Empowering patients with high myopia: The significance of education.

PURPOSE: To investigate the status of patient education among highly myopic individuals focusing on the presence, sources, content, timing of the education and impact on patients. METHODS: Self-reported data were collected through an online 13-item questionnaire consisting of open and multiple-choice questions. The questionnaire was sent to 250 highly myopic members of a patient organization in the Netherlands, of whom 128 (51%) responded. RESULTS: At least one acute event had occurred in 66% (84/128) of participants at the time of the questionnaire. Among all participants, 25% (32/128) had not received patient education regarding alarm symptoms for any of these events. Among those who had been informed, the ophthalmologist was the most frequent (57%, 73/128) source of information. Participants who visited the ophthalmologist annually were more frequently informed than participants without annual visits (53%, 26/49 versus 26%, 9/35, p = 0.002). Those not informed were more likely to have a more than 3 days patient delay (92%, 12/13). Doctors delay was also present; 26% (22/84) of the participants with alarm symptoms had to wait 2 or more days before the first appointment. Long-term consequences of myopia had been discussed with 102 participants (80%, 102/128), again with the ophthalmologist as the most frequent source (59%, 76/128). PERSPECTIVES: Many myopic individuals have not been educated about their increased risk of acute events, which can result in patient delay and serious consequences with respect to visual prognosis. These findings underscore the critical importance of integrating patient education across the entire ophthalmic care chain for myopia.

Analysis of human serum by liquid chromatography-mass spectrometry: improved sample preparation and data analysis.

Discovery of biomarkers is a fast developing field in proteomics research. Liquid chromatography coupled on line to mass spectrometry (LC-MS) has become a powerful method for the sensitive detection, quantification and identification of proteins and peptides in biological fluids like serum. However, the presence of highly abundant proteins often masks those of lower abundance and thus generally prevents their detection and identification in proteomics studies. To perform future comparative analyses of samples from a serum bank of cervical cancer patients in a longitudinal and cross-sectional manner, methodology based on the depletion of high-abundance proteins followed by tryptic digestion and LC-MS has been developed. Two sample preparation methods were tested in terms of their efficiency to deplete high-abundance serum proteins and how they affect the repeatability of the LC-MS data sets. The first method comprised depletion of human serum albumin (HSA) on a dye ligand chromatographic and immunoglobulin G (IgG) on an immobilized Protein A support followed by tryptic digestion, fractionation by cation-exchange chromatography, trapping on a C18 column and reversed-phase LC-MS. The second method included depletion of the six most abundant serum proteins based on multiple immunoaffinity chromatography followed by tryptic digestion, trapping on a C18 column and reversed-phase LC-MS. Repeatability of the overall procedures was evaluated in terms of retention time and peak area for a selected number of endogenous peptides showing that the second method, besides being less time consuming, gave more repeatable results (retention time: <0.1% RSD; peak area: <30% RSD). Application of an LC-MS component detection algorithm followed by principal component analysis (PCA) enabled discrimination of serum samples that were spiked with horse heart cytochrome C from non-spiked serum and the detection of a concentration trend, which correlated to the amount of spiked horse heart cytochrome C to a level of 5 pmol cytochrome C in 2 microl original serum.

Genetic association studies in complex disease: disentangling additional predisposing loci from associated neutral loci using a constrained - permutation approach.

In the process of genetically mapping a complex disease, the question may arise whether a certain polymorphism is the only causal variant in a region. A number of methods can answer this question, but unfortunately these methods are optimal for bi-allelic loci only. We wanted to develop a method that is more suited for multi-allelic loci, such as microsatellite markers. We propose the Additional Disease Loci Test (ADLT): the alleles at an additional locus are permuted within the subsample of haplotypes that have identical alleles at the predisposing locus. The hypothesis being tested is, whether the predisposing locus is the sole factor predisposing to the trait that is in LD with the additional locus under study. We applied ADLT to simulated datasets and a published dataset on Type 1 Diabetes, genotyped for microsatellite markers in the HLA-region. The method showed the expected number of false-positive results in the absence of additional loci, but proved to be more powerful than existing methods in the presence of additional disease loci. ADLT was especially superior in datasets with less LD or with multiple predisposing alleles. We conclude that the ADLT can be useful in identifying additional disease loci.

Genetical genomics: the added value from segregation.

The recent successes of genome-wide expression profiling in biology tend to overlook the power of genetics. We here propose a merger of genomics and genetics into 'genetical genomics'. This involves expression profiling and marker-based fingerprinting of each individual of a segregating population, and exploits all the statistical tools used in the analysis of quantitative trait loci. Genetical genomics will combine the power of two different worlds in a way that is likely to become instrumental in the further unravelling of metabolic, regulatory and developmental pathways.

Identification of the SAAT gene involved in strawberry flavor biogenesis by use of DNA microarrays.

Fruit flavor is a result of a complex mixture of numerous compounds. The formation of these compounds is closely correlated with the metabolic changes occurring during fruit maturation. Here, we describe the use of DNA microarrays and appropriate statistical analyses to dissect a complex developmental process. In doing so, we have identified a novel strawberry alcohol acyltransferase (SAAT) gene that plays a crucial role in flavor biogenesis in ripening fruit. Volatile esters are quantitatively and qualitatively the most important compounds providing fruity odors. Biochemical evidence for involvement of the SAAT gene in formation of fruity esters is provided by characterizing the recombinant protein expressed in Escherichia coli. The SAAT enzyme showed maximum activity with aliphatic medium-chain alcohols, whose corresponding esters are major components of strawberry volatiles. The enzyme was capable of utilizing short- and medium-chain, branched, and aromatic acyl-CoA molecules as cosubstrates. The results suggest that the formation of volatile esters in fruit is subject to the availability of acyl-CoA molecules and alcohol substrates and is dictated by the temporal expression pattern of the SAAT gene(s) and substrate specificity of the SAAT enzyme(s).

High frequency of interactions between lung cancer susceptibility genes in the mouse: mapping of Sluc5 to Sluc14.

Although several genes that cause monogenic familial cancer syndromes have been identified, susceptibility to sporadic cancer remains unresolved. Animal experiments have demonstrated multigenic control of tumor susceptibility. Recently, we described four mouse lung cancer susceptibility (Sluc) loci, the main effects of which are masked by their mutual interactions. Because such interactions can considerably affect the strategies for identification of cancer susceptibility genes in humans, it is necessary to establish whether they are common or rare. Here, we report the mapping of 10 additional Sluc loci and show that 13 of the 14 Sluc loci are involved in one or more interactions, demonstrating that interactions of tumor susceptibility genes are frequent and that they probably form complex networks.

Genetics of refractoriness to Plasmodium falciparum in the mosquito Anopheles stephensi.

We previously selected a line of the malaria vector mosquito Anopheles stephensi refractory (resistant) to the human malaria parasite Plasmodium falciparum, using in vitro infections with P. falciparum gametocytes. This report presents data on the genetic background of refractoriness. The results of F1-crosses and backcrosses show that refractoriness to P. falciparum in our A. stephensi line is autosomal and semi-dominant to susceptibility. The expression of refractoriness is apparently affected by a cytoplasmic factor. Interpretation of data from the crosses by quantitative trait locus analysis shows that one gene or two unlinked interacting autosomal genes, or groups of closely linked genes, are involved.

A mixture model approach to the mapping of quantitative trait loci in complex populations with an application to multiple cattle families.

A mixture model approach is presented for the mapping of one or more quantitative trait loci (QTLs) in complex populations. In order to exploit the full power of complete linkage maps the simultaneous likelihood of phenotype and a multilocus (all markers and putative QTLs) genotype is computed. Maximum likelihood estimation in our mixture models is implemented via an Expectation-Maximization algorithm: exact, stochastic or Monte Carlo EM by using a simple and flexible Gibbs sampler. Parameters include allele frequencies of markers and QTLs, discrete or normal effects of biallelic or multiallelic QTLs, and homogeneous or heterogeneous residual variances. As an illustration a dairy cattle data set consisting of twenty half-sib families has been reanalyzed. We discuss the potential which our and other approaches have for realistic multiple-QTL analyses in complex populations.

Dissection of a synthesized quantitative trait to characterize transgene interactions.

Six transgenic tobacco lines, each homozygous for the beta-glucuronidase (GUS) gene at a different locus, and wild type were selfed and intercrossed to evaluate GUS activity in all possible hemizygous, homozygous and dihybrid combinations of GUS alleles. The transgenic lines are characterized by their GUS activity (two low, three intermediate, one high), T-DNA complexity (four single-copy, two more complex single-locus) and the presence of the chicken lysozyme matrix-associated region (MAR) around the full T-DNA (two lines). Gene action and interaction was analyzed by weighted linear regression with parameters for additivity, dominance and epistasis. The analysis showed that each of the four single-copy lines acted fully additively. In contrast, the two complex single-locus lines showed classical single-locus overdominance and were epistatic dominant over all other GUS alleles. The latter is manifested in severe suppression of GUS activity in dihybrid lines, irrespective of the presence of MAR elements around the GUS gene. Such elements apparently do not protect against epistatic dominance. The quantitative data suggested that the epistatic dominance and overdominance are based on the same molecular mechanism. Our approach of a genetic analysis of quantitative variation in well-characterized transgenic lines provides a powerful tool to gain insight into complex plant traits.

QTL analysis of seed dormancy in Arabidopsis using recombinant inbred lines and MQM mapping.

The genetic differences for seed germination between two commonly used Arabidopsis thaliana ecotypes Ler and Col, both showing a low level of seed dormancy, were investigated. The analysis was performed with 98 recombinant inbred lines (RILs) derived from the cross between the two ecotypes, and these lines had previously been analysed for molecular marker composition by Lister and Dean (Norwich, UK). The analysis of germination was performed on seeds grown in three different maternal environments and each seed batch was tested in three different germination environments: in light, in darkness and in the presence of the gibberellin inhibitor paclobutrazol. Fourteen loci were identified using the multiple-QTL-model (MQM) procedure for mapping quantitative trait loci. At nine loci no significant interaction between the detection of the locus and environmental factors could be detected. However, three other distinct loci controlling the germination behaviour in the presence of the gibberellin inhibitor paclobutrazol had a much lower or no effect when germination was tested in water either in light or darkness. Two other loci affecting germination in darkness and/or light had practically no effect on germination in the presence of paclobutrazol.

Complex interactions of new quantitative trait loci, Sluc1, Sluc2, Sluc3, and Sluc4, that influence the susceptibility to lung cancer in the mouse.

Many complex traits, including susceptibility to lung cancer, are controlled by multiple genes--quantitative trait loci (QTLs). We facilitated the mapping of QTLs by making use of recombinant congenic strains (RCS), a system of mouse inbred strains in which the genetic complexity is reduced, and by applying MQM-mapping (multiple-QTL models or marker-QTL-marker), a multilocus method with an increased power of detecting of individual QTLs and interacting QTLs (epistasis). The mouse strain O20 develops significantly larger N-ethyl-N-nitrosourea induced lung tumours than mice of the RC strain OcB-9 (ref. 5); the latter share approximately 87.5% of their genes with strain O20 and 12.5% with strain B10.O20 (refs 6,7). QTL analysis of 222 (OcB-9 x O20) F2 mice revealed four new loci that influence susceptibility to lung cancer (Sluc genes). They are involved in two significant, partly counteracting interactions which mask their individual main effects: Sluc1 (on chromosome 19) interacts with Sluc2 (chromosome 2), and Sluc3 (chromosome 6) interacts with Sluc4 (chromosome 11). Together with the data of van Wezel et al. in the accompanying report, our results indicate that interactions between tumour susceptibility genes are a common phenomenon which complicates their mapping.

A general Monte Carlo method for mapping multiple quantitative trait loci.

In this paper we address the mapping of multiple quantitative trait loci (QTLs) in line crosses for which the genetic data are highly incomplete. Such complicated situations occur, for instance, when dominant markers are used or when unequally informative markers are used in experiments with outbred populations. We describe a general and flexible Monte Carlo expectation-maximization (Monte Carlo EM) algorithm for fitting multiple-QTL models to such data. Implementation of this algorithm is straightforward in standard statistical software, but computation may take much time. The method may be generalized to cope with more complex models for animal and human pedigrees. A practical example is presented, where a three-QTL model is adopted in an outbreeding situation with dominant markers. The example is concerned with the linkage between randomly amplified polymorphic DNA (RAPD) markers and QTLs for partial resistance to Fusarium oxysporum in lily.

The MAR-Mediated Reduction in Position Effect Can Be Uncoupled from Copy Number-Dependent Expression in Transgenic Plants.

To study the role of matrix-associated regions (MARs) in establishing independent chromatin domains in plants, two transgenes were cloned between chicken lysozyme A elements. These transgenes were the neomycin phosphotransferase (NPTII) gene under control of the nopaline synthase (nos) promoter and the [beta]-glucuronidase (GUS) gene controlled by the double cauliflower mosaic virus (dCaMV) 35S promoter. The A elements are supposed to establish an artificial chromatin domain upon integration into the plant DNA, resulting in an independent unit of transcriptional regulation. Such a domain is thought to be characterized by a correlated and position-independent, hence copy number-dependent, expression of the genes within the domain. The presence of MARs resulted in a higher relative transformation efficiency, demonstrating MAR influence on NPTII gene expression. However, variation in NPTII gene expression was not significantly reduced. The selection bias for NPTII gene expression during transformation could not fully account for the lack of reduction in variation of NPTII gene expression. Topological interactions between the promoter and A element may interfere with the A element as a domain boundary. In contrast, the GUS gene on the same putative chromatin domain showed a highly significant reduction in variation of gene expression, as expected from previous results. Surprisingly, no copy number-dependent GUS gene expression was observed: all plants showed approximately the same GUS activity. We concluded, therefore, that dCaMV 35S-GUS gene expression in mature tobacco plants is regulated by some form of dosage compensation.

Genotype-by-environment interaction in genetic mapping of multiple quantitative trait loci.

The interval mapping method is widely used for the genetic mapping of quantitative trait loci (QTLs), though true resolution of quantitative variation into QTLs is hampered with this method. Separation of QTLs is troublesome, because single-QTL is models are fitted. Further, genotype-by-environment interaction, which is of great importance in many quantitative traits, can only be approached by separately analyzing the data collected in multiple environments. Here, we demonstrate for the first time a novel analytic approach (MQM mapping) that accommodates both the mapping of multiple QTLs and genotype-by-environment interaction. MQM mapping is compared to interval mapping in the mapping of QTLs for flowering time in Arabidopsis thaliana under various photoperiod and vernalization conditions.

Partial resistance to late blight (Phytophthora infestans) in hybrid progenies of four South American Solanum species crossed with diploid S. tuberosum.

Resistant genotypes of the diploid tuber-bearing South American species Solarium arnezii x hondelmannii, S. berthaultii, S. leptophyes and S. microdontum were crossed with three diploid genotypes of S. tuberosum that varied in resistance and maturity type. The progenies were field tested for 2 years for resistance to a complex race of Phytophthora infestans. A wealth of genetic variation for resistance was found in most of the progenies. At least two susceptibility groups could be distinguished in some progenies of S. microdontum. This could be explained by the presence of several major resistance genes in the wild parent and, unexpectedly, in the susceptible parent SH 82-44-111. In most of the wild parents and in the susceptible parent SH 77-114-2988 there appeared to be minor resistance genes. General combining ability effects were predominant; small specific combining ability effects were detected in some crosses of S. microdontum. Gene action appeared dominant in some crosses.

Controlling the type I and type II errors in mapping quantitative trait loci.

Although the interval mapping method is widely used for mapping quantitative trait loci (QTLs), it is not very well suited for mapping multiple QTLs. Here, we present the results of a computer simulation to study the application of exact and approximate models for multiple QTLs. In particular, we focus on an automatic two-stage procedure in which in the first stage "important" markers are selected in multiple regression on markers. In the second stage a QTL is moved along the chromosomes by using the preselected markers as cofactors, except for the markers flanking the interval under study. A refined procedure for cases with large numbers of marker cofactors is described. Our approach will be called MQM mapping, where MQM is an acronym for "multiple-QTL models" as well as for "marker-QTL-marker." Our simulation work demonstrates the great advantage of MQM mapping compared to interval mapping in reducing the chance of a type I error (i.e., a QTL is indicated at a location where actually no QTL is present) and in reducing the chance of a type II error (i.e., a QTL is not detected).

High resolution of quantitative traits into multiple loci via interval mapping.

A very general method is described for multiple linear regression of a quantitative phenotype on genotype [putative quantitative trait loci (QTLs) and markers] in segregating generations obtained from line crosses. The method exploits two features, (a) the use of additional parental and F1 data, which fixes the joint QTL effects and the environmental error, and (b) the use of markers as cofactors, which reduces the genetic background noise. As a result, a significant increase of QTL detection power is achieved in comparison with conventional QTL mapping. The core of the method is the completion of any missing genotypic (QTL and marker) observations, which is embedded in a general and simple expectation maximization (EM) algorithm to obtain maximum likelihood estimates of the model parameters. The method is described in detail for the analysis of an F2 generation. Because of the generality of the approach, it is easily applicable to other generations, such as backcross progenies and recombinant inbred lines. An example is presented in which multiple QTLs for plant height in tomato are mapped in an F2 progeny, using additional data from the parents and their F1 progeny.

Reduced Position Effect in Mature Transgenic Plants Conferred by the Chicken Lysozyme Matrix-Associated Region.

Matrix-associated regions may be useful for studying the role of chromatin architecture in transgene activity of transformed plants. The chicken lysozyme A element was shown to have specific affinity for tobacco nuclear matrices, and its influence on the variability of transgene expression in tobacco plants was studied. T-DNA constructs in which this element flanked either the [beta]-glucuronidase (GUS) reporter gene or both reporter and selection gene were introduced in tobacco. The variation in GUS gene activity was reduced significantly among mature first-generation transgenic plants carrying the A element. Average GUS activity became somewhat higher, but the maximum attainable level of gene expression was similar for all three constructs. Transient gene expression assays showed that the A element did not contain general enhancer functions; therefore, its presence seemed to prevent the lower levels of transgene expression. The strongest reduction in variability was found in plants transformed with the construct carrying the A elements at the borders of the T-DNA. In this population, expression levels became copy number dependent. The presence of two A elements in the T-DNA did not interfere with meiosis.