DLA6, DLA7- Molecular Pathology

Question 1

Hereditary Hemochromatosis results from a mutation in the (1) gene and is inherited in (2) fashion. (T/F) hemochromatosis has 100% penetrance. The most common mutation with the highest risk with presence of two alleles is (4).

Answer 1

1- HFE
2- autosomal recessive
3- F, <100% penetrance
4- C282Y (90%)

Question 2

explain Hereditary Hemochromatosis testing through traditional PCR

Answer 2

(about 3 hrs)

• amplification of DNA: cyles of denature (95C), anneal (68C), elongate (72C)
• add series of Restriction Endonucleases –> DNA lengths vary depending on RFLP (restriction fragment length polymorphism)
• fragments are run on Gel Electrophoresis –> separate by size
• under UV light, compare to standard

Question 3

explain Hereditary Hemochromatosis testing through Real-time PCR

Answer 3

(about 20 mins)

• in the presence of a specific primer used to investigate a single base position –> add Nucleotides complementary to mutant and wild-type bases, label with fluorophores
• at different melting temperatures (varies based on mutation) measure fluorescence of both signals to determine homogeneity of either, or heterogeneity

Question 4

list some inherited diseases that can be diagnosed by DNA based techniques

Answer 4

fragile X syndrome, CF, sickle cell, hemophilia, Huntington’s, Neurofibromatosis, Factor V Leiden

Question 5

BRCA1 genes is found on (1)

BRCA2 gene is found on (2)

Answer 5

1- chr.17, long arm (q) [band 17q21]

2- chr.13, long arm (q) [band 13q12.3]

Question 6

list the two ways to test for BRCA mutations and why they are used

Answer 6

1- automated Sanger sequencing
2- Next Generation Sequencing (NGS)

-there is >200 mutation types for both BRCA genes, therefore it is pointless to use PCR to search for individual mutations since there isn’t any common ones

Question 7

compare Sanger and Next Gen. sequencing

Answer 7

• Sanger: use normal dNTPs and ddNTPs (dideoxyribonucleic acids) to stop simultaneous transcriptions at every base pair, sequence based on length (Gel electro.)
• NGS: template DNA mounted to slides, add florescent dNTPs, wash away the unbounded, sequenced by fluorescent machine

Question 8

list some tumor susceptibility genes that patients can be screened for

Answer 8

• BRCA1, BRCA2 = breast/ovarian cancer
• RET = MEN syndrome
• P53 = Li Fraumeni syndrome
• APC = adenomatous polyposis

Question 9

HNPCC, aka (1) or (2), is a diseased based on a mutation of (3) genes, most commonly the following: (4)

Answer 9

1- hereditary non-polyposis colon cancer
2- Lynch syndrome
3- DNA mismatch repair genes
4- MSH2, MLH1, hMSH6/hPMS2/EPCAM

Question 10

list the traits or characterizations of HNPCC

Answer 10

(lynch syndrome)

• young age of Dx (colon cancer)
• synchronous and metasynchronous tumors in R colon
• tumor development in endometrium, ovary, stomach, small bowel, ureter, kidney
• presence of any of the above in 1st degree relatives

Question 11

HNPCC is diagnosed by the presence of (1) in DNA, which are defined as (2). During testing for HNPCC, (3) is commonly tested for due to its prevalence in patients with colorectal cancer.

Answer 11

1/2- MSI (microsatellite instability)- repeated sequences of 1-6 NTs in length
3- BRAF mutation

Question 12

Patients with HNPCC have a (1)% risk of developing colorectal and endometrial cancer. If mutations are discovered, surveillance includes (2) and (3). Additional surgical options include (4).

Answer 12

1- 80%
2- colonoscopy every 1-3 yrs, start at 20-25 y/o
3- transvaginal US, endometrial aspiration
4- colectomy with consideration for hysterectomy / salpingo-oophorectomy at same time

Question 13

Besides BRCA mutations, (1) mutations are also common in breast cancer and indicate the (2) features on breast cancer prognosis. Testing is performed through either (3) or (4). Patients with overexpression of (1) are treated with (5).

Answer 13

1- HER2 (human epidermal growth receptor 2), overexpression
2- aggressive features and shorten disease-free survival
3- immunohistochemical staining
4- FISH (inc number of HER2 genes on chromosomes)
5- Trastuzumab (Herceptin)

Question 14

FISH techiniques are useful in detecting malignancies including the following:….

Answer 14

• HER2 (breast cancer)
• Urothelial Carcinomas (aneuploidy, rearrangement, deletion, amplification of chromosomal DNA)
• Brain tumors due to oligodendrogliomas (50-80%) from 1p or 19q deletions

Question 15

(1) oncogene mutations are common in colorectal cancer and other adenocarcinomas. Presence of (1) also prevents response to treatment that targets (2). (1) is detected by (3).

Answer 15

1- KRAS
2- EGFR (anti-EGFR treatment)
3- DNA sequencing techniques (Sanger or NGS)

Question 16

describe the 3 types of gene mutations seen in NSCLC

Answer 16

(non-small cell lung carcinomas: squamous cell carcinoma, adenocarcinoma)

• EGFR- treat with TKIs (tyrosine kinase inhibitors)
• KRAS
• ALK (anaplastic lymphoma kinase)- treat with ALK inhibitors, test through immunocytochemistry or FISH

Question 17

40-60% of melanomas have a (1) mutation which leads to (2) activation. Most of these are a (3) specific mutation. Presence of (1) indicates (4) tumor prognosis. (1) is detected by (5).

Answer 17

1- BRAF mutation
2- MAP kinase activation
3- V600E
4- aggressive tumor
5- Real-Time PCR, Sequencing techniques (Sanger, NGS)

Question 18

CML, aka (1), is due to (2) mutation occurring which results in (3) activation which drives (4) downstream. Diagnosis is detected by (5). Treatment is through (6).

Answer 18

1- chronic myeloid leukemia
2- chimeric BCR-ABL gene fusion
3- constitutive BCR-ABL TK activation
4- GF indep. proliferation (w/ complete differentiation) of myeloid progenitors => inc platelets and granulocytes
5- double FISH (translocation)
6- Imatinib (TK inhibitor)

Question 19

____ is a great technique for testing infectious diseases

Answer 19

PCR- viral and bacterial assays completed (track infectious outbreaks)

Question 20

identity determination techniques (ex. paternal testing) mainly includes….

Answer 20

1) VNTRs (variable number tandem repeats): 20-20000 nts long, considered DNA fingerprinting
2) STRs (short tandem repeats): multiple tetranucleotide repeats, PCR amplification
3) mitochondrial DNA (maternal testing)