryptic splice sites produces truncated or extended exons. Abnormal splicing
can have variable consequences.
• For a protein-coding gene, a loss of coding exon sequences (by exon skipping or exon
truncation) or a gain of coding exon sequences (by exon extension or intron retention) may
result in a frameshift in the translational reading frame at the RNA level.
• In that case, the introduction of a premature termination codon might induce RNA
degradation or produce a truncated protein.
• If there is no shift in the translational reading frame, pathogenesis may occur because of a
loss of key amino acids, or, for exon extension, by the inclusion of extra amino acids that
might destabilize a protein or impede its function.
• Intron retention in coding sequences would be expected to introduce a nonsense mutation
(because of the comparatively high frequency of termination codons in all three reading
frames).
EFFECT OF PARENTAL AGE AND SEX ON GERM-LINE MUTATION RATES
• Increased parental age often correlates with increased frequency of genetic disorders.
• For small-scale mutations there is often a higher frequency of de novo mutation in the male
germ line, and paternal age effects can be apparent.
• The frequency of de novo mutation can be expected to be high in gametes that have
undergone many cell divisions since originating from the zygote through a primordial germ
cell (the cells that are set aside in the early embryo to give rise to the germ line).
• That happens because DNA replication precedes each cell division, and mutations often
arise as a result of uncorrected errors in DNA replication.
• Two meiotic cell divisions are required to form oocytes and sperm cells, but the number of
preceding mitotic cell divisions required to produce the first meiotic cells is very different
between the two sexes.
• All the egg cells that will be available to a woman are formed before birth. By contrast, after
the onset of puberty in men, sperm are continuously being formed by the division of
spermatogonial stem cells.

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10
• The number of cell divisions required to produce gametes is therefore higher in men, and
especially so in older fathers.
• A small group of exceptional congenital disorders occur spontaneously at remarkably high
apparent rates, reaching 1 in 30,000 births for achondroplasia, with marked paternal age
effects and paternal germline transmission.
PATHOGENIC EXPANSION OF STRs To STRSTRSTR
• This is a novel mechanism of human gene mutations consisting of unstable, dynamic
mutations.
• There are over 20 repeat expansion diseases caused by expansion of short non-coding
tandem repeats or short tandem repeats in coding DNA.
• Unlike tandem mononucleotide or dinucleotide repeats, changes in the number of
trinucleotide repeats do not cause a translational frameshift, and small numbers of tandem
trinucleotide repeats may be tolerated and are translated to produce proteins with the
repetition of a single amino acid.
• Sometimes quite long runs of the same amino acid signify a functional domain.
• Some of the tandem repeat arrays are polymorphic; others are not. In the former case the
repeats may sometimes cause disease when the array expands in size above a critical
number of repeats.
• Different types of short tandem noncoding repeats can also undergo unstable expansion in
specific regions of the genome.
• Several are found in the promoter regions, 5’ and 3’ untranslated sequences, or introns of
genes; they are often composed of repeats with from three to six nucleotides and can result
in disease when expanded. In some other cases the sequences are in gene-poor regions
and do not cause disease.
• Trinucleotide repeats are polymorphic in the wild type. The normal range of repeats is 5-50,
the intermediate state (premutation) is 50-200, and the disease causing range is 200
upwards. These disease causing versions result in a loss-of-function effect.
• After a modest expansion in the normal number of repeats, a normal allele can change to a
premutation in which the expanded array of repeats is unstable but usually not big enough
to cause the disease associated with very large-scale expansion.
• Large-scale expansions of tandem oligonucleotide repeats underlie a proportion of
chromosome abnormalities known as fragile sites, specific regions of the genome that
appear as constrictions or gaps on the metaphase chromosomes prepared from cells
treated with certain chemicals to partly inhibit DNA replication.
• Fragile sites can be classified as common (present in virtually all individuals and thought to
be an intrinsic part of chromosome structure) or rare (occurring in a small percentage of
individuals—this category includes fragile sites resulting from a large expansion of tandem
noncoding repeats).
• Individuals with very high repeat numbers are more severely affected and develop
symptoms at an earlier age than those individuals who have a number of repeats that falls at
the lower end of the disease range.
• Because the mutation is dynamic, transmission down successive generations in a family can
lead to mutant alleles with an increasing repeat number and increasing disease severity in
successive generations (which has been referred to as anticipation).

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FRAGILE X-
SYNDROME
• Normal individuals have between 5-54 CGG repeats in the 5’UTR of the FMR1 gene.
• Alleles with from 55 to close to 200 CGG repeats in the 5ʹ UTR of the FMR1 gene are
premutations for fragile X syndrome (they do not cause this disease but are unstable and
can quickly expand from one generation to the next to reach the critical number of more
than 200 repeats associated with fragile X syndrome).
• Although fragile sites are often located in gene-poor regions, the rare fragile sites include
three examples—FRAXA (Xq27.3), FRAXE (Xq28), and FRA11B (11q23.3)—that are located
within or close to a gene and disturb its expression, causing disease.
• The fragile site involved is FRAXA at Xq27 and the pathogenesis results from loss of
function.
• The disease is more severe in males than females (recall males are hemizygous, and X-
inactivation needs to be considered).
• The FMR1 gene makes a regulatory RNA binding protein that is important for proper
synapse development and function (it works by binding certain target mRNAs, inhibiting
translation).
• Expansion to more than 200 and up to several thousand CGG repeats is known as a full
mutation. Individuals with this type of expansion reveal a fragile site (FRAXA) at Xq27 and
develop the fragile X syndrome.
• The most conspicuous feature is intellectual disability (often profound in affected males, but
mild in affected females); behavioural abnormalities are also common.
• The pathogenesis in fragile X syndrome results from loss of function—the expanded CGG
repeats are targets for cytosine methylation, resulting in hypermethylation of the region and
aberrant methylation in the upstream regulatory region, causing epigenetic silencing of
FMR1.
• Point mutations also occasionally cause fragile X syndrome by inactivating FMR1 with loss
of protein function. Alleles with 55–200 CGG repeats are premutation alleles—they do not
cause the classical fragile X syndrome, but they are unstable and can become full mutations
when transmitted to the next generation.
• Although carriers of a premutation allele do not have fragile X syndrome, they have been
proposed to be at risk of different diseases not obviously related to fragile X syndrome.
• A premutation from mother to child with between 70-90 CGG repeats expands to a full
mutation in one generation whereas the same from the father has almost no chance of
expanding to a full mutation.
• Repeats increase when transmitted by mother.
MYOTONIC DYSTROPHY TYPE 1
• Myotonic dystrophy is a multisystem disorder that notably includes progressive muscle

deterioration and myotonia (an inability to relax muscles after contraction).
• It affects the protein kinase encoded by the DMPK gene on chromosome 19.
• In type 1 the CTG in is affected and would produce very different polypeptide sequences.
The normal phenotype results when there are between 5-37 copies of this repeat.
• Animal studies suggest that the pathogenesis is exclusively due to an expression product
and is quite unrelated to the gene containing the expanded repeat (as shown by
constructing a mouse model of the disease simply by inserting an expanded CTG repeat
into a host actin gene).

CTGCTG
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COPYRIGHTS RESERVED SRP
BIBBEE
• Instead of being transported to the cytoplasm, the mutant RNAs accumulate within the
nucleus. The expanded repeats within the RNAs form very large hairpins that bind certain
RNA-binding proteins, and the mutant RNAs form small aggregates that are seen to be
discrete foci (or inclusions).
• In particular, proteins with a preference for binding CUG motifs are selectively bound,
notably MBNL1 and other members of the muscleblind family of splicing regulators.
• Because they bind in large quantities to the expanded CUG repeats, MBNL1 and related
proteins are effectively sequestered within the mutant RNA foci—they cannot get close to
their normal target mRNAs and so lose their ability to function normally.
• An additional effect is that the mutant RNAs or nuclear aggregates seem to induce the
increased production of another protein that binds to CUG repeats called CUGBP1 (CUG-
binding protein 1).
• Like MNBL1, CUGBP1 is a regulator of alternative splicing. The combined effect of reducing
the availability of the muscleblind family of splicing regulators and up regulating the
CUGBP1 splicing regulator causes misregulation of alternative splicing of multiple target
mRNAs including some key muscle proteins and various other proteins, causing disease.
• The phenotype can vary between affected individuals in families, but in a directional way.
DISEASES ASSOCIATED WITH REPEAT EXPANSION IN CODING REGION
• Many of our genes have arrays of tandem trinucleotide repeats in coding sequences.
• Unlike tandem mononucleotide or dinucleotide repeats, changes in the number of
trinucleotide repeats do not cause a translational frameshift, and small numbers of tandem
trinucleotide repeats may be tolerated and are translated to produce proteins with the
repetition of a single amino acid.
• Sometimes quite long runs of the same amino acid signify a functional domain, as in the
case of polyglutamine and polyalanine.
• Some of the tandem repeat arrays are polymorphic; others are not. In the case where they
are polymorphic, the repeats may sometimes cause disease when the array expands in size
above a critical number of repeats.
PATHOGENIC POLYALANINE EXPANSION
• Polyalanine regions are found in a small number of developmentally important transcription

factors, where they serve as a flexible nonpolar linker or spacer between two folded
domains.
• Individual polyalanine regions are often encoded by different alanine codons (notably GCG,
GCT, or GCA).
• They are generally not polymorphic, and a very modest increase in the number of alanine
codons can cause disease. These expansions DO NOT exhibit anticipation.
• Nine different polyalanine expansion diseases are known, all congenital disorders of
development except for a form of muscular dystrophy.
• According to the type of protein, the non-polymorphic polyalanine tract is normally 10–20
alanine residues long; pathogenesis occurs when the array is expanded by about a further
4–10 alanine residues on average.
• The congenital polyalanine malformations are thought to occur by loss of the transcription
factor function (expansion of the polyalanine array may induce aberrant protein folding or
interfere with the correct localization of the protein within the cell).

NOT FOR RESALE
10 MIN
UNSTABLE EXPANSION OF CAG REPEATS ENCODING POLYGLUTAMINE
• Unlike polyalanine, polyglutamine (often called poly(Q)) is highly polar. Moderately long
poly(Q) tracts are found in several proteins and are often involved in regulating the
transcription of certain target genes.
• Unlike polyalanine, poly(Q) arrays are typically specified by just one codon (CAG); they
show significant length polymorphism (possibly as a result of replication slippage).
• After a certain critical length has been reached, poly(Q) arrays can expand to very
significant lengths. The normal range is 10-35 and the disease causing range is 50-100.
CAG expansions are larger when transmitted through the father.
• At the DNA level, the expanded alleles are meiotically and somatically unstable (unlike
alleles encoding polyalanine expansions). That is, after a polyglutamine-coding sequence
has reached a critical length, the extended array of repeats tends to expand further and
faster than the smaller arrays.
• Unstable expansion of CAG repeats in coding sequences resembles the unstable expansion
of various types of short oligonucleotide repeat in noncoding sequences.
• They are sometimes described as dynamic mutations because the repeat length can
increase from one generation to the next (and sometimes from mother cell to daughter cell
in one individual).
• Increasing expansion of the repeats leads to increasing severity of the disease.
• Four major types of late-onset neurodegenerative disorder result from unstable CAG repeat
expansion producing an extended polyglutamine tract and in each case a build-up of toxic
products produced by the mutant allele is thought to underlie the disease.
• The effect of toxic intracellular products is particularly marked in neurons (which are
generally intended to be very long-lived and not replaced when lost).
• Depending on the disease, harmful consequences are apparent in different neural
components. Motor neurons in the brainstem and spinal cord are principally affected in
spinal bulbar muscular atrophy, for example, and neurons in different regions of the
cerebellum are affected in the different forms of spinocerebellar ataxia.
• The mutations have a gain of function effect and cause protein misfolding which then results
in aggregates.
MISPAIRED TANDEM REPEATS
• Many human genes and gene regions have significant arrays of long tandemly repeated
DNA sequences. This can include the repetition of exons, whole genes, and even multiple
genes (see Figure 2.12 on page 45 for examples). Tandem repeats within genes or
spanning coding sequences can predispose to a type of non-allelic homologous
recombination that can cause disease. Normally, recombination between homologous
chromosomes occurs after the chromosomes have paired up with their DNA sequences in
perfect alignment. However, local misalignment of the paired chromosomes is more likely to
occur in regions where there are highly similar tandem repeats—the DNA molecules of the
two chromatids can line up out of register. That is, the alignment is staggered and one or
more repeats on each chromatid do not pair up with their normal partner repeat on the
other chromatid. A subsequent recombination within the mismatched sequences is known
as unequal crossover (UEC) and results in one chromatid with an insertion (more tandem
repeats) and one with a deletion (fewer tandem repeats) An equivalent process can also
occur between sister chromatids, an unequal sister chromatid exchange (UESCE) (Figure
7.7). UEC and UESCE cause reciprocal exchanges between misaligned chromatids: one
NOT FOR RESALE
10 MEN
chromatid gains an extra DNA sequence, and the other loses an equivalent sequence.
Disease may result from a change in gene copy number, or through the formation of hybrid
genes that lack some of the functional gene sequence.
• Many human genes and gene regions have significant arrays of long tandemly repeated
DNA sequences. This can include the repetition of exons, whole genes, and even multiple
genes. Tandem repeats within genes or spanning coding sequences can predispose to a
type of non-allelic homologous recombination that can cause disease.
• Normally, recombination between homologous chromosomes occurs after the
chromosomes have paired up with their DNA sequences in perfect alignment.
• However, local misalignment of the paired chromosomes is more likely to occur in regions
where there are highly similar tandem repeats—the DNA molecules of the two chromatids
can line up out of register.
• That is, the alignment is staggered and one or more repeats on each chromatid do not pair
up with their normal partner repeat on the other chromatid.
• A subsequent recombination within the mismatched sequences is known as unequal
crossover (UEC) and results in one chromatid with an insertion (more tandem repeats) and
one with a deletion (fewer tandem repeats).
• An equivalent process can also occur between sister chromatids, an unequal sister
chromatid exchange (UESCE).
• UEC and UESCE cause reciprocal exchanges between misaligned chromatids: one
chromatid gains an extra DNA sequence, and the other loses an equivalent sequence.
• Disease may result from a change in gene copy number, or through the formation of hybrid
genes that lack some of the functional gene sequence.
• Just as with mispairing of tandem repeats, distantly spaced direct repeats can mispair when
chromatids are aligned within homologous chromosomes.
• Subsequent crossover at mismatched direct repeats results in deletions or duplications of
the intervening sequence.
• An equivalent type of exchange can occur between mispaired short direct repeats on the
same DNA strand (a form of intrachromatid recombination), and this can also lead to
deletion.
• Inverted repeats on a single chromatid can also mispair by looping out the intervening
sequence.
• Subsequent recombination at the mispaired sequences will produce an inversion of the
intervening sequence. An inversion like this can cause disease, for example by relocating
part of a gene, by disrupting the gene, or by separating a gene from important cis-acting
control elements.
• An instructive example is provided by haemophilia A: in about 50% of cases the cause is a
large inversion that disrupts the F8 gene, which makes blood clotting factor VIII.
i

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NOT FOR RESALE

THE HUMAN KARYOTYPE

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prometaphase chromosome preparations are chemically stained to reveal a pattern of

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CHROMOSOME BANDING NOMENCLATURE

• Human chromosome nomenclature is decided periodically by the International Standing

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• The essence of chromosome FISH is to fix chromosome preparations on microscopic

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Is parental consanguinity will strongly indicate autosomal recessive inheritance in a

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ORIGINS OF HUMAN GENETIC VARIABILITY

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THE SCALEOF HUMAN GENETIC VARIATION

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HOW GENETIC VARIATION RESULTS IN DISEASE

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• Myotonic dystrophy is a multisystem disorder that notably includes progressive muscle

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• Polyalanine regions are found in a small number of developmentally important transcription

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