Trinucleotide repeat disorders: Huntington DiseaseTrinucleotide repeat disorders: Huntington DiseaseYou MUST know material on course page objectives Review pages 217-220 in Gelerhter/Collins/Ginsburg text

“We used to think our fate is in the stars. Now we know, in large measure, our fate is in our genes.”

- James Watson

Types of Mutations

• Single base pair substitutionsSingle base pair substitutions• missense, nonsense, splice sitemissense, nonsense, splice site

• DeletionsDeletions

• DuplicationsDuplications

• InversionsInversions

• InsertionsInsertions

• Repeat ExpansionsRepeat Expansions

Outline of Lecture

• Overview of types of trinucleotide repeat disordersOverview of types of trinucleotide repeat disorders

• Huntington disease Huntington disease

• Molecular testing for trinucleotide repeat disordersMolecular testing for trinucleotide repeat disorders

• Ethical, legal, and social implications of predictive Ethical, legal, and social implications of predictive testingtesting

• Pathophysiology of trinucleotide disordersPathophysiology of trinucleotide disorders

• Discussion with visiting patient and her familyDiscussion with visiting patient and her family

Trinucleotide repeat: a type of short tandem repeat

• The size of repeat region varies between individuals and is polymorphic in normal individuals

• For some trinucleotide repeats, when the number of repeats exceeds a certain threshold, a neurological disease results

7 repeats

8 repeats

CAG

Timeline of Gene Discoveries for Trinucleotide Repeat Disorders

• 19911991 Fragile X MR syndrome, SBMAFragile X MR syndrome, SBMA

• 19921992 Myotonic dystrophyMyotonic dystrophy

• 19931993 Huntington disease, SCA1, FRAXE, DRPLA/HRHuntington disease, SCA1, FRAXE, DRPLA/HR

• 19941994 Machado-Joseph disease/SCA3Machado-Joseph disease/SCA3

• 19961996 SCA2, Friedreich ataxia, SCA6SCA2, Friedreich ataxia, SCA6

• 19971997 SCA7SCA7

• 19991999 SCA10, SCA5, SCA4SCA10, SCA5, SCA4

• 2000s2000s Other SCAs, Psychiatric disorders?Other SCAs, Psychiatric disorders?

Major Features of Most Trinucleotide Repeat Disorders

• Neurological/cognitive symptomsNeurological/cognitive symptoms

• Many are autosomal dominant with variable expressionMany are autosomal dominant with variable expression(exceptions: Friedreich ataxia (recessive); Spinobulbar muscular atrophy, (exceptions: Friedreich ataxia (recessive); Spinobulbar muscular atrophy,

Fragile X syndrome, FRAXE MR - X-linked recessive)Fragile X syndrome, FRAXE MR - X-linked recessive)

• Later age of onsetLater age of onset(exceptions: congenital myotonic dystrophy, Fragile X syndrome, FRAXE)(exceptions: congenital myotonic dystrophy, Fragile X syndrome, FRAXE)

• Meiotic and mitotic instability with some degree of anticipation in Meiotic and mitotic instability with some degree of anticipation in many of the conditionsmany of the conditions

Why Know About Trinucleotide Repeat Disorders?

• Over 15 genetic different disorders that cause a significant Over 15 genetic different disorders that cause a significant proportion of inherited neurological disease in adults and the proportion of inherited neurological disease in adults and the most common cause of inherited mental retardation in males most common cause of inherited mental retardation in males (Fragile X syndrome)(Fragile X syndrome)

• Molecular diagnosis is available for diagnostic confirmation, Molecular diagnosis is available for diagnostic confirmation, predictive testing, prenatal testing, preconception testing, and predictive testing, prenatal testing, preconception testing, and preimplantation diagnosis.preimplantation diagnosis.

• Genetic counseling issues are complex and important to Genetic counseling issues are complex and important to understand as are related ethical issuesunderstand as are related ethical issues

FMR1 in FRAXA Mental Retardation

• 17 exon FMR1 gene cloned in 199117 exon FMR1 gene cloned in 1991

• Highest FMR1 expression in neurons Highest FMR1 expression in neurons

and spermatogoniaand spermatogonia

• FMR1P associates with translating FMR1P associates with translating

ribososmes and is involved in ribososmes and is involved in

nucleocytoplasmic shuttlingnucleocytoplasmic shuttling

• Approximate repeat ranges:Approximate repeat ranges:• 6-45 CGGs (0-3 AGGs) 6-45 CGGs (0-3 AGGs) Unmethylated, Unmethylated, StableStable, Normal, Normal

• 46-60 CGGs (0-2 AGGs) 46-60 CGGs (0-2 AGGs) Unmethylated, Unmethylated, +/- Instability,+/- Instability, Normal Normal

• 60-200 CGGs 60-200 CGGs Unmethylated, Unmethylated, Premutation - UnstablePremutation - Unstable, Normal, Normal

• > 200 CGGs -> 200 CGGs - Methylated, no FMR1, UMethylated, no FMR1, Unstablenstable, , AffectedAffected

Myotonic

Dystrophy

• Common adult-onset muscular dystrophy (1 in 8000 in Caucasians, 1 in 475 in Quebec • Autosomal dominant - 19q13.2-.3

• Expansion of 3’ UTR CTG repeat in 15 exon myotonic dystrophy protein kinase gene (DMPK)

– 5 - 37 normal

– 50 - 90 mild - cataract, balding, limited muscle involvement, > 50 years

– 90 - 1000 classic muscle weakness, myotonia, cataracts, onset 20-30 years

– > 1000 often congenital,hypotonia, developmental delays

• Instability of repeats - 10% expansion, 3% contraction.

• Congenital DM always due to maternal expansion

Anticipation

Increasing severity and/or decreasing age of Increasing severity and/or decreasing age of onset of an inherited disease in successive onset of an inherited disease in successive

generations within a family.generations within a family.

• Friedreich Ataxia: Friedreich Ataxia:

– Autosomal recessive Autosomal recessive progressive neurological disorderprogressive neurological disorder, onset < , onset < 25 years with ataxia due to expansion of GAA repeat in 25 years with ataxia due to expansion of GAA repeat in intronintron of of FRATAXIN geneFRATAXIN gene

• Spinocerebellar Ataxia (many types):

– Autosomal dominant Autosomal dominant progressive neurological disorderprogressive neurological disorder characterized by ataxia usually in the 3rd or 4th decade due to characterized by ataxia usually in the 3rd or 4th decade due to expanded CAG repeat in expanded CAG repeat in coding exonscoding exons of SCA genes of SCA genes

• Myotonic Dystrophy:Myotonic Dystrophy:

– Autosomal dominant Autosomal dominant progressive neurological disorderprogressive neurological disorder with with variable expression and anticipation due to expansion of variable expression and anticipation due to expansion of 3’ UTR3’ UTR region of DMPK generegion of DMPK gene

• Fragile X syndrome:Fragile X syndrome:

– X-linked recessive X-linked recessive mental retardationmental retardation syndrome due to syndrome due to expansion of CGG repeat in expansion of CGG repeat in 5’ UTR5’ UTR region of FRAXA gene region of FRAXA gene

5’ UTR exon intron exon intron exon 3’ UTR

CGG

**FRAXAFRAXE

GAA

FA

CAG

**HDSCAsSMBA

DRLPA

CTG

MD

Trinucleotide repeat disorders can involve expansions of various repeats in coding and non-coding regions of the gene

Dr. George Huntington (1850 -1916)

• Became interested in hereditary chorea in 1871 • Wrote his seminal paper on this disease when he was 22 in 1872• Was a general practitioner - never on a medical faculty

Individual with Huntington Disease

Huntington Disease

• Average age of onset 40 years (range 2- >80 years); Average age of onset 40 years (range 2- >80 years); Progression over 10-25 years.Progression over 10-25 years.

• Movement disorderMovement disorder: choreic movements, twitching, balance : choreic movements, twitching, balance problems, tracking problems, slowing of voluntary problems, tracking problems, slowing of voluntary movement. In juvenile HD and in late stages of HD, rigidity movement. In juvenile HD and in late stages of HD, rigidity and dystonia. and dystonia.

• Cognitive dysfunctionCognitive dysfunction: problem solving, cognitive flexibility, : problem solving, cognitive flexibility, short term memory, visuospatial functioning; progression to a short term memory, visuospatial functioning; progression to a global subcortical dementiaglobal subcortical dementia

• Personality changes:Personality changes: depression, apathy, irritability, impulsive depression, apathy, irritability, impulsive behavior, affective disorders, rarely psychoses, increased behavior, affective disorders, rarely psychoses, increased alcohol use in early stages, increased suicide ratealcohol use in early stages, increased suicide rate

Incidence of Incidence of Huntington Disease per 100,000 PeopleHuntington Disease per 100,000 People

African BlacksAfrican Blacks 0.060.06

South African WhitesSouth African Whites 2.42.4

JapanJapan 0.380.38

FinlandFinland 0.50.5

Hong KongHong Kong 2.52.5

American BlacksAmerican Blacks 3-73-7

Western EuropeansWestern Europeans 77

American WhitesAmerican Whites 7-107-10

North SweedenNorth Sweeden 144144

Tasmania, AustraliaTasmania, Australia 174174

Moray Firth, Scotland*Moray Firth, Scotland* 560 (5 people in <1000)560 (5 people in <1000)

Zulia, VenezuelaZulia, Venezuela 700700

Woodrow Wilson Guthrie

Woody GuthrieDiagnosed in 1952, age 40 yearsDied15 years later at age 55 years

During 17 year career wrote more than 1,000 songs and left behind 2,500 lyrics. Near the end of his life he could only use “yes” and “no” cards to communicate.

Woody

Arlo

Abe

mood swingsbehavioral disturbances, hyperreflexia, memory impairment, increased clumsiness, impairment of voluntary movements, eye movement abnormalities

dysarthriachoreagait abnormalities

bradykinesia, rigidityglobal dementia, dystonia, dysphagia

incontinence, wasting, aspiration, bed riddendeath

Transitional

Early

Middle

Late

0-3

3-5

8-10

15-25

Phase Years Symptoms

Pathology of Huntington Disease

• Brain atrophy involving caudate nucleus and putamen with loss of Brain atrophy involving caudate nucleus and putamen with loss of striatal neurons and secondary atrophy of globus pallidusstriatal neurons and secondary atrophy of globus pallidus

• Dilation of lateral and third ventriclesDilation of lateral and third ventricles

• Additional atrophy throughout cortex, especially frontal and Additional atrophy throughout cortex, especially frontal and parietal lobesparietal lobes

• Loss of small neurons precedes larger neurons with neurons Loss of small neurons precedes larger neurons with neurons utilizing GABA and enkephalin or substance P preferentiallyutilizing GABA and enkephalin or substance P preferentially

• Fibrillary gliosisFibrillary gliosis

Huntington diseaseHuntington disease

Huntington Disease

• IT15/Huntingtin gene on 4p16.3 cloned in 1993IT15/Huntingtin gene on 4p16.3 cloned in 1993

• Disease mutation - CAG expansion in exon 1 Disease mutation - CAG expansion in exon 1 – Repeat numberRepeat number OutcomeOutcome

– 10-28:10-28: normal, no transmission of HDnormal, no transmission of HD

– 29-35:29-35: normal, paternal meiotic instabilitynormal, paternal meiotic instability

– 36-39:36-39: reduced penetrance (25%: 36 repeats, 90%: 39 repeats)reduced penetrance (25%: 36 repeats, 90%: 39 repeats)

– 40-100+:40-100+: will develop HD if person lives long enoughwill develop HD if person lives long enough

• Increased meiotic instability in males - Increased meiotic instability in males - Paternal transmission of Paternal transmission of expanded allele associated with over 3/4 of juvenile diseaseexpanded allele associated with over 3/4 of juvenile disease

• Encodes 348 kD huntingtin protein which is a target for caspase 3, a Encodes 348 kD huntingtin protein which is a target for caspase 3, a protease associated with neuronal apoptosisprotease associated with neuronal apoptosis

N N N HD N N

So the HD disease was cloned . . . now what?

• Provide precise, rapid diagnosis including prenatal Provide precise, rapid diagnosis including prenatal and predictive testingand predictive testing

• Improve molecular understanding of pathophysiologyImprove molecular understanding of pathophysiology• Increase ethical, psychosocial, and legal concernsIncrease ethical, psychosocial, and legal concerns• Improve medical managementImprove medical management• Develop novel, targeted therapiesDevelop novel, targeted therapies

HD DNA testing:

Diagnosis confirmationDiagnosis confirmation Prenatal diagnosisPrenatal diagnosis Predictive diagnosisPredictive diagnosis

psychosocial impactpsychosocial impact employment concernsemployment concerns insurance issuesinsurance issues

Molecular Detection of Trinucleotide Repeats: Determine the size of the repeat

• ‘‘Short’ Repeats (eg. HD):Short’ Repeats (eg. HD):– PCR based typing of alleles using primers directly PCR based typing of alleles using primers directly

flanking repeat region - With appropriate controls and flanking repeat region - With appropriate controls and size marker the size can be determined accuratelysize marker the size can be determined accurately

• Long repeats:determine size of the repeatLong repeats:determine size of the repeat– PCR and Southern blot analysisPCR and Southern blot analysis

• Methlylation status (eg. Fragile X)Methlylation status (eg. Fragile X)• ImmunoassaysImmunoassays

220

20

180

160

140

120

100

80

A,B B,D A,C A,D B,C B,D C,D A,B

ABC D

17244641

Predictive or Presymptomatic Testing

How should cost and benefit be defined?How should cost and benefit be defined? Many psychosocial issuesMany psychosocial issues Who has a right to be tested or not to be tested? Who has a right to be tested or not to be tested?

Who decides?Who decides? Who has a right to the results?Who has a right to the results? Probabilistic vs. DeterministicProbabilistic vs. Deterministic

susceptibility versus certainty of acquiring manifesting susceptibility versus certainty of acquiring manifesting symptoms of a disease or disordersymptoms of a disease or disorder

PUBLIC HEALTH CODE (EXCERPT) Act 368 of 1978PUBLIC HEALTH CODE (EXCERPT) Act 368 of 1978333.21072a 333.21072a

(1) A health maintenance organization shall (1) A health maintenance organization shall notnot require an enrollee or his or require an enrollee or his or her dependent or an asymptomatic applicant for coverage or his or her her dependent or an asymptomatic applicant for coverage or his or her asymptomatic dependent to do either of the following: asymptomatic dependent to do either of the following:

(a) Undergo genetic testing before issuing, renewing, or continuing a health (a) Undergo genetic testing before issuing, renewing, or continuing a health maintenance organization contract. maintenance organization contract.

(b) Disclose whether genetic testing has been conducted or the results of (b) Disclose whether genetic testing has been conducted or the results of genetic testing or genetic information. genetic testing or genetic information.

THE INSURANCE CODE OF 1956 (EXCERPT) Act 218 of 1956THE INSURANCE CODE OF 1956 (EXCERPT) Act 218 of 1956500.3407b500.3407b

(1) An expense-incurred hospital, medical, or surgical policy or certificate (1) An expense-incurred hospital, medical, or surgical policy or certificate delivered, issued for delivery, or renewed in this state shall not require an delivered, issued for delivery, or renewed in this state shall not require an insured or his or her dependent or an asymptomatic applicant for insurance insured or his or her dependent or an asymptomatic applicant for insurance or his or her asymptomatic dependent to do either of the following:or his or her asymptomatic dependent to do either of the following:

(a) Undergo genetic testing before issuing, renewing, or continuing the (a) Undergo genetic testing before issuing, renewing, or continuing the policy or certificate in this state. policy or certificate in this state.

(b) Disclose whether genetic testing has been conducted or the results of (b) Disclose whether genetic testing has been conducted or the results of genetic testing or genetic information.genetic testing or genetic information.

Positive outcomes: Predictive DNA Tests• Personal:Personal:

• Positively influence life decisions and long term planningPositively influence life decisions and long term planning

• Reduce morbidity and mortality by specific monitoring/surveillance, Reduce morbidity and mortality by specific monitoring/surveillance, interventional risk reduction medical or surgical care, and/or interventional risk reduction medical or surgical care, and/or lifestyle modificationslifestyle modifications

• Family:Family:• Inform own reproductive choicesInform own reproductive choices

• Enable informed health care choices of family membersEnable informed health care choices of family members

• Society:Society:• Improve medical care and health for populations at riskImprove medical care and health for populations at risk

• Help prioritize use of medical resourcesHelp prioritize use of medical resources

• Facilitate development of molecular based therapiesFacilitate development of molecular based therapies

HD Presymptomatic Testing:

Recommended Protocol(HDSA):Recommended Protocol(HDSA): genetic counselinggenetic counseling neurological evaluationneurological evaluation psychological/psychiatric evaluationpsychological/psychiatric evaluation DNA test if no concernsDNA test if no concerns identification of local support personidentification of local support person test results given in persontest results given in person follow-up visitsfollow-up visits no testing of presymptomatic minorsno testing of presymptomatic minors

Affected

Unaffected

Desirable Characteristics of Predictive Genetic Tests

• ““Negative” in unaffected individuals and people who Negative” in unaffected individuals and people who aren’t predisposed to the diseasearen’t predisposed to the disease– (eg. specific for the disease, low false positives)(eg. specific for the disease, low false positives)

• ““Positive” in affected individuals and those at increased Positive” in affected individuals and those at increased risk of the disease risk of the disease – (eg. sensitive for the disease, low false negatives)(eg. sensitive for the disease, low false negatives)

• ““Positive” test reflects prognosis and/or directs clinical Positive” test reflects prognosis and/or directs clinical managementmanagement

• Affordable, robust, reliable, reproducible!Affordable, robust, reliable, reproducible!

probabilistic probabilistic

NOTNOT

deterministicdeterministic

Predictive tests are….

Woody

Arlo - 54

AbeArlo is asymptomatic, what are his chances ofgetting Huntington Disease?What are Abe’s chances?

The Guthrie Family Humanitarian Award honors a scientist, researcher or medical leader who has demonstrated

compassion and concern for the care and support of people with Huntington's Disease

Chance that an Asymptomatic Individual at 50% Risk for Inheriting Huntington Disease decreases with age

AgeAge Risk(%)Risk(%)

2525 4949

3030 4848

3535 4646

4040 4343

4545 3838

5050 31

5555 2525

6060 1919

6565 1313

7070 66

UM MG Clinic Experience with Huntington Disease Testing

Asymptomatic

Prenatal

Symptomatic

UM MG Clinic Experience with Huntington Disease Testing

Positive

Negative

Intermediate

Again, genetic testing is a process, not just a laboratory procedure….

• Pre-testing evaluation, education, genetic Pre-testing evaluation, education, genetic counseling, and informed consentcounseling, and informed consent

• Laboratory analysisLaboratory analysis• Accurate interpretation of resultsAccurate interpretation of results• Follow-up must include psychosocial Follow-up must include psychosocial

support, education, and managementsupport, education, and management

Affected

Unaffected

Affected

Unaffected

Affected

Unaffected

+ DNA test

Predictive testing of minors for adult-onset conditions where there is no

preventative or curative treatment............

Only when there is an effective, curative, Only when there is an effective, curative, or preventive treatment that should be or preventive treatment that should be instituted early in life to achieve benefitinstituted early in life to achieve benefit

At an age where children can understand At an age where children can understand implications and make informed decisionsimplications and make informed decisions

Affected

Unaffected

Anticipation, Meiotic Expansion, and Parent of Origin Effects for Different Disorders

• HD HD paternal > maternal - mildpaternal > maternal - mild

• SCA1SCA1 paternal > maternal -mildpaternal > maternal -mild

• DRPLADRPLA paternal > maternal - mildpaternal > maternal - mild

• SMBASMBA paternal > maternal - mildpaternal > maternal - mild

• DMDM maternal >>> paternal - significantmaternal >>> paternal - significant

• FRAXAFRAXA maternal >>> paternal - significantmaternal >>> paternal - significant

CA G

Most CAG Trinucleotide Repeat Diseases:

• Autosomal dominant progressive neurological disorders with Autosomal dominant progressive neurological disorders with variable expression and reduced penetrancevariable expression and reduced penetrance

• Demonstrate mild meiotic instability that is paternal in originDemonstrate mild meiotic instability that is paternal in origin

• Associated with normal alleles of 5-34 repeats and disease alleles Associated with normal alleles of 5-34 repeats and disease alleles of 40-100 repeats with an unstable intermediate repeat rangeof 40-100 repeats with an unstable intermediate repeat range

• Demonstrate that age of onset, rapidity of progression, and severity Demonstrate that age of onset, rapidity of progression, and severity of disorder correlate with increasing repeat sizeof disorder correlate with increasing repeat size

• CAG expansion leads to gain of function “neurotoxic” mutationCAG expansion leads to gain of function “neurotoxic” mutation

Pathophysiology of Expanded Polyglutamine Tracts

• Knock out mice lack the neurological phenotype Knock out mice lack the neurological phenotype

• Heterozygous transgenic mutant mice with expanded Heterozygous transgenic mutant mice with expanded polyglutamine tracts exhibit neurological phenotype polyglutamine tracts exhibit neurological phenotype

• Cell loss is an apoptotic eventCell loss is an apoptotic event

• The ‘toxic fragment’ hypothesis, where proteins are cleaved into a The ‘toxic fragment’ hypothesis, where proteins are cleaved into a short toxic fragments with polyglutamine tracts that aggregate in short toxic fragments with polyglutamine tracts that aggregate in the nucleus, has been raised the nucleus, has been raised

• Association of CAG expansions with GAPDH suggests further Association of CAG expansions with GAPDH suggests further roles for regulation of cellular metabolismroles for regulation of cellular metabolism

• Key events regulating specificity of neuronal loss not understoodKey events regulating specificity of neuronal loss not understood

Potential Roles of Huntingtin

• A handful of huntintin interacting proteins have been A handful of huntintin interacting proteins have been described and suggest additional roles for the protein:described and suggest additional roles for the protein:

• HIP1HIP1 (homologous to yeast gene with cytoskeletal (homologous to yeast gene with cytoskeletal functions) with affinity to normal sized tractsfunctions) with affinity to normal sized tracts

• HIP2HIP2 which encodes an ubiquitin conjugating enzyme, which encodes an ubiquitin conjugating enzyme,

• HAP1HAP1 has affinity to larger polyglutamine tracts has affinity to larger polyglutamine tracts

• GADPHGADPH has direct affinity for polyglutamine tracts and is has direct affinity for polyglutamine tracts and is involved in several key cellular functionsinvolved in several key cellular functions

• EGF receptor complexEGF receptor complex where huntingtin binds to SH3 where huntingtin binds to SH3 domains of domains of Grb2Grb2 and and Ras-GAPRas-GAP suggesting some role in suggesting some role in the EGF signaling pathway the EGF signaling pathway

Drug trials to prevent/slowHD symptom progression

WorkAbility

FinancialAffairs

DomesticResponsibility

Daily livingskills

Where CareProvided

Stage Score Score Score Score Score

1 okay full full full full home

2 lower somehelp

full full full home

3 marginal majorhelp

impaired mildimpairment

mildimpairment

home

4 poor unable unable moderateimpairment

moderateimpairment

home/carefacility

5 unable unable unable severeimpairment

unable total carerequired

Assessing Functional Capacity

remacemide (dashed) vs no remacemide (solid)

Total functional capacity (TFC)

Functional assessment

Independence scale

Coenzyme Q10 (dashed) vs no Coenzyme Q10 (solid)

A. A normal nerve cell newly-injected with mutant HD genesB. The dying nerve cell with disappearance of the fingerlike processes C. Dying cells rescued by adding functional protein.

A B C

5’ UTR exon intron exon intron exon 3’ UTR

CGG

FRAXAFRAXE

GAA

FA

CAG

HDSCAsSMBA

DRLPA

CTG

MD

Trinucleotide repeat disorders are neurological disorders involving expansions of various repeats in coding and non-

coding regions of the gene

Summary

• Trinucleotide repeat disorders account for a large proportion of Trinucleotide repeat disorders account for a large proportion of inherited neurological and mental retardation conditionsinherited neurological and mental retardation conditions

• Sensitive and specific DNA based diagnosis may be used for Sensitive and specific DNA based diagnosis may be used for diagnostic, predictive, and prenatal testing if desireddiagnostic, predictive, and prenatal testing if desired

• Genetic counseling and education is useful for at-risk individuals Genetic counseling and education is useful for at-risk individuals to make informed choice about testing optionsto make informed choice about testing options

• Huntington Disease is an autosomal dominant later onset Huntington Disease is an autosomal dominant later onset progressive neurodegenerative disorder due to expansion of a progressive neurodegenerative disorder due to expansion of a CAG repeat in coding regionCAG repeat in coding region

• Guidelines for predictive and prenatal HD testing are well-Guidelines for predictive and prenatal HD testing are well-established and serve as prototype for predictive testing for adult established and serve as prototype for predictive testing for adult onset conditions onset conditions