Quantitative analysis of ethanolamine plasmalogen species in red blood cells using liquid chromatography tandem mass spectrometry for diagnosing peroxisome biogenesis disorders.

Plasmalogens are glycerophospholipids characterized by a vinyl-ether bond with a fatty alcohol at the sn-1 position, a polyunsaturated fatty acid at the sn-2 position, and a polar head at the sn-3 position, commonly phosphoethanolamine. Plasmalogens play crucial roles in several cellular processes. Reduced levels have been associated with Alzheimer's and Parkinson's disease progression. Markedly reduced plasmalogens are a classic feature of peroxisome biogenesis disorders (PBD) because plasmalogen synthesis requires functional peroxisomes. Particularly, severe plasmalogen deficiency is the biochemical hallmark of rhizomelic chondrodysplasia punctata (RCDP). Traditionally, plasmalogens are evaluated in red blood cells (RBCs) by gas-chromatography/mass-spectrometry (GC-MS), which cannot distinguish individual species. We developed a liquid-chromatography/tandem mass-spectrometry (LC-MS/MS) method to quantify eighteen phosphoethanolamine plasmalogens in RBCs to diagnose PBD patients, especially RCDP. Validation results showed a specific, robust, and precise method with broad analytical range. Age-specific reference intervals were established; control medians were used to assess plasmalogen deficiency in patients' RBCs. Clinical utility was also confirmed in Pex7 deficient mouse models recapitulating severe and milder RCDP clinical phenotypes. To our knowledge, this is the first attempt to replace the GC-MS method in the clinical laboratory. In addition to diagnosing PBDs, structure-specific plasmalogen quantitation could help understand disease pathogenesis and monitor therapy.

Expanding the genotypic and phenotypic landscapes of rhizomelic chondrodysplasia punctata type 3 (RCDP3) with two novel families, and a review of the literature.

Rhizomelic chondrodysplasia punctata (RCDP) are a group of peroxisomal disorders caused by plasmalogen synthesis defects. Patients with RCDP present with rhizomelic short stature, characteristic punctate epiphyseal calcifications, congenital cataracts, severe intellectual disability, seizures, and facial dysmorphism. Pathogenic variants in AGPS result in RCDP type 3 (RCDP3) which is an extremely rare disorder characterized by isolated ADHAPS deficiency. Six patients with RCDP3 have been identified, upto-date. We report two new patients with RCDP3 and their novel variants, c.154dupG (p.Ala52GlyfsTer6) and c.637+1G>A, in the AGPS gene. We also present a review of previously reported RCDP3 patients.

A new GNPAT variant of foetal rhizomelic chondrodysplasia punctata.

BACKGROUND: Rhizomelic chondrodysplasia punctata (RCDP) is a clinical entity resulting from defects of peroxisomal metabolism whose clinical phenotype is characterized by rhizomelia, calcified foci in periarticular cartilage, coronal lesions of vertebral bodies, cataracts and severe cognitive delay. Usually, survival does not exceed the first decade of life. Transmission is autosomal recessive and is related to mutations in the PEX7, GNPAT or AGPS. METHODS: A detailed description of the prenatal ultrasound signs of RCDP found in two successive pregnancies in a consanguineous couple is reported. Molecular genetic investigations included the study of the coding regions and the exon-intron junctions of the GNPAT (high-throughput amplification and sequencing performed with Roche NimbleGen SeqCap Target kit on Illumina platform); the confirmation test was carried out by amplification and Sanger sequencing with automatic capillary sequencer. RESULTS: In addition to the typical prenatal ultrasound signs described in the literature in association with RCDP, the presence of prefrontal oedema, never previously described, has been detected in both pregnancies. Moreover, genetic investigations have found a new splicing variant c.924+1G>A of the homozygous GNPAT. CONCLUSION: The role of mutation in the GNPAT suggests a likely association with the clinical phenotype.

The type-2 peroxisomal targeting signal.

The type-2 peroxisomal targeting signal (PTS2) is one of two peptide motifs destining soluble proteins for peroxisomes. This signal acts as amphiphilic α-helix exposing the side chains of all conserved residues to the same side. PTS2 motifs are recognized by a bipartite protein complex consisting of the receptor PEX7 and a co-receptor. Cargo-loaded receptor complexes are translocated across the peroxisomal membrane by a transient pore and inside peroxisomes, cargo proteins are released and processed in many, but not all species. The components of the bipartite receptor are re-exported into the cytosol by a ubiquitin-mediated and ATP-driven export mechanism. Structurally, PTS2 motifs resemble other N-terminal targeting signals, whereas the functional relation to the second peroxisomal targeting signal (PTS1) is unclear. Although only a few PTS2-carrying proteins are known in humans, subjects lacking a functional import mechanism for these proteins suffer from the severe inherited disease rhizomelic chondrodysplasia punctata.

Cervical Spine Deformities in Children With Rhizomelic Chondrodysplasia Punctata.

BACKGROUND: Cervical spine deformity in rhizomelic chondrodysplasia punctata (RCDP) has been described with different findings reported in the literature. However, available literature provides limited data from a few cases with magnetic resonance imaging (MRI) of the cervical spine. Our report describes the MRI findings in a group of children with RCDP, aiming to reach a better understanding of this pathology. METHODS: An Institutional Review Board-approved RCDP Registry was created at our institution with the goal of identifying pertinent medical issues over the lifespan of individuals with RCDP. Records of children within the registry were evaluated, and magnetic resonance images obtained between 2004 and 2015, were available for review. The levels of spinal canal stenosis were recorded and the severity of the stenosis was decided based on adults' parameters. Cord compression and myelomalacia were confirmed on the axial images. Sagittal lumbar spine magnetic resonance images were also evaluated when available, and the presence of tethered cord and fatty filum was recorded. RESULTS: Twenty-six children (15 boys and 11 girls) were identified in the RCDP Registry. Eleven children (6 boys and 5 girls) had sagittal MRI of the cervical spine available for review. Age at the time of MRI study was variable (1 wk to 32 mo). All patients except 1 had stenosis of the cervical spinal canal. Myelomalacia of the cord was noted only in this patient. CONCLUSIONS: This study suggests that, in children with RCDP, cervical spinal stenosis and cord compression are a real risk, and children with this diagnosis should have monitoring for these issues. Tethered cord is also a possible finding that needs to be evaluated. Full sagittal spine MRI is necessary to detect the possible deformities at the cervical and lumbar levels.

Rhizomelic Chondrodysplasia Punctata Type 1 Caused by a Novel Mutation in the PEX7 Gene.

Peroxisomes are involved in various metabolic reactions. Rhizomelic chondrodysplasia punctata (RCDP) type 1 is one of the peroxisomal biogenesis disorders caused by mutations in the PEX7 gene and is inherited in an autosomal recessive manner. We present a nine-year-old boy with skeletal abnormalities and dysmorphic facial appearance. The patient was born to parents who were first cousins. Very-long-chain fatty acids and pristanic acid levels were in the normal range, but an elevated phytanic acid level was detected by gas chromatography/mass spectrometry. The PEX7 gene was sequenced in the patient and his parents. A novel homozygous mutation, c.192delT (p.F64Lfs*10), was identified in the patient and was present in heterozygosity in both parents. In conclusion, the clinical presentation and peroxisome profile of the patient suggest that this novel mutation leads to RCDP type 1.

CUL4A-DDB1-Rbx1 E3 ligase controls the quality of the PTS2 receptor Pex7p.

Pex7p is the cytosolic receptor for peroxisomal matrix proteins harbouring PTS2 (peroxisome-targeting signal type-2). Mutations in the PEX7 gene cause RCDP (rhizomelic chondrodysplasia punctata) type 1, a distinct PTS2-import-defective phenotype of peroxisome biogenesis disorders. The mechanisms by which the protein level and quality of Pex7p are controlled remain largely unknown. In the present study we show that dysfunctional Pex7p, including mutants from RCDP patients, is degraded by a ubiquitin-dependent proteasomal pathway involving the CRL4A (Cullin4A-RING ubiquitin ligase) complex. Furthermore, we demonstrate that the degradation of dysfunctional Pex7p is essential for maintaining normal PTS2 import, thereby suggesting that CRL4A functions as an E3 ligase in the quality control of Pex7p. Our results define a mechanism underlying Pex7p homoeostasis and highlight its importance for regulating PTS2 import. These findings may lead to a new approach to Pex7p-based therapies for the treatment of peroxisome biogenesis disorders such as RCDP.

Peripheral nervous system plasmalogens regulate Schwann cell differentiation and myelination.

Rhizomelic chondrodysplasia punctata (RCDP) is a developmental disorder characterized by hypotonia, cataracts, abnormal ossification, impaired motor development, and intellectual disability. The underlying etiology of RCDP is a deficiency in the biosynthesis of ether phospholipids, of which plasmalogens are the most abundant form in nervous tissue and myelin; however, the role of plasmalogens in the peripheral nervous system is poorly defined. Here, we used mouse models of RCDP and analyzed the consequence of plasmalogen deficiency in peripheral nerves. We determined that plasmalogens are crucial for Schwann cell development and differentiation and that plasmalogen defects impaired radial sorting, myelination, and myelin structure. Plasmalogen insufficiency resulted in defective protein kinase B (AKT) phosphorylation and subsequent signaling, causing overt activation of glycogen synthase kinase 3ß (GSK3ß) in nerves of mutant mice. Treatment with GSK3ß inhibitors, lithium, or 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) restored Schwann cell defects, effectively bypassing plasmalogen deficiency. Our results demonstrate the requirement of plasmalogens for the correct and timely differentiation of Schwann cells and for the process of myelination. In addition, these studies identify a mechanism by which the lack of a membrane phospholipid causes neuropathology, implicating plasmalogens as regulators of membrane and cell signaling.

Rhizomelic chondrodysplasia punctata and cardiac pathology.

BACKGROUND: Rhizomelic chondrodysplasia punctata (RCDP) is an autosomal recessive peroxisomal disorder characterised by rhizomelia, contractures, congenital cataracts, facial dysmorphia, severe psychomotor defects and growth retardation. Biochemically, the levels of plasmalogens (major constituents of cellular membranes) are low due to a genetic defect in their biosynthesis. Cardiac muscle contains high concentrations of plasmalogens. Recently cardiac dysfunction was found in a mouse model for RCDP with undetectable plasmalogen levels in all tissues including the heart. This suggests the importance of plasmalogens in normal cardiac development and function. Congenital heart disease (CHD), however, has not been recognised as a major characteristic of RCDP. AIMS: We aimed to determine the prevalence of CHD found in RCDP patients as well as to describe genetic, biochemical and cardiac correlations. METHODS: We included 23 patients with genetically proven RCDP. The genetic, biochemical and physical data were evaluated. Echocardiograms were reviewed. RESULTS: Cardiac data were available for 18 patients. 12 (52%) had CHD. All twelve had type 1 RCDP and 11 (92%) had the PEX 7:c.875T>A mutation, of whom seven were homozygous (58%). Plasmalogen levels were significantly lower in the patients with CHD. Cardiac lesions included: septal defects (80% atrial), patent ductus arteriosus, pulmonary artery hypoplasia, tetralogy of Fallot and mitral valve prolapse (mostly older patients). CONCLUSIONS: The CHD prevalence among RCDP patients was at least 52%, significantly higher than among the normal population. Plasmalogen levels were significantly lower in patients with CHD. Routine cardiac evaluation should be included in the clinical management of RCDP patients.

The importance of ether-phospholipids: a view from the perspective of mouse models.

Ether-phospholipids represent an important group of phospholipids characterized by an alkyl or an alkenyl bond at the sn-1 position of the glycerol backbone. Plasmalogens are the most abundant form of alkenyl-glycerophospholipids, and their synthesis requires functional peroxisomes. Defects in the biosynthesis of plasmalogens are the biochemical hallmark of the human peroxisomal disorder Rhizomelic Chondrodysplasia Punctata (RCDP), which is characterized by defects in eye, bone and nervous tissue. The generation and characterization of mouse models with defects in plasmalogen levels have significantly advanced our understanding of the role and importance of plasmalogens as well as pathogenetic mechanisms underlying RCDP. A review of the current mouse models and the description of the combined knowledge gathered from the histopathological and biochemical studies is presented and discussed. Further characterization of the role and functions of plasmalogens will contribute to the elucidation of disease pathogenesis in peroxisomal and non-peroxisomal disorders. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of Peroxisomes in Health and Disease.

Defective lipid remodeling of GPI anchors in peroxisomal disorders, Zellweger syndrome, and rhizomelic chondrodysplasia punctata.

Many cell surface proteins in mammalian cells are anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). The predominant form of mammalian GPI contains 1-alkyl-2-acyl phosphatidylinositol (PI), which is generated by lipid remodeling from diacyl PI. The conversion of diacyl PI to 1-alkyl-2-acyl PI occurs in the ER at the third intermediate in the GPI biosynthetic pathway. This lipid remodeling requires the alkyl-phospholipid biosynthetic pathway in peroxisome. Indeed, cells defective in dihydroxyacetone phosphate acyltransferase (DHAP-AT) or alkyl-DHAP synthase express only the diacyl form of GPI-anchored proteins. A defect in the alkyl-phospholipid biosynthetic pathway causes a peroxisomal disorder, rhizomelic chondrodysplasia punctata (RCDP), and defective biogenesis of peroxisomes causes Zellweger syndrome, both of which are lethal genetic diseases with multiple clinical phenotypes such as psychomotor defects, mental retardation, and skeletal abnormalities. Here, we report that GPI lipid remodeling is defective in cells from patients with Zellweger syndrome having mutations in the peroxisomal biogenesis factors PEX5, PEX16, and PEX19 and in cells from patients with RCDP types 1, 2, and 3 caused by mutations in PEX7, DHAP-AT, and alkyl-DHAP synthase, respectively. Absence of the 1-alkyl-2-acyl form of GPI-anchored proteins might account for some of the complex phenotypes of these two major peroxisomal disorders.

Rhizomelic chondrodysplasia punctata type 1: report of mutations in 3 children from India.

Rhizomelic chondrodysplasia punctata is a rare autosomal recessive disorder characterized by stippled epiphyses and rhizomelic shortening of the long bones. We report 3 subjects of rhizomelic chondrodysplasia punctata from India and the PEX7 mutations identified in them. The common PEX7-L292X allele, whose high frequency is due to a founder effect in the northern European Caucasian population, was not identified in these patients. Instead, 2 novel alleles are described, including 64_65delGC, which was present on a single PEX7 haplotype and could represent a common allele in the Indian population.

Cervical spine stenosis in chondrodysplasia punctata.

Chondrodysplasia punctata (CDP) is a rare skeletal dysplasia characterized by stippled epiphyses during infancy. The frequency is probably underdiagnosed because of the large heterogeneity in this group. Many genotypic variations exist. Although cervical instability is commonly seen in many skeletal dysplasias, cervical spine stenosis associated with CDP is very rare. We report a boy with phenotypic features of brachytelephalangic chondrodysplasia punctata (BCDP) who had severe cervical spine stenosis successfully corrected by vertebrectomies of C6 and C7 with a fibular strut graft. We discuss the significance of this association.

Neuroimaging features in a neonate with rhizomelic chondrodysplasia punctata.

Rhizomelic chondrodysplasia punctata is a rare genetic disorder of peroxisomal metabolism that is characterized clinically by shortening of the proximal limbs, cataracts, a characteristic facial appearance, failure to thrive, and psychomotor retardation. This report describes a newborn with a severe phenotype whose neuroimaging showed pachygyria-polymicrogyria, severe spinal stenosis causing compression of the cervical cord and brainstem, and tethering of the spinal cord. Imaging of the brain and spinal cord in patients with this disorder may aid prognosis and guide management decisions.

The crucial step in ether phospholipid biosynthesis: structural basis of a noncanonical reaction associated with a peroxisomal disorder.

Ether phospholipids are essential constituents of eukaryotic cell membranes. Rhizomelic chondrodysplasia punctata type 3 is a severe peroxisomal disorder caused by inborn deficiency of alkyldihydroxyacetonephosphate synthase (ADPS). The enzyme carries out the most characteristic step in ether phospholipid biosynthesis: formation of the ether bond. The crystal structure of ADPS from Dictyostelium discoideum shows a fatty-alcohol molecule bound in a narrow hydrophobic tunnel, specific for aliphatic chains of 16 carbons. Access to the tunnel is controlled by a flexible loop and a gating helix at the protein-membrane interface. Structural and mutagenesis investigations identify a cluster of hydrophilic catalytic residues, including an essential tyrosine, possibly involved in substrate proton abstraction, and the arginine that is mutated in ADPS-deficient patients. We propose that ether bond formation might be orchestrated through a covalent imine intermediate with the flavin, accounting for the noncanonical employment of a flavin cofactor in a nonredox reaction.

MRI of the brain and cervical spinal cord in rhizomelic chondrodysplasia punctata.

BACKGROUND: The classic rhizomelic chondrodysplasia punctata (RCDP) phenotype involves a typical facial appearance, cataracts, skeletal dysplasia causing disproportionate somatic growth failure, microcephaly, and severe psychomotor defects. Biochemical abnormalities include impaired plasmalogen biosynthesis in all forms of RCDP and accumulation of phytanic acid in RCDP type 1. A subset of patients has a milder clinical and biochemical phenotype, with less severe neurologic impairment and an incomplete deficiency in plasmalogens. The impact of plasmalogen deficiency on neurologic function is severe, causing spasticity and mental defects, but its pathomechanism is still unknown. The authors specifically focused on myelination because myelin is rich in ethanolamine plasmalogens. OBJECTIVE: To define the neuroimaging characteristics of the genetic peroxisomal disorder RCDP. METHODS: Twenty-one MR images of the brain and cervical spine of 11 patients were evaluated and correlated with neurologic and biochemical profiles. RESULTS: No abnormalities on MRI were seen in the patients with a mild phenotype of RCDP, whereas delayed myelination, ventricular enlargement and increased subarachnoidal spaces, supratentorial myelin abnormalities, and cerebellar atrophy were observed in patients with the severe phenotype of both RCDP type 1 and 3. The severity of both the MRI abnormalities and the clinical phenotype is correlated with the plasmalogen level. CONCLUSIONS: The severe phenotype of rhizomelic chondrodysplasia punctata (RCDP) is accompanied by a specific pattern of both developmental and regressive MRI abnormalities. Plasmalogen levels seem to play an important role in the pathophysiology of CNS abnormalities in RCDP. Increased phytanic acid appears not to be the cause of cerebellar atrophy.

Multivoxel magnetic resonance spectroscopy in a rhizomelic chondrodysplasia punctata case.

A case of a 5-day-old newborn with rhizomelic chondrodysplasia punctata was investigated with multivoxel magnetic resonance spectroscopy, including chemical shift imaging maps, which disclosed a decrease in the choline peak and the choline signal intensity, respectively, in the right cerebral hemisphere. This is the second report of multivoxel magnetic resonance spectroscopy examination of the brain associated with rhizomelic chondrodysplasia punctata in the literature. Multivoxel magnetic resonance spectroscopy with chemical shift imaging maps has the advantage of obtaining more information in a short period of time, which shortens the duration of anesthesia and its associated risks and complications. We suggest that future efforts be directed to evaluating such patients with multivoxel magnetic resonance spectroscopy instead of single-voxel magnetic resonance spectroscopy.

[Case report: Rhizomelic chondrodysplasia punctata and foramen magnum stenosis in a newborn]. / Yenidoganda rizomelik kondrodisplazi punktata ve foramen magnum stenozu.

Chondrodysplasia punctata is a peroxisomal disorder which is a form of multiple epiphyseal dysplasia. It is characterized by calcifications of unossified cartilaginous epiphyseal centers during the first year of life. Severe autosomal recessive rhisomelic form shows bilateral proximal shortening of the upper and lower limbs with punctate epiphyseal calcifications. We report radiological findings of a patient with rhisomelic chondrodysplasia punctata. Magnetic resonance imaging showed foramen magnum stenosis that caused spinal cord compression.

Impaired neuronal migration and endochondral ossification in Pex7 knockout mice: a model for rhizomelic chondrodysplasia punctata.

Rhizomelic chondrodysplasia punctata is a human autosomal recessive disorder characterized by skeletal, eye and brain abnormalities. The disorder is caused by mutations in the PEX7 gene, which encodes the receptor for a class of peroxisomal matrix enzymes. We describe the generation and characterization of a Pex7 mouse knockout (Pex7(-/-)). Pex7(-/-) mice are born severely hypotonic and have a growth impairment. Mortality in Pex7(-/-) mice is highest in the perinatal period although some Pex7(-/-) mice survived beyond 18 months. Biochemically Pex7(-/-) mice display the abnormalities related to a Pex7 deficiency, i.e. a severe depletion of plasmalogens, impaired alpha-oxidation of phytanic acid and impaired beta-oxidation of very-long-chain fatty acids. In the intermediate zone of the developing cerebral cortex Pex7(-/-) mice have an increase in neuronal density. In vivo neuronal birthdating revealed that Pex7(-/-) mice have a delay in neuronal migration. Analysis of bone ossification in newborn Pex7(-/-) mice revealed a defect in ossification of distal bone elements of the limbs as well as parts of the skull and vertebrae. These findings demonstrate that Pex7 knockout mice provide an important model to study the role of peroxisomal functioning in the pathogenesis of the human disorder.