Molecular diagnosis in hereditary hemorrhagic telangiectasia: findings in a series tested simultaneously by sequencing and deletion/duplication analysis.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder characterized by a unique pattern of telangiectasia and arteriovenous malformations (AVMs). Mutations in one of two genes (ENG and ACVRL1) cause approximately 85% of cases. Genetic testing impacts clinical management because genotype/phenotype correlations exist, and early preventive screening for internal AVMs is recommended in affected individuals prior to the age at which a diagnosis can typically be made based on clinical criteria. We report 383 consecutive cases in which sequencing and large deletion/duplication analysis were performed simultaneously for endoglin (ENG) and activin-like receptor kinase 1 (ACVRL1). We report the first case of mosaicism in an affected individual and 61 novel mutations. We discuss the potential benefits of a diagnostic testing approach for HHT whereby ENG and ACVRL1 are analyzed simultaneously by sequencing and a method which detects large deletion/duplications, rather than by a sequential or reflex testing protocol. We report a case in which a deletion would probably have been missed if large deletion/duplication analysis was performed only if a suspected pathogenic mutation was not first identified by sequencing.

Optimal primer selection for clonality assessment by polymerase chain reaction analysis. III. Intermediate and high-grade B-cell neoplasms.

Previous studies have reported that low-grade B-cell lymphoproliferative disorders have variable B-cell monoclonality detection rates by polymerase chain reaction (PCR) analysis. For instance, monoclonal B-cell populations from chronic lymphocytic leukemia/small lymphocytic leukemia and mantle cell lymphoma are most often readily amplified with a single primer pair, whereas follicular lymphomas and plasma cell neoplasms require alternative strategies to approach these higher diagnostic sensitivity standards. Because most published reports have not focused on the variation in PCR B-cell monoclonality detection among subtypes of intermediate and high-grade B-cell neoplasms, additional information is necessary to determine primer selection strategies and identify problematic tumor subtypes within this group. The current investigation, the third part in a series, was aimed at documenting the efficiency of B-cell monoclonality detection by PCR in 71 aggressive B-cell neoplasms of various types using a comprehensive approach. A predetermined panel of primer sets was used in an algorithmic fashion. Specifically, all samples were analyzed with the standard VH-FRIII/JH assay previously shown to have the highest efficiency of monoclonality detection within low-grade B-cell lymphoproliferative disorders. Negative samples were further evaluated with primer sets in the following order until a positive result was observed, or all primer sets were used: (1) bcl-2/JH, (2) VH-FRI family specific/JH, and (3) VH-FRI consensus/JH. Forty-three (61%) of the 71 B-cell neoplasms evaluated with VH-FRIII/JH showed monoclonal B-cell populations. Sequential use of the three reserve primer sets in samples negative with this initial primer pair resulted in an overall improvement in PCR detection from 61% to 82% (58 of 71 specimens) (P < .001). The VH-FRI family specific assay identified B-cell monoclonality in 11 (73%) of these 15 specimens and was the most productive reserve primer set. Individual categories exhibited the following initial (I) and final (F) PCR detection rates: acute lymphoblastic leukemia/lymphoblastic lymphoma, 11 specimens (I = 91% to F = 91%); small noncleaved cell lymphoma, 14 specimens (I = 79% to F = 86% [P > .25]); diffuse large cell lymphoma, 33 specimens (I = 52% to F= 85% [P < .005]) and large cell, immunoblastic lymphoma, 13 specimens (I = 38% to F = 62% [P < .01]). The authors have shown that comprehensive PCR analysis is capable of detecting B-cell monoclonality in a significant proportion of samples from each subtype of intermediate and high-grade B-cell neoplasm. The VH-FRIII/JH assay was an adequate initial primer set, but required augmentation with the reserve PCR assays to attenuate the false negative rate and improve diagnostic sensitivity. The B-cell clonality PCR assay is optimally used as a screening tool and when used in this fashion, the more laborious and time-consuming restriction fragment-Southern blot hybridization (RF-SBH) method for IgH gene rearrangement detection may be limited to a relatively small proportion of PCR-negative aggressive B-cell neoplasms.

A rapid practical RT-PCR-based approach for the detection of the PML/RAR alpha fusion transcript in acute promyelocytic leukemia.

The t(15;17) and its molecular equivalent, PML/RAR alpha gene fusion, is strongly associated with acute promyelocytic leukemia (APL). Since treatment response to all-trans retinoic acid correlates directly with PML/RAR alpha, expeditious documentation is critical to patient care. We have designed an extremely rapid, practical, polymerase chain reaction (PCR)-based method using a rapid air thermal cycler to detect type A, B, and B-variant fusion patterns of PML/RAR alpha. We examined 15 cases of APL and 13 cases of leukemias other than APL with a nested reverse-transcription PCR assay. Three APL samples were type A, 11 were type B, and 1 was a B variant based on gel band patterns. PCR products exhibited positive probe hybridization signals and had sequences containing type A, B, or B-variant fusion patterns. PCR amplification of PML/RAR alpha was complete in 22 minutes, and the entire test required 4 1/2 hours. This method permits exceptional turnaround time and is an alternative to cytogenetics and slower PCR assays.

Comparison of automated short tandem repeat and manual variable number of tandem repeat analysis of chimerism in bone marrow transplant patients.

Hematopoietic chimerism can be monitored in bone marrow transplant patients at DNA polymorphic sites. In this study, allele detection and quantification by ethidium bromide-stained agarose gels were compared with automated fluorescent sizing on an artificially mixed system and on chimeric post-transplant whole blood and sorted cell populations. A panel of five variable number of tandem repeats (VNTRs) were amplified and quantified visually on an ethidium bromide-stained gel. The ten short tandem repeats (STRs) were amplified as a multiplex polymerase chain reaction (PCR) and fluorescently detected on a DNA sequencer. Fluorescent band intensities were converted to fluorescent peak areas for allele quantification. Using mixed DNA of different proportions, both STRs and VNTRs showed linearity and appeared equally sensitive. However, case studies showed STRs to be more sensitive (<5%) than VNTRs (<10%). The STRs more accurately quantified the minor DNA component at low concentrations.

Simultaneous detection of C282Y and H63D hemochromatosis mutations by dual-color probes.

BACKGROUND: Hemochromatosis is a common genetic disease, affecting one in every 200 individuals in the United States. A PCR assay was designed using fluorescent melting curve analysis to simultaneously detect the G845-->A (C282Y) and C187-->G (H63D) mutations. The G845-->A and C187-->G loci are distinguished by color, and mutant alleles are distinguished from wild type by probe melting temperature (Tm). METHODS AND RESULTS: The probe sets used two fluorophore pairs, fluorescein with LCRed 640 for G845-->A and fluorescein with LCRed 705 for C187-->G. The probes, complementary to the mutant allele, dissociate from the product at specific Tms. Wild-type alleles form mismatches with the probes, reducing the Tms by 6 degrees C (G845-->A) and 10 degrees C (C187-->G). One of 133 samples had a Tm shift 4 degrees C less than the wild-type Tm for the G845-->A locus. Sequencing confirmed the sample to be homozygous for G845-->A and heterozygous for a C-->A substitution at position 842 (C842-->A), substituting lysine for threonine. CONCLUSIONS: Multiplexing by color and Tm allows for simultaneous genotyping of each mutation. A novel base-pair alteration was detected in cis with a G845-->A mutation.

Quantification of HER2/neu gene amplification by competitive pcr using fluorescent melting curve analysis.

BACKGROUND: Molecular detection methods for HER2/neu gene amplification include fluorescence in situ hybridization (FISH) and competitive PCR. We designed a quantitative PCR system utilizing fluorescent hybridization probes and a competitor that differed from the HER2/neu sequence by a single base change. METHODS: Increasing twofold concentrations of competitor were coamplified with DNA from cell lines with various HER2/neu copy numbers at the HER2/neu locus. Competitor DNA was distinguished from the HER2/neu sequence by a fluorescent hybridization probe and melting curve analysis on a fluorescence-monitoring thermal cycler. The percentages of competitor to target peak areas on derivative fluorescence vs temperature curves were used to calculate copy number. RESULTS: Real-time monitoring of the PCR reaction showed comparable relative areas throughout the log phase and during the PCR plateau, indicating that only end-point detection is necessary. The dynamic range was over two logs (2000-250 000 competitor copies) with CVs < 20%. Three cell lines (MRC-5, T-47D, and SK-BR-3) were determined to have gene doses of 1, 3, and 11, respectively. Gene amplification was detected in 3 of 13 tumor samples and was correlated with conventional real-time PCR and FISH analysis. CONCLUSION: Use of relative peak areas allows gene copy numbers to be quantified against an internal competitive control in < 1 h.