Top

Journal of Neurodevelopmental Disorders

Published in:

Open Access 01-12-2013 | Research

Methotrexate treatment of FraX fibroblasts results in FMR1 transcription but not in detectable FMR1 protein levels

Authors: Cornelia Brendel, Benjamin Mielke, Merle Hillebrand, Jutta Gärtner, Peter Huppke

Published in: Journal of Neurodevelopmental Disorders | Issue 1/2013

Abstract

Background

Fragile X syndrome is caused by the loss of FMRP expression due to methylation of the FMR1 promoter. Treatment of fragile X syndrome patients’ lymphoblastoid cells with 5-azadeoxycytidine results in demethylation of the promoter and reactivation of the gene. The aim of the study was to analyze if methotrexate, an agent which also reduces DNA methylation but with less toxicity than 5-azadeoxycytidine, has therapeutic potential in fragile X syndrome.

Methods

Fibroblasts of fragile X syndrome patients were treated with methotrexate in concentrations ranging from 1 to 4 μg/ml for up to 14 days. FMR1 and FMRP expression were analyzed by quantitative PCR and western blotting.

Results

FMR1 mRNA was detected and levels correlated positively with methotrexate concentrations and time of treatment, but western blotting did not show detectable FMRP levels.

Conclusions

We show that it is possible to reactivate FMR1 transcription in fibroblasts of fragile X syndrome patients by treatment with methotrexate. However, we were not able to show FMRP expression, possibly due to the reduced translation efficacy caused by the triplet repeat extension. Unless FMR1 reactivation is more effective in vivo our results indicate that methotrexate has no role in the treatment of fragile X syndrome.

Available only for authorised users

Verkerk AJ, Pieretti M, Sutcliffe JS, Fu YH, Kuhl DP, Pizzuti A, Reiner O, Richards S, Victoria MF, Zhang FP, Zhang F, Eussen BE, van Ommen GB, Blonden LAJ, Riggins GJ, Chastain JL, Kunst CB, Galjaard HC, Caskey T, Nelson DL, Oostraa BA, Warren ST: Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell. 1991, 65: 905-914. 10.1016/0092-8674(91)90397-H.CrossRefPubMed

Oberle I, Rousseau F, Heitz D, Kretz C, Devys D, Hanauer A, Boué J, Bertheas MF, Mandel JL: Instability of a 550-base pair DNA segment and abnormal methylation in fragile X syndrome. Science. 1991, 252: 1097-1102. 10.1126/science.252.5009.1097.CrossRefPubMed

Yu S, Pritchard M, Kremer E, Lynch M, Nancarrow J, Baker E, Holman K, Mulley JC, Warren ST, Schlessinger D, Sutherland GR, Richards RI: Fragile X genotype characterized by an unstable region of DNA. Science. 1991, 252: 1179-1181. 10.1126/science.252.5009.1179.CrossRefPubMed

Naumann A, Hochstein N, Weber S, Fanning E, Doerfler W: A distinct DNA-methylation boundary in the 5′- upstream sequence of the FMR1 promoter binds nuclear proteins and is lost in fragile X syndrome. Am J Hum Genet. 2009, 85: 606-616. 10.1016/j.ajhg.2009.09.018.PubMedCentralCrossRefPubMed

Pieretti M, Zhang FP, Fu YH, Warren ST, Oostra BA, Caskey CT, Nelson DL: Absence of expression of the FMR-1 gene in fragile X syndrome. Cell. 1991, 66: 817-822. 10.1016/0092-8674(91)90125-I.CrossRefPubMed

Alisch RS, Wang T, Chopra P, Visootsak J, Conneely KN, Warren ST: Genome-wide analysis validates aberrant methylation in fragile X syndrome is specific to the FMR1 locus. BMC Med Genet. 2013, 14: 18-PubMedCentralCrossRefPubMed

De Boulle K, Verkerk AJ, Reyniers E, Vits L, Hendrickx J, Van Roy B, Van den Bos F, de Graaff E, Oostra BA, Willems PJ: A point mutation in the FMR-1 gene associated with fragile X mental retardation. Nat Genet. 1993, 3: 31-35. 10.1038/ng0193-31.CrossRefPubMed

Lugenbeel KA, Peier AM, Carson NL, Chudley AE, Nelson DL: Intragenic loss of function mutations demonstrate the primary role of FMR1 in fragile X syndrome. Nat Genet. 1995, 10: 483-485. 10.1038/ng0895-483.CrossRefPubMed

Hagerman RJ, Hull CE, Safanda JF, Carpenter I, Staley LW, O’Connor RA, Seydel C, Mazzocco MM, Snow K, Thibodeau SN, Kuhl D, Nelson DL, Caskey T, Taylor AK: High functioning fragile X males: demonstration of an unmethylated fully expanded FMR-1 mutation associated with protein expression. Am J Med Genet. 1994, 51: 298-308. 10.1002/ajmg.1320510404.CrossRefPubMed

10.

Pietrobono R, Tabolacci E, Zalfa F, Zito I, Terracciano A, Moscato U, Bagni C, Oostra B, Chiurazzi P, Neri G: Molecular dissection of the events leading to inactivation of the FMR1 gene. Hum Mol Genet. 2005, 14: 267-277.CrossRefPubMed

11.

Smeets HJ, Smits AP, Verheij CE, Theelen JP, Willemsen R, van de Burgt I, Hoogeveen AT, Oosterwijk JC, Oostra BA: Normal phenotype in two brothers with a full FMR1 mutation. Hum Mol Genet. 1995, 4: 2103-2108. 10.1093/hmg/4.11.2103.CrossRefPubMed

12.

Tabolacci E, Moscato U, Zalfa F, Bagni C, Chiurazzi P, Neri G: Epigenetic analysis reveals a euchromatic configuration in the FMR1 unmethylated full mutations. Eur J Hum Genet. 2008, 16: 1487-1498. 10.1038/ejhg.2008.130.CrossRefPubMed

13.

Chiurazzi P, Pomponi MG, Willemsen R, Oostra BA, Neri G: In vitro reactivation of the FMR1 gene involved in fragile X syndrome. Hum Mol Genet. 1998, 7: 109-113. 10.1093/hmg/7.1.109.CrossRefPubMed

14.

Pietrobono R, Pomponi MG, Tabolacci E, Oostra B, Chiurazzi P, Neri G: Quantitative analysis of DNA demethylation and transcriptional reactivation of the FMR1 gene in fragile X cells treated with 5-azadeoxycytidine. Nucleic Acids Res. 2002, 30: 3278-3285. 10.1093/nar/gkf434.PubMedCentralCrossRefPubMed

15.

Christman JK: 5-Azacytidine and 5-aza-2′-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene. 2002, 21: 5483-5495. 10.1038/sj.onc.1205699.CrossRefPubMed

16.

Kishi T, Tanaka Y, Ueda K: Evidence for hypomethylation in two children with acute lymphoblastic leukemia and leukoencephalopathy. Cancer. 2000, 89: 925-931. 10.1002/1097-0142(20000815)89:4<925::AID-CNCR28>3.0.CO;2-W.CrossRefPubMed

17.

Nesher G, Moore TL, Dorner RW: In vitro effects of methotrexate on peripheral blood monocytes: modulation by folinic acid and S-adenosylmethionine. Ann Rheum Dis. 1991, 50: 637-641. 10.1136/ard.50.9.637.PubMedCentralCrossRefPubMed

18.

Winter-Vann AM, Kamen BA, Bergo MO, Young SG, Melnyk S, James SJ, Casey PJ: Targeting Ras signaling through inhibition of carboxyl methylation: an unexpected property of methotrexate. Proc Natl Acad Sci U S A. 2003, 100: 6529-6534. 10.1073/pnas.1135239100.PubMedCentralCrossRefPubMed

19.

Pascale E, Battiloro E, Cimino Reale G, Pietrobono R, Pomponi MG, Chiurazzi P, Nicolai R, Calvani M, Neri G, D'Ambrosio E: Modulation of methylation in the FMR1 promoter region after long term treatment with L-carnitine and acetyl-L-carnitine. J Med Genet. 2003, 40: e76-10.1136/jmg.40.6.e76.PubMedCentralCrossRefPubMed

20.

Bear MF, Huber KM, Warren ST: The mGluR theory of fragile X mental retardation. Trends Neurosci. 2004, 27: 370-377. 10.1016/j.tins.2004.04.009.CrossRefPubMed

21.

Ashley CT, Wilkinson KD, Reines D, Warren ST: FMR1 protein: conserved RNP family domains and selective RNA binding. Science. 1993, 262: 563-566. 10.1126/science.7692601.CrossRefPubMed

22.

Gross C, Berry-Kravis EM, Bassell GJ: Therapeutic strategies in fragile X syndrome: dysregulated mGluR signaling and beyond. Neuropsychopharmacology. 2012, 37: 178-195. 10.1038/npp.2011.137.PubMedCentralCrossRefPubMed

23.

McConkie-Rosell A, Lachiewicz AM, Spiridigliozzi GA, Tarleton J, Schoenwald S, Phelan MC, Goonewardena P, Ding X, Brown WT: Evidence that methylation of the FMR-I locus is responsible for variable phenotypic expression of the fragile X syndrome. Am J Hum Genet. 1993, 53: 800-809.PubMedCentralPubMed

24.

Jacob RA, Gretz DM, Taylor PC, James SJ, Pogribny IP, Miller BJ, Henning SM, Swendseid ME: Moderate folate depletion increases plasma homocysteine and decreases lymphocyte DNA methylation in postmenopausal women. J Nutr. 1998, 128: 1204-1212.PubMed

25.

Rampersaud GC, Kauwell GP, Hutson AD, Cerda JJ, Bailey LB: Genomic DNA methylation decreases in response to moderate folate depletion in elderly women. Am J Clin Nutr. 2000, 72: 998-1003.PubMed

26.

Brouwer JR, Mientjes EJ, Bakker CE, Nieuwenhuizen IM, Severijnen LA, Van der Linde HC, Nelson DL, Oostra BA, Willemsen R: Elevated Fmr1 mRNA levels and reduced protein expression in a mouse model with an unmethylated Fragile X full mutation. Exp Cell Res. 2007, 313: 244-253. 10.1016/j.yexcr.2006.10.002.PubMedCentralCrossRefPubMed

27.

Antequera F, Boyes J, Bird A: High levels of de novo methylation and altered chromatin structure at CpG islands in cell lines. Cell. 1990, 62: 503-514. 10.1016/0092-8674(90)90015-7.CrossRefPubMed

28.

Jones PA, Wolkowicz MJ, Rideout WM, Gonzales FA, Marziasz CM, Coetzee GA, Tapscott SJ: De novo methylation of the MyoD1 CpG island during the establishment of immortal cell lines. Proc Natl Acad Sci U S A. 1990, 87: 6117-6121. 10.1073/pnas.87.16.6117.PubMedCentralCrossRefPubMed

Title: Methotrexate treatment of FraX fibroblasts results in FMR1 transcription but not in detectable FMR1 protein levels
Authors: Cornelia Brendel
Benjamin Mielke
Merle Hillebrand
Jutta Gärtner
Peter Huppke
Publication date: 01-12-2013
Publisher: BioMed Central
Published in: Journal of Neurodevelopmental Disorders / Issue 1/2013
Print ISSN: 1866-1947
Electronic ISSN: 1866-1955
DOI: https://doi.org/10.1186/1866-1955-5-23

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Methotrexate treatment of FraX fibroblasts results in FMR1 transcription but not in detectable FMR1 protein levels

Abstract

Background

Methods

Results

Conclusions

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Background

Methods

Results

Conclusions

Please log in to get access to this content

Other articles of this Issue 1/2013

Spontaneous and cued gaze-following in autism and Williams syndrome

Developmental patterns of DR6 in normal human hippocampus and in Down syndrome

Comparative DNA methylation among females with neurodevelopmental disorders and seizures identifies TAC1 as a MeCP2 target gene

Organization of brain networks governed by long-range connections index autistic traits in the general population

Whole-exome sequencing supports genetic heterogeneity in childhood apraxia of speech

Working memory deficits in high-functioning adolescents with autism spectrum disorders: neuropsychological and neuroimaging correlates