phop core fall 2016 intro to genetics and ocular development · 2017-09-08 · gross anatomy of the...

PHOP Core Fall 2016 Intro to Genetics and Ocular Development

D. C. Otteson PhD UHCO 1

Gross Anatomy of the Eye

Graw, Eye Development. (2010) inCurrent Topics in Developmental Biology (Volume 90) p. 343. 2

From the National Eye Institute http://www.nei.nih.gov/photo/

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Body Axis Terminology

Caudal(posterior)

Ventral

Dorsal

Medial

Lateral

Rostral(anterior)

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Eye-specific Axis Terminology

Dorsal / Superior

Ventral / Inferior

Posterior Anterior

Nasal

Temporal

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http://www.becomehealthynow.com/article/bodyembryo/789/1

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GastrulationInduction and cell migration forms three germ layers

Ectoderm

Mesoderm

Endoderm

Gastrulation initiates at posterior end

Figure from Developmental Biology by Scott Gilbert. Sinauer Associates, Inc.

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(From Martin, Neuroanatomy Text and Atlas, Elsevier press)

Neural Crest

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Neural tube segmentation: brain vesicles

(From Martin, Neuroanatomy Text and Atlas, Elsevier press)

Mouse: day 8 post fertilization day 10-11 post fertilizationHuman: week 4 week 5

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Growth of cranial nerves

Dekaban, A.S. and Sadowsky, D, Ann. Neurology, 4:345-356, 1978

3-vesiclestage 5-vesicle stage

Telencephalongrows

Cortical maturation and expansion

NOT TO SCALE !!!!

EYE

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Graw (2010) in Seminars in Developmental Biology 90: 343.

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Primary Eye Field in Anterior Neural PlateEyes develop from cells in anterior neural plate

Eye field contains cells competent to form eye structures

Not all cells will actually contribute to eye structures

Early genes important in specification of eye fields:

Pax6, Rax/Rx, Six3, Tbx3, Six6, Otx2, Lhx2, TllSpecies: Mouse E7 ~Human E17

http://www.med.unc.edu/embryo_images/ 12

Neurulation: Folding of Neural Plate

Neural plate grows rapidly

Eye fields in rostral neural plate expand

Eye fields separate at midline

Secreted sonic hedgehog (SHH) from notocord specifies ventral midline

http://www.med.unc.edu/embryo_images/

Species: Mouse E8 ~ Human E21View: Frontal

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PAX2 specifies ventral optic cup and optic stalkMutations: loss of ventral diencephalon, optic chiasm, ventral optic nerve coloboma, kidney and ear defectsHomozygous: lethal: no chiasm, no uritogenital tract

SHH secreted from notocord early embryoinduces ventral and midline structures in neural platerequired for separation of eye fieldsMutations: holoprosencephaly, cyclopia, midline facial defects, microcephaly

PAX6 specifies dorsal and lateral structuresneural retina, RPE, lens, cornea (+ non-ocular structures)Mutations: heterozygous: aniridia, glaucomahomozygous: (lethal) anophthalmia, failure to form nasal passage, brain defects

Separation of Eye FieldsSHH represses PAX6, induces PAX2

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Cells in Eye Fields Invaginate Forming Optic Grooves (a.k.a. optic sulci, optic pits)

Mouse E8.5 ~ Human E24 Fronto-Lateral

Mouse E8 ~ Human E22 View: Frontal


Neural tube, rat development showing optic vesicles

Zhang, Fu, Barnstable (2002) Molec. Neurobiol. 26:137-152.

Rat embryo, E11.

Rostral view of neural tubeHead ectoderm removed

Arrows show anterior neuropore

ov=optic vesicles

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Species: Mouse E8.5 to 9 (~Human E25)


Optic vesicle/Lens induction

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Mouse E 9 (~Human E28) Mouse E10 (~ Human E29)


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Species: Mouse E11 (~Human E36)View: Coronal

Lens placode invaginates to form lens vesicle

Invagination of optic vesicle forms bilayered optic cup

Lens vesicle pinches off surface ectoderm

Overlying ectoderm becomes cornea


Lens Vesicle and Optic Cup

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Species: Mouse E 11 (~Human E36)View: Coronal Cut


Retina/RPEPax6 (+Mitf in RPE)

Lens vesiclePax6, Sox2/3

Optic StalkPax2

Irido-pupillary MembraneFrom neural crest

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Lens DevelopmentPrimaryFiber elongation

SecondaryFiber elongation

Graw (2010) in Seminars in Developmental Biology 90: 343.

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A model of a genetic switch composed of SOX2/SOX3 and Pax6, which regulates initiation of lens development.

Kamachi Y et al. Genes Dev. 2001;15:1272-1286

©2001 by Cold Spring Harbor Laboratory Press 22

Lens maturationProliferation of cells at the equator Elongation of cells at bowFiber cells

nuclei are gone in central lenscells remain connected by gap junctions

Species: Human 8 Weeks http://www.med.unc.edu/embryo_images/ 23

Lens Fiber Cells

Song et al J Clin Invest. 2009;119(7):1837–1848

Scanning electron micrographs of bovine lens fiber cells

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induced by lens• outer epithelial layer forms from surface ectoderm • inner layers primarily from neural crest cells

Image of cornea from: T. Caceci (2001) Anatomy and Physiology of the Eye V2.0


Cornea

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RPE: Outer pigmented layer becomes relatively thinnerSingle cell layer RPE cells express PAX6 and MITF. Expression of MITF helps

specify RPE identity and this transcription factor directly regulates genes that are responsible for pigment formation in RPE.

RETINA: Inner, neural portion thickensPseudostratefied epitheliumDifferentiation begins at ~E11.5

Mouse Day E14 (~ Human 7 weeks) http://www.med.unc.edu/embryo_images/

RPE

Ventricle/Sub-retinalSpace

Retina

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The tunica vasculosa lentis From human fetus (a) Hyaloid artery (b) Posterior ciliary artery

From Duanes Ophthalmology 2006 Chapter 15: Lens, Kleiman and Worgul

Vasculature

Mouse(Gerhardt et al., 2003)

Superficial Plexus

Deep Plexus

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Iris and Ciliary Body Formation

Human ~15 Weekshttp://www.med.unc.edu/embryo_images/

Human 8 Weeks

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Ittner et al. Journal of Biology 2005 4:11

Neural Crest Derivatives in Eye

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Neural Crest

Ittner et al. Journal of Biology 2005 4:11 30

Human: 8 Weeks

Eyelids

•begin to form at end of embryonic period

•fuse at the start of 2nd trimester

•reopen at the beginning of the 3rd trimester

Human: 10 Weeks


Loci for Inherited Retinal Disease

RetNet; Stephen P Daiger PhDThe University of Texas Health Science Center, Houston, Texas

http://www.sph.uth.tmc.edu/RetNet

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genetics

DNA Replication and Inheritance

The basis of perpetuation of life and transmission of traits/genes

8-34

Inherited vs. Non-inherited Mutations

• Germ-line mutations lead to inherited mutations– Occurs in germ line tissue– If mutation passes into gametes (egg, sperm), it will be passed

on to next generation. • e.g. sickle cell anaemia• Retinitis pigmentosa,• Keratoconus

• Somatic mutations occur in somatic tissues – Population of identical cells derived from a single mutated

somatic cell is a clone– Often results in a patch of phenotypically mutant cells– Not inherited – Many tumours result from somatic mutation– Two-hit theory of cancer e.g. retinoblastoma

8-35

Heritable Genetic Changes

Single gene mutation Change in allele of a gene• Protein not expressed or• Protein non-functional or• Protein acquires novel/harmful/beneficial functions• Contributes to evolution

Chromosome mutation: multiple genes Changes in segments of chromosome Deletion, duplication, inversion, translocationLoss/duplication of whole chromosome

Epigenetic Changes

DNA modifications that can permanently change gene expression

Genes + Environment= Phenotype

genes environmentphenotype

Contributions from genotype/environment varies with disease

Some diseases have both genetic and environmental components

e.g. macular degeneration; cataract; heart disease

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8-37

Classifying Mutations : Phenotypic Aspects

• Morphological mutations– Visible/measurable change in phenotype– e.g. eye color; ocular coloboma

• Lethal mutations– Heterozygous: normal– Homozygotes: do not survive– e.g. Cystic Fibrosis= mutation in CFTR gene–

• Conditional mutations – Phenotype only presents under specific conditions – e.g. predisposition for developing disease: diabetes, heart

disease, cancer: can change life-style and reduce risk

• Biochemical mutations– Change in enzyme, biochemical pathway components– e.g. Albinism (inability to synthesize melanin) mutations in

Tyrosinase and other enzymes of melanin biosynthesis

8-38

Mutations: Mechanistic Aspects

• Silent mutations/Polymorphisms• Change in DNA sequence:

• doesn’t change sequence of protein (synonymous) – or • occurs in non-critical region of gene – Used for:– DNA “fingerprinting” – paternity analysis– genetic mapping (linkage analysis)

Consequences of point mutations

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Gametes get one copy of each chromosome Gene R is on a separate chromosome from A and B and segregates independently of A and B

All allelic combinations are found: AB +R AB + r ab + R ab + rTherefore, R is not linked to the A or B genes

Genes A and B are on the same chromosomeParental allelic combinations inherited together (AB or ab)A and B genes are linked

Independent assortment of chromosomes during meiosis

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Predicting Patterns of Inheritance: Punnett Square

Genotype:B/B B/b b/b 1 : 2 : 1

Phenotype:Black brown

3 : 1

diploid

diploid

haploid

Shown: two alleles of a single gene

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8-43

Mendelian Non-MendelianAutosomal EpigeneticSex-linked MitochondrialRecessive ImprintingDominant MultifactorialMonogenic

Other including complexSyndromicSemi-dominant/co-dominant Sporadic/spontaneousDigenic

Patterns of Inheritance

• Autosomal inheritance

– Based on the variation of single genes on regular chromosomes (autosomes)

• Sex-linked inheritance

– Based on the variation of single genes on sex-determining chromosomes

• Cytoplasmic inheritance

– Based on the variation of single genes on organelle’schromosomes (e.g. Mitochondrial)

Patterns of Inheritance

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Patterns of Inheritance: Recessive

Appearance of macula in Usher syndrome

Fundus pigmentation in Usher syndrome

Usher Syndrome I (human) USHIB mutations in MYO7A (Myosin7A)• Heterozygous (-/+) normal• Homozygous (-/-)

• profound congenital hearing impairment

• unintelligible speech• early retinitis pigmentosa (<10 yrs• vestibular dysfunction• defects in cilia (photoreceptor

connecting cilium; hair cells)


•How to detect this pattern in patients?

•Can you distinguish this from spontaneous mutation?

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Parents: heterozygous

(+/-)

normal

+CSNB

-

normal

+CSNB

-Genotypes: +/+ +/- -/-Ratios (genotype)PhenotypesRatios (phenotypes)


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Parents: heterozygous

(+/-)

normal

+CSNB

-

normal

++/+ -/+

CSNB

-+/- -/-

Genotypes: +/+ +/- -/-Ratios (genotype)PhenotypesRatios (phenotypes)

1 2 1Normal Normal mutant

13


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Parents:Heterozyous (+/-)

Homozygous (+/+)+ -

+

+Genotypes: +/+ +/- -/-Ratios (genotype)PhenotypesRatios (phenotypes)


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Parents:Heterozyous (+/-)

Homozygous (+/+)+ -

+ +/+ -/+

+ +/+ -/+Genotypes: +/+ +/- -/-Ratios (genotype)PhenotypesRatios (phenotypes)

1 1 0Normal Normal

1 0


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• Recessive genes are not major contributors in human genetic disease unless:

• highly consanguineous group• frequency of mutant allele in general population is

high• Difficult to determine if this is inherited or

spontaneous if only one affected individual


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Patterns of Inheritance: Dominant

• Dominant: Mutant allele is fully expressed and masks the expression of the allele.

• With true dominant, individuals heterozygous and homozygous for the mutated allele show the same phenotype.

• Haplo-insufficient: Absence of one allele results in intermediary phenotype; often referred to as semi-dominant

• Co-dominant: both alleles are fully expressed• e.g. blood groups (A, AB, B, O)

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genetics53

Patterns of Inheritance: DominantRetinitis Pigmentosa

Phenotype: • Constriction of the visual fields• Night blindness• Fundus changes• “bone spicule” lumps of pigment• Photoreceptor Degeneration• Variable age of onset

Multiple patterns of inheritance• Autosomal dominant• Autosomal recessive• X-linked

More than 3100 distinct mutations in 56 genes in have been identified(Daiger et al 2013 Clinical Genetics 84:132.)

From: www.stlukeseye.com/

genetics54

Male unaffected

Male affected

Female

unaffected

Female affected


genetics55


ParentsHeterozyous (+/-)

Homozygous (+/+) + +

+-


genetics56


1 1 0Normal Mutant Mutant

1 1 0


Homozygous (+/+) + +

+ +/+ +/+- +/- +/-


genetics57

Patterns of Inheritance: Semi-Dominant HaploinsufficientAniridia

• Mutations in transcription factor Pax6• Haploinsufficient• Homozygous lethal• Heterozygotes: anterior segment

malformations:• aniridia• corneal clouding with variable

iridolenticulocorneal adhesions • Peters anomaly (central corneal leukoma,

absence of the posterior corneal stroma and Descemet membrane, and a variable degree of iris and lenticular attachments to the central aspect of the posterior cornea)

• foveal hypoplasia • glaucoma• autosomal dominant keratitis

A normal eye is pictured above. Below is the eye of a child with aniridia, a congenital eye disorder. People born with the disease have no iris and are generally legally blind.CREDIT ANIRIDIA FOUNDATION

genetics58



+ -

+-

Patterns of Inheritance: Semi-Dominant / Haploinsufficient

genetics59


1 2 1Normal Mutant More severe

1 2 1


+ -

+ +/+ +/-- +/- -/-

Patterns of Inheritance: Semi-Dominant Haploinsufficient

genetics60

• Mutation on X chromosome

• Females: XX

• Males: XY

• Mutation/disease phenotype manifests in males

• Females are carriers, typically normal– But may manifest mosaic defects

• Generation skipping pattern

Patterns of Inheritance: X-linked

genetics61

Examples of X-linked Retinal Diseases

OA1 X-linked ocular albinism

RP23,RP6, X-linked Retinitis Pimentosa

RS1 Retinoschisis

OPA2 X-linked optic atrophy

NDP Norrie disease; familial exudative vitreoretinopathy

Colorblindness (mutations in red/green opsin genes):

OPN1SW protan (red deficient)

OPN1LW deutan (green deficient)

blue cone monochromacy (red & green deficient)

X-linked Diseases: X-inactivation

X chromosomes:Males have 1Females have 2

How to maintain same level of transcription (RNA levels) from X chromosome genes?

Random X-inactivation(Lyonization) in females

after Mary Lyon

e.g. coat color in cats 62

X-inactivation of B-gal transgene in retina

http://mentor.lscf.ucsb.edu/course/winter/mcdb101b/x-inactivation/xinactivation.h

X-inactivation in the cornea

John Westhttp://www.cip.ed.ac.uk/gallery/index.htm

Mutant phenotype present in males x* Y Carrier females may show mosaic defects Xx*Skips generations

carrier

XY Xx*

XX x*Y XY Xx* XY x*Y XX

XY Xx* Xx* Xx* Xx* XY x*Y XX XX x*Y Xx* Xx* XY


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genetics66

Genotypes: X*X XX X*Y XYRatios (genotype)PhenotypesRatios (phenotypes)

Normal*carrier

Normal Affected Normal

ParentsFather affected X* Y

X

X


genetics67


0 1 0 1

Normal*carrier


1 0 0 1

ParentsFather affected X* Y

X X*X XY

X X*X XY


genetics68


Normal*carrier


ParentsMother-carrier X Y

X*

X


genetics69


1 1 1 1

Normal*carrier


1 1 1 1

ParentsMother-carrier X Y

X* X*X X*Y

X XX XY


Mitochondrial diseases affecting the eye

8-70

Ophthalmic manifestations of mitochondrial diseases:cataract, retinopathy, optic atrophy, cortical visual loss, ptosis and ophthalmoplegia

• Leber’s Hereditary Optic Neuropathy (LHON)

• Kearns-Sayre Syndrome (KSS)

• Mitochondrial Encephalopathy, Lactic Acidosis Stroke (MELAS)

• Myoclonic Epilepsy and Ragged Red Fiber myopathy (MERRF)

genetics71

Patterns of Inheritance: MitochondrialLeber Optic Atrophy

• Presents in mid-life as acute or sub acute central vision loss leading to central scotoma and blindness

• Associated with many missense mutations in the mtDNA

• Mutations can act autonomously or in association with other mt mutations

• Final visual acuity can range from 20/50 to no light perception, depending on severity of the mutations

Leber Optic Neuropathy with temporal optic nerve pallor in both eyes.

http://www.revoptom.com/continuing_education/tabviewtest/lessonid/108143/

Healthy optic disc

http://www.intechopen.com/books/the-mystery-of-glaucoma/

8-72

http://www.usc.edu/dept/mda/180evolution/IMAGES/wmho.html

8-73

Patterns of Inheritance: Mitochondrial

Mitochondrial mutations: •inherited ONLY from the mother

•NEVER from the father

ROM1/ROM1; ROM1/ROM1; +/ROM1 ; +/ROM1;RDS/RDS +/RDS RDS/RDS +/RDS

ROM1/ROM1; ROM1/ROM1; +/ROM1; +/ROM1;RDS/+ +/+ RDS+ +/+

+/ROM1; ROM1/+; +/+; +/+;RDS/RDS +/RDS RDS/RDS +/RDS

ROM1/+; ROM1/+; +/+; +/+;+/RDS +/+ RDS/+ +/+

Mode of inheritance: multigenicROM1/+; RDS/+ x ROM1/+; RDS/+

ROM1; RDS ROM1; + +; RDS +; +

ROM1; RDS

ROM1; +

+; RDS

+;+

Genotypes 1:2:1:2:4:2:1:2:1Phenotypes: 1:3:2:1:9

phop core fall 2016 intro to genetics and ocular development · 2017-09-08 · gross anatomy of the...

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