Characterization of telomerase activity in the human oocyte and preimplantation embryo.
Telomerase, a ribonucleoprotein, has been described as an essential component of highly proliferative cells as it stabilizes the telomeres and avoids cellular senescence. The objective of this study was to modify the polymerase chain reaction-based telomeric repeat amplification protocol to detect telomerase activity in the single cell and to characterize the activity expressed in the human oocyte through to the blastocyst stage embryo. A comparative evaluation of telomerase activity and developmental stage was conducted using discarded or donated human oocytes and embryos. Telomerase activity was detected in all developmental stages evaluated from immature oocytes through to blastocyst stage embryos. Immature oocytes and blastocysts had similar levels of telomerase activity; however, both groups had significantly (P < 0.05) higher activity than zygote through to pre-morula stage embryos. Seventy-five thawed zygotes were cultured to day 3, biopsied by removing 1-2 cells, and the biopsied embryos were cultured to blastocyst stage. There was no difference (P < 0.05) in telomerase activity between cells biopsied from embryos that reached the blastocyst stage and cells from those that arrested in growth. This study has shown that human oocytes through to blastocyst stage embryos express telomerase activity, but that the level of telomerase activity in biopsied blastomeres, of the day 3 cleavage stage embryo, is not predictive of embryonic growth potential. (+info)
Characterization of relaxin binding in the uterus of the marmoset monkey.
The ovarian peptide hormone relaxin (RLX) plays an important role in the regulation of the endometrium both during the cycle and in early pregnancy. RLX interacts with specific receptors on endometrial stromal cells causing these to decidualize. In order to characterize the molecules with which RLX interacts in the primate uterus, a methodology based on a fully bioactive preparation of biotinylated porcine RLX was applied to cryosections of the uterus of female marmoset monkeys. Specific RLX binding was weakly detected in the proliferative phase in isolated endometrial stromal cells. In the secretory phase, the positively reacting cells increased in staining intensity and in number and also included some epithelial cells. Further increases occurred in pregnancy, but RLX binding in the endometrium decreased at the end of the cycle if pregnancy did not occur. The myometrium showed weak staining which did not vary through the cycle, but increased in pregnancy. Electrophoretic analysis of the RLX-binding moieties in these tissue sections indicated that a protein of approximately 40 kDa was the principal RLX-binding molecule, while minor specific bands were detectable at approximately 100 and approximately 200 kDa. The binding of biotinylated RLX could be specifically suppressed by co-incubation with unlabelled RLX, but not by insulin, IGF-I or biotin. This technique therefore allows the detection and molecular characterization of specific RLX binding in the primate uterus. In the marmoset monkey, the pattern of specific binding closely reflects the RLX-dependent physiology during implantation and early pregnancy, implying the probable involvement of a specific RLX receptor. (+info)
Influence of age and gender on the clinical expression of acute intermittent porphyria based on molecular study of porphobilinogen deaminase gene among Swiss patients.
BACKGROUND: Acute intermittent porphyria (AIP) is an inherited disorder in the heme biosynthetic pathway caused by a partial deficiency of porphobilinogen (PBG) deaminase. Clinically, AIP is characterized as acute neurovisceral attacks that are often precipitated by exogenous factors such as drugs, hormones, and alcohol. An early detection of mutation carriers is essential for prevention of acute attacks by avoiding precipitating factors. This study was aimed at analyzing genetic defects causing AIP among Swiss families to further investigate aspects concerning the clinical expression of the disease. MATERIALS AND METHODS: The PBGD gene of index patients from 21 Swiss AIP families was systematically analyzed by denaturing gradient gel electrophoresis of polymerase chain reaction (PCR) amplified DNA fragments and direct sequencing. RESULTS: Five new mutations insA503, del L170, T190I, P241S, and R321H, as well as three known mutations (R26H, R173Q and W283X) were detected. Twelve of the 21 index patients (57%) carried the prevalent mutation W283X previously found among the Swiss AIP population. Family-specific mutations were then screened among relatives of the index patients. Among the 107 studied individuals, 58 carried a PBGD gene mutation--30 were overt AIP patients and 28 were asymptomatic carriers. The apparent rate of overt disease in the study cohort was 52%, which is significantly higher than the previously reported penetrance of 10-20%. To further examine the clinical expression of AIP, the cumulative life-time risk was calculated among 58 mutation-positive individuals after stratifying for age. The result shows a linear increase of the percentage of the symptomatic patients with age, reaching up to 75% among carriers aged over 60. Moreover, statistical analysis of the gender distribution among patients and asymptomatic carriers indicated that the disease was more frequently expressed among females than males (Fisher's exact test two sided, p= (0.001). CONCLUSIONS: This comprehensive search for genetic defects in the PBGD gene confirmed the existence of a prevalent mutation W283X among Swiss AIP patients, as well as a number of family-private mutations. Genetic analysis laid a groundwork for further studies such as the effects of gender and age on the clinical expression of AIP. (+info)
BADGE, Beads Array for the Detection of Gene Expression, a high-throughput diagnostic bioassay.
Several methods are presently available for gene expression analysis. However, few of them are suitable for detection of moderate numbers of genes in thousands of samples with high speed and low cost. There is great demand for such a method for use in diagnostics and screening. To address this need, we have developed an assay for gene expression analysis using microspheres and a fluidic instrument made by Luminex. The assay is named Beads Array for the Detection of Gene Expression (BADGE). BADGE can monitor up to 100 genes in a single reaction, and it takes only 1 h to hybridize and <20 sec to read the results of all 100 genes in a sample for the detection process. For the genes detected in five independent replicate experiments, the standard deviation was <35% of the mean. We have monitored multiple pathogenesis-related genes simultaneously in chemical-treated and control Arabidopsis samples employing the BADGE assay. The data were compared with those obtained from an established technology, Affymetrix GeneChip. The changes in expression profiles were very similar. Our study showed that the BADGE assay was capable of profiling expression of multiple genes at affordable cost and rapid speed. (+info)
Detection of mutations in the dystrophin gene via automated DHPLC screening and direct sequencing.
BACKGROUND: Currently molecular diagnostic laboratories focus only on the identification of large deletion and duplication mutations (spanning one exon or more) for Duchenne Muscular Dystrophy (DMD) yielding 65% of causative mutations. These mutations are detected by an existing set of multiplexed polymerase chain reaction (PCR) primer pairs. Due to the large size of the dystrophin gene (79 exons), finding point mutations (substitutions, deletions or insertions of one or several nucleotides) has been prohibitively expensive and laborious. The aim of this project was to develop an effective and convenient method of finding all, or most, mutations in the dystrophin gene with only a moderate increase in cost. RESULTS: Using denaturing high performance liquid chromatography (DHPLC) screening and direct sequencing, 86 PCR amplicons of genomic DNA from the dystrophin gene were screened for mutations in eight patients diagnosed with DMD who had tested negative for large DNA rearragements. Mutations likely to be disease-causative were found in six of the eight patients. All 86 amplicons from the two patients in whom no likely disease-causative mutations were found were completely sequenced and only polymorphisms were found. CONCLUSIONS: We have shown that it is now feasible for clinical laboratories to begin testing for both point mutations and large deletions/duplications in the dystrophin gene. The detection rate will rise from 65% to greater than 92% with only a moderate increase in cost. (+info)
Rapid diagnostics: the detection of neuraminidase activity as a technology for high-specificity targets.
The accurate detection of influenza by clinical symptoms is challenging since multiple pathogenic viruses and bacteria mimic similar symptoms in a patient. With new and more effective influenza therapeutics available, there is a growing need for highly accurate and rapid diagnosis of influenza, particularly when the window of opportunity for proper treatment is measured in hours. A parallel technology, which is also used in the treatment of influenza, was developed for the rapid diagnosis of influenza by exploiting the enzymatic activity of influenza neuraminidase. This technology, which is called Pathozyme, offers the high specificity inherent from the conservation of the neuraminidase active site. The ZstatFlu test uses a small molecule derivative of sialic acid chemically coupled to a reporter group together with simple point-of-care reagents for directly detecting influenza from a patient specimen with high specificity. A second-generation platform technology using this neuraminidase detection system coupled with a more sensitive chemiluminescent reporter has been developed and formatted for reading on high-speed instant film. This modification resulted in a platform technology many-fold more sensitive than the former while maintaining its inherent high specificity. Preliminary data from a prototype tested during the mild 2000-2001 influenza season demonstrated that an optimized chemiluminescent test system could approach the accuracy of 14 day viral culture in a convenient 10-20 min test. This platform technology is currently being explored for the rapid detection of other pathogenic organisms where sensitivity, specificity and speed are essential in a point-of-care setting. (+info)
Molecular analysis of Spinocerebellar ataxias in Koreans: frequencies and reference ranges of SCA1, SCA2, SCA3, SCA6, and SCA7.
Spinocerebellar ataxias (SCAs) are a heterogeneous group of neurodegenerative disorders. CAG repeat expansions in the causative genes have been identified as the basic cause of several types of SCAs, and have been used for the diagnoses and classifications of patients with ataxia. In order to assess the frequency and CAG repeat size ranges of SCAs, and to establish an effective strategy for molecular diagnosis, we performed a molecular analysis of SCA1, SCA2, SCA3, SCA6, and SCA7 in 76 patients. These patients were as follows: 32 with dominant inheritance, 39 sporadic cases, and 5 with unknown family histories. The normal and affected CAG repeat size ranges were established at five SCA loci in Koreans, which was consistent with previous reports. The total prevalence of the five types of SCAs was 39.5% in the 76 patients with ataxia, regardless of their family history. It was 75.0% in the 32 families with a dominant inheritance. The most frequent type was SCA3 (15.8%), followed by SCA2 (14.5%). Both types combined formed 76.7% of the 30 patients with CAG expansions. SCA1, SCA6, and SCA7 were less frequent, affecting 3.9%, 2.6%, and 2.6% of the cases, respectively. This mutation spectrum is quite different from a previous report concerning Koreans, but is similar to the distributions that are seen in several ethnic populations worldwide. For a correct and effective diagnosis of SCAs, we suggest that a molecular diagnosis be undertaken, even in patients without a family history, as well as those with a family history. A stepwise approach is also recommended. Patients with ataxia should be tested for SCA2 and SCA3. Individuals testing negative should be tested for SCA1, SCA6, and SCA7. (+info)
Molecular diagnosis in haemophilia A.
Haemophilia A is the commonest cause of X-linked inherited bleeding disorder. Due to inadequate medical facility for management of the disease, the DNA based genetic diagnosis has assumed great importance. Ideally, the direct detection of mutations is the most accurate and reliable approach for carrier detection and prenatal diagnosis. However, mutation detection is possible only in limited number of cases. In majority of haemophiliacs, no common mutation is easily identifiable. The limitation has been over come by the use of linkage-based analysis using polymorphic DNA markers in the factor VIII gene. Some of these markers can be identified by restriction enzymes and are called RFLP markers. Other markers are a class of short tandem repeats sequences which result in differences in the number of CA repeats in different individuals. The combined use of these markers has made it possible to identify carriers and provide prenatal diagnosis in upto 95% of families having affected individuals. Therefore, the recurrence of the disease can be prevented to a great extent in the haemophilia A affected families. (+info)