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PubMed1992

Flow cytometric DNA analysis of vascular soft tissue tumors, including African endemic-type Kaposi's sarcoma.

Vascular tumors of the soft tissue display a wide spectrum of histologic features and biologic behavior. Flow cytometric DNA analysis was performed on 40 vascular tumors, including nine African endemic-type Kaposi's sarcomas, nine angiosarcomas, seven hemangiopericytomas, six glomus tumors, and nine capillary hemangiomas. Six of the nine angiosarcoma cases (67%) and one of the seven hemangiopericytomas cases (14%) were aneuploid. All benign vascular tumors and Kaposi's sarcomas were diploid. Clinically, five of the six angiosarcoma patients with aneuploidy died within 2 to 28 months, while the remaining patient, who had the smallest tumor (2 x 1 cm), survived more than 4 years after the initial diagnosis was made. All three angiosarcoma patients with diploidy died within 10 to 14 months. One hemangiopericytoma patient with aneuploidy died within 1 month. No cases of benign tumor recurred. These results suggest that most vascular tumors, which generally follow a benign clinical course, were diploid and that the majority of those with a poor outcome were aneuploid. However, flow cytometrically assessed DNA ploidy has no prognostic value in angiosarcomas or hemangiopericytomas.

Adolescent↗

PubMed1990

Flow cytometric DNA analysis of parathyroid tumors. Implication of aneuploidy for pathologic and biologic classification.

The previous cytometric studies on parathyroid tumors have provided conflicting data regarding the relationship between DNA content and histopathology, resulting from differences in technical methods and data analysis. This study measured nuclear DNA of parathyroid tumors by flow cytometry in fresh material and determined whether DNA aneuploidy really assists in making a pathologic diagnosis of carcinoma or not. From May 1987 through April 1989, 65 consecutive patients operated on for primary hyperparathyroidism had DNA analysis of the freshly excised parathyroid tumors. Three of the patients had metastatic lesions of parathyroid carcinoma in the lung, cervical lymph nodes, and lung and mediastinal lymph nodes, respectively. Pathologic classifications of the lesions from the other 62 patients were 54 adenomas, four carcinomas, and four hyperplasias. In all the latter patients, hyperplasia was associated with a multiple endocrine neoplasia syndrome. Unequivocal evidence of aneuploidy was found in all of the metastatic lesions and 60% of the primary lesions of the carcinomas, in 9% of the adenomas and in 50% of the hyperplasias. Therefore, parathyroid carcinomas were more apt to be aneuploid than were adenomas (P = 0.0015, both-sided testing). In each of the cases of aneuploid hyperplasia, a small aneuploid peak was found. The high incidence of aneuploidy in patients with multiple endocrine neoplasia type 1 may indicate the presence of clonal heterogeneity of hyperplastic glands and the presence of an abnormal subset of cells that have malignant potential. Cell distribution analysis did not provide any significant information beyond ploidy level. In conclusion, DNA flow cytometric analysis of DNA ploidy patterns is a valuable adjunct to the histopathologic diagnosis of parathyroid neoplasms.

Adenoma↗

PubMed1999

DNA analysis by microfabricated capillary electrophoresis device.

The LIGA (Lithographie Galvanoformung Abformung) process using synchrotron radiation lithography is applied to the microfabrication of capillary array electrophoresis (CAE) device. Laser-induced fluorescence detection system for the CAE device has been constructed by the modification of laser confocal fluorescence microscopy. DNA molecules were detected during migrating in the microchannels filled with polymer separation matrices under electric field to optimize the separation conditions for DNA analysis. Based on this observation, we demonstrated that microfabricated CAE device is realized the fast separation of DNA.

DNA↗

PubMed1989

DNA analysis in juvenile Huntington disease.

We report a 6-year-old boy with a positive family history of Huntington disease (HD), who presented with ambiguous neurological and psychiatric symptoms. The suspected diagnosis of HD was confirmed by DNA analysis. This new method is very helpful when the first signs of HD cannot be clearly interpreted.

Child↗

PubMed1992

Congenital 21-hydroxylase deficiency as a new deletion mutation. Detection in a proband during subsequent prenatal diagnosis by HLA typing and DNA analysis.

A child with 21-OH-def whose 9 weeks' pregnant mother was referred for prenatal diagnosis was found upon very careful histocompatibility testing to lack expression of any of his father's HLA antigens on his peripheral blood lymphocytes. The possibility of alternative paternity was considered to be extremely unlikely after additional genetic marker tests. The conclusion that the affected child's disease resulted from inheritance of a maternal CYP21B (21-OH) deletion and a de novo deletion in the paternal chromosome 6 segment that includes both the CYP21B (21-OH) and HLA genes was confirmed by subsequent DNA analysis using 21-OH, C4, DPB, and PCH6 probes. The presence of a heterozygous RFLP for DPB, the absence of a deletion for either CYP21B (21-OH) or C4 genes, and the presence of a paternal HLA antigen haplotype on the fetal cells additionally indicated that the fetus lacked the same deletion and could be predicted to be completely normal.

Adrenal Hyperplasia, Congenital↗

PubMed1993

Random amplified polymorphic DNA analysis of Trypanosoma cruzi strains.

DNA extracted from 32 isolates of Trypanosoma cruzi was subjected to polymerase chain reaction amplification using 4 arbitrary primers resulting in relatively complex DNA profiles that include polymorphic markers known as random amplified polymorphic DNAs (RAPDs). The RAPD profiles of 18 strains belonging to zymodeme 1 (Z1) collected from various regions of South America exhibited a consistant pattern and 59 (59%) of the bands produced were present in all Z1 strains. A similar level of consistency was seen in the number of bands shared between 5 Z2 strains, 4 ZB strains and 2 ZC strains. A phenetic analysis of the 5 most different Z1 strains based on band sharing showed that their interrelationships mirrored their geographical origin. Comparison of the RAPD profiles of strains from different zymodemes showed that less than 7% of bands of strains in one zymodeme are present in strains of another zymodeme. Analysis of band sharing using bands present in all strains of a given zymodeme showed ZB and ZC to be closely related and Z1 and Z2 to form distinct groups.

Animals↗

PubMed2005

[A case of long-term survival of a patient with infantile Alexander disease diagnosed by DNA analysis].

Alexander disease is a hereditary disorder of myelin degeneration. The pathological feature of the brain is the characteristic inclusion bodies in astrocytes called Rosenthal fibers. The major components of the Rosental fibers are known to be alpha B-crystallin and glial fibrillary acidic protein (GFAP). In recent years, reports have indicated mutations of the GFAP gene in patients with Alexander disease. The R239 mutation (R239C, R239H) tends to cause comparatively more severe conditions among the GFAP mutations. In this study. we examined a long-term survival case of a patient (age 25 years, 7 months) with infantile Alexander disease with an R239C mutation confirmed by DNA analysis. There are no past reports of subjects with the R239C mutation who had as prolonged a long-term survival as our case. Our subject's condition was not as severe as those with the R239H mutation. The clinical progress in those other reports also varied by case. The R239C mutation does not show as much correlation with the clinical presentation as the R239H mutation. We believe that factors such as the environment also play a part in the prognosis of the disease.

Adult↗

PubMed1994

[The importance of DNA analysis in the diagnosis of steroid 5-alpha-reductase deficiency].

The deficiency of steroid 5 alpha-reductase leads to the disturbances in sex differentiation that cause symptoms of male pseudohermaphroditism. The methods of DNA analysis used to diagnose mutations of steroid 5 alpha-reductase gene (SRD5A2) were presented and discussed. Within the group of 21 patients with the deficiency of steroid 5 alpha-reductase 2 described so far in literature, the analysis of SRD5A2 gene revealed two "major" deletions, one "minor" deletion, 16 point mutations (incl. 5 transitions and 11 transversions), in one case a mutation causing premature termination of translation and in one case mutation leading to defective splicing of the mRNA. The mutations localized in exons 2 and 5 cause a decrease in affinity of 5 alpha-reductase 2 to the substrate (testosterone), while the mutations in exons 1 and 4, a decrease in affinity to the coenzyme (NADPH).

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed1992

Prenatal diagnosis of bilirubin-UDP-glucuronosyltransferase deficiency in rats by genomic DNA analysis.

Hepatic bilirubin excretion requires UDP-glucuronosyltransferase-mediated glucuronidation. Patients with type I Crigler-Najjar syndrome and mutant rats (Gunn strain) inherit deficiency of UDP-glucuronyltransferase activity toward bilirubin as an autosomal recessive trait and, as a result, exhibit marked nonhemolytic unconjugated hyperbilirubinemia throughout postnatal life. Heterozygous carriers of the trait have normal serum bilirubin levels. Because of placental excretion of unconjugated bilirubin, type 1 Crigler-Najjar syndrome patients and Gunn rats are not jaundiced in utero, making prenatal diagnosis difficult. Here we report a diagnostic method in Gunn rats based on genomic DNA analysis for prenatal recognition of deficiency of UDP-glucuronyltransferase activity toward bilirubin in Gunn rats and identification of heterozygous carriers. We and others have shown that two distinct messenger RNA species (UDP-glucuronyltransferase activity toward bilirubin and the 3-methylcholanthrene-inducible phenol-UDP-glucuronyltransferase messenger RNA) in Gunn rat liver contain identical deletions of a single guanosine residue in their common 3' regions. Loss of the restriction site for the endonuclease BstNI, which results from this deletion, was used as the basis for a diagnostic test. Female heterozygous Gunn rats were mated with male homozygous Gunn rats. Genomic DNA was extracted from the chorionic aspect of placenta of 17-day fetuses or from leukocytes from normal rats, obligate heterozygotes and homozygous Gunn rats. The DNA was sequentially digested with the restriction enzymes EcoRI and BstNI and subjected to Southern-blot analysis with a double-stranded DNA probe for the common region of UDP-glucuronyltransferase activity toward bilirubin and the 3-methylcholanthrene-inducible UDP-glucuronyltransferase messenger RNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1983

A short-term in vitro test for tumour sensitivity to adriamycin based on flow cytometric DNA analysis.

A new method to test the sensitivity of tumour cells to chemotherapy is presented. Tumour cells were incubated in vitro on agar, and drug-induced cell cycle perturbation was monitored by flow cytometric DNA analysis. In the present study the method was applied to monitor the effect of adriamycin on an adriamycin-sensitive Ehrlich ascites tumour and two adriamycin-resistant tumours. Adriamycin caused a dose-related accumulation of tumour cells in the G2 + M phase in the sensitive tumour. Drug concentrations greater than or equal to 100-fold higher were required to induce similar changes in the resistant tumours. The dose level causing maximum accumulation in the G2 + M phase is suggested as a parameter for quantifying the sensitivity. The results indicate that the method can be extended to sensitivity testing of human tumours.

Animals↗

PubMedDate not supplied

Flow cytometric DNA analysis, and immunohistochemical p53, PCNA and histopathologic study in primary achalasia: preliminary results.

BACKGROUND/AIMS: Primary achalasia is a premalignant disorder of the esophagus. The studies for esophageal cancer pathogenesis may reveal early diagnosis of esophageal cancer. DNA aneuploidy, p53 mutations and cellular proliferation are important factors in cancer development. As far as we know, we have not encountered any study on these factors in achalasia. METHODOLOGY: We studied DNA ploidy by flow cytometry and p53 and PCNA index by immunohistochemical technique and studied histopathology in the esophageal mucosa of primary achalasia and control patients. RESULTS: DNA analysis revealed aneuploidy in 2 of 20 achalasia patients but none of the 18 control patients. Sixty-five percent of achalasia and 22% of normal patients showed p53 positivity (P < 0.05). We have found normal mucosa, basal cell hyperplasia-esophagitis and dysplasia in 13, 22 and 3 patients and p53 positivity in 2, 12 and 3 of these patients, respectively (P < 0.05). PCNA labeling indexes (as % +/- SD) were 34.8 +/- 12.2, and 28.4 +/- 9.3 in achalasia and control groups, respectively (P > 0.05). PCNA labeling index was 28.0 +/- 8.2 in p53(-) and 36.0 +/- 12.9 in p53(+) patients (P < 0.05). PCNA indexes were found 29.3 +/- 9.6 in normal histopathologic group, 31.8 +/- 13.4 in basal cell hyperplasia-esophagitis, and 41.7 +/- 6.5 in dysplasia group (P > 0.05). CONCLUSIONS: DNA aneuploidy, p53 positivity, and higher cellular proliferation index may have important role in the pathogenesis of esophageal cancer in primary achalasia.

Adult↗

PubMed1993

Proliferating cell nuclear antigen (PCNA), immunostaining and flow cytometric DNA analysis of renal cell carcinoma.

Nuclear PCNA immunoreactivity is found in the proliferating compartment of normal and tumor cells. The purpose of the present study was to examine immunohistochemical staining of PCNA in renal cell carcinoma and its relationship to tumor grading, flow cytometric DNA analysis and prognosis. The resected kidneys from 81 patients with renal cell carcinoma were used. Anti-PCNA monoclonal antibody was applied to paraffin sections to identify proliferating cells by the SAB technique. The proportion of PCNA-positive cells to negative cells was determined by counting the tumor cells stained by PCNA at 400x magnification and expressed as percentage of positive cells. In parallel with the PCNA immunostaining DNA flow cytometry was performed, using the cell suspension prepared from paraffin sections and stained with propidium iodide. 79 tumors were evaluable for PCNA immunohistochemistry and 54 tumors for DNA flow cytometry. Immunohistochemically, there was no significant difference in percentage of PCNA-positive cells between tumor grades G 1 and G 2 (1.4 +/- 1.3% in G 1 and 1.6 +/- 1.4% in G 2), while G 3 tumors contained high percentage of PCNA positive cells, the mean value being 7.6 +/- 5.3%. The percentage of PCNA-positive tumor cells was found to be correlated with histological grading of tumor and also with the S+G2+M phase fraction and the S phase fraction measured by flow cytometry (p < 0.01). The mean percentage of PCNA-positive tumor cells was higher in aneuploid tumors than in diploid tumors. There was no significant relationship between the percentage of PCNA positive cells and tumor staging. Sixty-one cases were available for survival analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Aneuploidy↗

PubMed2004

Solitary neurocysticercosis case caused by Asian genotype of Taenia solium confirmed by mitochondrial DNA analysis.

A Japanese woman presenting with neurologic symptoms was presumptively diagnosed with neurocysticercosis based on imaging findings. Hooklets in the scolex of the resected lesion were not confirmed through histopathological observation. However, the illness was confirmed by mitochondrial DNA analysis to be a solitary neurocysticercosis case caused by the Asian genotype of Taenia solium.

Animals↗

PubMed1996

Characterization of Serpulina hyodysenteriae isolates of serotypes 8 and 9 by random amplification of polymorphic DNA analysis.

A PCR-based DNA fingerprinting method termed RAPD (Random Amplification of Polymorphic DNA), or AP-PCR (for Arbitrary Primed PCR) was used to detect sequence diversity among reference strains and isolates of Serpulina hyodysenteriae. RAPD fingerprinting of 20 S. hyodysenteriae isolates of serotypes 8 or 9 from Quebec was generated with 2 different 10-base primers used independently. Reference strains and field isolates belonging to serotypes 8 or 9 revealed polymorphisms in RAPD fingerprints with both primers. Interspecies polymorphisms were observed by RAPD analysis of S. hyodysenteriae representing serotypes 1 to 9, S.innocens, and 5 other weakly beta-hemolytic intestinal spirochetes. A dendrogram based on the analysis of RAPD profiles of the strains tested with one of the primers (#17), permitted the clustering of these strains into 11 divisions. The predominance of particular RAPD profiles among S. hyodysenteriae isolates isolated from cases of swine dysentery in different herds suggested that certain S. hyodysenteriae types could be epidemiologically important. Our results indicate that RAPD could be used as a typing method for S. hyodysenteriae and as an epidemiological method for identifying spirochetes isolated from swine.

Base Sequence↗

PubMedDate not supplied

[Prognostic factors in neuroblastoma. Usefulness of DNA analysis using flow cytometry on paraffin-embedded material].

Nuclear DNA content was determined on paraffin blocks of 28 neuroblastic tumors retrieved from Surgical Pathology files. In 26 cases cytofluorimetric analysis for DNA content was satisfactory. 8 tumor were diploid, 12 were aneuploid and 6 tetraploid. All but one of the children with diploid neuroblastoma (NB) died after a survival ranging from 14 to 32 months. The only surviving child had a Stage I thoracic ganglioneuroblastoma (GNB) and was alive after 27 months. Conversely 11 of 12 patients with aneuploid tumors were alive with prolonged follow-up. Two children are alive after a period exceeding 146 months. The only exception in this group of aneuploid tumors was a child with Stage IV neuroblastoma of the adrenal gland with pleuro pulmonary metastasis who died after 6 months. In the group with tetraploid tumors 3 patients died (2 children with Stage III and IV died early while the third, with thoracic Stage II GNB, survived for 72 months). Two patients with tetraploid GNB Stage II and III are alive 65 and 72 months after diagnosis respectively. Another child with Stage IV adrenal NB is alive after 14 months but with metastatic spread to bones, bone marrow and lymph nodes. Cox analysis of the cases demonstrated ploidy as an independent prognostic factor. These results are in agreement with other molecular analysis linking near-ploidy of Neuroblastic tumors to poor prognosis. Ploidy, as detected by flow cytometry for nuclear DNA content, may represent an important prognostic factor in Neuroblastic tumors. The advantage of flow cytometry over other techniques of molecular analysis is represented by the simple methodology suitable for fixed and paraffin embedded tissue, even on retrieval material.

Adrenal Gland Neoplasms↗

PubMed1999

Phylogenetic analysis of Cryptosporidium isolates from captive reptiles using 18S rDNA sequence data and random amplified polymorphic DNA analysis.

Sequence alignment of a polymerase chain reaction-amplified 713-base pair region of the Cryptosporidium 18S rDNA gene was carried out on 15 captive reptile isolates from different geographic locations and compared to both Cryptosporidium parvum and Cryptosporidium muris isolates. Random amplified polymorphic DNA (RAPD) analysis was also performed on a smaller number of these samples. The data generated by both techniques were significantly correlated (P < 0.002), providing additional evidence to support the clonal population structure hypothesis for Cryptosporidium. Phylogenetic analysis of both 18S sequence information and RAPD analysis grouped the majority of reptile isolates together into 1 main group attributed to Cryptosporidium serpentis, which was genetically distinct but closely related to C. muris. A second genotype exhibited by 1 reptile isolate (S6) appeared to be intermediate between C. serpentis and C. muris but grouped most closely with C. muris, as it exhibited 99.15% similarity with C. muris and only 97.13% similarity with C. serpentis. The third genotype identified in 2 reptile isolates was a previously characterized 'mouse' genotype that grouped closely with bovine and human C. parvum isolates.

Animals↗

PubMed1990

DNA analysis and recombination in X-linked retinitis pigmentosa.

X-linked retinitis pigmentosa is a hereditary retinal degenerative disorder which has been localised to the proximal short arm of the X chromosome. Recent evidence suggests that the disorder is heterogeneous with two possible loci for the disease mutation. DNA analysis on the family presented in this paper showed that the mutation mapped to the more telomeric locus (RP3), thus enabling two flanking polymorphic DNA probes (XJ1.1 and M27 beta) to be used for the detection of female carriers in the family. In none of the carriers was a tapetal reflex (metallic sheen) observed.

Adult↗

PubMed1992

Gestational and nongestational trophoblastic tumors distinguished by DNA analysis.

In three patients in whom a diagnosis of gestational trophoblastic tumor was possible on the basis of pathology and elevated levels of serum human chorionic gonadotrophin, locus-specific minisatellite probes were used to identify restriction fragment length polymorphisms (RFLP) in DNA from the tumor, the patient, and her partner. On the basis of results from these studies, one tumor, originally diagnosed as a germ cell tumor, was reclassified as a gestational choriocarcinoma, whereas a second tumor, diagnosed as gestational choriocarcinoma, was shown to be of nongestational origin. In the third case, a diagnosis of gestational trophoblastic tumor was confirmed, but in this case the androgenetic origin of the tumor indicated that it was derived, not from the antecedent term pregnancy, but from a previous pregnancy with hydatidiform mole. This study clearly demonstrates the value of DNA analysis in the classification of tumors with trophoblastic differentiation.

Adult↗