Lemonde, Hugh A.; (2001) A study using tandem mass spectrometry and molecular biological techniques to facilitate the diagnosis of inborn errors of bile acid synthesis, with particular reference to Δ4-3-oxosteroid 5β-reductase deficiency. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Inborn errors of bile acid synthesis can be difficult to diagnose. This thesis describes the use of a multidisciphnary approach (including tandem mass spectrometry, gas chromatography-mass spectrometry and molecular biological techniques) to aid in the diagnosis of this group of disorders. Low-energy CID (collision induced dissociation) tandem mass spectrometry using a triple quadrupole mass spectrometer equipped with a FAB (fast atom bombardment) ion source is shown to provide a rapid and powerful method for the identification and characterisation of bile acids in urine. Examples are presented of the application of this technique for the identification of unusual bile acids in the urine of patients with inborn errors of bile acid synthesis and other cholestatic liver diseases. Unfortunately, severe liver disease (regardless of cause) can lead to the production of the abnormal metabolites that are also characteristic of one particular inborn error of bile acid synthesis - Δ4-3-oxosteroid 5β-reductase deficiency. At the onset of this study, no patient had been described with a definitive diagnosis of Δ4-3-oxosteroid 5β-reductase deficiency caused by a mutation of the 5β-reductase gene. Hence, a parallel molecular biological approach was considered pertinent for the conclusive diagnosis of patients postulated to have this disorder. Using reverse transcription and polymerase chain reaction (PCR) techniques, mRNA encoding for this gene was characterised from the liver biopsy of patient MS. This patient was found to be homozygous for a missense mutation causing a single amino acid substitution. The presence of this mutation was confirmed by genomic DNA analysis and the parents of the patient were shown to be heterozygous for the same mutation. A similar analysis of one hundred chromosomes revealed the absence of this mutation in a normal population. These findings have significant implications in the understanding, diagnosis and thus in the treatment of this disease.

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