BIO = 4

• UT4 focuses on the application of chromosome analysis techniques.

Objectives

• Understand metafase retrieval for karyotype analysis
• Select appropriate staining for each case
• Understand standard chromosome formulas
• Know major chromosomal alterations

Introduction

• Focuses on metafase obtention in chromosome spreads
• Discusses staining and banding methods for karyotype realization
• Addresses cytogenetic nomenclature standardization (ISCN)
• Outlines major chromosomal alterations

Basic Concepts

• Outlines key terminology definitions.

ADN (Deoxyribonucleic acid)

• Nucleic acid with genetic information that controls cell processes like embryogenesis, development, and reproduction.

ARN (Ribonucleic acid)

• Molecule that transmits information from DNA to cytoplasm for protein synthesis.

Base Pairs

• Opposite and complementary nucleotides in DNA/RNA strands, connected by hydrogen bonds.
• Adenine (A) pairs with thymine (T) in DNA, guanine (G) with cytosine (C) in DNA.
• In RNA, thymine is replaced by uracil (U); units used are kilobases (kb, 1000 base pairs) and megabases (Mb, million base pairs).

Chromatin

• DNA and proteins within the nucleus that form chromosomes
• Exists as either heterochromatin or euchromatin.

Heterochromatin

• Compacted chromatin structure throughout the cell cycle
• Remains condensed and transcriptionally inactive with a dark color.

Euchromatin

• Slightly compacted chromatin with high gene concentration
• Transcriptionally active with a light color.

Genes

• Genetic information units that encode DNA.

Genome

• Total genetic information of an organism/species
• The human genome has roughly 25,000 genes.

Alleles

• Different forms of a gene represented by letters
• Uppercase for dominant, lowercase for recessive
• Homozygous individuals have identical alleles, heterozygous have different alleles.

Genotype

• Allele combination for a trait
• Defined as an organism's gene set, constant throughout its existence.

Phenotype

• Observable genotype manifestation
• Can change due to environmental influence.

Chromosome

• Structure that condenses all nucleus content in eukaryotes with filamentous form
• Consists of DNA and proteins, carrying an individual's genetic information that appears in a constant amount
• Human species have 22 pairs plus 2 sex chromosomes, totaling 46 chromosomes.

Autosome

• Any non-sex chromosome
• Humans have 22 pairs of autosomes.

Mitosis

• Cell division that occurs in somatic and germ cells
• Produces two identical daughter cells

Meiosis

• Cell division that occurs during gamete formation and produces four haploid gametes (n) from a diploid (2n) germ cell

Metaphase

• Second phase of cell division
• Where chromosomes align in equatorial plane of the achromatic spindle

Chromatid

• Longitudinal unit of a duplicated chromosome
• Joined to sister chromatid by centromere.

Centromere

• Chromosome region that divides into two arms
• The location of the union to the fibers of the achromatic spindle during cell division.

Telomere

• Free end of linear chromosomes in eukaryotic cells
• Human telomere DNA comprises tandem sequence repeats of TTAGGG.

Karyotype

• Complete chromosomal makeup of an individual observed during mitosis duplication
• Graphic representation of chromosomes arranged in homologous pairs described by conventional nomenclature
• Indicates simplified diagram of metaphase chromosomes

Chromosome Spreading Techniques

• Allow a unique view of the complete genome
• Genetic study is incomplete without studying metaphase cells, despite detailed molecular analysis.
• Post optimal cell culture growth is sacrificed by scientists with two fundamental steps:
• Hypotonic shock and the inhibition of the achromatic spindle with colchicine.
• 1952: T.C. Hsu describes hypotonic shock; a solution with lower salt concentration causes cells to absorb water
• Chromosomes are separated without lysing the nucleus to be studied individually as a result.
• 1956: C.E. Ford & J.L. Hamerton modified Hsu/Pomerat technique, introducing colchicine to inhibit microtubule polymerization.
• Colcemid® now used, a less toxic synthetic analogue that halts mitosis stages.
• These discoveries greatly aided microscopic analysis by increasing metaphase cells and reducing chromosome overlap.
• 1956: J.H.Tjio & A. Levan first determine human chromosomal count at 46 (not 48).
• Late 1950s alterations numbered with associated syndromes:
  • Down Syndrome has 3 chromosomes 21
  • Klinefelter Syndrome is XXY.
  • Turner Syndrome consists of one X chromosome absent/incomplete.
• After Colcemid® incubation and hypotonic swelling, cells in metaphase are washed with Carnoy's solution (3:1 methanol/acetic acid) for chromosome fixation.
• Next, cells spread on slides that favor lysis, allowing chromosomes to stick to surface. Technique affected by atmospheric conditions.
• Chromosome spreading is crucial to monitor temperature/humidity.
• To assess chromosome spread quality involves use of phase-contrast microscope and evaluation of cell density, chromosome distribution/morphology.
• Cell density and dispersion on slides is a critical parameter.
• Moderate, uniform density helps drying/analysis; can be used to fix excess/insufficient cell counts; lack of mitotic cells can impede analysis.
• Chromosome distribution is second most important parameter with intent to have uniform dispersion, stretched chromosomes, and few overlaps.
• A result of errors of hypotonic shock or harvesting issues.
• Final evaluation is on chromosome morphology with phase-contrast microscope to evaluate banding/analysis.
• Best metaphases are uniformly distributed and less overlaps, dark, uniformly colored chromosomes with defined borders.
• There are four Chromosomal Preparation Categories based on the below criteria.

Chromosomal Preparation Categories

• Category I has low contrast with gray color and blurred chromosomes (plano/firmly fixated)
• Category II has medium contrast with light gray chromosomes and defined borders (ideal for FISH studies)
• Category III is dark chromosomes with well-defined borders (ideal for G-band chromosome banding)
• Category IV has too much contrast, refractant chromosomes (elevated/thick chromosomes with bad fixation)
• Chart shows common problems during chromosomal spreading solutions include:
  • Poor chromosomal dispersion, excessive contrast, encapsulated cytoplasm caused by rapid preparation drying: slow drying, increase humidity, cool preparation
  • Poor chromosomal dispersion, high cell density caused by high concentration: dilute cell suspension with Carnoy fixative
  • Poor chromosomal dispersion by hypotonic solution, un-swollen cells: Remake hypotonic solution
  • Poor chromosomal dispersion by cell fixation: Refix or soak Carnoy fixative 4°C until the next day
  • too dispered/broken metaphases/less contrast by Carnoy fixative stained/dirty: Use new methanol/acetic acid
  • too dispered/broken metaphases/less contrast by preperation drying slowly: Increase temperature decreased humidity by hipotomic solution over osmotic (lysis): Remake hypotomic solution

Staining and Banding Methods

• In 1970, T. Caspersson described chromosome banding techniques leading to specific banding patterns (qualitative jump in chromosome study).
• Banding techniques produce set of marks along chromosome length identifying chromosomes that aid in and improves evaluation, characterization, detection and localization
• Chromosome banding patterns remain that are identical cell-to-cell for tissues of individuals, in each species.
• Metaphases of 300, 400, 550, 700, 800 bands are obtained depending on chromosome extension level.
• Higher resolution offers more sub-bands and visualization.
• Facilitates:
• Chromosome evaluation
• Characterization of breakage points
• Detection of alterations
• Localization of specific genes

Staining Methods

• Conventional stains utilize Giemsa/Orcein for uniform, intense chromosome staining for count/order by centromere size.
• Individual chromosome identification limited by chromosome morphology causing structural anomalies.

Chromosome Banding Techniques

• Stains chromosomes discontinuously, defining transverse bands and generating specific banding patterns on each.

Banding Q

• First technique described Caspersson in 1970
• It uses quinacrine mustard stain and is visualized with fluorescence microscopy with a unique pattern of bright bands.
• Identification of chromosomes and strips is permitted, as well as the revelation of polymorphisms with the Q bands on chromosome 3,4,13,14,15,21,22 and Y
• It destains easily for other banding techniques with same material.

Banding G or GTG

• Common laboratory technique employing Giemsa or Wright for staining techniques.
• Preparations are viewed with a field microscope in 1-22 sex determining pairs (XX shown in diagram) resulting in optimal permanent staining .
• It can be used for micrograph processing for cartography banding which is transcribed from Q staining and appears brilliant using fluorescent dyes.

Banding R

• If chromosomes undergo a special staining to reverse dark/light bands.
• Preparations undergoing heating with high temperature/Giemsa staining/field microscopy or use chromomicin A/distamicina A & study fluorescence microscopy.
• Identification of chromosomes and band/extreme and poorly defined chromosome visualization is possible in way that is more accurate due to clearer distinctions (G or Q bands).

Binding C

• Specific circumstances use specific staining with banding - C to region of heterochromatin.
• Its use in a field microscope to evaluate polymorphism or inversion/dicentric.
• Allows for identification of the NOR bands and rearrangements with acrocentric bodies.

Nucleolar Organizer Region (NOR)

• Refers to satellite/areas where ribosomal gene clusters coding ribosomal RNA are found and associated with secondary construct. NOR bands occupy telomeres and short arm regions in acrocentric chromosomes in humans like 13,14,15,21 and 22.
• Properly stained chromosome spreads are analyzed by karyotyping (chromosome based analysis) with microphotography/software and banding with chromosomes or analysis depend on alteration sizes of the banding pattern.

Mutations and Polymorphisms

• Any normal sequence alteration of a gene in polymorphism

Qualifications for Polymorphisms

• Mutation has no adverse effect from normal alterations or can lead a altered phenotype expression or a disease expression
• Mutation can come from germinal/ alter gene expression or can come as a somatic expression not transmitting.
• Alteration can come from de novo expression in one expression or from lineage alteration.
• Genomic DNA variable exists among/within species with evolution or differential individuals

Somatic expressions

• Occur in a non inherited body
• Genetic mutations in specific cells are not generational bodies.

Mutation Classification

• Genomic with chromosome number change
• Chromosome with structure changes
• Gene/point implated in nucleotide change

Mutations Classification Averages

• Small mutations in unique DNA
• Mutations by exchange

Point and Gene Mutations

• Most representative which are classified by natural or genome levels as they occur in that level.

Classify By Gene Mutations

• Substitution by nucleotides where mRNA is removed along with translational regulation.
• Deletions or insertions occur in small nucleotide number with messenger RNA.

Mutations by Base Substitution

• Involes changing a DNA base (transitions or transversions) in coding/non-coding sequences (transitions > transversions) sub-classified (silent/missense/norecense)

Silent Based Mutation

• Occurs with code
• Changes the codon of the code

Transversion Based Mutation

• Can convert from a specific code to another

Mutations

• Alter protein code silences

Mutations by Change

• Produced by insertion/deletion of nucleotides in reading pattern, resulting in no frame of reference
• Diagram of insertion and deletion of bases in a DNA sequence provided.

In Pathogenesis Gene Mutation influenced factors

• mutation localization
• gene change/loss
• dominance
• the nature of heredity
• Proportion and nature of mutant hereditarity.

Chromosome Mutations

• ALTERATION TO CHROMOSOME AFFECTING HIGH LEVELS OF GENE VARIENCE and affecting somatic cells.

Human Body Chromosome:

• In bodies, chromosomes are constructed of different structural bodies:
• Women have XX sex chromosomes while men have XY.

Standard Formula:

• Men are set with both XX and XY with mutations occurring in somatic cells or organisms

Mosaic-ism

• Two genomes with different genetics from normal standards or expressions.

Germinal Expressions

• Mutations result in inheritance factors

Chromosomes Are A B C Structurally

• Structural anomalies are related to phenotypic expressions.
• Main anomalies detect prenatal (downsyndrome), resulting in molecular genetic development relating pathological genetic markers.

Anueloidism

• When bodies contain less than 100%.

Eumoloid

• When cells express more than 100%

Aneuploid

• Cell expression in total different to that of native states (absent or not) altering phenotypic expressions from genomic expressions altering phenotypic expressions from genomic expressions
• The alteration to autosomal in a chromosome is a result of abnormalities in the chromosomes
• Genomic alteration (5%) with gene mutations detected with trisomy

Triosomia

• When three copies appear

Monosomia

• When one copy appears

Poliploidia

• Ploidias where chromosomes have gene of numbers less than (n) in different types expressions like 3ns 4ns

Down Expressions Syndrome

• Where extra games are seen as X or Y sex-chromosomes.

Structural Alterations

• Loss detection or positions alteration (telomeres) as a result of rotation.
• Diagram of structural chromosomes shown
• Balanced affects living things that produce phenotypic imbalance but can be counter balanced and treated with correct solutions to achieve correct phenotypic expression but an imbalance here results in a loss here as deletion in fragments resulting in lack of genetic information
• Reciprocal means chromosomes transmit no homlogos.
• Insertion is segment transmission or alteration in cells with non functioning traits depending on whether the phenotype expression are functional and normal or not.
• Chromosomal organization are 3 fold: centromereal/size/morphology classified depending on the amount of genes shown with genes
  • Robertsonian transmissions/transmits/or translocation depends on the type and number of chromosomes.
  • Inversion depending on genes.
• Cytogene nomenclature is chromosome name with idiographic features (shown diagram as figure)

Naming Structure:

• Chromosome name (the number of chromosomes)
• Symbol
• Name with region
• Gene binding with structural and phenotypic expression
• Diagram shows naming structure with different functions.
• Depending on structure cells must bind depending on if expression is in high quality or no/ expression resolution in cells and depend on if it reaches high qualities.
• Chromosomes from the human body contain a continuous series called telomeres and have different shapes
• Inverted orientation based on regions
• These regions from telomeres are used as used by name..
• Region are numbered in sequence to find and express chromosomes for nomenclature of the structure.
• Binding depends with number with gene expression as it exists for each binding and gene (depending if with/without abnormalities) but this can depend gene regions within chromosome structure

Prenatal diagnosis

• Used to determine pregnancy in fetus chromosomal makeup . indication genetic.
• Study down genetic expressions.

Chromosomal expressions

• Study of the expression as two fold
• Biopsy

Prenatal genetic expressions

• To identify chromosome expressions

Amniotic fluid

• Used to identify genetic alteration that occur in the amniotic fluid or amniocentesis.

Diagnosis methods:

• Cytogenetic & cancer are intimate/related to genetic expressions
• Numeric Expressions -Structural expressions
• Genomic alteration-genes (translocation/transmission expressions) which is due to lack of tumor suppression with oncogenses but high levels can suppress genome production.
• Genomic (High levels cause tumor production with sub gene genetic activity).
• There's now also a mention about what's worth keeping in mind (A tener en cuenta)