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Gava, Alessandra (2009) Utilità della tecnica denaturing high performance liquid chromatography per la diagnosi di deficit di mevalonato chinasi nelle febbri periodiche di sospetta natura autoinfiammatoria. [Tesi di dottorato]

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Abstract (inglese)

Mevalonate Kinase Deficiency (MKD) is a rare autosomic recessive autoinflammatory disorder characterized by recurrent fever and musculoskeletal manifestations. MKD is due to mutations in the gene for mevalonate kinase (MK), located on chromosome 12q24, which cause variable degrees of enzyme deficiency. Although genetic investigation became essential for the diagnosis, especially in patient with normal IgD values in serum, the analysis of the sequence of the whole coding region is expensive: determination of mutations by sequencing can be made more efficient using techniques of pre-screening able to identify the mutated exon.
OBJECTIVES:
In this study we have developed a genetic investigation using Denaturing High Performance Liquid Chromatography (DHPLC), a pre-screening technique which is able to identify polymorphisms of the gene mvk in patients with autoinflammatory syndrome suspicion.
METHODS:
Starting from January 2007 we evaluated 10 healthy controls and 19 patients (mean age 33 years, range 13 – 61 years) affected by recurrent fevers and other clinical manifestations usually found in autoinflammatory syndromes and not correlated with infections or autoimmune disease. IgD level was measured in all patients.
Molecular testing was performed in DNA extracted from peripheral blood lymphocytes by standard methods. The 10 coding exons (from 2 to 11) of the mvk gene were analysed by means of denaturing high-performance liquid chromatography (DHPLC) using the Wave® Fragment Analysis system (Transgenomic). Primers for PCR amplifications, amplicon lengths and PCR conditions were designed in our laboratory. Melting temperature (Tm) were calculated with Navigator Software 1.7.0 (Transgenomic). Sequences of the same amplified fragments were determined by means of an automatic sequencer ABI PRISM 3130 Genetic Analyzer (APPLIED BIOSYSTEMS).
RESULTS:
IgD level was normal in 14 patients.
Healthy controls did not show any alteration of the DHPLC-profiles and of the DNA sequences. Twelve patients had at least one altered DHPLC-profile and these data have been confirmed by sequencing. In particular we detected the mutations S52N (patient 2-13-15), S135S (pt 13-16), D170D, c.632-18A>G(pt 4-5-6-10-12-17-19), c.78+61A>G (pt 6-11-19), c.885+24G>A (pt 6-19) already described in literature and in the database INFEVERS (http://fmf.igh.cnrs.fr/infevers).
The screening with DHPLC has allowed us to get the results in shorter time (10 hours/patient) and with lower cost (40 euro / patient) in comparison to direct sequencing (time: 25 hours / patient and cost: 150 euro / patient).
CONCLUSION:
High serum levels of IgD do not represent an essential marker for diagnosis of MKD, as already reported in literature. DHPLC is a rapid low cost technique in order to screen mutations in patients with MKD suspicion. Nevertheless, being an indirect tecnique, DHPLC is able to show a polymorphism in the DNA sequence but it is not able to identify the position, and this still remains a limiting factor for the use of the DHPLC as an exclusive technique for genetic analysis.
Twelve patients of our cohort carried at the same time D170D and c.632-18A>G: such event suggests that these SNPs could be in linkage disequilibrium and that such mutations could induce a diminution in the activity of the MK predisposing therefore to the MKD.

Abstract (italiano)

SCOPO DEL LAVORO
La Mevalonate Kinase Deficency (MKD), già nota come sindrome da Iper IgD (HIDS), è una rara malattia autoinfiammatoria di tipo autosomico recessivo caratterizzata da febbre ricorrente e disturbi muscoloscheletrici. La MKD è dovuta a mutazioni del gene mvk, mappato sul cromosoma 12q24, il quale codifica per l’enzima mevalonato chinasi (MK); polimorfismi nel gene mvk sono la causa di un’alterata attività enzimatica di MK.
L’individuazione della mutazione genica della mevalonato chinasi ha reso l’indagine genetica indispensabile per la diagnosi, soprattutto in pazienti con valori sierici di IgD normali, tuttavia il sequenziamento dell’intera regione codificante è costoso e la determinazione di mutazioni può essere resa più efficiente utilizzando tecniche di pre-screening in grado di identificare l’esone mutato. In questo studio è stata effettuata un’indagine genetica utilizzando l’analisi in DHPLC come tecnica di pre-screening per identificare le alterazioni nella regione codificante del gene mvk in pazienti con sospetta sindrome autoinfiammatoria.
MATERIALI E METODI:
A partire da gennaio 2007 sono stati presi in esame 19 pazienti (età media 33 anni, range 13 – 61 anni) seguiti presso la Cattedra e UOC di Reumatologia dell’Università di Padova per febbri ricorrenti non imputabili ad infezioni o a malattie autoimmuni e compatibili con sospetta MKD e 10 controlli sani. In ogni paziente è stato effettuato il dosaggio delle IgD nel siero. L’analisi del DNA, estratto da linfociti del sangue periferico, è stata effettuata mediante un protocollo di amplificazione per frammenti di 210-250 bp messo a punto da noi stessi, utilizzando primers specifici per le sequenze dei 10 esoni codificanti del gene mvk. I prodotti di amplificazione sono stati analizzati mediante DHPLC utilizzando il sistema Wave® Fragment Analysis (Transgenomic, Omaha, NE, USA). Le temperature di melting (Tm) sono state calcolate con Navigator Software 1.7.0 (Transgenomic). Le sequenze degli stessi frammenti amplificati sono state determinate mediante l’uso di un sequenziatore automatico ABI PRISM 3130 Genetic Analyzer (APPLIED BIOSYSTEMS, USA).
RISULTATI:
I valori sierici di IgD erano normali (<100 mg/L) in 14/19 pazienti. Nessun campione di controllo presentava alterazioni dei profili DHPLC e delle sequenze.
Dodici dei 19 pazienti esaminati presentavano almeno un profilo di DHPLC alterato e i dati sono stati confermati con il sequenziamento. Sono state evidenziate le mutazioni S52N (paziente 2-13-15), D170D e c.632-18A>G (paziente 4-5-6-10-12-17-19), S135S (pz 13-16) c.78+61A>G (pz 6-11-19), c.885+24G>A (pz 6-19) già descritte in letteratura e nel database INFEVERS (http://fmf.igh.cnrs.fr/infevers).
Lo screening con DHPLC ha permesso di ottenere i risultati in minor tempo (10 ore / paziente) e con costi inferiori (40 euro / paziente) rispetto alla tecnica di sequenziamento diretto (tempo: 25 ore / paziente , costi: 150 euro / paziente).
CONCLUSIONI:
Un elevato livello sierico di IgD non rappresenta più un marker per fare diagnosi di MKD, come già riportato in letteratura.
La DHPLC è una tecnica capace di effettuare uno screening rapido e a basso costo nell’analisi di mutazioni in pazienti con sospetta sindrome autoinfiammatoria legata al gene mvk.
Tuttavia, essendo una tecnica di analisi indiretta, è necessario considerare che la DHPLC aumenta l’efficienza dell’analisi ma non la sua sensibilità in quanto è in grado di evidenziare un polimorfismo di sequenza ma non la sua posizione.
Sette pazienti della nostra coorte sono risultati portatori delle varianti alleliche D170D e c.632-18A>G; tale evento suggerisce che questi SNP potrebbero essere in linkage disequilibrium e che tali mutazioni potrebbero indurre una diminuzione nell’attività della MK predisponendo quindi alla MKD.

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Tipo di EPrint:Tesi di dottorato
Relatore:Punzi, Leonardo
Dottorato (corsi e scuole):Ciclo 21 > Scuole per il 21simo ciclo > SCIENZE MEDICHE, CLINICHE E SPERIMENTALI > SCIENZE REUMATOLOGICHE
Data di deposito della tesi:01 Febbraio 2009
Anno di Pubblicazione:01 Febbraio 2009
Parole chiave (italiano / inglese):mevalonato chinasi, denaturing high performance liquid chromatography, malattie autoinfiammatorie
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/16 Reumatologia
Struttura di riferimento:Dipartimenti > Dipartimento di Medicina Clinica e Sperimentale
Codice ID:1905
Depositato il:01 Feb 2009
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I riferimenti della bibliografia possono essere cercati con Cerca la citazione di AIRE, copiando il titolo dell'articolo (o del libro) e la rivista (se presente) nei campi appositi di "Cerca la Citazione di AIRE".
Le url contenute in alcuni riferimenti sono raggiungibili cliccando sul link alla fine della citazione (Vai!) e tramite Google (Ricerca con Google). Il risultato dipende dalla formattazione della citazione.

1. Heberden W. Commentaries on History and Care of Disease. London: T. Payne, 1802: 151. Cerca con Google

2. Hull KM, Shoham N, Chae JJ, Aksentijevich I, Kastner DL. The expanding spectrum of systemic autoinflammatory disorders and their rheumatic manifestations. Current opinion in rheumatology 2003 Jan;15(1):61-69. Cerca con Google

3. Touitou I, Kone-Paut I. Autoinflammatory diseases. Best practice & research.Clinical rheumatology 2008 Oct;22(5):811-829. Cerca con Google

4. McDermott MF, Aksentijevich I, Galon J, McDermott EM, Ogunkolade BW, Centola M, et al. Germline mutations in the extracellular domains of the 55 kDa TNF receptor, TNFR1, define a family of dominantly inherited autoinflammatory syndromes. Cell 1999 Apr 2;97(1):133-144. Cerca con Google

5. Drenth JP, van der Meer JW. Hereditary periodic fever. The New England journal of medicine 2001 Dec 13;345(24):1748-1757. Cerca con Google

6. Galeazzi M, Gasbarrini G, Ghirardello A, Grandemange S, Hoffman HM, Manna R, et al. Autoinflammatory syndromes. Clinical and experimental rheumatology 2006 Jan-Feb;24(1 Suppl 40):S79-85. Cerca con Google

7. McDermott MF, Aksentijevich I. The autoinflammatory syndromes. Current opinion in allergy and clinical immunology 2002 Dec;2(6):511-516. Cerca con Google

8. Stankovic K, Grateau G. Auto inflammatory syndromes: Diagnosis and treatment. Joint Bone Spine, 2007 12;74(6):544-550. Cerca con Google

9. Ryan JG, Kastner DL. Fevers, genes, and innate immunity. Current topics in microbiology and immunology 2008;321:169-184. Cerca con Google

10. Bodar EJ, Drenth JP, van der Meer JW, Simon A. Dysregulation of innate immunity: hereditary periodic fever syndromes. British journal of haematology 2009 Feb;144(3):279-302. Cerca con Google

11. Miceli-Richard C, Lesage S, Rybojad M, Prieur AM, Manouvrier-Hanu S, Hafner R, et al. CARD15 mutations in Blau syndrome. Nature genetics 2001 Sep;29(1):19-20. Cerca con Google

12. van Duist MM, Albrecht M, Podswiadek M, Giachino D, Lengauer T, Punzi L, et al. A new CARD15 mutation in Blau syndrome. European journal of human genetics : EJHG 2005 Jun;13(6):742-747. Cerca con Google

13. Touitou I. The spectrum of Familial Mediterranean Fever (FMF) mutations. European journal of human genetics : EJHG 2001 Jul;9(7):473-483. Cerca con Google

14. Aksentijevich I, Galon J, Soares M, Mansfield E, Hull K, Oh HH, et al. The tumor-necrosis-factor receptor-associated periodic syndrome: new mutations in TNFRSF1A, ancestral origins, genotype-phenotype studies, and evidence for further genetic heterogeneity of periodic fevers. American Journal of Human Genetics 2001 Aug;69(2):301-314. Cerca con Google

15. Galon J, Aksentijevich I, McDermott MF, O'Shea JJ, Kastner DL. TNFRSF1A mutations and autoinflammatory syndromes. Current opinion in immunology 2000 Aug;12(4):479-486. Cerca con Google

16. Hull KM, Drewe E, Aksentijevich I, Singh HK, Wong K, McDermott EM, et al. The TNF receptor-associated periodic syndrome (TRAPS): emerging concepts of an autoinflammatory disorder. Medicine 2002 Sep;81(5):349-368. Cerca con Google

17. Aksentijevich I, Nowak M, Mallah M, Chae JJ, Watford WT, Hofmann SR, et al. De novo CIAS1 mutations, cytokine activation, and evidence for genetic heterogeneity in patients with neonatal-onset multisystem inflammatory disease (NOMID): a new member of the expanding family of pyrin-associated autoinflammatory diseases. Arthritis and Rheumatism 2002 Dec;46(12):3340-3348. Cerca con Google

18. Aksentijevich I, D Putnam C, Remmers EF, Mueller JL, Le J, Kolodner RD, et al. The clinical continuum of cryopyrinopathies: novel CIAS1 mutations in North American patients and a new cryopyrin model. Arthritis and Rheumatism 2007 Apr;56(4):1273-1285. Cerca con Google

19. Jesus AA, Silva CA, Segundo GR, Aksentijevich I, Fujihira E, Watanabe M, et al. Phenotype-genotype analysis of cryopyrin-associated periodic syndromes (CAPS): description of a rare non-exon 3 and a novel CIAS1 missense mutation. Journal of clinical immunology 2008 Mar;28(2):134-138. Cerca con Google

20. Mariathasan S, Weiss DS, Newton K, McBride J, O'Rourke K, Roose-Girma M, et al. Cryopyrin activates the inflammasome in response to toxins and ATP. Nature 2006 03/09/print;440(7081):228-232. Cerca con Google

21. Tschopp J, Martinon F, Burns K. NALPs: a novel protein family involved in inflammation. Nature reviews. Molecular cell biology 2003 02//print;4(2):95-104. Cerca con Google

22. Mariathasan S, Monack DM. Inflammasome adaptors and sensors: intracellular regulators of infection and inflammation. Nat Rev Immunol 2007 01//print;7(1):31-40. Cerca con Google

23. Dinarello CA. Mutations in cryopyrin: bypassing roadblocks in the caspase 1 inflammasome for interleukin-1beta secretion and disease activity. Arthritis and Rheumatism 2007 Sep;56(9):2817-2822. Cerca con Google

24. Dinarello CA. A signal for the caspase-1 inflammasome free of TLR. Immunity 2007 Apr;26(4):383-385. Cerca con Google

25. Martinon F, Tschopp J. Inflammatory Caspases: Linking an Intracellular Innate Immune System to Autoinflammatory Diseases. Cell, 2004 5/28;117(5):561-574. Cerca con Google

26. Brydges S, Kastner DL. The systemic autoinflammatory diseases: inborn errors of the innate immune system. Current topics in microbiology and immunology 2006;305:127-160. Cerca con Google

27. Gay NJ, Gangloff M. Structure and Function of Toll Receptors and Their Ligands. Annual Review of Biochemistry 2007 07/07;76(1):141-165. Cerca con Google

28. Gregersen PK. Modern Genetics, Ancient Defenses, and Potential Therapies. N Engl J Med 2007 March 22;356(12):1263-1266. Cerca con Google

29. Meylan E, Tschopp J, Karin M. Intracellular pattern recognition receptors in the host response. Nature 2006 Jul 6;442(7098):39-44. Cerca con Google

30. Bianchi ME. DAMPs, PAMPs and alarmins: all we need to know about danger. Journal of leukocyte biology 2007 Jan;81(1):1-5. Cerca con Google

31. McDermott MF. A common pathway in periodic fever syndromes. Trends in immunology 2004 Sep;25(9):457-460. Cerca con Google

32. Dinarello CA. The IL-1 family and inflammatory diseases. Clinical and experimental rheumatology 2002 Sep-Oct;20(5 Suppl 27):S1-13. Cerca con Google

33. Dinarello CA. The many worlds of reducing interleukin-1. Arthritis and Rheumatism 2005 Jul;52(7):1960-1967. Cerca con Google

34. Mathews RJ, Sprakes MB, McDermott MF. NOD-like receptors and inflammation. Arthritis research & therapy 2008 Nov 25;10(6):228. Cerca con Google

35. Andrei C, Margiocco P, Poggi A, Lotti LV, Torrisi MR, Rubartelli A. Phospholipases C and A2 control lysosome-mediated IL-1 beta secretion: Implications for inflammatory processes. Proceedings of the National Academy of Sciences of the United States of America 2004 Jun 29;101(26):9745-9750. Cerca con Google

36. Ferrari D, Pizzirani C, Adinolfi E, Lemoli RM, Curti A, Idzko M, et al. The P2X7 receptor: a key player in IL-1 processing and release. Journal of immunology (Baltimore, Md.: 1950) 2006 Apr 1;176(7):3877-3883. Cerca con Google

37. Romanovsky AA, Almeida MC, Aronoff DM, Ivanov AI, Konsman JP, Steiner AA, et al. Fever and hypothermia in systemic inflammation: recent discoveries and revisions. Frontiers in bioscience : a journal and virtual library 2005 Sep 1;10:2193-2216. Cerca con Google

38. Dinarello CA. Infection, fever, and exogenous and endogenous pyrogens: some concepts have changed. Journal of endotoxin research 2004;10(4):201-222. Cerca con Google

39. Gabay C, Kushner I. Acute-phase proteins and other systemic responses to inflammation. The New England journal of medicine 1999 Feb 11;340(6):448-454. Cerca con Google

40. Simon A, van der Meer JW. Pathogenesis of familial periodic fever syndromes or hereditary autoinflammatory syndromes. American journal of physiology.Regulatory, integrative and comparative physiology 2007 Jan;292(1):R86-98. Cerca con Google

41. Van Der Meer JM, Radl J, Meyer CLM, Vossen J, Van Nieuwkoop J, Lobatto S, et al. HYPERIMMUNOGLOBULINAEMIA D AND PERIODIC FEVER: A NEW SYNDROME. The Lancet, 1984 5/19;323(8386):1087-1090. Cerca con Google

42. Cuisset L, Drenth JP, Simon A, Vincent MF, van der Velde Visser S, van der Meer JW, et al. Molecular analysis of MVK mutations and enzymatic activity in hyper-IgD and periodic fever syndrome. European journal of human genetics : EJHG 2001 Apr;9(4):260-266. Cerca con Google

43. Ammouri W, Cuisset L, Rouaghe S, Rolland MO, Delpech M, Grateau G, et al. Diagnostic value of serum immunoglobulinaemia D level in patients with a clinical suspicion of hyper IgD syndrome. Rheumatology (Oxford, England) 2007 Oct;46(10):1597-1600. Cerca con Google

44. Haas D, Hoffmann GF. Mevalonate kinase deficiencies: from mevalonic aciduria to hyperimmunoglobulinemia D syndrome. Orphanet journal of rare diseases 2006 Apr 26;1:13. Cerca con Google

45. Houten SM, Wanders RJ, Waterham HR. Biochemical and genetic aspects of mevalonate kinase and its deficiency. Biochimica et biophysica acta 2000 Dec 15;1529(1-3):19-32. Cerca con Google

46. Greenwood J, Steinman L, Zamvil SS. Statin therapy and autoimmune disease: from protein prenylation to immunomodulation. Nature reviews.Immunology 2006 May;6(5):358-370. Cerca con Google

47. McTaggart SJ. Isoprenylated proteins. Cellular and molecular life sciences : CMLS 2006 Feb;63(3):255-267. Cerca con Google

48. Houten SM, Kuis W, Duran M, de Koning TJ, van Royen-Kerkhof A, Romeijn GJ, et al. Mutations in MVK, encoding mevalonate kinase, cause hyperimmunoglobulinaemia D and periodic fever syndrome. Nature genetics 1999 Jun;22(2):175-177. Cerca con Google

49. Drenth JP, Cuisset L, Grateau G, Vasseur C, van de Velde-Visser SD, de Jong JG, et al. Mutations in the gene encoding mevalonate kinase cause hyper-IgD and periodic fever syndrome. International Hyper-IgD Study Group. Nature genetics 1999 Jun;22(2):178-181. Cerca con Google

50. Milhavet F, Cuisset L, Hoffman HM, Slim R, El-Shanti H, Aksentijevich I, et al. The infevers autoinflammatory mutation online registry: update with new genes and functions. Human mutation 2008 Jun;29(6):803-808. Cerca con Google

51. Simon A, Mariman EC, van der Meer JWM, Drenth JPH. A founder effect in the hyperimmunoglobulinemia D and periodic fever syndrome. The American Journal of Medicine 2003 2/1;114(2):148-152. Cerca con Google

52. Mandey SH, Schneiders MS, Koster J, Waterham HR. Mutational spectrum and genotype-phenotype correlations in mevalonate kinase deficiency. Human mutation 2006 Aug;27(8):796-802. Cerca con Google

53. Houten SM, Frenkel J, Rijkers GT, Wanders RJ, Kuis W, Waterham HR. Temperature dependence of mutant mevalonate kinase activity as a pathogenic factor in hyper-IgD and periodic fever syndrome. Human molecular genetics 2002 Dec 1;11(25):3115-3124. Cerca con Google

54. Frenkel J, Rijkers GT, Mandey SH, Buurman SW, Houten SM, Wanders RJ, et al. Lack of isoprenoid products raises ex vivo interleukin-1beta secretion in hyperimmunoglobulinemia D and periodic fever syndrome. Arthritis and Rheumatism 2002 Oct;46(10):2794-2803. Cerca con Google

55. Mandey SH, Kuijk LM, Frenkel J, Waterham HR. A role for geranylgeranylation in interleukin-1beta secretion. Arthritis and Rheumatism 2006 Nov;54(11):3690-3695. Cerca con Google

56. Simon A, Cuisset L, Vincent MF, van Der Velde-Visser SD, Delpech M, van Der Meer JW, et al. Molecular analysis of the mevalonate kinase gene in a cohort of patients with the hyper-igd and periodic fever syndrome: its application as a diagnostic tool. Annals of Internal Medicine 2001 Sep 4;135(5):338-343. Cerca con Google

57. D'Osualdo A, Picco P, Caroli F, Gattorno M, Giacchino R, Fortini P, et al. MVK mutations and associated clinical features in Italian patients affected with autoinflammatory disorders and recurrent fever. European journal of human genetics : EJHG 2004 11/10/online;13(3):314-320. Cerca con Google

58. Kone-Paut I, Sanchez E, Le Quellec A, Manna R, Touitou I. Autoinflammatory gene mutations in Behcet's disease. Annals of the Rheumatic Diseases 2007 Jun;66(6):832-834. Cerca con Google

59. Gross E, Arnold N, Goette J, Schwarz-Boeger U, Kiechle M. A comparison of BRCA1 mutation analysis by direct sequencing, SSCP and DHPLC. Human genetics 1999 Jul-Aug;105(1-2):72-78. Cerca con Google

60. Gross E, Arnold N, Pfeifer K, Bandick K, Kiechle M. Identification of specific BRCA1 and BRCA2 variants by DHPLC. Human mutation 2000 Oct;16(4):345-353. Cerca con Google

61. Jones AC, Austin J, Hansen N, Hoogendoorn B, Oefner PJ, Cheadle JP, et al. Optimal temperature selection for mutation detection by denaturing HPLC and comparison to single-stranded conformation polymorphism and heteroduplex analysis. Clinical chemistry 1999 Aug;45(8):1133-1140 Cerca con Google

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