Peroxisomal Disorders and Their Mouse Models Point to Essential Roles of Peroxisomes for Retinal Integrity.

Peroxisomes are multifunctional organelles, well known for their role in cellular lipid homeostasis. Their importance is highlighted by the life-threatening diseases caused by peroxisomal dysfunction. Importantly, most patients suffering from peroxisomal biogenesis disorders, even those with a milder disease course, present with a number of ocular symptoms, including retinopathy. Patients with a selective defect in either peroxisomal α- or ß-oxidation or ether lipid synthesis also suffer from vision problems. In this review, we thoroughly discuss the ophthalmological pathology in peroxisomal disorder patients and, where possible, the corresponding animal models, with a special emphasis on the retina. In addition, we attempt to link the observed retinal phenotype to the underlying biochemical alterations. It appears that the retinal pathology is highly variable and the lack of histopathological descriptions in patients hampers the translation of the findings in the mouse models. Furthermore, it becomes clear that there are still large gaps in the current knowledge on the contribution of the different metabolic disturbances to the retinopathy, but branched chain fatty acid accumulation and impaired retinal PUFA homeostasis are likely important factors.

Familial low phospholipid-associated cholelithiasis resulting from an autosomal dominant ABCB4 mutation

Low phospholipid-associated cholelithiasis (LPAC) syndrome is characterized by early intrahepatic and symptomatic gallstones leading to cholangitis, acute pancreatitis and biliary colic. It has been associated with loss of function variants in the ABCB4 gene. ABCB4 encodes for a phospholipid translocator at the canalicular membrane of the hepatocyte, which "flops" phosphatidylcholine into bile. The autosomal recessive form is the most common, although autosomal dominant forms have also been described. We report the first family with autosomal dominant LPAC syndrome due to heterozygosity of the loss of function mutation c.2932T>C in ABCB4, identified by targeted next generation sequencing

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Familial low phospholipid-associated cholelithiasis resulting from an autosomal dominant ABCB4 mutation.

Low phospholipid-associated cholelithiasis (LPAC) syndrome is characterized by early intrahepatic and symptomatic gallstones leading to cholangitis, acute pancreatitis and biliary colic. It has been associated with loss of function variants in the ABCB4 gene. ABCB4 encodes for a phospholipid translocator at the canalicular membrane of the hepatocyte, which "flops" phosphatidylcholine into bile. The autosomal recessive form is the most common, although autosomal dominant forms have also been described. We report the first family with autosomal dominant LPAC syndrome due to heterozygosity of the loss of function mutation c.2932T>C in ABCB4, identified by targeted next generation sequencing.

Ether Lipid Deficiency in Mice Produces a Complex Behavioral Phenotype Mimicking Aspects of Human Psychiatric Disorders.

Ether lipids form a specialized subgroup of phospholipids that requires peroxisomes to be synthesized. We have previously detected that deficiency in these lipids leads to a severe disturbance of neurotransmitter homeostasis and release as well as behavioral abnormalities, such as hyperactivity, in a mouse model. Here, we focused on a more detailed examination of the behavioral phenotype of ether lipid-deficient mice (Gnpat KO) and describe a set of features related to human psychiatric disorders. Gnpat KO mice show strongly impaired social interaction as well as nestlet shredding and marble burying, indicating disturbed execution of inborn behavioral patterns. Also, compromised contextual and cued fear conditioning in these animals suggests a considerable memory deficit, thus potentially forming a connection to the previously determined ether lipid deficit in human patients with Alzheimer's disease. Nesting behavior and the preference for social novelty proved normal in ether lipid-deficient mice. In addition, we detected task-specific alterations in paradigms assessing depression- and anxiety-related behavior. The reported behavioral changes may be used as easy readout for the success of novel treatment strategies against ether lipid deficiency in ameliorating nervous system-associated symptoms. Furthermore, our findings underline that ether lipids are paramount for brain function and demonstrate their relevance for cognitive, social, and emotional behavior. We hereby substantially extend previous observations suggesting a link between deficiency in ether lipids and human mental illnesses, particularly autism and attention-deficit hyperactivity disorder.

A novel pathogenic variant of ATP-binding cassette subfamily B member 4 causing gallstones in a young adult.

The low phospholipid-associated cholelithiasis (LPAC) syndrome was reported in European adults with cholelithiasis and a mutation of the ATP-binding cassette subfamily B member 4 (ABCB4). The ABCB4 encodes multidrug resistance 3, which is a phospholipid translocator. Reduced phospholipid transport can lead to the formation of biliary cholesterol stones. Here, we describe a 31-year-old Japanese man diagnosed with recurrent biliary colic. Although he recovered quickly after endoscopic treatment for the most recent presentation, he had a family history of similar problems. His mother had required endoscopic treatment for choledocholithiasis and his maternal aunt had died at age 29 years because of liver failure (etiology unknown). We, therefore, performed genetic analysis, which revealed a heterozygous ABCB4C717S. LPAC syndrome was diagnosed and the patient has received ursodeoxycholic acid for 2 years with no recurrence. The same variant was identified in the patient's mother, who was subsequently found to have a left intrahepatic calculus requiring left-sided lobectomy. She has received ursodeoxycholic acid for 1 year with no recurrence. ABCB4C717S is a novel pathogenic variant, and this is the first patient diagnosed with LPAC syndrome in Japan. We should consider LPAC syndrome in young adults with recurrent cholesterol gallstones to ensure early therapy.

Expanding the mutational spectrum of the ABCB4 gene in inherited adult cholestatic liver disorders with four novel pathogenic variants.

Low phospholipid-associated cholelithiasis and intrahepatic cholestasis of pregnancy are two MDR3-related inherited liver disorders caused by biallelic or monoallelic ABCB4 loss-of-function variants. Low phospholipid-associated cholelithiasis is clinically characterized by the early onset of symptomatic cholelithiasis in young adults while intrahepatic cholestasis of pregnancy is a distinct clinical entity associated with adverse fetal outcomes. Of note, patients carrying ABCB4 sequence variations commonly exhibit phenotypic expression over a wide continuum due to environmental and hormonal contributing factors and genetic modifiers. Patients with an early diagnosis of MDR3-related diseases could benefit from ursodeoxycholic acid treatment in order to prevent acute and chronic complications as well as adverse pregnancy outcomes. We herein report five patients with an overlapping phenotype from low phospholipid-associated cholelithiasis to intrahepatic cholestasis of pregnancy, harboring five ABCB4 missense variants, four of which were novel. Our study highlights the phenotypic and genetic heterogeneity of inherited cholestatic liver diseases and also expands the mutation spectrum of ABCB4 sequence variations in adult cholestatic liver diseases.

Reduced muscle strength in ether lipid-deficient mice is accompanied by altered development and function of the neuromuscular junction.

Inherited deficiency in ether lipids, a subgroup of phospholipids whose biosynthesis needs peroxisomes, causes the fatal human disorder rhizomelic chondrodysplasia punctata. The exact roles of ether lipids in the mammalian organism and, therefore, the molecular mechanisms underlying the disease are still largely enigmatic. Here, we used glyceronephosphate O-acyltransferase knockout (Gnpat KO) mice to study the consequences of complete inactivation of ether lipid biosynthesis and documented substantial deficits in motor performance and muscle strength of these mice. We hypothesized that, probably in addition to previously described cerebellar abnormalities and myelination defects in the peripheral nervous system, an impairment of neuromuscular transmission contributes to the compromised motor abilities. Structurally, a morphologic examination of the neuromuscular junction (NMJ) in diaphragm muscle at different developmental stages revealed aberrant axonal branching and a strongly increased area of nerve innervation in Gnpat KO mice. Post-synaptically, acetylcholine receptor (AChR) clusters colocalized with nerve terminals within a widened endplate zone. In addition, we detected atypical AChR clustering, as indicated by decreased size and number of clusters following stimulation with agrin, in vitro. The turnover of AChRs was unaffected in ether lipid-deficient mice. Electrophysiological evaluation of the adult diaphragm indicated that although evoked potentials were unaltered in Gnpat KO mice, ether lipid deficiency leads to fewer spontaneous synaptic vesicle fusion events but, conversely, an increased post-synaptic response to spontaneous vesicle exocytosis. We conclude from our findings that ether lipids are essential for proper development and function of the NMJ and may, therefore, contribute to motor performance. Read the Editorial Highlight for this article on page 463.

Aberrant lung remodeling in a mouse model of surfactant dysregulation induced by modulation of the Abca3 gene.

The lipid transporter, ATP binding cassette class A3 (ABCA3), plays a critical role in the biogenesis of alveolar type 2 (AT2) cell lamellar bodies (LBs). A relatively large number of mutations in the ABCA3 gene have been identified in association with diffuse parenchymal lung disease (DPLD), the most common of which is a missense mutation (valine substitution for lysine at residue 292 (ABCA3E292V)) that leads to functional impairment of the transporter in vitro. The consequences of ABCA3E292V gene expression in vivo are unknown. To address this question, we developed mouse models expressing ABCA3E292V knocked-in to the endogenous mouse locus. The parental (F1) mouse line (mAbca3E292V) that retained an intronic pgk-Neo selection cassette (inserted in reverse orientation) (mAbca3E292V-rNeo) demonstrated an allele dependent extracellular surfactant phospholipid (PL) deficiency. We hypothesize that this PL deficiency leads to aberrant parenchymal remodeling contributing to the pathophysiology of the DPLD phenotype. Compared to wild type littermates, baseline studies of mice homozygous for the pgk-Neo insert (mAbca3E292V-rNeo+/+) revealed nearly 50% reduction in bronchoalveolar lavage (BAL) PL content that was accompanied by quantitative reduction in AT2 LB size with a compensatory increase in LB number. The phenotypic alteration in surfactant lipid homeostasis resulted in an early macrophage predominant alveolitis which peaked at 8 weeks of age. This was followed by age-dependent development of histological DPLD characterized initially by peribronchial inflammatory cell infiltration and culminating in both an emphysema-like phenotype (which included stereologically quantifiable reductions in both alveolar septal surface area and volume of septal wall tissue) plus foci of trichrome-positive collagen deposition together with substantial proliferation of hyperplastic AT2 cells. In addition to spontaneous lung remodeling, mABCA3E292V-rNeo mice were rendered more vulnerable to exogenous injury. Three weeks following intratracheal bleomycin challenge, mAbca3-rNeo mice demonstrated allele-dependent susceptibility to bleomycin including enhanced weight loss, augmented airspace destruction, and increased fibrosis. Removal of the rNeo cassette from mAbca3 alleles resulted in restoration of BAL PL content to wild-type levels and an absence of changes in lung histology up to 32 weeks of age. These results support the importance of surfactant PL homeostasis as a susceptibility factor for both intrinsic and exogenously induced lung injury/remodeling.

Pleiotropic effect of anionic phospholipids absence on mitochondrial morphology and cell wall integrity in strictly aerobic Kluyveromyces lactis yeasts.

Cardiolipin and phosphatidylglycerol are anionic phospholipids localized to the inner mitochondrial membrane. In this study, it is demonstrated by fluorescence and transmission electron microscopy that atp2.1pgs1Δ mutant mitochondria lacking anionic phospholipids contain fragmented and swollen mitochondria with a completely disorganized inner membrane. In the second part of this study, it was shown that the temperature sensitivity of the atp2.1pgs1Δ mutant was not suppressed by the osmotic stabilizer glucitol but by glucosamine, a precursor of chitin synthesis. The atp2.1pgs1Δ mutant was hypersensitive to Calcofluor White and caffeine, resistant to Zymolyase, but its sensitivity to caspofungin was the same as the strains with the standard PGS1 gene. The distribution of chitin in the mutant cell wall was impaired. The glucan level in the cell wall of the atp2.1pgs1Δ mutant was reduced by 4-8 %, but the level of chitin was almost double that in the wild-type strain. The cell wall of the atp2.1pgs1Δ mutant was about 20 % thinner than the wild type, but its morphology was not significantly altered.

Comprehensive biometric, biochemical and histopathological assessment of nutrient deficiencies in gilthead sea bream fed semi-purified diets.

Seven isoproteic and isolipidic semi-purified diets were formulated to assess specific nutrient deficiencies in sulphur amino acids (SAA), n-3 long-chain PUFA (n-3 LC-PUFA), phospholipids (PL), P, minerals (Min) and vitamins (Vit). The control diet (CTRL) contained these essential nutrients in adequate amounts. Each diet was allocated to triplicate groups of juvenile gilthead sea bream fed to satiety over an 11-week feeding trial period. Weight gain of n-3 LC-PUFA, P-Vit and PL-Min-SAA groups was 50, 60-75 and 80-85 % of the CTRL group, respectively. Fat retention was decreased by all nutrient deficiencies except by the Min diet. Strong effects on N retention were found in n-3 LC-PUFA and P fish. Combined anaemia and increased blood respiratory burst were observed in n-3 LC-PUFA fish. Hypoproteinaemia was found in SAA, n-3 LC-PUFA, PL and Vit fish. Derangements of lipid metabolism were also a common disorder, but the lipodystrophic phenotype of P fish was different from that of other groups. Changes in plasma levels of electrolytes (Ca, phosphate), metabolites (creatinine, choline) and enzyme activities (alkaline phosphatase) were related to specific nutrient deficiencies in PL, P, Min or Vit fish, whereas changes in circulating levels of growth hormone and insulin-like growth factor I primarily reflected the intensity of the nutritional stressor. Histopathological scoring of the liver and intestine segments showed specific nutrient-mediated changes in lipid cell vacuolisation, inflammation of intestinal submucosa, as well as the distribution and number of intestinal goblet and rodlet cells. These results contribute to define the normal range of variation for selected biometric, biochemical, haematological and histochemical markers.

LPAC syndrome associated with deletion of the full exon 4 in a ABCB4 genetic mutation in a patient with hepatitis C.

Low-phospholipid-associated cholelithiasis syndrome (LPAC) is associated with ABCB4 genetic mutation. ABCB4 encodes MDR3 protein, involved in biliary phosphatidylcholine excretion.Higher prevalence in women, biliary symptoms in young adults and ursodesoxycholic acid (UDCA) response are the main features. We report the case of a 48-year-old man with hepatitis C, genotype 1b, fibrosis F3, null responder to Peg-IFN-alpha-2b/ribavirin and nephritic colic. In 2011 he developed jaundice, pruritus and epigastric pain.He showed increased serum levels of AST, ALT, GGT, bilirubin and alpha-fetoprotein, and viral load (14,600,000 IU/mL). Pancreatic- CT, endoscopic ultrasonography and echo-Doppler showed noncirrhotic chronic liver disease. The episode resolved spontaneously and one year later he suffered a similar episode. UDCA was started with excellent response. An immunohistochemistry study and sequencing of ABCB4 did not find alteration. MLPA® technique detected heterozygous deletion of the full exon 4 confirming LPAC syndrome diagnosis.

Disorders of phospholipids, sphingolipids and fatty acids biosynthesis: toward a new category of inherited metabolic diseases.

We wish to delineate a novel, and rapidly expanding, group of inborn errors of metabolism with neurological/muscular presentations: the defects in phospholipids, sphingolipids and long chain fatty acids biosynthesis. At least 14 disorders have been described so far. Clinical presentations are diverse but can be divided into (1) diseases of the central nervous system; (2) peripheral neuropathies; and (3) muscular/cardiac presentations. (1) Leukodystrophy and/or iron deposits in basal ganglia is a common feature of phospholipase A2 deficiency, fatty acid hydroxylase deficiency, and pantothenate kinase-associated neurodegeneration. Infantile epilepsy has been reported in GM3 synthetase deficiency. Spastic quadriplegia with ichthyosis and intellectual disability are the presenting signs of the elongase 4 deficiency and the Sjogren-Larsson syndrome caused by fatty aldehyde dehydrogenase deficiency. Spastic paraplegia and muscle wasting are also seen in patients with mutations in the neuropathy target esterase gene. (2) Peripheral neuropathy is a prominent feature in PHARC syndrome due to α/ß-hydrolase 12 deficiency, and in hereditary sensory autonomic neuropathy type I due to serine palmitoyl-CoA transferase deficiency. (3) Muscular/cardiac presentations include recurrent myoglobinuria in phosphatidate phosphatase 1 (Lipin1) deficiency; cardiomyopathy and multivisceral involvement in Barth syndrome secondary to tafazzin mutations; congenital muscular dystrophy due to choline kinase deficiency, Sengers syndrome due to acylglycerol kinase deficiency and Chanarin Dorfman syndrome due to α/ß- hydrolase 5 deficiency. These synthesis defects of complex lipid molecules stand at the frontier between classical inborn errors of metabolism and other genetic diseases involving the metabolism of structural proteins.

Low phospholipid-associated cholestasis and cholelithiasis.

Low phospholipid-associated cholestasis and cholelithiasis (LPAC) is a genetic disorder characterized by cholesterol gallbladder and intrahepatic stones. It is caused by a mutation of the gene ABCB4, which encodes the canalicular protein ABCB4/MDR3, a flippase that plays an essential role in the secretion of phosphatidylcholine into bile. Failure of this protein leads to secretion of bile that is poor in phospholipids and, hence, highly lithogenic, with potent detergent properties. This, in turn, leads to cholangiocyte luminal membrane injury and biliary lesions causing cholestasis. The diagnosis should be suspected when at least two of the following criteria are present: onset of symptoms before the age of 40 years; recurrence of biliary symptoms (biliary colic, jaundice, cholangitis, acute pancreatitis) after cholecystectomy; presence of echogenic foci within the liver indicative of intrahepatic stones or biliary sludge; previous episode(s) of intrahepatic cholestasis of pregnancy; and family history of gallstones in first-degree relatives. Intrahepatic stones can be demonstrated by ultrasonography with color Doppler examination, computed tomography and magnetic resonance imaging with magnetic resonance cholangiography, and the diagnosis confirmed by ABCB4 genotyping. Therapy with ursodeoxycholic acid offers prompt relief of symptoms and usually prevents complications. In some cases, however, surgery may be necessary.

[44-year-old woman with elevated liver enzymes and a family history for cholelithiasis]. / 44-jährige Patientin mit unklarer Leberwerterhöhung und familiär gehäuftem Gallensteinleiden.

"Low phospholipid associated cholelithiasis" (LPAC) syndrome is an important differential diagnosis in younger patients with biliary symptoms after cholecystectomy and concomitant elevated serum liver tests. Typical symptoms include recurrence of biliary colics after cholecystectomy, echogenic material in the intrahepatic bile ducts, intrahepatic cholestasis of pregnancy or cholestasis under hormonal contraception and a family history of gallstone disease. Patients with LPAC syndrome can be successfully treated with ursodeoxycholic acid.

[Liver disease associated with hereditary defects of hepatobiliary transporters]. / Maladies hépatiques liées à des anomalies héréditaires de transporteurs hépatobiliaires.

The identification of biliary tranporters has enhanced our understanding of bile formation and some liver diseases. In this review, we first describe the main hepatobiliary transporters and their function. Then, some liver diseases related to mutations of biliary tranporters (FIC1/ATP8B1, BSEP/ABCB11, MDR3 /ABCB4 and MRP2/ABCC2) will be described with a focus on the pathological aspects. These diseases include progressive familial intrahepatic cholestasis (PFIC), benign recurrent intrahepatic cholestasis (BRIC), intrahepatic cholestasis of pregnancy, Dubin-Johnson's syndrome and low phospholipid associated cholelithiasis (LPAC).

Combined features of low phospholipid-associated cholelithiasis and progressive familial intrahepatic cholestasis 3.

Adenosine triphosphate-binding cassette, subfamily B, member 4 (ABCB4) gene alterations can cause two distinct clinical entities: progressive familial intrahepatic cholestasis type 3 (PFIC3) and low phospholipid-associated cholelithiasis (LPAC). Based on the findings in two siblings and a review of the literature, we aimed to identify determinants of disease phenotypic traits associated with ABCB4 gene alterations. Two siblings presented, before the age of 30 years, recurrent symptomatic cholelithiasis and extensive biliary fibrosis that progressed towards portal hypertension and liver failure necessitating liver transplantation. We analysed the sequence of the ABCB4 gene and immunolocalization of the protein in the liver. Sequence analysis of ABCB11, potentially involved in similar symptoms, was also performed. Two heterozygous non-synonymous variants of ABCB4 were found in both siblings. One of them (c.959C>T; p.Ser320Phe) was previously implicated in LPAC and the second one (c.2858C>A; p.Ala953Asp) in PFIC3. Both patients were also heterozygous for the ABCB11 variant Val444Ala, which predisposes to cholestatic disorders. ABCB4 was normally detected at the canalicular membrane of hepatocytes. The review of ABCB4 gene variants reported so far shows that the vast majority of variants causing PFIC3 and LPAC are distinct. Also as a general rule, homozygous variants cause PFIC3 while heterozygous variants lead to LPAC. Combined PFIC3 and LPAC phenotype is a rare clinical event, which may be determined by the coexistence of ABCB4 variants related to both phenotypes and also potentially to the ABCB11 variant. Thus, most of the patients presenting with LPAC are not at a particular risk of developing PFIC3 features in adulthood.

[Right upper quadrant abdominal pain and fever. Genetic phospholipid deficiency]. / Douleurs de l'hypochondre droit et fièvre.

A 35-year-old woman is referred for right upper quadrant abdominal pain and fever. She had a cholecystectomy for gallstones 4 years previously. An aunt has also had a cholecystectomy. Abdominal ultrasound examination shows numerous hyperechoic foci within the liver and some "comet tail" artifacts. Because of her age (less than 40), the recurrence of biliary symptoms after cholecystectomy, her family history and the intrahepatic hyperechoic foci, the diagnosis of low phospholipid-associated cholelithiasis (LPAC) syndrome is suspected and confirmed by the demonstration of a point mutation of the ABCB4 gene. Therapy by ursodeoxycholic acid (AUDC) is started. Symptoms improve and they disappear completely within a few weeks. The cause, pathophysiology, diagnosis and treatment are reviewed.