A-042 Assessment of Known and Newly Discovered IGF-1 Variants by a High-throughput Clinical Reference Laboratory

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Abstract Background The top-down approach to quantitate intact insulin-like growth factor 1 (IGF-1) by liquid chromatography coupled to high-resolution, accurate-mass, mass spectrometry (LC-HRAM-MS) is a highly specific and robust technique. The method also provides a unique opportunity to study point mutations represented as amino acid sequence variants in IGF-1. While physical consequences of most variants remain unknown, those in or close to the binding region of the IGF-1 protein (positions 30-62) may be pathogenic, while those at the C-terminus are likely benign. In addition, information about the presence of IGF-1 variants is critical for accurate estimation of growth hormone levels. In the current study, our goal was to assess the presence of new and known variants by targeted screening of a clinical population. Methods LC-HRAM-MS was used for the detection of the wild type (WT) IGF-1 and its variants in specimens submitted for IGF-1 testing. In total, 2,013,503 specimens were screened. A novel variant detection and identification (NVDI) approach was employed that uses variant group m/z, isotopic peak index (IPi), and relative retention time (rRT) to characterize IGF-1 variants. Deidentified specimens were analyzed with tandem mass spectrometry (MS/MS) and DNA sequencing to confirm variants. Results In the specimens that were analyzed, 0.44% had IGF-1 variants and 98% of these had amino acid substitutions in the C-terminal region. We identified 8 variants represented in the Exome Aggregation Consortium (ExAC) database (A67T, A70T, P66A, A67S, S34N, A38V, R36Q, and T4M), 2 previously reported variants (V44M and A67V), and 11 new variants (Y31H, S33P, R50Q, R56K, T41I, A62T, S34G, N26K, V44L, G7A, and R36W). The underlined variants listed were discovered in the current study and are first reported here. In total, 16 variants had amino acid substitutions in or close to the binding region of the IGF-1 protein, and these were found in 0.4% of patients with variants in the clinical population. Conclusions In the study population, almost all patients with IGF-1 variants had point mutations affecting the C-terminal amino acid sequence. Although these variants are likely benign, they could affect patient care if IGF-1 levels were underestimated by being based purely on WT concentrations. Pathogenic or possibly pathogenic variants occurred in a small but important fraction of the study population. The discovery of novel variants further validates this platform as a discovery engine for IGF-1 variants. We expect more variants to be discovered with more patients and broader criteria. Information about the presence of IGF-1 variants is critical for proper estimation of growth hormone levels. Patients with variants should have their physician informed to properly assess the patient’s condition. This collaboration between clinical laboratories and attending physicians may result in the characterization and better understanding of the role and effect of IGF-1 variants on growth disorders.