Suspected pathophysiologic mechanism
Cholesterol undergoing a series of enzymatic transformations is the primary source of bile acids in the human body. About 90% of bile acids are produced by a process that takes place only in the liver cells - called the classic pathway. Its final product under normal conditions is cholic acid and CDCA, which also play the role of negative feedback in the whole loop for the initial enzyme 7α-hydroxylase. However, things start to change when CYP27A1mutations occur. As they are a causative factor of sterol 27-hydroxylase dysfunction, it results in the inability to enzymatically process the intermediate product of the pathway - 7α-hydroxy-4-cholesten-3-one (7αC4).
Conversely, the lack of end products of the pathway impairs the negative feedback in the loop. This results in significant 7αC4 overproduction. Due to the lack of any other enzymatic option, it is converted mainly into cholestanol and to some degree into bile alcohols. As a result, excessive cholestanol and other redundant metabolites are deposited in lipophilic tissues. There is also an alternative way of metabolizing cholesterol in our bodies. However, it is responsible for only 10% of the process, and in addition, its final stages also take place in hepatocytes using 27-hydroxylase. The simplified enzymatic mechanism of dysfunction in bile acid synthesis is shown in the illustration below.
Diagnostic testing
Knowing the pathology involved in the cholesterol oxidation process allowed to replace the initial analyses of tissue deposits by determining cholestanol levels. Unfortunately, the limitations here were the technological possibilities of laboratories in various parts of the world. These specific differences also made it difficult to standardize the cholestanol/cholesterol ratio for particular age groups.
In the case of the classical course of the disease, in the presence of clinical symptoms, the level of cholestanol almost always exceeds the upper limit of the normal range (usually four to five-fold over average). However, there have been reports of a very mild course of the disease (subclinical) in which the level of cholestanol was not elevated.
Since cholestanol is an intermediate byproduct of the cholesterol pathway, it is evident that various factors will have an additional influence on this process. Examples include age-related changes in cholesterol absorption and individual differences in the gut microbiota. There have also been two reports of patients whose cholestanol (urine) was measured after propofol anesthesia, leading to abnormally high values. Moreover, chronic steroid therapy reduced cholesterol levels to normal in one case. It should be noted that increased cholestanol levels are not exclusive to CTX and can be seen in patients with familial hypercholesterolemia or sitosterolemia. Some studies described additional markers as even more suitable for diagnosing and monitoring therapy effectiveness, including mentioned intermediate 7αC4, and cholestane-3β,5α,6β-triol.
Problems related to diagnosis based on reliable evaluation of intermediates (markers) partially resolved in 1991 when the causative mutation for CTX was described. But, of course, it took time for genetic tests to become more widely available. Therefore, nowadays, if possible, biochemical CTX diagnosis should always be confirmed by finding the biallelic pathogenic CYP27A1mutations. This was additionally underlined in a 2020 scientific statement of the US National Lipid Association that genetic testing for CTX may directly affect patient care.
To enhance decision-making in testing for CTX, a team of Italian researchers developed the 'suspicion index', which allowed them to reduce the diagnostic uncertainty.
In the case of the diagnosis of CTX, it is essential to remember the necessity of carrying out the diagnostics in the patient's siblings since they can still be asymptomatic.
Illustration showing the simplified enzymatic mechanism of dysfunction in bile acid synthesis occurring in cerebrotendinous xanthomatosis.
Type of mutations
Currently, the CYP27A1is the only gene known to be responsible for CTX.
The CYP27A1gene is located in chromosome locus 2q35 and consists of 9 exons and 8 introns. Currently, over 114 pathological variants were described for all exons and introns (missense, nonsense, splice-site, and insertion/deletion mutations). The disease is inherited in an autosomal recessive pattern (homozygous and compound heterozygotes), and a loss-of-function mechanism is responsible for CTX.
In the available case reports, nearly 40% of mutations occurred in exons 6 and 8 (29% and 11% respectively). However, when distinguishing between the Asian and non-Asian populations, quite significant differences can be observed here. In the reports of Asian CTX patients, most pathological variants (48%) were present within exon 2 and 8. At the same time, mutations in these exons occurring in the Non-Asian CTX patients accounted only for around 14%.
Of course, there are population-specific and much more common variants. For the non-Asian population, the most common variants are c.1183C>T (16%), c.1184+1G>A (8%), c.1016C>T (7%), c.1263+1G>A (7%). For the Asian population the most common variants are c.1214G>A (18%), c.1421G>A (11%), c.1263+1G>A (9%), c.1420C>T (9%).
There are detailed reports of CYP27A1pathological variants occurring more likely in various populations. For example:
in China - c.420G>A, c.379C>T, c.1263+1G>A,
in Italy - c.646G>C, c.1183C>T, c.1184+1G>A,
in Japan - c.1214G>A, c.1421G>A, c.435G>T,
in Poland - c.1184+1G>A, and c.379C>T,
in the Netherlands - c.1016C>T, c.1183C>T, and c.1263+1G>A,
in Spain - c.1183C>T, and c.1213C>T.