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Flashcards in Genetics Deck (51):

specimen requirements:

sodium heparin
core biopsy
ffpe tissue acceptable for FISH unless it has been decalcified


specimen requirements:
G banding

Requires dividing cells to be able to examine chromosomes during metaphase


specimen requirements:

Cells need not be dividing


specimen requirements:

ACD tube or EDTA purple tube

if unsure of test to order up front request extraction of both dna and rna


specimen requirements:

used for detection of mutations within a gene


specimen requirements:

used to detect fusion transcripts resultsing from translocations (used most in molecular test in leukemias)


Reasons to do a cytogenetic study?

1. diagnose constituional disorders (trisomy 21)
2. add further diagnostic or prognostic info to an already diagnosed disorder (malignancy)


Why does cytogenetics require cells to be cultured?

so they can undergo mitosis

Lymphocytes are differentiated cells that normally do not undergo spontaneous cell division.
In culture process a mitogen is added to stimulate cells to replicate DNA and enter mitosis. THe lylmphocytes remain in culture 48-72 hours.


How can tissue cultures be synchronized?

By adding Methotrexate
 Blocks cells in S phase; this allows more of the cells in culture to enter and remain at S phase
 Thymidine releases the MTX block; cells then proceed synchronously to mitosis


How is mitosis stopped in metaphase?

Colcemid is added to prevent synthesis of spindle fibers (this stops cell in metaphase)

Best time to see and examine chromosomes


Constitutional chromosomal abnormalities

"Constitutional anomalies" herein refers to the chromosome inborn syndromes, such as trisomy 21, Turner syndromes, and others.


Acquired chromosomal abnormality

ACQUIRED: only one organ is involved, the other tissues being normal. The patient has a cancer of the affected organ.


What is the difference between a balanced vs unbalanced strucutral abnormality?

Translocations can be balanced (in an even exchange of material with no genetic information extra or missing, and ideally full functionality) or unbalanced (where the exchange of chromosome material is unequal resulting in extra or missing genes).


Is there an increased risk for miscarriage after amnio?



What is an alternative to amniocentesis?

first and second trimester screening for chromosome conditions


How does a screen work?

the raw values of serum analytes are converted to multiples of the median (MOM). How much the value deviates from the average for a given gestational age.

**A multiple of the median (MoM) is a measure of how far an individual test result deviates from the median. MoM is commonly used to report the results of medical screening tests, particularly where the results of the individual tests are highly variable.


What is the 2nd trimester quad screen?

Measures four analytes in maternal serum to adjust the age-related risk for:
trisomy 21
trisomy 18
open neural tube defects.


What are the benefits of first trimester screening?

– Patients get information earlier in pregnancy (CVS as a diagnostic option)
– More sensitive and specific than second trimester screening methodologies
– Better able to provide individualized risks in twin pregnancies.


What are serum/US biomarkers used for?

assign patients a specific risk on a spectrum

a THRESHOLD is identified to separate high risk and low risk populations and identify appropriate candidates for diagnostic testing


What are screening markers in a first trimester US?

nuchaltranslucency, nasal bone, tricuspid regurgitation, fronto‐ maxillary angle


What are screening markers in first trimester biochemistry?

PAPP‐A,free‐BhCG, total hCG, hyperglycosylated hCG, ADAM12


What are screening markers in second trimester biochemistry?

AFP,hCG,inhibin, estriol, ITA


What is the difference between amniocentesis and screening?

amnio: definitive diagnosis/exclusion, risk of miscarriage

screening: risk assessment only, no risk of miscarriage


what is cell free fetal DNA in maternal plasma and what are its characteristics?

• Fetal DNA is present in maternal plasma
• Usually comprises <10% of the total free DNA
in plasma, the remainder is maternal DNA.
• Generally small fragments (?derived from apoptotic cells at maternal/fetal interface)
• Very short half‐life (16 minutes). Rapidly cleared from maternal circulation after delivery.


How do you specifically analyze the fetal portion of cell free DNA when it usually constitutes <10% of the total cell free DNA?

1. Look for fetal alleles that are known to be absent in the mother

2. Analyze mRNA of placenta specific genes (quantitative PCR of polymorphic positions)


What are problems with the approach of looking at the fetal portion of cell free DNA to look for abnormalities?

• You need placenta specific‐genes
• These genes need to be in clinically relevant regions of the genome (i.e. chromosome 21)
• There must be polys within gene that are sufficiently heterozygous to be informative.
• You need to identify more than one poly that meets these requirements in case polys are not informative
• Assume equal expression from both alleles.


What is massively parallel sequencing?

• “shotgun sequencing” of all cell free DNA in maternal plasma
• No effort is made to distinguish fetal from maternal DNA
• Identify trisomies by detecting increased numbers of reads that map to chromosomes 13,18,or 21.


What is next generation sequencing?

1. whole genome is sheared into random DNA fragments
2. adaptors (contain PCR primer sequence that hybridizes to flow cell and molecular "bar codes") are ligated to both ends of fragments
3. The surface of the flow cell is coated in DNA polymers that are complementary to the adaptors
4. The number of reads mapping to each chromosome should be a DIRECT FUNCTION of HOW LARGE the chromosome is


If chromsome 21 contains less than 1% of the genome, then ____ of your reads shoudl map to chromosome21.



What is problematic about mapping reads onto chromosomes?

1.We can’t distinguish maternal from fetal reads
2.The absolute number of reads mapping to 21 will vary from run to run, so we care more about what proportion of the total reads map to 21.


What is the NCV?

z score or normalized chromosome value

a measure of how many standard deviations the value is from the median


 25 y.o. @ 20w5d gestation
 Normal quad screen
 Aneuploidy markers: echogenic
intracardiac focus (risk for DS 1/1444)
 R kidney dysplastic, multicystic
 Sent blood for NIPS -- c/w Tri 13, 21, monosomy 18

What are possible explanations?
Next steps?

 Bioinformatics issue (false positive)
 Baby has complex cytogenetic abnormality
 Maternal chromosome abnormality

Next steps:
Simultaneous chorionic villus sampling and amniocentesis
 Evaluate for confined placental mosaicism
 Maternal blood drawn for peripheral smear


What is therapy for t lymphoblastic leukemia?

 Typical therapy:
Aggressive induction chemo, prolonged maintenance chemo +/‐ hematopoietic stem cell tx
 Contraindicated due to pregnancy:
Methotrexate, asparaginase, bactrim prophylaxis

Ultimate therapy for pregnant pt:
 Transferred to Pediatric Oncology
 Modified COG AALL0434 protocol
 Cytarabine, vincristine, daunorubicin
 Omitted PEG asparaginase and MTX
 Planned duration 27 months


30-year-old female presents with a paternal family history of breast and ovarian cancer.. BRCA testing is performed and the test results are normal. What is the correct interpretation of this test result for this patient?

“4”- her test result does not change her risk assessment based upon family history---which in this case, would make “3” correct also---she should still have high risk screening for breast/ovarian cancer.


30-year-old female presents with a paternal family history of breast and ovarian cancer.. BRCA testing is performed and the test results are normal.

What are possible interpretations of a normal result in this scenario?

1. This family has a BRCA1/2 associated familial cancer syndrome, but current testing is not sensitive enough to identify the mutation in this family.
2. This family has a hereditary breast cancer syndrome that is not caused by mutations in the BRCA1/2 genes (i.e. Cowden or Li Fraumeni syndromes).
3. This family may have a BRCA1/2 mutation that can be identified by current technology and this young woman has not inherited the familial risk.


What is a more effective strategy to identify a familial mutation?

Test a family member to identify the familial mutation

If pt is tested by same methodology and familial BRCA1 mutation is absent the familial risk is excluded and patient is returned to population risk


Can genetic testing definitively exclude a familial risk?

ONLY if you have proven that the testing can positively identify the genetic risk in an affected family member


What is CF?

• Autosomal recessive disorder
• Common in Caucasian populations (both with and without Jewish ancestry)
• Relatively uncommon in the rest of the world.
• Caused by mutations in the CFTR gene


What is the incidence of carrier CFTR mutations in the general population?

• More than 1000 different mutations have been reported in the CFTR gene.
• 1/25 Caucasian individuals is a carrier of CF.
• 1/29 Ashkenazi Jewish individuals are carriers.


How do we effectively screen these populations when there are so many possible mutations?

Comprehensive gene sequencing

Focused mutation panel


What are the advantages and disadvantages of comprehensive gene sequencing?

•Comprehensive-will detect >99% of carriers

•Longer turnaround
•May generate results that are difficult to interpret (variants of uncertain significance).


What are the advantages and disadvantages of a focused mutation panel?

advantages: cheap, fast , exclude a handful of common muations, no variants of uncertain significance

disadvantages: will miss some carriers, may perform better in some populations vs others


How are mutations chosen for CF?

• Mutations must be clearly pathogenic.
• Mutations must account for >0.1% of disease alleles in the target population.
• Based upon these criteria, 25 mutations were initially included in the standard CF mutation panel.


When shoiud you choose the limited panel to test for CF?

Woman presents for prenatal care in the first trimester. She reports Caucasian ancestry and no known family history of CF. She requests general population carrier screening.


When should you choose the comprehensive test to test for CF?

Infant screens positive for cystic fibrosis on newborn screen. Initial sweat chloride test is abnormal, confirming the diagnosis. Testing by the standard mutation panel only reveals one mutation.


If you are using a CF carrier test to exclude familial mutations you must be sure ...

that the familial mutations are included in the panel that you are using!


What are hte two most common inherited thrombophilias in the caucasian population?

Factor V Leiden and prothrombin G20210A mutations are the two most common inherited thrombophilias in the Caucasian population.


What is gene heterozygosity?

• Heterozygosity means only one copy of the gene has a mutation
– Most common situation for all inherited thrombophilias.
– Each child has a 50% chance of inheriting the thrombophilia.
– Mutation is present in one parent, usually not both.
– Sibs have a 50% risk of also being heterozygous.


What is homozygosity?

• Homozygosity means that both copies of the gene have the same mutation.
– Sometimes seen for FV Leiden or prothrombin mutation.
– A homozygote will always pass ONE copy of the mutation to children, but not both.
– A homozygote got one mutation from each parent (both parents AT LEAST heterozygous).
– Siblings may be homozygous, heterozygous, or “normal.”


What is compound heterozygosity?

both copies of the gene have a mutation, but they are different mutations


What is doubly heterozygous?

Patient is heterozygous for two DIFFERENT thrombophilia risk factors
– Example-patient is heterozygous for factor V Leiden and prothrombin G20210A mutation.
– Because mutations are in different genes, it is possible that a child could inherit BOTH mutations, one mutation, or neither.
– Mutations may have both come from the same parent, or each parent may carry one mutation.