High-throughput urine screening for Smith–Lemli–Opitz syndrome and cerebrotendinous xanthomatosis using negative electrospray tandem mass spectrometry
Introduction
Testing urine samples for a wide range of metabolites is an important screening tool for the diagnosis of a number of genetic diseases affecting intermediary metabolism. The analysis of urinary amino acids and organic acids with techniques such as high-performance liquid chromatography, thin-layer chromatography, gas chromatography-mass spectrometry (GC-MS) and electrophoresis is well-established in many laboratories and forms the basis of a “urine metabolic screen”. Tandem mass spectrometry (TMS) methods, offering improved sensitivity and throughput, have also been applied to these classes of compounds, initially in dried blood spot samples [1] but more recently in urine samples [2]. TMS methods also offer the potential to screen for a wider range of metabolites than the older techniques. For example, acyl carnitines are important markers for a number of inborn errors of metabolism and their measurement can be incorporated into the same TMS urine analysis used for amino acids. Furthermore, the potential to screen a wider range of metabolites using TMS raises the possibility of diagnosing disorders that have not traditionally been considered detectable by a routine urine metabolic screen.
Two examples of this are Smith–Lemli–Opitz syndrome (SLOS, 7-dehydrocholeserol reductase deficiency, OMIM 270400) and cerebrotendinous xanthomatosis (CTX, sterol 27-hydroxylase deficiency, OMIM 213700). In SLOS, cholesterol biosynthesis is impaired (Fig. 1) and the resulting cholesterol deficiency results in congenital malformations and endocrine abnormalities [3]. The conversion of cholesterol to bile acids is impaired in CTX (Fig. 1) and this condition is associated with progressive neurological decline, cataracts and tendon xanthomatomas [4]. Currently, diagnosis of these disorders relies on recognition of a specific constellation of clinical features and the subsequent measurement of abnormal metabolites using specific and specialised techniques [5], [6]. Because the clinical and laboratory experience necessary to perform these functions resides in only a few specialist metabolic centers, it is likely that there is some ascertainment bias in the diagnosis of these disorders and that the diagnosis of some cases may be delayed or even missed. However, a number of the clinical features of the two disorders would be expected to prompt more general testing for inborn errors of metabolism with a urine metabolic screen even if SLOS or CTX was not specifically suspected. Addition of CTX and SLOS to the panel of disorders detected by routine urine metabolic screening would be useful for the early diagnosis and treatment of these disorders. This paper describes the development of a suitable high-throughput TMS urine screening technique for these two disorders that was easily incorporated into an existing TMS urine screening program for amino and organic acid disorders.
Section snippets
Urine samples
Urine samples were obtained from three patients with SLOS (all had typical clinical signs and were confirmed by increased plasma levels of 7- and 8-dehydrocholesterol), four patients with CTX (typical clinical signs and increased levels of urine bile alcohols determined by gas chromatography) and 1738 controls. The controls included samples from neonates, older children and adults, hospitalised patients and patients with abnormal liver function, ketoacidosis, and hypoglycaemia. These controls
Steroid profiles
Urine samples from SLOS and CTX patients were initially analysed in detail using a profiling technique that detects a variety of steroid, bile acid and bile alcohol conjugates. Fig. 2, Fig. 3 show the profiles obtained by precursor ion scanning of ions characteristic for sulfates (97 m/z) and glucuronides (75 m/z). The sulfate profile from a patient with SLOS (Fig. 2B) showed increases in the ions 411, 427 and 438 m/z, relative to the control. The 411 m/z ion is consistent with
Discussion
The diagnosis of SLOS is usually prompted by typical multiple congenital abnormalities such as microcephaly, syndactyly, hypospadias and ambiguous genitalia and is confirmed by the finding of increased levels of 8- and 7-dehydrocholesterol in plasma, usually using GC-MS techniques [6]. More recently, a number of abnormal urine steroids resulting from 7-dehydrocholesterol metabolism have been identified in SLOS using GC-MS [7] and have been used to perform prenatal diagnosis using maternal urine
Acknowledgments
Ms. Mary Eggington and Ms. Avantika Mishra are thanked for expert technical assistance. Mr Peter Sharp, Department of Genetic Medicine, Women's and Children's Hospital, Adelaide provided the urine samples from CTX patients. Dr. Stanley Korman, Hadassah Hospital, Israel, provided constructive advice regarding this manuscript.
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