ZFYVE19 deficiency: a ciliopathy involving failure of cell division, with cell death.

BACKGROUND AND AIMS: Variants in ZFYVE19 underlie a disorder characterised by progressive portal fibrosis, portal hypertension and eventual liver decompensation. We aim to create an animal model to elucidate the pathogenic mechanism. METHODS: Zfyve19 knockout (Zfyve19-/- ) mice were generated and exposed to different liver toxins. Their livers were characterised at the tissue, cellular and molecular levels. Findings were compared with those in wild-type mice and in ZFYVE19-deficient patients. ZFYVE19 knockout and knockdown retinal pigment epithelial-1 cells and mouse embryonic fibroblasts were generated to study cell division and cell death. RESULTS: The Zfyve19-/- mice were normal overall, particularly with respect to hepatobiliary features. However, when challenged with α-naphthyl isothiocyanate, Zfyve19-/- mice developed changes resembling those in ZFYVE19-deficient patients, including elevated serum liver injury markers, increased numbers of bile duct profiles with abnormal cholangiocyte polarity and biliary fibrosis. Failure of cell division, centriole and cilia abnormalities, and increased cell death were observed in knockdown/knockout cells. Increased cell death and altered mRNA expression of cell death-related signalling pathways was demonstrated in livers from Zfyve19-/- mice and patients. Transforming growth factor-ß (TGF-ß) and Janus kinase-Signal Transducer and Activator of Transcription 3 (JAK-STAT3) signalling pathways were upregulated in vivo, as were chemokines such as C-X-C motif ligands 1, 10 and 12. CONCLUSIONS: Our findings demonstrated that ZFYVE19 deficiency is a ciliopathy with novel histological features. Failure of cell division with ciliary abnormalities and cell death activates macrophages and may thus lead to biliary fibrosis via TGF-ß pathway in the disease.

Review of predicting protein stability changes upon variations.

Forecasting alterations in protein stability caused by variations holds immense importance. Improving the thermal stability of proteins is important for biomedical and industrial applications. This review discusses the latest methods for predicting the effects of mutations on protein stability, databases containing protein mutations and thermodynamic parameters, and experimental techniques for efficiently assessing protein stability in high-throughput settings. Various publicly available databases for protein stability prediction are introduced. Furthermore, state-of-the-art computational approaches for anticipating protein stability changes due to variants are reviewed. Each method's types of features, base algorithm, and prediction results are also detailed. Additionally, some experimental approaches for verifying the prediction results of computational methods are introduced. Finally, the review summarizes the progress and challenges of protein stability prediction and discusses potential models for future research directions.

Preoperative Platelet-Lymphocyte Ratio (PLR) as a prognostic inflammation biomarker in Asian HIV-infected patients with gastric cancer: a single-center study.

BACKGROUND: The serum systemic inflammation biomarkers have been established as predictors of prognosis in gastric cancer (GC) patients, but their prognostic value in human immunodeficiency virus (HIV)-infected patients with GC has not been well studied. This retrospective study aimed to evaluate the prognostic value of preoperative systemic inflammation biomarkers in Asian HIV-infected patients with GC. METHODS: We retrospectively analyzed 41 HIV-infected GC patients who underwent surgery between January 2015 and December 2021 at the Shanghai Public Health Clinical Center. Preoperative systemic inflammation biomarkers were measured and patients were divided into two groups based on the optimal cut-off value. Overall survival (OS) and progression-free survival (PFS) were measured using the Kaplan-Meier method and the log-rank test. Multivariate analysis of variables was performed using the Cox proportional regression model. As a comparison, 127 GC patients without HIV infection were also recruited. RESULTS: The median age of the 41 patients included in the study was 59 years, with 39 males and two females. The follow-up period for OS and PFS ranged from 3 to 94 months. The cumulative three-year OS rate was 46.0%, and the cumulative three-year PFS rate was 44%. HIV-infected GC patients had worse clinical outcomes compared to the normal GC population. The optimal cut-off value for preoperative platelet to lymphocyte ratio (PLR) was 199 in HIV-infected GC patients. Multivariate Cox regression analysis revealed that a low PLR was an independent predictor of better OS and PFS (OS: HR = 0.038, 95% CI: 0.006-0.258, P < 0.001; PFS: HR = 0.027, 95% CI: 0.004-0.201, P < 0.001). Furthermore, higher preoperative PLR in HIV-infected GC was significantly associated with lower BMI, hemoglobin, albumin, CD4 + T, CD8 + T, and CD3 + T cell counts. CONCLUSION: The preoperative PLR is an easily measurable immune biomarker that may provide useful prognostic information in HIV-infected GC patients. Our findings suggest that PLR could be a valuable clinical tool for guiding treatment decisions in this population.

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.

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.

MYO5B-associated diseases: Novel liver-related variants and genotype-phenotype correlation.

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.

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.

MicroRNA-21 regulates right ventricular remodeling secondary to pulmonary arterial pressure overload.

Right ventricular (RV) function is a critical determinant of survival in patients with pulmonary arterial hypertension (PAH). While miR-21 is known to associate with vascular remodeling in small animal models of PAH, its role in RV remodeling in large animal models has not been characterized. Herein, we investigated the role of miR-21 in RV dysfunction using a sheep model of PAH secondary to pulmonary arterial constriction (PAC). RV structural and functional remodeling were examined using ultrasound imaging. Our results showed that post PAC, RV strain significantly decreased at the basal region compared with t the control. Moreover, such dysfunction was accompanied by increases in miR-21 levels. To determine the role of miR-21 in RV remodeling secondary to PAC, we investigated the molecular alteration secondary to phenylephrine induced hypertrophy and miR21 overexpression in vitro using neonatal rat ventricular myocytes (NRVMs). We found that overexpression of miR-21 in the setting of hypertrophic stimulation augmented only the expression of proteins critical for mitosis but not cytokinesis. Strikingly, this molecular alteration was associated with an eccentric cellular hypertrophic phenotype similar to what we observed in vivo PAC animal model in sheep. Importantly, this hypertrophic change was diminished upon suppressing miR-21 in NRVMs. Collectively, our in vitro and in vivo data demonstrate that miR-21 is a critical contributor in the development of RV dysfunction and could represent a novel therapeutic target for PAH associated RV dysfunction.

A Molecular Mechanism Underlying Genotype-Specific Intrahepatic Cholestasis Resulting From MYO5B Mutations.

BACKGROUND AND AIMS: Progressive familial intrahepatic cholestasis (PFIC) 6 has been associated with missense but not biallelic nonsense or frameshift mutations in MYO5B, encoding the motor protein myosin Vb (myoVb). This genotype-phenotype correlation and the mechanism through which MYO5B mutations give rise to PFIC are not understood. The aim of this study was to determine whether the loss of myoVb or expression of patient-specific myoVb mutants can be causally related to defects in canalicular protein localization and, if so, through which mechanism. APPROACH AND RESULTS: We demonstrate that the cholestasis-associated substitution of the proline at amino acid position 600 in the myoVb protein to a leucine (P660L) caused the intracellular accumulation of bile canalicular proteins in vesicular compartments. Remarkably, the knockout of MYO5B in vitro and in vivo produced no canalicular localization defects. In contrast, the expression of myoVb mutants consisting of only the tail domain phenocopied the effects of the Myo5b-P660L mutation. Using additional myoVb and rab11a mutants, we demonstrate that motor domain-deficient myoVb inhibited the formation of specialized apical recycling endosomes and that its disrupting effect on the localization of canalicular proteins was dependent on its interaction with active rab11a and occurred at the trans-Golgi Network/recycling endosome interface. CONCLUSIONS: Our results reveal a mechanism through which MYO5B motor domain mutations can cause the mislocalization of canalicular proteins in hepatocytes which, unexpectedly, does not involve myoVb loss-of-function but, as we propose, a rab11a-mediated gain-of-toxic function. The results explain why biallelic MYO5B mutations that affect the motor domain but not those that eliminate myoVb expression are associated with PFIC6.

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.

NBAS disease: 14 new patients, a recurrent mutation, and genotype-phenotype correlation among 24 Chinese patients.

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.

Novel missense mutation in VPS33B is associated with isolated low gamma-glutamyltransferase cholestasis: Attenuated, incomplete phenotype of arthrogryposis, renal dysfunction, and cholestasis syndrome.

The typical phenotype of arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome involves three cardinal symptoms as the name describes, harboring biallelic mutations on VPS33B or VIPAS39. Except for ARC syndrome, low gamma-glutamyltransferase (GGT) cholestasis often implies hereditary hepatopathy of different severity; however, some remain undiagnosed. Several monogenic defects typically with multiorgan manifestations may only present liver dysfunction at times, such as DGUOK defect and AGL defect. Previously, four VPS33B mutated cases were reported without arthrogryposis, or with less severe symptoms and longer lifespan, indicating the possibility of incomplete ARC phenotype of isolated hepatopathy. So we retrospectively reviewed all patients with confirmed VPS33B/VIPARS39 defect in our center and identified three presenting isolated low-GGT cholestasis with intractable pruritus. Distinguished from others with typical ARC phenotype, these patients did not suffer the other two typical characteristics, survived much longer, and shared a novel missense VPS33B variation c.1726T>C, p.Cys576Arg, causing declined protein expression and abolished interaction with VIPAS39 in-vitro. Serum bile acid profiles of our VPS33B/VIPAS39 mutated patients revealed similar changes to primary defect of bile salt export pump, among which those with isolated cholestasis phenotype had a higher level of total secondary bile acids than that with typical ARC phenotype, indicating the partial residual function of VPS33B.

A novel mutation in EYA1 in a Chinese family with Branchio-oto-renal syndrome.

BACKGROUND: Branchio-oto-renal (BOR) syndrome is a dominant autosomal disorder characterized by phenotypes such as hearing loss, branchial fistulae, preauricular pits, and renal abnormalities. EYA1, the human homolog of the Drosophila "eye absent" gene on chromosome 8q13.3, is recognized as one of the most important genes associated with BOR syndrome. METHODS: The proposita in this study was a 5-year-old Chinese girl with hearing loss, bilateral otitis media with effusion, microtia, facial hypoplasia, palatoschisis, and bilateral branchial cleft fistulae. The girl's family members, except two who were deceased, agreed to undergo clinical examination. We collected blood samples from 10 family members, including six who were affected by the syndrome. Genomic DNA was extracted and subjected to Sanger sequencing. A minigene assay was performed to confirm whether splicing signals were altered. In addition, we performed western blotting to determine alterations in protein levels of the wild-type and mutant gene. RESULTS: Clinical tests showed that some of the family members met the criteria for BOR syndrome. The affected members harbored a novel heterozygous nonsense variation in exon 11 of EYA1, whereas no unaffected member carried the mutation at this position. Functional experiments did not detect abnormal splicing at the RNA level; however, western blotting showed that the mutated protein was truncated. CONCLUSIONS: This study reports a novel mutation associated with BOR syndrome in a Chinese family. We highlight the usefulness of genetic testing in the diagnosis of BOR syndrome. Thus, we believe that this report would benefit clinicians in this field.

Defects in myosin VB are associated with a spectrum of previously undiagnosed low γ-glutamyltransferase cholestasis.

Hereditary cholestasis in childhood and infancy with normal serum gamma-glutamyltransferase (GGT) activity is linked to several genes. Many patients, however, remain genetically undiagnosed. Defects in myosin VB (MYO5B; encoded by MYO5B) cause microvillus inclusion disease (MVID; MIM251850) with recurrent watery diarrhea. Cholestasis, reported as an atypical presentation in MVID, has been considered a side effect of parenteral alimentation. Here, however, we report on 10 patients who experienced cholestasis associated with biallelic, or suspected biallelic, mutations in MYO5B and who had neither recurrent diarrhea nor received parenteral alimentation. Seven of them are from two study cohorts, together comprising 31 undiagnosed low-GGT cholestasis patients; 3 are sporadic. Cholestasis in 2 patients was progressive, in 3 recurrent, in 2 transient, and in 3 uncategorized because of insufficient follow-up. Liver biopsy specimens revealed giant-cell change of hepatocytes and intralobular cholestasis with abnormal distribution of bile salt export pump (BSEP) at canaliculi, as well as coarse granular dislocation of MYO5B. Mass spectrometry of plasma demonstrated increased total bile acids, primary bile acids, and conjugated bile acids, with decreased free bile acids, similar to changes in BSEP-deficient patients. Literature review revealed that patients with biallelic mutations predicted to eliminate MYO5B expression were more frequent in typical MVID than in isolated-cholestasis patients (11 of 38 vs. 0 of 13). CONCLUSION: MYO5B deficiency may underlie 20% of previously undiagnosed low-GGT cholestasis. MYO5B deficiency appears to impair targeting of BSEP to the canalicular membrane with hampered bile acid excretion, resulting in a spectrum of cholestasis without diarrhea. (Hepatology 2017;65:1655-1669).

Comprehensive bile acid profiling in hereditary intrahepatic cholestasis: Genetic and clinical correlations.

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.

Splicing analysis of rare/novel synonymous or intronic variants identified in ABCB11 heterozygotes presenting as progressive intrahepatic cholestasis with low γ-glutamyltransferase.

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.

MicroRNA-34a Plays a Key Role in Cardiac Repair and Regeneration Following Myocardial Infarction.

RATIONALE: In response to injury, the rodent heart is capable of virtually full regeneration via cardiomyocyte proliferation early in life. This regenerative capacity, however, is diminished as early as 1 week postnatal and remains lost in adulthood. The mechanisms that dictate postinjury cardiomyocyte proliferation early in life remain unclear. OBJECTIVE: To delineate the role of miR-34a, a regulator of age-associated physiology, in regulating cardiac regeneration secondary to myocardial infarction (MI) in neonatal and adult mouse hearts. METHODS AND RESULTS: Cardiac injury was induced in neonatal and adult hearts through experimental MI via coronary ligation. Adult hearts demonstrated overt cardiac structural and functional remodeling, whereas neonatal hearts maintained full regenerative capacity and cardiomyocyte proliferation and recovered to normal levels within 1-week time. As early as 1 week postnatal, miR-34a expression was found to have increased and was maintained at high levels throughout the lifespan. Intriguingly, 7 days after MI, miR-34a levels further increased in the adult but not neonatal hearts. Delivery of a miR-34a mimic to neonatal hearts prohibited both cardiomyocyte proliferation and subsequent cardiac recovery post MI. Conversely, locked nucleic acid-based anti-miR-34a treatment diminished post-MI miR-34a upregulation in adult hearts and significantly improved post-MI remodeling. In isolated cardiomyocytes, we found that miR-34a directly regulated cell cycle activity and death via modulation of its targets, including Bcl2, Cyclin D1, and Sirt1. CONCLUSIONS: miR-34a is a critical regulator of cardiac repair and regeneration post MI in neonatal hearts. Modulation of miR-34a may be harnessed for cardiac repair in adult myocardium.

Novel NBAS mutations and fever-related recurrent acute liver failure in Chinese children: a retrospective study.

BACKGROUND: Underlying causes in Chinese children with recurrent acute liver failure (RALF), including liver crises less than full acute liver failure, are incompletely understood. We sought to address this by searching for genes mutated in such children. METHODS: Five unrelated Chinese boys presenting between 2012 and 2015 with RALF of unexplained etiology were studied. Results of whole exome sequencing were screened for mutations in candidate genes. Mutations were verified in patients and their family members by Sanger sequencing. All 5 boys underwent liver biopsy. RESULTS: NBAS was the only candidate gene mutated in more than one patient (biallelic mutations, 3 of 5 patients; 5 separate mutations). All NBAS mutations were novel and predictedly pathogenic (frameshift insertion mutation c.6611_6612insCA, missense mutations c.2407G > A and c.3596G > A, nonsense mutation c.586C > T, and splicing-site mutation c.5389 + 1G > T). Of these mutations, 3 lay in distal (C-terminal) regions of NBAS, a novel distribution. Unlike the 2 patients without NBAS mutations, the 3 patients with confirmed NBAS mutations all suffered from a febrile illness before each episode of liver crisis (fever-related RALF), with markedly elevated alanine aminotransferase and aspartate aminotransferase activities 24-72 h after elevation of body temperature, succeeded by severe coagulopathy and mild to moderate jaundice. CONCLUSIONS: As in other countries, so too in China; NBAS disease is a major cause of fever-related RALF in children. The mutation spectrum of NBAS in Chinese children seems different from that described in other populations.

A role for matrix stiffness in the regulation of cardiac side population cell function.

The mechanical properties of the local microenvironment may have important influence on the fate and function of adult tissue progenitor cells, altering the regenerative process. This is particularly critical following a myocardial infarction, in which the normal, compliant myocardial tissue is replaced with fibrotic, stiff scar tissue. In this study, we examined the effects of matrix stiffness on adult cardiac side population (CSP) progenitor cell behavior. Ovine and murine CSP cells were isolated and cultured on polydimethylsiloxane substrates, replicating the elastic moduli of normal and fibrotic myocardium. Proliferation capacity and cell cycling were increased in CSP cells cultured on the stiff substrate with an associated reduction in cardiomyogeneic differentiation and accelerated cell ageing. In addition, culture on stiff substrate stimulated upregulation of extracellular matrix and adhesion proteins gene expression in CSP cells. Collectively, we demonstrate that microenvironment properties, including matrix stiffness, play a critical role in regulating progenitor cell functions of endogenous resident CSP cells. Understanding the effects of the tissue microenvironment on resident cardiac progenitor cells is a critical step toward achieving functional cardiac regeneration.