exam 2 | part 3: chromosomal structures and mutations

allele

one of two or more alternative versions of a gene located at a specific position (locus) on a chromosome

what kind of organisms are we?

diploid organisms inheriting a haploid set of genes from each parent

how many bp of DNA do we have?

2.9 billion

how many chromosomes do we have?

46

range of chromosome length

48 million bp to 246 million bp

codominance

multiple alleles, when present, express equally

amorphic allele

no detectable gene product that will ever be produced by a given allele

phenotypic analysis

use microscopic or biochemical methods to identify phenotype

genotypic analysis

use cytogenetic or molecular methods to identify genotype

wild type

phenotype of typical or most frequent form of a species as it occurs in nature

variant

an alteration in the most common DNA nucleotide sequence

polymorphism

to the presence of two or more variant forms of a specific DNA sequence that can occur –usually in >1% of the population

mutation

an inherited or acquired change in DNA sequence

types of mutations

gene

chromosome

genome

gene mutations

affect single genes

usually, small changes in DNA sequence

what are gene mutations often referred to as?

molecular mutations

chromosome mutations

affect the structures of entire chromosomes

usually happens through movement of large chromosomal region

what are chromosome mutations often referred to as?

cytogenetic mutations

genome mutations

changes in the number of chromosomes

euploid

a cell or cell population with a normal complement of chromosomes

aneuploid

increased or decreased total number of chromosomes

what are genome mutations often referred to as?

cytogenetic mutations

centromere

site of attachment of chromosome to spindle apparatus

kinetochore

protein complex

what is the connection of the centromere made up of?

microtubules and kinetochore

what does the centromere consist of?

highly repetitive alpha satellite sequences

(aka alphoid DNA)

microscopically, how does a centromere appear?

as a constriction

location dictates chromosome morphology

centromere: long arm

q

centromere: short arm

p (petite)

metacentric chromosomes

centromere in center

submetacentric chromosomes

centromere positioned mid-way btwn p-arm’s telomeres and chromosome’s midpoint

acrocentric chromosomes

q arm to p arm ration ranges from 3:1 - 10:1

how is chromosome morphology defined by?

by location of centromere in relationship to p-arm and q-arm

chromosome classification: A and B

larger chromosomes

chromosome classification: C and D

medium-sized chromosomes

chromosome classification: E - G

short chromosomes

ways to visualize chromosomes

cytological stains: Wright and hematoxylin

Q-banding

forms when chromosomes are stained with quinacrine and quinacrine mustard

binds to AT rich regions, staining them

G-banding

Giemsa stain produces similar banding pattern as Q-banding

binds to AT rich regions, staining them

R-banding

a variation of Q and G banding

uses Giemsa, acridine orange, and harsher pretreatment gives inverse to Q/G banding

stains GC rich regions

C-banding

uses alkali treatment and Giemsa stain to create thick band at centromere

what do chromosome banding patterns facilitate?

detection of deletions, insertion, inversions, and other abnormalities

help in identification of distinct chromosomal locations

how does mapping of chromosomes help?

helps in identifying chromosome’s #, arms, regions, and bands/sub-bands

17q11.2

17 - chromosome #

q arm’s first region is the FIRST #1

second #1 represents you are in band 1, followed by subband 2

visualizing chromosomes via cytogenetic analysis

obtain sample

cells are grown in culture w/ phytohemagglutinin (induces mitosis)

cells enter metaphase and treated w/ colchicine (mitotic inhibitor, arrests cells at metaphase)

fix and stain

analysis

ways to detect genome and chromosomal mutations

karyotyping

fluorescence in situ hybridization

interphase FISH

metaphase FISH

karyotype

complete set of chromosomes in a cell

direct observation of metaphase chromosome structure

what are karyotypes useful for?

identifying large cytogenetic aberrations

translocations

exchange of genetic material between chromosomes

reciprocal translocations

similar sized parts of two chromosomes exchange

balanced translocation

exchange btwn chromosomes w/ no phenotypic effects - may affect offspring by not assorting properly during meiosis

Robertsonian translocation

movement of long arm of acrocentric chromosome to centromere of another acrocentric chromosome

chromosome abnormalities

deletion

insertion

inversions

isochromosome

ring chromosome

derivative chromosome

chromosome deletion

loss of chromosomal material

especially large deletions can be detected using karyotyping

chromosome insertion

duplication of regions

chromosome inversions

excision, flipping, and reconnecting chromosomes

types of chromosome inversions

pericentric

paracentric

pericentric chromosome inversion

include centromere in inverted region

paracentric chromosome inversion

involve sequences within one arm of the chromosome

isochromosome

metacentric chromosome that results from transverse splitting of the centromere

ring chromosome

deletion of genetic regions from ends to form a ring

derivative chromosome

translocated or rearranged parts from two or more chromosomes joined to normal chromosome

reporting karyotyping results

expressed as:

number of chromosomes per nucleus (normal is 46)

type of sex chromosomes (XX or XY)

followed by abnormalities

reporting karyotyping results example

normal karyotype: 46, XX

46, XX, del(7)(q13)

• indicates deletion in long arm q of chromosome 7 at region 1, band 3

fluorescence in situ hybridization (FISH)

targets specific sequences of chromosomes with fluorophore tagged probes

offers higher resolution than karyotyping for specific targets

• requires high degree of training due to 3D structures on slide

what is FISH limited to?

the regions complementary to the FISH probes

interphase FISH

circumvents cell culture step

cells are fixed, permeabilized, and exposed to a probe

what is interphase FISH used for?

prenatal samples, tumors, hematological malignancies

(easily brought into metaphase)

probes in interphase FISH

complementary to particular chromosome or chromosomal locus

• e.g a probe to any unique region on chromosome 22 should yield an image of two signals per nucleus – normal result

types of Down syndrome

trisomy 21

translocation

mosaic

trisomy 21

95% of people with Down syndrome

what can you use to diagnose Down syndrome?

interphase FISH

dual fusion probes in interphase FISH

mixture of two single probes, each labeled w/ fluorescent dye

binds to region spanning breakpoint of translocations

translocation will be seen as a signal from both probes

what is the purpose of dual fusion probes in interphase FISH?

sensitivity of FISH can be increased

how are break-apart probes in interphase FISH used?

used to decrease background signals and to identify translocation events where one chromosome can recombine with multiple partners

how are break-apart probes in interphase FISH designed?

designed to bind to intact chromosome flanking the translocation breakpoint

when translocation occurs, the two probes separate

types of probes in interphase FISH

dual fusion

break-apart

centromeric (CEN)

telomeric

centromeric probes (CEN) in interphase FISH

hybridize to highly repetitive alpha satellite sequences surrounding centromeres

can be combined with dual-color probes, making a tricolor probe

telomeric probes in interphase FISH

DNA probes specific to the telomers of all chromosomes

what are centromeric probes (CEN) in interphase FISH used for?

used to detect aneusomy of any chromosome

what are telomeric probes (CEN) in interphase FISH used for?

detection of structural abnormalities such as cryptic translocations or sub-telomeric deletions

metaphase FISH

allows for analysis of small regions in context of whole chromosomes not visible by regular chromosome banding

can distinguish all 23 chromosomes, used to detect abnormalities that affect multiple chromosomes

how does metaphase FISH work?

uses 200- to 700-kb fluorescent probes that bind to metaphase chromosomal regions or that illuminate whole chromosomes

uses whole chromosome paints by mixing combination of 5 fluorophores

comparative genome hybridization (CGH)

used to detect intrachromosomal amplifications or deletions

how does comparative genome hybridization (CGH) work?

uses labeled DNA from test and reference samples

• each is used as a probe on a normal metaphase chromosomal spread

test (sample) and reference DNA are labeled with diff fluorescent dyes

what dyes are the test and reference samples labeled with in comparative genome hybridization (CGH)?

(patient) test sample - Cy3 (green)

reference sample - Cy5 (red)

(normal samples should have equal signal from both dyes)

optimal genome mapping

newer clinical diagnostic technique that has higher resolution for mutations that karyotyping

used mainly prenatal testing and oncology

steps of optimal genome mapping

1. High MW DNA is purified and isolated

2. Flurophore attached at CTTAAG sites (found 14-15 times per 100kbp)

3. Stained DNA is fed to a fluorescent scope via electrophoresis – images taken on average of 200kbp

4. Images converted to virtual DNA Strands that are then mapped

what are limits to optimal genome mapping?

p-arms of acrocentric chromosomes, centromeres, telomeres being too small for reliable analysis

DiGeorge Syndrome

22q11.2 deletion syndrome (a microdeletion)

manifestations of DiGeorge Syndrome

congenital heart defects

immune deficiency

hypocalcemia

developmental delays

Pallister-Killian Syndrome

caused by duplication of short arms of chromosome 12

aka: tetrasomy 12p

forms an isochromosome 12p

manifestations of Pallister-Killian Syndrome

intellectual disability

hypotonia

high forehead w/ sparse scalp hair

hemophilia A (inversion type)

caused by inversion along factor VIII gene on X chromosome

disrupts gene expression

manifestations of hemophilia A

deficiency in coag factor VIII

bleeding tendencies

chronic myelogenous leukemia (CML)

caused by reciprocal translocation btwn chromosomes 9 and 22

aka: Philadelphia chromosome (not the ONLY cause of CML + seen in other hematological malignancies)

manifestations of chronic myelogenous leukemia (CML)

uncontrolled WBC proliferation

Down Syndrome (aneuploidy type)

most frequent cause is Trisomy 21

manifestations of Down Syndrome (aneuploidy type)

intellectual disability

congenital heart defects

Turner Syndrome

caused by monosomy X

some cases end up with Ringed X