Development of a device for rapid diagnosis of Down's syndrome.
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Chromosome Mapping↗
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[Identification of HLA class II polymorphism with molecular biology techniques].
All the biologically relevant HLA class II allelic variants can not be identified with conventional serological tissue typing techniques. During the past few years considerable advances have been made in HLA class II typing with molecular techniques now widely used in routine clinical tissue typing. The next few years are likely to see the development of tissue typing techniques based on the polymerase chain reaction (PCR), for use in acute transplantation. A new method for the rapid identification of genetic polymorphisms is described--allele-specific PCR amplification.
HLA-D Antigens↗
Genome-wide linkage disequilibrium in two Japanese beef cattle breeds.
There is little knowledge about the degree of linkage disequilibrium (LD) in beef cattle. This study aims to perform a genome-wide search for LD in Japanese Black and Japanese Brown beef cattle and to compare the level of LD between these two breeds. Parameter D' (the LD coefficient) was used as a measure of LD, and LD was tested for significance of allelic associations between syntenic and between non-syntenic marker pairs. Effects of breed, chromosome, genetic map distance and their interactions with D' were tested based on least squares analyses. Both breeds showed high levels of LD, which ranged over several tens of cM and declined as the marker distance increased for syntenic marker pairs. A rapid decline of the D' value was observed between markers that were spaced 5 and 20 cM apart. LD was significant in most cases for marker pairs <40 cM apart but was not significant between non-syntenic loci. The pattern of LD found in these two breeds was similar to that previously published for dairy cattle. The D' value between breeds was not significantly different (P > 0.05), but the interaction between breed and chromosome was highly significant (P < 0.001). Genetic selection seems to have caused the heterogeneity of the D' values among chromosomes within breed. These results indicate that LD mapping is a useful tool for fine-mapping quantitative trait loci of economically important traits in Japanese beef cattle.
Alleles↗
Testing for candidate gene linkage disequilibrium using a dense array of single nucleotide polymorphisms in case-parents studies.
The future of genetic studies of complex human diseases will rely more and more on the epidemiologic association paradigm, in particular the use of the transmission/disequilibrium test to detect linkage disequilibrium in a case-parents study. With the rapid progress in genomic studies, many single nucleotide polymorphisms will be identified and genotyped within a very short physical distance. Analyzing multiple single nucleotide polymorphisms within a candidate gene/region with Bonferroni correction for multiple transmission/disequilibrium tests will lead to a conservative test, and hence a power loss. I propose a new method, the "Adaptive PRIncipal COmponent Test" (APRICOT). The method has the following properties: (1) it does not need haplotype information; (2) it is nonparametric-it does not make specific assumptions about the population history or population structure; and (3) the calculation of the test statistic and the determination of its significance level are simple and straightforward. Monte-Carlo simulation reveals that adaptive principal component test maintains the nominal significance level under the null hypothesis of no linkage disequilibrium, even under complex situations of multiple ancestral haplotypes and structured populations. It provides a substantial power advantage over the conventional Bonferroni approach. The adaptive principal component test is a promising method for candidate gene testing using single nucleotide polymorphisms.
Genetic Markers↗
Genetic control of immunity to Trichinella spiralis. Donor bone marrow cells determine responses to infection in mouse radiation chimaeras.
Radiation chimaeras, prepared from NIH (rapid-responder) mice or from the F1 progeny of a cross between H-2 compatible B10.G (slow-responder) and NIH mice, were tested for their ability to respond to infection with the intestinal nematode parasite Trichinella spiralis. Mice reconstituted with bone marrow (BM) from NIH donors showed the rapid response characteristic of this strain, i.e. expelled worms from the intestine before day 12 of infection; those given BM from B10.G mice showed a show expulsion pattern, losing worms after day 12. There was no evidence that the environment of the recipient exerted any influence on the ability of the BM cells to express the response characteristic of the donor. When chimaeras were given immune mesenteric lymph node cells (IMLNC) from infected NIH donors there was successful adoptive transfer of immunity, resulting in an accelerated loss of worms. As before, the time course of the accelerated response was determined by the genotype of the BM used. These results confirm that genetic control of the process of worm expulsion is expressed at the level of a bone marrow-derived cell population and is independent of lymphocyte responsiveness. They further show that the factors involved are an inherent property of the cells concerned. The possibility that these cells are myeloid in nature is discussed.
Animals↗
[Microbiological diagnosis of tuberculosis].
The increased incidence of tuberculosis as well as the availability of new diagnostic testing methods clearly have various implications for the routine microbiology laboratory: samples must be sent to the microbiology lab for testing immediately after being taken and microscopically investigated the same day. In other countries, difficult to treat, multi-resistant Mycobacterium tuberculosis strains have occurred. Thus decisive hygienic measures must be taken early on in cases of highly infectious patients (i.e. patients with microscopically positive sputum). Liquid media (MB Check Roche, Bactec) as well as Löwenstein Jensen media must be inoculated in the lab. Liquid media allow both faster detection of certain atypical mycobacteria and increased accuracy. Classification of culturally established agents through commercial genetic probes (AccuProbe Mycobacterien) or with high pressure liquid chromatography is possible within hours when acid-fast rods are present. Time consuming identification by determination of biochemical and culture morphological characteristics should be reserved for reference labs. Today, rapid tests like analysis of tuberculostearic acid or polymerase chain reaction are already useful for special questions like ruling out tuberculous meningitis. In most cases, however, these rapid tests cannot replace identification of microbes with culture techniques.
AIDS-Related Opportunistic Infections↗
Studies on the role of cellular immunity and genetics in the etiology of rapidly progressing breast cancer in Tunisia.
It has been suggested that poussée évolutive (PEV) or rapidly progressing breast cancer (RPBC) represents a failure in the host immune system to control the proliferation of breast cancer cells. To evaluate this possibility, we have performed in vivo and in vitro assays of cellular immunity in Tunisian patients with breast cancer. Studies of delayed hypersensitivity using microbial antigens and in vitro including lymphocyte transformation tests and measurements of B and T cells indicated that RPBC patients had an immune response comparable to that of breast cancer patients without evidence of rapid progression. Normal Tunisians were more immunocompetent, however, an appeared to have a higher level of immune activity than normal individuals in the United States. In a second, independent series, an increased frequency of blood group A was found in RPBC patients, suggesting a genetic predisposition to this form of breast cancer. However HLA typing for A, B and DRW antigens revealed no specific RPBC-associated HLA antigen. Our studies clearly demonstrate that RPBC, or PEV, is not a reflection of immunodeficiency.
Breast Neoplasms↗
Genetically confirmed clinical Huntington's disease with no observable cell loss.
Huntington's disease (HD) results from neurodegeneration of the neostriatum. The mutation on chromosome 4 is an expansion in a triplet repeat (CAG)(n) located within the IT15 gene. Only six patients have been reported with clinical features of HD in association with limited neuropathology. Of these, only one has had the diagnosis confirmed by genetic (DNA) testing. We describe a patient with the clinical phenotype and genetically confirmed HD but unexpected limited neuropathology. The patient was seen because of aggressive behaviour and memory problems of two years duration. The differential diagnosis included HD although there was no family history. DNA testing was positive for the HD mutation. Clinical follow up three months later confirmed classic features of HD. Progression of the disease was rapid with death three years later. Neuropathology revealed a largely intact neostriatum with bilateral ischaemic damage and cell loss in the external globus pallidus. Such pathology alone could explain the clinical features of HD. This is only the second report of genetically confirmed clinically manifest HD with little evidence of HD neuropathology. There are several unusual features which could not have been predicted by the clinical picture, in particular the progressive course of bilateral ischaemic changes restricted to the external globus pallidus. The potential to miss other HD cases at post-mortem examination, and the implications of this for family members, are discussed.
Aggression↗
Genetic analysis of chemoresistance in primary murine lymphomas.
Understanding the basis of chemoresistance is a principal goal of molecular oncology. We have exploited a murine lymphoma model and retroviral gene transfer to rapidly generate a series of spontaneous tumors differing only in a gene of interest, and subsequently studied the impact of the test gene on the treatment sensitivity of tumors at their natural site. We demonstrate that the Bcl-2 oncoprotein produces multi-drug resistance when assessed in primary lymphomas in vivo. In contrast, this effect was dramatically reduced when the primary lymphomas were subjected to long-term culture, and completely missed in the standard clonogenic survival assay. This model highlights the importance of physiological test systems to address the complexity of clinical drug resistance and provides a novel strategy to evaluate compounds targeting specific genetic lesions.
Adaptation, Biological↗
Susceptibility testing. Phenotypic and genotypic tests for bacteria and mycobacteria.
Genotypic-based methods hold promise for the rapid and accurate detection or confirmation of antimicrobial resistance; however, phenotypic methods will continue to have an advantage when resistance to the same antimicrobial agent may be caused by several different mechanisms. The diversity of genetic mechanisms may exceed the capabilities of current molecular technology. Genotypic assays have the ability to detect resistance but not susceptibility. Although resutls can be obtained rapidly, many molecular methods are labor-intensive, expensive, and lack standardization. Clinical studies will be required to validate the genotypic approach to detection of antimicrobial resistance. Molecular assays are also at risk for false-positive results because of contamination of specimens by other specimens that carry the DNA targeted for the assay, or carryover of amplified target DNA (amplicons) from a previous PCR assay during sample preparation. Detection of certain genetic resistance loci in clinical specimens must be interpreted with caution, because organisms in normal flora may also harbor the same loci. All these factors must be taken into consideration when introducing a genotypic method in the clinical laboratory. Other considerations include cost, turnaround time, and assay performance. It must be emphasized that the bedside assessment of the patient should always be considered in addition to the results of antimicrobial susceptibility tests (whether phenotypic or genotypic) so that the best outcome is assured for the patient.
Anti-Bacterial Agents↗
High-throughput screening for GJB2 mutations--its clinical application to genetic testing in prelingual deafness screening for GJB2 mutations.
OBJECTIVES: Mutations in connexin26 (GJB2) are one of the most frequent causes of prelingual hearing impairment. Several different types of one-base deletions in exon2 were the most common type of GJB2 mutation regardless of ethnicity, including 35delG in American-European populations, 235delC in Japanese population and 167delT in Ashkenazi Jewish population. Various types of one-base substitutions were also considered to be causative mutations of GJB2 associated hearing impairment. This article describes a rapid and high-throughput screening procedure for the detection of one-base deletion/substitution in GJB2 with less invasive sampling procedure in the implication for the clinical application. METHODS: 53 hearing-impaired children and 50 healthy controls were admitted to take part in this study program. DNA samples obtained from buccal swab were used to amplify the exon2 of GJB2, and single run with an automated sequencer was used to identify the one-base deletion. Single-base substitutions were also screened by primer-extension procedure with dye terminators. The presence of both types of mutations was confirmed by direct sequence of the GJB2 exon2. RESULTS: Two of 50 controls (4%) included one-base deletion in GJB2 as heterozygote. 14 of 53 hearing impaired cases (26.4%) contained deletion in GJB2 either as homozygote (five cases) or heterozygote (nine cases) form. Sequencing analysis of whole exon2 of GJB2 identified all these deletions as 235delC. Primer-extension analysis revealed additional mutations with single base substitutions in three cases with compound heterozygote with 235delC. CONCLUSIONS: Rapid screening procedure of GJB2 can be potentially useful for the identification of prelingual deafness.
Chromosome Deletion↗
Rapid, random evolution of the genetic structure of replicating tobacco mosaic virus populations.
The effects of temperature and type of host on the evolution of variants in replicating populations of tobacco mosaic virus (TMV) originating from an in vitro RNA transcript of a cDNA clone were studied. The phenotypic markers measured were temperature-sensitive (ts) replication and induction of necrotic local lesions (nl) on Nicotiana sylvestris. The proportions of ts variants were about the same under all conditions tested, but changes in temperature or host had strong effects on proportions of nl variants. During replication at elevated temperatures in tobacco or tomato, percentages of nl variants were reduced rapidly to about one tenth their initial values. In contrast, when virus replicated at 25 degrees in tobacco, little change in the average proportion of nl variants was observed, and in tomato, only a slight average reduction occurred. Virus replicating at 25 degrees in Solanum nigrum or Petunia hybrida suffered effects similar to those observed in tobacco or tomato, respectively. However, at 25 degrees in Physalis floridana, there was strong selection against nl variants, which eventually reduced them to essentially undetectable levels. In all hosts tested at 25 degrees, surprisingly large, apparently random changes in the proportion of nl variants occurred in individual plants. These experiments showed that virus populations can evolve rapidly on a time scale of days, and that an element of randomness is an important component in the initiation of change.
Biological Evolution↗
Genetic basis of lethal junctional epidermolysis bullosa in an affected fetus: implications for prenatal diagnosis in one family.
Fetal skin biopsy at 20 weeks' gestation in a woman at risk for a child with the lethal skin-blistering disorder junctional epidermolysis bullosa (Herlitz) confirmed an affected fetus. Genomic DNA from the aborted fetus was examined for mutations in laminin 5, a macromolecule involved in adhesion at the dermal-epidermal junction, and a candidate protein in this condition. Polymerase chain reaction (PCR) amplification of exon 10 and parts of the flanking introns of the gene encoding the beta 3 chain of laminin 5 (LAMB3) and subsequent analysis by agarose gel electrophoresis showed a more slowly migrating band in the affected fetus compared with the normal control. Nucleotide sequencing of the abnormal PCR product revealed a homozygous 77 bp duplication within the exon, resulting in a premature termination codon 250 bp downstream from the 3' end of the duplication. Maternal DNA was heterozygous for the mutant and wild-type alleles. These findings illustrate the genetic basis of the skin disease in this case and also offer the prospects of a simple, rapid, and reliable first-trimester DNA-based prenatal, or even preimplantation, diagnostic test for future pregnancies in this family.
Adult↗
mRNA for cardiac calcium channel is expressed during development of skeletal muscle.
During the early development of skeletal muscle, cardiac isotypes of several contractile proteins are known to be transiently expressed. We report here that skeletal muscle developing in vivo, as well as primary cultures derived from skeletal muscle, express mRNA encoding the cardiac dihydropyridine-sensitive calcium channel. The mRNA is detectable at high concentration at the earliest stage tested in vivo and diminishes rapidly in concentration as myofibers mature. The concentration of the cardiac calcium channel mRNA also diminishes during the in vivo development of skeletal muscle in a genetically paralyzed mouse (mdg), indicating that muscle contractile activity is not necessary for the down-regulation. In contrast, mRNA for the skeletal muscle-specific calcium channel accumulates gradually in developing skeletal muscle. A similar temporal pattern of expression is also seen in primary cultures of skeletal myotubes. These results raise the question of whether the cardiac calcium channel may be functionally important during the early development of skeletal myofibers.
3T3 Cells↗
An evaluation of the possible carcinogenicity of bisphenol A to humans.
Bisphenol A (BPA) is a monomer component of polycarbonate plastics and epoxy resins. These resins are used in numerous consumer products, including food-contact plastics. There has been considerable scientific debate about the relevance to humans of reported estrogenic actions of BPA. Much less attention has been focused on the carcinogenic potential of BPA. The carcinogenic potential of BPA was assessed through a review of metabolic data, genetic toxicity studies, long-term toxicity/carcinogenicity studies, and estimates of consumer exposure. Following a weight-of-evidence approach as recommended by IARC and U.S. EPA, it was concluded that BPA is not likely to be carcinogenic to humans. The bases for this conclusion included: (a) the results of an NTP study which provided no substantive evidence to indicate that BPA is carcinogenic to rodents; (b) the lack of activity of BPA, at noncytotoxic concentrations, in standard in vitro genetic toxicity tests; (c) the lack of genotoxic activity of BPA in a GLP-compliant in vivo mouse micronucleus assay; and (d) the results of metabolism studies showing BPA is rapidly glucuronidated without evidence of formation of potentially reactive intermediates, except possibly at high doses that could saturate detoxication pathways. In addition, exposure assessment reveals that current use of BPA would result in only a trivial human exposure.
Animals↗
Critically unwell infants and children with mitochondrial disorders diagnosed by ultrarapid genomic sequencing.
PURPOSE: To characterize the diagnostic and clinical outcomes of a cohort of critically ill infants and children with suspected mitochondrial disorders (MD) undergoing ultrarapid genomic testing as part of a national program. METHODS: Ultrarapid genomic sequencing was performed in 454 families (genome sequencing: n = 290, exome sequencing +/- mitochondrial DNA sequencing: n = 164). In 91 individuals, MD was considered, prompting analysis using an MD virtual gene panel. These individuals were reviewed retrospectively and scored according to modified Nijmegen Mitochondrial Disease Criteria. RESULTS: A diagnosis was achieved in 47% (43/91) of individuals, 40% (17/43) of whom had an MD. Seven additional individuals in whom an MD was not suspected were diagnosed with an MD after broader analysis. Gene-agnostic analysis led to the discovery of 2 novel disease genes, with pathogenicity validated through targeted functional studies (CRLS1 and MRPL39). Functional studies enabled diagnosis in another 4 individuals. Of the 24 individuals ultimately diagnosed with an MD, 79% had a change in management, which included 53% whose care was redirected to palliation. CONCLUSION: Ultrarapid genetic diagnosis of MD in acutely unwell infants and children is critical for guiding decisions about the need for additional investigations and clinical management.
Humans↗
Rational identification of new antibacterial drug targets that are essential for viability using a genomics-based approach.
In the last two decades, the search for completely novel antibacterial agents has acquired a new sense of urgency due to the remarkable rise of antibiotic resistance among key bacterial pathogens. More recently, the advent of bacterial genomics has provided investigators with the data and bioinformatic tools to rationally identify novel antibacterial targets and the genome-scaled methodologies to validate them. Only 6 years have elapsed since the publication of the first complete bacterial genome sequence, but more than 50 complete microbial genome sequences are now available. This review will discuss the advantages and limitations of the existing bacterial genome dataset for the rational identification of novel antibacterial targets. Since the ability to rapidly identify essential genes where loss of function is coincident with loss of viability is the most important task of genomics-based target validation, essentiality testing methodologies (in which molecular genetic techniques are used to determine whether or not a gene product is required for viability of the parent cell) will be surveyed and their amenability to genome-scaled analysis assessed. Finally, we will discuss the impact of bacterial genomics to date on the development of novel and effective antibiotics.
Anti-Infective Agents↗
Antiviral drug resistance in human cytomegalovirus.
Drug-resistant cytomegalovirus (CMV) should be considered when viral shedding persists after several weeks of therapy. The problem is most likely to arise in the setting of a severely immunosuppressed host with continuing or relapsing disease. Not all treatment failure can be attributed to drug resistance. The testing of CMV isolates for drug resistance in cell culture is time-consuming and labor-intensive, but recent advances in understanding of the genetics of resistance have resulted in rapid genotypic assays for specific mutations in the viral UL97 phosphotransferase or UL54 DNA polymerase genes that can predict resistance and cross-resistance to specific drugs. This information may help in the selection of alternative therapy.
Antiviral Agents↗