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Evaluation of a DHPLC-based assay for rapid detection of RET germline mutations in Italian patients with medullary thyroid carcinoma.

Causative gain-of-function mutations of the RET tyrosine-kinase receptor gene have been reported in more than 95% of inherited cases of medullary thyroid carcinoma (MTC; OMIM# 155240). Most RET activating mutations are clustered in mutational "hot spots" in exons 10, 11, 13, 14, 15 and 16 and are usually detected by single-strand conformation polymorphism (SSCP) followed by direct sequencing. To improve sensitivity, time and costs of mutational screening we have developed a denaturing high performance chromatography (DHPLC) protocol, based on the detection of heteroduplex molecules by ion-pair reverse-phase liquid chromatography under partially denaturing conditions. The mutational screening of RET exons 10, 11, 13-16 was performed in a total of 111 subjects, including 45 MTC patients and 49 relatives with known RET mutations and 17 individuals, being at risk of hereditary MTC and carrying unknown RET alleles. Heteroduplex peaks with a distinct and reproducible DHPLC elution profile allowed the detection of both rare and common RET mutations. Overall, the DHPLC-based methodology showed a high level of sensitivity and accuracy, nearing 100%. Furthermore, our protocol showed the ability to identify: 1) all the mutated codons of RET located in the "hot spots" domain; 2) the different point mutations occurring in the same codon of RET gene; 3) less frequent or rare mutations; 4) polymorphisms. As such, it can be proposed as a relatively simple and highly accurate method for a rapid genetic testing for members of MTC families.

[Effect of bovine serum albumin and restriction enzyme on quality of DNA profile of tissues extracted from paraffin blocks and histological slides].

This paper reports the possibility of DNA profiling obtained from fixed and paraffin-embedded tissues or histological slides. The influence of using bovine serum albumin and restriction enzyme Hinfl on quality of DNA was analysed. The usability of those chemicals was estimated by examination of amplification results locus VWA, and multiplex PCR with the use commercial kit AmpFlSTR Identifiler (including 15 loci + amelogenine locus) and AmpFlSTR SEfiler kit (including 11 loci + amelogenine locus) Applied Biosystems. PCR products of VWA locus were separated by electrophoresis on polyacrylamide gels and visualized by silver staining. The products of loci of AmpFlSTR Identifiler and AmpFlSTR SEfiler were analyzed by capillary electrophoresis on an ABI Prism 310 sequencer. Results achieved with and without using bovine serum albumin and restriction enzyme Hinfl were compared and positive influence of those substances on DNA quality was demonstrated. Obtained results of purification DNA with the use of restriction enzyme were verified in paternity testing case.

Craniovertebral instability with spinal cord compression in a 17-month-old boy with Sly syndrome (mucopolysaccharidosis type VII): a surgical dilemma.

A case study with review of surgical technique in craniovertebral stabilization for young children with mucopolysaccharidosis.</AbstractText>To describe an interesting patient with a rare metabolic disorder and review surgical technique for craniovertebral instability in this rare patient population.</AbstractText>Craniovertebral instability has been reported in patients with mucopolysaccharidosis and poses a problem for spinal surgery because of the inherent metabolic disorder and age at presentation. We present the first case of craniovertebral instability and spinal cord compression occurring in Sly syndrome (mucopolysaccharidosis type VII) who is the youngest patient afflicted with this metabolic disorder to undergo craniovertebral stabilization.</AbstractText>A 17-month-old boy presented with inability to support his head, decreasing muscle strength in all extremities, distended abdomen, and shortness of breath. The patient was found to have a dilated cardiomyopathy, hepatosplenomegaly, abnormal hepatobiliary function, corneal clouding, and a questionable tracheal anomaly. Genetic testing provided a diagnosis of Sly syndrome, mucopolysaccharidosis type VII. Magnetic resonance imaging revealed focal stenosis with significant spinal cord compression at the craniovertebral junction. Neurologic examination revealed normal muscle volume with strength 3/5 in all extremities and significant weakness in the neck muscles with instability at the craniovertebral junction.</AbstractText>On a concerted preoperative medical clearance by pediatric intensive care, pediatric neuroanesthesia, pediatric cardiology, pediatric gastroenterology, and pediatric neurosurgery, the patient underwent occipital to C3 decompression and fusion with autogenous rib grafts. The patient was placed in a prefitted halo-vest after surgery and was neurologically intact.</AbstractText>This case demonstrates the heterogeneity of cervical spine deformities among the mucopolysaccharidosis syndromes and confirms the propensity for deposition of glycosaminoglycans at the craniovertebral junction. Further studies should investigate the etiology for this propensity of glycosaminoglycan deposition at the craniovertebral junction. We think that this case demonstrates that, with appropriate preoperative planning, these patients can undergo successful posterior cervical arthrodesis despite their age or metabolic defects.</AbstractText>

Nucleic acid amplification strategies for DNA microarray-based pathogen detection.

DNA microarray-based screening and diagnostic technologies have long promised comprehensive testing capabilities. However, the potential of these powerful tools has been limited by front-end target-specific nucleic acid amplification. Despite the sensitivity and specificity associated with PCR amplification, the inherent bias and limited throughput of this approach constrain the principal benefits of downstream microarray-based applications, especially for pathogen detection. To begin addressing alternative approaches, we investigated four front-end amplification strategies: random primed, isothermal Klenow fragment-based, phi29 DNA polymerase-based, and multiplex PCR. The utility of each amplification strategy was assessed by hybridizing amplicons to microarrays consisting of 70-mer oligonucleotide probes specific for enterohemorrhagic Escherichia coli O157:H7 and by quantitating their sensitivities for the detection of O157:H7 in laboratory and environmental samples. Although nearly identical levels of hybridization specificity were achieved for each method, multiplex PCR was at least 3 orders of magnitude more sensitive than any individual random amplification approach. However, the use of Klenow-plus-Klenow and phi29 polymerase-plus-Klenow tandem random amplification strategies provided better sensitivities than multiplex PCR. In addition, amplification biases among the five genetic loci tested were 2- to 20-fold for the random approaches, in contrast to &gt;4 orders of magnitude for multiplex PCR. The same random amplification strategies were also able to detect all five diagnostic targets in a spiked environmental water sample that contained a 63-fold excess of contaminating DNA. The results presented here underscore the feasibility of using random amplification approaches and begin to systematically address the versatility of these approaches for unbiased pathogen detection from environmental sources.

Adrenocortical hypertension.

Primary aldosteronism, congenital adrenal hyperplasia, Cushing's syndrome, glucocorticoid-remediable aldosteronism, and corticotropin-dependent forms of adrenal pathology can cause hypertension by excessive production of adrenocortical hormones. Although traditional biochemical assays continue to be used, genetic testing has simplified the diagnosis of glucocorticoid-remediable aldosteronism. Also, new interventional radiologic approaches for the diagnosis and treatment of corticotropin-dependent forms of Cushing's syndrome are available. Medical and surgical approaches, however, still remain viable options for treatment.

Recent developments in ovarian cancer genetics.

This review attempts to provide an update on recent research on inherited susceptibility to ovarian cancer. It covers articles mainly published in 2002 and 2003, with an emphasis on genetic counseling issues.</AbstractText>The major areas on which recent reports have focused include: (1) an expanded understanding of the BRCA1 and BRCA2 mutation spectrum and the frequencies of deleterious alleles in various ethnic groups; (2) investigations on how information is best transmitted to high-risk family members via genetic counseling; (3) an analysis of patient management changes based on genotype results; (4) social issues surrounding predictive testing for breast/ovarian cancer genes, including health insurance and discrimination concerns; and (5) an investigation into gynecologists' knowledge of ovarian cancer genetics, and their ability to provide genetic counseling for ovarian cancer to their patients. Preliminary reports from scientific meetings that have not yet been published in peer-reviewed journals are also discussed.</AbstractText>Recent developments in ovarian cancer genetics expand many of the areas that have been studied previously. A major focus of recent research has dealt with genetic counseling for families affected by hereditary breast and ovarian cancer.</AbstractText>

Screening for differential gene expression during the development of form-deprivation myopia in the chicken.

To use the technique of differential gene display to analyze changes in gene expression that occur during the development of and recovery from form-deprivation myopia.</AbstractText>The differential display-reverse transcriptase-polymerase chain reaction technique was used to detect cDNAs that are differentially expressed after 24 h (including 12 h in the light) after fitting with a diffuser to induce form-deprivation myopia. Messenger RNA levels were determined by quantitative Northern blotting in retinas after 11 days of form deprivation or in retinas where the diffusers had been removed the previous day.</AbstractText>Twenty-six differentially expressed genes were processed in our initial screen. Two of these, alphaB-crystallin and retinoic acid receptor-alpha, were studied further. Levels of alphaB-crystallin mRNA were increased on day 11 in retinas from form-deprived eyes relative to eyes of control chickens and were reduced to below those levels within 6 to 12 h after removal of the diffusers. Levels of retinoic acid receptor-alpha mRNA showed similar changes, except that after removal of the diffusers, the levels further increased.</AbstractText>The technique of differential gene display can be used to detect changes in gene expression during the regulation of eye growth. The response of alphaB-crystallin is particularly interesting because expression increases when eye growth is high and decreases when eye growth slows.</AbstractText>

Genetic polymorphisms of CYP1A1 in a Korean population.

Genetic polymorphisms in the coding exons of the CYP1A1 gene were analyzed in 100 Koreans. Three types of CYP1A1 polymorphisms, specifically G134A, G184C and A2455G, were identified with allelic frequencies of 18, 3, and 16%, respectively, and no linkage was observed among them. The novel G184C polymorphism identified in this study was associated with the mutation of an alanine residue at position 62 to proline. Other earlier-reported polymorphisms in the coding region of CYP1A1 were not detected.

Preimplantation HLA testing.

Preimplantation genetic diagnosis (PGD) has become an option for couples for whom termination of an affected pregnancy identified by traditional prenatal diagnosis is unacceptable and is applicable to indications beyond those of prenatal diagnosis, such as HLA matching to affected siblings to provide stem cell transplantation.</AbstractText>To describe preimplantation HLA typing, not involving identification of a causative gene, for couples who had children with bone marrow disorders at need for HLA-matched stem cell transplantation.</AbstractText><AbstractText Label="DESIGN, SETTING, AND PARTICIPANTS" NlmCategory="METHODS">HLA matching procedures conducted at a single site during 2002-2003 in an in vitro fertilization program for 9 couples with children affected by acute lymphoid leukemia, acute myeloid leukemia, or Diamond-Blackfan anemia requiring HLA-matched stem cell transplantation. In 13 clinical cycles, DNA in single blastomeres removed from 8-cell embryos following in vitro fertilization was analyzed for HLA genes simultaneously with analysis for short tandem repeats in the HLA region to select and transfer only those embryos that were HLA matched to affected siblings.</AbstractText>Results of HLA matching and pregnancy outcome.</AbstractText>As a result of testing a total of 199 embryos, 45 (23%) HLA-matched embryos were selected, of which 28 were transferred in 12 clinical cycles, resulting in 5 singleton pregnancies and birth of 5 HLA-matched healthy children.</AbstractText>This is the first known experience of preimplantation HLA typing performed without PGD for a causative gene, providing couples with a realistic option of having HLA-matched offspring to serve as potential donors of stem cells for their affected siblings.</AbstractText>

Nucleotide variation and recombination along the fourth chromosome in Drosophila simulans.

The fourth chromosome of Drosophila melanogaster and its sister species are believed to be nonrecombining and have been a model system for testing predictions of the effects of selection on linked, neutral variation. We recently examined nucleotide variation along the chromosome of D. melanogaster and revealed that a low average level of recombination could be associated with considerably high levels of nucleotide variation. In this report, we further investigate the variation along the fourth chromosome of D. simulans. We sequenced 12 gene regions evenly distributed along the fourth chromosome for a worldwide collection of 11 isofemale lines and 5 gene regions in a local population of 10 isofemale lines from South America. In contrast to predictions for regions of very low recombination, these data reveal that the variation levels in many gene regions, including an intron region of the ci gene, vary considerably along the fourth chromosome. Nucleotide diversity ranged from 0.0010 to 0.0074 in 9 gene regions interspersed with several regions of greatly reduced variation. Tests of recombination indicate that the recombination level is not as low as previously thought, likely an order of magnitude higher than that in D. melanogaster. Finally, estimates of the recombination parameters are shown to support a crossover-plus-conversion model.

Mitochondrial encephalomyopathies: diagnostic approach.

Mitochondrial diseases have extremely heterogeneous clinical presentations due to the ubiquitous nature of mitochondria and the dual genetic control of the respiratory chain. Thus, mitochondrial disorders can be multisystemic (mitochondrial encephalomyopathies) or confined to a single tissue, and they can be sporadic or transmitted by mendelian or maternal inheritance. Mendelian disorders are usually inherited as autosomal recessive traits, tend to present earlier in life, and usually "breed true" in each family. By contrast, mitochondrial DNA-related diseases usually start later and vary in their presentation within members of the same family. Precise diagnosis is often a challenge; we go through the traditional steps of the diagnostic process, trying to highlight clues to mitochondrial dysfunction in the family history, physical and neurological examinations, routine and special laboratory tests, and histo-chemical and biochemical results of the muscle biopsy. The ultimate goal is to reach, whenever possible, a definitive molecular diagnosis, which permits rational genetic counseling and a prenatal diagnosis.

Prenatal testing and pregnancy termination in Sri Lanka: views of medical students and doctors.

Prenatal diagnosis and pregnancy termination generate complex ethical issues. Surveys conducted in Sri Lanka among doctors and medical students in 1986 supported a change in law in favour of pregnancy termination when gross genetic defects are detected antenatally. A new generation of prenatal tests has focused attention again on the topic of termination and under what circumstances it might be legally done. The present survey contributes to the debate by means of a survey of doctors and medical students.</AbstractText>A self-administered questionnaire given to doctors and medical students.</AbstractText>Ninety three per cent of doctors and 81% of students accept pregnancy termination as an appropriate course of action if a gross genetic defect is detected antenatally, and 87% of doctors and 80% of students support a change in the law to allow termination of the pregnancy. The corresponding figures in previous surveys were 80%, 69% and 96%, 88% respectively.</AbstractText>A majority of doctors and medical students support a change in law in favour of liberalising pregnancy termination when a genetic defect is detected antenatally.</AbstractText>

An improved formulation of marker heterozygosity in recurrent selection and backcross schemes.

This report presents a theoretical formulation for predicting heterozygosity of a putative marker locus linked to two quantitative trait loci (QTL) in a recurrent selection and backcross (RSB) scheme. Since the heterozygosity at any given marker locus maintained in such a breeding programme reflects its map location relative to QTL, the present study develops the theoretical analysis of the QTL mapping method that recently appeared in the literature. The formulae take into account selection, recombination and finite population size during the multiple-generation breeding scheme. The single-marker and two-QTL model was compared numerically with the model involving two linked marker loci and two QTL. Without recombination interference, the two models predict the same expected heterozygosity at the linked marker loci, indicating that the model is valid for predicting marker heterozygosity maintained at any loci in an RSB breeding scheme. The formulation is demonstrated numerically for several RSB schemes and its implications in developing a likelihood-based statistical framework for modeling the RSB experiments are discussed.

Population subdivision and the Hudson-Kreitman-Aguade test: testing for deviations from the neutral model in organelle genomes.

The Hudson-Kreitman-Aguade (HKA) test is based on the prediction from the neutral theory that levels of polymorphism within a species and the divergence between two closely related species should be correlated. Population subdivision has been shown to alter both the amounts of polymorphism segregating within species and the rate of divergence between species, meaning that genomic regions with different population structures also differ in their divergence to polymorphism ratios. Population subdivision may hence hamper the utility of the HKA test for detecting deviations from the standard neutral model, especially for organelle genomes that often have different patterns of population structure compared with nuclear genes. In this paper, I show that population subdivision inflates the number of instances where the HKA test detects deviations from the neutral model. Using coalescent simulations I show that this bias is most apparent when population subdivision is strong and differs substantially between the loci included. However, if divergence time is large and population structure substantial even changes in the levels of polymorphism and divergence associated with differences in the effective population size between two loci is enough to substantially alter the number of significant outcomes of the HKA test. A dataset on cytoplasmic diversity in Sileine vulgaris and S. latifolia (Ingvarsson &amp; Taylor, 2002) is also reanalysed. The previous study had shown a marked excess of intraspecific polymorphism in both species. However, when effects of population subdivision were removed, ad hoc, levels of intraspecific polymorphism were no longer significantly different from neutral expectations, suggesting that population subdivision contributed to the observed excess of intraspecific polymorphism seen in both species of Silene.

Isolation and characterization of dinucleotide repeat microsatellites in Drosophila ananassae.

Drosophila ananassae is a cosmopolitan species with a geographic range throughout most of the tropical and subtropical regions of the world. Previous studies of DNA sequence polymorphism in three genes has shown evidence of selection affecting broad expanses of the genome in regions with low rates of recombination in geographically local populations in and around India. The studies suggest that extensive physical and genetic maps based on molecular markers, and detailed studies of population structure may provide insight into the degree to which natural selection affects DNA sequence polymorphism across broad regions of chromosomes. We have isolated 85 dinucleotide repeat microsatellite sequences and developed assay conditions for genotyping using PCR. The dinucleotide repeats we isolated are shorter, on average, than those isolated in many other Drosophila species. Levels of genetic variation are high, comparable to Drosophila melanogaster. The levels of variation indicate the effective population size of an Indonesian population of D. ananassae is 58,692 (infinite allele model) and 217,284 (stepwise mutation model), similar to estimates of effective population size for D. melanogaster calculated using dinucleotide repeat microsatellites. The data also show that the Indonesian population is in a rapid expansion phase. Cross-species amplification of the microsatellites in 11 species from the Ananassae, Elegans, Eugracilis and Ficusphila subgroups indicates that the loci may be useful for studies of the sister species, D. pallidosa, but will have limited use for more distantly related species.

Korean version of the diagnostic interview for genetic studies: Validity and reliability.

The Diagnostic Interview for Genetic Studies (DIGS), developed in 1994 by the National Institute of Mental Health (NIMH), was translated into Korean and tested for reliability and diagnostic validity. Concurrent validity was tested using the Structured Clinical Interview for DSM-IV (SCID) and clinical diagnoses in 53 patients, most of whom had either schizophrenia or bipolar disorder. Inter-rater reliability was tested in 24 patients. Test-retest reliability was also tested in 17 patients. Overall and specific diagnostic validity for the Korean version of DIGS (DIGS-K) was excellent for most diagnoses. Inter-rater and test-retest reliability for overall and specific diagnoses also ranged from fair to excellent. For schizoaffective disorder, the test-retest reliability of DIGS-K was in a fair range, although the level was lower than that of other diagnoses. However, its diagnostic validity and inter-rater reliability was below fair range. In conclusion, DIGS-K appears to be a reliable interview for major psychiatric disorders.

[From the conception of the PRINS to its coronation].

As a non-isotopic molecular cytogenetic technique, the primed in situ (PRINS) labelling reaction represents a major technological progress achieved in the past decade. It has become a routine technique for the microscopic visualization of specific DNA sequences in cells and nuclei and constitutes a good alternative to the fluorescence in situ hybridization (FISH) procedure. Among the multiple advantages that characterize the PRINS technique, specificity, rapidity, reliability, reproducibility, and cost-effectiveness can be mentioned. PRINS can be in addition associated with other techniques like FISH, indirect immunofluorescence, and nick translation. The most recent developments show the great potential of this technique. Now PRINS can be used to study single-copy genes and, consequently, can be routinely used to investigate deletions associated with microdeletion syndromes. Therefore, the PRINS technique has the potential to become a widely used molecular cytogenetic tool in clinics and research. This short review presents how the PRINS technique contributed to further the understanding of biological phenomena and describes the different possibilities and applications of the PRINS method in several biological and clinical fields (pre-implantation testing, prenatal, constitutional and oncologic genetic diagnosis).

A random-periods model for expression of cell-cycle genes.

We propose a nonlinear regression model for quantitatively analyzing periodic gene expression in studies of experimentally synchronized cells. Our model accounts for the observed attenuation in cycle amplitude by a simple and biologically plausible mechanism. We represent the expression level for each gene as an average across a large number of cells. For a given cell-cycle gene, we model its expression in each cell in the culture as following the same sinusoidal function except that the period, which in any individual cell must be the same for all cell-cycle genes, varies randomly across cells. We model these random periods by using a lognormal distribution. The variability in period causes the measured amplitude of the cyclic expression trajectory to attenuate over time as cells fall increasingly out of synchrony. Gene-specific parameters include initial amplitude and phase angle. Applying the model to data from Whitfield et al. [Whitfield, M. L., Sherlock, G., Saldanha, A. J., Murray, J, I., Ball, C. A., et al. (2002) Mol. Biol. Cell 13, 1977-2000], we fit the trajectories of 18 well characterized phase-marker genes and find that the fit does not suffer when a common lognormal distribution is assumed for all 18 genes compared with a separate distribution for each. We then use the model to identify 337 periodically expressed transcripts, including the 18 phase-marker genes. The model permits estimation of and hypothesis testing about biologically meaningful parameters that characterize cycling genes.

Testing the chromosomal speciation hypothesis for humans and chimpanzees.

Fixed differences of chromosomal rearrangements between isolated populations may promote speciation by preventing between-population gene flow upon secondary contact, either because hybrids suffer from lowered fitness or, more likely, because recombination is reduced in rearranged chromosomal regions. This chromosomal speciation hypothesis thus predicts more rapid genetic divergence on rearranged than on colinear chromosomes because the former are less porous to gene flow. A number of studies of fungi, plants, and animals, including limited genetic data of humans and chimpanzees, support the hypothesis. Here we reexamine the hypothesis for humans and chimpanzees with substantially more genomic data than were used previously. No difference is observed between rearranged and colinear chromosomes in the level of genomic DNA sequence divergence between species. The same is also true for protein sequences. When the gorilla is used as an outgroup, no acceleration in protein sequence evolution associated with chromosomal rearrangements is found. Furthermore, divergence in expression pattern between orthologous genes is not significantly different for rearranged and colinear chromosomes. These results, showing that chromosomal rearrangements did not affect the rate of genetic divergence between humans and chimpanzees, are expected if incipient species on the evolutionary lineages separating humans and chimpanzees did not hybridize.

The relevance of gene transfer to the safety of food and feed derived from genetically modified (GM) plants.

In 2000, the thematic network ENTRANSFOOD was launched to assess four different topics that are all related to the testing or assessment of food containing or produced from genetically modified organisms (GMOs). Each of the topics was linked to a European Commission (EC)-funded large shared cost action (see http://www.entransfood.com). Since the exchange of genetic information through horizontal (lateral) gene transfer (HGT) might play a more important role, in quantity and quality, than hitherto imagined, a working group dealing with HGT in the context of food and feed safety was established. This working group was linked to the GMOBILITY project (GMOBILITY, 2003) and the results of the deliberations are laid down in this review paper. HGT is reviewed in relation to the potential risks of consuming food or feed derived from transgenic crops. First, the mechanisms for obtaining transgenic crops are described. Next, HGT mechanisms and its possible evolutionary role are described. The use of marker genes is presented in detail as a special case for genes that may pose a risk. Furthermore, the exposure to GMOs and in particular to genetically modified (GM) deoxyribonucleic acid (DNA) is discussed as part of the total risk assessment. The review finishes off with a number of conclusions related to GM food and feed safety. The aim of this paper is to provide a comprehensive overview to assist risk assessors as well as regulators and the general public in understanding the safety issues related to these mechanisms.

Assessment of association between mitochondrial aldehyde dehydrogenase polymorphism and Alzheimer's disease in an older Korean population.

The mutant allele of mitochondrial aldehyde dehydrogenase (ALDH2(*)2) was found to be associated with Alzheimer's disease (AD) in a Japanese sample, interacting with the apolipoprotein E epsilon 4 allele (Apo E4).</AbstractText>In a community Korean population we sought to investigate associations between ALDH2 genotypes and the following outcomes: cognitive impairment, previous cognitive decline, dementia and AD.</AbstractText>Six hundred ninety community residents aged 65 or over were assessed for demographic characteristics, drinking behaviour, cognitive function, clinical diagnoses of dementia and AD, physical health status, and genotype (ALDH2 and Apo E).</AbstractText>There were no significant associations between the ALDH2(*)2 and any cognitive outcome, before or after adjustment for alcohol-related characteristics. These findings were consistent both in the non-drinkers and drinkers. Interaction between ALDH2 and Apo E was only found for one outcome (previous cognitive decline) at borderline levels of significance (P=0.058).</AbstractText>Overall, these findings in a community population did not support a substantial role for ALDH2 genotype in the aetiology of dementia.</AbstractText>

A haplotype of the methylenetetrahydrofolate reductase gene is protective against late-onset Alzheimer's disease.

Epidemiological studies have shown that elevated plasma homocysteine (Hcy) levels play an important role in the pathogenesis of Alzheimer's disease (AD). In spite of the evidence that a C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene elevates plasma Hcy levels, the impact of the C677T polymorphism on the development of AD is controversial. Here, we performed a genetic case-control study in a Japanese population to investigate whether three polymorphisms of the MTHFR gene, C677T (Ala222Val), A1298C (Glu429Ala), and A1793G (Arg594Gln), are associated with the development of late-onset AD (LOAD). In our study, the MTHFR gene had four major regional haplotypes: Haplotype A (677C-1298A-1793G), Haplotype B (677T-1298A-1793G), Haplotype C (677C-1298C-1793G), and Haplotype D (677C-1298C-1793A). The frequency of Haplotype C in LOAD was significantly lower than that in control group. Furthermore, the benefit conferred by the presence of at least one Haplotype C was stronger in LOAD patients who lacked the ApoE 4 allele (OR=0.293; 95% CI=0.115-0.744; P=0.010). The results indicate that Haplotype C of the MTHFR gene is protective against the development of LOAD.

DNA binding and antigene activity of a daunomycin-conjugated triplex-forming oligonucleotide targeting the P2 promoter of the human c-myc gene.

Triplex-forming oligonucleotides (TFO) that bind DNA in a sequence-specific manner might be used as selective repressors of gene expression and gene-targeted therapeutics. However, many factors, including instability of triple helical complexes in cells, limit the efficacy of this approach. In the present study, we tested whether covalent linkage of a TFO to daunomycin, which is a potent DNA-intercalating agent and anticancer drug, could increase stability of the triple helix and activity of the oligonucleotide in cells. The 11mer daunomycin-conjugated GT (dauno-GT11) TFO targeted a sequence upstream of the P2 promoter, a site known to be critical for transcription of the c-myc gene. Band-shift assays showed that the dauno-GT11 formed triplex DNA with enhanced stability compared to the unmodified TFO. Band shift and footprinting experiments demonstrated that binding of dauno-GT11 was highly sequence-specific with exclusive binding to the 11 bp target site in the c-myc promoter. The daunomycin-conjugated TFO inhibited transcription in vitro and reduced c-myc promoter activity in prostate and breast cancer cells. The daunomycin-conjugated TFO was taken up by cells with a distinctive intracellular distribution compared to free daunomycin. However, cationic lipid-mediated delivery was required for enhanced cellular uptake, nuclear localization and biological activity of the TFO in cells. Dauno-GT11 reduced transcription of the endogenous c-myc gene in cells, but did not affect expression of non-target genes, such as ets-1 and ets-2, which contained very similar target sequences in their promoters. Daunomycin-conjugated control oligonucleotides unable to form triplex DNA with the target sequence did not have any effect in these assays, indicating that daunomycin was not directly responsible for the activity of daunomycin-conjugated TFO. Thus, attachment of daunomycin resulted in increased triplex stability and biological activity of the 11mer GT-rich TFO without compromising its specificity. These results encourage further testing of this approach to develop novel antigene therapeutics.

Clinical germline genetic testing for melanoma.

Clinical genetic testing for mutations in CDKN2A (cyclin-dependent kinase inhibitor 2A), a melanoma susceptibility gene, is now available. The International Melanoma Genetics Consortium advocates that genetic testing for CDKN2A should be done only as part of a research protocol. Experience with genetic testing for other cancer-susceptibility genes indicates that CDKN2A testing has enormous potential for the prevention and detection of a deadly disease. However, clinicians need to understand the benefits and shortcomings of clinical CDKN2A testing so that it can be used advantageously. Here, we examine whether CDKN2A meets the recommendations of the American Society of Clinical Oncology (ASCO) for cancer-susceptibility genetic testing. Although genetic testing for hereditary melanoma should, whenever possible, occur within research protocols, it might be successfully done outside of research protocols if attention is paid to selection, education, and counselling needs of patients; valid test interpretation; and the changing of medical management in appropriate individuals.

Combination of porous hydroxyapatite and cationic liposomes as a vector for BMP-2 gene therapy.

The clinical significance of hydroxyapatite (HAP) as a bone substitute has become apparent in recent years and bone morphogenetic protein (BMP) a substance which induces bone has attracted much attention. In this study, a 1.2 cm diameter bone defects created on rabbit cranium were treated with the BMP-2 gene (cDNA plasmid) introduced with porous HAP after completion of hemostasis and the resultant bone formation was analyzed histopathologically. The amounts of bone formation was compared BMP-2 cDNA plasmids were not combined with cationic liposomes as a vector. Four groups of rabbits were compared. In the HAP group the cranial bone defect was treated with HAP containing 40 microg of liposomes and a dummy gene (PU). The BMP gene HAP group was treated with HAP soaked in liposomes and 10 microg of the BMP-2 gene. In addition, a group was treated with the gene without implanting HAP. Bone formation on the cranial defects was evaluated 3, 6 and 9 weeks after the operation, by X-ray and histopathological examinations. Three weeks after the operation there was vigorous bone formation in the cranial defect in the group which received the BMP-2 gene without HAP, and complete ossification was observed at 9 weeks. In the group which received HAP containing the BMP-2 gene, although new bone formation was evident surrounding the scaffold 3 weeks post-operation, the induced bone tissue did not fill all the pores of the scaffold even at 9 weeks post-operation. These results confirm the clinical usefulness of gene therapy for bone formation, using the BMP-2 gene combined with cationic liposomes as a vector. It is possible that the effects of administering the BMP-2 gene will be improved by specializing the microstructure of scaffold for gene therapy.

A sequencing-based typing method for HLA-DQA1 alleles.

Sequencing-based typing (SBT) is the most comprehensive method for characterizing human leukocyte antigen gene polymorphisms. Development of a SBT method for DQA1 is hampered because of a deletion of codon 56 in nearly half of the known DQA1 alleles. Sequence electropherograms of heterozygous samples comprising a deletion allele and a non-deletion allele display misalignment after codon 56 because of a three base-pair shift in the deletion allele. To overcome this problem, we have designed three group-specific primer sets to selectively amplify the deletion alleles from the nondeletion alleles. DNA samples are initially polymerase chain reaction (PCR)-typed using these primer sets along with an internal positive control primer set specific to growth hormone gene 1 (hGH1). The positive group-specific PCR reactions were selectively repeated without hGH1 control primers, and the amplicons were used as template in sequencing reactions. The sequence data were analyzed to obtain DQA1 types using ABI MatchTools software as well as the newly available Conexio Genomics Assign SBT Genotyping Software. The method was validated using a panel of reference DNA from the University of California, Los Angeles, International DNA Exchange Program. We conclude that the present SBT method is a technically simple and robust procedure to characterize the sequence polymorphisms in exon 2 of DQA1 gene.

HLA class II typing in newborns reveals a low frequency of the DRB1*04 allele and a high frequency of DRB1*11 allele in three regions of continental Italy.

As part of a longitudinal study aimed at defining the natural history of prediabetic autoimmunity and predicting the risk of future cases of type 1 diabetes, 3607 newborns from three regions of continental Italy (Lombardia, Liguria, and Lazio) were subjected to genetic testing to determine human leukocyte antigen-DRB1 (HLA-DRB1) and -DQB1 allele and phenotype frequencies. Polymerase chain reaction and immobilized sequence-specific oligonucleotide probe assays were used to identify ten DRB1 allele lineages and three DQB1 alleles. No major inter-regional differences emerged in the allelic distribution indicating homogeneous distribution of the HLA DRB1-DQB1 alleles among the three regions analyzed. Comparison of our data with those published for other Caucasian populations reveals that these three regions are characterized by a very low frequency of DRB1*04 (8%) and a high frequency of DRB1*11 (25%). The phenotype frequencies of HLA-DQB1*0302 and DQB1*0602 observed are also lower than those reported for other populations. Furthermore, the DRB1*04-DQB1*0302 haplotype was relatively infrequent in our population (5.3% of the newborns tested). These findings furnish a genetic "portrait" of the populations of the analyzed regions that will be useful not only for investigation of the genetic risk of type 1 diabetes mellitus in Italy but also for studies of other autoimmune diseases related to HLA genotypes.

Amyloid precursor protein gene analysis in familial Alzheimer's disease cases: a lack of mutations in exons 16 and 17.

The beta-amyloid precursor protein (APP) gene (on chromosome 21), Presenilin 1 (PS1) gene (on chromosome 14) and Presenilin 2 (PS2) gene (on chromosome 1) are responsible for autosomal dominant early-onset Alzheimer's disease (EOAD). Missense mutations in these genes cause abnormal APP processing with subsequent overproduction of amyloidogenic and toxic A beta (42 peptide. A mutational analysis of APP, PS1, and PS2 genes can be used for both symptomatic and presymptomatic genetic testing and counselling in familial Alzheimer's disease (FAD). To contribute to our knowledge on genetic background of Alzheimer's disease in Poland, we screened APP mutations in a sample of familial EOAD cases from Poznan region. We did not find pathogenic mutations within exons 16 and 17 of the APP gene. Our study confirmed that APP gene mutations account only for a very small portion of FAD.

The confluence of two clinical specialties: genetics and assisted reproductive technologies.

The confluence of genetic and reproductive technologies has significantly expanded the scope of available reproductive options. To ensure appropriate dissemination of information and timely referral for preconception counseling, nurses in all specialties must be aware of the clinical indications for advanced reproductive technologies.

A brief history of the political work of genetics.

The biological sciences have long been used to define distinctions between people and to define inequalities as a natural consequence of essential biological traits. Today, geneticists draw distinctions on the basis of genetic predispositions. Their population-based methods can reinforce stereotypes about race and ethnic differences, providing concepts, validated by science, through which group differences can be interpreted as biologically ordained. Cases suggest how genetic variants can be used in social policies as individuals are differentially treated, not on the basis of their individual condition, but because of predispositions attributed to their group.

Prenatal screening for birth defects: an update.

This article summarizes current second trimester prenatal screening methods, and describes recently developed first trimester screening tools. The advantages and potential pitfalls of first trimester screening are outlined.

The ethical, legal and psychosocial challenges of genetic testing: implications for primary medical care.

Recent advances in genetic information and technology have led to an explosion in molecular testing for mutations that cause human disease. Test results are likely to improve medical management and/or help individuals make important life and reproductive decisions. Unlike other laboratory testing, unique complexities and challenging psychosocial, legal and financial ramifications often accompany genetic tests. Some of the challenges associated with molecular testing and examples illustrating potential ethical, psychosocial and legal complexities are presented here.

Genetic susceptibility to breast and ovarian cancer: risk assessment, genetic counseling, and management issues.

Genetic testing for disease susceptibility genes is available for a large number of genetic disorders. With the expected shortage of trained genetic professionals to offer risk assessment, counseling, testing, and management to patients, primary care physicians will be asked to provide these services. In this review, we discuss the genetic basis of breast and ovarian cancer susceptibility and outline specific recommendations for clinical practice.

Thiopurine methyltransferase activity influences clinical response to azathioprine in inflammatory bowel disease.

<AbstractText Label="BACKGROUND &amp; AIMS" NlmCategory="OBJECTIVE">Genetic polymorphism in thiopurine methyltransferase (TPMT) activity may influence clinical responsiveness to azathioprine (AZA) therapy. Our aim was to determine if the measurement of erythrocyte TPMT enzyme activity could be used to optimize clinical responsiveness to AZA therapy in patients with inflammatory bowel disease (IBD).</AbstractText>A total of 142 consecutive patients were studied. Forty-one patients (32 with Crohn's disease [CD] and 9 with ulcerative colitis [UC]) were enrolled in a 4-month prospective nonrandomized study with AZA, and 101 (65 with CD and 36 with UC) were on either maintenance AZA or 6-mercaptopurine (6-MP). Erythrocyte TPMT activity and AZA metabolite levels were measured blinded to the clinical response.</AbstractText>The response rate after 4 months of continuous AZA therapy was 69% (9/13) in those patients with below-average (&lt;/=12 U/mL blood) TPMT activity, and 29% (8/27) in patients with enzyme activity levels &gt;12 U/mL blood (P &lt; 0.001). Patients with TPMT activity &lt;/=12 achieved a mean (SEM) erythrocyte 6-thioguanine ribonucleotide (6-TGn) level of 394 +/- 29 pmol/8 x 10(8) red blood cells (RBCs); higher than in patients with TPMT activity &gt;12 (218 +/- 28), despite similar mean (1.6 mg/kg/day) dosages of AZA (P &lt; 0.001). By multivariate logistic regression analysis, patients with a TPMT level &lt;15.3 U/mL blood were 6.2 times more likely to respond to AZA therapy. A 6-TGn level of &gt;292 pmol/8 x 10(8) RBCs was associated with a positive predictive value of clinical response of 85.7%.</AbstractText>Patients with higher than average TPMT activity (&gt;12) may remain refractory to conventional dosages of AZA, and may require high (&gt;292) 6-TGn levels. Prospective, randomized, controlled trials are needed to determine whether prior TPMT phenotype testing can be used to adjust the dose of AZA effectively to improve clinical response time and rate.</AbstractText>

Genetic and environmental factors influencing the human factor H plasma levels.

Factor H is a plasma protein that plays a critical role in the regulation of complement activation in fluid phase and on cellular surfaces. Over the years numerous reports have illustrated the association of factor H deficiencies with chronic renal and infectious diseases. Plasma levels of factor H show a five-fold range of variation in humans (116-562 microg/ml), which may also be relevant to disease susceptibility. To quantify the effects of the genetic and environmental factors responsible for the variation in the factor H plasma levels, we have applied variance-component methods to a family-based sample. Factor H plasma levels show an age-dependent increase ( P&lt;0.0001) and are decreased in smokers ( P&lt;0.0001). Interestingly, the heritability of the factor H trait is very high ( h(2)=0.62+/-0.07; P&lt;0.0001), indicating that 62% of the factor H phenotypic variance is due to the additive effects of genes. On this premise, we conducted a genome-wide linkage screen in order to identify genes regulating the factor H trait. Three genomic regions (1q32, 2p21-24 and 15q22-24) provided suggestive evidence of linkage (LOD scores 2.03, 2.15 and 2.00, respectively) with the plasma levels of factor H.

High-resolution genetic mapping of Xa27(t), a new bacterial blight resistance gene in rice, Oryza sativa L.

Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae ( Xoo) (Ishyama) Dye, is one of the serious diseases prevalent throughout Asia. In a previous study, a resistance ( R) locus was transferred from the tetraploid wild rice Oryza minuta to the cultivated rice species, Oryza sativa L. Here, we report the fine genetic mapping of the R locus, tentatively designated as Xa27(t). We performed disease evaluation with an Xa27(t) near-isogenic line, IRBB27, testing 35 Xoo strains collected from 11 countries. The Xa27(t) locus conferred a high level of resistance to 27 strains and moderate resistance to three strains. Resistance of the Xa27(t) gene was developmentally regulated in IRBB27 and showed semi-dominant or a dosage effect in the cv. CO39 genetic background. As a prelude to cloning Xa27(t), a molecular mapping strategy was employed with a large mapping population consisting of 3,875 gametes. Three molecular markers, M336, M1081, and M1059, closely linked to Xa27(t), were identified to facilitate the mapping of Xa27(t) to the long arm of chromosome 6. The Xa27(t) locus was confirmed by chromosome landing of M1081 and M1095 markers on the rice genome. Markers derived from the genomic sequence of O. sativa cv. Nipponbare were used to further saturate the Xa27(t) genomic region. Xa27(t) was finally located within a genetic interval of 0.052 cM, flanked by markers M964 and M1197, and co-segregated with markers M631, M1230, and M449.

Class discovery and classification of tumor samples using mixture modeling of gene expression data--a unified approach.

The DNA microarray technology has been increasingly used in cancer research. In the literature, discovery of putative classes and classification to known classes based on gene expression data have been largely treated as separate problems. This paper offers a unified approach to class discovery and classification, which we believe is more appropriate, and has greater applicability, in practical situations.</AbstractText>We model the gene expression profile of a tumor sample as from a finite mixture distribution, with each component characterizing the gene expression levels in a class. The proposed method was applied to a leukemia dataset, and good results are obtained. With appropriate choices of genes and preprocessing method, the number of leukemia types and subtypes is correctly inferred, and all the tumor samples are correctly classified into their respective type/subtype. Further evaluation of the method was carried out on other variants of the leukemia data and a colon dataset.</AbstractText>

Neonatal screening for glucose-6-phosphate dehydrogenase deficiency fails to detect heterozygote females.

We examined glucose-6-phosphate dehydrogenase (G6PD) deficiency in north-eastern Italian Caucasian neonates detected by neonatal screening, in order to measure the incidence of heterozygote females detected by neonatal screening, and to estimate the near-true total incidence. A total of 85,437 Caucasian neonates, born between January 2000 and December 2001, have been enclosed in the study. The total incidence of the disease, measured by fluorescent method, is 0.9 per thousand; the total incidence, calculated by Hardy-Weinberg law, is 4.8 per thousand. The frequency of missed females is 93% of total females expected with G6PD deficiency; most of them are very likely heterozygous females. The sensitivity of the fluorescent method might be not sufficient to detect all females. Since heterozygote females might develop the symptoms of G6PD deficiency later, these results suggest that the G6PD neonatal screening may not be helpful in preventing disease in females.

Multiplex allele-specific PCR assay for differential diagnosis of Hb S, Hb D-Punjab and Hb Tak.

Apart from hemoglobin (Hb) E, Hb D-Punjab [beta121(GH4)Glu-Gln] and Hb Tak [beta147Term-Thr] are the two most common beta-chain variants among the Asian population. These two Hb variants have similar alkaline electrophoretic mobilities and HPLC profiles as those of the Hb S [beta6(A3)Glu-Val]. Differential diagnosis of these clinically relevant hemoglobinopathies is therefore problematic. Direct detection of the beta-globin gene mutations would be another diagnostic alternative.</AbstractText>A simultaneous DNA diagnosis of the three Hb variants was developed based on the multiplex allele-specific polymerase chain reaction (PCR) approach. The method was validated on 10 carriers of Hb D-Punjab, 5 carriers of Hb Tak, 2 carriers of Hb S and 50 normal individuals of Thai origin.</AbstractText>The three abnormal Hbs could be correctly diagnosed with the simultaneous PCR approach, and a complete concordance with results using other established methods was obtained.</AbstractText>The multiplex allele-specific PCR approach developed should prove useful in complementing routine Hb analysis for differential diagnosis of these three common Hb variants and should facilitate a program of hemoglobinopathy screening in the region.</AbstractText>

Prenatal screening and the assessment of risk: the view from the other side.

Since the introduction, in 1984, of maternal serum screening for fetal aneuploidy, obstetrical practitioners and their patients have learned to cope with the challenges and limitations of risk estimation. In the instance where the "odds" are not entirely reassuring, the hazards of invasive, yet definitive, testing are weighed against the costs of uncertainty. Non-invasive prenatal screening has improved dramatically over the past 20 years, with early administration, high sensitivity, and low false positive rates as the benchmarks. With a wide array of tests at her disposal, the woman who chooses to undergo prenatal screening for aneuploidy presumably does so in the context of counselling and consent, and with the assurance that the tests offered, having evolved through the rigours of large-scale clinical trials, are as good as they can possibly be. Or does she? The following is a highly personal account of one woman's experience with prenatal screening, in particular, with the "Pandora's box" of ultrasonic soft markers for aneuploidy. The author challenges the experts in the field to ensure that all "advancements" meet the standards described above.

Anti-insulin activity in IgG-fractions from children with newly-diagnosed type 1 diabetes and negative for insulin autoantibodies.

Insulin autoantibodies (IAA) are often detected as the first humoral sign of beta-cell autoimmunity in prospective studies in young children with increased genetic risk of type 1 diabetes. After the appearance of IAA their level typically rise but seems to decline in many cases before the clinical presentation of type 1 diabetes. We hypothesized that the reason for the sudden drops in the levels of IAA could be the formation of immune complexes caused by binding of antibodies to free insulin in plasma. We studied whether isolation of the IgG-fraction and dissociation of immune complexes by acid treatment using protein A column results in the appearance of detectable IAA in those children with newly-diagnosed type 1 diabetes whose plasma samples test negative for IAA. IAA assay was performed in IgG-fractions and corresponding plasma samples from 17 children with type 1 diabetes and 23 unaffected children all testing negative for plasma IAA. The levels of IAA measured from IgG-fractions of diabetic children were higher than the levels of IAA measured from IgG-fractions in the control children (p = 0.004 in Mann-Whitney U-test). Forty-seven percent (8 out of 17) of newly-diagnosed patients negative for plasma IAA before IgG separation had increased levels of IAA in IgG-fractions and only 13% (3 out of 23) of controls. The levels of glutamate decarboxylase autoantibodies (GADA) did not differ between patients (n = 14) and controls (n = 21) negative for plasma GADA when measured in IgG-fractions. Our results suggest that formation of immune complexes results in false negative results in tests for IAA but not for GADA.

A gel electrophoresis method for detection of mitochondrial DNA mutation (3243 tRNA(Leu (UUR))) applied to a Norwegian family with diabetes mellitus and hearing loss.

Blood cells of selected patients from a large Norwegian family with maternally transmitted diabetes mellitus, hearing loss and muscular dysfunction were screened for possible A3243G mutation tRNA(Leu (UUR)) in mitochondrial DNA. We selected 7 patients from 3 of the 4 generations of the family and 10 unrelated healthy control subjects for mutation analysis using denaturing gradient gel electrophoresis (DGGE) and both manual and automated DNA sequencing. The A3243G mutation was found in peripheral blood cells of all 7 patients, but in none of the controls. The mutation was in the form of heteroplasmy and the amount of mutant DNA was found to be between 10% and 35% of total mtDNA in individual patients. This is the first report of a Norwegian family with maternally inherited diabetes and hearing loss carrying the A3243G mutation in mitochondrial DNA.

Cost-effectiveness in establishing hemophilia carrier detection and prenatal diagnosis services in a developing country with limited health resources.

The cost-effectiveness of carrier detection and prenatal diagnosis for hemophilia at the International Hemophilia Training Center, Bangkok, Thailand was studied. From 1991 to 2002, 209 females from 124 families with hemophilia A and B were included. There were 180 hemophilia A carriers and 29 hemophilia B carriers which could be classified into 78 obligate and 131 possible carriers. The phenotypic analysis for possible carriers involved the determination of levels of factor VIII or IX clotting activity (FVIII:C, FIX:C) and the ratio of FVIII:C and von Willebrand factor antigen. The result revealed that 49 females (37.4%) were diagnosed as carriers, 65 females (49.6%) were normal and 17 females (13%) were undetermined. Additional genotypic analysis was provided to 46 families with 74 females with obligate, proven or undetermined carriers within the reproductive life. The polymorphisms associated with factor VIII and IX genes were used including Bcl I for the factor VIII gene and combined use of Mse I, Sal I, Nru I, Hha I and Dde I for the factor IX gene. The informative rate was 59.4% (44/74). Consequently, 12 prenatal diagnoses for fetus at risk were performed. Sex determination was initially determined and followed by the diagnosis of hemophilia through informative gene tracking and/or the measurement of fetal levels of FVIII:C or FIX:C. The result revealed that 3 male fetuses were affected. The total cost of carrier detection and prenatal diagnosis that the families had to pay in the government hospital was 238,600 Baht (US dollars 5,965). It was compared to the estimated cost of minimal replacement therapy using lyophilized cryoprecipitate for the survival time of 30 years in one patient with hemophilia of 1,012,500 Baht (US dollars 25,312.5). The cost of prevention was much less than the replacement therapy. In conclusion, it is cost-effective to establish the service for carrier detection and prenatal diagnosis for hemophilia especially in developing countries with limited health resources.

Actor-network theory: a tool to support ethical analysis of commercial genetic testing.

Social, ethical and policy analysis of the issues arising from gene patenting and commercial genetic testing is enhanced by the application of science and technology studies, and Actor-Network Theory (ANT) in particular. We suggest the potential for transferring ANT's flexible nature to an applied heuristic methodology for gathering empirical information and for analysing the complex networks involved in the development of genetic technologies. Three concepts are explored in this paper--actor-networks, translation, and drift--and applied to the case of Myriad Genetics and their commercial BRACAnalysis genetic susceptibility test for hereditary breast cancer. Treating this test as an active participant in socio-technical networks clarifies the extent to which it interacts with, shapes and is shaped by people, other technologies, and institutions. Such an understanding enables more sophisticated and nuanced technology assessment, academic analysis, as well as public debate about the social, ethical and policy implications of the commercialization of new genetic technologies.

Learning from the voiceless.

This article is concerned with understanding what is at stake in the everyday lives of family members facing Huntington's Disease (HD). The methodological and analytical point of departure is German critical psychology, particularly the category of conduct of everyday life (Holzkamp, 1995; Dreier, 1999). Specifically, I address questions of accessing and understanding the conduct of everyday life of persons facing HD who are not visibly active with respect to this circumstance. The question of access is not merely about getting in touch with persons who are not known to the research, professional and HD communities, but also about the consequences of establishing contact with persons who have not made an entry into any of these public areanas themselves. The question of understanding is about developing an analysis from a first-person perspective on the personal conduct of everyday life that is not visibly active. The development of such an understanding has broader implications, not just for further research and health care practices, but importantly also for the prevailing moral and ethical demands made on persons living at risk of hereditary diseases.

Genetic testing and primary care: a new ethic for a new setting.

For several decades, clinical geneticists have espoused two key ethical principles, nondirectiveness and confidentiality. These principles made a great deal of sense in the highly personal and controversial setting of reproductive genetics. Now that clinical genetics has entered the primary care setting, clinicians are rethinking the strength of their commitment to these traditional norms and they are revamping their ethical priorities. Patients increasingly need advice about whether they should take genetic tests and whether and how they should respond to the test results. Patients also need to know about how this information will impact family members and whether other members of their family should be tested. Clinical geneticists may even consider breaking individual confidentiality in order to prevent harms to family members. Although clinical geneticists do not need to abandon nondirectiveness and confidentiality in this new setting, they may not strictly adhere to these principles in some circumstances in order to benefit patients and their families.

Insurance companies' access to genetic information: why regulation alone is not enough.

The background of this paper is the ongoing dismantling of the social insurance systems in favour of commercialisation and privatisation of insurances needed for illness, old age and premature death. This combined with the increased possibility of using genetic testing for differentiating personal insurance premiums has the potentiality of creating a 'genetic proletariat'--an uninsurable high-risk population. The common way of handling this problem in Sweden, and many other developed countries around the North Atlantic, has been to regulate insurance companies' right to ask for and use genetic information in various ways. There is a distinction between partial regulation (that allows insurance companies access to genetic information from genetic tests already made, sometimes only above a specified amount, but not to demand new tests) and total regulation (that forbids insurance companies to ask for or use any genetic information). I will argue that these forms of regulation probably will have adverse consequences given the dismantling of collective social insurance systems. If this is convincing, a better way to solve the problem of an uninsurable high-risk population (and other problems) is to resurrect the collective, obligatory insurance systems in which the individual risk profile does not constitute a basis for premium determination. Both arguments cast in terms of consequences and justice render support for this conclusion.

Differences in the frequency of the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene among the Lebanese population.

In view of its role in precipitating mild hyperhomocysteinemia as well as being a risk factor for vascular thrombosis, we investigated the frequency of the C677T mutation of the methylenetetrahydrofolate reductase (MTHFR) gene among 589 healthy Lebanese subjects by PCR-RFLP analysis (HinfI digestion) and compared them with those of other countries of Caucasian and non-Caucasian origin. The prevalence of the mutated homozygous (T/T) and heterozygous (C/T) C677T MTHFR genotype was 11.04% and 39.73%, respectively, giving an allele frequency of 0.309. While the prevalence of the T/T genotype was similar with respect to gender, higher prevalence was noted among Christian (13.08%) compared to Moslem (7.66%) subjects (P &lt; 0.001), and heterogeneity in its distribution was seen in the different Lebanese provinces, and was directly related to the Christian/Moslem composition of each province. The distribution of the MTHFR C677T in Lebanon is unique with regard to its higher occurrence among Christians compared to Moslems, adding to the existing body of literature on the heterogeneity of its prevalence and distribution.

[Gitelman's syndrome--a differential diagnosis in hypokalemia].

Gitelman's syndrome is a rare disease characterised by low levels of potassium and magnesium in the blood. It is caused by mutations in the gene encoding the thiazide-sensitive sodium chloride cotransporter in the distal collecting duct.</AbstractText>We present four patients (two brothers and two sisters) with Gitelman's syndrome and review the literature regarding the disease.</AbstractText>Gitelman's syndrome should be considered in patients with persistently low levels of potassium and magnesium. The diagnosis is confirmed by genetic testing.</AbstractText>

BRCA1 and sex ratio.

Two recent papers suggest distorted sex and transmission ratios associated with BRCA1 mutations. If real, these would provide novel insights into the normal biological function of this gene and have implications for genetic epidemiologic methods used to estimate penetrance. We addressed these observations in two settings: offspring of 283 mutation carriers and 471 mutation negative subjects from BRCA1/2 mutation-positive families with multiple cases of breast and ovarian cancer (NCI families); and relatives of 115 BRCA1/2 mutation carriers from the Washington Ashkenazi Study (WAS). The male:female ratio was below one in both BRCA1 (0.85, 95% CI 0.7-1.1 in NCI families; 0.90, 95% CI 0.6-1.4 in WAS) and BRCA2 families (0.77, 95% CI 0.5-1.3 and 0.80, 95% CI 0.5-1.2, in the NCI and WAS study groups, respectively). None of the sex ratios deviated significantly from one, and there was no significant difference between BRCA1 and BRCA2 families. The reduced sex ratio was due largely to the offspring of males, a distortion that is probably an artifact of ascertainment biases. Among adult daughters without breast or ovarian cancer born to mutation carriers, as expected, fewer than 50% were mutation carriers (39% in BRCA1 families and 44% in BRCA2 families). It is difficult, due to ascertainment biases, to draw firm conclusions regarding sex ratios in studies of a sex-limited phenotype. Nonetheless, these observations do not support the idea that BRCA1 mutation carriers have a lower ratio of male offspring than BRCA2 mutation carriers.

The prevalence and spectrum of alpha and beta thalassaemia in Guangdong Province: implications for the future health burden and population screening.

Thalassaemia is a good candidate disease for control by preventive genetic programmes in developing countries. Accurate population frequency data are needed for planning the control of thalassaemia in the high risk Guangdong Province of southern China.</AbstractText>In total, 13397 consecutive samples from five geographical areas of Guangdong Province were analysed for both haematological and molecular parameters.</AbstractText>There was a high prevalence of carriers of alpha thalassaemia (8.53%), beta thalassaemia (2.54%), and both alpha and beta thalassaemia (0.26%). Overall, 11.07% of the population in this area were heterozygous carriers of alpha and beta thalassaemia. The mutation spectrum of alpha and beta thalassaemia and its constitution were fully described in this area. This study reports the true prevalence of silent alpha thalassaemia in the southern China population for the first time. In addition, two novel mutations that give rise to alpha thalassaemia, one deletion resulting in beta thalassaemia, and a rare deletion (--(THAI) allele) previously unreported in mainland China were detected. The frequency of the most common mutation, the Southeast Asian type of deletion (--(SEA), accounting for 48.54% of all alpha thalassaemias) was similar to the total of two alpha(+) thalassaemia deletions (-alpha(3.7) and -alpha(4.2), accounting for 47.49% of alpha thalassaemia).</AbstractText>Both alpha and beta thalassaemia are widely distributed in Guangdong Province of China. The knowledge gained in this study will enable the projected number of pregnancies at risk to be estimated and a screening strategy for control of thalassaemia to be designed in this area.</AbstractText>

HER-2 testing in breast cancer using parallel tissue-based methods.

Testing for HER-2 oncogene in breast cancer has increased because of its role as a prognostic and predictive factor. Some advocate gene testing by fluorescence in situ hybridization (FISH) vs protein testing by immunohistochemistry as the method which most accurately evaluates and predicts response to the anti-HER-2 antibody, trastuzumab. However, critical examination of FISH on a screening basis has yet to be performed.</AbstractText>To determine the correlation between FISH and immunohistochemistry results by determining HER-2/neu gene status on tumor sections with indeterminate immunohistochemistry results (2+ score), confirm gene amplification on tumor sections with positive results (3+ score), and verify gene status on tumor sections with negative results (0 or 1+ score).</AbstractText><AbstractText Label="DESIGN, SETTING, AND PATIENTS" NlmCategory="METHODS">A quality control and quality assurance program for HER-2 testing by FISH, which used tumor specimens from 2963 patients (median age, 56 years) with breast cancer received from 135 hospitals and cancer centers in 29 states, was performed at a reference laboratory from January 1, 1999, to May 15, 2003. Every specimen evaluated by FISH was parallel tested with immunohistochemistry tests.</AbstractText>With FISH as the presumed standard testing method, the positive and negative predictive values and sensitivity and specificity of immunohistochemistry were calculated.</AbstractText>A total of 3260 clinical HER-2 tests by FISH were performed on 2963 serially referred breast cancer specimens. Of these, 2933 tests were successful and 2913 breast cancer specimens had both FISH and immunohistochemistry results available. With FISH as the standard testing method, the positive predictive value of positive immunohistochemistry score (3+) was 91.6%, and the negative predictive value of negative immunohistochemistry score (0 or 1+) was 97.2%. The sensitivity of immunohistochemistry tests, including tumor sections with scores of 2+ or 3+, was 92.6% and the specificity of immunohistochemistry tests with scores of 3+ was 98.8%. The FISH test had a significantly higher failure rate (5% vs 0.08%) and reagent cost (140 dollars vs 10 dollars), and longer testing (36 hours vs 4 hours) and interpretation times (7 minutes vs 45 seconds) vs immunohistochemistry tests.</AbstractText>A testing algorithm for HER-2 determination is most efficient by using immunohistochemistry as the method of choice, with FISH performed for cancers with indeterminate results (2+ score). Successful quality control and quality assurance programs are a prerequisite for such approaches.</AbstractText>

Familial aggregation of energy intake in children.

Uncompensated overnutrition promotes obesity, but the controls of children's eating behavior are poorly understood. Insights may be achieved by testing whether the eating patterns of children are associated with demographic variables or whether they aggregate among family members.</AbstractText>We tested whether children's total energy intake and macronutrient intake and their ability to compensate for earlier energy intake were associated with sociodemographic variables and anthropometric indexes. We also tested whether these behavioral traits aggregate among siblings.</AbstractText>Thirty-two sibling pairs aged 3-7 y consumed a multi-item lunch preceded by a low-energy (12.55 kJ) or high-energy (627.60 kJ) preload drink. Mixed-models regression tested the associations between children's energy intake, demographic variables, and anthropometric measures. An intraclass correlation coefficient quantified the family correlation of the measures of children's eating.</AbstractText>Children consumed significantly more total energy after consuming the low-energy preload ( +/- SD: 2237.39 +/- 1176.45 kJ) than after consuming the high-energy preload (1601.18 +/- 930.65 kJ). Compensation ability was unrelated to the children's age, sex, or ethnicity. Total energy and macronutrient intake, but not compensation propensity, were associated among siblings.</AbstractText>The familial association of total energy and macronutrient intakes, independent of anthropometric measures, suggests genetic or home environmental influences specific to these behaviors. Short-term energy compensation, although very accurate within this sample, showed no significant familial correlation.</AbstractText>

Review article: Osteoporosis and inflammatory bowel disease.

Studies using dual-energy X-ray absorptiometry have suggested a high prevalence of osteoporosis in inflammatory bowel disease. However, population-based data on fracture incidence suggest only a small increased risk of fracture amongst patients with inflammatory bowel disease compared with the general population. Therefore, it would be helpful to identify patients with inflammatory bowel disease at particularly high risk for fracture so that these risks might be modified or interventions might be undertaken. The data on calcium intake as a predictor of bone mineral density are conflicting. Although there are data suggesting that a one-time survey to determine current calcium intake will not help to predict bone mineral density in inflammatory bowel disease, persistently reduced calcium intake does appear to lead to lower bone mineral density. In the general population, body mass is strongly correlated with bone mineral density, which also appears to be true in Crohn's disease. Hence, subjects with inflammatory bowel disease and considerable weight loss, or who are obviously malnourished, could be considered for bone mineral density testing, and the finding of a low bone mineral density would suggest the need for more aggressive nutritional support. Although vitamin D is undoubtedly important in bone health, vitamin D intake and serum vitamin D levels do not correlate well with bone mineral density. Sex hormone deficiency can also adversely affect bone health, although a well-developed strategy for sex hormone measurements in patients with inflammatory bowel disease remains to be established. Ultimately, the determination of genetic mutations that accurately predict fracture susceptibility may be the best hope for developing a simplified strategy for managing bone health in inflammatory bowel disease. The therapy of osteoporosis in inflammatory bowel disease has been adapted from other osteoporosis settings, such as post-menopausal or corticosteroid-induced osteoporosis. To date, there remains no therapy proven to be efficacious in inflammatory bowel disease-related osteoporosis; however, calcium and vitamin D supplementation and bisphosphonates have their roles.

Classical phenotype of Laron syndrome in a girl with a heterozygous mutation and heterozygous polymorphism of the growth hormone receptor gene.

We describe here a 19 month-old girl with classical Laron syndrome (LS). Molecular analysis of the GH receptor gene in the patient and her parents was performed. The patient was found to be heterozygous for a mutation in exon 4 (R43X) and heterozygous for a polymorphism in exon 6 (Gly168Gly). Her mother was also heterozygous for R43X but homozygous for the polymorphism. In the father, a heterozygous polymorphism was found. Contrary to previous assumptions that only homozygous patients express the typical phenotype, this patient shows all the classical features of LS, despite being a heterozygote for a pathological defect.

HLA-DQA1 and -DQB1 alleles in Latino and African American children with diabetes mellitus.

Few studies have described the genetics of childhood diabetes mellitus (DM) in US minorities. High-risk DQA1 and DQB1 alleles (DQA1*0301, DQB1*0201, and DQB1*0302 in African Americans and Latinos, and DQA1 *0501 in African Americans) were identified from previous studies and tested in 45 African American and 26 Latino patients from the population-based Chicago Childhood Diabetes Registry, and in 50 healthy race-matched controls. Sixteen of the African American patients and three Latinos had youth-onset type 2 DM and were analyzed separately. In African Americans with type 1 DM, both DQA1*0102 and DQB1*0602 were protective (p &lt; 0.0001), and the susceptibility alleles DQA1*0301 and DQB1*0201 were more frequent than in controls (p &lt; 0.01). In Latinos, DQA1*0301 and DQB1*0302 were marginally increased in patients with DM1 compared to controls; no individual DQA1 or DQB1 allele was protective. Patients with DM1 were significantly more likely to carry one or two high-risk DQA1 alleles in both populations; they were also more likely than controls to carry at least one high-risk DQB1 allele. The odds ratio for the ability to form at least two high-risk DQA1-DQB1 heterodimers (cis and/or trans) was 7.9 (95% CI: 1.7-40.0) for African Americans and 5.7 (1.3-25.6) for Latinos with DM1. African American patients with DM2 were not statistically different from controls, and were less likely to carry four high-risk susceptibility alleles than patients with DM1 (p = 0.002). Many of the HLA-DQ associations previously documented in non-Hispanic White populations also are found in African Americans and Latinos with DM1, although some differences exist.

An unusual case of X-linked adrenoleukodystrophy with auditory processing difficulties as the first and sole clinical manifestation.

X-linked adrenoleukodystrophy (X-ALD) is characterized by demyelination that is associated with a deficient beta-oxidation of very long chain fatty acids. We report the unusual case of a male adult with X-ALD who was diagnosed at the age of 26 by a brain MRI performed because his brother had been diagnosed with a rapidly deteriorating form of X-ALD. His sole symptom was hearing difficulties in the presence of a normal audiogram since childhood. He has remained stable for seven years. Central auditory testing in our patient revealed severe deficits in several auditory processes. These findings correlated with involvement of the auditory pathway at the level of the trapezoid body, and posterior corpus callosum in particular, on his brain MRI. This case highlights not only the need for thorough audiological investigation of the patient who complains of hearing difficulties in the presence of a normal audiogram, but also that audiological investigations could be of value in the phenotypic evaluation of cases with adrenoleukodystrophy.

Post-conviction DNA testing: the UK's first 'exoneration' case?

The routine incorporation of forensic DNA profiling into the criminal justice systems of the United Kingdom has been widely promoted as a device for improving the quality of investigative and prosecutorial processes. From its first uses in the 1980s, in cases of serious crime, to the now daily collection, analysis and comparison of genetic samples in the National DNA Database, DNA profiling has become a standard instrument of policing and a powerful evidential resource for prosecutors. However, the use of post-conviction DNA testing has, until recently, been uncommon in the United Kingdom. This paper explores the first case, in England, of the contribution of DNA profiling to a successful appeal against conviction by an imprisoned offender. Analysis of the details of this case is used to emphasise the ways in which novel forms of scientific evidence remain subject to traditional and heterogeneous tests of relevance and credibility.

[Importance of genetic testing in couples with reproductive disorders].

To determine the prevalence of chromosomal aberrations in infertile couples undergoing in vitro fertilization (IVF).</AbstractText>Cytogenetic analysis of peripheral blood lymphocytes in the group of patients undergoing IVF. Detection of chromosomal aberrations in the fetuses after IVF.</AbstractText>Department of Medical Genetics and Fetal Medicine, Medical Faculty, Palack&#xfd; University and the University Hospital, Olomouc.</AbstractText>Cultivation of peripheral blood lymphocytes or fibroblasts of amniotic fluid. Using fluorescent in situ hybridization in cases of mosaicism.</AbstractText>Out of 638 patients undergoing treatment for male or female infertility, 595 had normal karyotype and 43 (6.8%) had abnormal karyotype. There were detected 9 (1.4%) cases of balanced chromosomal rearrangements, 2 (0.31%) cases of deletion of Y chromosome, 2 (0.31%) cases of inversion, 2 (0.31%) cases of marker chromosome, 5 (0.78%) cases of gonosomal aneuploidy (47,XXY) and 23 (3.65%) cases of gonosomal mosaicism--out of the 22 (3.5%) cases of low-level mosaicism. In the small group of pregnant patients after IVF investigated for the risk of genetic disorders included in our study (n = 60) the frequency of chromosomal abnormalities was 9 (15%).</AbstractText>Our data show that a high number of infertile couples is affected by chromosomal aberrations which occur more frequently in females than in males. It is caused by high frequency of low-level gonosomal mosaicism in the group of infertile women. Chromosomal analyses are highly recommended before each IVF procedure.</AbstractText>

Can flies help humans treat neurodegenerative diseases?

Neurodegenerative diseases are becoming increasingly common as life expectancy increases. Recent years have seen tremendous progress in the identification of genes that cause these diseases. While mutations have been found and cellular processes defined that are altered in the disease state, the identification of treatments and cures has proven more elusive. The process of finding drugs and therapies to treat human diseases can be slow, expensive and frustrating. Can model organisms such as Drosophila speed the process of finding cures and treatments for human neurodegenerative diseases? We pose three questions, (1) can one mimic the essential features of human diseases in an organism like Drosophila, (2) can one cure a model organisms of human disease and (3) will these efforts accelerate the identification of useful therapies for testing in mice and ultimately humans? Here we focus on the use of Drosophila to identify potential treatments for neurodegenerative diseases such as Huntington's and we discuss how well these therapies translate into mammalian systems.

Complex haplotypes of the PGC-1alpha gene are associated with carbohydrate metabolism and type 2 diabetes.

Peroxisome proliferator-activated receptor coactivator-1alpha (PGC-1alpha) is a transcriptional coactivator implicated in transcriptional programs of hepatic gluconeogenesis, oxidative phosphorylation, and insulin release by beta-cells. To study associations of the PGC-1alpha gene locus with carbohydrate metabolism and type 2 diabetes in humans, we identified several polymorphisms in the promoter region that were located in a haplotype block distinct from a second haplotype block containing part of intron 2 and extending beyond exon 13. Each block contained five common haplotypes. Oral glucose tolerance testing revealed associations of promoter haplotype combinations with 30- and 60-min postload plasma glucose levels, whereas haplotypes in both blocks were associated with indexes of beta-cell function. The associations of promoter haplotypes are supported by functional studies showing that some polymorphisms are located in transcription factor binding sites and affect transactivation in an allele-specific manner. By comparing patients with type 2 diabetes and control subjects, we observed borderline significant differences of four-loci haplotype distributions in the downstream haplotype block. Moreover, the haplotype that was associated with the strongest insulin response to glucose conferred the lowest risk of type 2 diabetes (P &lt; 0.01). Thus, the PGC-1alpha gene locus influences carbohydrate metabolism and contributes to type 2 diabetes in the population studied.

An oncologist-based model of cancer genetic counselling for hereditary breast and ovarian cancer.

We describe a multistep model of cancer genetic counselling designed to promote awareness, and disease surveillance and preventive measures for hereditary and familial breast and ovarian cancer.</AbstractText>Step T0 of the model entails information giving; this is followed by pedigree analysis and risk estimation (T1), risk communication and genetic testing (T2), and genetic test result communication (T3). User consent was required to proceed from one step to the next. Surveillance and preventive measures are proposed to at-risk users. Of the 311 subjects who requested cancer genetic counselling, consent data to each counselling step were available for 295: 93 were disease-free, 187 had breast cancer, 12 had ovarian cancer and three had breast plus ovarian cancer.</AbstractText>Consent was high at T0 (98.39%), T1 (96.40%) and T2 (99.65%). Consent decreased at the crucial points of counselling: T2 (87.71%) and T3 [genetic test result communication (85.08%), and extension of counselling to and testing of relatives (65.36%)].</AbstractText>The model fosters the user's knowledge about cancer and favours identification of at-risk subjects. Furthermore, by promoting awareness about genetic testing and surveillance measures, the algorithm enables users to make a fully informed choice of action in case of predisposing or familial cancer risk.</AbstractText>

A novel and accurate diagnostic test for human African trypanosomiasis.

Human African trypanosomiasis (sleeping sickness) affects up to half a million people every year in sub-Saharan Africa. Because current diagnostic tests for the disease have low accuracy, we sought to develop a novel test that can diagnose human African trypanosomiasis with high sensitivity and specificity.</AbstractText>We applied serum samples from 85 patients with African trypanosomiasis and 146 control patients who had other parasitic and non-parasitic infections to a weak cation exchange chip, and analysed with surface-enhanced laser desorption-ionisation time-of-flight mass spectrometry. Mass spectra were then assessed with three powerful data-mining tools: a tree classifier, a neural network, and a genetic algorithm.</AbstractText>Spectra (2-100 kDa) were grouped into training (n=122) and testing (n=109) sets. The training set enabled data-mining software to identify distinct serum proteomic signatures characteristic of human African trypanosomiasis among 206 protein clusters. Sensitivity and specificity, determined with the testing set, were 100% and 98.6%, respectively, when the majority opinion of the three algorithms was considered. This novel approach is much more accurate than any other diagnostic test.</AbstractText>Our report of the accurate diagnosis of an infection by use of proteomic signature analysis could form the basis for diagnostic tests for the disease, monitoring of response to treatment, and for improving the accuracy of patient recruitment in large-scale epidemiological studies.</AbstractText>

Optimising clinical practice in cancer genetics with cultural competence: lessons to be learned from ethnographic research with Chinese-Australians.

Hereditary cancer is about families, and clinicians and genetic counsellors need to understand the cultural beliefs of patients and families about cancer and inheritance. In the light of their kinship patterns Chinese-Australians were chosen for the present study, which aims to determine the explanatory models of inheritance, cancer, and inherited cancer, with a view to identifying the relationship between these culture-specific lay attributions and help-seeking behaviour, and to identify possible barriers to genetic counselling and testing. Qualitative ethnographically informed methodology involving semi-structured interview was used as a method to uncover latent beliefs held by the families who are represented by the subjects. In-depth interviews were conducted with 16 informants of Chinese ethnicity, who had been recruited through two major Sydney familial cancer clinics. We report the attributions of cancer in general, then on inheritance, kinship, genes and genetics and then focus on the way in which these beliefs come together around hereditary cancer. The majority of informants, despite high acculturation and belief in biomedical explanations about hereditary cancer, also acknowledged the influence of traditional family Chinese beliefs, where 'inheritance' and 'genetics' were related to retribution for ancestral misdeeds and offending ancestors. Extensive mismatch of attributes and beliefs were identified in those who attended the clinic and senior family members, creating barriers to optimal service utilisation. Three traditional patterns of beliefs were identified: (a) father and mother contributed in equal share to one's genetic makeup, linked to the ying-yang theory; (b) the dominance of life force (yang chi) and the shaping of genes were transmitted through the paternal line; and (c) natural and supernatural forces operated in the cause of hereditary cancer. The study provided guidance for clinical practice. Exploration and acknowledgement of family beliefs, regardless of cultural background and therefore avoiding stereotyping, can enable the clinician to work with the whole family-those who hold Western attributions, those who maintain traditional notions of genetics and inheritance, and those who incorporate both into their belief systems-and provide effective clinical services. Further ethnographic studies are needed, focusing on the Chinese groups who do not attend the clinic and those with lower acculturation and educational levels.

Integrating genetics as practices of primary care.

This study examines the responses of general practitioners (GPs) in Vic., Australia to an increased emphasis on genetics in primary care. A qualitative analysis of focus group interviews with GPs in regional and metropolitan areas and one focus group interview with genetics experts showed that despite the emphasis placed on genetics by the experts, GPs remained ambivalent to the routine integration of genetics into general practice. This response from GPs has been noted in several studies and is most commonly attributed to GPs' lack of knowledge about genetics. In this study we argue that a 'cognitive deficit' understanding of the problem excludes many of the factors that GPs regard as important in relating genetics to primary care. We show that GPs' ambivalence emerges from how they situate genetics within practices of patient care and in relation to what they regard as good patient management. We found that GPs respond most enthusiastically to genetics and genetic testing if they feel it changes their management in ways they consider of benefit to their patients. GPs have specialist skills in managing the heterogeneity of patient care and these skills enable them to situate genetics relative to the overall needs of patients. Preparing GPs to 'do' genetics when the need arises by finding ways to make genetics information available to them as the need arises would facilitate the integration of genetics as practices of primary care.

Human mitochondrial transcription factor B1 as a modifier gene for hearing loss associated with the mitochondrial A1555G mutation.

Phenotypic expression of the deafness-associated homoplasmic A1555G mutation in the mitochondrial 12S rRNA gene varies from profound congenital hearing loss to normal hearing. It has been shown that this variability in clinical expression in most patients is due to the complex inheritance of multiple nuclear-encoded modifier genes. Human mitochondrial transcription factor B1 (TFB1M) has been proposed as a candidate for being such a modifier, since it methylates adenine residues in the adjacent loop of the A1555G mutation in the 12S rRNA gene. Polymorphic markers within and adjacent to the TFB1M gene were genotyped in 214 individuals from 41 multiplex families with the A1555G mutation of Spanish, Italian, and Arab-Israeli origin. Multipoint non-parametric linkage analysis of all families combined revealed an NPL score of 1.7 (P = 0.05), and a Lod score of 1.4 (P = 0.04). Linkage disequilibrium by the Transmission Disequilibrium Test at D6S1577, a microsatellite adjacent to TFB1M, showed preferential non-transmission of an allele to affected individuals with chi2 = 8.76; P = 0.003. Sequence analysis of the coding region of the gene and testing of all intragenic SNPs did not reveal a putative causative mutation. These data provide suggestive evidence that TFB1M is a nuclear-encoded modifier gene for phenotypic expression of the A1555G mutation, and that the effect may occur through a regulatory or splicing mutation.

Analysis of 16 Y STR loci in the Finnish population reveals a local reduction in the diversity of male lineages.

We analysed samples of 400 Finnish males using nine Y-chromosomal short tandem repeat (STR) loci (minimal haplotype); for 200 of these subjects an additional seven Y-chromosomal STR loci were used. The geographical distribution of the observed haplotypes was determined from 200 individuals of known paternal origin within Finland. The observed number of alleles varied from 2 to 13 alleles per locus. A total of 146 minimal haplotypes were identified in our population sample. Interestingly, 90 (22.5%) individuals shared an identical haplotype. This haplotype was extremely frequent in the northern and eastern subpopulations of Savo, Pohjanmaa and Karjala (53, 42 and 37%, respectively). With the seven additional loci analysed in the sample of 200 individuals, 120 haplotypes were identified, and individuals sharing the most common haplotype decreased to 13.0%. However, in comparison to other European populations, the Finnish population showed decreased genetic diversity (GD) when the number of different minimal haplotypes in the population was divided by the sample size (36.5% in Finns versus 83.7% on average). Our results strongly support the earlier hypothesis of individual isolated Y-chromosomal lineages and population substructuring in Finland. For paternity testing, power of exclusion was 92% using minimal haplotype data, but including the seven additional loci this value increased to 97%.

A polymorphic locus in the intron 16 of the human angiotensin-converting enzyme (ACE) gene is not correlated with complex regional pain syndrome I (CRPS I).

Exaggerated neurogenic inflammation has been recognized to be one reason for many CRPS symptoms. Since angiotensin-converting enzyme (ACE) is a key enzyme for the termination of neurogenic inflammation, it has been selected as a candidate gene for CRPS predisposition. A previous report of an insertion/deletion (I/D) polymorphism in intron 16 within the ACE gene implicated an increased risk to develop CRPS I associated with the D allele. However, in the present study the D allele frequency was not increased in CRPS I cases (0.51 for D allele, 0.49 for I allele). Furthermore, there was no co-segregation of any genotype (DD, ID, II) with the CRPS phenotype in 12 selected familial CRPS I cases from six CRPS I families. In conclusion, the results presented herein render this particular ACE gene polymorphism unlikely to be a predisposing factor for CRPS I.

Genetic study of a large Chinese kindred with von Hippel-Lindau disease.

Von Hippel-Lindau (VHL) disease is a heraditary cancer syndrome caused by germline mutations of the VHL tumor on the suppressor gene. This study was to show the clinical characteristics of a large Chinese kindred with von Hippel-Lindau disease and to evaluate the role of the genetic test of VHL disease in the diagnosis of VHL disease and clinical screening of members of the VHL disease family.</AbstractText>DNA extracted from peripheral blood was amplified by PCR to three exons of the VHL gene in 27 members of a large kindred with VHL disease. PCR products were directly sequenced. The involvements of multi-organs in the kindred with VHL disease were confirmed by history taking and radiography.</AbstractText>Of 47 members in the four generations of the kindred, 18 members were diagnosed as having VHL disease. Clinical manifestations of 18 patients included: central nervous system (CNS) hemangioblastoma (5), renal cell carcinoma and CNS hemangioblastoma (3), renal cell carcinoma and retinal angioma (3), renal cell carcinoma and multiple pancreatic cysts (1), renal cell carcinoma and retinal angioma and multiple pancreatic cysts (2), renal cell carcinoma and CNS hemangioblastomas and multiple pancreatic cysts (1), and multiple pancreatic cysts and multiple renal cysts (1), multiple pancreatic cysts (2). The common lesions of the 18 patients were renal cell carcinoma (55.6%), CNS hemangioblastoma (50.0%), retinal angioma (27.8%), and multiple pancreatic cysts (38.9%). Among the 27 members who volunteered for genetic analysis, 15 members including 9 affected family patients and 2 asymptomatic patients and 4 carriers, who are still alive, presented a codon 78 from Asn to Ser change at nucleotide 446 (A--&gt;G) in exon 1. Four members were carriers with the same VHL gene mutation. Two asymptomatic patients were initially diagnosed by genetic testing and subsequently confirmed radiologically and surgically. Members without gene mutation had no clinical evidence of VHL disease.</AbstractText>The large Chinese kindred with VHL disease was classified as type I. The main characteristics in the kindred were higher incidence of renal cell carcinoma and lower incidence of retinal angioma. Genetic test plays an important role in early detecting asymptomatic patients and the carriers in clinical screening of members of the families with VHL disease. It is also important to prevent the transmission of VHL disease to their offsprings in the kindred.</AbstractText>

Selection for avian immune response: a commercial breeding company challenge.

Selection for immune function in the commercial breeding environment is a challenging proposition for commercial breeding companies. Immune response is only one of many traits that are under intensive selection, thus selection pressure needs to be carefully balanced across multiple traits. The selection environment (single bird cages, biosecure facilities, controlled environment) is a very different environment than the commercial production facilities (multiple bird cages, potential disease exposure, variable environment) in which birds are to produce. The testing of individual birds is difficult, time consuming, and expensive. It is essential that the results of any tests be relevant to actual disease or environmental challenge in the commercial environment. The use of genetic markers as indicators of immune function is being explored by breeding companies. Use of genetic markers would eliminate many of the limitations in enhancing immune function currently encountered by commercial breeding companies. Information on genetic markers would allow selection to proceed without subjecting breeding stock to disease conditions and could be done before production traits are measured. These markers could be candidate genes with known interaction or involvement with disease pathology or DNA markers that are closely linked to genetic regions that influence the immune response. The current major limitation to this approach is the paucity of mapped chicken immune response genes and the limited number of DNA markers mapped on the chicken genome. These limitations should be eliminated once the chicken genome is sequenced.

Non-major histocompatibility complex alloantigen genes affecting immunity.

An alloantigen is a genetically determined cell-surface molecule detected by specific antisera. An identifying letter has been assigned to each genetic locus responsible for the 12 distinct families of alloantigens: A, B, C, D, E, H, I, J, K, L, P, and R. The genes of each system segregate independently of the other systems, except that the A and E are very closely linked (0.5 centimorgans). Selection experiments over numerous generations have revealed distinct changes in gene frequency of the A-E alloantigens, suggesting immune responses associated with susceptibility to coccidiosis, response to immunizations with SRBC, and selection for size of the bursa of Fabricius. Immune response effects of the C system of alloantigen genes are indicated by distinct gene frequency changes following selection for response to SRBC, selection for size of bursa of Fabricius, and macrophage nitrite production after lipopolysaccharide (LPS) stimulation. Immune response effects of the D system of antigens are indicated by data from genetic selection for response to immunization with SRBC, selection for bursa size, and macrophage nitrite and cytokine interleukin (IL)-6 production following LPS stimulation. Immune response effects of the I system genes are indicated by distinct gene frequency changes in lines selected for bursa size and within family comparisons for macrophage nitrite and cytokine IL-6 production following LPS stimulation. Effects of the L system, consisting of only 2 alleles, are indicated by the gene frequency changes following selection for bursa size, direct comparison of genotypes within families for monocyte phagocytosis, susceptibility to coccidiosis, outcome of Rous sarcomas, and immune responses to SRBC and Brucella abortus. Genotypes of the P alloantigen system were directly compared within families of fully pedigreed chicks with significant differences for monocyte phagocytosis. An experimental procedure for simultaneously testing for immune responses of genotypes of 9 of the alloantigen systems (A, B, C, D, E, H, I, L, and P) has been established by producing test progeny from a single cross of parent lines segregating for genes of each of the systems.

Analysis of chicken TLR4, CD28, MIF, MD-2, and LITAF genes in a Salmonella enteritidis resource population.

Salmonella enteritidis is a foodborne pathogen that negatively affects both animal and human health. Genetic variations in response to pathogenic SE colonization or to SE vaccination were measured in a chicken resource population. Outbred broiler sires and 3 diverse, highly inbred dam lines produced 508 F1 progeny that were evaluated for either bacterial colonization after pathogenic SE inoculation or circulating antibody level after SE vaccination. Five candidate genes were selected for study, based on their biological function as possibly affecting response to SE: toll-like receptor 4 (TLR4), T-cell specific surface protein (CD28), macrophage migration inhibitory factor (MIF), MD-2, and lipopolysaccharide-induced tumor necrosis factor (TNF)-alpha factor (LITAF). Gene fragments were sequenced from the founder lines of the resource population. The LITAF and MIF genes were homozygous for all sires. Single nucleotide polymorphisms (SNP) were identified in 3 genes (TLR4, CD28, and MD-2) and were used to test for associations of sire SNP with SE response. Linear mixed models were used for statistical analyses. The CD28 broiler sire SNP was associated with both bacterial load in the cecum (P &lt; 0.003) and vaccine antibody response (P &lt; 0.05). The MD-2 SNP was associated (P &lt; 0.04) with the bacterial load in the spleen. The use of these SNP in these genes in marker-assisted selection may result in enhancement of disease resistance.

PKHD1 mutations in families requesting prenatal diagnosis for autosomal recessive polycystic kidney disease (ARPKD).

Autosomal recessive polycystic kidney disease (ARPKD) is one of the most common hereditary renal cystic diseases in children. The clinical spectrum ranges from stillbirth and neonatal demise to survival into adulthood. In a given family, however, patients usually display comparable phenotypes. Many families who lost a child with severe ARPKD desire an early and reliable prenatal diagnosis (PD). Given the limitations of antenatal ultrasound, this is only feasible by molecular genetics that became possible in 1994 when PKHD1, the locus for ARPKD, was mapped to chromosome 6p. However, linkage analysis might prove difficult or even impossible in families with diagnostic doubts or in whom no DNA of an affected child is available. In such cases the recent identification of the PKHD1 gene provides the basis for direct mutation testing. However, due to the large size of the gene, lack of knowledge of the encoded protein's functional properties, and the complicated pattern of splicing, significant challenges are posed by PKHD1 mutation analysis. Thus, it is important to delineate the mutational spectrum and the reachable mutation detection rate among the cohort of severely affected ARPKD patients. In the present study, we performed PKHD1 mutation screening by DHPLC in a series of 40 apparently unrelated families with at least one peri- or neonatally deceased child. We observed 68 out of an expected 80 mutations, corresponding to a detection rate of 85%. Among the mutations identified, 23 were not reported previously. We disclosed two underlying mutations in 29 families and one in 10 cases. Thus, in all but one family (98 percent;), we were able to identify at least one mutation substantiating the diagnosis of ARPKD. Approximately two-thirds of the changes were predicted to truncate the protein. Missense mutations detected were nonconservative, with all but one of the affected amino acid residues found to be conserved in the murine ortholog. PKHD1 mutation analysis has proven to be an efficient and effective means to establish the diagnosis of ARPKD.

Evidence for linkage between regulatory enzymes in glycolysis and schizophrenia in a multiplex sample.

Observations of impaired glucose regulation in schizophrenia are long-standing, although their pathological and etiological significance is uncertain. One approach to the issue that minimizes environmental variables (e.g., medication and diet) is to determine whether genes related to glucose regulation show genetic linkage to schizophrenia. We examined the potential role of glucose metabolism in schizophrenia through a genome scan of affection status in schizophrenia and an empirical method for deriving P-values. Data were utilized from the NIMH Genetics Initiative for Schizophrenia dataset, which comprises a total sample consisting of 71 pedigrees containing 218 nuclear families and 987 individuals. A genome scan with 459 markers spaced at an average of 10 cM intervals was conducted using the linkage analysis program Genehunter separately for European- and African-American groups. Enzymes that regulate glycolysis were identified and the genes regulating these enzymes were located through the Online Mendelian Inheritance in Man (OMIM) website. The focus in this study was on genes located near previously reported schizophrenia susceptibility regions. The genome-wide significance of these genes to schizophrenia was assessed using permutation testing. When results were adjusted for multiple testing within and across ethnic groups, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2; chromosome 1q32.2) achieved genome-wide significance (P = 0.04). In addition, hexokinase 3 (HK3; chromosome 5q35.3) was also suggestive of linkage (P = 0.09). For the European-American sample, PFKFB2 (1q32.2), hexokinase 3 (HK3; 5q35.3), and pyruvate kinase 3 (PK3; chromosome 15q23) achieved significance at the 0.05 level. None of the genes showed significance in the African-American sample. Our results provide further support for the view that genes that regulate glucose metabolism may also influence susceptibility to schizophrenia. More generally, they support the view that relationships between glucose dysregulation and schizophrenia are inherent to the disorder, and are not merely epiphenomena related to medication or other treatment factors.

Optimizing purebred selection for crossbred performance using QTL with different degrees of dominance.

A method was developed to optimize simultaneous selection for a quantitative trait with a known QTL within a male and a female line to maximize crossbred performance from a two-way cross. Strategies to maximize cumulative discounted response in crossbred performance over ten generations were derived by optimizing weights in an index of a QTL and phenotype. Strategies were compared to selection on purebred phenotype. Extra responses were limited for QTL with additive and partial dominance effects, but substantial for QTL with over-dominance, for which optimal QTL selection resulted in differential selection in male and female lines to increase the frequency of heterozygotes and polygenic responses. For over-dominant QTL, maximization of crossbred performance one generation at a time resulted in similar responses as optimization across all generations and simultaneous optimal selection in a male and female line resulted in greater response than optimal selection within a single line without crossbreeding. Results show that strategic use of information on over-dominant QTL can enhance crossbred performance without crossbred testing.

Lessons learned from establishing and evaluating indicators of the quality of measles surveillance in the United States, 1996-1998.

As part of a strategy to eliminate measles, 7 indicators were adopted in the United States in 1996 to ensure the quality of measles surveillance. This report summarizes the US experience with these indicators during 1996-1998. The indicators are compiled from data reported to the Centers for Disease Control and Prevention (CDC) during routine surveillance supplemented with information collected directly from states. Measles case investigations are generally thorough, and sufficient information is collected to control and monitor disease. A high proportion of measles cases are imported from other countries, suggesting that investigations are complete. For some states, the lag from disease onset to reporting is long, and the number of health department investigations of measleslike illnesses is low. Most of these investigations include laboratory testing of clinical specimens. Collection of measles virus specimens from cases for genetic analysis needs improvement. The CDC and health departments need to continue efforts directed at health care professionals to ensure the recognition, proper diagnostic workup, and reporting of measles.

[Osteogenesis imperfecta Type 1: a case presentation with a new mutation in gene COL1A1].

In a 4 year old girl the diagnosis osteogenesis imperfecta type I was suspected by following clinical criteria: four fractures after small trauma, intensive blue sclera, anomalies of dental enamel, macrocephalie with frontal bassing. Clinical diagnosis could be verified by moleculargenetic analysis, a newly recognized heterozygous point mutation (Arg420Stop) in the COL1A1-gene was found.

[cDNA-microarrays in cartilage research - functional genomics of osteoarthritis].

Functional genomics represents a new challenging approach in order to analyze complex diseases such as osteoarthritis on a molecular level. The characterization of the molecular changes of the cartilage cells, the chondrocytes, enables a better understanding of the pathomechanisms of the disease. In particular, the identification and characterization of new target molecules for therapeutic intervention is of interest. Also, potential molecular markers for diagnosis and monitoring of osteoarthritis contribute to a more appropriate patient management. The DNA-microarray technology complements (but does not replace) biochemical and biological research in new disease-relevant genes. Large-scale functional genomics will identify molecular networks such as yet identified players in the anabolic-catabolic balance of articular cartilage as well as disease-relevant intracellular signaling cascades so far rather unknown in articular chondrocytes. However, at the moment it is also important to recognize the limitations of the microarray technology in order to avoid over-interpretation of the results. This might lead to misleading results and prevent to a significant extent a proper use of the potential of this technology in the field of osteoarthritis.

Reproductive attitudes of couples having a child with cystic fibrosis in Brittany (France).

Cystic fibrosis (CF) has an incidence of one in 2,636 livebirths and a carrier rate of one in 26 inhabitants in Brittany. One objective of a major enquiry among parents having a CF child as well as CF adolescents and adults was to evaluate the reproductive behavior of 124 couples attending a CF care center. Knowledge of recurrence risk resulted in deciding against further progeny or in reducing the number of children (average number of children: 1.96; ideal mean number of children: 3.7). Thirty-five percent adopted or changed their method of contraception after the birth of their affected child, but the change was due to the birth of the CF child in only 14.3% of the couples. Prenatal diagnosis (PD) was favored by 95.1%, and 41.2% had used it; 68.6% were in favor of pregnancy interruption for CF and 76.2% would interrupt the pregnancy should PD reveal that their fetus had CF. All 123 respondents thought that genetic counseling was useful, but only 87.1% knew of its availability. Our results are quite different from those previously published. Although results could be population-specific, one cannot exclude the fact that they reflect a change of attitudes among parents, the other studies being much older.

[Congenital hypopituitarism: when should transcription factor gene screenings be performed?].

THE GENETIC ORIGIN INCREASINGLY INCRIMINATED: Congenital pituitary hormone deficiencies represent conditions of hypopituitarism resulting from abnormal pituitary ontogenesis. This group of genetically determined diseases has considerably widened with the development of molecular biology. Many transcription factors playing a role in pituitary development have been identified, and their mutations reported as causes of isolated or multiple pituitary hormone deficiencies. Isolated pituitary hormone deficiencies may affect somatotroph, gonadotroph, and corticotroph lineages. They result from mutations of the genes of hormones (such as growth hormone), of a factor that regulates their synthesis or secretion (such as TPIT for corticotrophics), or of their receptors (GHRH or GnRH receptor genes). Multiple (or combined) pituitary hormone deficiencies result in the concomitant or sequential onset of several anterior pituitary hormone deficiencies. They are due to mutations of transcription factors involved in the early steps of pituitary development (RIEG, HesX1, LHX4, LHX3, Prop1, POU1F1/Pit-1), and are associated with various phenotypes. FOR BETTER MANAGEMENT: Long-term follow-up of these patients and functional studies of the mutations identified in specialized research centers will help to determine phenotype-genotype correlations, hence providing a valuable help to the management of these orphan diseases.

In vitro transcription of Tomato spotted wilt virus is independent of translation.

Ongoing transcription in vitro of Tomato spotted wilt virus (TSWV) has previously been demonstrated to require the presence of reticulocyte lysate. This dependence was further investigated by testing the occurrence of transcription in the presence of two translation inhibitors: edeine, an inhibitor that still allows scanning of nascent mRNAs by the 40S ribosomal subunit, and cycloheximide, an inhibitor that completely blocks translation including ribosome scanning. Neither of these inhibitors blocked TSWV transcription initiation or elongation in vitro, as demonstrated by de novo-synthesized viral mRNAs with globin mRNA-derived leader sequences, suggesting that TSWV transcription in vitro requires the presence of (a component within) reticulocyte lysate, rather than a viral protein resulting from translation.

Low linkage disequilibrium indicative of recombination in foot-and-mouth disease virus gene sequence alignments.

We have applied tests for detecting recombination to genes of foot-and-mouth disease virus (FMDV). Our approach estimated summary statistics of linkage disequilibrium (LD), which are sensitive to recombination. Using the genealogical relationships, rate heterogeneity and mutation parameters estimated from individual sets of aligned gene sequences, we simulated matching RNA sequence datasets without recombination. These simulated datasets allowed for recurrent mutations at any site to mimic homoplasy in virus sequence data and allow construction of null distributions for LD parameters expected in the absence of recombination. We tested for recombination in two ways: by comparing LD in observed data with corresponding null distributions obtained from simulated data; and by testing for a negative relationship between observed LD between pairs of polymorphic nucleotide sites and inter-site distance. We applied these tests to six FMDV datasets from four serotypes and found some evidence for recombination in all of them.

Cigarette smoking, oncogenic human papillomavirus, Ki-67 antigen, and cervical intraepithelial neoplasia.

Although cigarette smoking has been identified as a cofactor for cervical neoplasia, it is not clear whether smoking exerts an early or late effect on the evolution of human papillomavirus (HPV)-related lesions. A case-control study of Washington State women who presented for routine gynecologic care from 1997 to 2001 was conducted. All women underwent cytologic testing and HPV DNA screening. Those with abnormal cytology findings or a positive oncogenic HPV test and a random sample of women negative on both tests were referred for colposcopically directed cervical biopsy with repeated testing. Among 461 women with oncogenic HPV were 181 controls with negative histology, 137 cases with histologically confirmed cervical intraepithelial neoplasia (CIN) grade 1 (CIN1), and 143 cases with histologically confirmed CIN grades 2-3 or higher (&gt;/= CIN2-3). Smoking information was obtained by questionnaire. Immunohistochemistry testing for Ki-67 was performed on a subset of biopsy specimens (n = 139). Smoking 10 or more cigarettes per day was associated with &gt;/= CIN2-3 (adjusted odds ratio = 2.6, 95% confidence interval: 1.3, 5.5) and CIN1 (adjusted odds ratio = 2.5, 95% confidence interval: 1.2, 5.3). Heavy smoking was positively associated with Ki-67 but not with repeated detection of oncogenic HPV. Since smoking was associated with both CIN1 and &gt;/= CIN2-3, cigarette by-products may affect the early evolution of HPV-related lesions, possibly by increasing the rate of cell turnover.

Does the addition of information on genotype improve prediction of the risk of melanoma and nonmelanoma skin cancer beyond that obtained from skin phenotype?

The authors quantified improvement in predicting cutaneous malignant melanoma, basal cell carcinoma, and squamous cell carcinoma of the skin made possible by information on common variants of the melanocortin-1 receptor gene (MC1R) in a 1998-1999 population-based case-control study of subjects aged 20-59 years of northern European ancestry in Tasmania, Australia. Melanin density at the upper inner arm was estimated by spectrophotometry. DNA samples were genotyped for five MC1R variants: Val60Leu, Asp84Glu, Arg151Cys, Arg160Trp, and Asp294His. Among controls (n = 267), variant carriers, versus noncarriers, had lower (p &lt; 0.01) mean melanin concentrations. Increased risk conferred by genotype was restricted mainly to those with the darkest skins: for subjects with at least 2% melanin, the odds of carrying each additional variant were higher for cutaneous malignant melanoma (n = 39; odds ratio = 1.45, 95% confidence interval: 0.87, 2.44), basal cell carcinoma (n = 35; odds ratio = 1.86, 95% confidence interval: 1.14, 3.02), and squamous cell carcinoma (n = 42; odds ratio = 2.67, 95% confidence interval: 1.50, 4.74) cases than for controls (n = 135). Adding MC1R information to prediction based on age, sex, and cutaneous melanin increased the area under the receiver operating characteristic curve by 1.4% (cutaneous malignant melanoma), 3.2% (basal cell carcinoma), or 2.0% (squamous cell carcinoma). The improvement in prediction was probably too small to be valuable in a clinical setting.

Signal transducer and activator of transcription 6 haplotypes and asthma in the Indian population.

In this paper, we report for the first time the results of an investigation on the association of signal transducer and activator of transcription 6 (STAT6) with asthma in the Indian population. A novel polymorphic CA-repeat in the proximal promoter region [R1] and a previously identified CA-repeat in the 5'-untranslated region [R3] were genotyped, and haplotypes [R1_R3] were generated using PHASE software. The 16 repeat allele at the R1 locus was positively associated (P = 0.01) with asthma. The 15 and 16 repeat alleles at the R3 locus were positively (P &lt; 10(-4)) and negatively (P &lt; 10(-5)) associated with asthma, respectively. Further, the 17_15 (P = 0.0031) and 16_15 (P = 0.001) haplotypes were found to be positively associated with asthma, whereas 17_14, 24_16, and 23_16 were negatively associated (P &lt; 10(-5)). It appears that the R3 and R1 loci together play a bigger role in asthma than either of them alone, and the R3 locus has a larger effect than the R1 locus. Although alleles at the R1 locus appeared to be associated with total serum immunoglobulin E level, the genotypes showed no association, and the R3 locus showed no effect. As no exonic variants of STAT6 are known as yet, repeat polymorphisms in the regulatory regions and their haplotypes could be important in deciphering the genetic role of STAT6 in asthma and atopy.

Gene-environment interactions and the complexity of human genetic diseases.

In the assessment of mortality and morbidity risk, the ability of family history and genetic test results to predict the age of occurrence, severity, and long-term prognosis of 'genetic' diseases is important. An increasing number of gene-gene and gene-environment interactions have been demonstrated in a number of monogenic Mendelian diseases. These interactions can significantly modify the clinical presentation (disease phenotype) of diseases previously regarded purely as 'genetic.' As a result, 'genetic' diseases can be positioned in a continuum between classic Mendelian and complex disease where the extremes, pure genetic or solely non-genetic, do not exist. The position of any given disease in this continuum is defined by three components: the major gene(s) contributing to the phenotype, the variability added by modifier genes and the significance of environmental factors influencing the phenotype. As the predictive value of genetic test results can be significantly influenced by additional genetic and environmental risk factors, a better understanding of these factors may influence the quantification of mortality and morbidity risk.

A rapid diagnostic method for a retrotransposal insertional mutation into the FCMD gene in Japanese patients with Fukuyama congenital muscular dystrophy.

Fukuyama-type congenital muscular dystrophy (FCMD) is characterized by congenital muscular dystrophy in combination with central nervous system (CNS) abnormalities. Differential diagnosis of FCMD from Duchenne and Becker muscular dystrophies (DMD/BMD) or other types of congenital muscular dystrophy is occasionally difficult, because of their phenotypic similarity. The gene (FCMD) responsible for FCMD at 9q31 was isolated in 1998. In Japan, most FCMD-bearing chromosomes (87%) have a 3-kb retrotransposal insertion into the 3'-untranslated region (UTR) of the gene that could be derived from a single ancestral founder. Nine non-founder mutations have been identified in Japanese FCMD patients. Severe phenotype was significantly more frequent in patients who were compound heterozygotes for a point mutation and the founder mutation, than in homozygotes for the founder mutation. We developed a PCR-based diagnostic method for a rapid detection of the retrotransposal insertion mutation. Using this system, we screened 18 FCMD patients, and found 16 homozygotes and two heterozygotes for the insertion. We also evaluated the carrier frequency in the normal Japanese population. Six of 676 persons were recognized as a heterozygous carrier. Furthermore, we found three homozygotes for the FCMD founder mutation among 97 patients who had been said to have probable DMD/BMD without any DMD mutations. On the other hand, there were no FCMD homozygotes but four heterozygous carriers among 335 patients with DMD mutations. The diagnostic method we developed will provide a rapid and reliable diagnosis of FCMD, which can bring important information in genetic counseling, such as the accurate mode of inheritance, recurrence risk and a life expectancy.

Maternal UPD(14) in the patient with a normal karyotype: clinical report and a systematic search for cases in samples sent for testing for Prader-Willi syndrome.

Maternal uniparental disomy for chromosome 14 causes a recognizable phenotype that has a number of consistent features, irrespective of the underlying chromosome abnormality. To illustrate this, we describe a patient with a 46,XX karyotype whose short stature, hypotonia, feeding problems, truncal obesity, early puberty, learning difficulties, and craniofacial characteristics led to a diagnosis of maternal (mat) UPD(14). No evidence is available to indicate how common mat UPD(14) in patients with a normal karyotype might be. Because of the similarity between Prader-Willi syndrome (PWS) and the mat UPD(14) phenotype in childhood, we systematically tested samples from 35 patients with normal karyotypes and an unexplained PWS-like phenotype referred to the Wessex Genetics Service. We sought to address the practical question should laboratories carry out tests for mat UPD(14) on all samples received for PWS testing when PWS testing gives negative results? None of the samples tested showed evidence of mat UPD(14). Routine screening of DNA from patients with possible PWS cannot be recommended on this basis.

RNA interference: historical overview and significance.

In the early 1990s, attempts to manipulate gene expression by researchers working in three different fields resulted in unanticipated gene silencing. Rather than ignoring such results, these researchers went on to document and further investigate the nature of such silencing, which was named "co-suppression" in plants, "quelling" in fungi, and "RNA interference" (RNAi) in nematodes. By the late 1990s, it was discovered that silencing could be initiated in this diverse set of organisms by exposing cells to double-stranded RNA (dsRNA), which directed the destruction of mRNAs containing similar sequences. Soon afterward, such dsRNA-mediated silencing was employed as a reverse genetic technique to analyze the functions of specific genes in a broad variety of organisms. Biochemical and genetic studies designed to uncover the components of the RNA silencing machinery identified a common core of proteins that serve to amplify the interfering RNA signal and direct endonucleolytic cleavage of target RNAs. A subset of silencing events may also direct DNA methylation of targeted genes. RNA silencing is thought to have evolved as a defense mechanism to suppress viral replication and transposon mobilization. However, additional functions involving the RNAi machinery have been uncovered, including posttranscriptional regulation of endogenous genes, and maintenance of structure and function of heterochromatin. Whereas many researchers have focused on understanding the natural biological functions of RNA silencing, others are testing its utility in antiviral and cancer therapies and in other biotechnological and biomedical applications.

Genetic analysis of soluble N-ethylmaleimide-sensitive factor attachment protein function in Drosophila reveals positive and negative secretory roles.

The N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein (SNAP) are cytosolic factors that promote vesicle fusion with a target membrane in both the constitutive and regulated secretory pathways. NSF and SNAP are thought to function by catalyzing the disassembly of a SNAP receptor (SNARE) complex consisting of membrane proteins of the secretory vesicle and target membrane. Although studies of NSF function have provided strong support for this model, the precise biochemical role of SNAP remains controversial. To further explore the function of SNAP, we have used mutational and transgenic approaches in Drosophila to investigate the effect of altered SNAP dosage on neurotransmitter release and SNARE complex metabolism. Our results indicate that reduced SNAP activity results in diminished neurotransmitter release and accumulation of a neural SNARE complex. Increased SNAP dosage results in defective synapse formation and a variety of tissue morphological defects without detectably altering the abundance of neural SNARE complexes. The SNAP overexpression phenotypes are enhanced by mutations in other secretory components and are at least partially overcome by co-overexpression of NSF, suggesting that these phenotypes derive from a specific perturbation of the secretory pathway. Our results indicate that SNAP promotes neurotransmitter release and SNARE complex disassembly but inhibits secretion when present at high abundance relative to NSF.

Data mining and multiparameter analysis of lung surfactant protein genes in bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD), the most common chronic lung disease in infancy, is influenced by a number of antenatal and postnatal risk factors and is mostly preceded by respiratory distress syndrome (RDS) in the newborn. Surfactant protein (SP-A, -B, -C and -D) gene variations may play a role in both BPD and RDS. An association study between these candidate genes and BPD was performed. A total of 365 preterm Finnish infants in a high-risk population with gestational age &lt;or=32 weeks were genotyped for all SP genes. A multiparameter analysis was performed using Agrawal's algorithm based data mining and conventional methods of statistical allelic association. In singletons and presenting multiples, the frequency of SP-B intron 4 deletion variant allele was increased in BPD versus controls (P=0.008, OR=2.0, 95%CI 1.2-3.4). The presence of the SP-B intron 4 deletion variant was a risk factor for BPD even when essential external confounding factors were included in the analyses. No other SP polymorphisms associated with BPD, and the SP-B intron 4 variation did not associate with RDS. Transcription Element Search Software predicted allele-specific differences at several putative transcription factor binding sites that may be important in SP-B regulation. The present multiparameter analysis demonstrates the presumable direct involvement of the SP-B intron 4 deletion variant allele as a genetic risk factor to BPD. We propose that two separate SP-B gene polymorphisms have a phenotypic significance via separate molecular mechanisms: the intron 4 length variation affecting transcriptional regulation, and the exonic Ile131Thr variation affecting post-translationally.

Prognostic analysis of E-cadherin gene promoter hypermethylation in patients with surgically resected, node-positive, diffuse gastric cancer.

Recent investigations have demonstrated that hypermethylation is a frequent mechanism for silencing tumor suppressor genes. This is a potentially reversible epigenetic change, and it is the target of a novel class of anticancer compounds with demethylating activity. Better understanding of the clinical implications of hypermethylation will allow the optimal planning of future trials with demethylating drugs. In this perspective, we investigated whether hypermethylation in the CDH1 promoter region is correlated with poor prognosis of patients with surgically resected, node-positive, diffuse gastric cancer.</AbstractText>Consecutive cases of diffuse gastric cancer were considered eligible for study entry. Additional inclusion criteria were radical surgery with a minimum of D1 lymphadenectomy, complete follow-up information, and availability of tumor specimens for methylation-specific PCR and immunohistochemistry analyses.</AbstractText>CDH1 promoter hypermethylation was found in 40 of 73 cases (54%), and it was significantly associated with worse prognosis. In patients with and without hypermethylation, the 5-year event-free survival rate was 30% and 62%, respectively, and the 5-year overall survival rate was 35% and 67%, respectively. CDH1 promoter hypermethylation retained its prognostic role for disease-free survival (P &lt; 0.001) and overall survival (P &lt; 0.001) in multivariate analysis. Immunohistochemistry showed a significant association between CDH1 methylation and E-cadherin expression (P &lt; 0.001).</AbstractText>This study shows adverse prognostic effect of CDH1 promoter hypermethylation in patients with diffuse gastric cancer. This form of cancer, and other types with frequent hypermethylation and silencing of critical tumor suppressor genes, would make appropriate targets for the testing of novel compounds with demethylating activity.</AbstractText>

Poxvirus vaccines for cancer and HIV therapy.

The poxviridae have a long history of causing disease in society, and their biological effects in humans and other mammals have been extensively studied. In the 1980s, genetic engineering techniques were applied to vaccinia in order to create replicating recombinant vectors that could express inserted genes encoding influenza virus proteins. In animal models, these recombinant viruses were able to deliver their foreign antigens to the immune system and elicit a specific adaptive immune response. Since then, improvements in our understanding of immunobiology, as well as technical advances in bioengineering, have led to the creation and clinical testing of a large number of recombinant poxviruses as candidate vaccines. Poxviruses can infect a broad range of cells, replicate with high efficiency and elicit strong immune responses - factors that make them especially well-suited as vaccines for the prevention and treatment of human immunodeficiency virus (HIV) and cancer. Both of these diseases are characterised by chronic antigen expression in the setting of focal or global deficits in the immune system that hamper the generation of protective immunity. This review traces the history of poxviruses as pathogens and immunogens, examines some of the approaches that have been taken to design poxviral vaccines for HIV and cancer and summarises the results of existing clinical trials of these vectors. In addition, the review aims to identify some of the factors that may shape the development of future therapies based on recombinant poxviruses.

Genetic analysis of a successful repatriation programme: giant Gal&#xe1;pagos tortoises.

As natural populations of endangered species dwindle to precarious levels, remaining members are sometimes brought into captivity, allowed to breed and their offspring returned to the natural habitat. One goal of such repatriation programmes is to retain as much of the genetic variation of the species as possible. A taxon of giant Gal&#xe1;pagos tortoises on the island of Espa&#xf1;ola has been the subject of a captive breeding-repatriation programme for 33 years. Core breeders, consisting of 12 females and three males, have produced more than 1200 offspring that have been released on Espa&#xf1;ola where in situ reproduction has recently been observed. Using microsatellite DNA markers, we have determined the maternity and paternity of 132 repatriated offspring. Contributions of the breeders are highly skewed. This has led to a further loss of genetic variation that is detrimental to the long-term survival of the population. Modifications to the breeding programme could alleviate this problem.

Human papillomavirus DNA presence of the upper respiratory tract mucosa of healthy children.

The papillomaviruses are a group of small DNA (8 kbp) viruses which induce papillomas in human and animals. Most of them can also transform epithelial cells of human and other vertebrates. Examinations of children with Recurrent Respiratory Papillomatosis (RRP), using PCR method with specific primers, revealed HPV type 6 or/and 11 in 98%-100% tissue samples. Little is known about HPV infection in upper respiratory tract of healthy children. The group of 201 healthy children (from 3 to 10 years old) was laryngologically examined. The smears from noses and throats were studied for the presence of HPV viruses DNA. PCR was performed as described by Tucker et al. 28.8% of children from the study group were HPV positive. The presence of HPV in respiratory tract in children is relatively high. "High risk" HPV are not observed in the respiratory tract in children. The Human Papillomavirus in children may be transmitted by direct contacts.

Association of anterior glottic webs with velocardiofacial syndrome (chromosome 22q11.2 deletion).

An association between anterior glottic webs and velocardiofacial syndrome (chromosome 22q11.2 deletion) has previously been noted in a number of case reports. Our objective was to determine if the presence of such webs warrants a high index of suspicion for this chromosome deletion. Study design and setting This study was carried out in the Division of Pediatric Otolaryngology-Head and Neck Surgery at Cincinnati Children's Hospital Medical Center. Chromosome 22q11.2 deletion status was determined for all patients endoscopically diagnosed with anterior glottic webs between July 1998 and December 2000. Families of patients who tested positive for the deletion were referred to the Cincinnati Children's Division of Human Genetics for additional evaluation and counseling.</AbstractText>Eleven of 17 patients (65%) with anterior glottic webs were positive for chromosome 22q11.2 deletion. Of these 11 patients, 5 showed subtle clinical manifestations of velocardiofacial syndrome and underwent genetic testing due only to the presence of a web. All 11 patients were diagnosed with velocardiofacial syndrome.</AbstractText>We strongly recommend that all patients diagnosed with anterior glottic webs undergo fluorescence in situ hybridization evaluation for this chromosome deletion.</AbstractText>

Results of a nationwide screening for Anderson-Fabry disease among dialysis patients.

Anderson-Fabry disease is possibly underdiagnosed in patients with end-stage renal disease. Nationwide screening was therefore undertaken for Anderson-Fabry disease among dialysis patients in Austria. Screening for alpha-galactosidase A (AGAL) deficiency was performed by a blood spot test. In patients with a positive screening test, AGAL activity in leukocytes was determined. Individuals with decreased leukocyte AGAL activity were subjected to mutation testing in the GLA gene. Fifty (90.9%) of 55 Austrian hemodialysis centers participated in this study; 2480 dialysis patients (80.1% of the Austrian dialysis population) were screened. In 85 patients, the screening test was positive (85 of 2480, 3.42%; women, 3.32%; men, 3.50%). Among these 85 patients, 4 men (in 3 of whom Anderson-Fabry disease was already known before screening) had a severely decreased and 11 subjects had a borderline low AGAL activity. Genetic testing revealed mutations associated with Fabry disease in all four men with severely decreased AGAL activity resulting in a prevalence of 0.161% for the entire study population. A nationwide screening of dialysis patients permitted detection of a hitherto unknown man with Anderson-Fabry disease. The overall prevalence among dialysis patients was at least ten times higher as compared with recent registry data. Screening programs among patients with end-stage renal disease, especially men, should be put in place to identify families with Anderson-Fabry disease who probably may benefit from specific clinical care, and perhaps from enzyme replacement therapy. In dialysis patients, however, there is no evidence to support enzyme replacement therapy at present.

Circulating immune complexes augment severity of antibody-mediated myasthenia gravis in hypogammaglobulinemic RIIIS/J mice.

Experimental autoimmune myasthenia gravis (EAMG) is severe in RIIIS/J mice, despite a significant B cell immunodeficiency and a massive TCR V beta gene deletion. Severity of EAMG in RIIIS/J mice is greater than MHC-identical (H-2(r)) B10.RIII mice, suggesting the influence of non-MHC genes as an EAMG-potentiating factor in this strain. To delineate the role of deleted TCR V beta genes in RIIIS/J mice, we obtained (RIIIS/J x B10.RIII)F(1) (V beta(b/c)) x RIIIS/J (V beta(c)) backcross mice using Mendelian genetic methods and immunized them with acetylcholine receptor. EAMG susceptibility was not elevated in mice with V beta(c) genotype having 70% V beta gene deletion. Next, we performed microarray analysis on 12,488 spleen cDNAs obtained from spleens of naive RIIIS/J and B10.RIII mice. In RIIIS/J mice, 263 cDNAs were overexpressed and 303 cDNAs were underexpressed greater than 2-fold, compared with B10.RIII mice. TCR gene expression was augmented, whereas NK receptor, C1q, and C3 gene expressions were diminished in RIIIS/J mice. RIIIS/J mice also had increased lymph node T cell counts, elevated serum anti-AChR Ab levels, and serum C3 and C1q-conjugated circulating immune complex levels. A direct correlation between increased serum C1q-conjugated circulating immune complex levels and disease severity was observed in RIIIS/J mice.

Rapid screening for HLA-B27 by a TaqMan-PCR assay using sequence-specific primers and a minor groove binder probe, a novel type of TaqMan trade mark probe.

HLA-B27 is strongly associated with ankylosing spondylitis (AS). As typing for HLA-B27 is routinely performed by serological methods, false-positive results can be generated. Therefore, several more accurate molecular methods have been developed for HLA-B27 genotyping. We describe a real-time PCR method for the detection of the HLA-B27 allele using sequence-specific primers (SSP) combined with a fluorogenic MGB probe (minor groove binder probe), a novel type of TaqMan probe. The MGB increases the melting temperature (T(m)) of the probe, allowing the use of shorter probes. Moreover, the use of a non-fluorescent quencher (NFQ) attached to the MGB probe improves the efficiency of fluorescence quenching, thus providing a very low fluorescent background. We tested this method on 150 subjects (41 HLA-B27 positive and 109 HLA-B27 negative) who underwent routine HLA-B27 serological testing by flow cytometry (FC). Serology and our TaqMan assay gave identical results in all cases and no false positive or negative results were observed. A graphical representation of fluorescence and normalized reporter signal (DeltaRn) values demonstrated that HLA-B27 positive and HLA-B27 negative samples formed two tight clusters making it possible to clearly differentiate between HLA-B27 positive and negative samples. This single tube PCR method for the detection of HLA-B27 should be particularly suitable for the routine analysis of large numbers of samples in the laboratory.

Autosomal dominant cerebellar ataxias: clinical features, genetics, and pathogenesis.

Autosomal dominant cerebellar ataxias are hereditary neurodegenerative disorders that are known as spinocerebellar ataxias (SCA) in genetic nomenclature. In the pregenomic era, ataxias were some of the most poorly understood neurological disorders; the unravelling of their molecular basis enabled precise diagnosis in vivo and explained many clinical phenomena such as anticipation and variable phenotypes even within one family. However, the discovery of many ataxia genes and loci in the past decade threatens to cause more confusion than optimism among clinicians. Therefore, the provision of guidance for genetic testing according to clinical findings and frequencies of SCA subtypes in different ethnic groups is a major challenge. The identification of ataxia genes raises hope that essential pathogenetic mechanisms causing SCA will become more and more apparent. Elucidation of the pathogenesis of SCA hopefully will enable the development of rational therapies for this group of disorders, which currently can only be treated symptomatically.