Toward Newborn Screening of Cerebrotendinous Xanthomatosis: Results of a Biomarker Research Study Using 32,000 Newborn Dried Blood Spots

PURPOSE: Cerebrotendinous xanthomatosis (CTX) is a treatable hereditary disorder caused by the deficiency of sterol 27- hydroxylase, which is encoded by the CYP27A1 gene. Different newborn screening biomarkers for CTX have been described, including 7α,12α-dihydroxy-4-cholesten-3-one ( 7α12αC4 ), 5b-cholestane-3 α, 7α,12α,25-tetrol glucuronide(GlcA-tetrol), GlcA-tetrol to tauro-chenodeoxycholic acid (t-CDCA) ratio (GlcA-tetrol/t-CDCA), and tauro- trihydroxycholestanoic acid (t-THCA) to GlcA-tetrol ratio (t-THCA/GlcA-tetrol ). We set out to evaluate these screening methods in a research study using 32,000–55,000 newborn dried blood spots (DBS). METHOD: Metabolites were extracted from DBS with methanol containing internal standard, which was then quantified by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). RESULTS: The measurement of 7α12αC4 was complicated by isobaric interferences and was discontinued after 2,033 samples. A total of 55,250 newborns were screened for the GlcA-tetrol/t-CDCA ratio, 32,737 of which had quantitative data on GlcA-tetrol. Only one newborn displayed both highly elevated GlcA-tetrol and a typical CTX biochemical profile. This newborn was interpreted as a CTX-affected patient as CYP27A1 gene sequencing identified two known pathogenic variants. CONCLUSION: The results indicate that both GlcA-tetrol and GlcA-tetrol/t-CDCA ratio are excellent CTX biomarkers suitable for newborn screening. By characterizing the relationship of GlcA-tetrol, t-CDCA, and t-THCA as secondary markers, 100% assay specificity can be achieved.