UoF

Prof. Dr. Sandro Sorbi

Benedetta Nacmias

Department of Neurology and Psychiatric Sciences

University of Florence

Viale Morgagni 85

50134 Florence

Italy

Phone: +39 055 4298 465

Fax: +39 055 4277 787

sorbi@neuro.unifi.it, nacmias@unifi.it

- The Neurology Research Unit (Florence University) is on of the main reference centres for the neurodegenerative disorders in our area for both the clinical and the molecular research. The unit focused its well documented experience especially on Alzheimer’s disease and other dementias.The principal fields of investigation are: the clinical neurological evaluation of patients with different kind of dementia, the neurogenetics through the molecular and cellular approach and the neuroimaging analysis. For these activities we collaborated with: Prof . Hyslop (Canada), one of the main expertise in the genetics of Alzheimer’s disease; Prof. Arendt (Germany) for biochemical studies on Alzheimer’s based upon cellular models; Prof. Heroldz (UK) for the neuroimaging studies. The last two persons were partners in projects founded by the European community (V° Framework). We are members of the Excellence Center “DENOTHE” for the study at molecular and clinical level of degenerative and chronic disorders

- The current research activities of the Neurology Research Unit are:

Neurogenetic investigations on Alzheimer’s disease and other dementias: screening for mutations in AD candidate genes (APP, Presenilin 1 and Presenilin 2, Tau), genotyping for ApoE polymorphism, analysis of sequence alterations on genetic risk factors for the disease through association studies using a case-control model; Radiological studies on Alzheimer’s diseases based on the neuroimaging investigations (MRI, fMRI, PET). Evaluation of neuroimaging data in  relation to genetic risk factors; Molecular analysis of Spinocerebellar ataxias; Studies on the genetic factors predisposing to longevity and influencing healthy-pathological aging; Management and maintenance of a bank devoted to process and store biological specimens (DNA, tissue biopsies, fibroblasts and lymploblastoid cell lines from over 4500 individuals) of neurological and psychiatric diseases.

- The Neurology Unit is equipped with:

Automated DNA sequencer Alf Express, 3 Air- flow cycler PCR, 1 gradient Themocycler (PCR), SSCP apparatus, 2 microcentrifuges, electrophoresis apparatus,  Biohazard laminar air flow hoods, 2 Hepa Filtered Cell Incubators, 1 liquid nitrogen storage system. In addition we have  1 dark room, 1 cold room, 1 room for cell cultures, 1 room for genetic analysis.

- the strategy

The diagnosis of Mild cognitive impairment (MCI) is still difficult to diagnose. In addiction the prognosis is extremely unpredictable, therefore there is growing attention  in studying the genetics of MCI. The research focused identify genes involved in the aetiology of MCI opens the opportunities for improvements in the diagnosis of MCI and the prediction of the course of disease patients. To date, the knowledge of the genes involved in MCI is still very restricted and confined to the genetics of Alzheimer’s disease (AD) and other dementias.

There is the need of large and carefully designed candidate gene studies aiming to discover new genetic factors involved in the risk and progression of MCI. The candidate gene method is a powerfull approach to find new genes involved in a multifactorial disease as MCI especially if it is realized with a multicentre effort. Modern molecular techniques allow the genotyping of large number of genes in a time and cost-effective way.  Given the heterogeneity of MCI we will probably identify a high number of low-risk genes, consequently expression profiles will clarify the regulation and indicate a possible role of genetic risk factors.  The discovery of genes involved in this complex disease may increase the understanding of the neuropathological mechanisms leading to MCI

- your possible contributions

 Workpackage Manager (WP-Manager)  for molecular and genetic WP.

- Description or contribution to significant parts from B1…B7

Introduction, rationale and state of the art

The field of aging and dementia is focusing on the characterization of the earliest stages of cognitive impairment. Recent research has identified a transitional state between the cognitive changes of normal aging and Alzheimer's disease (AD), known as mild cognitive impairment (MCI). MCI refers to the clinical condition between normal aging and AD in which persons experience memory loss to a greater extent than one would expect for age, yet they do not meet currently accepted criteria for clinically probable AD. When these persons are observed longitudinally, they progress to clinically probable AD at a considerably accelerated rate compared with healthy age-matched individuals. The boundary between normal aging and early or mild AD is an area of intense interest for theoretical and practical reasons because when these persons are observed longitudinally, they progress to clinically probable AD at a considerably accelerated rate compared with healthy age-matched individuals and they could represent an important model to study the development of the disease and the effect of potential therapeutic strategies. Basic research, such as the identification of secretase inhibitors and the development of an immunization model for the prevention of amyloid deposition, underscores the importance of developing techniques for early detection. Parallel with these endeavors, clinical research aimed at identifying the earliest signs of cognitive impairment has progressed.

The search of factors able to help to predict who are the persons with MCI who progress to dementia is obviously of great interest. It is obvious that the possible identification of MCI persons carrying one of the gene mutations known to cause AD or a risk polymorphism in a susceptibility gene, may represent an important factor to identify early AD within the MCI population.
Since 1991 genetic studies lead to the identification of several gene mutations and different polymorphisms that can either cause AD or significantly increase the risk of developing the disease.

The four genes for which the relation to AD is established are the amyloid precursor protein (APP), presenilin-1(PSEN1) and presenilin-2 (PSEN2) genes which are involved in rare autosomal dominant forms of AD, and the apolipoprotein E (APOE) gene, which is a common genetic risk factor for AD.

To date, no data have been published about the frequency of these mutations in patients with MCI, but different studies analized  asymptomatic carriers of APP mutations with the aim to characterize MCI and improve its diagnosis [Almkvist O, 2003]. Unfortunately, although these studies have been valuable, there are some important limiting factors to consider. Mutations in AD related genes are rare and accounts for approximately about 4-8 % of all patients with AD. This makes it difficult to study these mutations in AD and in particular in MCI, with sufficient statistical power, even in multi centre studies [Tol J, 1999; Almkvist O, 2002]. Moreover several mutations have been found in each gene which are characterized by different clinical phenotypes.

The APOE gene may be a more useful model for studies aiming to improve diagnostic criteria for MCI. According to recent data, APOE epsilon4 carrier status was associated with conversion to AD in older outpatients after controlling for known demographic and clinical risk factors, and APOE epsilon4 homozygosity was associated with increased risk of conversion to AD [Devanand DP,2005].

As MCI is more heterogeneous than AD, APOE or genes with a comparable effect by themselves are predicted to be not suitable for MCI diagnosis not  predicting cognitive decline or conversion to AD.

Besides the identification of specific causative mutations, it could be hypothesized that the analysis for genetic risk factors could provide important data for the identification of subject in early phase of AD. However, there may be alternative causes of MCI than AD including vascular pathology, diabetes, depression and other psychiatric conditions. As each of these disorders is in part of genetic origin, a very large number of different genes may underlie the pathology of MCI. Given the heterogeneity of the disease, most likely a large number of low-risk genes are involved and probably only the combination of the outcomes of multiple genes may be helpful in the diagnosis [Janssens AC, 2004].

Recent evidences suggest that  the combination of cDNA and protein arrays may provide a more global picture to study the sequential changes of distinctive gene expression patterns in the brain as a function of the progression of AD dementia.[ Ho L,. 2005]

The detection of all the loci responsible for the disease and the evaluation of the functional consequences of genetic changes and how they affect protein synthesis or alter metabolic activity, will allow us to comprehend the relation among the genes involved and thus the pathogenesis of this disorder.

Plan

The Group from Florence participates to the project through the identification of susceptibility and genetic gene factors of the progression in MCI subjects through the research of pathogenic mutations and polymorphisms in candidate genes implicated in Alzheimer's disease progression in persons with MCI. Moreover, we are planning to study the interactions and epistatic effects of AD-related genes and non-genetic, as environmental factors.

To perform this research we will identify and collect new families as well as sporadic patients affected by AD and MCI for an extensive mutation screenings to determine the exact contribution of different genes mutations to Alzheimer's disease pathology. Biological samples coming from the others participating centres will be analyzed for the same genetic markers.

Through molecular biology analysis and we will try to identify genetic markers co-segregating with the disease. Completion of these targets will allow the use of fully characterized biological material

- Analysis of pathogenetic mutations in candidate genes (PS-1, PS-2, APP) for initial screening. In addition we will perform the analysis of other susceptibility genes in order to study possible epistatic effect among them.

The mutations screening of genes implicated in the AD pathogenesis will be performed amplifying the coding exons by polymerase chain reaction (PCR), using specific primers near the polymorphic sequence followed by Single Strand Conformation Polymorphism (SSCP) technique.

When it should be possible, SSCP analyses of exons will be performed in the presence of positive control samples carrying known mutations.

In presence of aberrant SSCP patterns the PCR products are sequenced with an automated DNA sequencer (Alf Express; Pharmacia LKB. Uppsala, Sweden).

Sequence variations in PS-1, PS-2 and APPgenes are analyzed by restriction enzyme digestion of amplified products when they involve the creation or abolition of a restriction enzyme recognition site

- With regards to the study on susceptibility factors, we will study polymorphisms located on candidate genes using a case-control model. The study will be addressed to the following polymorphisms in genes that, although with conflicting results, have been considered potential risk factors for AD: PS-1 intronic polymorphism, a2-macroglobulin, the angiotensin converting enzyme, nitric oxide synthase, methylenetetrahydrofolate reductase, Cathepsin D, Cistatin C, Heat-Shock protein-8.

Moreover the research will be extended evaluating the role of other polymorphisms in other genes, identified in the future next years of the project, as potential risk factors for the AD-MCI pathogenesis. We will include genes involved in other MCI-related pathology such as ipertension and genes that are considered with an important role in the aetiology and prognosis of dementia (Van DUIJN, 2004).

Since APOE epsilon4 carrier status was associated with conversion to AD in older outpatients after controlling for known demographic and clinical risk factors, we will investigate if, in our large sample of European patients, the APOE epsilon4 carrier status by itself could predict cognitive decline or conversion to AD.

For this aim all the enrolled patients will be genotyped for ApoE polymorphism, thus confirming or not if APOE genotyping in patients with MCI may have a clinical applicability for prediction of outcome.

In addiction we will study also the interaction of the apolipoprotein E e4 allele and the other alleles and or genotypes associated to an increased susceptibility to the disease.

- In order to better define a more global picture of the evolution from MCI to dementia we are planning to study gene expression patterns of possible candidate genes through cDNA and protein arrays.

- Also we will be able to examine the area of gene- environment interaction an area of important new developments for studying the progression from MCI to AD. We will start analysing families carrying APP or Presenilins mutations that we have already collected in our laboratory. We will examine mutated patients who are starting with the first symptom of the disease.

We believe that rare mutations with major effects may have a great importance for prediction in clinical practice but  probably only the combination of test on large number of genes will be informative and will the be the right strategy for the genetic approach in the next future.

-Almkvist O, Axelman K, Basun H et al. Clinical findings in non demented mutation carriers predisposed to Alzheimer’s disease: a model of mild cognitive impairment. Acta Neurol Scan 2003; 179: 77–82.

-Tol J, Roks G, Slooter AJ, van Duijn CM. Genetic and environmental factors in Alzheimer’s disease. Rev Neurol (Paris) 1999; 155: S10–6.

-Almkvist O, Axelman K, Basun H et al. Conversion from preclinical to clinical stage of Alzheimer’s disease in carriers of the Swedish APP mutation. J Neural Transm 2002; 62: 117–25.

-Ho L, Sharma N, Blackman L, Festa E, Reddy G, Pasinetti GM.  From proteomics to biomarker discovery in Alzheimer's disease.Brain Res Brain Res Rev. 2005 Apr;48(2):360-9.

- Devanand DP, Pelton GH, Zamora D, Liu X, Tabert MH, Goodkind M, Scarmeas N, Braun I, Stern Y, Mayeux R.  Predictive utility of apolipoprotein E genotype for Alzheimer disease in outpatients with mild cognitive impairment.Arch Neurol. 2005 ;62(6):975-80.

-Janssens AC, Pardo MC, Steyerberg EW, van Duijn CM.Revisiting the clinical validity of multiplex genetic testing in complex diseases. Am J Hum Genet 2004; 74: 585–8.

- Van Duijn CM.  Prospects of genetic research of mild cognitive impairment.J Intern Med. 2004;256(3):235-9.

- Proposal for new WP (the WP-description will be about one page)

We propose to search of factors able to help to predict who are the persons with MCI who progress to dementia through the identification of specific causative sequence variations and the analysis for other susceptibility genetic factors as polymorphisms located in candidate genes. Moreover we will perform the identification of genetic risk factors involved in AD in order to allow the possible correlation with neuropsychological data and environmental factors.

3.3 Short description of the organisation (content of B5 < 10 lines)

The Neurology Research Unit (Florence University) is on of the main reference centres for the neurodegenerative disorders in our area for both the clinical and the molecular research. The unit focused its well documented experience especially on Alzheimer’s disease and other dementias. The principal fields of investigation are: the clinical neurological evaluation of patients with different kind of dementia, the neurogenetics through the molecular and cellular approach and the neuroimaging analysis.

3.4 CV of the key person and the partners (< 10 lines)

Prof. Sorbi  Sandro (male) was graduated in Medicine in 1978. Board certified in Neurology (1982), and in Psychiatry (1989), University of Florence. Certified in Neurorehabilitation, American Society of Neurorehabilitation (1992).Associate Professor of Neurology (1992).Professor of Neurology−University of Florence (2001). 1980−82−Physician Research Associate, Division of Chronic and Degenerative. 1983−92 Assistant Professor of Neurology, University of Florence

1997−Chief Neurologic Clinic−University of Florence

He is the leader of the group with more than 25 years experience in the field of dementia, involved in the clinical diagnosis of dementias and Alzheimer’s disease; study of genetic risk factors for Alzheimer’s disease; molecular analysis of Spinocerebellar ataxias, genetic factors for longevity. He has recently participated to 3 European and International projects and published more than 180 scientific articles in national and international journals.

Dr. Nacmias Benedetta (Biologist, female) is a Ph.D and a researcher working in the fields of neurogenetics with more than 10 years experience in the research of Alzheimer’s disease. She will coordinate all the genetic studies performed in our laboratory. Research interests are: mutation analysis and genetic factors for Alzheimer’s disease, eating disorders and associated to pathological and healthy aging.

Dr. Latorraca Stefania ( MD,  specialist in Neurology, female). She is in  the field of neurology since 15 years. His main interest is in the study of dementia and neurodegeneraticve disorders, through clinical assessment and neuropsychological evaluation of patients.

Dr. Cellini Elena, and Dr. Bagnoli Silvia  (Graduated in Biology, females) have  6 years of experience in the genetic and cellular studies on different neurodegenerative disorders.

Dr. Tedde Andrea (Graduated in Biology, PhD, male ), has 10 years of experience in analysis of genetic risk factors for Alzheimer’s disease and he introduced different molecular techniques for molecular diagnosis of  Alzheimer’s disease in our laboratory

UoF1. H. St. George  Hyslop, J. L. Haines, L. A. Farret, R. Polinski, C. Van Broeckhoven, A. Goate, D. R. Crapper McLachian, H. Orr, A.C. Bruni, S. Sorbi, I. Rainero, J. F. Foncin, D. Pollen, J. M. Cantu, R. Tupler, N.Voskresenskaya, R. Mayeux, J.Growdo, R. H. Myerst, J. Growdon, V. A. Fried, R. H. Myerst, L. Nee, H. Backhovens, J. J. Martin, M. Rossor, M. J.Owen, M. Mullan, M. E. Percy, H. Karlinsky, S. Rich,  L. Heston, M. Montesi, M. Mortilla, B. Nacmias, J. F.Gusella, J. A. Hardy (1990)- Genetic Linkage  Studies Suggest That Alzheimer's Disease is not a Single Homogeneus Disorder.- Nature 347:194-197 

UoF2. S. Sorbi, B. Nacmias, P. Forleo, S. Piacentini, L. Amaducci, L. Provinciali (1993)-.APP717 and Alzheimer's disease in Italy. Nature Genetics 4: 10

UoF3. S. Sorbi, , P. Forleo, S. Piacentini, R. Sherrington, E. Rogaev, P. St George Hyslop, L. Amaducci (1995)- Missense mutation of S182 gene in Italian families with early-onset Alzheimer's disease. The Lancet 436: 439-440

UoF4. E.I.Rogaev, R. Sherrington, E. Rogaeva, G. Levesque, M. Ikeda, Y. Liang, H. Chi, C. Lin, K. Holman, T. Tsuda, L. Mar, S. Sorbi, B. Nacmias, S. Piacentini, L. Amaducci, I Chumakov, D. Cohen, Lannfelt, P. E. Fraser, J. Rommenns, P. H. St George-Hyslop (1995)- Familial Alzheimer's disease in kindreds with missense  mutations in a gene on chromosome 1 related to the Alzheimer's disease type 3 gene. Nature 376: 775-778

UoF5. R.Sherrington, S. Froelich, S. Sorbi, D. Champion, H. Chi, E. A. Rogaeva, G. Levesque, E. I. Rogaev, C. Lin, Y. Liang, M. Ikeda, L. Mar, A. Brice, Y. Agid, M.E. Percy, F. Clerget-Darpoux, S. Piacentini, G. Marcon, B. Nacmias, L. Amaducci, T. Frebourg, L. Lannfelt, J. M: Rommens and P. H. St. George- Hyslop (1996)- Alzheimer's disease associated with mutations in presenilin 2 is rare and variably penetrant. Hum. Mol. Genet. 5  (7): 985-988

UoF6. Farrer L, ...., Sorbi S., B. Nacmias... and the ApoE and Alzheimer disease Meta Analysis      Consortium (1997)- Effects of Age, gender and ethnicity on the association between apolipoprotein E genotype and Alzheimer disease. JAMA 278:1349-1356

UoF7. Yu G, Nishimura M, Arawaka S, Levitan D, Zhang L, Tandon A, Song YQ, Rogaeva E, Chen F, Kawarai T, Supala A, Levesque L, Yu H, Yang DS, Holmes E, Milman P, Liang Y, Zhang DM, Xu DH, Sato C, Rogaev E, Smith M, Janus C, Zhang Y, Aebersold R, Farrer LS, Sorbi S, et al, St George-Hyslop P (2000). Nicastrin modulates presenilin-mediated notch/glp-1 signal transduction and betaAPP processing. Nature, 407:48-54.

UoF8. Nacmias B., Tedde A., Forleo P., Piacentini S., Guarnieri B. M., Bartoli A., Ortenzi L., Petruzzi C., Serio A., Marcon G, Sorbi S. (2001) Association Between 5-HT 2a Receptor Polymorphism and Psychotic Symptoms in Alzheimer’s Disease, Biological Psichiatry,  50: 472-475

UoF9. Tedde A, Nacmias B, Ciantelli M, Forleo P, Cellini E, Bagnoli S, Piccini C, Caffarra P, Ghidoni E, Paganini M, Bracco L, Sorbi S. (2003) Identification of new presenilin gene mutations in early-onset familial Alzheimer disease. Arch Neurol.;60(11):1541-4.

UoF10. Bagnoli S, Nacmias B, Tedde A, Guarnieri BM, Cellini E, Petruzzi C, Batoli A, Ortenzi L, Sorbi S. (2004) Brain-derived neurotrophic factor genetic variants are not susceptibility factors to Alzheimer's disease in Italy. Ann Neurol. ;55(3):447-8.

4. Contribution

description of your contribution fit for copy in the draft of the proposal.

For the outline of the Workpackages be so kind and use the template:

Workpackage description