Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.BACKGROUND:

Glaucoma is a leading cause of irreversible blindness. Pathogenic variants in the Myocilin gene (MYOC) cause juvenile open angle glaucoma (JOAG) in 8-36% of cases, and display an autosomal dominant inheritance with high penetrance. Molecular diagnosis is important for early identification as therapies are effective in minimizing vision loss and MYOC variants can be associated to severe glaucoma. MYOC variants are usually inherited, however a fifth of carriers do not report a family history. The occurrence of de novo MYOC variants is currently unknown.
CASE PRESENTATION:

In this study we investigated a 14 year old male Caucasian patient diagnosed with JOAG, and no family history of glaucoma. A novel probably deleterious MYOC:p.(Pro254Leu) variant was identified in the index case. This variant was not present in the parents or the siblings.
CONCLUSION:

This is the second report of a de novo MYOC variant in a sporadic case of JOAG and it is currently unknown if this mechanism occurs more frequently. This finding emphasizes the importance of screening individuals with JOAG for MYOC mutations irrespective of a negative family history

Burdon, Kathryn Penelope

Craig, Jamie E

Dubowsky, Andrew

Ridge, Bronwyn

Ruddle, Jonathan B

Souzeau, Emmanuelle

BMC Medical Genetics

Flinders Academic Commons

CASE REPORT Open AccessA novel de novo Myocilin variant in apatient with sporadic juvenile openangle glaucomaEmmanuelle Souzeau1*, Kathryn P. Burdon2, Bronwyn Ridge1, Andrew Dubowsky3, Jonathan B. Ruddle4and Jamie E. Craig1AbstractBackground: Glaucoma is a leading cause of irreversible blindness. Pathogenic variants in the Myocilin gene (MYOC)cause juvenile open angle glaucoma (JOAG) in 8–36 % of cases, and display an autosomal dominant inheritance withhigh penetrance. Molecular diagnosis is important for early identification as therapies are effective in minimizing visionloss and MYOC variants can be associated to severe glaucoma. MYOC variants are usually inherited, however a fifth ofcarriers do not report a family history. The occurrence of de novo MYOC variants is currently unknown.Case presentation: In this study we investigated a 14 year old male Caucasian patient diagnosed with JOAG, and nofamily history of glaucoma. A novel probably deleterious MYOC:p.(Pro254Leu) variant was identified in the index case.This variant was not present in the parents or the siblings.Conclusion: This is the second report of a de novo MYOC variant in a sporadic case of JOAG and it is currentlyunknown if this mechanism occurs more frequently. This finding emphasizes the importance of screeningindividuals with JOAG for MYOC mutations irrespective of a negative family history.Keywords: De novo variant, Juvenile open angle glaucoma, Genetic testing, Glaucoma, MyocilinBackgroundGlaucoma is one of the leading causes of irreversibleblindness affecting over 60 million individuals worldwide[1]. Primary open angle glaucoma (POAG, MIM 137760)is the most common type and is characterized by changesin the optic nerve head with corresponding visual fieldloss in the presence of an open anterior chamber angle[2]. Juvenile open angle glaucoma (JOAG) refers to ayounger age at diagnosis usually defined by an onsetbefore 30–40 years old and associated with a more severephenotype [3, 4]. Therapies for POAG aim at controllingintraocular pressure (IOP) and are usually effective inminimizing disease progression [5–7]. However, the earlystages are often asymptomatic and half of the cases re-main undiagnosed, making it challenging to implementtreatment before irreversible vision loss occurs.Pathogenic sequence variants in the MYOC gene (MIM601652) have been first described in association withJOAG in 1997 [8]. Since then, they have been consistentlyidentified in 2–4 % of adult-onset POAG [9, 10] and in8–36 % of JOAG [9, 11, 12] among different ethnicities.MYOC comprises three exons which encode a proteinconsisting of two major domains, an N-terminalmyosin-like domain and a C-terminal olfactomedin-likedomain [13]. Most disease causing variants are clus-tered within exon 3 in the olfactomedin domain [14].The pathophysiology is not fully understood but it hasbeen postulated that the accumulation of misfoldedproteins lead to endoplasmic reticulum stress, whichcompromises the trabecular meshwork cells regulatingthe IOP [15]. MYOC pathogenic variants are inheritedin an autosomal dominant fashion and are often associ-ated with high IOP, younger age at diagnosis and strongfamily history and can result in severe glaucoma andblindness if left untreated [9, 10, 16].* Correspondence: emmanuelle.souzeau@flinders.edu.au1Department of Ophthalmology, Flinders University, Flinders Medical Centre,Adelaide, AustraliaFull list of author information is available at the end of the article© 2016 Souzeau et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Souzeau et al. BMC Medical Genetics  (2016) 17:30 DOI 10.1186/s12881-016-0291-5The majority of MYOC carriers report a family historyof glaucoma, however sporadic cases still account for20 % of mutation carriers [9]. It is currently unknownwhether sporadic cases could be explained by de novovariants. In this study, we report a JOAG sporadic casewith a novel de novo MYOC variant, and discuss theoccurrence of de novo variants in MYOC associatedglaucoma and the implications for the patient and hisfamily.Case presentationClinical presentationThe pedigree of the family is shown in Fig. 1a. The indexcase and his family were referred to the Australian andNew Zealand Registry of Advanced Glaucoma (ANZRAG)through his treating ophthalmologist [17]. The probandwas a 14 year old Caucasian male patient (II-1). He wasreferred to an ophthalmologist following a routine optom-etrist review for his glasses prescription which revealedhigh IOP. Following examination, he was diagnosed withJOAG. His IOP at presentation were 31 mmHg in theright eye and 32 mmHg in the left. His vertical cup-to-disc ratio was 0.85 right and 0.8 left, and he had centralfield loss involving fixation in the right eye (HumphreyField Analyzer, Zeiss) (Fig. 2a). His visual acuity was20/20 in both eyes. His IOP was initially undercontrol with latanoprost and brimonidine/timolol.However he underwent bilateral trabeculectomiesfollowing his most recent IOP which were 40 mmHg.Optic nerve appearances and retinal nerve fiber layerloss (Spectralis®, Heidelberg Engineering) are depictedin Fig. 2b and c. His parents and two siblings hadnormal eye examinations.Genetic testingGenetic testing was performed through the NationalAssociation of Testing Authorities (NATA) accreditedlaboratories of SA Pathology at the Flinders MedicalCentre in Adelaide, Australia. The proband was sequencedfor the 3 coding exons of the MYOC gene as previouslydescribed [9]. A heterozygous substitution of Thymine forCytosine at nucleotide 761 of the MYOC exon 3 codingsequence was identified (MYOC:c.761C > T), encoding amissense substitution of Proline to Leucine at position 254(p.(Pro254Leu)) (Fig. 1b). No other variants were identi-fied in the MYOC gene of the proband. JOAG can also beassociated with CYP1B1 variants [18]. The coding regionof the CYP1B1 gene was sequenced to exclude othercausative genes. No disease-causing variants were identi-fied in CYP1B1.The p.(Pro254Leu) variant is novel since it was absentfrom the MYOC Database (www.myocilin.com), NCBIdbSNP (www.ncbi.nlm.nih.gov/SNP/), and the ExomeAggregation Consortium (http://exac.broadinstitute.org/)which comprises exome sequence data spanning 60 706unrelated individuals. A search of the scientific literaturealso failed to identify any reference to this variant. How-ever, a recent study reported a MYOC variant at thesame residue p.(Pro254Arg) in a patient with JOAG andhis affected mother [19]. SIFT and Polyphen-2 bothpredicted this variant to be deleterious, with sequencealignment demonstrating this position to be highlyconserved among vertebrates and other olfactomedindomain-containing proteins (Fig. 1c). MYOC is a wellcharacterized gene and codon position 254 resides in thecore hydrophobic β-sheet belt of the olfactomedindomain, which is important in protein-protein interac-tions and is sensitive to aggregation in the presence ofsubstitutions [20]. The p.(Pro254Leu) variant is likelypathogenic based on bioinformatics prediction, invariantconservation of this residue, and characterization of theprotein structure. MYOC disease-causing variants can beassociated with severe glaucoma and blindness [9]. Inthe view of the genetic result and the patient’s mostrecent IOP, bilateral trabeculectomies were performed tobetter control his IOP and minimize damage on hisoptic nerves.This variant was not detected in either parent of theindex case (Fig. 1b). The marker profile comparisonusing the AmpFLSTR® Identifiler® PCR Amplification Kitconfirmed a profile consistent with the proband beingthe biological child of the stated parents, indicatingp.(Pro254Leu) has arisen de novo in the proband. A denovo MYOC pathogenic variant, p.(Val251Ala), has beenpreviously reported once in a JOAG case [21]. Interest-ingly, this variant was located three amino acids fromp.(Pro254Leu) which was identified in this study.While the occurrence of de novo pathogenic variantsin the genome vary considerably based on genomic loca-tion, they are estimated to be common and have beenlinked to many sporadic diseases [22]. Conditions withdominant inheritance and modest fitness effect are morecommonly inherited than caused by de novo variants,and this is the situation for MYOC associated glaucomawhich is usually inherited. For example, a founder effectwith an origin prior to the European settlement ofAustralia has been suggested for the most commonMYOCdisease-causing variant, p.Gln368Ter, in some families[23]. However, we previously reported that 20 % of MYOCcarriers do not report a family history of the disease [9].Although this may be explained by individuals not beingaware of a diagnosis in their families, or relatives beingundiagnosed, it is possible that variants occur de novo insome families. MYOC variants are often identified in olderindividuals with parents usually unavailable for testing,making it difficult to evaluate whether variants are inher-ited or sporadic. This case is the second report of a de novoMYOC variant, emphasizing that a sporadic variant shouldSouzeau et al. BMC Medical Genetics  (2016) 17:30 Page 2 of 5ABCFig. 1 (See legend on next page.)Souzeau et al. BMC Medical Genetics  (2016) 17:30 Page 3 of 5be considered when evaluating the likelihood of MYOCvariants in cases with no family history of JOAG or POAG.De novo variants arise either in the germline or duringembryogenesis. If present in the germline cells of one par-ent, they can represent a recurrence risk in siblings of thevariant carrier. We have previously shown that MYOCgenetic testing is important for early identification of at-risk individuals and appropriate interventions to minimizeirreversible vision loss [9, 24]. To exclude a recurrencerisk resulting from germline mosaicism, both siblings ofthe proband were subsequently tested for the MYOCvariant. Our testing revealed that neither sibling carriedtheMYOC p.(Pro254Leu) variant, eliminating an inheritedrisk of developingMYOC associated glaucoma.ConclusionIn conclusion, we report a novel de novo MYOC variantconsidered pathogenic in a patient with sporadic JOAG.This is the second report of a MYOC de novo variant, andit is currently unknown if this mechanism occurs moreFig. 2 Clinical presentation of the index case. Glaucomatous defects in index case. a Visual field pattern deviation showing a superior arcuatedefect involving fixation in the right eye (Humphrey Field analyser, Zeiss). b Optic discs photos showing a right infratemporal notch and dischaemorrhage. c Optical coherence tomography showing inferior retinal nerve fibre layer loss more prominent in the right eye than the left asshown by the black arrow (Spectralis®, Heidelberg Engineering). RE: right eye, LE: left eye, TMP: temporal, SUP: superior, NAS: nasal, INF: inferior(See figure on previous page.)Fig. 1 Pedigree and genetic analysis. a Pedigree of the family. Round symbols indicate female; square symbols, male; fully filled symbols, open angleglaucoma; unfilled symbols, unaffected; arrow, proband; plus/minus, presence/absence of the MYOC:p.(Pro254Leu) variant. b Chromatogram showingthe presence of MYOC:c.761C > T, p.(Pro254Leu) sequence variant in individual II-1 at the top (affected) and its absence in individual I-1 at the bottom(unaffected). The black arrow marks the heterozygous variant. c. Alignment of MYOC protein sequences corresponding to residues 248 through 262(NP_000252.1), against different species, and of different human olfactomedin proteins. The residue of interest, p.(Pro254Leu), is highlighted in yellow.Reference sequences IDs of the genes/species aligned are shown in bracketsSouzeau et al. BMC Medical Genetics  (2016) 17:30 Page 4 of 5frequently. This case also highlights that MYOC testingshould not be restricted to individuals with a positive fam-ily history of glaucoma.ConsentEthics approval was obtained from the SouthernAdelaide and Flinders University Clinical ResearchEthics Committee. The study conformed to the tenets ofthe Declaration of Helsinski and follows the NationalHealth and Medical Research Council statement of ethicalconduct in research involving humans. Written informedconsents were obtained from each participating familymember. A copy of the written consent is available forreview by the Series Editor of this journal.AbbreviationsANZRAG: Australian and New Zealand Registry of Advanced Glaucoma;IOP: intraocular pressure; JOAG: juvenile open angle glaucoma;MYOC: Myocilin; NATA: National Association of Testing Authorities;POAG: primary open angle glaucoma.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsES participated in the design of the study, interpreted the data and draftedthe manuscript. KPB participated in the design of the study and criticallyrevised the manuscript. BR recruited the family in the study and criticallyrevised the manuscript. AD carried out the molecular genetic studies,interpreted the results and critically revised the manuscript. JBR performedophthalmological examination of the patients and critically revised themanuscript. JEC participated in the design of the study and critically revisedthe manuscript. All authors read and approved the final manuscript.AcknowledgmentsThis project has been supported by The RANZCO Eye Foundation(www.eyefoundation.org.au, Sydney, Australia), Glaucoma Australia(www.glaucoma.org.au) and is currently funded by the Australian NationalHealth and Medical Research Council (NHMRC) Centers of Research ExcellenceGrant 1023911 (2012–2016). Jamie E Craig is an NHMRC Practitioner Fellow andKathryn P Burdon is supported by an NHMRC Research Fellowship.Author details1Department of Ophthalmology, Flinders University, Flinders Medical Centre,Adelaide, Australia. 2Menzies Institute for Medical Research, University ofTasmania, Hobart, Australia. 3SA Pathology, Flinders Medical Centre, Adelaide,Australia. 4Centre for Eye Research Australia, University of Melbourne, RoyalVictorian Eye & Ear Hospital, Melbourne, Australia.Received: 12 September 2015 Accepted: 8 April 2016References1. Quigley HA, Broman AT. The number of people with glaucoma worldwidein 2010 and 2020. Br J Ophthalmol. 2006;90(3):262–7.2. Wolfs RC, Klaver CC, Ramrattan RS, van Duijn CM, Hofman A, de Jong PT.Genetic risk of primary open-angle glaucoma. Population-based familialaggregation study. Arch Ophthalmol. 1998;116(12):1640–5.3. Wiggs JL, Damji KF, Haines JL, Pericak-Vance MA, Allingham RR. Thedistinction between juvenile and adult-onset primary open-angle glaucoma.Am J Hum Genet. 1996;58(1):243–4.4. Turalba AV, Chen TC. Clinical and genetic characteristics of primary juvenile-onset open-angle glaucoma (JOAG). Semin Ophthalmol. 2008;23(1):19–25.5. Heijl A, Leske MC, Bengtsson B, Hyman L, Hussein M. Reduction ofintraocular pressure and glaucoma progression: results from the EarlyManifest Glaucoma Trial. Arch Ophthalmol. 2002;120(10):1268–79.6. The Advanced Glaucoma Intervention Study (AGIS): 7. The relationshipbetween control of intraocular pressure and visual field deterioration.TheAGIS Investigators. Am J Ophthalmol. 2000;130(4):429-440.7. The effectiveness of intraocular pressure reduction in the treatment ofnormal-tension glaucoma. Collaborative Normal-Tension Glaucoma StudyGroup. Am J Ophthalmol. 1998;126(4):498-505.8. Stone EM, Fingert JH, Alward WL, et al. Identification of a gene that causesprimary open angle glaucoma. Science. 1997;275(5300):668–70.9. Souzeau E, Burdon KP, Dubowsky A, et al. Higher prevalence of myocilinmutations in advanced glaucoma in comparison with less advanced diseasein an Australasian disease registry. Ophthalmology. 2013;120(6):1135–43.10. Fingert JH, Heon E, Liebmann JM, et al. Analysis of myocilin mutations in1703 glaucoma patients from five different populations. Hum Mol Genet.1999;8(5):899–905.11. Shimizu S, Lichter PR, Johnson AT, et al. Age-dependent prevalence ofmutations at the GLC1A locus in primary open-angle glaucoma. Am JOphthalmol. 2000;130(2):165–77.12. Wiggs JL, Allingham RR, Vollrath D, et al. Prevalence of mutations in TIGR/Myocilin in patients with adult and juvenile primary open-angle glaucoma.Am J Hum Genet. 1998;63(5):1549–52.13. Green CM, Kearns LS, Wu J, et al. How significant is a family history ofglaucoma? Experience from the Glaucoma Inheritance Study in Tasmania.Clin Experiment Ophthalmol. 2007;35(9):793–9.14. Hewitt AW, Craig JE, Mackey DA. Complex genetics of complex traits: thecase of primary open-angle glaucoma. Clin Experiment Ophthalmol. 2006;34(5):472–84.15. Anholt RR, Carbone MA. A molecular mechanism for glaucoma:endoplasmic reticulum stress and the unfolded protein response. TrendsMol Med. 2013;19(10):586–93.16. Hewitt AW, Mackey DA, Craig JE. Myocilin allele-specific glaucomaphenotype database. Hum Mutat. 2008;29(2):207–11.17. Souzeau E, Goldberg I, Healey PR, et al. Australian and New Zealand Registryof Advanced Glaucoma: methodology and recruitment. Clin ExperimentOphthalmol. 2012;40(6):569–75.18. Souzeau E, Hayes M, Zhou T, et al. Occurrence of CYP1B1 Mutations inJuvenile Open-Angle Glaucoma With Advanced Visual Field Loss. JAMAOphthalmol. 2015;133(7):826–33.19. Yang Y, Shi Y, Huang X, et al. Identification of a novel MYOC mutation in aChinese family with primary open-angle glaucoma. Gene. 2015;571(2):188–93.20. Donegan RK, Hill SE, Freeman DM, et al. Structural basis for misfolding inmyocilin-associated glaucoma. Hum Mol Genet. 2015;24(8):2111–24.21. Kuchtey J, Chowdhury UR, Uptegraft CC, Fautsch MP, Kuchtey RW. A de novoMYOC mutation detected in juvenile open angle glaucoma associated withreduced myocilin protein in aqueous humor. Eur J Med Genet. 2013;56(6):292–6.22. Veltman JA, Brunner HG. De novo mutations in human genetic disease. NatRev Genet. 2012;13(8):565–75.23. Baird PN, Craig JE, Richardson AJ, et al. Analysis of 15 primary open-angleglaucoma families from Australia identifies a founder effect for theQ368STOP mutation of myocilin. Hum Genet. 2003;112(2):110–6.24. Souzeau E, Glading J, Keane M, et al. Predictive genetic testing experiencefor myocilin primary open-angle glaucoma using the Australian and NewZealand Registry of Advanced Glaucoma. Genet Med. 2014;16(7):558–63.Souzeau et al. BMC Medical Genetics  (2016) 17:30 Page 5 of 5

A novel de novo Myocilin variant in a patient with sporadic juvenile open angle glaucoma

Emmanuelle Souzeau

Kathryn P. Burdon

Bronwyn Ridge

Andrew Dubowsky

Jonathan B. Ruddle

Jamie E. Craig

Springer - Publisher Connector

CASE REPORT Open Access
A novel de novo Myocilin variant in a
patient with sporadic juvenile open
angle glaucoma
Emmanuelle Souzeau1*, Kathryn P. Burdon2, Bronwyn Ridge1, Andrew Dubowsky3, Jonathan B. Ruddle4
and Jamie E. Craig1
Abstract
Background: Glaucoma is a leading cause of irreversible blindness. Pathogenic variants in the Myocilin gene (MYOC)
cause juvenile open angle glaucoma (JOAG) in 8–36 % of cases, and display an autosomal dominant inheritance with
high penetrance. Molecular diagnosis is important for early identification as therapies are effective in minimizing vision
loss and MYOC variants can be associated to severe glaucoma. MYOC variants are usually inherited, however a fifth of
carriers do not report a family history. The occurrence of de novo MYOC variants is currently unknown.
Case presentation: In this study we investigated a 14 year old male Caucasian patient diagnosed with JOAG, and no
family history of glaucoma. A novel probably deleterious MYOC:p.(Pro254Leu) variant was identified in the index case.
This variant was not present in the parents or the siblings.
Conclusion: This is the second report of a de novo MYOC variant in a sporadic case of JOAG and it is currently
unknown if this mechanism occurs more frequently. This finding emphasizes the importance of screening
individuals with JOAG for MYOC mutations irrespective of a negative family history.
Keywords: De novo variant, Juvenile open angle glaucoma, Genetic testing, Glaucoma, Myocilin
Background
Glaucoma is one of the leading causes of irreversible
blindness affecting over 60 million individuals worldwide
[1]. Primary open angle glaucoma (POAG, MIM 137760)
is the most common type and is characterized by changes
in the optic nerve head with corresponding visual field
loss in the presence of an open anterior chamber angle
[2]. Juvenile open angle glaucoma (JOAG) refers to a
younger age at diagnosis usually defined by an onset
before 30–40 years old and associated with a more severe
phenotype [3, 4]. Therapies for POAG aim at controlling
intraocular pressure (IOP) and are usually effective in
minimizing disease progression [5–7]. However, the early
stages are often asymptomatic and half of the cases re-
main undiagnosed, making it challenging to implement
treatment before irreversible vision loss occurs.
Pathogenic sequence variants in the MYOC gene (MIM
601652) have been first described in association with
JOAG in 1997 [8]. Since then, they have been consistently
identified in 2–4 % of adult-onset POAG [9, 10] and in
8–36 % of JOAG [9, 11, 12] among different ethnicities.
MYOC comprises three exons which encode a protein
consisting of two major domains, an N-terminal
myosin-like domain and a C-terminal olfactomedin-like
domain [13]. Most disease causing variants are clus-
tered within exon 3 in the olfactomedin domain [14].
The pathophysiology is not fully understood but it has
been postulated that the accumulation of misfolded
proteins lead to endoplasmic reticulum stress, which
compromises the trabecular meshwork cells regulating
the IOP [15]. MYOC pathogenic variants are inherited
in an autosomal dominant fashion and are often associ-
ated with high IOP, younger age at diagnosis and strong
family history and can result in severe glaucoma and
blindness if left untreated [9, 10, 16].
* Correspondence: emmanuelle.souzeau@flinders.edu.au
1Department of Ophthalmology, Flinders University, Flinders Medical Centre,
Adelaide, Australia
Full list of author information is available at the end of the article
© 2016 Souzeau et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Souzeau et al. BMC Medical Genetics  (2016) 17:30 
DOI 10.1186/s12881-016-0291-5
The majority of MYOC carriers report a family history
of glaucoma, however sporadic cases still account for
20 % of mutation carriers [9]. It is currently unknown
whether sporadic cases could be explained by de novo
variants. In this study, we report a JOAG sporadic case
with a novel de novo MYOC variant, and discuss the
occurrence of de novo variants in MYOC associated
glaucoma and the implications for the patient and his
family.
Case presentation
Clinical presentation
The pedigree of the family is shown in Fig. 1a. The index
case and his family were referred to the Australian and
New Zealand Registry of Advanced Glaucoma (ANZRAG)
through his treating ophthalmologist [17]. The proband
was a 14 year old Caucasian male patient (II-1). He was
referred to an ophthalmologist following a routine optom-
etrist review for his glasses prescription which revealed
high IOP. Following examination, he was diagnosed with
JOAG. His IOP at presentation were 31 mmHg in the
right eye and 32 mmHg in the left. His vertical cup-to-
disc ratio was 0.85 right and 0.8 left, and he had central
field loss involving fixation in the right eye (Humphrey
Field Analyzer, Zeiss) (Fig. 2a). His visual acuity was
20/20 in both eyes. His IOP was initially under
control with latanoprost and brimonidine/timolol.
However he underwent bilateral trabeculectomies
following his most recent IOP which were 40 mmHg.
Optic nerve appearances and retinal nerve fiber layer
loss (Spectralis®, Heidelberg Engineering) are depicted
in Fig. 2b and c. His parents and two siblings had
normal eye examinations.
Genetic testing
Genetic testing was performed through the National
Association of Testing Authorities (NATA) accredited
laboratories of SA Pathology at the Flinders Medical
Centre in Adelaide, Australia. The proband was sequenced
for the 3 coding exons of the MYOC gene as previously
described [9]. A heterozygous substitution of Thymine for
Cytosine at nucleotide 761 of the MYOC exon 3 coding
sequence was identified (MYOC:c.761C > T), encoding a
missense substitution of Proline to Leucine at position 254
(p.(Pro254Leu)) (Fig. 1b). No other variants were identi-
fied in the MYOC gene of the proband. JOAG can also be
associated with CYP1B1 variants [18]. The coding region
of the CYP1B1 gene was sequenced to exclude other
causative genes. No disease-causing variants were identi-
fied in CYP1B1.
The p.(Pro254Leu) variant is novel since it was absent
from the MYOC Database (www.myocilin.com), NCBI
dbSNP (www.ncbi.nlm.nih.gov/SNP/), and the Exome
Aggregation Consortium (http://exac.broadinstitute.org/)
which comprises exome sequence data spanning 60 706
unrelated individuals. A search of the scientific literature
also failed to identify any reference to this variant. How-
ever, a recent study reported a MYOC variant at the
same residue p.(Pro254Arg) in a patient with JOAG and
his affected mother [19]. SIFT and Polyphen-2 both
predicted this variant to be deleterious, with sequence
alignment demonstrating this position to be highly
conserved among vertebrates and other olfactomedin
domain-containing proteins (Fig. 1c). MYOC is a well
characterized gene and codon position 254 resides in the
core hydrophobic β-sheet belt of the olfactomedin
domain, which is important in protein-protein interac-
tions and is sensitive to aggregation in the presence of
substitutions [20]. The p.(Pro254Leu) variant is likely
pathogenic based on bioinformatics prediction, invariant
conservation of this residue, and characterization of the
protein structure. MYOC disease-causing variants can be
associated with severe glaucoma and blindness [9]. In
the view of the genetic result and the patient’s most
recent IOP, bilateral trabeculectomies were performed to
better control his IOP and minimize damage on his
optic nerves.
This variant was not detected in either parent of the
index case (Fig. 1b). The marker profile comparison
using the AmpFLSTR® Identifiler® PCR Amplification Kit
confirmed a profile consistent with the proband being
the biological child of the stated parents, indicating
p.(Pro254Leu) has arisen de novo in the proband. A de
novo MYOC pathogenic variant, p.(Val251Ala), has been
previously reported once in a JOAG case [21]. Interest-
ingly, this variant was located three amino acids from
p.(Pro254Leu) which was identified in this study.
While the occurrence of de novo pathogenic variants
in the genome vary considerably based on genomic loca-
tion, they are estimated to be common and have been
linked to many sporadic diseases [22]. Conditions with
dominant inheritance and modest fitness effect are more
commonly inherited than caused by de novo variants,
and this is the situation for MYOC associated glaucoma
which is usually inherited. For example, a founder effect
with an origin prior to the European settlement of
Australia has been suggested for the most commonMYOC
disease-causing variant, p.Gln368Ter, in some families
[23]. However, we previously reported that 20 % of MYOC
carriers do not report a family history of the disease [9].
Although this may be explained by individuals not being
aware of a diagnosis in their families, or relatives being
undiagnosed, it is possible that variants occur de novo in
some families. MYOC variants are often identified in older
individuals with parents usually unavailable for testing,
making it difficult to evaluate whether variants are inher-
ited or sporadic. This case is the second report of a de novo
MYOC variant, emphasizing that a sporadic variant should
Souzeau et al. BMC Medical Genetics  (2016) 17:30 Page 2 of 5
AB
C
Fig. 1 (See legend on next page.)
Souzeau et al. BMC Medical Genetics  (2016) 17:30 Page 3 of 5
be considered when evaluating the likelihood of MYOC
variants in cases with no family history of JOAG or POAG.
De novo variants arise either in the germline or during
embryogenesis. If present in the germline cells of one par-
ent, they can represent a recurrence risk in siblings of the
variant carrier. We have previously shown that MYOC
genetic testing is important for early identification of at-
risk individuals and appropriate interventions to minimize
irreversible vision loss [9, 24]. To exclude a recurrence
risk resulting from germline mosaicism, both siblings of
the proband were subsequently tested for the MYOC
variant. Our testing revealed that neither sibling carried
theMYOC p.(Pro254Leu) variant, eliminating an inherited
risk of developingMYOC associated glaucoma.
Conclusion
In conclusion, we report a novel de novo MYOC variant
considered pathogenic in a patient with sporadic JOAG.
This is the second report of a MYOC de novo variant, and
it is currently unknown if this mechanism occurs more
Fig. 2 Clinical presentation of the index case. Glaucomatous defects in index case. a Visual field pattern deviation showing a superior arcuate
defect involving fixation in the right eye (Humphrey Field analyser, Zeiss). b Optic discs photos showing a right infratemporal notch and disc
haemorrhage. c Optical coherence tomography showing inferior retinal nerve fibre layer loss more prominent in the right eye than the left as
shown by the black arrow (Spectralis®, Heidelberg Engineering). RE: right eye, LE: left eye, TMP: temporal, SUP: superior, NAS: nasal, INF: inferior
(See figure on previous page.)
Fig. 1 Pedigree and genetic analysis. a Pedigree of the family. Round symbols indicate female; square symbols, male; fully filled symbols, open angle
glaucoma; unfilled symbols, unaffected; arrow, proband; plus/minus, presence/absence of the MYOC:p.(Pro254Leu) variant. b Chromatogram showing
the presence of MYOC:c.761C > T, p.(Pro254Leu) sequence variant in individual II-1 at the top (affected) and its absence in individual I-1 at the bottom
(unaffected). The black arrow marks the heterozygous variant. c. Alignment of MYOC protein sequences corresponding to residues 248 through 262
(NP_000252.1), against different species, and of different human olfactomedin proteins. The residue of interest, p.(Pro254Leu), is highlighted in yellow.
Reference sequences IDs of the genes/species aligned are shown in brackets
Souzeau et al. BMC Medical Genetics  (2016) 17:30 Page 4 of 5
frequently. This case also highlights that MYOC testing
should not be restricted to individuals with a positive fam-
ily history of glaucoma.
Consent
Ethics approval was obtained from the Southern
Adelaide and Flinders University Clinical Research
Ethics Committee. The study conformed to the tenets of
the Declaration of Helsinski and follows the National
Health and Medical Research Council statement of ethical
conduct in research involving humans. Written informed
consents were obtained from each participating family
member. A copy of the written consent is available for
review by the Series Editor of this journal.
Abbreviations
ANZRAG: Australian and New Zealand Registry of Advanced Glaucoma;
IOP: intraocular pressure; JOAG: juvenile open angle glaucoma;
MYOC: Myocilin; NATA: National Association of Testing Authorities;
POAG: primary open angle glaucoma.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
ES participated in the design of the study, interpreted the data and drafted
the manuscript. KPB participated in the design of the study and critically
revised the manuscript. BR recruited the family in the study and critically
revised the manuscript. AD carried out the molecular genetic studies,
interpreted the results and critically revised the manuscript. JBR performed
ophthalmological examination of the patients and critically revised the
manuscript. JEC participated in the design of the study and critically revised
the manuscript. All authors read and approved the final manuscript.
Acknowledgments
This project has been supported by The RANZCO Eye Foundation
(www.eyefoundation.org.au, Sydney, Australia), Glaucoma Australia
(www.glaucoma.org.au) and is currently funded by the Australian National
Health and Medical Research Council (NHMRC) Centers of Research Excellence
Grant 1023911 (2012–2016). Jamie E Craig is an NHMRC Practitioner Fellow and
Kathryn P Burdon is supported by an NHMRC Research Fellowship.
Author details
1Department of Ophthalmology, Flinders University, Flinders Medical Centre,
Adelaide, Australia. 2Menzies Institute for Medical Research, University of
Tasmania, Hobart, Australia. 3SA Pathology, Flinders Medical Centre, Adelaide,
Australia. 4Centre for Eye Research Australia, University of Melbourne, Royal
Victorian Eye & Ear Hospital, Melbourne, Australia.
Received: 12 September 2015 Accepted: 8 April 2016
References
1. Quigley HA, Broman AT. The number of people with glaucoma worldwide
in 2010 and 2020. Br J Ophthalmol. 2006;90(3):262–7.
2. Wolfs RC, Klaver CC, Ramrattan RS, van Duijn CM, Hofman A, de Jong PT.
Genetic risk of primary open-angle glaucoma. Population-based familial
aggregation study. Arch Ophthalmol. 1998;116(12):1640–5.
3. Wiggs JL, Damji KF, Haines JL, Pericak-Vance MA, Allingham RR. The
distinction between juvenile and adult-onset primary open-angle glaucoma.
Am J Hum Genet. 1996;58(1):243–4.
4. Turalba AV, Chen TC. Clinical and genetic characteristics of primary juvenile-
onset open-angle glaucoma (JOAG). Semin Ophthalmol. 2008;23(1):19–25.
5. Heijl A, Leske MC, Bengtsson B, Hyman L, Hussein M. Reduction of
intraocular pressure and glaucoma progression: results from the Early
Manifest Glaucoma Trial. Arch Ophthalmol. 2002;120(10):1268–79.
6. The Advanced Glaucoma Intervention Study (AGIS): 7. The relationship
between control of intraocular pressure and visual field deterioration.The
AGIS Investigators. Am J Ophthalmol. 2000;130(4):429-440.
7. The effectiveness of intraocular pressure reduction in the treatment of
normal-tension glaucoma. Collaborative Normal-Tension Glaucoma Study
Group. Am J Ophthalmol. 1998;126(4):498-505.
8. Stone EM, Fingert JH, Alward WL, et al. Identification of a gene that causes
primary open angle glaucoma. Science. 1997;275(5300):668–70.
9. Souzeau E, Burdon KP, Dubowsky A, et al. Higher prevalence of myocilin
mutations in advanced glaucoma in comparison with less advanced disease
in an Australasian disease registry. Ophthalmology. 2013;120(6):1135–43.
10. Fingert JH, Heon E, Liebmann JM, et al. Analysis of myocilin mutations in
1703 glaucoma patients from five different populations. Hum Mol Genet.
1999;8(5):899–905.
11. Shimizu S, Lichter PR, Johnson AT, et al. Age-dependent prevalence of
mutations at the GLC1A locus in primary open-angle glaucoma. Am J
Ophthalmol. 2000;130(2):165–77.
12. Wiggs JL, Allingham RR, Vollrath D, et al. Prevalence of mutations in TIGR/
Myocilin in patients with adult and juvenile primary open-angle glaucoma.
Am J Hum Genet. 1998;63(5):1549–52.
13. Green CM, Kearns LS, Wu J, et al. How significant is a family history of
glaucoma? Experience from the Glaucoma Inheritance Study in Tasmania.
Clin Experiment Ophthalmol. 2007;35(9):793–9.
14. Hewitt AW, Craig JE, Mackey DA. Complex genetics of complex traits: the
case of primary open-angle glaucoma. Clin Experiment Ophthalmol. 2006;
34(5):472–84.
15. Anholt RR, Carbone MA. A molecular mechanism for glaucoma:
endoplasmic reticulum stress and the unfolded protein response. Trends
Mol Med. 2013;19(10):586–93.
16. Hewitt AW, Mackey DA, Craig JE. Myocilin allele-specific glaucoma
phenotype database. Hum Mutat. 2008;29(2):207–11.
17. Souzeau E, Goldberg I, Healey PR, et al. Australian and New Zealand Registry
of Advanced Glaucoma: methodology and recruitment. Clin Experiment
Ophthalmol. 2012;40(6):569–75.
18. Souzeau E, Hayes M, Zhou T, et al. Occurrence of CYP1B1 Mutations in
Juvenile Open-Angle Glaucoma With Advanced Visual Field Loss. JAMA
Ophthalmol. 2015;133(7):826–33.
19. Yang Y, Shi Y, Huang X, et al. Identification of a novel MYOC mutation in a
Chinese family with primary open-angle glaucoma. Gene. 2015;571(2):188–93.
20. Donegan RK, Hill SE, Freeman DM, et al. Structural basis for misfolding in
myocilin-associated glaucoma. Hum Mol Genet. 2015;24(8):2111–24.
21. Kuchtey J, Chowdhury UR, Uptegraft CC, Fautsch MP, Kuchtey RW. A de novo
MYOC mutation detected in juvenile open angle glaucoma associated with
reduced myocilin protein in aqueous humor. Eur J Med Genet. 2013;56(6):292–6.
22. Veltman JA, Brunner HG. De novo mutations in human genetic disease. Nat
Rev Genet. 2012;13(8):565–75.
23. Baird PN, Craig JE, Richardson AJ, et al. Analysis of 15 primary open-angle
glaucoma families from Australia identifies a founder effect for the
Q368STOP mutation of myocilin. Hum Genet. 2003;112(2):110–6.
24. Souzeau E, Glading J, Keane M, et al. Predictive genetic testing experience
for myocilin primary open-angle glaucoma using the Australian and New
Zealand Registry of Advanced Glaucoma. Genet Med. 2014;16(7):558–63.
Souzeau et al. BMC Medical Genetics  (2016) 17:30 Page 5 of 5


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A novel de novo Myocilin variant in a patient with sporadic juvenile open angle glaucoma

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