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2,337,700 |
[Prenatal diagnosis of spinal muscular atrophy (SMA) -- indications, restrictions, interpretation of results].
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Proximal spinal muscular atrophy of childhood and adolescence (SMA) is a genetic autosomal recessive disease. Caused in 96.5% by deletion in the SMN1 gene. Owing to the homogeneity of the molecular defect. Secondary prophylaxis can readily be offered to families at risk of SMA.</AbstractText>Prenatal diagnosis of SMA was carried out in a group of 50 families. Which were divided into two subgroups: with high and relatively low genetic risk of SMA. In all, 55 prenatal tests were performed in the period 1998-2003.</AbstractText>In the first group including 45 families at high genetic risk, 9 of the 50 tests were positive (18%). The diagnosis of SMA was tentative in 7 cases from this group and it was based only on the clinical picture (the affected children are not alive, therefore DNA samples are not available). Prenatal examination in 1 of these 7 families showed the SMA genotype. In the second subgroup including 5 families with relatively low genetic risk of SMA in none of the studies was there a biallelic deletion of exon 7 in the SMNI gene. Mainly parents of children with a severe form of SMA and having no healthy offspring asked for prenatal examination (73% of the families).</AbstractText>Prenatal diagnosis could be offered to families even if the diagnosis of SMA was not genetically verified. The negative result should he then interpreted individually. Until the carrier test will not he introduced to routine procedures. prenatal diagnosis can be also offered to families at relatively low risk of SMA.</AbstractText>
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2,337,701 |
Diagnostic testing by CFTR gene mutation analysis in a large group of Hispanics: novel mutations and assessment of a population-specific mutation spectrum.
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Characterization of CFTR mutations in the U.S. Hispanic population is vital to early diagnosis, genetic counseling, patient-specific treatment, and the understanding of cystic fibrosis (CF) pathogenesis. The mutation spectrum in Hispanics, however, remains poorly defined. A group of 257 self-identified Hispanics with clinical manifestations consistent with CF were studied by temporal temperature gradient electrophoresis and/or DNA sequencing. A total of 183 mutations were identified, including 14 different amino acid-changing novel variants. A significant proportion (78/85) of the different mutations identified would not have been detected by the ACMG/ACOG-recommended 25-mutation screening panel. Over one third of the mutations (27/85) occurred with a relative frequency >1%, which illustrates that the identified mutations are not all rare. This is supported by a comparison with other large CFTR studies. These results underscore the disparity in mutation identification between Caucasians and Hispanics and show utility for comprehensive diagnostic CFTR mutation analysis in this population.
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2,337,702 |
A novel method for creating artificial mutant samples for performance evaluation and quality control in clinical molecular genetics.
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The lack of readily available, patient-derived materials for molecular genetic testing of many heterozygous or rare disorders creates a major impediment for laboratory proficiency and quality control procedures. The paucity of clinically derived mutation-positive samples could be surmounted if it were possible to construct artificial samples containing mutations of interest that would sufficiently resemble natural human samples. Such samples could then function as acceptable and realistic performance evaluation challenges and quality control reagents for recipient laboratories. Using the cystic fibrosis gene (CFTR) as a prototype, we have devised and executed experiments designed to generate unique DNA samples that could be used for these purposes. We used site-directed mutagenesis to generate mutations of interest in plasmid DNA derived from common bacterial artificial chromosome sources containing the cystic fibrosis transmembrane conductance receptor gene. CFTR mutations G85E and 1078delT were chosen to represent mutations in the original American College of Medical Genetics-recommended population-screening panel of 25 mutations. DNA samples containing predetermined concentrations and ratios of wild-type and mutated plasmids, bacterial artificial chromosomes of interest, and nonhuman genomic carrier DNA were characterized and tested in-house and in a group of nine pilot testing laboratories using a variety of technical platforms. The results indicate that these constructs, containing CFTR mutations in heterozygous and homozygous states, can serve as valid and accessible materials for quality assurance, including performance evaluation, proficiency testing, and assay quality control.
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2,337,703 |
Long polymerase chain reaction-based fluorescence in situ hybridization analysis of female carriers of X-linked chronic granulomatous disease deletions.
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Chronic granulomatous disease (CGD) is a rare inherited disorder in which antimicrobial activity of phagocytes is impaired due to the lack of reactive oxygen species, or oxidative burst, produced by NADPH oxidase. The X-linked form of CGD, representing approximately 70% of all cases, is caused by mutations in the cytochrome b beta subunit (CYBB) gene, which maps to chromosome Xp21.1. CYBB encodes the gp91-phox protein, a necessary component in the NADPH oxidase pathway. A wide variety of mutations have been identified in X-linked CGD patients, all of which lead to deletion of the functional protein and no oxidative burst activity. The mutations vary from single nucleotide substitutions to deletions of the entire gene. In this article, we report a mutation detection method for probands of female relatives at risk for carrier status of large deletions of the CYBB gene. Through fluorescent in situ hybridization of metaphase chromosomes, we were able to consistently distinguish carriers from noncarriers using polymerase chain reaction-derived, labeled DNA specific for exons 2 to 13 of the CYBB region at Xp21.1.
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2,337,704 |
Isolation and characterization of a rice mutant hypersensitive to Al.
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Rice (Oryza sativa L.) is a highly Al-resistant species among small grain crops, but the mechanism responsible for the high Al resistance has not been elucidated. In this study, rice mutants sensitive to Al were isolated from M(3) lines derived from an Al-resistant cultivar, Koshihikari, irradiated with gamma-rays. Relative root elongation was used as a parameter for evaluating Al resistance. After initial screening plus two rounds of confirmatory testing, a mutant (als1) was isolated from a total of 560 lines. This mutant showed a phenotype similar to the wild-type plant in the absence of Al. However, in the presence of 10 microM Al, root elongation was inhibited 70% in the mutant, but only 8% in the wild-type plant. The mutant also showed poorer root growth in acid soil. The Al content of root apices (0-1 cm) was much lower in the wild-type plant. The sensitivity to other metals including Cd and La did not differ between the mutant and the wild-type plants. A small amount of citrate was secreted from the roots of the mutant in response to Al stress, but there was no difference from that secreted by the wild-type plant. Genetic analysis of F(2) populations between als1 and wild-type plants showed that the Al-resistant seedlings and Al-sensitive seedlings segregated at a 3 : 1 ratio, indicating that the high sensitivity to Al in als1 is controlled by a single recessive gene. The gene was mapped to the long arm of chromosome 6, flanked by InDel markers MaOs0619 and MaOs0615.
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2,337,705 |
Effects of AAV-2-mediated aspartoacylase gene transfer in the tremor rat model of Canavan disease.
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The tremor rat is a spontaneous epilepsy model with a seizure phenotype caused by a deletion in the aspartoacylase (ASPA) gene. The absence of ASPA expression in these animals results in undetectable levels of enzyme activity and the accumulation of the substrate N-acetyl-aspartate (NAA) in brain, leading to generalized myelin vacuolation and severe motor and cognitive impairment. In support of human gene therapy for CD, recombinant adeno-associated viral vector (AAV-2) expressing ASPA was stereotactically delivered to the tremor rat brain and effects on the mutant phenotype were measured. AAV-ASPA gene transfer resulted in elevated aspartoacylase bioactivity compared to untreated mutant animals and elicited a significant decrease in the pathologically elevated whole-brain NAA levels. Assessment of motor function via quantitative rotorod testing demonstrated that rats injected with AAV-ASPA significantly improved on tests of balance and coordinated locomotion compared to animals receiving control vectors. This study provides evidence that AAV-2-mediated aspartoacylase gene transfer to the brain improves biochemical and behavioral deficits in tremor rat mutants (tm/tm) and supports the rationale of human gene transfer for Canavan disease.
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2,337,706 |
Molecular screening for microdeletions at 9p22-p24 and 11q23-q24 in a large cohort of patients with trigonocephaly.
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Trigonocephaly is a rare form of craniosynostosis characterized by the premature closure of the metopic suture. To contribute to a better understanding of the genetic basis of metopic synostosis and in an attempt to restrict the candidate regions related to metopic suture fusion, we studied 76 unrelated patients with syndromic and non-syndromic trigonocephaly. We found a larger proportion of syndromic cases in our population and the ratio of affected male to female was 1.8 : 1 and 5 : 1 in the non-syndromic and syndromic groups, respectively. A microdeletion screening at 9p22-p24 and 11q23-q24 was carried out for all patients and deletions in seven of them were detected, corresponding to 19.4% of all syndromic cases. Deletions were not found in non-syndromic patients. We suggest that a molecular screening for microdeletions at 9p22-p24 and 11q23-q24 should be offered to all syndromic cases with an apparently normal karyotype because it can potentially elucidate the cause of trigonocephaly in this subset of patients. We also suggest that genes on the X-chromosome play a major role in syndromic trigonocephaly.
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2,337,707 |
Communication about genetic testing in families of male BRCA1/2 carriers and non-carriers: patterns, priorities and problems.
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This qualitative interview study explored the way in which information about predictive BRCA1/2 testing and its implications for children is disseminated within the families of at-risk men who undergo genetic testing. Twenty-nine in-depth interviews were carried out with family members [male patients (n = 17), their partners (n = 8) and adult children (n = 4)]. These explored the following themes: experiences of cancer and genetic testing, decision-making about testing and the communication of test results and genetic information within the immediate family. The interviews revealed that both male patients and their partners perceive themselves, rather than health professionals, as responsible for disclosing information about genetic testing and genetic risks to their children. Parents described three different communication strategies for the disclosure of genetic information to their children: complete openness, limited disclosure and total secrecy. The adoption of a particular communication strategy was justified in terms of children's rights to information vs their parental duties to protect their children from anxiety-provoking information. Some of the problems arising from the adoption of different disclosure patterns are identified and the implications for clinical practice are discussed.
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2,337,708 |
Psychological and genetic counseling implications for adolescent daughters of mothers with breast cancer.
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Adolescent daughters of women with breast cancer (BC) are themselves at risk for heritable BC. Although some preliminary evidence suggests this group is at an increased risk for emotional problems, evidence is limited to studies with small samples and no comparison groups. This study examined psychological and family functioning, health attitudes and beliefs about genetic risks in adolescent females. A case-comparison design was used to compare 55 mother-daughter pairs in which the mother had been treated for BC (BC group) to 55 families from the general population (GP). Participants completed an assessment battery measuring perceptions of personal risk for BC and attitudes about gene testing for BC susceptibility, family functioning, and adolescent psychological adjustment. Based on manova, no significant differences were found between the two groups on measures of the psychological functioning. However, BC group adolescents reported significant (p < 0.01) worries about their future health and genetic risk for BC. About 68% of BC adolescents compared with 12% of GP adolescents reported being moderately to greatly concerned about their susceptibility to genetic mutations. Further, 85% of BC group adolescents believed they were susceptible to BC compared with 10% of GP adolescents. The results indicated no evidence of emotional, behavioral, or familial distress in these families. However, BC adolescents have significant worries about their future health. The results of this study demonstrate the need to develop a comprehensive model of care where accurate information about genetics and health risks can be provided. The adolescents also need support to help them cope and communicate with their mothers their worries about BC.
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2,337,709 |
Demented flies? Using Drosophila to model human neurodegenerative diseases.
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The success of biomedical research in the past few decades has led to dramatic improvements in human health and, as a result, increased life expectancy. An unexpected consequence, however, has been an increase in the number of age-related diseases and, in particular, neurodegenerative diseases. Despite their prevalence, a therapeutic void exists in part due to an incomplete understanding of the biochemical pathogenesis of these diseases. A powerful method that can be used to understand the basic mechanisms underlying neurodegenerative diseases is to generate animal models based on manipulating the expression of single genes that are disease causative. This approach has been facilitated by the fact that many neurodegenerative diseases are inherited as autosomal dominant traits such that expression of the mutant gene in a model organism might be expected to recapitulate the disease. During the past few years, the fruit fly, Drosophila melanogaster, has emerged as a powerful tool to model human neurodegenerative diseases. Here, we describe the various approaches utilized to create fly models of human neurodegenerative disease, and how they can aid in our understanding of disease pathogenesis and facilitate drug discovery and testing.
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2,337,710 |
DNA-based genetic testing is rising steeply in a national health care system with open access to services: a survey of genetic test use in Germany, 1996-2002.
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The extent to which the fast-growing body of genetic knowledge is transferred into everyday clinical practice has nowhere been assessed in a systematic way. Available quantitative analyses of DNA-based genetic test provision and uptake rates are all concerned with specific test programs. The German health-care system is ideally suited for a more general approach, because it is highly flexible regarding access to services, thus permitting quick adjustments to sudden changes in particular subfields of medicine such as genetic testing. We have measured the amount of genetic service provision in Germany between 1996 and 2002 by making use of the central database of the German national health-care system and by inquiring with private health insurance. We can document a three-fold increase of DNA-based testing in the time period 1996-2002, whereas cytogenetic analyses and genetic counseling have remained constant. The growing body of genetic knowledge does indeed seem to be transferred into medical practice at an increasing rate, and the uptake rates are largely in proportion to test offers. DNA-based testing appears to be focussed on disease-associated germ-line alterations.
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2,337,711 |
Risk assessment for inherited susceptibility to cancer: a review of the psychosocial and ethical dimensions.
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The objective of this study was to conduct a broad-based systematic review of social, ethical, and legal considerations associated with genetic cancer risk assessment technologies (CaRATs). This paper focuses on psychosocial and ethical issues. Search results were limited to papers published in English, French, or German from January, 1990, to May, 2003. A quality assessment tool was developed and applied to retrieved papers. Application of the quality assessment tool resulted in 77 of 247 qualitative and quantitative primary research papers being reviewed and synthesized. A broad range of issues were addressed and grouped into content areas. Despite a large literature addressing psychosocial and ethical issues associated with CaRATs, many existing studies are not adequate to inform decision-makers and stakeholders. Careful policy analysis, as in some of the economic analyses reviewed here, is important to bridge this gap.
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2,337,712 |
Predictive testing for hereditary nonpolyposis colorectal cancer: subjective perception regarding colorectal and endometrial cancer, distress, and health-related behavior at one year post-test.
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This study evaluated illness representations, distress, and health-related behavior one year after disclosure of a predictive genetic test result for hereditary nonpolyposis colorectal cancer (HNPCC) in 36 carriers and 36 noncarriers. Post-test, no significant differences between carriers and noncarriers were found in perceived risk and perceived seriousness of colorectal and endometrial cancer or in perceived control over endometrial cancer. Confidence in the controllability of colorectal cancer by means of medical examinations was higher for carriers than noncarriers post-test. Mean levels of distress (cancer-specific distress, state anxiety, psychoneuroticism) were within normal ranges and none of the participants had an overall pattern (on all scales) of clinically elevated levels of distress. Carriers had significantly higher cancer-related distress one year posttest than noncarriers. In both groups, colorectal cancer-related distress decreased. Noncarriers additionally showed decreased endometrial cancer-related distress and state anxiety. Within the year after testing, none of the noncarriers had a colonoscopy and all carriers where adherent to the recommendations regarding colorectal cancer screening. Although interview data delineated individually different problems specifically related to predictive testing (e.g., worry, difficulties in relation to other relatives, burden of regular follow-up), generally, predictive testing for HNPCC does not seem to induce major psychological problems. Moreover, the presented data are promising regarding the impact on health-related behavior.
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2,337,713 |
Knowledge, attitudes, and utilization of BRCA1/2 testing among women with early-onset breast cancer.
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A total of 2,400 questionnaires were mailed to members of two mid-Atlantic breast cancer awareness/support groups to investigate the association between attitudes, knowledge, and use of BRCA1/2 testing among women with early-onset breast cancer. Of the 493 (21%) questionnaires returned, 406 respondents had a diagnosis of breast cancer, of whom 248 were diagnosed prior to age 50 and included in the analyses. Eighty-three percent (206/248) of these women had heard of BRCA1/2 testing and 12.5% (31/248) had undergone BRCA1/2 testing. Among women who had heard of BRCA1/2 testing, women who had been tested were younger (p = 0.03), more likely to have a college education (p = 0.03), more likely to have a family member who had undergone BRCA1/2 testing (p = 0.005), and had greater knowledge, more positive attitudes, and fewer negative attitudes about BRCA1/2 testing (p = 0.02, p = 0.004, and p = 0.004, respectively). In this sample, knowledge regarding BRCA1/2 testing is high, but uptake of genetic testing is low. Lack of information regarding how genetic testing might alter health-care decisions and fear about the genetic testing procedure, its costs, and possible false-positive results are associated with low uptake of genetic testing. Further education regarding these specific points may enhance the use of genetic testing.
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2,337,714 |
Automatic scanning of interphase FISH for prenatal diagnosis in uncultured amniocytes.
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Fluorescence in situ hybridization (FISH) of uncultured amniocytes using chromosome-specific DNA probes offers the opportunity for rapid aneuploidy screening. Between 80 and 95% of all chromosomal disorders expected in the second trimester of pregnancy can be discovered within 24 hr if DNA probes specific for chromosomes 21, 18, 13, X, and Y are used. Rapid results are crucial for clinical decision-making and are helpful in decreasing the anxiety level in most patients. One of the major factors that have been preventing the rapid FISH test from being broadly incorporated into the clinical setting is the limited staff in the cytogenetics laboratories. The present study demonstrates the use of an automated scanning system (Duet, BioView Ltd. Rehovot, Israel) for analyzing FISH in uncultured amniocytes. Fifty-six amniotic fluid samples were evaluated in parallel by karyotyping, manual FISH analysis, and automatic FISH scanning. Automatic scanning provided accurate results compared to both manual FISH scoring and karyotype analysis. The correlation between automatic and manual FISH scanning was found to be very high (r = 0.9, p < 0.0001). The availability of automation for aneuploidy screening in amniotic fluid samples will enable offering this test to a broader patient population while providing fast and reliable results.
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2,337,715 |
APC germ-line mutations in southern Spanish patients with familial adenomatous polyposis: genotype-phenotype correlations and identification of eight novel mutations.
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Familial adenomatous polyposis (FAP) is a disease characterized by the presence of hundreds of adenomatous polyps in the colon and rectum which, if not treated, develop into colorectal cancer. FAP is an autosomal dominantly inherited disorder caused by mutation in the APC gene. The aim of this study was to search for germ-line mutations of the APC gene in unrelated FAP families from southern Spain. By direct sequencing of all APC gene exons, we found the mutation in 13 of 15 unrelated FAP families studied. We identified eight novel mutations: 707delA (exon6), 730_731delAG (exon7), 1787C-->G and 1946_1947insG (exon14), 2496delC, 2838_2839delAT, 2977A-->T, and 3224dupA (exon15). Two patients presented de novo germ-line mutations. Genotype-phenotype correlations for extraintestinal and extracolonic manifestations were studied. Intrafamilial phenotypic variability was observed in two families with mutations in exon/intron boundary, probably due to alternative splicing.
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2,337,716 |
Facioscapulohumeral muscular dystrophy type 1A in northwestern Tuscany: a molecular genetics-based epidemiological and genotype-phenotype study.
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Facioscapulohumeral muscular dystrophy type 1A (FSHD1A) is an autosomal dominant inherited disorder characterized by early involvement of facial and scapular muscles with eventual spreading to pelvic and lower limb muscles. A high degree of clinical variability with respect to age at onset, severity, and pattern of muscle involvement, both between and within families, is present. For this reason, diagnosis of FSHD1A can be sometimes difficult and molecular diagnosis is then necessary. A clinical and molecular genetic-based epidemiological investigation has been carried out in the territory of northwestern Tuscany in central Italy to calculate the prevalence rate of FSHD1A as of March, 2004. The molecular diagnosis has been based on the detection of large deletions of variable size of kpnI repeat units on chromosome 4q35. Results have been compared to those of a previous study conducted in the same area in 1981 (in the premolecular diagnosis era). The minimum prevalence rate was 4.60 x 10(-5) inhabitants, a value four times higher compared to our previous study. No significant correlation between fragment size and clinical severity has been observed. This study confirms in an Italian population a prevalence rate of FSHD1A similar to that observed in other populations. Furthermore, it underlines the usefulness of routine adoption of the genetic testing in confirming clinical suspicion of FSHD1A as well as in correctly diagnosing atypical and otherwise misclassified cases.
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2,337,717 |
Phenotypic heterogeneity in patients with Gaucher disease and the N370S/V394L genotype.
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Correlation between genotype and phenotype in Gaucher disease is limited. It is known that the most common mutation N370S is protective of neurological involvement, but for the V394L mutation, described as the fifth most common among Ashkenazi Jews, little data are available. This study reports all known patients from a large referral clinic and from the international registry with Gaucher disease who are documented to have the N370S/V394L genotype. Of 476 patients in the Gaucher Clinic, 7 patients (2.0%) had the N370S/V394L genotype; of 2,836 patients in the registry, there were 14 patients (0.8%) with this genotype. There was an overlap of 3 patients, making a total of 18 patients, reflecting the rarity of this genotype among the studied cohorts. Most of these patients had mild disease; only 8 patients required specific enzyme therapy, none was splenectomized. Only 3 patients had skeletal involvement, but other baseline parameters were very diverse. Although genotype-phenotype correlation in this case may be difficult, because the V394L mutation when seen in a compound heterozygote with a null allele results in neuronopathic disease, one cannot conclude that this mutation is protective of neuronopathic disease and hence this is important for counseling of at-risk populations.
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2,337,718 |
The 35delG mutation in the connexin 26 gene (GJB2) associated with congenital deafness: European carrier frequencies and evidence for its origin in ancient Greece.
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The 35delG mutation in the connexin 26 gene (GJB2) at the DFNB1 locus represents the most common mutation in Caucasian patients with genetic sensorineural deafness. This new meta-analysis concerns published carrier frequencies of the 35delG mutation in 27 populations for 6,628 unrelated individuals in Europe and in the Middle East; the mean carrier frequency of the mutation is 1.9%. Compared on a regional basis, the most elevated carrier frequency value is of 1 individual carrier in 31 in southern Europe. It is probable that the 35delG mutation originated in ancient Greece and was subsequently propagated in other Mediterranean countries (especially in Italy) during recent historical times.
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2,337,719 |
Detection of rearrangements in the NF2 gene using semi-quantitative multiplex fluorescent PCR.
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Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder that predisposes to the development of bilateral vestibular schwannomas (sometimes associated with schwannomas at other locations), meningiomas, and ependymomas. Point mutations that inactivate the NF2 tumor suppressor gene, located in 22q12, have been found in 45-85% of NF2 patients; in addition, large genomic deletions can be found. To evaluate the presence of genomic NF2 rearrangements, we have developed a fluorescent semiquantitative multiplex PCR method. Briefly, short fragments corresponding to the 17 exons, the promoter region, and the 3' end of the NF2 gene were co-amplified by PCR using dye primers. An additional fragment, corresponding to another gene used as an internal control, was systematically amplified in each multiplex PCR. Initially, we validated the method by using monosomic 22q and trisomic 22 samples. The fluorescent multiplex PCR method was then used to analyze 21 NF2 individuals in which single-strand conformational polymorphism (SSCP) analysis and/or direct sequencing had revealed no NF2 point mutations; we were able to detect two deletions and one duplication in NF2 in 3 patients. In conclusion, the method we developed could easily be applied in detecting NF2 deletions and duplications. Discovering genomic duplications is invaluable because they are probably the most difficult molecular alterations to detect with conventional methods and, as a consequence, might be an underestimated cause of NF2.
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2,337,720 |
Microarray-based detection of mannose-binding lectin 2 (MBL2) polymorphisms in a routine clinical setting.
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The assessment of allelic variants in the human mannose-binding lectin 2 (MBL2) gene is of great clinical importance in newborns or immune-suppressed patients at high risk for a variety of infections. Here, we present a study on the genotyping accuracy of a DNA microarray-based on-chip PCR method suited for the detection of five different polymorphisms in the MBL2 gene. We tested 153 genomic DNA samples, prepared from archival blood spots on Guthrie cards, for the presence of allelic variants in the human MBL2 gene by the on-chip PCR method and compared the obtained results of three variants to standard DNA capillary sequencing. The genotyping power of the described assay was readily comparable to DNA sequencing (453/459 correct genotype calls in 153 DNA samples; 98.7% accuracy), mainly due to intrinsic technical benefits of microarrays such as high number of test replicates and automated data analysis. This study demonstrates, for the first time, the accuracy and reliability of a microarray-based on-chip PCR genotyping assay for measuring allelic variants in a routine clinical setting.
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2,337,721 |
[Pharmacogenetics in future medical care -- implications for patients and physicians].
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Since public acceptance of pharmacogenetic testing will largely influence their implementation in routine medical care, common implications of pharmacogenetic testing from patients', physicians' and scientists' perspective are reviewed.</AbstractText>Broad literature review (MEDLINE; MeSH terms: Pharmacogenetics, Delivery of Health Care, Ethics, Attitude, Patient Acceptance of Health Care) of empirical and theoretical studies describing psychological, family-related, social, and ethical consequences of pharmacogenetic testing to describe relevant aspects for further empirical studies.</AbstractText>Apart from anticipated benefit, acceptance of pharmacogenetic testing might be influenced by the following: Expectation of negative psychosocial consequences, fear of discrimination or violation of privacy. Due to its great complexity, understanding of test results and explanation of their impact pose new challenges for physician-patient relations. Since most studies are conceptual, empirical studies exploring attitudes of patients/physicians and determining medical as well as economic value of pharmacogenetic testing are imperative.</AbstractText>
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2,337,722 |
Small molecule and monoclonal antibody therapies in neurooncology.
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The prognosis for most patients with primary brain tumors remains poor. Recent advances in molecular and cell biology have led to a greater understanding of molecular alterations in brain tumors. These advances are being translated into new therapies that will hopefully improve the prognosis for patients with brain tumors.</AbstractText>We reviewed the literature on small molecule targeted agents and monoclonal antibodies used in brain tumor research and brain tumor clinical trials for the past 20 years.</AbstractText>Brain tumors commonly express molecular abnormalities. These alterations can lead to the activation of cell pathways involved in cell proliferation. This knowledge has led to interest in novel anti-brain-tumor therapies targeting key components of these pathways. Many drugs and monoclonal antibodies have been developed that modulate these pathways and are in various stages of testing.</AbstractText>The use of targeted therapies against brain tumors promises to improve the prognosis for patients with brain tumors. However, as the molecular pathogenesis of brain tumors has not been linked to a single genetic defect or target, molecular agents may need to be used in combinations or in tandem with cytotoxic agents. Further study of these agents in well-designed cooperative clinical trials is needed.</AbstractText>
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2,337,723 |
The RB protein family in retinal development and retinoblastoma: new insights from new mouse models.
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The Rb gene was isolated almost 20 years ago, but fundamental questions regarding its role in retinal development and retinoblastoma remain. What is the normal function of RB protein in retinogenesis? What is the cell-of-origin of retinoblastoma? Why do retinoblastoma tumors have recurrent genetic lesions other than Rb inactivation? Why is retinoblastoma not induced by defects in cell cycle regulators other than Rb? Why is the retina so sensitive to Rb loss? Recently developed conditional Rb knockout models provide new insight into some of these issues. The data suggest that RB protein may not control the rate of progenitor division, but is critical for cell cycle exit when dividing retinal progenitors differentiate into postmitotic transition cells. This finding focuses attention on the ectopically dividing transition cell, rather than the progenitor, as the cell-of-origin. Cell-specific analyses in the RB-deficient retina reveal that ectopically dividing photoreceptors, bipolar and ganglion cells die, but amacrine, horizontal and Muller cells survive and stop dividing when they terminally differentiate. Rare amacrine transition cells escape cell cycle exit and generate tumors. These data suggest that post-Rb mutations are required to overcome growth arrest associated with terminal differentiation, rather than apoptosis as previously suggested. To explain why perturbing cell cycle regulators other than RB does not initiate retinoblastoma, we speculate that mutations in other components of the RB pathway perturb cell cycle arrest, but only RB loss triggers genome instability in retinal transition cells, which may be critical to facilitate post-Rb mutations necessary for transformation. Cell-specific differences in the effect of Rb loss on genome stability may contribute to the tremendous sensitivity of retinal transition cells to tumorigenesis. The new mouse models of retinoblastoma will be invaluable for testing these possibilities.
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2,337,724 |
Dual targeting of topoisomerase IV and gyrase to reduce mutant selection: direct testing of the paradigm by using WCK-1734, a new fluoroquinolone, and ciprofloxacin.
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Quinolones that act equally against DNA gyrase and topoisomerase IV are a desirable modality to decrease the selection of resistant strains. We first determined by genetic and biochemical studies in Staphylococcus aureus that the primary target enzyme of WCK-1734, a new quinolone, was DNA gyrase. A single mutation in gyrase, but not topoisomerase IV, caused a two- to fourfold increase in the MIC. Studies with purified topoisomerase IV and gyrase from S. aureus also showed that gyrase was more sensitive than topoisomerase IV to WCK-1734 (50% inhibitory concentration, 1.25 and 2.5 to 5.0 microg/ml, respectively; 50% stimulation of cleavage complex formation, 0.62 and 2.5 to 5.0 microg/ml, respectively). To test the effect of balanced activity of quinolones against the two target enzymes, we measured the frequency of selection of mutants with ciprofloxacin (which targets topoisomerase IV) and WCK-1734 alone and in combination. With the combination of ciprofloxacin and WCK-1734, each at its MIC, the ratio of frequency of mutants selected was significantly lower than that with each drug alone at two times their respective MICs. We further characterized resistant strains selected with the combination of ciprofloxacin and WCK-1734 and found evidence to suggest the existence of novel mutational mechanisms for low-level quinolone resistance. By use of a combination of differentially targeting quinolones, this study provides novel data in direct support of the paradigm for dual targeting of quinolone action and reduced development of resistance.
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2,337,725 |
Factor V Leiden and its effect on children with cardiac pathology.
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Factor V Leiden thrombophilia, formed by a genetic mutation, slows the body's anticoagulation response to clot formation. First described in 1994, factor V Leiden mutation has been identified as a contributing risk factor for venous thromboembolism. Although venous thromboembolism is considered rare in the pediatric population, Factor V Leiden has implications for certain at-risk pediatric patients.
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2,337,726 |
Accuracy of revised Bethesda guidelines, microsatellite instability, and immunohistochemistry for the identification of patients with hereditary nonpolyposis colorectal cancer.
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The selection of individuals for hereditary nonpolyposis colorectal cancer (HNPCC) genetic testing is challenging. Recently, the National Cancer Institute outlined a new set of recommendations, the revised Bethesda guidelines, for the identification of individuals with HNPCC who should be tested for microsatellite instability.</AbstractText>To establish the most effective and efficient strategy for the detection of MSH2/MLH1 gene carriers.</AbstractText><AbstractText Label="DESIGN, SETTING, AND PATIENTS" NlmCategory="METHODS">A prospective, multicenter, nationwide study (the EPICOLON study) in 20 hospitals in the general community in Spain of 1222 patients with newly diagnosed colorectal cancer between November 1, 2000, and October 31, 2001.</AbstractText>Microsatellite instability testing and MSH2/MLH1 immunostaining in all patients regardless of age, personal or family history, and tumor characteristics. Patients whose tumors exhibited microsatellite instability and/or lack of protein expression underwent MSH2/MLH1 germline testing.</AbstractText>Effectiveness and efficiency of both microsatellite instability testing and immunostaining, either directly or previous selection of patients according to the revised Bethesda guidelines, were evaluated with respect to the presence of MSH2/MLH1 germline mutations.</AbstractText>Two hundred eighty-seven patients (23.5%) fulfilled the revised Bethesda guidelines. Ninety-one patients (7.4%) had a mismatch repair deficiency, with tumors exhibiting either microsatellite instability (n = 83) or loss of protein expression (n = 81). Germline testing identified 11 mutations (0.9%) in either MSH2 (7 cases) or MLH1 (4 cases) genes. Strategies based on either microsatellite instability testing or immunostaining previous selection of patients according to the revised Bethesda guidelines were the most effective (sensitivity, 81.8% and 81.8%; specificity, 98.0% and 98.2%; positive predictive value, 27.3% and 29.0%, respectively) to identify MSH2/MLH1 gene carriers. Logistic regression analysis confirmed the revised Bethesda guidelines as the most discriminating set of clinical parameters (odds ratio, 33.3; 95% confidence interval, 4.3-250; P = .001).</AbstractText>The revised Bethesda guidelines constitute a useful approach to identify patients at risk for HNPCC. In patients fulfilling these criteria, both microsatellite instability testing and immunostaining are equivalent and highly effective strategies to further select those patients who should be tested for MSH2/MLH1 germline mutations.</AbstractText>
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2,337,727 |
Oligonucleotide microarray for HLA-DRB1 genotyping: preparation and clinical evaluation.
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In this study, HLA-DRB1 gene was genotyped by using the microarray technique. Oligonucleotide probes were designed based on partial sequences of various genotypes of HLA-DRB1, and were fixed on a silylated slide to form a microarray. The second exon of HLA-DRB1 gene in the extracted genomic DNA samples was amplified and labelled by means of polymerase chain reaction (PCR); then it was hybridized to the microarray. The microarray was scanned, and the result was analysed in order to determine the genotypes of HLA-DRB1 of the tested sample. A total of 1574 of 1592 clinical samples had accordant results of genotypes in either microarray assay or PCR-SSP assay; 8 of 10 samples that had inconsistent results of genotypes were proved to be microarray-assay reliable by confirmation of DNA sequencing. It is concluded that microarray is an alternative reliable method for HLA-DRB1 genotyping.
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2,337,728 |
High-resolution genotyping of HLA-DQA1 in the GoKinD study and identification of novel alleles HLA-DQA1*040102, HLA-DQA1*0402 and HLA-DQA1*0404.
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In order to achieve high-resolution HLA-DQA1 genotyping, it is necessary to identify polymorphisms in exons 1, 2 and 3. We present a high-resolution sequence-based typing (SBT) strategy for genotyping exons 1, 2 and 3 of the polymorphic HLA-DQA1 locus. This method is an improvement upon previously presented methods, because it utilizes the minimum number of SSP-PCR assays to obtain clear DNA sequence in both the forward and reverse directions of all three exons. All known HLA-DQA1 alleles are resolved with the exception of HLA-DQA1*010101 and HLA-DQA1*010102 for which the distinguishing polymorphism is located in exon 4 and does not result in an amino acid change. This method has enabled our laboratory to identify three new HLA-DQA1 alleles - HLA-DQA1*040102, HLA- DQA1*0402 and HLA-DQA1*0404 - in the Genetics of Kidneys in Diabetes (GoKinD) study population. Additionally, we present single-allele amplification methods, which identify the coding sequences of HLA-DQA1 exons 1, 2, 3, intron 2 and 300 bp of the HLA-DQA1 promoter (QAP). This study, also describes the QAP for most of the known HLA-DQA1 alleles, three HLA-DQA2 promoter sequences and the intron 2 sequences for HLA-DQA1*040101, HLA-DQA1*040102, HLA-DQA1*0402 and HLA-DQA1*0404.
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2,337,729 |
HIV vaccine rationale, design and testing.
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A central obstacle to the design of a global HIV vaccine is viral diversity. Antigenic differences in envelope proteins result in distinct HIV serotypes, operationally defined such that antibodies raised against envelope molecules from one serotype will not bind envelope molecules from a different serotype. The existence of serotypes has presented a similar challenge to vaccine development against other pathogens. In such cases, antigenic diversity has been addressed by vaccine design. For example, the poliovirus vaccine includes three serotypes of poliovirus, and Pneumovax presents a cocktail of 23 pneumococcal variants to the immune system. It is likely that a successful vaccine for HIV must also comprise a cocktail of antigens. Here, data relevant to the development of cocktail vaccines, designed to harness diverse, envelope-specific B-cell and T-cell responses, are reviewed.
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2,337,730 |
Molecular biology of channelopathies: impact on diagnosis and treatment.
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Channelopathies are genetically determined ion channel alterations that lead to acute and transient symptoms in subjects who otherwise appear to be normal. This article reviews the recent progression of biomolecular studies that have clarified the mechanisms by which gene mutations may result in alterations of excitable tissues responsible for episodic neurological, neuromuscular and cardiac disorders, defined as channelopathies. The development of technologies capable of testing pharmacological agents in vitro on mutated channels expressed in cell lines makes it possible to define a more rational use of the available drugs acting on ion channels, and to design new molecules specifically targeted to known channel dysfunctions and new ones that could be identified by future genetic studies.
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2,337,731 |
The impact of genomics on E-health.
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The Human Genome Project (HGP) and e-Health are two fundamental changes that will alter the way we approach human health and life. These two scientific and societal forces will inevitably impact each other. This paper not only explores the ways that the HGP will change health care but also investigates the ways that e-Health systems will be influenced by the genomic data. The Electronic Medical Record (EMR) is discussed at length, including the probable impact on the laboratory, pharmacy, computerized provider order entry (CPOE), and other components. Thirteen points of a possible genomic future involving the EMR are presented. The genomic impact on other e-Health systems includes those at all levels of data: population, disease, patient, tissue and organ banks, cellular and for specific genes. The genomic impact on consumer health systems is explored, including Internet consumer information resources and the movement for direct-to-consumer genetic testing. The paper concludes that technology and trends of e-Health will enable the upcoming revolution caused by the health implications of the research emanating from the Human Genome Project.
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2,337,732 |
Germline BHD-mutation spectrum and phenotype analysis of a large cohort of families with Birt-Hogg-Dubé syndrome.
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Birt-Hogg-Dubé syndrome (BHD), a genodermatosis characterized by multiple hamartomas of the hair follicle (fibrofolliculoma), predisposes individuals to an increased risk of developing renal neoplasms and spontaneous pneumothorax. Previously, we localized the BHD locus (also known as FLCN) to chromosome 17p11.2 by linkage analysis and subsequently identified germline mutations in a novel gene in probands from eight of the nine families with BHD in our screening panel. Affected members of five of the families inherited an insertion/deletion of a cytosine in a C8 tract in exon 11. This mutation was also identified by exon 11 screening in probands from 22 of 52 additional families with BHD and therefore represents a hypermutable "hotspot" for mutation in BHD. Here, we screened the remaining 30 families from this large BHD cohort by direct sequence analysis and identified germline BHD mutations in 84% (51/61) of all families with BHD recruited to our study. Mutations were located along the entire length of the coding region, including 16 insertion/deletion, 3 nonsense, and 3 splice-site mutations. The majority of BHD mutations were predicted to truncate the BHD protein, folliculin. Among patients with a mutation in the exon 11 hotspot, significantly fewer renal tumors were observed in patients with the C-deletion than those with the C-insertion mutation. Coding-sequence mutations were not found, however, in probands from two large families with BHD whose affected members shared their family's BHD-affected haplotype. Of the 53 families with BHD whose members inherited either a germline mutation or the affected haplotype, 24 (45%) had at least one member with renal neoplasms. Three families classified with familial renal oncocytoma were identified with BHD mutations, which represents the first disease gene associated with this rare form of renal neoplasm. This study expands the BHD-mutation spectrum and evaluates genotype-phenotype correlations among families with BHD.
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2,337,733 |
A family with early-onset and rapidly progressive X-linked spinal and bulbar muscular atrophy.
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Spinal and bulbar muscular atrophy (SBMA) is an X-linked, late-onset neuroendocrine disorder resulting from an expansion of a CAG repeat in the androgen receptor gene. Reported here is a detailed phenotypic study in a series of seven patients from the same family with SBMA with 50 to 54 CAG repeats, juvenile onset (mean age at onset 13 years [8 to 15 years]), and rapid progression leading to compromised ambulation in the mid-20s.
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2,337,734 |
A novel mutation (G114V) in the prion protein gene in a family with inherited prion disease.
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Inherited prion diseases are characterized by mutations in the PRNP gene encoding the prion protein (PrP). We report a novel missense mutation in the PRNP gene (resulting in a G114V mutation in PrP) in members of a Uruguayan family with clinical and histopathologic features of prion disease. Affected individuals were characterized by an early age at onset, initial neuropsychiatric symptoms, late dementia with prominent pyramidal and extrapyramidal symptoms, and long disease duration.
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2,337,735 |
EGFR tyrosine kinase domain mutations in human gliomas.
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Gefitinib is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor effective in patients with lung cancer with mutations in exons 19 and 21 of the EGFR tyrosine kinase domain. In this study, the authors tested the presence of such mutations in 95 gliomas including glioblastomas, anaplastic oligodendrogliomas, and low-grade gliomas. No mutation was found, which suggests that the biology of EGFR in gliomas is different from lung cancer and that this may be a factor in the resistance of glioblastomas to gefitinib.
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2,337,736 |
Mild glycine encephalopathy (NKH) in a large kindred due to a silent exonic GLDC splice mutation.
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Classic neonatal-onset glycine encephalopathy (GE) is devastating and life threatening. Milder, later onset variants have been reported but were usually sporadic and incompletely defined.</AbstractText>To determine the clinical and biochemical phenotype and molecular basis of mild GE in nine children from a consanguineous Israeli Bedouin kindred.</AbstractText>Genomic DNA was screened for GLDC, AMT, and GCSH gene mutations. GLDC expression in lymphoblasts was studied by Northern blot and reverse transcriptase PCR analysis.</AbstractText>Clinical features included hypotonia, abnormal movements, convulsions, and moderate mental retardation with relative sparing of gross motor function, activities of daily living skills, and receptive language. Aggression and irritability were prominent. CSF-to-plasma glycine ratio was mildly to moderately elevated. All nine patients were homozygous and their parents heterozygous for a novel, translationally silent GLDC exon 22 transversion c.2607C>A. Lymphoblast GLDC mRNA levels were considerably reduced. Three aberrantly spliced cDNA species were identified: exon 22 and exon 22 to 23 skipping, and insertion of an 87-base pair cryptic exon. Homozygosity for c.2607C>A was also identified in an unrelated but haplotypically identical patient with an unusually favorable outcome despite severe neonatal-onset GE. Mutation analysis enabled prenatal diagnosis of three unaffected and one affected pregnancies.</AbstractText>The mutation in this kindred led to missplicing and reduced GLDC (glycine decarboxylase) expression. The 4 to 6% of normally spliced GLDC mRNA in the patients may account for their relatively favorable clinical outcome compared with patients with classic glycine encephalopathy.</AbstractText>
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2,337,737 |
Screen of the IMPDH1 gene among patients with dominant retinitis pigmentosa and clinical features associated with the most common mutation, Asp226Asn.
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To determine the frequency of mutations in IMPDH1 among patients with autosomal dominant retinitis pigmentosa (RP), to characterize the clinical features of patients with the Asp226Asn mutation in this gene, and to compare these features with those found among patients with selected dominant mutations in other RP genes.</AbstractText>The coding sequence and the adjacent flanking intron sequences of all 14 coding exons were sequenced in 183 unrelated patients with dominant RP. The clinical findings evaluated included visual acuity, refractive error, visual field area measured with the Goldmann perimeter, final dark-adaptation threshold, full-field electroretinogram (ERG) amplitudes, cataract, and funduscopic bone spicule pigmentation.</AbstractText>The mutation Asp226Asn was identified in 6 of the 183 unrelated patients with RP. One patient carried the novel, possibly pathogenic, change Lys238Glu. There was approximately a 100-fold variation in ERG amplitudes among patients of similar age with the Asp226Asn mutation. Patients had similar reductions of rod-plus-cone 0.5-Hz ERG amplitude and cone 30-Hz ERG amplitude. For a given amount of remaining visual field, there was a larger ERG amplitude in IMPDH1-carrying patients (average 0.5-Hz ERG/visual field ratio = 9.5 nV/deg(2)) compared with groups of patients with the RP1 mutation Arg677End (2.8 nV/deg(2)), the rhodopsin (RHO) mutation Pro23His (5.1 nV/deg(2)), or the RHO mutation Pro347Leu (1.7 nV/deg(2)).</AbstractText>IMPDH1 mutations account for approximately 2% of cases of dominant RP in North America. The most frequent mutation, Asp226Asn, appears to cause at least as much loss of rod function as cone function. Patients with this form of RP retain, on average, two to five times more ERG amplitude per unit of remaining visual area than patients with three other forms of dominant RP.</AbstractText>
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2,337,738 |
Nuclear and mitochondrial genes mutated in nonsyndromic impaired hearing.
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Half of the cases with congenital impaired hearing are hereditary (HIH). HIH may occur as part of a multisystem disease (syndromic HIH) or as disorder restricted to the ear and vestibular system (nonsyndromic HIH). Since nonsyndromic HIH is almost exclusively caused by cochlear defects, affected patients suffer from sensorineural hearing loss. One percent of the total human genes, i.e. 300-500, are estimated to cause syndromic and nonsyndromic HIH. Of these, approximately 120 genes have been cloned thus far, approximately 80 for syndromic HIH and 42 for nonsyndromic HIH. In the majority of the cases, HIH manifests before (prelingual), and rarely after (postlingual) development of speech. Prelingual, nonsyndromic HIH follows an autosomal recessive trait (75-80%), an autosomal dominant trait (10-20%), an X-chromosomal, recessive trait (1-5%), or is maternally inherited (0-20%). Postlingual nonsyndromic HIH usually follows an autosomal dominant trait. Of the 41 mutated genes that cause nonsyndromic HIH, 15 cause autosomal dominant HIH, 15 autosomal recessive HIH, 6 both autosomal dominant and recessive HIH, 2 X-linked HIH, and 3 maternally inherited HIH. Mutations in a single gene may not only cause autosomal dominant, nonsyndromic HIH, but also autosomal recessive, nonsyndromic HIH (GJB2, GJB6, MYO6, MYO7A, TECTA, TMC1), and even syndromic HIH (CDH23, COL11A2, DPP1, DSPP, GJB2, GJB3, GJB6, MYO7A, MYH9, PCDH15, POU3F4, SLC26A4, USH1C, WFS1). Different mutations in the same gene may cause variable phenotypes within a family and between families. Most cases of recessive HIH result from mutations in a single locus, but an increasing number of disorders is recognized, in which mutations in two different genes (GJB2/GJB6, TECTA/KCNQ4), or two different mutations in a single allele (GJB2) are involved. This overview focuses on recent advances in the genetic background of nonsyndromic HIH.
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2,337,739 |
XLMR in MRX families 29, 32, 33 and 38 results from the dup24 mutation in the ARX (Aristaless related homeobox) gene.
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X-linked mental retardation (XLMR) is the leading cause of mental retardation in males. Mutations in the ARX gene in Xp22.1 have been found in numerous families with both nonsyndromic and syndromic XLMR. The most frequent mutation in this gene is a 24 bp duplication in exon 2. Based on this fact, a panel of XLMR families linked to Xp22 was tested for this particular ARX mutation.</AbstractText>Genomic DNA from XLMR families linked to Xp22.1 was amplified for exon 2 in ARX using a Cy5 labeled primer pair. The resulting amplicons were sized using the ALFexpress automated sequencer.</AbstractText>A panel of 11 families with X-linked mental retardation was screened for the ARX 24dup mutation. Four nonsyndromic XLMR families - MRX29, MRX32, MRX33 and MRX38 - were found to have this particular gene mutation.</AbstractText>We have identified 4 additional XLMR families with the ARX dup24 mutation from a panel of 11 XLMR families linked to Xp22.1. This finding makes the ARX dup24 mutation the most common mutation in nonsyndromic XLMR families linked to Xp22.1. As this mutation can be readily tested for using an automated sequencer, screening should be considered for any male with nonsyndromic MR of unknown etiology.</AbstractText>
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2,337,740 |
Safety enhancement of a specialized power assisted tricycle for a child with osteogenesis imperfecta type III.
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A child in the community of Laramie, Wyoming was born with Osteogenesis Imperfecta which is a genetic disorder that limits the physical abilities, size, and strength of the child. A customized power assisted tricycle was developed, which offered a unique opportunity to serve multiple purposes in his childhood development. This tricycle will ultimately provide him with the opportunity to gain muscle mass, strength, coordination, and confidence. The tricycle was completed as a senior design project in 2002, funded by the National Science Foundation, Biomedical Engineering Program and research to Aid Persons with Disabilities Program and University of Wyoming, College of Engineering Undergraduate Design Project to Aid Wyoming Persons with Disabilities. Unfortunately, the tricycle did not provide the necessary features to allow him to ride the tricycle safely. For this reason the tricycle was redesigned to include many different redundant safety systems which allows the tricycle to be safe for the child's use. Being funded by the same grant, new systems were added to the tricycle. A panic kill switch, automatic brakes, numerous redundant velocity sensors, tip over prevention circuitry, a redesigned operating system, a battery recharge port, and other systems were added, allowing for the tricycle to provide a high level of safety. A great deal of testing and sound design practices have been taken into consideration throughout the addition of these systems. Without these improvements, the child would not have the opportunity to use the tricycle to help with his development.
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2,337,741 |
Statistical analysis of microarray data.
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Microarrays promise dynamic snapshots of cell activity, but microarray results are unfortunately not straightforward to interpret. This article aims to distill the most useful practical results from the vast body of literature available on microarray data analysis. Topics covered include: experimental design issues, normalization, quality control, exploratory analysis, and tests for differential expression. Special attention is paid to the peculiarities of low-level analysis of Affymetrix chips, and the multiple testing problem in determining differential expression. The aim of this article is to provide useful answers to the most common practical issues in microarray data analysis. The main topics are pre-processing (normalization), and detecting differential expression. Subsidiary topics include experimental design, and exploratory analysis. Further discussion is found at the author's web page (http://discover.nci.nih.gov --> Notes on Microarray Data Analysis).
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2,337,742 |
Molecular genetics of familial renal cell carcinoma syndromes.
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RCC represents a group of clinically and genetically diverse diseases. Familial RCC syndromes, although rare, provide an invaluable model to study the molecular mechanisms of renal carcinogenesis. Many causative oncogenes and tumor suppressor genes have been identified and it is now possible to identify the affected individuals and carriers by genetic testing. Understanding of the molecular pathways of these genes will have a significant impact on the diagnosis and treatment of familial and sporadic RCC.
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2,337,743 |
Effect of cytokines and chemokines (TGF-beta, TNF-alpha, IL-6, IL-10, MCP-1, RANTES) gene polymorphisms in kidney recipients on posttransplantation outcome: influence of donor-recipient match.
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Posttransplantation alloantigen-dependent and alloantigen-independent processes are both mediated by cytokines and chemokines. Recently cytokines and chemokines, as well as their receptors, have been shown to be highly polymorphic. The cytokine and chemokine gene polymorphisms are associated with variable production, activity, expression, or ligand-receptor affinity. The aim of our study was to analyze the relation between selected cytokine and chemokine gene polymorphisms in kidney donors and recipients as a function of donor-recipient match and posttransplantation outcome. Polymorphisms transforming growth factor-beta (TGF-beta); tumor necrosis factor-alpha (TNF-alpha); interleukin (IL)-6, and IL-10; monocyte chemoattractant protein-1 (MCP-1); and RANTES (regulated upon activation, normal T-cell expressed and secreted) genes were determined using DNA polymerase chain reaction technology in 268 healthy volunteers, 345 kidney transplant recipients (1997 to 1999), and 298 cadaveric donors. Patients were followed up for 4 to 6 years. The distribution of alleles of selected genes was identical in control subjects, cadaveric donors, and recipients. Low TGF-beta production in both the donor and recipient genotypes was associated with risk for early rejection (6 months) and worse graft function at 4 years. The only tendency for worse graft outcome was observed among donor-recipient combinations mismatched for TGF-beta genotype. Genetic determination of TNF-alpha and IL-10 production was associated with delayed graft function and rejection. IL-6 gene polymorphisms had no effect on the incidence of early acute rejections, but was associated with worse 5-year outcomes. Determinations of MCP-1 overproduction and RANTES-109 TT allele were associated with significant deterioration of graft function. Our data support the hypothesis that the strength of the alloimmune response after transplantation is in part genetically determined. Donor-recipient matching of cytokine gene polymorphisms has a marginal effect.
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2,337,744 |
Genetic dissection of the zebrafish retinal stem-cell compartment.
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In a large-scale forward-genetic screen, we discovered that a limited number of genes are required for the regulation of retinal stem cells after embryogenesis in zebrafish. In 18 mutants out of almost 2000 F2 families screened, the eye undergoes normal embryonic development, but fails to continue growth from the ciliary marginal zone (CMZ), the post-embryonic stem-cell niche. Class I-A mutants (5 loci) display lower amounts of proliferation in the CMZ, while nearly all cells in the retina appear differentiated. Class I-B mutants (2 loci) have a reduced CMZ with a concomitant expansion in the retinal pigmented epithelium (RPE), suggesting a common post-embryonic stem cell is the source for these neighboring cell types. Class II encompasses three distinct types of mutants (11 loci) with expanded CMZ, in which the progenitor population is arrested in the cell cycle. We also show that in at least one combination, the reduced CMZ phenotype is genetically epistatic to the expanded CMZ phenotype, suggesting that Class I genes are more likely to affect the stem cells and Class II the progenitor cells. Finally, a comparative mapping analysis demonstrates that the new genes isolated do not correspond to genes previously implicated in stem-cell regulation. Our study suggests that embryonic and post-embryonic stem cells utilize separable genetic programs in the zebrafish retina.
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2,337,745 |
Expression mapping using a retroviral vector for CD8+ T cell epitopes: definition of a Mycobacterium tuberculosis peptide presented by H2-Dd.
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Identification of CD8+ T cell epitopes is important because detection of specific CD8+ T cells after infection or immunization requires prior knowledge of epitope specificity. Furthermore, identification of CD8+ T cell epitopes permits the development of specific preventive and therapeutic approaches to both infections and tumors. Thus far, CD8+ T cell epitopes have been identified either using an overlapping peptide library covering an entire protein, or using algorithms designed to identify likely peptides that bind to major histocompatibility complex (MHC) class I molecules. The synthesis of overlapping peptides can be prohibitively expensive, and the algorithm programs used to predict CD8+ T cell epitopes are not always accurate. Here we describe a retroviral expression system that specifically allows longer polypeptides and shorter peptides to be expressed in the cytoplasm, and thereby to be processed onto class I MHC molecules. T cells from mice that were immunized with a DNA vaccine encoding MPT-51 were probed against MHC-compatible cell lines retrovirally transduced with overlapping gene fragments encoding 120-140 amino acids of the MPT-51 molecule. After further testing of shorter peptide sequences, we identified a CD8+ T cell epitope using cell lines expressing a relatively small number of algorithm-predicted candidate epitopes. We found that one of the requirements for cell surface display of the 20-mer peptide was the need for cotranslational ubiquitination. The restriction molecule was identified as Dd following transduction with MHC class I genes followed by transduction with the oligonucleotide encoding the epitope. The retroviral expression system described here is cost-effective, particularly if the target molecule is large, and could be adapted to identifying T cell epitopes recognized in infectious disease and against tumor cell antigens.
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2,337,746 |
Molecular basis for para-Bombay phenotypes in Chinese persons, including a novel nonfunctional FUT1 allele.
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The para-Bombay phenotype is characterized by H-deficient or H-partially deficient red blood cells (RBCs) in persons who secrete ABH antigens in their saliva. The studies that determined the genotypes for two Chinese individuals with the para-Bombay phenotype are described.</AbstractText>RBC phenotypes were characterized by conventional serologic methods. Exons 6 and 7 of the ABO gene were amplified, as well as the entire coding region for FUT1 and FUT2, with four independence polymerase chain reactions (PCRs) from genomic DNA. PCR products were excised, purified from agarose gels, and sequenced directly. Mutations of FUT1 were identified by TOPO cloning sequencing.</AbstractText>For both individuals, RBC ABO genotypes correlated with ABH substances in their saliva. One individual (a patient) had two heterozygous mutations of FUT1 by direct DNA sequencing, namely, a C-->T heterozygous mutation at position 293(C293T) and AG heterozygous deletion (CAGAGAG-->CAGAG) at position 547 to 552. These two mutations were confirmed to be compound heterozygotes; that is, each mutation was determined to be on a separate homologous chromosome by TOPO cloning sequencing. The FUT2 genotype was Se(357)Se(357). The other individual (a blood donor) had an AG deletion at position 547 to 552 homozygous allele in FUT1. The FUT2 genotype was Se(357)Se(357,385). C293T mutation can cause Thr/Met at amino acid position 98. AG deletion at position 547 to 552 caused a reading frameshift and a premature stop codon.</AbstractText>A novel nonfunctional FUT1 allele C293T was identified in a person with the para-Bombay phenotype. This rare H-deficient phenotype may result from different nonfunctional alleles.</AbstractText>
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2,337,747 |
A factorial experiment for optimizing the PCR conditions in routine genotyping.
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Although most PCRs would produce proper PCR products when first tried, a general optimization is required to yield the best results. This optimization is often achieved by changing one factor at a time. However, this may lead to suboptimal results, since interactions between conditions are difficult to detect with this approach. In the present study, we describe the factorial optimization of PCR conditions for microsatellite genotyping, by introducing small systematic variations in conditions during the genotyping process. The hypothesis was that small changes will not affect genotyping results, but will provide information about the optimality of current conditions. The conditions to vary were the concentrations of buffer, MgCl(2), dNTPs, primers, Taq polymerase and DNA, the annealing temperature (T(a)) and the number of cycles. We show that, by a 2(8) factorial experiment, it is possible to identify not only the factors responsible for obtaining good results, but also those responsible for bad results. However, the condition leading to the highest signals is not necessarily the best operational condition. The best operational condition is minimally sensitive to random pipetting fluctuations and yields reliable genotypes as well.
|
2,337,748 |
The MELISSA pilot plant facility as as integration test-bed for advanced life support systems.
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The different advances in the Micro Ecological Life Support System Alternative project (MELISSA), fostered and coordinated by the European Space Agency, as well as in other associated technologies, are integrated and demonstrated in the MELISSA Pilot Plant laboratory. During the first period of operation, the definition of the different compartments at an individual basis has been achieved, and the complete facility is being re-designed to face a new period of integration of all these compartments. The final objective is to demonstrate the potentiality of biological systems such as MELISSA as life support systems. The facility will also serve as a test bed to study the robustness and stability of the continuous operation of a complex biological system. This includes testing of the associated instrumentation and control for a safe operation, characterization of the chemical and microbial safety of the system, as well as tracking the genetic stability of the microbial strains used. The new period is envisaged as a contribution to the further development of more complete biological life support systems for long-term manned missions, that should be better defined from the knowledge to be gained from this integration phase. This contribution summarizes the current status of the Pilot Plant and the planned steps for the new period.
|
2,337,749 |
Prevalence of five previously reported and recurrent BRCA1 genetic rearrangement mutations in 20,000 patients from hereditary breast/ovarian cancer families.
|
Many rearrangement mutations in the BRCA1 gene have been identified. It is becoming clear that some of these mutations are prevalent, and therefore their detection is necessary in order for clinical genetic tests to have high sensitivity. Published information on particular rearrangements is frequently limited to a single patient, small groups of patients, or patients of a particular ethnicity. The objectives of this work included characterizing the prevalence of five specific rearrangement mutations in a large North American patient population. A mutation-specific multiplex PCR assay was used for determining the prevalence of five BRCA1 rearrangement mutations that previously had been reported to occur in unrelated patients. The mutation status of these rearrangements, which came from 20,712 patients at high risk for hereditary breast and/or ovarian cancers who had submitted specimens for clinical genetic testing, is presented. The results, obtained from 2,634 mutation carriers, showed a 6-kb duplication of exon 13, identified in 53 patients (2.01%); a 26-kb deletion encompassing exons 14-20, detected in seven patients (0.27%); a 510-bp deletion of exon 22, detected in 5 patients (0.19%); and a 3.4-kb deletion of exon 13, detected in one patient (0.04%). A previously reported 7.1-kb deletion of exons 8-9 was not found. The high frequency of the exon 13 duplication makes it the fourth most prevalent mutation in these patients. These results provide an accurate picture of the prevalence of these mutations in hereditary breast/ovarian cancer patients undergoing genetic testing in North America.
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2,337,750 |
Human leukocyte antigen matching in unrelated donor hematopoietic cell transplantation.
|
Hematopoietic cell transplantation (HCT) from unrelated donors is a curative therapy for many malignant and nonmalignant blood disorders. The success of unrelated HCT is influenced by the degree of human leukocyte antigen (HLA) compatibility between the donor and patient. When donor matching for HLA alleles is feasible, overall transplant outcome is superior. The presence of donor-recipient mismatching is associated with increased risk of post-transplant complications including graft rejection, acute and chronic graft-versus-host disease (GVHD), and mortality; these risks are increased with multiple HLA mismatches. For the majority of patients who lack HLA-matched unrelated donors, current research is focused on the identification of permissible HLA mismatches. The influence of nongenetic factors on the tolerability of HLA mismatching has recently become evident, demonstrating a need for the integration of both genetic and nongenetic variables in donor selection.
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2,337,751 |
Screening candidate genes for mutations in patients with hypogonadotropic hypogonadism using custom genome resequencing microarrays.
|
The purpose of this study was to determine the consistency of calling single nucleotide polymorphisms (SNPs) by custom genome resequencing microarrays compared with capillary DNA sequencing.</AbstractText>Amplified genomic DNA from 23 patients with hypogonadotropic hypogonadism was hybridized to microarrays containing 30 kilobases of sequence from 6 different candidate genes. Capillary DNA sequencing was performed in 10 patients.</AbstractText>For 10 patients with > or =90% of bases called, 49 SNPs in 5 of 6 genes were identified. Of the 490 bases, 75 were ambiguous (read as "N"), and 415 were able to be called an A, C, G, or T. Of 415 called, 401 (96.6%) sequences were confirmed by DNA sequencing. All homozygotes (285/285) were called identically, while sequence from 89.2% (116/130) of heterozygotes agreed by both methods. The level of agreement between microarray calls and capillary DNA sequencing demonstrated substantial accuracy.</AbstractText>Custom genome resequencing microarrays are highly consistent with capillary sequencing in calling individual bases in genomic DNA from patients with human disease.</AbstractText>
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2,337,752 |
Analysis of double-stranded RNA-induced apoptosis pathways using interferon-response noninducible small interfering RNA expression vector library.
|
We have developed an original vector library that allowed us to exploit the phenomenon of RNA interference but also allowed us to avoid the confounding effects of the interferon response. In the present work, we used our library of small interfering RNA expression vectors to examine the genes involved in apoptosis that was induced by double-stranded RNA. To our surprise, screening of our library revealed two novel double-stranded RNA-induced apoptotic pathways, a JNK/SAPK-mediated mitochondrial pathway and an ERK2-related pathway, both of which appeared to be independent of the serine-threonine protein kinase-dependent caspase pathway. We also found that MST2 and protein kinase Calpha both activated the pro-apoptotic signal mediated by ERK2. The results of our screening analysis suggested the utility of large scale screenings with libraries of small interfering RNA expression vectors.
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2,337,753 |
Sleep-wake states in transgenic mouse models overexpressing the human beta-amyloid precursor protein.
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Studies testing the amyloid hypothesis and recent advances in mouse molecular genetic technologies have played a critical role in improving our understanding of Alzheimer's disease (AD). Mouse models of AD currently available show only some of the characteristic neuropathology in human AD. Studies have demonstrated, however, that these models are excellent tools for characterizing different aspects of the molecular pathology of AD and the neurobiological basis for the clinical heterogeneity in AD. The present discussion focuses on behavioral and physiological data obtained in transgenic (Tg) mice overexpressing the mutant human beta-amyloid precursor protein (hbetaAPP). This mouse model exhibits memory and neurophysiological deficits at ages preceding amyloid-beta-peptide (Abeta) plaque formation that worsened with age and Abeta plaque formation. In spite of these findings, very little emphasis has been placed on characterizing the neurobiological basis of the diverse neuropsychiatric symptoms that are also observed in AD, including sleep disturbances. Taking into consideration the relationship between memory processes and sleep, the use of animal models of AD as a preclinical bioassay has the potential to characterize the neural substrates mediating clinical manifestations of AD, such as sleep-wake states, and contribute to the development of treatments for early stages of AD.
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2,337,754 |
Clinico-pathologic findings in medullary cystic kidney disease type 2.
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Medullary cystic kidney disease type 2 is an uncommon autosomal dominant condition characterized by juvenile onset hyperuricemia, precocious gout and chronic renal failure progressing to end-stage renal disease in the 4th through 7th decades of life. A family suffering from this condition is described. The patient in the index case presented with renal insufficiency as a child. A renal biopsy revealed tubular atrophy, and immunohistochemical staining of the tissue for uromodulin (Tamm Horsfall protein) revealed dense deposits in renal tubular cells. Genetic testing revealed a single nucleotide mutation (c.899G>A) resulting in an exchange of a cysteine residue for tyrosine (C300Y). Medullary cystic kidney disease type 2 (also known as uromodulin-associated kidney disease) likely represents a form of endoplasmic reticulum storage disease, with deposition of the abnormal uromodulin protein in the endoplasmic reticulum, leading to tubular cell atrophy and death.
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2,337,755 |
Induction and inhibition of the Th2 phenotype spread: implications for childhood asthma.
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The interactions between genetic and environmental factors play a major role in the development of childhood asthma. We hypothesized that a pre-existing Th2/asthmatic response can promote Th2 responses to newly encountered Ags (i.e., phenotype spread). To test this hypothesis, we developed a mouse model in which the requirements for the induction and inhibition of phenotype spread to a clinically relevant neo-allergen (i.e., ragweed) were investigated. Our results indicate that 1) phenotype spread to the neo-allergen can be induced only within the first 8 h after a bronchial challenge with the first Ag (OVA); 2) Th2 differentiation of naive CD4(+) T cells occurs in bronchial lymph nodes; 3) trafficking of naive CD4(+) T cells to local lymph nodes and IL-4 produced by OVA-activated Th2 cells play essential roles in the differentiation of naive CD4(+) T cells to Th2 cells; and 4) suppression of the production of chemokines involved in the homing of naive CD4(+) T and Th2 cells to bronchial lymph nodes by a TLR9 agonist inhibited phenotype spread and abrogated the consequent development of experimental asthma. These findings provide a mechanistic insight into Th2 phenotype spread and offer an animal model for testing relevant immunomodulatory interventions.
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2,337,756 |
[Identification of PCR markers associated with cytoplasmic male sterility in Brassica oleracea var Botrytis].
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The homology-based candidate gene method was used to identified the specific PCR markers linked to cytoplasmic male sterility (CMS) in cauliflower( Brassica oleracea var botrytis.). Searching the DNA and protein data-base of NCBI , correlative genes or open reading frames were identified . Analysis of biosoft, based on the conservative regions ,five primers were designed . Among them, only primer P9/P10 produced a 313- bp specific fragment. Identified by individual plant testing , analysis of RT-PCR and dot blot ,this fragment was only existed in CMS cauliflower knxd612. Analysis of the sequence indicated it was high homologous(98%) with orf138 of Ogura CMS radish. Primary result suggested that the cytoplasmic type of CMS cauliflower knxd612 may belong to Ogura type. This research offered a good foundation to further investigate the CMS mechanism of cauliflower in molecular level.
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2,337,757 |
[A family history of congenital fibrosis of the extraocular muscle with autosomal dominant inheritance].
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To discover novel disease genes, a family with congenital fibrosis of the extraocular muscle was studied by a follow-up investigation, eye examinations and histo-pathological examination. There were fifteen cases suffering from congenital general fibrosis syndrome in four generations. They have congenital blepharoptosis, head tilt, chin lift, primary gaze fixed in a hypo- and exotropic position. The diagnosis is confirmed with positive forced duction testing in the affected eye. Furthermore, fibrosis of the extraocular muscles and hyaline degeneration was confirmed by histo-pathological examination. Except for different levels of restriction of the eyeball movements, other eye symptoms in positive patients are substantially identical. The genetic analysis showed that this disease was caused by autosomal dominant inheritance. The pedigree may be precious resource candidate for discovering disease gene related with congenital fibrosis of the extraocular muscle.
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2,337,758 |
An autosomal genomic screen for dementia in an extended Amish family.
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Apolipoprotein E (APOE) is the only universally confirmed susceptibility gene for late-onset Alzheimer disease (LOAD), although many loci are believed to modulate LOAD risk. The genetic homogeneity of isolated populations, such as the Amish, potentially provide increased power to identify LOAD susceptibility genes. Population homogeneity in these special populations may reduce the total number of susceptibility genes contributing to the complex disorder, thereby increasing the ability to identify any one susceptibility gene. Dementia in the Amish is clinically indistinguishable from LOAD in the general population. Previous studies in the Amish demonstrated a significantly decreased frequency of the APOE-4 susceptibility allele, but significant familial clustering of dementia [M.A. Pericak-Vance, C.C. Johnson, J.B. Rimmler, A.M. Saunders, L.C. Robinson, E.G. D'Hondt, C.E. Jackson, J.L. Haines, Alzheimer's disease and apolipoprotein E-4 allele in an Amish population, Ann. Neurol. 39 (1996) 700-704]. These data suggested that a genetic etiology independent of APOE may underlie the dementia observed in this population. In the present analysis, we focused on a large, multiplex, inbred Amish family (24 sampled individuals; 10 of whom are affected). We completed a genomic screen to identify novel LOAD loci (n=316 genetic markers), using both model-dependent "affecteds-only" analysis (dominant and recessive) and model-independent affected relative pair analysis. Interesting results (lod>1.5 or p<0.01) were obtained for markers on eight chromosomes (2q, 5q, 6q, 7p, 8p, 8q, 11p, 18p, 18q, and 19q). The highest overall score was a multipoint lod score of 3.1 on chromosome 11p. Most regions we identified were not previously detected by genomic screens of outbred populations and may represent population-specific susceptibilities to LOAD. These loci are currently under further investigation in a study of LOAD including additional Amish families.
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2,337,759 |
[Mutation detection of mismatch repair genes in hereditary nonpolyposis colorectal cancer by denaturing high-performance liquid chromatography].
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To establish DHPLC method in detecting mutations of mismatch repair genes, hMLH1 and hMSH2, and to identify germline mutations of hMLH1 and hMSH2 in HNPCC kindreds fulfilling Chinese HNPCC criteria.</AbstractText>Fourteen peripheral blood DNA samples from 14 unrelated HNPCC probands fulfilling Chinese HNPCC criteria were obtained respectively. PCR amplified 35 exons of two main mismatch repair genes, hMLH1 and hMSH2. DHPLC followed by DNA sequencing was used to detect and confirm mutations.</AbstractText>a total of 41 colorectal cancers and 19 extracolonic tumors were developed in 14 HNPCC kindreds, and gastric cancer was the most common extracolonic tumor type. Twelve single nucleotide changes were identified by DHPLC in 14 probands. Among them, three were missense mutations, one was a nonsense mutation. Other single nucleotide changes included five single nucleotide polymorphisms, two intron single nucleotide changes, one synonymous mutation. hMLH1 EXON19 CODON749 TAC-->TAG (Tyr-->X), hMSH2 EXON12 CODON629 CAA-->CGA (Gln-->Arg) and hMSH2 EXON15 CODON839 CAT-->CGT (His-->Arg) were new discovered mutations.</AbstractText>(1) DHPLC was considered to be highly effective, convenient technique with consistent results for the mutation detection of hMLH1 and hMSH2 genes. (2) Valid mutations of hMLH1 and hMSH2 genes were identified in about one-third HNPCC kindreds fulfilling Chinese HNPCC criteria and missense mutation was the most common mutational types in this cohort of families.</AbstractText>
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2,337,760 |
Adenomas in young patients: what is the optimal evaluation?
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Colorectal adenomas are a known risk factor for colorectal cancer. The prevalence of colorectal adenomas among individuals under age 40 and the clinical implications of finding a single adenoma in a young individual have not been defined. Until the most recent revision of the Bethesda Guidelines, having one or more adenomas diagnosed at age <40 was an indication for evaluation for hereditary nonpolyposis colorectal cancer (HNPCC). In an effort to explore the association of young-onset adenomas with HNPCC, Velayos et al. tested adenomas from 34 subjects aged 18-39 for pathologic features of HNPCC. Finding that none of the young-onset adenomas demonstrated features of microsatellite instability (MSI) or loss of mismatch repair protein expression by immunohistochemistry (IHC), the authors conclude that the yield of such testing is low, and support the decision to exclude young-onset adenomas from the Revised Bethesda Guidelines for HNPCC. However, this study also revealed that MSI and IHC failed to detect abnormalities in half of the adenomas from control subjects with identified MLH1 and MSH2 mutations. These findings highlight the limitations of current molecular techniques for examining adenomas as an initial screen for HNPCC and the need for further studies evaluating the optimal genetic and clinical evaluation of patients with young-onset adenomas.
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2,337,761 |
Low rate of microsatellite instability in young patients with adenomas: reassessing the Bethesda guidelines.
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Screening adenomas for microsatellite instability (MSI) in patients younger than 40 yr of age has been recommended by the Bethesda Guidelines as a means of identifying patients at risk for hereditary nonpolyposis colorectal cancer (HNPCC). We sought to determine the rate of MSI in adenomas removed from individuals under 40 yr of age over a 5-yr period in a university general gastroenterology practice.</AbstractText>We identified patients between 18 and 39 yr of age with endoscopically removed adenomatous colorectal polyps. Patients with polyposis syndromes, inflammatory bowel disease, or colorectal carcinoma were excluded. A three-generation family history was obtained via telephone interview. Endoscopic and histology reports were reviewed. Adenomas were tested for MSI using the BAT26 and BAT40 microsatellite markers, and expression of the MSH2 and MLH1 proteins was assessed by immunostaining.</AbstractText>A total of 34 patients had 46 adenomas removed endoscopically. Out of 34 patients, 14 (41%) had a family history of colorectal cancer and 3 were from Amsterdam criteria positive families. A total of 28 of 46 adenomas (61%) were distal to the splenic flexure. Polyps ranged in size from 2 to 20 mm and averaged 6.6 mm. Five polyps (11%) were tubulovillous adenomas, and the remainder were tubular adenomas. None of the polyps were serrated adenomas and none demonstrated high-grade dysplasia. Among the 40 adenomas available for testing, none demonstrated MSI using either BAT26 (0/40) or BAT40 (0/21), nor did any of the polyps tested demonstrate loss of either MSH2 or MLH1 expression (0/16).</AbstractText>Screening adenomas from patients younger than 40 yr of age for MSI was ineffective in identifying potentially new cases of HNPCC. New strategies that improve on the current clinical and molecular screening methods should be developed so that at-risk individuals can be identified and referred for germline testing before developing their first cancer.</AbstractText>
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2,337,762 |
DNA identification of fetal cells isolated from cervical mucus: potential for early non-invasive prenatal diagnosis.
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To develop a reliable method to isolate fetal cells for genetic diagnosis.</AbstractText>Aspiration of cervical mucus from pregnant women in the first trimester.</AbstractText>Pregnant women were recruited before an elective termination of pregnancy.</AbstractText>Sixty pregnant women (7-10 weeks of gestation).</AbstractText>Fetal cells were isolated from aspirated cervical mucus of pregnant women using a combination of enzymatic digestion, fluorescent immunohistochemistry, micromanipulation and single-cell DNA allelic profiling.</AbstractText>The isolation and identification of fetal cells.</AbstractText>The transformation of the tenacious cervical mucus into a single-cell suspension enabled the isolation and identification of fetal cells by fluorescent immunohistochemistry. Confirmation of fetal origin was accomplished by single-cell DNA allelic profiling alongside known maternal cells.</AbstractText>This novel non-invasive method is rapid and efficient with results attainable within 24 hours as early as seven weeks of gestation. The technique would offer earlier reassurance and the option of first trimester therapeutic abortions to both high and low risk pregnant women.</AbstractText>
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2,337,763 |
Genetics and assisted reproduction technology.
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In the past 20 years, a significant improvement has been shown in the treatment for infertility in both women and men through the development of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Only donated sperm could be previously used for treatment; now oocytes can also be donated. Furthermore, the combination of IVF and ICSI with advanced genetic methods has made preimplantation genetic diagnosis possible for many genetic conditions. These methods enable genetic testing of the early human embryo by using only a single cell, one blastomere biopsied from the embryo, as the sample from which the diagnosis of many chromosome rearrangements and other inherited diseases can be made. It has also been established that a considerable proportion of infertility is caused by genetic defects, which have several implications for infertility treatment. The purpose of this review is to give a concise introduction on how genetics is involved in assisted reproduction technology to specialists who may not be working in this particular field of gynecology, but who would need some knowledge of this for proper care of their patients.
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2,337,764 |
External quality assurance of DNA testing for thrombophilia mutations.
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Because of the potential implications of results of genetic analyses of thrombophilic mutations, laboratories must undertake stringent internal quality control measures and participate in external quality assurance (QA) programs. A small number of external QA surveys of thrombophilic defects have been conducted across a large number of molecular laboratories and generally have indicated favorable levels of correct responses. The Royal College of Pathologists of Australasia QA program has conducted external QA testing of factor V Leiden G1691 A, prothrombin G20210A, and MTHFR C677T gene mutations for the past 5 years, including 133 DNA samples in 10 multilaboratory surveys. Of 3,799 responses, the overall success rate was 98.63%; the poorest individual sample result was 15% incorrect for a homozygous factor V Leiden sample. Success rates in identifying specific mutations were 98.13% for factor V Leiden, 98.84% for prothrombin G20210A, and 99.3% for the MTHFR C677T mutation. Among responding laboratories, 51% (20/39) made at least 1 error; 3 of 39 laboratories were responsible for 46% of all errors (24/52). Although encouraging, these data underscore the need for ongoing participation of molecular diagnostic laboratories in external QA programs to ensure the provision of quality genetic testing services.
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2,337,765 |
Connexin mutation testing of children with nonsyndromic, autosomal recessive sensorineural hearing loss.
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The etiology of hearing loss is heterogeneous and falls into the two broad categories of genetic and environmental. In the genetic subgroup, 70% are non syndromic. Fifty percent of nonsyndromic sensorineural deafness is due to a mutation in the connexin 26 gene. This article presents the detection rate of connexin mutations in a multiethnic Canadian population.</AbstractText>A study of patients with nonsyndromic hearing loss seen over a period of 2 years who had connexin 26 mutation testing.</AbstractText>Nine of the 18 patients had connexin 26 mutations.</AbstractText>The majority of our patients with connexin 26 mutations had moderate to profound hearing loss. Testing for connexin mutations should be standard care because it accounts for a large proportion of individuals with nonsyndromic hearing loss. Reasons for testing include ruling out a syndromic cause, predicting moderate to profound hearing loss, and the need for language intervention, cochlear implants, and genetic counselling.</AbstractText>
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2,337,766 |
Autosomal dominant hereditary spastic paraplegia: DHPLC-based mutation analysis of SPG4 reveals eleven novel mutations.
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We set up a new denaturing high-performance liquid chromatography (DHPLC)-based protocol to screen patients with autosomal dominant hereditary spastic paraplegia (AD-HSP) for mutations in SPG4. Six patients had a complicated form and 49 a pure HSP phenotype. We also analyzed 19 unrelated patients presenting with an HSP phenotype (pure in 17 and complicated in two subjects) but no clear family history, as such patients may be cases of dominant inheritance with low penetrance. The overall frequency of SPG4 mutations in our study of HSP (in which prior linkage data were unavailable) was 32.4%, rising to 46.9% when only pure AD-HSP patients were considered. This figure falls well within the range reported in different populations. Rather as expected, the clinical data available for the patients did not support an easy genotype-phenotype correlation. Moreover, the clinical picture was not influenced by the length of the predicted residual gene product. As well as identifying novel variants in SPG4, this study constitutes the molecular characterization of the largest cohort of Italian AD-HSP patients studied to date. In addition, it provided an efficient, cost-effective, and reliable detection protocol for mutational screening of SPG4, which might facilitate medical genetic counseling.
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2,337,767 |
Identification and characterization of three large deletions and a deletion/polymorphism in the CFTR gene.
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Cystic fibrosis (CF) is mainly caused by small molecular lesions of the CFTR gene; mutation detection methods based on conventional PCR do not allow the identification of all CF alleles in a population and large deletions may account for a number of these unidentified molecular lesions. It is only recently that the availability of quantitative PCR methodologies made the search for large gene rearrangements easier in autosomal diseases. Using a combination of different methods, nine of the 37 unidentified CF alleles (24%) were found to harbor large deletions in our cohort of 1600 CF alleles. Three are new deletions, and we report the breakpoints of the previously described EX4_EX10del40kb deletion. An intronic deletion polymorphism affecting intron 17b was also found on almost 1% of "normal" chromosomes. Examination of the breakpoint sequences confirmed that intron 17b is indeed a hot spot for deletions, and that most of these rearrangements are caused by non-homologous recombination.
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2,337,768 |
Detection of heterozygous deletions and duplications in the MECP2 gene in Rett syndrome by Robust Dosage PCR (RD-PCR).
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Fifty to eighty percent of Rett syndrome (RTT) cases have point mutations in the gene encoding methyl-CpG-binding protein-2 (MECP2). A fraction of MECP2 negative classical RTT patients has large heterozygous deletions. Robust Dosage PCR (RD-PCR) assays were developed as a rapid, convenient and accurate method to detect large heterozygous deletions and duplications. A blinded analysis was performed for 65 RTT cases from Portugal by RD-PCR in the coding exons 2-4 of the MECP2 gene. Neither the patients with point mutations nor the non-classical RTT patients without point mutation had a deletion or duplication. One of remaining eight female patients with classical RTT without point mutation had a heterozygous deletion. This is the first report of a deletion spanning the entire MECP2 gene. The deletion was confirmed by Southern blotting analysis and the deletion junction was localized 37 kb upstream from exon 1 and 18 kb downstream from exon 4. No duplications were detected. Our results suggest that RD-PCR is an accurate and convenient molecular diagnostic method.
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2,337,769 |
Complete, gene-specific siRNA libraries: production and expression in mammalian cells.
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Short interfering RNAs (siRNAs) are widely used to silence the expression of specific genes. Current practice for designing effective siRNAs is to use algorithms based on sequence-efficacy correlations; however, there are many highly effective sequences that these algorithms do not anticipate. To ensure that the best siRNAs are identified, all possible gene-specific siRNA sequences of appropriate lengths should be screened in cell culture. Synthesizing and testing all such sequences individually is costly. A potentially much easier alternative is to prepare a mixture of all these sequences (a gene-specific library), express them in cells, select cells having the desired phenotype, and identify the siRNA contained within the selected cells. Here we describe two new methods for preparing and expressing such libraries. The first uses cloned Dicer or RNase III to digest gene-specific RNA duplexes to siRNAs, which are then converted to the corresponding DNA sequences by attaching RNA primers and performing reverse transcription-PCR. The second method involves partial DNase I digestion of gene-specific DNA, purification of a 20-30-bp fraction, and amplification by attaching DNA adapters followed by PCR. DNA libraries specific for TNF-alpha, DsRed, and part of the hepatitis C virus genome, generated by methods, were inserted into siRNA expression vectors between convergent human U6 and H1 promoters. Randomly selected clones from each library together with vectors expressing the corresponding target genes were cotransfected into 293FT cells and assayed for target gene inhibition. About 10%-20% of siRNAs represented in these libraries show significant inhibition of their target genes. Most of these inhibitory sequences are not predicted by existing algorithms.
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2,337,770 |
Gain-of-function amino acid substitutions drive positive selection of FGFR2 mutations in human spermatogonia.
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Despite the importance of mutation in genetics, there are virtually no experimental data on the occurrence of specific nucleotide substitutions in human gametes. C>G transversions at position 755 of FGF receptor 2 (FGFR2) cause Apert syndrome; this mutation, encoding the gain-of-function substitution Ser252Trp, occurs with a birth rate elevated 200- to 800-fold above background and originates exclusively from the unaffected father. We previously demonstrated high levels of both 755C>G and 755C>T FGFR2 mutations in human sperm and proposed that these particular mutations are enriched because the encoded proteins confer a selective advantage to spermatogonial cells. Here, we examine three corollaries of this hypothesis. First, we show that mutation levels at the adjacent FGFR2 nucleotides 752-754 are low, excluding any general increase in local mutation rate. Second, we present three instances of double-nucleotide changes involving 755C, expected to be extremely rare as chance events. Two of these double-nucleotide substitutions are shown, either by assessment of the pedigree or by direct analysis of sperm, to have arisen in sequential steps; the third (encoding Ser252Tyr) was predicted from structural considerations. Finally, we demonstrate that both major alternative spliceforms of FGFR2 (Fgfr2b and Fgfr2c) are expressed in rat spermatogonial stem cell lines. Taken together, these observations show that specific FGFR2 mutations attain high levels in sperm because they encode proteins with gain-of-function properties, favoring clonal expansion of mutant spermatogonial cells. Among FGFR2 mutations, those causing Apert syndrome may be especially prevalent because they enhance signaling by FGF ligands specific for each of the major expressed isoforms.
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2,337,771 |
Do we need genomic research for the prevention of common diseases with environmental causes?
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Concerns have been raised about the value of genomic research for prevention and public health, especially for complex diseases with risk factors that are amenable to environmental modification. Given that gene-environment interactions underlie almost all human diseases, the public health significance of genomic research on common diseases with modifiable environmental risks is based not necessarily on finding new genetic "causes" but on improving existing approaches to identifying and modifying environmental risk factors to better prevent and treat disease. Such applied genomic research for environmentally caused diseases is important, because 1) it could help stratify disease risks and differentiate interventions for achieving population health benefits; 2) it could help identify new environmental risk factors for disease or help confirm suspected environmental risk factors; and 3) it could aid our understanding of disease occurrence in terms of transmission, natural history, severity, etiologic heterogeneity, and targets for intervention at the population level. While genomics is still in its infancy, opportunities exist for developing, testing, and applying the tools of genomics to clinical and public health research, especially for conditions with known or suspected environmental causes. This research is likely to lead to population-wide health promotion and disease prevention efforts, not only to interventions targeted according to genetic susceptibility.
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2,337,772 |
[Rapid genetic screening of Leber's hereditary optic neuropathy with mtDNA G11778A mutation by AS-PCR with whole blood].
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Rapid Genetic Screening of Leber's hereditary optic neuropathy (LHON) with mtDNA G11778A mutation by allele-specific polymerase chain reaction (AS-PCR) with whole blood.</AbstractText>Whole blood with anticoagulant was used as a template of AS-PCR for the analysis of LHON with mtDNA G11778A point mutation. The amplified DNA fragment was directly observed by electrophoretogram with ethidium bromide stained.</AbstractText>The accuracy was 100% by using this method in 24 blood samples tested, and the specific of PCR of which used whole blood as template was better than one of the purified mtDNA. The reliability of the method for screening of LHON with mtDNA G11778A mutation was checked by double-blind test in 22 blood samples.</AbstractText>This method does not need purified DNA from blood and only required one step of PCR. Thus, it is very simple, rapid and accurate for the clinical genetic screening of LHON with mtDNA G11778A point mutation.</AbstractText>
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2,337,773 |
Gene delivery to embryonic stem cells.
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Since the establishment of embryonic stem (ES) cells and the identification of tissue-specific stem cells, researchers have made great strides in the analysis of the natural biology of such stem cells for the development of therapeutic applications. Specifically, ES cells are capable of differentiating into all of the cell types that constitute the whole body. Thus, ES cell research promises new type of treatments and possible cures for a variety of debilitating diseases and injuries. The potential medical benefits obtained from stem cell technology are compelling and stem cell research sees a bright future. Control of the growth and differentiation of stem cells is a critical tool in the fields of regenerative medicine, tissue engineering, drug discovery, and toxicity testing. Toward such a goal, we present here an overview of gene delivery in ES cells, covering the following topics: significance of gene delivery in ES cells, stable versus transient gene delivery, cytotoxicity, suspension versus adherent cells, expertise, time, cost, viral vectors for gene transduction (lentiviruses, adenoviruses, and adeno-associated viruses, chemical methods for gene delivery, and mechanical or physical gene delivery methods (electroporation, nucleofection, microinjection, and nuclear transfer).
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2,337,774 |
Association between the TRAX/DISC locus and both bipolar disorder and schizophrenia in the Scottish population.
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The Translin-associated factor X/Disrupted in Schizophrenia 1 (TRAX/DISC) region was first implicated as a susceptibility locus for schizophrenia by analysis of a large Scottish family in which a t(1;11) translocation cosegregates with schizophrenia, bipolar disorder and recurrent major depression. We now report evidence for association between bipolar disorder and schizophrenia and this locus in the general Scottish population. A systematic study of linkage disequilibrium in a representative sample of the Scottish population was undertaken across the 510 kb of TRAX and DISC1. SNPs representing each haplotype block were selected for case-control association studies of both schizophrenia and bipolar disorder. Significant association with bipolar disorder in women P=0.00026 (P=0.0016 in men and women combined) was detected in a region of DISC1. This same region also showed nominally significant association with schizophrenia in both men and women combined, P=0.0056. Two further regions, one in TRAX and the second in DISC1, showed weaker evidence for sex-specific associations of individual haplotypes with bipolar disorder in men and women respectively, P<0.01. Only the association between bipolar women and DISC1 remained significant after correction for multiple testing. This result provides further supporting evidence for DISC1 as a susceptibility factor for both bipolar disorder and schizophrenia, consistent with the diagnoses in the original Scottish translocation family.
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2,337,775 |
Successful unmanipulated haploidentical bone marrow transplantation from an HLA 2-locus-mismatched mother for Wiskott-Aldrich syndrome after unrelated cord blood stem cell transplantation.
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The authors describe a boy with Wiskott-Aldrich syndrome (WAS) who was diagnosed immediately after birth using flow cytometric and genetic analysis. At 1 year of age he received unrelated cord blood stem cell transplantation (UCBSCT); however, the sex chromosomes of the peripheral blood mononuclear cells showed that the recipient type was over 70%. This rate gradually increased to over 90% after immunosuppressant therapy was discontinued. Clinical manifestations, including high fever, graft-versus-host disease (GVHD)-like eruptions, and signs of infection recurred. Results of flow cytometric and genetic analysis of mononuclear cells from the boy's mother were normal with no mutation. Three months after UCBSCT, he received an unmanipulated HLA-haploidentical 2-locus-mismatched bone marrow transplant (BMT) from his mother. The prophylaxis against GVHD was tacrolimus and short-term methotrexate. Hematopoietic reconstitution was rapid and fluorescence in situ hybridization analysis revealed sustained engraftment. Grade II acute GVHD developed but improved rapidly with the administration of methylprednisolone. The patient is progressing well and displays complete chimerism 2 years after the BMT. This case suggests that unmanipulated haploidentical BMT from the mother might be feasible not only for malignant disease but also for immunodeficiency disease patients who urgently need stem cell transplants and have no HLA-identical donors.
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2,337,776 |
Estimating haplotype relative risks on human survival in population-based association studies.
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Association-based linkage disequilibrium (LD) mapping is an increasingly important tool for localizing genes that show potential influence on human aging and longevity. As haplotypes contain more LD information than single markers, a haplotype-based LD approach can have increased power in detecting associations as well as increased robustness in statistical testing. In this paper, we develop a new statistical model to estimate haplotype relative risks (HRRs) on human survival using unphased multilocus genotype data from unrelated individuals in cross-sectional studies. Based on the proportional hazard assumption, the model can estimate haplotype risk and frequency parameters, incorporate observed covariates, assess interactions between haplotypes and the covariates, and investigate the modes of gene function. By introducing population survival information available from population statistics, we are able to develop a procedure that carries out the parameter estimation using a nonparametric baseline hazard function and estimates sex-specific HRRs to infer gene-sex interaction. We also evaluate the haplotype effects on human survival while taking into account individual heterogeneity in the unobserved genetic and nongenetic factors or frailty by introducing the gamma-distributed frailty into the survival function. After model validation by computer simulation, we apply our method to an empirical data set to measure haplotype effects on human survival and to estimate haplotype frequencies at birth and over the observed ages. Results from both simulation and model application indicate that our survival analysis model is an efficient method for inferring haplotype effects on human survival in population-based association studies.
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2,337,777 |
Simultaneous classification and feature clustering using discriminant vector quantization with applications to microarray data analysis.
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In many applications of supervised learning, automatic feature clustering is often desirable for a better understanding of the interaction among the various features as well as the interplay between the features and the class labels. In addition, for high dimensional data sets, feature clustering has the potential for improvement in classification accuracy and reduction in computational complexity. In this paper, a method is developed for simultaneous classification and feature clustering by extending discriminant vector quantization (DVQ), a prototype classification method derived from the principle of minimum description length using source coding techniques. The method incorporates feature clustering with classification performed by fusing features in the same clusters. To illustrate its effectiveness, the method has been applied to microarray gene expression data for human lymphoma classification. It is demonstrated that incorporating feature clustering improves classification accuracy, and the clusters generated match well with biological meaningful gene expression signature groups.
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2,337,778 |
A literature based method for identifying gene-disease connections.
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We present a statistical method that can swiftly identify, from the literature, sets of genes known to be associated with given diseases. It offers a comprehensive way to treat alias symbols, a statistical method for computing the relevance of the gene to the query, and a novel way to disambiguate gene symbols from other abbreviations. The method is illustrated by finding genes related to breast cancer.
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2,337,779 |
VTE Risk assessment - a prognostic Model: BATER Cohort Study of young women.
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BACKGROUND: Community-based cohort studies are not available that evaluated the predictive power of both clinical and genetic risk factors for venous thromboembolism (VTE). There is, however, clinical need to forecast the likelihood of future occurrence of VTE, at least qualitatively, to support decisions about intensity of diagnostic or preventive measures. MATERIALS AND METHODS: A 10-year observation period of the Bavarian Thromboembolic Risk (BATER) study, a cohort study of 4337 women (18-55 years), was used to develop a predictive model of VTE based on clinical and genetic variables at baseline (1993). The objective was to prepare a probabilistic scheme that discriminates women with virtually no VTE risk from those at higher levels of absolute VTE risk in the foreseeable future. A multivariate analysis determined which variables at baseline were the best predictors of a future VTE event, provided a ranking according to the predictive power, and permitted to design a simple graphic scheme to assess the individual VTE risk using five predictor variables. RESULTS: Thirty-four new confirmed VTEs occurred during the observation period of over 32,000 women-years (WYs). A model was developed mainly based on clinical information (personal history of previous VTE and family history of VTE, age, BMI) and one composite genetic risk markers (combining Factor V Leiden and Prothrombin G20210A Mutation). Four levels of increasing VTE risk were arbitrarily defined to map the prevalence in the study population: No/low risk of VTE (61.3%), moderate risk (21.1%), high risk (6.0%), very high risk of future VTE (0.9%). In 10.6% of the population the risk assessment was not possible due to lacking VTE cases. The average incidence rates for VTE in these four levels were: 4.1, 12.3, 47.2, and 170.5 per 104 WYs for no, moderate, high, and very high risk, respectively. CONCLUSION: Our prognostic tool - containing clinical information (and if available also genetic data) - seems to be worthwhile testing in medical practice in order to confirm or refute the positive findings of this study. Our cohort study will be continued to include more VTE cases and to increase predictive value of the model.
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2,337,780 |
Lhermitte-Duclos disease: a report of 31 cases with immunohistochemical analysis of the PTEN/AKT/mTOR pathway.
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Lhermitte-Duclos disease (LDD) is a rare cerebellar tumor associated with Cowden disease (CD) and germline mutations in the PTEN gene. To further define these relationships, we reviewed clinical and pathologic findings in 31 LDD cases and analyzed the status of the PTEN pathway in 11 of them. We hypothesized that the granule cell hypertrophy in LDD is secondary to activation of mammalian target of rapamycin (mTOR), a downstream effector in the PTEN/AKT pathway and a major regulator of cell growth. Histopathologically, in addition to the classical findings of LDD, we observed prominent vascular proliferation and vacuolization of the white matter in many of the lesions. Four patients met diagnostic criteria for CD, and many of the remaining patients had some clinical features of CD. Immunohistochemical analysis showed high levels of phospho-AKT and phospho-S6 in the large ganglionic cells forming the lesions, indicating activation of the PTEN/AKT/mTOR pathway and suggesting a central role for mTOR in the pathogenesis of LDD. These data support recommendations for genetic testing and screening for CD in patients with LDD and suggest a novel therapy for LDD through pharmacologic inhibition of mTOR.
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2,337,781 |
Rethinking the 'right not to know'.
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The idea that an individual has a 'right not to know' genetic information about himself or herself is entrenched in both the policy sphere and the genetic counselling ethos. In this paper, I interrogate this idea of a 'right not to know', questioning particularly its status as a right. I identify the conception of rights that seems to underlie the posited 'right not to know' as a conception of rights in which they are prioritised non-outweighable interests. Turning to a series of hypothetical situations in which the 'right not to know' is invoked, I argue that the 'right not to know' is not a right consistent with such a conception. The 'right not to know' can only exist in a framework of rights in which they lack significant moral weight. Thus, considering the gravity of rights in popular understanding, I suggest caution in continuing use of the phrase 'right not to know' in the context of genetic testing. This investigation suggests that structuring the debate around genetic ignorance in terms of the 'right not to know' is not conducive to moving forward ethical thinking in this area.
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2,337,782 |
Association testing with Mendel.
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This report presents an overview of association testing strategies from a user's perspective, with particular attention to the capabilities of the computer program Mendel. Association testing is driven by the nature of the study sample, the nature of the disease trait, and the kind of markers employed. The practicing statistician must also choose whether to conduct parametric or nonparametric tests. Because of the complexities involved, Mendel offers users several analysis options. The different options are tied together by shared input and output conventions and a shared language for defining models. Mendel also features new statistics and theory found in no other genetics software. The most important innovations include: association testing by penetrance estimation, expansion of matched-pair designs to permutation unit designs, and a rigorous implementation of the measured genotype approach for quantitative trait loci. This report explains how Mendel imputes allele counts and conducts both asymptotic and permutation tests in the measured genotype framework.
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2,337,783 |
Recommendations for animal DNA forensic and identity testing.
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Genetic analysis in animals has been used for many applications, such as kinship analysis, for determining the sire of an offspring when a female has been exposed to multiple males, determining parentage when an animal switches offspring with another dam, extended lineage reconstruction, estimating inbreeding, identification in breed registries, and speciation. It now also is being used increasingly to characterize animal materials in forensic cases. As such, it is important to operate under a set of minimum guidelines that assures that all service providers have a template to follow for quality practices. None have been delineated for animal genetic identity testing. Based on the model for human DNA forensic analyses, a basic discussion of the issues and guidelines is provided for animal testing to include analytical practices, data evaluation, nomenclature, allele designation, statistics, validation, proficiency testing, lineage markers, casework files, and reporting. These should provide a basis for professional societies and/or working groups to establish more formalized recommendations.
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2,337,784 |
The hemochromatosis C282Y allele: a risk factor for hepatic veno-occlusive disease after hematopoietic stem cell transplantation.
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Hepatic veno-occlusive disease (HVOD) is a serious complication of hematopoietic stem cell transplantation (HSCT). Since the liver is a major site of iron deposition in HFE-associated hemochromatosis, and iron has oxidative toxicity, we hypothesized that HFE genotype might influence the risk of HVOD after myeloablative HSCT. We determined HFE genotypes in 166 HSCT recipients who were evaluated prospectively for HVOD. We also tested whether a common variant of the rate-limiting urea cycle enzyme, carbamyl-phosphate synthetase (CPS), previously observed to protect against HVOD in this cohort, modified the effect of HFE genotype. Risk of HVOD was significantly higher in carriers of at least one C282Y allele (RR=3.7, 95% CI 1.2-12.1) and increased progressively with C282Y allelic dose (RR=1.7, 95% CI 0.4-6.8 in heterozygotes; RR=8.6, 95% CI 1.5-48.5 in homozygotes). The CPS A allele, which encodes a more efficient urea cycle enzyme, reduced the risk of HVOD associated with HFE C282Y. We conclude that HFE C282Y is a risk factor for HVOD and that CPS polymorphisms may counteract its adverse effects. Knowledge of these genotypes and monitoring of iron stores may facilitate risk-stratification and testing of strategies to prevent HVOD, such as iron chelation and pharmacologic support of the urea cycle.
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2,337,785 |
Detection of 677CT/1298AC "double variant" chromosomes: implications for interpretation of MTHFR genotyping results.
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Genotyping 37,026 individuals as part of a thrombophilia evaluation, we determined and analyzed the genotypic frequencies of the 677CT and 1298AC mutations in the methylenetetrahydrofolate reductase (MTHFR) gene.</AbstractText>The 677CT and 1298AC mutations in the MTHFR gene were determined by either a laboratory-developed test involving PCR amplification and restriction digestion utilizing the ABI 3100 capillary electrophoresis apparatus (Applied Biosystems Inc) or by using an Analyte Specific Reagent (ASR) supplied by Third Wave Technologies. The genotype for three specimens with triple variant MTHFR mutations were confirmed by DNA sequencing on the ABI 3100 capillary electrophoresis apparatus.</AbstractText>The MTHFR frequencies of the 677CT/1298AA, 677CC/1298AC, 677CT/1298AC, 677CC/1298AA, 677TT/1298AA, 677CC/1298CC, 677TT/1298AC, and 677CT/1298CC genotypes were 0.228, 0.208, 0.198, 0.153, 0.122, 0.088, 0.0005, and 0.0003, respectively.</AbstractText>Individuals containing double variant MTHFR mutations on one allele (cis) cannot be distinguished between compound heterozygotes (trans) for 677CT and 1298AC mutations in routine clinical testing, a genotype associated with thrombophilia. Such patients could be inappropriately counseled for being at high risk for thrombotic episodes. Until information regarding prevalence and the clinical consequences of this double variant (cis) allele becomes available, caution should be used in interpreting the genotyping results of compound heterozygosity for 677CT and 1298AC.</AbstractText>
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2,337,786 |
Detection and calibration of microdeletions and microduplications by array-based comparative genomic hybridization and its applicability to clinical genetic testing.
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Genome-wide telomere screening by fluorescence in situ hybridization (FISH) has revealed that approximately 6% of unexplained mental retardation is due to submicroscopic telomere imbalances. However, the use of FISH for telomere screening is labor intensive and time consuming, given that 41 telomeres are interrogated. We have evaluated the use of array-based Comparative Genomic Hybridization (aCGH) as a more efficient tool for identifying telomere rearrangements.</AbstractText>In this study, 102 individuals with unexplained mental retardation, with either normal or abnormal FISH results, were selected for a blinded retrospective study using aCGH. Results between the two methodologies were compared to ascertain the ability of aCGH to be used in a clinical diagnostics setting.</AbstractText>We detected 100% of all imbalances previously identified by FISH (n = 17) and identified two additional abnormalities, a 10q telomere duplication and an interstitial duplication of 22q11. Interphase FISH analysis verified all abnormal array results. We also demonstrated that aCGH can accurately calibrate the size of telomere imbalances by using an array with "molecular rulers" for the telomeric regions of 1p, 16p, 17p, and 22q.</AbstractText>This study demonstrates that aCGH is an equivalent methodology to telomere FISH for detecting submicroscopic deletions. In addition, small duplications that are not easily visible by FISH can be accurately detected using aCGH. Because aCGH allows simultaneous interrogation of hundreds to thousands of DNA probes and is more amenable to automation, it offers an efficient and high-throughput alternative for detecting and calibrating unbalanced rearrangements, both of the telomere region, as well as other genomic locations.</AbstractText>
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2,337,787 |
Fragile X syndrome carrier screening in the prenatal genetic counseling setting.
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To document our experience with fragile X carrier screening.</AbstractText>In this study, 29,103 women with no known or suspected family history of fragile X syndrome were offered fragile X carrier screening during their prenatal genetic counseling visit. Screening acceptance was analyzed by referral indication, carrier frequencies documented, and prenatal outcome data presented.</AbstractText>Overall, 7.9% accepted carrier screening. The premutation frequency was 1 in 382, and the intermediate allele frequency was 1 in 143.</AbstractText>Fragile X screening is a desirable option for some women seeking prenatal genetic counseling and should be made available to this population.</AbstractText>
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2,337,788 |
What's the message? Interpretation of an uninformative BRCA1/2 test result for women at risk of familial breast cancer.
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To test the "false-reassurance hypothesis," which suggests that women who receive an uninformative BRCA1/2 test result may incorrectly conclude that they no longer have an elevated risk, with possible harmful consequences for adherence to breast surveillance guidelines.</AbstractText>A prospective questionnaire design was used to compare 183 women with an uninformative BRCA test result (94 affected and 89 unaffected) with 41 proven BRCA mutation-carriers and 49 true negatives before and after BRCA1/2 test disclosure.</AbstractText>After DNA-test disclosure, test applicants differed from each other with regard to their perception of the likelihood of carrying a deleterious gene (P < 0.0001). The BRCA mutation carriers reported the highest perceived likelihood and the true negatives reported the lowest. Compared to the predisclosure measures, women who received an uninformative DNA test result reported a lower perceived risk after disclosure (P < 0.0001), suggesting a relatively high level of reassurance because of the test result. However, after DNA-test disclosure, only 12 women concluded that the risk of carrying a mutation was nonexistent, and perceived likelihood was significantly associated with the pedigree-based risk assessment (P = 0.0001). Moreover, despite the significant decrease in perceived likelihood for uninformative women, intention to obtain mammograms did not change (P = 0.71); it remained at the same almost optimal level as for BRCA mutation carriers.</AbstractText>No support was found for the suggestion that the nature of uninformative test results is often misunderstood. Moreover, an uninformative test result did not affect the positive mammography intentions of both affected and unaffected women.</AbstractText>
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2,337,789 |
Attitudes about genetic testing and genetic testing intentions in African American women at increased risk for hereditary breast cancer.
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To evaluate attitudes about the benefits, limitations, and risks of genetic testing for BRCA1 and BRCA2 (BRCA1/2) mutations and explore testing intentions in African American women at increased risk for hereditary breast cancer.</AbstractText>Attitudes and intentions were evaluated by telephone in African American women (n = 74) at moderate and high risk for having a BRCA1/2 mutation.</AbstractText>Attitudes about the benefits of genetic testing were endorsed at a higher rate relative to limitations and risks; however, only 30% of respondents indicated that they would definitely have testing. In regression analysis, women most likely to be considering testing were those with fatalistic beliefs about cancer and those who believed they had a BRCA1/2 mutation. Women who had two or more affected relatives were also most likely to be considering testing. Women who had a personal history of cancer and those who believed they were at high risk for developing breast cancer were most likely to report greater limitations and risks. Pros scores were higher among women older than age 50 and those who were unemployed.</AbstractText>Although African American women at moderate and high risk for BRCA1/2 mutations report favorable attitudes about genetic testing, interest in testing may be limited. Women affected with cancer and those who believe they are at a higher risk for developing breast cancer may be most concerned about the negative consequences of testing. Increased attention may need to be given to beliefs about genetic testing and testing motivations during genetic counseling with African American women.</AbstractText>
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2,337,790 |
Use of an educational computer program before genetic counseling for breast cancer susceptibility: effects on duration and content of counseling sessions.
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Patients seeking genetic testing for inherited breast cancer risk are typically educated by genetic counselors; however, the growing demand for cancer genetic testing will likely exceed the availability of counselors trained in this area. We compared the effectiveness of counseling alone versus counseling preceded by use of a computer-based decision aid among women referred to genetic counseling for a family or personal history of breast cancer.</AbstractText>We developed and evaluated an interactive computer program that educates women about breast cancer, heredity, and genetic testing. Between May 2000 and September 2002, women at six study sites were randomized into either: Counselor Group (n = 105), who received standard genetic counseling, or Computer Group (n = 106), who used the interactive computer program before counseling. Clients and counselors both evaluated the effectiveness of counseling sessions, and counselors completed additional measures for the Computer Group. Counselors also recorded the duration of each session.</AbstractText>Baseline characteristics did not differ significantly between groups. Participants and counselors both rated the counseling sessions as highly effective, whether or not the sessions were preceded by computer use. Computer use resulted in significantly shorter counseling sessions among women at low risk for carrying BRCA1/2 mutations. In approximately half of the sessions preceded by clients' computer use, counselors indicated that clients' use of the computer program affected the way they used the time, shifting the focus away from basic education toward personal risk and decision-making.</AbstractText>This study shows that the interactive computer program "Breast Cancer Risk and Genetic Testing" is a valuable adjunct to genetic counseling. Its use before counseling can shorten counseling sessions and allow counselors to focus more on the clients' individual risks and specific psychological concerns. As the demand for counseling services increases, a program such as this can play a valuable role in enhancing counseling efficiency.</AbstractText>
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2,337,791 |
Rare mutations predisposing to familial adenomatous polyposis in Greek FAP patients.
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Familial Adenomatous Polyposis (FAP) is caused by germline mutations in the APC (Adenomatous Polyposis Coli) gene. The vast majority of APC mutations are point mutations or small insertions/deletions which lead to truncated protein products. Splicing mutations or gross genomic rearrangements are less common inactivating events of the APC gene.</AbstractText>In the current study genomic DNA or RNA from ten unrelated FAP suspected patients was examined for germline mutations in the APC gene. Family history and phenotype were used in order to select the patients. Methods used for testing were dHPLC (denaturing High Performance Liquid Chromatography), sequencing, MLPA (Multiplex Ligation - dependent Probe Amplification), Karyotyping, FISH (Fluorescence In Situ Hybridization) and RT-PCR (Reverse Transcription - Polymerase Chain Reaction).</AbstractText>A 250 Kbp deletion in the APC gene starting from intron 5 and extending beyond exon 15 was identified in one patient. A substitution of the +5 conserved nucleotide at the splice donor site of intron 9 in the APC gene was shown to produce frameshift and inefficient exon skipping in a second patient. Four frameshift mutations (1577insT, 1973delAG, 3180delAAAA, 3212delA) and a nonsense mutation (C1690T) were identified in the rest of the patients.</AbstractText>Screening for APC mutations in FAP patients should include testing for splicing defects and gross genomic alterations.</AbstractText>
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2,337,792 |
Molecular diagnostics by microelectronic microchips.
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Molecular diagnostics is being revolutionized by the development of highly advanced technologies for DNA and RNA testing. One of the most important challenges is the integration of microelectronics to microchip-based nucleic acid technologies. The specific characteristics of these microsystems make the miniaturization and automation of any step of a molecular diagnostic procedure possible. This review describes the application of microelectronics to all the processes involved in a genetic test, particularly to sample preparation, DNA amplification and sequence variation detection.
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2,337,793 |
Evidence-based prenatal care: Part I. General prenatal care and counseling issues.
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Effective prenatal care should integrate the best available evidence into a model of shared decision making. Pregnant women should be counseled about the risks of smoking and alcohol and drug use. Structured educational programs to promote breastfeeding are effective. Routine fetal heart auscultation, urinalysis, and assessment of maternal weight, blood pressure, and fundal height generally are recommended, although the evidence for these interventions is variable. Women should be offered ABO and Rh blood typing and screening for anemia during the first prenatal visit. Genetic counseling and testing should be offered to couples with a family history of genetic disorders, a previously affected fetus or child, or a history of recurrent miscarriage. All women should be offered prenatal serum marker screening for neural tube defects and aneuploidy. Women at increased risk for aneuploidy should be offered amniocentesis or chorionic villus sampling. Counseling about the limitations and risks of these tests, as well as their psychologic implications, is necessary. Folic acid supplementation beginning in the preconception period reduces the incidence of neural tube defects. There is limited evidence that routine use of other dietary supplements may improve outcomes for the mother and infant.
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2,337,794 |
Therapeutic potential of insulin-like growth factor-1 in patients with diabetes mellitus.
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Insulin-like growth factor-1 (IGF-1) and its receptors share considerable homology with insulin and insulin receptors, and their respective signaling pathways interact at the post receptor level. While the growth hormone (GH)-IGF-1 axis principally regulates tissue growth and differentiation, insulin exerts it primary effects on fuel metabolism. However, these two endocrine systems interact at multiple levels and in diabetes mellitus the GH-IGF-1 axis is grossly disturbed, with increased secretion of GH, reduced plasma levels of IGF-1, and complex tissue-specific changes in IGF binding proteins (IGFBPs). These observations have given rise to the view that GH-IGF-1 axis dysfunction, particularly low plasma levels of circulating IGF-1, probably play a significant role in several aspects of the pathophysiology of diabetes mellitus, including insulin resistance and poor glycemic control, and may also influence the development of microvascular complications. The availability of recombinant human IGF-1 (rhIGF-1; mecasermin), used either alone or in combination with insulin, has led to experimental studies and clinical trials in humans testing these hypotheses. These studies have examined the impact of subcutaneous rhIGF-1 injections on sensitivity and metabolic parameters. In patients with type 1 and 2 diabetes mellitus, insulin sensitivity is significantly improved, insulin requirements are reduced, and glycemic control of dyslipidemia is generally improved in short-term studies. rhIGF-1 is a particularly attractive possibility in patients with type 2 diabetes mellitus, where insulin resistance is the fundamental problem. Some patients with genetic syndromes of severe insulin resistance also benefit from treatment with rhIGF-1, which can bypass blocks in the insulin signaling pathway. The common adverse effects reported for rhIGF-1 are dose-related and include edema, jaw pain, arthralgia, myalgia, hypotension, injection site pain, and less commonly, Bell's palsy and raised intracranial pressure. Although disturbance of the GH-IGF-1 axis participates in the development of diabetic complications, the functional consequences of the complex changes in IGFBP expression at the tissue level are uncertain, and it is not known whether systemic IGF-1 therapy or other manipulations of the GH-IGF-1 axis would be helpful or harmful. Experimentally, IGF-1 has a protective effect on neuropathy, and could find an application in the healing of neuropathic ulcers. The potential benefits of IGF-1 therapy in diabetes mellitus have yet to be realised.
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2,337,795 |
Evolutionary innovation in the vertebrate jaw: A derived morphology in anuran tadpoles and its possible developmental origin.
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The mouthparts of anuran tadpoles are highly derived compared to those of caecilians or salamanders. The suprarostral cartilages support the tadpole's upper beak; the infrarostral cartilages support the lower beak. Both supra- and infrarostral cartilages are absent in other vertebrates. These differences reflect the evolutionary origin of a derived feeding mode in anuran tadpoles. We suggest that these unique cartilages stem from the evolution of new articulations within preexisting cartilages, rather than novel cartilage condensations. We propose testing this hypothesis through a search for similarities in the development of the suprarostral and infrarostral cartilage articulations and of the primary jaw joint. In Xenopus, the gene zax is expressed in a region corresponding to the infrarostral cartilage. This gene is related to the bapx1-gene, which regulates jaw joint development. Further investigation of these genes, as well as other genes with joint-related functions, in anuran craniofacial development may provide a connection between the morphological diversity seen in the vertebrate head and the corresponding diversity in genetic regulatory processes. We believe that the evolution of larval jaws in anurans may shed light on the general evolutionary mechanisms of how new articulations, not only in the jaw region, could have arisen in the vertebrate skull.
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2,337,796 |
Cystic fibrosis carriers have higher neonatal immunoreactive trypsinogen values than non-carriers.
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Following cystic fibrosis (CF) neonatal screening implementation, a high frequency of heterozygotes has been reported among neonates with elevated immunoreactive trypsinogen (IRT) and normal sweat chloride levels. We studied the relationship between normal IRT values and CF heterozygosity: 10,000 neonates were screened for CF by IRT measurement and tested for 40 CF mutations; the 294 carriers detected were coupled with newborns negative to the same genetic testing, and the two groups' IRT levels compared. Heterozygotes had higher IRT levels than their controls (mean 35.32 vs. 27.58 microg/L, P<0.001). Even within normal trypsinogen range, the probability of being a CF carrier increases with neonatal IRT concentration.
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2,337,797 |
Characterization of iduronate-2-sulfatase gene-pseudogene recombinations in eight patients with Mucopolysaccharidosis type II revealed by a rapid PCR-based method.
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Various types of complex genetic rearrangements involving the iduronate-2-sulfatase (IDS) and its homologous pseudogene (IDS2, IDSP1) have so far been reported as the cause of Mucopolysaccharidosis type II (MPS2 or MPS II; Hunter syndrome). When using conventional mutational analyses, the occurrence in intronic regions of these rearrangements can be misleading. Here, we describe a rapid PCR-based method set up to detect possible gene/pseudogene recombinations among a series of Italian male patients who had negative results in the mutation analysis of the IDS gene. Our approach selected eight unrelated patients showing recombinations. The characterization of the proximal regions containing the breakpoints in the eight patients identified four different rearrangements due to both inversion and conversion events. Comparison of our data with previous publications confirmed that the recombinations between the IDS gene and the IDS2 pseudogene result from separate events, considering their occurrence at different positions within the same "hotspot" genomic region in unrelated patients. The RT-PCR analysis of the available cDNAs pointed out the different effects of similar rearrangements on the expression of the IDS gene. This method can be utilized effectively in the absence of the patients' cDNA, as well as for carrier detection among female family members. This advantageous approach reduces costs, is less time-consuming, and requires a smaller DNA quantity in comparison to the Southern blot hybridization technique often utilized for such complex rearrangements.
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2,337,798 |
Quantitative analysis of SMN1 and SMN2 genes based on DHPLC: a highly efficient and reliable carrier-screening test.
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Autosomal recessive spinal muscular atrophy (SMA) is a common, fatal neuromuscular disease caused by homozygous absence of the SMN1 gene in approximately 94% of patients. However, a highly homologous SMN2 gene exists in the same chromosome interval, centromeric to SMN1, and hampers detection of SMN1. We present a new, rapid, simple, and highly reliable method for detecting the SMN1 deletion/conversion and for determining the copy numbers of the SMN1 and SMN2 genes by DHPLC. We analyzed SMN1/SMN2 gene exon 7 deletion/conversion by DHPLC. A total of 25 patients with spinal muscular atrophy lacking the SMN1 gene as well as 309 control individuals from the general population and the family members of patients with SMA were analyzed. By DHPLC analysis, we could detect the SMA-affected cases efficiently just by recognizing an SMN2-only peak. Furthermore, after specific primer amplification and adjustment of the oven temperature, all of the SMA carriers with an SMN1/SMN2 ratio not equal to 1 could be identified unambiguously by this simple and efficient detection system. To calculate the total SMN1/SMN2 gene dosages further, we developed a specific multiplex competitive PCR protocol by simultaneously amplifying the CYBB gene (X-linked), the KRIT1 gene (on chromosome arm 7q), and the SMN1/SMN2 gene ratio by DHPLC. By applying this technique, we could successfully designate all of the genotypes with different SMN1/SMN2 gene copy numbers, including equal and unequal amounts of SMN1 and SMN2. We demonstrated that DHPLC is a fast and reliable tool for detection of carriers of SMA.
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2,337,799 |
Genomic rearrangements in the BRCA1 and BRCA2 genes.
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Mutations in the BRCA1 and BRCA2 genes predispose women to breast and ovarian cancer. BRCA1 and BRCA2 are 83 and 86 kb long, with coding sequences of 5.7 and 10.2 kb, scattered over 22 and 26 coding exons, respectively. The large majority of the alterations identified in these genes are point mutations and small insertions/deletions. However, an increasing number of large genomic rearrangements are being identified, especially in BRCA1. This review gives a brief overview of the techniques used to screen the BRCA1 and BRCA2 genes for large rearrangements, and describes those for which the breakpoints have been characterized. The principal mechanisms that are thought to lead to their formation, founder effects, and recombination hotspots, are also discussed.
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