Genetic Basis of Diseases and Inheritance
Introduction to Genetics
Cytogenetics:
Focuses on the study of the structure and function of chromosomes.
Examines chromosome behavior during both somatic (body cell) and germline (sex cell) division.
Molecular Genetics:
Involves the study of the structure and function of genes at a molecular level.
Investigates how genes are transferred from one generation to the next.
Importance and Applications of Cytogenetics
Definition: The study of human chromosomes in relation to health and disease.
Key Laboratory Diagnostic Procedure in:
Prenatal Diagnosis: To identify genetic abnormalities in a fetus before birth by studying chromosome samples.
Intellectual Disability and Multiple Birth Defects: Helps diagnose conditions caused by abnormal chromosomes, such as Huntington's disease.
Abnormal Sexual Development: Used to identify disorders resulting from abnormal sexual chromosomes, like Klinefelter's syndrome.
Infertility or Multiple Miscarriages: Investigations for underlying chromosomal causes.
Malignancies & Hematologic Disorders: Essential in the study and treatment of conditions such as Leukemia.
Advancements: New techniques offer increased resolution for chromosome studies.
Chromosomes
Fundamental Role: Carry genetic material.
Heredity: Each pair of homologous chromosomes consists of one paternal chromosome and one maternal chromosome.
Cell Division: The intact set of chromosomes is passed on to each daughter cell during every mitotic division.
Structure of Chromosomes
Chromosomes undergo continuous coiling and folding to achieve their compact structure.
Primary Coiling: Refers to the DNA double helix itself.
Secondary Coiling: The DNA double helix wraps around basic proteins called histones, forming structures known as nucleosomes. Thus, DNA + histones = nucleosomes.
Tertiary Coiling: Many nucleosomes further condense to form a chromatin fibre.
Higher-Order Coiling: Chromatin fibres form long loops on non-histone proteins and then undergo even tighter coiling to ultimately form a visible chromosome.
Procedures to Study Chromosomes
Two primary procedures are discussed:
Karyotype
Chromosomal Microarray (CMA)
Karyotyping
Definition: The systematic study of the entire set of chromosomes in a cell, focusing on their structure and function.
Procedure:
Draw 10 to 20 ml of blood.
Add phytohemagglutinin to stimulate lymphocytes (white blood cells) to divide by mitosis.
Incubate the blood sample at 37^\circ\text{C} for 2 to 3 days.
Add Colcemid to the culture for 1 to 2 hours to arrest mitosis at the metaphase stage.
Centrifuge the cells to concentrate them.
Add a low-salt solution to eliminate red blood cells and cause the lymphocytes to swell.
Transfer the cells to a tube containing a fixative.
Drop the fixed cells onto a microscope slide.
Stain the slide with Giemsa (or other stains).
Examine the stained cells under a microscope.
Digitize chromosome images and process them to construct a karyotype.
Phase of Study: Chromosomes are typically studied during metaphase because they are highly condensed and visible, allowing for clear observation of their size, shape, and centromere position.
Method of Arrest: Colcemid is used to stop cell division at metaphase by inhibiting the formation of spindle fibres.
Cell Type Selection: White blood cells (lymphocytes) are used for karyotyping because, unlike red blood cells, they possess a nucleus and therefore chromosomes.
Centromeric Position and Arm Length
The position of the centromere classifies chromosomes based on their arm lengths:
Metacentric: The centromere is located exactly in the middle, resulting in two arms of equal length.
Submetacentric: The centromere is positioned between the middle and the end of the chromosome, but closer to the middle, leading to arms of unequal length.
Acrocentric: The centromere is situated very near one end of the chromosome, producing one very short arm and one long arm.
Telocentric: The centromere is located at the telomere (the absolute end) of the chromosome, though this type is not typically found in humans.
Chromosomal Classification in Humans
Human chromosomes are organized into 23 pairs.
Autosomes: 22 pairs of non-sex chromosomes, numbered from 1 to 22 according to decreasing length.
Sex Chromosomes: 1 pair of chromosomes that determine an individual's sex.
Female: Two X chromosomes (XX).
Male: One X and one Y chromosome (XY).
Chromosome Banding
Purpose: Chemical treatment of chromosomes to reveal characteristic patterns of horizontal bands, similar to bar codes.
Specificity: Each chromosome exhibits a unique banding pattern, distinct from all other chromosomes.
Mechanism of Band Formation:
DNA is looped around nucleosome protein complexes.
Some looped segments of DNA condense more tightly and are closer together, forming regions called domains.
These closely condensed domains tend to absorb more stain, appearing darker than regions where DNA loops are more loosely arranged.
Types of Banding Patterns: A variety of treatments produce different banding patterns:
Q-bands (Quinacrine Mustard Fluorescence Bands):
Produced using quinacrine mustard, which fluoresces.
Bright bands are primarily composed of DNA rich in adenine (A) and thymine (T).
Dull bands are rich in guanine (G) and cytosine (C).
G-bands (Giemsa Bands):
Giemsa is the most commonly used stain in human cytogenetic analysis.
Stains regions rich in adenine (A) and thymine (T).
Correspond closely to Q-bands.
R-bands (Reverse Bands):
Essentially the reverse pattern of G-bands.
R-bands are rich in guanine (G) and cytosine (C).
The AT-rich regions are selectively denatured by heat, leaving the GC-rich regions intact to stain.
Particularly useful for analyzing the structure of chromosome ends, which typically stain lightly with G-banding.
C-bands (Centromeric Heterochromatin Bands):
Stains areas of heterochromatin, which is highly packed and repetitive DNA.
Specifically useful in humans for staining the centromeric regions of chromosomes and other regions containing constitutive heterochromatin.
Indicating Gene Segment Position on a Chromosome
Gene locations are described using maps, primarily falling into two categories:
Cytogenetic Location
Molecular Location
Cytogenetic Location
Basis: Based on the distinctive banding patterns created by staining chromosomes with specific chemicals.
Nomenclature: Describes the position of a particular band on a stained chromosome.
Chromosomes have two arms separated by the centromere:
p-arm: The short arm (from the French 'petit').
q-arm: The long arm.
Example Notation: 17\text{q}12
17: Refers to chromosome number 17.
\text{q}: Indicates the long arm.
12: Specifies region 1, band 2. (Note: Sub-bands can be further denoted, e.g., 17\text{q}12.5 where .5 is the sub-band).
Further Example: For the CFTR gene with a chromosomal location of 7\text{q}31.2:
Chromosome number: 7
Arm: Long arm (q)
Region number: 3
Band number: 1
Sub-band number: 2
Molecular Location
Basis: Determined by the precise sequence of DNA building blocks (base pairs) that constitute the chromosome.
Precision: Provides a much more precise localization of a gene compared to cytogenetic location.
Other Types of Karyotyping
Spectral Karyotype (SKY):
A technique where each chromosome is "painted" with a unique fluorescent color.
Offers a more convenient and precise analysis compared to conventional karyotyping for detecting complex rearrangements.
Fluorescence In-Situ Hybridization (FISH):
Involves the use of a DNA or RNA "probe" (a short, labeled segment of nucleic acid) designed to bind to complementary genetic material within cells or tissue.
Allows for the visualization of specific DNA sequences or entire chromosomes.
Genetic Disorders Detected by Karyotyping (Chromosomal Abnormalities)
Chromosomal abnormalities can be classified into two main types:
Numerical Abnormalities: Involve a different (abnormal) number of chromosomes.
Structural Abnormalities: Involve changes within the structure of one or more chromosomes.
Common Numerical Abnormalities
Monosomy: The presence of only 1 copy of a particular chromosome instead of the normal 2.
Triploidy: An individual has 3\text{N} (three complete sets of the genome) chromosomes, instead of the normal two sets.
Tetraploidy: An individual has 4\text{N} (four complete sets of the genome) chromosomes.
Trisomy: The presence of 3 copies of a specific chromosome instead of the normal 2.
Structural Abnormalities
Deletion: A segment or portion of a chromosome is missing.
Duplications: A segment or portion of a chromosome is repeated or duplicated, resulting in extra copies of genes.
Inversion: A segment of a chromosome breaks off, flips 180^\circ, and reattaches, meaning the genes within that segment are in reverse order.
Substitutions (Insertions): A portion of a chromosome from one location gets inserted into another location on the same or a different chromosome.
Translocations: Portions of two different chromosomes are exchanged. This can be balanced (no net gain or loss of genetic material) or unbalanced.
Chromosomal Microarray (CMA)
Definition: A powerful molecular technique used to detect submicroscopic changes (gains or losses of genetic material) in chromosomes that are often too small to be seen by conventional karyotyping.
Advantages over Conventional Karyotype:
Enhanced Detection: Can detect submicroscopic gains and losses of chromosomal material.
Faster Turnaround Time: Provides results more quickly.
Culture-Independent: Ability to obtain results from cells that do not grow well in culture, expanding its applicability.
Resolution Comparison (Karyotype vs. CMA):
CMA offers significantly greater resolution.
Leads to an approximately 15\% increase in diagnoses of previously undiagnosed chromosomal abnormalities.
Limitations of CMA:
CMA does not detect balanced chromosomal rearrangements (e.g., balanced translocations or inversions) as there is no net gain or loss of genetic material.