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Exome sequencing (ES) has identified biallelic kinesin family member 12 (KIF12) mutations as underlying neonatal cholestatic liver disease. We collected information on onset and progression of this entity. Among consecutively referred pediatric patients at our centers, diagnostic ES identified 4 patients with novel, biallelic KIF12 variants using the human GRCh38 reference sequence, as KIF12 remains incompletely annotated in the older reference sequence GRCh37. A review of these and of 21 reported patients with KIF12 variants found that presentation with elevated serum transaminase activity in the context of trivial respiratory infection, without clinical features of liver disease, was more common (n = 18) than manifest cholestatic disease progressing rapidly to liver transplantation (LT; n = 7). Onset of liver disease was at age <1 year in 15 patients; LT was more common in this group. Serum gamma‐glutamyl transpeptidase activity (GGT) was elevated in all patients, and total bilirubin was elevated in 15 patients. Liver fibrosis or cirrhosis was present in 14 of 18 patients who were biopsied. The 16 different pathogenic variants and 11 different KIF12 genotypes found were not correlated with age of onset or progression to LT. Identification of biallelic pathogenic KIF12 variants distinguishes KIF12‐related disease from other entities with elevated GGT. [ABSTRACT FROM AUTHOR]

Heat shock protein member 8 (HSPA8) is one of the most abundant chaperones in eukaryotic cells, but its biological roles in bladder cancer (BC) are largely unclear. First, we observed that HSPA8 was abundant in both cell lines and tissues of BC, and the HSPA8‐high group had poorer T stages and overall survival (OS) than the HSPA8‐low group in the TCGA patients. Next, when we knocked down HSPA8 in BC cells, the growth and migration abilities were significantly decreased, the apoptosis rates were significantly increased, and the Ki67 fluorescence intensity was decreased in BC cells. Moreover, caspase 3 was significantly decreased with overexpression of HSPA8 in BC cells. After that, a machine learning prognostic model was created based on the expression of HSPA8 by applying LASSO Cox regression in TCGA and GEO patients. The model indicated that the low‐risk (LR) group with BC had better tumour stages, lymphovascular invasion, and OS than the high‐risk (HR) group. Additionally, the risk score was demonstrated to be an independent risk factor for the prognosis of BC by univariate and multivariate Cox analyses. Moreover, the HR group showed a greater rate of TP53 mutations and was mostly enriched in the ECM‐receptor interaction pathway than the LR group. Importantly, lower CD8+ T‐cell and NK cell infiltration, higher immune exclusion scores, higher expression of PD‐L1 and CTLA4 and poorer immune checkpoint therapy effects were found in the HR group. These findings demonstrated how crucial HSPA8 plays a role in determining the prognosis of bladder cancer. [ABSTRACT FROM AUTHOR]

Background and Aims: A recent study suggested that administration of ursodeoxycholic acid (UDCA) at dosages usually employed clinically may reduce rates of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection. A recent surge of SARS‐CoV‐2 omicron infection in China allowed study of whether UDCA administration reduced susceptibility to SARS‐CoV‐2 infection in children with liver disease. Methods: Through WeChat groups, a questionnaire was distributed to families (n = 300) in which a child had been admitted to our liver service in the past 5 years. Among the families/households in which someone was infected with SARS‐CoV‐2, the proportion in which a child taking UDCA was infected was compared with the proportion in which a child not taking UDCA was infected. Results: Of the 300 questionnaire answers, 280 (93.3%) were valid. SARS‐CoV‐2 infection was confirmed in 226 families (80.7%): 146 children were taking UDCA (10‐20 mg/kg/day) and 80 children were not taking UDCA. SARS‐CoV‐2 infection was confirmed in 95 children taking UDCA (65.1%) and in 51 children not taking UDCA (63.8%) (p = 0.843); SARS‐CoV‐2 infection was suspected in 23 children taking UDCA (15.8%) and in 11 children not taking UDCA (13.8%) (p = 0.687). Conclusions: These results indicate that UDCA administration does not reduce susceptibility to SARS‐CoV‐2 infection in children with liver disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background & Aims: Biallelic pathogenic variants in MYO5B cause microvillus inclusion disease (MVID), or familial intrahepatic cholestasis (FIC). The reported FIC patients are scarce and so the genotype‐phenotype correlation has not been fully characterised. This study aimed to report more MYO5B‐associated FIC patients and correlate genotypes to phenotypes in more detail. Methods: The phenotype and genetic data of 12 newly diagnosed MYO5B‐associated (including 11 FIC) patients, as well as 118 previously reported patients with available genotypes, were summarised. Only patients with biallelic MYO5B variants were enrolled. Nonsense, frameshift, canonical splice sites, initiation codon loss, and single exon or multiexon deletion were defined as null MYO5B variants. Results: Phenotypically, 50 were isolated MVID, 47 involved both liver and intestine (combined), and 33 were isolated FIC (9 persistent, 15 recurrent, 3 transient, and 6 un‐sub‐classified) patients. The severity of intestinal manifestation was positively correlated to an increased number of null variants (ρ = 0.299, P =.001). All FIC patients carried at least one non‐null variant, and the severity of cholestasis was correlated to the presence of a null variant (ρ = 0.420, P =.029). The proportion of FIC patients (16/29, 55%) harbouring missense/in‐frame variants affecting the non‐motor regions of MYO5B was significantly higher than that of MVID (3/25, 12%, P =.001) and combined patients (3/31, 10%, P =.000). 10 of the 29 FIC patients harboured missense/in‐frame variants at the IQ motifs comparing to none in the 56 MVID and combined patients (P =.000). Conclusions: The phenotype of MYO5B deficiency was associated with MYO5B genotypes, the nullity or the domain affected. [ABSTRACT FROM AUTHOR]

Background & Aims: ABCB11 deficiency presenting in infancy is believed generally to manifest as persistent/progressive cholestasis. We describe a group of patients with biallelic ABCB11 variants whose disorder manifested as transient neonatal cholestasis (TNC). Methods: Neonatal intrahepatic cholestasis in 68 children (31 males) with biallelic predictedly pathogenic variants (PPV) in ABCB11 was classified as transient (TNC group, n = 23, 11 males), intermittent (benign recurrent intrahepatic cholestasis [BRIC] group, n = 3, 1 male) or persistent/ progressive (progressive familial intrahepatic cholestasis [PFIC] group, n = 42, 19 males). Clinical, genetic and bile salt export pump (BSEP) expression information was correlated with outcomes. Results: The median onset age of jaundice was 3 days (birth to 2 months) for the TNC group and 10.5 days (birth to 3 months) for the PFIC group (P =.034). The median length of follow‐up of TNC patients was 44 months (12 months‐168 months). At presentation, hepatobiliary‐injury biomarker values were similar between the groups (P >.05). TNC patients (17/23) more often than PFIC patients (20/42, P =.041) harboured biallelic non‐null variants (predicted not to terminate translation prematurely). TNC patient livers (7/7) more often than PFIC patient livers (5/16, P =.005) expressed immunohistochemically detectable BSEP. Kaplan‐Meier analysis showed better prognosis for patients with BSEP expression (P =.009). Too few BRIC patients were available for statistical study. Conclusions: Neonatal cholestasis associated with biallelic PPV in ABCB11 can resolve temporarily or persistently in one third of cases. Resolution is more likely in patients with biallelic non‐null PPV or with liver BSEP expression. [ABSTRACT FROM AUTHOR]

Aim: Neuroblastoma amplified sequence (NBAS)‐associated disease has a wide phenotypic spectrum, including infantile liver failure syndrome type 2 (ILFS2, OMIM #616483), short stature with optic nerve atrophy and Pelger–Huët anomaly (SOPH) syndrome (OMIM #614800), and a combined phenotype overlapping ILFS2 and SOPH syndrome. The mutation spectra of NBAS and its genotype–phenotype correlation among Chinese were not clear. Methods: Clinical and genetic data were retrospectively collected from the medical charts of patients with biallelic NBAS mutations, as well as from Chinese patients in previously published reports. Results: Fourteen new patients were identified, including 10 novel mutations: c.648‐1G>A, c.2563_c.2577+5del/p.His855_Gln859del, c.3115C>T/p.Gln1039Ter, c.3284G>A/p.Trp1095Ter, c.2570C>T/p.Ala857Val, c.6859G>T/p.Asp2287Tyr, c.1028G>A/p.Ser343Asn, c.1177_1182delinsAGATAGA/p.Val393ArgfsTer2, c.3432_3435dupCAGT/p.Ala1146GlnfsTer14, and c.680_690dupACTGTTTCAGC/p.Phe231ThrfsTer35. All 14 patients presented as fever‐triggered liver injury, including nine patients that satisfied the criteria of acute liver failure (ALF) in whom c.3596G>A/p.Cys1199Tyr occurred five times. Nine patients had extrahepatic manifestations including short stature, skeletal abnormalities, intellectual disability, ophthalmic abnormalities, low levels of serum immunoglobulins, facial dysmorphism, and cardiac abnormalities. Ten other Chinese patients were collected through a review of published works. Genotype–phenotype analysis in 24 Chinese patients revealed that the percentage of ALF patients with variants in the Sec39 domain was significantly higher than that in the C‐terminal (100% vs. 12.5%, P = 0.000), and the percentage of multi‐organ/system involvement in patients with variants in the Sec39 domain was significantly lower than that in the C‐terminal (40% vs. 100%, P = 0.0128). Conclusions: We reported 14 new patients, 10 novel mutations, and a unique recurrent mutation. Correlation analysis indicated that the domain of missense and non‐frameshift insertion/deletion mutations in NBAS protein is related to phenotype among Chinese patients. [ABSTRACT FROM AUTHOR]

Background & Aims: In about 20% of children with cholestasis and normal or low serum gamma‐glutamyltransferase (GGT) activity, no aetiology is identified. We sought new genes implicated in paediatric hepatobiliary disease. Methods: We conducted whole‐exome sequencing in 69 children evaluated at our centre from 2011 to 2018 who had low‐GGT cholestasis and in whom homozygous/compound heterozygous predictedly pathogenic variants (PPVs) in ATP8B1, ABCB11, NR1H4, MYO5B or TJP2 were not found. Clinical records and findings on light microscopy and transmission electron microscopy of liver biopsy materials were reviewed. Results: In seven patients from seven unrelated families, biallelic PPVs (10 in total) were found in USP53, recently associated with intrahepatic cholestasis. Seven variants were classified as pathogenic: one canonical splicing, c.569 + 2T > C, and six nonsense or frameshifting: c.169C > T (p.Arg57Ter), c.581delA (p.Arg195GlufsTer38), c.831_832insAG (p.Val279GlufsTer16), c.1012C > T (p.Arg338Ter), c.1426C > T (p.Arg476Ter) and c.1558C > T (p.Arg520Ter). Three were likely pathogenic: c.297G > T (p.Arg99Ser), c.395A > G (p.His132Arg) and c.878G > T (p.Gly293Val). In all patients, jaundice began at age <7 months. Cholestasis was transient, with documented resolution of hyperbilirubinaemia in all (oldest patient now aged 5 years) except one, who was lost to follow‐up. Light microscopy identified intralobular cholestasis, giant‐cell change of hepatocytes and perisinusoidal‐perihepatocytic and portal‐tract fibrosis. Ultrastructural study revealed elongated hepatocyte‐hepatocyte tight junctions. One patient was deaf. Conclusion: USP53 interacts with the tight junction constituent TJP2. TJP2 mutation can cause low‐GGT intrahepatic cholestasis, with elongated hepatocyte‐hepatocyte tight junctions, as well as deafness. Our findings extend a preliminary report of USP53 disease and indicate that USP53 mutation may generate a partial phenocopy of TJP2 disease. [ABSTRACT FROM AUTHOR]

Abstract: Background & Aims: Genetic defects causing dysfunction in bile salt export pump (BSEP/ABCB11) lead to liver diseases. ABCB11 mutations alter the bile acid metabolome. We asked whether profiling plasma bile acids could reveal compensatory mechanisms and track genetic and clinical status. Methods: We compared plasma bile acids in 17 ABCB11‐mutated patients, 35 healthy controls and 12 genetically undiagnosed cholestasis patients by ultra‐high‐performance liquid chromatography/multiple‐reaction monitoring‐mass spectrometry (UPLC/MRM‐MS). We developed an index to rank bile acid hydrophobicity, and thus toxicity, based on LC retention times. We recruited 42 genetically diagnosed hereditary cholestasis patients, of whom 12 were presumed to have impaired BSEP function but carried mutations in genes other than ABCB11, and 8 healthy controls, for further verification. Results: The overall hydrophobicity indices of total bile acids in both the ABCB11‐mutated group (11.89 ± 1.07 min) and the undiagnosed cholestasis group (11.46 ± 1.07 min) were lower than those of healthy controls (13.69 ± 0.77 min) (both p < 0.005). This was owing to increased bile acid modifications. Secondary bile acids were detected in patients without BSEP expression, suggesting biliary bile acid secretion through alternative routes. A diagnostic panel comprising lithocholic acid (LCA), tauro‐LCA, glyco‐LCA and hyocholic acid was identified that could differentiate the ABCB11‐mutated cohort from healthy controls and undiagnosed cholestasis patients (AUC=0.946, p < 0.0001) and, in non‐ABCB11‐mutated cholestasis patients, could distinguish BSEP dysfunction from normal BSEP function (9/12 vs 0/38, p < 0.0000001). Conclusions: Profiling of plasma bile acids has provided insights into cholestasis alleviation and may be useful for the clinical management of cholestatic diseases. [ABSTRACT FROM AUTHOR]

Aim: The aim of this study was to analyze the pathogenicity of rare/novel synonymous or intronic variants identified in ABCB11 heterozygotes presenting as progressive intrahepatic cholestasis with low γ‐glutamyltransferase. Methods: The enrolled variants were identified in ABCB11 between October 2009 and June 2016. The effects on pre‐RNA splicing were analyzed by in silico tools and minigene splicing assay. Results: There were three intronic (c.908 + 5G > A, c.2815‐8A > G, and c.612‐15_‐6del10bp) and two synonymous (c.1809G > A, p.K603 K and c.2418C > T, p.G806G) variants with unknown significance identified in ABCB11 of five ABCB11 heterozygotes. Parental studies were carried out for four patients, and revealed that the variants with unknown significance were compound heterozygous with other pathogenic variants. The five variants with unknown significance had minor allele frequency <0.1% or were absent from controls, and had positive prediction results by in silico tools. The effects on pre‐RNA splicing were further confirmed by minigene splicing assay. c.908 + 5A caused abnormal splicing in at least 78.5 ± 3.8% of products using a cryptic splice site (ss) 22 nucleotides (nt) upstream of the wild‐type (WT) 5′ss. Seven nucleotides of intron 22 upstream of the WT 3'ss was retained for all products from c.2815‐8G. c.612‐15_‐6del caused exon 8 skipping in 24.8 ± 7.7% of products, and 55 nt of exon 8 downstream of the WT 3′ss removal in remaining products. c.1809A led to exon 15 skipping. c.2418 T removed exon 20 and 62 nt of exon 21 downstream of the WT 3′ss by using a cryptic ss. Conclusions: We successfully identified five pathogenic synonymous or intronic variants with some common features. These features might help to choose the right variant for further functional assay. [ABSTRACT FROM AUTHOR]

Background The significance of ABCB11 variants have been studied in some cholestatic diseases, but this is not clear in transient neonatal cholestasis ( TNC). The aim of the present study was to explore the association between ABCB11 variants and TNC. Methods This was a case-control study. A total of 192 children with TNC referred to a tertiary referral hospital in eastern China were enrolled as subjects, and 196 healthy children were selected as controls. Part of the promoter and exons of the ABCB11 gene were sequenced directly. The single nucleotide polymorphism ( SNP) site of V444A was tested using fluorescent quantitative polymerase chain reaction. Potential consequences of variants were predicted using bioinformatics software. The biochemistry indices were compared between the patients with or without possibly pathogenic variants/mutations. Results Twenty-eight variants, including 14 novel ones, were detected. Four novel, possibly pathogenic mutations (I416I, K436N, R928Q and IVS7+5G>A) were detected in six subjects. The γ-glutamyltransferase level of these six was lower than in the others ( P = 0.054). The genotype distribution of the four common SNP sites, V444A, A535A, A865V and A1082A, was not significantly different between TNC patients and controls. Conclusions Approximately 3% of TNC cases can be attributed to ABCB11 mutations. [ABSTRACT FROM AUTHOR]

Background: Progressive familial intrahepatic cholestasis type 2 (PFIC2) is a severe autosomal recessive liver disorder of childhood that can cause cholestasis and progress to end-stage liver disease. ABCB11 gene mutations causing PFIC2 have been reported in some population groups, but not in mainland Chinese., Aims: To elucidate the existence of and characterize ABCB11 gene mutations in mainland Chinese with progressive intrahepatic cholestasis and low gamma glutamyltransferase (GGT)., Methods: Twenty-four children presenting with progressive intrahepatic cholestasis and low GGT were admitted to a tertiary paediatric hospital in eastern China from January 2004 to July 2007. All encoding exons and flanking areas of the ABCB11 gene were sequenced. Hepatic histopathology results were obtained by review of the medical record., Results: Twelve novel mutations of ABCB11 gene were found in seven patients: three nonsense mutations, six missense mutations, two splicing mutations and one intronic mutation. Giant cell transformation of hepatocytes was demonstrated in all the four patients with ABCB11 mutations and four of 12 patients without mutations in coding sequences of ABCB11 gene who received liver needle biopsy., Conclusions: ABCB11 gene mutations play an important role in Chinese patients with progressive intrahepatic cholestasis and low GGT. The characteristics of ABCB11 gene mutations in Chinese are different from other population groups. Histological examination may be helpful in diagnosis of PFIC2.