Bile Acid Synthesis Disorders in Japan: Long-Term Outcome and Chenodeoxycholic Acid Treatment

We encountered 7 Japanese patients with bile acid synthesis disorders (BASD) including 3β-hydroxy-Δ5-C27-steroid dehydrogenase/isomerase (3β-HSD) deficiency (n = 3), Δ4-3-oxosteroid 5β-reductase (5β-reductase) deficiency (n = 3), and oxysterol 7α-hydroxylase deficiency (n = 1) over 21 years between 1996 and 2017. We aimed to clarify long-term outcome in the 7 patients with BASD as well as long-term efficacy of chenodeoxycholic acid (CDCA) treatment in the 5 patients with 3β-HSD deficiency or 5β-reductase deficiency. Diagnoses were made from bile acid and genetic analyses. Bile acid analysis in serum and urine was performed using gas chromatography–mass spectrometry. Clinical and laboratory findings and bile acid profiles at diagnosis and most recent visit were retrospectively obtained from medical records. Long-term outcome included follow-up duration, treatments, growth, education/employment, complications of treatment, and other problems. Medians with ranges of current patient ages and duration of CDCA treatment are 10 years (8 to 43) and 10 years (8 to 21), respectively. All 7 patients, who had homozygous or compound heterozygous mutations in the HSD3B7, SRD5B1, or CYP7B1 gene, are currently in good health without liver dysfunction. In the 5 patients with CDCA treatment, hepatic function gradually improved following initiation. No adverse effects were noted. We concluded that CDCA treatment is effective in 3β-HSD deficiency and 5β-reductase deficiency, as cholic acid has been in other countries. BASD carry a good prognosis following early diagnosis and initiation of long-term CDCA treatment.

In Europe and the USA, primary bile acid treatment using oral cholic acid (CA) is the first-line therapy for patients with 3β-HSD deficiency and 5β-reductase deficiency [14][15][16]. We have treated 5 patients with 3β-HSD deficiency or 5β-reductase deficiency using oral chenodeoxycholic acid (CDCA; Chino) because CA has not been available for clinical use in Japan.
A schematic representation of bile acid biosynthetic pathways and locations of enzymatic defects, as well as effects of CA and CDCA, is provided in Supplementary Figures S1 and S2, respectively.
Here, we report the long-term outcomes and bile acid profiles in 7 Japanese patients with BASD. We also describe efficacy of long-term CDCA treatment in the 5 patients with 3β-HSD deficiency or 5β-reductase deficiency.

Patients
All 7 patients were previously reported. Diagnoses were made from bile acid profiles and genetic analyses by Sanger sequencing. These patients included 3 with 3β-HSD deficiency, 3 with 5β-reductase deficiency, and 1 with oxysterol 7α deficiency [8][9][10][11]13]. Two patients with 5β-reductase deficiency, initially found to have only a heterozygous mutation in the SRD5B1 gene [9,10], subsequently were re-analyzed by targeted next-generation sequencing [17] that identified compound heterozygous mutations in the SRD5B1 gene. Patient characteristics and laboratory results at diagnosis, gene mutations, and diagnosis are shown in previous reports [7][8][9][10][11]13]. Our study protocols were approved by the ethics committees at Kurume University and Nagoya City University Graduate School of Medical Sciences. Written informed consent was obtained from enrolled patients or their parents.

Long-Term Clinical and Laboratory Findings
Clinical and laboratory findings at the most recent visit were retrospectively obtained from medical records. Longterm outcome included follow-up duration, treatments, growth, education/employment, complications of treatment, and other problems. Growth was evaluated by height and weight, expressed in terms of standard deviation (SD) [18]. Other assessments included serum GGT, alanine aminotransferase (ALT), direct bilirubin (D. Bil), TBA using 3α-hydroxysteroid dehydrogenase enzyme method, and also tests for liver failure, fibrosis, or tumor such as complete blood count, coagulation factors, serum mac-2 binding protein glycosylation isomer, serum alpha-fetoprotein, and abdominal ultrasonography.

Bile Acid Analysis
In bile acid analysis, serum and urine samples were collected and stored below − 20 °C until analysis. Concentrations of individual bile acids in the urine were corrected for the creatinine (Cr) concentration and expressed as μmol/ mmol Cr. By comparison with control standard samples that we synthesized to represent specific unusual bile acids such as 3β-hydroxy-Δ 5 , 3-oxo-Δ 4 , and allo-bile acids, which occur in BASD, we analyzed bile acids in the patients' urine and serum using gas chromatography-mass spectrometry (GC-MS) with monitoring of selected ions. Specifically, we selectively monitored characteristic ionic fragments of methyl ester-dimethylethylsilyl ether-methoxime derivatives of bile acids after enzymatic hydrolysis (choloylglycine hydrolase, 30 units) and solvolysis (sulfatase, 150 units; Sigma, St. Louis, MO, USA), as described previously [19]. We had good recovery rates for all bile acids including 3β-hydroxy-Δ 5 -, 3-oxo-Δ 4 -, and all-bile acids, which were measured by GC-MS. After treatment by hydrolysis, solvolysis, ion-exchange purification, and methoxime-methyl ester-dimethylethylsilyl ether derivatization, recovery rates were 73.5 to 136.4%.
Using the above analytic method, we performed bile acid analysis in the enrolled patients at the time of diagnosis and follow-up evaluations including the most current visit. Table 1 summarizes the current clinical and laboratory features including long-term outcome for the 7 patients as well as their genetic mutations. Ages at the most recent visit ranged from 8 and 43 years (median, 10). Five patients, including 3 with 3β-HSD deficiency and 2 with 5β-reductase deficiency, had received long-term CDCA treatment (median, 10 years; range, 8 to 21). One patient with 5β-reductase deficiency was treated with ursodeoxycholic acid (UDCA) only during her first year, while early Table 1 Current clinical and laboratory findings in 7 patients with BASD BASD bile acid synthesis disorders, GGT γ-glutamyltransferase, ALT alanine aminotransferase, D. Bil direct bilirubin, TBA serum total bile acids using 3α-hydroxysteroid dehydrogenase enzyme method, SD standard deviation, 3β-HSD 3β-hydroxy-Δ 5 -C 27 -steroid dehydrogenase/isomerase, M male, yr years, n.d. not done, CDCA chenodeoxycholic acid, F female, 5β-reductase Δ 4 -3-oxosteroid 5β-reductase, oxysterol 7α oxysterol 7α-hydroxylase. Patients 4 and 5 were re-analyzed in this study by targeted next-generation sequencing, while other patients were analyzed by Sanger sequencing as previously reported in Refs [8,10,11,13]  1 3

Long-Term Outcome
in life another patient who had oxysterol 7α deficiency required liver transplantation, which was successful. At most recent assessment, all patients were healthy without any treatment complications or other problems. Additionally, laboratory results and images showed normal results and no evidence of liver failure, cirrhosis, liver tumor, or biliary stones. Further supporting data included liver stiffness measurements by transient elastography as well as determinations of serum mac-2 binding protein glycosylation isomer and alpha-fetoprotein, which yielded normal results in all patients except that transient elastography was not performed for Patients 2 and 7. We did not perform follow-up liver biopsy in the patients with 3β-HSD deficiency and 5β-reductase deficiency because no symptoms or laboratory findings suggested liver dysfunction. Patient 7 with oxysterol 7α deficiency underwent liver biopsy 10 years after liver transplantation, with the specimen showing no liver fibrosis. All patients developed normally, falling within ± 2 SD for height and body weight. All patients received a general education, and all 2 were employed upon reaching adulthood. Tables 2 and 3, respectively, summarize bile acid profiles before and after CDCA treatment in 5 patients including 3 patients with 3β-HSD deficiency and 2 with 5β-reductase deficiency who had undergone long-term CDCA treatment.

Bile Acid Profiles in the 5 Patients with CDCA Treatment for a Long Term
In the 5 CDCA-treated patients, percentages of unusual bile acids among serum and urinary TBA at the time of diagnosis were 87.5% or more except in serum from Patient 5 (Tables 2 and 3). Concentrations of unusual bile acids in serum and urine ranged from 2.4 to 25.1 μmol/L and 16.3 to 123.9 μmol/mmol Cr, respectively. During CDCA treatment in the 5 patients, percentages of serum unusual bile acids among TBA decreased, while percentages of urinary unusual bile acids among TBA were more than 58.8% (Tables 2  and 3). Concentrations of serum and urinary unusual bile acids during CDCA treatment decreased to 0.1 to 5.7 μmol/L and 2.0 to 13.9 μmol/mmol Cr, respectively.
Only small amounts of primary bile acids were present at diagnosis, while CDCA became predominant after treatment.

Bile Acid Profiles in the Patient with 5β-Reductase Deficiency Who Had No CDCA Treatment
One patient (Patient 4) with 5β-reductase deficiency who had never received CDCA showed jaundice at 4 weeks of age, which progressively worsened until 3 months of age, becoming deeply icteric with pale stools and dark urine. UDCA treatment (5 mg/kg/day) was started, after which serum bilirubin and aminotransferase concentrations gradually decreased (Supplementary Figure S3). When excessive 3-oxo-Δ 4 bile acids were detected (Supplementary Table S1), we recommended primary bile acid treatment for a suspected BASD. Her parents declined, so we increased the dose of UDCA to 10 mg/kg/day. By 11 months of age, serum bilirubin and aminotransferase concentrations were within the normal range (Supplementary Figure S3). Untreated after her first year, Patient 4 has been in good health without liver dysfunction at follow-up visits from 15 months of age to the present, although hyper-3-oxo-Δ 4 bile aciduria has continued with UDCA treatment. In a recent bile acid analysis (Supplementary Table S1), serum and urinary TBA and unusual bile acids (3-oxo-Δ 4 bile acids) appeared to be gradually decreasing. However, the percentage of unusual bile acids (3-oxo-Δ 4 bile acids) among serum and urinary TBA remained high.

Discussion
We identified long-term clinical and laboratory findings and bile acid profiles in 7 Japanese patients with BASD including 5 patients treated with CDCA and 1 with UDCA; the other patient underwent liver transplantation. All patients had improved liver dysfunction, freedom from treatment complications, normal growth and development, and absence of psychosocial problems. Accordingly, the longterm outcomes of these Japanese patients with BASD have been good. In Europe and the USA, 3β-HSD deficiency is about eight times more common than 5β-reductase deficiency [15,16,20]. In Saudi Arabia, 2.7% of patients with cholestasis in infancy were found to have BASD [21]. In Japan, our bile acid analyses in 1010 patients with cryptogenic cholestasis beginning in infancy between 1996 and 2017 showed a BASD prevalence of 0.7%, with equal numbers of cases for 3β-HSD deficiency and 5β-reductase deficiency. Our experience as well as previous reports from East Asia suggests that 3β-HSD deficiency is less prevalent in East Asia than in Europe or the USA [8-10, 13, 22, 23]. On the other hand, oxysterol 7α deficiency may be more prevalent in East Asia [11,24,25].
CA (5 to 15 mg/kg/day) has been approved in Europe and the USA for treating patients with 3β-HSD deficiency and 5β-reductase deficiency, making it the primary bile acid therapy for these BASD in most of the West [14][15][16]20]. In particular, CA has a very good therapeutic effect in 3β-HSD deficiency. CDCA has also been described as a treatment for 3β-HSD deficiency and 5β-reductase deficiency [26,27]. In Japan, CDCA is used instead of CA for treatment of BASD, since the latter is not available for clinical use in Japan; the same is true for China [28]. We have treated with as low-dose CDCA (4.0 to 7.8 mg/kg/day) as possible because CDCA is more hepatotoxic than CA [29]. Additionally, CDCA is contraindicated during pregnancy. We now recommend CA treatment during pregnancy.
Before CDCA treatment in our 3 patients with 3β-HSD deficiency, unusual bile acids accounted for 35.8% to 87.5% and 93.7% to 98.5% of serum and urinary bile acids, respectively; for our 2 patients with 5β-reductase deficiency, these respective percentages were 54.7% to 91.6% and 98.4% to 99.7% (Tables 2 and 3). During treatment, percentages of unusual bile acids among serum TBA decreased; in urine, however, these percentages remained high (Tables 2 and 3). However, the concentration of unusual bile acids in urine clearly decreased. In our study, details of bile acid dynamics in hepatocytes are not known, but liver function in our patients improved. This suggests that CDCA treatment suppressed synthesis of unusual bile acids, while the remaining unusual bile acids were excreted via the kidneys. During CDCA treatment, the decreasing concentrations of unusual bile acids in serum and urine resulted from a negative feedback effect by CDCA upon hepatocytic CYP7A1. Primary bile acids (CA and CDCA) down-regulate their own biosynthesis, including pathways that produce toxic unusual bile acids, through activation of the farnesoid X receptor, which represses transcription of the CYP7A1 gene encoding cholesterol 7α-hydroxylase, the rate-limiting enzyme for bile acid synthesis (Supplementary Figure S2) [15]. Concentrations of unusual bile acids in serum decreased except in Patient 3, and hepatic function improved. Given the renal excretion, evaluation of unusual bile acids during CDCA treatment requires bile acid analysis in serum and urine by GC-MS rather than simply determining the percentage of unusual bile acids among urinary TBA. Both concentrations and percentages of unusual bile acids in serum obtained by this analytic method are necessary.
Bile acid analysis showed disappearance of unusual bile acids immediately after liver transplantation in our patient with oxysterol 7α deficiency [11]. Recent reports described that oxysterol 7α deficiency can be treated with CDCA treatment [30,31]. Since liver function rapidly deteriorates in oxysterol 7α deficiency, early diagnosis and initiation of CDCA therapy are particularly urgent.
The somewhat enigmatic Patient 4 in Table 1 was diagnosed with 5β-reductase deficiency based on bile acid profiles and genetic analysis. Based on parental wishes, she was treated only with UDCA, which was discontinued at 12 months of age when her liver function had fully recovered and she was free of symptoms. With no treatment, this healthy state has continued through the most recent followup at 16 years. However, based on bile acid profiles, we believe that this patient should receive primary bile acid treatment. We speculate that her favorable course might be related to the small amounts of allo-bile acids, such as allo-cholic acid and allo-chenodeoxycholic acid, detected in serum and urine (Supplementary Table S1). Allo-bile acids presumably are synthesized from 3-oxo-Δ 4 -bile acids, which normally would be transformed to 5β-bile acids by 5β-reductase. Fortunately, the toxicity of allo-cholic acid is only on a par with that of CA [32]. Allo-bile acids also are efficiently excreted into the bile via transport systems apart from the bile salt export pump, such as multidrug resistance protein 2 [32,33]. This would account for the very limited quantities of allo-bile acids in the patient's serum and urine. For this reason, fat-soluble vitamin deficiency and other symptoms have not occurred since discontinuation of treatment. In general, however, allo-bile acids in excess of 3-oxo-Δ 4 -bile acids can cause significant liver dysfunction.
In summary, we believe that 3β-HSD deficiency, which is not common in East Asia, should be adequately treatable with CDCA or CA given that disease progression is slow. In contrast, 5β-reductase deficiency and oxysterol 7α deficiency rapidly progress to liver cirrhosis or cholestatic liver failure in infancy. We therefore believe that in infancy, prompt treatment with CDCA or CA is needed for patients with 5β-reductase deficiency. To our knowledge, this is the first report of long-term outcome and efficacy of CDCA treatment in a group of patients with BASD from East Asia.
In conclusion, we believe that CDCA treatment is useful for treating BASD including 3β-HSD deficiency and 5β-reductase deficiency, as has been shown for CA. BASD have a good prognosis following early diagnosis and CDCA treatment.