Bile Acid Synthesis Defect Research Articles

Diagnostic yield and novel candidate genes by next generation sequencing in 166 children with intrahepatic cholestasis.

Cholestatic liver disease is a leading referral to pediatric liver transplant centers. Inherited disorders are the second most frequent cause of cholestasis in the first month of life. We retrospectively characterized the genotype and phenotype of 166 participants with intrahepatic cholestasis, and re-analyzed phenotype and whole-exome sequencing (WES) data from patients with previously undetermined genetic etiology for newly published genes and novel candidates. Functional validations of selected variants were conducted in cultured cells. Overall, we identified disease-causing variants in 31% (52/166) of our study participants. Of the 52 individuals, 18 (35%) had metabolic liver diseases, 9 (17%) had syndromic cholestasis, 9 (17%) had progressive familial intrahepatic cholestasis, 3 (6%) had bile acid synthesis defects, 3(6%) had infantile liver failure and 10 (19%) had a phenocopy of intrahepatic cholestasis. By reverse phenotyping, we identified a de novo variant c.1883G > A in FAM111B of a case with high glutamyl transpeptidase (GGT) cholestasis. By re-analyzing WES data, two patients were newly solved, who had compound heterozygous variants in recently published genes KIF12 and USP53, respectively. Our additional search for novel candidates in unsolved WES families revealed four potential novel candidate genes (NCOA6, CCDC88B, USP24 and ATP11C), among which the patients with variants in NCOA6 and ATP11C recapitulate the cholestasis phenotype in mice models. In a single-center pediatric cohort, we identified monogenic variants in 22 known human intrahepatic cholestasis or phenocopy genes, explaining up to 31% of the intrahepatic cholestasis patients. Our findings suggest that re-evaluating existing WES data from well-phenotyped patients on a regular basis can increase the diagnostic yield for cholestatic liver disease in children.

Product Validation and Stability Testing of Pharmacy Compounded Cholic Acid Capsules for Dutch Patients with Rare Bile Acid Synthesis Defects

Bile acid synthesis defects (BASDs) comprise a group of rare diseases that can be severely disabling. Bile acid supplementation with 5 to 15 mg/kg cholic acid (CA) has been hypothesized to decrease endogenous bile acid production, stimulate bile secretion, and improve bile flow and micellar solubilization, thereby improving the biochemical profile and potentially slowing down disease progression. Currently, CA treatment is unavailable in the Netherlands, and CA capsules were compounded by the Amsterdam UMC Pharmacy from CA raw material. This study aims to determine the pharmaceutical quality and stability of the pharmacy compounded CA capsules. Pharmaceutical quality tests were performed on 25 mg and 250 mg CA capsules according to general monographs of the European Pharmacopoeia 10th ed. For the stability study, the capsules were stored under long-term conditions (25 °C ± 2 °C/60% ± 5% RH) and accelerated conditions (40 °C ± 2 °C/75% ± 5% RH). Samples were analyzed at 0, 3, 6, 9 and 12 months. The findings demonstrate that the pharmacy compounded CA capsules within a range of 25-250 mg that complied with the European regulations in regard to product quality and safety. The pharmacy compounded CA capsules are suitable for use in patients with BASD, as clinically indicated. With its simple formulation, pharmacies are provided a guidance on product validation and stability testing when commercial CA capsules are unavailable.

Substrate Specificity and the Direction of Transport in the ABC Transporters ABCD1-3 and ABCD4.

The ATP-binding cassette (ABC) transporters are one of the largest families of membrane-bound proteins and exist in almost all living organisms from eubacteria to mammals. They transport diverse substrates across membranes utilizing the energy of ATP hydrolysis as a driving force and play an essential role in cellular homeostasis. In humans, four ABC transporters classified as subfamily D have been identified. ABCD1-3 are localized to peroxisomal membranes and involved in the transport of various acyl-CoAs from the cytosol to the peroxisomal lumen. ABCD4 functions on the lysosomal membranes and transports vitamin B12 (cobalamin) from lysosomes into the cytosol. The mutation of genes encoding ABCD1, ABCD3, and ABCD4 are responsible for genetic diseases called X-linked adrenoleukodystrophy, congenital bile acid synthesis defect 5, and cobalamin deficiency, respectively. In this review, we summarize the targeting mechanism and physiological functions of the ABCD transporters and discuss insights that have been obtained on the transport mechanism based on disease-causing mutations and cryo-electron microscopy (EM) structural studies.

Neonatal cholestasis – A case report on congenital bile acid synthetic defect type 4 and severe anemia

Congenital defects of bile acid synthesis are rare disorders that cause progressive liver dysfunction. We present a case of alpha methylacyl-CoA racemase (AMACR) deficiency with non-spherocytic hemolytic anemia who presented with rapidly progressive severe cholestasis and liver failure with normal gamma-glutamyl transferase levels. After extensive investigation, he was found to have AMACR deficiency with HBB gene mutation associated with non-spherocytic hemolytic anemia possibly explaining the severity of the disease. To the best of our knowledge, a similar association has not been reported so far.

Congenital Bile Acid Synthesis Defect Type 3 With Severe Neonatal Cholestasis

Congenital bile acid synthesis defect type 3 is a rare metabolic liver disease with only eight patients reported in literature. We describe clinical, pathological and molecular features for a ninth patient. A 4-month-old infant presented to us with conjugated hyperbilirubinemia. His liver biopsy revealed giant cell change, steatosis, and activity with diffuse fibrosis. Immunostaining with bile salt export pump showed preserved canalicular pattern and γ-glutamyl transferase 1 staining showed unusual complete membranous pattern. Genetic workup revealed homozygous single base pair duplication in exon 3 of the CYP7B1 gene. He succumbed to liver disease at 7months of age.

The Liver Biopsy in Neonatal Cholestasis: Just a Cherry on Top?

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Pharmacy Compounded Medicines for Patients With Rare Diseases: Lessons Learned From Chenodeoxycholic Acid and Cholic Acid.

Patients with rare diseases are often confronted with the fact that effective medicines are unavailable or simply not being developed. This situation jeopardizes the health of a large population of vulnerable patients with rare diseases. Pharmacy compounded formulations can provide a safe alternative when authorized treatments are unavailable or unsuitable. Practical guidelines on how to develop and implement pharmacy compounded formulations for patients with rare diseases are limited. The aim of this article is to provide guidance for when and how to apply pharmacy compounded formulations for patients with rare diseases. This is illustrated with two challenging examples: the development and implementation of pharmacy compounding of 1) chenodeoxycholic acid (CDCA) capsules for patients with cerebrotendinous xanthomatosis (CTX) and 2) cholic acid (CA) capsules for patients with rare bile acid synthesis defects (BASD). All critical steps of the development of CDCA and CA capsules are explained and summarized in a practical guideline.

Liver Transplantation for Infantile Liver Failure Secondary to Oxysterol-7 Alpha Hydroxylase Deficiency

Bile acid synthesis defects (BASDs) are rare inborn errors of metabolism, causing neonatal hepatitis. These disorders are difficult to diagnose, especially in resource-limited settings where extensive laboratory and genetic analysis are not always feasible. A delay in diagnosis and replacement therapy where possible often leads to progressive liver cell failure. We report an infant with BASD (oxysterol-7 alpha hydroxylase deficiency) who had decompensated chronic liver disease and underwent successful living donor liver transplantation.

Peroxisomal ABC Transporters: An Update.

ATP-binding cassette (ABC) transporters constitute one of the largest superfamilies of conserved proteins from bacteria to mammals. In humans, three members of this family are expressed in the peroxisomal membrane and belong to the subfamily D: ABCD1 (ALDP), ABCD2 (ALDRP), and ABCD3 (PMP70). These half-transporters must dimerize to form a functional transporter, but they are thought to exist primarily as tetramers. They possess overlapping but specific substrate specificity, allowing the transport of various lipids into the peroxisomal matrix. The defects of ABCD1 and ABCD3 are responsible for two genetic disorders called X-linked adrenoleukodystrophy and congenital bile acid synthesis defect 5, respectively. In addition to their role in peroxisome metabolism, it has recently been proposed that peroxisomal ABC transporters participate in cell signaling and cell control, particularly in cancer. This review presents an overview of the knowledge on the structure, function, and mechanisms involving these proteins and their link to pathologies. We summarize the different in vitro and in vivo models existing across the species to study peroxisomal ABC transporters and the consequences of their defects. Finally, an overview of the known and possible interactome involving these proteins, which reveal putative and unexpected new functions, is shown and discussed.

The expression and purification of human steroid hormone processing enzyme CYP7B1 and the characterization of its interactive network

The cytochrome P450 monooxygenase CYP7B1 is an integral membrane protein at the endoplasmic reticulum (ER) membrane and catalyzes the hydroxylation of 7-alpha position of the steroid backbone, generating a series of important steroids such as 7α-hydroxy dehydroepiandrosterone and 7α-hydroxy pregnenolone. The disruption of CYP7B1 function could lead to abnormal oxysterols levels in neurons and the liver, and thus result in diseases such as hereditary spastic paraplegia and congenital bile acid synthesis defect. CYP7B1 was found to play a critical role in the biochemical network for biosynthesis/modification of steroids which involves hydrogenation/dehydrogenation, hydroxylation, oxygenation reaction at various carbon positions of the steroid backbone. Although a biologically-related enzyme, CYP7A1, has been successfully purified and structurally characterized, little was known about the structure and catalyzing mechanism for CYP7B1. To understand the structural basis of CYP7B1 functions, recombinant expression systems were screened for high-level expression and purification. Humans CYP7B1 was successfully expressed in the baculovirus/insect cell system and purified with its redox cofactor, indicating a natural folding for CYP7B1 samples. When challenged with detergents of various lengths of hydrophobic side chains, purified CYP7B1 proteins showed moderate stability but failed to maintain the natural folding upon the “harsh” detergent such as octyl glucoside. To explore the interactive proteins for CYP7B1, a pulldown-mass spectrometry assay using purified CYP7B1 was conducted and identified multiple ER-resident enzymes including cytochrome P450 reductase CYPOR as interactive proteins for CYP7B1, as well as other redox enzymes such as cytochrome b5 reductase and cytochrome b5 family members, indicating the existence of a physical interactive network at ER compromising cytochrome P450 and b5 family members and their reductases. Further efforts were conducted to confirm molecular interaction from mass-spectral assay and the in vitro macromolecular interaction assay showed the association between CYP7B1 and CYPOR. The CYP7B1-CYPOR protein complex was purified upon the optimization of co-expression system and in vitro assembly protocols, implying a catalytic complex might exist in ER membrane comprising CYP7B1, CYPOR, and other redox enzymes. Our results showed that human cytochrome P450 protein CYP7B1 could be purified in its cofactor-bound form and bind to cytochrome P450 reductase CYPOR to form a stable complex resistant to solubilization by detergent. Further studies should be conducted to map the components and interactions in the redox-enzyme complex at ER and determine the structural basis for the electron transfer therein.

Clinical characteristics and gene variants of patients with infantile intrahepatic cholestasis

Objective To explore the clinical characteristics and genetic findings of patients with infantile intrahepatic cholestasis. Methods The clinical data were collected in children who were admitted to the Department of Gastroenterology in Children's Hospital, Capital Institute of Pediatrics from June 2017 to June 2019 and were suspected of inherited metabolic diseases. Next generation sequencing based on target gene panel was used for gene analysis in these children. Sanger sequencing technology was used to verify the genes of the members in this family. Results Forty patients were enrolled. Pathogenic gene variants were identified in 13 patients (32%), including SLC25A13 gene variation in 3 patients who were diagnosed with citrin deficiency, JAG1 gene variation in 3 patients who were diagnosed with Alagille syndrome, ABCB11 gene variation in 3 patients who were diagnosed with progressive familial intrahepatic cholestasis type 2, HSD3B7 gene variation in 1 patient who was diagnosed with congenital bile acid synthesis defect type 1, AKR1D1 gene variation in 1 patient who was diagnosed with congenital bile acid synthesis defect type 1, NPC1 gene variation in 1 patient who was diagnosed with Niemann-Pick disease, and CFTR gene variation in 1 patient who was diagnosed with cystic fibrosis. Conclusions The etiology of infantile intrahepatic cholestasis is complex. Next generation sequencing is helpful in the diagnosis of infantile intrahepatic cholestasis.

Pediatric Cholestatic Liver Disease: Review of Bile Acid Metabolism and Discussion of Current and Emerging Therapies.

Cholestatic liver diseases are a significant cause of morbidity and mortality and the leading indication for pediatric liver transplant. These include diseases such as biliary atresia, Alagille syndrome, progressive intrahepatic cholestasis entities, ductal plate abnormalities including Caroli syndrome and congenital hepatic fibrosis, primary sclerosing cholangitis, bile acid synthesis defects, and certain metabolic disease. Medical management of these patients typically includes supportive care for complications of chronic cholestasis including malnutrition, pruritus, and portal hypertension. However, there are limited effective interventions to prevent progressive liver damage in these diseases, leaving clinicians to ultimately rely on liver transplantation in many cases. Agents such as ursodeoxycholic acid, bile acid sequestrants, and rifampicin have been mainstays of treatment for years with the understanding that they may decrease or alter the composition of the bile acid pool, though clinical response to these medications is frequently insufficient and their effects on disease progression remain limited. Recently, animal and human studies have identified potential new therapeutic targets which may disrupt the enterohepatic circulation of bile acids, alter the expression of bile acid transporters or decrease the production of bile acids. In this article, we will review bile formation, bile acid signaling, and the relevance for current and newer therapies for pediatric cholestasis. We will also highlight further areas of potential targets for medical intervention for pediatric cholestatic liver diseases.

Bile acid synthesis defect (5B-reductase deficiency) a rare cause of cholestasis in an infant

Bile acid synthesis defect (5B-reductase deficiency) a rare cause of cholestasis in an infant

Cholic acid for primary bile acid synthesis defects: a life-saving therapy allowing a favorable outcome in adulthood

BackgroundOral cholic acid (CA) replacement has been shown to be an effective therapy in children with primary bile acid synthesis defects, which are rare and severe genetic liver diseases. To date there has been no report of the effects of this therapy in children reaching adulthood. The aim of the study was to evaluate the long-term effectiveness and safety of CA therapy.MethodsFifteen patients with either 3β-hydroxy-Δ5-C27-steroid oxidoreductase (3β-HSD) (n = 13) or Δ4–3-oxosteroid 5β-reductase (Δ4–3-oxo-R) (n = 2) deficiency confirmed by mass spectrometry and gene sequencing received oral CA and were followed prospectively.ResultsThe median age at last follow-up and the median time of follow-up with treatment were 24.3 years (range: 15.3–37.2) and 21.4 years (range: 14.6–24.1), respectively. At last evaluation, physical examination findings and blood laboratory test results were normal in all patients. Liver sonograms were normal in most patients. Mean daily CA dose was 6.9 mg/kg of body weight. Mass spectrometry analysis of urine showed that excretion of the atypical metabolites remained low or traces in amount with CA therapy. Liver fibrosis scored in liver biopsies or assessed by elastography in 14 patients, after 10 to 24 years with CA therapy, showed a marked improvement with disappearance of cirrhosis (median score < F1; range: F0-F2). CA was well tolerated in all patients, including five women having 10 uneventful pregnancies during treatment.ConclusionsOral CA therapy is a safe and effective long-term treatment of 3β-HSD and Δ4–3-oxo-R deficiencies and allows affected children to reach adulthood in good health condition without the need for a liver transplantation.

Electron Microscopy Can Still Have a Role in the Diagnosis of Selected Inborn Errors of Metabolism.

Many anatomic pathology laboratories no longer have electron microscopy facilities. A retrospective review of autopsies was performed to identify cases of inborn errors of metabolism (IEM) and determine the contribution of electron microscopy in making the diagnosis in those cases. Over a period of 17 years, there were 900 perinatal and pediatric autopsies. There were 7 cases (1%) of IEM, including 4 cases of Pompe disease, 1 case of I-cell disease, 1 case of bile acid synthesis defect, and 1 case of mitochondrial disease (Leigh syndrome). Electron microscopy was important in the diagnosis of I-cell disease and Pompe disease in our series. This technique enabled a prenatal diagnosis to be made from a chorionic villus biopsy in 2 cases with a positive family history. In less developed countries where upfront genetic testing may be too expensive and may need international referral, electron microscopy can still be useful for diagnosis of IEM, providing an affordable alternative with a more rapid turnaround time compared to gene mutation analysis or enzyme assay. Results can be used both for patient management and as a screen for which cases might benefit from genetic testing.

Bile Acid Synthesis Defect and Hyperinsulinism

Congenital defects of bile acid synthesis are rare disorders that cause progressive liver dysfunction. Prolonged neonatal hyperinsulism (PNH) is a separate entity that leads to persistent hypoglycemia secondary to stress. We present a 4-month-old infant who presented with liver failure secondary to a bile acid synthesis defect. The patient's liver failure resolved with oral cholic acid therapy. This patient also developed PNH, which slowly resolved over time. This case illustrates a possible relationship between cholestatic liver failure and PNH. This relationship may help define specific stressors that increase the likelihood of developing PNH.

Clinical feature and genetic analysis of a family affected by congenital bile acid synthesis defect type 2: identification of 2 novel mutations in AKR1D1 gene

Congenital bile acid synthesis defect type 2 (CBAS2) is an autosomal recessive disorder caused by biallelic mutations of AKR1D1 gene, which encodes the Δ4-3-oxo-steroid 5β-reductase. Cholestatic jaundice is the main clinical manifestation, accompanied by malabsorption of fat and fat-soluble vitamins. This paper reported the clinical and genetic features of a CBAS2 patient definitely diagnosed by AKR1D1 genetic analysis. An 8-month-old male infant was referred to the hospital with the complaint of jaundiced skin and sclera over 7 months. On physical examination, growth retardation and malnutrition were discovered besides mild jaundice of the skin and sclera. The liver was palpable 8 cm below the right subcostal margin with medium texture, and the spleen was not enlarged. On liver function test, elevated levels of bilirubin (predominantly conjugated bilirubin) and transaminases were detected, but serum total bile acids and γ-glutamyl transpeptidase levels were within the normal ranges. Liver histopathologic analysis showed disorganized bile ducts, obvious multinucleated giant cells, significant cholestasis in hepatocytes, together with portal and interstitial fibrosis and lymphocytic infiltration. Via next generation sequencing analysis and Sanger sequencing confirmation, the infant proved to be a compound heterozygote of the AKR1D1 variants c.579+2delT and c.853C>T(p.Q285X), two novel mutations originated from his mother and father, respectively. CBAS2 was thus definitely diagnosed, and chenodeoxycholic acid was given orally. As a result, the abnormal liver function and hepatomegaly were improved gradually. On a follow-up 3 months later, a soft liver was palpable 2.5 cm below the right subcostal margin, and all liver function indices recovered to normal ranges.

Improved synthesis of glycine, taurine and sulfate conjugated bile acids as reference compounds and internal standards for ESI–MS/MS urinary profiling of inborn errors of bile acid synthesis

Bile acid synthesis defects are rare genetic disorders characterized by a failure to produce normal bile acids (BAs), and by an accumulation of unusual and intermediary cholanoids. Measurements of cholanoids in urine samples by mass spectrometry are a gold standard for the diagnosis of these diseases. In this work improved methods for the chemical synthesis of 30 BAs conjugated with glycine, taurine and sulfate were developed.Diethyl phosphorocyanidate (DEPC) and diphenyl phosphoryl azide (DPPA) were used as coupling reagents for glycine and taurine conjugation. Sulfated BAs were obtained by sulfur trioxide-triethylamine complex (SO3-TEA) as sulfating agent and thereafter conjugated with glycine and taurine. All products were characterized by NMR, IR spectroscopy and high resolution mass spectrometry (HRMS). The use of these compounds as internal standards allows an improved accuracy of both identification and quantification of urinary bile acids.

Tendon xanthomas: Not always familial hypercholesterolemia

Tendon xanthoma are most commonly associated with Familial Hypercholesterolemia, but the differential diagnosis includes sitosterolemia and cerebrotendinous xanthomatosis (CTX). The case presented here is of a 48-year old male with large tendon xanthomas attributable to CTX. CTX is a rare, recessive disorder caused by mutations in the CYP27A1 gene. The resultant defect in bile acid synthesis leads to cholestanol deposition in different tissues in the body, including tendons. CTX is associated with neurologic symptoms and a reduced life expectancy. Treatment consists of bile acid supplementation in combination with a statin. When patients present with tendon xanthomas and FH is ruled out, clinicians should consider CTX as a possible diagnosis.

Novel Mutations in the 3β-hydroxy-Δ5-C27-steroid Dehydrogenase Gene (HSD3B7) in a Patient with Neonatal Cholestasis

Bile acid synthetic defect (BASD) is a rare category of genetic disorders that are responsible for approximately 2% of persistent cholestasis in infants.[1] Until date, four enzymes responsible for congenital defects of bile acid synthesis (CBAS) have been identified. 3β-hydroxy-∆5-C27-steroid dehydrogenase (3β-HSD), the deficiency of which can cause CBAS1 (OMIM No. 607765), is encoded by the gene HSD3B7 and works in the second step of transforming the steroid into primary bile acids.