Eye Disease

Molecular Eye Disease

Mouse mutants affecting eye development and the health status of ocular tissues (cornea, lens, retina, optic nerve) are being characterized in a functional, morphological and molecular manner. In course of our ongoing ENU-mutagenesis screen (in cooperation with the HMGU-Institute of Experimental Genetics we are picking up mouse variants by



slit lamp analysis: general appearance of the eye and transparency of the cornea and lens


laser interference biometry: various eye size parameters


funduscopy: retinal degeneration


virtual optic drum: general properties of the visual system


Based upon the underlying mutation and the responsible phenotype, mouse mutations are grouped:

Mouse mutations affecting early eye development

In course of our ongoing ENU-mutagenesis screen (in cooperation with the HMGU-Institute of Experimental Genetics) new small-eye mutants were characterized as Pax6 alleles (Graw et al., 2005). Using the Pax6Aey18 allele with the inactive homeodomain, we could demonstrate the key role of the Pax6 paired domain in brain development (Haubst et al., 2004). In the pancreas, this mutant mouse line showed a nearly complete absence of glucagon-positive alpha-cells, reduced beta-cell area and disorganized islets; the proportion of ghrelin-positive epsilon-cells was expanded (Dames et al., 2010) strongly suggesting Pax6 mutants as models in diabetes research.

Our group was involved in the characterization of two Pitx3 mutants (aphakia, eyeless) suffering as homozygotes from small eyes without lenses (Semina et al., 2000; Rosemann et al., 2010) . Since Pitx3 is expressed also in the brain, particularly in the mesencephalic dopamine system (Smidt et al., 2004), systematic phenotype analysis in the German mouse clinic demonstrated in the homozygous eyeless mutants several behavioral changes, including reduced forelimb grip strength and increased nociception. Based upon this phenotypic analysis, the eyeless mutant should be considered as a genetic model for Parkinson’s disease.

Moreover, we identified a new mutant because of its small eyes, but there is no obvious variation between heterozygotes and homozygous mutants, which are viable and fully fertile. Detailed histological analysis during embryonic development demonstrated a lens vesicle without any primary fiber cells at E12.5. The lateral regions of the lens vesicle are thickened indicating the beginning of the secondary fiber cell formation. However, because of the missing primary fibers they cannot continue in their development remaining the lens vesicle empty till E15.5. The arrest of lens development at the lens vesicle stage has also major consequences for other ocular tissues: the cornea is thickened and opaque. The retina and the iris grow unlimited leading to closure of the anterior segment by the iris. The retina is folded; its photoreceptor layer is formed, however, the other cell types are severely affected and even the optic nerve does not develop properly. The mutation was mapped to chromosome 10; it affects a gene encoding a connexin-like protein (referred to as Gjf1; G->T point mutation at cDNA position 96, R32Q). It is the first report on Gjf1 in eye development; it is expressed in the posterior part of the lens vesicle, where the primary fiber elongation starts. This mouse mutant offers a new functional candidate gene for microphthalmia-related disorders at the corresponding locus on human chromosome 6 q24 (Puk et al., 2008).

Gene / Allele

Mutation

Phenotype

Own References

Fgf10Aey17S110T: 55 new aaSlit eye, loss of Harderian glandPuk et al., 2009
GjfAey12Arg32Glnmicrophthalmia, cataractPuk et al., 2008
Pax6ADD480Intron 8 G->A, splicingmicrophthalmiaGraw et al. 2005
Haubst et al. 2004
Dames et al., 2010
Pax6Aey11Gln215stopmicrophthalmia
Pax6Aey18Intron 5aG->A, splicing, 86aamicrophthalmia
Pitx3akDeletions in promotermicrophthalmia, no lens, loss of dopaminergic neurons in substantianigraSemina et al. 2000
Smidt et al., 2004
Pitx3eyl139Gly: 121 new aamicrophthalmia, no lens, loss of dopaminergic neurons in substantia nigraRosemann et al., 2010

Mapped mutations with unknown affected genes

Gene / Allele

Phenotype

Own References

Aey69Anophthalmia; mapped to chromosome 3
Aey80Microphthalmia; mapped to chromosome 2, suggested new allele of Pax6; in testing
Cat3vaoMicrophthalmia with vacuolated lens; mapped to chromosome 10Löster et al., 1997
Cat3vlMicrophthalmia with vacuolated lens; mapped to chromosome 10
KTA48Microphthalmia, kinky tail and white spots; mapped to chromosome 12

Reviews by Jochen Graw on eye development:
Graw, J.: Eye Development. Curr. Top. Dev. Biol. 90C, 2010, 343-386
Graw, J.: The genetic and molecular basis of congenital eye defects. Nature Rev. Genet. 4, 2003, 876-888

Mouse mutations affecting the axial length of the eye

In course of our ongoing ENU-mutagenesis screen (in cooperation with the HMGU-Institute of Experimental Genetics mutants were characterized because of their changed axial length of the eye. Using the AC master (Zeiss), we are able to identify very precisely changes in the corneal thickness, the anterior chamber depth, the lens size and the size of the vitreous body. A prerequisite is that all ocular tissues are clear at the time of investigation; however, several mutants suffering from slightly smaller lenses in young animals develop cataract by increasing age. The method has been described in detail in Puk et al., 2006.

IDG-mouse mutants affecting the axial length of the eye

Gene / Allele

Mutation

Phenotype

Own Reference

Col8a2Aca23Gly257AspThinner cornea, enlarged anterior chamberPuk et al., 2009
Cryba2Aca30Ser47ProSmall lens, age-related cataractPuk et al., 2011
Fgf9Aca12Small lensPuk et al., in revision
Lim2Aca47Small lens, age-related cataractPuk et al., in prep.

Mouse mutations causing lens opacities (cataracts)

During different mutagenesis screens, we identified a broad variety of mouse mutants suffering from congenital hereditary lens opacities (referred to as cataracts). Even if the cataractous phenotype differs (polar, nuclear, lamellar, total), there is no genotype-phenotype correlation possible. The genes, which are targeted by the mutations, are Gja8 (coding for connexin50), Cryaa (coding for αA-crystallin), and some of the Cryb/Cryg superfamily (coding for β- and γ-crystallins). For a long time, the crystallins have been considered to be expressed in a lens-specific manner. However, we have demonstrated Crybb2 expression in the brain (Ganguly et al., 2008). Moreover, progress in high-throughput technologies demonstrated that several crystallins are expressed also in other organs, particularly in the brain (Allen Brain Atlas). A review about “crystallins: cataract and beyond” was published recently (Graw 2009). Most of these mutations were identified using a slit lamp; currently we are establishing a screening method based upon the Scheimpflug principle allowing a quantification of the lens opacity.

Mouse cataract mutations

Gene / Allele

Mutation

Phenotype

Own Refernces

CryaaAey7Val124GluNuclear opacityGraw et al., 2001
Cryba1Aey3Trp168Arg splicingProgressive opacityGraw et al., 1999
Crybb2Aey2Val187GluProgressive cataractGraw et al., 2001
Crybb2O377A->T in intron 5, add. 19aa in exon 6Progressive cataractGanguly et al., 2008
CrygatolTrp43Argtotal cataract with vacuoles (severe)Graw et al., 2004
Cryga1NeuAsp77Glydiffuse nuclear opacity (medium)Klopp et al., 1998
CrygbNopc.450ins4 del11; trunc. proteinnuclear opacity (medium)Klopp et al., 1998
CrygcADD4297Val37GluCortical and yellow cataractunpublished
CrygcChl3ΔG141,R142total and lamellar opacity (medium)Graw et al., 2002
CrygcMNU8Trp157stoptotal opacity with vacuoles (medium)Graw et al., 2004
CrygdENU4011Leu45Procloudy nuclear opacity (medium)Graw et al., 2004
CrygdAey4Val76Aspnuclear and cortical cataract (medium)Graw et al., 2002
CrygdENU910Ile90Phediffuse total cataract (mild)Graw et al., 2004
CrygdK10Tyr144stoptotal cataract (medium)Graw et al., 2004
CrygeAey1c.1A->T; new protein (13 kDa)nuclear cataract (medium)Graw et al., 2001
CrygeAey6V76Dcataractunpublished
CrygeENU418Ile4: 153 new aanuclear and lamellar cataract (medium) intron 1: A66G, no splicing of intron 1Graw et al., 2002
CrygeZ2Ile4: 119 new aatotal lamellar cataract (medium) deletion nt 12-21Graw et al., 2004
CrygeNzC89delT; Phe30: 96 new aanuclear & zonular cataract (medium)Klopp et al., 2001
CrygeADD15306Leu45Prozonular cataract (medium)Graw et al., 2004
CrygeENU449Val126Metcapsular opacity (mild)Graw et al., 2004
CrygetTyr144stoptotal opacity (severe)

Klopp et al., 1998

CrygeNslarge deletion in exon 3suture cataract (mild)Unpublished
CrygfRopVal38Gluradial opacity (medium)Graw et al., 2002
Gja8Aey5Val64AlaProgressive cataract (nuclear and zonular)Graw et al., 2001


Mapped mutations with unknown affected genes

RCO-015                     Lamellar cataract; recessive;
homozygous females (?) steril;
mapped to chromosome 7
             

Reviews on mouse cataracts by Jochen Graw
Graw, J.: Mouse Models for Cataracts. J. Genet. 88, 2009, 469-486
Graw, J.: Congenital hereditary cataracts. Intern. J. Dev. Biol. 48, 2004, 1031-1044

Mouse mutations affecting the retina or the optic nerve

In course of our ongoing ENU-mutagenesis screen (in cooperation with the HMGU-Institute of Experimental Genetics mutants affecting the retina or the optic nerve were characterized because of their altered fundus, affected electroretinography (Dalke et al., 2004) or by changes in the virtual optokinetic drum. A prerequisite for all these methods is that all ocular tissues are clear at the time of investigation. Currently, we are going to establish a novel screen for alterations in the retina based upon optical coherence tomography (OCT).

IDG-mouse mutants affecting retina and optic nerve

Gene / AllelePhenotypeOwn Reference
Rs144TNJretinoschisisJablonski et al., 2005

Mapped mutations with unknown affected genes

Ali30Degeneration at the optic nerve head, mapped to chromosome 3
C3H-sightedERG phenotype, mapped to chromosome 11Hölter et al., 2008
Fun6Optic nerve coloboma, mapped to chromosome 19
Fun22Spotted fundus, mapped to chromosome 5

Reviews on mouse mutations affecting the retina by Jochen Graw:
Dalke, C., Graw, J.: Mouse mutants as models for congenital retinal disorders. Exp. Eye Res. 81, 2005, 503-512 

The mice are available either from the breeding colony (please contact Jochen Graw via email: ) or from the sperm archive (please contact the European Mouse Mutant Archive, EMMA; www.emmanet.org).
We are running also the vision screen within the German Mouse Clinic (GMC www.helmholtz-muenchen.de/en/ieg/gmc/). Here we can use more sensitive instruments like


Scheimpflug camera: quantification of the
transparency of the cornea and lens

Optical coherence tomography: imaging of the retina.

Further topics:

Cataracts and ionizing radiation

The lens of the eye is recognized as one of the most radiosensitive tissues in the human body, and it is known that cataracts can be induced by acute doses of less than 2 Gy of low-LET ionizing radiation and less than 5 Gy of protracted radiation. Although much work has been carried out in this area, the exact mechanisms of radiation cataractogenesis are still not fully understood. In particular, the question of the threshold dose for cataract development is not resolved. The combined results of recent mechanistic and human studies regarding induction of cataracts by ionizing radiation indicate that the threshold for cataract development is certainly less than was previously estimated, of the order of 0.5 Gy. For a recent review see Ainsbury et al. (2009). Jochen Graw was also involved as advisor into the corresponding discussion of the German Commission on Radiation Protection (Group of Risk Analysis) (Strahlenschutzkommission, Risiko-Ausschuss); the discussions have been summarized in “Strahleninduzierte Katarakte. Empfehlung der Strahlenschutzkommission mit wissenschaftlicher Begründung, 2009“.

Rat models for eye diseases

Additionally, three rat cataract mutants have been imported and are currently under investigation. The most important one was received from G. Wildner (University Eye Clinic Munich, Germany) and provisionally referred to as white eye (we). In cooperation with the HMGU-Institute of Pathology it turned out immediately that the cataracts precede other disorders, in particular multiple neuroendocrine malignancies, which appear within the first year of life. The endocrine neoplasia is characterized by bilateral adrenal pheochromocytoma, multiple extra-adrenal pheochromocytoma, bilateral medullary thyroid cell neoplasia, bilateral parathyroid hyperplasia, and pituitary adenoma. The recessive cat rat was imported from Mol/Belgium; the CNHL rat was found spontaneously within a colony of learned helplessness (B. Vollmayr, Central Institute of Mental Health, Mannheim, Germany).

Rat cataract mutants

Gene / AlleleMutationPhenotypeOwn Reference
Cdkn1bwec.520-528dup G177: 42 new aaCataract with multiple neuroendocrine tumors (MENX)Fritz et al., 2002
Piotrowska et al., 2004
Pellegata et al., 2006
Shyla et al., 2010

Mapped mutations with unknown affected genes

catCataract (recessive); mapped to chromosome 1
CNHLCataract (mapping in progress)

Human mutations causative for cataracts

ased upon our experience with hereditary congenital cataracts in mouse, we established a cooperative network with a few eye clinics in Germany (Gießen, Göttingen, Regensburg), Canada (Toronto) and India (Chennai, Amritsar and Manipal; the cooperation with the Indian groups was supported for a long time by an Indo-German Contract [BMBF-IND-03]). The table indicates the phenotypes and the molecular lesions of the human cataract mutations identified by our lab within this cooperation.

Human mutations causative for congenital cataracts

Gene

Mutation

Phenotype

Our references

CRYAA Arg21LeuCongenital dominant cataractGraw et al., 2006
CRYAA Gly98ArgProgressive cataractSanthiya et al., 2006
CRYBA4 Leu69ProCongenital lamellar cataractBillingsley et al., 2006
CRYBB2 Trp59CysTotal cataractSanthiya et al., 2010
CRYBB2 Asp128ValCongenital dominant cataractPauli et al., 2007
CRYBB2 Trp151CysCentral nuclear cataractSanthiya et al., 2004
CRYGCArg168Trplamellar cataractSanthiya et al., 2002
CRYGD Pro23Thrlamellar cataractSanthiya et al., 2002
CRYGDArg77SerAnterior-polar coronary cataractRoshan et al., 2010
CRYGD Trp156Stopcentral nuclear cataractSanthiya et al., 2002
GJA3Thr19MetPosterior-polar cataractSanthiya et al., 2010
GJA8I247MRare polymorphism, no cataract-causing mutationGraw et al., 2009
GJA8C767insG; 255Ile: 123 new aaSchmidt et al., 2008

Churchill, A., Graw, J.: Clinical and Experimental Advances in Congenital and Paediatric Cataracts. Phil. Transact. Royal Soc. Ser. B. 366, 2011, 1234-1249.

Cataracts and ionizing radiation

The lens of the eye is recognized as one of the most radiosensitive tissues in the human body, and it is known that cataracts can be induced by acute doses of less than 2 Gy of low-LET ionizing radiation and less than 5 Gy of protracted radiation. Although much work has been carried out in this area, the exact mechanisms of radiation cataractogenesis are still not fully understood. In particular, the question of the threshold dose for cataract development is not resolved. The combined results of recent mechanistic and human studies regarding induction of cataracts by ionizing radiation indicate that the threshold for cataract development is certainly less than was previously estimated, of the order of 0.5 Gy. For a recent review see Ainsbury et al. (2009). Jochen Graw was also involved as advisor into the corresponding discussion of the German Commission on Radiation Protection (Group of Risk Analysis) (Strahlenschutzkommission, Risiko-Ausschuss); the discussions have been summarized in “Strahleninduzierte Katarakte. Empfehlung der Strahlenschutzkommission mit wissenschaftlicher Begründung, 2009“.