Your nurse knocks on the door interrupting your examination of a child. On the phone is a lab technician from another state who reports that a newborn screen reveals a positive test for galactosemia in one of your patients. The patient, you recall, is the first child of a young professional couple. Reviewing her chart you are reminded that there were no perinatal complications. The child was discharged home from the hospital at just under 48 hours of age, he appeared well with only a 3% weight loss and mild facial jaundice upon follow-up at day 4 of life. His parents reported no breast feeding difficulties and, despite their concern for the number of hours their new baby slept, the new family appeared to be thriving.
Genetic testing has received much attention in the press, with interest focusing on the ethics and repercussions of genetic information. Despite this attention, most people do not realize that for the past twenty years, almost all newborns in the United States have been screened for a number of genetic diseases. Almost all newborn screen tests are quantitative tests for the presence or absence of metabolic or endocrine molecules. When the concentration of the tested molecule is greater or less than a level determined by the reference lab, the test is reported as positive. In order to provide 100% sensitivity for disease, the level of positive detection must be adjusted to a point at which specificity may be quite poor. Classical galactosemia affects roughly 1:59,000 infants born in the US. In 1994, 10,210 infants tested positive on newborn screening for galactosemia. Of these, 54 infants were confirmed to have the disease (positive predictive value 0.53%). Improvements in mass spectroscopy are greatly increasing the number of inborn errors of metabolism that can be efficiently screened in all newborn infants.
Metabolic screening represents one type of genetic testing. For diseases such as sickle cell disease and cystic fibrosis, in which the disease gene and disease alleles are known, very specific tests based on DNA are used. Unlike the quantitative screens for inborn errors, DNA-based tests have virtually 100% specificity. These tests provide binary answers (yes/no) to the hypothesis "does this particular unique region of DNA exist in this patient?" With this knowledge, we can understand the current limitations of DNA-based genetic testing.
1. A single gene must be discovered and sequenced, which when altered, produces a recognizable disease state.
2. The various disease-sequence alterations must be catalogued.
3. For every detectable alteration, a single unique test must be created and performed.
Since sickle cell disease is caused by a single diseased allele in all patients affected, a single PCR (polymerase chain reaction) test can diagnose it with near perfect sensitivity and specificity. On the other hand, some diseases such as Duchenne muscular dystrophy or osteogenesis imperfecta, may result from one of hundreds of different possible alleles. In these cases, DNA-based testing is not practical. Cystic fibrosis represents a middle ground, in which hundreds of disease alleles exist, but only a handful produce the vast majority of illness in select populations. In European descendants, the delta F508 allele represents 70% of disease alleles in that gene pool and four additional alleles represent another 10-20%. For this population, a handful of PCR based tests can be performed on a patient's blood which that cover the vast majority of possible alleles.
As more disease alleles are discovered, more tests can be run to determine if each allele is present in a given patient. Currently, most tests are PCR-based and involve duplicating small parts of a patient's genome in sufficient quantities to be detected on a gel by fluoroscopy or radioactivity. In the late 1990s, DNA chip technology was invented with the power to perform hundreds of thousands of DNA-based experiments simultaneously on a single patient. A DNA chip is a grid of hundreds to hundreds of thousands of individual matching tests mass produced on silicon wafers the size of a dime. Each matching test involves a small unique section of single stranded nucleic acid which is glued to the wafer in a specific grid position. A patient's DNA or RNA is extracted from blood, broken up into short strands, made single stranded (in the case of DNA) and then used to bathe the DNA chip. Sections of patient DNA or RNA which closely match specific test sequences on the chip bind and are detected. This technology has the potential to increase the sensitivity of DNA-based genetic testing as disease alleles continue to be discovered.
There are several other methods to detect genetic based illnesses, including the visual inspection of chromosomes, the augmented inspection of chromosomes using fluorescent antibodies, and multiple methods of detecting the presence, absence and relative quantity of proteins.
A person's DNA essentially remains unaltered from the moment his/her mother's ovum and father's sperm join, until the day that the last nucleus in his/her body is destroyed. In theory, the information to predict susceptibility to all genetically based disease is available in zygotes and ancient human remains. As science progresses and discovers how genetic information predicts disease states, the ethical debates over how best to use the information must also progress. Currently, the AAP recommends:
1) The introduction of new newborn screening tests only if identification provides clear benefit to the child, the diagnosis can be confirmed after a positive screening test result, and treatment and follow-up are available for each infant tested.
2) Informed parental consent.
3) No screening of healthy children for the detection of disease-carriers (children with potential to pass on an inherited disease to their children) except in the case of certain prenatal screening tests in well informed teens.
4) No testing for adult onset illness until the child is an adult and is able to make informed decisions for him/herself.
Since the discovery that genetic information can predict disease states, people have been afraid that this information might be used in a discriminatory manner. One such fear is the possibility that insurance companies might use genetic test results to increase rates or even deny coverage to individuals with genetic susceptibility to expensive illnesses. So far, these fears are only speculative. A panel of lawyers, genetic counselors, and geneticists reported (at the 1999 meeting of the American Society of Human Genetics) that they had been unable to identify any cases of discrimination by health insurers (6).
Simply stated, gene therapy is medicine practiced with a nucleic acid based pharmacy. The patient is given nucleic acids in order to modify a pathologic pattern of protein expression. One form of gene therapy, bone marrow transplant, is currently in widespread use. The treatment for refractory leukemia involves massive chemotherapy to destroy all cancerous cells that are then replaced by a population of cells with "normal" cell cycle regulation. The more classical definition of gene therapy requires the modification of protein expression in existing cells. Only a handful of individuals have undergone this experimental form of therapy. These therapies are based on the idea of utilizing a virus as a vector to implant new genetic information into a patient's cells. Thousands of viruses have evolved to efficiently insert their DNA or RNA into specific human cell lines for the purpose of turning the cells into viral copy machines. In creating vectors, scientists remodel viruses, retaining the machinery to identify and infect specific cells (adenoviruses which preferentially affect respiratory epithelium, lentiviruses which preferentially attach to T-cells, and herpes viruses which recognize neurons) but change the genetic material which the virus inserts into the cells.
The inserted nucleic acid can take any of the following forms: 1) RNA can be used to immediately translate functional proteins. 2) RNA or DNA can be engineered to be the non-coding ("anti-sense") sequence of a specific pathologic mRNA so as to produce functionless double stranded mRNA. 3) Ribozymes (RNA with the enzymatic ability to degrade specific mRNAs) can be introduced to degrade pathologic mRNA. 4) DNA can be inserted in the middle of a disease gene to turn off pathologic production. 5) Sections of DNA can be inserted permanently into the target cell to restore the production of proteins, or alter the degree to which proteins are expressed by the cell. Despite the elegance of these theories, no disease state has been "cured" using gene therapy in a human patient as of this writing. The failure of gene therapy to date, is attributable to both vector and nucleic acid design, which are limited by our rudimentary knowledge of basic cell and molecular biology.
The dream of the Human Genome Project, that one day patients will provide a drop of blood, a scraping of cheek cells, or a hair follicle and be provided with a set of probabilities of acquiring all disease states and a range of treatment options based on targeted gene therapy, is far from being realized. However, in the early 1990s the Human Genome Project's basic goal of sequencing the entire genome also appeared a pipe dream which was projected to run over time and budget. In 2000, five years ahead of projections, the first working draft sequence of a human genome was completed. Noninvasive, perfect tests will never become available for all diseases and silver bullets filled with DNA will not rid the world of illness, but it is certain that the power of nucleic acids to diagnose, treat, and prevent illness will become significantly greater in the coming decades.
Questions
1. True/False: Current newborn screening can diagnose a handful of inborn errors of metabolism like Galactosemia?
2. What are the limitations of DNA based genetic testing?
3. Why is it not currently ethical to test a 7 year old girl for the BRCA1 (breast cancer 1 gene) mutations even if early breast cancer runs in her family?
4. Currently, what is the most widely used form of gene therapy?
5. What is the function of a gene therapy vector?
6. Describe the various methods of introducing nucleic acids into a cell to alter disease states.
References
1. American Academy of Pediatrics, Committee on Bioethics. Ethical issues with genetic testing in pediatrics. Pediatrics 2001;107:1451-1455.
2. American Academy of Pediatrics, Committee on Genetics. Molecular genetic testing in pediatric practice: a subject review. Pediatrics 2000;106:1494-1497.
3. Collins F S, McKusick VA. Implications of the human genome project for medical science. JAMA 2001;285:540-544.
4. Kaji EH, Jeiden JM. Gene and stem cell therapies. JAMA 2001;285:545-550.
5. Kwon C, Farrel PM. The magnitude and challenge of false-positive newborn screening test results. Arch Pediatr Adolesc Med 2000;54:714-719.
6. Nowlan W. A rational view of insurance and genetic discrimination. Science 2002;297:195-196.
Answers to questions
1. False. Newborn screening is not diagnostic. Rather, it is a screen for illness with VERY poor specificity, which, if positive, must be followed with a more specific diagnostic test.
2. Sequence knowledge of the disease locus and mutant alleles and the 1:1 correlation of test to disease allele. For disease conditions with multiple mutant alleles, all possibilities must be specifically tested.
3. The disease does not affect the patient until adulthood when she can make her own decisions. There is no effective prophylactic treatment which can be offered to her as a child which will prevent the illness before she reaches adulthood. Testing may be appropriate for a 17 year old who is desiring pregnancy, has the consent of her parents, and who plans to make the decision to become pregnant based on the information of the test.
4. Bone marrow transplant.
5. Vectors transport engineered nucleic acids (DNA or RNA) into existing human cells.
6. 1) DNA based: Insertion of intact functional gene. Insertion of intact functional promotor or exons to correct production. Insertion of DNA for the purposes of disrupting expression of a gene. Insertion of single stranded DNA for the purposes of binding to mRNA and preventing translation. 2) RNA based: Insertion of RNA to be reverse transcribed and incorporated into DNA. Insertion of RNA to be translated immediately. Insertion of RNA ribozyme to destroy mRNA. Insertion of anti-sense RNA to prevent translation of mRNA.