Biallelic loss-of-function variants of SLC12A9 cause lysosome dysfunction and a syndromic neurodevelopmental disorder.

PURPOSE: Pathogenic variants of FIG4 generate enlarged lysosomes and neurological and developmental disorders. To identify additional genes regulating lysosomal volume, we carried out a genome-wide activation screen to detect suppression of enlarged lysosomes in FIG4-/- cells. METHODS: The CRISPR-a gene activation screen utilized sgRNAs from the promoters of protein-coding genes. Fluorescence-activated cell sorting separated cells with correction of the enlarged lysosomes from uncorrected cells. Patient variants of SLC12A9 were identified by exome or genome sequencing and studied by segregation analysis and clinical characterization. RESULTS: Overexpression of SLC12A9, a solute co-transporter, corrected lysosomal swelling in FIG4-/- cells. SLC12A9 (NP_064631.2) colocalized with LAMP2 at the lysosome membrane. Biallelic variants of SLC12A9 were identified in 3 unrelated probands with neurodevelopmental disorders. Common features included intellectual disability, skeletal and brain structural abnormalities, congenital heart defects, and hypopigmented hair. Patient 1 was homozygous for nonsense variant p.(Arg615∗), patient 2 was compound heterozygous for p.(Ser109Lysfs∗20) and a large deletion, and proband 3 was compound heterozygous for p.(Glu290Glyfs∗36) and p.(Asn552Lys). Fibroblasts from proband 1 contained enlarged lysosomes that were corrected by wild-type SLC12A9 cDNA. Patient variant p.(Asn552Lys) failed to correct the lysosomal defect. CONCLUSION: Impaired function of SLC12A9 results in enlarged lysosomes and a recessive disorder with a recognizable neurodevelopmental phenotype.

Severe acute liver disease in adults: Contemporary role of histopathology.

Liver biopsies have consistently contributed to our understanding of the pathogenesis and aetiologies of acute liver disease. As other diagnostic modalities have been developed and refined, the role of biopsy in the management of patients with acute liver failure (ALF), acute-on-chronic liver failure (ACLF) and acute hepatitis, including acute liver injury (ALI), has changed. Liver biopsy remains particularly valuable when first-line diagnostic algorithms fail to determine aetiology. Despite not being identified as a mandatory diagnostic tool in recent clinical guidelines for the management of ALF or ACLF, many centres continue to undertake biopsies given the relative safety of transjugular biopsy in this setting. Several studies have demonstrated that liver biopsy can provide prognostic information, particularly in the context of so-called indeterminate hepatitis, and is extremely useful in excluding conditions such as metastatic tumours that would preclude transplantation. In addition, its widespread use of percutaneous biopsies in cases of less severe acute liver injury, for example in the establishment of a diagnosis of acute presentation of autoimmune hepatitis or confirmation of a probable or definite drug-induced liver injury (DILI), has meant that many centres have seen a shift in the ratio of specimens they are receiving from patients with chronic to acute liver disease. Histopathologists therefore need to be equipped to deal with these challenging specimens. This overview provides an insight into the contemporary role of biopsies (as well as explant and autopsy material) in diagnosing acute liver disease. It outlines up-to-date clinical definitions of liver injury and considers recent recommendations for the diagnosis of AIH and drug-induced, autoimmune-like hepatitis (DI-AIH).

Clinicopathologic and MRI features of combined hepatocellular-cholangiocarcinoma in patients with or without cirrhosis.

BACKGROUND AND AIMS: Differences in combined hepatocellular-cholangiocarcinomas (cHCC-CCAs) arising in high-risk patients with or without liver cirrhosis have not been elucidated. This study aimed to compare the clinicopathologic and imaging characteristics of cHCC-CCAs in patients with or without cirrhosis and to determine the prognostic factors for recurrence-free survival (RFS) after curative resections of single cHCC-CCAs. METHODS: This retrospective study included 113 patients with surgically resected single cHCC-CCAs who underwent preoperative magnetic resonance imaging from January 2008 to December 2019 at two tertiary referral centres. Clinical, pathologic and imaging features of tumours were compared in high-risk patients with or without cirrhosis. Imaging features were assessed using the Liver Imaging Reporting and Data System (LI-RADS) version 2018. RFS and associated factors were evaluated using Cox proportional hazards regression analysis, Kaplan-Meier analysis and log-rank test. RESULTS: cHCC-CCAs arising from cirrhotic livers had a smaller mean tumour size (2.9 cm vs. 4.5 cm; P < .001) and were more frequently categorized as LR-5 or 4 (41.2% vs. 20.0%; P = .024) than those arising from non-cirrhotic livers. In multivariable analysis, a tumour size of > 3 cm (hazard ratio [HR], 2.081; 95% confidence interval [CI], 1.180-3.668; P = .011) and the LR-M category (HR, 2.302; 95% CI, 1.198-4.424; P = .012) were independent predictors associated with worse RFS. CONCLUSIONS: The tumour size and distribution of LI-RADS categories of cHCC-CCAs differed in high-risk patients with or without cirrhosis. And LR-M category was a worse prognosis predictor after curative resections than LR-5 or 4 category.

TPRG1-AS1 induces RBM24 expression and inhibits liver cancer progression by sponging miR-4691-5p and miR-3659.

BACKGROUND & AIMS: Noncoding RNAs (ncRNAs) play critical roles in hepatocellular carcinoma (HCC) progression. Here, by performing RNA-sequencing (RNA-Seq) profiling, we sought to identify novel ncRNAs that potentially drive the heterogeneous progression of liver cancers. METHODS: RNA-Seq profiles were obtained from 68 HCC specimens and 10 samples of adjacent non-tumour liver tissues. The functional significance of the potential driver ncRNAs was evaluated by cell experiments. RESULTS: TPRG1-AS1 was identified as a potential driver noncoding RNA that promotes heterogeneous liver cancer progression. TPRG1-AS1 induced tumour suppressor RNA-binding motif protein 24 (RBM24), suppressing tumour growth by activating apoptotic tumour cell death. In addition, we report that TPRG1-AS1 acts as a competing endogenous RNA (ceRNA) for RBM24, sponging miR-4691-5p and miR-3659 to interfere with their binding to RBM24. CONCLUSIONS: We suggest that TPRG1-AS1 is a novel ceRNA sponging miR-4691-5p and miR-3659, resulting in RBM24 expression and suppression of liver cancer growth. Our results provide new insights into the functions of ncRNAs in heterogeneous HCC progression.

Data set for the reporting of intrahepatic cholangiocarcinoma, perihilar cholangiocarcinoma and hepatocellular carcinoma: recommendations from the International Collaboration on Cancer Reporting (ICCR).

Optimal patient management benefits from comprehensive and accurate pathology reports that contribute to cancer staging and prognostication. Proforma reports are used in many countries, but these vary in their structure and implementation. The International Collaboration on Cancer Reporting (ICCR) is an alliance formed by the Royal College of Pathologists of Australasia, the Royal College of Pathologists of the United Kingdom, the College of American Pathologists, the Canadian Partnership Against Cancer the European Society of Pathology and the American Society of Clinical Pathology (ASCP), with the aim of developing an evidence-based reporting data set for each cancer site. It is argued that this should reduce the global burden of cancer data set development and reduplication of effort by different international institutions that commission, publish and maintain standardised cancer reporting data sets. The resultant standardisation of cancer reporting will benefit not only those countries directly involved in the collaboration but also others not in a position to develop their own data sets. We describe the development of a cancer data set by the ICCR expert panel for the reporting of the main malignant liver tumours: intrahepatic cholangiocarcinoma, perihilar cholangiocarcinoma and hepatocellular carcinoma and present the 'required' and 'recommended' elements to be included in the report with an explanatory commentary. This data set incorporates definitions and classifications in the most recent World Health Organisation (WHO) publication on hepatic malignancies (4th edition) and the recently published tumour-node-metastasis (TNM)8 staging system. Widespread adoption and implementation of this data set will enable consistent and accurate data collection, comparison of epidemiological and pathological parameters between different populations, facilitate research and ultimately result in better patient outcomes.

A model organism pipeline provides insight into the clinical heterogeneity of TARS1 loss-of-function variants.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed, essential enzymes that complete the first step of protein translation: ligation of amino acids to cognate tRNAs. Genes encoding ARSs have been implicated in myriad dominant and recessive phenotypes, the latter often affecting multiple tissues but with frequent involvement of the central and peripheral nervous systems, liver, and lungs. Threonyl-tRNA synthetase (TARS1) encodes the enzyme that ligates threonine to tRNATHR in the cytoplasm. To date, TARS1 variants have been implicated in a recessive brittle hair phenotype. To better understand TARS1-related recessive phenotypes, we engineered three TARS1 missense variants at conserved residues and studied these variants in Saccharomyces cerevisiae and Caenorhabditis elegans models. This revealed two loss-of-function variants, including one hypomorphic allele (R433H). We next used R433H to study the effects of partial loss of TARS1 function in a compound heterozygous mouse model (R432H/null). This model presents with phenotypes reminiscent of patients with TARS1 variants and with distinct lung and skin defects. This study expands the potential clinical heterogeneity of TARS1-related recessive disease, which should guide future clinical and genetic evaluations of patient populations.

Predictive modeling provides insight into the clinical heterogeneity associated with TARS1 loss-of-function mutations.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed, essential enzymes that complete the first step of protein translation: ligation of amino acids to cognate tRNAs. Genes encoding ARSs have been implicated in myriad dominant and recessive phenotypes, the latter often affecting multiple tissues but with frequent involvement of the central and peripheral nervous system, liver, and lungs. Threonyl-tRNA synthetase (TARS1) encodes the enzyme that ligates threonine to tRNATHR in the cytoplasm. To date, TARS1 variants have been implicated in a recessive brittle hair phenotype. To better understand TARS1-related recessive phenotypes, we engineered three TARS1 missense mutations predicted to cause a loss-of-function effect and studied these variants in yeast and worm models. This revealed two loss-of-function mutations, including one hypomorphic allele (R433H). We next used R433H to study the effects of partial loss of TARS1 function in a compound heterozygous mouse model (R433H/null). This model presents with phenotypes reminiscent of patients with TARS1 variants and with distinct lung and skin defects. This study expands the potential clinical heterogeneity of TARS1-related recessive disease, which should guide future clinical and genetic evaluations of patient populations.

Early on-treatment change in liver stiffness predicts development of liver-related events in chronic hepatitis B patients receiving antiviral therapy.

BACKGROUNDS/AIMS: Monitoring fibrosis is mandatory for detailed prognostification in patients with chronic liver disease. We developed optimized cut-offs for liver stiffness (LS) values, based on the histological subclassification of cirrhosis, and investigated whether early on-treatment changes in LS values can predict long-term prognosis in patients with hepatitis B virus (HBV)-related advanced liver fibrosis receiving antiviral therapy. METHODS: Between 2005 and 2008, 103 patients with F3 or F4 fibrosis on liver biopsy were enrolled prospectively. Cirrhosis was subclassified into three groups (F4A, F4B and F4C) according to Laennec system. The primary end-point was occurrence of liver-related event (LRE), including decompensation, hepatocellular carcinoma and liver-related death. RESULTS: Suggested LS cut-offs for predicting F4B-FC (vs. F3-F4A) and F4C (vs. F3-F4B) were 11.6 and 18.2 kPa respectively. As proportions of patients with LRE occurrence increased according to histological subclassifications stage F3-4A vs. F4B-4C (7.4% vs. 17.1%) and stage F3-4B vs. F4C (13.8% vs. 18.8%), they also increased according to LS cut-off value of 11.6 kPa (5.9% vs. 23.1%) and 18.2 kPa (9.8% vs. 33.3%) respectively (all P < 0.05). Similarly, according to stratified LS values (<11.6, 11.6-18.2 and ≥18.2 kPa), overall incidence of LREs and each constituent event increased significantly (all P < 0.05). In addition, the observed changes in LS values between baseline and 6 months of follow-up showed significant correlations with LRE development. CONCLUSIONS: Stratified LS values based on Laennec system and dynamic changes in LS values on follow-up may be helpful in assessing risk of LREs in subjects with HBV-related advanced liver fibrosis receiving antiviral therapy.

Serum markers for necroinflammatory activity in patients with chronic viral hepatitis and normal or mildly elevated aminotransferase levels.

BACKGROUND: Reports on the usefulness of serum markers for predicting liver necroinflammation are limited. The aim of this study was to determine the serum markers that predict significant inflammation in patients with chronic hepatitis B (CHB) and C (CHC) and normal or mildly elevated serum aminotransferase levels. METHODS: Two hundred twenty-seven patients with CHB or CHC with normal or mildly elevated serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (≤60 IU/L) were enrolled in this study. Significant inflammation was defined as inflammatory grade ≥3 activities using the Batt-Ludwig scoring system. The correlation between liver histology and serum markers of liver inflammation was analysed. RESULTS: Forty-eight (21.1%) and eight patients (3.5%) had grade 3 and 4 inflammation respectively. Univariate analysis revealed that age, platelet coun, and AST, ALT, γ-glutamyl transpeptidase, alkaline phosphatase, hyaluronic acid, haptoglobin, apolipoprotein A1 and procollagen III N-terminal peptide levels were significantly different between the patients with and without significant inflammation. There were no significant differences in the cytokeratin-18 fragment levels between the two groups. On the basis of multivariate analysis, the AST and apolipoprotein A1 levels and stage of fibrosis were highly predictive of significant inflammation. Using AST and apolipoprotein cut-off values ≥44 IU/L and ≤100 ng/ml, respectively, the presence of significant inflammation was predicted with high specificity (96.5%) and with a negative predictive value of 76.3%. CONCLUSION: The AST and apolipoprotein A1 levels were shown to be independent predictors of significant inflammatory activities in patients with CHB and CHC and normal or mildly elevated aminotransferase levels.

Predictive factors for long-term survival in patients with clinically significant portal hypertension following resection of hepatocellular carcinoma.

BACKGROUND: Hepatic resection for hepatocellular carcinoma (HCC) is not currently recommended for patients with clinically significant portal hypertension (PHT); however, recent studies have shown similar post-operative outcomes between patients with and without clinically significant PHT. AIM: To clarify the post-operative prognostic relevance of clinically significant PHT in Child-Pugh A cirrhotic patients. METHODS: A total of 100 Child-Pugh A cirrhotic patients who underwent curative resection of HCC were eligible for this analysis. Patients were divided into two groups: PHT group (n=47) and non-PHT group (n=53). RESULTS: Clinicopathological variables showed no significant differences except for prothrombine time. Liver-related complications were significantly higher in the PHT group (P=0.015), and the 5-year overall survival rate was significantly higher in the non-PHT group (78.7 vs. 37.9%, P<0.001). The proportion of patients who died because of complications of cirrhosis was significantly higher in the PHT group (P=0.001). Multivariate analysis indicated that the presence of clinically significant PHT was the most powerful adverse prognostic factor for overall survival. Multivariate analysis of the 47 patients with clinically significant PHT indicated that gross vascular invasion and non-single nodular type were poor prognostic factors. The 5-year survival rate of patients with single nodular type and without gross vascular invasion (n=17) was 78.4%. CONCLUSIONS: In Child-Pugh A cirrhotic patients, the presence of clinically significant PHT was significantly associated with post-operative hepatic decompensation and poor prognosis after resection of HCC. However, in patients with clinically significant PHT, those with single nodular tumours lacking gross vascular invasion may be good surgical candidates.

CRISPR knockout screen implicates three genes in lysosome function.

Defective biosynthesis of the phospholipid PI(3,5)P2 underlies neurological disorders characterized by cytoplasmic accumulation of large lysosome-derived vacuoles. To identify novel genetic causes of lysosomal vacuolization, we developed an assay for enlargement of the lysosome compartment that is amenable to cell sorting and pooled screens. We first demonstrated that the enlarged vacuoles that accumulate in fibroblasts lacking FIG4, a PI(3,5)P2 biosynthetic factor, have a hyperacidic pH compared to normal cells'. We then carried out a genome-wide knockout screen in human HAP1 cells for accumulation of acidic vesicles by FACS sorting. A pilot screen captured fifteen genes, including VAC14, a previously identified cause of endolysosomal vacuolization. Three genes not previously associated with lysosome dysfunction were selected to validate the screen: C10orf35, LRRC8A, and MARCH7. We analyzed two clonal knockout cell lines for each gene. All of the knockout lines contained enlarged acidic vesicles that were positive for LAMP2, confirming their endolysosomal origin. This assay will be useful in the future for functional evaluation of patient variants in these genes, and for a more extensive genome-wide screen for genes required for endolysosome function. This approach may also be adapted for drug screens to identify small molecules that rescue endolysosomal vacuolization.