A: Mass spectrum of a Bruton Tyrosyne Kinase peptide. B: Tandem mass spectrum of the tallest peak in panel A. C: Chromatogram (left) and MRM spectrum (right) for BTK 545:558 peptide.
The Hahn laboratory develops and validates assays that have important clinical applications for screening, diagnosing and monitoring diseases. We have been exploring a variety of new technologies and methods, including spectrophotometry, traditional and next-generation DNA sequencing and tandem mass spectrometry.
The Hahn laboratory has developed a mitochondrial respiratory chain assay for the diagnosis of mitochondrial disorders in skin fibroblasts and muscle tissues (Kramer et al., 2005; Oglesbee et al., 2006). This test, which is performed in only a few specialized centers worldwide, is a highly complex assay and is poorly standardized in the method, reagents and reference range among the clinical laboratories. The Hahn lab, in collaboration with the Sedensky laboratory and Morgan laboratory, has developed a valuable tool for the proficiency testing for the comparison of the assay as a quality improvement. This method is based on the mitochondria purified from C. elegans strains specifically deficient in one of the respiratory complex’s activities that can serve as a reference positive control (Chen et al., 2011).
Our team has unique expertise in developing and validating small molecule analysis using tandem mass spectrometry. A method for quantification of CoQ10 in WBC was developed that can be utilized to detect defects in this coenzyme pathway or in respiratory chain complexes in a subset of mitochondrial disorders, utilizing the blood cells instead of muscle to avoid invasive biopsies (Hahn et al., 2012; Vasta et al., 2011). Other mass spectrometry-based protocols have been developed in the Hahn laboratory, including pyridoxine dependent epilepsy (Jansen et al., 2014; Jung et al., 2013; Sadilkova et al., 2009).
While these tests involved the assay of small molecules accumulated in the serum, detection of defective proteins in a complex sample mixture such as dried blood spots by mass spectrometry has great potential for diagnosing genetic disorders. The Hahn laboratory has pioneered this new approach by targeting protein biomarkers for Wilson disease and primary immunodeficiencies (deWilde et al., 2008; Kerfoot et al., 2012). An example of this type of analysis is shown in the figure above. Signature peptides of a defined protein (in this case BTK) are selectively analyzed and quantified by tandem mass spectrometry which allows detecting a patient with absent or reduced protein of interest.
Currently, we are developing methods to screen or diagnose patients with Wilson disease and cystinosis. New protocols utilize specific antibodies in order to enrich the peptides of interest from biological samples. This will ultimately allow analysis of proteins from small patient samples that are collected non-invasively. This approach has the potential to be applied to dried blood spots for newborn screening.
Our laboratory has developed and implemented 24 traditional single-gene sequencing clinical tests and a next-generation DNA sequencing clinical test (nuclear mitome test) for mitochondrial disorders (DaRe et al., 2013; Hahn, 2012; Vasta and Hahn, 2013; Vasta et al., 2012; Vasta et al., 2009). The latter, targeting over 450 nuclear genes associated with mitochondrial disorders by next-generation sequencing (NGS), has been highly utilized in a clinical setting with promising results in which we might be able to avoid invasive procedures such as muscle biopsy and general anesthesia, particularly for patients at risk of developing serious complications (DaRe et al., 2013). Our laboratory is currently investigating the panel test for hereditary myopathy and brain development genes by NGS.
Sihoun Hahn, MD, PhD, is a principal investigator at Seattle Children’s Research Institute. He is a professor of pediatrics and adjunct professor of medicine at the University of Washington. He also directs Seattle Children’s Biochemical Genetics Program and Biochemical Genetics Laboratory. He came to Seattle Children’s in 2006 after spending six years at the Mayo Clinic as a professor of laboratory medicine and pathology, pediatrics and medical genetics, and as co-director of the Biochemical Genetics Laboratory.
His work has focused on developing a population screening method for Wilson disease, a genetic disease in which the body cannot excrete copper properly, leading to its accumulation in various organs including the liver and brain (Hahn, 2014; Bennett et al., 2013; Bennett and Hahn, 2011; deWilde et al., 2008; Hahn et al., 2002; Horslen and Hahn, 2010; Kroll et al., 2006; Perri et al., 2005). His other funded projects focus on developing peptide fingerprinting analysis by tandem mass spectrometry for various genetic disorders.
Hahn hopes to improve clinical practice through integrated laboratory testing true translational research. He remains a great believer in prevention: Identifying illness and providing interventions to patients before symptoms appear is always preferable to treating developed disease.
Hahn serves as a member of the Wilson Disease Association’s medical advisory committee, and of the Washington State Newborn Screening Advisory Committee. In 2009, he received the American College of Medical Genetics Foundation/Luminex Award.