Background

Cerebrotendinous xanthomatosis (CTX) is a rare genetic disorder of bile acid synthesis that can cause progressive neurological damage and premature death. Detection of CTX in the newborn period would be beneficial since an effective treatment is available. We previously described a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) test with potential to screen newborn dried bloodspots (DBS) for CTX. We report here modifications to the methodology and application of the modified test to analysis of DBS from a CTX-affected and unaffected newborns.

Methods

The testing methodology utilizes keto derivatization to enable sensitive LC-ESI-MS/MS measurement of elevated 7α,12α-dihydroxy-4-cholesten-3-one (7α12αC4) in CTX newborn DBS. We report here method modifications, including use of a DBS extraction procedure used in newborn screening laboratories and a reduced analysis time of 2 min per sample.

Results

Rapid isotope-dilution LC-ESI/MS/MS quantification of the ketosterol bile acid precursor 7α12αC4 provides a test that could readily discriminate a CTX positive newborn DBS sample (with a concentration of 104.4 ng/ml) from unaffected newborn samples (with a mean concentration of 4.1 ± 3.4 ng/ml; range 0.2–15.6 ng/ml, n = 39) analyzed in a blinded manner.

Conclusions

We provide additional evidence suggesting 7α12αC4 may be a promising test marker to screen newborn DBS for CTX. Early detection and intervention through newborn screening would greatly benefit those affected with CTX, preventing morbidity and mortality.

Cerebrotendinous xanthomatosis (CTX; OMIM# 213700 ) is an autosomal recessive childhood-to-adult onset leukodystrophy associated with deficient sterol 27-hydroxylase (CYP27A1), an enzyme important in conversion of cholesterol to the bile acids cholic and chenodeoxycholic acid (CDCA). CTX is difficult to diagnose; for most affected individuals it is not clinically obvious at birth, although neonatal cholestatic jaundice may be a presenting sign in some infants [1] and [2] . While symptoms of CTX may present in childhood and adolescence, the disorder is often not recognized until symptoms have progressed. A mean age of first symptom onset ranging between 14 and 19 years old and a mean delay in diagnosis ranging between 17–19 years has been reported [3] , [4] and [5] . Symptoms in children can include diarrhea, juvenile cataracts and developmental delay [3] . Adolescent-to-adult onset symptoms may include tendon and cerebral xanthomas. In 95–97% of patients neurological symptoms have developed at the time of diagnosis [4] and [5] ; these may include cognitive impairment, cerebellar signs (for example ataxia) and pyramidal signs (for example spasticity). As the disorder progresses patients can become incapacitated with motor dysfunction, with premature death often occurring due to advancing neurological deterioration. Although only around three hundred cases of CTX have been described worldwide [6] , relatively large series of patients have been identified by physicians familiar with the disorder, suggesting CTX may often be under or misdiagnosed.

A simple, effective oral therapy for CTX is available in the form of CDCA, the main bile acid deficient in CTX. Treatment with CDCA has been shown to normalize the biochemical phenotype and halt progression of disease in most cases [7] and [8] . Generally treatment of patients with advanced neurological disease does not reverse the impairment [8] . A recent study in a cohort of 16 CTX patients demonstrated those who began CDCA treatment after age 25 years were significantly more limited in ambulation and more cognitively impaired compared to those who started treatment earlier [9] . Therefore it is essential to diagnose and treat CTX as early as possible.

As the mean age of CTX diagnosis is currently estimated as between 35 and 37 years old [3] , [4] and [5] , we believe screening newborns for CTX will be the best way to achieve early identification and intervention for this disorder. Although CTX fulfills the majority of criteria required for a disorder to be screened for in newborns, there has been no suitable test available to screen newborn dried bloodspots (DBS) for CTX. Blood testing for diagnostic confirmation of CTX is routinely performed using gas chromatography–mass spectrometry (GC–MS) measurement of 5α-cholestanol which is elevated in affected individuals [10] and [11] . A focus of our research has been to develop high-throughput amenable electrospray ionization-tandem mass spectrometry (ESI-MS/MS) based blood tests with utility to screen DBS for CTX [12] and [13] . We recently described LC-ESI-MS/MS methodology that utilizes keto moiety derivatization to enable highly sensitive isotope dilution quantification of 7α,12α-dihydroxy-4-cholesten-3-one (7α12αC4) in DBS from CTX affected adults and newborns. Quantification of 7α12αC4 provided improved discrimination between CTX affected and unaffected samples compared to 5α-cholestanol, such that it could serve as an improved DBS test for CTX [14] . We demonstrated for the first time that elevated 7α12αC4 could be measured in DBS obtained from CTX affected newborns [14] . With the limitation that we were only able to retrieve and analyze two stored newborn DBS, there appeared to be no overlap between concentrations of 7α12αC4 in DBS from CTX affected and unaffected newborns [14] . We describe here modifications to the testing methodology that make it more amenable for use in newborn screening laboratories and provide further test validation data generated through the blinded analysis of an additional CTX positive newborn DBS, along with unaffected newborn DBS.

The CTX positive newborn DBS used to perform the blinded analysis was obtained through the Dutch Newborn Screening Program with the consent of the newborn's parents. Anonymized residual DBS from unaffected Dutch newborns were also used with approval of the Dutch National Institute for Public Health and the Environment (RIVM, Bilthoven, The Netherlands; responsible for the Dutch Newborn Screening Program) and the OHSU Institutional Review Board (IRB). Adult CTX positive DBS were collected from participants enrolled in an IRB-approved study at OHSU. Written informed consent was obtained for all OHSU study participants. De-identified DBS samples submitted to the Sterol Analysis Diagnostic laboratory at OHSU were used with IRB approval. For all CTX-positive samples diagnostic confirmation was performed by mutation analysis of CYP27A1 . Residual de-identified whole blood was provided by the Oregon Clinical and Translational Research Institute (OCTRI).

Authentic 7α,12α-dihydroxy-4-cholesten-3-one (7α12αC4) and 7α-hydroxy-4-cholesten-3-one-d₇ (7αC4-d₇) were from Toronto Research Chemicals (Toronto, Ontario). Methanol, water and acetonitrile (LC-MS grade) were from Burdick and Jackson (Muskegon, MI). Formic acid (90%) was J.T. Baker brand. Glacial acetic acid (99.99%), hydrazine and oxalic acid were from Sigma-Aldrich (St Louis, MO). Quaternary amonoxy (QAO) reagent ( O -(3-trimethylammoniumpropyl) hydroxylamine) bromide is commercially available as Amplifex™ Keto reagent from http://www.sciex.com . The QAO-d₃ reagent was provided by SCIEX. Protein Saver 903 filter-paper was obtained from Whatman (Miami, FL).