Defining pathogenicity of NOTCH2 variants for diagnosis of Alagille syndrome type 2 using a large cohort of patients.

BACKGROUND AND AIMS: Alagille syndrome (ALGS) type 2 caused by mutations in NOTCH2 has genotypic and phenotypic heterogeneity. Diagnosis in some atypical patients with isolated hepatic presentation could be missed. METHODS: Using 2087 patients with paediatric liver manifestations, NOTCH2 allele frequencies, in-silico prediction, protein domains and clinical features were analysed to define the pathogenicity of NOTCH2 variants for diagnosis of ALGS type 2. RESULTS: Among 2087 patients with paediatric liver manifestations, significantly more NOTCH2 variants were absent in gnomAD in patients with elevated γ-glutamyltransferase (GGT) (p = .041). Significantly more NOTCH2 variants which were absent in gnomAD were located in protein functional domains (p = .038). When missense variants were absent in gnomAD and predicted to be pathogenic by at least three out of seven in-silico tools, they were found to be significantly associated with liver manifestations with elevated GGT (p = .003). Comparing this to patients with likely benign (LB) variants, the patients with likely-pathogenic (LP) variants have significantly more liver manifestations with elevated GGT (p = .0001). Significantly more patients with LP variants had extra-hepatic phenotypes of ALGS compared with those patients with LB variants (p = .0004). CONCLUSION: When NOTCH2 variants are absent in gnomAD, null variants and missense variants which were predicted to be pathogenic by at least three in-silico tools could be considered pathogenic in patients with high GGT chronic liver diseases.

Association of novel TMEM67 variants with mild phenotypes of high gamma-glutamyl transpeptidase cholestasis and congenital hepatic fibrosis.

TMEM67 (mecklin or MKS3) locates in the transition zone of cilia. Dysfunction of TMEM67 disrupts cilia-related signaling and leads to developmental defects of multiple organs in humans. Typical autosomal recessive TMEM67 defects cause partial overlapping phenotypes, including abnormalities in the brain, eyes, liver, kidneys, bones, and so forth. However, emerging reports of isolated nephronophthisis suggest the possibility of a broader phenotype spectrum. In this study, we analyzed the genetic data of cholestasis patients with no obvious extrahepatic involvement but with an unexplained high level of gamma-glutamyl transpeptidase (GGT). We identified five Han Chinese patients from three unrelated families with biallelic nonnull low-frequency TMEM67 variants. All variants were predicted pathogenic in silico, of which p. Arg820Ile and p. Leu144del were previously unreported. In vitro studies revealed that the protein levels of the TMEM67 variants were significantly decreased; however, their interaction with MKS1 remained unaffected. All the patients, aged 7-39 years old, had silently progressive cholestasis with elevated GGT but had normal bilirubin levels. Histological studies of liver biopsy of patients 1, 3, and 5 showed the presence of congenital hepatic fibrosis. We conclude that variants in TMEM67 are associated with a mild phenotype of unexplained, persistent, anicteric, and high GGT cholestasis without typical symptoms of TMEM67 defects; this possibility should be considered by physicians in gastroenterology and hepatology.

Biallelic loss-of-function ZFYVE19 mutations are associated with congenital hepatic fibrosis, sclerosing cholangiopathy and high-GGT cholestasis.

BACKGROUND: For many children with intrahepatic cholestasis and high-serum gamma-glutamyl transferase (GGT) activity, a genetic aetiology of hepatobiliary disease remains undefined. We sought to identify novel genes mutated in children with idiopathic high-GGT intrahepatic cholestasis, with clinical, histopathological and functional correlations. METHODS: We assembled a cohort of 25 children with undiagnosed high-GGT cholestasis and without clinical features of biliary-tract infection or radiological features of choledochal malformation, sclerosing cholangitis or cholelithiasis. Mutations were identified through whole-exome sequencing and targeted Sanger sequencing. We reviewed histopathological findings and assessed phenotypical effects of ZFYVE19 deficiency in cultured cells by immunofluorescence microscopy. RESULTS: Nine Han Chinese children harboured biallelic, predictedly complete loss-of-function pathogenic mutations in ZFYVE19 (c.314C>G, p.S105X; c.379C>T, p.Q127X; c.514C>T, p.R172X; c.547C>T, p.R183X; c.226A>G, p.M76V). All had portal hypertension and, at liver biopsy, histopathological features of the ductal plate malformation (DPM)/congenital hepatic fibrosis (CHF). Four children required liver transplantation for recurrent gastrointestinal haemorrhage. DPM/CHF was confirmed at hepatectomy, with sclerosing small-duct cholangitis. Immunostaining for two primary-cilium axonemal proteins found expression that was deficient intraluminally and ectopic within cholangiocyte cytoplasm. ZFYVE19 depletion in cultured cells yielded abnormalities of centriole and axoneme. CONCLUSION: Biallelic ZFYVE19 mutations can lead to high-GGT cholestasis and DPM/CHF in vivo. In vitro, they can lead to centriolar and axonemal abnormalities. These observations indicate that mutation in ZFYVE19 results, through as yet undefined mechanisms, in a ciliopathy.

TJP2 hepatobiliary disorders: Novel variants and clinical diversity.

To assess the spectrum of pediatric clinical phenotypes in TJP2 disease, we reviewed records of our seven patients in whom intrahepatic cholestasis was associated with biallelic TJP2 variants (13; 12 novel) and correlated clinical manifestations with mutation type. The effect of a splicing variant was analyzed with a minigene assay. The effects of three missense variants were analyzed with protein expression in vitro. Our patients had both remitting and persistent cholestasis. Three exhibited growth retardation. Six responded to treatment with cholestyramine, ursodeoxycholic acid, or both. Two had cholecystolithiasis. None required liver transplantation or developed hepatocellular or cholangiocellular malignancy. None manifested extrahepatic disease not attributable to effects of cholestasis. The variant c.2180-5T>G resulted in exon 15 skipping with in-frame deletion of 32 amino acid residues in TJP2. The three missense variants decreased but did not abolish TJP2 expression. Patients with truncating or canonical splice-site variants had clinically more severe disease. TJP2 disease in children includes a full clinical spectrum of severity, with mild or intermittent forms as well as the severe and minimal forms hitherto described. Biallelic TJP2 variants must be considered in children with clinically intermittent or resolved intrahepatic cholestasis.

Severe Neonatal Cholestasis in Cerebrotendinous Xanthomatosis: Genetics, Immunostaining, Mass Spectrometry.

OBJECTIVES: Cerebrotendinous xanthomatosis (CTX) is caused by defects in sterol 27-hydroxylase (CYP27A1, encoded by CYP27A1), a key enzyme in the bile acid synthesis pathway. CTX usually presents as neurologic disease in adults or older children. The rare reports of CTX manifest as neonatal cholestasis assess the cholestasis as transient, with patient survival. Our experience differs. METHODS: Homozygous or compound heterozygous CYP27A1 mutations were detected in 8 neonatal cholestasis patients by whole exome sequencing, panel sequencing, or Sanger sequencing. Their clinical and biochemical data were retrospectively reviewed. Immunostaining for CYP27A1 was conducted in liver of 4 patients. Mass spectrometry was used to analyze patients' urine samples. RESULTS: All 8 infants had severe cholestasis. Five died from, or were transplanted for, liver failure; 3 cleared their jaundice eventually. Marking for CYP27A1 was weak or absent in 3 of the 4 patient specimens. Mass spectrometry of urine revealed a predominance of sulfated and doubly conjugated (sulfated-glucuronidated) bile alcohols. No patient harbored a putatively pathogenic mutation in genes other than CYP27A1 that have been implicated in cholestatic liver disease. CONCLUSIONS: CTX manifest as neonatal cholestasis has a bile acid profile different from CTX manifest in later life, and thus may be overlooked. Immunostaining, mass spectrometry of urine, and genetic studies can support one another in making the diagnosis. A substantial proportion of CTX patients with severe neonatal cholestasis may die or need liver transplantation. CTX manifest in infancy as severe cholestasis warrants further investigation of biochemical diagnostic criteria and best management.