1 Chromosomes and chromosome rearrangements Cytogenetics is the study of genetics by visualizing...

1

Chromosomes and chromosome rearrangements

Cytogenetics is the study of genetics by visualizing chromosomes. This area of research is germane to several areas of biological research.

Cytogenetics has been fundamental to understanding the evolutionary history of a species (for example, although the Chimp and the human are morphologically very different, at the level of the chromosome (and DNA sequence) they are extremely similar.

H = humanC= chimpG = GorillaO = Orang utang

2

Chromosomes are classified by size, centromere position and banding pattern:

Shown below is the human karyotype (description of the chromosome content of a given species)

Karyotype is the chromosome description of length, number, morphology.

Karyotype analysis is extremely important in medicine. Alternations in karyotypes are linked to birth defects and many human cancers.

Metacentric- centromere in the middle

Acrocentric- centromere off center

telocentric centromere at one end

Karyotype

3

Banding patterns

Specialized stains produce unique banding patterns along each chromosome. Banding patterns are extremely useful for detecting abnormalities in chromosome structure.

For many of the chromosome stains- the molecular basis of the banding patterns is unclear. Nonetheless these techniques remain fundamental in many areas of genetic research

4

MU to bp

Genetic maps are based on recombination frequencies and describe the relative order and relative distance between linked genes.

Remember genes reside on chromosomes.

So what we would like to know is where are the genes located on the chromosomes

22% Rf = 22MU

What does this mean in terms of chromosomes and DNA?

5

Physical maps

Physical maps provide information concerning the location of genes on chromosomes

Where are the genes on chromosomes?

Cytological studies have been successfully used to map genes to specific regions of a chromosome.

For example in Drosophila in some cells the chromosomes become highly replicated and exhibit very characteristic banding patterns:

6

In situ hybridization

Salivary glands

Squash on slide

Denature/Stain polytene chromosomes

label gene probe (you can only use this method if you have the gene cloned

Hybridize probe to polytene chromosomes

Autoradiography

7

Drosophila X chr tip

This map is actually very crude. The Drosophila genome consists of about 165 Mega base pairs (165 million bp). This region represents a small fraction (5 to 10 million base pairs).

Chromosome loss

8

Chromosome instability- Elevated gain or loss of complete chromosomes

Frequent in tumorsFrequent in in vitro fertilized embryos

Gross chromosomal rearrangements during in vitro fertilization. 40% of embryos carried entire chromosome imbalance

Gain or loss of segments of chromosomes

CNV- copy number variation of chromosome segments5% of individuals genome displays CNV

Synuclein gene CNV is involved in Parkinsons

Many cancers- malignant cells most often gain additional copies of chromosome segments- genes in these segments are mis-expressed or mis-express other genes)

Microarray hybridization of DNA from tissues of identical twins- differences at several loci seen (Bruder et al 2008)

Microarray hybridization of DNA from different tissues of single individual (Piotrowski et al., 2008)

Gross chromosomal rearrangements during in vitro fertilization55% of embryos carried terminal imbalance (sub-telomere loss)(Vanneste et al., 2009) -microarray based screen of IVF 35 embryo

9

Gross chromosomal changes

The Cri du chat syndrome in humans is a result of a deletion in the short arm of chromosome 5. This was determined by comparing banding patterns with normal and Cri du Chat individuals

Types of chromosome rearrangements that can be studied by karyotype analysis:

GROSS CHROMOSOMAL CHANGES

Deletions, Duplications, Inversions, Translocations

10

DDIT

Translocation

A____B____C________D____E____F

Normal Chromosome

Deletions (deficiency)

A____B____C________D____F

Duplications

A____B____C________D____E____E____F

Inversions

A____B____C________E____D____F

A____B____C________D____E____F

A____B____C________D____L

H____I____J________K____L

H____I____J________K____E____F

11

Deletions

Deletions are often detected cytologically by comparing banding patterns between the normal and the partially deleted chromosomes

Deletedsegment

46,XX, del(1)(q24q31)Female with a deletion of chromosome 1 on the long arm (q) between bands q24 to q31.

Chromosome nofemale deletion chromosome1 Band

12

In many instances deletions are too small to be detected cytologically. In these instances genetic criteria are used.

Since deletions remove a contiguous set of genes, there is a high probability that an essential gene will be deleted. Therefore deletions will survive as heterozygotes and not homozygotes.

Normal

Homologous deletion(Lethal?)

Heterologous deletion(NOT Lethal)

13

In many instances deletions are too small to be detected cytologically. In these instances genetic/molecular techniques are used.

Since cytological deletions remove a contiguous set of genes, there is a high probability that an essential gene will be deleted. Therefore deletions will survive as heterozygotes and not homozygotes.

A____B________C____D

A____B________C____D

Normal

A____________C____D

A____________C____D

Homologous deletion(Lethal?)

A____________C____D

A____B________C____D

Heterologous deletion(NOT Lethal)

14

In individuals heterozygous for the deletion, pairing is disrupted in the regions surrounding the deletion. Therefore recombination is also significantly reduced in these regions.

A deletion on one homologue unmasks recessive alleles on the other homologue. The effect is called pseudo-dominance.

A+_____B+_____C+___________D+

A+_____B+_____C+___________D+

Normal

Normal

15

Consequences of deletions

B+A+____/ \_____C+___________D+

A+___________C+___________D+

In individuals heterozygous for the deletion, pairing is disrupted in the regions surrounding the deletion. Therefore recombination is also significantly reduced in these regions.

A deletion on one homologue unmasks recessive alleles on the other homologue. The effect is called pseudo-dominance.

A+____b______c____________D+

A+___ _____C+___________D+

Genotype

A+_____B+_____C+___________D+

A+_____B+_____C+___________D+

Normal

A+_____b______c____________D+

A+_____B+_____C+___________D+

Normal

16

Deletions in X

Females in Drosophila XX

Males in Drosophila XY or XO

Deletion series phenotype

17

Deletions in X

Females in Drosophila XX

Males in Drosophila XY or XO

Deletion series phenotype

sick

dead

sick

18

Changes in chromosome structure

Deletions:

1. Homozygosity for large deletions results in lethality- even the smallest cytologically defined deletions take out tens of 1,000's of bps and are likely to remove essential genes.

2. Organisms can tolerate heterozygosity for small but not large deletions. The reason for this is not entirely clear and is placed under the rubric of disrupting the overall ratio of gene products produced by the organism

19

Deletion mapping

Deficiency mapping or deletion mapping: This provides a means of rapidly mapping a new mutation

A deficiency or deletion is the loss of a contiguous series of nucleotides

ATGATCGGGCCCATCAAAAAAAAAAAATCATCCCCCGGGG

DELETION

ATGATCGGGCCCATC CATCCCCCGGGG

ATGATCGGGCCCATC|CATCCCCCGGGG

Defined deficiencies are very useful for mapping genes

20

Deficiency mapping

Say we have 6 sites defined by point mutations withinthe rosy gene

---1-----2-----3-----4-----5-----6

----------------------------------

---------2------------------------

---------------------4------------

--------------DDDDDDDDDD----------

Can we get intragenic recombinants that will restorenormal rosy gene?

ry2 and ry4? Y

ry2 and the deletion? Y

ry4 and the deletion N

Say we isolate a new ry mutation you call it ry(zany)

You cross it to the deletion and do not find anyRecombinantsWhere does ry(z) map

21

Deficiency mapping

Say we have 6 sites defined by point mutations withinthe rosy gene

---1-----2-----3-----4-----5-----6

----------------------------------

---------2------------------------

---------------------4------------

--------------DDDDDDDDDD----------

Can we get intragenic recombinants that will restorenormal rosy gene?

ry2 and ry4? YES

ry2 and the deletion? YES

ry4 and the deletion? No

Say we isolate a new ry mutation you call it ry(z)

You cross it to the deletion and do not find anyrecombinantsWhere does ry(z) map?

22

Deficiency mapping

Generate a heterozygote

Gene point mutant/deletion mutant

Ask if you get intragenic recombinants

Heterozygote will be pseudodominant

The single point mutation will be observed over the deletion

23

Multiple deficiencies

Specific deletions can define a series of regions within a gene

---1-----2-----3-----4-----5-----6----7----8--

----------------------------------------------

DDDDDDDDDDDDDDDDDDDDDDDDDDDDD-----------------

------------------DDDDDDDDDDDDDDDDDDDDDD------

These two deletions define 4 regions within the gene

I II III IV

Now say a newly isolated mutation does not producenormal recombinants with both deletions

To which region does it map?

Gene

24

Multiple deficiencies

Multiple deletions can define a series of regions within a gene

---1-----2-----3-----4-----5-----6----7----8--

----------------------------------------------

DDDDDDDDDDDDDDDDDDDDDDDDDDDDD-----------------

------------------DDDDDDDDDDDDDDDDDDDDDD------

These two deletions define 4 regions within the gene

I II III IV 4-7 + - - + 1-5 - - + +

+ = If a mutation maps to this region, normal recombinant flies are produced

- = If a mutation maps to this region, normal recombinant flies are NOT produced

Now say a newly isolated white mutation does not producenormal recombinants with both deletions

To which region does it map?

Gene

25

Duplications

Individuals bearing a duplication possess three copies of the genes included in that duplication.

In general, for a given chromosomal region, organisms tolerate duplications much better than deletions.

46,XY, dup(7)(q11.2q22)

Male with a duplication of chromosome 7 on the long arm (q) between bands 11.2 to 22

A____B____C________D____E____F

A____B____C________D____E____E____F

normal

Duplication

26

Tandem duplications

This is a case in which the duplicated segment lies adjacent to the original chromosomal segment

A B C D ------ A B C B C B C B C D

Once a tandem duplication arises in a population, even more copies may arise because of asymmetrical pairing at meiosis.

Remember when the homologs pair during prophase of meiosis I, they line up base-pair for base pair. Duplications lead to mistakes in this pairing mechanism

27

Proper pairing:

A____B____C____B____C____D____E

Inappropriate pairing:

A____B____C____B____C____D____E

A____B____C____B____C____D____EA____B____C____B____C____D____E

A____B____C____B____C____D____E

A____B____C____B____C__-----------__D____EA____B____C____B____C__-----------__D____E

A____B____C____B____C____D____E

28

Tandem duplications expand by mistakes in meiosis during pairing

A

B

C B C D

A

BC B C D

E

E

a

b cb

c

d

a

b cb cd

e

e

A

B C

B C D E

a b c

b c

d e

29

A B C B C D

A B C B C D

A

B C BC

D

A B C B C D

A B C B C B C D

A B C D

A B C B C D

A

B CB

CD

What happens if you get a crossover after mis-pairing?

30

The four meiotic products of a crossover between regions B and C:

A-B-C-B-C-D-E

A-B-C-D-E

A-B-C-B-C-B-C-D-E

A-B-C-B-C-D-E

This process may repeat itself many times, such that a small fragment of the genome is repeated 10,000 times.

31

An example of this is near the centromeres of the Drosophila genome:

If you look at the DNA sequence in this region it consists of small 5-10 bp sequences (AATAC)n repeated 1,000s of times. It is believed to have arisen from unequal crossing over.

Junk DNA

Selfish DNA Conserved. Important?!

Heterochromatin

Repetitive DNA- cell does not like it- They try to reduce recombination of repetitive DNA by packaging the DNA with proteins to form heterochromatin- cold spots of recombination along the chromosome

32

Duplications provide additional genetic material capable of evolving new function. For example in the above situation if the duplication for the B and C genes becomes fixed in the population- the additional copies of B and C are free to evolve new or modified functions.

This is one explanation for the origin of the tandemly repeated globin genes in humans. Each of these has a unique developmental expression pattern and provides a specialized function.

The hemoglobin in fetus has a higher affinity for oxygen since it acquires its oxygen from maternal hemoglobin via competition

33

Two distinct globin chains (each with its individual heme molecule) combine to form hemoglobin. One of the chains is designated alpha. The second chain is called "non-alpha". The fetus has a distinct non-alpha chain called gamma. After birth, a different non-alpha globin chain, called beta, pairs with the alpha chain. The combination of two alpha chains and two non-alpha chains produces a complete hemoglobin molecule.

The genes that encode the alpha globin chains are on chromosome 16. Those that encode the non-alpha globin chains are on chromosome 11. The alpha gene complex is called the "alpha globin locus", The non-alpha complex is called the "beta globin locus". The expression of the alpha and non-alpha genes is closely balanced by an unknown mechanism. Balanced gene expression is required for normal red cell function. Disruption of the balance produces a disorder called thalassemia.

The closely linked globin genes may have originally arisen from tandem duplication.

34

Alternatively some duplicated genes accumulate mutations and are no longer expressed (these are akin to junked cars along the highway).

These are known as pseudogenes. One of the genes in the hemoglobin cluster is a pseudogene.

-G-A-*--

pseudogene

Unequal crossing over among the tandemly repeated hemoglobin gene cluster is the explanation for some inherited blood diseases.

Hemoglobin lepore

35

Alternatively some duplicated genes accumulate mutations and are no longer expressed (these are akin to junked cars along the highway).

The beta-globin gene cluster in humans contains 6 genes, called epsilon (an embryonic form), gamma-G, gamma-A (the gammas are fetal forms), pseudo-beta-one (an inactive pseudogene), delta (1% of adult beta-type globin), and beta (99% of adult beta-type globin. Gamma-G and gamma-A are very similar, differing by only 1 amino acid.

These are known as pseudogenes. One of the genes in the hemoglobin cluster is a pseudogene.

-G-A-*--

pseudogene

Unequal crossing over among the tandemly repeated hemoglobin gene cluster is the explanation for some inherited blood diseases.

Hemoglobin lepore anemia

-G-A---G-A--

-G-A-

36

•If mispairing in meiosis occurs, followed by a crossover between delta and beta, the hemoglobin variant Hb-Lepore is formed.• This is a gene that starts out delta and ends as beta. Since the gene is controlled by DNA sequences upstream from the gene, Hb-Lepore is expressed as if it were a delta. That is, it is expressed at about 1% of the level that beta is expressed.• Since normal beta globin is absent in Hb-Lepore, the person has severe anemia.

***

37

Inversion

Chromosomes in which two breaks occur and the resulting fragment is rotated 180 degrees and reinserted into the chromosome.

Inversions involve no change in the amount of genetic material and therefore they are often genetically viable and show no abnormalities at the phenotypic level.

Gene fusions may occur

Inversions are defined as to whether they span the centromere

Pericentric inversions span the centromere:

A B C D E

A B D C E

A C B D E

In a pericentric inversion one break is in the short arm and one in the long arm. Therefore an example might read 46,XY,inv(3)(p23q27).

A paracenteric inversion does not include the centromere and an example might be 46,XY,inv(1)(p12p31).

Paracentric inversions do not span the centromere:

38

Homologs which are heterozygous for an inversion have difficulties pairing in meiosis.

During pairing homologous regions associate with one another. Consequently individuals heterozygous for an inversion will form a structure known as an inversion loop.

Crossover within inverted region?

A---B---C---D---E---F---GA’--B’---C’---D’--E’---F’---G’

A B CF G

D E

A’ B’ C’ ‘F G’

D’ E’

A---B---C---D---E---F---GA’--B’---C’---E’---D’--F’---G’

39

The consequence of crossover within a paracentric inversion

a-b-c d-e f-g

a-b-c e-d f-g

a-b-c f-gd-e

During meiosis, pairing leads to formation of an inversion loopThis is a problem if crossing over occurs within the inversion

A-B-0-C-D-E’-C’--0--B’-A’ dicentric-fragmentation

G-F-E-D’-F’-G’ acentric- no segregation

A B CF G

D E

A’ B’ C’ ‘F G’

D’ E’

40

a-b-c f-g

During meiosis, pairing leads to formation of an inversion loopThis is a problem if crossing over occurs within the inversion

A-B-C-D-0-E’-C’-B’-A’ fragment

G-F-E-0-D’-F’-G’ fragment

The consequence of crossover within a pericentric inversion (one that spans the centromere).

a-b-c d-e f-g

d-e

a-b-c e-d f-g

A B CF G

D E

A’ B’ C’ ‘F G’

D’ E’

41

•Paracentric inversion crosses over with a normal chromosome, the resulting chromosomes are an acentric, with no centromeres, and a dicentric, with 2 centromeres. •The acentric chromosome isn't attached to the spindle, so it gets lost during cell division, and the dicentric is usually pulled apart (broken) by the spindle pulling the two centromeres in opposite directions. These conditions are lethal.

•Pericentric inversion crosses over with a normal chromosome, the resulting chromosomes are duplicated for some genes and deleted for other genes. (They do have 1 centromere apiece though). •The gametes resulting from these do not produce viable progeny.• •Thus, either kind of inversion has lethal results when it crosses over with a normal chromosome. •The only offspring that survive are those that didn't have a crossover or crossed over in regions outside the inversion. •Thus when you count the offspring you only see the non-crossovers, so it appears that crossing over has been suppressed.

42

What are the consequences of crossing-over in an individual homozygous for an inversion?

Genotype for normal individual

A B 0 C D E F G

A B 0 C D E F G

Genotype of an individual heterozygous for an inversion:

A B 0 C D E F G

A B 0 C F E D G

Genotype of an individual homozygous for an inversion:

A B 0 C F E D G

A B 0 C F E D G

43

Translocations

A segment from one chromosome is exchanged with a segment from another chromosome.

Chromosome 1

A B C D E F----------------------0---------------------------------------------0-----------------------A B C D E F

Chromosome 2

O P Q R S T----------------------0---------------------------------------------0-----------------------O P Q R S T

Reciprocal translocation

A B C D S T----------------------0-----------------------

O P Q R E F----------------------0-----------------------

This is more specifically called a reciprocal translocation and like inversions (and unlike duplications and deficiencies) no genetic material is gained or lost in a reciprocal translocation.

44

long arms of chromosome 7 and 21 have broken off and switched places. So you can see a normal 7 and 21, and a translocated 7 and 21.This individual has all the material needed, just switched around (translocated), so they should have no health problems. However there can be a problem when this person has children.

Remember that when the gametes are made, each parent gives one of each chromosome pair. What would happen if this person gave the normal seven and the 21p with 7q attached?

There are three copies of 7q instead of two. And there is only one copy of 21q

t(11;18)(q21;q21) translocation between chromosomes 11 and 18 at bands q21 and q21

Philadelphia chromosome: t(9;22)(q34;q11).

45

As with inversions, individuals heterozygous for a reciprocal translocation will exhibit abnormalities in chromosome pairing

Notice this individual has the normal amount of genetic material

(two copies of each gene).

However it is rearranged.

If the translocated fragment contains a centromere, you could get dicentri and acentric chromosomes

How will translocated chromosomes pair in meiosis?

A B C D E F----------------------0-----------------------

O P Q R S T----------------------0-----------------------

----------------------0-----------------------A B C D S T

----------------------0-----------------------O P Q R E F

46

These chromosomes will follow Mendel's rule of independent of assortment. In this instance one must focus on the centromere

There are three possible patterns of segregation.

A B C D

E

F

A B C D

S

T

S

T

R Q P O

N1

E

F

R Q P O

T1

T2 N2

Homologous regions associate with one another.

Normal Pairing of 10 chromosomes in maize

Chr8-9 translocation

47

Alternate segregation:

ｷ N1 and N2 segregate to one pole

ｷ T1 and T2 segregate the other pole

These gametes have the normal haploid gene content: one copy of each gene and are normal

Adjacent segregation:

ｷ N1 and T1 segregate to one pole

ｷ T2 and N2 segregate to the other pole

These gametes are anueploid: they are missing some genes and duplicated for other genes.

Adjacent segregation

ｷ N1 and T2 segregate to one pole

ｷ N2 and T1 segregate to one pole

Therefore, in a translocation heterozygote, some of the gametes are viable and some are inviable.

48

Reciprocal translocations result in genes that are known to map to different chromosomes but behave as linked genes.

Under normal circumstances genes E and R assort independently because they are on different chromosomes. However in a translocation they will behave as closely linked genes and segregate together.

49

Translocations (and inversion) breakpoints sometimes disrupt an essential gene. That is the break occurs in the middle of a gene.

In fact because of this, a number of specific translocations are causally associated with specific human cancers.

The inherited disease Duchenne muscular dystrophy was mapped through a translocation that specifically disrupted this gene.

50

1

Karyotypes and chromosome rearrangements

The Philadelphia chromosome:This is a translocation involving chromosome 9 and 22Individuals bearing this chromosome develop chronic myelogenous leukemia.First example of a chromosome translocation associated with a human disease.

51

Glevec and the Philadelphia chromosome

Abl is a tyrosine kinase.Function of the normal BCR gene product is not clear.

In chronic myelogenous leukemia, the Philadelphia chromosome leads to a fusion protein of abl with bcr (breakpoint cluster region), termed bcr-abl.

This is now a continuously active tyrosine kinase.

Glevec inhibits the abl protein of cancer and non-cancer cells but cells normally have additional redundant tyrosine kinases which allow them to continue to function. Tumourogenesis however is entirely dependent on Bcr-Abl and so these cells get inactivated.

52

abl/bcr Fusion protein Chronic myelogenous and acute lymphotic leukemia

ALK/NPM Fusion Large cell lymphomas

HER2/neu Fusion Breast and cervical carcinomas

MYH11/CBFB Fusion Acute myeloid leukemia

ML/RAR Fusion Acute premyelocytic leukemia

ERG/TMPRSS2 Fusion prostate cancer

Gene fusion -prostate cancer -ERG merges with a prostate-specific gene called TMPRSS2. ERG is a transcription factors