Variable expressivity of osteogenesis imperfecta in a Brazilian family due to p.G1079S mutation in the COL1A1 gene

Osteogenesis imperfecta (OI) is a Mendelian disease with genetic heterogeneity characterized by bone fragility, recurrent fractures, blue sclerae, and short stature, caused mostly by mutations in COL1A1 or COL1A2 genes, which encode the pro-alpha 1(I) and pro-alpha 2(I) chains of type I collagen, respectively. A Brazilian family that showed variable expression of autosomal dominant OI was identified and characterized. Scanning for mutations was carried out using SSCP and DNA sequence analysis. The missense mutation c.3235G>A was identified within exon 45 of the COL1A1 gene in a 16-year-old girl diagnosed as having OI type I; it resulted in substitution of a glycine residue (G) by a serine (S) at codon 1079 (p.G1079S). The proband's mother had the disease signs, but without bone fractures, as did five of nine uncles and aunts of the patient. All of them carried the mutation, which was excluded in four healthy brothers of the patient's mother. This is the first description in a Brazilian family with OI showing variable expression; only one among seven carriers for the c.3235G>A mutation developed bone fractures, the most striking clinical feature of this disease. This finding has a significant implication for prenatal diagnosis in OI disease.

Analysis of MLL2 gene in the first Brazilian family with Kabuki syndrome

Most patients with Kabuki syndrome (KS) are the only person in their family with the condition. However, familial cases of KS have been described showing evidence that this syndrome can be inherited as a dominant trait with variable expressivity. We report on two related individuals with facial findings characteristic of KS. The proposita had arched eyebrows, long and upward slanting palpebral fissures, cleft lip and palate, retromicrognathia, brachydactyly of hands and feet, stubby fingers, nail hypoplasia, and prominent finger pads. Her mother had eyebrows with dispersed lateral half, long and upward slanting palpebral fissures, retrognathia, abnormal and posteriorly rotated ears, prominent finger pads, brachydactyly of feet, learning difficulties, and psychomotor development delay. DNA sequencing revealed a novel missense mutation in the MLL2 gene in both the proposita and her mother. The mutation (p.R5432Q) was found in the exon 51, within the SET domain of the gene, which confers methyltransferase activity on the protein. Therefore, the epigenetic and transcriptional regulatory properties of this protein may be altered and this suggests that the mutation is the cause of phenotype observed in both the patient and her mother. The clinical signs and the molecular evidence in this family further support the notion that KS is an autosomal dominant condition with variable expressivity. To our knowledge this is the first report of a Brazilian family with recurrence of this syndrome. (C) 2012 Wiley Periodicals, Inc.

Flavonoid interactions with human transthyretin: Combined structural and thermodynamic analysis

Transthyretin (TTR) is a carrier protein involved in human amyloidosis. The development of small molecules that may act as TTR amyloid inhibitors is a promising strategy to treat these pathologies. Here we selected and characterized the interaction of flavonoids with the wild type and the V30M amyloidogenic mutant TTR. TTR acid aggregation was evaluated in vitro in the presence of the different flavonoids. The best TTR aggregation inhibitors were studied by Isothermal Titration Calorimetry (ITC) in order to reveal their thermodynamic signature of binding to TTRwt. Crystal structures of TTRwt in complex with the top binders were also obtained, enabling us to in depth inspect TTR interactions with these flavonoids. The results indicate that changing the number and position of hydroxyl groups attached to the flavonoid core strongly influence flavonoid recognition by TTR, either by changing ligand affinity or its mechanism of interaction with the two sites of TTR. We also compared the results obtained for ITRwt with the V30M mutant structure in the apo form, allowing us to pinpoint structural features that may facilitate or hamper ligand binding to the V30M mutant. Our data show that the TTRwt binding site is labile and, in particular, the central region of the cavity is sensible for the small differences in the ligands tested and can be influenced by the Met30 amyloidogenic mutation, therefore playing important roles in flavonoid binding affinity, mechanism and mutant protein ligand binding specificities. (C) 2012 Elsevier Inc. All rights reserved.

Analysis of energetically biased transcripts of viruses and transposable elements

RNA interference (RNAi) is a natural endogenous process by which double-stranded RNA molecules trigger potent and specific gene silencing in eukaryotic cells and is characterized by target RNA cleavage. In mammals, small interfering RNAs (siRNAs) are the trigger molecules of choice and constitute a new class of RNA-based antiviral agents. In an efficient RNAi response, the antisense strand of siRNAs must enter the RNA-induced silencing complex (RISC) in a process mediated by thermodynamic features. In this report, we hypothesize that silent mutations capable of inverting thermodynamic properties can promote resistance to siRNAs. Extensive computational analyses were used to assess whether continuous selective pressure that promotes such mutations could lead to the emergence of viral strains completely resistant to RNAi (i.e., prone to transfer only the sense strands to RISC). Based on our findings, we propose that, although synonymous mutations may produce functional resistance, this strategy cannot be systematically adopted by viruses since the longest RNAi-refractory sequence is only 10 nt long. This finding also suggests that all mRNAs display fluctuating thermodynamic landscapes and that, in terms of thermodynamic features, RNAi is a very efficient antiviral system since there will always be sites susceptible to siRNAs.

GH-Releasing Hormone Receptor Gene: A Novel Splice-Disrupting Mutation and Study of Founder Effects

Background: Mutations in GH-releasing hormone receptor gene (GHRHR) are emerging as the most common cause of autosomal recessive isolated GH deficiency (IGHD). Objective: To search for GHRHR mutations in patients with familial or sporadic IGHD and to investigate founder effects in recurring mutations. Methods: The coding region of GHRHR was entirely amplified and sequenced from DNA of 18 patients with IGHD (16 unrelated) with topic posterior pituitary lobe on MRI. Haplotypes containing promoter SNPs and microsatellites flanking GHRHR were analyzed in patients with c.57+1G>A (IVS1+1G>A) mutation of our previously published kindred and also a Brazilian patient and 2 previously reported Japanese sisters with c. 1146G>A (p.E382E) mutation. Results: A novel homozygous intronic GHRHR c.752-1G>A (IVS7-1G>A) mutation, predicting loss of the constitutive splice acceptor site, was identified in two siblings with IGHD. A compound heterozygous c.[57+1G>A];[1146G>A] and a heterozygous c.527C>T (p.A176V) were found in two sporadic cases. Haplotype analysis provided evidence for a founder effect for the c.57+1G>A mutation and independent recurrence for the c.1146G>A mutation. Conclusion: We report a novel splice-disrupting mutation in GHRHR in 2 siblings and provide evidence that all c.57+1G>A (IVS1+1G>A) mutant chromosomes have the same haplotype ancestor, indicating the occurrence of a founder effect in Brazilian patients with IGHD. Copyright (C) 2012 S. Karger AG, Basel

Conformation analysis of a surface loop that controls active site access in the GH11 xylanase A from Bacillus subtilis

Xylanases (EC 3.2.1.8 endo-1,4-glycosyl hydrolase) catalyze the hydrolysis of xylan, an abundant hemicellulose of plant cell walls. Access to the catalytic site of GH11 xylanases is regulated by movement of a short beta-hairpin, the so-called thumb region, which can adopt open or closed conformations. A crystallographic study has shown that the D11F/R122D mutant of the GH11 xylanase A from Bacillus subtilis (BsXA) displays a stable "open" conformation, and here we report a molecular dynamics simulation study comparing this mutant with the native enzyme over a range of temperatures. The mutant open conformation was stable at 300 and 328 K, however it showed a transition to the closed state at 338 K. Analysis of dihedral angles identified thumb region residues Y113 and T123 as key hinge points which determine the open-closed transition at 338 K. Although the D11F/R122D mutations result in a reduction in local inter-intramolecular hydrogen bonding, the global energies of the open and closed conformations in the native enzyme are equivalent, suggesting that the two conformations are equally accessible. These results indicate that the thumb region shows a broader degree of energetically permissible conformations which regulate the access to the active site region. The R122D mutation contributes to the stability of the open conformation, but is not essential for thumb dynamics, i.e., the wild type enzyme can also adapt to the open conformation.

Somatotroph pituitary adenoma with acromegaly and autosomal dominant polycystic kidney disease: SSTR5 polymorphism and PKD1 mutation

A 39-year-old woman with autosomal dominant polycystic kidney disease (ADPKD) presented with acromegaly and a pituitary macroadenoma. There was a family history of this renal disorder. She had undergone surgery for pituitary adenoma 6 years prior. Physical examination disclosed bitemporal hemianopsia and elevation of both basal growth hormone (GH) 106 ng/mL (normal 0-5) and insulin-like growth factor (IGF-1) 811 ng/mL (normal 48-255) blood levels. A magnetic resonance imaging scan disclosed a 3.0 cm sellar and suprasellar mass with both optic chiasm compression and left cavernous sinus invasion. Pathologic, cytogenetic, molecular and in silico analysis was undertaken. Histologic, immunohistochemical and ultrastructural studies of the lesion disclosed a sparsely granulated somatotroph adenoma. Standard chromosome analysis on the blood sample showed no abnormality. Sequence analysis of the coding regions of PKD1 and PKD2 employing DNA from both peripheral leukocytes and the tumor revealed the most common PKD1 mutation, 5014_5015delAG. Analysis of the entire SSTR5 gene disclosed the variant c.142C > A (p.L48M, rs4988483) in the heterozygous state in both blood and tumor, while no pathogenic mutations were noted in the MEN1, AIP, p27Kip1 and SSTR2 genes. To our knowledge, this is the fourth reported case of a GH-producing pituitary adenoma associated with ADPKD, but the first subjected to extensive morphological, ultrastructural, cytogenetic and molecular studies. The physical proximity of the PKD1 and SSTR5 genes on chromosome 16 suggests a causal relationship between ADPKD and somatotroph adenoma.

A novel DAX1/NR0B1 mutation in a patient with adrenal hypoplasia congenita and hypogonadotropic hypogonadism

We report a case of adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH) due to a novel DAX1 mutation. A 19-month-old boy with hyperpigmentation and failure to thrive came to our service for investigation. Three brothers of the patient had died due to adrenal failure, and a maternal cousin had adrenal insufficiency. Adrenoleukodystrophy was excluded. MRI showed normal pituitary and hypothalamus. Plasma hormone evaluation revealed high ACTH (up to 2,790 pg/mL), and low levels of androstenedione, DHEA-S, 11-deoxycortisol, and cortisol. At 14 years of age the patient was still prepubescent, his weight was 43.6 kg (SDS: -0.87) and his height was 161 cm (SDS: -0.36), with normal body proportions. In the GnRH test, basal and maximum values of LH and FSH were respectively 0.6/2.1 and < 1.0/< 1.0 U/L. Molecular investigation identified a novel mutation that consists of a deletion of codon 372 (AAC; asparagine) in exon 1 of DAX1. This mutation was not found in a study of 200 alleles from normal individuals. Prediction site analysis indicated that this alteration, located in the DAX1 ligand-binding domain, may damage DAX1 protein. We hypothesize that the novel (p.Asp372del) DAX1 mutation might be able to cause a disruption of DAX1 function, and is probably involved in the development of AHC and HH in this patient. Arq Bras Endocrinol Metab. 2012;56(8):496-500

Different approaches to identify markers for meat quality and production traits in pigs: analysis of the transcriptome in post mortem skeletal muscle and investigation of candidate genes

Due to the growing attention of consumers towards their food, improvement of quality of animal products has become one of the main focus of research. To this aim, the application of modern molecular genetics approaches has been proved extremely useful and effective. This innovative drive includes all livestock species productions, including pork. The Italian pig breeding industry is unique because needs heavy pigs slaughtered at about 160 kg for the production of high quality processed products. For this reason, it requires precise meat quality and carcass characteristics. Two aspects have been considered in this thesis: the application of the transcriptome analysis in post mortem pig muscles as a possible method to evaluate meat quality parameters related to the pre mortem status of the animals, including health, nutrition, welfare, and with potential applications for product traceability (chapters 3 and 4); the study of candidate genes for obesity related traits in order to identify markers associated with fatness in pigs that could be applied to improve carcass quality (chapters 5, 6, and 7). Chapter three addresses the first issue from a methodological point of view. When we considered this issue, it was not obvious that post mortem skeletal muscle could be useful for transcriptomic analysis. Therefore we demonstrated that the quality of RNA extracted from skeletal muscle of pigs sampled at different post mortem intervals (20 minutes, 2 hours, 6 hours, and 24 hours) is good for downstream applications. Degradation occurred starting from 48 h post mortem even if at this time it is still possible to use some RNA products. In the fourth chapter, in order to demonstrate the potential use of RNA obtained up to 24 hours post mortem, we present the results of RNA analysis with the Affymetrix microarray platform that made it possible to assess the level of expression of more of 24000 mRNAs. We did not identify any significant differences between the different post mortem times suggesting that this technique could be applied to retrieve information coming from the transcriptome of skeletal muscle samples not collected just after slaughtering. This study represents the first contribution of this kind applied to pork. In the fifth chapter, we investigated as candidate for fat deposition the TBC1D1 [TBC1 (tre-2/USP6, BUB2, cdc16) gene. This gene is involved in mechanisms regulating energy homeostasis in skeletal muscle and is associated with predisposition to obesity in humans. By resequencing a fragment of the TBC1D1 gene we identified three synonymous mutations localized in exon 2 (g.40A>G, g.151C>T, and g.172T>C) and 2 polymorphisms localized in intron 2 (g.219G>A and g.252G>A). One of these polymorphisms (g.219G>A) was genotyped by high resolution melting (HRM) analysis and PCR-RFLP. Moreover, this gene sequence was mapped by radiation hybrid analysis on porcine chromosome 8. The association study was conducted in 756 performance tested pigs of Italian Large White and Italian Duroc breeds. Significant results were obtained for lean meat content, back fat thickness, visible intermuscular fat and ham weight. In chapter six, a second candidate gene (tribbles homolog 3, TRIB3) is analyzed in a study of association with carcass and meat quality traits. The TRIB3 gene is involved in energy metabolism of skeletal muscle and plays a role as suppressor of adipocyte differentiation. We identified two polymorphisms in the first coding exon of the porcine TRIB3 gene, one is a synonymous SNP (c.132T> C), a second is a missense mutation (c.146C> T, p.P49L). The two polymorphisms appear to be in complete linkage disequilibrium between and within breeds. The in silico analysis of the p.P49L substitution suggests that it might have a functional effect. The association study in about 650 pigs indicates that this marker is associated with back fat thickness in Italian Large White and Italian Duroc breeds in two different experimental designs. This polymorphisms is also associated with lactate content of muscle semimembranosus in Italian Large White pigs. Expression analysis indicated that this gene is transcribed in skeletal muscle and adipose tissue as well as in other tissues. In the seventh chapter, we reported the genotyping results for of 677 SNPs in extreme divergent groups of pigs chosen according to the extreme estimated breeding values for back fat thickness. SNPs were identified by resequencing, literature mining and in silico database mining. analysis, data reported in the literature of 60 candidates genes for obesity. Genotyping was carried out using the GoldenGate (Illumina) platform. Of the analyzed SNPs more that 300 were polymorphic in the genotyped population and had minor allele frequency (MAF) >0.05. Of these SNPs, 65 were associated (P<0.10) with back fat thickness. One of the most significant gene marker was the same TBC1D1 SNPs reported in chapter 5, confirming the role of this gene in fat deposition in pig. These results could be important to better define the pig as a model for human obesity other than for marker assisted selection to improve carcass characteristics.

Charakterisierung der Vinorin-Synthase aus Rauvolfia serpentina durch Reinigung, Expression, Mutation und Kristallisation

ABSTRACTDie vorliegende Arbeit befasste sich mit der Reinigung,heterologen Expression, Charakterisierung, molekularenAnalyse, Mutation und Kristallisation des EnzymsVinorin-Synthase. Das Enzym spielt eine wichtige Rolle inder Ajmalin-Biosynthese, da es in einerAcetyl-CoA-abhängigen Reaktion die Umwandlung desSarpagan-Alkaloids 16-epi-Vellosimin zu Vinorin unterBildung des Ajmalan-Grundgerüstes katalysiert. Nach der Reinigung der Vinorin-Synthase ausHybrid-Zellkulturen von Rauvolfia serpentina/Rhazya strictamit den fünf chromatographischen TrennmethodenAnionenaustauschchromatographie an SOURCE 30Q, HydrophobeInteraktionen Chromatographie an SOURCE 15PHE,Chromatographie an MacroPrep Ceramic Hydroxyapatit,Anionenaustauschchromatographie an Mono Q undGrößenausschlußchromatographie an Superdex 75 konnte dieVinorin-Synthase aus 2 kg Zellkulturgewebe 991fachangereichert werden.Das nach der Reinigung angefertigte SDS-Gel ermöglichte eineklare Zuordnung der Protein-Bande als Vinorin-Synthase.Der Verdau der Enzymbande mit der Endoproteinase LysC unddie darauffolgende Sequenzierung der Spaltpeptide führte zuvier Peptidsequenzen. Der Datenbankvergleich (SwissProt)zeigte keinerlei Homologien zu Sequenzen bekannterPflanzenenzyme. Mit degenerierten Primern, abgeleitet voneinem der erhaltenen Peptidfragmente und einer konserviertenRegion bekannter Acetyltransferasen gelang es, ein erstescDNA-Fragment der Vinorin-Synthase zu amplifizieren. Mit derMethode der RACE-PCR wurde die Nukleoidsequenzvervollständigt, was zu einem cDNA-Vollängenklon mit einerGröße von 1263 bp führte, der für ein Protein mit 421Aminosäuren (46 kDa) codiert.Das Vinorin-Synthase-Gen wurde in den pQE2-Expressionsvektorligiert, der für einen N-terminalen 6-fachen His-tagcodiert. Anschließend wurde sie erstmals erfolgreich in E.coli im mg-Maßstab exprimiert und bis zur Homogenitätgereinigt. Durch die erfolgreiche Überexpression konnte dieVinorin-Synthase eingehend charakterisiert werden. DerKM-Wert für das Substrat Gardneral wurde mit 20 µM, bzw.41.2 µM bestimmt und Vmax betrug 1 pkat, bzw. 1.71 pkat.Nach erfolgreicher Abspaltung des His-tags wurden diekinetischen Parameter erneut bestimmt (KM- Wert 7.5 µM, bzw.27.52 µM, Vmax 0.7 pkat, bzw. 1.21 pkat). Das Co-Substratzeigt einen KM- Wert von 60.5 µM (Vmax 0.6 pkat). DieVinorin-Synthase besitzt ein Temperatur-Optimum von 35 °Cund ein pH-Optimum bei 7.8.Homologievergleiche mit anderen Enzymen zeigten, dass dieVinorin-Synthase zu einer noch kleinen Familie von bisher 10Acetyltransferasen gehört. Alle Enzyme der Familie haben einHxxxD und ein DFGWG-Motiv zu 100 % konserviert. Basierendauf diesen Homologievergleichen und Inhibitorstudien wurden11 in dieser Proteinfamilie konservierte Aminosäuren gegenAlanin ausgetauscht, um so die Aminosäuren einer in derLiteratur postulierten katalytischen Triade(Ser/Cys-His-Asp) zu identifizieren.Die Mutation aller vorhandenen konservierten Serine undCysteine resultierte in keiner Mutante, die zumvollständigen Aktivitätsverlust des Enzyms führte. Nur dieMutationen H160A und D164A resultierten in einemvollständigen Aktivitätsverlust des Enzyms. Dieses Ergebniswiderlegt die Theorie einer katalytischen Triade und zeigte,dass die Aminosäuren H160A und D164A exklusiv an derkatalytischen Reaktion beteiligt sind.Zur Überprüfung dieser Ergebnisse und zur vollständigenAufklärung des Reaktionsmechanismus wurde dieVinorin-Synthase kristallisiert. Die bis jetzt erhaltenenKristalle (Kristallgröße in µm x: 150, y: 200, z: 200)gehören der Raumgruppe P212121 (orthorhombisch primitiv) anund beugen bis 3.3 Å. Da es bis jetzt keine Kristallstruktureines zur Vinorin-Synthase homologen Proteins gibt, konntedie Struktur noch nicht vollständig aufgeklärt werden. ZurLösung des Phasenproblems wird mit der Methode der multiplenanomalen Dispersion (MAD) jetzt versucht, die ersteKristallstruktur in dieser Enzymfamilie aufzuklären.

Genetic analysis of tumour initiation and progression in a Drosophila model of epithelial cancer

Neoplastic overgrowth depends on the cooperation of several mutations ultimately leading to major rearrangements in cellular behaviour. The molecular crosstalk occurring between precancerous and normal cells strongly influences the early steps of the tumourigenic process as well as later stages of the disease. Precancerous cells are often removed by cell death from normal tissues but the mechanisms responsible for such fundamental safeguard processes remain in part elusive. To gain insight into these phenomena I took advantage of the clonal analysis methods available in Drosophila for studying the phenotypes due to loss of function of the neoplastic tumour suppressor lethal giant larvae (lgl). I found that lgl mutant cells growing in wild-type imaginal wing discs are subject to the phenomenon of cell competition and are eliminated by JNK-dependent cell death because they express very low levels of dMyc oncoprotein compared to those in the surrounding tissue. Indeed, in non-competitive backgrounds lgl mutant clones are able to overgrow and upregulate dMyc, overwhelming the neighbouring tissue and forming tumourous masses that display several cancer hallmarks. These phenotypes are completely abolished by reducing dMyc abundance within mutant cells while increasing it in lgl clones growing in a competitive context re-establishes their tumourigenic potential. Similarly, the neoplastic growth observed upon the oncogenic cooperation between lgl mutation and activated Ras/Raf/MAPK signalling was found to be characterised by and dependent on the ability of cancerous cells to upregulate dMyc with respect to the adjacent normal tissue, through both transcriptional and post-transcriptional mechanisms, thereby confirming its key role in lgl-induced tumourigenesis. These results provide first evidence that the dMyc oncoprotein is required in lgl mutant tissue to promote invasive overgrowth in developing and adult epithelial tissues and that dMyc abundance inside versus outside lgl mutant clones plays a key role in driving neoplastic overgrowth.

Identifizierung einer Mutation im Exon 2 des C1q-B-Ketten-Gens als Ursache eines hereditären C1q-Defekts mit Ausbildung einer SLE-ähnlichen Symptomatik

Komplementdefizienzen gehen mit einer erhöhten Infektionsanfälligkeit gegenüber bestimmten Krankheitserregern in den ersten Lebensjahren (MBL-Defizienz) und darüber hinaus (C1q- und anderen Komplementdefizienten) einher. Dies unterstreicht die Rolle des Komplementsystems als effektiver Abwehrmechanismus in der Übergangsphase zwischen Verlust des „mütterlichen Nestschutzes“ und Ausreifung der eigenen „erworbenen“ Immunität. Das Auftreten von Autoimmunerkrankungen wie dem SLE-ähnlichen Syndrom bei Defizienzen des Klassischen Weges beleuchten zusätzliche Funktionen des Komplementsystems während der Ausreifung der erworbenen Immunität und als wesentlicher Effektor in der Erkennung apoptotischer Zellen und deren Eliminierung aus dem System.rnHereditäre C1q-Defizienzen gehen mit einer hohen Wahrscheinlichkeit mit einem SLE-ähnlichen Syndrom einher. Sie stellen unter den Defizienzen des Komplementsystems eines Seltenheit dar, ihr klinisches „Gesicht“ ist umso eindrucksvoller. Sie sind von der funktionellen C1q-Defizienz im Rahmen eines erhöhten „turnover“ und in der Folge einer C1q-Autoantokörperbildung abzugrenzen. Ursächlich ist ihnen eine Mutation in einem der drei C1q-Gene, die auf dem Chromosom 1 lokalisiert sind. Homozygote Mutationsträger können den Defekt nicht ausgleichen und zeigen eine C1q-Defizienz mit Verlust der gesamthämolytischen Aktivität CH50. Häufungen treten bei Nachkommen von Geschwister- und Verwandtschaftsehen auf.rnrnIn dieser Arbeit wird der Fall einer Patientin mit einem schweren, frühkindlich einsetzenden, SLE-ähnlichen Syndrom aufgearbeitet. Als Ursache für eine Erkrankung konnte ein hereditärer C1q-Defekt, ohne immunologischem Nachweis eines C1q oer LMQ-C1q, identifiziert werden. Da sich keine der vorab beschriebenen Mutatonsmuster bei der Patientin detektieren ließ, erfolgte die Sequenzierung aller drei C1q-Gene. Dadurch ließ sich ein neues Mutationsmuster darstellen.rnrnDie in dieser Arbeit vorgestellte Mutation unterscheidet sich von den bislang beschriebenen Mutationen dadurch, dass es sich nicht um eine Punktmutation, sonder um eine Deletion von 29 Basen (c283_311) im Exon 2 des C1q-B-Ketten-Gens mit einhergehendem Rasterschub und vorzeitigem Stop-Codon (pMet95TrpfsX8) handelt. Durch die Analyse der Eltern und Geschwister der betroffenen Patientin konnte der Vererbungsweg dargestellt werden. Zudem gelang es die Mutation im Rahmen einer Pränataldiagnostik bei einem „ungeborenen“ Geschwisterkind auszuschließen.rn

High sensitivity analysis of BRAF mutations in neoplastic and non-neoplastic thyroid lesions

The clonal distribution of BRAFV600E in papillary thyroid carcinoma (PTC) has been recently debated. No information is currently available about precursor lesions of PTCs. My first aim was to establish whether the BRAFV600E mutation occurs as a subclonal event in PTCs. My second aim was to screen BRAF mutations in histologically benign tissue of cases with BRAFV600E or BRAFwt PTCs in order to identify putative precursor lesions of PTCs. Highly sensitive semi-quantitative methods were used: Allele Specific LNA quantitative PCR (ASLNAqPCR) and 454 Next-Generation Sequencing (NGS). For the first aim 155 consecutive formalin-fixed and paraffin-embedded (FFPE) specimens of PTCs were analyzed. The percentage of mutated cells obtained was normalized to the estimated number of neoplastic cells. Three groups of tumors were identified: a first had a percentage of BRAF mutated neoplastic cells > 80%; a second group showed a number of BRAF mutated neoplastic cells < 30%; a third group had a distribution of BRAFV600E between 30-80%. The large presence of BRAFV600E mutated neoplastic cell sub-populations suggests that BRAFV600E may be acquired early during tumorigenesis: therefore, BRAFV600E can be heterogeneously distributed in PTC. For the second aim, two groups were studied: one consisted of 20 cases with BRAFV600E mutated PTC, the other of 9 BRAFwt PTCs. Seventy-five and 23 histologically benign FFPE thyroid specimens were analyzed from the BRAFV600E mutated and BRAFwt PTC groups, respectively. The screening of BRAF mutations identified BRAFV600E in “atypical” cell foci from both groups of patients. “Unusual” BRAF substitutions were observed in histologically benign thyroid associated with BRAFV600E PTCs. These mutations were very uncommon in the group with BRAFwt PTCs and in BRAFV600E PTCs. Therefore, lesions carrying BRAF mutations may represent “abortive” attempts at cancer development: only BRAFV600E boosts neoplastic transformation to PTC. BRAFV600E mutated “atypical foci” may represent precursor lesions of BRAFV600E mutated PTCs.

Biochemical analysis of the phosphatase domain of the human soluble epoxide hydrolase (sEH)

Die lösliche Epoxidhydrolase (sEH) gehört zur Familie der Epoxidhydrolase-Enzyme. Die Rolle der sEH besteht klassischerweise in der Detoxifikation, durch Umwandlung potenziell schädlicher Epoxide in deren unschädliche Diol-Form. Hauptsächlich setzt die sEH endogene, der Arachidonsäure verwandte Signalmoleküle, wie beispielsweise die Epoxyeicosatrienoic acid, zu den entsprechenden Diolen um. Daher könnte die sEH als ein Zielenzym in der Therapie von Bluthochdruck und Entzündungen sowie diverser anderer Erkrankungen eingesetzt werden. rnDie sEH ist ein Homodimer, in dem jede Untereinheit aus zwei Domänen aufgebaut ist. Das katalytische Zentrum der Epoxidhydrolaseaktivität befindet sich in der 35 kD großen C-terminalen Domäne. Dieser Bereich der sEH s wurde bereits im Detail untersucht und nahezu alle katalytischen Eigenschaften des Enzyms sowie deren dazugehörige Funktionen sind in Zusammenhang mit dieser Domäne bekannt. Im Gegensatz dazu ist über die 25 kD große N-terminale Domäne wenig bekannt. Die N-terminale Domäne der sEH wird zur Haloacid Dehalogenase (HAD) Superfamilie von Hydrolasen gezählt, jedoch war die Funktion dieses N-terminal Domäne lange ungeklärt. Wir haben in unserer Arbeitsgruppe zum ersten Mal zeigen können, dass die sEH in Säugern ein bifunktionelles Enzym ist, welches zusätzlich zur allgemein bekannten Enzymaktivität im C-terminalen Bereich eine weitere enzymatische Funktion mit Mg2+-abhängiger Phosphataseaktivität in der N-terminalen Domäne aufweist. Aufgrund der Homologie der N-terminalen Domäne mit anderen Enzymen der HAD Familie wird für die Ausübung der Phosphatasefunktion (Dephosphorylierung) eine Reaktion in zwei Schritten angenommen.rnUm den katalytischen Mechanismus der Dephosphorylierung weiter aufzuklären, wurden biochemische Analysen der humanen sEH Phosphatase durch Generierung von Mutationen im aktiven Zentrum mittels ortsspezifischer Mutagenese durchgeführt. Hiermit sollten die an der katalytischen Aktivität beteiligten Aminosäurereste im aktiven Zentrum identifiziert und deren Rolle bei der Dephosphorylierung spezifiziert werden. rnrnAuf Basis der strukturellen und möglichen funktionellen Ähnlichkeiten der sEH und anderen Mitgliedern der HAD Superfamilie wurden Aminosäuren (konservierte und teilweise konservierte Aminosäuren) im aktiven Zentrum der sEH Phosphatase-Domäne als Kandidaten ausgewählt.rnVon den Phosphatase-Domäne bildenden Aminosäuren wurden acht ausgewählt (Asp9 (D9), Asp11 (D11), Thr123 (T123), Asn124 (N124), Lys160 (K160), Asp184 (D184), Asp185 (D185), Asn189 (N189)), die mittels ortsspezifischer Mutagenese durch nicht funktionelle Aminosäuren ausgetauscht werden sollten. Dazu wurde jede der ausgewählten Aminosäuren durch mindestens zwei alternative Aminosäuren ersetzt: entweder durch Alanin oder durch eine Aminosäure ähnlich der im Wildtyp-Enzym. Insgesamt wurden 18 verschiedene rekombinante Klone generiert, die für eine mutante sEH Phosphatase Domäne kodieren, in dem lediglich eine Aminosäure gegenüber dem Wildtyp-Enzym ersetzt wurde. Die 18 Mutanten sowie das Wildtyp (Sequenz der N-terminalen Domäne ohne Mutation) wurden in einem Expressionsvektor in E.coli kloniert und die Nukleotidsequenz durch Restriktionsverdau sowie Sequenzierung bestätigt. Die so generierte N-terminale Domäne der sEH (25kD Untereinheit) wurde dann mittels Metallaffinitätschromatographie erfolgreich aufgereinigt und auf Phosphataseaktivität gegenüber des allgemeinen Substrats 4-Nitophenylphosphat getestet. Diejenigen Mutanten, die Phosphataseaktivität zeigten, wurden anschließend kinetischen Tests unterzogen. Basiered auf den Ergebnissen dieser Untersuchungen wurden kinetische Parameter mittels vier gut etablierter Methoden berechnet und die Ergebnisse mit der „direct linear blot“ Methode interpretiert. rnDie Ergebnisse zeigten, dass die meisten der 18 generierten Mutanten inaktiv waren oder einen Großteil der Enzymaktivität (Vmax) gegenüber dem Wildtyp verloren (WT: Vmax=77.34 nmol-1 mg-1 min). Dieser Verlust an Enzymaktivität ließ sich nicht durch einen Verlust an struktureller Integrität erklären, da der Wildtyp und die mutanten Proteine in der Chromatographie das gleiche Verhalten zeigten. Alle Aminosäureaustausche Asp9 (D9), Lys160 (K160), Asp184 (D184) und Asn189 (N189) führten zum kompletten Verlust der Phosphataseaktivität, was auf deren katalytische Funktion im N-terminalen Bereich der sEH hindeutet. Bei einem Teil der Aminosäureaustausche die für Asp11 (D11), Thr123 (T123), Asn124 (N124) und Asn185 (D185) durchgeführt wurden, kam es, verglichen mit dem Wildtyp, zu einer starken Reduktion der Phosphataseaktivität, die aber dennoch für die einzelnen Proteinmutanten in unterschiedlichem Ausmaß zu messen war (2 -10% and 40% of the WT enzyme activity). Zudem zeigten die Mutanten dieser Gruppe veränderte kinetische Eigenschaften (Vmax allein oder Vmax und Km). Dabei war die kinetische Analyse des Mutanten Asp11  Asn aufgrund der nur bei dieser Mutanten detektierbaren starken Vmax Reduktion (8.1 nmol-1 mg-1 min) und einer signifikanten Reduktion der Km (Asp11: Km=0.54 mM, WT: Km=1.3 mM), von besonderem Interesse und impliziert eine Rolle von Asp11 (D11) im zweiten Schritt der Hydrolyse des katalytischen Zyklus.rnZusammenfassend zeigen die Ergebnisse, dass alle in dieser Arbeit untersuchten Aminosäuren für die Phosphataseaktivität der sEH nötig sind und das aktive Zentrum der sEH Phosphatase im N-terminalen Bereich des Enzyms bilden. Weiterhin tragen diese Ergebnisse zur Aufklärung der potenziellen Rolle der untersuchten Aminosäuren bei und unterstützen die Hypothese, dass die Dephosphorylierungsreaktion in zwei Schritten abläuft. Somit ist ein kombinierter Reaktionsmechanismus, ähnlich denen anderer Enzyme der HAD Familie, für die Ausübung der Dephosphorylierungsfunktion denkbar. Diese Annahme wird gestützt durch die 3D-Struktur der N-terminalen Domäne, den Ergebnissen dieser Arbeit sowie Resultaten weiterer biochemischer Analysen. Der zweistufige Mechanismus der Dephosphorylierung beinhaltet einen nukleophilen Angriff des Substratphosphors durch das Nukleophil Asp9 (D9) des aktiven Zentrums unter Bildung eines Acylphosphat-Enzym-Zwischenprodukts, gefolgt von der anschließenden Freisetzung des dephosphorylierten Substrats. Im zweiten Schritt erfolgt die Hydrolyse des Enzym-Phosphat-Zwischenprodukts unterstützt durch Asp11 (D11), und die Freisetzung der Phosphatgruppe findet statt. Die anderen untersuchten Aminosäuren sind an der Bindung von Mg 2+ und/oder Substrat beteiligt. rnMit Hilfe dieser Arbeit konnte der katalytischen Mechanismus der sEH Phosphatase weiter aufgeklärt werden und wichtige noch zu untersuchende Fragestellungen, wie die physiologische Rolle der sEH Phosphatase, deren endogene physiologische Substrate und der genaue Funktionsmechanismus als bifunktionelles Enzym (die Kommunikation der zwei katalytischen Einheiten des Enzyms) wurden aufgezeigt und diskutiert.rn

Analysis of the immunological and functional features of the Neisserial Heparin Binding Antigen (NHBA)

Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein ubiquitously expressed by genetically diverse Neisseria meningitidis strains and is an antigen of the multicomponent protein-based 4CMenB vaccine, able to induce bactericidal antibodies in humans and to bind heparin-like molecules. The aim of this study is to characterize the immunological and functional properties of NHBA. To evaluate immunogenicity and the contribution of aminoacid sequence variability to vaccine coverage, we constructed recombinant isogenic strains that are susceptible to bactericidal killing only by anti-NHBA antibodies and engineered them to express equal levels of selected NHBA peptides. In these recombinant strains, we observed different titres associated with the different peptide variants. These recombinant strains were then further engineered to express NHBA chimeric proteins to investigate the regions important for immunogenicity. In natural strains, anti-NHBA antibodies were found to be cross-protective against strains expressing different peptides. To investigate the functional properties of this antigen, the recombinant purified NHBA protein was tested in in vitro binding studies and was found to be able to bind epithelial cells. The binding was abolished when cells were treated specifically with heparinase III, suggesting that the interaction with the cells is mediated by heparan sulfate proteoglycans (HSPG). Mutation of the Arg-rich tract of NHBA abrogated the binding, confirming the importance of this region in mediating the binding to heparin-like molecules. In a panel of N. meningitidis strains, the deletion of nhba resulted in a reduction of adhesion with respect to each isogenic wild type strain. Furthermore, the adhesion of the wild-type strain was prevented by using anti-NHBA polyclonal sera, demonstrating the specificity of the interaction. These results suggest that NHBA could be a novel meningococcal adhesin contributing to host-cell interaction. Moreover, we analysed NHBA NalP-mediated cleavage in different NHBA peptides and showed that not all NHBA peptides are cleaved.