ASCP Molecular Diagnostics 1 — Questions and Answers

Question 1: In real-time PCR (qPCR), what does the Ct (cycle threshold) value represent?

The total number of PCR cycles run
The cycle at which fluorescence crosses a threshold above background (Correct answer)
The melting temperature of the amplicon
The concentration of primers used

Correct answer: The cycle at which fluorescence crosses a threshold above background

The Ct value is the PCR cycle number at which the fluorescence signal from a sample crosses a predefined threshold above background noise. Lower Ct values indicate more target DNA present. Higher Ct values indicate less target DNA.

In qPCR, fluorescent signal from SYBR Green intercalation or TaqMan probe hydrolysis is measured each cycle. The threshold is set in the exponential phase of amplification. Ct values are inversely proportional to initial template quantity: each 1-cycle decrease represents approximately twice as much target. Standard curves allow absolute quantification. Ct above 40 is generally considered negative. Applications include viral load quantification for HIV, HCV, and CMV, gene expression analysis, HER2 amplification testing, and minimal residual disease monitoring.

Question 2: Which molecular technique is used to detect chromosomal translocations such as t(9;22) (BCR-ABL) in CML?

Southern blotting
FISH and RT-PCR (Correct answer)
Western blotting
DNA microarray only

Correct answer: FISH and RT-PCR

Both FISH (fluorescence in situ hybridization) and RT-PCR (reverse transcription PCR) are used to detect t(9;22). FISH detects the chromosomal rearrangement; RT-PCR detects the BCR-ABL1 fusion mRNA and is used for quantitative minimal residual disease monitoring.

The Philadelphia chromosome t(9;22)(q34;q11.2) fuses the BCR gene on chromosome 22 to the ABL1 gene on chromosome 9, creating BCR-ABL1 fusion. FISH uses dual-fusion or break-apart probes to visualize the translocation in interphase nuclei for initial diagnosis. RT-PCR detects fusion mRNA such as p210 in CML or p190 in ALL; quantitative RT-PCR monitors treatment response and detects minimal residual disease. Imatinib resistance mutations including T315I are detected by sequencing.

Question 3: BRCA1 and BRCA2 are tumor suppressor genes involved in DNA repair. Mutations in these genes primarily increase the risk of:

Colorectal cancer only
Breast and ovarian cancer (Correct answer)
Leukemia
Thyroid cancer only

Correct answer: Breast and ovarian cancer

Pathogenic variants in BRCA1 and BRCA2 confer significantly elevated lifetime risk of breast cancer (approximately 70%) and ovarian cancer (approximately 44% for BRCA1, approximately 17% for BRCA2). BRCA2 also elevates pancreatic and male breast cancer risk.

BRCA1 and BRCA2 encode proteins involved in homologous recombination repair of double-strand DNA breaks. BRCA1 on 17q21: approximately 72% lifetime breast and 44% ovarian cancer risk. BRCA2 on 13q12: approximately 69% breast and 17% ovarian cancer risk, plus elevated pancreatic and prostate cancer risk. BRCA1 and BRCA2 mutations are found in approximately 5–10% of all breast cancers but approximately 15% of triple-negative breast cancers. PARP inhibitors such as olaparib exploit synthetic lethality in BRCA-deficient tumors.

Question 4: Which of the following best describes next-generation sequencing (NGS)?

Sequencing one DNA fragment at a time using chain termination
Massively parallel sequencing of millions of DNA fragments simultaneously (Correct answer)
DNA hybridization to a microarray
PCR amplification without sequencing

Correct answer: Massively parallel sequencing of millions of DNA fragments simultaneously

NGS (also called massively parallel sequencing) sequences millions of short DNA fragments simultaneously, providing high throughput and depth of coverage unavailable with Sanger (first-generation) sequencing.

NGS workflow: DNA fragmentation, library preparation with adapter ligation, cluster amplification, sequencing by synthesis, and bioinformatics alignment with variant calling. Platforms include Illumina (reversible dye terminators), Ion Torrent (pH changes), and Oxford Nanopore (ionic current). Applications include whole genome sequencing, whole exome sequencing, targeted gene panels for oncology or hereditary disease, pharmacogenomics, RNA-seq, and metagenomics. Depth of coverage of 100 to 1000 times determines sensitivity for low-frequency somatic mutations.

Question 5: Methylation-specific PCR (MSP) is used to detect which epigenetic modification?

Histone acetylation
DNA cytosine methylation at CpG sites (Correct answer)
RNA splicing variants
Protein phosphorylation

Correct answer: DNA cytosine methylation at CpG sites

MSP detects DNA methylation at CpG dinucleotides. Bisulfite treatment converts unmethylated cytosines to uracil (read as thymine after PCR), while methylated cytosines are protected. MSP primers distinguish methylated from unmethylated alleles.

CpG methylation (5-methylcytosine) in promoter regions silences gene expression. Aberrant promoter hypermethylation silences tumor suppressor genes in cancer: MLH1 in colorectal cancer, MGMT in glioblastoma, and BRCA1 in sporadic breast cancer. Clinical applications include: MGMT methylation predicting temozolomide response in glioblastoma, MLH1 methylation distinguishing sporadic from Lynch syndrome MSI-H colorectal cancers, and LINE-1 methylation as a global hypomethylation marker.

Question 6: Fragile X syndrome is caused by expansion of which mutation in the FMR1 gene?

Point mutation (missense)
CGG trinucleotide repeat expansion in the 5' UTR (Correct answer)
Gene deletion of entire FMR1
Translocation involving chromosome X

Correct answer: CGG trinucleotide repeat expansion in the 5' UTR

Fragile X syndrome results from more than 200 CGG trinucleotide repeats in the 5' UTR of FMR1, causing hypermethylation and gene silencing. Normal is 5–44 repeats; premutation is 55–200 repeats; full mutation is above 200 repeats.

FMR1 (Fragile X Mental Retardation 1) encodes FMRP, an RNA-binding protein essential for synaptic plasticity. Normal: below 45 CGG repeats. Intermediate or gray zone: 45–54 repeats. Premutation: 55–200 repeats — FMR1 mRNA is overproduced but protein is reduced; clinical presentations include premature ovarian insufficiency (FXPOI) and fragile X tremor-ataxia syndrome (FXTAS). Full mutation: above 200 repeats leads to promoter hypermethylation, FMR1 silencing, no FMRP, and resulting intellectual disability, autism features, and macroorchidism. Detection uses PCR for small expansions and Southern blot for full mutations.

ASCP Molecular Diagnostics 1

In real-time PCR (qPCR), what does the Ct (cycle threshold) value represent?