Novel germline variants identified in the inner mitochondrial membrane transporter TIMM44 and their role in predisposition to oncocytic thyroid carcinomas

Familial Non-Medullary Thyroid Carcinoma (fNMTC) represents 3–7% of all thyroid tumours and is associated with some of the highest familial risks among all cancers, with an inheritance pattern compatible with an autosomal dominant model with reduced penetrance. We previously mapped a predisposing locus, TCO (Thyroid tumour with Cell Oxyphilia) on chromosome 19p13.2, for a particular form of thyroid tumour characterised by cells with an abnormal proliferation of mitochondria (oxyphilic or oncocytic cells). In the present work, we report the systematic screening of 14 candidate genes mapping to the region of linkage in affected TCO members, that led us to identify two novel variants respectively in exon 9 and exon 13 of TIMM44, a mitochondrial inner membrane translocase for the import in the mitochondria of nuclear-encoded proteins. These variants were co-segregating with the TCO phenotype, were not present in a large group of controls and were predicted to negatively affect the protein (exon 9 change) or the transcript (exon 13 change). Functional analysis was performed in vitro for both changes and although no dramatic loss of function effects were identified for the mutant alleles, subtler effects might still be present that could alter Timm44 function and thus promote oncocytic tumour development. Thus we suggest that TIMM44 should be considered for further studies in independent samples of affected individuals with TCO

A Deficiency of Ceramide Biosynthesis Causes Cerebellar Purkinje Cell Neurodegeneration and Lipofuscin Accumulation

Sphingolipids, lipids with a common sphingoid base (also termed long chain base) backbone, play essential cellular structural and signaling functions. Alterations of sphingolipid levels have been implicated in many diseases, including neurodegenerative disorders. However, it remains largely unclear whether sphingolipid changes in these diseases are pathological events or homeostatic responses. Furthermore, how changes in sphingolipid homeostasis shape the progression of aging and neurodegeneration remains to be clarified. We identified two mouse strains, flincher (fln) and toppler (to), with spontaneous recessive mutations that cause cerebellar ataxia and Purkinje cell degeneration. Positional cloning demonstrated that these mutations reside in the Lass1 gene. Lass1 encodes (dihydro)ceramide synthase 1 (CerS1), which is highly expressed in neurons. Both fln and to mutations caused complete loss of CerS1 catalytic activity, which resulted in a reduction in sphingolipid biosynthesis in the brain and dramatic changes in steady-state levels of sphingolipids and sphingoid bases. In addition to Purkinje cell death, deficiency of CerS1 function also induced accumulation of lipofuscin with ubiquitylated proteins in many brain regions. Our results demonstrate clearly that ceramide biosynthesis deficiency can cause neurodegeneration and suggest a novel mechanism of lipofuscin formation, a common phenomenon that occurs during normal aging and in some neurodegenerative diseases

Mutations in the UBIAD1 Gene, Encoding a Potential Prenyltransferase, Are Causal for Schnyder Crystalline Corneal Dystrophy

Schnyder crystalline corneal dystrophy (SCCD, MIM 121800) is a rare autosomal dominant disease characterized by progressive opacification of the cornea resulting from the local accumulation of lipids, and associated in some cases with systemic dyslipidemia. Although previous studies of the genetics of SCCD have localized the defective gene to a 1.58 Mbp interval on chromosome 1p, exhaustive sequencing of positional candidate genes has thus far failed to reveal causal mutations. We have ascertained a large multigenerational family in Nova Scotia affected with SCCD in which we have confirmed linkage to the same general area of chromosome 1. Intensive fine mapping in our family revealed a 1.3 Mbp candidate interval overlapping that previously reported. Sequencing of genes in our interval led to the identification of five putative causal mutations in gene UBIAD1, in our family as well as in four other small families of various geographic origins. UBIAD1 encodes a potential prenyltransferase, and is reported to interact physically with apolipoprotein E. UBIAD1 may play a direct role in intracellular cholesterol biochemistry, or may prenylate other proteins regulating cholesterol transport and storage

Prenylation Inhibition-Induced Cell Death in Melanoma: Reduced Sensitivity in BRAF Mutant/PTEN Wild-Type Melanoma Cells.

While targeted therapy brought a new era in the treatment of BRAF mutant melanoma, therapeutic options for non-BRAF mutant cases are still limited. In order to explore the antitumor activity of prenylation inhibition we investigated the response to zoledronic acid treatment in thirteen human melanoma cell lines with known BRAF, NRAS and PTEN mutational status. Effect of zoledronic acid on proliferation, clonogenic potential, apoptosis and migration of melanoma cells as well as the activation of downstream elements of the RAS/RAF pathway were investigated in vitro with SRB, TUNEL and PARP cleavage assays and videomicroscopy and immunoblot measurements, respectively. Subcutaneous and spleen-to-liver colonization xenograft mouse models were used to evaluate the influence of zoledronic acid treatment on primary and disseminated tumor growth of melanoma cells in vivo. Zoledronic acid more efficiently decreased short-term in vitro viability in NRAS mutant cells when compared to BRAF mutant and BRAF/NRAS wild-type cells. In line with this finding, following treatment decreased activation of ribosomal protein S6 was found in NRAS mutant cells. Zoledronic acid demonstrated no significant synergism in cell viability inhibition or apoptosis induction with cisplatin or DTIC treatment in vitro. Importantly, zoledronic acid could inhibit clonogenic growth in the majority of melanoma cell lines except in the three BRAF mutant but PTEN wild-type melanoma lines. A similar pattern was observed in apoptosis induction experiments. In vivo zoledronic acid did not inhibit the subcutaneous growth or spleen-to-liver colonization of melanoma cells. Altogether our data demonstrates that prenylation inhibition may be a novel therapeutic approach in NRAS mutant melanoma. Nevertheless, we also demonstrated that therapeutic sensitivity might be influenced by the PTEN status of BRAF mutant melanoma cells. However, further investigations are needed to identify drugs that have appropriate pharmacological properties to efficiently target prenylation in melanoma cells

Expression and regulation of phospholipase D in the human keratinocyte cell line HaCaT

AbstractThe generation of lipid second messengers via phosphatidylcholine (PC)-specific phospholipase D (PLD) has emerged as an important step leading to transduction of extracellular signals. In the present investigation the expression of human cytosolic PLD isoenzymes in the immortalized human keratinocyte cell line HaCat was determined. At the mRNA level we found the expression of hPLD1b and for the first time in human cells also the expression of hPLD2. For further analysis of enzyme expression at the protein level, hPLD1 peptide fragments were synthesized and specific antibodies were generated (rabbit) to be used for detection of hPLD1 in Western blot experiments. Furthermore, small G-proteins were found to be involved in the regulation of PLD activity in HaCaT cells using the guanine nucleotide analogue GTPγS