Genetics - Ophthalmology

The structure of chromosomes
• Chromosomes: are coiled up strands of DNA
- Each metaphase chromosome is composed of two sister chromatids connected
by a centromere
- Centromeres are responsible for movement of chromosomes at cell division
- The ends of chromosomes are protected by telomeres, which are tandem
repeats of a hexameric DNA sequence, ending in a 3 ′ - single-stranded
overhanging sequence of 50–400 nucleotides, which loops back on itself to
form the T-loop. Telomeres prevent abnormal fusion between chromosomes,
protect the ends of chromosomes from degradation, ensure complete DNA
replication, and have a role in chromosome pairing during meiosis.

Definitions
- Locus: location of a specific gene on a chromosome
- Allele: One of the alternative versions of a gene at a given location (locus)
along a chromosome
An individual inherits two alleles for each gene, one from each parent.
If the two alleles are the same, the individual is homozygous for that gene.
If the alleles are different, the individual is heterozygous.

• Chromatin: complex of DNA + Histone ocatmers + acidic non-histone
proteins
• Nucleosomes : is the basic structural unit of chromatin. Composed of :
Segments of the DNA double helix wrap around histone octamers. These histone
cores are linked by spacer segments of DNA
• Solenoid: Long strings of nucleosomes are compacted into a helical structure,
each turn of the solenoid containing about six nucleosomes.
The solenoids themselves are arranged into loops attached to a central scaffold.
Octamer of histone protein, contains a high proportion of positively charged
amino-acid residues, can form ionic bonds with the negatively charged DNA

• Nucleotides: molecules, that when combined, form the structural units of DNA
and RNA. [RNA and DNA are polymers are created by long chains of nucleotides]
Composition of nucleotide:
Sugar molecule that is attached to a phosphate group and a nitrogen-containing base.
Linkage:
- 3'-5' phosphodiester bonds: link adjacent nucleotides in a polynucleotide chain
- Hydrogen bonds link pairs of bases

Nucleotide
The nitrogenous bases are:
Purines: Adenine, Guanine
Pyrimidines: Thymine or Cytosine

DNA
Only 3% of nuclear DNA codes for protein*

Less than 10% of the DNA in the genome encodes for genes.
There are 3 classes of DNA in the human genome:
1. Single copy DNA 75%: in which the nucleotide sequence is represented only
once per haploid genome
2. Dispersed repetitive DNA families 15%: repetitive sequences that are
dispersed throughout the genome, there are two major families in this group:
- Alu family
- L1 family
3. Satellite DNA: repeat sequences tend to be arranged in tandem, in a head to
tail fashion, and are often found in specific sites such as centromeres or
telomeres.

Genes are made up of DNA. Each chromosome contains many genes.

Chromosomes in human
Human cells contain 46 chromosomes (diploid)
22 pairs of autosomal chromosomes (numbered 1 to 22 in order of decreasing size) + one
pair of sex chromosome (XX or XY)

Stages of gene expression
1. Transcription: RNA polymerase initiates transcription from the "non-coding"
or "antisense" strand of the DNA.
Synthesis of the primary RNA transcript (which includes introns) proceeds in a
5'-3' direction beyond the position on the chromosome that corresponds to the 3'
end of the mature RNA.
A "CAP" structure is then added to the 5' end of the RNA and a polyA tail is
added after cleavage of the 3' end downstream of the coding information.
2. RNA processing and splicing: removal of the introns from the RNA transcript
takes place in the nucleus.
Then the mature mRNA is transported to the cytoplasm.

Stages of gene expression
3. Translation: tRNA molecules (each specific for an amino acid) transport
amino acids from the cytoplasm to the mRNA template located on ribosomes, so
adding to the polypeptide chain.
Each set of three bases in the mRNA constitutes a codon.
There are 64 (43) possible triplet combinations and as there are only 20 amino
acids, most amino acids are specified by more than one codon.
Translation is always initiated at a codon specifying methionine, so establishing a
reading frame for the mRNA.
The ribosome then moves along the mRNA three base pairs at a time until a stop
codon is reached.
The completed polypetide is then released.

The cell cycle
Cell enters interphase when a mitotic cell division is complete
Interphase consists of: G1, S phase and G2
- G1 (Gap 1): Variable duration (depending on cell type).
- S phase: Stage of DNA synthesis during which each chromosome replicates to
form two daughter chromatids.
- G2 (Gap 2): cell undergoes further enlargement, is ended by mitosis.
In some cell lines G1 may be only a few hours
In other cells, such as neurones and red blood cells, do not divide once they are
fully differentiated and are permanently stopped in a gap phase known as G0.

Mitosis
Process by which a cell divides into two daughter cells, each of which receives a
complete set of genetic information.
Stages:
1. Prophase: chromosomes condense, the nucleolus disappears and the mitotic
spindle begins to form.
2. Pro-metaphase: When the nuclear membrane breaks up, with subsequent
dispersal of the chromosomes within the cell. Kinetochores located at the
centromeres facilitate attachment of the chromosomes to the micro tubules of the
mitotic spindle.

Mitosis
3. Metaphase the chromosomes are maximally condensed and arranged in the
equatorial plane of the cell.
4. Anaphase: when the chromosomes separate at the centromere and the two
chromatids move to opposite poles of the cell.
5. Telophase: reformation of the nuclear membranes and decondensation of the
chromosomes, accompanied by cytokinesis (division of the cytoplasm).

Mieosis
Process by which a primary spermatocyte or oocyte divides to form two cells
(gametes), each with a haploid number of chromosomes.

Mieosis
Meiosis I: is a reductive division and has several similarities with mitosis.
1. Prophase I (is considerably more involved than that of mitosis).
A. Leptotene: chromosomes become visible
B. Zygotene: Pairing of homologous chromosomes along their entire length, a
process of synapsis that precisely aligns corresponding DNA sequences.
C. Pachytene: The homologous chromosomes now appear as bivalents + crossing
over occurs (exchange of homologous segments between non-sister chromatids)
D. Diplotene: The homologous chromosomes then begin to repel one another,
although they remain attached at points known as chiasmata.
These chiasmata are thought to mark the sites of cross over.

2. Metaphase I
3. Anaphase I: The most error prone phase in meiosis.
During anaphase I the bivalents separate independently of each other, so the
paternal and maternal set of chromosomes are sorted into a random combination.
Meiosis II is NOT preceded by an S phase and is similar to mitotic cell division.
Further segregation of paternal and maternal alleles may occur during meiosis II,
depending on whether they have been involved in a cross over during meiosis I.

Mutations
Any permanent change in the DNA.
The change may be:
1. Genome mutations: The most common, with a frequency of 10-2 per cell
division. They are the result of chromosomal mis-segregation.
2. Chromosome mutations: are the result of chromosomal rearrangements, and
have a frequency of 6 x 10 −4 per cell division.
3. Gene mutations are the least common form of mutation in the human genome,
with a frequency of 10-10 per base pair per cell division.
Although the DNA polymerase introduces one incorrect nucleotide every 107 base pairs,
DNA repair enzymes recognize and repair 99.9% of all replication errors.

Mind Map: Gene Mutations
1. Nucleotide substitutions:
A. Missense mutations
B. Nonsense mutations
C. RNA splicing mutations
2. Deletions and insertions:
A. Frame-shift mutations
B. Codon insertions or deletions
C. Gene deletions and duplications
3. Misalignment and pairing

Gene mutations
1. Nucleotide substitutions: can take the form of:
A. Missense mutations: point mutations  amino acid substitutions that alter the
"sense" of the coding strand of the gene, as in the haemoglobinopathies
B. Non-sense mutations: inclusion of a premature stop codon  premature
termination of translation e.g. in the neurofibromatosis type 1 gene
C. RNA splicing mutations (=Splice site mutation): which alter the sites;
involved with the recognition of intron/exon (acceptor sites) or exon/intron
(donor sites) boundaries  faulty RNA splicing.

Gene Mutations
2. Deletions and insertions: can take the form of:
A. Frameshift mutations: in which the reading frame of translation will be altered
by a deletion or an insertion when the number of bases involved is NOT a
multiple of three
B. Codon insertions or deletions: The bases involved are a multiple of three
C. Gene deletions and duplications, mutations that tend to occur in identical or
near identical DNA sequences.

Gene Mutations
3. Misalignment and pairing out of register may occur in both meiosis and
mitosis.
Recombination between mispaired chromosomes or sister chromatids  gene
deletion or duplication.
This unequal crossing over is responsible for the variation in the number and
structure of red and green visual pigment genes between normal individuals and
males with defective red or green colour vision.
DNA can also be damaged by extrinsic influences such as ultraviolet or ionising
radiation, and chemical processes such as deamination or depurination.

Chromosomal abnormalities
Causes:
1. Abnormal chromosome number (aneuploidy) : The most common 
e.g Down syndrome  , Turner syndrome, …….
2. Abnormal structure.

1. Abnormal chromosome number (aneuploidy) : The most common 
3-4% of all clinically recognized pregnancies.
Mechanism:
A. Nondisjunction in meiosis I   Gamete with 24 chromosomes containing
both the maternal and paternal members of the pair.
B. Nondisjunction in meiosis II  when die? gamete will have both copies of
either the paternal or maternal chromosome.

2. Abnormal structure of chromosome:
d.t. a breakage in the chromosome followed by an abnormal recombination
Types:
A. Balanced: if the recombination produces a chromosome with a normal
amount of genetic information
B. Unbalanced: if the recombination produces a chromosome with additional or
missing information.

Balanced Unbalanced
Amount of genetic
information
normal Abnormal
Phenotype Normal Abnormal
Changes occur • Inversions
• Translocations
• Deletions
• Duplications
• Ring chromosomes
• Isochromosomes

Chromosomal abnormalities: Balanced

Chromosomal abnormalities: Unbalanced

A. Balanced rearrangements:
NO abnormal phenotype, but can pose a threat to subsequent generations because
carriers are more likely to produce unbalanced gametes.
Examples:
1. Inversions: a chromosome sustains two breaks and the segment inverts before
rejoining the chromosome.
- Pericentric inversion: If the inverted segment includes the centromere
- Paracentric inversion: If both breaks occur in the same arm of a chromosome

2. Translocations: Exchange of chromosome segments between non-
homologous chromosomes.
- Reciprocal translocation: reciprocal exchange of the broken-off segments “the
total number of chromosomes is unchanged”
- Robertsonian translocation: rearrangement that involves two acrocentric
chromosomes that fuse near the centromere, with subsequent loss of the short
arms. Although the balanced karyotype has only 45 chromosomes (including the
translocation chromosome), the phenotype is invariably unaffected as the short
arms of all five pairs of acrocentric chromosomes have multiple copies of genes
for ribosomal RNA. Therefore deletion of two short arms is not deleterious to the
carrier.

Chromosomal abnormalities: Unbalanced
Unbalanced rearrangements:  Abnormal phenotype.
Types:
1. Deletions: may be terminal or interstitial
2. Duplications: Less harmful than deletions
3. Ring chromosomes: chromosome undergoes two breaks and the broken ends
unite
4. Isochromosomes: chromosomes that have one arm missing and the other
duplicated.
The clinical effect depends on the size of the deleted segment and the number
and function of the genes it coded for

Autosomal dominant (A.D) trait with 100% penetrance
Autosomal dominant traits account for more than 50% of all mendelian phenotypes.??
Affected children usually have one unaffected parent and one who is
heterozygous for the mutation (As the prevalence of the abnormal gene is low
compared with that of normal alleles)
Any child with such parents will have a 50% chance of inheriting the trait.

Autosomal dominant (A.D) trait with 100% penetrance

No skipped generations = each affected person having an affected parent
Except ?
- If fresh mutation in the gamete of a phenotypically normal parent, disorder is
not expressed (non-penetrant) or is expressed very mildly.
In AD disorders, Homozygotes are more severely affected than heterozygotes
in most autosomal dominant disorders, except is Huntington’s disease.

Autosomal recessive (A.R) trait
A.R. disorders are expressed only in homozygotes and account for
approximately 1/3 of the recognized mendelian phenotypes.
The most common scenario is the mating of two carriers.

- Sex: Both males and females are equally affected.
- Consanguinity:
Consanguinity between parents of an affected person is more likely if the gene
responsible for the condition is rare in the general population.
Consanguinity in previous generations is usually irrelevant

Parents Risk to offspring
Genotype Phenotype
Carrier x carrier = R/r x R/r ¼ R/R, ½ R/r, ¼
r/r
¾ Unaffected,
¼ affected
Carrier x affected: R/r x r/r ½ R/r, ½ r/r ½ Unaffected,
½ affected*
Affected x affected: r/r x r/r All r/r All affected
* The mating of a carrier and an affected homozygote leads to a quasi-dominant inheritance
pattern, with 50% of the siblings being affected. This pattern can be distinguished from a
true autosomal dominant pedigree as it rarely persists over more than two generations.

X inactivation = Lyonization
The principle of X inactivation was first put forward by Mary Lyon in 1961-2.
Lyon hypothesis (has three main points):
1. In somatic cells of female mammals: Only one X chromosome is active & the
second is inactive and appears in interphase cells as a Barr body.
(The exact mechanism of this inactivation is unknown)
2. Inactivation occurs from 3 days (16-64 cell stage) after fertilization and is
normally complete by the end of the first week
3. In any somatic cell, inactivation is purely random (the inactive X may be the
paternal or maternal X);
however, all the clonal descendants of that cell will have the same inactive X.

X inactivation
X inactivation explains “dosage compensation”: Quantity of a product
produced by a single allele in a male is equivalent to that produced by a pair of
alleles in a female.

X inactivation
NOT all of the condensed X chromosome is inactive; some segments, such as
the distal region of the short arm, remain active.
Females are mosaics with respect to their X-linked genes, that is, they have
two cell populations in which one or the other X chromosome is the active one.
In carrier females, the proportion of cells in which a particular allele is active is
variable  considerable phenotypic variability in X-linked disorders (ranging
from a normal individual to a full manifestation of the defect), that is, a
manifesting heterozygote.

X-linked recessive trait
The gene responsible for X-linked recessive traits is transmitted from an affected
male through all his daughters.
[The gene is never transmitted directly from father to son]
- Any son of a carrier female will have a 50% chance of inheriting the trait.

X-linked recessive trait
X-linked recessive traits are much more common in males, females may be
affected in the following situations:
1. Homozygous females, for example the daughter of an affected father and a
carrier mother
2. Manifesting heterozygotes
The genes responsible for X-linked recessive disorders are exposed to selection that may
be complete or partial, depending on the fitness of the genotype, and therefore tend to be
lost.
In cases where the mutation is genetically lethal (e.g. Duchenne muscular dystrophy),
selection will mitigate against the persistence of the trait and new mutations rather than
inheritance from a carrier mother account for a significant proportion of casts.

X-linked dominant trait
Genetic disorder is located on the X chromosome, and only one copy of the allele
is sufficient to cause the disorder when inherited from a parent who has the
disorder.
X-linked dominant traits do NOT necessarily affect males more than females
(unlike X-linked recessive traits)
Pedigree:
- Affected male  affects all the daughters but none of the sons
- Affected female  Like in autosomal dominant traits.
Example: hypOphosphataemic rickets (Vitamin D resistant rickets)

Mitochondrial inheritance
mDNA is circular (NOT straight) double-stand DNA*

Each cell contains multiple mitochondria, mitochondrion contains 2-10 DNA
molecules
Both normal & mutant mDNA can coexist = heteroplasmy
1. The proportion of normal to mutant mDNA: will fluctuate through the
process of replicative segregation.
2. The proportion of normal to mutant mDNA: required to produce a
disease phenotype = threshold effect,
and varies from organ to organ and person to person. This explains why,
although carrier females will pass on the defect to all their children, not all of
them will be manifestly affected.

mDNA contains 37 genes, all of which are essential for normal mitochondrial
function
- 30 genes provide instructions for making enzymes involved in oxidative
phosphorylation
- 7 genes provide instructions for making molecules called transfer RNA (tRNA)
and ribosomal RNA (rRNA)
NO introns in mitochondrial DNA (mtDNA)

All our mitochondrial DNA is maternally inherited, because the mitochondria
of the sperm are lost after fusion with the egg.
Therefore in mitochondrial inheritance an affected mother passes the defect to all
her children, but only her daughters will transmit the trait to subsequent
generations.

Diseases related to mDNA changes:
• Leber hereditary optic neuropathy
• Neuropathy, ataxia, and retinitis pigmentosa (NARP)
• Progressive external ophthalmoplegia (Kearns-Sayre syndrome)

mtDNA has a mutation rate 10-17 times higher than that of the nuclear genome d.t. :
1. DNA polymerase misincorporation: beta & gamma DNA polymerases are
highly inaccurate, and a substitution rate of 1 in 3-8000 base pairs causes random
misincorporations
2. Oxidative stress: DNA damage d.t. superoxide radicals, hydrogen peroxide
and hydroxyl radicals.
Mitochondria use 90% of cellular oxygen and in addition mtDNA is not bound to
histones, and is therefore more susceptible to damage by these oxidised species
3. Alkylating agents: some of these agents have been shown to modify the
mtDNA five times more efficiently than nuclear DNA in vitro

mtDNA has a mutation rate 10-17 times higher than that of the nuclear genome d.t. :
4. The "DNA repair repertoire" of mtDNA is underdeveloped in comparison
with that of nuclear DNA.
A rudimentary excision repair mechanism is able to prevent mutations, but the
mitochondrial polymerase cannot proofread and remove certain modified bases
5. The mtDNA is devoid of introns, so a random mutation is more likely to
strike a coding DNA sequence.

Molecular cloning
Restriction endonucleases are enzymes that recognise specific sequences at
double-stranded DNA and cleave the DNA at or near these recognition sites.
DNA ligase catalyses the formation of phosphodiester bonds on each strand, so
creating a "recombinant" DNA molecule.
These sites tend to be palindromes, that is they read the same 5' to 3' on both strands.
Their discovery has been instrumental in the development of molecular cloning, as they
enabled DNA to be broken up into a characteristic and reproducible collection of
fragments.
These fragments will all have identical single stranded sticky ends; any two DNA
fragments produced by a particular restriction endonuclease digest can therefore be joined
together.

Molecular cloning
A vector: is a DNA molecule into which the gene or DNA fragment of interest is
cloned.
They can replicate autonomously in a host such as bacterial or yeast cells.
Commonly used vectors include:
- Plasmids
- Bacteriophages
- Cosmids.

Molecular cloning
(1) Plasmids: are circular, double-stranded DNA molecules that replicate extra-
chromosomally in bacteria or yeast. Cloning into plasmids is a standard
technique for the analysis of short DNA molecules. 
(2) Bacteriophage lambda: is a bacterial virus that replicates during growth in
E-coli and produces large numbers of infectious double-stranded DNA viruses.
These eventually kill the host cell, which ruptures and releases the
bacteriophages. As about one-third of the bacteriophage genome is non-essential
it can be replaced by other DNA sequences, making it ideal for cloning pieces of
human DNA up to 20 kb
(3) Cosmids: are essentially plasmids that use infectious bacteriophage lambda
particles to process and introduce large DNA fragments (up to 50 kb) into
bacterial cells.

Molecular cloning
Complementary DNA (cDNA) is a synthetic single-strand DNA that is copied
directly from RNA by reverse transcriptase.
cDNA has several advantages over genomic DNA:
1. It contains NO introns nor non-coding sequences
2. mRNA isolated from specific tissues will be a good source of clones expressed
preferentially in that tissue.
cDNA library
Single-stranded cDNA can be converted to a double-stranded molecule, which can then be
ligated into a vector to create a cDNA library encompassing all the mRNA transcripts from
that cell type or tissue.

Molecular cloning
Probes: are cloned DNA or RNA molecules that can be used to detect
homolgous sequences in nucleic acids.
Nucleic acid hybridization is a technique used to find and analyse specific DNA
or RNA fragments;
Single-stranded nucleic acids are mixed with a specific probe in conditions that
promote the formation of double- stranded nucleic acid.
The probe only hybridises to its complementary strand sequence in the DNA or
RNA sample, which is now marked by a radioactive tag.
They are often labelled with a radioactive tracer
to facilitate detection and analysis.

Biochemical and
molecular biological
techniques
o PCR
o Southern blot
o Nourthern blot
o Western blot

Definitions
- Hybridization: Process of establishing sequence-specific interaction between
two or more complementary strands of nucleic acids into a single complex
‫تهجين‬=‫بعض‬ ‫مع‬ ‫يتحدوا‬ ‫مختلفين‬ ‫مصدرين‬ ‫من‬ ‫جزئين‬
- Denaturation: process by which double-stranded DNA unwinds and
separates into single strand
‫بعض‬ ‫عن‬ ‫يفصلوا‬ ‫اتنين‬
- Annealing: Complementary sequences of single-stranded DNA or RNA to
pair to form a double-stranded polynucleotide
‫اتنين‬ ‫يبقى‬ ‫واحد‬

Biochemical and molecular biological techniques
The process of extracting nucleic acids from cells
It relies on:
- Their different solubility when compared to other cellular constituents
or
- On their binding to synthetic resins.

Biochemical and molecular biological techniques
1. Restriction enzymes are endonucleases that cleave DNA on recognizing a
specific sequence.
[These sequences are between four and six base sequences long]*
Thus, a six basepair cutter will produce many fragments with an average length of 4 kb. Since
the genome is random, the fragment sizes will be variable about a mean.
2. Separation of these fragments by gel electrophoresis.
Fragments are loaded into wells in a tray of agarose gel, across which an electric
field is applied.
DNA is negatively charged  will move towards the positive charge, with small
fragments migrating more quickly than large fragments.
Ultraviolet light is used to visualize DNA after staining with ethidium bromide.

The polymerase chain reaction (PCR)
It can amplify a single molecule of DNA million fold in 20 cycles  enabling
the detection & analysis of specific gene sequences without cloning, and without
the need for Southern or Northern blotting.
Advantage of PCR over Southern or Northern blotting:
- Faster
- Less expensive
- More sensitive
- Less technically demanding
- It requires only a fraction of the genomic DNA or RNA for analysis
The PCR made a revolution in the analysis of DNA and RNA
The identification of a single sequence is possible

The polymerase chain reaction (PCR)

PCR procedure
PCR consists of a series of 20–40 repeated temperature changes “cycles”
1. Target DNA, two oligonucleotide primers and heat stable DNA polymerase are
placed in a tube.
2. Denaturation: The mixture is heated to just below 100°C  DNA is
denatured to form single-strand nucleic acids
3. Annealing step: The reaction temperature is lowered to 50–65 °C
Allowing annealing of the primers to the single-stranded DNA template  allow
for hybridization of the primer to the strand.
The polymerase binds to the primer-template hybrid and begins DNA formation.

PCR procedure
4. Elongation step: Taq polymerase (type of DNA polymerase)  synthesizes a
new DNA strand complementary to the DNA template strand
5. Continue for 20-40 cycles
RNA samples can also be amplified
Complementary DNA (cDNA) is produced using a reverse transcriptase enzyme. PCR
primers are then added with DNA polymerase, and one of these oligonucleotides primes
the synthesis of the second cDNA strand. The double-stranded DNA can then be
amplified as above.

PCR
PCR is highly specific & highly sensitive*
The high sensitivity is one of its major drawbacks !!
becasuse of the major risk of false positive reaction caused by
contamination

Southern blotting
Used for detection of a specific DNA sequence in DNA sample
It is NOT able to detect single base mutations.

Technique of southern blotting
(1) Genomic DNA is digested by restriction enzyme
(2) The fragments of DNA generated are separated on the basis of size by agarose
gel electrophoresis
(3) The DNA is then denatured with a strong base to form complementary single-
strand nucleic acid strands. These single strands are transferred to a nitrocellulose
or nylon filter paper by blotting or capillarity
(4) A radiolabelled probe (single-stranded DNA) and the filter paper are
incubated together, allowing the probe to hybridise with matching fragments on
the filter
(5) Unbound probe is washed off and the filter is exposed to x-ray film, to reveal
the positions to which the probe hybridised.

Northern blotting
This is a technique used to study the expression of genes
Molecules of RNA (or isolated mRNA) are separated by electrophoresis
Single base mutations can be detected using allele-specific oligonucleotides (ASOs).
These probes are synthesized from individual nucleotides and correspond to a known segment of a
particular gene; they are usually around 10—20 base pairs in length. These short probes can be
manufactured to correspond with either the normal or an imperfect DNA sequence, hence their
ability to detect such small mutations.
However, if false negative results are to be avoided, the outcome of ASO analysis should be
interpreted with caution, as not all mutant genes at a given locus have exactly the same mutation.
Northern blotting is the RNA equivalent of Southern blotting.
But, RNA can NOT be cleaved by restriction enzymes; cellular DNA or purified
mRNA is therefore separated by agarose gel electrophoresis and transferred to a
nylon or nitrocellulose membrane

Western blotting
This is the protein equivalent of the above techniques.
Gel electrophoresis is used to separate the denatured proteins by mass. These are then transferred
to a nitrocellulose membrane and probed for using a specific fluorescently labelled antibody,
which binds only to the protein under investigation.
NO Eastern blotting

MCQs (to be added later in its place)
Karyotype
Systemic display chromosomes from a single somatic cell
• 23 pairs
NOT obtained from:
Sperms & germ cells (contain 23 chromosomes only)
RBCs (No nucleus)

MCQs (to be added later in its place)
• Isochromosome: abnormal chromosome created by deletion of one arm or
duplication of other arm* (Chua)
• Genocopy: different non-allelic genotypes that result in a similar
phenotype
• Autosome: chromosomes other than sex chromosomes (22 pairs in
humans)
• Synteny: presence of genes on the same chromosomes
• Heteroplasmy: presence of 2 or more different populations of
mitochondria within a cell.

Gene therapy
Gene therapy has already been attempted with some success
Examples :
- Leber’s congenital amaurosis.
- Inhalation of the CFTR gene in a disabled adenovirus proved temporarily
successful in cystic fibrosis patients.
- The adenosine deaminase gene has successfully been inserted into the bone-
marrow cells of children with subacute combined immunodeficiency (SCID)

Gene therapy ???
Pre-requisites are that the gene concerned and its control elements must be fully
characterized and cloned.
Accessible target cells must be identified that have a reasonable and productive
life span.
Finally the chosen vector must be efficient and safe

Gene therapy
Methods used for gene sequence introduction:
1. Viruses have been manipulated to use their inherent ability to insert genetic
material into their host cells.
2. Non-viral delivery methods include the introduction of naked DNA directly
into the cell and the use of liposome-mediated DNA transfer.
More recently
Stem cells utilize their pluripotency and multipotency to replace
defective organ cells.
These are already commonplace for some haematological conditions such
as leukaemia.
The hope is that soon structures in which the cells do not multiply, such as
the RPE, might be replaced using stem cells.

Genetics - Ophthalmology

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