The course requires the typical knowledge of the basic course of the three years Genetics.

Lessons are Frontal

Introduction to the course.

Types of polymorphisms in the genomes.

Minisatellites, microsatellites.

DNA fingerprinting, instability of microsatellites.

The Slippage-misalignment model.

Neurodisorders for aberrant expansion of triplet microsatellites.

Single nucleotide polymorphisms (SNPs), micro-insertions, micro-deletions. Discovery methods:

high-resolution melting, single strand conformation polymorphisms, Sanger’s sequencing reaction.

Genotyping. DOT BLOT, PCR-RFLP (restriction fragment length polymorphism), ASO-PCR/ARMS (amplification refractory mutation system).

Genotyping of SNPs: oligonucleotide ligation assay (OLA), MALDI-TOF, TaqMan Allelic Discrimination Assay. Microarrays for genotyping.

The original method: Single base extension (SBE) - Arrayed Primer Extension Assay (APEX).

Genotyping with Illumina BeadArray. The Bead decoding.

Genotyping by hybridization. Affymetrix GeneChip. PM and MM set probes.

Segmental Duplications. Mandatory and optional. Mechanisms of formation: unequal crossing-over, whole-genome duplications, chromosomal rearrangements.

The loci of CYP2D6, GSTM1, GSTT1, and TP53 as example of mandatory and polymorphic duplications and interstitial deletions in the human genome. The example of CYP2D6 in the metabolism of antidepressants and other drugs.

Genotyping of polymorphic interstitial deletions and small insertions. The example of GSTT1, GSTM1. Analysis of interstitial deletions within the gene of the Duchenne Muscolar Distrophy.

Analysis by gel electrophoresis of PCR products, analysis by Multiplex Ligation-dependent Probe Amplification (also called Multiplex Oligonucleotide Ligation Assay), analysis by TaqMan assay (Real-time quantitative PCR).

Comparative Genomic Hybridization (Classical CGH).

BAC arrayCGH, tiling BAC arrayCGH.

SNP array CGH.

Interstitial inversions. The example of 900Kbps inversion polymorphisms within 17q21.31 and susceptibility to mental retardation.

The forces shaping allele frequencies in populations.

How human genome is organized. Satellite DNA, Sat I, II, III, alphoid sequences, beta-sau, organization of centromeric heterochromatin, G and Q bands.

Telomeric Minisatellites, multicopy genes (functional RNAs, duplicated genes, pseudogenes, processed pseudogenes). Retrotransposons: LINEs (LINE-1), SINEs (Alu dimer), Endogenous retroviruses, virus-like elements. Mechanisms of retrotransposition. DNA transposons. Mechanism of transposition with and without transposon duplication.

Mapping mendelian traits. The first example: Duchenne’s Muscolar Dystrophy. Cloning by subtraction (Kunkel’s method).

Examples of genes causative for various types of neurological disorders detected by SNP-arrays in micro-interstitial deletions.

Linkage analysis. Principles.

Example of LOD score calculation.

Two-points mapping, multi-point mapping. The multipoint LOD score. The first high density map of genetic markers (CHLC, CEPH).

Mapping homozygosity traits exploiting the autozygosity mapping. Chromosomal segments “Identitcal by Descent”  (IBD). The “Identity by State” (IBS). Example of calculation of a LOD score in the offspring of second cousins.

Candidate region identified with autozygosity mapping. Narrowing the candidate region exploiting a common ancestor in closed populations. Specific examples (cystic fibrosis, Nijmegen Breakage Syndrome, literature, see slides).

Fine mapping in pedegrees (dominant model, an example).

Identification of the candidate region by the use of ENU-mutagenized mice. Mice helping to discover the gene for human diseases: the examples of Waardenburg syndrome, and mice shaker-2.

The basics of the positional cloning. Pitfalls in linkage studies.

After the human genome project: gene predictions, prioritizing genes for mutation scan. The examples of Retinitis pigmentosa, marfan syndrome, Beals’s syndrome, Wilson’s disease, Menkes’s disease. Mutation screening of exons or cDNAs? The example of Haemophilia Factor VIII. 

Possible landscapes following mutation screening:

1) Good correspondence between genotype and phenotype. Carriers/homozygotes must show the phenotype, healthy people within family should not be carriers or homozygotes.

2) Verify if the variant is a simple polymorphism (Genebank).

3) Go for mutation screeening of the same gene on probands of other families-            (a) find the same mutation

(b) find a different mutation (in the same gene)

(c) find no mutations

4) Again: verify these mutations are not polymorphisms (Genebank)

5) Inferring a possible deleterious effect:

(a) evaluating the ORF

(b) use of in silico algorithms predicting the effect on the protein              

(c) using the conservation of the region by comparing with evolutionary distant organisms (orthologues) available in Genebank

Complex traits (non mendelian diseases).

Introduction to case-control association studies.

Hypothesis-driven case-control studies. Selection bias. Stratification bias.

The error alpha (type-I error). The error beta (power of the study).

Calculation of the Odd Ratio and the 95% confidence intervals. Examples of the genetics models (linear, dominant, recessive).

From candidate genes (hypothesis driven studies) to genome-wide associations studies (GWAS, hypothesis generating studies).

The Manhattan plots. The problems with multiple testing: the Bonferroni’s correction. A design multistep for a powerful and cost-effective GWAS.

SNO-Microarray: the choice of the correct SNPs: the haplotype tagging SNPs. Linkage disequilibrium (LD) and the calculation of the r2. The forces shaping the LD. The hapmap project (www.hapmap.org). Example of extraction of htSNPs.

The correct interpretations of the results: association is not causation. 

- -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

The course will focus on various diseases of the CNS with underlying genetic defects to illustrate genotype-phenotype correlations and experimental and clinical stage approaches to improve the disease outcome. The visual system will be often used as a paradigm to illustrate concepts and tools to which other genetic diseases of the CNS will be compared to obtain general concepts. Lectures involving specific CNS areas (i.e. the callosal body; motor areas pertinent to the mirror system; the hippocampus etc.) will be introduced by summaries of the main anatomical organization, relevant morphological features, basic nomenclature and position in the brain general context as well as fundamental of physiology. Indications for usage of web-based tools to navigate the brain will be provided. Various topics will comprise an experimental part illustrating modern methodological tools for the study of the CNS. Navigation and search of clinicaltrials.gov for the genetic diseases under study will be performed.

Specifically:

• General anatomy of the retina. Fundamental nomenclature. Inherited photoreceptor degenerations. From mutations to phenotype: known and (many) unknown cellular pathways to photoreceptor death starting from hundreds of mutations in 66 different genes. Retinitis Pigmentosa. The examples of rhodopsin and phosphodiesterase mutations. Retinal Pigment Epithelium, visual cycle and Leber Congenital Amaurosis. High photoreceptor efficiency and great genetic vulnerability .

• Gene therapy from bench to bedside. From RPE65 mutations to commercially available Luxturna®. Ongoing gene therapy and open clinical trials for Retinitis Pigmentosa, Leber Congenital Amaurosis, Achromatopsia. Subretinal injections, intravitreal injections, ocular and global immune responses.

• Viral Vectors for ocular gene therapy; limitations, targeting of specific cells. The undisclosed issue of inflammatory response to capsids. The problem of correcting  gain-of function mutations. Optogenetics to restore light sensitivity: state of the art of cell-specific promoters and extension to other genetic diseases. How to search and understand clinicaltrials.gov

• Alternative approaches to neuronal rescue: inhibition of apoptosis mediated by ceramide using small molecules. Manipulations of the environment to promote retinal rescue (environmental enrichment: definition, effects). Neurotrophins as neuroprotectants. Prostheses: Argus II and epiretinal prostheses. Subretinal approach. Expected recovery of vision. The cochlear implant, similarities and differences with retinal prosthesis. The artificial nose: applications.

• Alzheimer, Parkinson and thauopathies. General principles and latest discoveries on multiple roles of Tau (including the nuclear access). Antibody therapy to target specific cellular functions and localization. Rare examples of genetic patients as guidance for the discovery of mechanisms of these diseases.

• Protein targeting in neurodegenerative diseases by antibody design and delivery. The case of Alzheimer, alpha-beta amyloid accumulation and employment of conformational specific antibodies. In vivo targeting of intracellular organelles. The immune response in the CNS as a limiting factor for drug (or viral vectors; or protein;) delivery. The Blood brain barrier: consequences for pharmacological treatment and gene therapy to access to the brain. Ebola and Zika in the eye.

• Cell therapy for neurodegenerative diseases of genetic origin. Organoids as recent tools for cell-based repair therapy and diagnosis of genetic defects. Brain organoid. The brain in a dish. The eye in a dish. Experimental advantages, perspectives and limitations.  

• Hereditary CNS demyelinating diseases. Genotype-phenotype correlations in Krabbe disease (mutations in GALC gene and accumulation of  galactosylceramide);  leukoencephalopathy with vanishing white matter (mutations in the EIF2B5 gene). The case of ATAD3 deletions as substantial cause of pontocerebellar hypoplasia (PCH ).

• Connecting two parts of the brain. The callosal body organization and function. Complete Agenesis of the Callosal Body, pathophysiology and examples of underlying genetic defects. Mutations in Deleted in Colorectal Cancer gene and the multiple functions of the DCC protein. The callosal body: the largest commissural organ of myelinated fibers in the CNS.

• Regeneration and lack of regeneration in mammalian CNS. Central and peripheral myelin organization and molecular composition. Nogo, NgR, MAG. Inhibition of regeneration due to central myelin determinants. Reaction of injury in central and peripheral nerves. The case of dorsal root ganglia to study central and peripheral myelin effects in a single neuron. Possible involvement of  mutations of myelin inhibitor genes in schizophrenia.

• Manipulation of regenerative capacities of the CNS by interference with the microenvironment: peripheral nerve grafts, employment of olfactory ensheating glia. Mirror neurons and mirror system in monkeys and humans. Localization, basic physiological properties, possible implications in empathy and autism.

• Mirror system in birds. Correlation with articulated language. Mirror system dysfunctions in mammalians. Autism. Possible genetic causes. The TRIO gene and its effects on the development and regulation of the strength of connections between brain cells. Mutations in TRIO.

Text book (suggested):

"Genetica molecolare umana ", by Tom Strachan & Andrew P. Read (Zanichelli)

"Introduzione alla Genomica", by Greg Gibson & Spencer Muse (Zanichelli)

See

www.stefanolandi.eu

The exam is written (2 hours). An oral can be requested (optional) but it will change the final score of only + or - 1 point

Look at

e-learning

Moodle