[Fecal DNA analysis--future method for colorectal cancer screening?].
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Colorectal Neoplasms↗
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DNA analysis and parathyroid pathology.
The nuclear DNA content of 85 parathyroid glands (4 carcinomas, 39 adenomas, 21 secondary parathyroid hyperplasias, and 21 normal parathyroid glands) were determined by flow cytometric analysis. All normal parathyroid glands, 85% of the adenomas, and 83.3% of the secondary hyperplastic glands had DNA indices within values of 0.85-1.1. Paraffin-embedded fixed glands showed less DNA staining than that found with fresh or normal glands. Glands from patients with carcinoma showed DNA indices outside the normal DNA index range. When the percent of nuclei within the G0/G1 phase of the cell cycle was compared between the study groups, highly significant results were found. While patients with secondary hyperplasia showed a similar distribution to the normal glands studied, only 48% of primary adenomas showed over 80% of cells within the G0/G1 region. A clear subgroup of adenomas was defined with more rapidly cycling tetraploid cells, and showing classical adenoma pathology. This group showed negative correlation with gland weight, plasma calcium, and ionized calcium. These findings suggested that a different etiology of the disease process occurs between secondary hyperplasia and parathyroid adenoma. Such abnormal adenomas may form a group worthy of long-term follow-up.
Adenoma↗
Characterization of the lymphoproliferative diseases in the skin by DNA analysis.
Various samples from lymphoproliferative diseases in the skin were analyzed by Southern blotting technique with probes from the T cell receptor gene, immunoglobulin genes, and human T cell leukemia virus-I genome. Samples were taken from 10 mycosis fungoides (MF) patients, 1 parapsoriasis en plaque patient, 10 Adult T cell leukemia/lymphoma (ATL) patients, 1 cutaneous T cell lymphoma (CTCL) patient, 4 lymphomatoid papulosis (LP) patients, 4 B cell lymphoma patients, and 2 actinic reticuloid (AR) patients. In MF, the monoclonality of the T cells became detectable first in the skin when plaques develop to tumors then in lymph nodes, and finally in the blood lymphocytes, indicating this disease develops from local (skin) malignancy to systemic malignancy. In parapsoriasis en plaque, no monoclonality was detected in any sample. We could distinguish cutaneous ATL from the carrier state by detecting the T cell monoclonality and HTLV-I integration with these probes. One patient with CTCL showed detectable T cell monoclonality; 1 out of 4 patients with LP did the same. Four samples from patients with B cell lymphoma revealed detectable monoclonal rearrangement of immunoglobulin heavy and light chain genes. In AR, no monoclonality was detected in any sample. From these data, we conclude that DNA analysis is useful in determining the monoclonality, cell origin, and distribution of monoclonal cells from skin samples.
Antibodies, Monoclonal↗
Long-term storage of nuclear suspensions prepared from paraffin-embedded material for flow cytometric DNA analysis.
Nuclear suspensions were prepared from archival material of cancer biopsies. DNA content analysis by flow cytometry was performed after storage at -80 degrees C for 24 h and 6 months, compared to 24 h storage at +4 degrees C. No significant variations in CV or DNA indices were observed. Repeated freezing and thawing did not reduce the DNA histogram quality.
Cell Nucleus↗
16S ribosomal DNA analysis of the faecal lactobacilli composition of human subjects consuming a probiotic strain Lactobacillus acidophilus NCFM.
AIMS: The aims of this study were to evaluate the ability of exogenous Lactobacillus acidophilus strain NCFM to survive through the human gastro-intestinal (GI) tract, and to evaluate the selectivity of Rogosa SL medium for faecal lactobacilli. METHODS AND RESULTS: The composition of the faecal lactobacilli of 10 healthy subjects was monitored for two weeks prior to, two weeks during and two weeks after the administration of the Lact. acidophilus strain NCFM consumed with skim milk (daily dose 10(10) viable cells). Fresh faecal samples were collected, processed and cultured on Rogosa SL selective medium for lactobacilli enumeration. Colonies demonstrating various morphologies were identified and purified for 16S ribosomal DNA sequence analysis for speciation of colonial genotype. The species composition of cultivable faecal lactobacilli changed considerably during consumption of the strain NCFM. CONCLUSIONS: The probiotic Lact. acidophilus strain NCFM can survive through the human GI tract, but cannot colonize itself during the two-week consumption. Rogosa SL medium is selective for faecal lactobacilli. However, genetic analysis is required for colony speciation. SIGNIFICANCE AND IMPACT OF THE STUDY: It is demonstrated that continuous consumption is necessary to maintain a high population of the probiotic strain, and that the Rogosa SL medium is reliable.
Adult↗
Prognostic significance of morphological parameters and flow cytometric DNA analysis in carcinoma of the breast.
The prognostic significance of conventional TNM staging remains the standard for determining prognosis in breast carcinoma. The presence (or absence) of axillary lymph node metastases remains the single most important parameter for predicting patient outcome. The presence of regional lymph node metastases implies that the primary tumor has the capacity for successfully completing the steps of the metastatic cascade. However, the absence of regional lymph node metastases does not ensure that distant or systemic seeding of tumor cells has not occurred, only that it is less likely. Staging data appear to be refined by addition of several standard morphological parameters. Although there is considerable overlap and interaction between these factors, as well as with staging data, there is strong evidence that grade, necrosis, inflammatory cell "immune response," and possibly pattern of invasion and intravascular tumor each independently supplement staging information. Some data appear to have independent significance only when applied to specific patient subsets, raising serious question as to their biologic importance. Nevertheless, morphological data are subjective and susceptible to observer variation and have less statistical power in predicting patient outcome than staging data. It was initially thought that DNA analysis of breast cancer by flow cytometry might supplant morphological data in assessing tumor behavior. The following conclusions can now be drawn: (1) there is no clear association between aneuploidy and SPF and stage; (2) aneuploid tumors are associated with higher SPF and shorter disease-free survival while diploid-range tumors generally have lower SPF and longer disease-free survival; (3) aneuploid DNA content is significantly associated with markers of decreased morphological (grade) and biochemical (ER status) differentiation. Determination of S-phase fraction by FCM appears to be a rapid and potentially easy method for obtaining kinetic information on individual breast tumors, although the technology for improving the accuracy of SPF measurements is still under development (e.g., tumor cell gating, debris subtraction). SPF appears to be comparable to other kinetic measurements, such as TLI, and shows many of the same associations with morphological and clinical data as ploidy. This is due to the close association of ploidy and SPF. Which of these parameters is more important for predicting patient outcome has not been clearly defined. Additional technological refinements for determining SPF may result in a more prominent prognostic role for this measurement. Three problems have limited our ability to draw specific conclusions about the biologic significance of tumor ploidy and SPF.(ABSTRACT TRUNCATED AT 400 WORDS)
Breast Neoplasms↗
Spontaneous abortions and DNA ploidy. An application of flow cytometric DNA analysis in detection of non-diploidy in early abortions.
The value of DNA ploidy analysis utilizing paraffin-embedded materials in the detection of nondiploid gestations was examined in a year series of 217 spontaneous abortions in first trimester at a hospital. These placentas were histologically classified as follows: 19 hydropic placentas, six partial moles, three complete moles, and 189 nonhydropic placentas. Abnormal DNA content was found in 26 cases (12%). Twelve placentas (5.6%) were triploid, seven were tetraploid, and seven were nontriploid/tetraploid aneuploid. Of the triploid placentas, six were partial moles, one was a hydropic placenta, and the remaining five were nonhydropic. Since large mononuclear cells in villous mesenchyme were observed in the triploid and tetraploid as well as in the diploid hydropic placentas, in which category trisomic gestation was probable, these cells were not considered to be characteristic of trisomic placentas. Although placentas with hydropic villi had a significantly higher frequency of the nondiploid DNA content than nonhydropic placentas, with the exception of partial moles, no correlation between the histology of villi and DNA ploidy was observed. Flow cytometric analysis using paraffin-embedded materials appears to be of value for the retrospective study of spontaneous abortions, especially for assessing aneuploidy.
Abortion, Spontaneous↗
Grading of diffusely infiltrating astrocytomas by quantitative histopathology, cell proliferation and image cytometric DNA analysis. Comparison of 133 tumours in the context of the WHO 1979 and WHO 1993 grading schemes.
The aim of the study was to evaluate the applicability of quantitative histopathology as an aid for grading diffusely infiltrating astrocytomas. Primary astrocytomas were analysed for parameters (mean nuclear size, mitosis count, area fraction of endothelial cells and tumour necrosis, area fraction of nuclei, and Ki-67 (MIB-1) labelling index), which are closely related to the World Health Organization (WHO) 1979 and WHO 1993 grading criteria. All estimates correlated with the WHO histopathological grade and patient outcome. According to the receiver-operating characteristics curve, the presence of tumour necrosis and mitosis count (cut-off at 3 mitoses/mm2 of neoplastic tissue) showed the best sensitivity and specificity in separating patients with different survival. The multivariate survival analyses confirmed this result. A decision-tree model was constructed based on these two variables: twig I with less than 3 mitoses/mm2, twig II with equal or more than 3 mitoses/mm2 but no necrosis, and twig III with tumour necrosis. This model was found to be more strongly associated with survival than the WHO 1979 or WHO 1993 grading schemes. Low-malignancy astrocytomas (WHO grade II or twig I tumours) could be further divided into two prognostic categories by the image cytometric DNA analysis. The results put an emphasis on astrocytoma grading on mitosis counts (grade II vs. III) and tumour necrosis (grade III vs. IV). To standardize the sampling for mitosis counting, it is suggested that a parallel Ki-67 immunostaining be used for the identification of the most proliferative areas.
Astrocytoma↗
DNA analysis of a pedigree with myotonic dystrophy in Songjiang county, Shanghai.
OBJECTIVE: To make the molecular analysis of a pedigree with myotonic dystrophy (DM) in Songjiang county, Shanghai, and to observe the connection between CTG repeat number and clinical features. METHODS: In twenty-three individuals of a pedigree with DM, CTG trinucleotide repeat numbers located in the 3' untranslated region of a protein kinase gene (MTPK) on chromosome 19q13.2-3 were analyzed by using Long Expand TM Template PCR system. RESULTS: Four of eight clinical patients had expanded DM allele, the other four had no expanded CTG copies. Seven of eight suspicious DM cases had expanded CTG repeat numbers and were therefore genetically diagnosed as DM, and an asymptomatic individual was diagnosed as a doubted DM patient by DNA analysis. High risk of DM in six of seven individuals was ruled out, and a clinical doubted DM individual was ascertained a normal person by molecular analysis. A positive correlation was found beween early onset and expanded CTG repeat number in six parents (or grandparents)/child pairs, but in the pair II 2 /IV 7 CTG repeat numbers were reduced from 3100 in the grandmother to 175 in her grandson and there was no anticipation phenomenon. CONCLUSION: The measurement of CTG repeat number can help to ascertain the diagnosis of DM in clinical and preclinical patients, but some clinically- diagnosed DM patients might have normal CTG repeat numbers. Anticipation phenomena were common in the pedigree.
Adolescent↗
The relative prognostic value of flow cytometric DNA analysis and conventional clinicopathologic criteria in patients with operable rectal carcinoma.
The DNA histograms of resected rectal carcinomas from 121 patients were compared, along with a detailed clinicopathologic assessment of the same tumors, with the incidence of postresection tumor recurrence and patient survival over an extended period of 15-year follow-up. A poorer prognosis was found for patients with DNA aneuploid and DNA tetraploid rectal cancers as compared with patients with DNA diploid cancers. In addition, local tumor recurrence was twice as common among patients with DNA nondiploid rectal carcinomas. The DNA pattern of a rectal carcinoma was an independent prognostic variable in a Cox's multivariate analysis model. DNA nondiploid rectal carcinomas had a statistically significant increased incidence of vascular invasion, tumor fibrosis, and high Dukes' stage.
Aged↗
[A case of primary thyroid T-cell lymphoma with infiltration of lymphoma cells in peripheral blood: diagnosed by DNA analysis].
A 57-year-old woman came to our hospital with complaints of neck swelling and headache in 1991. She was diagnosed as having chronic thyroiditis in euthyroidism because she had a diffuse goiter with both antithyroglobulin antibody (TGHA) and antimicrosomal antibody (MCHA). In 1992, she complained of the rapid growth of her thyroid gland and a swallowing disturbance. Atypical lymphocytes were observed in 16.5% of leukocytes in peripheral blood and similar atypical cells were found in bone marrow. Although an ultrasound scan of the thyroid gland revealed a symmetrical enlargement without a pseudocystic appearance, cytological study with fine needle aspiration biopsy of the thyroid gland demonstrated an abundance of atypical lymphoid cells. A whole body scintigram with 67gallium citrate showed no significant accumulation except in the thyroid gland. With a diagnosis of suspected primary thyroid lymphoma, total thyroidectomy was performed. However the diagnosis of malignant lymphoma was not confirmed histologically. A study of lymphocytes subset with two-color flow cytometry, which was performed for both lymphocytes in peripheral blood and infiltrating lymphocytes in the resected thyroid gland, revealed abnormal increased CD4 positive T cells and decreased HLA-DR expression. Additionally, southern blot DNA analysis for abnormal lymphocytes using restriction enzymes, EcoRI and BamHI, demonstrated rearrangement of the T-cell antigen receptor, which indicates a monoclonal proliferation of lymphocytes. After total thyroidectomy, atypical lymphocytes in peripheral blood decreased, and circulating autoantibodies including TGHA and MCHA disappeared. From these data, this patient was finally diagnosed as having a primary T-cell lymphoma of the thyroid gland, which is a very rare type of thyroid lymphoma.(ABSTRACT TRUNCATED AT 250 WORDS)
Autoantibodies↗
Detection and identification of the Candida species by 25S ribosomal DNA analysis in the urine of candidal cystitis.
Candida species in clinical urine samples were identified directly by the newly developed method of PCR analysis on 25S ribosomal DNA (rDNA). Two dogs were referred to the Animal Medical Center, Nihon University School of Veterinary Medicine, Fujisawa, Kanagawa, Japan for the examination of chronic cystitis. Microscopic examination of urine samples from these dogs revealed yeast cells. Urine culture on Sabouraud's dextrose agar at 27 degrees C for 5 days produced white to cream colored colonies. The isolates were identifical to Candida albicans and C. parapsilosis by mycological examination, respectively. The nucleotide sequences of 25S ribosomal DNA from these urine isolates showed 99% similarity to those of a reference strain of Candida albicans or C. parapsilosis. The nucleotide sequences of 25S rDNA obtained directly from urine samples were also identical to C. albicans and C. parapsilosis, respectively. Confirming the results on the isolates cultured from the same urine samples. This PCR analysis method could be available for the direct identification of Candida species in urine samples within 2 days.
Animals↗
A case of infantile Alexander disease accompanied by infantile spasms diagnosed by DNA analysis.
Alexander disease (AD) is a rare leukodystrophy of the central nervous system of unknown etiology. AD is characterized by progressive failure of central myelination and the accumulation of Rosenthal fibers in astrocytes, and is inevitably lethal in nature. Symptomatically, AD is associated with leukoencephalopathy with macrocephaly, seizures, and psychomotor retardation in infants, and usually leads to death within the first decade. Its characteristic magnetic resonance imaging (MRI) findings have been described as demyelination predominantly in the frontal lobe. Moreover, dominant mutations in the GFAP gene, coding for glial fibrillary acidic protein (GFAP), a principal astrocytic intermediate filament protein, have been shown to lead to AD. The disease can now be detected by genetic diagnosis. We report the Korean case of an 8-month-old male patient with AD. He was clinically characterized due to the presence of psychomotor retardation, megalencephaly, spasticity, and recurrent seizures including infantile spasms which is a remarkable presentation. Demyelination in the frontal lobe and in a portion of the temporal lobe was demonstrated by brain MRI. Moreover, DNA analysis of peripheral blood showed the presence of a R239L mutation in the GFAP gene, involving the replacement of guanine with thymine.
Alexander Disease↗
Gene flow and population history in high dispersal marine invertebrates: mitochondrial DNA analysis of Holothuria nobilis (Echinodermata: Holothuroidea) populations from the Indo-Pacific.
The sea cucumber, Holothuria nobilis, has a long-lived planktotrophic larvae, and previous allozyme surveys have suggested that high dispersal is realized. In contrast, recent ecological studies indicate that dispersal is low. To reconcile these data, and to investigate the evolution of this Indo-Pacific species, we screened geographical variation in 559 bp of a mitochondrial gene (COI) in 360 samples from the Australasian region and La Réunion. Sequences from La Réunion differed by > 7% from others and may constitute another species. Haplotype diversity in other samples was high (0.942, SD = 0.007), but haplotypes were closely related (mean nucleotide diversity: 0.0075, SD = 0.0041). AMOVA, pairwise FST values and exact tests did not detect significant population structure. Nested clade analysis showed that one of two main clades was over-represented in west Australia, whereas the other was more common in the northern Great Barrier Reef. Isolation-by-distance was identified as the main determinant of population structure at several clade levels. Contiguous range expansion was inferred for evolutionary older clade levels and this may correspond to a late Pleistocene (88 000-193 000 years ago) population expansion inferred from haplotype mismatch distributions. Thus, the population genetic structures detected are likely to be formed prior to the last ice age, with some indications for high dispersal on shorter time scales.
Analysis of Variance↗
Flow cytometric DNA analysis after immunoselection of bladder tumor cells with monoclonal antibody DU83.21.
Adequate preservation of neoplastic cells and the elimination of interference by inflammatory cells in measuring tumor cell DNA content represent two important objectives necessary for accurate flow cytometric analysis of bladder carcinomas. An experimental model consisting of a mixture of cultured bladder carcinoma cells (T24) and human buffy-coat (BC) cells was used to evaluate various preservatives and an anti-bladder carcinoma monoclonal antibody (MoAb), DU83.21, for separating inflammatory cells (BC cells) from T24 cells. A final concentration of 25% ethanol was found to be the most effective preservative of several tested. After incubation with the MoAb DU83.21 and propidium iodide (DNA stain), the T24 cells could be separated from the BC cells, permitting accurate DNA analysis of the tumor cells. Application of this system to specimens from bladder cancer patients enhanced the detection and DNA analysis of the tumor cell populations.
Aneuploidy↗
An integrated nanoliter DNA analysis device.
A device was developed that uses microfabricated fluidic channels, heaters, temperature sensors, and fluorescence detectors to analyze nanoliter-size DNA samples. The device is capable of measuring aqueous reagent and DNA-containing solutions, mixing the solutions together, amplifying or digesting the DNA to form discrete products, and separating and detecting those products. No external lenses, heaters, or mechanical pumps are necessary for complete sample processing and analysis. Because all of the components are made using conventional photolithographic production techniques, they operate as a single closed system. The components have the potential for assembly into complex, low-power, integrated analysis systems at low unit cost. The availability of portable, reliable instruments may facilitate the use of DNA analysis in applications such as rapid medical diagnostics and point-of-use agricultural testing.
Costs and Cost Analysis↗
Mitochondrial DNA analysis reveals diverse histories of tribal populations from India.
We analyzed 370 bp of the first hypervariable region of the mitochondrial DNA (mtDNA) control region in 752 individuals from 17 tribal and four nontribal groups from the Indian subcontinent, to address questions concerning the origins, genetic structure and relationships of these groups. Southern Indian tribes showed reduced diversity and large genetic distances, both among themselves and when compared with other groups, and no signal of prehistoric demographic expansions. These results probably reflect enhanced genetic drift because of small population sizes and/or bottlenecks in these groups. By contrast, northern groups exhibited more diversity and signals of prehistoric demographic expansions. Phylogenetic analyses revealed that southern and northern groups (except northeastern ones) have related mtDNA sequences albeit at different frequencies, further supporting the larger impact of drift on the genetic structure of southern groups. The Indian mtDNA gene pool appears to be more closely related to the east Eurasian gene pool (including central, east and southeast Asian populations) than the west Eurasian one (including European and Caucasian populations). Within India, northeastern tribes are quite distinct from other groups; they are more closely related to east Asians than to other Indians. This is consistent with linguistic evidence in that these populations speak Tibeto-Burman languages of east Asian origin. Otherwise, analyses of molecular variance suggested that caste and tribal groups are genetically similar with respect to mtDNA variation.
Cluster Analysis↗
Ancient DNA analysis of human neolithic remains found in northeastern Siberia.
We successfully extracted DNA from a bone sample of a Neolithic skeleton (dated 3,600 +/- 60 years BP) excavated in northeastern Yakutia (east Siberia). Ancient DNA was analyzed by autosomal STRs (short tandem repeats) and by sequencing of the hypervariable region I (HV1) of the mitochondrial DNA (mtDNA) control region. The STR profile, the mitochondrial haplotype, and the haplogroup determined were compared with those of modern Eurasian and Native American populations. The results showed the affinity of this ancient skeleton with both east Siberian/Asian and Native American populations.
Base Sequence↗