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15.8: From Genetic Engineering and Genetic Modification - Biology

15.8: From Genetic Engineering and Genetic Modification - Biology


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By enabling us to focus on how genes and their regulation have evolved, these genomic, transcriptomic and proteomic technologies have vastly increased our knowledge of how cells work at a molecular level. We continue to add to our knowledge of disease process and in at least a few cases, how we can treat disease. The use of technologies to genetically modify organisms is more controversial, despite the best of human intentions. Some genetically modified organisms (GMOs) aim to increase food productivity to better feed the world. The introduction ‘beneficial’ genes into some GMOs have made:

  • drought-resistant crops to increase the range where major food crops can be grown.
  • pest-resistant crops to reduce reliance on environmentally toxic chemical pesticides.
  • herbicide-resistant crops that survive chemicals used to destroy harmful plants.

The quest for “improved” plant and animal varieties has been going on since before recorded history. Farmers have been cross-breeding cows, sheep, dogs, and crop varieties from corn to wheat, hoping to find faster growing, larger, hardier, (you name it) varieties. It is the manipulation of DNA (the essence of the genetic material itself) that is at the root of controversy. Controversy is reflected in opinions that GMO foods are potentially dangerous, and that their cultivation should be banned. However, the general consensus is that attempting to ban GMOs is too late! In fact, you are probably already partaking of some GMO foods without even knowing it. Perhaps the good news is that after many years of GMO crops already in our food stream, the emerging scientific consensus is that GMO foods are no more harmful than unmodified foods. The current debate is whether or not to label foods that are (or contain) GMO ingredients as genetically modified.

In an odd but perhaps amusing take on the discomfort some folks feel about GMOs, a startup company has genetically modified Petunias. When grown in water, their flowers are white, but when ‘watered’ with beer, they will produce pink flowers or purple flowers depending on how much beer they get (Check it out at Can Beautiful Flowers Change Face?). According to the company, they seek “to bring what it sees as the beauty of bioengineering to the general public” (and perhaps some profit as well?).

More recently, we have CRISPR and related tools that can precisely edit gene (in fact any DNA) sequences. And unlike the “quack medicines” of old, these tools have the real potential to cure disease, destroy disease-carrying vectors, cure cancer, improve crops and possibly alter the course of evolution. The speed with which one can accomplish such good (or evil) is truly awesome.


Integrating Genomics into Public Health Policy and Practice

This web page is archived for historical purposes and is no longer being maintained or updated.

by Laura M. Beskow, Marta Gwinn, and Mark A. Rothstein

Background

The Human Genome Project, an ongoing collaborative effort to unravel the mysteries of human DNA, has generated high expectations among scientists and the public. Rapid advances in human genetics and accompanying technologies (such as &ldquogene chips&rdquo) are expected to bring about major new developments in medicine and public health. Dr. Francis Collins, Director of the National Human Genome Research Institute, envisions a future where disease prevention and treatment advice are tailored to patients&rsquo genotypes, with such advice taking the form of more frequent or earlier medical surveillance, lifestyle or dietary modifications, or targeted drug therapy (1). Collins and McKusick predict that genetic assessment of individual disease risks and responses to drugs will reach mainstream health care as soon as the next decade (2).

Along with excitement about the prospect of tailored interventions, however, is some uncertainty. What do gene discoveries&mdashseemingly announced daily&mdashmean for public health? Until recently, the field of genetics had been confined largely to the realm of rare disorders caused by mutations in single genes. Even so, the public health community included genetics components in some of its work, experiencing noteworthy successes in birth defects prevention, newborn screening for inborn errors of metabolism, and development of genetic services capacity. Today, the mounting accomplishments of the Human Genome Project call for reassessment of the role of genomics in every condition of public health interest. Virtually all human disease results from the interaction between genetic susceptibility factors and the environment, broadly defined to include any exogenous factor&mdashchemical, physical, infectious, nutritional, social, or behavioral. This concept of gene-environment interaction may help explain why, for example, some health-conscious individuals with &ldquoacceptable&rdquo cholesterol levels suffer myocardial infarctions at 40 years of age, while others seem immune to heart disease despite years of smoking, poor diet, and lack of exercise. Unraveling the complex interplay between genes and environment will lead to better understanding of the biologic basis of disease and to new avenues for improving health and preventing disease. (We use the term &ldquogenomics&rdquo to denote this expanded view of genes and gene products within a whole system of genes and environmental factors.)

Fulfilling this promise represents an ambitious public health leadership agenda. An immense gap exists between the scientific products of the Human Genome Project and our ability to use genetic information to benefit health, and bridging this gap requires a wide range of public health activities. For example

  • Public health research is needed to translate information about genes and DNA sequences into knowledge about genetic susceptibility to disease and the interactions between these susceptibilities and modifiable risk factors.
  • The public health community must help formulate policies that promote the secure and appropriate use of genetic information.
  • Public health professionals must work with other healthcare sectors to ensure that valid genetic tests are available and accessible&mdashespecially in underserved populations&mdashand to ensure that people have access to proven interventions.
  • Public health has an important role in facilitating communication and education about genomics among all stakeholders, including health professionals the general public patients scientists policy makers and pharmaceutical, biotechnology, and insurance industry personnel.
  • Public health also has a crucial role in evaluating the impact and cost effectiveness of integrating genomics into health promotion and disease prevention programs.

Newborn screening, or more generally mandatory mass screening, is one paradigm for the integration of genomics into public health. Another is mandatory offering of genetic tests, such as laws in California (§125050-125110) requiring that all pregnant women (before a certain point in gestation) be provided information about prenatal screening for birth defects of the fetus. However, for common, complex diseases, the gene-environment interactions involved will most often increase a person&rsquos risk for disease but not definitively predict whether he or she has, or will get, the disease. Likewise, environmental interventions based on genotype may help reduce risk but not necessarily prevent or treat the disease. Thus, rather than mandatory screening, another paradigm for the integration of genomics into public health could be similar to that suggested by Dr. Collins, which is to provide individuals who wish to know with information about their personal genetic susceptibilities, together with tailored risk-reduction advice. Pharmacogenomics, the science of understanding the correlation between an individual&rsquos genetic make-up and his or her response to drug treatment, represents another potentially widespread application of DNA-based testing. Thus, while programs such as newborn screening will continue to be an important and valuable public health activity, other models may exist for future prevention programs involving genomics, particularly those focused on common, complex diseases.

Integrating genomics into public health research, policy, and practice raises many of the same legal issues discussed throughout this book. Although the addition of a genetic component to these activities does not necessarily change the fundamental legal considerations, society invests enormous power in the concept of genetics. Misplaced ideas of genetic determinism (a person&rsquos future is defined and fixed by his or her genetic makeup) and genetic reductionism (all traits, health problems, and behaviors are attributable to genetics) have significant negative implications for public health and prevention messages (3). In addition, genetics and its applications in the name of &ldquopublic health&rdquo bear the historical onus of eugenics, a movement that included racial hygiene laws in Nazi Germany as well as forced sterilization, antimiscegenation laws, and restrictive immigration policies in the United States and around the world (4). Early experiences with adult screening for sickle cell disease also portend issues that must be faced in the application of genetic knowledge (5). These programs were sometimes offered without providing proper education, consent, and follow-up and, as a result, carriers who were identified (and who had no risk of developing the disease) suffered stigmatization and discrimination (6).

Previous commentators have addressed ethical, legal, and social issues associated with genetic testing (7,8) and with genetic research (9,10). This chapter focuses on selected legal issues that arise with the integration of genomics into public health policy and practice.

Legal Authorities

The promise of genetic information is tempered by several concerns about its misuse and these concerns have been the subject of a variety of legislative activities. The National Conference of State Legislatures provides a regularly updated compilation of state genetic laws related to a number of issues, such as adoption, genetic engineering and cloning, criminal law and forensics, employment, insurance, research and medical testing, paternity, and privacy (11). Here we highlight as examples two categories of such legislative activities: those aimed at genetic discrimination in employment and those involving health insurance at state and federal levels. Concerns about discrimination in insurance and employment may be of particular public health importance because fear of discrimination may prevent individuals from seeking genetic counseling or testing that could benefit their health or from participating in valuable genetic research (12).

State Level

In 2000, the American Management Association surveyed 2,133 human resources managers about workplace medical testing of employees. When presented with a specific definition of genetic testing, only seven (0.3%) respondents answered that their firms performed such testing (13). Substantially greater proportions reported testing for susceptibility to workplace hazards and taking family medical histories (15.8% and 18.1%, respectively).

Over half the states have enacted laws prohibiting genetic discrimination in employment (11). All of these ban discrimination based on genetic test results, and all prohibit genetic discrimination in hiring, firing, or terms of employment. Some also cover information about genetic testing, family history, or inherited characteristics. For example, North Carolina (§95-28.1A) prohibits employment discrimination &ldquoon account of the person&rsquos having requested genetic testing or counseling services, or on the basis of genetic information obtained concerning the person or a member of the person&rsquos family.&rdquo The statute defines &ldquogenetic information&rdquo as &ldquoinformation about genes, gene products, or inherited characteristics that may derive from an individual or a family member.&rdquo Many of these laws also prohibit employers from requesting or requiring genetic information, performing genetic tests, or obtaining genetic information. In New York (§296.19(a)), for example, it is unlawful for employers to 1) directly or indirectly solicit, require, or administer a genetic test to a person as a condition or employment, or 2) buy or otherwise acquire the results or interpretation of an individual&rsquos genetic test results or to make an agreement with an individual to take a genetic test or provide genetic test results.

Twenty-nine states prohibit health insurers from seeking, requiring, or using genetic information to determine eligibility for insurance and 38 states forbid rating, canceling, or denying insurance on the basis of genetic information (11). Maryland law (Ins §27-909) does both, forbidding insurers, nonprofit health service plans, and health maintenance organizations from 1) using a genetic test, the results of a genetic test, genetic information, or a request for genetic services, to reject, deny, limit, cancel, refuse to renew, increase the rates of, affect the terms or conditions of, or otherwise affect a health insurance policy or contract and 2) requesting or requiring a genetic test, the results of a genetic test, or genetic information for determining whether to issue or renew health benefits coverage. State health insurance laws are preempted by the Employee Retirement Income Security Act (ERISA) (29 U.S.C. Chap. 18) to the extent that they attempt to regulate employer-based group health plans. Thus, the main value of the state laws is to prohibit discrimination in individual health insurance.

Hall and Rich (14) recently evaluated whether these types of laws reduce the extent of genetic discrimination by health insurers. From data collected at multiple sites, they found almost no well-documented cases of health insurers either asking for or using presymptomatic genetic test results in their underwriting decisions, either before or after these laws had been enacted or in states with or without these laws. They concluded, however, that such laws have made it less likely that insurers will use genetic information in the future and that, although insurers and agents are only vaguely aware of the laws, the laws have shaped industry norms and attitudes about the legitimacy of using this information. The authors also noted that the instances of adverse health insurance consequences they uncovered concerned payment for genetic services (e.g., genetic counseling, testing, prevention services) rather than the availability and pricing of health insurance. Payment for genetics-related services is an important barrier to access that genetic discrimination laws do not address.

Federal Level

Although a number of bills have been introduced over the last decade, no federal legislation has yet been passed directly related to genetic discrimination in individual insurance coverage or in employment. The 107 th Congress (2001&ndash02) introduced several such bills (e.g., H.R.602, S.318) that would prohibit health plans and insurers from discriminating on the basis of protected genetic information and also make discrimination because of protected genetic information unlawful. These bills define &ldquoprotected genetic information&rdquo as 1) information about an individual&rsquos genetic tests 2) information about genetic tests of family members of the individual or 3) information about the occurrence of a disease or disorder in family members.

Aside from specific legislation, however, other federal antidiscrimination laws apply to genetics. These include:

  • An executive order signed by President Clinton in February 2000 banning discrimination in federal employment on the basis of genetic information (15).
  • The Americans with Disabilities Act of 1990 (ADA) (Pub. L. 101-336), which covers individuals who have a physical or mental impairment that substantially limits a major life activity, have a record of such an impairment, or are regarded as having such an impairment. The Equal Opportunity Employment Commission (EOEC)has issued an interpretation of the ADA stating that entities that discriminate against individuals on the basis of genetic information are regarding those individuals as having impairments (16). This interpretation is not binding on the courts, however, and subsequent case law casts doubt on whether it would be upheld. In Sutton v. United Airlines, Inc. (17), the Supreme Court held that in determining the severity of an impairment under the ADA, the condition must be considered in its mitigated state, such as with eyeglasses or medications. Significantly, the Court reasoned that Congress intended the ADA&rsquos coverage to be limited to the 43 million Americans Congress estimated as having severe disabilities. According to the Court, if individuals with &ldquomitigated&rdquo impairments were included, the coverage would greatly exceed that figure. If similar reasoning were applied to asymptomatic individuals at genetically increased risk of disease, then the conclusion would be that they also are not covered under the ADA. In 2001, the EOEC settled its first court action challenging the use of workplace genetic testing when a U.S. railway company agreed to stop requiring genetic testing of employees who file claims for carpal tunnel syndrome (18).
  • The Health Insurance Portability and Accountability Act of 1996 (HIPAA) (Pub. L. 104-191) prohibits employer-based group health plans from using any health status-related factor, including genetic information, as a basis for denying or limiting eligibility for coverage or for charging an individual more for coverage (see Chapter 8). HIPAA also limits exclusions for pre-existing conditions and states explicitly that genetic information in the absence of a current diagnosis shall not be considered a pre-existing condition.

A key question in crafting legislative approaches that promote the appropriate use of genomics is whether genetic information should be dealt with separately or as part of measures intended to address health information more broadly. This controversy has important implications for the integration of genomics into public health surveillance activities.

Genetic Exceptionalism

The challenge of defining the term &ldquogenetic&rdquo is one of the conceptual difficulties arising from genetic exceptionalism&mdashthe practice of treating genetic information as different from other kinds of health information and affording it special privacy and security. Theoretically, anything from the results of a DNA test to routine observations about sex, eye color, and blood type could be classified as genetic information. Narrow legislative definitions (e.g., &ldquothe results of DNA analysis&rdquo) may not achieve desired policy goals, such as protecting individuals from genetic discrimination because they do not apply, for example, to family health history. On the other hand, broad definitions (e.g., &ldquoinformation about genes, gene products, or inherited traits&rdquo) may impede important medical and public health activities. Gostin and Hodge (19) present an analysis of the extent to which genetic information is the same as, or different from, other health information and conclude that it is not so different as to legally and ethically justify special status.

Michigan created the Michigan Commission on Genetic Privacy and Progress in 1997 to advise the governor and legislature on specific issues in genetics. In its final report (20), the Commission recommended that any legislation should consider genetics in the context of medical issues as a whole, and thus privacy protections should encompass all confidential medical information. It also recommended limiting legislation to areas in which professional standards and codes of ethics are insufficient to protect the public good and individual rights and avoiding legislation that inappropriately prohibits or hinders beneficial genetic testing and research.

In response to this report, Michigan passed a number of laws that addressed such issues as informed consent before performance of a genetic test (§333.17020), as well as genetic discrimination in employment (§37.1201) and insurance (§500.3407b). In these laws, &ldquogenetic test&rdquo is defined as &ldquothe analysis of human DNA, RNA, chromosomes, and those proteins and metabolites used to detect heritable or somatic disease-related genotypes or karyotypes for clinical purposes,&rdquo and &ldquogenetic information&rdquo is defined as &ldquoinformation about a gene, gene product, or inherited characteristic derived from a genetic test.&rdquo These definitions are similar to those suggested by other expert groups (8,21). Michigan does not, however, have laws that afford special privacy protections to genetic information, beyond laws already covering professional-patient interactions, research confidentiality, and general medical privacy. It also does not provide for property rights in genetic samples or information, although patients have rights to access medical information.

Public Health Surveillance

One of the primary functions of public health is public health surveillance. By collecting and analyzing information about disease outbreaks, epidemiologists can determine the likely cause of adverse health events and point the way for prevention and early intervention. As more becomes known about the relation between genetic and environmental factors in the etiology of complex disorders, obtaining genetic information from exposed and affected populations may be important as part of comprehensive public health surveillance. Regardless of whether the addition of genetic information to surveillance activities is considered qualitatively or merely quantitatively different, the issue is whether it is lawful for governmental agents to require the collection of this new information.

Legal challenges to government collection of health information usually involve constitutional claims such as illegal search and seizure, equal protection, and due process (see Chapter 7). Under all of these constitutional theories, the courts balance the public&rsquos interest in obtaining the health information against the individual&rsquos interest in preventing disclosure. An important consideration is the possible harm to the individual that could result from disclosure of private information. &ldquoThough the actual risk of social harm directly caused by surveillance is low, perceived risks (and higher actual threats arising in other settings) can create a context in which public health data collection is politically problematic or resisted by subjects&rdquo (22). Reducing public anxiety about disclosure of health information is an inexact enterprise, but the following measures undoubtedly would help:

  1. Enacting strong health privacy legislation that limits the uses of the information to public health purposes, secures the records from improper access by unauthorized parties, provides that the information must be kept and used in the least identifiable form consistent with public health purposes, and provides severe penalties for violations
  2. Educating the public about the existence and provision of such health privacy legislation and
  3. Enacting laws that prohibit unreasonable health-based discrimination by private and public sector entities.
Practice Considerations

The public health community has an important role to play in translating genetic discoveries into opportunities to improve health and prevent disease in ways that maximize the benefits of using genetic information, minimize the risks, and conserve healthcare resources. Law and policy related to public health programs and strategies, human resources, scientific and technical considerations, and consumer and financial interests will be important tools in carrying out this role. Newborn screening, professional licensure, and oversight of genetic tests are examples of areas in which the application of law and genomics already intersect.

Newborn Screening

Newborn screening for genetic disorders began in the 1960s, made possible by new technology for collecting blood samples and a simple, inexpensive laboratory test to screen for phenylketonuria (PKU). Because PKU screening was slow to become part of routine medical care, children&rsquos advocates pressed for state legislation that eventually led to newborn screening in all 50 states and the District of Columbia (23). Statewide screening programs were launched without a full assessment either of the validity of the screening test or of the utility of the dietary intervention to prevent mental retardation in children with PKU. Nevertheless, newborn screening for PKU is now generally acknowledged as a public health success.

Nearly all of the 4 million infants born in the United States each year are screened for PKU and from two to 10 other disorders. State health departments are responsible for carrying out newborn screening as mandated by state laws or regulations. In the absence of federal guidelines, the numbers and types of screening tests performed have varied from state to state and over time as tests have been added and subtracted from state laboratory screening panels (24). This lack of uniformity and the advent of new screening technology (25,26) led the federal Health Resources and Services Administration to ask the American Academy of Pediatrics to form a Newborn Screening Task Force. In August 2000, the Task Force published recommendations that called on federal and state public officials, healthcare providers, and advocacy groups to work together to develop up-to-date guidelines for newborn screening programs while addressing key ethical, legal, and social issues (27,28). These issues include informed consent, the confidentiality of screening results, the use of residual blood samples for research, and the need for heightened public and professional awareness of the capacity and limitations of newborn screening programs.

State policies on parental consent for newborn screening vary widely. Maryland has a voluntary newborn screening program, Wyoming requests informed consent, and Massachusetts has developed an informed consent process for a pilot study of newborn screening for cystic fibrosis (27). In all other states, newborn screening is mandatory, although most states permit parental refusal. The Task Force report recommended that &ldquoadditional approaches to informing and educating parents be studied further&rdquo (27).

Many state programs added newborn screening for sickle cell anemia in the 1980s, drawing renewed attention to the issue of confidentiality of screening test results (29). More recently, the growth of electronic databases for newborn screening and other public health records has added another dimension to this issue (30). Attempts to better coordinate and evaluate infant and child health programs have led to increased integration of information systems that were formerly largely independent. Data linkage and sharing require new methods for safeguarding confidentiality (27).

Residual samples from newborn screening programs have become recognized as a rich resource for research studies. These samples represent a truly population-based &ldquobiobank,&rdquo which can be used to develop new knowledge by identifying affected persons from medical records and retrieving their stored samples for testing (31). Even without personally identifying information, these samples are useful for population-based genotype frequency studies. However, no general consensus exists on the use of residual newborn screening samples for research. A policy statement published in 1996 outlined some of the issues and presented guidelines (32), but debate on this topic continues.

Professional Licensure

Ensuring a competent public and personal healthcare workforce is a vital service of public health. Because most health professionals were trained before the advances in genomics brought about by the Human Genome Project, few have the education or experience necessary to participate effectively in this rapidly emerging field. For example, Giardiello and colleagues (33) studied the clinical use of commercial APC gene testing for familial adenomatous polyposis (FAP). They found that only 18.6% of patients received genetic counseling before the test, and only 16.9% provided written informed consent. Physicians misinterpreted the test results in 31.6% of cases, providing patients with false assurance that they did not have FAP when in fact their results were inconclusive.

A number of efforts are ongoing to promote genetic education among clinical (33) and public health (34) professionals. A template of key data elements that should be made available to health professionals about a genetic test, for example in the form of a fact sheet, has also been proposed (35). However, as the number of DNA-based tests proliferate, one area that may receive increasing attention is licensure of genetic counseling professionals&mdashprofessionals who will aid people in making decisions about genetic testing, as well as help them interpret and respond to the results. The American Board of Genetic Counselors certifies genetic counselors, but medical billing processes typically prohibit reimbursement for unlicenced professionals. Thus, payors are billed for genetic counseling visits according to physician service codes, which are often poor indicators of the service delivered as well as more expensive than if billed directly (36).

The rationale for licensure of genetic counselors is several-fold. First, it may help protect public health and safety by defining the scope of practice, setting minimum standards for qualifications and conduct, and providing mechanisms for continuing education, performance monitoring, and disciplinary action. By restricting use of the title &ldquogenetic counselor,&rdquo licensure can also protect the public by helping it identify qualified professionals. Second, licensure may increase the supply of trained genetic counselors. Although the rapid commercialization of genetic tests may drive up the need for genetic counselors, actual demand may not equal this perceived need because of reimbursement constraints. Allowing direct reimbursement for genetic counseling services could help reinforce the legitimacy of the profession and attract high-quality candidates to the field. Finally, assuming that supply does in fact increase so that availability is not a barrier, licensure may facilitate access to genetic counseling services through insurance coverage and possibly through reduced costs because physician service codes are avoided.

California became the first state to enact a licensure bill (SB 1364) in September 2000, followed by Utah (SB 59) in March 2001 New York has licensure bills pending (AB2360, SB2471). One important issue for such legislation is: Who is eligible for licensure? In many cases, genetic counseling requires the ability to elicit and interpret family history provide information about the risks and benefits of genetic testing interpret and explain results and options and provide counseling, emotional support, and referral with regard to complex psychosocial issues. The California law calls for licensure of master&rsquos level genetic counselors and doctoral level clinical geneticists and restricts use of the title &ldquogenetic counselor&rdquo to those who have applied for and obtained a license. However, it allows for genetic counseling to be provided by a &ldquophysician, a certified advance practice nurse with a genetics specialty, or other appropriately trained licensed health care professional.&rdquo This highlights the importance of assuring that all health professionals have an appropriate level of genomics competence.

Licensure may be one important step in meeting the need for qualified genetic counseling professionals. At the same time, genetic counseling is traditionally founded on a &ldquolow throughput&rdquo model it is generally a time-intensive, one-on-one process, often oriented toward the analysis of family pedigrees for highly penetrant genetic mutations. Advances in genomics will bring about high throughput genetic testing for multiple, lower penetrance gene variants associated with increased risk for common diseases. A significant need, which public health professionals can help fill, will exist for innovative products using a variety to media to help raise general genomic literacy, as well as educational tools that can be used in primary care and other settings.

Oversight of Genetic Tests

The level of oversight of genetic tests has significant medical, social, ethical, legal, economic, and public policy implications, and the system of oversight can greatly affect individuals who undergo testing, who provide tests, and who develop tests (21). Genetic and nongenetic tests are accorded the same level of oversight, which occurs primarily through the Clinical Laboratory Improvement Amendments (CLIA) (42 C.F.R. 493), the Federal Food, Drug, and Cosmetics Act (21 U.S.C. 301), and during investigational stages, Federal Policy for the Protection of Human Subjects (45 C.F.R. 46, 21 C.F.R. 50 and 56). Most new genetic tests are developed and provided as clinical laboratory services, which are referred to as in-house tests or &ldquohome brews.&rdquo The Food and Drug Administration (FDA) has indicated that it has legal authority to regulate such tests as medical devices but has elected not to do so as a matter of enforcement discretion, in part because the number of such tests is estimated to exceed the agency&rsquos review capacity (37,38). However, the Secretary&rsquos Advisory Committee on Genetic Testing (SACGT), chartered in 1998 to advise the U.S. Department of Health and Human Services, has recommended that all new genetic tests be reviewed by FDA before they are used for clinical or public health purposes (21) and is developing additional recommendations to assist the FDA with this review.

SACGT also recommended that CLIA regulations be augmented to provide more specific provisions for ensuring the quality of laboratories conducting genetic tests. CLIA has requirements for certifying laboratories in such areas as cytology and microbiology, but a specialty category of genetics does not currently exist. A revision to CLIA has been proposed that would recognize a genetic testing specialty area and address issues related to accuracy and reliability of test results, informed consent, confidentiality, counseling, and clinical appropriateness (39).

Although these regulations and standards are being developed at a national level, state and local public health programs must be prepared to undertake additional activities to recommend when and how genetic information could be applied to improve health and prevent disease in their own communities. This involves assessing the state&rsquos own medical, epidemiologic, and economic data about diseases for which genetic tests are available the readiness and training of health professionals the adequacy of state laws to protect the public and ensure access laboratory proficiency and infrastructure capacity.

Emerging Issues

Given the rapidly evolving nature of genetic discovery, almost every issue could be classified as &ldquoemerging.&rdquo These include the commercialization and patenting of genetic materials, reproductive rights and decision making, human cloning, and genetic modification of food and microorganisms.

One emerging issue that could significantly affect environmental health, drug safety, and risk assessment is toxicogenomics. Toxicogenomics is the study of how genomes respond to environmental stressors. Scientists in this field are using powerful new tools, such as microarray and proteomics technologies, to assess changes in gene expression on a genomewide basis, providing a global perspective about how an organism responds to a specific stress, drug, or toxicant (40). According to the National Center for Toxicogenomics (40), toxicogenomics could help resolve three major scientific problems:

  1. Understanding biologic responses to environmental stressors, and identifying agents that are a significant risk to human health. Toxicologists rely largely on extrapolation from animal studies when predicting human responses to potential toxins. Toxicogenomics may help scientists gain insights into pathways of toxicity and their mechanisms, leading to better models for extrapolation, fewer animal studies, and faster conclusions.
  2. Improving exposure assessment. Use of mRNA signatures may make possible identification of the agent (class) and dose to which a person has been exposed. Protein markers could also be used to detect presymptomatic, environmentally induced disease. Thus, surveillance programs could be implemented in humans and animals in areas where exposure and/or contamination are suspected.
  3. Identifying susceptibility factors that influence an individual&rsquos response to environmental agents. This information could be used to predict interindividual variation in response to drugs or environmental toxicants.

These potential outcomes raise several legal issues. First, discussions about genetics and employment often focus on the possibility of employers excluding individuals from employment on the basis of predictive genetic information, for example information about future cancer risk, where the concern is excess healthcare costs. Toxicogenomics presents the possibility that individual risk could be identified before toxic exposure and used to protect worker health (41). Actions that might be taken in response to such information include increased medical monitoring to measure the early effects of exposure, more personal protection equipment or environmental controls to reduce exposure levels, and administrative controls such as limiting exposure times. Thus, the primary discrimination issue arising in these examples is whether the employer could require testing over employee objections. State laws vary on whether employers can lawfully require or even request that an individual take a job-related genetic test.

Another approach used in the beryllium industry (42) is for the employer to pay for the tests, which employees can take on a completely voluntary basis. The results are returned to the employee, who alone decides whether to accept any genetically heightened risk.

A second application of toxicogenomics is in setting environmental health regulations. Standards could differ from one location to the next on the basis of the genotypes of the population in the area, which may be correlated with race or ethnicity. For example, suppose a smelter is located adjacent to an Indian reservation. Also suppose evidence exists that individuals with a certain genetic marker are at increased risk from environmental pollutants, and that marker happens to be present at a very high rate in the members of the tribe. Such a scenario raises a number of questions: To what extent should genetic variation affect environmental standards? Should a new, more restrictive environmental standard be adopted for this particular area? Or should the same restrictive standard apply everywhere? Should individuals be urged to undergo susceptibility testing before locating in a certain area?

A significant challenge for toxicogenomics will be to reconcile the nondirective stance traditionally associated with genetics with the directiveness sometimes found in public health practice. Because of eugenics and other abuses in the first part of the 20th century, geneticists today offer patient-centered services that attempt to respect individual autonomy. By contrast, public health programs often focus on population rather than individual health, and some use governmental power to compel actions to protect health. Therefore, political and public support for integrating genomics into public health policies and programs will depend on accommodating the nondirectiveness that an ethical approach to genomics requires.

Conclusion

Advances in human genetics are expected to revolutionize medicine and public health, leading to new understandings of underlying disease processes&mdashincluding gene-environment interactions associated with common chronic diseases&mdashand to new avenues for prevention and treatment. Realizing this potential requires the integration of genomics into a wide range of public health research, policy, and practice activities. This integration does not give rise to fundamentally new or different legal challenges than those public health professionals generally encounter rather, genetic information adds a variable to the already complex interplay between medicine, public health, and health law.

Here we reviewed evolving legal authorities aimed at reducing the misuse of genetic information, including examples of state and federal legislative activities related to insurance and employment discrimination. These activities highlight the need to enact strong measures to protect better the privacy of all health information, while not impeding the core public health function of collecting and deploying information critical to the health of communities. Genetic information is already an integral part of public health practice in the area of newborn screening and, as we move beyond the realm of rare, single-gene disorders, the system of oversight for genetic tests and the need for widespread professional and public education about genomics present challenges for public health practitioners. Issues continue to emerge along with rapid advances in genetics and genetic technology, and these must be addressed as we work to understand the appropriate use of genetic information in medicine, public health, and society.

Acknowledgments

This project was supported under a cooperative agreement from the Centers for Disease Control and Prevention through the Association of Teachers of Preventive Medicine.


Introduction

Organoids are three-dimensional (3D) in vitro cultures derived from stem or progenitor cells, which can recapitulate the variety of cell types, architectural organization and function of their in vivo tissue counterparts [17, 64]. The first attempt of generating organs in vitro began in 1907 when Wilson demonstrated that dissociated sponge cells could reaggregate and self-organize to reform the whole organism [117]. Current attempts to generate organ-specific models grew from the work of Sasai and colleagues, who showed that three dimensional (3D) cerebral cortical tissue could be generated in vitro from pluripotent stem cells [26], as well as from the work of Clevers and colleagues, who generated gut organoids from adult intestinal stem cells [95]. These studies led to the classification of organoids into two main categories: pluripotent stem cell (PSC)-derived organoids and adult stem cell (AdSC)-derived organoids.

As there are already multiple reviews comparing these two categories [17, 43, 64], this chapter will provide only a short summary of the major distinguishing factors between PSC- and AdSC-derived organoids. In principle, PSC-derived organoids are grown from either embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), which we will collectively refer to as PSCs. These cells are first cultured in suspension in a defined medium to promote cell aggregation and directed differentiation [43]. Cell clusters are then embedded in a matrix that provides structural support, allowing the cells to organize into structures resembling the endogenous tissue. PSC-derived organoids can contain different cell types originating from the different germ layers (ectoderm, mesoderm, and endoderm). Since the first 3D cultures of the cerebral cortex [26], organoid differentiation protocols have been developed for generating models of various other tissues, based on the presence of specific signaling factors in the medium. Established murine PSC-derived organoids now include models of the optic cup [27], pituitary gland [105], inner ear [59], and thyroid gland [4, 63]. Human PSC-derived organoids include models of the brain [65], kidney [77, 106], small intestine [102], stomach [73], lung [25], liver [107], colon [79], and mammary gland [88].

AdSC-derived organoids, on the other hand, do not require directed differentiation, as they are grown from tissue-resident adult stem cells in a similar process to that used for the sponge cell reaggregation [117]. AdSCs are first extracted from the organ by tissue dissociation, then directed to form organoids in medium that supports their stem cell activity with an optimal growth factor combination. Examples of mouse AdSC-derived organoid cultures include the intestine [95], stomach [8, 104], liver [15, 41, 42, 45, 83], pancreas [15, 44], lung [67], endometrium [14], salivary gland [81], and taste bud [90]. Human AdSC-derived organoids have also been developed for the intestine [51, 96], liver [15, 41, 42], pancreas [15], endometrium [14, 109], fallopian tube [55], and prostate [53]. While generating organoids from AdSCs requires less time than from PSCs, the number of different cell types that can be generated from AdSCs is limited, as AdSC-derived organoids often only contain epithelial cells [43]. For this reason, they are useful for studying epithelial tissue maintenance and regeneration but not suitable for studies involving the interaction between different cell types, e.g., immune-epithelial interaction.

Since their development, organoids quickly became a popular model by bridging the gap between in vivo animal models, which are time-consuming to generate and costly to maintain, and in vitro two-dimensional cell culture systems, which lack 3D tissue organization and often contain cancer-associated genetic alterations. 3D organoid systems have been used for studying organ development [65] and host-pathogen interactions [20, 87]. They can also be used for disease modeling and therapy development, e.g., by using cancer and diseased tissues as starting materials for organoid formation [10, 11, 34, 65, 66, 96, 110]. Despite all these achievements, the ability to generate, repair, or introduce specific genetic mutations was needed for modeling monogenic disease and cancer, as well as for genome-wide screening and establishing reporter organoids.


Genetic engineering methods

There are currently multiple methods of genetic engineering that have been employed in organoids, opening a new field of research—organoid genetics. These methods enable specific modifications of the genomic DNA sequence. If the modifications are introduced in a coding sequence, they can lead to a specific change in the target protein, which can provide insight into the biological role of a specific residue or the protein itself. This process requires consideration of two major points: the genetic tools and the method of delivering them into the target cells.

Methods for delivery

There are currently two common methods of introducing gene-editing components into organoids: viral (e.g., retro/lentiviral or adenoviral transduction) and non-viral using naked DNA transfer (Fig. ​ (Fig.1). 1 ). Each method has its advantages and disadvantages, which will be briefly discussed here and summarized in Table ​ Table1. 1 . Choosing the appropriate delivery system requires consideration of the properties of the target cells, the size of the DNA fragment, and the required duration of gene expression.

Methods of generating organoids and genetic engineering with their possible applications. Organoids can be generated either from adult stem cells (AdSCs) or pluripotent stem cells (PSCs). AdSCs, extracted from the tissue of origin, can be cultured with the proper conditions to give rise to organoids that mimic the organ they derive from. PSC-derived organoids are grown from cell line of induced pluripotency or embryonic stem cells. Depicted on the left and right human figures are the types of organoids which have been generated with AdSCs or PSCs, respectively. Organoids can be modified with different genetic engineering methods such as CRISPR/Cas, transposase, or RNAi. These tools could be delivered with a non-viral approach such as lipofection or electroporation, or with a viral approach utilizing retrovirus, lentivirus, or adenovirus. The genetically edited organoids can be further utilized for various applications/fields of study including biological developmental models and translational/precision medicine

Table 1

Pros and cons of different methods of delivery

Can infect non-dividing cells

Infect dividing and non-dividing cells

Easy to achieve high viral titer

Efficient for any cell types and living organisms

Can introduce large constructs

Efficient in many cell types

Cannot infect non-dividing cells

Can induce immune response

Transgene size limited to 8 kb

Time-consuming for virus production

Issues with biosafety and mutagenesis

Transgene size limited to 8 kb

Time-consuming virus production

Issues with biosafety and mutagenesis

Transgene can be lost over divisions

Issues with biosafety and mutagenesis

Require extensive optimization

Potential cell damage/nonspecific transport to cells

Transient transgene expression

Retro- and lentiviral transfections utilize the viral machinery to induce stable integration of foreign genetic sequences whose expression can be consistently passed on to progenies [62]. However, retroviruses rely on the host cell cycle to integrate genetic information into the genome, thus cannot infect terminally differentiated, non-dividing cells. Furthermore, retrovirus infection requires high viral titer and can induce immune responses that may reduce the efficiency of genome integration [92, 101]. Lentiviruses have an adaptation that circumvents this limitation, and are thus commonly used for cells that are difficult to infect, such as immune cells or non-dividing cells [21]. However, with both retro- and lentiviruses, integration preferentially occurs in transcriptionally active sites, which can adversely affect the expression of host genes. Moreover, both viral vectors can only accommodate a maximum DNA insert of about 8 kb, which covers most cDNAs, but not all [21, 46].

The adenoviral method avoids permanent integration by remaining episomal after transfection and is effective in both dividing and non-dividing cells [101]. It is also easy to generate high virus titers for higher expression of the introduced transgene. However, due to the lack of genomic integration, the introduced gene can be lost over multiple rounds of host cell division [114, 115].

Lastly, non-viral naked DNA transfer generally involves one of two delivery approaches: electroporation or lipofection. Electroporation utilizes electrical pulses to transiently create openings in the cell membrane, allowing foreign DNA to enter the cell [32]. This method is usually efficient for many cell types and even in living organisms, and can also easily introduce large constructs into the cell. Nevertheless, electroporation requires a relatively expensive device and extensive pilot testing as the optimal parameters vary significantly for each device and cell type. Lipofection utilizes Lipofectamine or related lipid molecules that can form liposomes, encapsulating DNA and introducing it into the cell [97]. This method is relatively simple and usually efficient enough in many cells. However, the transgene expression is normally transient, and lipofection may affect cell survival.

Tools for genetic engineering

After deciding on a method of genetic delivery, it is important to consider the method of genetic editing. As there are many reviews comparing the different tools for genetic engineering [19, 56, 57, 120], we will only present a short summary of important methods that have been used to genetically modify organoids: RNA interference (RNAi), CRISPR/Cas9, retro/lentiviruses, and transposons (Fig. ​ (Fig.1 1 and Table ​ Table2 2 ).

Table 2

Pros and cons of gene-editing techniques

Effective in all mammalian somatic cells

No prior genetic manipulation needed

Introduce specific modification to target sequence

Prone to off-target effects

Random insertion can disrupt transcriptionally active genes

Difficult to perform on large scale

Susceptible to immune reaction

Possible off-target effects

The RNAi system utilizes the cell’s own machinery to silence expression of specific genes. In this system, synthesized RNAi sequences, either short-hairpin RNAs (shRNAs) or short-interfering RNAs (siRNAs), form complementary pairs with the mRNAs of the target gene to promote degradation or translational silencing and thereby suppress the protein expression of the target mRNA. This method is effective in all mammalian somatic cells and no prior genetic manipulation is necessary [28, 39]. shRNAs can be delivered into cells with various vectors such as retro-/lentiviruses, adenoviruses, plasmids, and transposons. [120]. However, RNAi is only a knockdown system, has lower efficacy, and is prone to off-target effects.

A useful choice for stable gene expression is transposon-based systems, e.g., PiggyBac and Sleeping Beauty, which can stably introduce the gene of interest into the host genome for long-term expression. Both PiggyBac and Sleeping Beauty use the 𠇌ut-and-paste” mechanism to 𠇌ut” the genetic sequence flanked by a specific terminal inverted repeat from one locus and “paste” it into another [46]. However, this random insertion sometimes occurs in an active gene, which can lead to unexpected effects on the host cell.

Since 2012, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have been widely adapted for sequence-specific editing in both prokaryotic and eukaryotic cells in vitro [16, 18, 35, 50, 71]. The system was first discovered in bacteria, giving them adaptive resistance to bacteriophage infections [9]. The CRISPR/Cas9 system was then further engineered with two components: Cas9 endonuclease and single-guide RNA (sgRNA or gRNA), where a spacer sequence binds to a complementary sequence of DNA (protospacer sequence) and guides Cas9 to a specific target. A DNA target containing both the protospacer sequence and the protospacer adjacent motif (PAM) forms a target for the Cas9 gRNA complex to introduce a double-strand break (DSB). The PAM sequence differs for the different Cas9 and Cas12a (Cpf1) endonucleases derived from different bacteria species, thus enabling a broad range of applications [18, 89, 121]. Following cleavage by the Cas9 nuclease, the DSB can be repaired by either homology-directed repair (HDR), which requires a template for precise, high-fidelity repair or by non-homologous end joining (NHEJ), where the blunt ends are re-ligated together [13]. Repair by HDR following a supplied template allows researchers to introduce specific sequence changes into target genes [33]. However, this process is inefficient and requires the cell to be in S phase of the cell cycle for the repair to occur [47]. Template plasmid must also be cloned with homology arms specific to each gene, thus increasing the work effort. Alternatively, DNA repair can occur by nucleotide insertions or deletions introduced by NHEJ which generate frameshifts mutations, leading to inactivation of the target gene. As NHEJ is often viewed as error-prone, it is not used for precise targeted mutation. However, recent work by Artegiani et al. [6] adapted the NHEJ for generation of fast knock-ins in various human organoids. This method removes the effort required for homology arm cloning as knock-in DNA is cloned into a self-cleaving plasmid containing a non-human sequence which is recognized by sgRNA. The authors could show more efficient knock-in generation as compared to HDR even with TP53 inhibition which was suggested to improve HDR efficiency in human pluripotent stem cells [6, 48].

Further advances in CRISPR/Cas9 development also target increasing efficiency of different Cas enzymes to detect a broader PAM sequence range [41, 42] or nearly remove the PAM sequence constraint completely [113]. Modification of the Cas9 endonuclease by fusing inactivated Cas9 nickase to cytidine deaminase to generate base editors [36, 40, 60]. This introduced new tools to generate precise base changes in organoids [37, 112].

Combining organoid technology with the various genetic editing techniques provides a new platform for organoid genetics and organoid-based disease modeling. The following chapter provides further details on the applications of gene editing with the CRISPR/Cas9 system in various AdSC- and PSC-derived organoids.


Introduction

Huntington's disease (HD MIM 143100), a familial neurodegenerative disorder characterized by progressive movement disorder, cognitive decline, and psychiatric disturbances, is caused by an expanded CAG glutamine codon repeat in HTT, which encodes huntingtin [1𠄳]. This genetic defect was originally mapped to chromosome 4p16.3 using linkage analysis in part in Venezuelan families from an HD population cluster whose generous participation in this fundamental HD genetic research also contributed, along with many North American and European families also carrying a CAG expansion mutation, to the discovery of HTT [3𠄶]. The HTT repeat is polymorphic in the normal population [7], but inheritance of 㸵 CAGs can lead to HD, while repeats of 㹀 CAGs are fully penetrant within a normal lifespan [8,9]. Expanded repeats show frequent germline instability so that HD individuals may not have a CAG repeat length identical to their transmitting parent [10�]. Rare de novo cases of HD are generated sporadically in the population by intergenerational expansion of a normal length CAG repeat [13,14]. The length of the inherited HTT CAG repeat is the primary determinant of the age at onset of diagnostic neurological signs, accounting for

65% of the variance in this phenotype, with longer repeats leading on average to earlier clinical manifestations [7,15�]. Individuals with biallelic HD mutations (i.e., HD homozygotes) have been reported and support complete dominance with respect to the pathogenic mechanism leading to disease manifestations since the age at onset of an individual with two expanded HTT CAG repeats is comparable to that of a heterozygote with the longer of the two repeats [7,19]. Interestingly, based upon one report, despite an age at onset similar to HD heterozygotes, individuals with two expanded HTT CAG repeats may display more rapid decline in functional capacity [20]. Although the timing of disease onset is CAG repeat size-dependent, the duration of manifest disease (i.e., the length of time from onset to death) is not [21], suggesting that progression to death involves different tissues or mechanisms than those leading to clinical onset. The relationship between CAG repeat size and HD clinical onset has played an important role in guiding 1) the generation of animal models [22�], 2) design/interpretation of molecular studies [27], and 3) identification of genetic modifiers [28].

The extensive accumulated experimental data and complete dominance of the mutant allele are most consistent with a gain-of-function mechanism, which has prompted exploration of mutant allele-specific interference with HTT expression as a treatment option [29]. Although highly promising in model systems, gene-targeting approaches are just now beginning human trials of safety and efficacy. An alternative strategy for the identification of therapeutic targets is to capitalize on observations from humans with HD to discover naturally occurring modifiers of disease. Although CAG length accounts for a significant proportion of the variance in HD age at onset [15,16,18], each CAG repeat size in the HD population is associated with a wide range of onset ages [7]. Since individual differences between the observed age at clinical onset and the age at clinical onset expected based upon the size of the mutation (i.e., residual age at onset) are partially heritable [30,31], both genetic and environmental factors are thought to influence the timing of onset in addition to CAG length [32]. The HD-associated CAG expansion sits on diverse haplotypes [33�], reflecting multiple independent mutation events on different polymorphic chromosome backbones. However, none of the most frequent disease chromosome haplotypes is associated with a difference in age at onset [34], arguing that HD is not commonly modified by a common cis genetic factor at HTT. Similarly, age at diagnostic motor onset of HD is not influenced by the length of the normal CAG repeat [7,32], or by the presence of a second mutant allele [7], suggesting that heritable variance in age at onset for a given CAG repeat is largely due to unlinked trans factors [39]. To identify these, we performed an initial genome-wide association (GWA) analysis in HD subjects of European descent that revealed the first set of genome-wide significant loci associated with residual age at onset of motor symptoms. Briefly, there were two independent onset modification signals on chromosome 15 [28], one modification signal on chromosome 8 [28], and one modification signal on chromosome 3 [40]. Two of the modifier loci acted to delay onset and two acted to hasten onset [28,40,41]. These findings established the proof-of-principle that the pathogenesis of HD can be altered prior to emergence of clinical disease. Consistent with single SNP analysis results, pathway analysis supported a role for DNA repair/maintenance pathways in modifying the age at onset of HD, suggesting that somatic size changes of the CAG repeat may play a role in modification of the HD pathogenic process that leads to diagnostic clinical signs [28]. Together, these observations demonstrated that genetic factors in humans are capable of modifying the rate of HD pathogenesis prior to diagnosis and point to an approach based upon such human observations for therapeutic targeting to delay HD onset.

Most genome-wide analyses to date have focused on Europeans, providing important but potentially limited insights into the genetic contribution to disease risk and normal traits (GWAS Catalog https://www.ebi.ac.uk/gwas/). Our initial European HD GWA study [28] was highly successful in revealing significant modifier loci of relatively strong effect based upon a smaller sample size than most common disease genetic risk studies (GWAS Catalog https://www.ebi.ac.uk/gwas/). However, we might have missed genetic modifiers not present in Europeans. Here, we have extended our genetic analysis to the Venezuelan HD cluster of non-European HD subjects to: 1) determine the full sequence of HTT on the chromosome bearing the CAG expansion mutation 2) to investigate its origin, and 3) to determine whether modification of age at onset in this Venezuelan population is associated with specific naturally occurring genetic factors.


Abstract

Abstract Cholesterol metabolism in macrophages from atherosclerosis-prone C57BL/6J mice was compared with that in macrophages from atherosclerosis-resistant C3H/HeN mice. Plasma total cholesterol levels of both types of mice were significantly increased, but HDL cholesterol level was increased only in C3H/HeN mice when a high-cholesterol diet (1% cholesterol) was fed for 5 weeks. After incubation of macrophages from male and female mice on the high-cholesterol diet with β-VLDL for 24 hours, cholesterol content in macrophages from C57BL/6J was approximately 1.5- to 2.0-fold higher than in those from C3H/HeN mice. [ 3 H]Cholesterol oleate–β-VLDL incorporation into macrophages from C57BL/6J mice on the high-cholesterol diet was greater than incorporation into those from C3H/HeN mice. The release of [ 3 H]cholesterol from macrophages from C57BL/6J mice on the high-cholesterol diet was one seventh that from macrophages from C57BL/6J mice on the basal diet or that from macrophages from C3H/HeN mice on the basal or high-cholesterol diet. Acid cholesterol esterase activity was almost the same in macrophages from any group. Acyl CoA:cholesterol acyltransferase activity in macrophages from C57BL/6J mice on the high-cholesterol diet increased compared with that from macrophages from C57BL/6J mice on the normal diet. Neutral cholesterol esterase activity in macrophages from C57BL/6J mice was about half of that in macrophages from C3H/HeN mice independent of the type of diet. There were no sex differences in these metabolisms. Considered with our previous data, these results suggested that a high-cholesterol diet may cause metabolic changes to accumulate cholesterol ester in macrophages from C57BL/6J mice in accordance with genetic abnormalities.

C57BL/6J mice on a high-cholesterol diet are known to be very susceptible to atherosclerosis. 1 2 3 4 5 However, C3H/HeN mice on a high-cholesterol diet for 1 year were resistant to the formation of atherosclerotic lesions. 3 4 5 After C57BL/6J mice had consumed a high-cholesterol diet, HDL-C levels were decreased at 4 weeks 4 and atheroscelotic lesions were observed at 7 weeks, and these lesions continued to grow until all mice had large atheromatous plaques in the aorta and coronary arteries. 5 Foam cells apparently derived from macrophages in typical fatty lesions were observed overlying acellular areas containing cellular debris in C57BL/6J mice. 1 2 However, the HDL-C level of C3H/HeN mice did not change with a high-cholesterol diet and they did not develop atheromatous lesions. 4 Nevertheless, it is uncertain whether the difference of the response of plasma HDL levels is sufficient to explain the difference in atherogenicity between the two strains of mice. This prompted us to clarify the genetic difference of lipid metabolism at the cellular level.

We have reported the mechanism of foam cell formation by macrophages by use of β-VLDL. 6 7 That is, macrophages incorporate β-VLDL 8 and accumulate cholesterol ester as lipid droplets in cells. Cholesterol metabolism regulating cholesterol ester accumulation in macrophages acts as follows. 9 First, cholesterol ester in β-VLDL is hydrolyzed by acid cholesterol esterase in lysosomes. 10 11 Then the product, free cholesterol, is reesterified by ACAT and stored as cholesterol ester in intracellular lipid droplets. 12 13 After that, the reesterified cholesterol is hydrolyzed by neutral cholesterol esterase, 14 15 16 and finally free cholesterol is released from the cells. It is hypothesized that the imbalance of incorporated cholesterol and released cholesterol induces cholesterol ester accumulation and thereby foam cell formation. Each enzyme activity affects this imbalance. To further elucidate the genetic difference between C57BL/6J and C3H/HeN mice at the cellular level, we also investigated β-VLDL–C metabolism in macrophages.

Because it has also been reported that female C57BL/6J mice were more susceptible to atherosclerosis than male mice (ie, that female mice developed more and larger-sized lesions than males 4 ), we also compared β-VLDL–C metabolism in male and female mouse macrophages.

Methods

Materials

Cholesterol [ 14 C]oleate (2.1 GBq/mmol), [ 3 H]cholesterol oleate (3.0 GBq/mmol), and [1- 14 C]oleoyl-CoA (1.5 GBq/mmol) were purchased from New England Nuclear Corp. DMEM was from Nissui Pharmaceutical Co, Ltd. Male and female C57BL/6J and C3H/HeN mice were from Japan Clea.

Treatment of Animals

Mice at 6 weeks of age were fed basal diet (Oriental Kobo) or basal diet containing 1% cholesterol (high-cholesterol diet) for 5 weeks (the composition of each diet is shown in Table 1 ). We designated C57BL/6J mice C57 and C3H/HeN mice C3H. The groups were C57 fed the basal diet (C57-B), C57 fed the high-cholesterol diet (C57-H), C3H fed the basal diet (C3H-B), and C3H fed the high-cholesterol diet (C3H-H).

Preparation of Macrophages

Peritoneal exudate macrophages were harvested 4 days after injection of 1 mL thioglycollate medium (DIFCO). 17 Cells were plated at a density of 2×10 6 cells/mL in DMEM supplemented with 10% FBS (DMEM/10% FBS). After 4 hours of adherence, the cells were washed and cultured overnight. The cells were then used for the experiments.

Determination of Total Cholesterol, LDL-C+VLDL-C, and HDL-C Levels in Plasma

Isolation of HDL was determined after the LDL and VLDL had been precipitated out with the precipitation reagent containing 555 μmol/L phosphotungstic acid and 25 mmol/L MgCl2, pH 2.5. 18 The precipitation reagent (100 μL) was added to 50 μL of plasma, then the mixture was incubated for 10 minutes at room temperature and centrifuged twice, for 2 minutes each time, at 14 000g. The precipitated residue contained apoB-containing lipoproteins (LDL and VLDL) and the supernatant contained HDL. The HDL fraction was determined for analysis of cholesterol within 2 hours by enzymatic method by use of a kit that detected both free and esterified cholesterol (Determiner TC 555 Kyowa Medex Co, Ltd). Total cholesterol was determined with the same kit. LDL-C plus VLDL-C was determined by subtraction of HDL-C from total cholesterol.

Preparation of Reconstituted [ 3 H]Cholesterol Oleate Into β-VLDL

β-VLDL (d<1.006) was isolated from serum of cholesterol-fed rabbits by ultracentrifugation for 16 hours. 8 Incorporation of [ 3 H]cholesterol oleate into β-VLDL was done essentially by the method of Brown et al 19 : 1 GBq [ 3 H]cholesterol oleate was added with 1 mL DMSO. The mixture was sonicated for 30 seconds, then 2 mL plasma density buffer (0.154 mol/L NaCl, 1 mmol/L EDTA, and 10 mmol/L Tris-HCl, pH 7.4, 0.01% NaN3) was added and the mixture was resonicated for 30 seconds. It was then added dropwise to 6 mL β-VLDL (10 mg total cholesterol/mL) in 3 minutes. The solution was incubated for 8 hours at 37°C and then dialyzed against 3 L plasma density buffer for 10 hours. After the dialysis, the solution was centrifuged for 16 hours at 105 000g. The top layer was used as [ 3 H]cholesterol oleate–incorporated β-VLDL. The specific activity was about 1×10 7 dpm/mg total cholesterol.

Incorporation of [ 3 H]Cholesterol Oleate–β-VLDL by Macrophages

Macrophages (2×10 6 cells/well) were plated in 12-well plates and incubated for certain times in 0.75 mL of DMEM/10% FBS containing 200 μg [ 3 H]cholesterol oleate–β-VLDL (5×10 6 dpm). After incubation, the cells were washed three times with DMEM/10% FBS and their radioactivity was measured with a scintillation counter. Furthermore, to determine the free [ 3 H]cholesterol released from the cells during incubation, organic solvent (chloroform:methanol, 2:1) was added to the medium and lipids were extracted from the chloroform layer. The lipids were applied to thin-layer chromatographs. 20 The radioactivity in the free cholesterol fraction was then counted. The total uptake was the sums of intracellular radioactivity and free [ 3 H]cholesterol radioactivity in the medium. 7

Release of [ 3 H]Cholesterol From Macrophages Loaded With [ 3 H]Cholesterol Oleate–β-VLDL

Macrophages (2×10 6 cells) were plated in 12-well plates and incubated for 24 hours in 1 mL DMEM/10% FBS containing 200 μg [ 3 H]cholesterol oleate–β-VLDL. The cells were then washed three times with DMEM/10% FBS. These macrophages were incubated further in 2 mL DMEM/10% FBS. At the times indicated in Fig 1 , 0.4 mL of the medium was removed and its radioactivity was measured with a scintillation counter. 7

Assay of Acid and Neutral Cholesterol Esterase Activities 5

Macrophages (2×10 7 cells) were washed three times with PBS and suspended in 1 mL 10 mmol/L Tris-HCl (pH 7.4) containing 0.25 mol/L sucrose. The cells were then sonicated twice for 15 seconds and used as the enzyme solution. The reaction mixture contained, in addition to the enzyme solution, 0.5 mmol/L cholesterol oleate, 0.37 MBq cholesterol [ 14 C]oleate, 0.5 mmol/L phosphatidic acid, and 100 mmol/L Tris-HCl, pH 7.4, for neutral cholesterol esterase or 0.5 mmol/L phosphatidylcholine and sodium acetate buffer, pH 4.0, for acid cholesterol esterase in a total volume of 200 μL. The incubation was carried out at 37°C for 1 hour. The [ 14 C]oleate released was extracted by a modification of the method of Belfrage and Vaughan. 21 In brief, the reaction was stopped with 3.25 mL chloroform/methanol/heptane (1.42:1.25:1.00) and then 1 mL 0.1N NaOH was added. The radioactivity in the water phase was measured.

Assay of ACAT Activity

ACAT activity of the above enzyme solution was assayed by the method of Gillies et al 20 with [1- 14 C]oleoyl-CoA without exogenous cholesterol.

Analyses

Intracellular cholesterol content was measured as follows 7 : the washed cells in each well were treated with 1 mL hexane/isopropanol (2:1), and the organic solvent was evaporated. The pellet was dissolved in 100 mL methanol, and the total and free cholesterol contents in the methanol solution were assayed enzymatically by use of Determiner TC 555 and Determiner FC 555 kits. The cholesterol ester content was taken as the difference between the total and free cholesterol contents.

Protein concentration was determined with a kit by use of Bradford’s method (Bio-Rad, Protein Assay) .

Significance was analyzed by Student’s t test.

Results

Effect of a High-Cholesterol Diet on Total Cholesterol Levels in the Plasma of Mice

Plasma lipid levels are shown in Table 2 . The total cholesterol level of female C57-B mice was less than the levels of the other three groups. HDL-C levels of C57-B mice of both sexes were less than those of C3H-B mice of both sexes. Moreover, the level in male C57-B mice was higher than that in female C57-B mice. The LDL-C+VLDL-C level in male C57-B mice was higher than the levels in the other three groups. The ratio of HDL-C to LDL-C+VLDL-C was in the order male C3H-B>female C3H-B>female C57-B>male C57-B.

After consumption of a high-cholesterol diet, total cholesterol and LDL-C+VLDL-C levels were increased in all groups. However, HDL-C levels in C57-H mice of both sexes did not change statistically, whereas those in C3H-H mice of both sexes increased. The ratios of HDL-C to LDL-C+VLDL-C were much less in all groups on the high-cholesterol diet than in those on the basal diet. Nevertheless, the ratio in C57-H mice was about half or less than that in C3H-H mice.

Cholesterol Metabolism in Male Mouse Macrophages

First, to clarify the effect of the high-cholesterol diet on cholesterol ester accumulation in macrophages from male mice, we measured the cholesterol ester content after incubation with or without β-VLDL (Fig 1 ). The cholesterol content was very low without β-VLDL, and there were hardly any differences among the four groups. However, after incubation with β-VLDL, the cholesterol ester content of macrophages of all the groups increased greatly. That in C57-B macrophages was almost the same as in C3H-B and C3H-H macrophages, but that in C57-H macrophages was twofold higher than in C57-B macrophages.

Next, we investigated the incorporation of β-VLDL into macrophages in male mice by using [ 3 H]cholesterol oleate–β-VLDL (Fig 2 ). The incorporation of [ 3 H]cholesterol oleate–β-VLDL into C57-H macrophages was increased 1.3-fold compared with C57-B macrophages at 12 and 36 hours. The levels of incorporation into C57-B, C3H-B, and C3H-H macrophages were quite similar.

Next, we examined the release of [ 3 H]cholesterol into the medium from macrophages loaded with [ 3 H]cholesterol oleate–β-VLDL. Fig 3 shows the ratio of radioactivity released into the medium to total incorporated radioactivity in macrophages as a percentage. The amount increased in a time-dependent manner in all macrophages. However, the amount from the C57-H macrophages was remarkably low it was about one sixth the amounts from the macrophages of the other three groups at 12 hours. The above results indicated that C57-H macrophages accumulate cholesterol ester by taking up a large amount of β-VLDL and hardly releasing free cholesterol from the cells.

We then measured the enzyme activities involved in intracellular cholesterol metabolism (Figs 4 , 5 , and 6 ). It is known that there are three important enzymes involved in lipid accumulation in macrophages the first is acid cholesterol esterase (Fig 4 ), the second ACAT (Fig 5 ), and the third neutral cholesterol esterase (Fig 6 ). Acid cholesterol esterase activities in male mice (Fig 4 ) did not change among the four groups of macrophages. The ACAT activity of C57-B macrophages from male mice (Fig 5 ) was about one third that of C3H-B macrophages. However, the activity of C57-H macrophages increased by more than threefold compared with that of C57-B macrophages. The activity of C3H-H macrophages was not changed. The neutral cholesterol esterase activities of C57-B and C57-H macrophages in male mice (Fig 6 ) were about half those of C3H-B and C3H-H macrophages. The enzyme activity was not significantly changed by high-cholesterol diet in the two strains of mice. These results thus strongly suggested the involvement of genetic regulation in these enzyme activities and their response to a high-cholesterol diet.

Comparison of Cholesterol Metabolism Between Male and Female Mice

β-VLDL–C metabolism in macrophages was almost the same in male and female mice in both strains. Cholesterol ester content in female C57-H macrophages incubated with β-VLDL was highest among all the groups of macrophages and 1.6-fold higher than that in C57-B macrophages (Fig 1 ). The release of [ 3 H]cholesterol from female C57-H macrophages was the lowest, being only one sixth those of other macrophages (Fig 3 ). Absolute and relative enzyme activities in male and female mouse macrophages were almost the same (Figs 3 , 4 , and 5 ).

However, a slight sex difference was observed in the incorporation of [ 3 H]cholesterol oleate–β-VLDL. That into female C57-H macrophages was 1.3-fold greater than that into female C57-B at 36 hours of incubation. The incorporation of [ 3 H]cholesterol oleate–β-VLDL into female C3H-B macrophages was two thirds of that into female C57-B macrophages. The incorporation into female C3H-H macrophages was almost the same as that into female C3H-B macrophages. The above results indicated that there were no remarkable sex differences in terms of lipid metabolism in macrophages.

Discussion

It has been reported that total cholesterol levels did not differ greatly between C57 and C3H strains either with basal chow (4% fat) or atherogenic diet (15% fat, 1.25% cholesterol, and 0.5% cholic acid) and that HDL-C levels were decreased only in the C57 strain with atherogenic diet. 3 4 In this study we observed a difference in HDL-C levels between C57 and C3H strains either with basal chow or high-cholesterol diet. Despite such a difference, the ratios of HDL-C to LDL-C+VLDL-C were very low in all groups of mice fed the high-cholesterol diet compared with those fed the basal diet. Accordingly, we observed differences in the characteristics of macrophages between the two strains of mice. The results suggested that there are genetic differences in the cholesterol metabolism at a cellular level between the two strains with high-cholesterol feeding.

Five weeks after the start of the high-cholesterol diet, male and female C57-H macrophages showed the characteristic of accumulating more cholesterol ester by increased β-VLDL incorporation and decreased release of free cholesterol from the cells compared with other macrophages (Figs 2 and 3 ). Enzyme changes were in accordance with this metabolism. That is, ACAT activity was induced only in macrophages of C57 mice by feeding of a high-cholesterol diet. However, neutral cholesterol esterase activity was not induced by such a diet in C57 macrophages. These activities are quite similar to those reported in rat thioglycollate-elicited peritoneal macrophages, rabbit atherosclerotic lesion cells, 6 and blood monocyte–derived macrophages from rabbits fed a high-cholesterol diet. 22 All these macrophages easily accumulate cholesterol ester in the cells. Therefore, it could be concluded that C57 mouse macrophages possess the feature of accumulating more cholesterol ester and forming foam cells, and that this may contribute to the formation of atheroma when loaded with lipoproteins.

However, macrophage cholesterol metabolism in C3H-H macrophages was almost the same as that in C3H-B macrophages, and cholesterol ester content in C3H-H macrophages incubated with β-VLDL was less than that in C57-H macrophages. These results suggested that macrophages from C3H mice did not change their cholesterol metabolism in response to a high-cholesterol diet. The C3H macrophages had a high activity of cholesterol metabolism, like rat and rabbit alveolar macrophages 6 and THP-1–derived macrophages treated with macrophage colony–stimulating factor 7 ie, C3H-H macrophages preserved high ACAT activity and high neutral cholesterol esterase activity. These macrophages (C3H-B and C3H-H) must have a high capacity to catabolize exogenous cholesterol and to release cholesterol from the cells, and these cells accumulate less cholesterol ester. These properties may explain why the C3H mouse is resistant to atherosclerosis induced by an atherogenic diet.

Of the various differences, the most remarkable one between C57 and C3H mice was the activity of neutral cholesterol esterase in normal diet– and high-cholesterol diet–fed mice (Fig 6 ). Probably this enzyme, in combination with ACAT, is critical for the release of cholesterol from macrophages. The lack of induction of this enzyme coupled with induction of ACAT in C57 mice by cholesterol feeding leads to a low level of release and accumulation of cholesterol ester. In the case of C57-B mice, the release of cholesterol was high despite low neutral cholesterol esterase activity. Under conditions of low ACAT activity, release of cholesterol depending on both acid and neutral cholesterol esterase could be stimulated. 23

Female C57 mice become atherosclerotic more easily than male mice. When testosterone was administered to female C57 mice, atherosclerotic lesions decreased. 4 In this study, total cholesterol and HDL-C levels in female C57-B mice were significantly lower than those in male C57-B mice (Table 2 ). However, the ratio of HDL-C to LDL-C+VLDL-C was higher in female C57-B mice than in the males. After high-cholesterol diet feeding, both total cholesterol and LDL-C+VLDL-C levels increased in both sexes. Absolute HDL-C level and the ratio of HDL-C to LDL-C+VLDL-C were not significantly different between both sexes of C57-H mice. These results suggested that plasma lipids do not explain sex differences in terms of atherogenicity. In addition, there was no difference in β-VLDL–C metabolism between male and female mouse macrophages, indicating that our results cannot explain sex differences at the cellular level either. Other factor(s) related to sex may contribute to the atherogenicity of female C57 mice together with abnormalities of plasma lipid and macrophage lipid metabolism.

In summary, our findings lead to the conclusion that enzyme activities in cellular lipid metabolism and their responses to a high-cholesterol diet are genetically regulated in C57 and C3H mice.


CReasPy-Cloning: A Method for Simultaneous Cloning and Engineering of Megabase-Sized Genomes in Yeast Using the CRISPR-Cas9 System

Over the past decade, a new strategy was developed to bypass the difficulties to genetically engineer some microbial species by transferring (or "cloning") their genome into another organism that is amenable to efficient genetic modifications and therefore acts as a living workbench. As such, the yeast Saccharomyces cerevisiae has been used to clone and engineer genomes from viruses, bacteria, and algae. The cloning step requires the insertion of yeast genetic elements in the genome of interest, in order to drive its replication and maintenance as an artificial chromosome in the host cell. Current methods used to introduce these genetic elements are still unsatisfactory, due either to their random nature (transposon) or the requirement for unique restriction sites at specific positions (TAR cloning). Here we describe the CReasPy-cloning, a new method that combines both the ability of Cas9 to cleave DNA at a user-specified locus and the yeast's highly efficient homologous recombination to simultaneously clone and engineer a bacterial chromosome in yeast. Using the 0.816 Mbp genome of Mycoplasma pneumoniae as a proof of concept, we demonstrate that our method can be used to introduce the yeast genetic element at any location in the bacterial chromosome while simultaneously deleting various genes or group of genes. We also show that CReasPy-cloning can be used to edit up to three independent genomic loci at the same time with an efficiency high enough to warrant the screening of a small (<50) number of clones, allowing for significantly shortened genome engineering cycle times.

Keywords: CRISPR-Cas9 Saccharomyces cerevisiae genome cloning genome editing genome transplantation mycoplasma.


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Diseases, syndromes, or other abnormal conditions caused by mutations in one or more genes, or by chromosomal alterations.

An alteration in the nucleotide sequence of the genome of an organism.

A medical condition that is present at or before birth. These conditions, also referred to as birth defects, can be acquired during the fetal stage of development or from the genetic make up of the parents.

Refers to the relationship between two versions of a gene. Individuals receive two versions of each gene, known as alleles, from each parent. If the alleles of a gene are different, one allele will be expressed it is the dominant gene. The effect of the other allele, called recessive, is masked.

A person or other organism that has inherited a recessive allele for a genetic trait or mutation but usually does not display that trait or show symptoms of the disease.

A special type of cell division in sexually-reproducing organisms used to produce the gametes, such as sperm or egg cells. It involves two rounds of division that ultimately result in four cells with only one copy of each chromosome.

The failure of homologous chromosomes or sister chromatids to separate properly during cell division.

A threadlike structure of nucleic acids and protein found in the nucleus of most living cells, carrying genetic information in the form of genes.

A mature haploid male or female germ cell which is able to unite with another of the opposite sex in sexual reproduction to form a zygote.

The union of the sperm cell and the egg cell. Also known as a fertilized ovum, the zygote begins as a single cell but divides rapidly in the days following fertilization. After this two-week period of cell division, the zygote eventually becomes an embryo.

A medical procedure used primarily in prenatal diagnosis of chromosomal abnormalities and fetal infections as well as for sex determination. In this procedure, a small amount of amniotic fluid, which contains fetal tissues, is sampled from the amniotic sac surrounding a developing fetus.

Biological molecules that lower amount the energy required for a reaction to occur.

An experimental technique that uses genes to treat or prevent disease.

A sequence of nucleotides in DNA or RNA that codes for a molecule that has a function.

The smallest unit of life, consisting of at least a membrane, cytoplasm, and genetic material.

A class of biological molecule consisting of linked monomers of amino acids and which are the most versatile macromolecules in living systems and serve crucial functions in essentially all biological processes.

A carrier genetically engineered to deliver a gene. Certain viruses are often used as vectors because they can deliver the new gene by infecting the cell.


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