Ethical challenges at the beginning of the millennium.

In the mid-1980s, the Centers for Disease Control and Prevention (CDC) recognized the need to study genetic risk factors for common diseases such as diabetes and heart disease. To take advantage of a rare opportunity to obtain a nationally representative, population-based sample to study genetic risk factors, the CDC collected and stored DNA as part of the Third National Health and Nutrition Examination Survey (NHANES III). At the time, the methods for studying these risk factors in large epidemiologic studies were not available. However, in the midst of planning for NHANES III, a revolution was occurring in the field of genetics. The resulting changes would provide a means to realize the goal of explaining why some people are more susceptible than others to risks such as elevated cholesterol or exposure to carcinogens. During this period, ethicists were increasingly asking questions about the safety and risks for participants in genetic research. Was genetic research different from other research? Were new rules for obtaining informed consent for genetic research needed, or should our methods of obtaining informed consent be equally rigorous for all research? When collection of the NHANES III DNA bank was complete in 1994, the CDC and the National Institutes of Health (NIH) held a workshop to address these questions. The published recommendations of this workshop stimulated a national debate that resulted in a significant change in the way genetic epidemiologic research is done in the United States including not only stored biologic specimens but data collected for one purpose but used for another. In 1999, the National Bioethics Advisory Commission (NBAC) published recommendations for the ethical use of human biological materials. The recommendations of the NBAC and policies and practices of the CDC about informed consent for research on stored tissue samples will serve as models for future epidemiologic research. The problems that were recognized in the national debate that ensued and the solutions that followed will affect the way we gain access to biological specimens and data in the 21st century. Published in 2001 by John Wiley & Sons, Ltd.

Prevalence of C282Y and H63D mutations in the hemochromatosis (HFE) gene in the United States.

CONTEXT: Population-based estimates of the prevalence of disease-associated mutations, such as hemochromatosis (HFE) gene mutations, are needed to determine the usefulness of genetic screening. OBJECTIVE: To estimate the prevalence of the HFE mutations C282Y and H63D in the US population. DESIGN: Cross-sectional population-based study of samples in the DNA bank from phase 2 of the Third National Health and Nutrition Examination Survey conducted from 1992 to 1994. SETTING AND PARTICIPANTS: Genotyped samples of cells from a total of 5171 participants, cross-classified by sex, age, and race/ethnicity in the analysis. MAIN OUTCOME MEASURES: Estimates of the prevalence of C282Y and H63D mutations. RESULTS: The prevalence of C282Y homozygosity is estimated to be 0.26% (95% confidence interval [CI], 0.12%-0.49%); 1.89% (95% CI, 1.48%-2.43%) for H63D homozygosity; and 1.97% (95% CI, 1.54%-2.49%) for compound heterozygosity. The prevalence estimates for C282Y heterozygosity (C282Y/wild type) are 9.54% among non-Hispanic whites, 2.33% among non-Hispanic blacks, and 2.75% among Mexican-Americans. The prevalence estimates of the C282Y mutation in the US population are 5.4% (95% CI, 4.7%-6.2%) and 13.5% (95% CI, 12.5%-14.8%) for the H63D mutation. CONCLUSIONS: Estimates of prevalence of HFE mutations are within the expected range for non-Hispanic whites and blacks but the estimated prevalence of the C282Y mutation among Mexican-Americans is less than expected. Mutation data now need to be linked to clinically relevant indices, such as transferrin saturation level.

Risks associated with genetic testing: health insurance discrimination or simply business as usual?

Doctors can test patients for mutations that put them at high risk for hereditary forms of such diseases as breast and ovarian cancer. Even though the opportunities for disease prevention using genetic testing will increase with time, the risks of testing, which include insurance loss and employment discrimination, currently make testing problematic. Some have proposed making use of genetic information in health insurance underwriting illegal. But in the current system of risk-based underwriting, it is hard to imagine that insurance companies will willingly forego access to a growing body of information on risk. If the American public chooses to limit the use of genetic information, a reassessment of current laws or methods of paying for health care will be needed.

BRCA1 and BRCA2 gene mutations and risk of breast cancer. Public health perspectives.

CONTENT: Breast cancer is the most common cancer and the second most common cause of cancer death among U.S. women. In 1998, about 178,700 new cases will be diagnosed and 43,500 women will die from the disease. Mutations in the BRCA1 gene, which was cloned in 1994 and is located on chromosome 17q, have been identified as causes of predisposition to breast, ovarian, and other cancers. A second breast cancer gene, BRCA2, has been localized to chromosome 13q. Using inferential procedures, the overall carrier frequency of BRCA1 gene mutations has been estimated at 1 in 500 in the general U.S. population. Recent studies have indicated that the carrier frequency of a specific BRCA1 allele, the 185delAG mutation, may be as high as 0.8% to 1% among women of Ashkenazi Jewish descent. CONCLUSIONS: Due to the proliferation of laboratories offering genetic tests for breast cancer susceptibility, their appropriate use in public health needs careful scrutiny. Several issues are raised when such genetic tests are considered for population-based prevention programs for breast cancer. Public health agencies, such as the Centers for Disease Control and Prevention, are important to monitoring and evaluating genetic testing done outside of research protocols. If genetic tests for breast cancer are to be incorporated into future prevention programs, evaluation is needed of whether the testing can have the intended effect.

Increased risk for familial ovarian cancer among Jewish women: a population-based case-control study.

Jewish women have been reported to have a higher risk for familial breast cancer than non-Jewish women and to be more likely to carry mutations in breast cancer genes such as BRCA1. Because BRCA1 mutations also increase women's risk for ovarian cancer, we asked whether Jewish women are at higher risk for familial ovarian cancer than non-Jewish women. To determine the effects of 1) Jewish religion and 2) ovarian cancer in a first-degree relative on women's risk for epithelial ovarian cancer, we used data from a population-based, case-control study conducted in 8 geographic regions in the United States from 1980 through 1982. The study group included 471 cases and 4,025 controls. Jewish women were more likely to have familial ovarian cancer than non-Jewish women [odds ratio (OR) = 8.4, 95% confidence interval (CI) = 2.6-28]. The risk of having ovarian cancer appeared to be greater in Jewish women having a first-degree relative with ovarian cancer (OR = 8.81, 95% CI = 2.02-38.23) than in non-Jewish women having a first-degree relative with ovarian cancer (OR = 3.01, 95% CI = 1.61-5.64), but differences between Jewish and non-Jewish women were not statistically significant. Jewish women with no first-degree relative with ovarian cancer had no increased risk for ovarian cancer (OR = 1.27, 95% CI = 0.74-2.91) compared to non-Jewish women. These results suggest that Jewish women may have a higher rate of familial ovarian cancer than non-Jewish women, but because the results are based on a small number of Jewish women with familial ovarian cancer, the results need to be confirmed in larger studies.

Comparison of effect estimates from a meta-analysis of summary data from published studies and from a meta-analysis using individual patient data for ovarian cancer studies.

To determine the relative merits of two quantitative methods used to estimate the summary effects of observational studies, the authors compared two methods of meta-analysis. Each quantified the relation between oral contraceptive use and the risk for ovarian cancer. One analysis consisted of a meta-analysis using summary data from 11 published studies from the literature (MAL) in which the study was the unit of analysis, and the second consisted of a meta-analysis using individual patient data (MAP) in which the patient was the unit of analysis. The authors found excellent quantitative agreement between the summary effect estimates from the MAL and the MAP. The MAP permits analysis 1) among outcomes, exposures, and confounders not investigated in the original studies, 2) when the original effect measures differ among studies and cannot be converted to a common measure (e.g., slopes vs. correlation coefficients), and 3) when there is a paucity of studies. The MAL permits analysis 1) when resources are limited, 2) when time is limited, and 3) when original study data are not available or are available only from a biased sample of studies. In public health epidemiology, data from original studies are often accessible only to limited numbers of research groups and for only a few types of studies that have high public health priority. Consequently, few opportunities for pooled analysis exist. However, from a policy view, MAL will provide answers to many questions and will help in identifying questions for future investigation.

Assessment of reproductive disorders and birth defects in communities near hazardous chemical sites. I. Birth defects and developmental disorders.

Members of the workgroup on birth defects and developmental disorders discussed methods to assess structural anomalies, genetic changes and mutations, fetal and infant mortality, functional deficits, and impaired fetal and neonatal growth. Tier 1 assessments for all five adverse reproductive outcomes consist of questionnaires and reviews of medical records rather than laboratory testing of biologic specimens. The work-group members noted a role for neurodevelopmental testing and for limited genetic studies, such as karyotyping in Tier 2 assessments. Emerging methodologies to identify chromosomal aberrations, DNA adducts, and repair inhibition were reserved for Tier 3.

Informed consent for genetic research on stored tissue samples.

OBJECTIVE: To develop recommendations for obtaining adequate informed consent in the future when gathering tissue samples that may be used for genetic studies and defining the circumstances under which it is necessary to obtain further consent if tissue samples already in hand are to be used for such research. PARTICIPANTS: Scientists, ethicists, lawyers, and consumers selected by the National Center for Human Genome Research and the Centers for Disease Control and Prevention to represent a wide array of opinions. EVIDENCE: Statutes, regulations, and cases and articles on law and ethics. CONSENSUS PROCESS: Initial workshop, followed by circulation of several drafts of this document with opportunities for comment by workshop participants and others as well as smaller meetings involving participants with widely differing views. CONCLUSION: Genetic research using stored tissue samples poses an array of benefits and risks to individuals, researchers, and society. As a result, the workshop participants conclude that (1) informed consent is required for all genetic research using linkable samples unless conditions for limitation or waiver are met; (2) informed consent is not required for genetic research using anonymous samples but may be considered if identifiers are to be removed from currently linkable samples; (3) institutional review boards could usefully review all protocols that propose to use samples for genetic research; and (4) further work regarding these issues is warranted.

On combining dose-response data from epidemiological studies by meta-analysis.

Using data from a meta-analysis of the effects of oestrogen replacement therapy on the development of breast cancer, we compared alternative methods for combining dose-response slopes from epidemiological studies. We evaluated issues related both to summarizing data from single studies and to combining results from multiple studies. Findings related to the analysis of individual dose-response studies include: (1) a method of weighing studies that gives greater influence to dose-response slopes that conform to the linear relation of relative risk to duration can lead to large differences in calculated weights as a function of non-linearity; (2) a regression model using a variable-intercept resulted in a mean dose-response slope that increased as much as threefold when compared with the values obtained with a zero-intercept model. When combining results from multiple studies, we found: (1) calculating standard errors of mean dose-response slopes by methods that allow for both among-study and within-study variability (a random-effects type model) gave values different from a method that assumes homogeneity and equal within-study precision (a fixed-effects model); (2) the random-effects model gives mean and standard error results most similar to a bootstrap resampling method as increasing heterogeneity is observed (however, this model could give biased mean estimates compared with the bootstrap method); (3) a components-of-variance model compares favourably with the bootstrap and is easier to apply than the random-effects model. Based on these findings, we recommend the use of methods which incorporate heterogeneity to guard against underestimating the standard error. However, caution is urged because bias in point estimates can occur if extreme heterogeneity is present. Two other observations affect the interpretation of data combined from multiple studies. First, inclusion into a model of quality scores assigned by blinded reviewers had little effect on the mean dose-response slope and its standard error. Second, the number of studies required to achieve desired statistical power, varies with effect size.

Breast cancer risk and duration of estrogen use: the role of study design in meta-analysis.

Recent meta-analyses of studies of the risk of breast cancer associated with hormone replacement therapy agree that little risk is associated with ever-use or short-term use of estrogen replacement. These analyses disagree, however, about the effect of long-duration estrogen use. To understand differences in the findings among the meta-analyses of the effect of long-term use, we investigated the source of heterogeneity among the included studies. We analyzed subgroups by source of controls (community vs hospital), study design (case-control vs follow-up), and types of estrogen. We also examined the effect of modeling assumptions: that before women began estrogen use, those who chose to use estrogen replacement (1) were, or (2) were not, at substantially different risk from those who chose not to use estrogen. We found a small increase in risk in all subgroups of studies except those that used hospital controls. From a homogeneous group of case-control studies using community controls that analyzed the effect of conjugated equine estrogens, we estimated that the risk of breast cancer after 10 years of estrogen use increased by at least 15% and up to 29%.

Chronic renal effects in three studies of men and women occupationally exposed to cadmium.

We measured sensitive indicators of renal damage in three different populations occupationally exposed to cadmium, and examined the degree of variation in damage and the relative sensitivity of different types of indicators. The three studies included (1) men exposed in a cadmium recovery plant, (2) men exposed in a nickel/cadmium battery plant, and (3) women exposed in the latter plant. The indicators of renal damage were urinary proteins in three categories: (1) the high molecular weight enzymes alanine aminopeptidase (AAP) and N-acetyl-beta-D-glucosaminidase (NAG), (2) the intermediate molecular weight protein albumin (ALB), and (3) the low molecular weight proteins retinol-binding protein (RBP) and beta 2-microglobulin (B2M). These tests indicate that exposed groups with higher urine cadmium levels had varying degrees of renal damage. All exposed groups showed evidence of renal damage when compared with their respective control groups. A higher percentage of elevated protein levels was noted in the exposed group of Study 1 than in the exposed groups of Studies 2 and 3. In Study 1, the means of all five protein levels and ALB, RBP, and B2M fractional clearances were significantly elevated in the group with higher urine cadmium concentrations when compared with the groups with lower urine cadmium concentrations. Highly significant dose-response relationships for all of the urinary protein tests, including fractional clearances, were found. All of the tests were more sensitive in detecting evidence of subclinical renal damage than serum creatinine, a commonly used indicator of renal function. The order of test sensitivity in men was determined by considering three factors: (1) the magnitude of the correlation coefficient between the test and the urine cadmium concentration in the study with the most advanced damage, (2) the relative cadmium level predicted by the dose-response model at which there is a 10% chance of observing an elevated test value, and (3) the ability of the tests to detect renal effects in the population with less advanced damage. The tests in order of decreasing sensitivity in men are ALB, AAP, NAG, RBP approximately B2M. The women with higher urine cadmium levels in Study 3 had a higher percentage of elevated AAP and NAG values when compared with the control group.

Considerations when measuring urinary albumin: precision, substances that may interfere, and conditions for sample storage.

The measurement of small but abnormal amounts of albumin in urine is important in evaluating kidney disease in people with diabetes mellitus, hypertension, or possible adverse health effects from exposure to nephrotoxins. Routine laboratory methods for measuring albumin are not sensitive enough to measure the amounts that are significant in urine (less than 30 mg/L). In our laboratory we used three immunoassays for measuring urinary albumin: enzyme-linked immunosorbent assay (EIA), radioimmunoassay (RIA), and immunoturbidimetric assay (IT). We calculated the CVs of the three methods, investigated potential interfering substances at three times their normal concentrations, and stored urine under different conditions to find the best way to protect the sample until assay. The potential interferents we checked were transferrin, urea, beta 2-microglobulin, retinol-binding protein, creatinine, kappa and lambda light chains, IgG, hemoglobin, ketone, and glucose. The stability study involved two study temperatures (-20 and -70 degrees C) and four treatments (centrifuging or filtering, before or after storage). We found the following: the RIA had the lowest CV; the results from the interference study showed no interference from normal physiological concentrations of the substances investigated; storage at -70 degrees C regardless of the treatment should be adequate to prevent loss of albumin immunoreactivity.

Osteocalcin and bone alkaline phosphatase in the serum of women with liver disease.

To identify the types of liver disease in which osteopenia is a prominent feature and to understand the mechanisms of bone loss, bone mineral density was measured in the lumbar spine and hip, bone alkaline phosphatase, osteocalcin, and biochemical markers of calcium homeostasis were measured in 42 women, aged 33 to 52, with chronic liver disease and in 299 healthy women of similar age. In control women, bone alkaline phosphatase and osteocalcin correlated negatively with bone density at all sites (p less than 0.05). In women with liver disease, osteocalcin correlated negatively with bone density in the lumbar spine (p less than 0.007), whereas bone alkaline phosphatase did not correlate with bone density at any site. Bone alkaline phosphatase correlated positively with osteocalcin in control women (p = 0.001) and negatively with osteocalcin in women with liver disease (p = 0.03). Serum bone alkaline phosphatase in women with liver disease was increased significantly over serum bone alkaline phosphatase of control women, probably because of decreased clearance owing to defective function or decreased numbers of hepatic asialoglycoprotein receptors. Bone density was lower in the lumbar spines and hips of women with primary sclerosing cholangitis, primary biliary cirrhosis, and chronic active hepatitis or fibrosis without cirrhosis than in the lumbar spine and hips of control women. However, the differences were not significant, possibly because of the small sample size. It is concluded that, in liver disease, osteocalcin is a more reliable marker of osteoblastic function than bone alkaline phosphatase. Although our results show that bone density may decrease in women with cholestatic liver disease, larger studies are needed to determine the degree of osteopenia.

A meta-analysis of the effect of estrogen replacement therapy on the risk of breast cancer.

To quantify the effect of estrogen replacement therapy on breast cancer risk, we combined dose-response slopes of the relative risk of breast cancer against the duration of estrogen use across 16 studies. Using this summary dose-response slope, we calculated the proportional increase in risk of breast cancer for each year of estrogen use. For women who experienced any type of menopause, risk did not appear to increase until after at least 5 years of estrogen use. After 15 years of estrogen use, we found a 30% increase in the risk of breast cancer (relative risk, 1.3; 95% confidence interval [CI], 1.2 to 1.6). The increase in risk was largely due to results of studies that included premenopausal women or women using estradiol (with or without progestin), studies for which the estimated relative risk was 2.2 (CI, 1.4 to 3.4) after 15 years. Among women with a family history of breast cancer, those who had ever used estrogen replacement had a significantly higher risk (3.4; CI, 2.0 to 6.0) than those who had not (1.5; CI, 1.2 to 1.7).