TRM6/61 connects PKCα with translational control through tRNAi(Met) stabilization: impact on tumorigenesis.

Accumulating evidence suggests that changes of the protein synthesis machinery alter translation of specific mRNAs and participate in malignant transformation. Here we show that protein kinase C α (PKCα) interacts with TRM61, the catalytic subunit of the TRM6/61 tRNA methyltransferase. The TRM6/61 complex is known to methylate the adenosine 58 of the initiator methionine tRNA (tRNAi(Met)), a nuclear post-transcriptional modification associated with the stabilization of this crucial component of the translation-initiation process. Depletion of TRM6/61 reduced proliferation and increased death of C6 glioma cells, effects that can be partially rescued by overexpression of tRNAi(Met). In contrast, elevated TRM6/61 expression regulated the translation of a subset of mRNAs encoding proteins involved in the tumorigenic process and increased the ability of C6 cells to form colonies in soft agar or spheres when grown in suspension. In TRM6/61/tRNAi(Met)-overexpressing cells, PKCα overexpression decreased tRNAi(Met) expression and both colony- and sphere-forming potentials. A concomitant increase in TRM6/TRM61 mRNA and tRNAi(Met) expression with decreased expression of PKCα mRNA was detected in highly aggressive glioblastoma multiforme as compared with Grade II/III glioblastomas, highlighting the clinical relevance of our findings. Altogether, we suggest that PKCα tightly controls TRM6/61 activity to prevent translation deregulation that would favor neoplastic development.

Role of allelic variants Gly972Arg of IRS-1 and Gly1057Asp of IRS-2 in moderate-to-severe insulin resistance of women with polycystic ovary syndrome.

To assess the role of insulin receptor, insulin receptor substrate (IRS)-1, and IRS-2 genes in insulin resistance, we explored the genomic DNA in women with polycystic ovary syndrome (PCOS) and a variable degree (mean +/- SE) of insulin resistance (homeostasis model assessment index for insulin resistance [HOMA(IR)] 3.2 +/- 0.6, n = 53; control subjects 1.56 +/- 0.34, n = 102) using direct sequencing. Whereas no novel mutations were found in these genes, gene-dosage effects were found on fasting insulin for the Gly972Arg IRS-1 variant and on 2-h plasma glucose for the Gly1057Asp IRS-2 variant. The Gly972Arg IRS-1 variant was more prevalent in insulin-resistant patients compared with non-insulin-resistant individuals or control subjects (39.3 vs. 4.0 and 16.6%, P < 0.0031, respectively). A multivariate model that included BMI as a variable revealed significant effects of the Gly1057Asp IRS-2 variant on insulin resistance (P < 0.016, odds ratio [OR] 7.2, 95% CI 1.29-43.3). HOMA(IR) was higher in carriers of both IRS variants than in those with IRS-2 mutations only or those with wild-type variants (6.2 +/- 2.3, 2.8 +/- 0.5, and 1.8 +/- 0.2, respectively; P < 0.01), and it was significantly associated with this genotype (P < 0.0085, OR 1.7, 95% CI 1.09-2.99). We conclude that polymorphic alleles of both IRS-1 and IRS-2, alone or in combination, may have a functional impact on the insulin-resistant component of PCOS.

Conservation among Old World Leishmania species of six physical linkage groups defined in Leishmania infantum small chromosomes.

We have characterised 49 DNA probes specific for each of the six smallest chromosomes in Leishmania infantum and have examined the allocation of these probes in the molecular karyotypes of the other Old World Leishmania species Leishmania donovani, Leishmania major, Leishmania tropica and Leishmania aethiopica. These 49 probes define 6 physical linkage groups in the molecular karyotypes of various strains of L. infantum. 40 of these probes hybridise in the other Old World Leishmania species and show a remarkably conserved linkage pattern. No interchromosomal exchange nor fusion could be detected. Thus, in spite of the chromosomal size polymorphisms, the general structure of the genome seems to be conserved in the six smallest chromosomes among Old World Leishmania species. This structural genomic homogeneity should be helpful for mapping studies of any Old World Leishmania genomes.

Identification and sequence analysis of arginine vasopressin mRNA in normal and Brattleboro rat aortic tissue.

Arginine vasopressin (AVP), a hormone of the hypothalamic-pituitary axis, was also localized in peripheral tissues. To explore AVP precursor gene expression at the vascular level, we have investigated gene transcripts by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing in aortic tissue of normal rat and in the particular genetic condition of the homozygous (di/di) Brattleboro rat strain suffering from diabetes insipidus. In these rats, a gene deletion induces an unprocessed AVP precursor in the hypothalamus with undetectable immunoreactive AVP, in contrast to the detection of immunoreactive material at the vascular level. In normal rats, using primers complementary to exon 1 and 3 of the AVP neurophysin precursor gene, RT-PCR and sequencing revealed transcripts of the expected size from aorta, mesenteric artery and hypothalamus with normal, authentic sequences. Removal of aortic endothelium severely reduced the amounts of transcripts, suggesting their main endothelial origin. In Brattleboro rats, transcripts of similar size were obtained from aorta and hypothalamus and sequencing revealed the homozygous deletion (deltaG316) in both tissues, identical to that found in genomic DNA (deltaG1864). While sequence data from normal rats provide the first direct evidence for the presence of AVP precursor transcripts in rat aortic tissue, identification of the deleted sequence of transcripts in Brattleboro rat aorta suggests that tissue-specific mechanisms are operating for the expression of vasopressin neurophysin precursor in peripheral vascular tissue compared with the hypothalamus.

Identification by RT-PCR and immunolocalization of arginine vasopressin in rat pancreas.

Arginine vasopressin (AVP), a hormone of the hypothalamic pituitary axis, has been described in several peripheral tissues, including pancreas. To demonstrate the ectopic synthesis of AVP at the pancreatic level, we explored the expression of the AVP-neurophysin-II (AVP-NP-II) precursor gene by reverse-transcriptase polymerase chain reaction (RT-PCR) and sequencing and attempted to localise the peptide by immunocytochemistry in normal rat pancreas. Primers designed at the 3' and 5' ends of the AVP-NP-II gene, RT-PCR, and automatic sequencing of PCR products from rat pancreas revealed transcripts of the predicted size with an identical sequence to those from the hypothalamus. In addition, AVP antiserum revealed immunoreactive material of perivascular localisation. These data provide the first direct evidence for the presence of AVP transcripts in rat pancreatic tissue, whereas concurrent immunodetection of this hormone offers further support for the potential role of ectopic AVP in local regulation of the secretory activity of the pancreas.

[Insulin resistance: from clinical diagnosis to molecular genetics. Implications in diabetes mellitus]. / La résistance à l'insuline: du diagnostic clinique à la génétique moléculaire. Implications dans le diabète sucré.

Insulin resistance is observed in several diseases such as non insulin dependent diabetes mellitus (NIDDM) or polycystic ovarian syndrome (PCOS). To understand genetic determinism of this abnormality we have developed a multidisciplinary approach including selection of phenotypes with insulin resistance confirmed in vivo by minimal model of Bergman and characterization of cellular defects in insulin action on circulating erythrocytes and monocytes. Exploration of variability in candidate genes by direct sequencing in some genetic syndromes of severe insulin resistance and acanthosis nigricans (mainly the Type A syndrome) revealed mutations of the insulin receptor gene associated with major defects in insulin binding or kinase activity. In other rare genetic syndromes or patients affected by NIDDM or PCOS defects appear to be located at post-receptor level, where IRS (insulin receptor substrate) genes are the most attractive candidates. Prevalence of some allelic variants suggested a potential role of IRS genes in insulin resistance, although their involvement in the pathogenesis of NIDDM remains controversial. Genotype-phenotype correlations in first degree relatives of an index case caring the Type A syndrome, suggested that association of allelic variants of IRS-1 and IRS-2 with insulin receptor mutations contribute, by synergistic effects, to phenotypic expression of defects in signal transduction. These mechanisms through genetic epistasis, involving several genes in insulin action, fit better with the polygenic nature of current forms of NIDDM and represent a good model in the study of pathogenesis of insulin resistance.

Mutation in the gene for connexin 30.3 in a family with erythrokeratodermia variabilis.

Erythrokeratodermia variabilis (EKV) is an autosomal dominant keratinization disorder characterized by migratory erythematous lesions and fixed keratotic plaques. All families with EKV show mapping to chromosome 1p34-p35, and mutations in the gene for connexin 31 (Cx31) have been reported in some but not all families. We studied eight affected and three healthy subjects in an Israeli family, of Kurdish origin, with EKV. After having mapped the disorder to chromosome 1p34-p35, we found no mutations in the genes for Cx31, Cx31.1, and Cx37. Further investigation revealed a heterozygous T-->C transition leading to the missense mutation (F137L) in the human gene for Cx30.3 that colocalizes on chromosome 1p34-p35. This nucleotide change cosegregated with the disease and was not found in 200 alleles from normal individuals. This mutation concerns a highly conserved phenylalanine, in the third transmembrane region of the Cx30.3 molecule, known to be implicated in the wall formation of the gap-junction pore. Our results show that mutations in the gene for Cx30.3 can be causally involved in EKV and point to genetic heterogeneity of this disorder. Furthermore, we suggest that our family presents a new type of EKV because of the hitherto unreported association with erythema gyratum repens.

Identification of two novel insulin receptor mutations, Asp59Gly and Leu62Pro, in type A syndrome of extreme insulin resistance.

To elucidate genetic determinants of insulin resistance, we investigated insulin receptor (IR) and insulin receptor substrate-1 (IRS-1) genes, in vitro IR function and in vivo insulin sensitivity in a family with Type A syndrome. Two missense IR mutations (Asp59Gly and Leu62Pro) found in the proband, resulted in reduction by 90% of insulin binding to erythrocytes, decreased receptor autophosphorylation and a dramatic reduction of insulin sensitivity. The proband and mother were heterozygote for Gly972Arg IRS-1 variant. Asp59Gly mutation, also carried by proband's brother with no consequence on insulin sensitivity, was inherited from the mother who is diabetic and insulin resistant and Leu62Pro was from the father. We conclude that severity of insulin resistance in the proband may be explained by the genetic condition of compound heterozygote for IR mutations while severe insulin resistance in the mother raises the possibility that other genetic factors, like IRS-1 polymorphisms, may contribute to the phenotypic expression of IR mutations.

Refinement of genetic localization of the Alström syndrome on chromosome 2p12-13 by linkage analysis in a North African family.

Alström syndrome is a rare autosomal recessive disorder characterized by retinal pigment degeneration, neurogenic deafness, infantile obesity, hyperlipidemia, and non-insulin-dependent diabetes mellitus. While the disease-related gene remains unknown, studies of the genetic isolate of French Acadians provisionally locate the Alström syndrome on chromosome 2p12-13 within a 14.9-cM interval. To confirm this finding in another ethnic population and refine the candidate region we investigated by linkage analysis a consanguineous family of North African origin, in which three of seven siblings displayed all major neurological and metabolic features of Alström syndrome. Genotyping was performed on an ABI377 DNA automatic sequencer and LOD scores were obtained with the Fastlink program. Five markers previously investigated in French Acadians confirmed the involvement of the candidate region, although pairwise LOD scores were of poor significance (Zmax = 2.9). To further confirm homogeneity and refine the candidate region, 20 additional markers were investigated. Haplotype analysis and allele segregation revealed that affected children shared a single haplotype and were homozygous for the eight most centromeric markers (D2S291-D2S2114), over a 6.1-cM interval. Significative multipoint LOD scores (Zmax = 3.96) were obtained between markers D2S2110/145 and D2S286. Two clusters of known genes are present in this refined region of chromosome 2p, the most attractive candidate being the hexokinase II gene. However, except for several known polymorphisms, no mutations were detected in the coding region of this gene. In conclusion, the location of Alström syndrome on chromosome 2p12-13 is confirmed, reducing the genetic interval to 6.1 cM.

Mutations in the gene encoding SLURP-1 in Mal de Meleda.

Mal de Meleda (MDM) is a rare autosomal recessive skin disorder, characterized by transgressive palmoplantar keratoderma (PPK), keratotic skin lesions, perioral erythema, brachydactyly and nail abnormalities. We report the refinement of our previously described interval of MDM on chromosome 8qter, and the identification of mutations in affected individuals in the ARS (component B) gene, encoding a protein named SLURP-1, for secreted Ly-6/uPAR related protein 1. This protein is a member of the Ly-6/uPAR superfamily, in which most members have been localized in a cluster on chromosome 8q24.3. The amino acid composition of SLURP-1 is homologous to that of toxins such as frog cytotoxin and snake venom neurotoxins and cardiotoxins. Three different homozygous mutations (a deletion, a nonsense and a splice site mutation) were detected in 19 families of Algerian and Croatian origin, suggesting founder effects. Moreover, one of the common haplotypes presenting the same mutation was shared by families from both populations. Secreted and receptor proteins of the Ly-6/uPAR superfamily have been implicated in transmembrane signal transduction, cell activation and cell adhesion. This is the first instance of a secreted protein being involved in a PPK.