Author Archives: ppretorius

Yan and John to present at Student Seminar on Thursday (17.March) at11:45 in 2-501

Yan and John  will be presenting Student Seminar this week in 2-501 BSB at 11:45. I have had both speakers provide background on their talk.  Please note the earlier start time, this is to allow time for Tinaa Tootle and Sarit Smolikove to solicit feedback on the Genetics web site.   Hope to see you there, this is a Student Seminar not to miss!

Background for Yan’s talk

Functional Analysis of Cep290

Purpose: CEP290 is a large, multi-domain protein implicated in several cilia related syndromic disorders including Meckel-Gruber, Joubert, Senor-Loken and Bardet-Biedl Syndrome (BBS). Moreover, CEP290 is the most frequently mutated gene underlying the non-syndromic blinding disorder, Leber’s congenital amaurosis (LCA). The Purpose of the current study is to characterize the function of various CEP290 domains and to characterize a zebrafish model aimed at progressing towards future therapy for patients with CEP290 LCA.

Methods:  We generated several truncated CEP290 protein fragments, which comprise several domains of the whole protein. We tagged these fragments with GFP and examined their localization in 293T and RPE cells. We also evaluated the physical interaction between these truncated proteins and other cilia proteins by co-immunoprecipitation. To determine the in vivo function of cep290 in the zebrafish, we examined the effects of cep290 knockdown using an antisense oligonucleotide (Morpholino, MO) designed to generate an altered cep290 splice product that models a common LCA mutation.

Results: The N-terminal and C-terminal fragments of CEP290 both localize to the centrosome. The N-terminus  interacts with NPHP2 and NPHP5, whereas the C-terminus interacts with MKS1. Histological analysis of the retina of cep290 knockdown zebrafish revealed no gross morphological defects; however, knockdown of cep290 results in a statistically significant reduction in visual function. We tested whether truncated CEP290 proteins could rescue the cep290 MO knockdown phenotype in zebrafish by co-injection of RNA with the cep290 MO. We demonstrate that vision impairment caused by disruption of cep290 can be rescued by expressing the N-terminus protein fragment.

Background for John’s talk

Genetic Fine Mapping of Metabolic Syndrome in Lyon Hypertensive Rats

Lyon Hypertensive rats (LH) is an established animal model for metabolic syndrome. Previous research has demonstrated that the chromosome 17 of LH harbours QTLs for several metabolic syndrome phenotypes, including hypertension and dyslipidemia. In order to more accurately select candidate genes for the LH phenotype, we are using SNP discovery to perform fine mapping of the established major haplotypic region on chromosome 17. In this presentation I will discuss the current progress of this investigation.

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Leah to present at Student Seminar on Thursday (17.Feb) at12:00 in 2-501

Leah will be presenting Student Seminar this week in 2-501 BSB at 12:00. I have Leah to provide a brief overview for her talk. Hope to see you there!

Background for Leah’s talk

The role of Irf6 in keratinocyte migration

Mutations in Interferon Regulatory Factor (IRF6) cause two orofacial clefting syndromes, Van der Woude (VWS) and Popliteal Pterygium (PPS).  In addition to an oral defect, recent data show that VWS patients are more likely to have wound complications after corrective cleft surgery than patients with isolated cleft.  Thus we hypothesize that Irf6 is critical to wound closure.  To test this, we used keratinocytes from Irf6 null mice.  We created in vitro scratches and observed a significant delay in scratch closure.  To determine the cause of delay, we examined the cellular structure of Irf6-/- keratinocytes by staining for filamentous actin and found that these cells have more stress fibers.  In addition, we examined cellular adhesion by staining Irf6-/- keratinocytes for E-cadherin and vinculin. We found that these cells form junctions, but cell-cell contacts appear in an abnormal pattern, and there appear to be more focal adhesions.  Finally, we analyzed proliferation of Irf6-/- keratinocytes and found no deficiency.  Together, our data suggests Irf6-/- keratinocytes exhibit characteristics of less motile cells.  Thus, we speculate that the defect in Irf6-/- keratinocyte scratch closure is due to aberrant migration and/or adhesion, and that cellular proliferation is not inhibitory to closure.  Broadly, these data suggest that Irf6 is necessary for events critical to proper cutaneous wound healing.

Thesis Seminar: Kacie Meyer

Ph.D. Thesis Seminar: Kacie Meyer

Adviser: Tom Wassink

Date:  Wednesday, 20.January 2011

Time: 2pm

Place: 2117 MERF

Amber and Alissa to present at Student Seminar on Thursday (16.December) at 12:30 in 2-501 BSB

Amber and Alissa will be presenting Student Seminar this week in 2-501 BSB at 12:30. I have asked the speakers to provide a brief overview for their talks. Hope to see you there!

Background for Amber’s talk

Understanding the Role of Topoisomerase 2 in Chromosome Interactions

Homologous chromosomes display associations in many organisms.  Many of these interactions lead to changes in gene expression, as exemplified by processes of transvection, mammalian X-inactivation, and imprinting.  Drosophila serves as an excellent model to study pairing interactions because homologous chromosomes are fully aligned in all cells.  One gene required for these homolog interactions is Topoisomerase II (Top2; Williams et al. 2007).  Top2 encodes an ATP dependent homodimeric enzyme that generates double stranded breaks (DSBs) to change DNA topology.

The catalytic cycle of Top2 depends on a number of functional domains.  The ATPase domain is a member of the GHKL superfamily (Gyrase, Hsp90, CheA-type histidine Kinase, and MutL) that is common to all type II topoisomerases [Schoeffler and Berger 2008].  This domain forms a clamp structure that captures a DNA segment (often referred to as the G segment).  Adjacent to the ATPase domain is a transducer domain that signals to the ATPase domain that the G segment is bound and cut.  Top2 cleavage requires an acidic triad of amino acids within the TOPRIM domain that binds Mg+2 and interacts with the active site tyrosine of the winged helix domain (WHD) of the partner subunit.  DNA cleavage occurs when the active site tyrosine covalently attaches to the 5’ end.  As such, DNA cleavage by Top2 does not generate unprotected DNA ends, preventing activation of a DNA checkpoint.  DNA binding is aided by the tower domain that lies next to the WHD.  The C-terminus continues in a coiled-coil domain important for DNA segment release (known as T segment release).

Top2 is important in many biological processes, including DNA replication, recombination, chromosome condensation, transcription, and the segregation of sister chromatids in mitosis and meiosis [reviewed in Nittiss 2009].  Top2 is a target for many chemotherapeutic drugs because enzyme-mediated DNA damage causes cells to undergo apoptosis.  To date, published Top2 studies in Drosophila have used RNAi or chemical inhibitors to understand the function of the enzyme in vivo. To expand our understanding of the role of Top2 in chromosome pairing, we conducted a genetic screen to isolate germ line mutations in Top2.  Fourteen recessive lethal alleles were identified from a screen of three thousand chromosomes.  Molecular analyses of these alleles uncovered single or multiple base pair substitutions within each mutant line.  These included changes that generated premature stop codons or missense mutations within previously identified functional domains.  Even though flies carrying each missense allele in trans to a deficiency produce inviability, hetero-allelic combinations of several missense alleles produce live flies.  Current studies are directed at using the viable hetero-allelic combinations to examine chromosome structure and function during development.  These data suggest that the collection of Top2 mutations will provide a resource for understanding chromosome functions that require homolog interactions.

Background for Alissa’s talk

A NOVEL ROLE FOR A CDC42 EFFECTOR PROTEIN IN XENOPUS DEVELOPMENT AND NEUROGENESIS

Many cellular functions, such as axis determination and cell movement, are dependent on the asymmetrical distribution of proteins and RNAs.  In Xenopus laevis, several maternal mRNAs essential for normal development are localized to the oocyte vegetal cortex, and our lab has used microarrays to identify numerous additional localized RNAs.  In this work we characterize one of these cortex-enriched transcripts, cep4l.  CEP4L belongs to a family of Cdc42 effector proteins (CEPs) that bind Cdc42 and related small GTPases, which can regulate the actin cytoskeleton.  Using in situ hybridization we found that cep4l is expressed in the oocyte vegetal cortex and throughout embryonic development.  The embryonic expression pattern includes migratory cell populations during gastrulation and neurulation, and neural regions in older embryos.  To begin to ascertain the function of cep4l in development we misexpressed cep4l RNA in embryos.  We found that this overexpression causes convergent extension defects and induces ectopic neuronal marker expression, indicating a role in neurogenesis. Structure-function analysis using deletion mutant constructs show a role for the CRIB domain, a conserved GTPase binding domain.  Experiments to identify upstream and downstream pathways indicate a potential role for FGF.  Co-expression studies with FGF8a, which also induces ectopic neurons, demonstrate an enhancement of the neurogenic phenotype. We also present loss of function data showing a role for cep4l in normal axial and nervous system development, as well as a requirement for FGF8a-induced neurons.  Although the roles of small GTPases in cell migration and adhesion are well-characterized, our results suggest novel roles for these proteins and their effectors in neurogenesis and early development.

Thesis Seminar: Pamela Pretorius

Ph.D. Thesis Seminar:  Pamela Pretorius

Advisers:  Diane Slusarski and Val Sheffield

Time:  1:00pm

Place:  Braley Auditorium (Pomerantz Family Pavilion)

 

 

 

Elucidating a role for BBS3 in syndromic and non-syndromic retinal disease

Bardet-Biedl Syndrome (BBS, OMIM) is a syndromic form of retinitis pigmentosa, characterized by retinal degeneration, obesity, learning disabilities, congenital abnormalities and increased incidences of hypertension and diabetes. Individuals with BBS are blind by the third decade of life.  At least fourteen genes are reported to individual cause BBS.  This thesis focuses on BBS3, with the overall goal of characterizing the function of BBS3 in terms of both syndromic and non-syndromic retinal degeneration.

A member of the Ras family of small GTP-binding proteins, BBS3 is postulated to play a role in vesicular transport.  A second highly conserved transcript of BBS3, BBS3L, is expressed in the mouse and zebrafish eye.   Histological analysis of Bbs3L knockout mice at 9 months reveals disorganization of the inner segments, indicative of retinal degeneration. To further evaluate the functional effects of BBS3 deficiency in the eye, an antisense oligonucleotide (Morpholino) approach was utilized to knockdown bbs3 gene expression in zebrafish. Consistent with an eye specific role, knockdown of bbs3L results in mislocalization of the photopigment green cone opsin and reduced visual function, but not abnormalities of the Kupffer’s vesicle or delays in intracellular trafficking of melanosomes, both cardinal features of BBS in the zebrafish. To dissect the individual functions of BBS3 and BBS3L, BBS3 or BBS3L RNA was co-injected with the bbs3 morpholinos.  BBS3L RNA, but not BBS3 RNA, restores green opsin localization and vision.  Moreover, only BBS3 RNA is sufficient to rescue melanosome transport.  Together these data demonstrate that BBS3L is necessary and sufficient for retinal function and organization.

This work was extended to humans by characterizing the A89V missense mutation in BBS3 identified in retinitis pigmentosa patients.  To evaluate the in vivo function of the A89V missense mutation in non-syndromic retinal degeneration and BBS, rescue experiments were performed in the zebrafish.  Unlike BBS3L RNA, BBS3L A89V RNA does not rescue the vision defect seen with loss of bbs3 in zebrafish; however, BBS3 A89V RNA suppresses melanosome transport delays.  These data demonstrate that the mutation identified in patients with non-syndromic retinal degeneration is critical and specific for the vision defect.

Ji and Erin to present at Student Seminar on Thursday (18.November at 12:30 in 2-501 BSB

Ji and Erin will be presenting Student Seminar this week in 2-501 BSB at 12:30. I have asked the speakers to provide a brief overview for their talks. Hope to see you there!

Background for Ji’s talk

Transcriptome-wide identification of alternative polyadenylation events regulated by epithelial splicing factor ESRP1

The translation of messenger RNA (mRNA) is strongly influenced by regulatory elements in the 3’ untranslated region (3’ UTR). Different isoforms of 3’ UTR can be generated due to multiple polyadenylation (polyA) sites within a single mRNA. The differential usage of alternative polyA sites in different biological or physiological conditions is called APA switch. In this study, we aim to investigate the regulation of APA switch events by an epithelial-cell-specific RNA binding protein — Epithelial Splicing Regulatory Protein 1 (ESRP1) (OMIM). ESRP1 can regulate diverse types of alternative splicing events and control a genome-wide alternative splicing network during the epithelial-to-mesenchymal transition (EMT). However, whether ESRP1 can regulate APA switch events has not been investigated before.

To study the effects of ESRP1 on alternative splicing and APA switch events, we ectopically expressed a cDNA for mouse ESRP1 in the MDA-MB-231 mesenchymal human breast cancer cell line by viral transduction. Using high-throughput mRNA sequencing (RNA-Seq), we generated a total of 256 million 76bp RNA-Seq reads on cells with ESRP1 overexpression or empty vector control. In order to identify APA switch events upon ESRP1 overexpression, we developed a computational pipeline to quantify the usage of individual polyA sites using RNA-Seq data. Using this pipeline, we identified hundreds of genes with APA switch events after ESRP1 overexpression. We also found consistent APA switch patterns by comparing RNA-Seq data to Affymetrix Human Exon Array ST 1.0 data for the same samples. These results show that ESRP1 regulates polyadenylation of specific mRNAs and could contribute to the generation of 3’ UTR isoforms during EMT.

Background for Erin’s talk

A Hyperactive piggyBac DNA Transposon Achieves Persistent Gene Transfer

Nonviral vector systems are used increasingly in gene targeting technologies and as tools for gene transfer applications.  The piggyBac (PB) transposon represents an alternative integrating vector for in vivo gene transfer.  To date, there are no reports of gene transfer to mammals using the hyperactive piggyBac (PB7) transposon system.  We hypothesized that this system could achieve persistent gene transfer to the liver when administered systemically.  Here we report that the PB7 transposase generated higher transposition efficiency than a codon-optimized transposase in human liver cells.  Following hydrodynamic tail-vein injection, we show that firefly luciferase expression driven by a liver-specific promoter persisted up to eight months in mice even following partial hepatectomy.  This finding is consistent with expression from an integrated transgene.  We extended these studies and delivered the human α1 anti-trypsin (hAAT) gene to murine liver along with PB7 and achieved stable hAAT expression in serum.  To determine the integration pattern, we mapped 853 integration sites in human cells.  We found an altered pattern of integration than previously reported for insect PB.  Our results support the PB transposon combined with the hyperactive PB7 transposase is an efficient integrating vector system for in vitro and in vivo applications.

Lily and Danielle to present at Student Seminar on Thursday (23.September at 12:30 in 2-501 BSB

Lily and Danielle will be presenting Student Seminar this week in 2-501 BSB at 12:30 (please note the room change). I have asked the speakers to provide a brief overview for their talks. Hope to see you there!

Background for Lily’s talk

EVALUATING THE ROLE OF PRICKLE MUTATIONS IN HUMAN PROGRESSIVE MYOCLONIC EPILEPSY

Epilepsy is a complex genetic brain disorder that is characterized by seizures and afflicts 50 million people worldwide. Over 35 dominant genes in consanguineous families cause epilepsy but the role of autosomal recessive genes remains largely unexplored. Progressive Myoclonic Epilepsy (PME) is a subset of epilepsy characterized by neurodegeneration, myoclonus, and generalized seizures. A number of genes identified in consanguineous families such as CSTB, MALIN & LAFORIN are responsible for 80% of PME cases, but the remaining causes of the 20% remain unknown. Bassuk et. al identified mutations in Prickle1 and Prickle2 responsible for PME in the general population. Prickles are expressed in the developing nervous system and mature neurons, involved Planar Cell Polarity and are players in Wnt-mediated signaling; a cascade likely critical in neurodevelopment and neurodegeneration. The involvement of Malin, Laforin, and Prickles in Wnt signaling suggests that Wnt could be an anti-epileptic target. Co-Immunoprecipitation assays carried out to verify interactions between wild-type Prickle1 and members of the Wnt pathway (Disheveled, Smurf, Rest, etc) confirmed some of these Interactions. Future experiments include characterizing the effect of Prickle mutations on protein-protein interactions and neuronal differentiation. Findings from these studies would clarify molecular mechanisms by which Prickle mutations alter neuronal function and help identify novel therapeutic targets.

Background for Danielle’s talk

Identifying CNVs from SNP Arrays that Influence Schizophrenia Susceptibility

Schizophrenia (OMIM) is a serious mental disorder that strikes in early adulthood and has heritability estimates up to 80%.  It is a life-long disability without a cure and many attempts have been made to identify genetic components, but the complexity of the disease confounds these efforts.  I will identify genome-wide copy-number variants (CNVs) in a sample set and determine whether such CNVs are characterized by a common phenotypic profile.  I will run DNA on Affymetrix Genome-Wide Human SNP Array 6.0 microarrays which provide signal intensity data that can be analyzed with Affymetrix Genotyping Console 3.0 to provide copy number data.  To date we have analyzed 124 probands and 61 controls.  CNVs discussed in this abstract were confirmed by qPCR and were not found to be present in controls.  In four subjects we identified a deletion that encompasses the entire gene  (OMIM) which is located at 15q11.2.  CYFIP1 functions in synaptic plasticity, brain development, regulation of axonal and dendritic outgrowth and development and maintenance of neuronal structure.  It also interacts with fragile-X mental retardation protein (FMRP).  Of the four subjects we identified this deletion in, three are patients and one is the mother of one.  This means that we have a case where the deletion was transmitted from the mother to the offspring.  The inherited deletion is 627 kb in size and the other two are 975 kb and 581 kb, respectively.  Both genes reported here have been reported in other studies and both inherited and spontaneous mutations and both are believed to act in a gene dose dependent manner.  Preliminary results in our genome-wide analysis of copy number variation in schizophrenia have identified promising candidates for disease etiology.  Future directions include analysis and validation of CNVs in the remainder of the sample set.

Thesis Seminar: Janine Martin

Ph.D. Thesis Seminar:  Janine Martin

Time:  1:00pm

Place:  2117 MERF

Developing RNAi Therapy for DYT1 Dystonia

DYT1 (OMIM) is the most common inherited dystonia, a disabling movement disorder with no effective treatment caused by a dominantly inherited mutation in the protein torsinA.  Our group and others have previously demonstrated the therapeutic potential of RNAi for DYT1 dystonia by achieving potent shRNA-mediated silencing of mutant torsinA in cultured cells. In this project we had two main aims: one- to see if we could identify phenotypes that could be utilized to evaluate therapeutic efficacy in preclinical trials and two- to determine whether RNAi therapy could be successfully employed to treat DYT1 murine models.  In an effort to identify novel phenotypes we examined whether the presence of mutant torsinA was sufficient to induce changes in transcriptional regulation, miRNA expression or produced an abnormal motor behavior in DYT1 murine models.

During our studies we found that abnormal accumulation of mutant torsinA at the nuclear envelope was not sufficient to induce transcriptional dysregulation in vitro or in vivo, despite its interaction with nuclear envelope proteins. We did find several miRNAs that had significantly altered expression in the cerebellum which may implicate a role for this brain region in DYT1 dystonia.  And finally we found no gross motor abnormalities in heterozygous DYT1 knockin (KI) mice or in a novel DYT1 transgenic model indicating that these mice may better embody nonmanifesting DYT1 carriers.

Our preclinical trial to test the feasibility of employing RNAi therapy in DYT1 murine models presented some unexpected results.  We found that intrastriatal injections of AAV2/1 vectors expressing different shRNAs, whether targeting torsinA expression or mismatched controls, resulted in significant toxicity with progressive weight loss, motor dysfunction and animal demise.  Toxicity was not observed in animals that received control AAV2/1 encoding no shRNA.  Interestingly, the different genetic background of both mouse models influenced toxicity, being earlier and more severe in 129/SvEv than C57BL/6 mice, perhaps explained by the lower levels of exportin5 expression observed in 129/SvEv mice.  In conclusion, our studies demonstrate that expression of shRNA in the mammalian brain can lead to lethal toxicity.  Furthermore, the genetic background of rodents modifies their sensitivity to this form of toxicity, a factor that should be taken into consideration in the design of preclinical therapeutic RNAi trials.

In summary, the studies completed in this thesis contribute important information to the fields of dystonia pathogenesis and therapeutics, and more broadly pertain to the development of therapeutic gene silencing for neurological disease.

Matt and Jonah to present at Student Seminar on Thursday (26.August) at noon in 2-501 BSB

Matt and Jonah will be presenting Student Seminar this week in 2-501 BSB at noon. I have asked the speakers to provide a brief overview for their talks. Hope to see you there!

Background for Matt’s talk

Francisella tularensis Infects and Grows in Small Airway Epithelial Cells: a new target in early infections.

Francisella tularensis is a gram negative facultative intracellular pathogen and is the causative agent of tularemia.  Though tularemia is not a common disease, its low infective dose and high mortality rate for untreated infections has classified F. tularensis as a class A select agent that could be utilized as a bioterrorism weapon.  Our lab studies F. tularensis by using various genetic tools to study many of the unique traits of the pathogen.  I will be discussing recent work, which demonstrates that small airway epithelial cells can be infected by F. tularensis.  These cells may play an initial role in an infection as they are likely to be the first cells that F. tularensis comes in contact with in a pneumonic infection. This work adds to the understanding of the route of infection and sets up an important model for studying early infection.

Background for Jonah’s talk

Myxococcus xanthus is a gram-negative soil bacterium that has a complex life cycle. Under starvation conditions, cells aggregate to form spore-containing fruiting bodies which persist until nutrients or suitable bacterial prey become available leading to germination. M. xanthus employs greater than 120 two-component systems, including eight chemosensory signaling pathways in order to regulate its complex developmental program as well as aspects of gene expression, predation, and even carotenoid production. Chemosensory pathways are specialized chemotaxis-like two component systems that enable the cell to respond appropriately to its environment. This occurs through a sensor domain that propagates the signal via a conformational change leading to the phosphorylation of a sensor kinase. The phosphoryl group is transferred to a response regulator, which will prompt the appropriate response.

My research focuses on the genetic characterization of two laboratory strains of M. xanthus, DZ2 and DK1622. A genome wide comparison of the two strains revealed a number of small nucleotide polymorphisms (SNPs). We thus wish to elucidate the physiological impact of these sequence differences in the two strains. The majority of these SNPs occur in annotated genes of unknown function, hypothetical genes and phage genes clustered in approximately 60kb. However, four SNPs occur in three previously characterized genes yielding significant amino acid substitutions. Two SNPs are in frzE, the kinase for the Frz system, which regulates reversal frequency, vegetative swarming, and directed swarming during development. A third SNP occurs in epsK, within a exopolysaccharide (EPS) biosynthesis gene cluster.  EPS is required for M. xanthus motility, fruiting body development and cell-cell adhesion. The fourth SNP occurs in a gene encoding a scaffolding protein within a chemosensory system involved in pilus-based motility. We hypothesize that one or more of these SNPs is the cause of the phenotypic differences seen between DZ2 and DK1622 and the objective is to test this hypothesis through genetic manipulation of the two strains to mimic each other.

Tryphena and Elizabeth to present at Student Seminar on Thursday (12.August) at noon in 1-107 BSB

Tryphena and Elizabeth will be presenting Student Seminar this week in 1-107 BSB at noon. I have asked the speakers to provide a brief overview for their talks. Hope to see you there!

Background for Tryphena’s talk

“Examining the Lystbg-J Mutation to Extend Genetic Pathways of Exfoliation Syndrome”

Exfoliation syndrome (XFS, OMIM) is a common age-related disorder characterized by the pathological accumulations of fibrillar exfoliative material in the anterior chamber of the eye. Patients with XFS can go on to develop exfoliative glaucoma, potentially a result of an accumulation of exfoliative material at the drainage structures of the eye. Human eyes with XFS exhibit a striking pattern of Marcel-like iris transillumination defects. The same pattern is recapitulated in mice containing the Lystbg-J mutation.  We have found that the molecular basis of the bg-J mutation is a three base pair deletion in the WD40 encoding region of the Lyst gene. However, the function of the Lyst gene in XFS disease and glaucoma remains unknown.

Previously, it has been shown that the LYST WD40 domain interacts with CSNK2B. Our lab confirmed this interaction with a GST pull-down experiment. Interestingly, LYSTbg-J completely disrupts the interaction with CSNK2B. Further experiments have suggested CSNK2B as an important candidate in the pathogenesis of XFS. Here, my goal is to further examine how LYST may influence CSNK2B in this iris disease. In this seminar, I will discuss my progress towards determining the underlying genetic pathways of LYST’s involvement in XFS and glaucoma.

Background for Elizabeth’s talk

“Pursuit of etiologic variants for cleft lip and palate following successful genome wide association study”

Nonsyndromic cleft lip and/or palate (NSCL/P) is a complex disorder caused by the interaction of multiple genetic and environmental factors.  Various genetic approaches, including linkage studies and candidate gene association studies have been performed with relatively little success in identifying major contributors to NSCL/P.  In 2009, four genome wide association studies (GWAS) were completed.  As a result, there are now several candidate genes and loci with compelling statistical and/or biological support which include IRF6, FOXE1, MAFB, and a region of 8q24.  We participated in the most recent GWAS which identified several novel loci, including one near ABCA4 on 1p22.1.

Mutations in ABCA4 have been associated with a range of retinal disorders including Stargardt disease, cone-rod dystrophy, retinitis pigmentosa, as well as age-related macular degeneration.  I have sequenced this gene and completed expression studies, which do not support a role for this gene in the clefting.  A case can be made for looking at either of the two flanking genes: GCLM, a player in glutathione synthesis, and ARHGAP29, a Rho-GTPase associated protein.  At seminar I will discuss the progress towards identifying the gene and etiologic variants that contribute to clefting at the 1p22.1 locus.

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