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Genetics Fellowship > 2-2 Molecular Genetic techniques > Flashcards

2-2 Molecular Genetic techniques Flashcards

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1
Q

what are the mechanisms of disease?

A
  • loss of function
      • in Autosomal recessive
    • PKU
  • Gain of function
    • in AD
  • Haploinsufficiency
    • normal protein but in LOW ammount (not enough to prevent disease)
    • AD
  • dominant negative
    • a bad protein interferes with function despite a normal protein present
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2
Q

what is PCR?

A

technique to amplify DNA

1) DENATURE heat up DNA ( so strands separate) 94 degrees
2) ANNEAL primers - cool down (50 degrees)
3) extend primers (72 degrees celcius

each PCR doubles DNA.

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3
Q

when was PCR invented and who invented it?

A

In 1983 by Dr.Kary Mullis and Dr.Suzane Hart

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4
Q

what is the most common form of short-limbed dwarfism?

A

achondroplasia

remember dwarf but short extremities

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5
Q

what is the inheritance for achondroplasia?

what % of the cases have NEW mutations?

A

autosomal dominant

80% have new mutations

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6
Q

which is the gene for achondroplasia?

what is the inheritance?

are mutations new or passed along?

what AA change occurs in the mutation?

A

FGFR3 fibroblast growth factor receptor 3 (FGFR3)

autosomal dominant

80% have new mutations

the mutation is a p.Gly380Arg glycine for Arginine

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7
Q

are the achondroplasia mutations coming from mom or dad?

A

always from the father

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8
Q

which is the most mutable nucleotide ever?

A

the one in achondroplasia mutation.

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9
Q

what is a restriction enzime?

AKA?

A
  • AKA endonucleases
  • its an enzyme that cuts DNA at restriction
  • restriction sites arespecific recognition sites
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10
Q

what is transversion and what is transition ?

A
  • transition: point mutation that changes a
    • purine for another purine (A-G) or
    • pyrimidine to another pyrimidine (C-T)
  • Transversion: point mutation that changes
    • purine for pyrimidine
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11
Q

what do we use the restriction enzimes for?

A
  • to cut DNA
  • to digest DNA
  • there are specific restriction enzymes depending on the mutation that you want to check for
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12
Q

what was the diagnostic technique before PCR?

whyit didn’t work?

was it similar?

A

Southern blot

took a lot of DNA

took a lot of time

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13
Q

what is ethidium bromide?

A

substance that intercalates between DNA strands

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14
Q

does everyone with sickle cell have the same mutation?

what kind of mutation is it?

what nucleotide is changed?

A
  • yes everyone has the
  • Glu-Val
  • missense (changing AA)
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15
Q

what is complementary?

A

its a high poutput test

spots the mutatinos

has specific probes

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16
Q

what is ASO? what is it used for?

A

ASO

allele

specific oligonucleotides

used in Complementary technique diagnosis

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17
Q

which is the most common autosomal recessive disease in caucasians?

what is the distribution in othe rpopulations?

A

Cystic Fibrosis

1 in 25 caucasians

1 in 45 hispanics

1 in 62 AA

1 in 150 asians

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18
Q
  1. where is the mutation for Cystic fibrosis?
  2. is it a large gene?
  3. what does it encode for?
  4. how many mutations on this gene cause CF?
A
  1. 7q31
  2. CF gene has 27 exons (pretty large)
  3. encondes for transmembrane glycoproteins
  4. over 1500 mutations described!!!! (think that RP has ~200 mutaitons described)
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19
Q

interpret the following mutations:

  • deltaF508
  • p.W1282*
  • 3120+1G
A
  • deltaF508 is a NON-frameshift mutation (delta deleted 3 nucleotides)
  • p.W1282 is a NONSENSE mutation (the * means stop codon created)
  • 3120+1G IS SPLICING MUTATION
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20
Q

are de novo mutations more common in AD or AR?

A

they are more common in AD for rare in AR diseases

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21
Q

what is OLA?

oligonucleotide Ligation Assay

A

using OLA probes to diagnose genetic diseases by electorphoresis

22
Q

what are the indications to do DNA testing in CF?

A
  • phenotype-genotype correlation
    • for example, there are some phenotypes with pancreatic cancer
  • help with diagnosis when chloride sweat test is not conclussive
  • allow prenatal diagnosis
  • male infertility testing
23
Q

if you see a boy in a wheel chair with hypertrophic leg muscles specially the calfs waht dg should you consider?

A

duchene’s muscular dystrophy

24
Q

what duchene muscular dystrophy?

what is the inheritance

what % are de novo mutations?

which is the gene?

is it large or small gene?

A
  • severe muscular dystrophy
    • muscle waekness begins age of 4
    • progressive
    • first in the pelvis and upper legs
    • later in arms
    • unable to walk by age 12
    • muscles are replaced ny FAT so muscles look boggy (que musculazos!)
  • scoliosis is common
  • x-linked disorder
    • 1/3 are de novo mutation
  • Gene is the Dystrophin gene
    • HUGE hene 79 exons!
    • large deletions
    • 15%
25
Q
A
26
Q

what is another type of muscular dystrophy besides Duchene?

what are the similarities and differences?

A

Becker muscular dystrophy

  • Becker and duchene
    • affect skeletal muscles and cardiac muscle (both have cardiopathy)
    • almost only in MALES
    • SAME GENE (but different mutations)
    • dystrophin gene (called DMD)
  • Differences:
    • duchene appears EARLIER and progresses FASTER
      • appear in early childhood
      • by teens ar wheelchair dependent
    • Becker appears in teens and goes very slow
    • Becker and Duchene have HUGE DELETIONS
    • Becker’s deletions are “in frame” so you have abnormal but STILL FUNCTIONAL PROTEIN
    • Duchene has out-of-frame deletions
  • Affects 1 in 5K
  • X-linked recessive
27
Q

what is the genetic difference between duchene and becker muscular dystrophy if both are due to mutations in the same gene?

A
  • Becker and duchene
    • affect skeletal muscles and cardiac muscle (both have cardiopathy)
    • almost only in MALES
    • SAME GENE (but different mutations)
    • dystrophin gene (called DMD)
  • Differences:Duchene appears EARLIER and progresses FASTER
    • appear in early childhood
    • by teens ar wheelchair dependent
    • Becker appears in teens and goes very slow
    • Becker and Duchene have HUGE DELETIONS
    • Becker’s deletions are “in frame” so you have abnormal but STILL FUNCTIONAL PROTEIN
    • Duchene has out-of-frame deletions
28
Q

A couple has a boy with duchenes dystrophy, they ask you what are the chances that the next offspring will have the disease. what do you tell them?

A

duchenes MD is an x-linked recessive disease

  • it will appear only in males
  • there is 50% chances to pass it on the next child
    • but to be a male there is another 50%
    • so the chance to have an affected boy is 1 in 6
  • BUT remember this could have been a de-novo mutation
    • 1/3 of x-linked disease are caused by de-novo mutations
29
Q

what is LMPA?

A

multiplex ligation-dependent probe amplification (MLPA)

(separates amplification products

  • compares with same sex control
  • its the best for deletion/duplication analysis
30
Q

what is myotonic dystrophy and

what kind of mutation happens on it?

A
  • its a multisystem disorder that affects
    • skeletal muscle
    • smooth muscle
    • heart
    • endocrine system
    • OCULAR
      • ptosis
      • ophthalmoplegia
      • decreased vision
      • CHRISTMAS TREE cataract
      • maculopathy and pigmentary retinopathy
  • mutation is a REPEAT mutation
    • trinucleotide REPEAT
    • autosomal dominant
31
Q

what are the ocular manifestations of Myotonic dystrophy

A

OCULAR

  • ptosis
  • ophthalmoplegia
  • decreased vision
  • CHRISTMAS TREE cataract
  • maculopathy and pigmentary retinopathy
32
Q

what genetic testing can you do for myotonic dystrophy?

remember this is a nucleotide REPEAT mutation

what gene?

what chromosome?

what is the repeat?

how many repeats you see?

A
  • gene is DMPK
  • chromosome 19
  • CTG repeat in 3’ untranslated region
  • affected people have more than 50 repeats!!
33
Q

what is anticipation?

what kind of mutation causes anticipation?

what does that mean?

A
  • anticipation is when the disease appear at EARLIER age OR with MORE severity in the next generations
  • when you see this is likely due to REPEAT expansion siorder or DYNAMIC MUTATION
  • you can dg with Souther blot or if you want to go faster…. you can use:
    • TRIPLET PRIMED PCR
34
Q

what is the process to sequence DNA?

A

we need:

  • DNA primer
  • DNA template
  • DNA Polumerase enzyme
  • nucleotides
  • put all in a pie and the in to the sequenzer
  • laser detects created electrophorectogram
35
Q

what is sanguer sequencing?

A
  • its a method to sequence DNA
    • uses selective incorportaion of chain-terminating nucleotides
    • by DNA polymerase
  • its a gold standard test but only for
    • small deletions or duplications
    • does not detect mosaicism
36
Q

what technology is used for the smart panels?

A

next generation sequencing

37
Q

what is whole exome sequencing?

what % of the genome is captured with this?

what % of exons?

A
  • genetic test
  • to sequence all protein coding genes (exomes)
    • 1st selects all exons
      • exons are 1-2 % n (30CM)
    • then sequences them
    • using high-throughput technology
  • This approach is cheaper than checking all the genome
  • WES is good for MENDELIAN DISEASES
38
Q

what is a DNA microarray?

A
  • its microscopic DNA attached to solid surface that has small containers
  • each container has 10-12 picomoles of DNA sequence (known as probes)
  • used to measure gene expression
39
Q

what is picomole?

A

pico means one TRILLION

40
Q

what is SNP array?

A

is a type of DNA microarray used to detect polymorphisms within a population.

SNP = single nucleotide polymorphism

There are 85 million SNPs identified

41
Q

when to use SNP array vs WES?

A
  • WES is good for
    • rare mendelian diseases
    • because it checks all genetic variants in one person
  • SNP array
    • good for common diseases
    • detects genetic variants common in a population
42
Q

what % of genome is EXONS?

A

just 1-2%

43
Q

what % of exons are targeted in WES?

A

80%

44
Q

what % of WES is successful in identifying the mutation?

A

about 30%

45
Q

what % of WES are successful ?

what are the reasons of WES failure?

A

30% successful

  • reasons for failure
    • genes has not been identified
    • exon has not been identified
    • problem is in non-coding regions
      *
46
Q

what is super important to do before doing WES and why?

A
  • DO genetic counseling because
  • you may find surprises and you need to know if they want to know:
    • carrier of AR condition
    • mutations related with LATE-ONSET diseases
    • susceptibility alleles
    • non-paternity
    • VOUS
    • false positives and negatives
47
Q

1) can you test a NORMAL minor for a late-onset disorder?

for ex, can you test a normal girl for the BRCA gene if there is strong family hx of breast cancer?

2) what if you do WES and you find a disease causing gene such as BRCA?

A
  • NOOOOO
    • only at age 18 they can decide
    • so children only tested if they are diseases happening at the moment
  • if something is found on WES you are supposed to tell them
48
Q

what is the charcot-marie tooth disease?

what is the tooth problem?

A
  • its a progressive distal weakness and sensory loss
  • caused by degeneration of peripheral nerve
  • there is no Tooth problem but it was described by Dr.Tooth
49
Q

when and why are panels convenient?

A

for diseases that have multiple genes and multiple phenotypes

for example RP or the maculopathies

50
Q

a patient has AR condition

parents deny consanguinity

gene sequence shows he is homozygoud, how to explain?

A

due to ISODIZOMY (one parent is carrier and gave two copies)

could be de-novo but that is unlikely in AR

51
Q

when to use SNPs?

A
  • in mendelian disorders with variable expresitivity such as ABCA4
  • in linkage analysis
    • determine location of gene associated with phenotype
52
Q

are de-novo mutations more common in AR or AD?

A

in AD

AD-de-novo.

Genetics Fellowship (22 decks)

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