Short tandem repeat markers in diagnostics: what's in a repeat?
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Base Sequence↗
Biomedical subjects
M G J Tilanus
Publications and source records attributed to M G J Tilanus.
High level of chromosome 15 aneuploidy in head and neck squamous cell carcinoma lesions identified by FISH analysis: limited value of beta2-microglobulin LOH analysis.
In cancer research, loss of heterozygosity (LOH), defined by microsatellite markers, is frequently used in the identification of gene loss. Especially, genomic alterations in the human leukocyte antigen (HLA) genes and the beta2-microglobulin (beta2m) gene on chromosome 15 are of interest regarding their function in the immune system. Because LOH analysis detects any allelic imbalance and not just allelic loss, we evaluated the LOH analysis in 11 head and neck squamous cell carcinoma (HNSCC) lesions using fluorescence in situ hybridization (FISH). The 11 tumors were selected out of 53 HNSCC lesions based upon beta2m LOH analysis and beta2m expression. Centromere 1 and 15 FISH were developed to determine the chromosome 15 copy number. Sequence-based mutation analysis of beta2m was conducted on tumors without beta2m expression; no mutations in the coding sequences were found. For five HNSCC lesions with LOH and beta2m expression, centromere 15 FISH indicated gain rather than loss. In the majority of the 11 HNSCC lesions, FISH showed centromere 1 and 15 heterogeneity throughout the tumor. Moreover, FISH indicated a more complex chromosome 1 and 15 distribution than could be concluded from microsatellite LOH analysis. Our results show that microsatellite LOH analysis does not represent the beta2m gene copy number and support the results obtained from comparative genomic hybridization (CGH) studies. Conclusions on genomic alterations in tumors cannot be based on LOH data only but depend on the results of immunohistochemical staining, FISH, and CGH.
Allelic Imbalance↗
Quality control in diagnostic molecular pathology in the Netherlands; proficiency testing for patient identification in tissue samples.
AIMS: To describe the evolution of proficiency testing for molecular diagnostic pathology with respect to determining unambiguously the patient identity of tissue samples by microsatellite analysis. METHOD: Four rounds of quality control exchanges of samples from different patients were sent with the purpose of identifying the correct origin of these samples. The samples were either paraffin wax embedded sections on glass, sections in tubes, or isolated DNA. Blinded samples were distributed to all participating laboratories. No restrictions to the method and short tandem repeat markers used for identification were imposed. RESULTS: In four subsequent rounds the number of participating laboratories increased from three to 10. The numbers of samples tested increased in time from five to 12. The microsatellite markers used by the different laboratories showed little overlap. In the first three rounds, in which isolated DNA was provided, all samples were accurately classified irrespective of the microsatellite markers used. In the last round, which also included paraffin wax embedded sections, a small number of laboratories experienced problems, either with amplification or incorrect classification of a few samples. CONCLUSION: Proficiency testing was useful, and showed country wide high quality and correct identification of (patient) samples with molecular techniques for diagnostic purposes.
DNA↗
Nonlethal transfusion associated graft-versus-host disease in a severe combined immunodeficient patient.
An X-linked severe combined immunodeficient (SCID) patient received a nonirradiated erythrocyte transfusion and developed transfusion-associated graft-versus-host disease (TAGVHD), which was controllable with high-dose corticosteroids. Haplo-identical SCT was performed, after a myeloablative conditioning regimen. At day +26, he developed GVHD. Chimerism studies revealed DNA of the erythrocyte transfusion donor (ETD) and recipient only. Because of early nonengraftment and the presence of alloreactive T cells of ETD origin, the patient was treated with an immunosuppressive conditioning regimen followed by a second SCT from the same donor. While tapering immunosuppression, he again developed mild GVHD, and DNA of ETD and bone marrow donor origin were both present. On cyclosporin, the ETD-DNA signal finally disappeared. High-resolution HLA typing revealed haplo-identity between BMD, ETD and the patient, which might have contributed to the relative mild course of the TAGVHD.
Adrenal Cortex Hormones↗
Prognostic factors in localised prostate cancer with emphasis on the application of molecular techniques.
Prostate cancer is the most prevalent malignancy in males in the Western world and the second leading cause of male cancer death. Prostate specific antigen (PSA) based screening and case finding leads to identification of early stage prostate cancer. It is often difficult to discriminate between patients that need curative treatment and those that can be managed conservatively. Prognostic factors are used to make this clinical decision. Based on the classification proposed by the American College of Pathologists and the World Health Organisation, selected prognostic factors in prostate cancer are described. Clinical applicable factors are stage, grade and serum PSA. Prognostic factors that are not routinely used (for various reasons) are ploidy, histological type and cancer volume in needle biopsies. All other factors (including circulating tumour cells, angiogenesis, growth factors, proliferation rate, apoptosis, nuclear morphometry, neuroendocrine differentiation, loss of chromosomal regions, tumour suppresser genes and adhesion molecules) are promising as prognostic factor although currently their use in clinical decisions is not recommended. The role of these factors in prostate cancer growth and their predictive value are discussed. The rapid developments in molecular techniques allow assessment of structure or function of thousands of genes in a prostate biopsy sample. We expect that molecular characterisation of tumour material will become a clinically important tool to predict prognosis in patients with localised prostate cancer.
Biomarkers, Tumor↗
P53 tumor suppressor gene mutations in laryngeal cancer and in recurrent disease following radiation therapy.
In this study we performed p53 sequencing based mutation analysis in laryngeal cancers and matched recurrent disease following irradiation. The question is if irradiation affects the DNA and introduces or deletes mutations so that p53 cannot be used as a clonal marker anymore. P53 mutations were identified in fresh-frozen laryngectomy specimens with either primary laryngeal cancers, treated by surgery and irradiation post-operative with local failure during follow-up, or with recurrent laryngeal cancers following primary irradiation. In 21 tumors the p53 status was analyzed by direct sequencing full-length mRNA through RT-PCR. DNA sequencing analysis of exons 2 through 11 was performed when RNA isolation could not be performed. The marker mutation identified in this way was detected by DNA sequencing of the corresponding exon in formalin-fixed deparaffinized tumor biopsy samples in respectively matched recurrent disease following surgery and irradiation or primary tumor before irradiation. DNA sequencing analysis of the corresponding exon of peripheral blood leukocytes excluded the presence of germline mutations or polymorphisms. In 16 out of 21 tumors (71%), a mutation was identified. Fifteen of these marker mutations were detected in the matched tumor biopsy sample (94%). The only case lacking the marker mutation probably was a second primary tumor. We conclude that we find no direct evidence for induction or loss of p53 mutations following irradiation. Consequently, p53 may be used as a diagnostic tool when histological examination fails, for example in discriminating between the presence of a second primary tumor in the same area versus recurrent disease.
Aged↗
HLA-A*3306, a novel variant in the Indian population.
We describe a new HLA-A allele, A*3306, which was identified by sequencing based typing (SBT) in an individual of Indian origin. A*3306 is similar to A*3303, with a difference at position 228 (A to G). This difference leads to an amino-acid change at codon 52 from Ile (ATA) to Met (ATG). Until now this position has been considered conserved.
Base Sequence↗
HLA class II sequence-based typing in normal Saudi individuals.
We have adopted a system that combines low resolution PCR-SSP followed by sequence-based typing (SBT) to analyze HLA-DRB1, -DPB1 and -DQB1 alleles in the Saudi population. The SBT method was used to identify HLA class II alleles in Saudis for the first time. Nineteen HLA-DRB1 alleles in currently recognized subtypes of the DRB locus were detected. DR1 and DR9 were not encountered. SBT did not detect diversity within the DR7 and DR10 alleles. Sixteen HLA-DQB1 and 10 HLA-DPB1 alleles were identified. This study represents the first molecular report on the HLA class II allele frequency in the population of Saudi Arabia.
Arabs↗