Eugène Apert is the name of the French pediatrician who gave his name in 1906 to Apert syndrome (AS), one of the more grievous of childhood maladies. "Apert syndrome," observed clinical molecular geneticist Andrew Wilkie, at England's Oxford University, "is a very easily recognizable condition. One in 70,000 live births are afflicted with Apert syndrome. They have a distortion in their appearance in the skull, which leads to a number of significant physical problems. Most visible is webbing [syndactyly] of fingers and toes.

"Not the toes but the digits," he continued, "are separated surgically. But theirs' is not just simple webbing like flaps of skin between digits. In fact, the bone is involved as well, so that the bones in the fingertips are fused together. Surgically it's more challenging than just snipping a piece of skin apart. AS is a significant pediatric problem."

Wilkie, who holds an endowed chair in pathology at Oxford, is senior author of a paper in the current issue of Science, dated Aug. 1, 2003. Its title: "Evidence for selective advantage of pathogenic FGFR2 [fibroblast growth factor receptor type 2] mutations in the male germ line."

"What we were trying to do," Wilkie explained, "was to quantitate accurately the proportions of a number of different known or anticipated molecular species of amplification products in a mixture. We knew roughly what we were expecting to find there, but what we wanted to do was to say in any given sample what those proportions were. And to put that question into perspective, we were fortunate enough to come across the technique called pyrosequencing. From a technological point of view this Science paper is all about the pyrosequencing approach. It solved the problem we'd been having trouble with. It's a technology that has been invented and is marketed by a Swedish company called Pyrosequencing AB. A couple of people from [the company] are co-authors on our paper.

"Pyrosequencing is a method for sequencing short sections of DNA where you anticipate, roughly, what sequences are going to be present. The thing that's so different about it is that you add nucleotides one at a time. And the output that you're measuring is actually a flash of light. That's connected with the clever chemistry that the Swedish inventors have built into their pyrosequencing. When they add a nucleotide, they have pyrophosphate released. What they do is convert the amount of pyrophosphate that's produced into a read-out of the flash of light."

Light Flash Quantitates It All

"And that's what is measured. What I would say from a biotech point of view," Wilkie told BioWorld Today, "is that this particular invention, pyrosequencing, was absolutely brilliant.

"Apert syndrome has arisen as a new mutation in the child, where the question is analyzing the samples from their parents: Has AS come from mother or from father?' From prior research we determined that these mutations always come from the father, without exception. Clearly, whatever the problem, it's something about when you make sperm rather than how you make eggs.

"If you look at the ages of parents of children with AS," Wilkie noted, "they tend to be a bit older than the national average. So there's an age factor here to have samples of sperm from men at a whole range of different ages, from 20s to 70s. Thus, we could actually have a look at this age effect, and see whether the levels of mutation in sperm did indeed change with the age of the donor. That turns out to be the case - positive correlations between the age of the donor and the level of mutation in his sperm. This is what we call a gain of function, with the AS mutation in a souped-up form of the growth-factor receptor.

"The paradoxical benefit," Wilkie went on, "is that for the cell in which the mutation arises, that cell is now at a great advantage compared with its neighbors. Its population expands in the testis. What's it doing there? Stem cells provide the long-lived memory cells that have a copy of this genetic information that is used for making sperm. When a stem cell divides, normally what's happened is that it makes exactly one stem cell and one other cell, which goes on to make sperm and is finally lost from the testis. That's the way to maintain a stable population over years and years of male spermatogenesis.

"What if one time out of 10 instead of that stem cell making one stem cell and one other cell that makes the sperm, imagine the stem cell when it divides makes two stem cells that have the Apert syndrome mutation instead of one. And after another 10 divisions you'd have four stem cells, and as time goes on slowly from that one stem cell you've got a built-up population of stem cells. Normally, you'd get one in every cell generation, one stem cell the offspring of the original stem cell. So that's the advantage: Over the course of time that stem cell has increased in the population compared with its neighbors.

"A subsequent line of four cells is growing in a way that's not completely under the control of its neighbors. That gives rise to cancer, so we're interested in seeing whether we can find any links between this process that we're seeing between these mutations and processes that cause testicular cancer."

Genetic Counseling: A Real-time Application

"This is of great concern in genetic counseling where a child has an identified mutation and you test both the parents for it and neither has the mutation. Then they have another pregnancy and the second child turns out to have the selfsame mutation. One of the key findings from this research is that such a process is very unlikely to occur with AS in that the individual mutations never reach the levels in the testes whereby there would be any significant likelihood of a second child also being affected with another genetic disorder. The highest level of mutation causing AS that we'd ever measured in a sperm sample is one in 6,000."