Self-Classified Topic Area(s): Cases in Clinical MS
Correlation of Genotyping and Phenotyping with the IGF-1 assays for Growth Hormone disorders
Ravinder J Singh Mayo Clinic
Ravinder Singh, PhD (Presenter) Mayo Clinic
Presenter Bio: Dr. Singh studies the application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to clinical laboratory analysis. Many of the methods that Dr. Singh developed are now considered reference methods. They have subsequently been utilized for method standardization efforts as well as to establish clinical disease correlates, which he has published with his collaborators.
Relevant Financial Disclosures
(within past 24 months, reported on Sep 02, 2025)
No relevant financial relationship(s) to disclose.
Abstract
Introduction:
Low or increased serum insulin-like growth factor 1 (IGF-1)1 concentrations might indicate growth hormone (GH) deficiency or overproduction, respectively. The rate of false positives is also a big concern by the clinicians with the current reference ranges. Currently, IGF-1 is mostly measured by automated immunometric assays. However, reagent availability issues prompted us to evaluate LC-MS with high-resolution accurate mass measurement (HRAM) as an alternative method.
Objectives: Validation of mass spec assay for IGF-1 and expanding its use in detecting pathological and non pathological mutations.
Methods:
For all samples and QCs, we added 400 μL of 7:1 ethanol/1 N HCl to 100 μL of human serum and 25 μL of internal standard to precipitate excess proteins and dissociate IGF-1 from binding proteins. Following 30 min incubation at room temperature, 90 μL of 1.5 M Tris was added to neutralize the solution, which was then centrifuged at 3000 rpm for 10 min. This was followed by 30 min incubation at −20 °C and centrifugation for 10 min at 3000 rpm. The resulting supernatant then underwent LC-MS analysis. Data collection, clinical correlation, and interpretation of thousands of samples have been performed in the lab.
Results:
We have identified common variants of IGF-1, A70T-IGF1 and A67T-IGF1, of unknown clinical significance by our new LC-MS HRAM assay, which cannot be distinguished from wild-type by the current market-leading immunoassay. Our method has also identified V44M pathogenic mutations in a few patients. One reason for doing this is that a “missed” pathogenic variant would give the clinically correct result for the wrong reason, which might result in a failed opportunity for definitive genetic family testing. The other reason is that several of the variants are either nonpathogenic or of uncertain significance, leading to a potentially falsely low IGF-1 result being reported.
Conclusions:
When known pathogenic mutations exist or discordant results with established immunoassays are discovered, variant protein sequences should be considered and evaluated. When mass spectrometric assays give unexpectedly low results, one should contemplate the possibility of the presence of variants and scan for them.
= Discovery stage. (19.79%, 2022)
= Translation stage. (37.97%, 2022)
= Clinically available. (42.25%, 2022)
