GLC1A mutations point to regions of potential functional importance on the TIGR/MYOC protein.
OBJECTIVE
The aim of this study was to screen affected members of glaucoma families for mutations in the Trabecular Meshwork Inducible Glucocorticoid Response (TIGR) gene also known by the name myocilin (MYOC) or by combined names such as TIGR/MYOC. Our primary objectives were (1) to identify mutations responsible for glaucoma in members of three families for which we have shown linkage between chromosome 1 GLC1A-region markers and the primary open angle glaucoma (POAG) phenotype, and (2) to determine the relationship of these and other mutations to key points of predicted function and structure of the TIGR/MYOC protein.


METHODS
DNA sequence determination was used to identify sequence changes in sections of the TIGR/MYOC gene that were PCR-amplified from genomic DNA from the probands of three previously-reported GLC1A juvenile-onset POAG families, UM:JG1, UM:JG3, UM:GL57, and unmapped family UM:JG5. Allele-specific oligonucleotide hybridization was used to screen for the identified mutations in PCR-amplified DNA from individual members of each pedigree and from a panel of 11 additional juvenile glaucoma family probands, 42 adult POAG family probands, and 43 normal individuals. Computerized algorithms were used to identify functional motifs and predict structures of normal and mutant forms of the protein.


RESULTS
Sequence changes were found that alter amino acids in the olfactomedin-like domain near the carboxy terminal end of the TIGR protein in affected members of families UM:JG1 (Pro370Leu), UM:JG3 (Val426Phe), UM:GL57 (Glu323Lys) and UM:JG5 (Gly252Arg). Co-segregation of glaucoma and Pro370Leu, Val426Phe, and Gly252Arg in known GLC1A families suggests that these are mutations. Although the Gly252Arg substitution observed in UM:JG5 is non-conservative, it was not possible to distinguish whether it is a mutation or a polymorphism. None of the sequence changes described in these families were observed in other juvenile glaucoma cases in this study, nor in any of the POAG or phenotypically normal individuals tested here. Analysis of amino acid sequence changes resulting from mutations described in this and other works demonstrate localization of many mutations in the vicinity of predicted functional motifs in the olfactomedin-like domain. Identification of rat latrophilin (LPH1/CIRL) as a new member of the olfactomedin-like protein family to which TIGR/MYOC belongs suggests that the region of olfactomedin homology is a protein domain that can occur in different protein contexts.


CONCLUSIONS
Location of mutations described in this and previous work suggests that some specific predicted protein motifs in the olfactomedin-like domain may be important to TIGR/MYOC function. In some cases, the role of TIGR/MYOC in the etiology of glaucoma may result from alteration of the sequences recognized by modifying enzymes such as casein kinase II. In other cases altered protein folding may affect access of enzymes to their target sequences on TIGR/MYOC. Although modifications and structures discussed here are predicted rather than proven, they provide a useful theoretical framework for design of subsequent experiments. Alterations to protein folding and predicted modification motifs cannot explain the pathogenic mechanisms of all of the known TIGR/MYOC glaucoma mutations.
Department of Ophthalmology, The University of Michigan Medical School, Ann Arbor, MI 48105, USA.
F W
FW
Rozsa
S
S
Shimizu
P R
PR
Lichter
A T
AT
Johnson
M I
MI
Othman
K
K
Scott
C A
CA
Downs
T D
TD
Nguyen
J
J
Polansky
J E
JE
Richards
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D001483:Base Sequence; D003598:Cytoskeletal Proteins; D016326:Extracellular Matrix Proteins / Q000235:genetics; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D005260:Female; D008040:Genetic Linkage; D005902:Glaucoma, Open-Angle / Q000235:genetics*; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008297:Male; D008875:Middle Aged; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010375:Pedigree; D017433:Protein Structure, Secondary; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D000099179:Myocilin
D003598:Cytoskeletal Proteins; D016326:Extracellular Matrix Proteins; D005136:Eye Proteins; D006023:Glycoproteins; D000099179:Myocilin; C081451:olfactomedin