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 two mutated copies of the gene are one mutated copy of a gene is sufficient test selection required to cause the disease to cause the disease healthy heterozygous parents transmit 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 horizontal transmission (multiple for diseases non reducing fitness, the affected individuals in one generation) mutation is transmitted vertically from negative or ambiguous results positive results can also present in sporadic cases one affected parent to 50% offspring diagnostic reassessment? implications for management, X-linked further genetic testing? prenatal diagnosis, 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 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) café-au-lait spots, axillary freckles Early onset pure cutaneous neurofibromas cerebellar atrophy • AT (ATM) optic gliomas, cranial tumours, other MRI lesions • AOA1 (APTX) • SCA29 (ITPR1) … AT AOA1 ITPR1 Leigh syndrome (mitochondrial) Joubert syndrome onset < 1 year congenital ataxia reproductive risks, carrier testing and prenatal diagnosis severe neurological variable organ involvement specific treatments, therapeutic trials picture typical “molar tooth sign” ++ severe disorders with full penetrance typical basal ganglia NBIA lesions Neuronal Ceroid Lipofuscinoses 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 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: a specific mutation causes most cases of a disease (e.g. Friedreich’s Ataxia Æ GAA expansion in the FXN gene) rationale and procedure of the test standardized genetic testing, high sensitivity inheritance, natural history, and management of the disease implications for the health and reproductive choices of the relatives genetic and/or molecular heterogeneity (commonest!) potential benefits, disadvantages, and consequences for the future health, employment, and insurance prospects of the individual The same disease can be caused by different mutations and different mutational mechanisms involving one or even many genes referral for psychosocial support when indicated ataxia-telangiectasia (ATM, very large gene, many mutations) results, especially when positive, should also be rendered in the 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!!!) 13 14 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). 15 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