Cerebrotendinous xanthomatosis (CTX, OMIM 213700) is a rare autosomal recessive disorder associated with deficiency of the CYP27A1 enzyme (sterol 27-hydroxylase), encoded by the CYP27A1 gene1,2. The presentation of clinical features in CTX is complex, with heterogeneous symptoms, variable severity and evolution of complications, and diverse differences in progression of disease even within the same family1,2. In a series of 43 cases in the United States, the mean age at diagnosis was 32 years3. In these patients, 53% experienced chronic diarrhea in childhood, 74% had cognitive impairment in childhood adolescence, 70% were diagnosed with premature cataracts in adolescence, 77% developed tendon xanthomas by the age of 25 years, and 81% experienced neurologic disease beginning in the third or fourth decade of life (range 20-40 years of age)3. Patients with CTX as a group are likely underdiagnosed and many patients often remain undiagnosed for more than 10 to 20 years after onset of initial symptoms, resulting in a significant burden of often irreversible neurologic disease4,5. Tools that may help mitigate underdiagnosis and late diagnosis include greater awareness of the disease amongst health care providers, availability of newborn screening and access to sensitive genetic and biochemical testing.

The accepted primary blood biomarker used for the diagnosis of CTX is an abnormally elevated cholestanol concentration. Accumulation of cholestanol in the central nervous system in CTX was first described by Menkes, Schimschock and Swanson in 19686. In 1971 it was proposed by Salen that a biochemical diagnosis of CTX could be established through measurement of an elevated plasma cholestanol concentration7. As a result of those early findings more than 50 years ago, cholestanol elevation has been regarded since as the main diagnostic biomarker for CTX. Conversely, identification of a normal concentration of cholestanol in plasma was considered definitive for exclusion of CTX.

Clinical tools to assist in establishing a diagnosis of CTX generally include measurement of cholestanol. For example, a clinical suspicion index for CTX initially proposed by Mignarri et al, recommends measurement of elevated plasma cholestanol for diagnosis of CTX in cases with suspicion index scores ≥100 (a score ≥200 is a formal indication for molecular analysis of the CYP27A1 gene)4. Additional diagnostic criteria for CTX were proposed by Sekijima et al in 2018, which also included measurement of elevated cholestanol for confirmatory diagnosis8. A recently published expert consensus study on diagnosing, treating and managing patients with CTX reported that genetic analyses and/or determination of serum (or plasma) cholestanol levels should be used for diagnostic confirmation of CTX9. All expert panelists indicated that patients with CTX always have elevated serum cholestanol at diagnosis and that measuring serum cholestanol is the diagnostic biomarker of choice. In addition, different groups have proposed screening of groups at high risk for CTX using elevated blood cholestanol as a screening biomarker. For example, Fernández-Eulate et al recently proposed prospective cholestanol screening for identification of CTX among patients with juvenile-onset unexplained bilateral cataracts10.

Additional biomarkers or biochemical signatures for CTX have been identified as useful for diagnosis of CTX11. For example, elevated levels of cholesterol precursors (including 7- and 8-dehydrocholesterol, lathosterol and lanosterol) can be present in CTX10. Although serum cholesterol is normal in most CTX patients, these biomarkers of hepatic and whole-body cholesterol “de-novo” synthesis are increased in untreated patients, indicating an increased rate of cholesterol synthesis in CTX12. In addition, metabolic end-products of the CYP27A1 enzyme, in particular chenodeoxycholic acid (CDCA), are deficient in CTX. The CYP7A1 enzyme mediated 7α-hydroxylation of cholesterol (a rate-limiting step in bile acid synthesis), is subject to diminished repression in patients with CTX due to the lack of CDCA, which results in a high rate of bile acid precursor synthesis and elevated levels of bile acid pathway intermediates in plasma and other tissues (see Figure 1), including 7α-hydroxy-4-cholesten-3-one (7αC4) and 7α,12α-dihydroxy-4-cholesten-3-one (7α12αC4)13,14. In CTX elevated 7α12αC4 is metabolized to generate elevated levels of C27 bile alcohols2 (Figure 1). Bile alcohol biomarkers are highly elevated in CTX and are predominately present as bile tetrol glucuronides in the blood and bile pentol glucuronides in urine15,16.

To facilitate the clinical use of 7αC4 and 7α12αC4 biomarkers, our group developed quantitative isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for 7αC4 and 7α12αC4 and reported normal concentration ranges as well as typical concentration ranges in the blood of untreated CTX patients with high cholestanol concentrations (see reference ranges in Table 1 and refer to13,14. Elevated 7αC4 was proposed in 2010 by our group as a sensitive marker for diagnosis of CTX and for monitoring of the efficacy of CTX treatment with bile acids14

Our group also developed a quantitative isotope-dilution LC-MS/MS assays for the predominant bile tetrol glucuronide, 5β-cholestane-3α,7α,12α,25-tetrol-3-O-β-D-glucuronide (25-tetrol glucuronide), present in bile and in blood15,16 (method modified from17), and the predominant bile pentol, 5β-cholestane-3α,7α,12α,23S,25-pentol (23S-pentol), present in urine18 (for which glucuronide conjugates are cleaved prior to the measurement of total 23S-pentol)19. We have reported normal blood and urine bile alcohol concentrations, as well as typical concentration reference ranges in untreated CTX patients with high cholestanol concentrations (see reference ranges in Table 1 and refer to17,19).

Here, we describe three new cases of genetically confirmed CTX in association with extensor tendon xanthomas (and in some cases other clinical CTX features) with normal to high normal levels of plasma cholestanol. Full biochemical characterization was performed for these CTX cases with bile acid precursor and bile alcohol biomarkers measured, including on treatment of these patients with CDCA.