Over the past several months we have made continued progress in evaluating various gene candidates which may play a role in AHC. I will highlight this progress in the order they were specifically presented in the initial grant proposal, and in the same format as that contained in the prior progress report, in order to better help the committee assess our progress toward the outlined goals. We are pleased with our recent progress, and think that the speed and efficiency of evaluating new gene candidates in our laboratory and in collaboration with others has greatly increased our excitement over the past few months.
As per our ongoing goals, work has continued throughout the project period to continue to increase and expand this database at the national and international levels, including a successful clinical meeting for families in Boston in which we were able to interview and examine a number of new and returning families. This meeting led to renewed excitement about our progress and strengthening of affiliations with our collaborators in several exciting new directions: 1) It solidified our interaction with our colleagues in Boston in the Gusella laboratory, who after meeting the families, returned to the lab with renewed enthusiasm and much more rapid progress in helping us to evaluate new gene candidates, although their primary focus remains to help us further investigate a family with a balanced chromosome translocation, who we continue to believe will be instrumental in uncovering a genetic cause for AHC. We have some preliminary data that there may be a “position effect” of this translocation rather than a direct disruption of a single gene. That data is preliminary and will require several additional months to perform the necessary investigations needed to prove this hypothesis. 2) It provided the first opportunity for our collaborator Dr. Joanna Jen (UCLA) to spend an extensive amount of time becoming more familiar with the clinical features of AHC patients and helping to strengthen our ongoing collaboration in the laboratory expanding evaluation of new gene candidates; she and Dr. Baloh have recently identified a new gene candidate, a glutamate transporter, which they have now implicated as causing the disorder in a single child in California affecting by a disorder, which although not identical to AHC, shares many features, including alternating hemiplegia and epilepsy. This gives us a new direction in evaluating a whole new group of gene candidates which could play a role in AHC. 3) We established a new and critical relationship with the parent of a recently diagnosed daughter with AHC, who has been instrumental in bringing to our attention a potential new treatment direction in AHC. This has allowed us to develop new relationships with a group of physicians in Toronto with whom we intend to collaborate in investigating such new treatment directions outside of the current grant proposal. As always, I am greatly encouraged and continually amazed by the tremendous energy demonstrated by the families to help support this effort, and provide us with the very important information requested to continue this work. Phenotypic information obtained for individuals and families with AHC clearly continue to help to guide choices about possible candidate genes, and help to define subgroups of children who have AHC with more or less severe symptoms, or some atypical features which may provide additional clues to help us better understand this disease. As I have previously noted, I believe that ongoing funding for maintenance of the database and funding of these interim meetings to update families and provide a critical point of interaction focused only on AHC remains critically important to the success of this project. We have reached a turning point in getting additional collaborators to participate, and the scientific strength of the symposia continues to improve.
Specific Aim 1. Genome wide linkage analysis and further characterization of resulting loci in selected kindreds with more than one affected individual with AHC:
As mentioned in our last update, we identified a possible locus on chromosome 1p which we are currently pursuing in more detail. This locus may be shared in 3 kindreds (families) with 2 or more affected children. Maximum lod score at this region is 2.74, which is likely to be significant. In addition to further narrowing this locus using selected markers in the region that are increasingly closer together, we have also begun to examine selected promising gene candidates from this region, including the gene SLC1A7, a glutamate transporter. We continue to pursue sequencing of additional gene candidates in this region.
Also as mentioned in our last update, glutamate transporters have been been of increased interest to us as candidate genes for AHC in the past year, due to the identification of mutations in a glutamate transporter in a child with features of AHC, although clearly also with additional features not entirely consistent with AHC. Glutamate transporters play an important role in protecting neurons against glutamate toxicity in the central nervous system. Glutamate toxicity is known to contribute greatly to neuronal death in individuals who have a stroke, for instance. We know that regulation of glutamate levels is critical to the health of many types of nerve cells in the brain and spinal cord. Selective blocking of glutamate receptors can help to reduce the size and severity of stroke in animal models. Up-regulation of the transporters in the cells supporting the neurons ( known as glial cells), may help protect motor neurons in degenerative diseases affecting those nerve cells, like amyotrophic lateral sclerosis, or ALS. In addition, selective blocking of these receptors can help to reduce the size and severity of stroke in animal models. This new interest in the potential role of glutamate in this disorders has contributed to a potential new direction in therapy which will be pursued in collaboration with our colleagues in Toronto, as touched on above.
Work is ongoing to further narrow the locus on chromosome 6p by picking markers that are increasingly closer together to help narrow the critical region of interest, and we have begun analysis of gene candidates in this area.
Specific Aim 2. Analysis of loci and genes which present appealing candidates based on shared phenotypic similarities with AHC in other human diseases or in animal models, or due to their known role in cell membrane excitability, ion transport.
This work continues, but with a new major breakthrough to report: we have identified a calcium channel mutation in one of our families with two affected children with AHC. Neither parent seems to carry the mutation, leading us to assume the mutation could have arisen in the germ cells, or eggs or sperm, without the parents being affected. This has renewed our feeling that calcium channels continue to be important to screen in this disorder, in spite of their large size, and the obstacles and expense it entails. Work is currently in progress to perform studies in additional unaffected siblings in this family, and to further determine whether this particular calcium channel mutation may play a role in symptoms in other children with AHC. We hope to begin to prepare this work for publication within the next few months, once the necessary additional studies are completed. Only time will tell the importance of this observation.
In addition, as touched on above, we have shared a plate of 96 samples from patients with the sporadic form of AHC, and are now working closely in tandem with the Jen/Baloh laboratory to evaluate glutamate transporters in AHC patients.
2a. Linkage, SNP and haplotype analysis at candidate AHC loci using multiplex AHC families: 2b. Selective high throughput screening of non-linked candidate genes in familial and sporadic patients:
As mentioned above, we are pursuing several new directions with regard to candidate loci, include the known ATPases and glutamate transporters, using our familial cases to help include or exclude candidates.
3. Selective cytogenetic analysis of sporadic cases, or more detailed analysis using techniques designed to identify the precise location of an abnormality in cases in which a specific cytogenetic abnormality has already been identified.
a) Cytogenetic analysis of selected sporadic patients in whom dysmorphic features might suggest an underlying cytogenetic disorder which could be an important clue to the molecular pathogenesis:
Based on our phenotyping data, we have identified a subset of patients with shared dysmorphic features and in some cases with more severe affection status or neurocutaneous abnormalities (unusual birthmarks or areas of patch skin hyperpigmentation). These patients have an increased probability of having a chromosomal deletion or rearrangement as the cause of their disorder. We have used high resolution karyotyping and fluorescent in-situ cytogenetic techniques to begin to investigate a subgroup of the patients. Tests performed in this screen include high resolution karyotype (to the 650 band level), and telomere FISH studies. We have performed such testing so far in half a dozen selected patients; of this group, we have identified a new cytogenetic abnormality in one patient. The significance of this abnormality is not yet known, but may be a valuable clue for finding disruption of one or more genes which play a causative role in AHC. We have continued our work in examining this patient via a novel approach using a gene chip which has recently become available to identify potential small deletions across the genome.
b) Further high resolution FISH mapping studies in AHC patients in which a specific cytogenetic abnormality has already been identified:
We continue our work in collaboration with Dr. Kim Hyung-Goo and his colleagues in the Gusella lab at Harvard. We have maintained monthly correspondence regarding progress, and have examined 4 additional gene candidates since our last progress. They have identified and narrowed the critical region on chromosome 3p, and have confirmed our prior localization of the exact breakpoint on 9q. They have shared with us the details of this region, and both we and now the Harvard laboratory have begun actively screening candidate genes in this region in a collaborative fashion. I have sent them a set of 50 sporadic AHC samples to help speed this screening process.
AHCF Progress Report Swoboda Neurogenetics Laboratory