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Molecular and Cellular Princples of Medicine > Unit 7 - Diagnostic Technologies > Flashcards

Unit 7 - Diagnostic Technologies Flashcards

1
Q

what is FISH? what does it do?

A

fluoresence in situ hybridization

  • molecular probes are hybridized to Xm, then observed in fluorescent microscope
  • goal is to determine if a gene, specific mutation, or particular Xmal rearrangement is present/absent
  • probes used must be well characterized and specific to locus being examined
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2
Q

what phase of mitosis to cells need to be in for FISH?

A

metaphase or interphase

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3
Q

how are FISH slides prepared?

A

just like in karyotype analysis

  • DNA is denatured, and fluorescently labeled ss probe hybridizes to Xmal DNA
  • the rest of the DNA is counterstained with another fluoroschrome so you can see the entire Xmal complement
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4
Q

what will a person with deletions and without deletions look like on FISH? what about in newer preps that have 2 probes?

A

no deletion: 2 signals, one of each Xm
-2 probes: 4 signals (both test and control)

deletion: only 1 probe
- 2 probes: 3 signals (2 controls, 1 test)
- done b/c sometimes the testing doesn’t work well

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5
Q

what are the parameters of the FISH probe?

A

locus and Xm specific

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6
Q

what are the 3 basic types of FISH?

A
  1. repeat sequences
  2. single copy DNA
    - subtelomere FISH
  3. Xm painting
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7
Q

repeat sequence FISH probes

A

usually isolated from telomere or centromere regions

  • centromere used in Xm enumeration
  • true telomere probe recognizes 6 base repeats present at ends of all Xm, and will confirm presence/absence of telomeric regions
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8
Q

unique sequence/single copy probes

A

isolated from cloned DNA of disease-causing gene or fragment of DNA
-used to identify presence/absence of gene, gene region, or Xmal rearrangement of interest

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9
Q

subtelomere FISH probes

A

DNA sequences from distal ends of Xm in regions proximal to actual telomere regions

  • coding regions ajacent to telomeres are gene rich
  • DNA used must be unique to Xm and to the specific arm of the Xm
  • short arms (p) are green, long arms (q) are red
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10
Q

what kind of cases are tubtelomere FISH used for?

A

known cryptic translocations/deletion

-link unexplained mental retardation and autism (3-5%)

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11
Q

Xmal painting probes and their use

A

WCP (whole Xm paints) are cocktail of many unique DNA fragments from along entire length of Xm

  • following hybridization, the entire Xm fluoresces
  • most useful in identifying complex rearrangements or marker Xm (if abnormal Xm with extra material or unknown origin)
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12
Q

multi-color FISH

A

type of Xm painting to detect multiple Xm with one hybridization
-have 3 colors for target sequence, control sequence, and counter stain
-

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13
Q

what are some cons of using FISH?

A

probes don’t cover entire deletion (just “critical” region)

  • for a 3 MB deletion, probe might only be 10 KB
  • a deletion may be present that cannot be detected by FISH probe designated for that disease, so do not throw out diagnosis just because of FISH
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14
Q

how can you choose which FISH to do?

A

cannot screen all Xm or loci, so must maximize results

  • if you think you know the disease, start there (unique sequence)
  • if karyotype analysis has given you Xm, use that info (whole Xm paint or unique sequence that will identify a particular region of Xm
  • use dlinical information (developmental delay may be associated with subtelomeric microdeletion)
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15
Q

contiguous gene syndromes

A
  • regions in genome with clusters of closely associated genes whose normal functions are generally unrelated
  • deletion or duplication of that region causes multiple phenotypic abnormalities
  • size of deletion and number of genes affected may vary from person to person
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16
Q

what are deletions in the following contiguous gene syndromes?

  1. WAGR
  2. Miller-Dieker/Lissencephaly
  3. Williams syndrome
  4. VCFS
A
  1. 11p (short)
  2. 17p (short)
  3. 7q (long)
  4. 22q (long)
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17
Q

what is WAGR?

A

11p deletion: Wilms tumor + Aniridia + Genitourinary + Retardation
-can have deletion encompassing any combination of adjacent genes

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18
Q

what is Williams syndrome?

A

7q deletion
-associated with deletion of elastin gene in ~3 adjacent genes
-coarse hair/skin, lack of aorta flexibility, supravalvular aortic stenosis, skeletal/joint limitations, renal anomalites
-usually low IQ, bad math skills, but good with music
-outgoing and friendly, with blue sclera and stellate iris
-

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19
Q

what is VCFS?

A

2nd most common syndrome (first is Down)

  • interstitial 3 MB deletion on Xm 22, though specific genes are unknown
  • hypotonia, short stature
  • cleft lip and/or palate, facial anomalies, conductive hearing loss
  • cardiac anomalies, weak immune system
  • learning disabilities, difficulty feeding at birth
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20
Q

how is the VCFS deletion “interesting”?

A

repeated sequences that flank the gene

  • during meiosis, homologous Xm should pair evenly, but since the repeats have similar sequences, the deletions/duplications occur
  • VCFS patients get deletions
  • reciprocal duplication 22q syndrome get the larger, duplicated Xm
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21
Q

what is microduplication 22q syndrome?

A

reciprocal to VCFS (get the larger duplicated Xm during meiosis)
-has a completely different phenotype

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22
Q

why is the VCFS phenotype variable, and have parents that are much more mildly affected with the same deletion?

A

combo of alleles inherited by affected child is different from either parent

  • so in the parent with the affected Xm, the complement may take over for what is lacking
  • the other parent may give an Xm that doesn’t complement the Xm, and cause VCFS in the child
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23
Q

what is gene chip technology?

A

microarrays for comparitive genomic hybridization

  • most commonly gene arrays (look specifically at DNA sequences) or expression arrays (look at gene products)
  • Xm arrays also done
24
Q

what do gene arrays look for?

A

gene polymorphisms, mutations, and copy number variations (CNVs)

  • green signal = excess of reference DNA, so a deletion in test DNA
  • red signal = excess of test DNA, so duplication in test DNA
  • black signal = median expression
  • will not detect balanced rearrangements
25
Q

what have various studies determined should be the first tier of study in unexplained cases of developmental delay, intellectual disability, autism spectrum disorders, and multiple congenital anomalies?

A

CMA (Xmal microarray)

26
Q

compare karyotype, molecular diagnostics, FISH, and microarray technologies

A

karyotype: relatively large (>3Mb) numerical and structural abnormalities
- genome wide

MD: well defined, specific, very small (1-300 bp) mutations; targeted testing

FISH: well defined, specific, medium (10 Kb - 10 Mb) mutations; targeted testing

CMA: generalized genome-wide screen for small (1Kb) to large mutations; will not detect balanced rearrangements

DNAs: high resolution for targeted regions to detect mutations to single base level

27
Q

what test should you order if there is a known genetic syndrome (aneuploidy or structural)

A

karyotype and FISH

28
Q

what test should you order if there is a clinical feature suggestive of a genetic defect without a clear association with a known syndrome

A

microarray and DNA sequencing

29
Q

what test should you order if there is a disease with known molecular mutation (CF, DMD, JAK2)

A

molecular diagnostics

30
Q

what test should you order if there is a known mutation > 10 Kb?

A

FISH and molecular diagnostics

31
Q

what test should you order if there is a balanced rearrangement?

A

karyotype
FISH
molecular
DNA sequencing

32
Q

what test should you order if there is a developmental delay, autism spectrum disorder, or MCA?

A

microarray

33
Q

what is included in a genomic screen looking for cryptic anomalies?

A

microarray

DNA sequencing

34
Q

what test should you order if there is mosaicism?

A

FISH

microarray

35
Q

what test should you order if there is a UPD

A

molecular diagnostics

36
Q

what test should you order if there is a cosanguinity/idenity by descent?

A

microarray

37
Q

what are the Xm in Prader Willi syndrome?

A

Xm 15 problems w/ no paternal contribution

  • paternal microdeletion
  • maternal disomy
  • paternal imprinting failure (such that both Xm are “maternal”)
38
Q

what are the Xm in Angelman syndrome?

A

Xm 15 problems w/ no maternal contribution

  • maternal microdeletion
  • paternal disomy
  • maternal imprinting failure (such that both Xm are “paternal)
39
Q

what is disomy? uniparental disomy? the different types of UD?

A

disomy = presence of 2 Xm
UD: inheritance of Xm or Xms from 1 parent to exclusion of other
-isodomy = duplication of 1 Xm (lack of heterozygosity)
-heterodisomy = 2 different Xm from same parent

40
Q

what is and how does “zygote resuce” cause Xmal abnormalities?

A

if it senses trisomy or monosomy, will either delete Xm or make a copy of the single Xm

41
Q

how can a male carrier and a female non-carrier have a child with cystic fibrosis?

A

uniparental isodomy

  • mother doesn’t donate that one Xm, or it’s deleted, so only the father Xm is passed on
  • zygote rescue will copy the father’s carrier Xm so that the child will get cystic fibrosis
42
Q

what are the effects of zygote rescue on trisomic Xm?

A

1/3 - uniparental heterodisomy (will take away the sole Xm from one parent)
2/3 - biparental heterodisomy (normal)

43
Q

what is imprinting?

A
  • the differential modification of maternal and paternal genetic contributions to zygote resulting in differential expression of parental alleles during development and in adult (important epigenetic mechanism)
  • high frequency of developmental genes
  • probably important in early development of the zygote
44
Q

male VS female imprinting effect

A

for some genes or Xmal regions, it may be important to have maternal + paternal contribution

  • not all genes or all Xm
  • usually associated with methylation (epigenetic modification)
  • imprinting usually lasts only one generation
  • change occurs at meiosis
45
Q

epigenetic methylation

A

addition of CH3 to cytosine residues in DNA

  • can occur within a single gene or a group of adjacent genes
  • can occur over a portion of a single Xm
  • can occur over the full length of one or more Xm
  • pattern of methylation is different between males and females
  • occurs in X-inactivation or imprinting
46
Q

what is meiotic imprinting?

A

gamete Xm are “reimprinted” as maternal or paternal Xm (since they previously had 1 set of paternal and 1 set of maternal)

47
Q

what is imprinting failure?

A

when meiotic imprinting fails, meaning one parent’s gamete Xm are still divided into paternal and maternal Xm

48
Q

what are the Prader Willi and Angleman syndrome-related genes?

A

SNRPN, necdin, and UBE3A

  • in PW: only maternal Xm, with genes SNRPN and nectin inactive and only UBE3A active
  • in A: only paternal Xm, with UBE3A inactive, and SNRPN and necdin active
49
Q

what is epigenetics?

A

study of heritable changes in gene function that are not caused by change in DNA sequence

  • modification of transcription that alters gene expression, and thus phenotype
  • -can be stably transmitted through cell division
  • -in certain situations, can be reset or re-initiated
  • normal process required for normal cell function
  • change in epigenetic effects can result in up- or down-regulation of genes and this can result in disease
50
Q

epigenetic development

A

stem cells retain ability to differentiate into any cell type

  • as organism develops, differentiation occurs resulting in different cell types with different functions
  • specific patterns of genes must be active while others are inactivated to create specific tissue and organ phenotypes
  • mech include DNA methylation, histone modification, remodeling of chromatin structure
51
Q

what do transcription factors do?

A

bind to DNA and alter gene transcription

  • can act as an activator or repressor
  • bind specifically to enhancer or promoter regions of DNA adjacent to specific gene
52
Q

how are miRNA involved in epigenetics?

A

small, non-coding RNAs

  • bind to mRNA to regulate gene expression
  • can prevent translation or interfere with translation process
  • down-regulation of miRNA caused by hypermethylation at miRNA promoters is reported in many tumors
  • present targets for therapy and drug development, as miRNA differ in diseases and disease phases
53
Q

how are miRNA related to

  • leukemia?
  • Alzheimer’s?
  • breast cancer?
A
  1. miR-15a and miR-16-1 are down-regulated
  2. down-regulation of miR-107
  3. miR-21 is up-regulated in breast cancer
54
Q

how is epigenetics related to human disease?

A
  1. cancer (breast, ovarian, pancreatic, melanoma, leukemia, lymphoma)
  2. auto-immune disorders (arthritis, diabetes, MS)
  3. neurodevelopmental disease (Rett, Coffin-Lowry)
  4. neurological and neurodegenerative disease (fragile X, Alzheimer, Prader-Willi, Angelman, Parkinson, Huntington)
  5. agin
55
Q

proto-oncogenes VS tumor suppressor genes

A

PO: hypomethylation may result in over-expression of genes

TS: hypermethylation may inactivate necessary regulatory genes

56
Q

what is Rett syndrome and its cause?

A

neurodevelopmental disorder, primarily affecting females

  • normal early development, followed by arrested development, then regression
  • disrupts motor functions (problems with control of hands and feet)
  • intellectual disability
  • loss of speech
  • seizures
  • variable phenotype; appears to be partially dependent on frequency of mutant alleles that are inactivated (X-linked Xq28)
57
Q

what is MECP2?

A

transcription factor that activates or represses transcription

  • normal function is required for maturation of neurons and normal development
  • involved in Rett syndrome

Molecular and Cellular Princples of Medicine (42 decks)

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