[2S] UNIT 5 Molecular Diagnosis of Chromosomal Disorders Flashcards
T/F: Linear DNA undergoes process of compacting to ensure that the very long sequences of DNA fit inside the cells
T
are made up of supercoiled strands of DNA around histone octamers
Chromosomes
The DNA is duplicated and transmitted via _____ or _____ to the next cell generation
mitosis or meoisis
cell division for somatic cells
Mitosis
cell division for gametes
Meoisis
■ Darkly staining
■ Composed of DNA repeating sequences
Heterochromatic Bands
■ Light staining
■ Contains many protein encoding genes
■ Non-repetitive sequences
Euchromatic Bands
T/F: The bulk of chromosomes are primarily euchromatin which are coding for any protein
F; The bulk of chromosomes are primarily heterochromatin which are not coding for any protein
● Nucleosomes pack tightly together
● TRFs cannot bind DNA
● Genes are not expressed
Methylation of DNA and Histones: Heterochromatin
● Loose packing of nucleosomes
● TRFs bind DNA
● Genes are expressed
Histone Acetylation: Euchromatin
■ Less compact, evenly spaced
■ Makes DNA segments available for
transcription, and later to translation
Euchromatin
■ Tightly packed together
■ Transcription factors cannot readily access the DNA sequences; play lesser role in transcription and translation
Heterochromatin
47, XXY
Klinefelter Syndrome
45, X
Turner Syndrome
47, XX, +21
Trisomy 21 : Down Syndrome
47, XY +13
47, XX +13
Trisomy 13 : Patau Syndrome
47, XY +18
47, XX +18
Trisomy 18 : Edward Syndrome
The offspring of a generation wherein the condition manifested and was used as a basis for tracing of the inheritance of traits from previous generations
Proband (↖)
Represents the chromosomal aberrations that are present in offsprings
Pedigree Analysis
CHROMOSOMAL DISORDERS
● One mutated allele caused the disease
● Each person usually has one affected parent
● Appears in every generation of an affected family (Vertical)
Autosomal Dominant
CHROMOSOMAL DISORDERS
● Approximately half of everybody
● Males and females affected
● All Generations
Autosomal Dominant
CHROMOSOMAL DISORDERS
● Two mutated alleles needed to cause the disease
● Parents are usually unaffected heterozygotes
● Not typically seen in every generation (Horizontal)
Autosomal Recessive
CHROMOSOMAL DISORDERS
● Rare
● Skips generations
● Males and females affected
● Consanguinity
Autosomal Recessive
CHROMOSOMAL DISORDERS
Marfan Syndrome
Achondroplasia
Huntington Disease
Myotonic Dystrophy
Freckles
Polydactylism
Autosomal Dominant
CHROMOSOMAL DISORDERS
● Females are more frequently affected than males
● No male-to-male transmission
X-Linked Dominant
CHROMOSOMAL DISORDERS
Beta-Thalassemia
Cystic Fibrosis
Homocystinuria
Congenital Adrenal Hyperplasia
Maple Syrup Urine
PKU
Tay Sach
Autosomal Recessive
CHROMOSOMAL DISORDERS
● Males are more frequently affected
than females
● Both parents of an affected daughter must be carriers
● Fathers cannot pass X-linked traits
to their sons
X-Linked Recessive
CHROMOSOMAL DISORDERS
● Some females can have it
● All Generations
● Males get it from affected mothers and give it to their daughters
X-Linked Dominant
CHROMOSOMAL DISORDERS
Rett Syndrome
Hypophosphatemia
X-Linked Rickets
Incontinienta Pigmenti
X-Linked Dominant
CHROMOSOMAL DISORDERS
● Rare
● Males predominantly have it
● Generally skips generations
● Y-Linked
● Males generally get it from unaffected mothers
X-Linked Recessive
CHROMOSOMAL DISORDERS
Hemophilia
Duchenne Muscular Dystrophy
Red-Green Colorblindness
X-Linked Ichthyosis
X-Linked Recessive
CHROMOSOMAL DISORDERS
Leber’s Hereditary Optic Neuropathy (LHON)
Heteroplasmy
Mitochondrial
CHROMOSOMAL DISORDERS
● Only females can pass on to their children (Maternal Inheritance)
● Both males and females can be affected
● Can appear in every generation of a family
Mitochondrial
CHROMOSOMAL DISORDERS
● All males all the time
● All generations
Y-Linked
CHROMOSOMAL DISORDERS
Every child of affected mother is affected
Mitochondrial
Manifest even though there is only one dominant trait
Dominant
Both traits should be recessive for it to manifest
Recessive
GENE MUTATION VS CHROMOSOMAL MUTATION: Alteration
Nucleotide sequence of a gene
Gene Mutation
Mitochondrial can be transmitted only through ______
placenta
GENE MUTATION VS CHROMOSOMAL MUTATION: Caused by errors in
DNA Replication
Mutagens
UV & Chemicals
Gene Mutation
GENE MUTATION VS CHROMOSOMAL MUTATION: Alteration
Chromosome structure or number
Chromosomal Mutation
GENE MUTATION VS CHROMOSOMAL MUTATION: Affected Gene
Single affected gene
Gene Mutation
GENE MUTATION VS CHROMOSOMAL MUTATION
Lethal
Chromosomal Mutation
GENE MUTATION VS CHROMOSOMAL MUTATION
Low influence
Gene Mutation
GENE MUTATION VS CHROMOSOMAL MUTATION: Affected gene
Multiple affected gene
Chromosomal Mutation
GENE MUTATION VS CHROMOSOMAL MUTATION: Disease
● Sickle Cell Anemia
● Hemophilia
● CF
● Tay-sachs
● Cancers
Gene Mutation
GENE MUTATION VS CHROMOSOMAL MUTATION: Diseases
Aneuploidies/Polyploidies
● Klinefelter Syndrome
● Turner Syndrome
● Down Syndrome
Chromosomal Mutation
CHROMOSOMAL ABERRATIONS IN HUMAN DISORDERS
Part of a chromosome is deleted
Deletion
CHROMOSOMAL ABERRATIONS IN HUMAN DISORDERS
Portion of a chromosome is duplicated
Duplication
CHROMOSOMAL ABERRATIONS IN HUMAN DISORDERS
Interchange of genetic material between non homologous chromosomes
Translocation
CHROMOSOMAL ABERRATIONS IN HUMAN DISORDERS
Interchange of genetic material between two nonhomologous chromosomes
Reciprocal Translocation
CHROMOSOMAL ABERRATIONS IN HUMAN DISORDERS
The fusion of the long arms of two acrocentric chromosomes and loss of their short arms
Robertsonian Translocation
CHROMOSOMAL ABERRATIONS IN HUMAN DISORDERS
Portion of a chromosome is inverted
Inversion
CHROMOSOMAL ABERRATIONS IN HUMAN DISORDERS
The inverted segment includes the centromere
Pericentric Inversion
CHROMOSOMAL ABERRATIONS IN HUMAN DISORDERS
The inverted segment is located on one arm of the chromosome
Paracentric Inversion
CHROMOSOMAL ABERRATIONS IN HUMAN DISORDERS
Both arms of a chromosome have fused together as a ring
Ring
CHROMOSOMAL ABERRATIONS IN HUMAN DISORDERS
A chromosome that has two identical arms because of duplication of one arm of the chromosome
Isochromosome
CHROMOSOMAL ABERRATIONS IN HUMAN DISORDERS
Mirror-image of one arm of a chromosome
Isochromosome
CHROMOSOMAL ABERRATIONS IN HUMAN DISORDERS
Abnormal chromosome that has two centromeres
Dicentric Chromosome
CHROMOSOMAL ABERRATIONS IN HUMAN DISORDERS
Abnormal number of chromosomes
Aneuploidy
CHROMOSOMAL ABERRATIONS IN HUMAN DISORDERS
Presence of only one of two homologous chromosome in a diploid organism (e.g. Human)
Monosomy
CHROMOSOMAL ABERRATIONS IN HUMAN DISORDERS
Inheritance of two pairs of homologous chromosome from one parent and no copy from the other parent
Uniparental Disomy
CHROMOSOMAL ABERRATIONS IN HUMAN DISORDERS
Existence of three copies of a homologous chromosome
Trisomy
CHROMOSOMAL ABERRATIONS IN HUMAN DISORDERS
Existence of four copies of a homologous chromosome
Tetrasomy
CHROMOSOMAL ABERRATIONS IN HUMAN DISORDERS
The state of having a single (non-homologous) set of chromosomes
Monoploidy
CHROMOSOMAL ABERRATIONS IN HUMAN DISORDERS
Having three sets of chromosomes instead of two
Triploidy
CYTOGENETICS AND MOLECULAR METHODS FOR MUTATION DETECTION
Detecting numerical and gross structural aberrations
Karyotype
CHROMOSOMAL ABERRATIONS IN HUMAN DISORDERS
Is equivalent to simultaneous duplication and deletion of genetic material
Isochromosome
CYTOGENETICS AND MOLECULAR METHODS FOR MUTATION DETECTION
● Detecting trisomies, monosomies, and microdeletions
● Detects mosaicism
FISH
CYTOGENETICS AND MOLECULAR METHODS FOR MUTATION DETECTION
● Detects copy number variations of genetic material
● Used only for losses and gains
CGH
CYTOGENETICS AND MOLECULAR METHODS FOR MUTATION DETECTION
● Restriction fragments are separated by electrophoresis
● Requires mutation in restriction site
RFLP (Restriction Fragment Length Polymorphism)
CYTOGENETICS AND MOLECULAR METHODS FOR MUTATION DETECTION
Amplification of more than one target simultaneously
Multiplex PCR
CYTOGENETICS AND MOLECULAR METHODS FOR MUTATION DETECTION
Amplification using external and internal primer sets
Nested PCR
CYTOGENETICS AND MOLECULAR METHODS FOR MUTATION DETECTION
Amplification of RNA
RT-PCR (Reverse Transcription PCR)
CYTOGENETICS AND MOLECULAR METHODS FOR MUTATION DETECTION
● It is based on a combination of PCR, transcription, and translation
● Detects translation-terminating mutations
● Missense mutations are not detected
PTT (Protein Truncation Test)
CYTOGENETICS AND MOLECULAR METHODS FOR MUTATION DETECTION
● It is based on ligation of two flanked primers annealed with target sequences
● Detects all base exchanges
OLA (Oligonucleotide Ligation Assay)
Chromosomes 1,3,16,19,20
Metacentric
Chromosomes 13,14,15,21,22,Y
Acrocentric
KARYOTYPING
T/F: High resolution banding needs fixation before the chromosomes are fully compacted
T
INDICATIONS
● Suspected chromosome abnormality
● Sexual disorders
Karyotyping
BANDING TECHNIQUES
Casperson et al.
Q (Quinacrine)
INDICATIONS
● Multiple congenital anomalies and/or developmental
retardation
● Undiagnosed learning disabilities
Karyotyping
INDICATIONS
Infertility or multiple miscarriage, stillbirth and malignancies
Karyotyping
Chromosomes whose size has
condensed and whose diameter is increased are used for chromosome banding studies after fixing the stage
Metaphase Chromosomes
BANDING TECHNIQUES
Summer et al.
G (Giemsa)
BANDING TECHNIQUES
Linde & Laursen
C (Centromeric)
BANDING TECHNIQUES
Matsui & Sasaki
N (NOR)
BANDING TECHNIQUES
ID of all chromosomes and bands; reveals polymorphisms on chromosomes 3, 4, 13, 14, 15, 21, 22, and Y; easily destained for sequential staining
BANDING TECHNIQUES
● ID of all chromosomes and bands
● Permanent stain
● Simple photography
G-Banding
BANDING TECHNIQUES
● ID of all chromosomes and bands
● Visualization of ends of chromosomes and small positive R-bands
R-Banding
BANDING TECHNIQUES
● ID of all chromosomes and bands
● ID of inactive late-replication X chromosome
Replication Banding
BANDING TECHNIQUES
● ID of all centromeric and distal Y heterochromatin
● Reveals polymorphisms including heterochromatin inversions
● Evaluation of ring and dicentric chromosomes
C-Banding
BANDING TECHNIQUES
● ID of active NOR
● Reveals polymorphisms and rearrangements of acrocentric chromosomes
NOR Banding
BANDING TECHNIQUES
● ID of centromeric heterochromatin regions of chromosomes: 1,9,15,16,Y
● Useful in evaluation of chromosome 15-derived markers
DA-DAPI Staining
BANDING TECHNIQUES
● Giemsa stain
● AT-rich regions stain darker than GC-rich regions
G-Banding
BANDING TECHNIQUES
Quinacrine fluorescent dye stains AT-rich regions
Q-Banding
BANDING TECHNIQUES
● Banding pattern is opposite
G-banding
● GC-rich regions stain darker than AT-rich regions
R-Banding
ANEUPLOIDY
One copy instead of a diploid number (2N-1)
Monosomics
BANDING TECHNIQUES
● Stains heterochromatic regions
close to the centromeres
● Usually stains the entire long arm of the Y chromosome
C-Banding
ANEUPLOIDY
Gain of an extra copy of chromosome (2N+1)
Trisomics
BANDING TECHNIQUES
● Denaturation with barium hydroxide followed by giemsa
● The dark bands represent heterochromatin near the centromere
C-Banding
ANEUPLOIDY
Condition where there is lack of both the normal chromosome for a pair of species; humans with this condition typically don’t survive (2N-2)
Nullisomics
ANEUPLOIDY
Gain of extra pair of chromosome (2N+2)
Tetrasomics
POLYPLOIDY
Gain of an extra set of chromosome (3N); resulting to 69 chromosomes rather than the normal 46
Triploid
POLYPLOIDY
Gain of two extra sets of chromosome (4N); resulting to 92 chromosomes rather than 46
Tetraploid
del q15
Prader Willi Syndrome
A cytogenetic technique that uses fluorescent probes that bind specifically to a part of chromosomes complementary to its sequence
Fluorescence In Situ Hybridization (FISH)
Useful in detecting and mapping the presence or absence of particular DNA sequences within chromosomes
Fluorescence In Situ Hybridization (FISH)
Applied to provide specific localization of genes on chromosome
Fluorescence In Situ Hybridization (FISH)
● Rapid Diagnosis of trisomies and microdeletions is acquired using specific probes
● Check the cause of
○ Trisomies
○ Microdeletion syndromes
○ etc.
Fluorescence In Situ Hybridization (FISH)
METAPHASE VS INTERPHASE FISH
Gold standard and routinely done
Metaphase FISH
METAPHASE VS INTERPHASE FISH
Done on cultured cells
Metaphase FISH
METAPHASE VS INTERPHASE FISH
Allows direct visualization of chromosomes and exact position of signals
Metaphase FISH
METAPHASE VS INTERPHASE FISH
Useful in the detection of
structural changes in the genome; for molecular analysis
Metaphase FISH
METAPHASE VS INTERPHASE FISH
May also be done on
uncultured specimens
Interphase FISH
METAPHASE VS INTERPHASE FISH
Advantageous in the rapid screening of many nuclei for prenatal diagnosis and newborn studies; detection of genetic abnormalities
Interphase FISH
METAPHASE VS INTERPHASE FISH
Also, beneficial in the study of samples with a low mitotic index, such as most solid tumors
Interphase FISH
METAPHASE VS INTERPHASE FISH
Major disadvantage is the inability to detect unknown structural chromosomal changes
Interphase FISH
METAPHASE VS INTERPHASE FISH: Samples
Amniocytes
PBS
Bone Marrow Aspirate / Direct Harvest
Interphase FISH
METAPHASE VS INTERPHASE FISH
Amniocytes
Chronic Villous Cells
Lymphocytes
Cells from bone marrow aspirates or solid tumors
Fibroblasts
Metaphase FISH
Special FISH technique (dual probes) applied in detecting all genomic imbalances
Comparative Genomic Hybridization (CGH)
A Fluorescent microscope equipped with a CCD camera and an image analysis system are used for evaluation.
Comparative Genomic Hybridization (CGH)
APPLICATION
Applied in detecting all genomic imbalances
Comparative Genomic Hybridization (CGH)
APPLICATION
Determine copy number alterations of genome in cancer and those cells whose karyotype is hard or impossible to prepare/analyze
Comparative Genomic Hybridization (CGH)
APPLICATION
Basics of the technique: comparison of total genomic DNA of the given sample DNA (e.g tumor DNA) with total genomic DNA of normal cells
Comparative Genomic Hybridization (CGH)
APPLICATION
Identical amount of both tumor and normal DNAs is labeled with 2 different fluorescent dyes
○ Mixture is added and hybridized to a normal lymphocyte metaphase slide
Comparative Genomic Hybridization (CGH)
