-Fluorescent in situ hybridization (FISH).

Fluorescent in situ hybridization (FISH)

Using Fluorescent-labelled DNA probes which are designed that are complementary to the DNA sequences being assessed, to detect and localize the presence or absence of specific DNA sequence on Chromosome.

Steps:

ü Chromosomes are immobilized and denatured on a microscope slide and exposed to a solution containing a fluorescently labelled probe specific to a specific chromosomal region.

ü After hybridization (the formation of a double strand of DNA from complementary single strands), the slide is washed and examined microscopically.

ü Where the probe has hybridized, fluorescent spots are seen over the relevant chromosome.

For example, if a child were suspected of having 22q11 deletion syndrome, FISH using a 22q11-specific probe would show only one pair of fluorescent spots, rather than two.

Types of FISH probes:

ü  Centromeric (aneuploidy)

ü  Telomeric (subtelomeric rearrangements)

ü  Sequence specific (microdeletions)

ü  Whole chromosome paint (complex rearrangements)

ü  Reverse painting (to identify origin of unidentified chromosomal material)

Useful for microdeletion syndromes.

- From gene to protein!

 

Human genes and protein synthesis

Ü Human genes: They are units of DNA sequences that containing information which determine the composition of an RNA molecule and most often translated to a protein.

Ü Every three nucleotides "triplet" represent a single codon, coding for a particular amino acid. Some codons act as a “start” signal, whereas others serve as “stop” signals that terminates translation .

Protein synthesis steps (From gene to protein) 

1. DNA replication

ü  Replication is semiconservative, i.e. each daughter molecule receives one strand from the parent DNA molecule.

ü  Unwinding proteins, DNA-directed RNA polymerase, DNA polymerase and ligase are also required.

ü  DNA polymerases synthesize the new strand in a 5' to 3' direction.

ü  Discontinuous replication, i.e. one or both DNA strands may be synthesized in pieces known as Okazaki fragments, which are then linked together to yield a continuous DNA chain.

2. Transcription

ü  Synthesis of complete RNA molecules from DNA by the enzyme RNA polymerase.

ü  Transcription yields three types of RNA: mRNA, tRNA and rRNA.

ü  Each nucleotide in the mRNA is complementary to one in the DNA template.

3. Post-transcriptional processing: the initial transcript is edited to remove introns and splice exons together by means of an RNA-protein complex called the spliceosome. Multiple different transcripts may be produced (alternative splicing).

- Gene switch off/ on!

   Gene switch on/off

  

˗  Each cell expresses (turns on) only a fraction of its genes. The rest of the genes are repressed (turned off).

˗  The process of turning genes on and off is known as gene regulation.

˗  Gene regulation is an important part of normal development.

˗  Genes are turned on and off in different patterns during development to make a brain cell look and act different from a liver cell or a muscle cell, for example.

˗  Gene regulation can occur at any point during gene expression, but most commonly occurs at the level of transcription (when the information in a gene’s DNA is transferred to mRNA).

- Numerical Chromosomal Aberrations.

Numerical Chromosomal Aberrations:

Aneuoploidy

A chromosome profile with fewer or greater than the normal diploid number where the total is not a multiple of 23.

n  If an extra chromosome is present (presence of three copies of the chromosome) : trisomy for that particular chromosome e.g. trisomy 21, trisomy 18, trisomy 13.

Trisomies are the most common numerical chromosomal anomalies found in humans.

Most autosomal aneuploidies are fatal in utero.

Three are survivable to term: Trisomy 13, 18 and 21.

Children with trisomy 21 (Down’s syndrome) are believed to have the most favorable outcome, as chromosome 21 has fewer genes than the other autosomes (it is short and has a low gene density)

Mechanism:  Non disjunction (Failure of the two chromosomes of one pair to disjoin).

n  If a chromosome is missing: monosomy for that chromosome e.g. Turnersyndrome (monosomy X chromosome).

Mechanism:

ü  Non-dysjunction.

ü  Anaphase lag; failure of the chromosome to move quickly to be incorporated in the daughter cell, so it will be lost. 

- Genetic Counseling.

Genetic Counseling

Definition: A process of communication and education which deals with estimating the risk of developing and/or transmitting a genetic disorder.

Goals of Genetic Counseling:

The consultand الذى يطلب الاستشارة should be provided with information and support which enables him to understand:

1.     The medical diagnosis and its consequences as: prognosis and possible treatment.

2.     Mode of inheritance of the disorder and the risk of developing or transmitting it.

3.     To make their own decisions about choices or options available for managing a particular disease and those for dealing with genetic testing and reproduction and the following risks.:

ü  Recurrence risk in future children

ü  Recurrence risk in near relatives

ü  Recurrence risk in children of unaffected brothers & sisters

A primary goal of genetic counselling is to provide information to allow for greater autonomy and choice in reproductive decisions and other areas of personal life. Avoiding additional cases of genetic disease in a family may be a consequence of genetic counselling but is not the primary aim.

Indications

1.     Maternal age > 35 years

2.     Known or suspected hereditary condition in the family

3.     Hx of child with birth defect or MR

4.     Hx of recurrent abortion

5.     Exposure to known or suspected teratogens

6.     consanguinity