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PubMed2025

Utility of Plasma Cell-free Chromatin Immunoprecipitation to Detect Cardiac Allograft Rejection.

BACKGROUND: Antibody-mediated rejection (AMR) remains the major risk factor for allograft loss across all solid organ transplantation. Unfortunately, its diagnosis relies on biopsy, an invasive gold standard that often sample unaffected allograft tissue leading to missed diagnosis. Plasma donor-derived cell-free DNA (dd-cfDNA) is noninvasive biomarker that has high sensitivity but low specificity for AMR diagnosis. This proof-of-concept study assessed the utility of cell-free chromatin immunoprecipitation (cfChIP) as a surrogate for gene expression to detect cardiac AMR and the associated pathobiology. METHODS: The discovery GRAfT multicenter cohort of heart transplant patients (NCT02423070) identified AMR, acute cellular rejection (ACR), and stable controls based on biopsy and ddcfDNA results. Plasma cfChIP-sequencing was performed to identify peaks, associated genes and pathobiological pathways. Plasma from an external cohort (GTD, NCT01985412) was also analyzed to verify pathways identified. Digital droplet PCR (ddPCR) assays targeting differential regions were constructed to test the diagnostic performance of cfDNA to detect AMR/ACR from stable controls (rejection-specific assays) or AMR from ACR (AMR-specific assays). RESULTS: The cohort included 21 AMR, 28 ACR, and 45 stable controls from GRAfT and GTD, and 23 healthy controls. cfChIP detected expected active genes, including housekeeping genes and gene targets of transplant immunosuppressive drugs but not inactive genes. Unsupervised clustering of the discovery GRAfT cohort assigned 95% of samples correctly as AMR, ACR or stable control. Differential analysis identified pathobiological pathways of AMR such as neutrophil degranulation and complement activation. The pathways were consistent in GTD samples. Rejection-specific assays detected AMR/ACR from controls with AUC of 0.78 - 0.95. AMR-specific assays detected AMR from ACR with AUC of 0.71 - 0.85, sensitivities of 0.73 - 0.94 and specificities of 0.73 - 0.80. CONCLUSION: This study provides valuable preliminary data supporting the use of cfChIP to detect AMR and the associated pathobiological pathways.

Allograft rejection↗
PubMed2026

High-Sensitivity ctDNA Analysis Uncovers Relevant Signals Missed by NGS in Pancreatic Cancer.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) carries high mortality despite multimodal therapy, and improved biomarkers are needed to guide perioperative care. This study evaluated the prognostic significance of Kirsten rat sarcoma virus (KRAS)-mutant circulating tumor DNA (ctDNA) detected by next-generation sequencing (NGS) and digital droplet PCR (ddPCR) in localized PDAC. EXPERIMENTAL DESIGN: In this prospective cohort study (2020-2024), patients with localized PDAC undergoing neoadjuvant chemotherapy (NAC) were enrolled across multiple sites within Northwestern Medicine. Blood samples for ctDNA were assessed at diagnosis, after NAC, and after resection using tumor-agnostic NGS and ddPCR targeting KRAS G12D/V/R mutations. Overall survival (OS) was assessed using Kaplan-Meier analysis. RESULTS: The cohort included 106 patients. At diagnosis, KRAS ctDNA was detected in 17.2% (17/99) by NGS and 64.9% (63/97) by ddPCR. Detection by both platforms was associated with shorter OS, with the higher-sensitivity ddPCR assay providing greater prognostic discrimination by identifying additional patients with poor outcomes not captured by NGS (NGS median OS 11.2 vs. 30.5 months, P < 0.001; ddPCR median OS 24.7 vs. 70.9 months, P = 0.004). Stratified by detection method, median OS was shortest in patients with ctDNA detected by both NGS and ddPCR (10.9 months), longest in those not detected by either platform (40.7 months), and intermediate in patients detected only by ddPCR (26.9 months; P < 0.001). CONCLUSIONS: In localized PDAC, KRAS-mutant ctDNA detected by NGS or ddPCR was associated with worse survival. ddPCR identified additional patients missed by NGS. Integrating ddPCR with NGS ctDNA measures may improve perioperative risk stratification, although validation is needed before clinical implementation.

Humans↗
PubMed2003

Expression of growth hormone receptor and its mRNA in hepatic cirrhosis.

AIM: To investigate the expression of growth hormone receptor (GHR) and mRNA of GHR in cirrhotic livers of rats with the intension to find the basis for application of recombinant human growth hormone (rhGH) to patients with liver cirrhosis. METHODS: Hepatic cirrhosis was induced in Sprague-Dawley rats by administration of thioacetamide intraperitoneally for 9-12 weeks. Collagenase IV was perfused in situ for isolation of hepatocytes. The expression of GHR and its mRNA in cirrhotic livers was studied with radio-ligand binding assay, RT-PCR and digital image analysis. RESULTS: One class of specific growth hormone-binding site, GHR, was detected in hepatocytes and hepatic tissue of cirrhotic livers. The binding capacity of GHR (R(T), fmol/mg protein) in rat cirrhotic liver tissue (30.8+/-1.9) was significantly lower than that in normal control (74.9+/-3.9) at the time point of the ninth week after initiation of induction of cirrhosis (n=10, P<0.05), and it decreased gradually along with the accumulation of collagen in the process of formation and development of liver cirrhosis (P<0.05). The number of binding sites (X10(4)/cell) of GHR on rat cirrhotic hepatocytes (0.86+/-0.16) was significantly lower than that (1.28+/-0.24) in control (n=10, P<0.05). The binding affinity of GHR among liver tissue, hepatocytes of various groups had no significant difference (P>0.05). The expression of GHR mRNA (riOD, pixel) in rat cirrhotic hepatic tissues (23.3+/-3.1) was also significantly lower than that (29.3+/-3.4) in normal control (n=10, P<0.05). CONCLUSION: The growth hormone receptor was expressed in a reduced level in liver tissue of cirrhotic rats, and lesser expression of growth hormone receptors was found in a later stage of cirrhosis. The reduced expression of growth hormone receptor was partly due to its decreased expression on cirrhotic hepatocytes and the reduced expression of its mRNA in cirrhotic liver tissue.

Acetamides↗
PubMed2006

Integrated polymerase chain reaction chips utilizing digital microfluidics.

This study reports an integrated microfluidic chip for polymerase chain reaction (PCR) applications utilizing digital microfluidic chip (DMC) technology. Several crucial procedures including sample transportation, mixing, and DNA amplification were performed on the integrated chip using electro-wetting-on-dielectric (EWOD) effect. An innovative concept of hydrophobic/hydrophilic structure has been successfully demonstrated to integrate the DMC chip with the on-chip PCR device. Sample droplets were generated, transported and mixed by the EWOD-actuation. Then the mixture droplets were transported to a PCR chamber by utilizing the hydrophilic/hydrophobic interface to generate required surface tension gradient. A micro temperature sensor and two micro heaters inside the PCR chamber along with a controller were used to form a micro temperature control module, which could perform precise PCR thermal cycling for DNA amplification. In order to demonstrate the performance of the integrated DMC/PCR chips, a detection gene for Dengue II virus was successfully amplified and detected. The new integrated DMC/PCR chips only required an operation voltage of 12V(RMS) at a frequency of 3 KHz for digital microfluidic actuation and 9V(DC) for thermal cycling. When compared to its large-scale counterparts for DNA amplification, the developed system consumed less sample and reagent and could reduce the detection time. The developed chips successfully demonstrated the feasibility of Lab-On-a-Chip (LOC) by utilizing EWOD-based digital microfluidics.

Dengue Virus↗
PubMed1994

Aspects of nonrandom turnover involved in the concerted evolution of intergenic spacers within the ribosomal DNA of Drosophila melanogaster.

Polymerase chain reaction (PCR)-amplified, sequenced, and digitally typed intergenic spacers (IGSs) of the ribosomal (r)DNA in D. melanogaster reveal unexpected features of the mechanisms of turnover involved with the concerted evolution of the gene family. Characterization of the structure of three isolated IGS length variants reveals breakage "hot spots" within the 330-base-pair (bp) subrepeat array found in the spacers. Internal mapping of variant repeats within the 240-bp subrepeat array using a novel digital DNA typing procedure (minisatellite variant repeat [MVR]-PCR) shows an unexpected pattern of clustering of variant repeats. Each 240-bp subrepeat array consists of essentially two halves with the repeats in each half identified by specific mutations. This bipartite structure, observed in a cloned IGS unit, in the majority of genomic DNA of laboratory and wild flies and in PCR-amplified products, has been widely homogenized yet is not predicted by a model of unequal crossing over with randomly placed recombination breakpoints. Furthermore, wild populations contain large numbers of length variants in contrast to uniformly shared length variants in laboratory stocks. High numbers of length variants coupled to the observation of a homogenized bipartite structure of the 240-bp subrepeat array suggest that the unit of turnover and homogenization is smaller than the IGS and might involve gene conversion. The use of PCR for the structural analysis of members of the rDNA gene family coupled to digital DNA typing provides powerful new inroads into the mechanisms of DNA turnover affecting the course of molecular evolution in this family.

Animals↗
PubMed2001

Quantitative assessment of the expression of melanoma-associated antigens by non-competitive reverse transcription polymerase chain reaction.

The assessment of tumor-associated antigens (TAA) recognized by T lymphocytes is a prerequisite for diagnosis and immunotherapy of melanoma. Different reverse transcription-polymerase chain reaction (RT-PCR) protocols allowing the quantification of the TAA mRNA expression in the solid tumor or the detection of circulating melanoma cells have been described. We have recently shown a positive correlation between the amount of specific product formed by RT-PCR and the staining intensity in immunohistochemical analysis of the corresponding sample. Here we describe a quantification procedure based on the direct digitization of the PCR products after separation on ethidium bromide-stained agarose gels, followed by computer-assisted densitometry. To standardize our method, we examined the linear range of the densitometric quantification procedure as reflected by the correlation of signal intensity to the amount of the corresponding DNA. As an internal measure for the so-termed cDNA in the different samples after RNA isolation and reverse transcription, a beta-actin PCR was introduced. Subsequently, we chose four sets of primers for the melanoma-associated antigens MAGE1, tyrosinase, Melan A/MART-1 and gp100/Pmel17 and performed PCR analysis over a range of cycle numbers. In each case, the amplification rate remained constant up to at least 26 cycles under the respective conditions. Plotting the logarithm of the amount of product against the cycle number yields a slope that equals the logarithm of the amplification rate. The amount of starting material can be determined from the intercept with the ordinate. In summary, the method introduced in the present work allows the quantification of TAA in melanoma which might be important for the monitoring of disease. Technically the method is sound and sensitive, avoids post-PCR manipulations and can be performed with the standard equipment of a molecular biology laboratory. It can be applied also to other solid tumors and leukemias.

Antigens, Neoplasm↗
PubMed1993

Allele-specific MVR-PCR analysis at minisatellite D1S8.

Minisatellite variant repeat mapping by the polymerase chain reaction (MVR-PCR) provides a digital approach to DNA typing of great potential use both in forensic medicine and, by mapping single alleles, for exploring allelic variability and mutation processes at minisatellites. The MVR haplotypes of single alleles can be determined either from physically separated alleles or by pedigree analysis of digital diploid codes generated from both alleles simultaneously. We now show that single alleles can be rapidly mapped from total genomic DNA using allele-specific PCR primers directed to polymorphic sites in the DNA flanking the minisatellite. This approach can also be used to dissect mixed DNA samples such as those often encountered in forensic DNA analysis.

Alleles↗
PubMed1997

Reverse transcriptase-polymerase chain reaction assays for prostate cancer.

RT-PCR, if targeted against a prostate-specific marker, can be a highly specific and sensitive assay to detect the presence of occult prostate cancer cells at sites far distant from the primary tumor. The low false-positive rate (0.8%) observed when combining most published studies and the high rate of detection of prostate cells in metastatic patients (88%) found in well-defined clinical studies address the basic requirements of a clinically effective diagnostic modality. Additionally, all reports indicate that RT-PCR positivity for PSA or PSMA increases with increased stage of prostate cancer, suggesting that RT-PCR assays may have value in staging the patient presumed to have clinically localized disease. Indeed, data from two institutions have shown the unique contribution that RT-PCR technology might lend to this most vexing problem. With the suggestive data already published, we remain optimistic that molecular staging will become a reality in the future. After all, the variability in techniques of RT-PCR (as well as differences in targets and samples assayed) used by the various laboratories now investigating this method could themselves be responsible for many of the differences reported. There is no question that standardization among laboratories is essential before clinical utility of molecular staging can be proved. Indeed, this endeavor represents the major aim of the RT-PCR consortium in the United States. Nevertheless, at our own medical center, we are struck by our finding that if a preoperative RT-PCR for PSA is negative, the patient can be assured of a successful operative outcome in 88% of cases. We also are impressed by the data showing that if the serum PSA level is greater than 10 ng/mL and the RT-PCR assay is positive, 90% of patients undergoing radical surgery have adverse pathology reports (and, in fact, have already experience operative failure in nearly half the cases). There is increasing consensus that RT-PCR assays afford prognostic information that is also unique. Three institutions have similar data comparing operative or preoperative RT-PCR strategies (employing all three potential sample sources) with treatment outcome. This may suggest the validity of RT-PCR as a staging modality or, alternatively, suggest that a truly metastatic phenotype is identified in patients with circulating PSA-synthesizing cells, or in patients harboring PSA-synthesizing cells in node or marrow tissues. In either case, RT-PCR appears already to provide information not available before the use of this technology. In fact, in the only series comparing RT-PCR with other, more established predictors (PSA, Gleason score) in a multivariate analysis, RT-PCR status provided the best prognostic information. Prostate manipulation does, in fact, appear to affect the RT-PCR assay. Although digital rectal examination and cystoscopy do not seem sufficiently invasive to release prostate cells into the circulation, a small number of individuals undergoing transrectal ultrasound biopsy will experience this phenomenon. This observation provides a cautionary note relative to timing of sample collection for RT-PCR assays. Furthermore, a significant fraction of patients undergoing prostate manipulation during radical surgery may experience shedding of prostate cells into the operative field and release of some into the peripheral circulation. This has not been universally observed and, even if true, may not share the significance of spontaneous RT-PCR positivity (before prostatic manipulation). In the one instance, a potentially metastatic phenotype is responsible for RT-PCR positivity, whereas in the other instance, PSA or PSMA synthesizing cells may have no metastatic potential at all. Further studies on the biologic half-life of such cells are required before any clinical judgement can be made regarding this phenomenon. In summary, there appears to be consensus that RT-PCR technology can differentiate controls from patients with metas

Humans↗
PubMed2005

Digital single-nucleotide polymorphism analysis for allelic imbalance.

Digital single-nucleotide polymorphism (SNP) analysis is developed to amplify a single template from a pool of DNA samples, thereby generating the amplicons that are homogeneous in sequence. Different fluorophores are then applied as probes to detect and discriminate different alleles (paternal vs maternal alleles or wild-type vs mutant), which can be readily counted. In this way, digital SNP analysis transforms the exponential and analog signals from conventional polymerase chain reaction (PCR) to linear and digital ones. Digital SNP analysis has the following advantages. First, statistical analysis of the PCR products becomes available as the alleles can be directly counted. Second, this technology is designed to generate PCR products of the same size; therefore, DNA degradation would not be a problem as it commonly occurs when microsatellite markers are used to assess allelic status in clinical samples. Last, digital SNP analysis is designed to amplify a relatively small amount of DNA samples, which is available in some clinical samples. Digital SNP analysis has been applied in quantification of mutant alleles and detection of allelic imbalance in clinical specimens and it represents another example of the power of PCR and provides unprecedented opportunities for molecular genetic analysis.

Allelic Imbalance↗
PubMed2004

Minisatellites and MVR-PCR for the individual identification of parasite isolates.

In recent years, a wide variety of biochemical and molecular typing systems have been employed in the study of parasite diversity aimed at investigating the level of genetic diversity and delineating the relationships among different species and subspecies. Parasite sequence-specific polymerase chain reaction (PCR)-based genotyping systems are among the most useful tools employed to date, because they can be applied to very small quantities of host-contaminated parasite material and, using repeated loci such as mini- and microsatellites, allow the identification and tracking of individual strains as well as the determination of allele and genotype frequencies in populations. Although minisatellites have been used very successfully to study parasite populations, in particular Trypanosoma brucei populations, there are some technical problems involved in the use of these markers. For example, minisatellite alleles tend to vary in a quasi-continuous fashion, making unambiguous allele identification difficult. The development of minisatellite variant repeat (MVR) mapping by the polymerase chain reaction (MVR-PCR) as a digital approach to DNA typing has overcome many of the drawbacks of minisatellite length analysis. The system assays the dispersion patterns of MVRs within minisatellite alleles, producing an easily interpretable code for each allele. This technique not only allows unequivocal allele identification but also reveals cladistic information that can be used to determine the possible genetic relationships among the different strains and subspecies. The MVR mapping technique has been applied successfully to minisatellites in the parasite Plasmodium falciparum to uniquely identify strains, and more extensively in Trypansoma brucei, where it was used to determine population structure and to examine the relationships among T. brucei subspecies, providing evidence for multiple origins of human infectively. In this chapter, the methods for genotyping of T. brucei parasites using both minisatellite allele length and MVR mapping are described in full and can be easily adapted to apply to mini-satellites in other parasites.

Animals↗
PubMed1992

[Initial experience with digital luminescence radiography in the radiological diagnosis of the visceral cranium].

The purpose of this study was the evaluation of digital luminescence radiography (PCR; Philips Computed Radiography) used in routine radiological analysis of the facial skull. Besides the standard radiographs additional PCR shots were taken in 16 cases. The subjective evaluation was carried out by three different people using clinical criteria. PCR films showed a significant advantage compared to standard radiographs for the above mentioned applications.

Evaluation Studies as Topic↗
PubMed1993

[The hand in hematologic diseases].

Radiography of the hand often reflects the picture of generalized diseases, affecting both the muscolo-skeletal system and the others. Some of the most common hematologic disorders may be detected in roentgenograms of the hand, especially in the anemias, but also in plasma cell dyscrasias and proliferative malignant diseases (i.e., lymphomas and leukemias). On the basis of their experience, the authors have reviewed and discussed the radiographic "pattern" of the hand in several hematologic conditions (i.e., anemias; thalassemias; sickle-cell disease; lymphomas; multiple myeloma; etc.), and their pathogenesis. Radiographies of both the hands, in antero-posterior view, were performed using Kodak-Min R film; xeroradiography was performed--in the same projection--using Rank Xerox plate, developed always in "positive mode" in 125 Rank Xerox System, conditioned with contrast "D", for emphasizing osseous details. Recently, digital radiography--employing PCR system--has substituted xeroradiography, because of its well-known properties and diagnostic advantages: in this manner, changes in bone and soft tissue are demonstrated on the same image, with augmentation of diagnostic information, with reduced dose to patient. In our series, hand is always involved (100% of cases) in thalassemias: lesions are characterized by diffuse osteopenia (washed out melted appearance), with widening of bone marrow space, diaphyseal convex aspect of the long bones, thinning of the cortex, and cyst-like changes (rain drops). Lesions disappear completely after the hypertransfusion regimen (HTR). Following chelation therapy, lesions of the wrist and hand are similar to those described in rickets and/or scurvy. Sometimes, the hand is characteristically affected in sickle-cell disease--particularly in the so called hand-foot syndrome--as "cone-deformity". In multiple myeloma incidence of hand involvement is 2.9%: lesions reflect general abnormalities observed in other skeletal sites, and they consist in multiple well-circumscribed lytic lesions. In amyloidosis, poorly defined radiolucent areas may be discovered. In non-Hodgkin lymphoma, incidence of hand involvement is less frequent, approximately 0.2%: the radiographic pattern is aspecific (mottled lytic lesions), sometimes simulating multiple myeloma and/or leukemias. In hemophilia, swelling of soft tissues, around the interphalangeal joint, related to intra-articular and/or per-articular hematoma, is observed. The other conditions reflect general radiographic features of anemias, which are of three main types: 1. the over active marrow (i.e., polycythemia); 2. the infarction of bone (i.e., sickle-cell disease); 3. non-specific findings, resulting from chronic illness (delays of maturation; dwarfism; osteopenia; tendency to infection).

Adolescent↗
PubMed1998

Hypervariable digital DNA codes for human paternal lineages: MVR-PCR at the Y-specific minisatellite, MSY1 (DYF155S1).

We describe the first haploid minisatellite, the human Y chromosome-specific locus, MSY1. It consists of an array of 48-114 AT-rich 25 bp repeats of at least five different variant types. A minisatellite variant repeat PCR (MVR-PCR) system gives Y-specific DNA codes, with a virtual heterozygosity of 99.9%, making MSY1 by far the single most variable locus on the Y. African populations contain the most diverged MSY1 structures. MSY1 is the only Y-chromosomal system where the characteristics of large numbers of mutations can be studied in detail: it provides a uniquely powerful tool both for the investigation of mutation in a haploid system, and for the dating of paternal lineages.

Africa↗
PubMed1997

Detection of thymidylate synthase gene expression levels in formalin-fixed paraffin embedded tissue by semiquantitative, nonradioactive reverse transcriptase polymerase chain reaction.

We describe a new, simple and reliable semiautomated strategy for quantifying mRNA from archival specimens by using oligo(dT)25 paramagnetic beads and the reverse-transcriptase polymerase chain reaction (PCR) coupled with quantitative digital image analysis (Q-DIA). To evaluate the experimental conditions, we examined thymidylate synthase (TS) gene expression in mRNA isolated from both flash-frozen and formalin-fixed paraffin-embedded human biopsy samples using biopsy material obtained from 2 patients prior to chemotherapy with 5-fluorouracil. Following the electrophoretic separation of the PCR products through a 20% polyacrylamide gel, quantitation of the perimeters of the silver-nitrate-stained PCR products will be done by Q-DIA using a video frame-grabber board attached to a CCD camera using Image-Pro+ software. Validation of this approach will involve a comparison of the observed gene expression levels to TS protein levels obtained by tissue homogenization assays of TS, tetrahydrofolate, 5,10-methylenetetrahydrofolate, 2'-deoxyuridine-5'-monophosphate and 5-fluoro-2'-deoxyuridylate (FdUMP), by established [3H]FdUMP ligand-binding assays. The novelty of this method is that it offers a low-cost means whereby Q-DIA is performed directly from the gel to rapidly and accurately determine the level of TS gene expression, which is standardized against the beta-actin housekeeping gene. In the protocol described herein, gene expression studies can be done quickly and without the use of radioactive substances in both normal clinical samples shock frozen at the time of surgical excision and in formalin-fixed paraffin-embedded archival samples, which are commonly available in all hospital pathology departments. To demonstrate the utility of this method, mRNA was extracted from both nonpathological and tumor biopsies originating from both types of material from the same patients. TS gene expression in the flash-frozen and archival materials was compared to the level of TS intracellular enzyme activity in the same samples and a correlation of 89 and 80% between the shock-frozen and archival material relative to TS intracellular enzyme activity levels was observed. These findings suggest that routine semiautomated quantitative analysis of rare mRNA transcripts, e.g. TS, from archival material can be applied for retrospective studies.

DNA, Complementary↗
PubMed2000

Quantification of relative mRNA expression in the rat brain using simple RT-PCR and ethidium bromide staining.

We developed a protocol for quantification of relative gene expression using reverse transcription-polymerase chain reaction (RT-PCR) without the use of radioisotopes, special equipment or extra nucleotide fragments, such as competitors. The relative gene expression of GABA(A) receptor beta(1) subunit (GABA(A)Rbeta(1)) and phospholipase C beta(4) subtype (PLCbeta(4)) in rat cerebrum and cerebellum were determined by comparing the ratio of PCR products generated by linear amplification of the target cDNA segments and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA segment as a reference. The density of PCR products was measured from digitized images of photographs of ethidium-bromide-stained agarose gels. The linear region of PCR amplification was within the linear range (from 0.3 to 12 ng DNA in a single band) of the detection system. The accuracy of the present method was <2-fold difference in gene expression in a single determination and a 1.5-fold difference was statistically significant after repeated measurements. The estimated relative expression of PLCbeta(4) was significantly higher in cerebellum than cerebrum, and that of GABA(A)Rbeta(1) was the same in these two regions. Using the present method, it is possible to quantify several different subunits and subtypes of known ion channel, neurotransmitter receptor and intracellular signaling enzyme gene families.

Animals↗
PubMed1995

A comparative study of karyotypes of muntjacs by chromosome painting.

We have used a combination of chromosome sorting, degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), chromosome painting and digital image capturing and processing techniques for comparative chromosome analysis of members of the genus Muntiacus. Chromosome-specific "paints" from a female Indian muntjac were hybridised to the metaphase chromosomes of the Gongshan, Black, and Chinese muntjac by both single and three colour chromosome painting. Karyotypes and idiograms for the Indian, Gongshan, Black and Chinese muntjac were constructed, based on enhanced 4', 6-diamidino-2-phenylindole (DAPI) banding patterns. The hybridisation signal for each paint was assigned to specific bands or chromosomes for all of the above muntjac species. The interspecific chromosomal homology was demonstrated by the use of both enhanced DAPI banding and comparative chromosome painting. These results provide direct molecular cytogenetic evidence for the tandem fusion theory of the chromosome evolution of muntjac species.

Animals↗
PubMed2006

A single amino acid exchange shifts the serological reactivity of the novel HLA-B*4442 allele product from HLA-B44 to HLA-B21.

A novel HLA-B (human leukocyte antigen-B) allele, HLA-B*4442, was identified both in a Czech patient with leukaemia and in his mother. The presence of a novel allele was initially suspected because conflicting results were obtained by serological and DNA typing techniques. The HLA typing using the polymerase chain reaction-sequence-specific primers (PCR-SSP) at the two-digit level indicated an allele belonging to the HLA-B*44 group, whereas serological typing indicated HLA-B21. Typing with PCR-sequence-specific oligonucleotides (PCR-SSO) resulted in a unique reaction pattern that could not be assigned to a known allele, PCR-SSP typing at the four-digit level did not match any known B*44 allele, either. The sequencing-based typing of the HLA-B locus then revealed the novel B*4442 allele that is identical with B*4405 except a single C-->G nucleotide exchange at position 572. This exchange results in an amino acid substitution from serine to tryptophan at position 167 of the expressed HLA-B protein. The B21 serological reactivity of the novel B*4442 allele product was confirmed by employing an additional serological panel of typing sera. Our findings support previous reports claiming that serine at the position 167 in the alpha-2 domain of the HLA-B protein is a major determinant of the HLA-B44(12) serological epitope.

Alleles↗
PubMed2006

Four-digit allele genotyping of the HLA-A and HLA-B genes in Japanese patients with Behcet's disease by a PCR-SSOP-Luminex method.

The present study represents the first four-digit allele genotyping of HLA-A and -B in Japanese Behcet's disease (BD) patients and controls using a new genotyping method (named the PCR-SSOP-Luminex method) to determine the association of certain HLA-A or -B alleles with BD. Peripheral blood lymphocytes were collected from 180 Japanese BD patients and 170 healthy controls. The genotype frequency of HLA-B*5101 was significantly increased in the patients (61.7%) as compared with the controls (15.9%) (Pc = 1 x 10(-16), OR = 8.5). When we recalculated the phenotype frequencies after excluding the HLA-B*51-positive patients and controls to account for the effects of the linkage disequilibrium and the abundance of the HLA-B*51 allele, the frequencies of HLA-A*2602 and HLA-B*3901 had a weak association in the patient group without HLA-B*51 as compared with the control group without HLA-B*51 (A*2602; Pc = 0.130, OR = 4.3, B*3901; Pc = 0.099, OR = 3.5). This study confirmed on the basis of using a new and more accurate genotyping method that Japanese BD patients have a strong primary association with HLA-B*5101. The significant increase of HLA-A*2602 and B*3901 in the patient group without HLA-B*51 suggests that these two alleles might also have some secondary influence on the onset of BD.

Alleles↗