Molecular and clinical characterization of a series of patients with childhood-onset lysosomal acid lipase deficiency. Retrospective investigations, follow-up and detection of two novel LIPA pathogenic variants.

BACKGROUND AND AIMS: Childhood/Adult-onset Lysosomal Acid Lipase Deficiency (LAL-D) is a recessive disorder due to loss of function variants of LAL, the enzyme which hydrolyses cholesteryl esters, derived from internalized apoB containing lipoproteins. The disease is characterized by multi-organ involvement including the liver, spleen, intestine and cardiovascular system. The aim of this study was the clinical and molecular characterization of 14 (13 unrelated) previously unreported patients with childhood-onset LAL-D. METHODS: Data collected included clinical and laboratory investigations, liver imaging, liver biopsy and LIPA gene analysis. The response to lipid-lowering medications, liver transplantation and enzyme replacement therapy (ERT) was reported for some patients. RESULTS: LAL-D was suspected at 4.4 ± 3.3 years of age for the presence of hepatomegaly, elevated serum transaminases and hypercholesterolemia, and was confirmed by liver biopsy/imaging and LAL assay. The follow up period ranged from 3 to 40 years (mean 7.8 ± 4.0 years in 13 cases). Patients treated with statins with or without ezetimibe showed 28% reduction of plasma LDL-cholesterol without a tangible effect on liver enzymes; some patients receiving ERT showed normalized lipoprotein profile and transaminase levels. The common c.894G > A variant was observed in homozygosity or compound heterozygosity in 10 patients. We found seven previously reported variants: p.(Trp140*), p.(Arg218*), p.(Gly266*), p.(Thr288Ile), p.(Leu294Ser), p.(His295Tyr) and p.(Gly342Arg) and two novel variants: p.(Asp345Asn), affecting the LAL catalytic triad, and c.229+3A > C, affecting splicing. Homozygosity for p.(Thr288Ile) or c.229+3A > C was associated with a severe phenotype. CONCLUSIONS: This study provides additional data on the features of childhood-onset LAL-D and describes two novel pathogenic variants of the LIPA gene.

Deubiquitinase YOD1: the potent activator of YAP in hepatomegaly and liver cancer.

Advances in the understanding of the Hippo signaling as a key regulatory pathway of proliferation and apoptosis have provided mechanical insights for controlling organ size and tumorigenicity. Recently, much attention has been directed to the regulation of LATS1/2 (large tumor suppressor) kinases that phosphorylate YAP/TAZ, a transcriptional co-activator in the Hippo pathway, and control the level and nuclear localization of YAP/TAZ. In our recent work, we showed that deubiquitinase YOD1 stabilizes ITCH, and facilitates ITCH-mediated LATS1/2 ubiquitination and degradation, resulting in increased YAP/TAZ level. Furthermore, we found that the YOD1- ITCH-LATS1/2-YAP/TAZ signaling axis is controlled by the differential expression of miR-21 in a cell-density-dependent manner. Using a transgenic mouse model, we showed that the inducible expression of YOD1 enhances the proliferation of hepatocytes and leads to hepatomegaly in a YAP/TAZ-activitydependent manner. Moreover, a strong correlation was observed between YOD1 and YAP expression in liver cancer patients. Overall, our data suggest that YOD1 is a novel regulator of the Hippo pathway, and thereby a potential therapeutic target for liver cancer. [BMB Reports 2017; 50(6): 281-282].

Vertebral compression fractures as the initial presentation of AL amyloidosis: case series and review of literature.

The clinical presentation of AL amyloidosis is highly variable. In this series, we describe five cases of AL amyloidosis with vertebral compression fractures as initial presentation. All five patients had evidence of bone marrow replacement on magnetic resonance imaging and bone marrow biopsies demonstrating diffuse interstitial amyloid deposition. Hepatomegaly and elevated liver enzymes, consistent with liver involvement with amyloidosis, were also seen in each case. All five patients responded well to anti-plasma cell chemotherapy, with normalization of serum free light chain levels, reduction in alkaline phosphatase and improvement in pain and functional status. Although rare, AL amyloidosis should be considered in the differential diagnosis of selected patients with spontaneous vertebral compression fractures. Moreover, there seems to be an association of vertebral compression fractures with liver involvement in AL amyloidosis.

Novel LIPA mutations in Mexican siblings with lysosomal acid lipase deficiency.

Lysosomal acid lipase (LAL) deficiency is an under-recognized lysosomal disease caused by deficient enzymatic activity of LAL. In this report we describe two affected female Mexican siblings with early hepatic complications. At two months of age, the first sibling presented with alternating episodes of diarrhea and constipation, and later with hepatomegaly, elevated transaminases, high levels of total and low-density lipoprotein cholesterol, and low levels of high-density lipoprotein. Portal hypertension and grade 2 esophageal varices were detected at four years of age. The second sibling presented with hepatomegaly, elevated transaminases and mildly elevated low-density lipoprotein and low high-density lipoprotein at six months of age. LAL activity was deficient in both patients. Sequencing of LIPA revealed two previously unreported heterozygous mutations in exon 4: c.253C>A and c.294C>G. These cases highlight the clinical continuum between the so-called Wolman disease and cholesteryl ester storage disease, and underscore that LAL deficiency represents a single disease with a degree of clinical heterogeneity.

Defective adipose tissue development associated with hepatomegaly in cathepsin E-deficient mice fed a high-fat diet.

Cathepsin E is an intracellular aspartic proteinase, which is predominantly distributed in immune-related and epithelial cells. However, the role of the enzyme in adipose tissues remains unknown. In this study, we investigated the characteristics of cathepsin E-deficient (CatE(-/-)) mice fed a high-fat diet (HFD), as a mouse model of obesity. HFD-fed CatE(-/-) mice displayed reduced body weight gain and defective development of white adipose tissue (WAT) and brown adipose tissue (BAT), compared with HFD-fed wild-type mice. Moreover, fat-induced CatE(-/-) mice showed abnormal lipid accumulation in non-adipose tissues characterized by hepatomegaly, which is probably due to defective adipose tissue development. Detailed pathological and biochemical analyses showed that hepatomegaly was accompanied by hepatic steatosis and hypercholesterolemia in HFD-induced CatE(-/-) mice. In fat-induced CatE(-/-) mice, the number of macrophages infiltrating into WAT was significantly lower than in fat-induced wild-type mice. Thus, the impaired adipose tissue development in HFD-induced CatE(-/-) mice was probably due to reduced infiltration of macrophages and may lead to hepatomegaly accompanied by hepatic steatosis and hypercholesterolemia.

Tuberous sclerosis complex-1 deficiency attenuates diet-induced hepatic lipid accumulation.

Non-alcoholic fatty liver disease (NAFLD) is causally linked to type 2 diabetes, insulin resistance and dyslipidemia. In a normal liver, insulin suppresses gluconeogenesis and promotes lipogenesis. In type 2 diabetes, the liver exhibits selective insulin resistance by failing to inhibit hepatic glucose production while maintaining triglyceride synthesis. Evidence suggests that the insulin pathway bifurcates downstream of Akt to regulate these two processes. Specifically, mTORC1 has been implicated in lipogenesis, but its role on hepatic steatosis has not been examined. Here, we generated mice with hepatocyte-specific deletion of Tsc1 to study the effects of constitutive mTORC1 activation in the liver. These mice developed normally but displayed mild hepatomegaly and insulin resistance without obesity. Unexpectedly, the Tsc1-null livers showed minimal signs of steatosis even under high-fat diet condition. This 'resistant' phenotype was reversed by rapamycin and could be overcome by the expression of Myr-Akt. Moreover, rapamycin failed to reduce hepatic triglyceride levels in models of steatosis secondary to Pten ablation in hepatocytes or high-fat diet in wild-type mice. These observations suggest that mTORC1 is neither necessary nor sufficient for steatosis. Instead, Akt and mTORC1 have opposing effects on hepatic lipid accumulation such that mTORC1 protects against diet-induced steatosis. Specifically, mTORC1 activity induces a metabolic shift towards fat utilization and glucose production in the liver. These findings provide novel insights into the role of mTORC1 in hepatic lipid metabolism.

Targeted deletion of liver glucose-6 phosphatase mimics glycogen storage disease type 1a including development of multiple adenomas.

BACKGROUND AND AIMS: Glycogen storage disease type 1a (GSD1a) is an inherited disease caused by a deficiency in the catalytic subunit of the glucose-6 phosphatase enzyme (G6Pase). GSD1a is characterized by hypoglycaemia, hyperlipidemia, and lactic acidosis with associated hepatic (including hepatocellular adenomas), renal, and intestinal disorders. A total G6pc (catalytic subunit of G6Pase) knock-out mouse model has been generated that mimics the human pathology. However, these mice rarely live longer than 3 months and long-term liver pathogenesis cannot be evaluated. Herein, we report the long-term characterization of a liver-specific G6pc knock-out mouse model (L-G6pc(-/-)). METHODS: We generated L-G6pc(-/-) mice using an inducible CRE-lox strategy and followed up the development of hepatic tumours using magnetic resonance imaging. RESULTS: L-G6pc(-/-) mice are viable and exhibit normoglycemia in the fed state. They develop hyperlipidemia, lactic acidosis, and uricemia during the first month after gene deletion. However, these plasmatic parameters improved after 6 months. L-G6pc(-/-) mice develop hepatomegaly with glycogen accumulation and hepatic steatosis. Using an MRI approach, we could detect hepatic nodules with diameters of less than 1 mm, 9 months after induction of deficiency. Hepatic nodules (1 mm) were detected in 30-40% of L-G6pc(-/-) mice at 12 months. After 18 months, all L-G6pc(-/-) mice developed multiple hepatocellular adenomas of 1-10 mm diameter. CONCLUSIONS: This is the first report of a viable animal model of the hepatic pathology of GSD1a, including the late development of hepatocellular adenomas.

Deficiency of 5-hydroxyisourate hydrolase causes hepatomegaly and hepatocellular carcinoma in mice.

With the notable exception of humans, uric acid is degraded to (S)-allantoin in a biochemical pathway catalyzed by urate oxidase, 5-hydroxyisourate (HIU) hydrolase, and 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase in most vertebrate species. A point mutation in the gene encoding mouse HIU hydrolase, Urah, that perturbed uric acid metabolism within the liver was discovered during a mutagenesis screen in mice. The predicted substitution of cysteine for tyrosine in a conserved helical region of the mutant-encoded HIU hydrolase resulted in undetectable protein expression. Mice homozygous for this mutation developed elevated platelet counts secondary to excess thrombopoietin production and hepatomegaly. The majority of homozygous mutant mice also developed hepatocellular carcinoma, and tumor development was accelerated by exposure to radiation. The development of hepatomegaly and liver tumors in mice lacking Urah suggests that uric acid metabolites may be toxic and that urate oxidase activity without HIU hydrolase function may affect liver growth and transformation. The absence of HIU hydrolase in humans predicts slowed metabolism of HIU after clinical administration of exogenous urate oxidase in conditions of uric acid-related pathology. The data suggest that prolonged urate oxidase therapy should be combined with careful assessment of toxicity associated with extrahepatic production of uric acid metabolites.

A critical role for ceramide synthase 2 in liver homeostasis: II. insights into molecular changes leading to hepatopathy.

We have generated a mouse that cannot synthesize very long acyl chain (C22-C24) ceramides (Pewzner-Jung, Y., Park, H., Laviad, E. L., Silva, L. C., Lahiri, S., Stiban, J., Erez-Roman, R., Brugger, B., Sachsenheimer, T., Wieland, F. T., Prieto, M., Merrill, A. H., and Futerman, A. H. (2010) J. Biol. Chem. 285, 10902-10910) due to ablation of ceramide synthase 2 (CerS2). As a result, significant changes were observed in the sphingolipid profile of livers from these mice, including elevated C16-ceramide and sphinganine levels. We now examine the functional consequences of these changes. CerS2 null mice develop severe nonzonal hepatopathy from about 30 days of age, the age at which CerS2 expression peaks in wild type mice, and display increased rates of hepatocyte apoptosis and proliferation. In older mice there is extensive and pronounced hepatocellular anisocytosis with widespread formation of nodules of regenerative hepatocellular hyperplasia. Progressive hepatomegaly and noninvasive hepatocellular carcinoma are also seen from approximately 10 months of age. Even though CerS2 is found at equally high mRNA levels in kidney and liver, there are no changes in renal function and no pathological changes in the kidney. High throughput analysis of RNA expression in liver revealed up-regulation of genes associated with cell cycle regulation, protein transport, cell-cell interactions and apoptosis, and down-regulation of genes associated with intermediary metabolism, such as lipid and steroid metabolism, adipocyte signaling, and amino acid metabolism. In addition, levels of the cell cycle regulator, the cyclin dependent-kinase inhibitor p21(WAF1/CIP1), were highly elevated, which occurs by at least two mechanisms, one of which may involve p53. We propose a functional rationale for the synthesis of sphingolipids with very long acyl chains in liver homeostasis and in cell physiology.

Deficiency of mitochondrial ATP synthase of nuclear genetic origin.

We present clinical and laboratory data from 14 cases with an isolated deficiency of the mitochondrial ATP synthase (7-30% of control) caused by nuclear genetic defects. A quantitative decrease of the ATP synthase complex was documented by Blue-Native electrophoresis and Western blotting and was supported by the diminished activity of oligomycin/aurovertin-sensitive ATP hydrolysis in fibroblasts (10 cases), muscle (6 of 7 cases), and liver (one case). All patients had neonatal onset and elevated plasma lactate levels. In 12 patients investigated 3-methyl-glutaconic aciduria was detected. Seven patients died, mostly within the first weeks of life and surviving patients showed psychomotor and various degrees of mental retardation. Eleven patients had hypertrophic cardiomyopathy; other clinical signs included hypotonia, hepatomegaly, facial dysmorphism and microcephaly. This phenotype markedly differs from the severe central nervous system changes of ATP synthase disorders caused by mitochondrial DNA mutations of the ATP6 gene presenting mostly as NARP and MILS.

Overexpression of protein kinase C-{epsilon} in the mouse epidermis leads to a spontaneous myeloproliferative-like disease.

Protein kinase C (PKC)-epsilon, a Ca(2+)-independent, phospholipid-dependent serine/threonine kinase, is among the PKC isoforms expressed in mouse epidermis. We reported that FVB/N transgenic mouse lines that overexpress (8- or 18-fold) PKC-epsilon protein in basal epidermal cells and cells of the hair follicle develop papilloma-independent squamous cell carcinoma (SCC) elicited by 7,12-dimethylbenz(a)anthracene initiation and 12-O-tetradecanoylphorbol-13-acetate-promotion or by repeated ultraviolet radiation exposures. The susceptibility to the development of SCC was proportional to the level of expression of the PKC-epsilon transgene. We now report that PKC-epsilon FVB/N transgenic mice (line 215) that overexpress in epidermis approximately 18-fold PKC-epsilon protein more than their wild-type littermates spontaneously develop a myeloproliferative-like disease (MPD) in 100% of PKC-epsilon transgenic mice. The MPD was characterized by an excess of neutrophils and eosinophils, resulting in invasion of almost all vital organs of the mouse by 6 months of age. On gross examination these mice present with splenomegaly, hepatomegaly, and severe lymphadenopathy. Examination of the bone marrow revealed almost complete effacement by neutrophils, eosinophils, and their precursors. Furthermore, the spleen and lymph nodes were enlarged and exhibited marked extramedullary hematopoiesis. Complete pathological analysis of the second PKC-epsilon transgenic mouse (line 224) that expresses approximately eightfold PKC-epsilon protein more than their wild-type littermates revealed no remarkable findings in any of the affected organs as seen in line 215. However, peripheral blood analyses of PKC-epsilon transgenic mice indicated significant increases of neutrophils in the circulating blood in both PKC-epsilon transgenic lines. To determine whether there was an imbalance of cytokines in PKC-epsilon transgenic mice (line 215), resulting in aberrant myelopoiesis, we analyzed 17 cytokines in the peripheral blood. This analysis indicated that interleukin-5, interleukin-6, and granulocyte-colony stimulating factor were up-regulated as a function of age. The transgene PKC-epsilon was not detected in any of the affected organs (bone marrow, liver, spleen, lung) We suggest that overexpression of PKC-epsilon in the epidermis may lead to the induction of specific cytokines that may, in a paracrine mechanism, perturb normal hematopoiesis in bone marrow resulting in a granulocytic skew toward that of neutrophils and eosinophils. The susceptibility of PKC-epsilon transgenic mice to the induction of SCC and the spontaneous development of MPD are unrelated.

[Gaucher's disease: pathogenesis, diagnosis and therapy]. / Gaucher-kór: patogenezis, diagnózis, kezelés.

Gaucher's disease is the most common lysosomal storage disorder. Gene defect leads to deficiency or decreased activity of glucocerebrosidase followed by the accumulation of glucosylceramide. Most frequently hepatosplenomegaly, anemia, skeletal and hematological abnormalities are present. Different types are known based on the clinical findings. Recently used enzyme replacement therapy seems to eliminate bone marrow transplantation and has favourable effects on symptoms and outcome. Development of gene therapy (reintroduction of missing DNA sequence) hints the possibility of real causal therapy of the disease.

[Hereditary fructose intolerance (HFI) as cause of isolated gamma GT rise in a 5-year old boy with hepatomegaly]. / Hereditäre Fruktoseintoleranz (HFI) als Ursache einer isolierten gamma GT-Erhöhung bei einem 5-jährigen Jungen mit Hepatomegalie.

The diagnosis of HFI is easily missed during childhood. It should be suspected in children presenting with hepatomegaly and an isolated increase in GGT. A carefully taken nutritional history forms the basis of the diagnosis of HFI which can be confirmed by molecular analysis with a sensitivity of > 95%. I.v. fructose tolerance tests and liver biopsies often can be omitted.

Biochemical effects of vinyl chloride monomer on the liver of occupationally exposed workers.

We investigated the effects of vinyl chloride monomer exposure on the liver of 86 workers by measuring beta-glucuronidase, arylsulfatase A, adenosine deaminase, 5'-nucleotidase and routine liver function enzymes in the sera of the workers. In 21 of them, three or more of these parameters were raised, with a significant decrease in the level of blood glutathione and a significant increase in the enzyme activity level of glutathione S-transferase. Of these 21 workers, 14 had fatty liver infiltration, 8 of whom were also suffering from liver enlargement. Also, 4 workers had liver enlargement without fatty infiltration and 3 had enlarged spleens. The study highlights the need for vigilance in environmental monitoring and medical surveillance of workers exposed to this chemical.

A novel deficiency of mitochondrial ATPase of nuclear origin.

We report a new type of fatal mitochondrial disorder caused by selective deficiency of mitochondrial ATP synthase (ATPase). A hypotrophic newborn from a consanguineous marriage presented severe lactic acidosis, cardiomegaly and hepatomegaly and died from heart failure after 2 days. The activity of oligomycin-sensitive ATPase was only 31-34% of the control, both in muscle and heart, but the activities of cytochrome c oxidase, citrate synthase and pyruvate dehydrogenase were normal. Electrophoretic and western blot analysis revealed selective reduction of ATPase complex but normal levels of the respiratory chain complexes I, III and IV. The same selective deficiency of ATPase was found in cultured skin fibroblasts which showed similar decreases in ATPase content, ATPase hydrolytic activity and level of substrate-dependent ATP synthesis (20-25, 18 and 29-33% of the control, respectively). Pulse-chase labelling of patient fibroblasts revealed low incorporation of [(35)S]methionine into assembled ATPase complexes, but increased incorporation into immunoprecipitated ATPase subunit beta, which had a very short half-life. In contrast, no difference was found in the size and subunit composition of the assembled and newly produced ATPase complex. Transmitochondrial cybrids prepared from enucleated fibroblasts of the patient and rho degrees cells derived from 143B. TK(-)human osteosarcoma cells fully restored the ATPase activity, ATP synthesis and ATPase content, when compared with control cybrids. Likewise, the pattern of [(35)S]methionine labelling of ATPase was found to be normal in patient cybrids. We conclude that the generalized deficiency of mitochondrial ATPase described is of nuclear origin and is caused by altered biosynthesis of the enzyme.

Defects of the mitochondrial respiratory chain complexes in three pediatric cases with hypotonia and cardiac involvement.

Three children displaying hypotonia, cardiac involvement and defects of the mitochondrial respiratory chain complexes are reported. The first case showed severe neonatal hypotonia, failure to thrive, hepatomegaly, dilation of the right cardiac cavities, profound lactic acidosis and amino aciduria. The boy died at the age of 7 weeks. In the second case hypotonia, severe cardiomyopathy, cyclic neutropenia, lactic acidosis and 3-methylglutaconic aciduria occurred. The boy died at the age of 27 months. The third case presented at the age of 16 months as an acute hypokinetic hypertrophic cardiomyopathy with transient hypotonia and mild lactic acidosis. Spontaneous clinical remission occurred. In all cases muscle biopsy was performed. Morphological studies failed to show ragged-red fibers but there was lipid storage myopathy and decreased cytochrome c oxidase activity. Biochemical studies confirmed the cytochrome c oxidase deficiency in muscle in all cases. It was associated with complex I III deficiency in case 1 and with severe deficits of all respiratory chain complexes in case 2. Post-mortem studies in case 1 indicated that complex IV was reduced in the liver but not in the heart and quantitative analysis of mtDNA revealed a depletion in muscle. Cases 1 and 2 shared some clinical features with fatal infantile myopathy associated with cytochrome c oxidase deficiency, while case 3 displayed a very unusual clinical presentation. The histochemical enzyme reaction of cytochrome c oxidase is useful for the diagnosis of mitochondrial myopathy because ragged-red fibers may be lacking. Finally, biochemical measurement of the different mitochondrial respiratory chain complexes is required because multiple defects are frequent and occasionally related to mtDNA depletion.

Liver structure and function in print workers exposed to toluene.

An unresolved controversy is whether exposure to organic solvents in the workplace causes hepatotoxicity. From a medical surveillance study of 289 printing factory employees who were exposed primarily to toluene, we identified eight workers who had persistently abnormal serum transaminase and/or alkaline phosphatase values. The eight men were generally healthy and gave no history of taking medications or of drinking ethanol to excess. None was obese or diabetic. Six patients had hepatomegaly based on physical examination. All eight had mild elevations (less than 2 to 3 times the upper value of normal) of serum transaminases [alanine (ALT) and aspartate aminotransferase (AST)]. However, there was a marked increase in the ratio of ALT/AST (mean = 1.61). In each case, liver biopsy revealed mild, pericentral fatty change. Our results, consistent with those previously published by some others, suggest that pericentral fatty liver with mild "reactive hepatitis" is the most likely diagnosis in workers exposed to solvents for whom common causes of mild liver test abnormalities have been excluded. An increased ALT/AST ratio may represent a convenient, previously unrecognized indicator of this condition.

[Cholesteryl ester storage disease in the liver (author's transl)]. / Cholesterinester-Speicherkrankheit der Leber.

Cholesteryl ester storage disease is a rare disorder if cholesterol metabolism characterized by excessive hepatic storage of cholesteryl esters. The underlying defect probably is decreased activity of alpha-naphtyl-acetatesterase, a lysosomal acid lipase. The leading symptom in the early stage is a pronounced enlargement of the liver without subjective complaints or other changes in the physical status. Diagnosis can be established by liver biopsy, which will yield characteristic findings, and by exclusion of other storage disease. Histologic examination of the biopsy specimen will show lipid droplets in hepatic parenchymal cells, vacuolated Kupffer cells and focal accumulation of foamy cells. Biochemical analysis of the biopsy specimen will show an increased content of cholesteryl esters. Investigation of media of culture fibroblasts will show decreased activity of lysosomal alpha-naphtyl-acetat-esterase. In addition adrenal calcification has to be ruled out, in order to exclude wolman's disease. The prognosis of the disease will depend upon the degree of liver insufficiency, since lympho-histiocytic infiltration as a secondary phenomenon may lead to septal fibrosis and cirrhosis in children or adolescents in most cases. A case report is given of a 13-year old girl suffering from this disease.