Screening for carriers of cystic fibrosis--a general practitioner's perspective.

The identification of the gene for cystic fibrosis has led to the possibility of population based screening for carriers of cystic fibrosis to identify couples at risk of having an affected child. Pilot studies have shown that screening is feasible and does not cause untoward anxiety, though the uptake of testing varies considerably with the setting and method of invitation. Screening offered at times when individuals (and health professionals) perceive it as directly relevant will probably gradually become established in the United Kingdom. This review examines the role of general practice in genetic carrier screening as exemplified by cystic fibrosis. General practice has a pivotal role from the beginning in providing individuals and couples with information, facilitating testing of patients'' relatives and of carriers identified by screening elsewhere (such as antenatal clinics), and offering testing in the context of reproduction. Screening for the cystic fibrosis gene will probably be followed by other genetic screening programmes.     

A new United States Pharmacopeia (USP) Chapter 1046: cell and gene therapy products

The United States Pharmacopeia (USP) has published the first draft of a new information chapter on cell and gene therapy products in January, 2000. The chapter discusses the manufacturing and testing cell and gene therapy products. It is intended for people working in the field as well as for pharmacists and clinicians who would like more information on this subject. This article outlines the background of USP and explains how it became involved with cell and gene therapy and what an information chapter is. It details the subjects covered by the chapter and the philosophy behind the chapter. This draft will be revised based on comments received from the public and then published as a revision subject to further comments before it becomes an official chapter in a supplement to USP 24-NF 19.     

Context, ethics and pharmacogenetics

Most of the literature on pharmacogenetics assumes that the main problems in implementing the technology will be institutional ones (due to funding or regulation) and that although it involves genetic testing, the ethical issues involved in pharmacogenetics are different from, even less than, 'traditional' genetic testing. Very little attention has been paid to how clinicians will accept this technology, their attitudes towards it and how it will affect clinical practice. This paper presents results from interviews with clinicians who are beginning to use pharmacogenetics and explores how they view the ethics of pharmacogenetic testing, its use to exclude some patients from treatment, and how this kind of testing fits into broader debates around genetics. In particular this paper examines the attitudes of breast cancer and Alzheimer's disease specialists. The results of these interviews will be compared with the picture of pharmacogenetics painted in the published literature, as a way of rooting this somewhat speculative writing in clinical practice.     

Considerations in the U.S. Environmental Protection Agency's testing approach for mutagenicity.

OPP: This paper provides the rationale and support for the decisions the OPP will make in requiring and reviewing mutagenicity information. The regulatory requirement for mutagenicity testing to support a pesticide registration is found in the 40 CFR Part 158. The guidance as to the specific mutagenicity testing to be performed is found in the OPP's Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals (referred to as the Subdivision F guideline). A revised Subdivision F guideline has been presented that becomes the current guidance for submitters of mutagenicity data to the OPP. The decision to revise the guideline was the result of close examination of the version published in 1982 and the desire to update the guidance based on developments since then and current state-of-the-science. After undergoing Agency and public scrutiny, the revised guideline is to be published in 1991. The revised guideline consists of an initial battery of tests (the Salmonella assay, an in vitro mammalian gene mutation assay and an in vivo cytogenetics assay which may be either a bone marrow assay for chromosomal aberrations or for micronuclei formation) that should provide an adequate initial assessment of the potential mutagenicity of a chemical. Follow-up testing to clarify results from the initial testing may be necessary. After this information as well as all other relevant information is obtained, a weight-of-evidence decision will be made about the possible mutagenicity concern a chemical may present. Testing to pursue qualitative and/or quantitative evidence for assessing heritable risk in relation to human beings will then be considered if a mutagenicity concern exists. This testing may range from tests for evidence of gonadal exposure to dominant lethal testing to quantitative tests such as the specific locus and heritable translocation assays. The mutagenicity assessment will be performed in accordance with the Agency's Mutagenicity Risk Assessment Guidelines. The mutagenicity data would also be used in the weight-of-evidence consideration for the potential carcinogenicity of a chemical in accordance with the Agency's Carcinogen Risk Assessment Guidelines. In instances where there are triggers for carcinogenicity testing, mutagenicity data may be used as one of the triggers after a consideration of available information. It is felt that the revised Subdivision F guideline will provide appropriate, and more specific, guidance concerning the OPP approach to mutagenicity testing for the registration of a pesticide. It also provides a clearer understanding of how the OPP will proceed with its evaluation and decision making concerning the potential heritable effects of a test chemical.(ABSTRACT TRUNCATED AT 400 WORDS)     

A database for the analysis of immunity genes in Drosophila: PADMA database

While microarray experiments generate voluminous data, discerning trends that support an existing or alternative paradigm is challenging. To synergize hypothesis building and testing, we designed the Pathogen Associated Drosophila MicroArray (PADMA) database for easy retrieval and comparison of microarray results from immunity-related experiments (www.padmadatabase.org). PADMA also allows biologists to upload their microarray-results and compare it with datasets housed within PADMA. We tested PADMA using a preliminary dataset from Ganaspis xanthopoda-infected fly larvae, and uncovered unexpected trends in gene expression, reshaping our hypothesis. Thus, the PADMA database will be a useful resource to fly researchers to evaluate, revise, and refine hypotheses.     

A database for the analysis of immunity genes in Drosophila

While microarray experiments generate voluminous data, discerning trends that support an existing or alternative paradigm is challenging. To synergize hypothesis building and testing, we designed the Pathogen Associated Drosophila MicroArray (PADMA) database for easy retrieval and comparison of microarray results from immunity-related experiments (www.padmadatabase.org). PADMA also allows biologists to upload their microarray-results and compare it with datasets housed within PADMA. We tested PADMA using a preliminary dataset from Ganaspis xanthopoda-infected fly larvae, and uncovered unexpected trends in gene expression, reshaping our hypothesis. Thus, the PADMA database will be a useful resource to fly researchers to evaluate, revise, and refine hypotheses.     

Differences between African Americans and Whites in their attitudes toward genetic testing for Alzheimer's disease

The possibility of predictive genetic testing for Alzheimer's disease (AD) has prompted examination of public attitudes toward this controversial new health-care option. This is the first study to examine differences between Whites and African Americans with regard to: (1) interest in pursuing genetic testing for AD, (2) reasons for pursuing testing, (3) anticipated consequences of testing, and (4) beliefs about testing. We surveyed a convenience sample of 452 adults (61% white; 39% African American; 78% female; mean age = 47 years; 33% with family history of AD). Both racial groups indicated general interest in predictive genetic testing for AD, viewed it as having many potential benefits, and believed it should be offered with few restrictions. However, in comparison to whites, African Americans showed less interest in testing (p < 0.01), endorsed fewer reasons for pursuing it (p < 0.01), and anticipated fewer negative consequences from a positive test result (p < 0.001). These preliminary findings show important distinctions between whites and African Americans in their attitudes toward genetic testing for AD. These differences may have implications for how different racial and ethnic groups will respond to genetic testing programs and how such services should be designed. Future research in real-life testing situations with more representative samples will be necessary to confirm these racial and cultural differences in perceptions of genetic testing.     

Attitudes toward genetic testing among the general population and relatives of patients with a severe genetic disease: a survey from Finland.

In the present study we explore the attitudes of the Finnish population toward genetic testing by conducting a questionnaire study of a stratified sample of the population as well as of family members of patients with a severe hereditary disease, aspartylglucosaminuria (AGU). The questionnaire evaluated attitudes toward gene tests in general and also respondents' preparedness to undergo gene tests for predictive testing, carrier detection, prenatal diagnosis, and selective abortion, in theoretical situations. The results of the study indicate that both the Finnish population in general and family members of AGU patients have a favorable attitude toward genetic testing. However, a commonly expressed reason against testing was that test results might lead to discrimination in employment or insurance policies. Based on the responses, we predict that future genetic testing programs will most probably be met with a high acceptance rate by the Finnish population.     

THE CASE FOR CONTINUING PRIMARY CERVICAL SCREENING WITH CYTOLOGY RATHER THAN HPV TESTING

Infection by certain types of human papilloma virus is now recognised as the main factor for cervical cancer. About 50% of cancers contain HPV 16 and over 95% contain one or more of the high‐risk types (16, 18, 31, 33, 35, 45, 51, 52, 56, 58). Several studies have shown that HPV testing is considerably more sensitive than conventional cytology. However it is not as specific, especially for younger women. These studies will be reviewed and overall comparisons with cytology will be made. Data from the 11 000 women HART study will be presented which suggest that for women aged 30 or over, a rational strategy would be to use HPV testing as the sole primary screening test, reserving cytology for the triage of HPV positive women. The results of this study indicate that HPV positive cytology negative women can be safely managed by repeat testing at one year. This reverses the current role of HPV testing as a triage test for women with borderline (ASCUS) cytology. Use of liquid‐based collection media will facilitate the transition to this new approach to screening. A detailed algorithm will be presented.     

THE CASE FOR CONTINUING PRIMARY CERVICAL SCREENING WITH CYTOLOGY RATHER THAN HPV TESTING

Infection by certain types of human papilloma virus is now recognised as the main factor for cervical cancer. About 50% of cancers contain HPV 16 and over 95% contain one or more of the high-risk types (16, 18, 31, 33, 35, 45, 51, 52, 56, 58). Several studies have shown that HPV testing is considerably more sensitive than conventional cytology. However it is not as specific, especially for younger women. These studies will be reviewed and overall comparisons with cytology will be made. Data from the 11 000 women HART study will be presented which suggest that for women aged 30 or over, a rational strategy would be to use HPV testing as the sole primary screening test, reserving cytology for the triage of HPV positive women. The results of this study indicate that HPV positive cytology negative women can be safely managed by repeat testing at one year. This reverses the current role of HPV testing as a triage test for women with borderline (ASCUS) cytology. Use of liquid-based collection media will facilitate the transition to this new approach to screening. A detailed algorithm will be presented.     

Mass spectrometry based approach for identification and characterisation of fluorescent proteins from marine organisms

We present here a new analytical strategy for identification and characterisation of fluorescent proteins from marine organisms. By applying basic proteomics tools it is possible to screen large sample collections for fluorescent proteins of desired characteristics prior to gene cloning. Our methodology which includes isolation, spectral characterisation, stability testing, gel-based separation and mass spectrometric identification was optimised on samples collected during the Danish Galathea 3 expedition. Four corals of the Fungia, Sarcophyton and Acropora species emitting green fluorescence were tested. Each of the fluorescent extracts behaves differently under denaturing conditions but complete fluorescence loss was not observed. Optimised electrophoretic conditions yielded effective separation of active fluorescent proteins in both 1DE and 2DE. Mass spectrometric analysis of the proteins in the fluorescent spots excised directly from unstained 2DE gels provides sequence information that might be sufficient to design degenerate primers for gene cloning. Identified fluorescent proteins are in agreement with the coral species determined by visual examination of the samples. The presented methodology is a viable alternative to direct gene cloning for the discovery of novel fluorescent proteins and will be further validated on other samples collected during the Galathea 3 expedition.     

Exploiting the complete yeast genome sequence

The completion of the genome sequence of the budding yeast Saccharomyces cerevisiae marks the dawn of an exciting new era in eukaryotic biology that will bring with it a new understanding of yeast, other model organisms, and human beings. This body of sequence data benefits yeast researchers by obviating the need for piecemeal sequencing of genes, and allows researchers working with other organisms to tap into experimental advantages inherent in the yeast system and learn from functionally characterized yeast gene products which are their proteins of interest. In addition, the yeast post-genome sequence era is serving as a testing ground for powerful new technologies, and proven experimental approaches are being applied for the first time in a comprehensive fashion on a complete eukaryotic gene repertoire.     

Molecular diagnostics: hurdles for clinical implementation

In the coming years, molecular diagnostics will continue to be of critical importance to public health worldwide. It will facilitate the detection and characterization of disease, as well as monitoring of the drug response, and will assist in the identification of genetic modifiers and disease susceptibility. A wide range of molecular-based tests is available to assess DNA variation and changes in gene expression. However, there are major hurdles to overcome before the implementation of these tests in clinical laboratories, such as which test to employ, the choice of technology and equipment, and issues such as cost-effectiveness, accuracy, reproducibility, personnel training, reimbursement by third-party payers and intellectual property. At present, PCR-based testing predominates; however, alternative technologies aimed at reducing genome complexity without PCR are anticipated to gain momentum in the coming years. Furthermore, development of integrated chip devices ("lab-on-a-chip") should allow point-of-care testing and facilitate genetic readouts from single cells and molecules. Together with proteomic-based testing, these advances will improve molecular diagnostic testing and will present additional challenges for implementing such testing in health care settings.     

The Salmonella typhimurium/mammalian microsomal assay. A report of the U.S. Environmental Protection Agency Gene-Tox Program

The Salmonella assay has been in use for almost 15 years and can be defined as a routine test for mutagenicity and for predicting potential carcinogenicity. It detects the majority of animal carcinogens and consequently plays an important role in safety assessment. The test is also routinely used as the frontline screen for environmental samples (complex mixtures) isolated from air, water and food. This role will continue to remain an area of growth as or because sample volumes associated with these testing areas are generally very limited and more extensive testing is generally impossible. While this test, like all others, has some limitations, it is recommended that it be regularly included in all genetic testing batteries.     

1种用于检测抑制基因功能的酵母株系的建立

酵母被广泛用于分子生物学中基因功能的检测。为扩大酵母株系UCC419在抑制基因活性检测方面的应用,本研究通过向UCC419株系中导入用特殊引物扩增出的包含标记基因TRP1的PCR片段,利用同源重组将UCC419中的筛选标记基因LEU2敲除,并同时插入TRP1,新建立的株系命名为UCC419m（m：modi-fied）。UCC419m为TRP1筛选、leu2突变型菌株,其它基因型均同UCC419。给UCC419m中转入携带LEU2的质粒pDEST32检测是否能恢复其表现型,同时转入不携带LEU2的质粒pDEST22作为阴性对照,将转化子在不含LEU2与URA3的培养基中培养,结果显示,携带LEU2质粒pDEST32的转化子能够在LEU2与URA3缺陷型培养基上正常生长,而不携带LEU2质粒pDEST22的转化子不能生长。本研究结果表明,成功建立了一种适用于基于Invitrogen载体的抑制基因活性检测或从文库中筛选抑制基因的酵母菌株。     

在杂交作物分子育种中利用普通核雄性不育的几个可能途径

由一对隐性基因控制的普通核雄性不育性遗传方式能够满足对植物最佳雄性不育系选育的要求,是水稻等作物杂种优势利用的极好遗传工具。如果能解决其不育系繁殖问题,将优于现有的其他杂种优势利用方式。克隆出普通核雄性不育性的可育基因,通过叶绿体转化,将核雄性不育性可育基因向普通核雄性不育株细胞质转移,创造普通核雄性不育株的保持系;通过种子成熟后表达的启动子;和以位点特异性重组技术为基础的基因开关以及化学诱导启动子的利用,都可能繁殖出100％不育株率的普通核雄性不育系,创造普通核雄性不育性利用的新途径,对植物杂种优势利用产业有十分重要的意义。     

Construction of a cDNA library of Aphis gossypii Glover for use in RNAi

Aphis gossypii Glover is an important insect pest that functions as a viral vector and mediates approximately 45 different viral diseases. As part of a strategy for control of A. gossypii, we investigated the functions of genes using RNAi. To this end, a cDNA library was constructed for various genes and for selecting appropriate targets for RNAi mediated silencing. The cDNA library was constructed using the Gateway cloning system with site‐specific recombination of bacteriophage λ. It was used to carry out single step cloning of A. gossypii cDNAs. As a result, a cDNA library with a titer of 8.4 × 10⁶ was constructed. Since the sequences in this library carry att sites, they can be cloned into various binary vectors. This library will be of value for various studies. For later screening of selected genes, it is planned to clone the library into virus‐induced gene silencing (VIGS) vectors, which makes it possible to analyze gene function and allow subsequent transfection of plants. Such transfection experiments will allow testing of RNAi‐induced insecticidal activity or repellent activity to A. gossypii, and result in the identification of target genes. It is also expected that the constructed cDNA library will be useful for analysis of gene functions in A. gossypii.     

在杂交作物分子育种中利用普通核雄性不育的几个可能途径

由一对隐性基因控制的普通核雄性不育性遗传方式能够满足对植物最佳雄性不育系选育的要求,是水稻等作物杂种优势利用的极好遗传工具。如果能解决其不育系繁殖问题,将优于现有的其他杂种优势利用方式。克隆出普通核雄性不育性的可育基因,通过叶绿体转化,将核雄性不育性可育基因向普通核雄性不育株细胞质转移,创造普通核雄性不育株的保持系;通过种子成熟后表达的启动子;和以位点特异性重组技术为基础的基因开关以及化学诱导启动子的利用,都可能繁殖出100%不育株率的普通核雄性不育系,创造普通核雄性不育性利用的新途径,对植物杂种优势利用产业有十分重要的意义。