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Original Articles: Hepatology

Severe Neonatal Cholestasis in Cerebrotendinous Xanthomatosis: Genetics, Immunostaining, Mass Spectrometry

Gong, Jing-Yu; Setchell, Kenneth D.R.; Zhao, Jing‡,§; Zhang, Wujuan; Wolfe, Brian; Lu, Yi‡,§; Lackner, Karolin||; Knisely, A.S.||; Wang, Neng-Li; Hao, Chen-Zhi‡,§; Zhang, Mei-Hong; Wang, Jian-She‡,§

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Journal of Pediatric Gastroenterology and Nutrition: November 2017 - Volume 65 - Issue 5 - p 561-568
doi: 10.1097/MPG.0000000000001730
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Abstract

What Is Known

  • Cerebrotendinous xanthomatosis usually presents as neurologic disease in adults or older children.
  • Neonatal cholestasis associated with cerebrotendinous xanthomatosis is believed rare, and is generally assessed as transient, with patient survival.

What Is New

  • That cerebrotendinous xanthomatosis underlies cholestasis in some neonates is easily overlooked, perhaps because bile acid profiles may differ from those in older children or adolescents.
  • We describe new candidate biomarkers for cerebrotendinous xanthomatosis manifest as neonatal cholestasis.

Cerebrotendinous xanthomatosis (CTX; OMIM 213700), an autosomal-recessive disease with a possibly underestimated prevalence of 1:40,000 to 1:400,000 (1), is caused by mutations in CYP27A1 (OMIM 606530) (2,3), encoding CYP27A1 (sterol 27-hydroxylase) (4,5). Reduced synthesis of the primary bile acids (BAs), cholic acid (CA), and chenodeoxycholic acid (CDCA) (6), leads classically to accumulation of C27 sterol intermediates, excreted in urine and bile as predominantly bile alcohol (BAlc) glucuronides (7–9).

CTX patients usually present with dementia, motor dysfunction, tendon xanthoma, cataracts, and diarrhea, often as teenagers (10,11). These result from tissue deposits of cholesterol, cholestanol, and sterol intermediates (12,13). Oral administration of CDCA is standard therapy for CTX (14–16), with neurologic improvement in some but not all patients (17). CA, recently approved for treatment of other genetic disorders of BA synthesis (18,19), also is effective in CTX (20). Ursodeoxycholic acid (UDCA) is not (21). Survival is related to age at diagnosis (22,23). The important options in diagnosis are urinary BA profiling and gene sequencing, although blood spot analysis has been proposed (24).

In 2000, a preliminary description of CTX manifest in infancy as cholestatic liver disease (20), not previously associated with sterol 27-hydroxylase deficiency, was presented (25). Several reports since have confirmed that CTX can be associated with hepatobiliary injury in infancy (20,26–28).

Here we describe 8 infants with genetically confirmed CTX presenting as severe cholestasis; 5 died from liver failure or were transplanted. The urine was enriched in BAlc sulfates rather than in BAlc mono-glucuronides, the classical biomarkers for CTX.

PATIENTS AND METHODS

Patients—Histories and Clinical Assessments

From January 2013 to August 2016, 8 patients (Table 1), 2 boys (patients 1 and 8) and 6 girls (patients 2–7), were referred to Children's Hospital of Fudan University and Jinshan Hospital, Fudan University, for investigation of neonatal conjugated hyperbilirubinemia was eventually confirmed as associated with CTX by demonstration of homozygous or compound heterozygous CYP27A1 mutations (detailed below). These 8 infants were identified among 734 evaluated for conjugated hyperbilirubinemia; of the 734, 93 underwent urinary analysis of BA profile and 413 underwent genetic screening.

T1
TABLE 1:
Demographic data and clinical information

All 8 subjects were born at term, following an uneventful pregnancy, to nonconsanguine parents with unremarkable histories. Four patients were “only children”. Patient 3 had 1 healthy older brother; another had died aged 6 months, with chronic cholestasis and frequent diarrhea. Patient 5 was the product of a couple's sixth pregnancy: 3 had ended in spontaneous abortion and the mother had undergone an induced abortion. The patient's older sister was healthy. The mother of patient 6 had had a spontaneous abortion. Patient 7 had a 6-year-old healthy sister.

Three patients (2, 3, and 5) had undergone laparoscopic cholangiography, at ages 3 months 27 days, 1 month 7 days, and 2 months 16 days, respectively, that showed patent and well-developed bile ducts. Liver biopsy specimens were obtained simultaneously. On evaluation at our institutions, all were active and well developed, and no facial or limb abnormalities were evident. Additional clinical information is listed in Table 1.

Substantial conjugated hyperbilirubinemia and markedly increased serum aminotransferase activity were uniformly present, as was only slightly elevated serum gamma-glutamyltransferase activity (Table S1). Routine immunoassay found mild to marked hypercholanemia.

No serologic evidence of infection with hepatitis A, B, C, and E viruses, toxoplasma, rubella virus, herpes simplex virus, Epstein-Barr virus, human immunodeficiency virus, or syphilis was found in any patient. Thyroid function test results, serum carnitine and acylcarnitine profiles, and urine organic acid profiles were unremarkable in all. Blood amino acid profiles determined by tandem mass spectrometry were unremarkable except for varying degrees of hypermethioninemia in patients 1 to 3 and 5 and for hyperthreoninemia and hypertyrosinemia in patient 7. Abdominal imaging studies confirmed hepatomegaly or hepatosplenomegaly in all (Table 1).

Histologic Study—Light Microscopy

Specimens of liver obtained at laparoscopic cholangiography (patients 2, 3, and 5, wedge biopsy, ages as above) or transplantation (patient 7, 8 months 14 days) were routinely fixed in formalin and processed into paraffin. Sections cut at 4u and picked up on glass slides were stained with hematoxylin/eosin and by periodic acid/Schiff technique with and without diastase predigestion (Shanghai). Parallel sections were deparaffinized, subjected to heat-induced antigen retrieval (CC1, Ventana Medical Systems, Tucson, AZ) and immunostained for CYP27A1 (EPR7529 rabbit monoclonal antibody, Abcam, Cambridge, UK; 1:100 dilution), with diaminobenzidine as chromogen and hematoxylin as counterstain, using a UView Detection kit and a Benchmark Ultra Immunostainer (both Ventana; Graz). Nonlesional human liver incidentally resected at partial hepatectomy (Graz) served as an immunostaining control.

Mass Spectrometry Analysis of Urine Bile Acid Profiles

In patients 1 to 7, urine was collected before starting any BA therapy, or at least 5 days after pausing UDCA administration, for analysis by fast atom bombardment ionization-mass spectroscopy (FAB-MS, Cincinnati Children's Hospital Medical Center) to screen for inborn errors in the cholesterol-BA biosynthetic pathway (18,29–31) at ages of 4 months 22 days, 1 month 22 days, 6 months 5 days, 3 months 28 days, 2 months 25 days, 14 months 4 days, and 11 months 22 days (∼3 months after liver transplantation) respectively, and repeated in patient 2 at age 6 months 10 days.

BAs were extracted from urine samples (5 mL) by liquid-solid extraction using cartridges of octadecylsilane-bonded silica (32,33). The extracts were analyzed directly by FAB-MS on an Autospec Q magnetic sector mass spectrometer (Waters, Milford, MA) and negative ion mass spectra were acquired as described (18,31). Ions representing BAs, BAlcs, and their metabolites, including those typically found in patients with BA synthesis disorders, are comprehensively listed elsewhere (18).

Sample extracts were also subjected to analysis by electrospray ionization tandem mass spectrometry (Premier API triple quadruple mass spectrometer; Waters) (34) with collision-induced dissociation (CID) to obtain additional qualitative data to confirm structural identity of specific conjugate species of bile alcohols. Samples were introduced by direct flow injection without the use of chromatography (31).

Sequencing of CYP27A1 and Other Genes Implicated in Cholestatic Disorders

With the approval of the Ethics Committees of Jinshan Hospital (2014-13-01, 2014-07-01) or the Children's Hospital of Fudan University (178) and with written informed parental consent, 1.5 mL of peripheral blood was obtained from patients and parents. Patient 1 was identified by whole exome sequencing after urinary BA spectrum analysis prompted special attention to genes involved in the BA synthesis pathway. Patients 4 and 5 were diagnosed by urinary BA spectrum analysis, and confirmed by Sanger sequencing of CYP27A1. Panel sequencing of >60 genes implicated in cholestasis (35) was carried out for other patients.

Possible mutations were verified by Sanger sequencing of another PCR fragment in the patients, as were in the parents of the patients who harbored 2 heterozygous mutations in CYP27A1. NM_000784.3 was used as a reference. Pathogenicity of exonic mutations was assessed using MutationTaster software (http://www.mutationtaster.org/).

RESULTS

Patients—Management and Outcomes

Although UDCA had been given to patients 1 to 3 and 5 to 8 before a diagnosis was established, clinical-biochemistry markers of hepatobiliary injury and function worsened in all but patients 5 and 6 (Table S1). Patients 1 to 3 and 7 had received ganciclovir for cytomegalovirus infection before referral. CDCA (31 mg twice a day, 8 mg/kg bw/d) was given to patient 2 from the age of 6 months 11 days, but with no response. Patient 4 received an extract of animal bile containing CA as a major ingredient (http://baike.haosou.com/doc/7875985–8150080.html), 33 mg twice a day (8.8 mg/kg bw/d), for the last 2 months of life, ending in death with ascites and liver failure. Patient 5 received the same bile extract (CA at 50 mg twice a day, 12.5 mg/kg bw/d) from aged 7 months. Review at the age of 11 months found jaundice to have disappeared, liver and spleen to be of normal size, and clinical biochemistry test result abnormalities to be nearly resolved. Patient 6 received UDCA (80 mg twice a day, 14 mg/kg bw/d) from age 5 months 7 days till age 13 months, when values for biomarkers of hepatobiliary injury had returned to normal ranges. CDCA therapy (50 mg twice a day, 8 mg/kg bw/d) commenced at the age of 14 months after the diagnosis of CTX was made; at this writing, biomarker values continue in normal ranges. Patient 7 underwent liver transplantation at the age of 8 months 14 days, and since has been well, with normal liver function. Patient 8 received UDCA without effect; the parents then withdrew all medication, after which spontaneous improvement occurred. Table 1 summarizes the patients’ clinical details. Table S1 summarizes their clinical-biochemistry test results and coagulation biomarkers. Overall, 4 patients died, 1 patient underwent a liver transplant, and in 3 patients cholestasis has resolved.

Histologic Study—Histopathologic Findings

In all 3 patients (2, 3, and 5) who underwent liver biopsy, light microscopy found intralobular cholestasis (hepatocellular and canalicular), lobular disarray, and giant-cell transformation of hepatocytes, with some necrotic hepatocytes. Scant steatosis was noted. Portal tract fibrosis and ductular reaction, without bile plugs in portal-tract bile ducts, also were uniformly present. Other abnormalities were not appreciated. The explanted liver of patient 7 was deeply bile stained. Cirrhosis and chronic hepatitis with moderate activity accompanied changes like those seen in patients 2, 3, and 5. In control liver, cytoplasm marked in a finely granular pattern on immunostaining for CYP27A1. No marking was detected in liver of patients 3 and 5. Finely granular cytoplasmic marking was present in hepatocytes of patient 2; pale, blushlike, weak cytoplasmic marking was present in hepatocytes of patient 7 (Fig. 1).

F1
FIGURE 1:
Liver biopsy and hepatectomy materials immunostained for CYP27A1 (antibody and chromogen, see text; hematoxylin counterstain). A, adult control, 400×. B, Patient 2, 200×. C, patient 7, 400×. Granular cytoplasmic marking is found in both A and B. Weaker, blush-like marking is seen in C. Note giant cell, left, and rosette of hepatocytes with bile plug (arrow), right, in C.

Mass Spectrometry Analysis of Urine Bile Acid Profiles—Absence of Usual Primary Bile Acid Conjugates, Prominence of Sulfated and Sulfated/Glucuronidated BAlc Conjugates

FAB-MS of urine was performed on 7 of the 8 patients. Urine from patient 8 was unavailable for analysis and from patient 7 was available only after liver transplantation (data not shown); Figure 2 compares negative ion FAB-MS mass spectra of the urine from patients 1 to 6 with that of a patient with CTX (18,36) manifest after childhood. The 6 spectra were uniformly remarkable for the absence of ions for the primary BA conjugates common to patients with chronic cholestasis (18). Unique and intense ions (m/z 481, 515, 607, and 657) consistent sulfates and sulfate/glucuronide double-conjugates of BAlc were observed in patients 1 to 5 rather than the diagnostically significant ions from BAlc glucuronides (m/z 611, 627, 643) usually observed for CTX patients (18,36). The interpretation of these mass spectra and the structural confirmation of these conjugates by CID are described in Supplementary Data 1 and Figure S1. Elevated levels of hydroxylated cholesterol sulfate (m/z 481), that is, an oxysterol sulfate and tetrahydroxy—(m/z 515) and pentahydroxy—(m/z 531) BAlc sulfates were excreted. The ions of m/z 607 were assigned as a cholestenetriolone glucuronide and m/z 657 a double conjugate (sulfate/glucuronide) of cholestenediol (Fig. S1). These atypical ions, rather than those for monoglucuronides of BAlcs, were present in the mass spectrum of all but 1 patient. Remarkably, an FAB-MS urine analysis from patient 5, 2 years after the initial diagnosis of CTX featured the BAlc monoglucuronides typical of CTX patients as teenagers or adults, a shift encountered before (26).

F2
FIGURE 2:
Negative ion FAB-MS spectra of the urine from 6 infants with genetically confirmed sterol 27-hydroxylase (CYP27A1) deficiency accompany a diagnostically significant mass spectrum typical for CTX. The urine FAB-MS mass spectrum for patient 5, collected 2 years after initial diagnosis of CYP27A1 deficiency, is also shown. Ions shown in boxes represent bile alcohol conjugates that are of diagnostic significance.

FAB-MS analysis of urine of patient 2 at age 1 month 22 days before hospital admission was unremarkable (data not shown). When mutations in CYP27A1 were found urine sampled 5 days after withdrawal of BA therapy (age 6 months 10 days) provided an atypical mass spectrum differing from that of classical CTX; the major ions were m/z 657, 481, and 607. Ions at m/z 464, 498, and 514 were interpreted as residual to BA therapy. Although spectra in patients 4 and 5 had some of the ions observed in classical CTX, the atypical ions of m/z 481, 607, and 657 were also prominent. In all cases CID spectra obtained by electrospray ionization-mass spectrometry confirmed these BAlcs as sulfates rather than monoglucuronides.

Sequencing of CYP27A1 and Other Genes Implicated in Cholestatic Disorders—Clinically Pathogenic Mutations Identified Only in CYP27A1

Patient 4 was a homozygote for a CYP27A1 mutation; the other patients were compound heterozygotes (Table 2). In total, 8 mutations were identified, with 2 nonsense (c.346C>T and c.886C>T), 1 frameshift (c.1221delA), 1 typical splicing site (c.1263+1G>A), and 4 missense (c.379C>T, c.389T>A, c.1214G>A, and c.1420C>T). Mutation c.379C>T was seen in 6 patients and c.1420C>T in 3 patients; the 6 other mutations were each present in 1 copy in individual different patients. The mutations c.346C>T, c.389T>A, c.886C>T, and c.1221delA are novel. The others are described (37,38). c.346C>T, c.886C>T, and c.1221delA are all predicted to result in nonsense-mediated mRNA decay. c.389T>A is predicted to replace methionine by lysine in position 130 of sterol 27-hydroxylase; the methionyl residue there is highly conserved among different species (Fig. S2). The mutation is recorded in neither the Exome Aggregation Consortium database nor the 1000 Genome Project database, and is assessed as disease causing. Sequencing in the healthy brother of patient 3 and the healthy sister of patient 5 revealed a monoallelic c.379C>T mutation in CYP27A1 in each. Table S2 presents the variations from canonical sequence found in genes other than CYP27A1 that have been implicated in cholestatic liver disease.

T2
TABLE 2:
CYP27A1 mutations, deduced effects, and CYP27A1 expression on immunostaining (“marking”)

DISCUSSION

Although CTX is generally a diagnosis assigned in the second or third decade, it has been detected in several pediatric patients (39,40) and in 4 infants (20,25–28). These few observations have led CTX manifest in infancy as cholestasis to be considered both rare and of low morbidity. Our experience in 8 infants with genetically documented CTX and cholestasis suggests that morbidity in infants with cholestatic CTX may be severe, and that role of CYP27A1 mutations in neonatal cholestasis may often be overlooked due to the different urine BA profiles determined when cholestatic from those in adults with CTX. We also propose that a set of unusual ions detected on mass spectrometry of urine BAs in neonatal cholestasis may both suggest the diagnosis of CTX and mark an adverse prognosis.

Several reports (41–44) have given rise to the notion that when CTX presents as cholestasis in early infancy its course is relatively benign, with spontaneous resolution. Close reading, however, reveals that 1 of the 3 subjects of these case reports died in infancy (27), and that the brother of another died aged 13 months of cholestatic liver disease of undetermined etiology (26). Although review of descriptions of patients with CTX diagnosed in later life indeed had identified 6 with histories of spontaneously resolving neonatal cholestasis (26), the subject cohort, composed of “survivors,” could not include patients with CTX in whom infantile cholestatic liver disease had proved fatal. Among our 8-patient cohort, 4 died and 1 was transplanted; a brother of 1 died aged 6 months of cholestatic liver disease of undetermined etiology. We propose that CTX manifest as neonatal cholestasis can have a relatively adverse prognosis.

Early-onset cytomegalovirus infection was hypothesized to contribute to the death of a patient with CTX and cholestatic liver disease (27). Four of our patients were cytomegalovirus infected and received ganciclovir therapy. Whether this triggered manifestation of CTX as cholestatic hepatitis in infancy is matter for speculation. Of interest in 1 reported infant with CTX is that the onset of cholestasis and hepatomegaly was associated with the initiation of CDCA therapy (28). Our relatively small sample size makes it difficult to identify possible genetic triggers, or “co-insults” in our patients, viz., mutations in genes associated with cholestatic liver disease that may predispose to manifestation of CTX as cholestatic hepatitis in infancy. We found no variants that could definitely be implicated in the early clinical manifestation of cholestasis (Table S2). Comparison with patients whose CTX assumed a noncholestatic pattern, with diagnosis only in later life, is of course required.

In clinical and laboratory terms, our patients resembled the subjects of the 3 reports of infantile CTX presenting as neonatal cholestasis (20,26,27); early-onset jaundice and hepatosplenomegaly accompanied substantially elevated transaminase values and serum gamma-glutamyltransferase activity that did not rise pari passu with serum conjugated bilirubin values. These features suggested ABCB11 disease (45), TJP2 disease (46), or a disorder of BA synthesis or conjugation (47). At least in patient 3 serum total BA concentrations were, however, higher than usual for disorders of BA synthesis or conjugation—as high as 169 μmol/L, a value that often disqualifies a patient from mass spectrometry screening for atypical urinary BAs.

Even when urinary BA profiles are determined, however, abnormalities such as those present in our patients and described in earlier case reports may not be recognized as signaling CTX. In patient 1, initial FAB-MS spectra were interpreted not as diagnostic for CTX, but instead as possibly resulting from a novel disorder of BA synthesis. The urine of adults with sterol 27-hydroxylase deficiency contains substantial amounts of side chain polyhydroxylated BAlc glucuronides (7–9), whereas primary BA concentrations are low or undetectable. BAlc glucuronide concentrations also are elevated in infantile cholestasis associated with CTX. In our patients, urine sample volumes did not suffice to take exact structural elucidation of the BAlcs beyond confirmation of their conjugation states. Whether the preferential sulfation of BAlcs is ontogenic, that is, a function of increased sulfation in early life, as is typical for steroid hormones (48,49), or contributes per se to cholestatic liver disease via altered BA synthesis and metabolism is speculation. Ions at m/z 481, 607, and 657 appeared to be associated with the poorest outcome and could mark poor prognosis. In patient 5, who survived the neonatal period with this atypical metabolic pattern, the sulfated species disappeared: At the age of 2 years, mass spectroscopy revealed exclusively BAlc monoglucuronides (Fig. 2), classical for CTX. Of note is that patient 6, whose urinary BA profile was classical for CTX, with predominance of BAlc monoglucuronides and lack of BAlc sulfates (Fig. 2), is alive and well with primary BA therapy.

Laboratory diagnosis of CTX has historically required demonstration of an increased plasma cholestanol: cholesterol ratio (50), although in some cases this is not entirely reliable. These analyses are specialized, time-consuming, complex, and outside the scope of most routine clinical laboratories. The alternative of mass spectrometry also is specialized, complex, and restricted in availability. It, however, permits the rapid and definitive identification of CTX using small samples of urine (18,36) to demonstrate abundant ions of BAlc monoglucuronides in children and adults (m/z 611, 627, and 643; Fig. 2)—and now, in infancy, to demonstrate high levels of BAlc sulfates, new biomarkers for CTX. Worth emphasis, perhaps, is that because urine, as a concentrated ultrafiltrate of blood plasma, is enriched in the abnormal metabolites of interest, it is more suitable for mass spectrometry studies than are plasma or serum.

These new biomarkers for CTX appear both diagnostically and prognostically significant. They were not uniformly present (patient 6 lacked them; her BA profile was that considered classic in adults), and they may be present only temporarily, as in patients 2 and 5. When identified, however, they were associated with the poor outcome of death or need for liver transplantation. They must be evaluated in infants with liver dysfunction of various defined etiologies before they can be considered specific for CTX. As usual, reciprocal complementation between genetic and mass spectrometry analyses offers best results.

In 2 of our patients immunostaining for CYP27A1 found no expression. This observation suggests that immunohistopathologic study may allow diagnosis in settings that make genetic or FAB-MS analyses impracticable, as when only archival liver tissue is at hand. Of interest is correlation in patients 2 and 7 of mutations in CYP27A1 with hepatocellular immunoreactivity for CYP27A1 (Fig. 1). Patient 2 was a compound heterozygote for the CYP27A1 mutations c.1214G>A and c.379C>T. Patient 7 is a compound heterozygote for the CYP27A1 mutations c.1420C>T and c.379C>T. The mutation c.379C>T also was present in patients 3 and 5, neither of whom expressed immunohistochemically demonstrable CYP27A1. We infer that both the c.1214G>A mutation and the c.1420C>T mutation in CYP27A1 impair CYP27A1 function without ablating CYP27A1 expression.

Early diagnosis of CTX is thought desirable because primary BA substitution is likely to slow the accumulation of function-impeding abnormal metabolites in various tissues, thereby reducing the severity of disease. Two children diagnosed with CTX in infancy, of whom 1 had neonatal cholestasis (20,25), have received CA continuously and have prospered, as has an additional affected sibling diagnosed by genetic studies in infancy and without neonatal cholestasis. Intellectual function and social adaptation are adequate in all, including the oldest, now an adult (personal communication, 2016; Prof P. McKiernan, Children's Hospital of Pittsburgh, Pittsburgh, PA). Diagnosis in infancy thus should be a goal.

We conclude that CTX should be considered as a disorder that underlies cholestatic “neonatal hepatitis” even when BA and BAlc profiles generated by mass spectrometric analysis do not match those in adults with CTX. To lower the threshold, or to enlarge the criteria, for CYP27A1 analysis in infants with cholestatic hepatitis, given the variability in BA profiles associated with CTX, appears worthwhile, using the mutually supportive triple approach of BA profiling, immunohistochemical study, and genetic analysis.

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Keywords:

bile acid synthesis defect; bile alcohol; CYP27A1; mass spectrometry; next-generation sequencing

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