Update on newborn dried bloodspot testing for cerebrotendinous xanthomatosis: An available high-throughput liquid-chromatography tandem mass spectrometry method

摘要

Abstract BackgroundCerebrotendinous 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.MethodsThe 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.ResultsRapid 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.ConclusionsWe 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. Abbreviations CTX , cerebrotendinous xanthomatosis ; ESI-MS/MS , liquid chromatography-electrospray ionization-tandem MS ; DBS , dried bloodspots ; 7α12αC4 , 7α,12α-dihydroxy-4-cholesten-3-one ; CDCA , chenodeoxycholic acid ; GC–MS , gas chromatography–mass spectrometry ; IRB , institutional Review Board ; QAO , quaternary amonoxy ; MRM , multiple reaction monitoring ; LLOQ , lower limit of quantification ; S/N , signal-to-noise ; RSD , relative standard deviation ; QCs , quality control samples Keywords Leukodystrophy ; CYP27A1 ; Bile acid synthesis ; Ketosterols ; Newborn screening ; LC-ESI-MS/MS prs.rt("abs_end"); 1. Introduction 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 spectromet