 Research
CGS Research Project Descriptions
I. SEGREGATION ANALYSES, GENETIC LINKAGE STUDIES AND POSTIONAL
CLONING OF GENES ASSOCIATED WITH INHERITED HUMAN DISEASES AND
SUSCEPTABILITIES (CGS projects 1-10)
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 Center for Genomic Sciences (CGS), Drs. J. Christopher Post and Garth Ehrlich, and has been awarded NIH
funding for 11 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 with Dr. Joseph Donfack 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 (CGS projects 11-18)
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 (CGS projects 19-22)
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.
V BIOFILM RESEARCH
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