We have described three patients from a multiple consanguineous family with severe liver disease. In addition, patient 2 had severe diarrhoea requiring parenteral nutrition, and other features of THES including hair amino acid abnormalities which are found in these patients. As part of the clinical investigations sequencing of TTC37, mutations in which were known to cause THES, was undertaken but revealed no abnormalities. A combination of genome wide linkage scan and whole exome sequencing was performed to look for other genetic causes of liver disease in this family. All three of the patients were found to have a homozygous mutation in AKR1D1, consistent with a treatable bile acid biosynthesis disorder. Further bile acid analysis and successful treatment of patient 3 confirmed the initial diagnosis. Furthermore, patient 2 was found to have an additional disease causing mutation in SKIV2L, that was only recently described to cause THES.
Bile acid synthesis disorders can normally be identified by measurement of bile acids in the urine using liquid secondary ionization mass spectrometry . However, the presence of exogenous bile acids, such as ursodeoxycholic acid (UDCA), may mask the ability to detect a bile acid synthetic defect because UDCA and primary bile acids, being stereoisomers, have the same molecular weight. The FAB-MS analysis of the urine from patient 1, performed in 2003, revealed very high concentrations of dihydroxy-bile acid conjugates derived from the administered UDCA. There was no evidence for substantial amounts of Δ4-3-oxo bile acids that characterize a deficiency in Δ4-3-oxosteroid 5β-reductase , and the mass spectrum was not consistent with this bile acid disorder . More comprehensive GC-MS analysis performed 8 years later on this original urine sample, and only after the identification of the mutation in AKR1D1, showed that Δ4-3-oxo bile acids, while present, accounted for <2% of the total bile acids in urine, and the 5α-reduced metabolite, allo-cholic acid also synthesized in patients with Δ4-3-oxosteroid 5β-reductase deficiency was also found in traces , because the urine was dominated by UDCA and its metabolites (data not shown). Δ4-3-Oxo bile acids are normal metabolites of the urine of neonates and are consistently found in cases of advanced liver disease [7–10]. Cholic or chenodeoxycholic acid are normally also detected in the urine however in our case they were not identified. 5β-reduced bile acids were identified in the urine of patient 1 which are presumed to be metabolites of UDCA (a 5β-cholanoic acid) and its metabolites. Thus, the more comprehensive GC-MS analysis of the urine of patient 1 would support the diagnosis of a Δ4-3-oxosteroid 5β-reductase deficiency, with the complementary molecular confirmation of a mutation in AKR1D1 in this patient.
Bile acid synthesis disorders account for approximately 2% of idiopathic forms of liver disease but are important to diagnose as they can be treated by supplementation of primary bile acids, which is highlighted by this report. Indeed the new affected sibling of Patient 2 responded well to treatment with Cholic acid.
In conclusion we have extended the clinical features of Δ4-3-oxosteroid 5β-reductase enzyme deficiency as severe hypoglycaemia has not been previously reported. We also highlight the importance of performing urinary bile acid analysis in the absence of UDCA therapy in combination with genetic analysis. We also expand the mutational spectrum of SKIV2L and provide the first report of a combined phenotype of THES and Δ4-3-oxosteroid 5β-reductase enzyme deficiency. Combining the technique of whole genome linkage mapping and whole exome sequencing creates a powerful tool to elucidate the molecular basis of uncharacterized genetic disorders.
Patient consent and ethics approval
This study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the local Research Ethics Committees. All participants provided written informed consent for the collection of samples and subsequent analysis. In the case of children, consent was obtained from the patient’s guardian, parent or next of kin.