Clinical genetic testing

Variant interpretation and

Genetic counselling for

Parkinson disease

Anneke Kievit

Department of Clinical Genetics

ErasmusMC Rotterdam

Introduction

• Ins, outs of genetic testing in PD

• Different genetic tests

• Diagnostic genetic testing

Variant interpretation

Segregation analysis

Incidental findings

Diagnostic yield

Pitfalls in genetic testing

• Predictive testing

• Reproductive options and testing

(prenatal/ preimplantation genetic testing)

• Genetic counselling

• Conclusions

Patients interest and knowledge PD

genetics/testing

• High interest in genetic testing (75-95% ) (Falcone et al. 2011; Gupte et al. 2015; Scuffham et al. 2014;Maloney et al, 2018)

• Low level of genetic knowledge (43-65%), despite positive attitudes toward PD genetic testing (Falcone et al. 2011; Sakanaka et al. 2014; Scuffham et al. 2014)

• 50% PD patients know about presymptomatic and prenatal testing; 70% would take a presymptomatic test if the test was available because of partnership and family (Jacobs et al, 2001)

Doctors interest and knowledge PD

genetics/testing

Questionnaire to 146 movement disorders specialists to assess knowledge and attitudes about genetic testing for PD (Alcalay et al, 2020), 48% completed the questionnaire

41% not referred any PD patients for genetic testing in the last year,

>80% reported referring fewer than 11 patients over the same period, due to lack of insurance coverage/cost to the patient and lack of perceived utility.

52 is mean level of comfort in respondents' own ability to genetically counsel on GBA and LRRK2

60% clinicians correctly answered questions about the inheritance and penetrance of GBA and LRRK2

need to increase knowledge and reduce practical barriers to genetic counselling and testing in PD

Sporadic, familial or monogenic PD

• PD was considered a sporadic disorder;10-15% patients positive familyhistory.

• Risk to develop PD increases with age, affecting

1% of the population more than age 55 years,

3% more than age 75 years, and

5% more than age 85 years.

• In sporadic PD, risk to 1st degree relatives PD is 3-7% over lifetime

• 10% familial PD and 5% sporadic PD is monogenic

• Most influential risk factor: positive family history

Important to investigate the family history construct 3 generation pedigree: insight in different possible inheritance patterns

Pedigree

Symbols

Inheritance patterns

Autosomal Dominant Autosomal Recessive X-linked Recessive Mitochondrial

Single gene test

(Sanger sequencing,

MLPA)

Next Generation

Sequencing (NGS)

Whole exome sequencing

(WES)

Whole genome

sequencing (WGS)

1 gene 10-200 25.000 genes (2% genome) Whole genome

Gene panels Gene panels/open exome One test fits all

Good coverage Good coverage Coverage variable Bioinformatic challenge

Del/dup detection Del/dup detection limited

No incidental findings No incidental findings Incidental findings Incidental findings

Genetic tests

Is a genetic test different?

A genetic test result may have consequences now or in the future for yourself but also for youroffspring, siblings and other relatives, because you share part of your DNA with your relatives

1/2 1/8

1/2 1/4

1/2 1/4

1/4

Why genetic testing?

Molecular diagnosis

• confirms of clinical diagnosis or helps in diagnostic process (often less invasive, less expensive and more accurate than other test methods)

• may offer information on prognosis/ prevention of or targeted monitoring for secondary complications known

• may give information on inheritance pattern

risk for offspring and reproductive options

risk for relatives and testing options

• of rare movement disorders (Manganese transporter defect, CTX, NPC, glutaricaciduria) offers options for therapy or collaboration to clinical trials (GBA1; LRRK2)

Different genetic tests

• Diagnostic genetic test:

genetic test to find/confirm cause of the disease

• Reproductive genetic carriertest:

genetic test in a healthy person in order todefine the risk of a genetic disease in offspring

• Predictive genetic test:

genetic test in a healthy person to define thegenetic status of a late onset disease

Diagnostic genetic test

European guideline genetic testing

affected patient PD (EFNS, 2013)

Restricted testing: no specific treatment/ only for counselling patient and relatives

• SNCA point mutations and gene multiplications: AD families with early- or late-onset PD

• LRRK2, known pathogenic variants in AD families with clinical picture of typical PD and in sporadic patients, known LRRK2 founder mutations in appropriate populations

• GBA mutations in typical PD with or without a positive family history, limited to the known founder mutations in the appropriate populations

• Parkin, PINK1 and DJ-1 genes in typical PD and AR family history with onset < 50 years and sporadic cases with onset

Diagnostic genetic testing?

• Positive familyhistory

• Variable and/or complex phenotype, slowly progressivedisease

• Early age at onset

• Ethnicity (Ashkenazy, North African Berber

• Anticipation

• Consanguinity

• Choice of the diagnostic test depends on clinical situation

• Sanger 1 or 2 candidate genes, if negative: WES movementdisorder panel or skip Sanger

• Consanguinity SNP array tolook for ROH candidategene(s)

• If there is ID and dymorphismsSNP array to look for CNV

SNP array for ROH may lead you to

the diagnosis 41 yr old male, negative FH, consanguineousparents

Normal until 6 months, ID, dystonia, hypertonia, swallowingproblems, progressivecomplaints in night

Array: no CNV, 37Mb ROH with

35 AR genes: SPR gene in one of the largestROH’s

DRD was clinically compatibleSPR mutationanalysis: homozygous pathogenic SPR variant c.655C>T, p.(Arg219*)

Dopa responsive dystonia, due to sepiapterinreductase deficiency good effect on DOPA

Parents both carrier of variant

SNP array for CNV may lead you to

the diagnosis

28 year old male referred for neurodegeneration with brain iron accumulation and negative WES NBIA panel

Early mild motor and intellectual disability, dysmorphic featuresprogressive deterioration cognitive function, dysmorphic features, movement disorder with dystonia, tremor and sensory ataxia

SNP array 3.3Mb de novo loss of 8p11.21p11.1, that deleted several OMIM genes, including SLC20A2, RNF170 and KAT6A

CT-scan showed clear symmetric calcifications in the basal ganglia, compatible with primary familial brain calcification/Fahr disease

RNF170 and KAT6A are respectively associated with a sensory ataxia and ID and dysmorphisms (Say-Barber Biesecker-Young variant of Ohdo syndrome)

Yield diagnostic WES

• In literature (Kumar et al, 2013; Fogel et al, 2014,

Nemeth et al 2012, Deng et al, 2018, Montaut et al, 2018):

5-15% Parkinson’s disease

18-26% Ataxia

26% Hereditary Spastic Paraplegia

• In ErasmusMC Rotterdam:

20% movement disorders (ID 40%, deafness 30%, retinal diseases 60%)

Test single person, panel based, usually no open exome because of low yield and risk of incidental findings; only open exome with use of other affected relatives or in trio with parents

• Important to re-evaluate patient after 2-3 yrs if test is negative or if class 3 or 4 variant is found

Classification of pathogenicity of a variant

Class Probability of pathogenicity

1 Benign,1%

2 Likely benign, 1-5%

3 Variant of uncertain significance (VUS), 5-95%

4 Likely pathogenic, 95-99%

5 Pathogenic, >99%

Pathogenicity of a variant

• Previous observation of a variant: population reference databases (Gnomad, Exac), allele frequencies

• Conservation in evolution: might implicate pathogenicity

• Effect on protein: stop/frameshift: high impact; synonymus coding: low impact; tools to predict pathogenitcity (Polyphen, SIFT, mutationtaster

• Effect on splicing

• Results of earlier interpretation: LOVD/HGMD

• Inheritance match: OMIM

• Relation to genotypic spectrum

• Relation to phenotypic spectrum: be aware of variation

• Co-segregation: de novo occurence strong evidence pathogenicity

Co-segregation analysis variants

• Classification of a variant of unknown siginificance (VUS)

Class 3 or 4 variant shared by affected relatives and not found

in unaffected relatives, large family needed, willing to cooperate,

also clinical examination/information needed

• Confirmation of molecular diagnosis and

Inheritance pattern. Defining alleles of class 4 or 5 variants

(AR disease)

+ + +

+ + + + + - -

4 5 4/5

- - - + +

AR confirmed AR not confirmed

4/54/5

Pretest counselling genetic testing

Genetic counselling is offered to support patients in clarifying gaps of knowledge regarding PD genetics as well as the risks, benefits, and limitations of genetic testing and to support them in their decision making process (Resta et al, (2006)

Information:

Genetic testing may result in:

• Cause of the genetic disease

• Possible/unknown cause of genetic disease (further tests needed): VUS co-segregation relatives, additional clinical examination patient/relatives

• No significant findings (for now)

• Genetic information on other diseases Incidental/secondary findings

Incidental findings genetic testing (ACMG, 2013)

• Risk depends upon the genetic test used (high in open exome; low in NGS/panel based WES)

• Clinical utility: actionable versus non actionable

• Ask pretest patients preference (all findings or only actionable ones)

• Responsibility of clinician to provide comprehensive pre- and post-test counseling

• Clinicians should be familiar with the basic attributes and limitations of clinical sequencing, know which genes are in the panel

Incidental findings

Positive

• genetic predisposition to disease that can be treated/prevented

• carrier of recessive condition; important for reproductive choices

Negative

• untreatable conditions

• leading to problems with health insurance/employers/ mortgage

• uncertainty regarding the manifestation of the disease

Pitfalls genetic testing

Missed in WES

• Mutations in mitochondrial DNA

• Structural chromosome variants: translocations or inversions

• Triplet repeat disorders

• Copy nummer variants

• Mutations in introns

• "Uniparental disomie"

• Control sequences:m mutations in microRNAs (intronic)

• Epistatic interactions (gene-gene)

• Epigenetic changes

Posttest counseling

Discuss the result of the molecular test

Result confirms clinical diagnosis

• Discuss test result

• Reproductive options

• Inform relatives if they are at risk

Result negative

Discuss test result (does not exclude genetic form PD)

Re-evaluation after 2-3 yrs

Result class 3 or 4

Organise segregations studies

Reverse phenotyping

Discuss in multidisciplinary team

Importance of multidisciplinary team

• For optimal clinical care

• Multidisciplinary team: neurologist, clinical geneticist, moleculargeneticist, researcher/scientist

• To discuss

• Pathogenicity of a variant,

• Possibility of functional studies, research project

• Possibility of further co-segregation studies, open WES

• Reverse phenotyping: clinical re-evaluation or further work up of patient and/or family members

Reproductive genetic carriertest

Reproductive genetic carriertest

• Genetic test in a healthy person in order to define the risk of a genetic disease in offspring (AR/XR conditions)

• Test the partner of patient with an AR PD due to Parkinvariants

• Risk partner carrier heterozygous Parkin variant and carriers small risk of PD

• Discuss reproductive options if carrier

Reproductive options

• Accept the risk and have children

• Refrain from having children

• Adoption/fostering

• Donor sperm insemination/egg donation

• Invasive prenatal diagnosis

• Preimplantation genetic diagnosis

Invasive Prenatal Diagnosis (PND)

• Chorionvillussampling: fetal cells from chorion,

10-14 wks GA, miscarriage risk 0.2%;

TOP vacuum aspiration

• Amniocentesis; fetal cells from amniotic fluid,

>15 wks GA, miscarriage risk 0.1%;

TOP clinical deliverey

• Genetic counseling, psychosocial care, MD team

• Request for PND/PGD for PD in NL is limited;

• Less interest in prenatal genetic testing, with only 56% expressing interest in knowing if their unborn child inherited a PD mutation (Maloney et al, 2018)

Preimplantation Genetic Diagnosis (PGD)

• IVF/ICSI procedure,

• Single cell DNA diagnosis on embryo of 3-5 days

• Embryo transfer of embryo withfavourable test result

• Pregnancy in 25%

• No PGD for PD yet in NL (often forHD and SCA’s)

Predictive genetic test

Predictive genetic test

• Neurological examination/imaging/biomarkers can also be a predictive test

• Pre- and posttest counseling and psychosocial care

-information about the test and the consequences for the individual and relatives,

*Social: Insurance issues (life- and occupational disability) with a threshold above which insurance companies may ask result genetic test

*Psychological: burden of knowing, symptom searching and new uncertainty when and how, survivor guild

*Genetic: risk for children/siblings; reproductive options

*Medical: become a patient

-help in the decision process to make an autonomous choice,

-prepare for the test result

-offer help after the testresult to cope with it and offer help to inform relatives

• No specific guideline for predictive testing in PD, predictive testing not mentioned in EFNS guideline; use guideline for predictive testing in Huntington disease (Macleod et al, 2013)

Why predictive testing if therapy is no option?

• end uncertainty

• obtain control over the future (decisions about future-, relation- and familyplanning)

• inform offspring and relatives

• reproductive options

Predictive testing in minors?

• Not done usually, when the disease is non actionable

• Choice for predictive testing must must be autonomous, well informed

• Everyone has a right not to know

• Handle in best interest of child

• Social consequences (insurance/work problems), may increase in future

• Knowing genetic status may have psychological impact (child, relatives, others)

• Not testing may also have consequences

• Practical reason: future planning (school choices)

Predictive testing for Huntington disease

• Before predictive testing became possible: 57-84% at risk persons interested in test

• When predictive testing became possible: 24% at risk persons tested (1987-1997); high compared to othercountries (2-16%) (Maat-Kievit et al, 2000) 1st test untreatable late onset diosrder, worries if it was ethicaljustified, could result in anxiety, depression, relation problems, discrimination or surviviors guilt, usinginternational guideline

• Adverse effects after test: 2% carriers and 0.3% non carriers (Almqvist et al,1999)

pretest mental status predicts problems posttest better than the testresult

majority of those with adverse effect had psychiatric problems or were symptomatic

• 1-3 yrs after test carriers and non carriers showed less problems (Broadstock et al, 2000)

• 7-10 yrs after test carriers showed more problems (Timman et al, 2000)

• Effect predictive testing for late-onset neurodegenerative disorders on social and personal life (Cohn-Hokke et al, 2018)

no significant differences in jobs, financial situation, lifestyle, anxiety or depression between carriers, non carriers and at risk persons

Conclusions

• Parkinson disease is clinically and genetically very heterogenous: NGS/WES panels for dx

• Familyhistory is an important instrument to get insight in possible inheritance pattern

• Monogenic PD is suspected if PD is familial, variable and complex phenotype, early age at onset, ethnicity, anticipation, slowly progressive disease and consanguinity

• Pretest counseling very important (general information, informed consent, incidental findings)

• Choice of genetic test depends on clinical situation

• Importance of cosegregation studies, reverse phenotyping in class3 and 4 variants for diagnosis

• Be aware of limitations and pitfalls of genetic testing

• Be careful with predictive testing, especially in minors (refer to clinical geneticist)

• Inform about reproductive options for patients, inform relatives at risk (refer clinical geneticist)

• Discuss findings,problems and plans in multidisciplinary setting

• Important to re-evaluate patient after 2-3 yrs if test is negative/if class 3 or 4 variant is found

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