MAST BOLOGNA, 25-26 OCTOBER, 2016 DEPArray User Meeting Setup of a - - PowerPoint PPT Presentation

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MAST BOLOGNA, 25-26 OCTOBER, 2016 DEPArray User Meeting Setup of a DEPArray-Based Protocol for Sorting Single Neural Cells Potentially Carrying PI3K/AKT/mTOR Genes Mutations Valerio Conti, PhD Pediatric Neurology, Neurogenetics and

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MAST • BOLOGNA, 25-26 OCTOBER, 2016

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MAST • BOLOGNA, 25-26 OCTOBER, 2016

DEPArray™ User Meeting

Setup of a DEPArray-Based Protocol for Sorting Single Neural Cells Potentially Carrying PI3K/AKT/mTOR Genes Mutations

Valerio Conti, PhD Pediatric Neurology, Neurogenetics and Neurobiology Unit and Laboratories, Neuroscience Department, A Meyer Children’s Hospital, University of Florence, Florence, Italy. valerio.conti@meyer.it

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MAST • BOLOGNA, 25-26 OCTOBER, 2016

Main research areas of the Center of Excellence in Neurosciences, Children’s Hospital Meyer

Epileptic encephalopathies with seemingly non-lesional
rigin:
Phenotype definition
Genetic causes
Exploring functional consequences
Treatment & prevention
Epileptogenic malformations of cortical development:
Phenotype definition
Genetic causes
Exploring functional consequences
Treatment & prevention

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MAST • BOLOGNA, 25-26 OCTOBER, 2016

Main research areas of the Center of Excellence in Neurosciences, Children’s Hospital Meyer

Epileptic encephalopathies with seemingly non-lesional
rigin:
Phenotype definition
Genetic causes
Exploring functional consequences
Treatment & prevention
Epileptogenic malformations of cortical development:
Phenotype definition
Genetic causes
Exploring functional consequences
Treatment & prevention

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MAST • BOLOGNA, 25-26 OCTOBER, 2016

PI3K/AKT/MTOR pathway

From KEGG: http://www.genome.jp/kegg/kegg2.html

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MAST • BOLOGNA, 25-26 OCTOBER, 2016

PI3K/AKT/MTOR pathway and neurological diseases

Mutations in genes belonging to the PI3K/AKT/MTOR pathway have been associated

with a wide neurological phenotypic spectrum:

The number of clinical conditions in which developmental brain abnormalities, epilepsy,

intellectual disability, and autism are variably associated and that are caused by different somatic or constitutional mutations involving the PI3K/AKT/MTOR pathway is growing.

TSC1/TSC2: Tuberous Sclerosis;
AKT3, PIK3CA, MTOR, DEPDC5, NPRL2, NPRL3: Type Ib, IIa and IIb FCD, HME, MEG;
PTEN: MAEP;
PIK3CA: MPPH, MCAP, BPP with or without macrocephaly.

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MAST • BOLOGNA, 25-26 OCTOBER, 2016

Focal VS diffuse MCDs

FOCAL MALFORMATIONS (e.g. FCD) DIFFUSE MALFORMATIONS (e.g. DYSPLASTIC MEGALENCEPHALY) EPILEPSY SURGERY NGS STUDIES ON DYSPLASTIC BRAIN SPECIMENS

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MAST • BOLOGNA, 25-26 OCTOBER, 2016

The background noise issue…

Lodato et al., Science. 2015;350:94-8

Mosaic variants that arose during development and are causative for brain malformations in patients.

… each human cortical neuron has a profoundly distinctive genome, harboring as

many as 1458 to 1580 somatic single nucleotide variants, in addition to large copy number variants and occasional retro-element insertions …

… most of these variants reflect DNA damage during active transcription and are

related to physiological neuronal aging and neurodegenerative diseases…

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MAST • BOLOGNA, 25-26 OCTOBER, 2016

The mosaicism issue…

The capability to detect these mutations directly

depends from the percentage of cells carrying them.

High-throughput sequencing of DNA isolated from

thousands of cells from brain specimens, although useful, is unable to detect mutations present in small numbers o cells. SOMATIC MOSAICISM > 10%

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MAST • BOLOGNA, 25-26 OCTOBER, 2016

Target cytological subtypes

Autoptic control HME PQD Anti-p-RPS6 Dysplastic, cytomegalic neuron Balloon cell Normal cortical neuron

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MAST • BOLOGNA, 25-26 OCTOBER, 2016

DEPArray protocol setup

Fibroblasts cultures from a patient with BPP and macrocephaly carrying the Gly373Arg mosaic mutation in PIK3R2 (Mirzaa et al., Lancet Neurol. 2015) Single cells separation, WGA, mutation validation and genotype/phenotype correlation STEP 1 Evaluation of the phenotype in dysplastic brain specimens and genotype/phenotype correlation for a known mutation STEP 2 NGS analysis of dysplastic brain specimens for molecular diagnosis STEP 3

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MAST • BOLOGNA, 25-26 OCTOBER, 2016

Step 1: fibroblasts

HOECHST FITC (p-RPS6) MERGE BRIGHTFIELD HOECHST MERGE BRIGHTFIELD

Fibroblast_Cell Id = 985 Fibroblast_Cell Id = 2257 n = 6

FITC (p-RPS6)

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MAST • BOLOGNA, 25-26 OCTOBER, 2016

Step 2: dysplastic brain specimens

HOECHST FITC (p-RPS6) MERGE BRIGHTFIELD

Brain cell_Cell Id = 7892

HOECHST MERGE BRIGHTFIELD

Brain cell_Cell Id = 6760 n = 1

FITC (p-RPS6)

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MAST • BOLOGNA, 25-26 OCTOBER, 2016

Conclusions and future directions

The DEPArray has the capability of recognizing and

separating aberrant cells carrying mutations in genes of the PI3K/AKT/mTOR pathway.

Massive parallel sequencing protocol for analyzing

samples carrying known mutations.

Massive parallel sequencing studies in samples from

patients without molecular diagnosis.

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Neurobiology Lab Valentina Ariu Valentina Cetica Cristiana Pelorosso

Neurogenetics Lab Claudia Bianchini Elena Cellini Laura Chiti Dalila De Vita Davide Mei Elena Parrini Daniela Pucatti Domenico Rutigliano Simona Virdò Pediatric Neurology Unit Carmen Barba Emanuele Bartolini Sara Chiari Viola Doccini Carla Marini Francesco Mari Federico Melani Martino Montomoli Tiziana Pisano Anna Rosati

Prof. Renzo Guerrini

Center of Excellence in Neurosciences Meyer Children’s Hospital

Silicon Biosystems Alessandra Totaro Giulia Bregola