Monitoring Tacrolimus Concentrations in Whole Blood and Peripheral Blood Mononuclear Cells: Inter- and Intra-Patient Variability in a Cohort of Pediatric Patients

Tacrolimus (TAC) is a first-choice immunosuppressant for solid organ transplantation, characterized by high potential for drug-drug interactions, significant inter- and intra-patient variability, and narrow therapeutic index. Therapeutic drug monitoring (TDM) of TAC concentrations in whole blood (WB) is capable of reducing the incidence of adverse events. Since TAC acts within lymphocytes, its monitoring in peripheral blood mononuclear cells (PBMC) may represent a valid future alternative for TDM. Nevertheless, TAC intracellular concentrations and their variability are poorly described, particularly in the pediatric context. Therefore, our aim was describing TAC concentrations in WB and PBMC and their variability in a cohort of pediatric patients undergoing constant immunosuppressive maintenance therapy, after liver transplantation. TAC intra-PBMCs quantification was performed through a validated UHPLC–MS/MS assay over a period of 2–3 months. There were 27 patients included in this study. No significant TAC changes in intracellular concentrations were observed (p = 0.710), with a median percent change of −0.1% (IQR −22.4%–+46.9%) between timings: this intra-individual variability was similar to the one in WB, −2.9% (IQR −29.4–+42.1; p = 0.902). Among different patients, TAC weight-adjusted dose and age appeared to be significant predictors of TAC concentrations in WB and PBMC. Intra-individual seasonal variation of TAC concentrations in WB, but not in PBMC, have been observed. These data show that the intra-individual variability in TAC intracellular exposure is comparable to the one observed in WB. This opens the way for further studies aiming at the identification of therapeutic ranges for TAC intra-PBMC concentrations

Optimization of RT-QuIC for detection of seeding activity in preclinical blood samples from prion-infected sheep.

Contrasting the knowledge about prion diseases or TSEs in sheep, only a very limited number of strain typing studies are available in goats. Two cases deriv- ing from the zoonotic bovine BSE epidemic were however detected in goats. During 2004–2012, over 70 TSE goat brain samples were collected from seven European countries and evaluated for TSE type/strain variation. A selection of these materials was chosen for in-depth analysis based on various criteria: tissue quality, genotype, broad geographical distribution, potential type variation...

Genotype-phenotype relationships of truncating mutations, p.E297G and p.D482G in bile salt export pump deficiency

Background &amp; Aims: Bile salt export pump (BSEP) deficiency frequently necessitates liver transplantation in childhood. In contrast to two predicted protein truncating mutations (PPTMs), homozygous p.D482G or p.E297G mutations are associated with relatively mild phenotypes, responsive to surgical interruption of the enterohepatic circulation (siEHC). The phenotype of patients with a compound heterozygous genotype of one p.D482G or p.E297G mutation and one PPTM has remained unclear. We aimed to assess their genotype-phenotype relationship. Methods: From the NAPPED database, we selected patients with homozygous p.D482G or p.E297G mutations (BSEP1/1; n = 31), with one p.D482G or p.E297G, and one PPTM (BSEP1/3; n = 30), and with two PPTMs (BSEP3/3; n = 77). We compared clinical presentation, native liver survival (NLS), and the effect of siEHC on NLS. Results: The groups had a similar median age at presentation (0.7-1.3 years). Overall NLS at age 10 years was 21% in BSEP1/3 vs. 75% in BSEP1/1 and 23% in BSEP3/3 (p &lt;0.001). Without siEHC, NLS in the BSEP1/3 group was similar to that in BSEP3/3, but considerably lower than in BSEP1/1 (at age 10 years: 38%, 30%, and 71%, respectively; p = 0.003). After siEHC, BSEP1/3 and BSEP3/3 were associated with similarly low NLS, while NLS was much higher in BSEP1/1 (10 years after siEHC, 27%, 14%, and 92%, respectively; p &lt;0.001). Conclusions: Individuals with BSEP deficiency with one p.E297G or p.D482G mutation and one PPTM have a similarly severe disease course and low responsiveness to siEHC as those with two PPTMs. This identifies a considerable subgroup of patients who are unlikely to benefit from interruption of the enterohepatic circulation by either surgical or ileal bile acid transporter inhibitor treatment. Impact and implications: This manuscript defines the clinical features and prognosis of individuals with BSEP deficiency involving the combination of one relatively mild and one very severe BSEP deficiency mutation. Until now, it had always been assumed that the mild mutation would be enough to ensure a relatively good prognosis. However, our manuscript shows that the prognosis of these patients is just as poor as that of patients with two severe mutations. They do not respond to biliary diversion surgery and will likely not respond to the new IBAT (ileal bile acid transporter) inhibitors, which have recently been approved for use in BSEP deficiency.</p

Genotype-phenotype relationships of truncating mutations, p.E297G and p.D482G in bile salt export pump deficiency

Background & Aims: Bile salt export pump (BSEP) deficiency frequently necessitates liver transplantation in childhood. Homozygous p.D482G or p.E297G mutations are associated with relatively mild phenotypes, responsive to surgical interruption of the enterohepatic circulation (siEHC), in contrast to patients with two predicted protein truncating mutations (PPTM). The phenotype of patients with a compound heterozygous genotype of one p.D482G or p.E297G mutation and one PPTM has remained unclear. We aimed to assess their genotype-phenotype relationship. Methods: From the NAPPED database, we selected patients with homozygous p.D482G or p.E297G mutations (BSEP1/1; n=31), with one p.D482G or p.E297G, and one PPTM (BSEP1/3; n=30), and with two PPTMs (BSEP3/3; n=77). We compared presentation, native liver survival (NLS), and effect of siEHC on NLS. Results: The groups had a similar median age at presentation (0.7-1.3 years). Overall NLS at age 10 years was 21% in BSEP1/3 vs. 75% in BSEP1/1 and 23% in BSEP3/3 (P<0.001). Without siEHC in their follow-up, NLS of BSEP1/3 was similar to BSEP3/3 patients, but considerably lower than BSEP1/1 patients (at age 10 years: 38%, 30%, and 71%, resp; P=0.003). After siEHC, BSEP1/3 and BSEP3/3 patients had similarly low NLS, while this was much higher in BSEP1/1 patients (10 years after siEHC, 27%, 14%, and 92%, resp.; P<0.001). Conclusions: BSEP deficiency patients with one p.E297G or p.D482G mutation and one PPTM have a similarly severe disease course and low responsiveness to siEHC as patients with two PPTMs. This identifies a considerable subgroup of patients who are unlikely to benefit from interruption of the enterohepatic circulation by either surgical or ileal bile acid transporter inhibitor treatment

Genetic landscape of pediatric acute liver failure of indeterminate origin.

BACKGROUND AIMS Pediatric acute liver failure (PALF) is a life-threatening condition. In Europe, main causes are viral infections (12-16%) and inherited metabolic diseases (14-28%). Yet, in up to 50% of cases the underlying etiology remains elusive, challenging clinical management, including liver transplantation. We systematically studied indeterminate PALF cases referred for genetic evaluation by whole-exome sequencing (WES), and analyzed phenotypic and biochemical markers, and the diagnostic yield of WES in this condition. METHODS With this international, multicenter observational study, patients (0-18 y) with indeterminate PALF were analyzed by WES. Data on the clinical and biochemical phenotype were retrieved and systematically analyzed. RESULTS In total, 260 indeterminate PALF patients from 19 countries were recruited between 2011 and 2022, of whom 59 had recurrent PALF (RALF). WES established a genetic diagnosis in 37% of cases (97/260). Diagnostic yield was highest in children with PALF in the first year of life (46%), and in children with RALF (64%). Thirty-six distinct disease genes were identified. Defects in NBAS (n=20), MPV17 (n=8) and DGUOK (n=7) were the most frequent findings. When categorizing, most frequent were mitochondrial diseases (45%), disorders of vesicular trafficking (28%) and cytosolic aminoacyl-tRNA synthetase deficiencies (10%). One-third of patients had a fatal outcome. Fifty-six patients received liver transplants. CONCLUSION This study elucidates a large contribution of genetic causes in PALF of indeterminate origin with an increasing spectrum of disease entities. The high proportion of diagnosed cases and potential treatment implications argue for exome or in future rapid genome sequencing in PALF diagnostics