Novel mutations in the MYOC/GLC1A gene in a large group of glaucoma patients

Michels-Rautenstrauss, Karin; Mardin, Christian Y.; Wakili, Nina; Jünemann, Anselm; Villalobos, Luis; Mejía, Carlos A Isaza; Chavarría-Soley, Gabriela; Azofeifa, Jorge; Ozbey, Sevinc; Naumann, Gottfried O.H.; Reis, André; Rautenstrauß, Bernd

doi:10.1002/humu.9092

Cited by 24 publications

(18 citation statements)

References 27 publications

(40 reference statements)

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“…These variants are well tolerated in HEK-293T and NTM-5 cells (>56% of WT MYOC secretion in all instances). Whereas G434S MYOC has been found in one POAG patient, 49 Y471C MYOC has been found in two POAG patients 46,47 and is listed as having “unknown pathologic significance” on the UniProt website (available in the public domain at www.uniprot.org). The combination of small patient cohorts combined with our secretion results, begs the question if Y471C and G434S MYOC are truly pathogenic.…”

Section: Discussionmentioning

confidence: 99%

A Novel Luciferase Assay For Sensitively Monitoring Myocilin Variants in Cell Culture

Zadoo

Nguyen

Zode

et al. 2016

Invest. Ophthalmol. Vis. Sci.

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PurposePrimary open angle glaucoma–associated mutations in myocilin (MYOC) cause protein “nonsecretion,” rendering secreted MYOC difficult to detect using conventional techniques. This study focused on developing and using an assay that can quickly and easily detect mutant MYOC secretion.MethodsWe fused Gaussia luciferase (eGLuc2) to MYOC variants and expressed the constructs in HEK-293T and NTM-5 cells. Secreted and intracellular levels of MYOC eGLuc2 variants were evaluated by Western blotting and compared to untagged and FLAG-tagged MYOC constructs. Secreted and soluble intracellular MYOC eGLuc2 were measured by a GLuc assay. The secretion of nine additional MYOC mutants was assayed in conditioned media from transfected cells to test the applicability of the assay for monitoring other MYOC variants.ResultsMyocilin eGLuc2 behaved similarly to untagged and FLAG-tagged MYOC with respect to secretion, soluble intracellular levels, and in response to drug treatment. The GLuc assay could sensitively detect Y437H MYOC secretion 30 minutes after media change. Gaussia luciferase fused variants followed anticipated trends; nonpathogenic variants (D208E, G244V) were secreted at wild-type (WT) levels, whereas predicted disease-causing variants (C245Y, G246R, E300K, Y437H, I477N) demonstrated substantial secretion defects. Secretion defects caused by the C245Y, G246R, and Y437H mutations were partially rescued by permissive growth temperature. Interestingly, however, this increase in secretion was independent of newly synthesized protein.ConclusionsFusion of eGLuc2 to MYOC does not significantly change the behavior of MYOC. This newly developed MYOC reporter system can be used to study engineered MYOC variants and potentially to identify modulators of MYOC secretion and function.

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Section: Discussionmentioning

confidence: 99%

A Novel Luciferase Assay For Sensitively Monitoring Myocilin Variants in Cell Culture

Zadoo

Nguyen

Zode

et al. 2016

Invest. Ophthalmol. Vis. Sci.

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“…Similar to nearly all pathogenic MYOC mutations, the Val251Ala mutation is within the olfactomedin homology domain encoded by exon 3. A previous study reported a Val251Ala mutation, however, the nucleotide position of the mutation and the codon to amino acid conversion reported were erroneous [15]. Therefore, it is difficult to know if that variant was Val251Ala or Gly244Ala.…”

Section: Discussionmentioning

confidence: 99%

A de novo MYOC mutation detected in juvenile open angle glaucoma associated with reduced myocilin protein in aqueous humor

Kuchtey

Chowdhury

Uptegraft

et al. 2013

European Journal of Medical Genetics

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MYOC mutations were originally identified in patients with juvenile open angle glaucoma (JOAG). Cell culture and mouse studies suggest that MYOC mutations cause glaucoma through a dominant-negative effect on myocilin protein secretion. We tested this hypothesis with patient samples in this study. Glaucoma and control patients underwent complete ocular examination. DNA samples from glaucoma patients, and unaffected relatives and controls were used for DNA sequencing of MYOC. Aqueous humor (AH) samples from glaucoma and control patients were obtained at the time of surgery. Myocilin protein in AH was detected by quantitative Western blot analysis. A de novo Val251Ala mutation of MYOC was found to segregate with disease in a family with autosomal dominant JOAG. Myocilin protein was detected in all control AH samples but not was nearly undetectable in AH samples from a patient heterozygous for the Val251Ala mutation. Our results using human patient samples are consistent with a dominant-negative effect of pathogenic MYOC mutations on myocilin secretion.

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“…Information on MYOC variants in European POAG patients is presently limited (Cobb et al, 2002;Hulsman et al, 2002a;Michels-Rautenstrauss et al, 2002). In the present work, we investigated the genetic variation of the coding region of MYOC in 237 unrelated French POAG patients and 108 control subjects, using denaturing high performance liquid chromatography (DHPLC).…”

Section: Introductionmentioning

confidence: 99%

Myocilin analysis by DHPLC in French POAG patients: Increased prevalence of Q368X mutation

et al. 2003

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Primary open-angle glaucoma (POAG) is a prevalent optic neuropathy with complex genetics. A small number of patients carry a mutation in the coding region of the myocilin (MYOC) gene. The nature and the frequency of these mutations, however, vary substantially, notably with the age at onset and the ethnic origin of the patients. Here, we showed that denaturing high performance liquid chromatography (DHPLC) is an appropriate method for screening carriers of MYOC mutations. We have applied the method to a group of 237 POAG patients and 108 control subjects from France. Mutations were found in 17 (7.5%) patients and in none of the controls. A single mutation, Q368X (c.1102C>T), accounted for the majority (12/17) of these mutations, corresponding to a frequency of 5% among POAG patients, the highest ever reported for this mutation. Furthermore, analysis of allelic associations at closely linked microsatellite markers indicated that most, if not all, patients inherited Q368X from a same ancestor. Five other patients carried four distinct mutations, including N480K (c.1440C>A) (2 cases), I499F (c.1495A>T), G367R (c.1099G>A) and T438I (c.1313C>T), which is reported here for the first time. Altogether, MYOC mutations in French patients were associated with a significantly increased intraocular pressure at diagnosis. In addition, the age at diagnosis of patients with a mutation other than Q368X was significantly younger than that of Q368X carriers or of patients with a normal MYOC. Based on these observations, a screening strategy of MYOC mutations in French POAG patients is briefly outlined.

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Novel mutations in the MYOC/GLC1A gene in a large group of glaucoma patients

Cited by 24 publications

References 27 publications

A Novel Luciferase Assay For Sensitively Monitoring Myocilin Variants in Cell Culture

A Novel Luciferase Assay For Sensitively Monitoring Myocilin Variants in Cell Culture

A de novo MYOC mutation detected in juvenile open angle glaucoma associated with reduced myocilin protein in aqueous humor

Myocilin analysis by DHPLC in French POAG patients: Increased prevalence of Q368X mutation

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