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

Abstract

Cerebrotendinous xanthomatosis (CTX) is a progressive metabolic leukodystrophy. Early identification and treatment from birth onward effectively provides a functional cure, but diagnosis is often delayed. We conducted a pilot study using a two-tier test for CTX to screen archived newborn dried bloodspots (DBSs) or samples collected prospectively from a high-risk Israeli newborn population. All DBS samples were analyzed with flow injection analysis (FIA)-MS/MS, and 5% of samples were analyzed with LC-MS/MS. Consecutively collected samples were analyzed to identify CTX-causing founder genetic variants common among Druze and Moroccan Jewish populations. First-tier analysis with FIA-MS/MS provided 100% sensitivity to detect CTX-positive newborn DBSs, with a low false-positive rate (0.1-0.5%). LC-MS/MS, as a second-tier test, provided 100% sensitivity to detect CTX-positive newborn DBSs with a false-positive rate of 0% (100% specificity). In addition, 5β-cholestane-3α,7α,12α,25-tetrol-3-O-β-D-glucuronide was identified as the predominant bile-alcohol disease marker present in CTX-positive newborn DBSs. In newborns identifying as Druze, a 1:30 carriership frequency was determined for the c.355delC CYP27A1 gene variant, providing an estimated disease prevalence of 1:3,600 in this population. These data support the feasibility of two-tier DBS screening for CTX in newborns and set the stage for large-scale prospective pilot studies.

Keywords: bile acids and salts; diagnostic tools; inborn errors of metabolism; mass spectrometry; storage diseases.

Fig. 1.

LC-MS/MS analysis of CTGS present in CTX-positive DBSs. A, B: Extracted ion chromatograms for MRM detection of 5β-cholestane-25-tetrol-glucuronide standard spiked at 500 ng/ml (A) and 5,000 ng/ml (B) into whole blood and spotted onto filter paper [* is the authentic standard at 1.45 min retention time, black and red traces are product ions m/z 85 and 75, respectively]. C, D: Extracted ion chromatograms for MRM detection of CTGS in representative adult (C) and newborn (D) CTX-positive DBSs. The primary CTGS peak present co-chromatographs with 5β-cholestane-25-tetrol-glucuronide standard. Additional CTGS peaks elute at retention time 1.32, 1.41, and 1.55 min that are putative 5β-cholestane-3α7α,12α,23- and 24-tetrol glucuronides (25).

Fig. 2.

Determination of disease markers in newborn DBSs as a test for CTX. A: CTGS/tCDCA ratio determined using FIA-MS/MS (all newborn samples, n = 1,250; numeric data is provided in Table 2). Note that the FIA-MS/MS signal detected for tCDCA includes all dihydroxycholanoic acid isomer species present. B, C: The 5β-cholestane-25-tetrol-glucuronide/tCDCA ratio (B) and 7α12αC4 concentration (C) determined using LC-MS/MS (approximately 5% of unaffected newborn samples plus CTX-positive, identified carriers, cholestasis, and non-newborn PBD-ZSD samples, n = 98; numeric data is provided in Table 3).

Fig. 3.

Two-tier screening approach to screen newborn DBSs for CTX. If the second-tier biochemical positive result is confirmed by genetic testing, or the genetic testing result is ambiguous, families should be contacted for follow-up, which should provide diagnostic and medical evaluation, genetic counseling, and referral to all needed specialists.