Diagnosis of Barth syndrome using a novel LC‐MS/MS method for leukocyte cardiolipin analysis

Barth syndrome (BTHS) is an X-linked disorder characterised by cardiomyopathy, skeletal myopathy, growth retardation, neutropenia and 3-methylglutaconic aciduria. It is caused by mutations in the TAZ gene which codes for tafazzin, a protein with acyl transferase activity involved in synthesis of cardiolipin. Monolysocardiolipin (MLCL) is an intermediate in this process. Diagnosis of BTHS is difficult, as clinical and biochemical features are variable and numerous TAZ mutations have been described. These factors, together with lack of a straightforward diagnostic test are thought to have contributed to under-diagnosis of the condition. A novel method for cardiolipin analysis by reversed-phase ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) is reported which is less complicated and faster than previously described methods and uses a readily available sample type. The equipment, reagents and expertise required are found in most clinical laboratories performing metabolic investigations. Leukocytes were prepared from whole blood, phospholipids extracted and tetralinoleyl cardiolipin (CL4) and MLCL analysed by UPLC-MS/MS. Reference values were derived from analysis of 76 control and 23 BTHS samples as follows: CL4 in controls >132 (95 % CI 100-169), BTHS <30.2 (21.3-40.4) pmol/mg protein; MLCL/CL4 ratio in controls <0.006 (0.004-0.009) and >2.52 (1.51-4.22) in BTHS patients. We describe an improved method for CL4 and MLCL/CL4 analysis which can be incorporated into the routine work of a clinical biochemistry laboratory. It shows 100 % sensitivity and specificity for BTHS, making it a suitable diagnostic test.