Structural Chromosomal abnormalities

Question 1

Describe the normal human karyotype and define terms associated with structural abnormalities

Answer 1

The normal human karyotype consists of 46 chromosomes: 22 pairs of autosomes (non-sex chromosomes), numbered from 1 to 22, and one pair of sex chromosomes, which are either XX (in females) or XY (in males)

These chromosomes can be visualized and organized in a chart called a karyogram, where they are sorted by size and shape

Each chromosome has a short arm (p arm, for “petit”) and a long arm (q arm), which are separated by a constriction known as the centromere

When it comes to structural chromosomal abnormalities, there are several terms that are commonly used:

1) Deletion:

• This is when a segment of the chromosome is missing or deleted
• Large deletions can be cytogenetically visible, while smaller deletions (microdeletions) might require molecular techniques to be detected

2) Duplication:

• This refers to a condition where a segment of the chromosome is duplicated and thus appears more than once in the genome

3) Inversion:

• This is a situation where a segment of a chromosome has been reversed end to end
• An inversion occurs when a single chromosome undergoes breakage and rearrangement within itself

4) Translocation:

• This refers to a condition where the whole or a part of one chromosome is attached to another chromosome
• When parts of two non-homologous chromosomes are exchanged, it is termed reciprocal translocation

5) Ring Chromosome:

• This occurs when a chromosome’s arms are lost and the two ends fuse to form a ring-like structure

6) Isochromosome:

• An isochromosome is a chromosome that has lost one of its arms and replaced it with an exact copy of the other arm

Question 2

Critically compare reciprocal translocations and Robertsonian translocations

Answer 2

Reciprocal Translocation:

• Reciprocal translocation is a type of chromosomal abnormality where segments from two different non-homologous chromosomes have been exchanged
• This means that there is no net gain or loss of genetic material - the genetic content is the same, but it’s rearranged
• In terms of genetic consequences, most carriers of a balanced reciprocal translocation are healthy and do not have any visible signs or symptoms
• However, they may have an increased risk of creating gametes with unbalanced chromosome translocations, which can lead to disorders in the offspring
• E.g. spontaneous miscarriages, stillbirths, or genetic disorders such as Down syndrome

Robertsonian Translocation:

• Robertsonian translocation is a specific kind of chromosomal translocation involving two acrocentric chromosomes (chromosomes with the centromere near one end, specifically chromosomes 13, 14, 15, 21, and 22 in humans)
• In this translocation, the long arms of the two chromosomes fuse at the centromere, forming a single chromosome.
• The short arms, which carry little genetic material, are usually lost
• Most common chromosomal translocation in humans
• Similar to reciprocal translocations, carriers of a balanced Robertsonian translocation usually do not have any health problems related to the translocation
• However, they are at risk of having children with an unbalanced translocation, which can lead to chromosomal disorders
• E.g. Down syndrome if involving chromosome 21

Comparison:

• The main difference between the two lies in the chromosomes involved and the nature of the rearrangement
• Reciprocal translocations can occur between any two chromosomes and involve a simple exchange of segments
• Robertsonian translocations, on the other hand, occur only between specific chromosomes (acrocentric) and result in the formation of a single chromosome from two

Question 3

Explain the process of non-homologous end joining

Answer 3

Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA

Double-strand breaks can result from various types of damage such as ionizing radiation or certain chemicals, and they are also generated during normal processes such as V(D)J recombination, which generates diversity in B-cell and T-cell receptors in the immune system

1) Detection of Double-Strand Break (DSB):

• The double-strand break is first detected by a protein complex known as the Ku heterodimer, consisting of Ku70 and Ku80
• These proteins bind to the ends of the broken DNA strands

2) Recruitment of Additional Factors:

• Upon binding, Ku recruits a number of additional proteins to the site of the break
• One of the main proteins recruited is the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), forming the DNA-PK complex
• DNA-PK facilitates the recruitment and activation of other proteins such as Artemis and a complex of proteins including DNA Ligase IV, XRCC4, and XLF (also known as Cernunnos)

3) End Processing:

• The DNA ends at the break site may be processed by several enzymes to prepare them for ligation
• This can include trimming of overhanging strands, filling in of gaps, and removal of damaged bases or other modifications
• The Artemis nuclease, when activated by DNA-PK, can carry out some of these processing steps
• This part of the NHEJ process is quite flexible, and different types of end processing can occur depending on the nature of the DNA ends

4) Ligation:

• Finally, the DNA Ligase IV/XRCC4/XLF complex carries out the ligation step, joining the two ends of the broken DNA together
• This ligation is “non-homologous” because it does not rely on sequence homology to align the DNA ends

One important point to note about NHEJ is that it is error-prone. Because it does not use a homologous template to guide the repair, small insertions or deletions (indels) can occur at the site of the break

This is in contrast to the more accurate homologous recombination repair pathway, which is used when a homologous sequence is available (such as during the S and G2 phases of the cell cycle when a sister chromatid is present)

Question 4

Explain how unequal crossing over can result in deletions and duplications

Answer 4

Unequal crossing over during meiosis is an irregular event that results in one chromosome with a deletion and the other with a duplication of a particular chromosomal segment

This process involves a misalignment of homologous chromosomes during meiosis, specifically at the point of Prophase I where recombination or ‘crossing over’ takes place

Step-by-step overview of how it happens:

1) Misalignment of Homologous Chromosomes:

• During meiosis, the two homologous chromosomes pair up in a process called synapsis
• If there are repeated sequences on the two chromosomes, there’s a chance that one repeat on one chromosome could align with the other repeat on the other chromosome
• This can cause the chromosomes to misalign

2) Crossing Over:

• Once the chromosomes are aligned (even if misaligned), the process of crossing over occurs
• This is where the DNA molecules are broken and then reattached to the other chromosome at the point of crossing over
• The proteins involved in recombination don’t recognize that the chromosomes are misaligned and continue the process as if they were properly aligned

3) Resulting Abnormal Chromosomes:

• If crossing over occurs in the region where the chromosomes are misaligned, this results in one chromosome having a duplication (extra genetic material) and the other chromosome having a deletion (missing genetic material)

Question 5

Explain briefly how structural abnormalities may be detected using stained metaphase chromosomes, FISH and array-CGH

Answer 5

1) Stained Metaphase Chromosomes (Karyotyping):

• Karyotyping involves staining chromosomes during the metaphase stage of cell division, then photographing them under a microscope
• The chromosomes are sorted and arranged according to their size and banding pattern, producing a karyotype
• The banding pattern results from the staining and helps to identify any structural abnormalities such as deletions, duplications, inversions, or translocations
• However, this method only detects large-scale abnormalities and is not able to detect subtle or small-scale changes

**2) Fluorescent In Situ Hybridization (FISH)

• FISH uses fluorescent probes that bind to specific DNA sequences on the chromosomes
• By visualising these probes under a fluorescence microscope, scientists can detect the presence, absence, or location of the DNA sequences
• This technique can identify smaller-scale structural abnormalities than karyotyping, such as microdeletions or duplications, and can also detect abnormalities in specific genes
• It can also be used to detect the presence of specific translocations

**3) Array Comparative Genomic Hybridisation (array-CGH)

• Array-CGH is a powerful technique used to detect copy number variations (CNVs) throughout the entire genome
• In this method, differentially labelled patient and control DNA are hybridised to a microarray containing thousands of DNA probes from different regions of the genome
• The relative amount of patient to control DNA bound to each probe is measured, allowing the detection of duplications or deletions in the patient’s genome
• Array-CGH can detect much smaller changes than either karyotyping or FISH and provides a genome-wide view of structural changes