A 7-month-old female infant was referred to our hospital due to prolonged jaundice for approximately 4 months and growth retardation for 1.5 months. Her jaundice (yellow skin and sclera) drew the attention of her parents at the age of 3.3 months. A liver function test at the local hospital revealed elevated cholestatic indices, including gamma-glutamyl transpeptidase (GGT), direct bilirubin (Dbil) and total bile acid (TBA) (). Due to the prolonged jaundice and unresolved laboratory abnormalities, the infant was referred to another hospital at the age of 4 months, where magnetic resonance cholangiopancreatography revealed the dilatation of common hepatic and bile ducts. Laparoscopy was thus performed when the infant was aged 4.5 months, and an intraoperative cholangiography displayed biliary tree dilatation and filling defect in the common bile duct, along with an accessory hepatic duct (AHD) joining the cystic duct (). A cholecystectomy was subsequently performed, an intrabiliary thick plug in the color of dark green was removed, and bile duct irrigation and choledochostomy with T-tube drainage was carried out. Pathological analysis of the plug confirmed the diagnosis of inspissated bile syndrome (IBS). Thereafter, the infant’s jaundice subsided and the laboratory indices gradually improved (); the infant was discharged at the age of 5 months. Half a month later, nevertheless, a physical examination revealed that her body weight was 5.34 kg (−3.6 SD), her length was 59 cm (−3.2 SD) and her head circumference was 39 cm (−2.5 SD). Another anthropometric test at the age of 6 months revealed a weight of 6.0 kg (−2.5 SD) and a length of 62.0 cm (−2.2 SD). The growth retardation continued in the following month, and the infant was referred to our hospital at the age of 7 months for further evaluation, following the removal of the drainage T-tube.\nThe infant was born to a non-consanguineous couple after 37 weeks of uneventful gestation with a birth weight of 2.25 kg. On the first day after birth, the infant was admitted to hospital due to vomiting and respiratory distress. Contrast imaging of the upper digestive tract revealed the existence of esophageal atresia (EA) (), which was then resolved by a gastroesophagostomy under general anesthesia. The couple had experienced 2 pregnancies prior to this one, but both were aborted in the first trimester. Both parents appeared healthy, without any clinical symptoms or signs of CTLN2. There was no known family history of any genetic disease.\nPhysical examination at referral revealed a weight of 6.25 kg (−2.6 SD), a length of 63.0 cm (−3.3 SD) and a head circumference of 42 cm (−1.0 SD). There was no dysmorphic appearance, only a slightly chubby face. There was no evidence of jaundice (yellow skin and sclera) and there were no visible petechiae or ecchymoses. No pallor or cyanosis of the lips were observed. There was no tachypnea or dyspnea, and no stridor, wheezes, crackles or crepitus could be heard on auscultation of the both lungs. The heart sounds were normal without audible murmurs or arrhythmia. Upon abdominal inspection, no dilated veins or abdominal distention were observed. The liver was palpated with a soft edge 2 cm below the right costal margin in the mid-clavicular line. Her spleen was not palpable. A neurological examination revealed slightly reduced muscle tone. There was no neck stiffness, knee reflex was normal and there was no positivity for Brudzinski’s or Kernig’s sign.\nFollowing biochemical analysis, no abnormal liver function index was observed, although the serum GGT level was elevated, suggesting the existence of cholestasis, as shown in . Taking into consideration her prolonged jaundice, growth retardation and chubby face, and the 2 first-trimester miscarriages of her mother, SLC25A13 gene analysis was performed on the family to evaluate the possibility of a diagnosis of CD. A lactose-free and MCT-enriched therapeutic formula was subsequently introduced, while a diet rich in protein and lipids was also encouraged. A clinical following-up 4 months later revealed that the weight of the infant was 7.9 kg (−1.6 SD), her length was 70 cm (−1.5 SD) and her head circumference was 44 cm (−0.8 SD), along with a marked improvement (complete recovery) of the cholestatic indices, GGT, TBA and Dbil ().
An 18.5-year-old female was presented to the outpatient clinic due to overweight. Her height was 166.1 cm, her weight was 80.1 kg, and her body mass index (BMI) was 29.0 kg/m2. No clinical signs of hyperandrogenism or hypercortisolism were observed. The skin was normal; however, striae cutis distensae were seen. Neither hirsutism nor acanthosis nigricans was present. No indication of ichthyosis was noted. Pubertal development was complete, with breast stage B5, pubic hair stage PH5, menstrual periods were regular. Blood pressure was within the norm, at 125/80 mm Hg.\nThe patient fulfilled only two criteria for metabolic syndrome in adolescence by having disturbed glucose metabolism and abdominal obesity. Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) was increased to 6.9; oral glucose tolerance was pathological (2 h glucose concentration 165 mg/dl). Serum concentrations of high-density lipoprotein (HDL)-cholesterol (60 mg/dl) and triglycerides (108 mg/dl) were within the reference range, while cholesterol (232 mg/dl) and low-density lipoprotein (LDL)-cholesterol (186 mg/dl) were increased. The patient underwent a relatively successful lifestyle intervention, which enabled a stabilization of her BMI and a normalization of her oral glucose tolerance within 6 months.\nRoutine analysis of adrenal hormones revealed an extremely high serum DHEAS level, at 7,546 ng/ml (reference < 4,000; ). A second blood sample confirmed the DHEAS excess, with even a higher serum level, at 8,835 ng/ml. In contrast, serum DHEA was in the normal range, at 443 ng/dl (reference < 750). Serum ACTH (4.8 pmol/L), cortisol (22.7 μg/dl), androstenedione (10 nmol/L), 17-OH progesterone (70 ng/dl), testosterone (28 ng/dl), luteinizing hormone (LH; 5.7 UI/L), follicle-stimulating hormone (FSH; 1.7 IU/L), as well as thyroid-stimulating hormone (TSH; 1.57 mIU/L), and free thyroxine (fT4; 14 pmol/L) levels were all within the normal range.\nThe serum steroid sulfatome was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The high DHEAS serum level was confirmed. Interestingly, with the exception of cholesterol sulfate, several other sulfated steroids were found to be above the reference range, namely, pregnenolone sulfate, 17-hydroxypregnenolone sulfate, 16-α-hydroxy-DHEAS, androstenediol-3-sulfate, androsterone sulfate, and epiandrosterone sulfate (). In addition, the urinary steroid metabolome was delineated by gas chromatography-mass spectrometry (GC-MS) in a 24 h urinary sample. It revealed excessive excretion of DHEAS metabolites at 200% of the upper limit of the reference interval. Urinary analysis excluded any known enzyme defect of the adrenal steroid synthesis.\nFor exclusion of an autonomous hormone secretion by an adrenal or an ovarian tumor, administration of 0.5 mg dexamethasone four times daily on four consecutive days was performed which resulted in an efficient suppression of DHEAS and DHEA as well as their urinary metabolites. On day 5, the DHEAS level decreased to 1,199 ng/ml (13.6% of baseline; reference < 47.2%) and the DHEA level to 144 ng/dl (32% of baseline; reference < 38.9%) (). In addition, urinary cortisol metabolites were sufficiently suppressed during the dexamethasone test. Ultrasound and MRI scans of the adrenal glands and the ovaries were normal. No further deterioration in the patient's hormonal investigations was noted during the 4 year course of observation.\nFollowing the hypothesis that the high DHEAS level was likely to be hereditary, we sequenced the STS gene, which is responsible for the hydrolysis of aryl and alkyl steroid sulfates and catalyzes the conversion of DHEAS to DHEA (). Sequence analysis revealed a heterozygous single-base substitution (g.117217G>T) that results in a nonsense mutation at codon 173 (p.G173X). This mutation predicts a truncation of the carboxyl region of the STS enzyme that is implicated in substrate binding. The mutant allele contained two further variants within the fifth exon: a silent mutation (g.117309C>A) at codon 203 C → A (leucine), and a missense mutation at codon 464 G → T, which resulted in substitution of methionine for isoleucine (g.117465G>T). This nonsense mutation is not present in the Exome Variant Server. At 19 bp upstream in the genomic sequence of exon 10, we identified another C → T transition (g.207619C>T). The nonsense mutation p.G173X found in the patient was de novo as demonstrated by allele-specific amplification of the wild-type (wt) and mutant sequences (mut) of both parents' DNA samples. No partial gene deletion of the presumably intact allele outside exon 5 was detected by multiplex ligation-dependent probe amplification.\nBiological activity of STS was measured in the patient's blood leukocytes after addition of 1,2,6,7-3H-DHEAS. In our experimental setting, ~7 nmol DHEA was produced by 1 × 106 leukocytes per 4 h. The mean (SD) STS activity detected in patient's leukocytes was 107% in comparison to those of five healthy age-matched female controls. Therefore, normal hydrolytic activity of STS was revealed in the patient's blood leukocytes.\nNo clear explanation for the high levels of DHEAS connected to the STS gene structure and enzyme activity was found. Therefore, a defect of one or more transporter proteins was suggested (). On the one hand, a reduced steroid sulfate efflux activity in the liver would enhance serum levels; on the other hand, impaired steroid sulfate uptake from the circulation into peripheral target cells or hepatocytes would have the same effect. Prominent candidate efflux transporters were MRP2 and BCRP, both highly expressed at the canalicular membrane of hepatocytes and involved in the hepatobiliary elimination of many drugs and some endogenous substrates such as sulfated steroids (). However, as MRP2 deficiency would typically increase the levels of conjugated bilirubin in the plasma (Dubin–Johnson syndrome) () what was not seen in the patient, BCRP was the only candidate carrier for the efflux site. On the uptake site, several OATP and OAT carriers were on the list. However, as most of them show highly overlapping substrate specificities and redundant expression patterns (e.g., OATP1B1, OATP1B3, and OATP2B1 are expressed in the liver) (), it was supposed that a transport defect of one of these carriers could have been compensated by another member of the same carrier family. Therefore, we decided to select two carriers from the SLC10 carrier family, which are more unique in the sense of substrate specificity and tissue expression and which both show sodium-dependent transport of sulfated steroids (, ). In both carriers, genetic variants or mutations were described before with significantly reduced transport activity, associated with increased plasma levels of their substrates (–). However, as the determination of DHEAS serum levels is not included in standard diagnostics, elevated DHEAS levels may have been overseen in patients with such carrier defects. Using exon-spanning PCR, all exons of the abovementioned membrane transporters were sequenced. There were no relevant variants detected for the SLC10A1 and SLC10A6 genes. Sequence analysis revealed a heterozygous Q141K variant for BCRP. Interestingly, this variant has in its homozygous state previously been associated with reduced efflux transport activity ().