Assessment of MYCN amplification status in Tunisian neuroblastoma: CISH and MLPA combining approach

Background: Neuroblastoma [NB] shows a complex combination of genetic aberrations. Some of them represent poor genetic prognosis factors that require specific and intensive chemotherapy. MYCN amplification consists of the major bad outcome prognostic factor, it is indeed frequently observed in aggressive neuroblastomas. To date different methods are used for MYCN status detection

Objectives: The primary aim of our study was to provide a critical assessment of MYCN status using 2 molecular techniques CISH and MLPA. We also focused on the correlation between neuroblastoma genetic markers and patient's clinical course among 15 Tunisian patients

Methods: we developed a descriptive study that includes 15 pediatric Tunisian patients referred to our laboratory from 2004 to 2011. We reported the analysis of fresh and FFPE NB tumors tissues

Results:No significant correlation was found between COG grade and patients overall survival. Assessment of NMYC gene copy number by kappa statistic test revealed high concordance between CISH and MLPA tests [kappa coefficient = 0.02]

Conclusion: Despite misdiagnosing of MYCN status fewer than 5 copies, MLPA remains an effective molecular technique that enables a large panel of genomic aberrations screening. Thus combining CISH and MLPA is an effective molecular approach adopted in our laboratory. Our results allow pediatric oncologists to set up the first Neuroblastoma therapeutic strategy based on molecular markers in Tunisia

[Circadian clock genes expression in peripheral blood mononuclear cells of comatose patients]

Recently circadian clock genes have been identified in humans but information regarding their expression has remained very limited. The evaluation of circadian variations in the expression of clock genes in humans seems to be a major importance both from a fundamental point of view as a diagnostic and therapeutic perspective. In this context, several works including ours have described the fluctuation of clock genes. Describing rhythmic expression of clock genes in intensive care units patients during 24h and we tried to determine the effect of the absence of synchronizers such as light/ dark cycle on these rhythms. 15 patients received care in private room in intensive care units in the hospital Sahloul [5 comatous and 10 non comatous patients]. For RNA isolation we used peripheral blood mononuclear cells which represent an ideal material to investigate non-invasively the human clock at the molecular level. In the present study, we noticed that clock genes mRNA exhibit a circadian expression in comatose patients, while the rhythmicity of some studied genes disappeared in non-comatose patients. The disturbance of the rhythmic fluctuation of the clock genes could be the result of the effect of surgery on some biological rhythms as it could be explained by the lack of synchronizers in intensive care units such as light/dark cycle

Familial early-onset diabetes is not a typical MODY in several Tunisian patients

MODY [Maturity-onset diabetes of the young], a dominantly inherited form of early-onset diabetes, is clinically and genetically heterogeneous with more than ten genetic subtypes described worldwide. To evaluate the possible existence of MODY in 12 young diabetic Tunisian patients by searching for mutations in the most prevalent MODY genes. Twelve patients with diabetes in 2-to-3 generations, all diagnosed before age 31, were screened for mutations and deletions in HNF1A, HNF4A, INS, IPF1, NEUROD1 and GCK genes by Sanger sequencing and by Multiplex ligation-dependent probe amplification assay. The patients had no evidence of autoimmunity and a mean age at diabetes diagnosis of 25.66 +/- 3.96 years with severe overt diabetes [fasting glycaemia: 10.91 +/- 3.55 mmol/ l; HbA1c: 10.46 +/- 3.31%]. Two subjects were initially treated with insulin. On the ten initially treated with OHA or on diet, eight converted to insulin therapy [within 3 months to 20 years]. Molecular analysis showed only one missense HNF4A mutation [I453V] in one family. No mutations in the studied genes were detected in the other patients. A molecular defect in known MODY genes has been excluded in 11 patients with early-onset diabetes suggesting that other genetic causes may explain diabetes in these families. In such cases, new generation sequencing approaches may be well appropriate to identify specific molecular etiologies from extended families and to establish a strategy of molecular diagnostic of MODY in Tunisia