Defects of bile acid synthesis represent a group of very rare inherited metabolic disorders. While several conditions within this group typically present with neonatal cholestasis and hepatopathy, cerebrotendinous xanthomatosis (CTX) is the most widely recognised entity and is increasingly diagnosed. Advances in biochemical and genetic diagnostics have revealed a broader phenotypic spectrum of CTX than previously appreciated, suggesting that the disease may be substantially underdiagnosed.

The estimated frequency of CTX varies considerably depending on the data source. Orphanet reports a prevalence of approximately 1:50 000; however, this estimate appears to be largely based on early observations by Lorincz et al., who identified a pathogenic variant in a single patient and one heterozygous carrier among 115 control individuals [36]. More robust prevalence estimates were provided by Appadurai et al., who analysed ExAC population data (exome sequence data of around 60 000 individuals from the Exome Aggregation Consortium) and estimated the prevalence of CTX in the European population to range between 1:130 000 and 1:460 000 [37]. Subsequent analyses have demonstrated variable geographical distribution, with higher estimated prevalence in Asian populations (1:44 407–1:93 084) and markedly lower prevalence in Finland (1:3 388 767) [38]. Despite these estimates, fewer than 600 patients with CTX have been reported worldwide, strongly suggesting underdiagnosis. Data on the prevalence of CTX in Central and Eastern Europe are currently lacking. Based on the prevalence estimates reported for the European population [37], and considering the population size of Slovakia (approximately 5,4 million), a rough estimate would suggest the presence of at least 40 affected individuals in the country.

In our cohort, we present metabolomic, molecular genetic, and clinical findings that are consistent with previously published data on cerebrotendinous xanthomatosis. Our observations further illustrate the broad and variable phenotypic spectrum of the disease, which extends beyond the classical triad of neurological impairment, cataracts, and tendon xanthomas. Increased awareness of this clinical heterogeneity is essential for earlier recognition of CTX.

In the typical course of CTX, prolonged neonatal or cholestatic icterus is often described as one of the earliest manifestations and may be accompanied by hepatopathy, although progression to liver failure is rare [39, 40].

As observed in our cohort, neonatal hepatic manifestations are frequently overlooked or remain diagnostically unresolved in early medical records, which may delay subsequent consideration of CTX later in life.

Another early clinical feature that may raise suspicion of CTX is chronic diarrhoea, particularly when occurring in combination with cataracts. Diarrhoea typically begins in childhood and may represent the sole clinical manifestation for several years [19, 41]. The underlying mechanism remains incompletely understood. In addition to impaired bile acid synthesis, increased intestinal motility and accelerated intestinal transit, potentially mediated by bile alcohols, have been proposed. This hypothesis is supported by reports demonstrating a correlation between decreased bile alcohol excretion and resolution of diarrhoea [19].

Although urinary bile alcohol analysis was not available in our cohort, diarrhoea was also among the first symptoms to improve after treatment initiation, consistent with published observations.

A cataract is recognised as one of the typical signs of CTX and is reported in over 70% of patients [9]. Cataracts usually occur in the first to the second decade; however, in some patients, they may only be observed in adulthood. Cataracts usually form in both eyes [9, 42]. In our cohort, cataracts were among the most frequent clinical findings, documented in the majority of patients. The age at detection varied considerably, ranging from early childhood to adulthood. Notably, one patient underwent cataract surgery as early as 6 years of age, whereas in other individuals, cataracts were identified or treated in the third decade of life. In several cases, cataracts preceded the onset of neurological symptoms by many years.

Another hallmark of CTX is the presence tendon xanthomas. Their reported prevalence varies across published cohorts. While some studies have described tendon xanthomas in all affected individuals [43], other reports indicate a lower occurrence, approaching 70% [9, 14, 22]. Tendon xanthomas typically develop during the second to third decade of life in patients with CTX [44].

In our cohort, visible tendon xanthomas were observed in 3 out of 8 patients. Three patients were diagnosed at a young age, and tendon xanthomas may not yet have developed at the time of clinical evaluation. In two additional patients without clinically apparent xanthomas, broadened Achilles tendons were noted, and lipid deposits were subsequently identified by USG. These findings suggest that tendon involvement may be missed on routine physical examination when visible xanthomas are absent and subtle changes can be easily overlooked. We therefore propose that, in such cases, ultrasonography should be considered to assess tendon involvement.

Of neurological symptoms, the most prevalent, according to the literature, are motor disturbances and ataxia [45,46,47], but Parkinsonism, mild/moderate intellectual disability, poor school performance, and epilepsy are also reported [20, 22, 48]. Polyneuropathy is often subclinical and is usually classified as axonal, occasionally demyelinating, or mixed, which represents a common feature of the disease [45].

In our patients, the most frequent neurological manifestations included spastic paraparesis and gait disturbances with dysarthria, which were present in all adult patients. Some patients also exhibited polyneuropathy; however, this was mainly subclinical and diagnosed by EMG.

According to published data, psychiatric manifestations are reported only in a subset of patients with CTX. They usually occur in the context of organic dementia, and several cases of behavioural changes, often manifesting at a younger age, have been described. While uncommon, some patients may predominantly present with psychiatric symptoms, at least in the early course of the disease [14, 20]. There is also a reported case of a young adult with CTX masquerading as attention-deficit/hyperactivity disorder (ADHD) [49]. A purely psychiatric presentation appears to be rare.

During the first two decades of life, psychiatric manifestations such as behavioural changes or intellectual disability should prompt consideration of CTX in the diagnostic work-up and trigger a targeted search for additional disease-specific features. In our cohort, psychiatric symptoms were frequent and were observed in 7 of 8 patients. All adult patients exhibited psychiatric disturbances of varying severity. In one patient (Case 3), psychiatric manifestations represented the dominant clinical feature.

Skeletal involvement represents a recognised, although less frequently discussed, manifestation of CTX. Reduced bone mineral density and osteoporosis, predominantly affecting the vertebrae and long bones, have been reported and may clinically manifest as an increased risk of fractures [10, 50].

Of note, one adolescent patient in our cohort (Case 4) was diagnosed following metabolic assessment prompted by the combination of lens clouding and osteoporosis associated with scoliosis. In total, osteoporosis was documented in three patients in our cohort, including the adolescent case and two adult patients (Cases 1 and 5). Recurrent bone fractures were observed in one additional adult patient (Case 2), although osteoporosis was not formally documented in this individual.

The pathogenesis of skeletal involvement in CTX remains incompletely understood. Proposed mechanisms include impaired vitamin D metabolism, particularly reduced levels of 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 [10, 51]. Additional contributing factors may include decreased intestinal calcium absorption and increased calcium mobilisation from bone, leading to reduced bone mineral density [52].

Although our data do not allow definitive conclusions regarding the timing of onset of skeletal involvement, the occurrence of osteoporosis in both adolescence and adulthood in our cohort suggests that reduced bone density may represent an underrecognised manifestation of CTX. In clinical practice, the presence of osteoporosis, especially when combined with early-onset cataracts, should raise suspicion of CTX and prompt further diagnostic evaluation.

Several clinical and imaging findings observed in our cohort deviated from the classical presentation of CTX. In addition to the typical triad, some patients presented with early or predominant psychiatric manifestations, while tendon xanthomas were absent or only detectable by imaging in younger individuals. Furthermore, MRI findings showed variability across patients and included cerebral atrophy, white-matter abnormalities, and signal changes in the dentate nuclei described in several cases. These observations highlight that CTX may present with non-classical or incomplete phenotypes, which can contribute to diagnostic delay. Recognising such atypical features may therefore be crucial for earlier diagnosis and timely initiation of treatment.

Bile acid synthesis in humans proceeds through two major pathways, both of which require the activity of sterol 27-hydroxylase. In cerebrotendinous xanthomatosis, deficiency of this enzyme results in impaired synthesis of chenodeoxycholic acid and disruption of normal bile acid homeostasis. As a consequence, sterol intermediates accumulate and are shunted into alternative metabolic pathways, leading to increased production of cholestanol and bile alcohols, which are thought to contribute to tissue injury, particularly within the nervous system [7, 53,54,55].

Although formal diagnostic guidelines for CTX have not been universally established, current expert recommendations emphasize biochemical testing as a key component of the diagnostic process, with measurement of serum cholestanol representing the most widely used biochemical marker [15]. Similarly, in the Mignarri suspicion index, cholestanol testing is included as a central diagnostic parameter whenever CTX is clinically suspected [9]. However, the sensitivity of cholestanol as a diagnostic marker appears to be lower than previously assumed. Several CTX patients with normal or near-normal serum cholestanol concentrations have been reported [17, 18, 56]. and reduced cholestanol levels have been described in association with glucocorticoid therapy [57] or treatment with ezetimibe [17]. Conversely, elevated cholestanol concentrations have also been reported in conditions other than CTX, such as cholestatic icterus [58] or as a result of certain medications (e.g., propofol) [59], thereby reducing the specificity of cholestanol as a diagnostic biomarker. Additional biomarkers may therefore be useful for improving the sensitivity and specificity of biochemical diagnostics.

In our cohort, serum cholestanol were markedly elevated in all CTX patients, ranging from 46,3 to 105,3 µmol/l. In addition, serum 7-dehydrocholesterol was consistently elevated alongside cholestanol and showed a many-fold increase, approximately 30- to 100-fold (23,3–85,4 µmol/l), compared with reference values (Table 3).

Those two markers present clear biochemical separation between CTX patients and healthy controls, as illustrated in Fig. 1. The close association between elevated cholestanol and 7-dehydrocholesterol levels, together with their distinct separation from controls, suggests that these parameters reflect disturbances in cholesterol and bile acid synthesis and and may represent useful complementary indicators in the evaluation of the disease.

Scatter plot of serum cholestanol and 7-dehydrocholesterol concentrations in CTX patients (red) and healthy controls (blue). CTX patients form a distinct cluster clearly separated from controls, highlighting the diagnostic value of these biochemical markers. Both axes are shown on a logarithmic scale, ellipses indicate the approximate distribution of each group

A practical advantage is that both analytes can be determined within a single GC-MS analytical run. In contrast, some biomarkers require instrumentation that is less widely available, such as LC-MS/MS [60,61,62], or require analysis of additional biological matrices, such as urine in the case of bile alcohols [63, 64]. On the other hand, because our cohort did not include CTX patients with normal or near-normal serum cholestanol concentrations, it is not possible to confirm whether 7-dehydrocholesterol remains elevated in such individuals. Further studies are needed to assess the usefulness of this two-marker approach, which could represent a more robust biochemical strategy in the biochemical evaluation of CTX.

The relationship between age and cholestanol concentration remains controversial in the literature, with some authors reporting increasing levels from infancy to adulthood in healthy individuals, while others describe relatively stable concentrations across age groups. The highest cholestanol concentration was observed in the oldest patient at the time of diagnosis (42 years, Case 5), whereas the lowest value was measured in the youngest patient (5 years, Case 8). This observation should be interpreted with caution, as no consistent relationship between age and cholestanol concentration was identified, and interindividual variability appeared substantial. Similarly, no clear differences in either cholestanol or 7-dehydrocholesterol concentrations by age or sex were observed in our cohort.

A biochemically based CTX diagnosis must be confirmed by molecular genetic analysis of the CYP27A1 gene. Sequence analysis of all 9 exons and flanking intronic regions of the CYP27A1 gene is warranted as the first step in the molecular genetic diagnosis scheme, since most pathogenic variants of CTX have the character of point mutations. In cases where no point mutations are identified despite persistent metabolic suspicion, analysis for deletions or duplications should be considered, as such variants have also been reported in CTX patients.

All the variants established in our patients, except c.1263 + 5G > A, were previously described. Transition c.1263 + 5G > A is not found in any public database of CTX patients; however, the transversion c.1263 + 5G > T has been described as pathogenic variant associated with CTX [35, 65]. We classified the c.1263 + 5G > A variant as pathogenic based on several criteria. Its pathogenicity is supported by in silico prediction tools (VarSome), which classified it as a class 5 variant. The variant was absent from control population databases (gnomAD exomes) and was identified in a patient with typical biochemical findings, including markedly elevated serum cholestanol and 7-dehydrocholesterol levels.

Of note, the variant c.1183 C > T (p.Arg395Cys) is historically among the first CYP27A1 variants reported in CTX patients [33]. This variant has been identified in multiple populations [36, 38, 66], raising the possibility that it represents a mutational hotspot. In accordance with previous reports, we also observed poor genotype–phenotype correlation among siblings diagnosed with CTX in our cohort.