Genetic testing

Def: Genetic testing identifies changes in chromosomes, genes, or proteins.

Importance:  can confirm or rule out a suspected genetic condition or help determine a person’s chance of developing or passing on a genetic disorder.

More than 1,000 genetic tests are currently in use, and more are being developed.

Knowledge of the test result could lead to emotional and social problems in the child, altering both relationships within a family and expectation for the future in a variety of areas such as education, employment, and relationships.

For many types of genetic analysis, a negative test result must be interpreted with caution in case it is a false negative, i.e a genetic abnormality is actually present but was not detected by the test.

Pathological significance of detected genetic alteration may be unclear due to missense mutation and variable expression.

When preforming genetic testing, it is important to be aware that the diagnosis of a genetic disease will not only have implications for the affected individual but may have ramifications for the other family members, who may be at risk of being affected or of being carriers for the condition.

Several methods can be used for genetic testing:

·       Molecular genetic tests (or gene tests): study single genes or short lengths of DNA to identify variations or mutations that lead to a genetic disorder, it depends on base sequences on chromosome.

ü  Southern blot analysis

ü  The polymerase chain reaction (PCR)

ü  DNA sequencing

ü  Next generation sequencing (NextGen).

·       Cytogenetics: concerned with how the chromosomes relate to cell behavior, particularly to their behavior during mitosis and meiosis include: Karyotyping

·       Molecular cytogenetics: involves the combination of molecular biology and cytogenetics

ü  Fluorescent in situ hybridization (FISH)

ü Comparative genomic hybridization (CGH).

ü Array comparative genomic hybridization (also microarray-based comparative genomic hybridization, matrix CGH, array CGH, aCGH)

·       Biochemical genetic tests study the amount or activity level of proteins; abnormalities in either can indicate changes to the DNA that result in a genetic disorder.

The chromosomes could be sampled from:

˗   Peripheral blood lymphocytes                                     - Bone marrow cells

˗   Skin fibroblast                                                              - Solid tissue cells

˗   Amniotic fluid cells

Medical applications (indications of genetic testing, karyotyping, chromosomal culture, analysis) :

1.     Multiple congenital anomalies:

Birth defects > one developmental regions of the body

A.    Confirmation of a clinical diagnosis

B.    Estimate recurrence risk of future sibling

- Cell division.

 

Cell division

Two types of cell division: Mitosis and Meiosis

ü  Mitosis: occurs in somatic cells and results in cell duplication→ two diploid daughter cells which are genetically identical both to each other and the original parent cell.

 

ü  Meiosis: occurs in the germ cells of the gonads and is also known as ‘reduction division’ because it results in four haploid daughter cells, each containing just one member (homologue) of each chromosome pair, all genetically different.

-Karyotyping.

Karyotyping

Def: studying chromosomes in a form suitable for analysis during metaphase or Chromosomes are stained and visualized under a microscope.

Steps:

1.  As chromosomes are only visible microscopically in dividing cells (metaphase chromosomes)

2.     Chromosome analysis involves taking some non-dividing cells (usually peripheral blood lymphocytes)

3.     Culturing them to encourage cell division on certain media.

4.     Arrest of cellular division in metaphase.

5.     Adding hypertonic saline leading to bursting of cells & release of chromosomes.

6.     Staining which creates unique banding patterns on the chromosomes and subsequently examines the cells under microscopy for any extra or missing chromosomes, or large scale structural chromosomal abnormalities.

This method will miss any changes smaller than 5–10 megabases (5–10 million bases) in size, so is unsuitable for the detection of most microdeletions or duplications.

Along with array comparative genomic hybridization it is helpful in identifying changes in amount of genetic material – gene copy number variation (CNV) including duplications (increased copy number) and deletions (decreased copy number), so they are useful Investigate broad/complex phenotype.

-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).