In order to be included in the study, patients needed to have been diagnosed with either biallelic mutations in the mutated genes (found in underlying genomic mutations) or a dominant mutation in TREX1 or ADAR. Control samples were from a diverse population of individuals with no preexisting medical conditions and who were not taking medication during specimen collection. Additionally, data was collected from existing samples in which ISG activity was analyzed. 

Tulane University

The methodology for assessing interferon-related biomarkers in Aicardi-Goutières syndrome (AGS) involves defining a specific sample population with relevant mutations, conducting experimental procedures to measure the fold change of interferon-stimulated genes (ISGs) using RT-PCR, and performing statistical analysis to calculate and compare interferon scores against healthy controls.

Methods for Assessing Interferon-Related Biomarkers in AGS

Rice GI, Forte GM, Szynkiewicz M, Chase DS, Aeby A, Abdel-Hamid MS, Ackroyd S, Allcock R, Bailey KM, Balottin U, Barnerias C, Bernard G, Bodemer C, Botella MP, Cereda C, Chandler KE, Dabydeen L, Dale RC, De Laet C, De Goede CG, Del Toro M, Effat L, Enamorado NN, Fazzi E, Gener B, Haldre M, Lin JP, Livingston JH, Lourenco CM, Marques W Jr, Oades P, Peterson P, Rasmussen M, Roubertie A, Schmidt JL, Shalev SA, Simon R, Spiegel R, Swoboda KJ, Temtamy SA, Vassallo G, Vilain CN, Vogt J, Wermenbol V, Whitehouse WP, Soler D, Olivieri I, Orcesi S, Aglan MS, Zaki MS, Abdel-Salam GM, Vanderver A, Kisand K, Rozenberg F, Lebon P, Crow YJ. Assessment of interferon-related biomarkers in Aicardi-Goutières syndrome associated with mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, and ADAR: a case-control study. Lancet Neurol. 2013 Dec;12(12):1159-69. doi: 10.1016/S1474-4422(13)70258-8. Epub 2013 Oct 30. PMID: 24183309; PMCID: PMC4349523.

Assessment of interferon-related biomarkers in Aicardi-Goutières syndrome associated with mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, and ADAR: a case-control study

Sample Population

The 6 interferon stimulated genes (ISGs) were tested with 9 other ISGs and were selected for this experiment because they were highly expressed and easily differentiated from the controls. The median fold change of the ISGs compared to the median fold change of the control samples was used to construct an interferon score. The experiment included running the samples, the six ISGs and two internal controls, through the RT-PCR to measure the fold change of each ISG. The internal controls were housekeeping genes: HPRT1 and 18S. 

Procedure

Interferon (IFN) scores higher than 2 SD of the mean IFN scores of the controls were denoted as positive IFN signatures. Repeated samples were averaged and the mean value was used to determine the interferon signature. ISG values and interferon scores were plotted logarithmically and analyzed with parametric testing (t tests for two groups and ANOVA for more than 2 groups). Spearman rank correlation was used to compare age and CSF and serum interferon activity. 

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