Update on newborn dried bloodspot testing for cerebrotendinous xanthomatosis: An available high-throughput liquid-chromatography tandem mass spectrometry method

Abstract

BackgroundCerebrotendinous xanthomatosis (CTX) is a rare genetic disorder of bile acid synthesis that can cause progressive neurological damage and premature death. Detection of CTX in the newborn period would be beneficial since an effective treatment is available. We previously described a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) test with potential to screen newborn dried bloodspots (DBS) for CTX. We report here modifications to the methodology and application of the modified test to analysis of DBS from a CTX-affected and unaffected newborns.MethodsThe testing methodology utilizes keto derivatization to enable sensitive LC-ESI-MS/MS measurement of elevated 7α,12α-dihydroxy-4-cholesten-3-one (7α12αC4) in CTX newborn DBS. We report here method modifications, including use of a DBS extraction procedure used in newborn screening laboratories and a reduced analysis time of 2 min per sample.ResultsRapid isotope-dilution LC-ESI/MS/MS quantification of the ketosterol bile acid precursor 7α12αC4 provides a test that could readily discriminate a CTX positive newborn DBS sample (with a concentration of 104.4 ng/ml) from unaffected newborn samples (with a mean concentration of 4.1 ± 3.4 ng/ml; range 0.2–15.6 ng/ml, n = 39) analyzed in a blinded manner.ConclusionsWe provide additional evidence suggesting 7α12αC4 may be a promising test marker to screen newborn DBS for CTX. Early detection and intervention through newborn screening would greatly benefit those affected with CTX, preventing morbidity and mortality.

Epidemiology, diagnosis, and treatment of cerebrotendinous xanthomatosis (CTX)

“…Including CTX in universal newborn screening programs could allow early detection and intervention, and in light of the potential benefits of early recognition and treatment, should be added to newborn screening programs when a validated method is available. Rapid isotope-dilution LC-ESI/MS/ MS quantification of the ketosterol bile acid precursor, 7α,12α-dihydroxy-4-cholesten-3-one, in dried bloodspot samples of newborns effectively discriminates between CTX and unaffected newborns, suggesting that 7α,12α-dihydroxy-4-cholesten-3-one may be a useful test marker for screening (Bleyle et al 2016). A recent study evaluating a new dried blood spot screening assay for CTX based on different ratios between the accumulating cholestanetetrol glucuronide (tetrol) and specific bile acids/bile acid intermediates taurochenodeoxycholic acid (t-CDCA) and taurotrihydroxycholestanoic acid (t-THCA), showed that the tetrol:t-CDCA ratio had excellent separation between CTX patients and controls, Zellweger patients, and newborns with cholestasis, suggesting that this derived biomarker has the potential for use in newborn screening programs .…”

Newborn screening for cerebrotendinous xanthomatosis is the solution for early identification and treatment

“…Biochemical approaches to screen newborn dried bloodspots (DBSs) for CTX have recently been described (22)(23)(24). The defect in CTX results in the accumulation of bile acid precursors, including the ketosterols, 7-hydroxy-4-cholesten-3-one and 7,12-dihydroxy-4-cholesten-3-one (712C4).…”

“…The defect in CTX results in the accumulation of bile acid precursors, including the ketosterols, 7-hydroxy-4-cholesten-3-one and 7,12-dihydroxy-4-cholesten-3-one (712C4). DeBarber and colleagues have demonstrated that elevated 712C4 can be measured in CTX newborn DBSs using LC-MS/MS (22,23). Analysis of this marker can be used to discriminate CTX-positive from CTX-negative newborn DBSs (22,23), although the necessity for LC and the cost of ketosterol derivatization reagent limits the applicability of this methodology as a first-tier test.…”

“…DeBarber and colleagues have demonstrated that elevated 712C4 can be measured in CTX newborn DBSs using LC-MS/MS (22,23). Analysis of this marker can be used to discriminate CTX-positive from CTX-negative newborn DBSs (22,23), although the necessity for LC and the cost of ketosterol derivatization reagent limits the applicability of this methodology as a first-tier test. The ketosterol, 712C4, is metabolized to form bile alcohol species, such as 5-cholestane-3,7,12-triol, or is further hydroxylated to form 5-cholestane-tetrol isomers.…”

A newborn screening method for cerebrotendinous xanthomatosis using bile alcohol glucuronides and metabolite ratios

“…The detection of CTX newborn patients, however, is hampered by the absence of a suitable neonatal screening method in dried blood spots (DBSs). DeBarber and coworkers (13,14) have published a candidate, high-throughput, newborn screening method for CTX that is based on the quantification of a ketosterol intermediate in DBSs using an LC-ESI/MS/MS method. A potential disadvantage is that a derivatization step is required before analysis, complicating implementation into existing neonatal screening programs and at higher costs.…”