Flashcards in Unit 7 - Diagnostic Technologies Deck (57):
what is FISH? what does it do?
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
what phase of mitosis to cells need to be in for FISH?
metaphase or interphase
how are FISH slides prepared?
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
what will a person with deletions and without deletions look like on FISH? what about in newer preps that have 2 probes?
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
what are the parameters of the FISH probe?
locus and Xm specific
what are the 3 basic types of FISH?
1. repeat sequences
2. single copy DNA
3. Xm painting
repeat sequence FISH probes
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
unique sequence/single copy probes
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
subtelomere FISH probes
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
what kind of cases are tubtelomere FISH used for?
known cryptic translocations/deletion
-link unexplained mental retardation and autism (3-5%)
Xmal painting probes and their use
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)
type of Xm painting to detect multiple Xm with one hybridization
-have 3 colors for target sequence, control sequence, and counter stain
what are some cons of using FISH?
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
how can you choose which FISH to do?
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)
contiguous gene syndromes
-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
what are deletions in the following contiguous gene syndromes?
3. Williams syndrome
1. 11p (short)
2. 17p (short)
3. 7q (long)
4. 22q (long)
what is WAGR?
11p deletion: Wilms tumor + Aniridia + Genitourinary + Retardation
-can have deletion encompassing any combination of adjacent genes
what is Williams syndrome?
-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
what is VCFS?
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
how is the VCFS deletion "interesting"?
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
what is microduplication 22q syndrome?
reciprocal to VCFS (get the larger duplicated Xm during meiosis)
-has a completely different phenotype
why is the VCFS phenotype variable, and have parents that are much more mildly affected with the same deletion?
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
what is gene chip technology?
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
what do gene arrays look for?
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
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?
CMA (Xmal microarray)
compare karyotype, molecular diagnostics, FISH, and microarray technologies
karyotype: relatively large (>3Mb) numerical and structural abnormalities
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
what test should you order if there is a known genetic syndrome (aneuploidy or structural)
karyotype and FISH
what test should you order if there is a clinical feature suggestive of a genetic defect without a clear association with a known syndrome
microarray and DNA sequencing
what test should you order if there is a disease with known molecular mutation (CF, DMD, JAK2)
what test should you order if there is a known mutation > 10 Kb?
FISH and molecular diagnostics
what test should you order if there is a balanced rearrangement?
what test should you order if there is a developmental delay, autism spectrum disorder, or MCA?
what is included in a genomic screen looking for cryptic anomalies?
what test should you order if there is mosaicism?
what test should you order if there is a UPD
what test should you order if there is a cosanguinity/idenity by descent?
what are the Xm in Prader Willi syndrome?
Xm 15 problems w/ no paternal contribution
-paternal imprinting failure (such that both Xm are "maternal")
what are the Xm in Angelman syndrome?
Xm 15 problems w/ no maternal contribution
-maternal imprinting failure (such that both Xm are "paternal)
what is disomy? uniparental disomy? the different types of UD?
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
what is and how does "zygote resuce" cause Xmal abnormalities?
if it senses trisomy or monosomy, will either delete Xm or make a copy of the single Xm
how can a male carrier and a female non-carrier have a child with cystic fibrosis?
-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
what are the effects of zygote rescue on trisomic Xm?
1/3 - uniparental heterodisomy (will take away the sole Xm from one parent)
2/3 - biparental heterodisomy (normal)
what is imprinting?
-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
male VS female imprinting effect
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
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
what is meiotic imprinting?
gamete Xm are "reimprinted" as maternal or paternal Xm (since they previously had 1 set of paternal and 1 set of maternal)
what is imprinting failure?
when meiotic imprinting fails, meaning one parent's gamete Xm are still divided into paternal and maternal Xm
what are the Prader Willi and Angleman syndrome-related genes?
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
what is epigenetics?
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
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
what do transcription factors do?
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
how are miRNA involved in epigenetics?
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
how are miRNA related to
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
how is epigenetics related to human disease?
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)
proto-oncogenes VS tumor suppressor genes
PO: hypomethylation may result in over-expression of genes
TS: hypermethylation may inactivate necessary regulatory genes
what is Rett syndrome and its cause?
neurodevelopmental disorder, primarily affecting females
-normal early development, followed by arrested development, then regression
-disrupts motor functions (problems with control of hands and feet)
-loss of speech
-variable phenotype; appears to be partially dependent on frequency of mutant alleles that are inactivated (X-linked Xq28)