Talk outline

• How geneticists can help neuroradiologists • Genetic testing: when, which and why

• Genetic testing today Neurogenetics made easy • genome-wide strategies for mutation detection Enza Maria Valente, MD, PhD Dept of Molecular Medicine, University of Pavia • How neuroradiologists can help geneticists Neurogenetics, IRCCS Mondino Foundation • practical examples • the importance of collaboration

Whygeneticsisimportant?

for patients How geneticists can help improved diagnosis neuroradiologists prognostic indications indications for management and treatment Genetic testing: for families when, which and why assessment of recurrence risks early prenatal diagnosis testing relatives (e.g. carrier testing) new therapies, clinical trials etc… 4

A quick refresh about mendelian inheritance Decision-making flowchart for genetic testing

everything that is not acquired (such as infective, toxic, vascular, traumatic Indication to genetic testing etc…) MUST recognize a genetic basis pre-test genetic counselling and informed consent autosomal recessive autosomal dominant ƒ two mutated copies of the gene are ƒ one mutated copy of a gene is sufficient test selection required to cause the disease to cause the disease ƒ healthy heterozygous parents transmit ƒ for diseases reducing fitness, the laboratory selection both mutated copies of the gene to mutation usually occurs de novo during 25% offspring parental meiosis Æ sporadic cases GENETIC TESTING ƒ horizontal transmission (multiple ƒ for diseases non reducing fitness, the affected individuals in one generation) mutation is transmitted vertically from negative or ambiguous results positive results ƒ can also present in sporadic cases one affected parent to 50% offspring diagnostic reassessment? implications for management, X-linked further genetic testing? prenatal diagnosis, ƒ recessive: hemizygous male patients inherit one mutated copy of a gene on the X research testing? counselling for relatives chrom. from the heterozygous mother (healthy or minimally affected) – also de novo ƒ dominant: heterozygous females are affected while in hemizygous males the mutation post-test genetic counselling is lethal or gives rise to much more severe phenotypes – also de novo 5 Indications to diagnostic genetic testing Indications to diagnostic genetic testing

validation of diagnostic suspicion (clinical / imaging) diagnosis of conditions without clear clinical or imaging clues prognostic indications (genotype-phenotype correlates) differential diagnosis among distinct conditions with overlapping phenotypes Neurofibromatosis 1 (NF1 gene) ƒ café-au-lait spots, axillary freckles Early onset pure ƒ cutaneous neurofibromas cerebellar atrophy • AT (ATM) ƒ optic gliomas, cranial tumours, other MRI lesions • AOA1 (APTX) • SCA29 (ITPR1) … AT AOA1 ITPR1 Leigh syndrome (mitochondrial) Joubert syndrome ƒ onset < 1 year ƒ congenital ataxia reproductive risks, carrier testing and prenatal diagnosis ƒ severe neurological ƒ variable organ involvement specific treatments, therapeutic trials picture ƒ typical “molar tooth sign” ++ severe disorders with full penetrance ƒ typical basal ganglia ƒ NBIA lesions ƒ Neuronal Ceroid Lipofuscinoses ƒ Lysosomal storage disorders PLAN Infantile NCL Infantile NCL

Poretti et al, Neuropediatrics 2015; Vedolin et al, AJNR 2013

Genetic counselling Selection of test

genetic testing must be considered an integrated service, mutation(s) already identified in affected family members preceded and followed by specific genetic counseling ƒ genetic testing specifically targeted to confirm the familial mutation(s) an appropriately trained person (clinical or medical geneticist, genetic counsellor, or genetic nurse) offers the patient and his family accurate and genetic and molecular homogeneity comprehensive information on: ƒ a specific mutation causes most cases of a disease (e.g. Friedreich’s Ataxia Æ GAA expansion in the FXN gene) ƒ rationale and procedure of the test ƒ standardized genetic testing, high sensitivity ƒ inheritance, natural history, and management of the disease ƒ implications for the health and reproductive choices of the relatives genetic and/or molecular heterogeneity (commonest!) ƒ potential benefits, disadvantages, and consequences for the future health, employment, and insurance prospects of the individual ƒ The same disease can be caused by different mutations and different mutational mechanisms involving one or even many genes ƒ referral for psychosocial support when indicated ƒ ataxia-telangiectasia (ATM, very large gene, many mutations) ƒ results, especially when positive, should also be rendered in the ƒ Joubert syndrome (>40 genes known to date) context of genetic counselling

Genetic strategies: high resolution array

standard cytogenetics array studies (CNVs)

Genetic testing today

genome-wide strategies for Ordered pool of genomic sequences (oligos – CGHarray or SNPs – SNParray), which physical position on the genome is known mutation detection The whole genome is analyzed in a single experiment Resolution depends on the length of DNA probes and the average distance between two adjacent probes on the genome

on the same chip you can simultaneously test for: - copy number variations on the whole genome (dels-dups) -regionsof homozygosity by descent 12 Dandy Walker syndrome – chrom. 3q25 deletion Dandy Walker syndrome – chrom. 6p25 deletion

- heterozygous point mutations: Axenfeld-Rieder syndrome + mild CVH

- small deletions or duplications: CVH – mega cisterna magna

-large deletions: nearly all have DWS (but 1 normal!!!)

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Grinberg et al, Nat Genet 2004; Ferraris et al, OJRD 2013 Aldinger et al, Nature Genet 2009

Genetic strategies: next generation sequencing (NGS) Caution in interpretation of NGS results

linkage studies bioinformatics

high-throughput, cost- and time-effective simultaneous resequencing of mutation Æ permanent change polymorphism Æ variant bothconfusion replaced by large portions of the genome in the nucleotide sequence with frequency >1% assumed as pathogenic assumed as benign Current diagnostic applications: variant • target resequencing of panels of genes or clinical exome Æ hundreds of genes of our choice or about 4000-7000 genes known to cause inherited disorders • mutation screening in clinically and genetically heterogeneous disorders pathogenic likely of uncertain likely benign pathogenic significance (VUS) benign • whole exome sequencing Æ the coding regions of the >20.000 genes in our genome • novel gene identification (even in small families/sporadic) • «fishing» for diagnosis in patients with limited clinical-imaging clues with respect to a condition and inheritance pattern (e.g., c.1521_1523delCTT (p.Phe508del), pathogenic, cystic fibrosis, autosomal recessive).

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Incidental findings in NGS testing Joubert syndrome (>40 genes)

findings that have potential health or reproductive importance discovered in the course of conducting research but beyond the aims of the study

sequencing laboratories should actively seek examples: and report pathogenic variants in 56 genes - BRCA1-2: breast / ovarian cancer associated with 24 conditions, all with evidence - LDLR: hypercholesterolemia that early intervention can prevent or - PKP2: arithmogenic cardiomiopathy ameliorate severe adverse medical outcomes

Comprehensive pre- and post-test counselling is essential

18 Gene-phenotype correlates Ponto-Cerebellar Hypoplasias: clinical and genetic classification

NGS-based screening PCH GENE LOCUS CLINICAL FEATURES MKS1 in 400 JS probands EXOSC3, VRK1 9p13 Onset at birth 2% TMEM216 B9D1 TMEM237 other genes PCH1 1% 1% 1% 6% TSEN54, RARS2 14q32 axonal motor neuropathy CSPP1 INPP5E 2% JS + kidney 2% TSEN54 17q25 Onset at birth, dyskinesias / chorea OFD1 PCH2 TSEN2 3p25 vermis less hypoplastic than hemispheres RPGRIP1L 2% 3% TSEN34 19q13 (dragonfly appearance) -Pure JS KIAA0586 -JS + JATD + OFD 3% Onset at birth, optic atrophy negative PCH3 Not known 7q11-q21 TMEM67 hearing impairement JS + liver 6% 43%

CC2D2A PCH4/PCH5 TSEN54 17q25 Fetal onset, severe progression, myoclonus 6% PCH6 RARS2 6q15 Onset at birth, mitochondrial defects Variable AHI1 phenotypes 6% PCH7 TOE1 ambiguous genitalia CEP290 C5orf42 8% 9% PCH8 CHMP1A 16q24 onset at birth Pure JS (++ progressive microcephaly, ID, deafness retinopathy) CASK CASK Xp11 Dragonfly/butterfly, normal CC Cerebello- unspecific clinical features, death by age 10 oculo-renal -Pure JS PCH9 AMPD2 1p13 -OFDVI CC hypoplasia, «8» shape of vermis (axial cut) Slow onset, progressive spasticity/seizures PCH10 CPL1 11q12 Absent or delay speech 20

PCH screening – Italian cohort «Shrunken cerebellum»: non-progressive cerebellar atrophy

RARS2 VLDLR PPM2/ATP2B3 2% 2% 18 months • unique inclusion criterion: PCH 2% EXOSC3 9 Mild developmental delay confirmed on neuroimaging 5% 9 Cerebellar Ataxia • 61 probands analyzed TSEN54 neg 16% 39% • 44 genes 9 Normal cognitive development or mild intellectual deficit CASK 9 Clinical improvement in time Males: 34% mosaic loss of function 9 No progression of atrophy at severe missense neuroimaging Males: Females: Males: missense or splice het loss of function hemiz. loss of function several genes involved CASK mutations 6 years DD/ID progressive microcephaly most severe clinical presentation nystagmus variable PCH severe to profound DD seizures (not severe) moderate to severe DD/ID rapidly progressive microcephaly (short stature) very limited language cortical atrophy / hypomielination PMM2 (tremor, hypotonia) seizures, short stature, scoliosis severe intractable seizures (dysmorphism) sensory-neural hearing loss (congenital heart defects, contractures) Congenital Defect butterfly (also dragonfly) often lethal of Glycosilation IA: normal corpus callosum both atrophy and hypoplasia 21 22

Burglen 2012, Saitsu 2012, Moog 2015

The same genes cause late onset SCA and early onset SCAR … and things can get even more complicated!

R480W: dominant (de novo), congenital onset, intellectual impairment SCA5: dominant, late onset, non LOF

SCAR14: autosomal recessive, congenital onset, intellectual impairment, LOF 23 24 Same gene, multiple phenotypes = BRAT1 two brothers, 12 and 7 years old 9 Mild psychomotor delay, ID affected p.V214Gfs*189; WT p.T465T; WT 9 Ataxia Mother Father Proband sib CTRL 9 Nystagmus

9 Not progressive cerebellar atrophy p.V214Gfs*189; p.T465T p.V214Gfs*189; p.T465T How pediatric neuroradiologists Rigidity and multifocal seizure syndrome / lethal can help geneticists 9 Microcephaly, Rigidity 9 Multifocal seizures 9 Apnea and bradycardia 9 Death in infancy (3-21 m) 9 MRI: normal / Frontal hypoplasia / cerebro-cerebellar atrophy a few practical examples 9 milder, non lethal form reported in few cases the importance of collaboration

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Why neuroimaging is important for genetics? Peculiar imaging patterns aiding genetic diagnosis

• clinical features can be unspecific (especially at onset) and Pantothenate Kinase Associated Neurodegeneration (PKAN) indistinguishable among distinct disorders - early onset, rapid progression • the same disorder may present wide clinical heterogeneity - extrapyramidal signs (dystonia, parkinsonism), pyramidal signs - bulbar involvement • definite clear-cut correlations are emerging between certain - mental deterioration, dementia gene mutations and specific neuroimaging patterns - epilepsy, retinopathy, optic atrophy, acanthocythosis…

Imaging - atrophy of basal ganglia and substantia nigra - iron deposition in the basal ganglia a good neuroimaging classification is essential to: - address certain patients to specific genetic testing eye of the tiger sign - group homogeneous patients for identification of novel genes

100% correlation with PANK2 mutations

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Peculiar imaging patterns aiding genetic diagnosis Peculiar imaging patterns aiding genetic diagnosis

Tubulinopathies cerebellar agenesis + pancreatic agenesis • variable neurological presentation • malformations involving: • severe IUGR, microcephaly, death in infancy • basal ganglia • dysmorphisms • commissural tracts • neonatal diabetes, near absence of subcutaneous fat • cerebellum (+/- cysts), brainstem • marked cerebellar hypoplasia / agenesis • cerebral cortex (lys, pachigyria, PMG) Tubulin genes (dominant) PTF1A (recessive)

Chudley-McCullough Syndrome Poretti-Boltshauser syndrome • profound sensorineural hearing loss • ataxia, ID, language impairment • (mild motor/cognitive delay) • myopia or retinal dystrophy • cerebellar dysplasia • diffuse cerebellar dysplasia • hydrocephalus, partial CC agenesis • cerebellar cysts • frontal PMG, gray matter heterotopias • square-like IV ventricle • splayed SCP LAMA1 (recessive) GPSM2 (recessive) 29 30 Peculiar imaging patterns aiding genetic diagnosis

GPR56-related PMG

- hypotonia, cerebellar and pyramidal signs - severe ID - frontoparietal or diffuse polymicrogyria - cerebellar dysplasia with cysts the importance of - patchy white matter anomalies - thin corpus callosum GPR56 (recessive) - mild pontine hypoplasia collaborations

horizontal gaze palsy + progressive scoliosis

• congenital absence of horizontal gaze • severe progressive scoliosis • absence of decussation of corticospinal and somatosensory axonal tracts in the medulla

ROBO3 (recessive) 31 32

MRI pattern recognition for gene discovery MRI pattern recognition for gene discovery

2 unrelated sporadic patients with unspecific clinical features (DD, tetraparesis, 2 sisters with unspecific clinical features (DD, hyperkinetic movements) but identical and highly peculiar MRI pattern: tetraparesis, intellectual defect) and MRI pattern control patient 1 resembling a tubulinopathy WES of the two probands Æ pathogenic recessive mutations in GSX2 hypothalamic- tubulin genes all NEGATIVE Æ WES Æ midbrain fusion control Gsh2 ko mouse (E16.5) homozygous missense mutation in TTL gene – Tubulin Tyrosin Ligase

absence of putamina KO mouse model fully recapitulates CNS malformations seen in patients

absence of olfactory tracts Toresson et al, Development 2000 33 34

De Mori et al, Brain 2019 manuscript in preparation

Still hunting the causative genes… What can we do with other “mysterious” defects?

romboencephalosynapsis pontine tegmental cap dysplasia

• defects that are clinically and neuroradiologically heterogeneous Æ diagnosis is descriptive, nosology is scarce • very rare conditions, only few patients described • e.g. some primary brainstem anomalies, cerebellar cortical dysplasias, • extremely peculiar, well recognizable malformations “macrocerebellum” • diagnosis made on a neuroradiological basis • it is mandatory to homogeneously CLASSIFY these cases according to • no candidate genes, no shared chromosomal rearrangements strict criteria Æ similar defects may recognize a similar genetic basis • it is possible to speculate that sporadic cases are due to de novo (related to specific developmental pathways) mutations in a single gene or in genes involved in the same pathway • collaborations are important! 35 36 Networking is crucial!

IRCCS Besta IRCCS Mondino

IRCCS Medea Univ. Brescia Univ. Verona Univ. IRCCS Santa Padova Lucia, Univ. Sapienza, Univ. IRCCS Gaslini Cattolica, OPBG

IRCCS Stella Maris

Univ. Federico II

Univ. Messina

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