CGS Research Project Descriptions
I. SEGREGATION ANALYSES, GENETIC LINKAGE STUDIES AND POSTIONAL CLONING OF GENES ASSOCIATED WITH INHERITED HUMAN DISEASES AND SUSCEPTABILITIES
1) The Genetics of Hereditary Craniofacial Dysmorphologies and Related Syndromes
This program began in 1992 as a collaborative project between the future founders and directors of the Cellular for Genomic Sciences (CGS), Drs. J. Christopher Post and Garth Ehrlich, and has been awarded NIH funding for 17 years to date. Published works from this project include manuscripts in Gene, Nature Genetics and Human Molecular Genetics as well as invited talks at national and international meetings. This project began with the mapping by Dr. Robert Preston and cloning of the gene (FGFR2) for Crouzon syndrome and Jackson-Weiss Syndrome (Gorry, et al). These observations turned out to be a watershed event in the field of craniofacial dysmorphologies and within months, five other craniofacial syndromes had been mapped to FGFR2 or other FGFR genes. Based on this synergy, CGS hosted a world-wide symposium in Pittsburgh to bring together the research community in craniosynostoses.
CGS research led to the elucidation of the entire genomic structure of the FGFR2 gene with DNA sequence available for all intron-exon boundaries and a comparison with other FGFRs in human and mice. This was accomplished using a combination of genomic library screening, long-PCR, and automated DNA sequencing. Ongoing studies are designed to further characterize the promoter and enhancer elements of the FGFR2 gene. Preliminary data indicates control elements as far as 7 kb 5′ of the transcription start site as well as tissue-specific positive and negative cis-acting regulatory elements.
Together with our collaborator, Dr. Michael Cunningham (University of Washington, Seattle), we have developed a chimeric xenotransplant small animal model (nude rat) that faithfully recapitulates the cardinal features of craniosynostosis. We are currently exploiting this model to study the downstream effects of the dominant gain of function mutations associated with the point mutations in FGFR2 by screening sutural expressomes from normal and induced synostotic coronal sutures.
We are also investigating whether FGFR2 mutant osteoblasts can be used therapeutically to promote bony growth in cases of nonunion. (ENT, Plastic Surgery, Orthopedics, Pathology, Human Genetics)
2) The Genetics and Biochemistry of Hereditary Pancreatitis (HP)
This program was begun by Dr. Ehrlich in 1995 with the concept of identifying the genetic cause of HP. We successfully assembled the kindreds, mapped the gene to chromosome 7q, cloned the cationic trypsinogen gene as the cause of the disease, and constructed a theoretical model to explain the disease symptoms in less than one year. This work was highly editorialized, and the NIDDK held press releases about the importance of these discoveries and reported it to the United States Congress in their annual review. This work has resulted in over a dozen publications (including three rapid publications in Nature Genetics and Gastroenterology) and presentations around the world. Based upon the discoveries resulting from this work, CGS hosted an international meeting on the genetics of pancreatitis which was attended by scientists across the North American Continent and from Asia and Europe. The President of West China University of the Medical Sciences visited CGS to learn more about our HP work. (Gastroenterology, Surgery)
3) The Genetics of Vesicoureteral Reflux (VUR)
This project was begun as a collaboration between the CGS directors and Dr. Francis Schneck (University of Pittsburgh) in 1995 and was directed by Dr. Robert Preston. In addition, CGS has collaborated with a group headed by Dr. Mike Eccles in New Zealand with whom Dr. Ehrlich had previously worked on the construction of physical maps of human chromosome 10. This project is designed to physically map major genes for VUR. At this juncture in spite of having assembled several large families and some 40 sib pairs, no single locus has been unambiguously identified after performing multiple gene scans suggesting that the genetics may be quite complex and heterogeneous. (Pediatric Urology, Human Genetics)
4) The Genetics of Diffuse Abnormal Insertional Activity
This project was initiated in 1994 by Dr. Ehrlich in collaboration with Drs. Giuliani and Hurtt at the University of Pittsburgh. Dr. Giuliani had realized that several members of a family had the same abnormal EMG results. Dr. Hurtt prepared a pedigree which revealed an AD pattern of inheritance in a three generation family. IRB approval was obtained and bloods were collected from all affected and unaffected family members for DNA preparations. A genome-wide scan using microsatellite markers was performed and genetic linkage analyses revealed a single locus which demonstrated co-segregation with the phenotype through the family. This project is ready for a major NIH grant application. (Neurology, Neuropathology)
5) The Genetics of Hereditary Severe Infantile Gastroesophageal Reflux (GER)
This is a major project undertaken by CGS beginning in 1998. Dr. Post initially realized through his clinical ENT practice (Allegheny Pediatric ENT Associates) that severe pediatric GER was familial. His observations were in concordance with those of the GER patient support group (Pediatric/Adolescent Gastroesophageal Reflux Association) and this synthesis of ideas resulted in the establishment of a collaborative project between CGS and PAGER Directors, Beth Anderson and Caroline McGraw. In addition, we have established collaborations with pediatric gastroenterologist, Dr. Thomas Self and Dr. Ramamurti Chandra. This disorder is quite possibly the most common autosomal dominant disease of mankind, affecting >2% of all children. The laboratory aspects of this project, directed by Dr. Robert Preston and Dr. Fen Hu, included the performance of a genome-wide scan on a large family which resulted in the establishment of linkage to chromosome 13q14. These results were subsequently confirmed with several additional GER families and we obtained a multipoint, multifamily LOD of greater than 7. This work has been published in JAMA and Human Molecular Genetics. We are currently in the midst of a large-scale positional cloning effort and have to date identified over novel 150 SNPs within the linkage region which are being further analyzed. An NIH R01 supporting this work was funded as of July 1, 2000. (Pediatric GI, Pediatric ENT)
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6) Genes that make Humans Human
Drs. Ehrlich and Post have had a longstanding interest in genes associated with language development and cognition as it is this aspect of our behavior as a species which is uniquely human. To this end CGS scientists have been working for the past several years with Dr. Thomas Campbell of Children’s Hospital of Pittsburgh (CHP) to identify suitable study populations for genetic mapping projects. Recently, we have also added Dr. Larry Schrieber to our collaborative group and the three groups plan to apply for joint NIH funding in the future. Drs. Campbell and Schrieber are recognized as two of the premier phenotypers for language and speech disorders in the world today and Dr. Campbell, as Director of the Audiology and Speech Communications Department at CHP, has thousands of children in his clinical practice, many of whom who are already in study protocols. Drs. Ehrlich, Post and Schreiber were all invitees of the NIH’s NIDCD in 1997 to be part of a blue-ribbon panel that was charged with assessing whether or not the technology and phenotyping had progressed to the point where it was realistic to begin using genetic mapping methodologies to identify candidate loci for behavioral traits associated with communication. We have chosen apraxia of speech as our first target in this area and are working with the national patient support group to obtain individuals for phenotyping and genetic testing. (Audiology and Speech Communications)
7) Genetic Susceptibility to Chronic or Recurrent Otitis Media with Effusion
This project was initiated over a decade ago as an epidemiological study by Dr. Margaretha Casselbrant at Children’s Hospital of Pittsburgh who was comparatively evaluating monozygotic and dizygotic twins for concordance in terms of ear infection occurrence rates and severity. Several years ago CGS joined her on an NIH-funded project to begin laying the groundwork for performing an affected sib-pair genome-wide linkage analysis study. The theoretical basis for this project came from the observation that the monozygotic twins showed a significantly higher degree of concordance than did the monozygotic twins indicating a strong genetic component the subject of a JAMA article (Casselbrant, et al) and editorial (Post and Ehrlich). Ascertainment biases was eliminated as the entire study was done in a blinded manner with the clinical investigators not knowing the zygosity of the twins. To date we have collected blood specimens and immortalized cell lines from over 80 sets of twins, in addition Dr. Post is identifying other families through his clinical practice. (Pediatric ENT)
8) The Genetics of Dupuytren Contracture
Dupuytren contracture is a disease of the fascia in the hand that does not usually onset until the fifth or sixth decade of life and is most prevalent among the Xanthocroix peoples of Northern Europe. This project was begun in 1995 in collaboration with our Swedish colleague, Dr. Ake Nystrom. Dr. Nystrom, currently at the University of Nebraska in Omaha, Dr. Ehrlich and Dr. Hu have completed several specimen collection trips to Sweden to access informative Scandinavian families that Dr. Hu and Dr. Preston successfully used to identify a locus on chromosome 16. (Plastic Surgery)
9) Fine Mapping of a Locus for Ectrodactyly
This project was performed in collaboration with Dr. Charlie Richard using a large family from Turkey. We were able to reduce the size of the locus from >20 cM to less than 2 cM. (Psychiatry).
10) Identification of Susceptibility Loci for Recurrent Respiratory Papillomatosis
Dr. Farrel Buchinsky heads CGS’s most recent foray into human genetics by combining two prime CGS emphases, chronic infectious pathogens and genetic susceptibility. In the current study a large cohort of affected children and adults and their parents will be assembled for the performance of a genome-wide transmission disequilibrium scan through the auspices of the RRP Task Force. Dr. Buchinsky received a K08 award to investigate these issues. The first phase, patient ascertainment, cohort assembly and HPV typing, requires several years. As of January 2007, 200 patients and their available parents have been enrolled making this the biggest DNA repository of unique RRP cases ever. Initially, several candidate genes are being explored by genotyping scores of SNPs in each with the Sequenom MASSArray iPLEX. The next step is to conduct genome-wide typing at sufficiently high resolution to detect association by the transmission disequilibrium test; we shall use the Illumina BeadStation with a 500K chip.
II. GENE IDENTIFICATION STUDIES USING COMPARATIVE EXPRESSOME and COMPARATIVE GENOME TECHNOLOGIES
11) The Molecular Biology of a Fetal Model of Scarless Wound Healing
This program was begun in 1994 as a collaboration between Dr. Ehrlich and Dr. Joseph Dohar at Children’s Hospital of Pittsburgh to identify genes associated with scarless wound healing in fetal mammals with the long-term goal of being able to affect the wound healing process in adults to minimize or eliminate scar formation. Dr. Kathju has recently taken over direction of this project at CGS. He has been employing SS-RT-PCR and gene array technology to compliment earlier studies using a heavily controlled differential display strategy for the identification of genes expressed in fetal wound healing that are not expressed in adult wounds or normal fetal or adult tissues. To date we have identified over 100 candidate genes of which 50% are completely new. This work has been presented at multiple international meetings and has been published in Pediatric Surgery and Archives of Otolaryngology – Head and Neck Surgery. This project was one of the driving forces behind the setting up of a robotic array facility for comparative expressomics. Briefly, this technology provides a means to compare the levels of expressions for all genes transcribed in a given tissue under different environmental conditions. Dr. Kathju received a K08 grant from the NIH to further these studies. There is also the potential for pharmaceutical interest in this project as the financial outlook for biologicals that could reduce scarification have enormous commercial potential. (Plastic Surgery, Dermatology, Pediatric ENT)
12) Developmental Expressomics of Host and Pathogen during the Development of Chronic Supportive Otitis Media (Otorrhea)
Drs. Ehrlich, Post, Erdos, Antalis, Sayeed and Hu are studying the molecular biology underlying the shift in bacterial growth from a planktonic mode to the biofilm mode. This metabolic shift has been most fully characterized in vitro in Pseudomonas aeruginosa. Thus, we’ve chosen to study otorrhea (a P. aeruginosa-associated disease) as a model system to establish the methodologies to study the global changes in gene expression on a gene-by-gene basis in an in vivo situation. We have obtained specimens from a large number of closely spaced time points of both the bacteria and the host mucosa that will serve as probes to interrogate CGS constructed gene arrays to identify changes in both host and pathogen gene expression patterns. mRNA pools from all time points have been used in the generation of species-specific cDNA libraries that will be picked, arrayed, gridded and normalized for use in comprehensive hybridization screens. The individual time points RNAs will then be used to probe the libraries to discern which genes are differentially regulated at various time points. A data display system will be developed to visualize the transcriptional activity of the bacterial genome. (Pediatric ENT, Human Genetics)
13) Downstream Affects of Mutant FGFR2 Signaling
Dr. Kai Shen working with Drs. Ehrlich and Post are studying the downstream gene expression effects of FGFR2 constitutive signaling associated with the dominant gain-of-function mutations that we have identified in patients with craniofacial synostotic syndromes. This is being accomplished in collaboration with Dr. Michael Cunningham of the University of Washington, with whom we have developed a xenotransplant model in the nude rat. In this model, human osteoblast cell lines are placed below the developing calvarial sutures of weanling (6-8 week) rats. Mutant osteoblasts from patients with Crouzon and Apert syndrome induce premature suture closure, whereas osteoblasts from normal patients do not. Using global gene expression monitoring technology, we are identifying the genes that are involved in craniosynostosis. This comparative expressomics aspect of the craniofacial project forms the bulk of the proposal for Dr. Post’s NIDCD continuation grant. (Pediatrics, Maxillo-Facial Surgery, Human Genetics)
14) Host Mucosa Comparative Expressome Analysis in OME
This is a subproject of the Otitis Media program in which we will be using the assembled tools for comparative mRNA studies to look at the differences in mRNA expression patterns of host mucosa in the presence and absence of chronic and acute bacterial infections. (Human Genetics, Pathology)
15) Genomic Plasticity Among and Within Populations of Bacterial Pathogens
This project was initiated by Dr. Ehrlich in 1999 to investigate the degree of difference among various strains of various chronic bacterial pathogens. In 2000 Dr. Erdos was hired to run this program. To attack this problem, massive pooled genomic libraries of H. influenzae and P. aeruginosa were constructed, each from 10-12 low-passage clinical isolates. These libraries were arrayed and subjected to DNA sequencing and cross hybridizations. The data from these studies indicates that the average strain differs from each other strain by 10-15% and that the entire genome space for a bacterial species may be several times the genome of any single bacterium. This data has provided the engine behind the concepts of the supragenome, supravirulence factors, the Distributed Genome Hypothesis and the edifice of ‘Bacterial Plurality’.
16) The Difference in Gene Expression Changes between Responders and Non-Responders of Anti-MS Therapy
Dr. Thomas Scott (Neurology) and Drs. Ehrlich and Hu of CGS are investigating the differences in the changes in gene expression between responders and nonresponders of anti-multiple sclerosis therapy in a Biogen-supported clinical research project. PBLs are collected pre- and post-treatment from persons being put on standard therapy and then interrogated to identify changes and differences in gene expression patterns.
17) Development of Prognostic Markers for Identifying Colon Cancers with Metastatic Potential
Drs. Mark Roh (Surgery) and Ehrlich are using paired primary and liver mets from patients with colon cancer and comparing the gene expression profiles from these tumors from those that do not metastasize to identify prognostic markers for cancer metastasis.
III. INFECTIOUS DISEASE STUDIES
18) Otitis Media with Effusion as a Bacterial Biofilm Disease
This project had its root in 1990 when the CGS directors first met and has been continuously NIH funded by the NIDCD since 1993. The application of molecular diagnostics to the issue of chronic, but culturally-sterile middle-ear effusions, resulted in a major paradigm shift that Dr. Ehrlich developed that states that chronic bacterial infections, which are physiologically but not genetically, antibiotic resistant, result from a metabolic change from a planktonic form to a biofilm form. It is postulated that this changeover occurs through the coordinate induction and repression of different sets of contingency genes in a manner analogous to sporulation or metamorphosis. It is expected that the comparative gene expression studies detailed above will provide novel targets for the development of entirely new classes of antimicrobial compounds. Beginning in 1996, Dr. Bill Costerton, Director of the Center for Biofilm Engineering, and his team joined the project and have provided the imaging and proteomic aspects of this work. The work performed on this project to date has resulted in over three dozen publications and presentations including three articles in JAMA and dozens of national and international talks. In addition, the research completed to date has twice been highlighted by the NIDCD and has received direct comment from both the past and current NIDCD Directors in their summaries. This work was also chosen for presentation to NIDCD Council.
19) Association of Common Respiratory Pathogens with Sudden Infant Death Syndrome (SIDS)
This project was begun at the request of program personnel within the NIDCD and NIMCH and was funded by an NO1 contract and a Personal Services Contract to Dr. Ehrlich. In this study Drs. Post and Ehrlich worked with the Medical Examiner’s office for the State of Maryland, and the University of Maryland Brain and Tissue Bank run by Dr. Ron Zielke. These latter two institutions perform autopsies and collect specimens from all SIDS cases, respectively, within the State of Maryland. Dr. Zielke’s lab then shipped the specimens to CGS from SIDS deaths and other causes in a blinded manner for infectious pathogen analyses via multiplex PCR assays that were developed as part of the CGS effort to study otitis media. A database with over 8000 data points was supplied to NIH personnel for unblinding analyses indicated a significant association between SIDS and enteroviral infection.
20) Host-Pathogen Interactions During HIV-1 Infection
Drs. Ehrlich and Shankarappa have studied the mechanisms by which HIV-1 persists and evades the immune system in the face of a strong antibody response. The collaborative group has included: Drs. Peter Nara and Robert Garrity of the National Cancer Institute; Dr. Jerry Zack of UCLA; Drs. Rinaldo and Gupta at Pitt; and Dr. Mullins at Washington University. Three major approaches have been utilized. The first method involves characterizing the effect on the T-cell repertoire of HIV-1 infection in humans, chimpanzee, and SCID-HU mice using TCR-specific gene amplification systems (this approach established Koch’s postulates for HIV-1 and AIDS for the first time in a controlled laboratory environment). The second method involves performing longitudinal DNA sequencing of viral isolates from individuals with different rates of disease progression and looking for the appearance of sequence or structure motifs associated with disease progression. The third project involves selection of HIV-1 mutants that are neutralization resistant to monoclonal antibodies that could neutralize parental strains. This is then followed by cassette mutagenesis and site-directed mutagenesis, sequentially, to identify regions and bases at distant sites that changed coordinately with proximal mutations. The idea behind these experiments is to develop a set of rules which indicate that if the virus mutates at one site which other sites must also be mutated to maintain function. We argue that if a relatively complete set of rules could be developed, it should be possible to attack the virus with a cocktail of rationally designed aptamers or antibodies that target collectively the several sites which must change together to maintain function thereby boxing the virus in using what we know about how it evolves in response to the host immune response. These projects are extremely technically difficult, but have generated six significant scientific papers to date in J. Virology, Virology and AIDS and Human Retroviruses. These projects also have led to the publication of numerous technique papers over the past several years as much of the methodology to perform these studies had to be developed within the lab. One of the methodologies developed – the placement of restriction enzyme sites within coding regions using silent mutagenesis – has turned out to be very broadly applicable and we developed a shareware computer program to aid scientists in the performance of these studies which is available at the CGS website. The tabular forms of this work have been widely distributed by many of the molecular biological reagent vendors through inclusion in their catalog reference sections. This work was funded for years at the University of Pittsburgh through the NIH’s Multicenter AIDS Cohort Study (MACS) and an RO1 to Drs. Gupta and Ehrlich.
IV BIO-ENGINEERING PROJECTS
21) Development of Intelligent Implants to Control Biofilm Infections
Dr. J. William Costerton (Center for Biofilm Engineering, MSU), Dr. Qiao Lin (Carnegie Mellon University), and Drs. Ehrlich, Kathju, Sotereanos, and Erdos (CGS) head a large multi-institutional multidisciplinary program to develop and test auto-diagnostic/auto-therapeutic implantable joints as a means to combat the scourge of biofilms which can, if established on joint prostheses, result in severe morbidity and even limb amputation.