Before Mark Hughes begins to describe his research, he likes to bring genetics down to earth. "If I could break open one human cell and stretch out the DNA, and then type in 12-point font (a standard type size) the four letters of the genetic alphabet contained in that DNA, it would take up a 300-volume set of Encyclopedia Britannica." Continuing with the analogy, he described, "We can think of the chromosome as one of the volumes of that encyclopedia set. If you have too many books, you have problems like Down's syndrome or worse. If you have too few, you have severe birth defects."

Dr. Hughes says, "Technology often drives science, science drives medicine, and medicine is always pushing society into ethical corners."

Mark Hughes is in demand. As a clinician and researcher, he is requested by patients, consulted by fellow investigators, and sought by numerous medical and scientific groups for his work on pre-implantation diagnosis and single-cell analysis. The first gives parents the opportunity to have a successful pregnancy. The second is a tool for the first, and also a method with the potential to yield invaluable information about cancer and other gene-related diseases.

"I walk this line between the bench and the bedside, and that's my favorite niche," said Dr. Hughes, who holds both a PhD in biochemistry and an MD. His drive to blend laboratory and clinical work found a fitting companion in genetics research. "There's probably no other field of medicine that moves faster than genetics, where a gene is discovered one month, two months later you can test patients for it, and in two months more, we're already talking about drugs that are designed around that gene discovery and about gene therapies."

Pre-implantation diagnosis
Dr. Hughes' research encompasses a good deal of this encyclopedia of life. One of his major emphases is on single-cell analysis, which he portrays as a molecular spell-checker. "We want to be able to look for typographical errors as little as a single base change - a single letter in the four-letter genetic alphabet - and be able to do this very, very rapidly." 

To compound the task, he and the other members of his research team often have only one cell to analyze, and sometimes only one copy of a gene. "You have to push technologies like the polymerase chain reaction (which makes many, many copies of the DNA in a sample) to their theoretical and practical limits, and do it in a diagnostic setting where it truly matters what the result is, because there's a patient on the other end," he said. "That requires approaching 100 percent diagnostic accuracy. And we can do that." 
Their efforts on single-cell analysis garnered substantial attention through their work in pre-implantation diagnosis. A collaboration between Dr. Hughes' team and a group of investigators in England resulted in a healthy pregnancy for a couple whose genetic makeup posed a substantial risk for cystic fibrosis to any children they might have. "Our first tests resulted in the birth of a baby without cystic fibrosis. We published that work as the lead article in the New England Journal of Medicine."

The story behind this healthy infant and the many others since begins with a false pretense. "We pretend the couple is infertile, even though we know they're not. Most of the time the couple already has a child." With the help of gonadotrophins, which are hormones that stimulate the sex glands, the woman becomes super-ovulated and produces 10-12 eggs instead of the usual single egg. Doctors then remove the eggs, fertilize them outside the woman's body and let the pre-embryos grow in an incubator for three days.
Each egg cell divides into two cells, which divide again and again. "At the eight-cell stage, we go in with a tiny little needle, and biopsy one from each pre-embryo," Dr. Hughes explained. "Then we use our molecular spell-checker, and we scan that cell for the presence of a particular genetic mistake." If the couple carries the potential for cystic fibrosis, for example, the researchers exclusively scan for the genes that cause that disease. "This is not eugenic technology to try do some kind of newborn screening. This is for a family who's coming with a specific problem."

When the scanning is completed, the researchers know which pre-embryos are free of the disease in question, and transfer those back into the mother. 

"Now the woman can start her pregnancy with the knowledge that her baby will be healthy. We're eliminating the 15 weeks of high anxiety that come before she has an amniocentesis, and we're eliminating the only alternatives she would then have, which are potential pregnancy termination or bringing into the world another child who can't live because of this disease." 

Single-cell analysis
The single-cell analysis at the crux of the pre-implantation diagnosis is basically a "diagnostic dipstick," as Dr. Hughes calls it. The dipsticks are small pieces of glass dappled with tiny dots. When the researchers are looking for chromosome differences, they create dipsticks in which each dot is a separate chromosome. 

Dr. Hughes explained that normal cells have 23 pairs of chromosomes for a total of 46. The difference between males and females is that females carry two copies of the X chromosome, whereas males carry one X and one Y chromosome. "Let's say we have a normal 46 XY cell from a male, and an unknown cell that is going to turn out to be 47 XX plus 18. The 47 XX plus 18 means the cell is from a female, it has one too many chromosomes, and the extra chromosome is chromosome 18."

Preimplantation genetic diagnosis determined that these three embryos were free from sickle cell anemia, even though the parents were both carriers.  As reported in the New England Journal of Medicine, the embryo transfer resulted in a successful pregnancy and the birth of healthy twin girls.

After removing the DNA from each cell and amplifying it with polymerase chain reaction, they label it with either a red or a green dye. In this example, the female cell receives the red and the male receives the green. Then, they paint the dyes onto a glass slide. "Every place that the DNA is the same between the 46 XY and the 47 XX plus 18, you're going to get an intermediate color between the red and the green, which we let the computer pseudo-color blue. So most of the chromosomes will be blue, because they're the same."

Dr. Hughes is looking for the red and green markers, which the computer makes easier to spot by lining up like chromosomes. Because only one of the initial cells has a Y chromosome, that chromosome will show up green on the slide. The X chromosome paints red, however, because the initial female's cell has twice as many Xs as the male's. "In the female we have three 18s and in the normal male's cell we've got two, so the chromosome 18s will paint red." 

With this technique, researchers can instantly and readily spot chromosomal abnormalities in the pre-embryo's cell.

On the gene level
The same idea can be applied on the gene level by making dipsticks flecked with genes instead of chromosomes, Dr. Hughes said.

"We now have one of the largest collections of human genes that are available - about 27,500 of the 80-100,000 genes in the human genome. We've set up a collaboration with a company to spot these genes down onto glass, so that we can actually do grand canvassing of thousands of different genes," Dr. Hughes said. This technology allows them to compare individual cells and pinpoint even slight genetic differences. 

Its applications are widespread. Because this technique provides a quick determination of genetic differences between individual cells, he said cancer researchers and developmental biologists have become particularly interested. Developmental biologists want to know how a cell is able to turn on and off specific tasks over a person's lifespan. Cancer researchers want to know the genetic aspects of the disease.

"The problem with cancer has been that tumors are rarely pure. You've got carcinoma in there, you've got normal tissue in there, you've got tissue that's trying to become the tumor - you've got a lot of processes going on." An analysis of the DNA had been very confusing and difficult to untangle, Dr. Hughes said. With single-cell analysis, however, a researcher can compare two single cells from differently behaving areas of one tumor. "I can do single-cell analysis on them to see what different genes are being expressed, and those are the candidate genes for what's causing their cancer."

Similar to the chromosome example, the gene dipstick can pinpoint single gene duplications or deletions. "Let's say there's a cancer cell with a duplication out on the end of chromosome 3. The duplicated region will paint red or green instead of the normal blue. You can immediately see the problem in the cell. You would never have been able to see that before."

Another possibility for single-cell analysis is determining where genes are being expressed. "The same type of thing happens, except that you're painting expressed genes instead of the genomic DNA. Consequently, we're painting only the genes that are being read. So in a single cell, you can begin to get information about which genes are being turned on and off, and what exactly in the encyclopedia is key to this process." 

From bench to bedside
These procedures have great research potential, Dr. Hughes remarked. "There's tremendous excitement about this, and we're on the front end of it."

The full team behind this Wayne State University research comprises investigators in the pathology, biological sciences, and obstetrics and gynecology departments, and in the Karmanos Cancer Institute, the Institute of Chemical Toxicology, and the Center for Molecular Medicine and Genetics. 

A large team is necessary, because the field is immense. Asked Dr. Hughes, "What field goes from birth to death, males to females, covers all tissues of the body and covers just about every area of medicine? What else but genetics?"

Understanding the benefits of genetic technology
A worldwide argument is brewing that pits the benefits of genetics research against its potential misuse. For Dr. Mark Hughes, the benefits clearly come out on top. With the work he and a team of scientists and doctors are doing at Wayne State University, he sees the positive side of genetics research in the faces of new parents - parents who only months earlier looked upon pregnancy with trepidation rather than joy, with visions of a tragic past rather than a bright future. He also sees dozens upon dozens of advantages yet to come.

Dr. Hughes gives high-risk parents better odds at disease-free children.

Listening to the parents is the best way to understand the importance of genetics research, he said. "It's hard to sit down in my clinic office with a couple who has a pregnancy ongoing, and tell them that the ultrasound shows a problem. For them, that's pretty traumatic; all of a sudden their dreams are being shattered in real time." 

As difficult as that is, he said, he repeatedly faces tougher situations. "I think it's even harder to sit down with a couple who has a kid over in a children's hospital who is dying from Tay-Sachs disease or muscular dystrophy or cystic fibrosis or any number of thousands of other conditions. For them, they know this disease better than any doctor. This disease is in their family. Now, they want to have another child, but they don't want it to happen again. 

Dr. Hughes' work with pre-implantation diagnosis gives couples another option. Although the benefits can be quite spectacular, Dr. Hughes said that he and the research team don't offer this option as a routine clinical tool. In fact, Dr. Hughes still considers the option to be in the research stage. 

Beyond helping at-risk parents, the possibilities for genetics research are tremendously far-reaching. "I think most of the action is in canvassing large numbers of genes in toxicology and in cancer cases," he said. These investigations could tell researchers about specific genetic abnormalities that promote disease. 

Another important avenue of investigation involves diseases caused by multiple genes. Diseases like sickle cell anemia, hemophilia and Tay-Sachs are caused by a single base change - a difference in just one letter of thousands in a person's genetic code. "What, on the other hand, if you have 10 genes all interacting together or not interacting properly together that give you the clinical phenotype of schizophrenia or some other condition. You can't easily follow it down through a family. It's more complicated. These conditions become polynomial equations with lots of variables, and that's why you need these kinds of next-generation technologies."

Another potential impact of genetics research is the new knowledge genetics can yield about an individual's predisposition to a variety of diseases. "The goal would be to know about these conditions ahead of time, to head the problem off at the pass." He explained, "Perhaps we could take a little drop of blood and say, 'Well, you are at even-odds chance of getting lung and colon cancer, but we've got to worry about your ovaries, so we're going to customize your medical care to what you're at risk for.'" 

That information could be critical. "Right now, you go in to see the doctor, get patted on the back, and think everything's fine, while you've got a tumor the size of a marble in your colon. They didn't look for it, because it's not cost-effective to look at everybody. Now, you've got to wait until it's big enough to have symptoms, and then it's too late." With genetics testing, he said, the doctor would have the tip-off on a patient's predisposition. "It's incredibly cost-effective medicine."

To those who argue that too much knowledge about an individual's DNA is a bad thing, Dr. Hughes said it's a simple case of history repeating itself. 

"When I was in Washington, D.C., a few years ago, I went to the Library of Congress and asked for all the information they had on Christiaan Barnard and the first heart transplant. What a lesson."

After all of the positive news reports about the 1967 transplant had died down, the tide began to turn, Dr. Hughes summarized. The editorials and commentaries became negative, with predictions that transplants would become a "fountain of youth for the rich." He remarked, "When taken to its logical conclusion, they said that we were going to have body-parts stores on every corner. We were going to be decreased to our component parts, and our humanity was going to be lost to its components." 

Time has tempered those commentaries, Dr. Hughes noted. "Now, we routinely sign the back of our driver's licenses to donate organs."

Dr. Hughes proposed that the lesson from the transplant hysteria can be extended to genetics research. Now, he said, editorials and columns trumpet privacy breakdowns and ensuing employment, life insurance, even social discrimination based upon an individual's composition of DNA. "These doomsayers all will jump off the cliff together in great harmony and say we're all going down a dead-end pathway, whereas, in reality, people are not so dumb. Things find a way of sorting themselves out, and they're not so bad. It isn't the technology; it's society's understanding of it and how it should interplay with our civilization."

He added, "Technology often drives science, science drives medicine, and medicine is always pushing society into ethical corners."