Mass‐Spec Identification of Human Genetic Disease

Oxysterols are metabolites of cholesterol that play important roles in the brain, immune system, and liver. Mutations in four of seven genes that produce oxysterols cause learning defects, immunoglobulin abnormalities, and liver failure. To identify mutations in the remaining three genes, a mass spectrometry (MS)‐based method was developed to quantify sterols and oxysterols in small volumes (~200 μl) of human serum. Every procedural step was optimized, including organic solvents used to extract total lipids from serum, partial purification of sterols and oxysterols by aminopropyl silica chromatography, derivatization prior to gas chromatography‐MS, and resolution by ultra‐HPLC prior to quantification via isotope‐dilution electrospray or atmospheric pressure ionization MS. To test the method, levels of 60 sterols and oxysterols were determined in sera from 200 volunteers from the Cooper Aerobics Clinic in North Dallas. The power of the approach to detect mutations was illustrated by identification of an undiagnosed case of sitosterolemia (abnormal accumulation of plant sterols). Sterol and oxysterol levels are now being measured in ~3,500 individuals from the Dallas Heart Study, a cohort whose genomic DNAs and clinical histories are maintained on campus. Oxysterol biosynthetic and catabolic genes will be sequenced in individuals with oxysterol levels in the lowest and highest 5 th percentiles and the consequences of mutations identified determined from clinical histories.