Sirtuin1 single nucleotide polymorphism (A2191G) is a diagnostic marker for vibration-induced white finger disease
© Voelter-Mahlknecht et al.; licensee BioMed Central Ltd. 2012
Received: 25 April 2012
Accepted: 25 July 2012
Published: 1 October 2012
Vibration-induced white finger disease (VWF), also known as hand-arm vibration syndrome, is a secondary form of Raynaud’s disease, affecting the blood vessels and nerves. So far, little is known about the pathogenesisof the disease. VWF is associated with an episodic reduction in peripheral blood flow. Sirtuin 1, a class III histone deacetylase, has been described to regulate the endothelium dependent vasodilation by targeting endothelial nitric oxide synthase. We assessed Sirt1single nucleotide polymorphisms in patients with VWF to further elucidate the role of sirtuin 1 in the pathogenesis of VWF.
Peripheral blood samples were obtained from 74 patients with VWF (male 93.2%, female 6.8%, median age 53 years) and from 317 healthy volunteers (gender equally distributed, below 30 years of age). Genomic DNA was extracted from peripheral blood mononuclear cells and screened for potential Sirt1single nucleotide polymorphisms. Four putative genetic polymorphisms out of 113 within the Sirt1 genomic region (NCBI Gene Reference: NM_012238.3) were assessed. Allelic discrimination was performed by TaqMan-polymerasechainreaction-based allele-specific genotyping single nucleotide polymorphism assays.
Sirt1single nucleotide polymorphism A2191G (Assay C_25611590_10, rs35224060) was identified within Sirt1 exon 9 (amino acid position 731, Ile → Val), with differing allelic frequencies in the VWF population (A/A: 70.5%, A/G: 29.5%, G/G: 0%) and the control population (A/A: 99.7%, A/G: 0.3%, G/G: 0.5%), with significance levels of P < 0.001 (Mann–Whitney U test (two-tailed) P <0.001; F-exact t-test and Chi-square test with Yates correction (all two-tailed): P <0.0001). The heterogeneous A/G genotype in base pair position 2191 is significantly overrepresented in the VWF patient population when compared with healthy controls.
We identified theSirt1A2191Gsingle nucleotide polymorphism as a diagnostic marker for VWF.
KeywordsEpigenetics Hand-arm vibration syndrome (HAVS) Sirtuins Vibration-induced white finger disease (VWF)
Vibration-induced white finger disease (VWF) is an industrial injury that is triggered by the continued use of vibrating hand-held machinery. The disease is a widespread and officially recognized occupational disease affecting tens of thousands of employees. According to data that have been published by the Medical Research Council, around 2 million people in Britain are continuously subjected to potentially harmful levels of hand-arm vibration and around 300,000 people are anticipated to suffer from moderate to severe finger blanching (VWF) linked to such exposure, which may lead to considerable time off work, early retirement and considerable payouts from civil compensation schemes. In fact, a UK government fund that had been set up to cover claims by ex-coalminers who were exposed to the use of vibrating hand-held machinery had exceeded £100 million in payments by 2004.
The pathogenesis of this disease is currently unclear. Epigenetics is gaining increasing importance in the understanding of numerous diseases. Epigenetic pathways have recently been suggested to be important in the regulation of vascular gene expression in the pathophysiology of atherosclerosis[6, 7], the microvascular environment of tumors, cytokine-inducible gene expression in vascular endothelium, and in the developmental regulation of vascular remodeling[10, 11]. Chromatin-based regulatory mechanisms may therefore play a key role in the constitutive expression of endothelium-restricted genes.
A single nucleotide polymorphism (SNP) is defined as the difference between chromosomes in the base present at a particular site in the DNA sequence that naturally occurs within a population, and presents the most common type (90%) of genetic variation in humans. Hopefully, increasing knowledge of an individual’s SNP genotype may contribute to the assessment of disease susceptibility and individualized treatment modalities.
Based on structural and functional similarities, mammalian histone deacetylases (HDACs) are grouped into four categories. There are three classes of non-sirtuin HDACs, comprising the yeast HDACright parietal dorsal 3 homologs (class I HDACs); class II HDACs, which share a significant degree of homology with the yeast HDA1; and the most recently described class IV HDACs, which comprise HDAC11-related enzymes. There is one class of sirtuin HDACs (class III HDACs), which are homologs to the yeast Sir2 protein.
The yeast Sir2 protein has seven human homologs (SIRT1-7), which play a central role in epigenetic gene silencing, DNA repair and recombination, cell-cycle, microtubule organization, and in the regulation of aging.
SIRT1, which is a member of the Sir2 family of NAD+-dependent HDACs, deacetylates histone H3 lysines 9 and 14 and specifically histone H4 lysine-16, while it hydrolyzes one molecule of NAD+ for every lysine residue that is deacetylated[14, 15]. Derivatives of the yeast Sir2 HDAC share a common catalytic domain, which is highly conserved in organisms ranging from bacteria to humans and which is composed of two distinct motifs that bind NAD+ and the acetyl-lysine substrate, respectively[14–17]. SIRT1 is known to directly modify chromatin and to silence transcription, to modulate the meiotic checkpoint, and as a probable antiaging effect, to increase genomic stability and to suppress recombinant DNA recombination[18, 19]. While, for yeast Sir2, no targets are known apart from histones, SIRT1 has a large and still growing list of targets,including p53 and forkhead transcription factors, which are mammalian homologs of Daf-16 and which are known to function as sensors of the insulin signaling pathway[14, 18].
Results and discussion
Comparison of the allelic distribution of patients with vibration-induced white finger disease and healthy controls
dbSNP [Assay ID]
Patients with VWF
Allele A/A: 0/74
Allele A/T: 0/74
Allele T/T: 74/74
Allele A/A: 0/203
Allele A/T: 0/203
Allele T/T: 203/203
Allele A/A: 0/74
Allele A/T: 0/74
Allele T/T: 74/74
Allele A/A: 0/200
Allele A/T: 1/200
Allele T/T: 199/200
Allele C/C: 0/49
Allele C/T: 0/49
Allele T/T: 49/49
Allele C/C: 0/299
Allele C/T: 0/299
Allele T/T: 299/299
Allele A/A: 52/74
Allele A/G: 22/74
Allele G/G: 0/74
Allele A/A: 316/317
Allele A/G: 1/317
Allele G/G: 0/317
Allele A/A: 62/68
Allele A/G: 6/68
Allele G/G: 0/68
Allele A/A: 170/189
Allele A/G: 18/189
Allele G/G: 1/189
Allele A/A: 58/66
Allele A/G: 8/66
Allele G/G: 0/66
Allele A/A: 171/193
Allele A/G: 22/193
Allele G/G: 0/193
Sirt1 Intron 4
To date, little is known about the pathogenesis of VWF. It is however widely accepted that both the nervous and the vascular systems are affected and that vasoconstrictive effects dominate over vasodilatative effects during a vasospastic attack. In this process, endothelium-dependent and endothelium-independent mechanisms regulating the vascular tone may be distinguished. The endothelium-dependent vascular regulation is based on the interplay of competing vasoconstrictive and vasodilatative substrates, of which nitric oxide (NO), a factor that significantly depends on the activity of the endothelial isoform of NO synthase (eNOS, synonym: NOS3), is a key player.
There is increasing evidence that epigenetic mechanisms play a key role in a number of vascular disorders[10, 20]. SIRT1 and eNOS co-localize and co-precipitate in endothelial cells, and SIRT1 deacetylates eNOS, thus stimulating eNOS activity, which subsequently increases endothelial NO. Fully functional Sirt1 is essential in the regulation of endothelium-dependent vasodilation and may have profound effects not only on the control of the local vascular tone and systemic blood pressure, but also in the pathogenesis of VWF.
Our study shows an important, highly significant accumulation of the heterogeneous genotype of the Sirt1 SNP A2191G (Ile → Val) when compared with healthy controls (P <0.001).
We therefore claim the Sirt12191 A/G genotype to be a risk factor for VWF, and that it may be used as a biological marker to facilitate the identification of risk populations who are being considered for exposure to potentially hazardous hand-held vibrating machinery. This may prevent thousands of employees from experiencing physical pain and disability, in addition to long-lasting administrative fights for VWF compensation, which frequently end unsettled and in great frustration.
Patients and control group
Peripheral blood samples were obtained from 74 patients with VWF and from 317 healthy volunteers. Informed consent was given and the study was approved by the local ethics committee. The patient group was composed of 69 male (93.2%) and five female (6.8%) patients with a median age of 53 years, ranging from 29 to 74 years; the gender contribution is caused by a higher frequency on VWF in men and reflects the real world gender distribution of the disease. In the control group, genders were equally distributed (all below the age of 30 years). Genomic DNA was extracted from 5 mL peripheral EDTA (ethylenediaminetetraacetic acid)-blood with the FlexiGene DNA Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA quantification was carried out on a Nanodrop ND-1000 Spectrophotometer (Peqlab Biotechnologie GmbH, Erlangen, Germany). Samples were then stored in double diluted H2O at −20°C.
In silico analysis
Based on in silico expression analyses with the EST profile viewer (National Center for Biotechnology Information (NCBI)), a strong overexpression of Sirt1 was observed in vascular endothelial cells. In addition, we had observed increased Sirt1 expression levels in response to the NO liberator sodium nitroprusside in earlier analyses. Since VWF results from episodic reduction in peripheral blood flow in response to occupational exposure to hand-held vibrating machinery, and Sirt1 has been reported to promote endothelium-dependent vasodilation by targeting eNOS, we screened the Database of Single Nucleotide Polymorphisms (dbSNP; NCBI), the HapMap database and the Applied Biosystems database (TaqMan SNP Genotyping Assays, Applied Biosystems, Life Technologies Corporation, 5791 Van Allen Way, PO Box 6482 Carlsbad, California 92008 for potential Sirt1 SNPs in order to further elucidate the role of Sirt1 in the pathogenesis of VWF. We focused our search criteria on coding nonsynonymous SNPs, in addition to a Sirt1 intron 4 SNP and a Sirt1 promoter SNP. As the HapMap data are publicly released to dbSNP, we performed our search mainly via dbSNP and the Applied Biosystems database, which both included HapMap data.
Overview of the potential nonsynonymous Sirt1 SNPsin silico and analyzed
dbSNP [Assay ID]
5′Near Seq 30 bp
3′Near Seq 30 bp
Amino acid position
ancestral allele: T
ancestral allele: T
ancestral allele: T
ancestral allele: n.a.
ancestral allele: A
ancestral allele: A
This work was supported by institutional funds from the Saarland University Medical Center, Department of Internal Medicine, Division of Immunotherapy and Gene Therapy and the Johannes Gutenberg University Mainz, Medical School, Center for Preventive Medicine.
- Palmer KT, Coggon D, Bendall HE, Pannett B, Griffin MJ, Haward BM: Hand-transmitted vibration: occupational exposures and their health effects in Great Britain. 1999, Suffolk: HSE Books, Sudbury
- Bolognia JL, Jorizzo JL, Rapini RP: Dermatology: 2-Volume Set. 2007, St. Louis, MO: Mosby
- Taylor W: The vibration syndrome. 1974, London: Academic
- Dupuis H: Wirkung mechanischer Schwingungen auf das Hand-Arm-System. 1982, BAU Dortmund: Literaturanalyse
- Dupuis H, Riedel S: Handbuch der Arbeitsmedizin. Vibrationsbedingtes Vasospastisches Syndrom VS (BK 2104). Edited by: Konietzko J, Dupuis H. 1999, Landsberg: Ergomed Verlag, 3-4. 2
- Dong C, Yoon W, Goldschmidt-Clermont P: DNA methylation and atherosclerosis. J Nutr. 2002, 132: 2406S-2409S.PubMed
- Lund G, Andersson L, Lauria M, Lindholm M, Fraga MF, Villar-Garea A, Ballestar E, Esteller M, Zaina S: DNA methylation polymorphisms precede any histological sign of atherosclerosis in mice lacking apolipoprotein E. J Biol Chem. 2004, 279: 29147-29154. 10.1074/jbc.M403618200.View ArticlePubMed
- Kim M, Kwon H, Lee Y, Baek JH, Jang JE, Lee SW, Moon EJ, Kim HS, Lee SK, Chung HY, Kim CW, Kim KW: Histone deacetylases induce angiogenesis by negative regulation of tumor suppressor genes. Nat Med. 2001, 7: 437-443. 10.1038/86507.View ArticlePubMed
- Edelstein L, Pan A, Collins T: Chromatin modification and the endothelial-specific activation of the E-selectin gene. J Biol Chem. 2005, 280: 11192-11202. 10.1074/jbc.M412997200.PubMed CentralView ArticlePubMed
- Fish J, Matouk C, Rachlis A, Lin S, Tai SC, D’Abreo C, Marsden PA: The expression of endothelial nitric-oxide synthase is controlled by a cell-specific histone code. J Biol Chem. 2002, 280: 24824-24838.View Article
- Wu J, Iwata F, Grass J, Osborne CS, Elnitski L, Fraser P, Ohneda O, Yamamoto M, Bresnick EH: Molecular determinants of NOTCH4 transcription in vascular endothelium. Mol Cell Biol. 2005, 25: 1458-1474. 10.1128/MCB.25.4.1458-1474.2005.PubMed CentralView ArticlePubMed
- Collins FS, Brooks LD, Chakravarti A: A DNA polymorphism discovery resource for research on human genetic variation. Genome Res. 1998, 8: 1229-1231.PubMed
- Barnes MR: SNP and mutation data on the web - hidden treasures for uncovering. Comp Funct Genomics. 2002, 3: 67-74. 10.1002/cfg.131.PubMed CentralView ArticlePubMed
- Zschoernig B, Mahlknecht U: SIRTUIN 1: regulating the regulator. Biochem Biophys Res Commun. 2008, 376: 251-255. 10.1016/j.bbrc.2008.08.137.View ArticlePubMed
- Zschoernig B, Mahlknecht U: Carboxy-terminal phosphorylation of SIRT1 by protein kinase CK2. Biochem Biophys Res Commun. 2009, 381: 372-377. 10.1016/j.bbrc.2009.02.085.View ArticlePubMed
- Mahlknecht U, Voelter-Mahlknecht S: Chromosomal characterization and localization of the NAD + −dependent histone deacetylase gene sirtuin 1 in the mouse. Int J Mol Med. 2009, 23: 245-252.PubMed
- Voelter-Mahlknecht S, Mahlknecht U: Cloning, chromosomal characterization and mapping of the NAD-dependent histone deacetylases gene sirtuin 1. Int J Mol Med. 2006, 17: 59-67.PubMed
- Blander G, Guarente L: The Sir2 family of protein deacetylases. Annu Rev Biochem. 2004, 73: 417-435. 10.1146/annurev.biochem.73.011303.073651.View ArticlePubMed
- Vaquero A, Scher M, Lee D, Erdjument-Bromage H, Tempst P, Reinberg D: Human SirT1 interacts with histone H1 and promotes formation of facultative heterochromatin. Mol Cell. 2004, 16: 93-105. 10.1016/j.molcel.2004.08.031.View ArticlePubMed
- Tai S, Robb G, Marsden P: Endothelial nitric oxide synthase: a new paradigm for gene regulation in the injured blood vessel. Arterioscler Thromb Vasc Biol. 2004, 24: 405-412. 10.1161/01.ATV.0000109171.50229.33.View ArticlePubMed
- Mattagajasingh I, Kim CS, Naqvi A, Yamamori T, Hoffman TA, Jung SB, DeRicco J, Kasuno K, Irani K: SIRT1 promotes endothelium-dependent vascular relaxation by activating endothelial nitric oxide synthase. Proc Natl Acad Sci U S A. 2007, 104: 14855-14860. 10.1073/pnas.0704329104.PubMed CentralView ArticlePubMed
- Engel N, Mahlknecht U: Aging and anti-aging: unexpected side effects of everyday medication through sirtuin1 modulation. Int J Mol Med. 2008, 21: 223-232.PubMed
- Smigielski EM, Sirotkin K, Ward M, Sherry ST: dbSNP: a database of single nucleotide polymorphisms. Nucleic Acids Res. 2000, 28: 352-355. 10.1093/nar/28.1.352.PubMed CentralView ArticlePubMed
- International HapMap Consortium: The International HapMap Project. Nature. 2003, 426: 789-796. 10.1038/nature02168.View Article
- De La Vega FM, Dailey D, Ziegle J, Williams J, Madden D, Gilbert DA: New generation pharmacogenomic tools: a SNP linkage disequilibrium Map, validated SNP assay resource, and high-throughput instrumentation system for large-scale genetic studies. Biotechniques. 2002, 52: 48-50. 54
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.