Treating genetic disease: Expanding the options
Authors: Richard J. Thompson, Laura N. Bull
Journal: Hepatology (2015)

Potential conflict of interest: Dr. Thompson is on the speakers' bureau for and received grants from Shire. See Article on Page 558 Author names in bold indicate shared co‐first authorship. We practice in the postgenomic era of medicine. We now understand that genetic variants contribute to disease phenotypes in many ways. Complex diseases have multiple risk loci, and even Mendelian diseases can have modifiers. Clearly, the aim is to use this knowledge to better treat our patients. Understanding the genetic variants that contribute to any phenotype will help us to understand the pathophysiological processes involved. Such abnormalities, in known pathways, could be ameliorated or bypassed. Examples of these might be ursodeoxycholic acid (UCDA), which can decrease the consequences of reduced biliary phospholipids in multidrug resistance 3 deficiency, or nitisinone, which prevents accumulation of toxic intermediates in tyrosinemia. An obvious approach to genetic disease would be gene therapy; but this has had limited impact in liver disease thus far. So, can we direct our treatment to the mutant gene or protein underlying the disease? In this issue of Hepatology, Gonzales et al. have made progress in that direction, with particular respect to bile salt export protein (BSEP) deficiency.1 BSEP is encoded by ABCB11. Deleterious mutations in ABCB11 can result in BSEP deficiency, in which patients have reduced or no BSEP activity, resulting in impaired export of bile acids out of the liver, leading to cholestasis, and liver damage and malabsorption. BSEP deficiency varies in severity, depending, in part, on whether a patient's disease mutation(s) result in complete or partial loss of BSEP function. Patients with severe BSEP deficiency typically progress and require liver transplantation during childhood. They are also at significant risk of developing hepatocellular carcinoma (HCC) in their native liver.2 Patients with milder BSEP deficiency may do well for prolonged periods with medical treatment (especially ursodeoxycholate) and/or biliary diversion, and their risk of HCC may be lower, compared to those with severe BSEP deficiency.3 There are many different types of mutation that lead to disease; it is possible that some may be amenable to treatment based on the exact mechanisms of action. Whole gene deletion, for example, might only be amenable to replacement gene therapy. Attempts have been made to overcome the effects of protein truncating "stop" mutations.4 However, in most genes, a substantial proportion of disease‐causing mutations are described as "missense" mutations. In severe BSEP deficiency, 80% of patients have at least one missense mutation.2 Such mutations are often described using the change predicted to occur at the amino acid level (e.g., p.E297G). However, such nomenclature disguises a multitude of potential disease‐causing mechanisms. The actual mutation is, of course, in genomic DNA, where few effects are observed, though occasionally transcription may be affected. Once an RNA transcript is generated, this must be processed, most importantly through the splicing out of introns. It is clear that changes in coding regions can significantly disrupt this process. At least in vitro, measures can be taken to overcome these effects.5 Assuming mature messenger RNA is available and translation occurs, then a mutant protein would be expected to be made. BSEP is a membrane protein with 12 transmembrane spans. For this membrane topology to occur, the appropriate hydrophobic domains must be maintained. Once inserted in the membrane, the complex must be trafficked to the apical domain of the cell. This requires correct interaction with other membrane and cytoskeletal proteins. Last, once in the correct place in the membrane, the amino acid sequence must allow normal protein function to occur. It is clear that for proteins such as BSEP, most missense mutations actually result in either RNA processing or protein trafficking defects.5 For the latter, it is possible that the effects of sequence variants can be reduced though use of chaperone molecules. The work published in this issue by Gonzales et al. describes the use of a pharmacological chaperone drug, 4‐phenylbutyrate (4‐PB), in BSEP deficiency. This builds on previous studies of 4‐PB in this disease. Two single‐case studies have previously been reported, in which 4‐PB was administered to BSEP deficiency patients. The first such study focused upon administration of 4‐BP to patient 1 in the current study, with 19‐month follow‐up.6 In the second study, 4‐BP was administered to a BSEP deficiency patient for 6 months and then withdrawn.7 In both of these studies, in vitro data were presented indicating that the patients carried missense mutations resulting in impaired targeting of BSEP to the canalicular membrane, and that 4‐PB enhanced correct targeting. Also, in both case reports, improvement in pruritus and aspects of serum biochemistry tracked with consistent use of adequate doses of 4‐PB. In the current study, available data on effects of 4‐PB on BSEP deficiency are substantially increased, with both in vitro and clinical results presented.1 The 4 patients treated with 4‐PB each bear at least one missense mutation, in vitro canalicular targeting of which is increased upon administration of UCDA or 4‐PB; generally, the combination of UDCA and 4‐PB further enhanced correct targeting. The patients were treated with 4‐PB for between 3 and 50 months, although in the case of 2 patients, there were periods of noncompliance. This study provides substantial new evidence for the efficacy of 4‐PB in treatment of BSEP deficiency, in patients bearing mutations that result in protein with residual function, but abnormal localization. Although 4‐PB is termed a chaperone, there is still little evidence for the mechanisms underlying the benefits observed. Whereas more protein is evident at the cell surface, this might still reflect a relative failure of clearance, rather than a true chaperone effect.8 If use of 4‐PB is to become more common in BSEP deficiency patients, several issues remain to be addressed. For example, it is likely impractical to perform in vitro assays on every missense mutation, to determine whether it results in protein mistargeting responsive to 4‐PB, and whether that protein is still a capable bile acid transporter. Therefore, clinical decisions regarding whether to try 4‐BP in a patient will sometimes need to be made in the absence of such data. In such a case, upon which criteria will the decision be made? Perhaps patients with mutations in highly conserved amino acids with known roles in BSEP's function will not be considered good candidates, whereas those with mutations in less‐conserved residues will be? Furthermore, a challenge with 4‐PB is that it has an unpleasant taste and involves ingestion of a large number of pills. Will this limit its utility? In the current study, the patients ranged in age from 10 to 22 years. These patients are likely old enough to understand the utility of the medication, and indeed 2 of them restarted the medication voluntarily after periods of noncompliance, based upon their experience of decreased pruritus on 4‐PB. However, one would imagine that earlier use would be expected to produce better long‐term outcomes. Patients surviving into the second or third decade with their own liver are certainly not the most severely affected. Another important issue regarding BSEP deficiency is the increased risk of HCC. Whereas this risk appears greatest in patients with mutations likely to result in complete loss of BSEP function,2 monitoring for HCC in all patients with BSEP deficiency is essential. To what extent this risk is reduced by such treatment is obviously not yet known. 4‐PB may not be the best drug for the purpose to which it is put in the current studies. However, it does provide evidence that, in this disease, the potential exists for use of such drugs.