Medical genetics
Medical Genetics Unit
Department of Molecular Biology
University of Siena
Viale Bracci 2, 53100 Siena
Tel. + 39 0577 233303
Fax + 39 0577 233325
Principal investigator: Alessandra Renieri
| Team members | |
|---|---|
| Francesca Mari | Assistant Professor | Maria Antonietta Mencarelli | Medical Genetics Consultant |
| Francesca Ariani | Assistant Professor |
| Annabella Marozza | Medical Genetics Resident |
| Mirella Bruttini | Biologist |
| Maria Carmela Epistolato | Research Fellow |
| Mariangela Amenduni | PhD Student |
| Vittoria Di Sciglio | PhD Student |
| Gabriella Livide | PhD Student |
Introduction
TThe Medical Genetics Unit is involved in both clinical and basic research in human genetics. In particular, research interests focus on the molecular pathogenesis of mental retardation (Rett Syndrome and other forms of mental retardation), hereditary nephropathy (Alport Syndrome) and cancer (Retinoblastoma RB). The Unit has two ambulatories for genetic counseling and a laboratory equipped with the main items required for molecular biology activities (cell culture room, five PCR machines, a Real Time quantitative PCR machine, two stations for DHPLC analysis, two machines for automated fluorescent DNA sequencing, a microarray spotter, a microarray hybridization station, a Wash station, a hybridization oven and a microarray scanner). A large bank of DNA, cell lines and tissues from patients with Rett Syndrome, other forms of mental retardation and RB is maintained at the Medical Genetics laboratory. Each patient contributing to the collection has an accurate description of phenotype and genotype. The bank has a dedicated website (http://www.biobank.unisi.it) which is available to the scientific community.
Main Research Themes
Studies on retinoblastoma carcinogenesis
CIn recent years, we have collected 516 DNA samples from RB patients (176) and healthy relatives (340) through the activity of genetic counseling. We have collected 23 RB tissues from these patients, including two very rare samples also containing areas of retinoma (RN). With regard to inheritance, among the 176 probands, 36 have been classified as familial cases and 130 as sporadic cases (84 unilateral and 46 bilateral). We have performed a RB1 molecular analysis by DHPLC or direct sequencing, identifying 11 frameshift, 18 nonsense, 11 splice-site and 2 missense mutations. Patients have also been analyzed using MLPA and array CGH to investigate the presence of RB1 gross rearrangements, and 9 large deletions have been identified. Clinical and molecular information on RB patients, made anonymous through coding, are present in an on-line catalog available at http://www.biobank.unisi.it.
During the last years, our group has successfully used the Agilent platform for array-CGH studies on RB samples. In particular, we used this technique to analyze DNA isolated from retinoma and RB tissues in order to compare the chromosomal aberrations and identify somatic events accompanying malignancy. To accomplish these goals, we used a laser microdissection technique in order to distinguish the areas of RB, retinoma and normal tissues in Formalin-Fixed Paraffin-Embedded (FFPE) samples obtained after enucleation. This protocol creates problems due to the amount of DNA that can be obtained. In the case of a particularly low DNA yield, we used the GenomePlex Whole Genome Amplification (WGA) kit to pre-amplify paraffin-embedded archival specimens before array CGH experiments.
The group investigated, using array-CGH, a series of 18 RB (10 bilateral and 8 unilateral) and 2 RN samples to characterize genomic changes in the two lesions. In RB, a total of 64 rearrangements (47 gains and 17 losses) were detected. In accordance with previous data, recurrent imbalances were found on chromosomes 1, 2, 6, 13 and 16. In addition, three previously undescribed recurrent rearrangements were identified, two on chromosome 9 (9q22.2 and 9q33) and one on chromosome 11 (11q24.3). All these rearrangements indicated interesting candidates for RB progression. Bilateral cases showed a lower number of imbalances (mean, 1) compared to unilateral cases (mean, 7), with statistical significance (p = 0.002). The unilateral cases could be divided into low (< 4) and high level (> 7) chromosomal instability groups, the first group presented younger age at diagnosis (mean, 511 days) compared to the second one (mean, 1,606 days). Array-CGH in RN samples revealed different results. In one RN, ophthalmoscopically diagnosed as a benign lesion, no rearrangements were detected, while adjacent RB displayed seven aberrations. Differently, in the other RN, identified by retrospective histopathological examination, five genomic rearrangements were detected. Among these, three were in common with the adjacent RB (dup5q13.2, dup6p, dup8p23.1), while the remaining two (dup1q32.2 and dup13q31.2) were exclusively detected in RN and did not contain any gene. One rearrangement, dup5p, was RBspecific and included the SKP2 gene, an interesting candidate for tumor progression. The genomic profile therefore uncovered the different molecular nature of the lesions classified as 'retinomas' on the basis of their histopathological appearance.
Recent studies in the field of DNA methylation have lead to the awareness that epigenetic changes may represent an alternative or complementary mechanism to mutational events in tumor progression. In particular, methylation in the CpG islands in the promoter regions of a large number of tumor suppressor genes is observed in several human cancers (a). Methylation-specific MLPA has been recently described as a method that allows the simultaneous identification of epigenetic changes at multiple sites (b). Therefore, we decided to use this technique to characterize the role of epigenetic silencing in RB pathogenesis. We analyzed 10 RB samples, comparing the results to those obtained in normal retina (Table 1). Tumor tissues showed frequent hypermethylation of MGMT (7/10, 70%), MSH6 (6/10, 60%), CD44 (5/10, 50%), PAX5 (5/10, 50%) and GATA5 (3/10, 30%). Since these genes are involved in DNA repair (MSH6), cellular differentiation (PAX5 and GATA5), and cell-to-cell communication (CD44), their epigenetic silencing could play an important role in RB initiation and progression. In particular, aberrant methylation of these factors could play a key role in tumor development, especially in bilateral cases, where chromosomal imbalances are less frequently observed.
Table 1 - Summary of tumor samples analyzed for aberrant methylation. %met indicates the gene methylation percentage detected in 10 retinoblastoma samples (#1-10)
In order to test the hypothesis that RB phenotypic variability (age of onset, involvement of one/two eyes
and therapy necessary) may in part result from the variable function of genes involved in the cell cycle
and apoptosis, we investigated the effect of two functional polymorphisms, one in TP53 (Arg72Pro) and
the other in the promoter of MDM2 (SNP309 T>G), on the age of tumor diagnosis. We tailored specific
Pyrosequencing assays for the two SNPs and genotyped 90 RB patients with a characterized RB1
germline mutation (i.e. familial and bilateral cases) belonging to our collection. A descriptive analysis
showed an earlier age at diagnosis in patients with bilateral retinoblastoma than in those with unilateral
retinoblastoma (median age: 0.57 years versus 1.49 years, respectively, p < 0.001). Since age of onset
is often not exactly known, we considered bilaterality a more robust measure of the variable genetic
risk. A multivariate logistic regression model adjusted for age and gender showed the risk of bilateral
disease to be higher for splicing and missense mutations than for deletions, duplications, nonsense
and frameshift mutations, but not statistically significant (OR = 1.33, 95% CI 0.22-8.22). Regarding
MDM2 SNP309, the model revealed a significantly higher risk of bilaterality for the GG genotype than
TT (OR = 11.78, 95% CI 2.18-63.65), but was not significant for TG. As for TP53 R72P SNP, the risk of
bilaterality is not significant for the PP genotype (Figure 1). Our results suggest, for the first time, that
MDM2 and TP53 may be modifiers of RB as well.
Figure 1 - Graphical representation of multivariate logistic regression model for TP53 (SNP R72P) and MDM2 (SNP309 T>G) polymorphisms adjusted for age and gender. The time is expressed in days from birth. Log odds indicates the risk of bilaterality for each polymorphism
References
- Hegi ME, Diserens AC, Gorlia T, et al: MGMT gene silencing and benefit from temozolomide in
glioblastoma.
N Engl J Med 2005; 352(10): 997-1003. - Nygren AO, Ameziane N, Duarte HM, et al: Methylation-specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences. Nucleic Acids Res 2005; 33(14): 128.
Research Grants
| Anno | Funding Agency | Amount |
|---|---|---|
| 2007/2009 | Istituto Toscano Tumori | € 70,000 |
| 2007/2008 | Ministero dell'Istruzione, dell'Universitá e della Ricerca, FIRB 2005 | € 113,600 |
| 2007/2008 | University of Siena, PAR 2006 | € 10,000 |
Main collaborations
With units within ITT
- Department of Human Pathology and Oncology, University of Siena
- Retinoblastoma Referral Center, Department of Ophthalmology, University of Siena
- Department of Pediatrics, University of Siena
With other Italian and Foreign Institutions/Organizations
- Medical Genetics Unit, Azienda Ospedaliero Universitaria 'San Luigi', Orbassano (Torino)
- Institut de Recherches sur le Cancer de Lille (France)
Publications
- Sampieri K, Amenduni M, Papa FT, et al: Array comparative genomic hybridization in retinoma and retinoblastoma tissues. Cancer Sci 2009; 100: 465-71.
- Sampieri K, Mencarelli MA, Epistolato MC, et al: Genomic differences between retinoma and retinoblastoma.Acta Oncol 2008; 47: 1483-92.
- Caselli R, Speciale C, Pescucci C, et al: Retinoblastoma and mental retardation microdeletion syndrome: clinical characterization and molecular dissection by array CGH.J Hum Genet 2007; 52: 535-42.
- Sampieri K, Hadjistilianou T, Mari F, et al: Mutational screening of the RB1 gene in Italian patients with retinoblastoma reveals 11 novel mutations. J Hum Genet 2006; 51: 209-16.
