FISH

Question 1

Steps of FISH

Answer 1

denature, hybridize, analyze

Question 2

Steps of FISH

Answer 2

denature, hybridize, analyze

Question 3

Types of repetitive DNA probes

Answer 3

beta satellite, alpha satellite, classical satellite, telomere

Question 4

Types of unique DNA probes

Answer 4

microdeletion

Question 5

Types of paint probes

Answer 5

whole chromosome, arm specific, band specific

Question 6

Paint (whole chromosome paints, WCPs)

Answer 6

hybridize to unique sequences which cover the length of an entire chromosome
useful for studying: marker chromosomes, translocations, and aneuploidy in metaphase
Not useful for interphase

Question 7

Alpha satellite probes - centromere

Answer 7

hybridize to alpha-satellite DNA
give large, bright signal
useful for detecting aneuploidy

Question 8

Telomere probes

Answer 8

hybridize to DNA tandem repeat TTAGGG sequences at terminal ends of chromosomes
non-specific probes target this sequence
Subtelomeric probes bind to unique DNA sequences immediately proximal to telomere repeats

Question 9

Locus specific probes

Answer 9

identify specific gene or locus on a chromosome and hybridize to the target DNA at that location
Unique sequences in the genome

Question 10

Direct preparations

Answer 10

uncultured cells from blood or amniotic fluid sample
smears made from blood, buccal cells, bone marrow

Question 11

Paraffin embedded tissue sections

Answer 11

tumors
products of conception samples

Question 12

Chromosomes that use centromere probes in prenatal samples

Answer 12

x, y, 18

Question 13

Chromosomes that use probes on large arm to FISH prenatal samples

Answer 13

13, 21

Question 14

Structural abnormalities may produce a _________ or _________ during prenatal interphase FISH

Answer 14

false abnormal or false normal

Question 15

Percent of aneuploidies in interphase fish prenatally that are normal

Answer 15

0-10%

Question 16

Percent of aneuploidies in interphase fish prenatally that are mosaicism/abnormal inconclusive

Answer 16

11-60%

Question 17

Percent of aneuploidies in interphase fish prenatally that are abnormal

Answer 17

> 60%

Question 18

Limitations of prenatal interphase FISH

Answer 18

only common aneuploidies
20-35% of visible chromosome abnormalities will go undetected (mostly numerical and structural abnormalities of untested chromosomes or chromosomal regions)
Need karyotyping and metaphase FISH to verify abnormals
It is a screening

Question 19

Microdeletion syndromes

Answer 19

small recurring interstitial deletions each of which is associated with specific phenotype

Question 20

DiGeorge/Velocardiofacial Syndrome features

Answer 20

Facial (prominent nose with squared nasal root, small eyes, small ears with abnormal folding)
Abnormal palate (70%) (overt/submucous cleft)
Congenital heart disease (75%) (conotruncal malformations)
Learning problems (70-90%)
Hypocalcemia (40-50%)
Immune deficiencies
Psychiatric disorders

Question 21

Percent of 22q deletions that are de novo and recurrence risk

Answer 21

90%
<1%
risk for germline mosaicism unknown

Question 22

Percent of 22q deletions that are inherited and recurrence risk

Answer 22

10%
50%

Question 23

Limitations of FISH for 22q

Answer 23

can only detect very large deletions
some DiGeorge microdeletions are outside of the critical region

Question 24

Complementary duplications and deletions occur due to

Answer 24

NAHR (nonallelic homologous recombination)

Question 25

Counseling points for deletions

Answer 25

Most deletions are de novo Some occur secondary to unequal segregation of balanced parental translocation If the deletion is small and/or associated with mild phenotype, parent may also carry it Need parental karyotyping and/or FISH for clarity on mechanism of deletion

Question 26

Counseling for duplications

Answer 26

Most duplications are de novo If duplication is small and/or associated with mild phenotype, one of the parents may carry it (more common for duplications than deletions) Some occur secondary to unequal segregation of balanced parental rearrangement

Question 27

FISH can be used to

Answer 27

Confirm CNV Provide information about mechanism responsible for gain and/or loss

Question 28

FISH is performed before signing out CMA when

Answer 28

family history, G-band and/or CMA findings suggest presence of something other than likely pure interstitial deletion or tandem duplication mosaicism is identified

Question 29

Break apart FISH probes

Answer 29

To see if the locus are separated (if together, will show up yellow) Will have one yellow signal, one small green, and one small red Useful for rearrangements where there are multiple known partners

Question 30

Dual FISH probe

Answer 30

If moved, will have two yellow signals, one large red, and one large green

Question 31

Extra signal, single fusion FISH probe

Answer 31

If abnormal, will have one large green, one large red, one yellow, and one small extra red or green

Question 32

Most common mechanism for genome rearrangements

Answer 32

NAHR (non-allelic homologous recombination)

Question 33

NAHR is mediated by

Answer 33

LCRs (low copy repeat sequences)

Question 34

LCRs

Answer 34

aka - segmental duplications/duplicons/paralogous duplications at least 1 kb in length share >90% sequency identity Usually occur twice or a few times within genome preferential to pericentromeric and subtelomeric regions

Question 35

LCRs rearrangement frequency positively correlated to

Answer 35

increased length, close proximity, and high sequence identity between the segmental duplications mediating the rearrangement

Question 36

Tandem (direct) LCRs mediate

Answer 36

duplications and deletions (translocations)

Question 37

Inverted LCRs mediate

Answer 37

inversions (isochromosomes)

Question 38

Palindromic sequences

Answer 38

inverted sequences that are complementary to each other and capable of forming secondary structures

Question 39

AT-rich palindromes are present

Answer 39

within the breakpoint regions of both chromosomes

Question 40

Palindromic sequences mediate translocation through

Answer 40

NHEJ (non-homologous end joining)

Question 41

Replication Fork Stalling and Template Switching (FoSTeS)

Answer 41

replication fork invades various regions of same and different replication forks

Question 42

With a break-apart probe design for FISH, which of the following signals would you be most likely to see under a microscope if a balanced translocation involving this region has occurred

Answer 42

A yellow signal, a red signal, and a green signal

Question 43

Hereditary neuropathy with liability to pressure palsy (HNLPP) causes numbness, tingling, pain, and muscle weakness in the limbs. It is caused by a deletion on 17p12 in an area flanked by low copy repeats (Timmerman & Lupski, 2006). Based on this information, SELECT ALL of the following that are TRUE

Answer 43

Non-allelic homologous recombination is a mechanism leading to this deletion Charcot-Marie Tooth disease type 1A is the corresponding duplication in the 17p12 region

Question 44

A patient presents with clinical features of conotruncal malformation, hypocalcemia, and microphthalmia consistent with DiGeorge syndrome. However, FISH studies return a negative result. Which of the following reflects appropriate guidance for next steps

Answer 44

FISH testing may not be accurate for DiGeorge syndrome, these results should be confirmed using a microarray

Question 45

Pelizaeus-Merzbacher disease is characterized by complex duplications of the PLP1 gene involving three duplications flanking smaller segments with no copy number change. Which of the following methods of genetic rearrangement is most likely to explain this phenomenon

Answer 45

Replication Fork Stalling and Template Switching (FoSTeS)