Dissertations / Theses on the topic 'Novel mutation'

Background: Bernard-Soulier syndrome (BSS) is a severe congenital bleeding disorder characterized by thrombocytopenia, thrombocytopathy and decreased platelet adhesion. BSS results from genetic alterations of the glycoprotein (GP) Ib/IX/V complex. Methods: We report on a patient demonstrating typical BSS phenotype (thrombocytopenia with giant platelets, bleeding symptoms). However, BSS was not diagnosed until he reached the age of 39 years. Results: Flow cytometry of the patient’s platelets revealed absence of GPIb/IX/V receptor surface expression. In addition, immunofluorescence analysis of patient’s platelets demonstrated very faint staining of GPIX. A novel homozygous deletion comprising 11 nucleotides starting at position 1644 of the GPIX gene was identified using molecular genetic analysis. Conclusions: The novel 11-nucleotide deletion (g.1644_1654del11) was identified as causing the bleeding disorder in the BSS patient. This homozygous deletion includes the last 4 nucleotides of the Kozak sequence as well as the start codon and the following 4 nucleotides of the coding sequence. The Kozak sequence is a region indispensable for the initiation of the protein translation process, thus preventing synthesis of functional GPIX protein in the case of deletion
Hintergrund: Das Bernard-Soulier-Syndrom (BSS) ist eine angeborene Blutungsstörung, die mit Thrombozytopenie, Thrombozytopathie und verminderter Thrombozytenadhäsion assoziiert ist. BSS wird durch genetische Veränderungen des Glykoprotein(GP)-Ib/IX/V-Komplexes verursacht. Methoden: Wir berichten über einen Patienten mit typischem BSS-Phänotyp (Thrombozytopenie mit Riesenthrombozyten, Blutungssymptome). Dennoch wurde die Diagnose BSS erst im Alter von 39 Jahren gestellt. Ergebnisse: Die Durchflusszytometrie der Thrombozyten des Patienten ergab eine fehlende Oberflächenexpression des GPIb/IX/V-Rezeptors. Zusätzlich zeigten Immunfluoreszenz-Analysen der Thrombozyten eine nur sehr schwache Anfärbung von GPIX. In der molekulargenetischen Analyse wurde eine noch nicht bekannte homozygote Deletion von 11 Nukleotiden (beginnend an Position 1644 im GPIX-Gen) identifiziert. Schlussfolgerungen: Diese neue Deletion von 11 Nukleotiden (g.1644_1654del11) wurde als Ursache für die vermehrte Blutungsneigung bei dem BSS-Patienten identifiziert. Von der homozygoten Deletion betroffen sind die letzten 4 Nukleotide der Kozak-Sequenz sowie das Startkodon und weitere 4 Nukleotide des kodierenden Bereichs. Die Kozak-Sequenz ist unerlässlich für die Initiation der Translation in der Proteinbiosynthese, so dass die bei dem Patienten nachgewiesene Deletion die Synthese des funktionellen GPIX-Proteins verhindert
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Background: Bernard-Soulier syndrome (BSS) is a severe congenital bleeding disorder characterized by thrombocytopenia, thrombocytopathy and decreased platelet adhesion. BSS results from genetic alterations of the glycoprotein (GP) Ib/IX/V complex. Methods: We report on a patient demonstrating typical BSS phenotype (thrombocytopenia with giant platelets, bleeding symptoms). However, BSS was not diagnosed until he reached the age of 39 years. Results: Flow cytometry of the patient’s platelets revealed absence of GPIb/IX/V receptor surface expression. In addition, immunofluorescence analysis of patient’s platelets demonstrated very faint staining of GPIX. A novel homozygous deletion comprising 11 nucleotides starting at position 1644 of the GPIX gene was identified using molecular genetic analysis. Conclusions: The novel 11-nucleotide deletion (g.1644_1654del11) was identified as causing the bleeding disorder in the BSS patient. This homozygous deletion includes the last 4 nucleotides of the Kozak sequence as well as the start codon and the following 4 nucleotides of the coding sequence. The Kozak sequence is a region indispensable for the initiation of the protein translation process, thus preventing synthesis of functional GPIX protein in the case of deletion.
Hintergrund: Das Bernard-Soulier-Syndrom (BSS) ist eine angeborene Blutungsstörung, die mit Thrombozytopenie, Thrombozytopathie und verminderter Thrombozytenadhäsion assoziiert ist. BSS wird durch genetische Veränderungen des Glykoprotein(GP)-Ib/IX/V-Komplexes verursacht. Methoden: Wir berichten über einen Patienten mit typischem BSS-Phänotyp (Thrombozytopenie mit Riesenthrombozyten, Blutungssymptome). Dennoch wurde die Diagnose BSS erst im Alter von 39 Jahren gestellt. Ergebnisse: Die Durchflusszytometrie der Thrombozyten des Patienten ergab eine fehlende Oberflächenexpression des GPIb/IX/V-Rezeptors. Zusätzlich zeigten Immunfluoreszenz-Analysen der Thrombozyten eine nur sehr schwache Anfärbung von GPIX. In der molekulargenetischen Analyse wurde eine noch nicht bekannte homozygote Deletion von 11 Nukleotiden (beginnend an Position 1644 im GPIX-Gen) identifiziert. Schlussfolgerungen: Diese neue Deletion von 11 Nukleotiden (g.1644_1654del11) wurde als Ursache für die vermehrte Blutungsneigung bei dem BSS-Patienten identifiziert. Von der homozygoten Deletion betroffen sind die letzten 4 Nukleotide der Kozak-Sequenz sowie das Startkodon und weitere 4 Nukleotide des kodierenden Bereichs. Die Kozak-Sequenz ist unerlässlich für die Initiation der Translation in der Proteinbiosynthese, so dass die bei dem Patienten nachgewiesene Deletion die Synthese des funktionellen GPIX-Proteins verhindert.
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Primary aldosteronism (PA), a common form of secondary hypertension, is characterized by an excess autonomous aldosterone secretion. In a percentage ranging from a half to two thirds of the cases it is due to a surgically curable aldosterone-producing adenoma (APA) and in the rest to bilateral adrenal hyperplasia. The molecular mechanisms underlying aldosterone hypersecretion are unknown. Recent evidences suggest that amino acid residue substitutions in the selectivity filter of the Kir3.4 (KCNJ5) potassium channel may cause a constitutive aldosterone secretion from aldosterone-producing adenomas (APA). Such somatic mutations were also found to be associated with higher plasma aldosterone concentrations in the patients with an APA, thereby suggesting a causative role of the mutations in the development of APA and hyperaldosteronism. Hence, we performed a study with the aims to search for KCNJ5 mutations in APA patients referred to two Italian referral centers. Through this search we could also identify a novel KCNJ5-insT149 mutation out of the selectivity filter that was a fully characterized from the electrophysiological and phenotypic standpoint. APA samples (n=195) from consecutive patients with a conclusive diagnosis of APA were screened by high melting resolution curve for KCNJ5 mutations. We found that the mutations occurred in 24.6% of patients. These findings were confirmed by Sanger sequencing. The mRNA content of CYP11B2, but not of CYP11B1, and plasma aldosterone and, accordingly, the lateralization index were higher (P < 0.02) in the APA with the mutation than in the APA without such mutations. A novel c.446insAAC insertion resulting in the mutant protein KCNJ5-insT149 was identified In a patient presenting with severe drug-resistant hypertension. To functionally characterize this novel KCNJ5 channel mutation a mutated cDNA harbouring c.446insAAC insertion was generated by site-directed mutagenesis and transfected in mammalian cells. KCNJ3 cDNA was also transfected into the same cells to reproduce the tetrameric structure of the KCNJ3/KCNJ5 channel. CYP11B1, CYP11B2 and 17α-hydroxylase were localized in the adrenal gland of the mutated APA patient with immunohistochemistry and immunofluorescence. CYP11B2 mRNA levels and aldosterone concentrations were also measured to investigate the impact of the mutation on the secreting activity. By using a whole-cell patch clamp technique and molecular modeling we explored membrane Na+ and Ca2+ currents and created a 3D image of the insT149 KCNJ5 channel. Compared to wild type and mock-transfected HAC15 adrenocortical cells, those expressing the mutant KCNJ5 showed increased CYP11B2 expression and aldosterone secretion. Likewise HEK293 expressing the mutated KCNJ5-insT149 channel exhibited a 2-fold increase in intracellular Na+ and a substantial rise in intracellular Ca2+ caused by activation of voltage-gated Ca2+ channels. Hence, the novel KCNJ5 K+ channel mutation induces abnormal Na+ permeability, membrane depolarization, a rise in cytosolic Ca2+ and increased aldosterone synthesis. Thus, our findings support the concept that channelopathies involving the KCNJ5 K+ channel mechanistically account for constitutive secretion of aldosterone in human APA.
L’ iperaldosteronismo primario (PA) è la causa più frequente di ipertensione secondaria ed è caratterizzato da una secrezione elevata ed autonoma di aldosterone. Le due forme principali sono l’iperplasia surrenalica bilaterale e l’adenoma secernente aldosterone. I meccanismi molecolari alla base dell’ipersecrezione di aldosterone sono tuttora sconosciuti. Tuttavia recenti studi hanno dimostrato che sostituzioni amminoacidiche all’interno del filtro di selettività del canale del potassio Kir3.4 (KCNJ5 possono provocare una secrezione autonoma di aldosterone in adenomi producenti aldosterone (APA). Tali mutazioni somatiche sono associate ad alti livelli plasmatici di aldosterone nei pazienti con APA, suggerendo un ruolo causale di tali mutazioni nello sviluppo di APA e iperaldosteronismo. Pertanto abbiamo condotto uno studio in pazienti affetti da APA afferenti a due centri di riferimento italiani, effettuando lo screening per le mutazioni somatiche di KCNJ5, ed abbiamo individuato e caratterizzato la mutazione KCNJ5-insT149, mai descritta in precedenza. Mediante analisi ad alta risoluzione delle curve di melting per le mutazioni in KCNJ5 sono stati studiati 195 pazienti consecutive con una diagnosi conclusiva di APA. Il 24,6% dei pazienti presentava una mutazione nel filtro di selettività del KCNJ5, tale prevalenza è stata confermata mediante sequenziamento Sanger. Nei pazienti affetti da mutazione di KCNJ5 l’espressione genica di CYP11B2 (29,9 ± 7,4 vs 10,3 ± 3,6, P <0,02), ma non quella di CYP11B1, risultava superiore rispetto ai pazienti non affetti da mutazioni, lo stesso valeva per l’indice di lateralizzazione. In un paziente con ipertensione farmaco-resistente grave è stata identificata l’ inserzione c.446insAAC, che codifica per la proteina mutante KCNJ5-insT149. Per caratterizzare funzionalmente questa nuova mutazione, attaverso mutagenesi sito-diretta, è stato generato un cDNA codificante per il canale KCNJ5 mutato e trasfettato in cellule di mammifero. Il cDNA codificante KCNJ3 è stato transfettato insieme a quello per KCNJ5 in modo da riprodurre la struttura tetramerica del canale KCNJ3/KCNJ5. CYP11B1, CYP11B2 e 17α-idrossilasi sono stati rilevati attraverso tecniche di immunoistochimica e immunofluorescenza nella ghiandola surrenale del paziente. L’espressione genica di CYP11B2 e le concentrazioni di aldosterone sono stati misurati per studiare l'impatto della mutazione sull'attività secernente. Utilizzando la tecnica di “whole-cell patch clamp e modeling molecolare” abbiamo studiato le correnti trans-membrana di Na+ e Ca2+ e generato una immagine 3D del canale insT149 KCNJ5. Rispetto al wild type e alle cellule adrenocorticali HAC15, le cellule transfettate con KCNJ5-insT149 esprimevano alti livelli del gene CYP11B2 e mostravano un’aumentata produzione di aldosterone. Allo stesso modo cellule HEK293 che esprimono il canale KCNJ5-insT149 mutato mostravano un aumento pari a due volte di Na+ intracellulare e un aumento sostanziale di Ca2+ intracellulare in seguito all’ attivazione dei canali del Ca2+ voltaggio-dipendenti. Quindi, la nuova mutazione del canale del K+ KCNJ5 induce un’anomala permeabilità della membrana al Na+, depolarizzazione della membrana, un aumento di Ca2+ intracellulare e aumento della sintesi di aldosterone. I nostri risultati nel complesso supportano il concetto che le canalopatie che coinvolgono il canale del K+ KCNJ5 sono alla base della secrezione costitutiva di aldosterone in pazienti affetti da APA.

Objective: Description of the clinical, biochemical and genetic features of a Polish patient with familial glucocorticoid deficiency. Methods: Detailed clinical investigation, hormonal analysis and sequencing of the coding region of the melanocortin 2 receptor (MC2R) gene in this patient. Results: We report on a 3-month-old boy with familial glucocorticoid deficiency who presented at the age of 3 months with skin hyperpigmentation, muscle weakness, mild jaundice and constipation. Hormonal analyses revealed high ACTH and TSH serum concentrations, low serum cortisol concentration along with normal blood electrolytes. On hydrocortisone supplementation, the disease symptoms disappeared and the child recovered completely. His physical and mental development progresses normally. Genetic analysis disclosed a novel compound heterozygous MC2R mutation p.Leu46fs and p.Val49Met. Conclusion: The heterozygous p.Leu46fs mutation adds to the small number of MC2R nonsense mutations and is the first frameshift mutation within the first transmembrane domain of the receptor. According to molecular modeling the Val49Met mutation results in a structural change of the first transmembrane domain and in a potential novel interaction of the transmembrane domains I and VII
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Objective: Description of the clinical, biochemical and genetic features of a Polish patient with familial glucocorticoid deficiency. Methods: Detailed clinical investigation, hormonal analysis and sequencing of the coding region of the melanocortin 2 receptor (MC2R) gene in this patient. Results: We report on a 3-month-old boy with familial glucocorticoid deficiency who presented at the age of 3 months with skin hyperpigmentation, muscle weakness, mild jaundice and constipation. Hormonal analyses revealed high ACTH and TSH serum concentrations, low serum cortisol concentration along with normal blood electrolytes. On hydrocortisone supplementation, the disease symptoms disappeared and the child recovered completely. His physical and mental development progresses normally. Genetic analysis disclosed a novel compound heterozygous MC2R mutation p.Leu46fs and p.Val49Met. Conclusion: The heterozygous p.Leu46fs mutation adds to the small number of MC2R nonsense mutations and is the first frameshift mutation within the first transmembrane domain of the receptor. According to molecular modeling the Val49Met mutation results in a structural change of the first transmembrane domain and in a potential novel interaction of the transmembrane domains I and VII.
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Pain is an important physiological function, whose primary role is to preserve an organism’s integrity. Disruption of the nociception transduction chain results in the inability to perceive pain. Among these “painlessness” pathologies, Hereditary Sensory and Autonomic Neuropathy type V (HSAN V) is caused by the 661C>T transition in the ngf gene, resulting in the R100W missense mutation in mature Nerve Growth Factor (NGF), in keeping with the key role of this neurotrophin in the development of nociceptors and in their function in the adult . Homozygous HSAN V patients display indifference to noxious stimuli but, no cognitive deficits. In contrast, heterozygous carriers do not show an overt clinical phenotype and have been identified only through pedigree and genetic screening. Considering the particular features of HSAN V patients, I hypothesized that the R100W mutation might cause a dissociation between the actions of NGF on the central and peripheral nervous systems. To test this hypothesis and understand the mechanisms underlying the HSAN V phenotype, I generated a transgenic mouse line harboring the human 661C>T mutation in the human ngf gene. Homozygous NGFR100W/R100W mice were born normal, but failed to reach the first month of age. This early lethality could be due to reduced NGF bioavailability and, indeed, was rescued by continuous treatment, during development and the early postnatal life, with wild type NGF. In contrast, heterozygous NGFR100W/m mice grew normally but displayed impaired nociception, despite Dorsal Root Ganglia (DRGs) neurons being morphologically normal. On the other hand, skin innervation was reduced. The NGFR100W protein showed reduced capability to activate pain-specific signalling, paralleling its reduced ability to induce mechanical allodynia. Surprisingly, NGFR100W/m mice, unlike heterozygous mNGF+/- mice, showed no learning nor memory deficits, despite a reduction in secretion and brain levels of NGF. These results prove the hypothesized dissociation between the peripheral and central actions of NGF, prompting me to investigate if the R100W mutation might affect brain elaboration of pain. To address this issue, I used the fear conditioning test and found that NGFR100W/m mice, despite normal nociceptive responses to a painful conditioning stimulus, showed a deficit in learned fear. Strikingly, their innate fear responses were normal. This was accompanied by a reduced activation of brain regions involved in pain processing and in the generation of task-related motor responses. I also found a decreased density of CGRP-positive fibers in the amygdala, which can provide a mechanistic explanation of the reduced fear response. On the other hand, the expression of endogenous analgesic peptides, namely β-endorphin and oxytocin, was decreased in NGFR100W/m mice, suggesting a different set point of the homeostatic pain/analgesia system, as a consequence of a prolonged reduction of afferent pain signals. Consistent with these findings in mice, data collected in humans showed that heterozygous R100W carriers, despite having a normal pain threshold, had a decreased urgency to react to a painful stimulus, along with impaired ability to integrate sensory information with behavioral task requirements. Functional magnetic resonance imaging (fMRI) revealed, in accordance with mouse data, an altered processing of painful stimuli in brain areas involved in pain processing. These findings demonstrate an uncoupling of nociceptive signals from their central elaboration, leading to altered interpretation and meaning attributed to painful stimuli in human HSAN V carriers and heterozygous NGFR100W/m mice. In addition to clarify the role of NGF in transduction of nociceptive inputs, these data also demonstrate that NGF is at the center of a regulation system linking peripheral nociception to the brain processes responsible for constructing painful perceptions and pain-related memories. Moreover, the peculiar effects of NGFR100W could be exploited to open new avenues for treating conditions of chronic pain.

Acute myeloid leukemia (AML) is a genetically heterogeneous disease characterized by the accumulation of acquired somatic genetic abnormalities in hematopoietic progenitor cells. Recurrent chromosomal rearrangements are well-established diagnostic and prognostic markers. However, approximately 50% of AML cases have normal cytogenetics and have variable responses to conventional chemotherapy. Molecular markers have been begun to subdivide cytogenetically normal AML (CN-AML) and have been shown to predict clinical outcome. Despite these achievements, current classification schemes are not completely accurate and improved risk stratification is required. My overall objective was to identify specific gene expression and mutation signatures to define novel subclasses of CN-AML. I hypothesized that CN-AML would be separated into at least two or more subgroups. Gene expression and mutational profiles were established using RNA-Sequencing, clustering, de novo transcriptome assembly, and variant detection. I found the CN-AML could be separated into three groups, two of which had statistically significant survival differences (Kaplan-Meier analysis, log-rank test, p=9.75x10-³). Variant analysis revealed nine fusions that are not detectable via cytogenetic analysis and differential expression analysis identified a set of discriminatory genes to classify each subgroup. These findings contribute to the current understanding of the genetic complexity of AML and highlight gene fusion candidates for follow-up functional analyses.
Medicine, Faculty of
Medical Genetics, Department of
Graduate

Les moustiques sont colonisés par un virome très peu étudié. Comme les bactéries, le virome influence probablement la biologie et l'immunité des populations de moustiques vecteurs, mais les modèles expérimentaux sont rares. Nous avons récemment découvert deux nouveaux virus chez le virome des vecteurs sauvages du paludisme, anophèles et des colonies d’Anopheles coluzzii : Anopheles C virus (AnCV) et Anopheles cypovirus (AnCPV). L’un ou les deux virus sont présents dans toutes les colonies de laboratoire d’An. gambiae et An. coluzzii. La prévalence des virus varie en fonction des stades du moustique. L'abondance des deux virus est négativement corrélée chez les moustiques individuels. L'analyse fonctionnelle révèle l'implication des voies de signalisation immunitaire des moustiques sur la réplication du virus, avec une influence différentielle sur les deux virus. Un modèle expérimental a été développé pour l'infection d’AnCPV chez les anophèles non porteurs de ces virus, en utilisant du sang infecté afin d'étudier les réponses antivirales chez ces moustiques. Les séquences de l'AnCPV sont hautement polymorphiques chez les moustiques individuels, alors que l'AnCV est pratiquement dépourvue de mutations. AnCPV entraine une plus grande mortalité chez An. stephensi, mais certaines mutations semblent impliquées dans son adaptation à cette espèce. AnCPV peut être potentiellement transmis comme un arbovirus à travers un hôte mammifère à des moustiques non infectés, ce qui suggère une voie évolutive relativement simple. Le virome d’An. stephensi contient un chaq-like virus et un partiti-like virus. Ce dernier appartenant à la famille des Partitiviridae a des formes d’ADN
Mosquitoes are colonized by a little-studied natural virome. Like the bacterial microbiome, the virome also probably influences the biology and immunity of mosquito vector populations, but tractable experimental models are lacking. We recently discovered two novel viruses in the virome of wild Anopheles and in colonies of the malaria vector Anopheles coluzzii: Anopheles C virus and Anopheles cypovirus. One or both viruses are present in all tested laboratory colonies of An. coluzzii and An. gambiae. Viral abundance varies reproducibly during mosquito development. Relative abundance of the two viruses is inversely correlated in individual mosquitoes. Functional genomic analysis reveals the implication of mosquito immune signaling pathways on virus replication, with differential influence on the two viruses. An experimental model was developed for AnCPV infection of Anopheles by bloodmeal, in order to study mosquito antiviral responses. Sequences of AnCPV are highly polymorphic in individual mosquitoes, while AnCV is virtually devoid of variation. AnCPV is pathogenic to An. stephensi but some viral mutations seem to be involved in its adaption to this species. AnCPV can be transmitted like an arbovirus through a vertebrate host to uninfected mosquitoes, suggesting that the evolutionary pathway from vertical “insect specific” to infective blood transmission may be remarkably simple. The Anopheles stephensi virome harbors a chaq-like virus and partiti-like virus. This latter belonging to Partitiviridae is present in An. stephensi as DNA forms of the virus genome

Genetic analysis of Neurofibromatosis type 1 (NF1) may facilitate the identification of patients in early phases of the disease. Here, we present an overview of our diagnostic research spanning the last eleven years, with a focus on the description of 225 NF1 mutations, 126 of which are novel, found in a series of 605 patients (513 unrelated) in Italy. Between 2003 and 2013, 443 unrelated patients were profiled by DHPLC analysis of 60 amplicons derived from genomic NF1 DNA and subsequent sequencing of heterozygotic PCR products. In addition, a subset of patients was studied by MLPA to identify any duplications, large deletions or microdeletions present at the locus. Over the last year, 70 unrelated patients were investigated by MLPA and sequencing of 22 amplicons spanning the entire NF1 cDNA. Mutations were found in 70% of the 293 patients studied by DHPLC, thereby fulfilling the NIH criterion for the clinical diagnosis of NF1 (detection rate: 70%); furthermore, 87% of the patients studied by RNA sequencing were genetically characterized. Mutations were also found in 36 of the 159 patients not fulfilling the NIH clinical criteria. These data support the use of RNA-based methods for genetic analysis and provide novel information for relevant for improving the management of symptoms in oligosymptomatic patients.

Viruses that infect thermophilic Archaea are unique in both their structure and genetic makeup. The lemon-shaped fuselloviruses - which infect members of the order Sulfolobales, growing optimally at 80º C and pH 3 - are some of the most ubiquitous and best studied viruses of the thermoacidophilic Archaea. They provide a malleable and useful genetic tool for probing into the functions of their host, as well as the host responses to infection. Nonetheless, much about these viruses remains to be learned to further understand their morphological, genetic, and life cycle characteristics. In order to investigate these aspects of these Fuselloviridae, as well as their evolution, this work reports the isolation and characterization of a novel fusellovirus, Sulfolobus Spindle-shaped virus 10 (formerly SSV-L1). Genetic and genomic analyses highlight significant homology with both SSV8 and SSV9, as well as conservation of promoter elements within the Fuselloviridae. SSV10 encodes five ORFs with no homology within or outside of the Fuselloviridae, as well as a putatively functional Cas4-like ORF which may play a role in anti-CRISPR host evasion. Moreover, we demonstrate the ability of SSV10 to withstand mutation in a fashion consistent with mutagenesis in SSV1. Lastly, analysis of predicted protein structures from SSV10 provide new insights into virus-host interactions. These analyses help to expand our understanding of the viral life cycle while contextualizing the mutagenesis data presented in the following chapters.

An Escherichia coli strain carrying the phs mutation was isolated by other workers who were interested in determining the role of the Na /H antiport in pH homeostasis. The mutation was observed to cause impaired growth on L-glutamate amd melibiose and was also reported to cause a sensitivity to alkaline conditions due to an impairment in pH homeostasis. Since uptake of both glutamate and melibiose is energised by a Na⁺ gradient, these observations were interpreted as evidence that the phs mutation impairs Na⁺ /H⁺ antiport activity, and so by implication, that the Na⁺ /H⁺ antiport is involved in pH homeostasis in E. coli. In work for this thesis, evidence was accumulated that the phs mutation does not affect Na⁺ /H⁺ antiport activity. Proline uptake, which is energised by a Na⁺ gradient, was found to be normal in a mutant strain. Also, arabinose uptake via two different transport systems, neither of which is energised by a Na⁺ gradient, was found to be impaired by the mutation. K⁺ uptake by Na⁺ loaded cells, a phenomenon which is thought to give a measure of Na⁺ /H⁺ antiport activity, was not affected by the mutation. Studies on growth under alkaline conditions indicated that growth is not impaired by the mutation. Evidence was obtained however, that the phs mutation causes filamentation under these conditions. There is a possibility that this may be due to an effect on the penicillin-binding protein content of the cell envelope. Investigations of the effect of the mutation on pH homeostasis suggested that it affects either pH homeostasis, or the validity of the techniques used for determining the cytoplasmic pH. In view of the mapping of the mutation to the gene coding for the α-subunit of RNA polymerase (Rowland et al, 1985), the effects of the mutation on transcription were studied. It was found that the mutation dramatically impairs transcription of genes under the positive regulation of the AraC gene product. An examination of the effect of the mutation on transcription from a variety of different promotors showed that the transcription defect is highly selective. Some evidence was obtained that the mutation impairs the process of positive regulation. To test this, the effect of the mutation on transcription from AraC independent derivatives of an AraC dependent promoter was determined. In the absence of AraC, the mutation had no effect. In the presence of AraC, complex effects were observed. It was concluded that the mutation affects the process of positive regulation or CRP-cAMP mediated derepression of this promoter.

MAMLD1 is suggested to play a role in the development of 46,XY disorders of sexual development (46,XY DSD). So far, mutations in this gene have been detected in several cases of hypospadias with normal testosterone levels at birth. From in vitro studies it was concluded that Mamld1 might transiently affect testosterone synthesis during genital development. We describe the first MAMLD1 mutation in a 46,XY patient with complete gonadal dysgenesis. The novel MAMLD1 missense mutation (p.P677L) results in a severely reduced transactivation in vitro of the promoter of the MAMLD1 target gene HES3/Hes3. However, as knowledge about the functional role of MAMLD1 in gonadal development is limited, we suggest that additional factors (digenic or oligogenic cause) play a role in the development of complete gonadal dysgenesis in this patient.

We aimed to characterize well-know PIDs, novel genetic defects and immunopathological mechanisms in Brazilian patients with susceptibility to mycobacterial diseases. The patients developed different mycobacterial diseases and M. tuberculosis was the most frequent species. Molecular and genetic analysis revealed mutations in different genes: RAG1 (P1), CD40LG (P2, P3, P4), NEMO (P5), NCF1 (P6), TLR2 (P7) IL-12Rb2 (P8), IL-12Rb1 (P9), TLR10 (P10), DKC1(P11), SOCS-1(P12) and IRAK2 (P13). Finally, MDMs from patients phagocytose normally but were unable to appropriately control intracellular M. tuberculosis growth in comparison to MDMs from healthy subjects. We concluded that the Brazilian patients have heterogeneous mutations previously associated with susceptibility to mycobacterial diseases and novel genetic variations were identified suggesting novel PIDs. In addition, the inability of MDMs to control the intracellular growth of M. tuberculosis indicates this contributes to patients´ susceptibility to mycobacterial infections.
Objetivamos identificar novos defeitos genéticos e mecanismos imunopatológicos em pacientes brasileiros com suscetibilidade a infecções por micobactérias. Os pacientes foram investigados se portadores de imunodeficiencias previamente caracterizadas tais como SCID, deficiência de CD40L, MSMD, defeitos na sinalização via TLRs e CGD. A análise genética foi realizada por sequenciamento Sanger e \'\'whole exome sequencing\'\' para identificar possíveis novas imunodeficiências primárias. Além disso a função dos macrófagos dos pacientes foi avaliada. Infecções por diferentes espécies de micobactérias foram apresentadas pelos pacientes, sendo M. tuberculosis a espécie mais frequentemente identificada. Mutações em diferentes genes foram encontradas: RAG1 (P1), CD40LG (P2, P3, P4), NEMO (P5), NCF1 (P6), TLR2 (P7), IL-12Rb2 (P8), IL-12Rb1 (P9), IRAK2 (P10), SOCS-1 (P11) e TLR10 (P12). MDMs dos pacientes fagocitaram normalmente M. tuberculosis, porém reduzida capacidade em inibir o crescimento da M. tuberculosis foi observada. Concluímos que os pacientes estudados possuem defeitos moleculares heterogêneos e que os MDMs desses indivíduos apresentam falhas no controle do crescimento da M. tuberculosis. Nossos dados sugerem que esses são fatores subjacentes à susceptibilidade a infecções por micobactérias nesses indivíduos.

Presenilin-1 (PS1) mutations caused by the PSEN1 gene mutations are the major cause of early onset familial Alzheimer’s disease (EOFAD). Two Chinesespecific EOFAD related PS1 mutations, V97L and A136G, have been found. Studies suggested that V97L mutation lead to the overexpression of Aβ42 and tau hyperphosphorylation, which are the major hallmarks of Alzheimer’s disease (AD), while properties of A136G were unclear. Since calcium dysregulation was suggested to play an important role in AD, the research project investigated if V97L and A136G mutations also lead to altered endoplasmic reticulum (ER) 〖Ca〗^(2+) regulation. SH-SY5Y cells transduced with retrovirus carrying V97L mutant or A136 mutant PSEN1 were used as the experiment models. In Western blotting, while the PS1 expression level was unaffected in V97L mutant, the expression level was significantly lower in A136G mutant. In carbachol (CCh) perfusion experiment, V97L mutant was found to exaggerate ER 〖Ca〗^(2+) release when stimulated by higher concentration (30, 100 and 300 μM) CCh, while A136G mutant exaggerated ER Ca2+ release when stimulated by 30 μM and 300 μM CCh, but not 100 μM CCh. In 5% fetal bovine serum (FBS) perfusion experiment, both V97L and A136G mutants were found to sensitize 〖Ca〗^(2+) oscillation, which the sensitization effect of V97L was 3 folds of A136G. The results suggested that V97L mutation exaggerates ER 〖Ca〗^(2+) release, possibly via interaction with IP3R. However the results of A136G were inconclusive and contradicting, therefore further investigation is needed.
published_or_final_version
Physiology
Master
Master of Medical Sciences

The phenotypic effects of a novel X-chromosome linked mutation were studied in the offspring of male mice treated with the mutagen ethyl nitrosourea (ENU). Studies of X-chromosome inactivation patterns in females heterozygous for the mutation (ENU/+) has delineated four distinct cell lineages affected by the mutation, namely, B, pre-B and T lymphoctyes, erythrocytes and skeletal myocytes. The penetrance of the mutation depended on the age of mice, the cell lineage affected by the mutation and the stage of maturation of the cell lineage. Studies of B cells in females heterozygous for the mutation and the X-linked mutation, xid that affects B but not pre-B cells, indicated that the two mutations were not allelic. Factors influencing differences in X-chromosome inactivation between cells and hybrids and their relationship to alleles of the X-chromosome controlling element (Xce) in the ENU-mutant and parental strains were studied. It was not possible to identify the effects of the mutation on the immune system in functional terms either by flow cytometric analysis of leukocytes or after sensitisation to oxazolone. Results imply the mutation renders the affected cell lineages susceptible to competition with normal cells in the heterozygote, rather than there being any fundamental defect in cell function and that the mutation may be in a gene encoding a component of the cell cycle or controlling a maturation step.

Human congenital heart disease (CHD) is the most common cause of non-infectious neonatal death affecting 1-2% of live births (Hoffman and Kaplan, 2002). Treatment of CHD requires major surgery and quality of life is often significantly reduced despite treatment. With the discovery of single gene mutations that cause CHD in model animals (Lyons et al., 1995), the role of genetics in CHD has become appreciated. The genetic basis of CHD is poorly understood, with different members of the same family presenting with different types of CHD (Schott et al., 1998), suggesting the causes of CHD are multifactorial. Cardiogenesis is intimately associated with the establishment of the left-right (L-R) body axis, with the two processes sharing several important transcription factors. Heart looping, in which the heart turns dextrally, is the earliest physical manifestation of L-R asymmetry. L-R patterning disorders are associated with an increased risk of CHD; heterotaxy (in which L-R asymmetry is neither normal nor mirror image) accounts for about 3% of all CHD (Zhu et al., 2006).Investigating cardiogenesis and the causes of CHD necessitates the use of animal models, typically mice, chicks, zebrafish and Xenopus. Recently a strain of mouse with a mutation in a gene essential for cardiac development was isolated from an ENU mutagenesis screen (Kile et al., 2003) using mice carrying a balancer chromosome. It has been subsequently found that the most likely candidate gene codes for the protein Prpf8, an integral component of the spliceosome. The mutation is homozygous lethal, with homozygous mice having a grossly deformed heart, developmental delay and a high incidence of heart looping reversal, indicative of a L-R patterning disorder. In depth characterisation of homozygous mutant embryos revealed defects in the morphology of the embryonic node, nodal cilia and the expression pattern of L-R axis genes. We also investigated the expression of Prpf8 during embryogenesis, and the effect that the point mutation we found in our homozygous embryos has on splicing kinetics.

Introduction: The aims of this thesis was to explore novel data types in healthcare that could enhance epidemiology studies in epilepsy and to develop novel methods of analysing routinely collected linked healthcare data, unstructured free text in hospital clinic letters and genetic variation. Method: The SAIL Databank was used to source linked healthcare data for people with epilepsy across Wales to study the effects of epilepsy and social deprivation, coding of epilepsy in GP records and the educational attainment of children born to mothers with epilepsy. Hospital clinic letters from Morriston Hospital in Swansea were analysed using Natural Language Processing techniques to extract rich clinic data not typically recorded as part of routinely collected data. An automated pipeline was developed to predict the pathogenicity of Single Nucleotide Polymorphisms to prioritize potential disease-causing genetic variation in epilepsy for further in-vitro analysis. Results: Incidence and prevalence of epilepsy was found to be strongly correlated with increased social deprivation, however a 10 year retrospective follow-up study found that there was no increase in deprivation following a diagnosis of epilepsy, pointing to deprivation contributing to social causation of epilepsy rather than epilepsy causing social drift. An algorithm was developed to accurately source epilepsy patients from GP records. Sodium Valproate was found to reduce educational attainment in 7 year olds by 12%. A Natural Language Processing pipeline was developed to extract rich epilepsy information from clinic letters. A pipeline was created to predict pathogencity of epilepsy SNPs that performed better than commonly used software. Conclusion: This thesis presents novel studies in epilepsy using population level healthcare data, unstructured clinic letters and genetic variation. New methods were developed that have the potential to be applied to other disease areas and used to link different data types into routinely collected healthcare records to enhance further research.

Triple A syndrome, formerly known as Allgrove syndrome, is an autosomal recessive disorder characterized clinically by adrenal insufficiency, alacrima, achalasia, and neurological abnormalities. We report a 17-year-old boy presented to the endocrine clinic with delayed puberty and a 4-year’s history of fatigue and muscle weakness. He had achalasia, alacrima, and skin and mucosal hyperpigmentation. Hormonal assessment revealed isolated glucocorticoid deficiency. Clinical diagnosis of triple A syndrome was confirmed by sequencing the entire coding region including exon-intron boundaries of the AAAS gene. Analysis revealed a homozygous novel indel mutation encompassing intron 7 to intron 10 of the gene (g.16166_17813delinsTGAGGCCTGCTG; NG_016775). This is the first report of triple A syndrome in Jordan with a novel indel mutation and presenting with delayed puberty.

Mutations in the BRCA1 tumour suppressor gene are associated with significantly elevated risks of develop breast or ovarian carcinomas, up to 80% or 40% respectively by age 70. The anti-tumourigenic activity of the wild-type protein product is the result of participation in a number of distinct but overlapping protective pathways; DNA damage repair, transcription & splicing, cell cycle regulation, ubiquitination & regulation of apoptosis. Through modulating these cellular mechanisms, BRCA1 is able to participate in the preservation of genomic integrity. Consequently, cells lacking functional BRCA1 exhibit traits associated with genetic instability; gross chromosomal translocations, often involving multiple, non-homologous chromosomes; as well as deletions and/or amplifications of genetic material. Furthermore, BRCA1 deficient cells lack fully functional DNA repair processes and are sensitive to genotoxic agents. It has been demonstrated that BRCA1 acts principally as a scaffold protein, and mediates the formation of a number of complexes in the cell with unique functions. The BRCA1-associated factors in these complexes participate in the pathways described above, and ultimately contribute to BRCA1-mediated tumour suppression. Accordingly, it is reasonable to suggest that a significant proportion of BRCA1 function is a product of the factors with which it interacts. Protein interaction sites have been described across the entire length of the BRCA1 protein sequence, and some specific regions have been broadly associated with particular functions or pathways, for instance the C-terminus is associated with DNA damage response, whilst the N-terminus is required for ubiquitination. However, there is limited information as yet associating specific domains of the BRCA1 protein with preventing tumour formation or disease progression. In this study, we have attempted to assess the contribution of specific domains to BRCA1 function and tumour suppression through directly analysing the effect of pathogenic mutations on BRCA1 function and how phosphorylation of BRCA1 contributes to complex formation.

Hereditary Motor Sensory Neuropathy (HMSN), also known as Charcot-Marie-Tooth disease (MIM118300), is an heterogeneous group of Mendelian diseases which affects the peripheral motor and sensory nervous system (PNS). With a prevalence of about 1/2500 individuals, it is considered the most common inherited neuromuscular disorder. The clinical hallmarks include slow and progressive muscular weakness of distal limbs, peroneal atrophy and bilateral pes cavus, however they are often associated with other signs such as spastic paraplegia, ataxia, mental retardation and incontinence. Distal HMN disorders are a subgroup of HMSN characterized by a predominant motor involvement and minor or no sensory loss. To date, more than 50 genes have been detected for HMSN and 17 genes for dHMN, but many disease-genes have to be identified yet. This study aims to identify and, whenever possible, functionally characterise novel genes causing different forms of peripheral neuropathy. In order to achieve this objective, four families affected by complex HMSN or distal HMN and showing no mutations in known disease-genes were examined. The identification of novel genes was performed by using a strategy which integrates the traditional positional cloning approach with the next-generation whole-exome sequencing. In all the four families candidate linkage regions were identified by genome-wide linkage analysis, specifically for the recessive forms they were identified also by homozygosity mapping and identity-by-descent analysis using high-density SNPs arrays and STR markers. In one family a candidate-genes screening by Sanger sequencing was preferred for the small size of the critical region. In the remaining three families the whole-exome sequencing (WES) approach was adopted in order to inspect all coding variants within the previously identified linkage regions. Considering the advantage of having variants within the whole exome, known disease-related genes were also analysed and less stringent genome wide searches were even conducted. Moreover, coverage analysis was performed and poorly-covered exons were sequenced. The best candidate variants were selected by filtering and prioritization analyses which allowed to evaluate the putative pathogenicity of variants. For the variants which represented potential mutations, further confirmations were obtained by in silico analyses, control population screening, unrelated patients characterization and functional studies. This work represents the first step to demonstrate the pathogenicity of a candidate variant and dissect the mechanisms underlying peripheral neuropathies.
La neuropatia ereditaria sensitivo-motoria (HMSN), anche denominata malattia di Charcot-Marie-Tooth (MIM118300), costituisce un gruppo eterogeneo di disordini mendeliani che colpiscono il sistema nervoso periferico motorio e sensitivo. Tale malattia è considerata il disordine neuromuscolare ereditario più comune, con una prevalenza stimata di 1 caso su 2500 nella popolazione. I segni clinici distintivi includono una progressiva ipostenia e ipotrofia dei muscoli distali degli arti, un’atrofia peroneale e piede cavo bilaterale. Questo fenotipo clinico è di frequente associato ad altri sintomi, quali la paraparesi spastica, il ritardo mentale, atassia e disturbi urinari. Le neuropatie ereditarie motorie distali (dHMN) costituiscono un sottogruppo delle neuropatie ereditarie sensitive-motorie, caratterizzato da un prevalente coinvolgimento motorio e un minore o assente interessamento sensitivo. Ad oggi, più di 50 geni sono stati associati alle neuropatie ereditarie sensitivo-motorie e 17 alle forme motorie distali, tuttavia molti geni rimangono ancora sconosciuti. Il presente studio si prefigge di identificare e possibilmente caratterizzare dal punto di vista funzionale, nuovi geni causativi di varianti ereditarie di neuropatia periferica. A tale scopo sono state studiate quattro famiglie in cui erano presenti soggetti affetti da HMSN complesse o dHMN e nelle quali erano state escluse le mutazioni nei geni malattia già noti. Al fine di identificare nuovi geni causativi, in questo lavoro è stata adottata una strategia che va ad integrare l’approccio tradizionale di clonaggio posizionale con il sequenziamento next-generation dell’esoma. Nelle quattro famiglie oggetto di studio, mediante SNPs ad alta densità e marcatori microsatelliti, è stata svolta una analisi di linkage genome-wide e si sono identificate regioni candidate; inoltre per le forme recessive si è proceduto con il mappaggio per omozigosità e l’analisi di identità per discendenza (IBD). In una di queste famiglie, data la ridotta estensione della regione candidata, lo screening mutazionale dei geni candidati è avvenuto per mezzo del sequenziamento diretto. Nelle rimanenti tre famiglie, invece, è stato adottato l’approccio di sequenziamento next-generation dell’esoma, al fine di analizzare tutte le varianti presenti negli esoni codificanti delle regioni in linkage. Il vantaggio di avere un’informazione estesa all’intero esoma ha permesso un’analisi genome-wide meno stringente e la ricerca delle varianti nei geni malattia già noti. L’analisi dell’efficienza di copertura delle regioni candidate ha permesso invece di sequenziare gli esoni poco coperti di queste regioni. Un’approfondita e dettagliata analisi di filtraggio e prioritizzazione ha reso possibile valutare la possibile patogenicità delle varianti e di identificare le migliori candidate. A partire da queste, ulteriori validazioni sono state poi ottenute mediante studi in silico, screening di popolazione di controllo, caratterizzazione di pazienti non imparentati e studi funzionali. Tale studio costituisce il primo step fondamentale per identificare nuovi geni causativi e studiare nel dettaglio i meccanismi alla base delle neuropatie distali. A tale scopo screening genetici su coorti di pazienti e studi funzionali potranno far luce sull’effettivo coinvolgimento di questi nuovi geni candidati nelle neuropatie periferiche.

Familial history is the strongest risk factor for developing ovarian cancer (OC), and a significant contributor to breast cancer risk. Most hereditary breast cancers and OCs are associated with mutation of the tumor suppressor Breast and Ovarian Cancer Susceptibility Gene 1 (BRCA1). Studying risk-associated BRCA1 truncation mutations, such as the founder mutation 185delAG, may reveal signaling pathways important in OC etiology. Recent studies have shown novel BRCA1 mutant functions that may contribute to breast and OC initiation and progression independent of the loss of wtBRCA1. Previously, we have found that normal human ovarian surface epithelial (HOSE) cells expressing the 185delAG mutant, BRAT ( BRCA1 185delAG Amino Terminal truncated protein), exhibit enhanced chemosensitivity and up-regulation of the OC-associated serpin, maspin. In the current study, I identify an additional target of the BRAT mutation, matrix metalloprotease 1 (MMP1), a key player in invasion and metastasis. BRAT-expressing HOSE cells exhibit increased MMP1 messenger RNA (mRNA) by real time PCR and protein by Western blotting. Pro-MMP1 levels are also higher in conditioned media of BRAT-expressing cells and HOSE cell lines derived from BRAT mutation carriers. c-Jun is critical for BRAT-mediated MMP1 up-regulation, as siRNA knockdown diminishes MMP1 levels. Luciferase reporter constructs reveal that activator Protein 1 (AP1) sites throughout the distal end of the promoter contribute to BRAT-mediated MMP1 expression, and basal activity is mediated in part by an AP1 site at (-72). Reporters containing a single nucleotide polymorphism (SNP) associated with OC risk and progression yield increased activity that is further enhanced in BRAT cells. Interestingly, BRAT-mediated changes in chemosensitivity and gene regulation are not recapitulated in a normal breast epithelial or breast cancer cell model. This suggests tissue-specific mutant BRCA1 functions may contribute to breast and ovarian tissue specificity of BRCA1 mutation-associated cancer risk and also to differential breast and ovarian cancer risk and penetrance associated with specific mutations. Early molecular and cellular changes such as MMP1 up-regulation in the ovarian surface epithelium of BRCA1 mutation carriers may promote OC initiation and progression and represent a step forward on the continuum of cellular malignancy. Further investigation is warranted, as elucidating these early changes will aid in identification of potential screening and treatment strategies.

This research identifies a novel AE1 mutation in a newly-referred family where dRTA segregates with disease through a four-generation pedigree, as determined by DNA sequencing and specific restriction enzyme digestion. This mutation – AE1-M909T – lies within the last four residues of the tail, in a potential PDZ-binding domain. AE1-M909T expression in Xenopus oocytes confirmed preserved anion exchange function. To investigate the mechanism of disease, N-terminal GFP-tagged AE1 was stably expressed in a variety of cell types in HEK293 and non-polarised MDCK cells, wild-type (AE1wt) and AE1-M909T both reach the cell surface. In polarised MDCK cells, GFP-tagged AE1wt has normal basolateral residency. In marked contrast, the mutant protein is aberrantly targeted, appearing at the apical membrane in addition to preserved basolateral presence. Antibody-labelling assays demonstrated post-synthetic AE1wt traffic direct to the basolateral membrane without any apical passage. In contrast, AE1-M909T traffics directly to both cell surfaces, thus implying gain of an apical targeting signal (plus persistent basolateral signals): this non-polarised expression will cause failure of α-IC function and dRTA. This novel SLC41 mutation causes AE1 mistargeting through acquisition of an apical targeting signal. The results suggest that basolateral targeting motifs are concentrated upstream of the extreme C-terminus of AE1, but that this micro-domain is important for normal biosynthetic traffic to the cell surface, potentially through PDZ-based interactions.

The common human GNB3 825C > T variant, which is present in 50% of the world’s chromosomes, has previously been shown to predispose individuals to hypertension, cardiac and neural disorders. This variant causes the production of a stable and gain of function protein Gβ_3S- This thesis describes the discovery of a novel D153del mutation that produces an unstable, loss of function, protein Gβ_3D in the recessively inherited, retinopathy globe enlarged (rge) chickens. This thesis also demonstrates that the normal Gβ₃ downstream phosphorylation signalling pathways are significantly altered in a tissue specific manner in rge chicken organs and in a human GNB3 825TT lymphoblast cell line. In rge tissues expressing Gβ_3D protein, the cAMP induced GRK2 phosphorylation activity is significantly altered. Moreover MAPK1 (ERK2) phosphorylation is significantly decreased compared to normal tissues. In contrast human 825TT cell lines expressing the Gβ_3S protein, showed enhanced cAMP induced GRK2 and MAPK (ERK1 and ERK2) phosphorylation activity. These results confirm previous findings of 825C > T Gβ₃ studies, that Gβ_3S is indeed a hyper-activating structural variant, in contrast to the D153del Gp3D is a classical recessively inherited non-functional mutation.

À ce jour, STAT3 est le seul gène pour lequel des mutations dans les régions codantes sont à l'origine du syndrome hyper-immunoglobuline E autosomique dominant (AD-HIES). Cependant, l'étiologie génétique de 5% des individus répondant aux critères cliniques pour l'AD-HIES reste inconnue. En combinant le séquençage de l'exome entier et l'analyse de liaison génétique, nous avons identifié la première mutation hétérozygote STAT3 intronique, c.1282-89C>T, causant l'AD-HIES chez sept patients de la même famille. Cette mutation crée un nouvel exon au niveau de l'ADNc de STAT3 (D427ins17). Nous avons également identifié deux nouvelles mutations STAT3 non-sens; c.1552C>T conduisant à une protéine tronquée avec un codon stop dans le domaine de liaison de STAT3 (R518*) chez un patient souffrant de l'AD-HIES, et c.2091delT conduisant à une protéine tronquée avec un codon stop entraînant un décalage du cadre de lecture dans le domaine de transactivation de STAT3 (D698Tfs*9) chez deux apparentés atteints de la tuberculose. Dans un système de surexpression, les trois protéines STAT3 mutantes sont perte de fonction en terme de phosphorylation de la tyrosine, de liaison à l'ADN et d'activité transcriptionnelle. Dans les lymphocytes B des patients, les deux allèles non-sens ne sont pas exprimés alors que nous avons trouvé par spectrométrie de masse que l'allèle D427ins17 ne représente que 5 à 20% du STAT3 total dans les cellules du patient. L'activation des leucocytes du patient a démontré une faible réponse aux cytokines STAT3-dépendantes, comme d'autres patients avec l'AD-HIES. Dans un système de surexpression, nous avons montré que les allèles D427ins17 et D698Tfs*9 sont également dominants-négatifs, alors que l'allèle R518* est neutre. Ce travail démontre l'importance du séquençage des introns dans l'établissement du diagnostic génétique pour l'AD-HIES. De plus, l'étude de l'allèle D427ins17 suggère que les mutations provoquant l'AD-HIES peuvent exercer un effet dominant-négatif même lorsqu'elles sont exprimées à des niveaux significativement inférieurs à ceux de la protéine de type sauvage. En revanche, l'étude de l'allèle R518* suggère l'haploinsuffisance comme étant un autre mécanisme susceptible d'entraîner l'AD-HIES; cependant, l'identification et la caractérisation d'un plus grand nombre de mutations non-sens sont nécessaires avant de pouvoir tirer des conclusions définitives
To date STAT3 is the only gene in which variants in coding regions are known to cause Autosomal Dominant Hyper-Immunoglobulin E Syndrome (AD-HIES). Yet, the genetic etiology of 5% of individuals meeting the clinical criteria for AD-HIES remains unknown. Combining whole exome sequencing and genetic linkage analysis, we identified the first deep intronic heterozygous STAT3 mutation, c.1282-89C>T, causing AD-HIES in seven relatives. This mutation creates a new exon in the STAT3 cDNA (D427ins17). We also identified two novel nonsense STAT3 mutation; c.1552C>T leading to a truncated protein with a stop codon in the STAT3 linker domain (R518*) in a patient with AD-HIES, and c.2091delT leading to a truncated protein with a stop codon with a frameshift in STAT3 transactivation domain (D698Tfs*9) in two relatives with tuberculosis. Upon over-expression, the three mutant STAT3 proteins are loss of function in terms of tyrosine phosphorylation, DNA-binding, and transcriptional activity. In patient's B cells, R518* and D698Tfs*9 alleles are not expressed whereas we found by mass spectrometry that the D427ins17 allele only represents 5 to 20% of total STAT3 in the patient's cells. Activation of patient's leucocytes demonstrated a poor respond to STAT3-dependent cytokines, like other patients with AD-HIES. Upon overexpression, we show that D427ins17 and D698Tfs*9 are equally dominant-negative alleles, whereas R518* allele is neutral. This work emphasis the importance of intron sequencing in the establishment of genetic diagnostics in AD-HIES. Moreover, the study of the D427ins17 allele suggests that AD-HIES-causing mutations can exert their negative-dominance even when expressed at significantly lower levels than the wild-type protein. On the other hand, the study of R518* allele shed the light on haploinsufficiency as another possible mechanism causing AD-HIES; however, the identification and characterization of more nonsense mutations is necessary before drawing any firm conclusions

Il mieloma multiplo (MM) è una proliferazione maligna fatale delle plasmacellule (PC) secernenti anticorpi del midollo osseo (BM), caratterizzata da un ampio spettro clinico e da una profonda instabilità genomica. Per quanto riguarda in particolare il panorama mutazionale, recenti studi di sequenziamento di nuova generazione (NGS) nei pazienti con MM hanno indicato la mancanza di una mutazione pilota universale, ma diversi geni mutati ricorrenti appartenenti a percorsi chiave coinvolti nella malattia, come la via MAP-chinasi. La caratterizzazione del MM attualmente si basa sull'analisi mutazionale effettuata sugli aspirati di BM. Tuttavia, questo approccio potrebbe non catturare la presunta eterogeneità spaziale e genetica della malattia e imporre ostacoli tecnici che ne limitano il trasferimento nel laboratorio diagnostico di routine e clinico, riducendo anche la possibilità di un monitoraggio longitudinale dei marcatori molecolari della malattia. Ciononostante, recenti dati relativi a cancri solidi indicano che il DNA circolante nel sangue periferico (PB) può essere usato come fonte di DNA tumorale per fornire informazioni sulla massa tumorale, sulla malattia residua e sul genotipo tumorale, con ovvi vantaggi in termini di accessibilità. Sulla base delle osservazioni precedenti, gli obiettivi del progetto erano: (i) la valutazione dell'incidenza delle mutazioni nei geni “driver” del MM (KRAS, NRAS, TP53, BRAF, FAM46C e DIS3) in una coorte ampia e rappresentativa di pazienti a diversi stadi di discrasia plamacellulare (132 MM e 24 casi di leucemia plasmacellulare primaria (pPCL), entrambi all'esordio, e in 11 pazienti con PCL secondaria (sPCL)); (ii) il confronto del profilo mutazionale del DNA libero da cellule circolante (cfDNA) e del DNA derivato dal BM in una piccola serie di pazienti rappresentativa di diversi stadi clinici di tumori plasmacellulari. Nello specifico, abbiamo valutato, mediante NGS ad alta profondità, l'incidenza di mutazioni in BRAF (esoni 11 e 15), NRAS (esoni 2 e 3) e KRAS (esoni 2-4), DIS3 nei domini funzionali PIN (esoni 1-4) e RNB (esoni 10-18), TP53 (esoni 4-9) e FAM46C (esone 2). Complessivamente, la via MAPK è risultata affetta da mutazioni nel 60,1% dei pazienti (63,6% di quelli con sPCL, 59,8% di quelli con MM e 41,7% di quelli con pPCL). In particolare, il 12% dei pazienti è stato trovato mutato in BRAF, il 23,9% in NRAS e il 29,3% in KRAS. Mutazioni di DIS3 sono state riscontrate, rispettivamente, nel 18,5% dei pazienti con MM alla diagnosi, nel 25% delle pPCL e nel 30% delle sPCLs, con una maggior frequenza nei pazienti IGH-traslocati/non iperdiploidi. In FAM46C sono state identificate 24 mutazioni tumore-specifiche interessanti 18/162 (11%) pazienti. In particolare, la frequenza delle mutazioni di FAM46C era dell'11,7% nei MM di nuova diagnosi, e del 4,2% e del 20%, rispettivamente, nelle PCL primarie e secondarie. TP53 era mutato in 4/129 (3%) MM, 6/24 (25%) casi di pPCL e 2/10 (20%) di sPCL. Una frequenza simile associata all'aggressività della malattia (5%, 29,2% e 44% rispettivamente) è stata osservata per la delezione di TP53. In tutti i geni analizzati, le mutazioni coesistenti tendevano a verificarsi a diverse frequenze di varianti alleliche (VAFs), sostenendo in tal modo la presenza di subcloni tumorali. L'analisi longitudinale alla diagnosi e alla recidiva in un sottogruppo di 19 pazienti, che includeva casi di MM e PCL, ha rivelato diversi modelli di mutazione in BRAF/NRAS/KRAS. Abbiamo osservato la presenza di varianti clonali ad entrambe le tempistiche; l'acquisizione/espansione clonale delle varianti nel campione successivo; e persino la scomparsa di varianti a valori di VAF relativamente alti, ma sempre in concomitanza con l'emergenza/l’espansione clonale di un'ulteriore mutazione in un altro gene della via MAPK. Queste analisi longitudinali hanno evidenziato alcuni casi di aumento del carico di mutazione di DIS3 durante la progressione della malattia. Al contrario, in FAM46C abbiamo osservato una riduzione o scomparsa di tre mutazioni primarie con una VAF abbastanza costante nei casi all'esordio; invece, abbiamo notato l'acquisizione di mutazioni di TP53 in tre dei diciannove casi analizzati in recidiva. La maggior parte delle varianti di BRAF/DIS3/FAM46C nei casi mutati erano comparabilmente rilevabili a livello trascrizionale. Complessivamente, la scoperta che gli alleli mutati di FAM46C avevano un'espressione biologica rilevabile e, in alcuni casi, sembrava addirittura trascritta preferenzialmente, suggerisce che le mutazioni qui identificate possono avere implicazioni funzionali. Lo studio sul cfDNA come fonte accessibile di materiale genomico nei pazienti con MM è stato condotto su una serie di 28 pazienti con disordini plasmacellulari, dei quali abbiamo raccolto: (1) cfDNA isolato dal plasma; (2) DNA genomico (gDNA) tumorale da PC di BM CD138+ purificate, per scopi comparativi e (3) gDNA germinale estratto da granulociti da PB, per filtrare i polimorfismi. L'approccio “NGS CAPP-seq ultra-deep targeted” è stato eseguito per genotipizzare un pannello genico specificamente progettato per massimizzare l’identificazione delle mutazioni nei tumori plasmacellulari. Complessivamente, all'interno dei geni interrogati, 18/28 (64%) pazienti avevano almeno una mutazione somatica non sinonima rilevabile nel cfDNA. Coerentemente con lo spettro dei geni mutati nei disordini delle PC, la genotipizzazione del cfDNA nel plasma ha rivelato varianti somatiche di NRAS (25%); KRAS (14%); TP53, TRAF3 e FAM46C (11%, rispettivamente); CYLD e DIS3 (7%, rispettivamente); e BRAF e IRF4 (4%, rispettivamente). La genotipizzazione del cfDNA ha identificato correttamente il 72% delle mutazioni (n=28/39) rilevate nelle PC tumorali e, nel complesso, le VAFs nei campioni di plasma correlavano con quelle delle biopsie tumorali. Va notato che le rimanenti mutazioni non rilevate nel cfDNA avevano una bassa rappresentazione nelle PC midollari purificate (VAF mediana=2,5%). L'analisi ROC ha mostrato che la genotipizzazione di cfDNA aveva la massima sensibilità (100%) se le mutazioni erano rappresentate con una VAF> 5% nelle PC di BM purificate. I risultati del nostro studio confermano ed estendono le evidenze pubblicate in precedenza che l'attivazione del pathway delle MAPK è ricorrente nel MM, e la scoperta che essa è mediata da mutazioni in BRAF in una frazione significativa di pazienti ha implicazioni cliniche potenzialmente immediate. Inoltre, l'analisi del profilo di espressione genica in pazienti DIS3-mutati ha identificato una “signature” trascrizionale suggestiva per la riduzione della funzione dell’esosoma dell'RNA. I nostri dati supportano ulteriormente la rilevanza patologica delle mutazioni di DIS3 nelle discrasie plasmacellulari e suggeriscono che DIS3 possa rappresentare un potenziale gene soppressore del tumore in tali disordini. E 'anche rafforzata la crescente evidenza che FAM46C sia coinvolto nella patogenesi del MM come potenziale gene oncosoppressore, anche se il suo ruolo rimane da chiarire. I nostri risultati confermano anche che le mutazioni di TP53 sono rare nel MM all’esordio e rappresentano piuttosto un marker di progressione, analogamente alla del(17p); tuttavia, il loro verificarsi anche in assenza di delezioni supporta l'importanza della loro valutazione in pazienti con discrasia plasmacellulare, in termini sia di stratificazione del rischio sia di implicazioni terapeutiche. I nostri risultati dimostrano anche che la genotipizzazione di cfDNA è un approccio fattibile, non invasivo, in tempo reale in grado di rilevare mutazioni somatiche clonali e subclonali rappresentate in almeno il 5% degli alleli nelle PC tumorali, supportando così la consigliabile introduzione di questo metodo negli studi clinici che monitorano le discrasie plasmacellulari.
Multiple myeloma (MM) is a fatal malignant proliferation of antibody-secreting bone marrow (BM) plasma cells (PCs) characterized by a wide clinical spectrum and a profound genomic instability. Concerning in particular the mutational landscape, recent next generation sequencing (NGS) studies in MM patients indicated the lack of a universal driver mutation but several recurrently mutated genes belonging to key pathways involved in the disease, such as the MAP-kinase pathway. MM characterization currently depends on BM aspirates for mutational analysis. However, this approach might not capture the putative spatial and genetic heterogeneity of the disease, and imposes technical hurdles that are limiting its transfer in the routine and clinical grade diagnostic laboratory, restricting also the possibility of longitudinal monitoring of disease molecular markers. Nevertheless, recent data produced in solid cancers indicate that circulating tumor DNA into the peripheral blood (PB) can be used as source of tumor DNA to provide information about tumor mass, residual disease and tumor genotype, with obvious advantages in terms of accessibility. Based on the previous observations, the aims of the project were: (i) the evaluation of the incidence of mutations in MM driver genes (KRAS, NRAS, TP53, BRAF, FAM46C and DIS3) in a large and representative cohort of patients at different stages of PC dyscrasia (132 MM and 24 primary PC leukemia (pPCL) cases, both at onset, and 11 secondary PCL (sPCL) patients); (ii) the comparison of the mutational profiling of circulating cell free DNA (cfDNA) and BM derived DNA in a small series of patients representative of different clinical stages of PC tumors. Specifically, we evaluated, by means of deep NGS, the incidence of mutations in BRAF (exons 11 and 15), NRAS (exons 2 and 3) and KRAS (exons 2-4), DIS3 in the PIN (exons 1-4) and RNB (exons 10-18) functional domains, TP53 (exons 4-9) and FAM46C (exon 2). Overall, the MAPK pathway resulted affected in 60.1% of the patients (63.6% of those with sPCL, 59.8% of those with MM, and 41.7% of those with pPCL). In particular, 12% of patients were found mutated in BRAF, 23.9% in NRAS, and 29.3% in KRAS. DIS3 mutations were found, respectively, in 18.5% of MM patients at diagnosis, 25% of pPCLs and 30% of sPCLs, occurring more frequently in IGH-translocated/nonhyperdiploid patients. Twenty-four tumour-specific mutations were identified in FAM46C affecting 18/162 (11%) patients. In particular, the frequency of FAM46C mutations was 11.7% in newly diagnosed MM, 4.2% and 20% in primary and secondary PCL, respectively. TP53 was mutated in 4/129 (3%) MM, 6/24 (25%) pPCL, and 2/10 (20%) sPCL cases. A similar increase in prevalence associated with disease aggressiveness (5%, 29.2% and 44%, respectively) was observed for TP53 deletion. In all analyzed genes, co-existing mutations tended to occur at different variant allele frequencies (VAFs), thus supporting the occurrence of tumor subclones. Longitudinal analysis at diagnosis and relapse in a subset of 19 cases including both MM and PCL cases, revealed different mutation patterns in BRAF/NRAS/KRAS. We observed the presence of clonal variants at both time-points; the acquisition/clonal expansion of variants in the later sample; and even the disappearance of variants at relatively high VAF values, but always concurrently with the emergence/clonal expansion of an additional mutation in another gene of the MAPK pathway. These longitudinally analysis highlighted some instances of increasing DIS3 mutation burden during disease progression. On the contrary, in FAM46C we observed a reduction or disappearance of three primary mutations with a quite constant VAF in cases at onset; instead, we noticed the acquisition of TP53 mutations in three of the nineteen cases analyzed at relapse. The majority of BRAF/DIS3/FAM46C variants in mutated cases were comparably detectable at transcriptional level. Overall, the finding that FAM46C mutated alleles had detectable biological expression, and in some cases seemed to be even preferentially transcribed, suggests that the mutations identified herein may have functional implications. The study on cfDNA as an accessible source of genomic material in MM patients was based on a series of 28 patients with PC disorders, of whom we collecteded: (1) cfDNA isolated from plasma; (2) tumor genomic (g)DNA from CD138+ purified BM PCs for comparative purposes, and (3) germline gDNA extracted from PB granulocytes, to filter out polymorphisms. CAPP-seq ultra-deep targeted NGS approach was performed to genotype a gene panel specifically designed to maximize the mutation recovery in PC tumors. Overall, within the interrogated genes, 18/28 (64%) patients had at least one non-synonymous somatic mutation detectable in cfDNA. Consistent with the spectrum of mutated genes in PC disorders, plasma cfDNA genotyping revealed somatic variants of NRAS (25%); KRAS (14%); TP53, TRAF3 and FAM46C (11%, respectively); CYLD and DIS3 (7%, respectively); and BRAF and IRF4 (4%, respectively). cfDNA genotyping correctly identified 72% of mutations (n=28/39) discovered in tumor PCs and, overall, the VAFs in plasma samples correlated with those in tumor biopsies. Notably, the remaining mutations not discovered in cfDNA had a low representation in the purified BM PCs (median VAF=2.5%). ROC analysis showed that cfDNA genotyping had the highest sensitivity (100%) if mutations were represented with a VAF >5% in purified BM PCs. The results of our study confirm and extend previous published evidence that MAPK pathway activation is recurrent in MM, and the finding that it is mediated by BRAF mutations in a significant fraction of patients has potentially immediate clinical implications. Furthermore, gene expression profiling analysis in DIS3-mutated patients identified a transcriptional signature suggestive for impaired RNA exosome function. Our data further support the pathological relevance of DIS3 mutations in PC dyscrasia and suggest that DIS3 may represent a potential tumor suppressor gene in such disorders. It is also strengthened the growing evidence that FAM46C is involved in the pathogenesis of MM as a potential tumour suppressor, although its role remains to be clarified. Our outcomes also confirm that TP53 mutations are rare in MM at presentation and rather represent a marker of progression, similarly to del(17p); however, their occurrence even in absence of deletions supports the importance of their assessment in patients with PC dyscrasia, in terms of both risk stratification and therapeutic implications. Our results demonstrate as well that cfDNA genotyping is a feasible, non-invasive, real-time approach able to detect clonal and subclonal somatic mutations represented in at least 5% of alleles in tumor PCs, thus supporting the advisable introduction of this method in clinical trials monitoring PC dyscrasia.

Neuronal ceroid lipofuscinoses (NCL/Batten disease) are a group of fatal inherited neurodegenerative diseases that occur in many species including humans, sheep, dogs and cattle. Typical NCL symptoms include progressive loss of vision, regression of mental and motor development, epileptic seizures and premature death. Currently there is no effective treatment or cure for NCL, with the underlying disease mechanisms still poorly understood. Advances in molecular genetics in recent years have allowed the characterisation of hundreds of causative mutations and polymorphisms in at least 17 disease-causing genes across all species. For some species, research colonies have been established for studies relevant to the corresponding human NCL variants. Best characterised of all animal models for NCL is the New Zealand South Hampshire (SH) sheep which is a model for the human variant late-infantile form of NCL (vLINCL). Past studies have revealed the ovine CLN6 gene (CLN6) as a strong candidate gene for this disease in South Hampshire sheep however no disease-causing mutation was identified. The main objective of the present thesis is the identification and characterisation of the mutation responsible for NCL in the South Hampshire sheep. It was proposed that the mutation lies in the non-coding regions within or flanking the gene and that this mutation affects gene regulation. Bioinformatic tools were initially used to identify conserved non-coding sequences (CNCS) which are deemed potential regions of interest for regulatory mutations. Due to the limited ovine genome resource available when the study was commenced in 2006, CLN6 orthologous sequences from other species were initially used for identification of highly conserved regions. Of the five identified CNCS (5’ UTR, 3’UTR and introns 1, 2 and 6) the region upstream of CLN6 and intron 1 were considered priorities for sequencing. Given that the Sanger sequencing method was laborious and time-consuming, and that there was rapid development of technology; the Sanger sequencing approach was abandoned and Next-generation sequencing (NGS) methods utilised for the following studies. The 454 Pyrosequencing NGS technology was used to sequence the complete ovine Bacterial artificial chromosome (BAC) to generate an ovine reference sequence for mutation screening approaches. The first mutation screening approach, sequence capture and targeted sequencing approach failed; however, the second approach involving sequencing of long-range PCR (LR-PCR) products successfully identified the disease-causing mutation. LR-PCR amplification of 14 regions within the ovine genome region spanning the CLN6 and flanking sequences followed by SOLID sequencing-by-ligation NGS method identified the disease-associated mutation as a 402bp deletion and 1bp insertion in ovine CLN6, namely g.-251_+150del and g.+150_151insC. The mutation is predicted to lead to the deletion of the whole of exon 1 and the ATG start codon as well as flanking non-coding sequence. Identifying the disease-causing mutation for NCL in SH sheep provides the long-awaited confirmatory evidence that ovine CLN6 is the causative gene for NCL in SH sheep. Future research in this large animal model will allow for more effective strategies for developing therapeutic approaches for NCL in humans and further strengthens the invaluable role of this animal model for NCL studies.

Mutations affecting the sodium-chloride cotransporter (NCC) in the distal convoluted tubule of the nephron are causative of Gitelman'€™s syndrome (GS), a rare autosomal recessive disease characterized by electrolytic alterations similar to those induced by high dose thiazide treatment. Notably, the co-existence of hypomagnesemia and hypocalciuria is a feature of GS which is a distinguish hallmark from another hypokalemic renal tubulopathy, the Bartter'€™s syndrome (BS). Commonly GS is heterozygous compound with an estimated prevalence of 1:40000 and can be silent for years before the revealing in the early adulthood. Recognizing the genetic background is fundamental for the screening and the diagnosis of the disease, as recently studies showed that mutation affecting regulators of renal salt handling are underestimated in the general population. In a registry of BS/GS based at our University we discovered a novel point mutation (c.1204G>A which codify for an amino acid exchange G394D) in the sodium-chloride cotransporter NCC (SLC12A3) in a young woman with hypokalemia, hypomagnesemia and hypocalciuria associated to muscle pain and cramps. The present study aimed to investigate how this mutation affects NCC function by using a molecular biology approach and providing functional evidences. After a prior screening with bioinformatics tools predicting the possible pathogenicity of the mutation, were created different expression vectors with either the wild-type (wt-NCC) or the mutated G394D-NCC sequences. DNA and in-vitro transcribed RNA were afterwards transfected in a human embryonic kidney cells line (HEK293) and injected into oocytes deriving from Xenopus Laevis frog respectively. In transfected HEK 293 cells, wildtype NCC was detected by immunoblotting as two bands at approximately 130 kDa and 115 kDa corresponding to fully and core-glycosylated NCC, respectively. In contrast, G394D-NCC was seen as a single band at about 115 kDa only, suggesting an impaired maturation of the mutated protein. Similar findings were made in the oocyte expression system. Confocal microscopy on the oocytes, did also show a strong cell surface localization of wildtype NCC while mutated NCC was retained at intracellular compartments. Consistently, a decent thiazide-sensitive 22Na+ uptake into injected oocytes was only found for wildtype but not mutated NCC. Taken together all the findings in this study, a novel GS point mutation has been characterized to diminish NCC function by impairing trafficking of the protein to the cell surface. The absence of any mature glycosylation form of G394D-NCC suggests that the mutation impairs protein folding leading to a retention of NCC in the endoplasmic reticulum.
Le mutazioni che colpiscono il cotrasportatore per il sodio e cloro (NCC) nel tubulo contorto distale del nefrone, sono responsabili della sindrome di Gitelman (GS). Quest'€™ultima è una rara tubulopatia renale autosomica recessiva caratterizzata da alterazioni elettrolitiche simili a quelle indotte dal trattamento ad alte dosi con diuretici tiazidici. La co-presenza di ipomagnesemia e ipocalciuria è una delle caratteristiche di GS che la distinguono da un'altra tubulopatia renale ipokalemica, la sindrome di Bartter (BS). Generalmente, i soggetti affetti sono eterozigoti composti con una prevalenza stimata di 1 su 40000. La malattia può essere silente per anni prima di presentarsi nell'€™età  adulta. Riconoscerne la componente genetica è fondamentale per lo screening e la diagnosi. Recenti studi hanno di fatto dimostrato come le mutazioni a carico dei regolatori renali dell'€™omeostasi del sodio siano sottostimate nella popolazione generale. Nel nostro database universitario di pazienti BS/GS abbiamo riscontrato una nuova mutazione puntiforme (c.1204G>A che comporta lo scambio aminoacidico Gly394Asp) nel cotrasportatore del sodio e cloro NCC (SLC12A3) in una giovane donna con ipokaliemia, ipomagnesemia e ipocalciuria associati a dolori e crampi muscolari. Il presente studio ha lo scopo di investigare tramite un approccio biologico molecolare come questa mutazione influenzi la funzionalità  di NCC. Previo screening con softwares bioinformatici che predicono la possibile patogenicità  della mutazione, sono stati creati dei vettori di espressione contenenti le sequenze per NCC wild type e per NCC con mutazione G394D. Successivamente, le sequenze sono state trasfettate in una linea di cellule fetali umane ricombinate (HEK293) e in ovociti derivati da rane Xenopus Laevis. Nelle cellule trasfettate, l'€™immunoblotting di NCC wild-type ha dimostrato la presenza di due bande approssimativamente a 130 KDa e 115 KDa che corrispondono rispettivamente alla forma glicosilata e nativa della proteina. Al contrario, G394D-NCC presenta una sola banda a 115 KDa. Risultati simili sono stati ottenuti negli ovociti. In questi ultimi l'€™immunoistochimica ha inoltre mostrato una forte localizzazione di NCC wild-type presso la membrana, mentre NCC mutato rimane in compartimenti cellulari interni. Gli studi funzionali di uptake del sodio radioattivo (22Na+) hanno ulteriormente confermato che solo la proteina wild-type è in grado di riassorbire il sodio al contrario di G394D-NCC. I risultati di questo studio dimostrano come la nuova mutazione puntiforme inibisca la funzione di NCC a causa della diminuita capacità della proteina di raggiungere la superficie cellulare. L'assenza di una forma glicosilata matura di G394D-NCC suggerisce che la mutazione ne condizioni il folding e ne provochi la ritenzione nel reticolo endoplasmico ove vengo attivati processi di degradazione anticipata.

Familial Hypertrophic cardiomyopathy (HCM) causes ventricle walls to thicken and often leads to sudden death especially in adults. Mutations in the subfragment 2 (S2) of β-cardiac myosin are implicated in the genetic disorder. This S2 region is a coiled-coil rod region resulting from the dimeric form of myosin II. It has been proposed that an elastic quality allows normal S2 to absorb force during the powerstroke according to the sliding filament model. To test the flexibility of single molecules of S2 against levels of physiological force, the Gravitational Force Spectrometer (GFS) is being developed. This novel system employs a standard microscope on an equatorial mount that allows the spectrometer to be rotated freely in space. Stationary glass beads are attached to a microscope slide where the molecule is tethered between the stationary bead and a smaller mobile bead. The GFS is oriented so that the force of gravity can act on the mobile bead and so impart a small force to the tethered subfragment. Additionally, a video system in conjunction with ImageJ software makes a distance measurement of the molecule possible with a resolution of around 11 nm. The S2 can be stretched parallel or perpendicular to the coiled coil to elucidate different structural properties of the rod. This study is the first to show structural evidence that S2 in vertebrate skeletal myosin uncoils proportionally to physiological force loads. Because of this, the usefulness and promise of the novel GFS is highlighted, and the biological role of S2's flexibility can be directly commented on. If the dimer undergoes uncoiling at physiological force loads as shown, then it is reasonable to think that this might occur in nature in response to the stress of the powerstroke on a single molecule. This unwinding could be to absorb force as a mechanism to protect the muscle fiber.

Background: Primary aldosteronism (PA) is the most common endocrine form of secondary arterial hypertension with an estimated prevalence of ~ 11.2% in patients referred to specialized centers and 4% in primary care, but as high as 50% in patients with resistant hypertension. The two main forms of PA are: aldosterone-producing adenoma (APA), surgically curable with the removal of adenoma that is able to overproduce aldosterone, and bilateral adrenocortical hyperplasia (BAH), which needs drug therapy. About ~ 30% APAs have somatic mutations in KCNJ5 gene that encodes for the potassium channel (KIR3.4), which plays a crucial role in the maintenance of the cell membrane by pumping potassium (K+) out of the cells, thereby producing a negative membrane potential. The KCNJ5 channels that contain G151R, T158A, or L168R mutations cause membrane permeability to Na+, resulting in Na+ entry, cell depolarization, constitutive aldosterone production, and cell proliferation. The standard approach used to detect the mutations in KCNJ5 gene is sequencing of DNA extracted from adrenal tissue of hypertensive patients with lateralized excess in aldosterone production. However, DNA sequencing is a time consuming and rather expensive technique not feasible in all standard laboratories. Aims of our study were 1) to develop a strategy for genotyping DNA extracted from the adrenal tissue, which exploited a TAQ-MAN based PCR technology; 2) to evaluate if such Taq-Man-base technology allows detection of KCNJ5 mutations in the adrenal tissue and cf-DNA isolated from peripheral blood. Methodologic Approach: 1. Development of a novel strategy based on Taq-man probe to detect KCNJ5 mutations 2. Development of a protocol to isolate cf-DNA from blood collected in the inferior vena cava and adrenal veins. 3. Measurement of cf-DNA concentration and assessment of its fragmentation. 4. Identification of KCNJ5 mutation in cf-DNA Results: By applying the novel technology based on Taq-man probe we correctly identified 30 mutated patients in a cohort of 50 consecutive APA patients, with no misclassification. After isolating cf-DNA from the adrenal veins and inferior cava blood, and measuring its concentration, we evaluated cf-DNA fragmentation with the integrity index in 24 samples. Integrity index < 1 suggested that cf-DNA was released from the adrenal tissue through an apoptotic mechanism. HRM analysis of cf-DNA isolated from adrenal blood allowed us to identify the KCNJ5 mutation in the APA side. Conclusions and perspectives: The novel technology based on Taq-man probe allowed detection of all mutated patients in the examined cohort of APA patients, thus proving that this strategy could be used as an alternative to DNA sequencing. The results of our study also showed the feasibility to isolate cf-DNA from small amount of blood collected from the adrenal veins, and suggested that KCNJ5 mutations may be detected in cf-DNA. However, this approach needs to be verified for a larger population to determine feasibility and accuracy. If confirmed, analysis of cf-DNA isolated from the peripheral venous blood could be helpful for an early detection of KCNJ5 mutations and therefore for the selection of PA patients to be submitted to adrenal vein sampling. Furthermore, this strategy could be also useful for detecting KCNJ5 germline mutations responsible for the rare hereditary form of hyperaldosteronism FH-3.
Backgouund. L’Iperaldosteronismo primario (PA) è la forma endocrina più comune d’ipertensione arteriosa secondaria con una prevalenza stimata di circa il 4% nella popolazione generale e dell’11% nei pazienti che afferiscono ai centri di riferimento per l’ipertensione. Nei pazienti con ipertensione resistente la prevalenza del PA è stimata pari al 50%, mostrando che tale patologia non è così rara come ritenuto in passato. Le due forme principali di PA sono l’adenoma producente aldosterone (APA), caratterizzato da iperproduzione lateralizzata di aldosterone, e l’iperplasia surrenalica bilaterale (BAH). La distinzione tra le due forme è di cruciale importanza poiché la prima richiede terapia chirurgica, mentre la seconda terapia medica. Considerando che la rimozione dell’APA determina la correzione del quadro biochimico-clinico di PA e la cura o il miglioramento dell’ipertensione, il riconoscimento dell’APA è fondamentale per offrire una chance di guarigione dell’ipertensione o di miglioramento del controllo dei valori pressori ai pazienti che ne sono affetti. Il 40% degli APA presenta mutazioni somatiche nel gene KCNJ5 che codifica per il canale del potassio KIR 3.4. Questo canale gioca un ruolo fondamentale nel mantenimento del potenziale di membrana pompando il K+ al di fuori della cellula, provocando in tal modo un potenziale di membrana negativo. Allorquando il gene KCNJ5 contiene le mutazioni G151R, T158A o L168R il canale KIR 3.4 acquisisce capacità di condurre Na+ all’interno della cellula. Gli effetti della mutazione a livello della cellula sono depolarizzazione cronica, produzione costitutiva di aldosterone e proliferazione cellulare. L’approccio attualmente in uso per identificare tali mutazioni è il sequenziamento, secondo Sanger, del DNA estratto dal tessuto surrenalico rimosso durante surrenectomia nei pazienti con PA e iperproduzione lateralizzata di aldosterone. Il sequenziamento del DNA, tuttavia, è costosa richiede tempo e non è disponibile di routine in tutti i laboratori. Scopo generale dello studio è stato quello di sviluppare una strategia alternativa al sequenziamento che preveda l’uso delle sonde Taq-man in Real Time PCR (Q-PCR) per il rilevamento di mutazioni KCNJ5 nel tessuto surrenalico e nel DNA circolante (cell-free DNA, cf-DNA) isolato da sangue periferico. Lo sviluppo di questa metodologia potrebbe semplificare notevolmente l’identificazione delle mutazioni KCNJ5 negli APA e, infine, permetterne la detenzione nel DNA del sangue circolante. In sintesi, l’approccio metodologico include: sviluppo di una nuova strategia basata sull’utilizzo delle sonde Taq-man per rilevare le mutazioni nel gene KCNJ5. Sviluppo di un protocollo per isolare il cf-DNA da sangue delle vene surrenaliche e dalla vena cava inferiore. Misurazione della concentrazione del cf-DNA valutandone la sua frammentazione. Identificazione di mutazioni nel gene KCNJ5 a partire dal cf-DNA Risultati. Applicando la tecnologia sviluppata nel nostro laboratorio, basata sulle sonde Taq-man, sono stati identificati correttamente 30 pazienti mutati in una coorte di 50 pazienti APA consecutivi, senza errori di classificazione. Dopo aver isolato il cf-DNA dal sangue delle vene surrenaliche e dalla vena cava inferiore, e misurato la sua concentrazione, abbiamo valutato la frammentazione del cf-DNA in 24 campioni con l'indice di integrità. I bassi valori dell’indice d’integrità riscontrati nei cf-DNA isolati da sangue venoso surrenalico suggeriscono che la ghiandola surrenalica rilasci per apoptosi frammenti di DNA. L’analisi HRM dei cf-DNA isolati dal sangue delle vene surrenaliche di un paziente con un APA sinistro contenente la mutazione L168R ha permesso d’identificare correttamente la mutazione nel cf-DNA isolato dalla vena surrenalica sinistra. Conclusioni e prospettive. La tecnologia basata sulle sonde Taq-man ha permesso d’identificare, senza errori di misclassificazione, in una coorte di 50 pazienti con APA, tutti i 30 pazienti che presentano una mutazione del gene KCNJ5. Tale strategia, pertanto, potrebbe rappresentare un’alternativa alla ben più lunga e complessa tecnica basata sul sequenziamento del DNA. I risultati del nostro studio hanno anche mostrato che è possibile isolare il cf-DNA da esigue quantità di sangue raccolto dalle vene surrenaliche permettendo l’identificazione delle mutazioni KCNJ5 usando il cf-DNA tramite approccio combinato sonde Taq-man e analisi HRM. Quest’ultimo approccio, che prevede l’uso del cf-DNA richiede, tuttavia, conferma in un ampio numero di soggetti. La stessa strategia potrebbe anche essere impiegata in futuro per la rilevazione di mutazioni germinali KCNJ5 responsabili della nota forma ereditaria d’iperaldosteronismo FH-3.

L’œuvre romanesque d’Amin Maalouf propose un traitement particulier du personnage qui dépasse le cadre habituel de son analyse. Objet de toutes les attentions mais aussi de tous les débordements, le personnage reflète l’ensemble des problématiques qui opèrent à l’intérieur de la fiction. On trouve ainsi dans les romans d’Amin Maalouf une contamination, voire même une dissémination, du personnage vers d’autres instances. Ces instances le prolongent en peuplant, à leur tour, le roman et en se proposant d’y laisser une empreinte poétique.Les métamorphoses et les mutations successives, dont le roman fait l’objet, viennent relayer sa présence, lui octroyant une dimension nouvelle. En multipliant ses appartenances et ses origines, la fiction propose un voyage à travers les multiples facettes du personnage qui lui permettent de renouer avec sa nature originelle. Tourné vers le mouvement, aspirant à se redéfinir au travers des autres instances textuelles, le personnage s’impose comme un véritable agent romanesque. La thèse propose ainsi de révéler précisément les tenants de ces métamorphoses et de ces mutations romanesques, afin de mettre en lumière les infinis prolongements qui concourent à l’élaboration d’une poétique singulière
Amin Maalouf’s novels offer a special treatment of the character beyond the usual scope of its analysis. The object of all attention and excesses, the character reflects all issues operating within the fiction. Thus we find in Amin Maalouf’s fiction a sort of contamination, even a release of the character to other authorities. Throughout the novel these authorities extend adopt traits of the main character as personifications. They extend its presence their presence, occupy the fiction in their turn and try to leave a poetic mark.Metamorphoses and successive mutations, of which the novel is the subject, just relay its presence, giving it a new dimension. Increasing its appurtenances and its origins, fiction offers a journey through the many facets of character that allow it to return to its original nature. Facing movement, aspiring to redefine itself through other textual authorities, the character stands out as a true romantic agent.The thesis proposes to reveal precisely the proponents of these metamorphosis and mutations of fictions to highlight the infinite extensions that contribute to the development of a singular poetic

Variations in the expression of drug metabolizing enzymes of the Cytochrome P450 superfamily are a principal cause of atypical reactions to therapeutics. The molecular mechanisms by which the metabolizing enzymes of the drug are regulated, and the effects of genetic variation on this regulation, are not completely understood. Cytochrome P450 1A2 (CYP1A2) is one such enzyme. The APN mouse strain has low expression of the CYP1A2 enzyme, relative to the C3H/HeJ strain. It was hypothesized that this difference in expression of the CYP1A2 was occurring as a result of a single nucleotide polymorphism at the Cyp1a2 locus. This work has demonstrated that this variation in CYP1A2 expression occurs at the level of transcription and that a single nucleotide change 76 base pairs upstream of the transcriptional start site was critical for promoter function. The mechanism of constitutive CYP1A2 expression involves a previously unidentified cis-acting element in this region.