Amplification Of Microsatellite Markers Research Articles

Physiological and Genetic Variation of Hordeum vulgare L. and Triticum aestivum L. Lines Planted in Turkey

Hordeum vulgare L. (barley) and Triticum aestivum L. (wheat) are among the most valuable crops cultivated and planted in many regions including Turkey. These plants have wide range of adaptation ability and capacity; they represent high level of variation in terms of physiological, genetics and epigenetics parameters and characteristics. Physiological and genetic variations were investigated by relative water content (RWC) assays, measuring electroconductivity (EC) levels and amplification of microsatellite markers in 21 barley and 43 wheat lines. At least three drought sensitive and three drought resistant lines were detected in barley and wheat lines via RWC assays. RWC values were recorded between 0.05±0.013 and 0.55±0.003%. Similarly great variation was detected for EC values of both barley and also wheat lines. Minimum and maximum EC values were ranged from 4.00±0.06 μS cm-1 to 59.88±3.209 μS cm-1. Three microsatellite markers, Bmag0120, Bmag0306 and Bmag375, were targeted in barley genome. Similarly, Han18, Wmc506 and Wmc623 microsatellite markers were targeted in wheat genome. Among these markers only Bmag0120 and Han18 were amplified from each line’s genome by PCR and qPCR assays. In PCR and qPCR analysis homozygous and heterozygous lines were detected for Bmag0120 while each line was homozygous for Han18. Idiomorphic band size as 300 bp was detected in Han18 while it was ranged from 224 to 279 bp for Bmac0120 marker. Results showed that homozygous lines were drought resistant ones in barley lines whereas no correlation was found for wheat lines investigated in this study.

Genetic diversity of diploid and triploid Carassius gibelio (Bloch, 1782) from bisexual population

Event Abstract Back to Event Genetic diversity of diploid and triploid Carassius gibelio (Bloch, 1782) from bisexual population Karolina Kowalewska1*, Anna Przybył1, Natalia Jędrzejewska1, Edyta Kurantowicz1 and Alicja Boron1, 2 1 Department of Zoology, Faculty of Biology and Biotechnology, University of Warmia and Mazury in Olsztyn, Poland 2 University of Warmia and Mazury in Olsztyn, Poland The silver crucian carp Carassius gibelio is a cyprinid (Cyprinidae) species living in almost all available water bodies as it is the most invasive freshwater species in Europe (Tóth et al. 2005; Grabowska and Przybylski 2015). It spread due to its enormous resistance to adverse environmental conditions, plasticity of reproduction and the possibility of creating viable, better adopted hybrids. It reproduces sexually and clonally, through gynogenesis; females lay eggs, which are stimulated to develop by the sperm of males, including related species (Zhang et al. 2015). This species is an interesting object of research, also due to recently reported changes in the sex and ploidy structure occurring in its populations. Initially, in Europe, unisexual populations, consisting of triploid (3n) gynogenetic females were dominated. Recently, however, more diploid-triploid populations have been described with varying numbers of diploid and sometimes triploid males (Vetemaa et al. 2005; Šlimková et al. 2015). This trend is also visible in Poland, where females with single males and bisexuals populations were recorded (Boroń et al. 2011; Szabelska et al. 2017). The causes of changes in the sex ratio and the associated reproduction in C. gibelio populations are not precisely understood. Genetic diversity plays an important role in the survival of species, populations and in their response to environmental changes. Relatively large differences in the genetic variability of the C. gibelio populations can be explained by the dominance of different modes of reproduction (Zhigileva et al. 2015). For this reason, this preliminary study aimed to compare the genetic diversity of C. gibelio diploid and triploid males and females inhabiting the diploid-polyploid population in the Siemianówka Reservoir. This population is interesting because of the unclear origin of 3n males and also because of the lack of knowledge about the reproduction of triploids individuals. Materials for investigation, fin or muscle tissues were taken 10 each from diploid and triploid females and males. Animals were treated in accordance with the Act of 15 January 2015 on the protection of animals for scientific and educational purposes, according to the opinion (No. 20/2013/N) of the Local Ethics Committee of the University of Warmia and Mazury in Olsztyn, Poland. Genetic diversity was analyzed by random amplification of polymorphic DNA fragments (RAPD) and by microsatellite markers. The DNA was extracted with the modified phenol-chloroform method using a urea buffer and a phenol-chloroform-isoamyl alcohol mixture (24:25:1). Ten primers selected were tested as the most useful for the C. gibelio genetic diversity using the RAPD method (Tóth et al. 2005; Dong et al. 2013). Among them, we managed to achieve effective amplification for five primers at 52 °C. The ZJ6 starter was characterized by the biggest differentiation of obtained banding DNA profiles, but starters AR5 and ZJ7 also maintained a high diversity of obtained profiles. Some individuals were characterized by the similarity of the obtained RAPD profile. However, in order to be considered as clonal lines, it was necessary to conduct further analyzes using more specific molecular markers. Amplification of microsatellite markers was carried out using multiplex PCR with universal fluorochrome labeled primers (Li et al. 2017). The conducted research confirmed the high efficiency of amplification of the 16 microsatellite markers used. The obtained products were subjected to capillary electrophoresis in the ABI3130xl sequencer (Applied Biosystem) using the GeneScan LIZ-500 marker. All the loci were polymorphic, and a total of 148 alleles were identified. The most polymorphic was GF19 (14 alleles), while the MFW2 was the less polymorphic (three alleles). The lengths of the ranges for the tested primers and the number of alleles deviated from the literature data, but these differences may result primarily from species and population differences and the presence of C. gibelio hybrids. The relatively large genetic diversity of the triploid C. gibelio males demonstrated, seems to be confirmed by literature data indicating the possibility of sexual reproduction between triploid females and males. The obtained results using microsatellite sequences require comparison with similar data of progeny from experimental crosses between diploid and triploid C. gibelio females and males. The presented data will be used in planning further experiments. Acknowledgements We express our thanks to the Polish Angling Association in Bialystok and Mr. Andrzej Filinowicz for collecting fish and to Dr. Anna Leska for collected tissue. This work was carried out within the projects no. 12.610.006-300 and 12.620.029-500 of the UWM in Olsztyn, financed by Ministry of Science and Higher Education, Poland.

Identification and cross-species amplification of microsatellite markers derived from expressed sequence data of rose species

Genic SSR markers derived from public expressed sequence tags (ESTs) data are valuable and cost effective marker resources for genome mapping and diversity studies. Owing to their derivation from the transcribed regions which often have putative functions, these markers can be easily associated with desired trait. In the present study, 19 novel SSR markers were identified from 450 non redundant unigenes derived from 3,726 public ESTs of two rose species. Among SSRs, tri-repeats (61.3 %) were most abundant followed by di-repeat (29 %). Newly identified EST-SSR markers recorded significant homology with the known/putative proteins of Arabidopsis thaliana. The cross transferability to 12 rose species ranged from 63.2 to 100 %. Novel SSR loci found to be moderately to highly polymorphic with locus wise average number of alleles and polymorphism information content (PIC) were 4.1 and 0.33, respectively. Cloning and sequencing of EST-SSR size variant amplicons of marker locus Rches12 revealed that the variation in the number of SSR repeat-units was the main source of fragment polymorphism. The high polymorphic potential coupled with high cross-transferability rate demonstrates wider applicability of novel SSR markers in genetic diversity, genome mapping and evolutionary studies in various rose species.

Identification of salmonid fish using microsatellite markers with identical PCR-primers

A set of homologous primers were designed to provide the amplification of microsatellite markers in a variety of salmonid species. They can help to solve problems associated with the genetics and conservation biology of salmonids, viz., (1) species identification; (2) determination of interspecific hybrids; (3) genetic estimation of salmonid biodiversity in a watershed; (4) individualization of tissue samples, i.e., testing on their belonging to the same individual; and (5) identification of a fish’s origins.

An effective method for extraction and polymerase chain reaction (PCR) amplification of DNA from formalin preserved tissue samples of snow leopard

Formalin-preserved biological samples obtained from endangered species are valuable in assessing genetic diversity. To make use of snow leopard samples preserved in formalin over a period of two to seven years, we optimized the method of extracting DNA from these samples. We used (a) phenol chloroform : isoamyl alcohol, (b) the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Germany), (c) the Qiagen DNeasy Blood and Tissue Kit after treating the samples with NaOH for three days and (d) the Qiagen DNeasy Blood and Tissue Kit after treating the samples with phosphate buffered saline (PBS) for three days. The usefulness of the extracted DNA was assessed on the basis of mitochondrial (150 to 550 bp) and nuclear (95 to 229 bp) markers. There was no PCR amplification with the first two methods. The PCR amplification with the NaOH and PBS treatment had a success rate of 30 to 100% for both mitochondrial and nuclear markers. The PBS method is the best method for extraction of DNA from formalin-preserved samples of longer period (two to seven years) because of higher success rate in amplifying mitochondrial gene of ca. 550 bp (60%) than the NaOH method (28%). The overall amplification of microsatellite markers in such samples was also higher in samples treated with PBS (43 to 100%) than NaOH (0 to 100%). The PCR products obtained were confirmed through DNA sequencing to be of snow leopard origin. The optimized protocol will enable genetic studies to be conducted on tissue samples of other species that have been preserved in formalin. The protocol will be particularly useful for species that are elusive and from which it is difficult to collect fresh tissue samples.

Cross Species Amplification of Pennisetum glaucum Microsatellite Markers in Pennisetum purpureum and Genetic Diversity of Napier Grass Accessions

ABSTRACTNapier grass (Pennisetum purpureum Schum.) is an important forage crop in tropical areas although little is known about its genome information, and few molecular markers have been developed for this species. This work aimed to check the viability of cross‐species amplification of microsatellite markers between pearl millet (Pennisetum glaucum) and Napier grass and to evaluate the genetic diversity among Napier grass germplasm accessions. Fifty‐four microsatellite markers previously described in pearl millet were tested against Napier grass, and 30 markers (55.5%) showed successful cross‐amplification. From them, 18 microsatellite markers were selected to study the genetic diversity in the Embrapa Active Germplasm Bank of Napier Grass (Embrapa‐BAGCE). A total of 180 alleles were identified by these selected microsatellite markers in 107 Napier grass accessions and four pearl millet samples. The average similarity coefficient (Dice) calculated among the Embrapa‐BAGCE accessions was 0.651, ranging from 0.254 to 1.0. Some accessions showed similarity coefficients equal to one, indicating that they have common progenitors or that they might be the same accessions with different denomination. To our knowledge, this work is the first to describe microsatellite markers in Napier grass and represents a significant advance regarding the use of molecular markers in this species.

Diversity and specificity of microsatellites within Aspergillus section Fumigati

BackgroundMicrosatellites (or short tandem repeats, STRs) are the genetic markers of choice for studying Aspergillus fumigatus molecular epidemiology due to its reproducibility and high discrimination power. However, the specificity of these markers must be investigated in a group of isolates from closely related species. The aim of this work was to test a microsatellite-based PCR multiplex previously designed for A. fumigatus in a set of species belonging to section Fumigati, namely Aspergillus fumigatiaffinis, Aspergillus lentulus, Aspergillus novofumigatus, Aspergillus unilateralis, Aspergillus viridinutans, Neosartorya fischeri, Neosartorya hiratsukae, Neosartorya pseudofischeri and Neosartorya udagawae.ResultsThe reference A. fumigatus strain ATCC 46645 was easily genotyped in standard conditions showing a final electrophoretic profile of 8 expected peaks corresponding to each microsatellite locus. Inversely, no peaks were observed for all other species from section Fumigati, with an exception for marker MC6b in A. unilateralis. By screening the genome sequence of Neosartorya fischeri NRRL 181, the results showed that MC3, MC6a and MC7 might be employed for N. fischeri genotyping since these markers present several repeats of each motif. The accumulation of insertions and deletions was frequently observed in the genomic regions surrounding the microsatellites, including those where the A. fumigatus primers are located. The amplification of microsatellite markers in less stringent amplification conditions resulted in a distinct electrophoretic profile for species within section Fumigati.ConclusionsTherefore, the microsatellite-based PCR multiplex allow simple identification of A. fumigatus and, with a slight modification of temperature conditions, it also allows discriminating other pathogenic species within section Fumigati, particularly A. fumigatiaffinis, N. fischeri and N. udagawae.

Cross species amplification ability of novel microsatellites isolated from Jatropha curcas and genetic relationship with sister taxa

The present investigation was undertaken with an aim to check the ability of cross species amplification of microsatellite markers isolated from Jatropha curcas--a renewable source of biodiesel to deduce the generic relationship with its six sister taxa (J. glandulifera, J. gossypifolia, J. integerrima, J. multifida, J. podagrica, and J. tanjorensis). Out of the 49 markers checked 31 markers showed cross species amplification in all the species studied. JCDS-30, JCDS-69, JCDS-26, JCMS-13 and JCMS-21 amplified in J. curcas. However, these markers did not show any cross species amplification. Overall percentage of polymorphism (PP) among the species studied was 38% and the mean genetic similarity (GS) was found to be 0.86. The highest PP (24) and least GS (0.76) was found between J. curcas/J. podagrica and J. curcas/J. multifida and least PP (4.44) and highest GS (0.96) was found between J. integerrima/J. tanjorensis. Dendrogram analysis showed good congruence to RAPD and AFLP than nrDNA ITS data reported earlier. The characterized microsatellites will pave way for intraspecies molecular characterization which can be further utilized in species differentiation, molecular identification, characterization of interspecific hybrids, exploitation of genetic resource management and genetic improvement of the species through marker assisted breeding for economically important traits.

Transmission of Familial Mediterranean Fever Mutation After Bone Marrow Transplantation and Successful Treatment With Anakinra

Familial Mediterranean fever (FMF) is a recessively inherited autoinflammatory disorder characterized by recurrent episodes of fever accompanied by arthritis, peritonitis, pleuritis, pericarditis, or erysipelas-like erythematosus rash (1). The gene responsible for FMF, MEFV, is located on chromosome 16p13 (2) and encodes a 791-amino acid protein, known as pyrin or marenostrin, which is believed to regulate inflammation by modulating the caspase 1 or interleukin (IL)-1β pathway (3). Herein, we report the transmission of FMF mutations in a 22-year-old male patient who underwent bone marrow transplantation (BMT) for idiopathic aplastic anemia in May 2009 from his FMF-affected human leukocyte antigen (HLA)-identical 19-year-old brother. Informed written consent was obtained from the patient, not carrying any FMF mutations pre-BMT, concerning the risk of transmission of FMF. Conditioning regimen consisted of cyclophosphamide 200 mg/kg and antithymocyte globulin 10 mg/kg, each given intravenously in four doses. Graft-versus-host disease prophylaxis-associated cyclosporine 3 mg/kg was initiated intravenously on day 1 and methotrexate intravenously on days 1, 3, and 6. Engraftment was observed on day 19. On day 23, the patient presented fever and abdominal pain, resistant to broad-spectrum antibiotic therapy. C-reactive protein (CRP) was elevated to 229 mg/L. Thoracic and abdominal computed tomography was normal. Resolution of the febrile episode and normalization of CRP were achieved 24 hr after the administration of methylprednisolone at 0.5 mg/kg per day, intravenously. The patient was dismissed from hospital on day 48 under 0.4 mg/kg per day of oral corticosteroids and oral cyclosporine. Donor chimerism 100% was confirmed by amplification of microsatellite markers by polymerase chain reaction. On day 60, the patient presented with fever, abdominal pain, diarrhea, arthritis, erysipelas-like rash, and renal failure. CRP was increased to 150 mg/L. Cyclosporine was interrupted, and antibiotic treatment was administered. The patient was not receiving any corticosteroid treatment. He was dismissed after improvement, 15 days later. He consulted 6 days after dismissal for another similar episode. A treatment with colchicine 1 mg per day was initiated, and the patient was screened for FMF mutations that were positive for homozygous M694V mutation. On day 110, the patient presented one more FMF flare under colchicine treatment, as well as severe neutropenia (<200/mm3), which was attributed to colchicine. Furthermore, an ultrasound echocardiogram revealed a reduction in the ejection fraction from 62% to 30%. Taking into account, the persistent neutropenia (although granulocyte-colony stimulating factor treatment was given at a dose of 48 μg daily), as well as the recurrence of FMF flares, a treatment with anti-IL1 antagonist (anakinra) at a dose of 100 mg per day subcutaneously was initiated on day 120. Neutrophil count and CRP attained normal values 1 week after the cessation of colchicine. Renal function and left ventricular ejection fraction were also normalized 2 weeks after the discontinuation of colchicine. In a 4-month follow-up, neither FMF crisis nor any side effect of anakinra treatment has been observed. The patient continues to receive a daily subcutaneous injection of anakinra at a dose of 100 mg per day. This is to our knowledge, the first report of successful treatment with anakinra in a patient who acquired FMF mutation after allogeneic BMT and who presented intolerance and resistance to colchicine treatment. Transmission of FMF mutations after BMT has rarely been described (4). MEFV is almost exclusively expressed in monocytes and granulocytes. These two cell lineages are of donor origin after successful allogeneic BMT (5). Therefore, FMF mutations are potentially transferred through BMT. Oral daily treatment with colchicine is the most effective therapy for FMF. Nevertheless, bone marrow suppression, especially neutropenia, is a known side effect of colchicine (6,7). Moreover, side effects of colchicine could be reinforced by the concomitant administration of cyclosporine, which suppresses P-glycoprotein; therefore, increasing the intracellular concentration of colchicine and, which interacts with CYP3A4, thus decreasing the hepatic elimination of colchicine (8). Severe neutropenia, cardiomyopathy, and peripheral neuropathy have recently been described in the context of heart transplantation (9). Furthermore, 10% of patients are unresponsive to colchicine (10). Recent short reports demonstrate the efficacy of IL-1 receptor antagonist therapy (anakinra) in colchicine-resistant FMF patients (3, 11–13). Efficacy of anakinra in colchicine-resistant FMF patients after kidney transplantation has been recently reported (14). Moreover, IL-1 receptor antagonist has been evaluated without toxicity in allogeneic BMT in the prevention of graft-versus-host disease (15). Our observation supports the transmission of FMF mutations after allogeneic BMT and demonstrates that anakinra might be an efficacious and safe treatment in allotransplanted FMF patients with severe colchicine-induced neutropenia or intolerance or colchicine resistance. Anna D. Petropoulou1 Marie Robin1 Gérard Socié1 Lionel Galicier2 1Service Hématologie-Greffe Hôpital Saint-Louis Paris, France 2Service Immunologie Clinique Hôpital Saint-Louis Paris, France

The detection of aneuploidy and maternal contamination by QF-PCR in samples undergoing prenatal diagnosis for thalassemia in Southern China

Objectives To apply a simple and low-cost approach, which would easily and accurately detect both the common chromosomal abnormalities and maternal cell contamination (MCC) when invasive prenatal testing is performed for diagnosis of thalassemia. Study design Quantitative fluorescence-polymerase chain reaction (QF-PCR) was carried out by amplification of microsatellite markers using fluorescence-labelled primers, followed by quantitative analysis of the allele peaks on a genetic analyser. A multiplex of 11 primer pairs for loci on each of chromosomes 13, 18 and 21 was used. Results A total of 387 prenatal samples were tested. Five (1.3%) samples showed MCC, including two (0.7%, 2/289) amniotic fluid samples and three (3.1%; 3/98) chorionic villi samples. Of the 379 prenatal samples without MCC, QF-PCR assays detected two (0.5%) cases of trisomy 21, which were confirmed by traditional karyotyping, and missed one case of trisomy 2 mosaicism and one case of monosomy X. The case of trisomy 2 mosaicism was later found to be limited to the placenta, and the case of monosomy X was picked up by ultrasound. There was no clinically significant case that would have been missed if QF-PCR had been used as a stand-alone test instead of karyotyping when invasive prenatal testing was performed for diagnosis of thalassaemia. Conclusions The QF-PCR assay could allow simultaneous detection of aneuploidy and possible MCC in the fetal material. This is especially valuable when PCR-based techniques are used in the DNA analysis for thalassaemia. This strategy may be applied to prenatal diagnosis of other recessive disorders.

Detection of multiple paternity and sperm storage in a captive colony of the central Asian tortoise, Testudo horsfieldii

Reproductive success is a critical measure of an organism’s fitness. Determining reproductive success in vertebrates is confounded by the concealed mechanism and timing of fertilization (e.g., sperm competition and storage). To assess the relationship between observed mating behavior and reproductive success in the central Asian tortoise, Testudo horsfieldii Gray, 1844, we determined individual genotypes from a captive colony of adults and their offspring. We constructed a size-selected genomic library from T. horsfieldii and screened for polymorphic microsatellite markers. The screen resulted in identification of two novel microsatellite regions. Cross-species amplification of microsatellite markers using primers developed for the bog turtle, Glyptemys muhlenbergii (Schoepff, 1801), resulted in isolation of three additional polymorphic microsatellites for T. horsfieldii. The five loci, which have between 5 and 17 alleles and observed heterozygosities between 0.44 and 0.90, were used to determine the frequency of multiple paternity in the captive colony. We found evidence for multiple paternity in 27% of the clutches examined, as well as evidence for overwinter sperm storage and variance in adult male reproductive success. These data indicate that ample opportunity exists for sperm competition and female mate choice in T. horsfieldii.

Maternal cell contamination of prenatal samples assessed by QF-PCR genotyping

To establish the genotype of cultured cells from a cohort of amniotic fluid and chorionic villus samples, and compare this genotype with that obtained from uncultured material from the same sample, in order to assess the frequency and significance of maternal cell contamination of prenatal samples. Quantitative fluorescence-polymerase chain reaction (QF-PCR) was carried out by amplification of microsatellite markers using fluorescence-labelled primers, followed by quantitative analysis of the allele peaks on a genetic analyser. A multiplex of 12 primer pairs for four loci on each of chromosomes 13, 18 and 21 was used. A total of 307 prenatal samples were tested. Of the 254 amniotic fluid samples, 39.8% had some degree of bloodstaining, ranging from 5% bloodstaining in the cell pellet to heavily bloodstained fluid. Uncultured samples were tested by QF-PCR analysis and the cultured cells were tested by both QF-PCR and karyotype analysis. Of the samples, 90.2% had the same single genotype on direct and cultured material. Two samples (0.65%) were mosaic for an aneuploidy cell line. A second genotype, interpreted as maternal cell contamination, was identified in direct and/or cultured preparations in 9.1% of samples, 17.8% of which were not bloodstained. Seven amniotic fluid samples (2.8%) showed maternal cell contamination in cultured material. For heavily bloodstained amniotic fluid samples, a maternal blood specimen may help interpret the results of rapid trisomy testing, followed by confirmation of the fetal origin of cultured cells. QF-PCR analysis has established a higher incidence of maternal cell contamination of cultured amniocytes than previous reports; the presence of MCC (maternal cell contamination) in cultured cells from samples with no bloodstaining underlines the need for karyotype analysis of more than one XX culture.

Modified Rapid DNA Extraction Protocol for High Throughput Microsatellite Analysis in Wheat

ABSTRACTNew technology is allowing marker‐assisted selection to fulfill the promise of increasing efficiency of cultivar development. However, these techniques depend upon the ability to extract DNA from large populations of plants. The objective of this project was to develop a high‐throughput DNA extraction procedure without the need for greenhouse space or growing wheat (Triticum aestivum L.) plants. A sodium hydroxide rapid DNA extraction was modified for a 96‐well format to reduce costs. Seeds were germinated in 8‐well tissue culture plates, and 4‐d‐old seedling tissue was used to extract DNA by means of sodium hydroxide methodology. Approximately 1 μg of genomic DNA per 10 mg of tissue was isolated at a cost of about $0.10. The DNA quality was verified by amplification of microsatellite markers. Results were consistent with either fresh or stored tissue extracts. This technique allows one person to extract nearly 1000 storage‐stable DNA samples daily, while keeping costs at a minimum.

P53 allelic loss and outcome in patients with squamous cell carcinoma of the head and neck.

Although loss of heterozygosity (LOH) on the p53 locus is frequent in squamous cell carcinomas of the head and neck (SCCHN), controversy still remains regarding the prognostic significance of such an event. Eighty consecutive SCCHN were studied. DNA was extracted from matched sets of normal and tumour tissue and used for polymerase chain reaction amplification of microsatellite markers located in the p53 locus. LOH at the p53 locus was found in 39 (70%) of the 56 informative cases. No relationship was found between p53 LOH and age, site, T stage, N stage, disease stage, and histological differentiation. Recurrence occurred in 53% of the cases with LOH and in 58% of the cases without it (P = 0.28). Moreover, no statistically significant association was found between p53 LOH and disease-specific survival (log-rank P = 0.98). These data suggest that although LOH at the p53 locus is common in SCCHN, this finding is of little clinical significance.

Microsatellite Analysis of Microdissected Tumor Cells and 6p High Density Microsatellite Analysis in Head and Neck Squamous Cell Carcinomas with Down-Regulated Human Leukocyte Antigen Class I Expression

Down-regulated human leukocyte antigen (HLA) class I expression is frequently correlated with allelic loss at 6p21.3, which is the location of the HLA coding sequence, in head and neck squamous cell carcinomas (HNSCCs). Previously, we have demonstrated loss of heterozygosity (LOH) at 6p21.3 for at least one locus in 49% of the HNSCCs using 5 microsatellite markers spanning the 4 megabase HLA region. In the present study, the detection threshold (25%) to assign LOH was addressed by laser-assisted microdissection of tumor cells from tumors containing marginal loss. In addition, we describe high density microsatellite analysis of chromosome 6p21.3 in HNSCC with down-regulated HLA class I expression. The purpose of this study was to refine the identification of genetic alterations at 6p21.3 and to pinpoint allelic loss to individual HLA class I genes, using additional markers closely located to the HLA-A, -B, and -C loci and the transporter associated with antigen processing (TAP) genes. LOH analysis by amplification of microsatellite markers and subsequent fluorescent detection is a rapid and sensitive technique to predict HLA class I loss phenotypes in tumors. LOH can be identified at 25% relative signal reduction. Analysis of heterogeneous tumor samples and samples containing a small amount of tumor cells is facilitated by laser-assisted microdissection of tumor cells. In addition, we showed that accurate HLA LOH analysis requires application of microsatellite markers in close proximity to HLA class I and TAP genes.

Analysis of tetraploidElymusspecies using wheat microsatellite markers and RAPD markers

An analysis of Amplification fragment polymorphism of DNA from 27 accessions of 19 tetraploid Elymus species was carried out using 18 wheat microsatellite (WMS) primer pairs and 10 decamer primers. Ten WMS primer pairs produced multiple polymorphism on all accessions tested. Two independent phenograms, one based on WMS-PCR and one on RAPDs, separated the 19 tetraploid species into two main groups, viz., the SH genome species group and the SY genome species group. The results coincide with the genomic classification of these species and hence support previous studies showing that Elymus is not a monophyletic genus. The assays indicated that accessions within a species cluster together, which concurs with the morphological classification. Interspecific and intraspecific polymorphisms were detected by the WMS-PCR and RAPD analyses. Variation was observed among accessions of Elymus caninus. The WMS-PCR detected a much higher level of polymorphism than the RAPD analysis. WMSs seem to be more efficient markers than RAPD markers for studying the population diversity of Elymus species. The potential of cross-species amplification of microsatellite markers as an additional source for genetic analysis and applications in Elymus is discussed in the context of these results.

Loss of heterozygosity at chromosome segment Xq25-26.1 in advanced human ovarian carcinomas

To determine whether a tumor suppressor gene of importance to epithelial ovarian cancer resides on the X chromosome, we examined loss of heterozygosity (LOH) in 123 epithelial ovarian cancer cases. In 54 such cases, we examined LOH at 26 loci on the human X chromosome. In eight cases, we examined LOH in 14 loci and in 61 cases we examined LOH in 13 loci. Matched DNA samples from tumors and corresponding normal tissues were analyzed by polymerase chain reaction (PCR) amplification of microsatellite markers. Frequent losses were found in epithelial carcinomas at the Xq25-26.l region, including DXS1206 (34.5% loss in informative cases), DXS1047 (27.7%), HPRT (24.1%), and DXS1062 (33.3%). The minimum overlapping region of LOH was approximately 5 megabases (Mb), flanked by DXS1206 (Xq25) and HPRT (Xq26.1). The methylation status of the remaining allele of the androgen receptor gene in the tumors exhibiting LOH at the Xq25-26.1 region suggested that the loss was exclusively in the inactive X chromosome. We next determined whether a significant relationship exists between Xq LOH and other parameters, including histologic grade and/or clinical stage of the tumors and LOH at TP53. The Xq LOH had a significant association with grade 2 to 3 tumors at stages II to IV. Sixteen of 18 cases that showed Xq LOH revealed LOH at the TP53 locus, and 45% of tumors exhibiting LOH at TP53 showed Xq LOH. These results suggest that there may be a tumor suppressor gene or genes which escape inactivation of the X chromosome at Xq25-26.1, and that the loss of the gene(s) at Xq25-26.1 is frequently accompanied by loss of the TP53 or loss of another gene on chromosome 17. These losses may contribute to the progression from a well-differentiated to a more poorly differentiated state or to metastatic aggressiveness.

Correlations of allelic imbalance of chromosome 14 with adverse prognostic parameters in 148 renal cell carcinomas.

To investigate cumulative genetic alterations during development and progression of renal cell carcinoma (RCC), we examined DNAs that were isolated from 148 RCCs for allelic imbalance (AI) at four loci on chromosome arm 3p and at 26 loci on chromosome arm 14q by using polymorphic microsatellite markers and densitometric scanning. Because the analysis of solid tumor unbalanced rearrangements remains difficult due to the large proportion of cells that infiltrate from the stroma, we developed a method for the detection and quantification of AI between control and tumor samples by using polymerase chain reaction (PCR) amplification of microsatellite markers. This technique allows detection down to 20% of contaminating cells with good accuracy. We detected AI on 3p and 14q in 57 and 28% of RCC, respectively. A comparison of genetic changes with clinicopathological data showed that, in marked contrast to AI on 3p, AI on 14q was correlated significantly with the stage and grade of the tumors, with 56 and 58% of RCC in Stage IV and Grade 4, respectively, showing AI. Our results suggest that tumor suppressor genes on 3p, including the von Hippel-Lindau gene, may be involved in early steps of carcinogenesis in clear cell carcinoma and that AI on 14q may play an important role in the progression of clear cell and papillary chromophilic cell carcinomas. Loss of heterozygosity (LOH) on 14q may be a new prognostic factor in RCC. Despite the size of the series of tumors and the number of markers used, only rearrangements that involved the whole length of the long arm of chromosome 14 were observed in the present study. The localization of the putative tumor suppressor gene on 14q will require further investigation of RCC with structural rearrangements of 14q.

Correlations of allelic imbalance of chromosome 14 with adverse prognostic parameters in 148 renal cell carcinomas

To investigate cumulative genetic alterations during development and progression of renal cell carcinoma (RCC), we examined DNAs that were isolated from 148 RCCs for allelic imbalance (AI) at four loci on chromosome arm 3p and at 26 loci on chromosome arm 14q by using polymorphic microsatellite markers and densitometric scanning. Because the analysis of solid tumor unbalanced rearrangements remains difficult due to the large proportion of cells that infiltrate from the stroma, we developed a method for the detection and quantification of AI between control and tumor samples by using polymerase chain reaction (PCR) amplification of microsatellite markers. This technique allows detection down to 20% of contaminating cells with good accuracy. We detected AI on 3p and 14q in 57 and 28% of RCC, respectively. A comparison of genetic changes with clinicopathological data showed that, in marked contrast to AI on 3p, AI on 14q was correlated significantly with the stage and grade of the tumors, with 56 and 58% of RCC in Stage IV and Grade 4, respectively, showing AI. Our results suggest that tumor suppressor genes on 3p, including the von Hippel-Lindau gene, may be involved in early steps of carcinogenesis in clear cell carcinoma and that AI on 14q may play an important role in the progression of clear cell and papillary chromophilic cell carcinomas. Loss of heterozygosity (LOH) on 14q may be a new prognostic factor in RCC. Despite the size of the series of tumors and the number of markers used, only rearrangements that involved the whole length of the long arm of chromosome 14 were observed in the present study. The localization of the putative tumor suppressor gene on 14q will require further investigation of RCC with structural rearrangements of 14q. Genes Chromosom Cancer 17:215–224 (1996). © 1996 Wiley-Liss, Inc.