Abstract:
Homocystinuria is an inborn error of metabolism caused primarily by deficiency of the cystathionine -synthase enzyme (CBS). Measurement of methionine by expanded screening by electrospray-ionisation tandem mass spectrometry (ESI-MS/MS) has identified almost 800 false-positive tests for CBS deficiency in New Zealand since its launch in December 2006. However, no clinical cases have been identified. This thesis describes the development and evaluation of three biochemical methods and a molecular method to identify quality improvements that will reduce the high false-positive rate associated with CBS deficiency screening. A second-tier total homocysteine assay has been developed using LC-MS/MS (liquid chromatography-tandem mass spectrometry) technology and evaluated for accuracy, precision, analytical sensitivity, linearity and analyte recovery. UPLC-UV (ultra-performance liquid chromatography with ultraviolet detection) and enzymatic total homocysteine assay methods have also been investigated. In addition, a molecular genetic approach involving the development of a novel MALDI-TOF (matrix-assisted laser desorbtion/ionisation – time of flight) mass spectrometry assay targeting two common variant alleles in the CBS gene is also described in this study. Testing the LC-MS/MS total homocysteine assay on the population of newborns that had an initial positive screen excluded CBS deficiency from 98.8% thus potentially reducing the number of false positive tests from about 100 to 1-2 per year. . Targeted genotyping of two common variants using MALDI-TOF mass spectrometry detected 20% of the variant alleles in a group of known CBS deficient patients, thereby identifying 35% of the individuals known to have at least one variant allele in the CBS gene. The methods are evaluated based on their analytical results and their applicability to screening. This study demonstrates that implementation of a second-tier LC-MS/MS total homocysteine is likely to be the most effective method in reducing the high false-positive rate associated with screening for CBS deficiency using the current primary marker methionine.
