Medical Laboratory Science Review Harr 8.2 Molecular Diagnostics: Molecular Diagnostics

A FISH test is performed on a slide of peripheral blood leukocytes. Te test uses a dual fusion probe, consisting of a Spectrum Green labeled probe to the BCR 22 q11.2 locus, and a Spectrum Orange labeled probe to ABL 9q34. What disease is this test for? A. Chronic myelogenous leukemia B. Multiple myeloma C. Bladder cancer D. Tyroid cancer

A. Chronic myelogenous leukemia A Although all of these cancers involve chromosomal ploidy or gene rearrangement that can be detected by FISH, the BCR/ABL translocation is found in > 95% of CML and 25% of AML patients and in rare cases of chronic neutrophilic leukemia. There is a relationship between the type of leukemia and the portion of the BCR locus involved in the translocation. In CML, the major (M BCR) portion of the gene is involved, giving rise to a 210 dalton chimeric protein. In ALL, the minor (m BCR) portion is involved, giving rise to a 190 dalton chimeric protein. In CNL, an extended region beyond the M region, called the μ BCR region, is involved, giving rise to a 230 dalton chimeric protein. Also, variants in BCR/ABL exist, giving rise to different FISH patterns. Some variants are associated with essential thrombocythemia that occurs at disease onset in a small percentage of CML patients

In general, which statement best characterizes the relationship between white blood cells and light scattering in flow cytometry? A. Forward scatter is related to cell size and side scatter to granularity B. Forward scatter is related to nuclear density and side scatter to size C. Forward scatter is inversely related to size and side scatter is directly related to size D. Forward scatter is related to shape and side scatter to size

A. Forward scatter is related to cell size and side scatter to granularity A Forward scatter of light from a laser directed through the aperture of the cytometer is directly related to cell size. Right angular scatter (side scatter) is dependent upon the number of granules inside the cytoplasm. For example, small lymphocytes that are agranular have the lowest forward and side scatter and are easily identified as the cluster of cells closest to the bottom and left of the scatterplot.

A cell population is positive for surface markers CD45, CD3, CD4, and Tdt. Which type of leukocytes are these? A. Lymphocytes B. Granulocytes C. Monocytes D. Early myeloid precursors

A. Lymphocytes A CD45 is a panleukocyte marker and reacts with all white blood cells and precursors. CD3, CD4, and Tdt are markers for T lymphocytes. Typically, a panel of 12 or more antibodies is used to characterize the lineage and maturity of a cell population. The abbreviation CD stands for cluster of differentiation. Monoclonal antibodies with the same CD number recognize the same marker, although the specific moiety they react with may be different.

Which mechanism is responsible for retinoblastoma? A. Mutation of a tumor suppressor gene B. Mutation of a tyrosine kinase gene C. Activation of an oncogene D. Deletion of a gene encoding a GTPase activator

A. Mutation of a tumor suppressor gene A A mutation or deletion of a tumor suppressor gene such as p53, p14, or RB1 (the retinoblastoma gene) causes loss of a protein that inhibits mitosis and is associated with an increased risk of malignancy. Mutations of p53 occur frequently in several cancers, including lung, breast, liver, and colon cancer. RB1 mutations are associated primarily with retinoblastoma, a tumor of the retina occurring in young children. Although they may be inherited, mutations usually arise in somatic cells. Mutations that produce more active proteins with tyrosine kinase activity such as HER-2/neu are oncogenic because they stimulate the signal transduction pathway for mitosis. Likewise, a deletion of a GTPase activator is also oncogenic, since it permits higher levels of intracellular GTP, which is involved in the same pathway

Which statement best describes the relationship between HLA DNA typing and serological haplotypes? A. One or two bands are seen for each locus correlating to reactivity with a specific antigen or group of antigens B. HLA alleles cannot be related to HLA antigens because antisera specificity is unrelated to genetic polymorphism C. A single antibody specificity always corresponds to a single allele D. Not all HLA genes produce antigens recognized by antibodies

A. One or two bands are seen for each locus correlating to reactivity with a specific antigen or group of antigens A Antibodies to HLA antigens recognize determinants that may be shared by several polymorphisms. However, it is possible to correlate primer specificities to gene products that react with commercial HLA typing seras. For example, DR103 correlates with the primer recognizing DRB1*0103. On the other hand, alleles DRB3*010101-10, DRB3*0101-14, and DRB3*030101-03 will all react with antisera to DR52.

HLA typing can be done by which molecular method? A. PCR analysis using 96 well microtrays with allele or groups specific primers in each B. Restriction fragment length polymorphism testing C. Direct hybridization with WBCs on a peripheral blood film D. Fluorescent in situ hybridization reactions with peripheral blood lymphocytes

A. PCR analysis using 96 well microtrays with allele or groups specific primers in each A The DNA is extracted from peripheral blood leukocytes, added to the master mix, and an aliquot is transferred to each well of a 96-well plate. Each well contains a primer to a specific base sequence of one allele or allele group. Gel electrophoresis is performed after PCR to identify those wells that contain amplified products. Each well also contains a primer to a second nucleotide sequence, such as a region of the growth hormone gene that serves as a PCR internal control. Bands are stained with ethidium bromide and can be visualized by direct observation with a near ultraviolet light source.

Polymorphisms of the cytochrome p450 genes are important in identifying which condition? A. Poor drug metabolism B. Risk for primary biliary cirrhosis C. Progression of hepatitis C to hepatic cirrhosis D. Parentage in cases where HLA results are inconclusive

A. Poor drug metabolism A Pharmacogenetics (sometimes called pharmacogenomics) is the study of the role inheritance plays in the metabolism of drugs. Individual differences in drug metabolism can be attributed in part to polymorphisms in the genes coding for enzymes comprising the cytochrome p450 system. Of the more than 100 CYP genes, seven are principally involved in drug metabolism. Of these, CYP2D6, CYP2C9, CYP2C19, and CYP2A6 are polymorphic genes that account for metabolism of approximately 40% of drugs. Phenotypical expression varies with the locus involved. For the CYP2C19 locus that metabolizes several dozen drugs—including some tricyclic antidepressants, antiepileptics, and acid reflux inhibitors—persons who inherit one copy of the wild-type gene metabolize normally, whereas homozygotes or double heterozygotes for any of the seven polymorphisms metabolize poorly. For CYP2D6—which metabolizes tricyclic antidepressants, antipsychotics, antihypertensives, and several other drugs—heterozygotes with one wild-type gene have intermediate and those with no wild-type gene have poor drug metabolism. Persons with poor metabolic efficiency are at a greater risk of drug toxicity. On the other hand, their response to some antibiotics may be more positive.

What method is used to identify maternal cell contamination in amniocentesis and chorionic villus samples (CVS)? A. STR analysis B. FISH C. Microarray analysis D. MicroRNA (MiRNA) analysis

A. STR analysis A Maternal cell contamination can result in misinterpretation when performing genetic testing directly on uncultured CVS or amniotic fluid cells. FISH can identify maternal cell contamination if the fetus is male but not female. STR analysis using 5 loci can detect maternal cell contamination as little as 1%. A level of maternal contamination below 1% does not guarantee accuracy, but misinterpretation due to maternal contamination is unlikely. Negative genetic tests can be reported, but positive result should be confirmed using cultured cells. Maternal contamination is more common from CVS than amniotic fluid samples. MiRNAs are small RNA molecules that bind to mRNA and block their translation. There are about 500 miRNAs in human cells and their expression has been used to identify the tissue of tumor origin.

In flow cytometry, the term "gating" refers to: A. Selection of a subpopulation of cells to count B. Determining the fluorescent emission spectrum of cells of interest C. Interference caused by binding of more than a single antibody D. Selecting the appropriate counting aperture

A. Selection of a subpopulation of cells to count A In flow cytometry, cells can be divided into subpopulations based upon their light-scattering properties. Cells to be interrogated by the laser(s) are selected by identifying the area in which they appear on a scatterplot.

Which is the most common method used for parentage testing in the United States? A. Short tandem repeat analysis B. Nuclear DNA sequencing C. HLA DNA typing D. Mitochondrial DNA sequencing

A. Short tandem repeat analysis A DNA testing is the primary method of determining parentage because it is 100% accurate in exclusion and > 99.9 % accurate for inclusion of parentage. DNA testing is at least 10-fold more conclusive than the combination of HLA, blood group, and protein markers, and DNA samples can be tested prenatally, neonatally, and postmortem. Testing is performed on nuclear DNA because mitochondrial DNA is inherited exclusively from the mother. Rather than testing for base sequence variations within genes, DNA is tested for length polymorphisms. These are short base sequences within the introns that repeat. The number of times the sequence repeats is inherited as a trait. Short tandem repeats (STRs) are oligonucleotide sequences of four or five base pairs. Usually, 12 STR loci are amplified by PCR using specific oligonucleotide primers labeled with fluorescent dyes. The products are detected by capillary electrophoresis. The size of the fragments and their fluorescence determine which alleles are present.

What gene must be amplified in PCR to differentiate methicillin-resistant Staphylococcus aureus from methicillin-resistant coagulase-negative Staphylococcus? A. orfX B. mecA C. VanA D. iles-2

A. orfX A The mecA gene codes for resistance to methicillin in S. aureus and coagulase-negative Staphylococcus. In order to differentiate mecA-positive S. aureus from coagulase-negative Staphylococcus such as Staphylococcus epidermidis that are often present together in clinical specimens, the orfX gene specific for S. aureus is also amplified by multiplex PCR. A molecular beacon binds to the amplicon, causing fluorescence in real-time PCR, indicating the presence of both mecA and orfX products. The VanA gene codes for vancomycin resistance but is not found in S. aureus. The iles-2 gene codes for resistance to mupirocin in S. aureus

Which of the following thalassemias can be detected by PCR followed by blotting with a single specific oligonucleotide probe? A. α-Thalassemia B. Hemoglobin S/β-thalassemia C. β-Thalassemia D. Hemoglobin S/β-thalassemia

A. α-Thalassemia A α-Thalassemia carriers have a full or partial deletion of one or two of their four globin genes. Genotyping can determine whether two deletions are cis or trans and is performed by PCR using primers that are specific for the four most common deletions. β-Thalassemia may be caused by single base substitutions, deletions, or mutations in the flanking regions of the β-gene. Over 200 different mutations have been described, and 20 are relatively common. Microarray analysis is required to detect these.

How can cell proliferation be explained by the BCR/ABL gene rearrangement that occurs in the 9:22 translocation that causes the Ph1 chromosome of CML? A. It causes underexpression of p53 B. A hybrid protein is made that up-regulates the cell cycle C. Translocation induces a point mutation in the ABL oncogene D. ABL activates p23

B. A hybrid protein is made that up-regulates the cell cycle B Cancers are caused by genetic damage to cells that disrupt the cell cycle. Cell proliferation can be induced by under-expression of genes with tumor suppressor properties (e.g., p53) or overexpression of oncogenes (e.g., p21) that increases cell signaling, transcription, and mitosis. In CML, translocation of the ABL oncogene from chromosome 9 to the 3´ end of the BCR (breakpoint cluster region or area where recombination occurs) of chromosome 22 results in production of a hybrid BCR/ABL mRNA. This produces a chimeric protein with increased tyrosine kinase activity, causing the cell to enter G1. FISH can be used to identify cells with the BCR/ABL translocation. DNA probes specific for ABL and BCR are labeled with two different fluorescent dyes. In normal cells, each dye produces two colored spots (e.g., red and green) on chromosome pairs 9 and 22. If a BCR/ABL translocation is present, the probes bind next to each other, producing a spot of a different color (e.g., yellow).

In order to prove exclusion in DNA paternity testing, why must two genes be identified that must come from the biological father and did not? A. A single exclusion can result from laboratory error B. A single exclusion can result from germ line mutation within one locus being tested C. The biological father may be a blood relative to the alleged father D. The biological mother may be different than the purported mother

B. A single exclusion can result from germ line mutation within one locus being tested B Two exclusions are needed rather than one to be 100% certain of nonpaternity because of the rare possibility of a mutation having occurred in one of the loci being tested. Loci used for DNA testing are sufficiently polymorphic that the mother's sample is not necessary to determine paternity. Exclusion is based on the premise that the biological father must have at least one allele in common with the child at each locus.

Which of the following parameters are used to gate cells processed by the flow cytometer? A. Font surface fluorescence versus incident laser intensity B. Forward light scatter versus side scatter C. The ratio of light emitted at two different wavelengths D. Impedance amplitude versus background conductance

B. Forward light scatter versus side scatter B The gated population is selected by evaluating the scatterplot of forward light scattering (x axis) and right angular or side scatter (y axis). Cells falling within the specified limits are counted. For example, monocytes can be differentiated from neutrophils because the former have greater forward scatter and less side scatter.

How are cases of Duchenne's muscular dystrophy not detected by PCR usually confirmed? A. DNA sequencing B. Linkage analysis C. Macroarray analysis D. Dystrophin protein staining

B. Linkage analysis B The majority of gene deletions associated with Duchenne's muscular dystrophy is detected by PCR using multiple primers (multiplex PCR). The others are usually detected by indirect gene analysis. An alternative to testing for these mutations is linkage analysis. This process follows other genetic markers located near the disease gene so that crossing over is improbable. Linkage analysis for an X-linked disease or an autosomal recessive disease such as cystic fibrosis requires DNA from at least one affected family member. However, linkage analysis for an autosomal dominant disease such as Huntington's disease requires DNA from at least two family members. Next generation gene sequencing (several technologies that are alternatives to Sanger sequencing) have been used to identify deletions of the dystrophin gene that cannot be detected by PCR.

Which oncogene is involved in the etiology of Burkitt's lymphoma? A. ABL B. Myc C. Ras D. HER/neu

B. Myc B Burkitt's lymphoma is associated with a translocation involving the long arm of chromosome (8 on which the c-myc gene is located) with one of three immunoglobulin genes. The translocation most often involves the IgH gene on chromosome 14. The result is a hybrid mRNA that produces the c-myc protein whenever the immunoglobulin gene is transcribed. The c-myc protein is an activator of genes involved in mitosis.

Which method of DNA analysis is used most often to detect the hemoglobin S gene? A. FISH B. PCR followed by RFLP C. Cytogenetic analysis of chromosome 11 D. Labeled probe painting of chromosome 11

B. PCR followed by RFLP B The β-globin gene is located near the end of the short arm on chromosome 11 and consists of three exons and two introns constituting 1,600 base pairs. The substitution of valine for glutamic acid at position 6 of the protein is the result of a single-point mutation at position 6(A3) in exon 1, in which GAG is replaced by GTG. In hemoglobin C, the same codon is mutated but the substitution involves the preceding base at the 5´ end (GAG is changed to AAG). The hemoglobin S mutation alters the restriction site for MstII, preventing the enzyme from cutting the DNA. This causes production of a fragment that is 200 base pairs longer than seen for the normal β-gene. Most commonly, PCR is used to amplify a portion of the exon containing the S mutation, and MstII is used to digest the PCR product. Heterozygotes produce one normal and one longer band, whereas homozygotes produce a single band that is 200 base pairs longer than the normal amplicon. Alternatively, PCR is performed followed by Southern blotting, using specific oligonucleotide probes for hemoglobin A and S.

p21 is a GTP binding protein produced by which oncogene? A. RET B. Ras C. HER-2/neu D. N-Myc

B. Ras B All of the genes are oncogenes. RAS is a group of three genes that produce GTP-binding proteins, which activate transcription by up-regulating the signal transduction pathway of the cell. RAS is implicated in lung, breast, colon, and other carcinomas. It is measured by RT-PCR, which quantifies the amount of mRNA present in the malignant cells.

Approximately how may mutations have been identified in the gene coding for the cystic fibrosis trans membrane conductor regulator protein (CFTR)? A. 10 B. 100 C. 1,000 D. 10,000

C. 1,000 C The CFTR protein regulates the movement of chloride across the cell membrane, and a defect in this protein results in cystic fibrosis (CF). The CFTR gene is located on the long arm of chromosome 7 and consists of 27 exons spread over 230,000 bases. The most common mutation is a deletion of three base pairs that code for phenylalanine at position 508 of the protein, ∆F508. This mutation accounts for 70% of CF genes in Whites. It causes a severe form of CF involving pancreatic insufficiency. No single test can detect all possible CF carriers and a core panel consisting of 25 probes is recommended for initial screening. The core panel is used to screen for carriers of the CF gene and can detect more than 85% of CF mutations. Since two mutations are required to produce CF, the core panel can detect approximately 80% of CF.

Inheritance of BRCA1 or BRCA2 mutations increases the risk of breast and ovarian cancer by which mechanism? A. Oncogene production B. Transcription signaling by the mutant protein C. Deficient tumor suppressor function D. Chimeric protein production

C. Deficient tumor suppressor function C BRCA1 and BRCA2 are mutations of genes that produce tumor suppressor proteins. These down-regulate cell signaling events that lead to cell division. The mutations are inherited as autosomal dominant traits and are associated with > 85% lifetime risk at age 70 of developing breast cancer if one is found in a person with a positive family history.

The majority of cases of Duchenne's muscular dystrophy are caused by which type of genetic damage? A. Point mutation B. Insertion C. Deletion D. Trinucleotide repeats

C. Deletion C The dystrophin gene is approximately 2.5 million bases and has extensive sites at which both large and small deletions, insertions, and point mutations can occur. Approximately 60% of cases are caused by deletions that can be detected by the absence of one or more PCR products produced by the normal gene. The remaining 40% can be caused by microdeletions,point mutations, or insertions that are not usually detected by available primer sets

Which method is most useful for confirmation that a culture isolate is Group B streptococcus? A. Southern blotting B. Polymerase chain reaction C. Direct hybridization D. Probe capture assay

C. Direct hybridization C In direct hybridization, a specific labeled probe reacts directly with the sample. Since a colony or pure broth culture of a primary isolate represents the progeny of a single bacterium, there is no need for the use of Southern blotting. The quantity of DNA available for testing is sufficient, so that amplification methods such as PCR or probe capture hybridization are unnecessary. The colony or broth isolate is lysed, and a hybridization solution is used to promote denaturation. The sample is heated above the melting temperature, and a DNA probe is added that hybridizes with bacterial DNA or ribosomal RNA. The probe is conjugated to a chemiluminescent label. A reagent is added to neutralize the unbound probe, and H2O2 and NaOH are added to cause chemiluminescence. The signal is read in a luminometer and compared to a cutoff value. Such tests take approximately 1 hour to perform and most are 99%-100% sensitive and specific.

Which of the following genetic diseases is caused by an expanded trinucleotide repeat? A. Prader-Willi syndrome B. Angelman's syndrome C. Fragile X syndrome D. Williams' syndrome

C. Fragile X syndrome C Prader-Willi and Angelman's syndromes are most often caused by microdeletion, and Williams' syndrome is caused by a microdeletion in the gene coding for elastin. Fragile X syndrome, Huntington's disease, and myotonic dystrophy are examples of diseases caused by an expansion of trinucleotide repeats. Fragile X is so named because when cells from an affected individual are cultured in folate-deficient medium, the long arm of the X chromosome appears to have a break caused by deficient staining. The Xq27 region contains a CGG tandem sequence that can repeat up to 50 times in normal individuals. In fragile X syndrome, the repeat is extended and its length determines whether the affected persons will show mental retardation. Repeats of 50 to 230 times are associated with a carrier (premutation) state. During meiosis in females, the CGG repeat can undergo further expansion. The probability of this expansion increases with each generation. As the size of the repeat increases, so does the chance that it will cause methylation of the promoter for the FMR1 gene. The gene is needed for normal brain function and its underexpression results in mental retardation. Females in whom the premutation expands in size to a full mutation transmit the syndrome to all of their male and half of their female offspring

In situ hybridization (ISH) tests for human papilloma virus (HPV) using cervical smears differ from immunochemical staining of tissue in which regard? A. ISH has lower analytical sensitivity B. ISH has lower analytical specificity C. ISH differentiates subtypes more easily D. ISH differentiates cervical neoplasia from genital warts

C. ISH differentiates subtypes more easily C In situ hybridization using probes that anneal with specific subtypes of HPV are able to distinguish the subtype of virus most commonly responsible for sexually transmitted warts and associated with neoplasia. Positive reactions can be detected by light microscopy using probes conjugated to biotin. After the hybridization reaction, the slides are washed to remove the unbound probe, and streptavidin conjugated to horseradish peroxidase is added. Addition of hydrogen peroxide and aminoethylcarbazole results in the formation of a reddish-brown precipitate. Sensitivity is approximately 88% and specificity 99%, which is higher than for histochemical immunoperoxidase staining. HPV is present in normal-appearing cells as well as those demonstrating intraepithelial neoplastic lesions. However, persons testing positive for HPV types associated with cervical cancer such as type 16 are at higher risk for the disease.

An assay based on the principle of proteomics may be used for which of the following? A. Screening for colorectal cancer B. Screening for lung cancer C. Identifying malignant ovarian masses D. Identifying malignant breast tumors

C. Identifying malignant ovarian masses C Proteomics is the study of the proteome. Analogous to the genome, the proteome is the totality of proteins present within a cell or organism. Proteomic studies are aimed mainly at identifying protein signatures for various cancers. Serum is analyzed by time-of-flight mass spectroscopy and thousands of proteins are matched to identify those that can discriminate between cancerous and normal cells. A commercially available test based on proteomics is available for differentiating malignant from benign ovarian tumors. The test detects the presence of five proteins in serum linked to ovarian cancer, and uses multivariate statistical analysis to derive a number from 1-10 indicating the risk of cancer. It has a high sensitivity and negative predictive value.

What is the clinical significance of K-ras testing? A. K-Ras mutations make tumor cells more susceptible to chemotherapy B. K-Ras is a tumor suppressor gene and mutations are associated with increased lifetime risk of malignancy C. K-Ras mutations result in treatment resistance to growth factor receptor inhibitors D. K-Ras is used to identify the tissue of origin

C. K-Ras mutations result in treatment resistance to growth factor receptor inhibitors C K-Ras is a proto-oncogene that makes a GTP binding protein. When the protein is bound to GTP, it initiates a cascade of phosphorylation reactions leading to transcription. K-Ras is activated when an epidermal growth factor binds to the epidermal growth factor receptor (EGFR). EGFR is overexpressed in several cancers including colorectal, lung, and pancreatic cancer. These can be treated with EGFR inhibitors, but treatment resistance occurs if the cells have a K-Ras mutation because K-Ras is downstream of EGFR in the signaling pathway.

Fluorescent dyes most commonly conjugated to antibodies used in flow cytometry are: A. Fluorescein isothiocyanate and Texas red B. Calcofluor white and Texas red C. Phycoerythrin and fluorescein isothiocyanate D. Acridine orange and rhodamine

C. Phycoerythrin and fluorescein isothiocyanate C In flow cytometry, cells are mixed with a panel of specific antibodies that bind to surface antigens that characterize their lineage and maturation state. The antibodies are conjugated to fluorescent dyes that are excited by the laser. If light of the characteristic wavelength emitted by the fluorescent label is detected, then the cell bound the labeled antibody and is positive for the respective antigen. The two most frequently used dyes are fluorescein isothiocyanate (FITC) and phycoerythrin (PE). Since they emit green and red light, respectively, they can be differentiated in the same sample, allowing two antibodies to be tested simultaneously. Using more dyes such as PE-Texas Red allows for the simultaneous measurement of more markers. For example, different fluorescent dyes can be attached to latex beads in different proportions so that up to 100 combinations can be discriminated by the optics. This allows 100 different markers to be measured in the same sample simultaneously. Flow cytometry is used to measure specific plasma proteins and antibodies using fluorescent antibody-coated beads.

Which method of analysis is considered the most reliable means of detecting mutations of BRCA, p53, BRAF, and other genes linked to cancer? A. FISH B. Immunohistochemistry C. Sequencing D. STR analysis

C. Sequencing C Genes such as BRCA1 and BRCA2 are large and thousands of point mutations have been identified, making it impossible to perform an assay using DNA probes. When many mutations of a gene are possible, the most widely used detection method is sequencing. Next generation sequencing has made clinical applications affordable. The process involves creating a library of templates, copying them by PCR, separating the copied strands, and determining their base order by PCR using a reversible dye termination reaction. When a fluorescent-labeled nucleotide is added a laser determines its emission and the base is identified. The blocking group and fluorescent dye are removed and the process repeated for the next base added

Which statement about CF is accurate? A. A sweat chloride test is abnormal in all forms of CF B. Immunoreactive trypsin is deficient in all persons with CF C. Some CF mutations can cause male infertility with no other symptoms D. The CF genotype always predicts the severity of the disease

C. Some CF mutations can cause male infertility with no other symptoms C Serum immunoreactive trypsin is the recommended screening test for CF, but pancreatic insufficiency is not found in about 15% of CF cases. An abnormal result is confirmed by sweat chloride testing. Some infants may be too young for accurate sweat testing, and some mild forms of CF may give indeterminate results. DNA testing can be used in these cases. The CF genotype is not predictive of phenotype in most cases (an exception being ∆F508, which is almost always associated with pancreatic disease). CF mutations are responsible for about 75% of congenital bilateral absence of the vas deferens. Affected persons have at least one abnormal CF gene. Other than infertility, they are asymptomatic and may or may not have a sweat chloride level above 65 mmol/L.

A tissue sample for DNA analysis by PCR was processed for DNA by simple lysis and proteinase K digestion. The lysate was diluted 1:100 and its absorbance measured in an ultraviolet spectrophotometer at 260 nm and 280 nm. The absorbance ratio 260:280 was 1.2. What does this indicate? A. The DNA concentration is too high for PCR B. The DNA concentration is too low for PCR C. The sample contains too much protein D. The sample is optimal for PCR

C. The sample contains too much protein C The mass and purity of DNA affect the efficiency of DNA amplification. A common way to determine the amount of DNA recovered from a sample is to measure its absorbance at 260 nm, where the nitrogenous bases have an absorbance maximum. Based on the molar absorptivity of DNA at 260 nm, an absorbance of 1.0 equates to 50 ng/µL; thus, multiplying the absorbance by 50 and the dilution factor estimates the DNA concentration in ng/µL. However, proteins also absorb at 260 nm, and if present in sufficient amounts will cause overestimation of DNA and inhibit amplification. An absorbance maximum for protein is 280 nm owing to the phenolic rings of tyrosine and tryptophan. The absorbance ratio of 260:280 is a measure of protein contamination. If the ratio is below 1.7, then too much protein is present, and further purification is needed.

Which statement best describes real-time PCR testing for Mycobacterium tuberculosis? A. The test is positive only in cases of smear-positive and culture-positive infections B. The test has a sensitivity of > 99% on all specimen types when compared to culture C. The test can detect 85%-90% of smear-negative, culture-positive infections D. The test sensitivity is near 100% but specificity is approximately 80%

C. The test can detect 85%-90% of smear-negative, culture-positive infections C PCR detection is dependent on having at least a minimal number of organisms present in the specimen, and sensitivity is 90% or lower when the specimen is AFB smear negative but culture positive. Specificity of PCR is approximately 98%.

How can cell proliferation be explained by the BCL 2 translocation t(14;18) that occurs in up to 90% of persons with follicular B-cell lymphoma? A. p53 is underexpressed B. A hybrid protein is made that up-regulates the cell cycle C. Transcription of the BCL 2 oncogene is increased by the translocation D. The BCL 2 gene joins with the p21 gene, making it inactive

C. Transcription of the BCL 2 oncogene is increased by the translocation C In follicular B-cell lymphoma, relocation of the BCL oncogene next to the gene for the immunoglobulin heavy chain (IgH) occurs. The BCL oncogene product is a protein that inhibits apoptosis. When the cell transcribes the IgH gene, it produces the BCL 2 protein as well, which protects the cell from apoptosis. This translocation occurs in all cases of follicular B-cell lymphoma and can be identified using FISH with fluorescent-labeled DNA probes to IgH and BCL 2 genes.

Highest-resolution HLA typing is needed for which of the following transplants? A. Heart B. Liver C. Kidney D. Bone marrow

D. Bone marrow D Solid organ transplants require medium resolution of alleles belonging to HLA class I and class II genes. Bone marrow transplants require high-resolution typing. This involves identifying which allelic groups are present by medium-resolution testing, then sequencing of the PCR products to determine the exact alleles present.

Which of the following alleles has the highest frequency in the general population? A. ∆F508 (cystic fibrosis) B. Factor V-Leiden (hereditary thrombophilia) C. Prothrombin G20210A (hereditary thrombophilia) D. Methylene tetrahydrofolate reductase mutation C677T homocysteinemia

D. Methylene tetrahydrofolate reductase mutation C677T homocysteinemia D Methylene tetrahydrofolate reductase (MTHFR) mutation is a point mutation in which thymidine replaces cytosine at nucleotide 677 in the gene. This results in a codon that substitutes valine for alanine and results in an enzyme that is more heat sensitive. The enzyme converts 5,10 methylenetetrahydrofolate to 5-methyltetrahydrofolate (folate). The methyl group from the latter is transferred to homocysteine, forming methionine. In homozygotes (TT) with less than optimal dietary folate intake, deficiency of the enzyme reduces the availability of 5-methyltetrahydrofolate, causing the serum homocysteine to be increased. Such persons have an approximately threefold increased risk of coronary artery disease. In the general population, the C677T allele of MTHFR has a frequency of 30%. All of the alleles listed are of sufficiently high frequency to warrant screening of at-risk populations. The prothrombin G20210A allele has a frequency of approximately 2%, factor V-Leiden 5%, and ∆F508 approximately 3% (in Whites). Both factor V-Leiden and the prothrombin G20210A mutation result in proteins that increase the risk of thrombosis. The point mutation in factor V-Leiden results in a protein that is resistant to inactivation by protein C. The base substitution in G20210A (guanine to adenine at position 20210) results in increased transcription of the gene and overproduction of prothrombin

Which method is used to determine if the hemoglobin C gene is present in fetal cells? A. Chromosome painting B. FISH C. Restriction enzyme analysis D. PCR followed by blotting with a specific oligonucleotide probe

D. PCR followed by blotting with a specific oligonucleotide probe D The base substitution of hemoglobin C does not affect the MstII restriction site and is not visible by FISH or other tests that detect damage to larger areas of the chromosome. PCR is used to amplify the gene region involved, and the product is tested by Southern blotting using a label-specific oligonucleotide probe.

Which is the most sensitive method of minimal residual disease testing in chronic myelogenous leukemia? A. Karyotyping analysis B. FISH C. Flow cytometry D. RT-PCR

D. RT-PCR D RT-PCR measures the mRNA transcript of BCR/ABL using primers to the p210 and p230 transcripts. The procedure can be done using real-time PCR with a sensitivity of 1:100,000 cells far more sensitive than karyotyping and FISH that have sensitivities of around 1:100 and 1:1,000 cells, respectively. Flow cytometry can detect 1 malignant cell per 10,000 nonmalignant cells, but a panel of antibodies is required that can differentiate malignant from normal cells. RT-PCR can also be used to evaluate the response to treatment. A 3-log decrease in copy number indicates effective treatment.

Which method is most sensitive for detection of viral meningitis? A. Viral culture B. CSF WBC count C. Specific antibody testing of CSF for viral antigens D. Real-time RT-PCR

D. Real-time RT-PCR D Enterovirus is the most common cause of viral meningitis, accounting for more than 85% of cases. Viral culture is positive in 50%-70%, while the sensitivity of real-time PCR is above 95%. Enterovirus strains show significant homology at the 5' end, making it possible to detect several different enterovirus serotypes using a single primer pair. The PCR reaction is not inhibited by antiviral therapy and can be done in hours versus days for viral culture

Hereditary hemochromatosis is the result of which type of mutation? A. Nonsense mutation B. Microdeletion C. Translocation D. Single nucleotide substitution

D. Single nucleotide substitution D Hereditary hemochromatosis is an autosomal recessive disease with a frequency as high as 0.5% in the White population. The mutation occurs in the HFE gene on chromosome 6 and involves a single base that results in tyrosine substituting for cysteine in the HFE protein. The HFE protein down-regulates iron absorption. The mutant protein usually increases iron absorption by at least 100%. Homozygous HFE mutation (C28Y) accounts for approximately 80% of hereditary hemochromatosis. The remaining cases are caused by a single-point mutation at position 63 on the protein (H63D), which produces a milder increase in iron absorption. Genotype is determined by PCR using specific oligonucleotide probes to identify the products.

Which statement accurately describes the clinical utility of translocation testing in leukemia? A. Relapse is predicted by any new translocation occurring after treatment B. Specific translocations associated with a type of leukemia will occur in all cases C. Translocation products for each leukemia subtype are always the same D. Translocation is a sensitive way to identify surviving leukemic cells following treatment

D. Translocation is a sensitive way to identify surviving leukemic cells following treatment D Some translocations occurring after treatment are predictive of relapse. For example, a second translocation in a person with Philadelphia chromosome-positive CML occurs in the majority of persons preceding blast crisis. However, other translocations, such as the 15:22 translocation associated with M3 AML are seen during remission and are not associated with relapse. Some translocations occur with 100% or near 100% frequency, such as 9:22 in CML and 15:17 in M3 AML. However, others occur only in some affected persons. Translocations associated with a type of leukemia are not identical in all cases. For example, the 9:22 translocation associated with CML can give rise to transcripts of different length. RT-PCR can detect as few as 1 per 105 cells containing the translocation, making translocations useful markers for detecting cells that have escaped destruction following treatment.