[2S] UNIT 5 Molecular Diagnosis of Chromosomal Disorders Flashcards by JULIA CASSANDRA RECCION

T/F: Linear DNA undergoes process of compacting to ensure that the very long sequences of DNA fit inside the cells

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are made up of supercoiled strands of DNA around histone octamers

Chromosomes

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The DNA is duplicated and transmitted via _____ or _____ to the next cell generation

mitosis or meoisis

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cell division for somatic cells

Mitosis

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cell division for gametes

Meoisis

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■ Darkly staining
■ Composed of DNA repeating sequences

Heterochromatic Bands

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■ Light staining
■ Contains many protein encoding genes
■ Non-repetitive sequences

Euchromatic Bands

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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

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● Nucleosomes pack tightly together
● TRFs cannot bind DNA
● Genes are not expressed

Methylation of DNA and Histones: Heterochromatin

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● Loose packing of nucleosomes
● TRFs bind DNA
● Genes are expressed

Histone Acetylation: Euchromatin

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■ Less compact, evenly spaced
■ Makes DNA segments available for
transcription, and later to translation

Euchromatin

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■ Tightly packed together
■ Transcription factors cannot readily access the DNA sequences; play lesser role in transcription and translation

Heterochromatin

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47, XXY

Klinefelter Syndrome

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45, X

Turner Syndrome

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47, XX, +21

Trisomy 21 : Down Syndrome

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47, XY +13
47, XX +13

Trisomy 13 : Patau Syndrome

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47, XY +18
47, XX +18

Trisomy 18 : Edward Syndrome

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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 (↖)

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Represents the chromosomal aberrations that are present in offsprings

Pedigree Analysis

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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

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CHROMOSOMAL DISORDERS

● Approximately half of everybody
● Males and females affected
● All Generations

Autosomal Dominant

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CHROMOSOMAL DISORDERS

● Two mutated alleles needed to cause the disease
● Parents are usually unaffected heterozygotes
● Not typically seen in every generation (Horizontal)

Autosomal Recessive

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CHROMOSOMAL DISORDERS

● Rare
● Skips generations
● Males and females affected
● Consanguinity

Autosomal Recessive

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CHROMOSOMAL DISORDERS

Marfan Syndrome
Achondroplasia
Huntington Disease
Myotonic Dystrophy
Freckles
Polydactylism

Autosomal Dominant

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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

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