Assessment of fidelity and utility of the whole-genome amplification for the clinical tests offered in a histocompatibility and immunogenetics laboratory

Increasing emphasis on the use of molecular tests in a histocompatibility and immunogenetics laboratory (HIL) poses a potential problem of lack of sufficient DNA to perform multiple genetic analyses. In this study, we report the feasibility, fidelity and utility of multiple displacement amplification (MDA) method to perform whole-genome amplification (WGA) to generate DNA specimens that can be analyzed by multiple molecular techniques and can be used for different clinical tests offered by an HIL. The MDA-generated DNA when compared with the native DNA showed 100% congruency in genotyping of 37 genes/loci using multiple downstream molecular techniques: sequence-based typing and sequence-specific primer-based typing for 5 human leukocyte antigen (HLA) class I and II genes (HLA-A, B, C, DRB1 and DQB1), luminex-based sequence-specific oligonucleotide (SSO) genotyping for a panel of 16 killer immunoglobulin-like receptor (KIR) genes and automated fragment size analysis for a panel of 15 short tandem repeat (STR) loci and amelogenin gene. For post-allogeneic hematopoietic cell transplantation (HCT) chimerism analysis, MDA-generated DNA appeared useful for enriching pre-transplant DNA but not for enriching post-transplant chimeric DNA. Overall, our results show that MDA-based WGA could generate DNA of high yield and fidelity that can be used for various clinical tests and research purposes.     

Amelogenin self-assembly and the role of the proline located within the carboxyl-teleopeptide

A hallmark of biological systems is a reliance on protein assemblies to perform complex functions. We have focused attention on mammalian enamel formation because it relies on a self-assembling protein complex to direct mineral habit. The principle protein of enamel is amelogenin that self-assembles to form nanospheres. In mice, the principal amelogenin product is a 180 amino acid hydrophobic protein. The yeast two-hybrid assay has been used to demonstrate the importance of amelogenin self-assembly domains. We have generated specific variants of amelogenin to analyze contributions of individual amino acids to the self-assembly process. These amelogenin variants have been produced either by deleting carboxyl-terminal amino acids (to generate proteins that relate to the documented proteolytic products of mouse amelogenin) or by a site-directed mutagenesis approach. Assessment of variant amelogenins truncated at the carboxyl-terminal imply that the proline at position 169 of mouse amelogenin (M180) plays a significant role in amelogenin self-assembly. Site-directed mutagenesis of this particular proline, however, failed to disrupt the amelogenin self-assembly property. These conflicting data add to the complexity of protein-protein assembly mechanisms as they relate to the enamel matrix. Available data suggest a robustness of this enamel protein (amelogenin) that ensures a functional, even though mechanically less than optimal, enamel results despite either minor or major genetic errors to the amelogenin gene locus.     

Amelogenin Self-Assembly and the Role of the Proline Located within the Carboxyl-Teleopeptide

A hallmark of biological systems is a reliance on protein assemblies to perform complex functions. We have focused attention on mammalian enamel formation because it relies on a self-assembling protein complex to direct mineral habit. The principle protein of enamel is amelogenin that self-assembles to form nanospheres. In mice, the principal amelogenin product is a 180 amino acid hydrophobic protein. The yeast two-hybrid assay has been used to demonstrate the importance of amelogenin self-assembly domains. We have generated specific variants of amelogenin to analyze contributions of individual amino acids to the self-assembly process. These amelogenin variants have been produced either by deleting carboxyl-terminal amino acids (to generate proteins that relate to the documented proteolytic products of mouse amelogenin) or by a site-directed mutagenesis approach. Assessment of variant amelogenins truncated at the carboxyl-terminal imply that the proline at position 169 of mouse amelogenin (M180) plays a significant role in amelogenin self-assembly. Site-directed mutagenesis of this particular proline, however, failed to disrupt the amelogenin self-assembly property. These conflicting data add to the complexity of protein-protein assembly mechanisms as they relate to the enamel matrix. Available data suggest a robustness of this enamel protein (amelogenin) that ensures a functional, even though mechanically less than optimal, enamel results despite either minor or major genetic errors to the amelogenin gene locus.     

High-throughput copy number analysis of 17q23 in 3520 tissue specimens by fluorescence in situ hybridization to tissue microarrays

Analysis of allelic loss in archival tumor specimens is constrained by quality and quantity of tissue and by technical limitations on the number of chromosomal sites that can be efficiently evaluated in conventional analyses using polymorphic microsatellite markers. Newly developed array-based assays have the potential to yield genome-wide data from small amounts of tissue but have not been validated for use with routinely processed specimens. We used the Affymetrix HuSNP assay, composed of 1494 single nucleotide polymorphism sites, to compare allelic loss results obtained from both formalin-fixed and frozen breast tissue samples. Tumor cells were separated from normal epithelia and nonepithelial cells by dissection and bivariate cytokeratin/DNA flow sorting; normal breast cells from the same patient served as constitutive normal. Allele results from the HuSNP array averaged 96% reproducibility between duplicates and were concordant between the fixed and frozen normal samples. We also analyzed DNA from the same samples after whole-genome amplification (primer extension preamplification). Although overall signal intensities were lower, the genotype data from the primer extension preamplification material was concordant with genomic DNA data from the same samples. Results from genomic normal tissue DNA averaged informative single nucleotide polymorphism at 379 (25%) loci genome-wide. Although data points were clustered and some segments of chromosomes were not informative, our data indicated that the Affymetrix HuSNP assay could provide an efficient and valid genome-wide analysis of allelic imbalance in routinely processed and whole genome-amplified pathology specimens.     

PCR detection of Pneumocystis carinii in bronchoalveolar lavage specimens: analysis of sensitivity and specificity.

Although PCR detection of Pneumocystis carinii DNA has been described, little is known about the sensitivity or specificity of the assay in routine laboratory practice. We had the unique opportunity to use a bronchoalveolar lavage (BAL) specimen bank with samples for which the direct examination results for P. carinii were known. DNA purified from 129 selected specimens was amplified by using the primers described previously (A. E. Wakefield, F. J. Pixley, S. Banerji, K. Sinclair, R. F. Miller, E. R. Moton, and J. M. Hopkin, Mol. Biochem. Parasitol. 43:69-76, 1990). Of the 129 specimens, 37 were positive for P. carinii by direct examination. All 37 specimens were positive for P. carinii by PCR, yielding a 100% sensitivity and 100% negative predictive value for the assay. An additional 23 specimens were repeatedly positive for P. carinii by PCR but were not positive by direct examination. Review of the patient charts for these specimens with discordant results demonstrated that five of the patients were actually positive for P. carinii, as determined by either biopsy or examination of repeat or prior BAL specimens. A response to empiric therapy for P. carinii pneumonia was seen in an additional two patients. Of the remaining specimens, 8 produced no significant isolates other than P. carinii, while 12 contained culture-confirmed significant respiratory pathogens in addition to P. carinii (two fungal, nine bacterial, and one viral pathogen). Cytomegalovirus, which was of unknown significance, was isolated from 16 additional specimens. Overall, the specificity of the PCR assay was 79.3% compared to the results of direct examination. We hypothesized that the apparently poor specificity of the PCR assay was due to the increased sensitivity of the assay compared to that of direct examination. The sensitivity of the PCR assay was therefore assessed with BAL specimens containing P. carinii cysts. Serial dilutions of this preparation were evaluated by direct examination and PCR. PCR was found to be 100-fold more sensitive than direct examination, which detected one to two cysts per amplification. No false-positive results were detected in controls containing no DNA or by using target DNA from various fungal, viral, or bacterial respiratory pathogens. We conclude that PCR detection of P. carinii in BAL specimens is very sensitive and should be considered for patients whose specimens do not yield a diagnosis. The increased sensitivity of the PCR assay may help to identify those patients with low-titer infections who might benefit from directed antibiotic therapy for P. carinii and would otherwise be missed by direct examination alone.     

Murine enamelin: cDNA and derived protein sequences

Enamelin is the largest enamel protein. Recently we reported the characterization of a cDNA clone encoding porcine enamelin. The secreted protein has 1104 amino acids--over 6 times the length of amelogenin (173 amino acids) and almost 3 times the lengths of sheathlin (395 amino acids) and tuftelin (389 amino acids). Immunohistochemistry has shown that uncleaved porcine enamelin concentrates at the growing tips of the enamel crystallites while its cleavage products localize to rod and interrod enamel. Here we report the isolation and characterization of cDNA encoding murine amelogenin and demonstrate the tooth specificity of porcine enamelin. The murine clone is 4154 nucleotides in length and encodes a protein of 1274 amino acids. In the absence of post-translational modifications murine enamelin has an isotope averaged molecular mass of 137 kDa and an isoelectric point of 9.4. Multiple tissue Northern blot analyses detect porcine enamelin mRNA in developing teeth but not in liver, heart, brain, spleen, skeletal muscle and lung. Mouse and porcine enamelin share 61% amino acid identity and 75% DNA sequence identity. Mouse enamelin has 14 tandemly arranged copies of an 11 amino acid segment that is found only once in porcine enamelin.     

Evaluation of culture methods and a DNA probe-based PCR assay for detection of Campylobacter species in clinical specimens of feces

Campylobacter species are the leading agents of bacterial gastroenteritis in developed countries. In this study 320 specimens of feces from patients with symptoms of acute gastroenteritis were cultured for Campylobacter species by direct plating on modified charcoal cefoperazone deoxycholate agar and by enrichment in modified Preston broth, with or without blood added, for 48 h at 37 degrees C prior to plating. A 16S/23S PCR/DNA probe membrane-based colorimetric detection assay was evaluated on a subset of the feces (n = 127), including 18 culture-positive and 109 culture-negative specimens. DNA was extracted directly from the fecal specimens by using the QIAamp DNA stool Minikit for the DNA probe-based PCR assay (PCR/DNA probe assay). A second PCR/DNA probe assay based on the 16S rRNA gene in Campylobacter spp. was applied to all specimens that were culture negative, PCR/DNA positive on initial analysis. Campylobacter species were cultured in 20 of the 320 specimens. The 16S/23S PCR/DNA probe assay detected campylobacter DNA in 17 of 18 (94% sensitivity) culture-positive specimens and in 41 (38%) culture-negative specimens. The presence of campylobacter DNA in 35 of 41 culture-negative specimens was confirmed by the 16S PCR/DNA probe assay. DNA sequence analysis of seven 16S/23S PCR products and five 16S PCR products amplified from a selection of these specimens confirmed the presence of campylobacter DNA and more specifically Campylobacter jejuni, C. concisus, C. curvus, and C. gracilis DNA in these specimens. The molecular assays described in this study are rapid methods for the detection and identification of Campylobacter species in fecal specimens. The finding of Campylobacter spp. DNA in a large number of specimens of feces from patients with no other identified cause of diarrhea may suggest that Campylobacter spp. other than C. jejuni and C. coli may account for a proportion of cases of acute gastroenteritis in which no etiological agent is currently identified.     

Tuftelin mRNA is expressed in a human ameloblastoma tumor

RT-PCR, Southern blotting and DNA sequencing have established for the first time that tuftelin mRNA is expressed in human ameloblastoma tumor. The expression of amelogenin mRNA in ameloblastoma was also established, confirming earlier reports by Snead et al. These results corroborate, on a molecular level, the enamel organ epithelial origin of ameloblastoma. In view of the present results, it is interesting that previous studies have indicated that although ameloblastoma, a non-mineralized odontogenic tumor, transcribes amelogenin mRNA, amelogenin (and enamelin) proteins are not expressed in this tissue. However, in mineralizing odontogenic tumors, both these classes of proteins are expressed.     

Detection of herpes simplex virus DNA from cerebrospinal fluid by PCR and a rapid, nonradioactive hybridization technique.

A molecular assay for the detection of herpes simplex virus (HSV), including a novel, nonradioactive hybridization technique, was evaluated with a total of 123 cerebrospinal fluid specimens. After DNA extraction, specific HSV DNA sequences were amplified with digoxigenin-labeled primers derived from the DNA polymerase gene-coding region from HSV. Amplified products were detected by the Enzymun-Test DNA detection assay (Boehringer, Mannheim, Federal Republic of Germany), which uses biotinylated probes. Amplification with nonlabeled primers and then Southern blotting and nonradioactive detection of hybrids by the digoxigenin technique was the reference system. The sensitivities of the molecular assays were determined with 10-fold dilutions of plasmid pS4 with the SalI restriction fragment of the DNA polymerase gene obtained from the HSV type 1 strain Angelotti. The Enzymun assay was able to detect all of the 16 positive samples, giving 100% agreement with the Southern blot hybridization results. Optical density values were widely separated for the positive and negative groups of specimens. Ten copies of plasmid pS4 per microliter could be distinctly detected by the Enzymun assay. The cutoff was determined for the hybridization assay, and an equivocal zone was defined. The whole molecular assay including the Enzymun-Test DNA detection proved to be sensitive and easy to use. It may contribute to the rapid and safe detection of HSV DNA in cerebrospinal fluid.     

Simultaneous identification of mycobacterial isolates to the species level and determination of tuberculosis drug resistance by PCR followed by electrospray ionization mass spectrometry

Mycobacterium tuberculosis that is resistant to both isoniazid (INH) and rifampin (RIF) is spreading. It has become a public health problem in part because the standard culture methods used to determine the appropriate treatment regimen for patients often take months following the presumptive diagnosis of tuberculosis. Furthermore, the misidentification of nontuberculosis mycobacteria (NTM) in patients presumably suffering from tuberculosis results in additional human and health care costs. The mechanisms of resistance for several drugs used to treat Mycobacterium tuberculosis are well understood and therefore should be amenable to determination by rapid molecular methods. We describe here the use of PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) in an assay that simultaneously determines INH and RIF resistance in Mycobacterium tuberculosis and identifies and determines the species of NTMs. The assay panel included 16 primer pairs in eight multiplexed reactions and was validated using a collection of 1,340 DNA samples from cultured specimens collected in the New York City area, the Republic of Georgia, and South Africa. Compared with phenotypic data, the PCR/ESI-MS assay had 89.3% sensitivity and 95.8% specificity in the determination of INH resistance and 96.3% sensitivity and 98.6% specificity in the determination of RIF resistance. Based on a set of 264 previously characterized liquid culture specimens, the PCR/ESI-MS method had 97.0% sensitivity and 99.9% specificity for determination of NTM identity. The assay also provides information on ethambutol, fluoroquinolone, and diarylquinoline resistance and lineage-specific polymorphisms, to yield highly discriminative digital signatures potentially suitable for epidemiology tracking.     

HOPE-fixation enables improved PCR-based detection and differentiation of Mycobacterium tuberculosis complex in paraffin-embedded tissues

Standard PCR-based detection of mycobacterial DNA in paraffin-embedded specimens may lack sufficient sensitivity because of the degradation of nucleic acids caused by routinely used formalin fixation. Therefore, we set up an approach that aimed at improving the results by applying the novel HOPE-fixative in PCR-detection of mycobacteria in paraffin-embedded tissues. Comparison of PCR-results using DNA extracted from either HOPE- or formalin-fixed specimens in BCG-infected SCID-mice revealed a more than 100fold enhanced sensitivity for the HOPE-fixed material. Owing to the preservation of DNA from degradation in HOPE-fixed tissues, even differentiation within the M. tuberculosis complex was possible by spoligotyping. We therefore conclude that the HOPE-fixative is a useful tool for molecular pathology that enhances the sensitivity of PCR-based methods for the detection of pathogens in paraffin-embedded tissues compared to formalin-fixation. Owing to the better preserved DNA, improved differentiation of mycobacteria from archived materials is possible. These results promise new and a substantially wider range of possibilities in the field of molecular pathology.     

Use of FTA gene guard filter paper for the storage and transportation of tumor cells for molecular testing.

CONTEXT: Efficient methods of storing tumor specimens for molecular testing are needed in the modern surgical pathology laboratory. The FTA Gene Guard system is a novel method for the collection and room temperature storage of blood samples for DNA testing. The method uses index card-sized filter papers that provide an ideal medium on which to store tumor specimens for DNA testing. OBJECTIVE: To determine whether FTA filter paper can be used in the surgical pathology laboratory to store tumor cells for DNA testing. DESIGN: Cell suspensions were prepared from 60 surgical specimens, and DNA was extracted either immediately or after storage on FTA paper. The DNA extracted by each method was tested by polymerase chain reaction (PCR) for the beta-globin and interferon gamma genes, and the results were compared. Fifteen lymph node specimens stored on FTA paper were then tested for immunoglobulin heavy chain (IgH) gene rearrangement by PCR, and these results were compared with those obtained for immediately extracted DNA.SETTING: University medical center. RESULTS: The DNA extracted from cells stored on FTA paper performed as well in the PCR as the freshly extracted DNA in nearly all cases (>95%). The results of tests for IgH gene rearrangements showed 100% concordance between the 2 methods of DNA extraction.Conclusion.-Cells from surgical specimens can be stored on FTA paper for extended lengths of time, and DNA can be extracted from these cells for PCR-based testing. FTA filter paper is a reliable medium for the storage and/or transport of tumor cells for PCR-based DNA analysis.     

The unexpected detection of varicella-zoster virus in genital specimens using the Lyra™ Direct HSV 1+2/VZV Assay

BackgroundThe Lyra™ Direct HSV 1+2/VZV Assay is a moderately complex, multiplex PCR assay that qualitatively detects the presence of HSV 1, HSV 2, and VZV DNA in cutaneous and mucocutaneous specimens with a time-to-result of less than 60 min.ObjectivesTo report a one-year laboratory experience using Lyra assay for testing cutaneous and mucocutaneous specimens for HSV and VZV that resulted in the unexpected detection of VZV in 14 male and female genital specimens.Study designOver a one-year period, 2113 cutaneous and mucocutaneous specimens from male and female patients were submitted for testing using the Lyra assay. An unexpected 14 genital specimens were positive for the presence VZV DNA. Eleven of the 14 specimens were available for confirmatory testing using two alternative molecular methods and Sanger sequencing.ResultsFourteen male and female genital specimens were positive for the presence of VZV DNA. All of the 11 specimens (9 female and 2 male) that were available for confirmatory testing by the alternative molecular method and Sanger sequencing were confirmed as containing VZV DNA.ConclusionsUsing of the Lyra assay over a one-year time period, VZV DNA was detected in 126 specimens of which 14 (11.1%) were from male and female genital sites. This rare and unexpected finding suggests that the appearance of zoster lesions in the genital area may not be as uncommon as previously thought and that this finding would have considerable impact on patient counseling and public health considerations.     

Of mice and men: anatomy of the amelogenin gene

Mammalian enamel matrix is composed of two principal proteins, the enamelins and amelogenin. Recombinant complementary DNA (cDNA) molecules for the predominant mouse amelogenin have been identified, characterized by direct determination of the DNA sequence, and used as a specific hybridization probe. The spatial- and temporal-restricted pattern for amelogenin gene expression within developing mouse molars has been traced at the level of a single cell using in situ hybridization. The mouse genome has been shown to contain only one copy of the amelogenin (AMEL) gene which is not amplified or rearranged during ameloblast determination. In contrast, the human genome contains two copies of the AMEL gene, one residing on the X chromosome and one upon the Y chromosome. These observations, the availability of specific enamel gene probes coupled with the application of new techniques in molecular biology now afford unique opportunities for the analysis of the molecular basis of inherited defects of human enamel such as amelogenesis imperfecta. Recent advances towards obtaining a physical map and the complete nucleotide sequence for the human genome, as well as the documented developmental biology, defined genetics and transgenic capability of the mouse, suggest that mouse and man are the most relevant and potentially informative models for analysis of normal and abnormal enamel biomineralization.     

Detection of Helicobacter pylori DNA in Fecal Samples from Infected Individuals

Stool, gastric biopsy, and serum samples were collected from 22 subjects. DNA from stool was extracted, amplified, and hybridized with primers specific for the 16S rRNA gene of Helicobacter pylori. DNA from gastric biopsy specimens was analyzed similarly for comparison. Universal primers were used to confirm successful extraction of DNA from samples. Histologic, serologic, and DNA analyses were scored in a blinded fashion. Universal primer amplification verified successful DNA extraction from all stool and gastric tissue specimens. The gastric tissue DNA assay was positive for H. pylori in 11 of the 22 subjects, correlating completely with histologic and serologic results. Stool DNA was positive for H. pylori by our molecular assay in 8 of these 11 H. pylori-positive subjects. All subjects who were negative by histologic, serologic, and gastric tissue DNA analyses were also negative by stool DNA analysis. Compared to histology, serology, and gastric tissue DNA analyses, the sensitivity of our stool DNA assay was 73%, with a specificity of 100%.     

Identifying cross contaminants and specimen mix-ups in surgical pathology

Maintaining patient identity throughout laboratory processing is critical for any diagnostic pathology laboratory. It has also been listed as a main National Patient Safety Goal for the Joint Commission Laboratory Goals. Most pathology facilities have excellent quality assurance programs and initiatives that include multiple checkpoints to ensure that specimen identity is carefully maintained. Even in the best laboratory operations, however, the potential for specimen mix-up, cross contamination, floaters, or carry-over artifacts cannot be eliminated completely. The work-up and analysis of these cases can be challenging to the pathologist for a number of reasons, including the fact that there is no standardized approach. This review discusses a standardized approach that includes clinical correlation, laboratory correlation, and laboratory analysis, potentially including a molecular DNA fingerprinting analysis. The methods and techniques behind DNA fingerprinting for tissue and sample identity are also discussed in detail.     

Detection of supercoiled hepatitis B virus DNA and related forms by means of molecular hybridization to an oligonucleotide probe

A novel assay for supercoiled and other fully double-stranded forms of hepatitis B virus (HBV) DNA in blood is presented that utilizes molecular hybridisation to a radiophosphorous-labeled oligonucleotide probe. The probe [5'-d(ACGTGCAGAGGTGAAGCGA)] is complementary to the S(+)-strand sequence furthest downstream, at the end of the gap. We examined blood specimens from 137 healthy HBsAg-positive individuals, applying the probe to dots representing 2-3.5 ml serum or plasma. We found that supercoiled HBV is present in many HBV DNA-positive blood specimens albeit in small quantities. Of the 104 specimens that were positive for HBV DNA of any form, 53 annealed to the probe. Serial specimens from the same subject taken over a period of months showed that the proportion of supercoil to other HBV DNA forms was variable. The presence of supercoil HBV DNA was not closely correlated with the level of serum HBV DNA polymerase. The supercoil is an HBV DNA form that can persist in the liver in the presence or absence of other replicative intermediates. This assay may enable further characterization of the status of HBV infection.     

Detection of Herpes simplex virus DNA by real-time PCR

Molecular detection of herpes simplex virus (HSV) DNA is recognized as the reference standard assay method for the sensitive and specific diagnosis of central nervous system infections caused by HSV. In this study, a molecular assay based on real-time PCR on the LightCycler (LC) instrument was evaluated and compared with a home-brew molecular assay. The detection limit of the LC assay was determined with 10-fold dilutions of plasmid pS4 with the SalI restriction fragment of the DNA polymerase gene and with the First European Union Concerted Action HSV Proficiency Panel. A total of 59 cerebrospinal fluid (CSF) specimens were investigated for the comparative study. With plasmid pS4, the detection limit of the LC assay was found to be 10(4) copies per ml, i.e., 12.5 copies per run. When samples of the First European Union Concerted Action HSV Proficiency Panel were tested, 2x10(3) to 5x10(3) HSV type 1 genome equivalents (GE) per ml, i.e., 2.5 to 6.3 GE per run, could consistently be detected. There was a correlation between the LC assay and the home-brew assay in 55 of 59 specimens. In conclusion, the LC assay allows very rapid detection of HSV DNA in CSF. It was found to be laborsaving and showed sufficient sensitivity.