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PubMed1998

Identification of genetic loci implicated in the survival of Mycobacterium smegmatis in human mononuclear phagocytes.

A luminescence-based procedure that permits the rapid evaluation of the survival of mycobacteria within mononuclear phagocytes was developed and used to screen insertional mutants of Mycobacterium smegmatis for their ability to survive in human monocyte-derived macrophages. Among the 5000 mutants tested, eight mutants were identified that demonstrated impaired intracellular survival in human macrophages but that grew normally in the absence of cells. For each mutant, a portion of the gene interrupted by the transposition event was amplified by ligand-mediated PCR and sequenced. In all cases, the existence of homologous genes of as yet unknown function were identified in the Mycobacterium tuberculosis genome. Complementation of the mutant mycobacterial strains with cosmids containing the homologous loci from M. tuberculosis restored normal intracellular growth in three of the four mutants tested, supporting the idea that these loci contain genes that are important for intracellular survival. This study demonstrates the feasibility of directly screening mutant mycobacterial strains to identify genes coding for activities necessary for the intracellular survival in human mononuclear phagocytes, an important initial step in the identification of potential targets for new therapeutic agents.

Base Sequence↗
PubMed2005

Rapid evolution of expression and regulatory divergences after yeast gene duplication.

Although gene duplication is widely believed to be the major source of genetic novelty, how the expression or regulatory network of duplicate genes evolves remains poorly understood. In this article, we propose an additive expression distance between duplicate genes, so that the evolutionary rate of expression divergence after gene duplication can be estimated through phylogenomic analysis. We have analyzed yeast genome sequences, microarrays, and transcriptional regulatory networks, showing a >10-fold increase in the initial rate for both expression and regulatory network evolution after gene duplication but only an approximately 20% rate increase in the early stage for protein sequences. Based on the estimated age distribution of yeast duplicate genes, we roughly estimate that the initial rate of expression divergence shortly after gene duplication is 2.9 x 10(-9) per year, whereas the baseline rate for very ancient gene duplication is 0.14 x 10(-9) per year. Relative expression rate tests suggest that the expression of duplicate genes tends to evolve asymmetrically, that is, the expression of one copy evolves rapidly, whereas the other one largely maintains the ancestral expression profile. Our study highlights the crucial role of early rapid evolution after gene/genome duplication for continuously increasing the complexity of the yeast regulatory network.

Evolution, Molecular↗
PubMed2000

Molecular genetics of thyroid tumors and surgical decision-making.

The study of thyroid tumor genetics has great relevance to surgeons and facilitates understanding tumor pathogenesis, prediction of tumor behavior, and management decisions. The genes implicated can be broadly categorized as oncogenes or tumor-suppressor genes. The RET oncogene has well established roles in the development of both papillary (PTC) and medullary (MTC) thyroid carcinoma. Genetic screening for germline RET mutations in members of multiple endocrine neoplasia type II (MEN-II) families is now widely performed, and prophylactic thyroidectomy in gene carriers is advisable at an early age. Patients with apparently sporadic MTC can also be screened to rule out familial disease. The demonstration of a RET rearrangement in a patient's PTC may have prognostic significance, but as yet there are no management implications. The thyrotropin receptor (TSH-R) and Gsalpha become oncogenic through point mutation and are associated with the development of toxic thyroid adenomas. The ras oncogene is implicated in the early stages of development of several thyroid tumor types. Tumor-suppressor genes also have a role in thyroid tumor formation. The p53 gene appears to be involved in the process of transformation to the anaplastic phenotype and the PTEN gene in the development of follicular adenomas but not carcinomas. There is still limited evidence for the so called adenoma-carcinoma sequence of the thyroid follicular cell. Loss of heterozygosity studies have enabled identification of tumor-suppressor genes, and their findings suggests differences in the pathogenesis of PTCs compared with follicular cancers. Surgical decision-making will benefit from these basic molecular advances, which rapidly translates into improved patient management.

DNA, Neoplasm↗
PubMed2003

Rapid screening of multiple beta-globin gene mutations by real-time PCR on the LightCycler: application to carrier screening and prenatal diagnosis of thalassemia syndromes.

BACKGROUND: Hemoglobinopathies are priority genetic diseases for prevention programs. Rapid genotype characterization is fundamental in the diagnostic laboratory, especially when offering prenatal diagnosis for carrier couples. METHODS: As a model, we designed a protocol based on the LightCycler technology to screen for a spectrum of beta-globin gene mutations in the Greek population. Design was facilitated by dual fluorochrome detection and close proximity of many mutations. Three probe sets were capable of screening 95% of beta-globin gene mutations in the Greek population, including IVSII-745C-->G, HbS, Cd5-CT, Cd6-A, Cd8-AA, IVSI-1G-->A, IVSI-5G-->A, IVSI-6T-->C, IVSI-110G-->A, and Cd39 C-->T. RESULTS: The protocol, standardized by analysis of 100 beta-thalassemia heterozygotes with known mutations, was 100% reliable in distinguishing wild-type from mutant alleles. Subsequent screening of 100 Greek beta-thalassemia heterozygotes with unknown mutations found 96 of 100 samples heterozygous for 1 of the 10 mutations, although melting curves were indistinguishable for mutations HbS/Cd6 and IVSI-5/IVSI-1, indicating a need of alternative methods for definitive diagnosis. One sample demonstrating a unique melting curve was characterized by sequencing as Cd8/9+G. Three samples carried mutations outside the gene region covered by the probes. The protocol was 100% accurate in 25 prenatal diagnosis samples, with 14 different genotype combinations diagnosed. The protocol was also flexible, detecting five beta-globin gene mutations from other population groups (IVSI-1G-->T, IVSI-5G-->C, IVSI-116T-->G, Cd37 TGG-->TGA, and Cd41/42 -TCTT). CONCLUSIONS: The described LightCycler system protocol can rapidly screen for many beta-globin gene mutations. It is appropriate for use in many populations for directing definitive mutation diagnosis and is suited for rapid prenatal diagnosis with low cost per assay.

DNA Mutational Analysis↗
PubMed2001

The Danish 1905 cohort: a genetic-epidemiological nationwide survey.

OBJECTIVES: The authors studied nonagenarians, a rapidly growing age group whose cognitive and physical abilities have yet to be investigated systematically. METHODS: All Danes born in 1905 were invited to participate in a home-based 2-hour multidimensional interview, including cognitive and physical performance tests and collection of DNA, carried out by lay interviewers. Population-based registers were used to evaluate representativeness. RESULTS: There were 2,262 participants. A total of 1,632 (72%) gave a DNA sample. Participants and nonparticipants were highly comparable with regard to marital status, institutionalization, and hospitalization patterns, but men and rural area residents were more likely to participate. Six months after the survey began, 7.2% of the participants and 11.8% of the nonparticipants had died. DISCUSSION: Despite the known difficulties of conducting surveys among the extremely old, it was possible to conduct a nationwide survey, including collection of DNA, among more than 2,000 fairly nonselected nonagenarians using lay interviewers.

Aged↗
PubMed2003

Rapid response to artificial selection on flower size in Phlox.

Quantitative characters are often said to evolve rather slowly, taking many generations to exhibit appreciable differences among populations. We tested this notion experimentally by performing bi-directional selection on corolla diameter of plants from a wild population of Phlox drummondii for three generations. By monitoring flower size, tube length and stigma-anther proximity of flowers, we obtained the direct and indirect responses to selection, and calculated genetic correlations, realized and narrow sense heritabilities using offspring-mother regression. Realized heritability of flower size was high (0.83), whereas genetic correlations among traits were weak or not significant. The per-generation average of the response in corolla diameter was about 5%. We found that P. drummondii has a great capacity to respond rapidly to selection, and this capacity may be in part responsible for the observed high degree of differentiation within the species. We also concluded that rapid evolution of morphological floral traits is possible.

Analysis of Variance↗
PubMed2001

Simpson Golabi Behmel syndrome: progress toward understanding the molecular basis for overgrowth, malformation, and cancer predisposition.

Simpson Golabi Behmel syndrome (SGBS) is a complex congenital overgrowth syndrome with features that include macroglossia, macrosomia, and renal and skeletal abnormalities as well as an increased risk of embryonal cancers. Most cases of SGBS appear to arise as a result of either deletions or point mutations within the glypican-3 (GPC3) gene at Xq26, one member of a multigene family encoding for at least six distinct glycosylphophatidylinositol-linked cell surface heparan sulfate proteoglycans. As a class of molecules, heparan sulfate proteoglycans have been found to play essential roles in development by modulating cellular responses to growth factors and morphogens. Specifically, mutations in both the murine GPC3 gene and the Drosophila glypican, dally, have been found to modify cellular responses to bone morphogenetic proteins, providing important clues to the molecular basis of SGBS in humans. Despite these advances, there remains a paucity of information about the natural history of SGBS and optimal medical management strategies, and whether select mutations influence the SGBS phenotype and risk of cancer. To this end, an International SGBS Registry has been created and is being maintained to improve the clinical care and understanding of the pathogenesis of SGBS. Using an integrated approach employing epidemiology, molecular genetic characterization of specific GPC3 mutations, and the use of model organisms should rapidly expand the understanding of this complex disorder.

Abnormalities, Multiple↗
PubMed1996

Analysis of genetic differentiation of host races of the common cuckoo Cuculus canorus using mitochondrial and microsatellite DNA variation.

It has long been argued that populations of the parasitic common cuckoo Cuculus canorus consist of sympatric host-specific female races, each of which lays eggs that match, to varying degrees, those of their chosen hosts. We tested this hypothesis by comparing rapidly evolving DNA markers among the cuckoo chicks reared by the three most common hosts in the United Kingdom. Comparing cuckoos from different hosts, we found no significant differences in the number of repeats in the control region of the mtDNA nor in the allele frequencies of three microsatellite loci. Given that cuckoos parasitizing the three different hosts do lay different eggs, these results suggest that either: (i) egg-colour variation in cuckoos is facultative, which is unlikely; (ii) gene flow between races occurs because female cuckoos sporadically successfully parasitize alternative hosts; or (iii) the presumably neutral markers in this study have not have not tracked the rapid and/or recent evolution of host races in this species. Studies of the laying and mating patterns of female cuckoos in marked populations in the wild will help evaluate which of these interpretations is most likely.

Alleles↗
PubMed2004

Genetic analysis of Pseudomonas aeruginosa by enterobacterial repetitive intergenic consensus polymerase chain reaction (PCR) and arbitrarily primed PCR: gel analysis compared with microchip gel electrophoresis.

OBJECTIVES: To assess the applicability of a newly emerging microchip gel electrophoresis for rapid strain differentiation among clinical isolates of Pseudomonas aeruginosa, and to compare this technique with the traditional gel method for DNA separation. METHODS: One hundred clinical strains of P. aeruginosa obtained from a hospital in northwestern Ohio were tested for reactivity to 3 serotype-specific monoclonal antibodies by enzyme-linked immunosorbent assay. Twelve strains (4 from each serogroup) were selected for DNA analysis by polymerase chain reaction (PCR)-based, single primer DNA fingerprinting methods with 3 different primers: 1 enterobacterial repetitive intergenic consensus PCR and 2 arbitrarily primed PCRs. The PCR products were analyzed by agarose slab gel and microchip gel electrophoresis. RESULTS: Of the 100 clinical isolates tested, 39% (4%, 14%, and 21%) were found to be serotypes 0:3, 0:6, and 0:11, respectively. Twelve strains were chosen for DNA analysis by PCR. The PCR products were analyzed by agarose slab gel electrophoresis and on microchips to determine interspecies diversity. Both methods demonstrated that different serotypes exhibited different electrophoretic patterns. Two strains (clinical strains 6 and 7, serotype 0:6) showed identical patterns, indicating a high degree of relatedness. CONCLUSION: In all cases, there was concordance between the electrophoretic patterns detected by the two methods. The capability of conducting both PCR and microchip gel electrophoresis offers an opportunity for an automated and rapid method for genetic analysis and differentiation among strains of P. aeruginosa and other microorganisms.

Bacterial Typing Techniques↗
PubMed2000

A reliable screening test to identify adult carriers of the (--SEA) alpha zero-thalassemia deletion. Detection of embryonic zeta-globin chains by enzyme-linked immunosorbent assay.

Homozygous (--SEA) alpha zero-thalassemia deletion, the cause of up to 80% of fetal hydrops in Southeast Asia, is encountered in many other countries. Heterozygous carrier rates of the deletion in Southeast Asian populations range from 4% to 14%. The laboratory screening for adult carriers of (--SEA) and other alpha zero-thalassemia deletions currently rests primarily with microscopic detection of hemoglobin H inclusion bodies within erythrocytes (Hb H screen). This test is laborious and observer dependent and has poor sensitivity. We assessed a colorimetric enzyme-linked immunosorbent assay (ELISA) to detect embryonic zeta-globin chains in adult hemolysates as an alternative to detect (--SEA) alpha zero-thalassemia deletion carriers. Blood samples from 221 adults with a mean corpuscular volume less than 80 micron 3 (80 fL) were studied prospectively by currently accepted hemoglobin screening tests and ELISA. Suspected cases of alpha-thalassemia were confirmed by DNA-based diagnostics. ELISA was highly sensitive (1.0) and specific (0.94) for the detection of adult carriers of (--SEA) alpha zero-thalassemia deletion. The hemoglobin H screen had a sensitivity of 0.47 and specificity of 0.99. The zeta-globin ELISA proved simple to perform, rapid, and applicable to high volume or population-based screening programs.

Adult↗
PubMed2004

Preventing and managing antiretroviral drug resistance.

Development of resistance to antiretroviral drugs (ARVs) is a major impediment to optimum treatment of HIV-1 infection. Although resistance testing can help to select subsequent regimens when virologic failure occurs, cross-resistance, which affects all classes of ARVs, may make it more difficult to achieve optimum control of HIV. We have known for some time that our first choice of antiretroviral therapy offers the best chance to control HIV replication and that initial therapy should be selected with an eye on future options. Potency is the first line of defense against the development of resistance. Other factors that affect resistance development include: tolerability, potential for optimum adherence, and genetic and pharmacologic barriers to development of resistance. If resistance emerges, only a single drug may be affected initially, and a rapid change in ARVs may preserve the efficacy of other components. One cautionary note is that we can no longer assume that a patient's HIV is fully susceptible to all ARVs even in the initial regimen. Transmission of drug-resistant HIV means that the genetic composition may be that of an "experienced" virus with reduced susceptibility to ARVs. Resistance testing at the time of transmission is most likely to reveal this resistance, but over time the dominant genetic pattern may revert to wild-type, and be missed by resistance testing. Because "archived" resistant HIV may emerge quickly once treatment is initiated, we need to keep this in mind when selecting initial therapy.

Anti-HIV Agents↗
PubMed2003

Influence of transferable genetic determinants on the outcome of typing methods commonly used for Enterococcus faecium.

A variety of methods is used for a molecular typing of Enterococcus spp. and related gram-positive bacteria including macrorestriction analysis using pulsed-field gel electrophoresis (PFGE), ribotyping, rapid amplification of polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP). To test the influence of transferable determinants on the outcome of different typing methods commonly used for enterococci, we established a homogenous strain collection of 24 transconjugants resulting from filter matings with antibiotic-resistant Enterococcus faecium. As expected, AFLP, RAPD, and PFGE all identified our model bacteria as strongly related. However, distinct differences in the resolving and discriminatory power of the tested methods could be clearly addressed. In PFGE, 22 of 24 transconjugants possessed less than a three-band difference to the recipient pattern and would be regarded as strongly related. Three different RAPD PCRs were tested; in two reactions, identical patterns for all transconjugants and the recipient were produced. One RAPD PCR produced an identical pattern for 18 transconjugants and the recipient and a clearly different pattern for the remaining 6 transconjugants due to a newly appearing fragment resulting from acquisition of the tetL gene. AFLP clusters all transconjugants into a group of major relatedness. Percent similarities were highly dependent on the method used for calculating the similarity coefficient (curve-based versus band-based similarity coefficient). Fragment patterns of digested plasmids showed the possession of nonidentical plasmids in most transconjugants. PFGE still could be recommended as the method of choice. Nevertheless, the more-modern AFLP approach produces patterns of comparable discriminatory power while possessing some advantages over PFGE (less-time-consuming internal standards). Plasmid fingerprints can be included to subdifferentiate enterococcal isolates possessing identical macrorestriction and PCR typing patterns.

Anti-Bacterial Agents↗
PubMed1996

UHG-based mutation screening in type 2B von Willebrand's disease: detection of a candidate mutation Ser547Phe.

We have recently described a novel mutation screening technique for the diagnosis of type 2B von Willebrand's disease (vWD). Analysis involves the use of a synthetic universal heteroduplex generator (UHG). To test the validity of the technique, we have applied UHG screening to seven type 2B vWD patients of previously unknown genotype. Characteristic heteroduplex patterns for Arg543Trp and Val553Met mutations were found in three patients and one patient, respectively. A fifth patient gave a novel pattern and direct sequencing revealed a hitherto unreported candidate mutation (Ser547Phe) 8 bases downstream of an "identifier" deletion in the UHG molecule. The two remaining patients gave normal heteroduplex patterns; an Arg578Gln mutation was identified by PstI digestion in one individual and no mutation could be identified in the sequence covered by the UHG in the final patient. Using a combination of UHG technology and restriction analysis, over 85% of type 2B vWD patients can be rapidly diagnosed by genotype.

Amino Acid Sequence↗
PubMed2005

Regulation of parathyroid hypertensive factor secretion by vitamin D3 analogs in parathyroid cells derived from spontaneously hypertensive rats.

Parathyroid hypertensive factor (PHF) is a novel substance secreted by the parathyroid gland (PTG), which is elevated in 30-40% of all hypertensive patients; specifically, the low-renin subset. However, very little is known about the regulation of PHF secretion. Since the classical parathyroid regulator, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), may be elevated concurrent with or preceding the development of low-renin hypertension and elevated plasma PHF, we hypothesized that 1,25-(OH)2D3 would stimulate PHF release. To test this hypothesis, PTG organ and cell cultures, derived from spontaneously hypertensive rats (SHR) and the normotensive genetic control Wistar Kyoto (WKY) rats, were exposed to various vitamin D3 metabolites and PHF release measured by ELISA. 1,25-(OH)2D3 rapidly stimulated PHF release with enhanced sensitivity in SHR versus WKY cultures indicated by a leftward shift in the dose-response curve, whereas 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) had the converse effect. Vitamin D3 analog "BT," an agonist for the classical nuclear vitamin D receptor (1,25VDR(nuc)), was without effect suggesting a 1,25VDR(nuc)-independent mechanism and potential involvement of the plasma membrane-bound vitamin D receptor (1,25 D3-MARRS). Interestingly, protein expression of the 1,25 D3-MARRS was increased in SHR versus WKY parathyroid cells. In conclusion, these results support the idea that 1,25-(OH)2D3 may contribute to elevated plasma PHF in the SHR.

24,25-Dihydroxyvitamin D 3↗
PubMed1994

Results from a reference laboratory for prenatal diagnosis of sickle cell disorders in Cuba.

A nationwide programme for the prevention of sickle cell (SS and SC) disorders was initiated in Cuba in 1983. Couples at risk were identified by screening pregnant women and the partners of those who carry an abnormal haemoglobin, followed by genetic counselling and the offer of prenatal diagnosis. Prenatal diagnosis was performed in one laboratory, which had carried out 1068 prenatal tests for Hb SS and SC disorders by the end of 1992. The centralization of the service has permitted rapid identification and resolution of problems.

Amniocentesis↗
PubMed2006

Quantitative genetics of cortical bone mass in healthy 10-year-old children from the Fels Longitudinal Study.

The genetic influences on bone mass likely change throughout the life span, but most genetic studies of bone mass regulation have focused on adults. There is, however, a growing awareness of the importance of genes influencing the acquisition of bone mass during childhood on lifelong bone health. The present investigation examines genetic influences on childhood bone mass by estimating the residual heritabilities of different measures of second metacarpal bone mass in a sample of 600 10-year-old participants from 144 families in the Fels Longitudinal Study. Bivariate quantitative genetic analyses were conducted to estimate genetic correlations between cortical bone mass measures, and measures of bone growth and development. Using a maximum likelihood-based variance components method for pedigree data, we found a residual heritability estimate of 0.71 for second metacarpal cortical index. Residual heritability estimates for individual measures of cortical bone (e.g., lateral cortical thickness, medial cortical thickness) ranged from 0.47 to 0.58, at this pre-pubertal childhood age. Low genetic correlations were found between cortical bone measures and both bone length and skeletal age. However, after Bonferonni adjustment for multiple testing, rho(G) was not significantly different from 0 for any of these pairs of traits. Results of this investigation provide evidence of significant genetic control over bone mass largely independent of maturation while bones are actively growing and before rapid accrual of bone that typically occurs during puberty.

Bone Density↗
PubMed2005

Testing the hypothesis of recent population expansions in nematode parasites of human-associated hosts.

It has been predicted that parasites of human-associated organisms (eg humans, domestic pets, farm animals, agricultural and silvicultural plants) are more likely to show rapid recent population expansions than are parasites of other hosts. Here, we directly test the generality of this demographic prediction for species of parasitic nematodes that currently have mitochondrial sequence data available in the literature or the public-access genetic databases. Of the 23 host/parasite combinations analysed, there are seven human-associated parasite species with expanding populations and three without, and there are three non-human-associated parasite species with expanding populations and 10 without. This statistically significant pattern confirms the prediction. However, it is likely that the situation is more complicated than the simple hypothesis test suggests, and those species that do not fit the predicted general pattern provide interesting insights into other evolutionary processes that influence the historical population genetics of host-parasite relationships. These processes include the effects of postglacial migrations, evolutionary relationships and possibly life-history characteristics. Furthermore, the analysis highlights the limitations of this form of bioinformatic data-mining, in comparison to controlled experimental hypothesis tests.

Animals↗