Genetics-Molecular Diagnostics

Question 1

Mutations that are due to misaggregation of chromosomes during meiosis or mitosis

Answer 1

Genome mutations, this results in an abnormal number of chromosomes.

Question 2

Euploid

Answer 2

of chromosomes is a multiple of the haploid genome (our haploid genome is 23 and we are diploid w/46 chromosomes)

Question 3

Aneuploid

Answer 3

of chromosomes not a multiple of 23

Question 4

Mutation caused by chromosome breakage, unequal crossing over or nondisjunction error.

Answer 4

Deletion. There can be interstitial and terminal deletions.

Question 5

A middle-aged woman presents with refractory anemia and an elevated platelet count. Histologic analysis reveals hypercellular bone marrow. What genetic abnormality is causing her condition?

Answer 5

She has myelodysplastic syndrome. This happens as a result of del(5q).

Question 6

A single chromosome undergoes 2 breaks and is reconstituted with the segment between the breaks.

Answer 6

Inversion. There are pericentric inversions and paracentric inversions.

Question 7

Cancer associated with inversion of chromosome 16

Answer 7

Acute Myelogenous Leukemia

Question 8

Breakage of non homologous chromosomes with exchange of broken segments

Answer 8

Reciprocal translocation

Question 9

2 types of reciprocal translocations

Answer 9

1) Quantitative (regulatory element drives oncogene) 2) Qualitative (abnormal function due to fusion of 2 genes)

Question 10

Most common type of translocation in B-cell lymphomas?

Answer 10

Quantitative. Usually and Ig heavy, kappa or light chain linked with an oncogene.

Question 11

Translocation involved in Ewing sarcoma?

Answer 11

Qualitative. t(11;22). EWSR1:FLI1 translocation generates an aberrant transcription factor that drives tumor growth.

Question 12

Misdivision of the centromere during mitosis or abnormal homologous cross over.

Answer 12

Isochromes. 2 arms that are the same on one chromosome.

Question 13

Medulloblastoma mutation

Answer 13

Isochrome i(17)q (2 q arms on chromosome 17)

Question 14

Yolk sac tumor mutations

Answer 14

i(12p) (2 p arms on chromosome 12)

Question 15

Chromosome undergoes two breaks and the broken ends reunite

Answer 15

Ring chromosome

Question 16

Liposarcoma mutation

Answer 16

r(12) (12 has two breaks and the broken ends fuse to form a ring)

Question 17

Neuroblastoma mutation

Answer 17

Double minutes. (Small accessory chromosomes)

Question 18

3 types of point mutations

Answer 18

Missense (AA substitution), Nonsense (premature stop), RNA processing mutation (abnormal splice site, cap site and polyadenylation site)

Question 19

Gene mutations seen in polycythemia vera, essential thrombocytopenia, primary myelofibrosis and chronic myelogenous leukemia.

Answer 19

Missense mutation of JAK2 results in increased proliferation and survival of erythroid and myeloid cells, causing myeloproliferative neoplasms.

Question 20

Gene mutation commonly sen in colorectal carcinoma and non-small cell lung carcinoma. How do you treat?

Answer 20

KRAS point mutation causes abnormal signaling downstream from EGFR, can’t treat with EGFR therapies.

Question 21

A mutation that disrupts the triplet reading frame

Answer 21

Frameshift mutation from a deletion, insertion or replication.

Question 22

Mutation that commonly affects EGFR in non-small cell lung carcinoma.

Answer 22

Exon deletion that causes constitutive activation of the tyrosine kinase receptor and over activation of EGFR.

Question 23

Most important prognostic predictor in patients with acute myelogenous leukemia and a normal karyotype.

Answer 23

FLT3 duplications cause constitutive activation of a tyrosine kinase receptor and poor prognosis. NPM1 duplication in exon 12 = good prognosis. Note that some of the cytogenic abnormalities have a better prognosis and some have a worse prognosis.

Question 24

Common mutation that affects breast cancer

Answer 24

HER2 amplification. Part of erbB family of tyrosine kinases. Poor prognosis.

Question 25

How does FFPE affect molecular diagnostics?

Answer 25

“Formalin-fixed paraphin-embeded” It fragments DNA and RNA so you can’t find genes

Question 26

Hybridization for finding abnormal genes

Answer 26

Make a complementary probe for the gene you are looking for. The probe fluoresces so you can see the gene is present (Fluorescence in Situ Hybridization)

Question 27

3 types of FISH probes

Answer 27

1) Locus specific indicator probes (LSI), looks for specific genes 2) Chromosome enumeration probes (CEP), looks for centromeres 3) Chromosome painting probes, looks for specific chromosomes

Question 28

Chronic myelogenous leukemia mutation. How do you detect it?

Answer 28

t(9;22). This is the Philadelphia chromosome that has fusion of the bcr:abl gene, which drives proliferation of myeloid cells. You detect by dual color single fusion strategy (2 fluorescent probes are close to one another that are normally on different chromosomes)

Question 29

What FISH method is less likely to occur by chance when detecting translocations?

Answer 29

Dual color dual fusion. You put the marker across the break point of the chromosomes involved in the translocation.

Question 30

Putting the gene probe on both sides of a chromosome pair to assess for translocation

Answer 30

Dual color break-apart strategy

Question 31

Steps of PCR

Answer 31

Denature DNA, hybridize and anneal primers, bring to 72 degrees C so TAC polymerase can form an extension copy.

Question 32

Reverse transcription PCT

Answer 32

cDNA formed from mRNA template. DNA strand formed w/cDNA strand. Amplification of DNA.

Question 33

Using PCR as a modality monitor recurrent of lymphoma

Answer 33

Lymphoma can arise due to a fusion of 2 genes after their chromosomes break. You can make a fusion RNA transcript that shows the two genes right next to each other, indicating presence of the mutation

Question 34

How do you analyze PCR?

Answer 34

Gel electrophoresis, capillary electrophoresis (mass spec)

Question 35

Germline mismatch repair gene mutation seen in hereditary colon, endometrial and gastric cancers. How do you screen for it?

Answer 35

Lynch syndrome. Screen with microsatellite instability or immunohistochemistry testing.

Question 36

What are microsatellites?

Answer 36

Short tandem repeats of 1-6 DNA base pairs throughout the genome.

Question 37

What enzymes typically correct the little replication errors and mutations made by DNA polymerase? How does this relate to Lynch syndrome?

Answer 37

DNA MMR (mismatch repair enzymes). The enzyme has four proteins, MSH2, MLH1, PMS2 and MSH6. If any of these are mutated you get Lynch syndrome, which will manifest as microsatellite instability on PCR (varying sizes of different microsatellites)

Question 38

Real-time PCR

Answer 38

Running PCR and fluorescent tag emits light as it is released from the transcript by the polymerase. It will emit light in proportion to the amount of gene product that is present.

Question 39

Common uses of PCR for leukemia and lymphoma

Answer 39

Antigen receptor gene rearrangements. This tells you if the lymphoid cells are monoclonal or polyclonal or B-cells or T-cells.

Question 40

How can immunoglobulin gene rearrangement help to detect neoplastic B-cells.

Answer 40

Normal B cell IgH undergoes diverse rearrangement made of V,D,J and C segments. Tumor cells all inherit the same Ig coding sequence.