Toward newborn screening of cerebrotendinous xanthomatosis: results of a biomarker research study using 32,000 newborn dried blood spots

Genet Med. 2020 Oct;22(10):1606-1612. doi: 10.1038/s41436-020-0846-x. Epub 2020 Jun 10.


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), 5β-cholestane-3α,7α,12α,25-tetrol glucuronide (GlcA-tetrol), the ratio of GlcA-tetrol to tauro-chenodeoxycholic acid (t-CDCA) (GlcA-tetrol/t-CDCA), and the ratio of tauro-trihydroxycholestanoic acid (t-THCA) to GlcA-tetrol (t-THCA/GlcA-tetrol). We set out to evaluate these screening methods in a research study using over 32,000 newborn dried blood spots (DBS).

Methods: Metabolites were extracted from DBS with methanol containing internal standard, which was then quantified by ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS).

Results: The measurement of 7α12αC4 was complicated by isobaric interferences and was discontinued. A total of 32,737 newborns were screened based on the GlcA-tetrol concentration in DBS. GlcA-tetrol/t-CDCA and t-THCA/GlcA-tetrol ratios were also calculated. Newborns displaying both elevated GlcA-tetrol and GlcA-tetrol/t-CDCA ratio were considered to be screen positives. The t-THCA/GlcA-tetrol ratio was used to further distinguish CTX screen positives from Zellweger Spectrum Disorder (ZSD) screen positives. Only one newborn displayed both elevated GlcA-tetrol concentration in DBS 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 the 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.

Keywords: CYP27A1; biomarker; biomarker ratio; cerebrotendinous xanthomatosis; newborn screening.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Biomarkers
  • Chromatography, Liquid
  • Humans
  • Infant, Newborn
  • Neonatal Screening
  • Tandem Mass Spectrometry
  • Xanthomatosis, Cerebrotendinous* / diagnosis
  • Xanthomatosis, Cerebrotendinous* / genetics


  • Biomarkers