Oral pharmacological chaperone migalastat compared with enzyme replacement therapy in Fabry disease: 18-month results from the randomised phase III ATTRACT study.

BACKGROUND: Fabry disease is an X-linked lysosomal storage disorder caused by GLA mutations, resulting in α-galactosidase (α-Gal) deficiency and accumulation of lysosomal substrates. Migalastat, an oral pharmacological chaperone being developed as an alternative to intravenous enzyme replacement therapy (ERT), stabilises specific mutant (amenable) forms of α-Gal to facilitate normal lysosomal trafficking. METHODS: The main objective of the 18-month, randomised, active-controlled ATTRACT study was to assess the effects of migalastat on renal function in patients with Fabry disease previously treated with ERT. Effects on heart, disease substrate, patient-reported outcomes (PROs) and safety were also assessed. RESULTS: Fifty-seven adults (56% female) receiving ERT (88% had multiorgan disease) were randomised (1.5:1), based on a preliminary cell-based assay of responsiveness to migalastat, to receive 18 months open-label migalastat or remain on ERT. Four patients had non-amenable mutant forms of α-Gal based on the validated cell-based assay conducted after treatment initiation and were excluded from primary efficacy analyses only. Migalastat and ERT had similar effects on renal function. Left ventricular mass index decreased significantly with migalastat treatment (-6.6 g/m2 (-11.0 to -2.2)); there was no significant change with ERT. Predefined renal, cardiac or cerebrovascular events occurred in 29% and 44% of patients in the migalastat and ERT groups, respectively. Plasma globotriaosylsphingosine remained low and stable following the switch from ERT to migalastat. PROs were comparable between groups. Migalastat was generally safe and well tolerated. CONCLUSIONS: Migalastat offers promise as a first-in-class oral monotherapy alternative treatment to intravenous ERT for patients with Fabry disease and amenable mutations. TRIAL REGISTRATION NUMBER: NCT00925301; Pre-results.

Host conditioning and rejection monitoring in hepatocyte transplantation in humans.

BACKGROUND & AIMS: Hepatocyte transplantation partially corrects genetic disorders and has been associated anecdotally with reversal of acute liver failure. Monitoring for graft function and rejection has been difficult, and has contributed to limited graft survival. Here we aimed to use preparative liver-directed radiation therapy, and continuous monitoring for possible rejection in an attempt to overcome these limitations. METHODS: Preparative hepatic irradiation was examined in non-human primates as a strategy to improve engraftment of donor hepatocytes, and was then applied in human subjects. T cell immune monitoring was also examined in human subjects to assess adequacy of immunosuppression. RESULTS: Porcine hepatocyte transplants engrafted and expanded to comprise up to 15% of irradiated segments in immunosuppressed monkeys preconditioned with 10Gy liver-directed irradiation. Two patients with urea cycle deficiencies had early graft loss following hepatocyte transplantation; retrospective immune monitoring suggested the need for additional immunosuppression. Preparative radiation, anti-lymphocyte induction, and frequent immune monitoring were instituted for hepatocyte transplantation in a 27year old female with classical phenylketonuria. Post-transplant liver biopsies demonstrated multiple small clusters of transplanted cells, multiple mitoses, and Ki67+ hepatocytes. Mean peripheral blood phenylalanine (PHE) level fell from pre-transplant levels of 1343±48µM (normal 30-119µM) to 854±25µM (treatment goal ≤360µM) after transplant (36% decrease; p<0.0001), despite transplantation of only half the target number of donor hepatocytes. PHE levels remained below 900µM during supervised follow-up, but graft loss occurred after follow-up became inconsistent. CONCLUSIONS: Radiation preconditioning and serial rejection risk assessment may produce better engraftment and long-term survival of transplanted hepatocytes. Hepatocyte xenografts engraft for a period of months in non-human primates and may provide effective therapy for patients with acute liver failure. LAY SUMMARY: Hepatocyte transplantation can potentially be used to treat genetic liver disorders but its application in clinical practice has been impeded by inefficient hepatocyte engraftment and the inability to monitor rejection of transplanted liver cells. In this study, we first show in non-human primates that pretreatment of the host liver with radiation improves the engraftment of transplanted liver cells. We then used this knowledge in a series of clinical hepatocyte transplants in patients with genetic liver disorders to show that radiation pretreatment and rejection risk monitoring are safe and, if optimized, could improve engraftment and long-term survival of transplanted hepatocytes in patients.

The Role of ARF6 in Biliary Atresia.

BACKGROUND & AIMS: Altered extrahepatic bile ducts, gut, and cardiovascular anomalies constitute the variable phenotype of biliary atresia (BA). METHODS: To identify potential susceptibility loci, Caucasian children, normal (controls) and with BA (cases) at two US centers were compared at >550000 SNP loci. Systems biology analysis was carried out on the data. In order to validate a key gene identified in the analysis, biliary morphogenesis was evaluated in 2-5-day post-fertilization zebrafish embryos after morpholino-antisense oligonucleotide knockdown of the candidate gene ADP ribosylation factor-6 (ARF6, Mo-arf6). RESULTS: Among 39 and 24 cases at centers 1 and 2, respectively, and 1907 controls, which clustered together on principal component analysis, the SNPs rs3126184 and rs10140366 in a 3' flanking enhancer region for ARF6 demonstrated higher minor allele frequencies (MAF) in each cohort, and 63 combined cases, compared with controls (0.286 vs. 0.131, P = 5.94x10-7, OR 2.66; 0.286 vs. 0.13, P = 5.57x10-7, OR 2.66). Significance was enhanced in 77 total cases, which included 14 additional BA genotyped at rs3126184 only (p = 1.58x10-2, OR = 2.66). Pathway analysis of the 1000 top-ranked SNPs in CHP cases revealed enrichment of genes for EGF regulators (p<1 x10-7), ERK/MAPK and CREB canonical pathways (p<1 x10-34), and functional networks for cellular development and proliferation (p<1 x10-45), further supporting the role of EGFR-ARF6 signaling in BA. In zebrafish embryos, Mo-arf6 injection resulted in a sparse intrahepatic biliary network, several biliary epithelial cell defects, and poor bile excretion to the gall bladder compared with uninjected embryos. Biliary defects were reproduced with the EGFR-blocker AG1478 alone or with Mo-arf6 at lower doses of each agent and rescued with arf6 mRNA. CONCLUSIONS: The BA-associated SNPs identify a chromosome 14q21.3 susceptibility locus encompassing the ARF6 gene. arf6 knockdown in zebrafish implicates early biliary dysgenesis as a basis for BA, and also suggests a role for EGFR signaling in BA pathogenesis.

Blood ammonia and glutamine as predictors of hyperammonemic crises in patients with urea cycle disorder.

PURPOSE: The aim of this study was to examine predictors of ammonia exposure and hyperammonemic crises in patients with urea cycle disorders. METHODS: The relationships between fasting ammonia, daily ammonia exposure, and hyperammonemic crises were analyzed in >100 patients with urea cycle disorders. RESULTS: Fasting ammonia correlated strongly with daily ammonia exposure (r = 0.764; P < 0.001). For patients with fasting ammonia concentrations <0.5 upper limit of normal (ULN), 0.5 to <1.0 ULN, and ≥1.0 ULN, the probability of a normal average daily ammonia value was 87, 60, and 39%, respectively, and 10.3, 14.1, and 37.0% of these patients, respectively, experienced ≥1 hyperammonemic crisis over 12 months. Time to first hyperammonemic crisis was shorter (P = 0.008) and relative risk (4.5×; P = 0.011) and rate (~5×, P = 0.006) of hyperammonemic crises were higher in patients with fasting ammonia ≥1.0 ULN vs. <0.5ULN; relative risk was even greater (20×; P = 0.009) in patients ≥6 years old. A 10- or 25-µmol/l increase in ammonia exposure increased the relative risk of a hyperammonemic crisis by 50 and >200% (P < 0.0001), respectively. The relationship between ammonia and hyperammonemic crisis risk seemed to be independent of treatment, age, urea cycle disorder subtype, dietary protein intake, or blood urea nitrogen. Fasting glutamine correlated weakly with daily ammonia exposure assessed as 24-hour area under the curve and was not a significant predictor of hyperammonemic crisis. CONCLUSION: Fasting ammonia correlates strongly and positively with daily ammonia exposure and with the risk and rate of hyperammonemic crises, suggesting that patients with urea cycle disorder may benefit from tight ammonia control.

Convergent patterns of association between phenylalanine hydroxylase variants and schizophrenia in four independent samples.

Recessive mutations in the phenylalanine hydroxylase (PAH) gene predispose to phenylketonuria (PKU) in conjunction with dietary exposure to phenylalanine. Previous studies have suggested PAH variations could confer risk for schizophrenia, but comprehensive follow-up has not been reported. We analyzed 15 common PAH "tag" SNPs and three exonic variations that are rare in Caucasians but common in African-Americans among four independent samples (total n = 5,414). The samples included two US Caucasian cohorts (260 trios, 230 independent cases, 474 controls), Bulgarian families (659 trios), and an African-American sample (464 families, 401 controls). Analyses of both US Caucasian samples revealed associations with five SNPs; most notably the common allele (G) of rs1522305 from case-control analyses (z = 2.99, P = 0.006). This SNP was independently replicated in the Bulgarian cohort (z = 2.39, P = 0.015). A non-significant trend was also observed among African-American families (z = 1.39, P = 0.165), and combined analyses of all four samples were significant (rs1522305: chi(2) = 23.28, 8 d.f., P = 0.003). Results for rs1522305 met our a priori criteria for statistical significance, namely an association that was robust to multiple testing correction in one sample, a replicated risk allele in multiple samples, and combined analyses that were nominally significant. Case-control results in African-Americans detected an association with L321L (P = 0.047, OR = 1.46). Our analyses suggest several associations at PAH, with consistent evidence for rs1522305. Further analyses, including additional variations and environmental influences such as phenylalanine exposure are warranted.

2-methylbutyryl-CoA dehydrogenase deficiency in Hmong infants identified by expanded newborn screen.

In April 2000, the Wisconsin Newborn Screening Program implemented tandem mass spectrometry (MS/MS) technology to expand the newborn screening panel from 13 to 48 disorders, the majority of which are inborn errors of metabolism. Among other tests, this technology measures the acylcarnitine profile from blood spots collected from infants at 24 to 48 hours of age. During the first 5.75 years of expanded screening, 27 infants were identified with elevated C5-acylcarnitine concentrations, an unexpectedly high number for any inborn error of metabolism. For these infants, elevated C5-acylcarnitines suggested a diagnosis of isovaleric acidemia (IVA), a metabolic defect of leucine metabolism. Subsequent testing showed that the infants did not have isovaleric acidemia, but did have 2-methylbutyryl-CoA dehydrogenase deficiency or 2-MBAD deficiency, a newly described defect of isoleucine metabolism. (An official abbreviation has not been established for this disorder. Other abbreviations include SBCADD, 2-MBG, and 2-MBCD deficiency.) All but 1 of the 27 infants identified with 2-MBAD deficiency are offspring of Hmong parents. To date, those diagnosed with the disorder in the Hmong community have been largely asymptomatic, though further research is needed to determine whether newborns with 2-MBAD deficiency are at risk for neurodevelopmental disorders.

REMOVED: Short-chain acyl-CoA dehydrogenase gene mutation (319 C>T) presents with clinical heterogeneity and is candidate founder mutation in Ashkenazi Jewish population.

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