Over the study period, 16 patients (11 male) with ∆4-3-oxo-R deficiency from 13 families were included (Table 1). Consanguinity was reported in two families (15%). One patient was diagnosed in absence of any symptom due to familial screening (Patient D2), whereas all other patients had symptoms from infancy (median 2 months of age, IQR 1.0–3.5, range 1–5) (Table 2). Jaundice was present in 14 out of 16 patients (87%), with pale stools in 6 patients (38%). Three patients (19%) presented with bleedings, including 2 with cerebral hemorrhage, due to profound vitamin K-dependent coagulopathy (PT < 10%, with complete correction following vitamin K supplementation). Eight patients (50%) and 2 patients (13%) displayed clinical hepatomegaly and splenomegaly respectively (Table 3). One patient (Patient A2) exhibited ascites at UDCA initiation and two patients presented with liver failure (Patient A2: PT 35% of control value, Factor V 39% of control value; Patient G1: PT 39% of control value). All 14 patients with jaundice had elevation of aspartate aminotransferase (AST, median 355 IU/L, IQR 267–778), alanine aminotransferase (ALT, median 464 IU/L, IQR 356–724) and conjugated bilirubin (median 128 µmol/L, IQR 68–199) levels in serum (Table 4). As expected, serum γGT (median 45 IU/L, IQR 31–48) and BA levels (median 6 µmol/L, range 2–9) were normal (Table 4), as well as plasma cholesterol level (median 4.1 mmol/L, range 3.3–4.8, n = 12). Plasma fat-soluble vitamins levels were low in 7 out 12 patients for vitamin A (median 174 µg/L, IQR 128–262, N < 200), 11 out of 12 for 25-hydroxy-vitamin D (median 8.6 µg/L, IQR 5.4–15.3, N < 20) and 7 out of 10 for vitamin E (median 3.7 mg/L, IQR 1.7–5.1, N < 5). Ten patients underwent a liver biopsy before CA initiation and liver histology showed cholestasis in 8 patients (80%), giant cell transformation of hepatocytes in 8 patients (80%), mild ductular reaction in 7 patients (70%), portal or lobular inflammation in 6 patients (60%) and fibrosis in 7 patients (70%; F1 n = 3; F2 n = 2; F3, n = 1; F4 n = 1) (Table 5). No patient presented with histological signs of biliary obstruction. Six patients had elastography evaluation before CA initiation, with values corresponding to fibrosis levels ranging from F0 to F4 (Table 5). Fourteen patients had urinary BA analysis at diagnosis, with massive increase of 3-oxo-∆4 derivatives (median 89% of total urinary BA, IQR 68–94%), quantified in 6 patients (median 68 µmol/mmol of creatinine, range 3–717) (Fig. 1). All patients had biallelic pathogenic variants in the AKR1D1 gene (Table 1 and Additional file 1: Table 1), corresponding to classes 4 or 5 of the American College of Medical Genetics (ACMG) classification [26]. Most of them were missense variants (22 out of 26 alleles), some of them being recurrent such as p.Arg261Cys/His (4 families), p.Asp81Val (3 families), p.Arg266Gln (2 families) and p.Arg307Cys (2 families). All patients except the one diagnosed through familial screening received oral UDCA therapy for a median duration of 2.8 months (IQR 2.0–6.2, range 1–153) before receiving CA. Jaundice disappeared in only 6 out of 14 patients with UDCA treatment (Table 3). Serum liver tests improved in 8 patients out of 14 with UDCA treatment and normalized in only 5 patients (Table 4). Nonetheless, in the two patients with liver failure at UDCA initiation serum liver tests did not improve and two further patients developed liver failure while being treated with UDCA (Patient F1: PT 56% of control value; Patient M1: 59% of control value).

Evolution of 3-oxo-∆4 derivatives in urine of patients before (n = 6) and after (n = 13) cholic acid treatment. Data are presented as median ± interquartile range, with a logarithmic scale. Of note, among the 13 patients with quantification of 3-oxo-∆4 derivatives at last follow-up, six also had quantification at baseline. Statistical analysis was performed using non-parametric two-tailed Mann–Whitney test. CA = cholic acid

All patients eventually received oral CA therapy at a median age of 8.1 months (IQR 5.3–26.9, range 3.1–159) once the diagnosis was confirmed (Table 2). Of note, one patient (Patient E1) was initially diagnosed as undetermined cholestasis and treated with oral UDCA for 13 years until a diagnosis of ∆4-3-oxo-R deficiency was made. The initial median dose of oral CA was 7.4 mg/kg/d (IQR 5.8–9.6). Within 6–12 months of CA therapy, jaundice and liver biochemistry normalized in all patients (Tables 3 and 4) and liver failure resolved in patients A2, F1, G1 and M1. The dose of CA was adapted to weight gain during growth and to urinary BA analysis in order to maintain low 3-oxo-∆4 derivatives, but was usually not increased above 8–10 mg/kg/d as long as serum liver tests remained normal. At last follow-up, patients received a median dose of 8.3 mg/kg/d (IQR 7.4–9.5) and one patient (patient A2) also received concomitant UDCA (200 mg/d) (Table 2). After a median CA replacement therapy of 4.5 years (IQR 2.9–6.8, range 1.1–24), all patients were alive with their native liver. All patients had normal physical examination and abdominal ultrasonography, except patient E1 who still exhibited hepatosplenomegaly (Table 3). Of note, this patient was diagnosed and started on CA therapy at 13 years of age. All patients displayed normal AST, ALT, γGT and bilirubin levels in serum (Table 4). Serum alpha-fetoprotein was slightly elevated in the youngest patient (Patient D2, 18 µg/L at 18 months of age (N < 10)) and normal in all other patients tested (n = 10). No patient displayed nodules on liver ultrasonography. Unlike patients with 3β-HSD deficiency [11, 12], no gallstones or kidney cysts were reported in our cohort. Four patients had a liver biopsy in the follow-up and liver histology showed no significant cholestasis or inflammation. Liver fibrosis scored in these biopsies was either stable or improved (F1: n = 2; F1/F2: n = 1; F3, n = 1) compared to previous liver biopsies (Table 5). Liver fibrosis assessed at last follow-up by liver elastography in nine patients showed values corresponding to a Metavir F0 score in 8 patients and F1 in one patient (Table 5). Of note, in patient G1, no evaluation of fibrosis was performed in the follow-up, but this patient displayed normal clinical examination and normal serum liver tests at last follow-up.

No patient had failure to thrive with a median Z-score for age of + 0.3 (IQR − 0.7 to + 1.3, range − 1 to + 4.6, n = 13) and − 0.2 (IQR − 0.5 to + 0.4, range − 0.9 to + 2.0, n = 13) for weight and height at last follow-up respectively. At last follow-up, no patient received vitamin supplementation beyond what is recommended for the general population. Plasma levels of vitamin A or E were within normal ranges in all evaluated patients (n = 13 and 14 respectively), whereas 2 patients out of 14 had a mild 25-hydroxy-vitamin D deficiency. All patients had urinary BA analysis at last follow-up. These analyses showed a significant reduction in 3-oxo-∆4 derivatives in all patients that were nonetheless still detectable at low levels in most patients (trace amounts). The metabolites were quantified at last follow-up in 13 patients, including the 6 who had quantification at baseline, with a median value of 5.5 µmol/mmol of creatininuria (IQR 3–12, range 0–56) corresponding to a decrease of 12-fold compared to the initial median level (Fig. 1).

The treatment with oral CA was well tolerated without severe adverse events. One patient (Patient L1) exhibited pruritus with weight loss 12 months after CA therapy initiation in absence of CA overdosing and with normal serum liver tests including serum bile acids. This event spontaneously resolved during follow-up. All patients had a normal quality of life and one patient (patient A2) had an uneventful pregnancy with CA treatment (already reported in (12)).