Apert syndrome is a rare congenital disorder characterized by malformation of the skull, face, hands and feet. The cranial malformations, termed craniosynostosis, are a particular hallmark of the disorder and result in part from the fibrous joints of the skull (sutures) fusing prematurely during development. The underlying genetic cause of this autosomal dominant disorder was identified as mutations in fibroblast growth factor receptor 2 (FGFR2). Numerous studies in model organisms have therefore focused on the effect of these mutations on the function of cells in the vicinity of sutures. Joan Richtsmeier from Pennsylvania State University, USA, and colleagues took a new approach to understanding this condition in their recent study in BMC Developmental Biology, where they examined morphological changes in the overall 3D landscape of the cranium in two mouse models for Apert syndrome. Richtsmeier explains more about what their results revealed and the implications for human craniofacial diseases.
What was the main goal when you started this research?
I have been studying craniosynostosis since my PhD thesis. The main goal at that time was to try to understand how postnatal growth of the skull in children with Apert and Crouzon syndrome differed from patterns of typically developing children. I was able to demonstrate that postnatal cranial growth was different from typical growth in these syndromes, using what were at the time cutting-edge quantitative tools and longitudinal data (head x-rays). But it was clear that the shapes of the heads of these children were not typical at birth, and so it was critical to obtain prenatal data in order to understand how growth patterns contributed to the morphology that is obvious in newborns with these conditions. This cannot be done in humans, but it can be done in mice.
Also, sutures, or the ‘seams’ between bones of the skull, have been thought of as ‘growth centers’ for a long time. However, it was our idea that the suture might not be driving everything and that growth occurs on all surfaces of bones of the skull, and so we wanted to characterize that growth. It turns out that we were right, head shape is different before sutures close in animals carrying the mutations and change in form occurring on all surfaces of all bones contribute to the growth pattern in both typical and abnormal growth. We did find that differences in suture patency (whether the suture remains open or closed and for how long) also contribute significantly to the growth pattern, especially in the face. So if patterns of growth of all skull bones are different and suture closure patterns (timing and order of closure) are different in mice carrying these mutations, then we need to think more generally when developing therapies.
What are the benefits of studying these mutations in a mouse model?
The obvious benefit is that we can study biological processes and measure the resulting phenotypes at any point during prenatal development. Here we studied the later stages of prenatal development, but colleagues have shown amazing things by looking at earlier stages of development and, of course, what happens later is in many ways dependent upon what happens earlier in development. For the very specific event of premature suture closure, this occurs prenatally. It would be difficult, actually impossible, to observe and score the timing of suture closure (normal or abnormal) in humans and yet, with these mice, we can do this at the anatomical level by visualizing the sutures using micro computed tomography (3D x-rays) or at the mechanistic level by using immunohistochemistry or other approaches to see what the cells are doing as the sutures close.
Your study examined two different FGFR2 mutations known to cause Apert syndrome in humans. What are the main differences between these two mutations?
These two mutations are on the same gene occupying neighboring amino acids. In humans, these two mutations are responsible for approximately 99 percent of all cases of Apert syndrome. Once these mutations were discovered and the causative mutation in people with Apert syndrome could be precisely identified, clinicians began to accumulate data in an attempt to understand which disease traits were more common or more pronounced in individuals carrying one mutation or the other. From this work, it was concluded that individuals carrying the FGFR2 S252W mutation showed relatively more severe anomalies of the head and a higher incidence of cleft palate, while individuals who carried the FGFR2 P253R mutation showed relatively more profound limb anomalies.
In a previous study, we were able to demonstrate that features of the head were more strongly affected in mice carrying the mouse version of the Fgfr2 S252W mutation relative to those carrying the Fgfr2 P253R mutation. In our study published in BMC Developmental Biology, we were interested in further defining these differences and demonstrating how the two mutations affect cranial growth differently. We propose that this is due to differential primary effects of the mutations and differences that have to be made in the ‘adjustment’ among affected cranial tissues during growth.
How do you control for variation between individuals with your measurements?
As an evolutionary biologist, variation is the property in which I’m most interested, but it is very, very hard to study correctly, especially using three-dimensional coordinates of biological points on our specimens as primary data. In order to measure variation of biological objects in three dimensions, typically investigators pick a reference object (for example, a specific individual or the sample mean) and rotate and translate and scale data from all individuals so that they are superimposed onto the same coordinate system. Mathematically, the coordinate data representing the form of all individuals in the sample are overlaid one on top of the other and then stretched and shrunk along coordinate axes until the ‘best fit’ among them is attained. This is the most common way that biologists deal with this type of analysis but there is a problem in that the ‘best fit’ (the precise mathematical rule by which measures are minimized when the stretching and shrinking is done) is arbitrarily chosen and is not informed by the available data. We take an alternate approach: we start with three dimensional coordinate data and then estimate all unique linear distances among the original three dimensional coordinates of biological landmarks. Using those data to analyze our forms, we skip the step of superimposition, which in the case of a Generalized Procrustes fit tends to spread variation evenly across the objects of study. This is the reason why we are able to precisely locate the differences in form and differences in growth among the organisms we study. We have developed statistical tests that use confidence intervals to test for differences in form and in growth, so that we can provide information about the localized variation within samples.
How closely do the results in mice mimic what is seen in humans?
Pretty darn well for organisms whose last common ancestor existed 65 million years ago! We lean on evolutionary theory when we use mice to study human disease. We know that the appearance and individual features of mice are very different from humans, but we also know that many genes are conserved across mammals and the proximate functions of most of these genes are likely to be conserved as well. Evolutionary developmental biology has shown us that conservation of the patterns of development of complex structures (limbs, brains, hearts) implies that the genetic programs that specify structural design might also be conserved. This is why we can ‘recreate’ a complex genetic insult known in humans to cause a specific outcome and see similar outcomes in mice.
What are the implications for human health based on this work?
I think the implications are really important in terms of managing these and other craniofacial diseases that are caused by genetic mutations. Currently, the only option for people with Apert syndrome is rather significant reconstructive surgery, sometimes successive planned surgeries that occur throughout infancy and childhood, and into adulthood. These surgeries are necessary to restore function to some cranial structures and to provide a more typical morphology for some of the cranial features. But, if what we found in mice is analogous to the processes at work in humans with Apert syndrome, then we need to decide whether or not a surgical approach that we know is necessary, is also sufficient. If it is not, in at least in some cases, then we need to be working towards therapies that can replace or further improve surgical outcome. However, this is a very, very difficult problem because we have found, at least in mice, that these Fgfr2 mutations also change the size and shape of non-skeletal structures of the head. So the mutation initiates processes that are persistent, continually affecting shapes and shape changes during growth, in both skeletal and non-skeletal tissues.
Where will your research go from here?
I have great collaborators and a great group of really smart people in my lab, so there is no telling where we will go. I have always been interested in the relationship between development and evolution and these mouse models provide an ideal ‘laboratory’ for the formulation of hypotheses having to do with normal variation and variation that occurs at the ends of the normal distribution. That ‘normal distribution’ of organismal form changes as evolution proceeds, but many of the processes that underlie the production of structures remain. These processes just get ‘tweaked’ to happen more often during development, or at a faster pace, or in a different tissue, or to slow down or to only occur in certain environments. Being able to study what these mutations do across many cell types and tissues at different times during development, and how these various changes combine to produce changes in the overall appearance of the organism provides a wonderful place in which to formulate great questions. In terms of this particular disease, I would love to contribute to finding one of the key mechanisms that that would lead to improving the lives of people with craniosynostosis and other craniofacial disorders.