Overlapping and Distinct Features of Cardiac Pathology in Inherited Human and Murine Ether Lipid Deficiency.

Inherited deficiency in ether lipids, a subgroup of glycerophospholipids with unique biochemical and biophysical properties, evokes severe symptoms in humans resulting in a multi-organ syndrome. Mouse models with defects in ether lipid biosynthesis have widely been used to understand the pathophysiology of human disease and to study the roles of ether lipids in various cell types and tissues. However, little is known about the function of these lipids in cardiac tissue. Previous studies included case reports of cardiac defects in ether-lipid-deficient patients, but a systematic analysis of the impact of ether lipid deficiency on the mammalian heart is still missing. Here, we utilize a mouse model of complete ether lipid deficiency (Gnpat KO) to accomplish this task. Similar to a subgroup of human patients with rhizomelic chondrodysplasia punctata (RCDP), a fraction of Gnpat KO fetuses present with defects in ventricular septation, presumably evoked by a developmental delay. We did not detect any signs of cardiomyopathy but identified increased left ventricular end-systolic and end-diastolic pressure in middle-aged ether-lipid-deficient mice. By comprehensive electrocardiographic characterization, we consistently found reduced ventricular conduction velocity, as indicated by a prolonged QRS complex, as well as increased QRS and QT dispersion in the Gnpat KO group. Furthermore, a shift of the Wenckebach point to longer cycle lengths indicated depressed atrioventricular nodal function. To complement our findings in mice, we analyzed medical records and performed electrocardiography in ether-lipid-deficient human patients, which, in contrast to the murine phenotype, indicated a trend towards shortened QT intervals. Taken together, our findings demonstrate that the cardiac phenotype upon ether lipid deficiency is highly heterogeneous, and although the manifestations in the mouse model only partially match the abnormalities in human patients, the results add to our understanding of the physiological role of ether lipids and emphasize their importance for proper cardiac development and function.

Structural Characterization and Quantitation of Ether-Linked Glycerophospholipids in Peroxisome Biogenesis Disorder Tissue by Ultraviolet Photodissociation Mass Spectrometry.

The biological impact of ether glycerophospholipids (GP) in peroxisomal disorders and other diseases makes them significant targets as biomarkers for diagnostic assays or deciphering pathology of the disorders. Ether lipids include both plasmanyl and plasmenyl lipids, which each contain an ether or a vinyl ether bond at the sn-1 linkage position, respectively. This linkage, in contrast to traditional diacyl GPs, precludes their detailed characterization by mass spectrometry via traditional collisional-based MS/MS techniques. Additionally, the isomeric nature of plasmanyl and plasmenyl pairs of ether lipids introduces a further level of complexity that impedes analysis of these species. Here, we utilize 213 nm ultraviolet photodissociation mass spectrometry (UVPD-MS) for detailed characterization of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) plasmenyl and plasmanyl lipids in mouse brain tissue. 213 nm UVPD-MS enables the successful differentiation of these four ether lipid subtypes for the first time. We couple this UVPD-MS methodology to reversed-phase liquid chromatography (RPLC) for characterization and relative quantitation of ether lipids from normal and diseased (Pex7 deficiency modeling the peroxisome biogenesis disorder, RCDP) mouse brain tissue, highlighting the ability to pinpoint specific structural features of ether lipids that are important for monitoring aberrant lipid metabolism in peroxisomal disorders.

A Pex7 Deficient Mouse Series Correlates Biochemical and Neurobehavioral Markers to Genotype Severity-Implications for the Disease Spectrum of Rhizomelic Chondrodysplasia Punctata Type 1.

Rhizomelic chondrodysplasia punctata type 1 (RCDP1) is a peroxisome biogenesis disorder caused by defects in PEX7 leading to impairment in plasmalogen (Pls) biosynthesis and phytanic acid (PA) oxidation. Pls deficiency is the main pathogenic factor that determines the severity of RCDP. Severe (classic) RCDP patients have negligible Pls levels, congenital cataracts, skeletal dysplasia, growth and neurodevelopmental deficits, and cerebral hypomyelination and cerebellar atrophy on brain MRI. Individuals with milder or nonclassic RCDP have higher Pls levels, better growth and cognitive outcomes. To better understand the pathophysiology of RCDP disorders, we generated an allelic series of Pex7 mice either homozygous for the hypomorphic allele, compound heterozygous for the hypomorphic and null alleles or homozygous for the null allele. Pex7 transcript and protein were almost undetectable in the hypomorphic model, and negligible in the compound heterozygous and null mice. Pex7 deficient mice showed a graded reduction in Pls and increases in C26:0-LPC and PA in plasma and brain according to genotype. Neuropathological evaluation showed significant loss of cerebellar Purkinje cells over time and a decrease in brain myelin basic protein (MBP) content in Pex7 deficient models, with more severe effects correlating with Pex7 genotype. All Pex7 deficient mice exhibited a hyperactive behavior in the open field environment. Brain neurotransmitters analysis of Pex7 deficient mice showed a significant reduction in levels of dopamine, norepinephrine, serotonin and GABA. Also, a significant correlation was found between brain neurotransmitter levels, the hyperactivity phenotype, Pls level and the severity of Pex7 genotype. In conclusion, our study showed evidence of a genotype-phenotype correlation between the severity of Pex7 deficiency and several clinical and neurobiochemical phenotypes in RCDP1 mouse models. We propose that PA accumulation may underlie the cerebellar atrophy seen in older RCDP1 patients, as even relatively low tissue levels were strongly associated with Purkinje cells loss over time in the murine models. Also, our data demonstrate the interrelation between Pls, brain neurotransmitter deficiencies and the neurobehavioral phenotype, which could be further used as a valuable clinical endpoint for therapeutic interventions. Finally, these models show that incremental increases in Pex7 levels result in dramatic improvements in phenotype.

Clinical, biochemical, and molecular characterization of mild (nonclassic) rhizomelic chondrodysplasia punctata.

Rhizomelic chondrodysplasia punctata (RCDP) is a heterogenous group of disorders due to defects in genes encoding peroxisomal proteins required for plasmalogen (PL) biosynthesis, specifically PEX7 and PEX5 receptors, or GNPAT, AGPS and FAR1 enzymes. Most patients have congenital cataract and skeletal dysplasia. In the classic form, there is profound growth restriction and psychomotor delays, with most patients not advancing past infantile developmental milestones. Disease severity correlates to erythrocyte PL levels, which are almost undetectable in severe (classic) RCDP. In milder (nonclassic) forms, residual PL levels are associated with improved growth and development. However, the clinical course of this milder group remains largely unknown as only a few cases were reported. Using as inclusion criteria the ability to communicate and walk, we identified 16 individuals from five countries, ages 5-37 years, and describe their clinical, biochemical and molecular profiles. The average age at diagnosis was 2.6 years and most had cataract, growth deficiency, joint contractures, and developmental delays. Other major symptoms were learning disability (87%), behavioral issues (56%), seizures (43%), and cardiac defects (31%). All patients had decreased C16:0 PL levels that were higher than in classic RCDP, and up to 43% of average controls. Plasma phytanic acid levels were elevated in most patients. There were several common, and four novel, PEX7, and GNPAT hypomorphic alleles in this cohort. These results can be used to support earlier diagnosis and improve management in patients with mild RCDP.

Oral administration of a synthetic vinyl-ether plasmalogen normalizes open field activity in a mouse model of rhizomelic chondrodysplasia punctata.

Rhizomelic chondrodysplasia punctata (RCDP) is a rare genetic disorder caused by mutations in peroxisomal genes essential for plasmalogen biosynthesis. Plasmalogens are a class of membrane glycerophospholipids containing a vinyl-ether-linked fatty alcohol at the sn-1 position that affect functions including vesicular transport, membrane protein function and free radical scavenging. A logical rationale for the treatment of RCDP is therefore the therapeutic augmentation of plasmalogens. The objective of this work was to provide a preliminary characterization of a novel vinyl-ether synthetic plasmalogen, PPI-1040, in support of its potential utility as an oral therapeutic option for RCDP. First, wild-type mice were treated with 13C6-labeled PPI-1040, which showed that the sn-1 vinyl-ether and the sn-3 phosphoethanolamine groups remained intact during digestion and absorption. Next, a 4-week treatment of adult plasmalogen-deficient Pex7hypo/null mice with PPI-1040 showed normalization of plasmalogen levels in plasma, and variable increases in plasmalogen levels in erythrocytes and peripheral tissues (liver, small intestine, skeletal muscle and heart). Augmentation was not observed in brain, lung and kidney. Functionally, PPI-1040 treatment normalized the hyperactive behavior observed in the Pex7hypo/null mice as determined by open field test, with a significant inverse correlation between activity and plasma plasmalogen levels. Parallel treatment with an equal amount of ether plasmalogen precursor, PPI-1011, did not effectively augment plasmalogen levels or reduce hyperactivity. Our findings show, for the first time, that a synthetic vinyl-ether plasmalogen is orally bioavailable and can improve plasmalogen levels in an RCDP mouse model. Further exploration of its clinical utility is warranted.This article has an associated First Person interview with the joint first authors of the paper.