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PubMed2003

Multiplex PCR design strategy used for the simultaneous amplification of 10 Y chromosome short tandem repeat (STR) loci.

The simultaneous amplification of multiple regions of a DNA template is routinely performed using the polymerase chain reaction (PCR) in a process termed multiplex PCR. A useful strategy involving the design, testing, and optimization of multiplex PCR primer mixtures will be presented. Other multiplex design protocols have focused on the testing and optimization of primers, or the use of chimeric primers. The design of primers, through the close examination of predicted DNA oligomer melting temperatures ( T(m)) and primer-dimer interactions, can reduce the amount of testing and optimization required to obtain a well-balanced set of amplicons. The testing and optimization of the multiplex PCR primer mixture constructed here revolves around varying the primer concentrations rather than testing multiple primer combinations. By solely adjusting primer concentrations, a well-balanced set of amplicons should result if the primers were designed properly. As a model system to illustrate this multiplex design protocol, a 10-loci multiplex (10plex) Y chromosome short tandem repeat (STR) assay is used.

Base Sequence↗

PubMed2006

Gene-based analysis suggests association of the nicotinic acetylcholine receptor beta1 subunit (CHRNB1) and M1 muscarinic acetylcholine receptor (CHRM1) with vulnerability for nicotine dependence.

Based on our previously identified linkage regions for nicotine dependence (ND), we selected six and five single nucleotide polymorphisms (SNPs) in the muscarinic cholinergic receptor subtype M1 (CHRM1) and nicotinic cholinergic receptor beta1 (CHRNB1), respectively, to determine the association of the two genes with ND in a total of 2,037 subjects from 602 nuclear families of either African-American (AA) or European-American (EA) origin. Individual SNP- and/or haplotype-based analyses indicated that the CHRNB1 was significantly associated with ND, which was assessed by smoking quantity (SQ), the Heaviness of Smoking Index (HSI), and the Fagerström Test for ND (FTND), in both ethnic samples. The association of rs2302763 in the CHRNB1 was significant with adjusted SQ in the EA sample after correction for multiple testing (P=0.013). Haplotype A-T-A formed by SNPs rs2302765, rs2302762, and rs9217 in the CHRNB1 was significantly associated with the high risk allele for all the three ND measures (minimum P=0.009, 0.006, and 0.008 for SQ, HSI and FTND, respectively) in the AA sample while haplotype A-T-A formed by rs2302765, rs2302763, and rs9217 was significantly positively associated with ND (minimum P=0.005, 0.016, and 0.016 for SQ, HSI and FTND, respectively) in the EA sample. The CHRM1 exhibited significant protective associations of haplotype C-C-A-T-G-G formed by all six SNPs of this gene with at least one ND measure in the AA sample after Bonferroni correction (minimum P=0.008, 0.013, and 0.009 for SQ, HSI and FTND, respectively), but no significant association was found in the EA sample. The significant associations, together with their location of linked region to ND, suggest that the CHRNB1 and CHRM1 are likely candidates for further investigation.

Adult↗

PubMed2004

Haplotype analysis and identification of genes for a complex trait: examples from schizophrenia.

For more than a decade there has been intensive research into the genetic etiology of schizophrenia, yet it is only recently that the first findings of promising genes associating with the disorder have been reported. Linkage analyses in families collected from different populations have provided relatively well defined genomic loci. These have been typically followed by fine mapping studies using single nucleotide polymorphisms (SNPs). A number of analysis programs have been produced to test SNPs and their haplotypes for association. Typically association has been established to specific haplotypes representing an allelic variant of the corresponding gene. The inherent problem of multiple testing in the analysis of haplotypes needs to be addressed fully, to determine if any of these recent findings can be considered as confirmed susceptibility genes for schizophrenia. However, informative haplotypes have provided a way to define allelic variants of genes associated with schizophrenia in numerous study samples, and are a useful tool in characterizing the extent of allelic diversity of putative schizophrenia susceptibility genes within different populations.

Chromosome Mapping↗

PubMed2005

Why might clinicians in Malawi not offer HIV testing to their patients?

In order to identify reasons clinicians in Malawi might not offer HIV testing to patients, a cross-sectional descriptive postal census with telephone and fax follow-up was conducted. Proportions were calculated for each reason given for not offering HIV testing. Multiple logistic regression was used to determine whether responses differed by demographic characteristics. The response rate was 54.3%. The five most common reasons for not offering HIV testing were: (1) inadequate training in HIV counselling (50.3%); (2) perception that the patient is not ready (49.5%); (3) no indication (35.6%); (4) testing facilities unavailable (35.4%); and (5) insufficient time (28.9%). Differences in reasons for not offering HIV tests amongst clinician groups were few. Thus, development of one general programme based on common reasons identified in this study could be effective in increasing HIV testing rates. Further investigation of the identified reasons should be undertaken to facilitate programme development.

AIDS Serodiagnosis↗

PubMed1979

Chemical profiles of disease.

Chemical profiles have been used successfully in teaching the interpretation of laboratory test values. The profiles have a visual impact that assists in making associative and correlative observations about disease processes. Profiles are most useful in attempts to integrate multiple test abnormalities in a given disease process. Representative profiles are illustrated and discussed as examples of the effectiveness of this mode of analysis of clinical laboratory data in laboratory medicine.

Adult↗

PubMed2002

No evidence for minority male mating advantage in wild type strains of Drosophila ananassae tested in multiple-choice experiments.

Minority male mating advantage was tested in wild type strains of Drosophila ananassae through multiple-choice experiments. Mating success of two types of flies present in five different ratios was scored by direct observation in an Elens-Wattiaux mating chamber. We found no evidence for minority male mating advantage in wild type strains of D. ananassae. The relative mating success of two types of females was also compared in the multiple-choice experiments at different ratios; there was no evidence for a rare female effect. Further, there was similarity in the results of experiments employing different methods. The total number of homogamic and heterogamic matings was obtained by combining the data (all five ratios) from each experiment. Homogamic matings were significantly more frequent than heterogamic ones, which demonstrates preferential mating between males and females of the same strain; this was also supported by a lower isolation estimate. There was also a significant difference in the degree of mating preference between the two strains.

Animals↗

PubMed1996

The acceptability of voluntary HIV antibody testing in the United States: a decade of lessons learned.

OBJECTIVE: As the benefits of early diagnosis of HIV increase, US adults are more likely to be offered HIV counseling and testing in settings where they may not seek testing. Rates and determinants of counseling and testing acceptance in these settings are poorly understood. DESIGN: We reviewed articles and abstracts published from 1985 to 1995 which addressed rates or determinants of counseling and testing acceptance in facilities that provide perinatal, family planning, gynecology, sexually transmitted disease (STD) and drug treatment services, hospitals, and prisons. Data reflected testing experience of more than 240,000 adults. RESULTS: Acceptance rates varied widely (3-100%), even within settings of the same type. Acceptance was generally higher (> 50%) among persons at high risk for acquiring or transmitting the infection (e.g., STD patients, pregnant women at high risk) than among low-risk persons. Factors associated with high acceptance rates included the client's perception of HIV risk, acknowledging risk behaviors; confidentiality protections; presenting counseling and testing as 'routine' rather than optional; and the provider's belief that counseling and testing will benefit the client. Factors associated with low acceptance rates included prior HIV testing, fears about coping with results, and explicit informed consent. CONCLUSIONS: To institute and evaluate counseling and testing programs for persons who do not specifically seek testing, multiple determinants of acceptance must be considered. Practices that protect confidentiality, endorse counseling directed to a client's unique circumstances, and highlight the medical and social benefits of testing are likely to promote acceptance. Acceptance of counseling and testing offered nonroutinely to the numerous Americans who have been previously tested or are at low risk is likely to be low.

Acquired Immunodeficiency Syndrome↗

PubMed1999

A study of five candidate genes in Parkinson's disease and related neurodegenerative disorders. European Study Group on Atypical Parkinsonism.

OBJECTIVE: To determine whether reported genetic association of polymorphisms in the CYP2D6, CYP1A1, N-acetyltransferase 2 (NAT2), DAT1, and glutathione s-transferase M1 (GSTM1) genes with PD were evident in a population of 176 unrelated patients with sporadic PD and to extend these findings to other disease groups (familial PD [n = 30], ALS [n = 50], multiple system atrophy [n = 38], progressive supranuclear palsy [n = 35], and AD [n = 23]). METHODS: A combination of allele-specific PCR and analysis of restriction fragment length polymorphisms were performed. RESULTS: We genotyped 1,131 individuals. After matching each patient with a control subject by age, sex, ethnicity, and geographic origin, there was no association of any allele/genotype with any of the six disease groups. There was an increased frequency of NAT2 slow acetylators in the ALS group compared with controls (70% versus 50%; OR 2.33 [95% CI, 1.03 to 5.30]), but this was not significant after adjusting for multiple testing. CONCLUSIONS: This is one of the most extensive candidate gene studies performed in PD and the first time that some of these loci have been studied in multiple system atrophy and progressive supranuclear palsy. In contrast with previous studies, we found no role for these polymorphisms in the etiology of PD, ALS, multiple system atrophy, progressive supranuclear palsy, or AD.

Aged↗

PubMed2004

Determination of antibiotic resistance and resistance plasmids of clinical Enterococcus species.

To determine the antibiotic resistance pattern and resistance plasmids, we studied 23 antibiotic-resistant clinical isolates of Enterococcus spp. which caused infection in Bayindir-Ankara Hospital, Turkey. Biochemical and physiological identification tests were applied by the Vitek system and compared with the results of protein profiles by SDS-PAGE. From 23 isolates, 20 were identified as E. faecalis, 2 as E. faecium and 1 as E. gallinarum. Twenty four antibiotics belong to 10 different groups were used in susceptibility tests. Multiple antibiotic resistance was determined in 10 of 23 Enterococcus spp. Overall resistance to the used antibiotics was 47.3% and low level resistance was 16.6%. Among the isolates tested, 8.7% demonstrated high level gentamicin resistance, 17.4% demonstrated high level streptomycin resistance, and 43.5% demonstrated penicillin resistance. High level vancomycin resistant Enterococcus spp. rate was 34.8%, and 60.9% exhibited low level resistance to vancomycin and teicoplanin. They contain plasmids which varied in numbers between 1 and 11 and the plasmid sizes ranged from 2.08 to 56.15 kb. In curing experiments with acriflavine, two different plasmids were shown in different molecular sizes of 33.49 and 13.6 kb while the first determined glycopeptide and penicillin resistance, the second one determined either glycopeptide or penicillin resistance in two different E. faecalis strains. On the other hand, a 22.58 kb plasmid, determining kanamycin resistance, was detected in an E. faecium strain. After the curing experiments, an elimination of 37.17 and 44.47 kDa protein bands was shown in E. faecium EFA1 and E. faecalis EFA13 in SDS-PAGE, respectively. This survey indicates the increase of antibiotic-resistant enterococci, especially to vancomycin in our hospital isolates.

Anti-Bacterial Agents↗

PubMed2003

Double checking the cross-check principle.

The cross-check principle was proposed by Jerger and Hayes over 20 years ago. Actually, the cross-check principle consists of a concept and a specific test protocol. The concept, that one test result confirms another test result, is still in use. Their specific protocol is essentially forgotten. The cross-check protocol they described, however, differs from more traditional test protocols. From the design of their cross-check protocol we can extract two unique testing strategies. The first strategy requires agreement between both tests before a decision can be made. This strategy can significantly improve testing performance; however, the "cost" of this strategy is a population of individuals for whom no decision is possible because the two tests disagree. The cross-check protocol uses a third test when the first two tests disagree. Essentially, the cross-check protocol employs an intermediate criterion, the second unusual strategy. Somewhat surprisingly, this intermediate criterion has the ability to simultaneously increase protocol hit rate and decrease protocol false alarm rate relative that of the individual tests in the protocol. The more traditional protocol criteria cannot do this. Each strategy offers some interesting and important advantages and should be considered by audiologists when using multiple tests.

Audiology↗

PubMed1992

[Quality of day time sleep in the multiple sleep latency tests in patients with narcolepsy, obstructive sleep apnea and psychogenic hypersomnia].

The multiple sleep latency test (MSLT) has proved to be a useful diagnostical tool for patients complaining of excessive daytime sleepiness (EDS). The intention of the present study was to investigate the structure of MSLT naps and in particular sleep spindle and k-complex density in three different groups of EDS patients. MSLT was performed at 8 a.m., 10 a.m. 12 a.m., 2. p.m. and 4 p.m.. Each recording lasted 20 minutes and was not stopped even if sleep occurred before 20 min. Sleep was scored visually. Spindle and k-complex density was determined per minute of S2 sleep. Statistical analysis used ANOVA. Each of the three groups consisted of 15 patients. Diagnosis of narcolepsy, sleep apnea, of EDS due to a psychiatric disorder has been confirmed subsequently. There were 5 female and 10 male narcoleptics (mean age: 43.9 +/- 10.9 years), 2 female and 13 male obstructive sleep apnea patients (mean age: 53.9 +/- 10.9 years) and 7 female and 8 male patients complaining of EDS, in whom a psychiatric disorder was diagnosed (mean age: 38.8 +/- 13.8 years). Narcoleptics sent more than half of the recording time of 100 min asleep (52.9%). Apnea patients slept 41.3% and psychogenic EDS patients 22.7%. The proportion of sleep stages 1 and 2 in narcoleptics (S2/S1 = 1:1) was clearly different from the other two (apnea patients: S2/S1 = 4:1; psychogenic EDS patients: S2/S1 = 3:1). 18.5% of the naps contained stage REM and during the afternoon naps 0.9% of S3 in the narcoleptics. Neither REM nor S3 was observed in the others.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed2004

Quantitative oligonucleotide microarray fingerprinting of Salmonella enterica isolates.

We report on a genome-independent microbial fingerprinting method using nucleic acid microarrays for microbial forensics and epidemiology applications and demonstrate that the microarray method provides high resolution differentiation between closely related microorganisms, using Salmonella enterica strains as the test case. In replicate trials we used a simple 192 probe nonamer array to construct a fingerprint library of 25 closely related Salmonella isolates. Controlling false discovery rate for multiple testing at alpha = 0.05, at least 295 of 300 pairs of S.enterica isolate fingerprints were found to be statistically distinct using a modified Hotelling T2 test. Although most pairs of Salmonella fingerprints are found to be distinct, forensic applications will also require a protocol for library construction and reliable microbial classification against a fingerprint library. We outline additional steps required to produce such a protocol.

Bayes Theorem↗

PubMed2002

Preterm premature rupture of the membranes is associated with a reduction in neonatal respiratory distress syndrome.

OBJECTIVE: The purpose of this study was to evaluate the effect of preterm premature rupture of the membranes on the frequency of respiratory distress syndrome among singleton pregnancies that are complicated with preterm delivery. STUDY DESIGN: We performed a retrospective analysis of singleton pregnancies that were delivered between 24 and 34 weeks of gestation. Patients were categorized on the basis of membrane integrity into two groups: ruptured versus intact. All patients received prophylactic antibiotics and a single course of antenatal betamethasone. Data were analyzed with the Student t test, the chi(2) test, and the Fisher exact test. Multiple logistic regression analysis was performed to examine the effect of possible confounding variables that were considered risk factors for respiratory distress syndrome. Probability values of <.05 for all two-tailed tests were considered significant. RESULTS: A total of 366 patients were included (99 patients in the preterm premature rupture of the membranes group and 267 patients in the intact membranes group). Patients were delivered at 30.7 +/- 2.9 and 30.1 +/- 2.7 (mean +/- SD) weeks of gestation, with birth weights of 1620 +/- 594 and 1417 +/- 501 g, respectively. The frequency of respiratory distress syndrome in the neonate was significantly lower in the preterm premature rupture of the membranes group than in their intact counterparts (17% vs 39%, P <.001). Multiple logistic regression analysis confirmed that preterm premature rupture of the membranes (odds ratio, 0.16; 95% CI, 0.08-0.34) was independently associated with a reduction in the frequency of respiratory distress syndrome. CONCLUSION: In the clinical setting of delivery before 34 weeks of gestation, preterm premature rupture of the membranes is associated with a significant decrease in the frequency of neonatal respiratory distress syndrome.

Adult↗

PubMed1998

Second trimester maternal dimeric inhibin-A in the multiple-marker screening test for Down's syndrome.

The aim of this study was to evaluate the additional value of dimeric inhibin-A serum concentration in second trimester multiple-marker screening tests for pregnancies affected by Down's syndrome. We anticipated that second trimester maternal serum dimeric inhibin-A concentrations would be altered in pregnancies complicated by fetal Down's syndrome and that dimeric inhibin-A would perform better than one of the three substances analysed in the multiple-marker screening test currently in use. A total of 1156 serum samples were screened for dimeric inhibin-A in parallel with the routine classic triple test screening programme performed on a random obstetric population. Classic triple test performance was compared with detection rates obtained after substitution of unconjugated oestriol by inhibin-A and with the performance of inhibin-A and alpha-fetoprotein alone. Absolute dimeric inhibin-A maternal serum concentrations of Down's syndrome pregnancies were indeed significantly higher than those of normal pregnancies in our screened population. The performance of dimeric inhibin-A in combination with the multiple-marker screening test, however, is limited because of its strong correlation with intact human chorionic gonadotrophin.

Algorithms↗

PubMed2001

Statistical power when testing for genetic differentiation.

A variety of statistical procedures are commonly employed when testing for genetic differentiation. In a typical situation two or more samples of individuals have been genotyped at several gene loci by molecular or biochemical means, and in a first step a statistical test for allele frequency homogeneity is performed at each locus separately, using, e.g. the contingency chi-square test, Fisher's exact test, or some modification thereof. In a second step the results from the separate tests are combined for evaluation of the joint null hypothesis that there is no allele frequency difference at any locus, corresponding to the important case where the samples would be regarded as drawn from the same statistical and, hence, biological population. Presently, there are two conceptually different strategies in use for testing the joint null hypothesis of no difference at any locus. One approach is based on the summation of chi-square statistics over loci. Another method is employed by investigators applying the Bonferroni technique (adjusting the P-value required for rejection to account for the elevated alpha errors when performing multiple tests simultaneously) to test if the heterogeneity observed at any particular locus can be regarded significant when considered separately. Under this approach the joint null hypothesis is rejected if one or more of the component single locus tests is considered significant under the Bonferroni criterion. We used computer simulations to evaluate the statistical power and realized alpha errors of these strategies when evaluating the joint hypothesis after scoring multiple loci. We find that the 'extended' Bonferroni approach generally is associated with low statistical power and should not be applied in the current setting. Further, and contrary to what might be expected, we find that 'exact' tests typically behave poorly when combined in existing procedures for joint hypothesis testing. Thus, while exact tests are generally to be preferred over approximate ones when testing each particular locus, approximate tests such as the traditional chi-square seem preferable when addressing the joint hypothesis.

Animals↗

PubMed2006

Statistical methods for the detection of spatial clustering in case-control data.

In this paper, I develop new approaches for the detection of spatial clustering in case-control data. One method is based upon drawing Thiessen polygons around each control. It is unnecessary to actually draw or compute the boundaries of the polygons; it is sufficient to count, for each control, the number of cases that are closer to that control than to any other control. A second method is similar to the Cuzick-Edwards method, which is based on counts of cases that are among the k-nearest neighbours of cases, but is instead based upon the number of cases within a specified distance of cases. These first two methods are global methods in the sense that they provide a single statistic that measures the degree of spatial clustering. The third method suggests a local statistic, for tests of the null hypothesis of no spatial clustering around a prespecified focus. The method is based upon the cumulative chi2 test, which is typically used to test whether cases are more prevalent than expected around a prespecified location. This is also extended to the case where all observational locations are considered as potential cluster locations and multiple testing is carried out. Each of the new methods is illustrated using data on childhood leukaemia and lymphoma cases in North Humberside.

Bayes Theorem↗

PubMed2002

Parkinson's disease and apolipoprotein E: possible association with dementia but not age at onset.

Idiopathic Parkinson's disease (PD) is an age-dependent, neurodegenerative condition frequently associated with dementia. Although it is predominantly a sporadic disease, 20-30% of cases are familial, suggesting a complex mode of inheritance. Apolipoprotein E (APOE) allele epsilon4 has been associated with familial and sporadic late-onset senile dementia of the Alzheimer's type. To investigate the role of this gene in the development of dementia associated with PD and age at onset of PD, we evaluated the frequency of APOE gene polymorphism in a sample of PD patients with (n=118) and without (n=167) a family history, as well as matched normal controls (n=96). The PD sample was categorized according to age at onset and presence or absence of dementia. Kaplan-Meier survival analysis was used to plot genotype-specific age at onset distribution curves. Allele frequencies of APOE in PD patients with and without a family history and normal controls were not significantly different. APOE genotypes were also similar between the groups. However, the frequencies of epsilon4 allele and epsilon4/- genotype in the PD group with dementia were more than twofold higher than in normal controls, and the differences were statistically significant. There were no differences in the allele and genotype frequencies of the APOE gene between PD groups with different age at onset. The familial PD had significantly earlier age at onset than sporadic PD (Log-rank test, P=0.027). The age at onset distribution curves for different genotype groups were similar, and their differences were not statistically significant (P=0.38). After the Bonferroni's correction for multiple tests, the positive results are not significant at the P<0.05 level. We conclude that APOE does not play an important role in susceptibility to PD or age at onset of PD, but may play a role in dementia associated with PD in our sample.

Adult↗

PubMed2005

A testing framework for identifying susceptibility genes in the presence of epistasis.

An efficient testing strategy called the "focused interaction testing framework" (FITF) was developed to identify susceptibility genes involved in epistatic interactions for case-control studies of candidate genes. In the FITF approach, likelihood-ratio tests are performed in stages that increase in the order of interaction considered. Joint tests of main effects and interactions are performed conditional on significant lower-order effects. A reduction in the number of tests performed is achieved by prescreening gene combinations with a goodness-of-fit chi2 statistic that depends on association among candidate genes in the pooled case-control group. Multiple testing is accounted for by controlling false-discovery rates. Simulation analysis demonstrated that the FITF approach is more powerful than marginal tests of candidate genes. FITF also outperformed multifactor dimensionality reduction when interactions involved additive, dominant, or recessive genes. In an application to asthma case-control data from the Children's Health Study, FITF identified a significant multilocus effect between the nicotinamide adenine dinucleotide (phosphate) reduced:quinone oxidoreductase gene (NQO1), myeloperoxidase gene (MPO), and catalase gene (CAT) (unadjusted P = .00026), three genes that are involved in the oxidative stress pathway. In an independent data set consisting primarily of African American and Asian American children, these three genes also showed a significant association with asthma status (P = .0008).

Black or African American↗