Collected DNA Samples Research Articles

Assessment of a rapid-cycle PCR assay for the identification of the recurrent c.3421C>T mutation in the ABCC6 gene in pseudoxanthoma elasticum patients

Pseudoxanthoma elasticum (PXE) is a heritable disorder of the connective tissue. Mutations in the ABCC6 gene could be linked to this disease and, just recently, the c.3421C>T mutation was also associated with a high risk of coronary artery disease. We have now developed new real-time PCR assays for the accurate and rapid determination of the c.3421C>T genotype. Using our new assay, we analyzed the presence of the c.3421C>T mutation in the largest collection of DNA samples from unrelated German PXE patients (n=64) and in a control cohort (n=910). For assay setup, two sets of samples with known genotype for the c.3421C>T mutation were analyzed over a period of 14 days. Results were confirmed by restriction endonuclease mapping, sequence-specific PCR and DNA sequencing. In order to ensure that no further mutations or deletions interfered with the c.3421C>T genotyping, we scanned the exon 24 of the ABCC6 gene by DHPLC and investigated the presence of the ABCC6del23–29 deletion in all patients. The assay has been set up on a group of patients with known genotype and validated on 64 PXE patients. In this group four PXE patients (6.3%) were found to be homozygous and 25 (39.0%) to be heterozygous carriers of the c.3421C>T mutation. The common ABCC6del23–29 deletion, possibly interfering with genotype determination, was searched and excluded. Furthermore, two novel mutations in the ABCC6 gene could be identified in two patients. The novel mutations c.3389C>T and c.3341G>A did not interfere with our new assay. Our new c.3421C>T genotyping assays can be used for the rapid identification of this frequent mutation in PXE patients and of the recently newly proposed cardiac risk factor in young patients with myocardial infarcts of unknown origin.

KEY ISSUES IN GENETIC EPIDEMIOLOGY: LESSONS FROM A UK BASED EMPIRICAL STUDY

1. Introduction Population-based genetic research aims to study gene-disease associations and genetic polymorphisms. By combining genetic samples (taken from blood or tissue) with medical, genealogical and lifestyle information, researchers hope to better understand disease causation and to develop improved methods of diagnosis and treatment. Such studies are a growing area of international research (Austin, et al 2003). In 2004, the world's largest proposed gene bank, UK BioBank (UKBB), plans to start collecting DNA samples from 500,000 British men and women aged between 45 and 69. As one might expect, however, gene banks raise a number of ethical, legal, and social issues. A key question for policy-makers and practitioners is how to create a climate in which potential health benefits can be realized without harming the interests of donors. In order to be successful, biobanks must achieve high rates of participation. Thus, a central question concerns the acceptability of databases to the public (Lowrance 2001). This paper addresses critical issues in DNA banking by using the UK-based North Cumbria Community Genetics Project (NCCGP) as a case study. The NCCGP was a collaboration between the University of Newcastle and Westlakes Research Institute in north-west England. From January 1996 until April 2003, pregnant women in West Cumbria were informed of the project by their local community mid-wife and were asked to provide written consent prior to giving birth. The NCCGP collected blood and tissue samples from the umbilical cord of newborn babies, maternal blood samples (from 1999) and personal health information derived from questionnaires (Chase et al 1998). The samples were frozen and stored for future use, so that the NCCGP can provide a resource of DNA samples for other researchers to use in genetic epidemiological studies (which so far include investigations on heart disease, breast cancer, and neural tube defects) (1). The NCCGP enjoyed a high response rate. Nearly ten thousand samples were collected, which means that nearly 90% of the pregnant women approached agreed to provide umbilical cord samples and maternal blood specimens (Chase et al 2000). However, only 60% of those approached completed the 'mother's questionnaire' (a health and lifestyle questionnaire for the woman and her partner) as well as donating samples. Such response rates could be thought as curious, given the ethical concerns often raised about DNA banking, such as informed consent, confidentiality and security of data, the potential for misuse and abuse of samples, third party access, feedback to donors (House of Lords 2001). Our study, funded by the Wellcome Trust, sought to compare and contrast the perceptions and attitudes of those who have agreed to donate samples to the North Cumbria Community Genetics Project (NCCGP) with those who have declined. This has provided much needed empirical data to add to normative discussions of why people do and do not donate. It was based on the premise that we know little about the views of those who have actually been asked to donate to a genetic database and that policy-makers and researchers would benefit from an investigation that opened up the 'black box' of the social processes that lie between the request to donate and the collection of samples and information. As well as addressing original questions regarding donation and participation, our research enables us to address a wider set of issues including the nature of 'informed consent', 'altruism', and 'benefit-sharing'. The fieldwork for our study involved semi-structured qualitative interviews with forty-three women who donated tissue samples to the NCCGP, seven who refused, seven NCCGP team members, two members of the NCCGP's Ethics Advisory Group, two focus group discussions involving ten community midwives, and three members of local community groups that opposed the NCCGP when plans for it were first announced in the early 1990s. …

Assessment of a rapid-cycle PCR assay for the identification of the recurrent c.3421C>T mutation in the ABCC6 gene in pseudoxanthoma elasticum patients.

Pseudoxanthoma elasticum (PXE) is a heritable disorder of the connective tissue. Mutations in the ABCC6 gene could be linked to this disease and, just recently, the c.3421C>T mutation was also associated with a high risk of coronary artery disease. We have now developed new real-time PCR assays for the accurate and rapid determination of the c.3421C>T genotype. Using our new assay, we analyzed the presence of the c.3421C>T mutation in the largest collection of DNA samples from unrelated German PXE patients (n=64) and in a control cohort (n=910). For assay setup, two sets of samples with known genotype for the c.3421C>T mutation were analyzed over a period of 14 days. Results were confirmed by restriction endonuclease mapping, sequence-specific PCR and DNA sequencing. In order to ensure that no further mutations or deletions interfered with the c.3421C>T genotyping, we scanned the exon 24 of the ABCC6 gene by DHPLC and investigated the presence of the ABCC6del23-29 deletion in all patients. The assay has been set up on a group of patients with known genotype and validated on 64 PXE patients. In this group four PXE patients (6.3%) were found to be homozygous and 25 (39.0%) to be heterozygous carriers of the c.3421C>T mutation. The common ABCC6del23-29 deletion, possibly interfering with genotype determination, was searched and excluded. Furthermore, two novel mutations in the ABCC6 gene could be identified in two patients. The novel mutations c.3389C>T and c.3341G>A did not interfere with our new assay. Our new c.3421C>T genotyping assays can be used for the rapid identification of this frequent mutation in PXE patients and of the recently newly proposed cardiac risk factor in young patients with myocardial infarcts of unknown origin.

Genetic testing for familial Mediterranean fever in Austria by means of reverse-hybridization teststrips.

Familial Mediterranean fever (FMF) is an autosomal-recessive, inflammatory disorder characterized by short, recurrent attacks of fever, accompanied by pain in the abdomen, chest, or joints and erysipelas-like erythema. Its most severe complication is progressive amyloidosis, leading to end-stage renal failure. FMF predominantly affects Turks, Arabs, Armenians, and Sephardic Jews, with carrier rates reported as high as 1 in 5, but it has been observed in lower frequencies throughout the Mediterranean area (1). It is caused by several mutations within the marenostrin/pyrin-encoding gene MEFV on chromosome 16p13.3, which differently affect the severity of the disease phenotype and the risk of developing renal amyloidosis (2)(3)(4). Although established clinical criteria for FMF exist (5), many patients remain undiagnosed because of rather nonspecific symptoms; therefore, molecular genetic analysis could substantially improve early and correct diagnosis of FMF and allow initiation of lifelong prophylactic treatment of affected individuals with colchicine (6). Aiming at a simple but powerful first-line screening tool for FMF genotyping, we have set up a reverse-hybridization, teststrip-based assay (FMF StripAssay) for the simultaneous detection of 12 MEFV mutations: E148Q in exon 2, P369S in exon 3, F479L in exon 5, and M680I (G/C), M680I (G/A), I692del (2076–2078), M694V, M694I, K695R, V726A, A744S, and R761H in exon 10. For this purpose, we collected DNA samples from individuals who had previously been typed positive for one of these mutations and used them to generate recombinant plasmid clones for the 12 mutant alleles (TOPO TA Cloning Kit; Invitrogen). After confirming the presence of mutations by DNA sequencing, we used these plasmid clones as homozygous reference samples to determine suitable reverse-hybridization probes. We synthesized a series of candidate 15- to 25mer oligonucleotides, selected from the FMF databank sequence (GenBank accession no. AF111163) and encoding all wild-type or mutant alleles, and immobilized …

Ethical, Legal, and Social Issues of Genome Research — New Phase of Genome Research Desperately Requires Social Understanding and Safeguards on the Use of Medical Records and Other Personal Information

AbstractFor Abstract see ChemInform Abstract in Full Text.

Spectrum of mutations in the arylsulfatase A gene in a Canadian DNA collection including two novel frameshift mutations, a new missense mutation (C488R) and an MLD mutation (R84Q) in cis with a pseudodeficiency allele

We describe three novel mutations in the human arylsulfatase A gene in three patients with MLD, an autosomal recessive lysosomal storage disorder. An insertion, 2590_2591insCCCC in exon 8 and a deletion, 752_758delGCCGGCC, in exon 3 will both result in frameshifts. A mutation in exon 8, 2566T → C, results in a missense mutation C488R, disrupting an unusual cysteine-knot at the C-terminal end of the protein. All three mutations are heterozygous with previously documented mutations. A previously reported mutation, R84Q was identified on a pseudodeficiency allele. These mutations are part of a heterogeneous spectrum of mutations found in a collection of DNA samples from MLD patients from across Canada and the USA.

Preempting genetic discrimination and assaults on privacy: report of a symposium.

At a symposium in June, 2002, biomedical researchers, clinicians, legal experts, policymakers, and representatives of the insurance industry and the advocacy community gathered to address issues of genetic privacy and discrimination; and to identify research, legal, and policy gaps needing to be filled. They concluded that over the next decade, as more genetic information becomes available and the public becomes more aware of individual risks, concerns about privacy and discrimination will become increasingly important. Documented cases of genetic discrimination are rare and largely anecdotal, yet individuals with genetic conditions harbor significant fears about discrimination. Current laws enacted to protect individuals from workplace and insurance discrimination offer some measure of protection, but leave many unfilled gaps. Moreover, the use of genetic information in potentially discriminatory ways is not limited to employment and insurability. Existing laws do little to protect people seeking life, disability, or long-term care insurance. And the courts have used genetic information in a wide variety of cases including paternity, criminal, and tort (personal injury) cases. Genetic information that might jeopardize an individual's right to privacy may also be obtained in the course of research studies, including through the collection of DNA and tissue samples. The insurance industry, State and Federal agencies, and the advocacy community are all making efforts to address some of these gaps through legislation and education of clinicians, the public, and policy makers.

Ethical, legal, and social issues of genome research--new phase of genome research desperately requires social understanding and safeguards on the use of medical records and other personal information

This article provides an overview of the use of human materials and information (human subject) in the new phase of pharmacological research and development in the current context, especially as it relates to the progress of the human genome project. In a sense, humanity has been drastically reduced to an array of DNA sequences that can be universally used in comparing living things. Pharmacological studies now acquire a unique status in bridging chemical substances to human body function. To perform the full activity of the nature of pharmacology, it requires both genotype and personal information, i.e. medical records and life style information, as research resources. In the UK, the Medical Research Council, the Wellcome Trust, and the Department of Health had started to plan UK Biobank for promoting and supporting the new stage of medical and pharmacological research and development. UK Biobank will collect DNA samples, medical records, and life style information of 500,000 people between the age range of 45 to 69 years old. It will follow the changes in health status of the participants for more than 10 years. The Biobank will provide researchers chances to correlate the genotypic traits to phenotypic ones, i.e. common diseases. In relation to the secondary use of medical records in health research, National Health Service (NHS) initiated a new strategy on the governance of patient information. These movements clearly demonstrated the indispensable nature of infrastructures for promoting and supporting pharmacological and medical research. We discuss on the necessary policies in constructing the Japanese infrastructure.

Infrequent mutation of the tumour-suppressor gene Smad4 in early-stage colorectal cancer.

Smad4 is a candidate tumour-suppressor gene identified recently on chromosome 18q21.1. Both alleles are inactivated in nearly one-half of pancreatic carcinomas, but its role in the tumorigenesis of other tumours is still unknown. The aim of this study was to investigate the potential involvement of the Smad4 locus in early-stage colorectal cancers (stages I–III) in tumour samples from a randomised multicentre trial. Of a large collection of DNA samples, 73 with a loss of one allele of the Smad4 gene were analysed for the presence of point mutations in the remaining gene. Patients, from whom biopsies were isolated, were part of a previous randomised multicentre study of the Swiss Group for Clinical Cancer Research on the benefit of adjuvant chemotherapy (SAKK study 40/81). Mutation analysis was restricted to the highly conserved C-terminal domain (exons 8, 9, 10 and 11) of Smad4, using PCR and single-strand conformational variant analysis. Two of the 73 patients (3%) with loss of one allele of Smad4 had a point mutation in the remaining allele. These results indicate that whereas Smad4 point mautations are prevalent in pancreatic carcinoma, they are infrequent in early stages (I–III) of colorectal cancer.

EBNA-1 sequence variations reflect active EBV replication and disease status or quiescent latency in lymphocytes.

EBV infects most of the global population, but only a small percentage of infected individuals develop EBV-associated malignancies. Host and viral factors may play a role in determining the clinical outcome. Because EBNA-1 functions as an oriP binding protein and links the episomal viral DNA to metaphase chromosomes, variation of EBNA-1 has been suggested to contribute to determining the tissue tropism of EBV and the development of various EBV-associated diseases. Five subtypes have been described, according to the amino acid at residue 487: P-ala (B95-8 prototype), P-thr (aa 487 ala to thr), V-val, V-pro and V-leu. Other studies, however, concluded that EBNA-1 sequence variation simply reflects the geographical distribution of EBV. To clarify these possibilities, we collected DNA samples from healthy individuals and patients with various EBV-associated diseases in Taiwan for PCR amplification and DNA sequencing. The results indicate that: 1) V-val EBNA-1 was detected in patients with nasopharyngeal carcinoma (NPC) and other EBV-associated malignant diseases; 2) the prototype P-ala strain was detected only in peripheral blood lymphocytes; 3) mixed populations of different subtypes of N-terminal and C-terminal sequences were observed in samples from one patient with nasopharyngeal carcinoma, one with T lymphoma and one with infectious mononucleosis sample; 4) intermediate variations between P-ala and V-val were observed in T-lymphoma, Hodgkin disease and infectious mononucleosis samples; and 5) in comparison with the major sequences identified in healthy carriers, the EBNA-1 sequences in peripheral lymphocytes from nasopharyngeal carcinoma were mixed types in 4 of 5 patients, implying increasing frequency of V-val might correlate with the progression of nasopharyngeal carcinoma.

Outline of disease gene hunting approaches in the Millennium Genome Project of Japan.

To capture the potentially enormous and vital opportunity in the post sequence era of human genome research, the Japanese government on December 19, 1999 drew up the basic outlines of the 5-year Millennium Genome Project (MGP). The “Disease Gene” Team of MGP aims to establish genome-based personalized medical care and to seed a revolutionary drug development inspired by the new information and technology, which the genome research provides. Five disease categories have been chosen as project targets: dementia, cancer, diabetes, hypertension and asthma, which are manifesting a major impact on the health and welfare of the rapidly graying Japanese society. These so-called “common diseases” are etiologically multifactorial, and the genetic components, if any, are considered polygenic with relatively high disease allele frequencies in the population. In the first year, 2000, a consortium or “Subteam” was established for each disease category, and several hypothesis-driven candidate gene approaches were launched. In 2001, a complementary strategy of disease gene hunting, a statistics-based genome-wide approach, was initiated. The Disease Gene Team decided to employ a genome-wide, gene-based SNP scan through close collaboration with another arm of MGP, the “Human Genome Variation” Team, which discovered 194, 393 SNPs as of December 25, 2002 from genome of the Japanese people. Each Subteam reached a consensus regarding eligibility criteria and research protocols, which were reviewed by the institutional review boards in accordance with the new Ethics Guidelines For Human Genome/Genetic Analysis Research promulgated by the government on April 1, 2001. The Subteams organized a multi-institutional consortium, which in 2001 collaborated in the collection of quality germline DNA samples and clinical and life-style related information. In 2002, the third year of MGP, the genome scan started at two typing centers using the high-throughput SNP typing system established by RIKEN. The premise and prospect of the approach will be discussed.(Communicated by Takashi SUGIMURA, M. J. A., Feb. 12, 2003)

Race, distributive justice and the promise of pharmacogenomics: ethical considerations.

Pharmacogenomics has emerged in the popular press as a key vehicle ushering in a new era of personalized medicine. Often described in utopian terms, gene-sequencing technology is predicted to result in the creation of a new line of therapeutics tailored to individual genetic signatures. In the absence of cost-effective, ubiquitous genome scanning tests, it may be more accurate to describe the next wave of genomic medicine as population-based rather than one focused on individual differences. Although the completion of the Human Genome Project seemed to confirm the fallacy of a genetic basis of 'race', the use of race in understanding human genetic variation has become a central focal point in the development of tools in genomic research in medicine. Despite the often repeated statement that humans share 99.9% of their genetic makeup, the growing number of privately and publicly funded cell repositories collecting DNA samples from racially identified populations reflects the increasing salience of the relationship between race and genes. Research on the ethical implications of identifying race in pharmacogenomics research has thus far, been fairly limited. As the field surges ahead, it is critical to examine the use of race in pharmacogenomics research and its attendant benefits and potential harm to individuals and groups.

Depression Plus Anger Prompts Search for Genetic Link

Back to table of contents Previous article Next article Clinical & Research NewsFull AccessDepression Plus Anger Prompts Search for Genetic LinkJoan Arehart-TreichelJoan Arehart-TreichelSearch for more papers by this authorPublished Online:6 Dec 2002https://doi.org/10.1176/pn.37.23.0042aCould there be a subtype of depression characterized by irritability and anger—what the ancient Greeks called “the yellow, choleric, bile type of personality”?Roy Perlis, M.D.: “I think we may be onto genes that cross criteria in DSM-IV.”Maurizio Fava, M.D., a professor of psychiatry at Harvard Medical School, thinks that there might be. In fact, a decade or so ago, he described “depression with anger attacks.” Moreover, one of the young psychiatric researchers working with him—Roy Perlis, M.D., an instructor in psychiatry at Harvard Medical School—is getting a study under way to probe for a gene or genes that might underlie this kind of depression.Perlis announced this effort at the 14th Annual Scientific Symposium of the National Alliance for Research on Schizophrenia and Depression, held in New York City in October.So far, Perlis explained, he and his colleagues have collected DNA samples from some 150 subjects with either major depression alone or with major depression accompanied by anger attacks, and they plan on ultimately collecting such samples from 300 such individuals. They will then probe the samples to see whether they can find a gene or genes that are peculiar to major depression accompanied by anger attacks.“I think we may be onto genes that cross criteria in DSM-IV,” he said. “For instance, bipolar patients who are depressed often show anger.”Someone in the audience asked Perlis whether more men than women experience depression with anger outbursts, since men have higher levels of testosterone than do women and thus tend to be more aggressive. On the contrary, Perlis replied—more women than men seem to experience them. However, when men do experience depression with anger outbursts, he added, they tend to be more violent than women.Someone else asked whether Perlis and his coworkers planned to measure the testosterone levels of their subjects. While he responded no, he said that it was a good idea.Perlis likewise reported that he and his coworkers are collaborating with a group of researchers at Massachusetts General Hospital to investigate whether brain-imaging data might distinguish persons with major depression and anger from those with just major depression.The type of research that Perlis and his colleagues are pursuing is similar to that being pursued by some other psychiatric investigators today, Lewis Judd, M.D., chair of psychiatry at the University of California at San Diego, pointed out at the symposium. In other words, they are attempting to identify subtypes of depression that to date have not been recognized by the psychiatric community. ▪ ISSUES NewArchived

Patents and public health. European institutions are challenging Myriad Genetics's patent monopoly on the brca1 gene.

An increasing number of international research and governmental institutions are challenging several gene patents, arguing that the patent holders’ absolute control of diagnostic methods is not in the public's best interests. Most notably, the Institut Curie, a cancer research centre in Paris, is leading the fight against Myriad Genetics, a US biotechnology company that plans to install a monopoly on all genetic work associated with the breast and ovarian cancer predisposition gene brca1 . The critics of Myriad's wide‐ranging patent rights maintain that the company's absolute control not only prohibits further research on the diagnosis of and therapies against breast cancer, but also has a detrimental effect on public health. Physician examining mammographs. ![][1] > Critics of Myriad's wide‐ranging patent rights maintain that it not only prohibits further research on diagnostics and therapies but also has a detrimental effect on public health Since the European Commission adopted a directive allowing human genes to be patented in July 1998, many such patents have been granted and indeed challenged, including the battles over the insulin, relaxin and hematopoietin genes. In 2001, seven years after Myriad Genetics first identified the sequence of brca1 , the European Patent Office (EPO) granted the company three patents covering all potential diagnostic and therapeutic applications based on the gene's sequence. Several European research centres and associations quickly contested the first two patents in an attempt to fight Myriad's monopoly. More recently, in August 2002, the Institut Curie, the Institut Gustave‐Roussy, the Assistance‐Publique‐Hopitaux de Paris together with almost all European genetics societies and many scientific institutions and governments turned up the heat and filed a joint opposition notice to the third patent. As this protects the isolated gene and the corresponding protein, and includes all imaginable future therapeutic uses, such as gene therapy and screening of drugs or transgenic animals, … [1]: /embed/graphic-1.gif

The Australian Twin Registry.

The Australian Twin Registry (ATR), established in the late 1970s, is a volunteer registry of over 30,000 pairs of Australian twins of all zygosity types and ages unselected for their health or medical history. The ATR does not undertake research itself but acts as facilitator, providing an important national and international resource for medical and scientific researchers across a broad range of disciplines. Its core functions are the maintenance of an up-to-date database containing basic contact details and baseline information, and the management of access to the resource in ways that enhance research capacity within Australia while protecting the rights of twins. The ATR has facilitated more than 200 studies using a variety of designs, including classic biometrical twin and twin family studies, co-twin control studies, intervention studies, longitudinal studies, and studies of issues relevant specifically to twins. These have yielded more than 300 peer-reviewed publications to date. Areas of major research include studies of behavior, musculoskeletal conditions, teeth and face patterns, cardiovascular risk factors, substance abuse, and risk factors for melanoma and breast cancer. Extensive longitudinal data are available for around 10,000 pairs. DNA samples have been obtained from more than 6000 twins. Considerable efforts are devoted to maintaining the commitment of registry members and recruitment. The ATR hopes to secure funding to expand its activities, including the systematic collection of DNA samples, so that it can continue to play a major role in the development of twin research and contribute to the annotation of the human genome.

The Australian Twin Registry

The Australian Twin Registry (ATR), established in the late 1970s, is a volunteer registry of over 30,000 pairs of Australian twins of all zygosity types and ages unselected for their health or medical history. The ATR does not undertake research itself but acts as facilitator, providing an important national and international resource for medical and scientific researchers across a broad range of disciplines. Its core functions are the maintenance of an up-to-date database containing basic contact details and baseline information, and the management of access to the resource in ways that enhance research capacity within Australia while protecting the rights of twins. The ATR has facilitated more than 200 studies using a variety of designs, including classic biometrical twin and twin family studies, co-twin control studies, intervention studies, longitudinal studies, and studies of issues relevant specifically to twins. These have yielded more than 300 peer-reviewed publications to date. Areas of major research include studies of behavior, musculoskeletal conditions, teeth and face patterns, cardiovascular risk factors, substance abuse, and risk factors for melanoma and breast cancer. Extensive longitudinal data are available for around 10,000 pairs. DNA samples have been obtained from more than 6000 twins. Considerable efforts are devoted to maintaining the commitment of registry members and recruitment. The ATR hopes to secure funding to expand its activities, including the systematic collection of DNA samples, so that it can continue to play a major role in the development of twin research and contribute to the annotation of the human genome.

Integration of DNA sample collection into a multi-site birth defects case-control study.

Advances in quantitative analysis and molecular genotyping have provided unprecedented opportunities to add biological sampling and genetic information to epidemiologic studies. The purpose of this article is to describe the incorporation of DNA sample collection into the National Birth Defects Prevention Study (NBDPS), an ongoing case-control study in an eight-state consortium with a primary goal to identify risk factors for birth defects. Babies with birth defects are identified through birth defects surveillance systems in the eight participating centers. Cases are infants with one or more of over 30 major birth defects. Controls are infants without defects from the same geographic area. Epidemiologic information is collected through an hour-long interview with mothers of both cases and controls. We added the collection of buccal cytobrush DNA samples for case-infants, control-infants, and their parents to this study. We describe here the methods by which the samples have been collected and processed, establishment of a centralized resource for DNA banking, and quality control, database management, access, informed consent, and confidentiality issues. Biological sampling and genetic analyses are important components to epidemiologic studies of birth defects aimed at identifying risk factors. The DNA specimens collected in this study can be used for detection of mutations, study of polymorphic variants that confer differential susceptibility to teratogens, and examination of interactions among genetic risk factors. Information on the methods used and issues faced by the NBDPS may be of value to others considering the addition of DNA sampling to epidemiologic studies.

The leatherback turtle, Dermochelys coriacea, exhibits both polyandry and polygyny.

The leatherback turtle (Dermochelys coriacea) is an endangered species, and world-wide populations are declining. To understand better the mating structure of this pelagic and fragile species, we investigated paternity in nearly 1000 hatchlings from Playa Grande in Parque Marino Nacional Las Baulas, Costa Rica. We collected DNA samples from 36 adult female leatherbacks and assessed allele frequency distributions for three microsatellite loci. For 20 of these 36 females, we examined DNA from hatchlings representing multiple clutches, and in some cases assessed up to four successive clutches from the same female. We inferred paternal alleles by comparing maternal and hatchling genotypes. We could not reject the null hypothesis of single paternity in 12 of 20 families (31 of 50 clutches), but we did reject the null hypothesis in two families (eight of 50 clutches). In the remaining six families, the null hypothesis could not be accepted or rejected with certainty because the number of hatchlings exhibiting extra nonmaternal alleles was small, and could thus be a result of mutation or sample error. Successive clutches laid by the same female had the same paternal allelic contribution, indicating sperm storage or possibly monogamy. None of 20 females shared the same three-locus genotype whereas there were two instances of shared genotypes among 17 inferred paternal three-locus genotypes. We conclude that both polyandry and polygyny are part of the mating structure of this leatherback sea turtle population.

The American Cancer Society Cancer Prevention Study II Nutrition Cohort

BACKGROUND Large-scale, prospective cohort studies have played a critical role in discovering factors that contribute to variability in cancer risk in human populations. Epidemiologists and volunteers at the American Cancer Society (ACS) were among the first to establish such cohorts, beginning in the early 1950s and continuing through the present, and these ACS cohorts have made landmark contributions in many areas of epidemiologic research. METHODS AND RESULTS The Cancer Prevention Study II Nutrition Cohort was established in 1992 and was designed to investigate the relation between diet and other lifestyle factors and exposures and the risk of cancer, mortality, and survival. The cohort includes over 84,000 men and 97,000 women who completed a mailed questionnaire in 1992. New questionnaires are sent to surviving cohort members every other year to update exposure information and to ascertain new occurrences of cancer; a 90% response rate was achieved for follow-up questionnaires in 1997 and 1999. Reported cancers are verified through medical records, registry linkage, or death certificates. The cohort is followed actively for all cases of incident cancer and for all causes of death. Through a collaborative effort among ACS national and division staff, volunteers, and the American College of Surgeons, blood samples were collected from a subgroup of 40,000 cohort members and are in storage at a central repository for future investigation of dietary, hormonal, genetic, and other factors and cancer risk. Collection of DNA samples from buccal cells in an additional 50,000 cohort members is underway currently and will be completed in 2002. CONCLUSIONS This new cohort of both men and women promises to be particularly valuable for the study of cancer occurrence, mortality, and survival as they relate to obesity and weight change, physical activity at various points in life, vitamin supplement use, exogenous hormone use, other medications (such as aspirin and nonsteroidal anti- inflammatory drugs) and cancer screening modalities. Cancer 2002;94:2490–2501. © 2002 American Cancer Society. DOI 10.1002/cncr.101970

Lack of association of the two polymorphisms in alpha-2 macroglobulin with Alzheimer disease.

Alzheimer disease is a complex neurodegenerative disorder that is characterized by cognitive decline and distinct neuropathology. The gene for alpha-2 macroglobulin (A2M) on chromosome 12 has two polymorphisms that, in some cases, are associated with Alzheimer disease. We examined these two polymorphisms in our families in the DNA Bank (a collection of DNA samples from families with Alzheimer disease in Texas). Using both association studies and sib transmission/disequilibrium tests, we found no association of the two polymorphisms with the disease in our collection. In addition, we did not find an association of the disease with the two polymorphisms in A2M in a small subset of National Institute of Mental Health (NIMH) families. Thus, our studies do not support the A2M polymorphisms as a risk factor for Alzheimer disease.