In order to understand how a pathogenic change in a gene causes disease, it is necessary to recognize how pathogenic mutations could affect a protein structure-function, protein-protein interactions in protein networks and how these changes could be associated with clinical parameters describing the disease phenotype. We approached this problem computationally using molecular modeling to simulate the effect of pathogenic changes on protein stability and to suggest a new parameter quantifying the impact of missense pathogenic change at molecular level. We were analyzing data for several inherited diseases to verify this approach. Gene mutations that encode retinoschisin (RS1) cause X-linked retinoschisis (XLRS), a form of juvenile macular and retinal degeneration that affects males. Initially we implied molecular modeling of retinoschisin with functional analysis of 27 mutations from 60 XLRS patients from the NEI clinic or literature and demonstrated that mutational change in RS1 protein structure affects the phenotype of XLRS measured from electroretinogram (ERG). Further verification was performed recently with a new dataset sharing ERG information on 15 missense mutations for 38 patients from Moorfields Eye Hospital (London, UK). These results support an idea that causing missense variants might be divided in two classes of severe and less severe changes which are respectively associated with severe and less severe XLRS phenotype characterized by ERG. Choroideremia (CHM) is an X-linked form of retinal degeneration characterized by slowly progressive atrophy of the retinal pigment epithelium (RPE), photoreceptors, and choroid. The CHM gene encodes Rab escort protein 1 (REP-1), which is involved in trafficking of Rab proteins in the cell. To date, the majority of reported mutations in the CHM gene cause a complete loss of REP-1 and its function. We have analyzed 10 pathogenic mutations from the NEI clinic causing choroideremia. To analyze the effect of the mutations, the 3D structure of human REP-1 and the proteins associated with REP-1 function (Rab-7 and Rab geranylgeranyl transferase) were modeled using sequence homology to rat proteins and docked together to form a hypothetical oligomeric complex of human proteins. In silico analysis of the missense mutation L550P suggests that the P550 destabilizes the -structural elements, and the REP-1 tertiary structure. Truncation and deletion mutants are associated with a partial or total loss of the REP-1 essential activity and protein-protein interactions as predicted by the computational analysis of the structure and stability of these protein products. The partial or complete loss of protein was confirmed by the Western blot analysis of protein from mononuclear cells and FB from the CHM patients. Most CHM mutations with the exception of the L550P mutation are linked to complete degradation of the rep-1 protein product and exclude REP-1 from isoprenylation cycle. PAX2/Pax2 is a developmentally-regulated transcriptional factor gene expressed in the ventral optic cup &stalk of the developing eye. Mutations of PAX2 cause the papillorenal syndrome (PRS) in humans. We characterize a novel mouse model of PRS due to a missense mutation in Pax2, &characterize the molecular mechanism of 2 previously-reported human missense mutations located near the mouse mutation. In this project I modeled the structure of the DNA-PAX2 complex and performed an analysis of structural changes of Pax2/PAX2 due to genetic mutations. A mouse T74A mutation and two human mutations, (p.G75S and p.dup73ET) are expected to disrupt critical hydrogen bonds in the Pax2 homeodomain. Reduced Pax2/PAX2 protein stability might suggest a molecular mechanism responsible for the PRS phenotype. To report the clinical and molecular phenotype associated with 2 novel, de novo, CRX mutations causing dominant LCA. Patients underwent detailed ophthalmic examination, full field ERGs and retinal imaging. We modeled a novel CRX mutation, c.G264T (p.K88N), which reduced transactivation to 10% of wild type (WT) protein activity, respectively. This mutant is expressed in vitro at levels comparable to WT protein. However, unlike WT, coexpression of mutant c.G264T with NRL drastically reduced amounts of both the proteins. Mutation significantly reduces baseline CRX activity and partially mislocalized. The c.G24T mutant acts in a dominant-negative-like fashion with NRL. In our collaborative work with Dr. Iwata we shown that mutant WDR36 directly affects axon growth of retinal ganglion cells leading to progressive retinal degeneration in mice. Molecular modeling was applied to simulate mouse Wdr36 protein structure and to calculate structural changes caused by three different mutations that were introduced in transgenic mouse models. This is the first demonstration of an association between gene mutations in Wdr36 and changes in a phenotype observed in a mammalian system. Oculocutaneous albinism type 1 (OCA1) is an autosomal recessive disorder characterized by absence of pigment melanin eyes, hair and skin. Pathogenic mutations causing OCA1 decrease or abolish the melanogenic pathway by affecting tyrosinase enzymatic activitythe rate limiting step in pigment production. We modeled structure of mouse tyrosinase and predicted the effect of missense mutations R77L and H420R causing OCA1 with severe (OCA1a) and less severe (OCA1b) phenotypes, respectively. From this modeling the missense change H420R is likely affects enzymatic function and the L77R might interfere with tyrosine binding. This modeling agree with recent experiments in Dr. Brooks laboratory demonstrated that the chemical blocking a tyrosine degradation pathway show no effect in Tyr(c-2J/c-2J) mouse model (OCA1a) and cause the albino phenotype change in Tyr(c-h/c-h) mouse model (OCA1b). The stabilizing role tyrosine was proven experimentally by Dr. Dolinska in cell culture by demonstration of an increase in the steady state level of R77L and H420R mutants in the presence of tyrosine.
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