Lecture 5-7: Cytogenetics

cytogenetics

looks at numerical and structural abnormalities in chromosomes

molecular genetics

looks at any type of abnormality in DNA and RNA down to a single base pair

epigenetics

alteration to dna that does not affect eh sequence but affects the regulation of expression of DNA

molecular cytogenetics

combination of cyto and molecular

uses both techniques

walther flemming

created the earliest cytogenetic technique by staining chromatin (later named chromosome)

early description of mitosis

number of chromosomes prior to 1955 and after

prior = 48

in 1955= 46

cytogenetics definition

study of chromosomes, their structure adn inheritance

cytogenetic disorder

can be detected by cytogenetics

increase as maternal and paternal age increases

chromosome spread

first step of cytogenetics analysis

chromosome spread steps / needs

cells must be capable of rapid growth and division in culture

grown for a few days then arrested in metaphase

which cells are typically used for chromosome spreads

white blood cells

acrocentric

centromere near telomeres

submetacentric

slightly off centre centromere

metacentric

centromere in the middle

telocentric

centromere at very end of chromosome, not present in humans

what makes acrocentric chromosomes unique

have satellite pieces of DNA attached by “stalks” containing rRNA genes and repetitive sequences

which chromosomes are acrocentric

13, 14, 15, 21, 22

why do rearrangements of the p arm of acrocentric chromosomes have little phenotypic effect

they are rRNA repeats, lots of redundancy

banded chromosome staining

chromosome is treated with chemicals creating dark/light bands

banded chromosome staining allows for

identification of individual chromosomes and identification of numerical and large structural abnormalities

non banded chromosome staining

specific staining of chromosomes, structures, or sequences

giemsa (G) banding

binds to AT rich regions which have fewer genes

banded chromosome staining

cytogenetic banding nomenclature

chromosome number, p or q arm, group number from centromere out, subregions

which stages of mitosis show more bands

prophase > prometaphase > metaphase

G banding

each dark staining or light staining band is given a number starting from the centromere to telomeres

euchromatin

loosely packed, available for transcription

heterochromatin

densely packed - unavailable for transcription

constitutive heterochromatin

always densely packed

facultative heterochromatin

may be unpacked to become euchromatin

C banding

stains centromeres

treatment with strong alkali followed by giemsa

non banded staining

fluorescence in situ hybridization (FISH)

non banded staining

dna probes specific for individual chromosomes, chromosomal regions, or genes

crosses boundary between molecular and cytogenetics

multicolour FISH

non banded staining

simultaneously use probes with different coloured fluorescence

spectral karyotyping

non banded staining

each homologous pair of chromosomes has its own fluorescent colour

large chromosomal alterations

quantitatively alter gene expression, does not affect the function of the protein

deviation of level of expressed copies leads to altered phenotype

aneuploidy

abnormal chromosomeal number

structural abnormalities

chromosome break and abnormal rejoin

can be balanced or unbalanced

unbalanced vs balanced chromosomes

unbalanced = gain or loss in genetic info

balanced = no genetic info missing or in excess

most common aneuploidy

x and y chromosomes

do numerical or structural abnormalities quantitatively alter gene expression

numerical, structural may do the same

normal gene dosage

2

how do abnormalities alter phenotype

increase or decrease in gene dosage

international system for human cytogenetic nomenclature

description of chromosomes at metaphase

describes chromosome abnormalities

heteroploidy

any chromosome number other than 46

euploidy

multiple of n

missing or extra entire chromosome compliments

triploidy

3n, 69 chromosomes

fertilization by 2 sperm

tetraploidy

nondisjunction event early in development

what determines phenotypic consequenes

timing of nondisjunction events

what is the most common human chromosome disorder

aneuploidy

what is the most common type of aneuploidy

trisomy

less phenotypically severe than monosomy

most common trisomies and why

21, 18, 13

they have the least amount of genes

exception to monosomies are always lethal

x chromosome

meiotic nondisjunction

failure of a pair of chromosomes to disjoin during one of the meiotic divisions

nondisjunction in meiosis II

two normal gametes, gamete with extra chromosome from the same source (grandmaternal or grandpaternal), gamete with none

nondisjunction in meiosis I

two gametes with no chromosome

two with extra chromosome, each parental origin is different

when are chromosome structural abnormalities a concern

when it occurs in germline cells

how common are chromosome structural abnormalities

less common than aneuploidy

spontaneous at low frequency

phenotypes of unbalanced rearrangments depend on

size of the region and the genes within and if there are breakpoints interrupting a gene

effects of unequal crossing over in meiosis

one chromosome with a deletion, the other with a duplication

why unequal crossing over in meiosis happens

repetitive DNA

marker chromosome

extra chromosomal material that can form a ring structure

centromeric in origin

larger markers origin

may contain material from p and q arms

partial trisomy

phenotype varies

isochromosomes

result of improper disjunction in meiosis II

one arm is missing, the other is duplicated (2p or 2q arms)

balanced rearrangements effect

usually no phenotypic effect

effect more likely in progeny

balanced - inversions

dna inverted between two break points

paracentric inversion

loop formed by inverted chromosome pairing with homolog in meiosis I

generates 2 balanced and 2 inviable gametes (one with 2 chromosomes and one with no centromeres)

pericentric inversions

loop formed by inverted chromosome pairing with homolog in meiosis I

generates 2 balanced and 2 unbalanced gametes

balanced - translocations

reciprocal exchange of chromosome segments between two non-homologous chromosomes

generally phenotypically normal

consequences in next generation of gametes

quadrivalent

formed in meiosis I

4 chromosomes with translocations pair up

alternate segregations of quadrivalent

normal or balanced gametes

adjacent-1 segregations of quadrivalent

unbalanced gametes

adjacent-2 segregations of quadrivalent

unbalanced gametes

robertsonian translocations

translocations between two acrocentric chromosomes where p arms are lost

fusion of 2 q arms

balanced

why are robertsonian translocations considered balanced

short arms of all acrocentric chromosomes have multiple copies rRNA genes

pregnancy outcomes with high mortality

autosomes, unbalanced rearrangements

pregnancy outcomes with low mortality

sex chromosomes, balanced rearrangements

phenotypes of trisomy depend on

dosage of genes on the missing or extra chromosome

phenotypic consequences due to dosage of genes

improper quantities of proteins

most common liveborn trisomy

trisomy 21

how is down syndrome confirmed

by karyotype

phenotypic consequence of down syndrome

reduced lifespan

cognitive deficits, stereotypical physical characteristics

down syndrome frequency increases at what age and why

maternal age of 35

maternal meiosis is paused in prophase I then paused, period of stasis is where chromosome pairs up, chance of a nondisjunction increases the older the egg

why is majority of individuals with down syndrome born to mothers under 35

younger women have higher birth rates

down syndrome can be caused by

meiotic disjunction during meiosis I

mosaicism

mutations occur during development causing an individual to be a mosaic of normal and abnormal cells

mitotic nondisjunction

phenotypic consequences of mosaicism depend on

location and timing of mutation

mosaicism trisomy 21

phenotype may be milder, highly variable

depends on timing and location of nondisjunction

chromosomal abnormalities leading to down syndrom

trisomy 21 mostly

robertsonian translocation often but not always chromosome 14 (extra extra copy of chromosome 21 q arm)

genomic imprinting

different expression of the maternal and paternal alleles at a given locus

genomic imprinting caused by

epigenetic, alter chromatin state (gene dosage - what is available for transcription)

epigenetics

heritable, reversible alterations to DNA which do not alter the DNA sequence

when is gene dosage determined

when a germline is established

cytosine methylation

causes genomic imprinting

silences teh gene

prader-willi and angelman syndromes

both caused by the same deletion, depends on which chromosome (parental or maternal) contains the deletion

chromosome 15

imprinting centre

controls which gene is silenced and which is active, controls imprinting

prader-willi syndrome genes are maternal or paternal

paternal

angelman syndrome genes are maternal or paternal

maternal

angelman syndrome

deletion from maternal chromosome

what happens in a normal chromosome 15 resulting in no PWS or AS

PWS is not expressed on maternal, but AS expressed on maternal

what happens when there is a deletion encompassing both PWS and AS

depends on which chromosome it occurs on

deletion on maternal results in angelman syndrome

what determines if AS or PWS is expressed

if methylated in a male pattern, AS genes are not expressed resulting in AS

vice versa