Angelo Notarangelo

MYO6, the Human Homologue of the Gene Responsible for Deafness in Snell’s Waltzer Mice, Is Mutated in Autosomal Dominant Nonsyndromic Hearing Loss

Mutations in the unconventional myosin VI gene, Myo6, are associated with deafness and vestibular dysfunction in the Snells waltzer (sv) mouse. The corresponding human gene, MYO6, is located on chromosome 6q13. We describe the mapping of a new deafness locus, DFNA22, on chromosome 6q13 in a family affected by a nonsyndromic dominant form of deafness (NSAD), and the subsequent identification of a missense mutation in the MYO6 gene in all members of the family with hearing loss.

Sedlin Controls the ER Export of Procollagen by Regulating the Sar1 Cycle

A Tight Squeeze During intracellular transport, the export of procollagen from the endoplasmic reticulum is intriguing because procollagen is too large to fit into conventional coat protein complex II (COPII)–coated transport vesicles. Recent work has implicated the receptor TANGO1 in procollagen export. Now, Venditti et al. (p. 1668) report that TANGO1 recruits Sedlin—also known as TRAPPC2, a homolog of the yeast TRAPP subunit Trs20—and helps to allow COPII-coated carriers to grow large enough to incorporate procollagen. Sedlin, the product of the gene mutated in spondyloepiphyseal dyplasia tarda, acts to expand cargo containers to fit bulky procollagen. Newly synthesized proteins exit the endoplasmic reticulum (ER) via coat protein complex II (COPII) vesicles. Procollagen (PC), however, forms prefibrils that are too large to fit into typical COPII vesicles; PC thus needs large transport carriers, which we term megacarriers. TANGO1 assists PC packing, but its role in promoting the growth of megacarriers is not known. We found that TANGO1 recruited Sedlin, a TRAPP component that is defective in spondyloepiphyseal dysplasia tarda (SEDT), and that Sedlin was required for the ER export of PC. Sedlin bound and promoted efficient cycling of Sar1, a guanosine triphosphatase that can constrict membranes, and thus allowed nascent carriers to grow and incorporate PC prefibrils. This joint action of TANGO1 and Sedlin sustained the ER export of PC, and its derangement may explain the defective chondrogenesis underlying SEDT.

High Specificity of Quantitative Methylation-Specific PCR Analysis for MGMT Promoter Hypermethylation Detection in Gliomas

Normal brain tissue from 28 individuals and 50 glioma samples were analyzed by real-time Quantitative Methylation-Specific PCR (QMSP). Data from this analysis were compared with results obtained on the same samples by MSP. QMSP analysis demonstrated a statistically significant difference in both methylation level (P = .000009 Mann Whitney Test) and frequencies (P = .0000007, Z-test) in tumour samples as compared with normal brain tissues. Although QMSP and MSP showed similar sensitivity, the specificity of QMSP analysis was significantly higher (93%; CI95%: 84%–100%) as compared with MSP (64%; 95%CI: 46%–82%). Our results suggest that QMSP analysis may represent a powerful tool to identify glioma patients that will benefit from alkylating agents chemotherapy.

Homocysteine levels in amniotic fluid. Relationship with birth-weight.

Hyperhomocysteinemia could play a similar role in the placenta to that played in adults at risk of thrombosis. Moreover, hyperhomocysteinemia in women is described to be associated with the birth of small for gestational age (SGA) newborns, although there are discrepancies on this issue. To date, there is no biochemical marker predictive of SGA in a given pregnancy. We verified the presence of a relationship between homocysteine in amniotic fluid at mid-pregnancy and birth-weight. Amniotic fluid was obtained from 459 healthy women undergoing midtrimester amniocentesis (17.1 +/- 1.2 weeks) because of maternal age. Homocysteine levels were measured in 434 (10 twin) pregnancies. In addition, femur length (FL) and biparietal diameter (BPD) were measured. Outcome of pregnancy was recorded. 233 (53.7%) foetuses were males, 201 (46.3%) females. The mean homocysteine concentration was 1.04 +/- 0.72 microM, (95% C.I. 0.43-2.41). An univariate analysis showed the presence of an association with gestational age, FL, BPD. A multiple linear regression showed that homocysteine levels were significantly associated with FL (p < 0.001) and BPD (p = 0.011). After excluding twin pregnancies, 31 newborns (7.3%) were classified as SGA. Mean birth-weight was 2390 g in SGA, whereas it was 3360 g in 393 adequate for gestational age (AGA) newborns (p < 0.001). The adjusted mean level of homocysteine was significantly lower in AGA (1.01 microM; 95% C.I: 0.94-1.08) than that recorded in pregnancies resulting in a SGA (1.29 microM; 95% CI: 1.05-1.51; p = 0.03). In a large setting, these data provide reference values for homocysteine in amniotic fluids. Moreover, they suggest that homocysteine levels in amniotic fluids may be higher in pregnancies with a SGA newborn.

Translocation of the proto-oncogene Bcl-6 in human glioblastoma multiforme

Bcl-6 translocation is a genetic alteration that is commonly detected in Primary Central Nervous System Lymphoma. The role of this protein in cerebral tumors is unclear. In this study we investigated Bcl-6 translocation and its transcriptional and translational levels in formalin-fixed, paraffin-embedded cerebral tissue sections from glioblastoma (GBM), low-grade glioma (Astrocytoma grade II and III), and meningioma patients, and correlated them with apoptotic processes and p53 and caspase-3 expression. The results showed a frequency of 36.6% of Bcl-6 translocation in GBM patients and a decreased expression in low-grade glioma patients, correlated with the severity of the disease. Bcl-6 translocation induced an overexpression of both Bcl-6 protein and messenger in GBM, inhibiting apoptotic processes and caspases 3 expression. On the contrary, in low-grade gliomas and meningiomas Bcl-6 expression was reduced, resulting in an increase of apoptotic processes. Finally, p53 expression levels in brain tumors were comparable to Bcl-6 levels. Overall, these data demonstrate, for the first time, that the Bcl-6 gene translocates in GBM patients and that its translocation and expression are correlated with apoptosis inhibition, indicating a key role for this gene in the control of cellular proliferation. This study offers further insights into glioblastoma biology, and supports Bcl-6 as a new diagnostic marker to evaluate the disease severity.

Abstract 65: Regulation of KEAP1 expression by promoter methylation in malignant gliomas and association with patient's outcome

In light with the view that KEAP1 loss of function may impact tumour behavior and modify response to chemotherapeutical agents, we sought to determine whether KEAP1 gene is epigenetically regulated in malignant gliomas. We developed a Quantitative Methylation Specific PCR (QMSP) assay to analyze 86 malignant gliomas and 20 normal brain tissues. The discriminatory power of the assay was assessed by Receiving Operating Characteristics (ROC) curve analysis. The AUC value of the curve was 0.823 (95%CI: 0.764-0.883) with an optimal cut off value of 0.133 yielding a 74% sensitivity (95%CI: 63%-82%) and an 85% specificity (95%CI: 64%-95%). Bisulfite sequencing analysis confirmed QMSP results and demonstrated a direct correlation between percentage of methylated CpGs and methylation levels (Spearmans Rho 0.929, P=0.003). Remarkably, a strong inverse correlation was observed between methylation levels and KEAP1 mRNA transcript in tumour tissue (Spearmans Rho -0.656 P=0.0001) and in a cell line before and after treatment with 5-azacytidine (P=0.003). RECPAM multivariate statistical analysis studying the interaction between MGMT and KEAP1 methylation in subjects treated with radiotherapy and temozolomide (n=70), identified three prognostic classes of glioma patients at different risk to progress. While simultaneous methylation of MGMT and KEAP1 promoters was associated with the lowest risk to progress, patients showing only MGMT methylation were the subgroup at the higher risk (HR 5.54, 95% CI 1.35-22.74). Our results further suggest that KEAP1 expression is epigenetically regulated. In addition we demonstrated that KEAP1 is frequently methylated in malignant gliomas and a predictor of patients outcome.