At a Glance
Name: Holmes Morton
Title: Director, Clinic for Special Children
Professional Background: 1989 present, director, Clinic for Special Children, Lancaster, Penn.
Education: 1983 MD, Harvard University, Boston, Mass.; 1979 BS, Trinity College, Hartford, Conn.
Name: Erik Puffenberger
Title: Lab Director, Clinic for Special Children
Professional Background: 1998 present, lab director, Clinic for Special Children, Lancaster, Penn.
Education: 1996 PhD, human genetics, Case Western Reserve University; 1987 BSc, Swarthmore College.
Among the revolving motifs on Affymetrix's website is a photo of two Amish children with the tag line "move genetics to the clinic."
The clinic in this case is the Clinic for Special Children, a 16-year-old non-profit based in Lancaster County, Penn., that has benefited recently by using Affy's technology in its diagnostic and pediatric services for the Amish and Mennonite communities in Pennsylvania Dutch country.
According to statement from Affy last week, the CSC is making progress, including a recent discovery profiled in the American Journal of Medical Genetics that links a disease suffered by two Mennonite children to Salla disease a rare syndrome leading to early physical impairment and mental retardation primarily found in Finland.
To learn more about how a rural clinic is putting the fairly new technology to use, BioArray News spoke with clinic director and founder Holmes Morton, as well as CSC's lab director Erik Puffenberger, last week.
It appears from you work that there is a significant rate of hereditary diseases in the "Plain" populations. How prominent are these diseases and are there in any that are specifically prominent?
HM: In the Amish and Mennonite populations there have historically been recognized to be a few disorders that are more common than in the general population. These are recessive disorders that are not absent from the general population but are less common. So that is explained by what we call "founder effect," which is that when people came here 300 years ago they brought with them genes from Europe, recessive genes that were introduced into this population, and over the past 16 generations some of those genes have distributed in the population in a way that they've become more common. That's called genetic drift. It's usually caused by inbreeding, but it really has more to do with the ways genes distribute themselves after a bottleneck introduction of a few genes into a rapidly expanding population.
For example, glutaric aciduria, which is the disorder among the Amish that I came out here to study, in the general population it occurs in about one in 20,000 children. In this population it's about one in 500. At the same time, cystic fibrosis, which occurs in our population about one in 2,000 children, is in the Amish population absent. So whether that gene for CF didn't come in with the founders, or whether it just disappeared from the population, the way some names disappear from a population, we don't know, but we know that there's an uneven distribution among genes in the population.
Has anybody joined the population in this time?
HM: Probably about 100 years ago when cultural differences weren't as great as they are now, before television, some people did marry into the Amish population. There are French Huguenots that married into the population. Some Native Americans came into the population genetically. But largely it has been a gene pool that has grown from 24 founders in the Amish; the Mennonite population was actually a little larger maybe about 150 people founded the Mennonite population.
Now there have been migrations into the population since then, both from Europe and from other settlements that were in Ohio and the Midwest and were established later by different groups of people that came over in the mid-1800s and established some of the large populations in the Midwest and some of those people have migrated back into Lancaster County and in a sense contributed different genes into the pool in Lancaster County from outside. It's complicated.
Sounds like a lot of information to sift through …
HM: I think one of the mistakes people make when they think about the presence of these conditions within the population [is that] they assume that these are some strange genes that only happen to the Amish or the Mennonite. That is not true at all. These are gene mutations that came from Europe, and the mutations are still found in Europe and the United States. So the disorders that we study and treat here, we hear from patients all over the world with these disorders. These are not unique to the Amish.
Why do people contribute to the clinic, and what value does your research have outside of Lancaster County?
HM: I think the contributions to the clinic come either from people who are interested in these communities because of their way of living, culture, curiosity there is a great curiosity about the Amish and the Mennonite people around the country and the world. I think beyond that, many of the contributions come from families from around the country and around the world who have families with the diseases we treat. And they know how difficult it is to find care for them.
I think the most important thing is that these are not just conditions unique to these cultures but what we learn in terms of treating maple syrup urine disease [a condition in which the body's unmetabolized amino acids are passed in the urine giving it a distinctive maple syrup odor], or glutaric aciduria here, is helpful to the kids in Israel, to the kids in Holland, to the children in India, [and] Brazil. As we learn to take care of the children here in these populations we apply that to other children all over the world. The reason that these people are interesting medically and genetically is not because they are different but because they are the same. But for technical reasons it is easier to find genetic disorders, it is easier to find the cause of disease [in this population].
How aware is the local community of these studies and are they supportive of them?
HM: They are very aware. They are out there raising money for us and bringing their children here. You probably could not go to a gathering of Amish people anywhere in the United States and talk about the clinic and somebody wouldn't know about it. Historically, both the Amish and the Mennonite people have been the most important populations for the study of human genetics. They are pretty knowledgeable about genetics [as well]. They have these genealogies that show how disease runs through their families.
How difficult was it for you to master the Affy technology?
EP: We just have gotten our own system here and so I have personally not been running the arrays myself. The DNA samples have been set out to the Translational Genomics Institute in Arizona. We are just getting to the point where we are going to start running them right here in the clinic. The Affy scanner, fluidics station all the hardware is here. We are just waiting for the training session.
Have you ever used microarray technology in the past?
EP: No, we haven't. Obviously there's been a lot of work and people have been using them for many years both with the genotyping platforms and the expression platforms. Now most of our work at least initially will be with genotyping. We have been using the Affymetrix 10K GeneChips with at least 10,000 SNPs on them. And those have worked very well for the studies we want to do. 100K will be useful for some of the studies that we haven't started yet, but we will be interested in moving towards those studies in the future of more complex disorders. But the 10Ks have provided us with more than enough information to map these diseases in this population. So we are the beneficiaries of unique populations here that allow us to do mapping studies very efficiently.
The microarray technology is nice because it just speeds up what normally would have taken years to accomplish using microsatellite markers in a laboratory. The arrays give us much more information for less money and faster.
What about this latest discovery? I believe you linked one disease in the Mennonite population to Salla disease.
EP: We did that with only two individuals. You can imagine that [with] gene mapping there are two critical parameters, and what we have relied on in the past is lots of patients. So, if you want to do a mapping study with the old microsatellite markers you may look at 300 to 500 markers throughout the genome. But because you have fewer markers you need a lot of patients to get good statistical results.
The alternative is to use a whole lot of markers and use fewer samples from patients. What we've been able to do is basically show that we can take two infected individuals and look at their DNA and identify a disease chain just based on those two individuals. That's very powerful, because you can imagine that lots of the diseases we work with are rare disorders. We don't have lots of patients. So it can be very difficult to map these genes. This technology allows us to tease out that little bit of DNA [we need] and identify it with very small numbers of patients and in a very fast way.
We've got a number of projects that we are working on right now. Some have worked out. The Salla disease is one that worked out very well and we finished very quickly. And there are a number of disorders that we are working on and hopefully should be getting some papers out shortly.
There's one concerning Pretzel Syndrome [a disorder characterized by skeletal deformities and malformations of the brain, heart, and other organs].
EP: That's one that's coming up soon. We are actively working on that now. I doubt it will get [published] in 2005. I believe it will happen in 2006. That was an interesting one because it highlighted the pitfalls of using these microarrays as well because we used seven patients in that case and we could not map the disease. We could not find the region. It turns out that it maps to a region where there's very poor coverage on the array. There's a region in the chromosome where there's only one SNP and four megabases of DNA. So that's very poor coverage actually. And our gene happens to map there. It was very difficult to map, except for the fact that we got very lucky. The single SNP that was in that four megabases of DNA was deleted in all of our patients. It turns out that this particular condition is caused by a 7,000-basepair deletion. And our SNP happened to be in those 7,000 bases that were deleted. So we got very lucky that we found it that way. But we were not so lucky because the coverage was bad.
I read that you are planning a new study to map undiagnosed disorders in the Amish and Mennonite populations and that it will involve more than 200 children. Can you tell me more about that study?
EP: Dr. Morton has been out here for 17 to 18 years seeing patients. And over that time we've added up all the patients we've seen with developmental delays. And there are about 200 of them. And that's removing the children that already have biochemical disorders where we've already identified the mutation. So we removed that group and we still have about 200 children who have been seen here at the clinic who have unexplained developmental delay. What we are planning on doing is taking DNA samples on all of them and genotyping them. The idea is to get the pediatricians that are here to sit down and rank the kids by which children seem to be most similar to one another. We'll start to put them together based on their clinical problems and then look at their genotypes to see if the children that are most similar phenotypically share any unusual pieces of DNA in the genome. It will be a way that we can hopefully tease out some additional single-gene disorders that cause developmental delay here in the population.
Again, we benefit from the population because we have a finite number of discrete mutations that are in the population because they have a finite number of founders.