Borut Peterlin borut.peterlin@guest.arnes.si

Genomic biomarkers in human disease

Modern Molecular- biochemical Markers in Clinical and Experimental Medicine - 2019

7– 9 November 2019, Prague

Genomics for healthcare

Genome medicine

Diagnosis of rare diseases

Personalized medicine

67 % Disease

panels

Clinical geneticist Center for Mendelian Genomics

Clinical Institute of Medical Genetics

Family doctors

Medical specialties

Medical specialties

Several familial MS cases

Primary MS – malignant melanoma family

Sporadic MS cases

Presentation outline

Human Genome initiatives

1.11.2019

Public health challenge

Primary service

S/T service

Specialized (international)

Cummulative point prevalence of Rare diseases

3.5-5.9% EJHG 2019, PMID:31527858

Diagnostic approach

Panels Exomes Genomes

Trio design HPO target definition

+

Medical interpretation of NGS data

NGS in the diagnosis of rare diseases

History Physical & examination

Key features -> Clinical diagnosis

Definite diagnosis

Final diagnosis

Creativity in NGS diagnostics

Clinical/differential diagnosis

Clinical symptoms & signs

Gene target

G1

G2

Gn

Ge

Classification of genetic variants

Genetics in Medicine 2015

AJHG 2016

There is remarkable convergence in bioinformatic techniques,

but medical interpretation and reporting are areas that require

further development by many groups.

Brownstein et al. Genome Biology 2014, 15:R53

Alzheimer disease Frontotemporal

dementia Parkinson’

s

Parkinsonism

Hallucination

Dementia

Language impairment

Personality changes

Parkinsonism Dementia

Bradykinesia

Personality changes

APP

PSEN

1

PSEN

2

SORL

1

C9Orf7

2

GRN MAPT

CHMP2

B

SNCA

LRRK2 PRKN

PINK1

Apathy Disinhibition

Dementia

PSEN

1

TREM

2

LRRK

2

Seizures

Language impairment

Agitation

Confusion

TREM

2

TOMM4

0

Resting tremor

Rigidity

Parkinsonism

Postural instability

PARK7

Alzheimer disease Frontotemporal

dementia Parkinson’

s

Parkinsonism

Hallucination

Dementia

Language impairment

Personality changes

Parkinsonism Dementia

Bradykinesia

Personality changes

APP

PSEN

1

PSEN

2

SORL

1

C9Orf7

2

GRN MAPT

CHMP2

B

SNCA

LRRK2 PRKN

PINK1

Apathy Disinhibition

Dementia

PSEN

1

TREM

2

LRRK

2

Seizures

Language impairment

Agitation

Confusion

TREM

2

TOMM4

0

Resting tremor

Rigidity

Parkinsonism

Postural instability

PARK7

Alzheimer disease Frontotemporal

dementia Parkinson’

s

Parkinsonism

Hallucination

Dementia

Language impairment

Personality changes

Parkinsonism Dementia

Bradykinesia

Personality changes

APP

PSEN

1

PSEN

2

SORL

1

C9Orf7

2

GRN MAPT

CHMP2

B

SNCA

LRRK2 PRKN

PINK1

Apathy Disinhibition

Dementia

PSEN

1

TREM

2

LRRK

2

Seizures

Language impairment

Agitation

Confusion

TREM

2

TOMM4

0

Resting tremor

Rigidity

Parkinsonism

Postural instability

PARK7

Genetic heterogeneity of pediatric neurologic disorders

Developmental delay

(1629 genes)

Epilepsy (870 genes) Metabolic

disorders (615 genes)

Brain malformation

disorders (377 genes)

Neuronal migration disorders

(377 genes)

Hereditary myopathies (237 genes)

Hereditary ataxia

(201 gene)

Neuromuscular disorders

(220 genes)

Intellectual disability

(161 genes)

Hereditary neuropathies (161 genes)

Epileptic encephalopathies

(116 genes)

Leukodystrophies (116 genes)

Myasthenias (56 genes) Microcephaly/po

ntocerebellar hypoplasia (57

genes)

NBIA (15

genes)

Hereditary spastic

paraplegia (54 genes)

Hereditary dystonia

(67 genes) Basal ganglia calcification (27 genes)

Hereditary neurodegeneration

(18 genes) Rett and Rett-Like

syndromes (23 genes)

High genetic heterogeneity of referrals to exome sequencing Over 1000 variants in 618 distinct genes reported in pediatric neurologic conditions

Diagnosis of rare diseases is challenging

• > 7000 diseases

• > 5200 genes

• Clinical & Genetic heterogeneity

• Rarity -> Lack of diagnostic expertise

EUCERD 2014 report

Case 3&4: No obvious hypothesis

Symptoms and signs • Female 14 years • Severe developmental delay • Epilepsy • Corpus callosum hypoplasia • Microcephaly with Brachycephaly • Hearing impairment • Strabismus • Thin, fragile nails • Decreased lacrimation

Differential diagnosis

Chromosome 1p36 deletion syndrome Sotos Dodge syndrome Pallister Killian syndrome Aspargine synthetase deficiency

• Female 52 years • Carpal tunnel syndrome • Mild intellectual disability • Dysplasia of metacarpal bones • Small stature • Hearing impairment • irregular menstrual cycle • Hypothyroidism

Symptoms and signs

Differential diagnosis

Pseudohypoparthyroidism 45 X syndrome Acrodysostosis

Frameshift variant in POGZ gene POGZ:NM_015100.3:c.2771delC

POGZ:NM_015100.3:c402_409dupTGCCAATC

White Sutton syndrome

White Sutton syndrome

Epilepsy Corpus callosum hypoplasia Dysplastic nails Decreased lacrimation

Carpal tunnel syndrome Dysplasia of metacarpal bones Irregular menstrual cycle Hypothyroidism

Autism Visual problems GIT problems

Case 3

Case 4

Full clinical picture

Diagnostic yield / approach

67 % Disease panels

Exome sequencing & interpretation

Disease

Phenotypes

Symptom 1

Sign 1

Symptom 3

Symptom 2

Sign 2

Gene candidates

Gene candidates

Gene candidates

Gene candidates

Gene candidates

Gene candidates

Gene candidates

Diagnostic yield / approach

67 % Disease panels

21 % New diagnosis

67 % Disease panels

21 % New diagnosis

New associations

Reinterpretation

Whole Genome Sequencing

Patient with suspected Angelman like syndrome

2014

Negative report

No mutation found in

the exome sequencing data.

Reinterpretation of exome sequencing data

2015

New data in the literature

We conclude that the identified

EEF1A2 variant presents a likely

cause of the referral clinical

presentation.

2016

Establishment of a diagnosis

Clinical geneticist Center for Mendelian Genomics

Clinical Institute of Medical Genetics

Family doctors

Medical specialties

Medical specialties

Clinical pathway for NGS testing at KIMG

Clinical pathway for patient referral

02

03

04

01

28%

18%

14%

DIrectly Neurologist to laboratory

Combination Combination of approaches

Team

Clinical geneticist

35%

Prioritisation of NGS in diagnostic algorithm

46% First tier diagnostic diagnostic tool

After traditional diagnostic workup 54%

Referring physician Molecular geneticist MD +

Bioinformatician Clinical geneticist

Different specialities

Pathway for NGS diagnostics at KIMG

Diagnostic yield: KIMG experience

Positive

41,2% Negative

45,0%

VUS

13,8%

WGS

+ 0,2%

New diagnostic alghorithms

Primary prevention of genetic diseases

US/biochemical NIPT

Down syndrome Congenital anomalies Inherited diseases

Karyotyping

QF-PCR

aCGH

Next generation sequencing

Targeted testing

AC/CVS PGD

Preconceptional genetic testing

Preimplantation genetic diagnosis (PGD)

Polar body biopsy Cleavage stage biopsy Blastocyst biopsy

Blastocentesis

Structural rearrangement by NGS

• Mother 46,XX,t(4;5)(p15.3;p15.1)

• Father 46,XY

Disease

Location

Eiberg 1985

Reverse genetics – positional cloning

Gene Rommens 1989

Description of new syndrome & gene

Identification of new genes

Disease

Gene

Clinical exome Whole exome

sequencing

Whole mitochondrial

genome sequencing

Sequencing > 5000

known genes associated

with human diseases

Sequencing of all

predicted human genes –

20 000

Sequencing of the whole

mitochondrial genome

Whole genome

sequencing

Sequencing of the whole

human genome

A boy with prematurely aged appearance, growth retardation, hypoplastic terminal phalanges with features of premature aging

included reduced subcutaneous fat, wrinkled skin, scant hair

Suspected Petty syndrome, Petty type (gene not known yet)

Fotografija: Irena Lesjak, delo.si

Pat

ien

t M

oth

er

Fat

her

SLC25A24 p.Arg217His PPP1R13B p.Thr391fs

Exome sequencing in the patient

Matchmaking in a progeria syndrome with yet

unknown causative gene (all identified cases share the same de novo variant)

French case Our case

Italian case Spanish case

Germany (multiple cases)

All patients shared

de novo variants affecting

Arg217 residue in

SLC25A24 protein

G A

wt/wt wt/wt

PArg217His/wt

wt/wt wt/wt

P

wt/wt

P

wt/wt

Arg217Cys/wt

wt/wt wt/wt

PArg217His/wt

Arg217His/wt

French familySlovenian family

Spanish family* Italian family

G A C G G G A C G G

G A Y G G

G A C G G G A C G GG A

C/T

G A Y G GC/T

* results from single base extension assay

denotes sample availability for testingP denotes familial proband

G A

G A Y G GC/T

G A C G GG A C G G

Writzl et al, AJHG, 2017.

p.Arg217Cys or p.Arg27His Causes Significant Structural Changes within the SLC25A24 Transmembrane Domain

A boy with prematurely aged appearance, growth retardation, hypoplastic terminal phalanges with features of premature aging

included reduced subcutaneous fat, wrinkled skin, scant hair

Suspected Petty syndrome, Petty type (gene not known yet)

Fotografija: Irena Lesjak, delo.si

Patient with seizures, polymicrogyria and developmental delay of unknown etiology

A de novo missense variant in SCN3A gene

Although SCN1A and SCN2A have been reported, the role of de novo SCN3A variants in epilepsy was unknown at the time (early 2016)

Variant of uncertain significance in a gene of unknown significance

46

Our case

Several cases with de novo SCN3A

variants and an overlapping phenotype

New York case

UK cases (several)

Washington case

Germany case

Pennsylvania case

Monogenic vs. complex (polygenic + environment) disorders

G

Clinical Matcmaking

51

French

case

Our

case

nature genetics • volume 33 • january 2003 Neuroscience Letters 650 (2017) 25–32

Conclusion: Poor evidence for association

NR4A2 role suggested but unconfirmed

High genetic heterogeneity of referrals to exome sequencing Since introducing NGS we reported over 3000 variants in 1124 distinct genes

Economic innovation & sustainability

0

100

200

300

400

500

600

700

2011 2012 2013 2014 2015 2016 2017

Solved cases VUS Negative result

Diagnostics abroad Diagnostic exome sequencing

• Clinical pathways – NGS in health system

• Diagnostic approach – exome sequencing & interpretation

• Diagnostic algorithms

• Funding

• Telegenetics

• Identification of Mendalian disorders in health system

• Routine diagnostics meets research

• “Future” personalized medicine

Genomic inovations in the Slovene Health System

Genome screening

Setting the scene for personalized genomic medicine

• Primary purpose of carrier screening

• Carrier screening panels

• Evidence

• Timing of screening

• Information and support

• Informed consent

• Voluntary participation

• Quality of services

• Maximizing quality of care

• Professionals and public education and dialogue

• Governance

Family history screening in primary care

13%

Family history tool into the primary health system

Clinical geneticist Center for Mendelian Genomics

Clinical Institute of Medical Genetics

Medical specialties

Would you like to know if you/partner are carriers for genetic predisposition which might affect your child?

1- not at all, 5-very much

Slovenian Genomic Variability db

Coding variants • 164,301 SNVs & Indels

• 79,800 SNVs & Indels / patient

• 78,146 rare variants

• 22,724 rare variants not in GnomAD

• 43.7 rare variants / patient

1. CEMGPdb

2. Variant Interpretation Platform

3. New genes for human diseases

“Future” Personalized Medicine

54 %

3 %

Preconceptional genetic screening Screening for preventable AO disorders

• 293 genes associated with drug response • 24 known actionable pharmacogenetic variants • 61 variants with PharmaGKB 2A or 2B support • 308 novel potentially actionable variants

Monogenic contribution to complex disorders

Parkinson’s disease

Multiple sclerosis

Alzheimer’s disease

Ataxias Choreas

ALS

G

G ?

G

G

G

Genetic predisposition for MS

Common variants

“Missing heritability”

Rare variants

Shared environment

Individual environment

Ristori G et al., Ann Neurol 2006, 59: 27–34

IMSGC, Cell 2018, 175, 1679–1687

Several familial MS cases

Primary MS – malignant melanoma family

Sporadic MS cases

Searching for monogenic forms and disturbed pathways in Multiple sclerosis

The role of inflammasome in MS

Are mutations in the GJB1 gene risk factor for multiple sclerosis?

No other mutation in the GJB1

gene In our cohort of familial

and sporadic MS patients

No increased burden in panel of

genes for demyelinating

neuropathies

Human endogenous viruses

HERVs

Coding region

HERV insertions as risk factors for MS

HERV-K insertions in the introns of

RASGRF2 and PTPRN2 genes

are a heritable risk factor for MS

Thank you!