Diagnostic method for myopathy

A myopathy and lipid technology, which is applied in the field of diagnosis of myopathy induced by statins, can solve the problems that there are no diagnostic methods or clinical tests for diagnosing non-symptomatic myopathy, and the risk of myopathy cannot be estimated in patients.

Inactive Publication Date: 2009-09-02
ZORA BIOSCIENCES OY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, there are no diagnostic methods or clinical tests for the diagnosis of asymptomatic myopathies
A

Method used

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  • Diagnostic method for myopathy
  • Diagnostic method for myopathy
  • Diagnostic method for myopathy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1. Gene expression analysis

[0054] gene expression

[0055] According to the instructions given, use Human-6 Expression Bead Chip Analysis Microarray experiments were performed on 46,000 known genes, candidate genes and splice variants (Illumina, San Diego, CA, USA). Biopsy samples were homogenized using an Ultra-Turrax (IKA Turrax T8 / S8N-5G, IKA-Werke, Staufen, Germany). Total RNA was extracted using TRIzol (#15596-018, Invitrogen Corporation, Carlsbad, CA). DNase treatment and secondary RNA purification were performed using Qiagen kits (#74106 and #79254, Qiagen GmbH, Hilden, Germany) according to the given instructions.

[0056] A 200 ng aliquot of total RNA from each sample was amplified as cDNA using Ambion's Illumina RNA Amplification Kit (Cat. No. 11755, Ambion, Inc., Austin, TX, USA) according to the instructions. Perform an overnight (14h) in vitro transcription (IVT) reaction from cDNA to cRNA containing biotin-11-dUTP (PerkinElmer, Cat. No. PC ...

Embodiment 2

[0060] Example 2.RT-PCR analysis

[0061] Based on hierarchical cluster analysis (as described in Example 1), 20 genes were selected for further RT-PCR controls to identify a gene expression-based fingerprint of the effect of statins on human skeletal muscle.

[0062] Microarray expression results recorded in the simvastatin group were validated by real-time quantitative TaqMan PCR (n=5, one case did not have enough muscle RNA for PCR). Use previously purified cRNA as a starting material for cDNA synthesis. 1000 ng-18 μl aliquots of cRNA were mixed with 1 μl Promega random primer (C1181, Promega U.S., Madison, WI, USA) and incubated at +70°C for 10 minutes. The following reagents were added to bring the total reaction volume to 25 μl: 1 μl of 10 μM dNTP mix (F09892, Applied Biosystems, Foster City, CA, USA), 1 μl of Promega M-MLV reverse transcriptase 200 U / μl (M3682) and 4 μl of M-MLV Reverse Transcription (RT) 5X Reaction Buffer. Finally, incubation was performed in the f...

Embodiment 3

[0065] Example 3. Lipidomic analysis of plasma

[0066] Aliquots of internal standard mixture (10ml) containing 11 lipid classes and 0.05M NaCl (10ml) were added to plasma samples (10ml) and lipids were extracted with chloroform / methanol (2:1, 100ml) . After vortexing (2 minutes), standing (1 hour) and centrifugation (10,000 RPM, 3 minutes), the lower layer was separated and a standard mixture (10 ml) containing 3 labeled standard lipids was added to the extract (inner Standards and external standards are listed in the appendix). The order of samples for LC / MS analysis was determined randomly.

[0067] Using Waters Q-Tof Premier mass spectrometer combined with Acquity ultra-high performance liquid chromatography (LC) TM (UPLC) to analyze lipid extracts. The column was maintained at 50°C and was an Acquity UPLC™ BEH C18 10 x 50mm containing 1.7mm particles. Binary solvent system (binary solvent system) includes: A. Water (1% 1M NH 4 Ac, 0.1% HCOOH); B. LC / MS grade (Rathbu...

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Abstract

This invention provides a diagnostic method for determining statin induced myopathy. The method is especially applicable for determining warnings, early signs and aiso symptomatic myopathy. The method inciudes coilecting a lipidomic profile from a biological sample such as blood or serum and comparing the obtained lipidomic profile to reference iipidomic markers. The reference iipidomic markers have been established by combining a pro-inflammatory muscle tissue gene expression profile with a iipidomic profile associated with high dosage statin treatment. This invention aiso relates to a kit for performing a method for determining statin induced myopathy.

Description

technical field [0001] The present invention relates to a diagnostic method for determining statin-induced myopathy. This method is especially useful for identifying warnings, early signs, and symptomatic myopathies. The method includes collecting a lipid biomarker profile and comparing it to reference lipidomic markers. The method also includes stoichiometric modeling and statistical analysis of the biomarker profile. The invention also relates to a kit for carrying out the diagnostic method for determining statin-induced myopathy. Background technique [0002] High levels of blood cholesterol are one of the main risk factors for atherosclerosis and cardiovascular disease. Clinically, elevated cholesterol levels can be reduced with 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (collectively known as statins). Statins have been shown in numerous clinical trials to be effective in lowering cholesterol blood levels. A large number of different statins currently exis...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N33/92G01N33/49G06F19/00
Inventor 雷约·拉克索宁马泰·奥伦西克泰尔霍·莱赫蒂迈基汉努·派瓦
Owner ZORA BIOSCIENCES OY
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