Anophthalmia, hearing loss, abnormal pituitary development and response to growth hormone therapy in three children with microdeletions of 14q22q23

BACKGROUND: Microdeletions of 14q22q23 have been associated with eye abnormalities and pituitary defects. Other phenotypic features in deletion carriers including hearing loss and response to growth hormone therapy are less well recognized. We studied genotype and phenotype of three newly identified children with 14q22q23 deletions, two girls and one boy with bilateral anophthalmia, and compared them with previously published deletion patients and individuals with intragenic defects in genes residing in the region. RESULTS: The three deletions were de novo and ranged in size between 5.8 and 8.9 Mb. All three children lacked one copy of the OTX2 gene and in one of them the deletion involved also the BMP4 gene. All three patients presented partial conductive hearing loss which tended to improve with age. Analysis of endocrine and growth phenotypes showed undetectable anterior pituitary, growth hormone deficiency and progressive growth retardation in all three patients. Growth hormone therapy led to partial catch-up growth in two of the three patients but just prevented further height loss in the third. CONCLUSIONS: The pituitary hypoplasia, growth hormone deficiency and growth retardation associated with 14q22q23 microdeletions are very remarkable, and the latter appears to have an atypical response to growth hormone therapy in some of the cases

Etude transcriptomique du métabolisme central et de sa variabilité chez la levure Kluveromyces lactis: Comparaison de la souche de référence et d'une souche industrielle

Diplôme : Dr. d'UniversiteKluyveromyces lactis is one of the most important yeast in cheeses. It is of double interest: fundamental and industrial. Indeed, in spite of its close phylogenetic proximity to the classical model Saccharomyces cerevisiae, its physiology is very different. Its weaker fermentative activity makes of it a model of choice for recombinant proteins production and its ability to metabolize the lactose justifies its utilization for dairy industry. Although K. lactis is the most studied non-conventional yeast, many aspects of its physiology remained to be understood, what we undertook thanks to the data of the partial sequencing of its genome. This work started with the construction of a DNA microarray representing 482 K. lactis genes, involved mainly in the main cellular functions. The comparison of gene expression between the reference strain and an industrial strain in the presence of glucose and/or lactose evidenced a variability that was not detectable at the molecular level. We observed in particular changes in the expression of the genes involved in glucose and lactose transport, in the glycolysis, the Pentose Phosphate Pathway, the Tricarboxylic Acids Cycle, the ethanol metabolism and the amino-acids metabolism, which were perfectly correlated with the physiological data made in parallel. Our results show a very different sensitivity to the glucose repression between both strains, which may be linked to a difference in the sugar transport and may reflect the way by which K. lactis adapts to its environment.Kluyveromyces lactis est l'une des espèces de levures majoritaires dans les fromages. Elle présente un intérêt à la fois fondamental et industriel. En effet, malgré sa proximité relative du modèle classique Saccharomyces cerevisiae sa physiologie est très différente. Sa plus faible activité fermentaire en fait un organisme de choix pour la production de protéines recombinantes et sa capacité à métaboliser le lactose justifie son utilisation en industrie laitière. Bien que K. lactis soit la levure non conventionnelle la plus étudiée, de nombreux aspects de sa physiologie restaient à éclaircir, ce que nous avons entrepris grâce aux données du séquençage partiel de son génome. Ce travail a débuté par l'élaboration d'une puce à ADN représentant 482 gènes de K. lactis, impliqués dans les principales fonctions cellulaires. La comparaison de l'expression des gènes entre la souche de référence et une souche industrielle en présence de glucose et/ou de lactose a mis en évidence une variabilité indétectable du point de vue moléculaire. Nous avons observé en particulier des changements d'expression des gènes impliqués dans le transport du glucose et du lactose, la glycolyse, la voie des pentoses phosphate, le cycle de Krebs, le métabolisme de l'éthanol et le métabolisme des acides aminés, parfaitement corrélées aux données physiologiques effectuées en parallèle. Nos résultats montrent notamment une sensibilité à la répression par le glucose totalement différente entre les deux souches, qui pourrait être liée à une différence de transport des sucres et refléter la façon dont K. lactis s'adapte à son environnement

Etude transcriptomique du métabolisme central et de sa variabilité chez la levure Kluveromyces lactis: Comparaison de la souche de référence et d'une souche industrielle

Diplôme : Dr. d'UniversiteKluyveromyces lactis is one of the most important yeast in cheeses. It is of double interest: fundamental and industrial. Indeed, in spite of its close phylogenetic proximity to the classical model Saccharomyces cerevisiae, its physiology is very different. Its weaker fermentative activity makes of it a model of choice for recombinant proteins production and its ability to metabolize the lactose justifies its utilization for dairy industry. Although K. lactis is the most studied non-conventional yeast, many aspects of its physiology remained to be understood, what we undertook thanks to the data of the partial sequencing of its genome. This work started with the construction of a DNA microarray representing 482 K. lactis genes, involved mainly in the main cellular functions. The comparison of gene expression between the reference strain and an industrial strain in the presence of glucose and/or lactose evidenced a variability that was not detectable at the molecular level. We observed in particular changes in the expression of the genes involved in glucose and lactose transport, in the glycolysis, the Pentose Phosphate Pathway, the Tricarboxylic Acids Cycle, the ethanol metabolism and the amino-acids metabolism, which were perfectly correlated with the physiological data made in parallel. Our results show a very different sensitivity to the glucose repression between both strains, which may be linked to a difference in the sugar transport and may reflect the way by which K. lactis adapts to its environment.Kluyveromyces lactis est l'une des espèces de levures majoritaires dans les fromages. Elle présente un intérêt à la fois fondamental et industriel. En effet, malgré sa proximité relative du modèle classique Saccharomyces cerevisiae sa physiologie est très différente. Sa plus faible activité fermentaire en fait un organisme de choix pour la production de protéines recombinantes et sa capacité à métaboliser le lactose justifie son utilisation en industrie laitière. Bien que K. lactis soit la levure non conventionnelle la plus étudiée, de nombreux aspects de sa physiologie restaient à éclaircir, ce que nous avons entrepris grâce aux données du séquençage partiel de son génome. Ce travail a débuté par l'élaboration d'une puce à ADN représentant 482 gènes de K. lactis, impliqués dans les principales fonctions cellulaires. La comparaison de l'expression des gènes entre la souche de référence et une souche industrielle en présence de glucose et/ou de lactose a mis en évidence une variabilité indétectable du point de vue moléculaire. Nous avons observé en particulier des changements d'expression des gènes impliqués dans le transport du glucose et du lactose, la glycolyse, la voie des pentoses phosphate, le cycle de Krebs, le métabolisme de l'éthanol et le métabolisme des acides aminés, parfaitement corrélées aux données physiologiques effectuées en parallèle. Nos résultats montrent notamment une sensibilité à la répression par le glucose totalement différente entre les deux souches, qui pourrait être liée à une différence de transport des sucres et refléter la façon dont K. lactis s'adapte à son environnement

Transcriptomic Analysis of Extensive Changes in Metabolic Regulation in Kluyveromyces lactis Strains

Genome-wide analysis of transcriptional regulation is generally carried out on well-characterized reference laboratory strains; hence, the characteristics of industrial isolates are therefore overlooked. In a previous study on the major cheese yeast Kluyveromyces lactis, we have shown that the reference strain and an industrial strain used in cheese making display a differential gene expression when grown on a single carbon source. Here, we have used more controlled conditions, i.e., growth in a fermentor with pH and oxygen maintained constant, to study how these two isolates grown in glucose reacted to an addition of lactose. The observed differences between sugar consumption and the production of various metabolites, ethanol, acetate, and glycerol, correlated with the response were monitored by the analysis of the expression of 482 genes. Extensive differences in gene expression between the strains were revealed in sugar transport, glucose repression, ethanol metabolism, and amino acid import. These differences were partly due to repression by glucose and another, yet-unknown regulation mechanism. Our results bring to light a new type of K. lactis strain with respect to hexose transport gene content and repression by glucose. We found that a combination of point mutations and variation in gene regulation generates a biodiversity within the K. lactis species that was not anticipated. In contrast to S. cerevisiae, in which there is a massive increase in the number of sugar transporter and fermentation genes, in K. lactis, interstrain diversity in adaptation to a changing environment is based on small changes at the level of key genes and cell growth control

Intraspecific gene expression variability in the yeast Kluyveromyces lactis revealed by micro-array analysis.

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Sub1 and Maf1, Two Effectors of RNA Polymerase III, Are Involved in the Yeast Quiescence Cycle

<div><p>Sub1 and Maf1 exert an opposite effect on RNA polymerase III transcription interfering with different steps of the transcription cycle. In this study, we present evidence that Sub1 and Maf1 also exhibit an opposite role on yeast chronological life span. First, cells lacking Sub1 need more time than wild type to exit from resting and this lag in re-proliferation is correlated with a delay in transcriptional reactivation. Second, our data show that the capacity of the cells to properly establish a quiescent state is impaired in the absence of Sub1 resulting in a premature death that is dependent on the Ras/PKA and Tor1/Sch9 signalling pathways. On the other hand, we show that <i>maf1Δ</i> cells are long-lived mutant suggesting a connection between Pol III transcription and yeast longevity.</p></div

Sub1 is required for an optimal reactivation of Pol III transcription when cells are exiting quiescence.

<p>(<b>A</b>) Microarray hybridization. Expression ratios of a subset of Pol III-transcribed genes in the <i>sub1Δ</i> mutant and the wild type strain grown in YPD, at day10 stationary phase (S) and after 1 or 6 h post-inoculation into fresh medium are shown. Results for the indicated genes are presented according to the red-green color scale. (<b>B</b>) Northern blot analysis. Total RNA isolated from the indicated strains grown in YPD, at day5 (S) or after 1 to 8 h post-inoculation into fresh medium was hybridized with probes designed to reveal both pre-tRNA^Ileu and tRNA^Ileu, or 5S rRNA. (<b>C</b>) Real-time PCR analysis. The expression of tDNA^iMet, tDNA^Leu or tDNA^Ileu genes in the indicated strains grown in YPD at day5 (S) or after 1 to 3 h post-inoculation into fresh medium was determined by real-time PCR. Relative gene expression levels were calculated using 5S rRNA as an internal control since its levels did not significantly change over time in all strains. Standard deviations were calculated from two independent preparations of cDNA.</p

Reactivation of transcription upon exit from quiescence.

<p>(<b>A</b>) Day4 cultures of the indicated strains grown in SD4x were re-inoculated into fresh medium. RNA synthesis by Pol III (5S rRNA, tRNAs) or Pol I (25S, 18S, 5.8S rRNAs) was monitored over time by metabolic labelling with ³H-uracil (³H). For 25S and 18S rRNAs, a shorter exposure (SE) is shown. Total RNA loaded was visualized by staining with ethidium bromide (EtBr). (<b>B</b>) The expression of the 35S rRNA gene and of <i>RPS6A</i>, <i>ACT1</i> or <i>IMD2</i> genes, three of Sub1 genomic targets, in the indicated strains grown in YPD, at day5 (S) and after 1 to 6 h post-inoculation into fresh media was determined by real-time PCR. Expression levels were normalized to those of the 5S rRNA gene. Standard deviations were calculated from two independent preparations of cDNA. As expected from the repressive role of Sub1 on <i>IMD2</i> gene expression, a higher expression level of <i>IMD2</i> gene was detected in the absence of Sub1.</p