Pediatric Patients with Sitosterolemia: Next-Generation Sequencing and Biochemical Examination in Clinical Practice.

Here, we report the pediatric cases of sitosterolemia, a rare autosomal-recessive genetic disorder, characterized by high concentrations of plant sterols in blood and heterogeneity manifestations. All three patients (two girls aged 2 and 6 years old, and one boy aged 14 years old) were initially diagnosed with hypercholesterinemia. Next-generation sequencing (NGS) revealed homozygous (p.Leu572Pro/p.Leu572Pro) and compound (p.Leu572Pro/p.Gly512Arg and p.Leu572Pro/p.Trp361*) variants in the ABCG8 gene that allowed for the diagnosis of sitosterolemia. Two patients whose blood phytosterol levels were estimated before the diet demonstrated high levels of sitosterol/campesterol (69.6/29.2 and 28.3/12.4 µmol/L, respectively). Here, we demonstrate that NGS-testing led to the proper diagnosis that is essential for patients' management. The variant p.Leu572Pro might be prevalent among patients with sitosterolemia in Russia.

Clinical Characterization of Alagille Syndrome in Patients with Cholestatic Liver Disease.

Alagille syndrome (ALGS) is a multisystem condition characterized by cholestasis and bile duct paucity on liver biopsy and variable involvement of the heart, skeleton, eyes, kidneys, and face and caused by pathogenic variants in the JAG1 or NOTCH2 gene. The variable expressivity of the clinical phenotype and the lack of genotype-phenotype correlations lead to significant diagnostic difficulties. Here we present an analysis of 18 patients with cholestasis who were diagnosed with ALGS. We used an NGS panel targeting coding exons of 52 genes, including the JAG1 and NOTCH2 genes. Sanger sequencing was used to verify the mutation in the affected individuals and family members. The specific facial phenotype was seen in 16/18 (88.9%). Heart defects were seen in 8/18 (44.4%) patients (pulmonary stenosis in 7/8). Butterfly vertebrae were seen in 5/14 (35.7%) patients. Renal involvement was detected in 2/18 (11.1%) cases-one patient had renal cysts, and one had obstructive hydronephrosis. An ophthalmology examination was performed on 12 children, and only one had posterior embryotoxon (8.3%). A percutaneous liver biopsy was performed in nine cases. Bile duct paucity was detected in six/nine cases (66.7%). Two patients required liver transplantation because of cirrhosis. We identified nine novel variants in the JAG1 gene-eight frameshift variants (c.1619_1622dupGCTA (p.Tyr541X), c.1160delG (p.Gly387fs), c.964dupT (p.C322fs), c.120delG (p.L40fs), c.1984dupG (p.Ala662Glyfs), c.3168_3169delAG (p.R1056Sfs*51), c.2688delG (p.896CysfsTer49), c.164dupG (p.Cys55fs)) and one missense variant, c.2806T > G (p.Cys936Gly). None of the patients presented with NOTCH2 variants. In accordance with the classical criteria, only six patients could meet the diagnostic criteria in our cohort without genetic analysis. Genetic testing is important in the diagnosis of ALGS and can help differentiate it from other types of cholestasis.

Clinical Presentation of a Patient with a Congenital Disorder of Glycosylation, Type IIs (ATP6AP1), and Liver Transplantation.

The congenital disorder of glycosylation type IIs (ATP6AP1-CDG; OMIM# 300972) is a rare X-linked recessive complex syndrome characterized by liver dysfunction, recurrent bacterial infections, hypogammaglobulinemia, and defective glycosylation of serum proteins. Here, we examine the case of a 1-year-old male patient of Buryat origin, who presented with liver dysfunction. At the age of 3 months, he was hospitalized with jaundice and hepatosplenomegaly. Whole-exome sequencing identified the ATP6AP1 gene missense variant NM_001183.6:c.938A>G (p.Tyr313Cys) in the hemizygous state, which was previously reported in a patient with immunodeficiency type 47. At the age of 10 months, the patient successfully underwent orthotopic liver transplantation. After the transplantation, the use of Tacrolimus entailed severe adverse effect (colitis with perforation). Replacing Tacrolimus with Everolimus led to improvement. Previously reported patients demonstrated abnormal N- and O-glycosylation, but these data were collected without any specific treatment. In contrast, in our patient, isoelectric focusing (IEF) of serum transferrin was performed only after the liver transplant and showed a normal IEF pattern. Thus, liver transplantation could be a curative option for patients with ATP6AP1-CDG.

A Clinical Case of a Homozygous Deletion in the APOA5 Gene with Severe Hypertriglyceridemia.

Background: Hypertriglyceridemia (HTG) is one of the most common forms of lipid metabolism disorders. The leading clinical manifestations are pancreatitis, atherosclerotic vascular lesions, and the formation of eruptive xanthomas. The most severe type of HTG is primary (or hereditary) hypertriglyceridemia, linked to pathogenic genetic variants in LPL, APOC2, LMF1, and APOA5 genes. Case: We present a clinical case of severe primary hypertriglyceridemia (TG level > 55 mmol/L in a 4-year-old boy) in a consanguineous family. The disease developed due to a previously undescribed homozygous deletion in the APOA5 gene (NM_052968: c.579_592delATACGCCGAGAGCC p.Tyr194Gly*68). We also evaluate the clinical significance of a genetic variant in the LPL gene (NM_000237.2: c.106G>A (rs1801177) p.Asp36Asn), which was previously described as a polymorphism. In one family, we also present a different clinical significance even in heterozygous carriers: from hypertriglyceridemia to normotriglyceridemia. We provide evidence that this heterogeneity has developed due to polymorphism in the LPL gene, which plays the role of an additional trigger. Conclusions: The homozygous deletion of the APOA5 gene is responsible for the severe hypertriglyceridemia, and another SNP in the LPL gene worsens the course of the disease.

Hepatic glycogen synthase (GYS2) deficiency: seven novel patients and seven novel variants.

Glycogen storage disease type 0 (GSD 0) is an autosomal recessive disorder of glycogen metabolism caused by mutations in the GYS2 gene manifesting in infancy or early childhood and characterized by ketotic hypoglycemia after prolonged fasting, and postprandial hyperglycemia and hyperlactatemia. GSD 0 is a rare form of hepatic glycogen storage disease with less than 30 reported patients in the literature so far.DNA samples of 93 Russian patients with clinical diagnoses of hepatic GSDs were collected and analyzed by next-generation sequencing custom target panel and by direct sequencing. Seven new GSD 0 patients with variable phenotypes were found showing 10 variants. Seven variants are novel.We present seven new GSD 0 patients with variable phenotypes. Overall, 10 different mutant alleles of the GYS2 gene were found. Seven of them are novel: c.214delC, c.845delT, c.1644C>A, c.205T>A, c.929G>T, c.1169G>C and c.1703C>A. Three of the novel variants were annotated as pathogenic and likely pathogenic; four other variants have an uncertain significance.The current results expand the spectrum of known mutations in GYS2 and suggest that phenotypes of GSD 0 are more variable and less specific than the reported ones. SYNOPSIS: Seven new patients with glycogen storage disease type 0 were found using next-generation sequencing and seven novel variants of GYS2 gene were annotated.

Pharmacokinetics, Safety, and Efficacy of Glecaprevir/Pibrentasvir in Adolescents With Chronic Hepatitis C Virus: Part 1 of the DORA Study.

The pangenotypic regimen of glecaprevir and pibrentasvir (G/P) is approved to treat adults with chronic hepatitis C virus (HCV) infection and has yielded high cure rates in adults in clinical trials. Approved treatment options for pediatrics may include ribavirin. A pangenotypic regimen for pediatric patients remains an unmet need. DORA is an ongoing phase 2/3, nonrandomized, open-label study evaluating the pharmacokinetics (PK), safety, and efficacy of G/P in pediatric patients with chronic HCV. This analysis includes Part 1 of the study, conducted in adolescent patients 12-17 years of age given the adult regimen of G/P (300 mg/120 mg) once daily for 8-16 weeks according to the indication durations used in adults. Patients were either treatment naïve or experienced with interferon-based regimens. The primary PK endpoint was steady-state exposures for glecaprevir and pibrentasvir; the primary efficacy endpoint was sustained virologic response 12 weeks after treatment (SVR12). The secondary efficacy endpoints were on-treatment virologic failure, relapse, and reinfection. Safety and tolerability were monitored. Part 1 enrolled 48 adolescent patients infected with genotypes 1, 2, 3, or 4, of whom 47 were administered G/P. All 47 patients (100%) achieved SVR12. No on-treatment virologic failures or relapses occurred. PK exposures of glecaprevir and pibrentasvir were comparable to exposures in adults. No adverse events (AEs) led to treatment discontinuation, and no serious AEs occurred. Conclusion: Adolescent patients with chronic HCV infection treated with G/P achieved a comparable exposure to adults, 100% SVR12 rate, and safety profile consistent with that in adults. This pangenotypic regimen demonstrated 100% efficacy within the adolescent population in as little as 8 weeks of treatment.

A kinetic assay of total lipase activity for detecting lysosomal acid lipase deficiency (LAL-D) and the molecular characterization of 18 LAL-D patients from Russia.

Laboratory diagnostics of lysosomal acid lipase deficiency (LAL-D), a rare disorder associated with LIPA alterations, are based on the evaluation of LAL activity. In dry blood spots (DBS) submitted for LAL-D diagnostics (the screening cohort) over a two-year period or obtained from a cohort of retrospective LAL-D patients, we measured: (1) LAL activity using a two-reaction assay with 4-methylumbelliferone palmitate (4-MU-Palm) and Lalistat-2, a specific LAL inactivator; (2) total lipase (TL) activity by a 1-hour kinetic 4-MU-Palm cleavage reaction (no Lalistat-2). The TL activity was expressed as the area under the kinetic curve after 1 hour (TL-AUC1h) of the reaction and presented as the median (min-max). LAL activity was reduced in 30/537 individuals from the screening cohort, among which LIPA sequencing revealed six patients and one carrier. Overall, 16 (89%) individuals among six novel and 12 retrospective LAL-D patients carried at least one c.894G>A mutation (six were homozygous). The TL-AUC1h in nonLAL-D specimens with normal LAL activity (n = 90) was unambiguously higher (9471 [4015-23 585] RFU*h/punch) compared to LAL-D patients, including six new and nine retrospective patients (1810 [357-2608] RFU*h/punch). Importantly, in 13/15 examined nonLAL-D specimens with reduced LAL activity the TL-AUC1h was above a threshold of 2652 RFU*h/punch. Applying this threshold, the TL-AUC1h index discriminated all LAL-D patients (100% sensitivity) and 103/105 nonLAL-D specimens (98% specificity). Given that there is no need for Lalistat-2 and two parallel enzymatic reactions in conjunction with high sensitivity and specificity, the kinetic assay seems to be practical for LAL-D screening. SYNOPSIS: Lysosomal acid lipase deficiency responsible for Wolman disease and cholesterol ester storage disease could be reliably detected using a kinetic assay of total lipase activity with a fluorogenic substrate.