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.

Alkaptonuria in Russia: mutational spectrum and novel variants.

Alkaptonuria is a rare genetic disease caused by mutations in HGD gene. Here we report the results of genetic and biochemical analysis of 49 Russian patients with alkaptonuria. One of the common variants c.481G > A; p.(Gly161Arg) comprising 72.4% of identified alleles was found in 45 of 49 patients in our cohort, which is probably the highest frequency of this variant worldwide. 9 novel variants were found: 6 missense, 2 splicing and 1 loss of start-codon. For missense variants we performed bioinformatic analysis, protein 3D-modeling and molecular dynamics simulations, which strongly suggest their pathogenic effect. For the rare synonymous variant c.753C > T; p.(Gly251Gly), which was found in 3 cases and predicted to activate cryptic splice site, we performed the detailed functional analysis on patient's cDNA and minigene assay and confirmed its pathogenicity.

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.

Oxysterol/chitotriosidase based selective screening for Niemann-Pick type C in infantile cholestasis syndrome patients.

BACKGROUND: Niemann-Pick disease type C (NP-C) is an inherited neurodegenerative disease (1 per 100 000 newborns) caused by NPC proteins impairment that leads to unesterified cholesterol accumulation in late endosomal/lysosomal compartments. To date the NP-C diagnostics is usually based on cholesterol detection in fibroblasts using an invasive and time-consuming Filipin staining and we need more arguments to widely introduce oxysterols as a biomarkers in NP-C. METHODS: Insofar as NP-C represents about 8% of all infant cholestases, in this prospective observational study we tried to re-assess the specificity plasma oxysterol and chitotriosidase as a biochemical screening markers of NP-C in children with cholestasis syndrome of unknown origin. For 108 patients (aged from 2 weeks to 7 years) the levels of cholestane-3ß,5α,6ß-triol (C-triol) and chitotriosidase (ChT) were measured. For patients with elevated C-triol and/or ChT the NPC1 and NPC2 genes were Sanger-sequenced and 47 additional genes (from the custom liver damage panel) were NGS-sequenced. RESULTS: Increased C-triol level (> 50 ng/ml) was detected in 4 (of 108) infants with cholestasis syndrome of unknown origin, with following molecular genetic NP-C diagnosis for one patient. Plasma cholesterol significantly correlates with C-triol (p < 0.05). NGS of high C-triol infants identified three patients with mutations in JAG1 (Alagille syndrome) and ABCB11 (Byler disease) genes. Increased ChT activity was detected in 8 (of 108) patients with various aetiologies, including NP-C, Byler disease and biliary atresia. CONCLUSION: Combined analysis of ChT activity and C-triol levels is an effective method for identifying NP-C.

Early onset lysosomal acid lipase deficiency presenting as secondary hemophagocytic lymphohistiocytosis: Two infants treated with sebelipase alfa.

Two unrelated infants were diagnosed with and initially treated for hemophagocytic lymphohistiocytosis (HLH), but progressed to cholestasis and liver failure. Early onset lysosomal acid lipase deficiency (EO-LAL-D) was suspected due to lymphocytes with cytoplasmic vacuolation and/or adrenal calcifications and confirmed by enzymatic and genetic analysis. Enzyme replacement therapy with sebelipase alfa was implemented, but both children died, despite initial improvement. Since this inborn error of metabolism progresses rapidly in infants, early diagnosis is crucial, and appropriate treatment should be started as soon as possible. The authors suggest that the diagnosis of EO-LAL-D should be considered in infants with symptoms of HLH.