Emanuele Bellacchio

AHI1 gene mutations cause specific forms of Joubert syndrome–related disorders

Joubert syndrome (JS) is a recessively inherited developmental brain disorder with several identified causative chromosomal loci. It is characterized by hypoplasia of the cerebellar vermis and a particular midbrain‐hindbrain “molar tooth” sign, a finding shared by a group of Joubert syndrome–related disorders (JSRDs), with wide phenotypic variability. The frequency of mutations in the first positionally cloned gene, AHI1, is unknown.

NF1 Gene Mutations Represent the Major Molecular Event Underlying Neurofibromatosis-Noonan Syndrome

Neurofibromatosis type 1 (NF1) demonstrates phenotypic overlap with Noonan syndrome (NS) in some patients, which results in the so-called neurofibromatosis-Noonan syndrome (NFNS). From a genetic point of view, NFNS is a poorly understood condition, and controversy remains as to whether it represents a variable manifestation of either NF1 or NS or is a distinct clinical entity. To answer this question, we screened a cohort with clinically well-characterized NFNS for mutations in the entire coding sequence of the NF1 and PTPN11 genes. Heterozygous NF1 defects were identified in 16 of the 17 unrelated subjects included in the study, which provides evidence that mutations in NF1 represent the major molecular event underlying this condition. Lesions included nonsense mutations, out-of-frame deletions, missense changes, small inframe deletions, and one large multiexon deletion. Remarkably, a high prevalence of inframe defects affecting exons 24 and 25, which encode a portion of the GAP-related domain of the protein, was observed. On the other hand, no defect in PTPN11 was observed, and no lesion affecting exons 11-27 of the NF1 gene was identified in 100 PTPN11 mutation-negative subjects with NS, which provides further evidence that NFNS and NS are genetically distinct disorders. These results support the view that NFNS represents a variant of NF1 and is caused by mutations of the NF1 gene, some of which have been demonstrated to cause classic NF1 in other individuals.

Familial transposition of the great arteries caused by multiple mutations in laterality genes

Background The pathogenesis of transposition of the great arteries (TGA) is still largely unknown. In general, TGA is not associated with the more common genetic disorders nor with extracardiac anomalies, whereas it can be found in individuals with lateralisation defects, heterotaxy and asplenia syndrome (right isomerism). Objective To analyse genes previously associated with heterotaxy in order to assess mutations in familial TGA unassociated with other features of laterality defects. Methods Probands of seven families with isolated TGA and a family history of concordant or discordant congenital heart disease were screened for mutations in the ZIC3, ACVR2B, LEFTYA, CFC1, NODAL, FOXH1, GDF1, CRELD1, GATA4 and NKX2.5 genes. Results Mutation analysis allowed the identification of three sequence variations in two out of seven TGA probands. A FOXH1 (Pro21Ser) missense variant was found in a proband who was also heterozogous for an amino acid substitution (Gly17Cys) in the ZIC3 gene. This ZIC3 variant was also found in another family member with a second sequence variation (Val150Ile) in the NKX2.5 gene homeodomain who was affected by multiple ventricular septal defects. A second proband was found to harbour a splice site variant (IVS2-1G→C) in the NODAL gene. Conclusions The present study provides evidence that some cases of familial TGA are caused by mutations in laterality genes and therefore are part of the same disease spectrum of heterotaxy syndrome, and argues for an oligogenic or complex mode of inheritance in these pedigrees.

The PINK1 phenotype can be indistinguishable from idiopathic Parkinson disease.

Mutations in the PINK1 gene cause autosomal recessive parkinsonism characterized by early onset and a variable phenotypic presentation. A patient homozygous for the Ala168Pro mutation has been fully characterized clinically. Apart from onset at age 39 years and the excellent and sustained response to levodopa, all clinical and laboratory features, including SPECT and assessment of autonomic function, were indistinguishable from typical idiopathic Parkinson disease.

ATM gene alterations in chronic lymphocytic leukemia patients induce a distinct gene expression profile and predict disease progression

Background The genetic characterization of chronic lymphocytic leukemia cells correlates with the behavior, progression and response to treatment of the disease. Design and Methods Our aim was to investigate the role of ATM gene alterations, their biological consequences and their value in predicting disease progression. The ATM gene was analyzed by denaturing high performance liquid chromatography and multiplex ligation probe amplification in a series of patients at diagnosis. The results were correlated with immunoglobulin gene mutations, cytogenetic abnormalities, ZAP-70 and CD38 expression, TP53 mutations, gene expression profile and treatment-free interval. Results Mutational screening of the ATM gene identified point mutations in 8/57 cases (14%). Multiplex ligation probe amplification analysis identified six patients with 11q deletion: all of them had at least 20% of deleted cells, analyzed by fluorescent in situ hybridization. Overall, ATM point mutations and deletions were detected in 14/57 (24.6%) cases at presentation, representing the most common unfavorable genetic anomalies in chronic lymphocytic leukemia, also in stage A patients. Patients with deleted or mutated ATM had a significantly shorter treatment-free interval compared to patients without ATM alterations. ATM-mutated cases had a peculiar gene expression profile characterized by the deregulation of genes involved in apoptosis and DNA repair. Finally, definition of the structure of the ATM-mutated protein led to a hypothesis that functional abnormalities are responsible for the unfavorable clinical course of patients carrying these point mutations. Conclusions ATM alterations are present at diagnosis in about 25% of individuals with chronic lymphocytic leukemia; these alterations are associated with a peculiar gene expression pattern and a shorter treatment-free interval.

Sgk1 activates MDM2-dependent p53 degradation and affects cell proliferation, survival, and differentiation

Serum and glucocorticoid regulated kinase 1 (Sgk1) is a serine–threonine kinase that is activated by serum, steroids, insulin, vasopressin, and interleukin 2 at the transcriptional and post-translational levels. Sgk1 is also important in transduction of growth factors and steroid-dependent survival signals and may have a role in the development of resistance to cancer chemotherapy. In the present paper, we demonstrate that Sgk1 activates MDM2-dependent p53 ubiquitylation. The results were obtained in RKO cells and other cell lines by Sgk1-specific RNA silencing and were corroborated in an original mouse model as well as in transiently and in stably transfected HeLa cells expressing wild-type or dominant negative Sgk1 mutant. Sgk1 contributes to cell survival, cell-cycle progression, and epithelial de-differentiation. We also show that the effects of Sgk1 on the clonogenic potential of different cancer cells depend on the expression of wild-type p53. Since transcription of Sgk1 is activated by p53, we propose a finely tuned feedback model where Sgk1 down-regulates the expression of p53 by enhancing its mono- and polyubiquitylation.

TRIB3 R84 Variant Is Associated With Impaired Insulin-Mediated Nitric Oxide Production in Human Endothelial Cells

Background—In the endothelium, insulin promotes nitric oxide (NO) production, through the insulin receptor/IRS-1/PI3-Kinase/Akt/eNOS signaling pathway. An inhibitor of insulin action, TRIB3, has recently been identified which affects insulin action by binding to and inhibiting Akt phosphorylation. We have recently described a Q84R gain-of-function polymorphism of TRIB3 with the R84 variant being associated with insulin resistance and an earlier age at myocardial infarction. Methods and Results—To investigate the TRIB3 R84 variant impact on endothelial insulin action, we cultured human umbilical vein endothelial cells (HUVECs) naturally carrying different TRIB3 genotypes (QQ-, QR-, or RR-HUVECs). TRIB3 inhibitory activity on insulin-stimulated Akt phosphorylation and the amount of protein which was coimmunoprecipitable with Akt were significantly greater in QR- and RR- as compared to QQ- HUVECs. After insulin stimulation, Akt and eNOS activation as well as NO production were markedly decreased in QR- and RR- as compared to QQ-HUVECs. TRIB3 molecular modeling analysis provided insights into the structural changes related to the polymorphisms potentially determining differences in protein-protein interaction with Akt. Conclusions—Our data demonstrate that the TRIB3 R84 variant impairs insulin signaling and NO production in human endothelial cells. This finding provides a plausible biological background for the deleterious role of TRIB3 R84 on genetic susceptibility to coronary artery disease.

Mutation screening of the DYT6/THAP1 gene in Italy

Mutations in the THAP1 gene on chromosome 8p21‐p22 (DYT6 locus) have been recently reported as causative of autosomal dominant primary torsion dystonia (PTD) in four Amish–Mennonite families and in 12 additional probands of different ancestry. We sequenced the THAP1 gene in 158 patients with DYT1‐negative PTD who had onset of symptoms below 30 years and/or positive family history. One sporadic Greek male patient, aged 57 years, was found to carry a novel heterozygous missense variant in THAP1 exon 3 (p.Cys170Arg), of likely pathogenic significance. This subject first presented with right writers cramp at age of 10 years and, subsequently, developed left arm dystonia and an extremely severe left laterocollis, without further spreading to other body districts. Our findings expand the genotypic spectrum of THAP1 and strengthen the association with upper body involvement, including the cranial and cervical districts that are usually spared in DYT1‐PTD.

Human Papillomavirus-16 E7 Interacts with Glutathione S-Transferase P1 and Enhances Its Role in Cell Survival

Background Human Papillomavirus (HPV)-16 is a paradigm for “high-risk” HPVs, the causative agents of virtually all cervical carcinomas. HPV E6 and E7 viral genes are usually expressed in these tumors, suggesting key roles for their gene products, the E6 and E7 oncoproteins, in inducing malignant transformation. Methodology/Principal Findings By protein-protein interaction analysis, using mass spectrometry, we identified glutathione S-transferase P1-1 (GSTP1) as a novel cellular partner of the HPV-16 E7 oncoprotein. Following mapping of the region in the HPV-16 E7 sequence that is involved in the interaction, we generated a three-dimensional molecular model of the complex between HPV-16 E7 and GSTP1, and used this to engineer a mutant molecule of HPV-16 E7 with strongly reduced affinity for GSTP1.When expressed in HaCaT human keratinocytes, HPV-16 E7 modified the equilibrium between the oxidized and reduced forms of GSTP1, thereby inhibiting JNK phosphorylation and its ability to induce apoptosis. Using GSTP1-deficient MCF-7 cancer cells and siRNA interference targeting GSTP1 in HaCaT keratinocytes expressing either wild-type or mutant HPV-16 E7, we uncovered a pivotal role for GSTP1 in the pro-survival program elicited by its binding with HPV-16 E7. Conclusions/Significance This study provides further evidence of the transforming abilities of this oncoprotein, setting the groundwork for devising unique molecular tools that can both interfere with the interaction between HPV-16 E7 and GSTP1 and minimize the survival of HPV-16 E7-expressing cancer cells.

Are MYO1C and MYO1F associated with hearing loss

The role of myosins in the pathogenesis of hearing loss is well established: five genes encoding unconventional myosins and two genes encoding nonmuscle conventional myosins have so far been described to be essential for normal auditory function and mutations in these genes associated with hearing impairment. To better understand the role of this gene family we performed a mutational screening on two candidate genes, MYO1C and MYO1F, analyzing hundreds of patients, affected by bilateral sensorineural hearing loss and coming from different European countries. This research activity led to the identification of 6 heterozygous missense mutations in MYO1C and additional 5 heterozygous missense mutations in MYO1F. Homology modelling suggests that some of these mutations could have a potential influence on the structure of the ATP binding site and could probably affect the ATPase activity or the actin binding process of both myosins. This study suggests a role of the above mentioned myosin genes in the pathogenesis of hearing loss.