PLP1 Mutations Research Articles

Epidemiological, clinical, and genetic landscapes of hypomyelinating leukodystrophies

To determine the epidemiological, clinical, and genetic characteristics of congenital hypomyelinating leukodystrophies, including Pelizaeus-Merzbacher disease (PMD), we conducted a nationwide epidemiological survey in Japan. A two-step survey targeting all medical institutions specializing in pediatric neurology and childhood disability (919 institutes) in Japan was performed. Detailed information was collected for 101 patients (86 males and 15 females) with congenital hypomyelinating leukodystrophies. The prevalence of congenital hypomyelinating disorders was 0.78 per 100,000 people (0-19 years old), and the incidence was 1.40 per 100,000 live births. Molecular testing was performed in 75 % of patients, and PLP1 gene abnormalities were observed in 62 %. The incidence of PMD with PLP1 mutations was estimated to be 1.45 per 100,000 male live births and that for congenital hypomyelinating disorders with unknown cause to be 0.41 per 100,000 live births. Patients with PLP1 mutations showed a higher proportion of nystagmus and hypotonia, both of which tend to disappear over time. Our results constitute the first nationwide survey of congenital hypomyelinating disorders, and provide the epidemiological, clinical, and genetic landscapes of these disorders.

Insertion of proteolipid protein into oligodendrocyte mitochondria regulates extracellular pH and adenosine triphosphate

Proteolipid protein (PLP) and DM20, the most abundant myelin proteins, are coded by the human PLP1 and non-human Plp1 PLP gene. Mutations in the PLP1 gene cause Pelizaeus-Merzbacher disease (PMD) with duplications of the native PLP1 gene accounting for 70% of PLP1 mutations. Humans with PLP1 duplications and mice with extra Plp1 copies have extensive neuronal degeneration. The mechanism that causes neuronal degeneration is unknown. We show that native PLP traffics to mitochondria when the gene is duplicated in mice and in humans. This report is the first demonstration of a specific cellular defect in brains of PMD patients; it validates rodent models as ideal models to study PMD. Insertion of nuclear-encoded mitochondrial proteins requires specific import pathways; we show that specific cysteine motifs, part of the Mia40/Erv1 mitochondrial import pathway, are present in PLP and are required for its insertion into mitochondria. Insertion of native PLP into mitochondria of transfected cells acidifies media, partially due to increased lactate; it also increases adenosine triphosphate (ATP) in the media. The same abnormalities are found in the extracellular space of mouse brains with extra copies of Plp1. These physiological abnormalities are preventable by mutations in PLP cysteine motifs, a hallmark of the Mia40/Erv1 pathway. Increased extracellular ATP and acidosis lead to neuronal degeneration. Our findings may be the mechanism by which microglia are activated and proinflammatory molecules are upregulated in Plp1 transgenic mice (Tatar et al. (2010) ASN Neuro 2:art:e00043). Manipulation of this metabolic pathway may restore normal metabolism and provide therapy for PMD patients.

Neurochemistry in shiverer mouse depicted on MR spectroscopy

To evaluate the neurochemical changes associated with hypomyelination, especially to clarify whether increased total N-acetylaspartate (tNAA) with decreased choline (Cho) observed in the thalamus of msd mice with the plp1 mutation is a common finding for hypomyelinating disorders. We performed magnetic resonance imaging (MRI) and proton MR spectroscopy ((1) H-MRS) of the thalamus and cortex of postnatal 12-week shiverer mice devoid of myelin basic protein (mbp), heterozygous and wild-type mice with a 7.0T magnet. Luxol Fast Blue staining and immunohistochemical analysis with anti-Mbp, Gfap, Olig2, and NeuN antibodies were also performed. In the thalamus, decreased Cho and normal tNAA were observed in shiverer mice. In the cortex, tNAA, Cho, and glutamate were decreased in shiverer mice. Histological and immunohistochemical analysis of shiverer mice brains revealed hypomyelination in the thalamus, white matter, and cortex; astrogliosis and an increased number of total oligodendrocytes in the white matter; and a decreased number of neurons in the cortex. The reduction of Cho on (1) H-MRS might be a common marker for hypomyelinating disorders. A normal tNAA level in the thalamus of shiverer mice might be explained by the presence of mature oligodendrocytes, which enable neuron-to-oligodendrocyte NAA transport or NAA catabolism.

A novel mutation in PLP1 causes severe hereditary spastic paraplegia type 2

Hereditary spastic paraplegia (HSP) type 2 is a proteolipid protein (PLP1)-related genetic disorder that is characterized by dysmyelination of the central nervous system resulting primarily in limb spasticity, cognitive impairment, nystagmus, and spastic urinary bladder of varying severity. Previously reported PLP1 mutations include duplications, point mutations, or whole gene deletions with a continuum of phenotypes ranging from severe Pelizaeus–Merzbacher disease (PMD) to uncomplicated HSP type 2. In this manuscript we report a novel PLP1 missense mutation (c.88G>C) in a family from Argentina. This mutation is in a highly conserved transmembrane domain of PLP1 and the mutant protein was found to be retained in the endoplasmic reticulum when expressed in vitro. Due to the variable expressivity that characterizes these disorders our report contributes to the knowledge of genotype–phenotype correlations of PLP1-related disorders.

PLP1 duplication at the breakpoint regions of an apparently balanced t(X;22) translocation causes Pelizaeus–Merzbacher disease in a girl

PLP1 (proteolipid protein1 gene) mutations cause Pelizaeus-Merzbacher disease (PMD), characterized by hypomyelination of the central nervous system, and affecting almost exclusively males. We report on a girl with classical PMD who carries an apparently balanced translocation t(X;22)(q22;q13). By applying array-based comparative genomic hybridization (a-CGH), we detected duplications at 22q13 and Xq22, encompassing 487-546 kb and 543-611 kb, respectively. The additional copies were mapped by fluorescent in situ hybridization to the breakpoint regions, on the derivative X chromosome (22q13 duplicated segment) and on the derivative 22 chromosome (Xq22 duplicated segment). One of the 14 duplicated X-chromosome genes was PLP1.The normal X chromosome was the inactive one in the majority of peripheral blood leukocytes, a pattern of inactivation that makes cells functionally balanced for the translocated segments. However, a copy of the PLP1 gene on the derivative chromosome 22, in addition to those on the X and der(X) chromosomes, resulted in two active copies of the gene, irrespective of the X-inactivation pattern, thus causing PMD. This t(X;22) is the first constitutional human apparently balanced translocation with duplications from both involved chromosomes detected at the breakpoint regions.

Evidence for disease penetrance relating to CNV size: Pelizaeus–Merzbacher disease and manifesting carriers with a familial 11 Mb duplication at Xq22

The potential causes for the incomplete penetrance of Pelizaeus-Merzbacher disease (PMD) in female carriers of PLP1 mutations are not well understood. We present a family with a boy having PMD in association with PLP1 duplication and three females who are apparent manifesting carriers. Custom high-resolution oligonucleotide array comparative genomic hybridization (aCGH) and breakpoint junction sequencing were performed and revealed a familial complex duplication consisting of a small duplicated genomic interval (∼56 kb) and a large segmental duplication (∼11 Mb) that resulted in a PLP1 copy number variation gain. Breakpoint junction analysis implicates a replication-based mechanism underlying the rearrangement formation. X-inactivation studies (XCI) showed a random to moderate advantageous skewing pattern in peripheral blood cells but a moderate to extremely skewed (≥90%) pattern in buccal cells. In conclusion, our data show that complex duplications involving PLP1 are not uncommon, can be detected at the level of genome resolution afforded by clinical aCGH and duplication and inversion can be produced in the same event. Furthermore, the observation of three manifesting carriers with a large genomic rearrangement supports the contention that duplication size along with genomic content can be an important factor for penetrance of the PMD phenotype in females.

Molecular Genetic Analysis of the PLP1 Gene in 38 Families with PLP1-related disorders: Identification and Functional Characterization of 11 Novel PLP1 Mutations

BackgroundThe breadth of the clinical spectrum underlying Pelizaeus-Merzbacher disease and spastic paraplegia type 2 is due to the extensive allelic heterogeneity in the X-linked PLP1 gene encoding myelin proteolipid protein (PLP). PLP1 mutations range from gene duplications of variable size found in 60-70% of patients to intragenic lesions present in 15-20% of patients.MethodsForty-eight male patients from 38 unrelated families with a PLP1-related disorder were studied. All DNA samples were screened for PLP1 gene duplications using real-time PCR. PLP1 gene sequencing analysis was performed on patients negative for the duplication. The mutational status of all 14 potential carrier mothers of the familial PLP1 gene mutation was determined as well as 15/24 potential carrier mothers of the PLP1 duplication.Results and ConclusionsPLP1 gene duplications were identified in 24 of the unrelated patients whereas a variety of intragenic PLP1 mutations were found in the remaining 14 patients. Of the 14 different intragenic lesions, 11 were novel; these included one nonsense and 7 missense mutations, a 657-bp deletion, a microdeletion and a microduplication. The functional significance of the novel PLP1 missense mutations, all occurring at evolutionarily conserved residues, was analysed by the MutPred tool whereas their potential effect on splicing was ascertained using the Skippy algorithm and a neural network. Although MutPred predicted that all 7 novel missense mutations would be likely to be deleterious, in silico analysis indicated that four of them (p.Leu146Val, p.Leu159Pro, p.Thr230Ile, p.Ala247Asp) might cause exon skipping by altering exonic splicing elements. These predictions were then investigated in vitro for both p.Leu146Val and p.Thr230Ile by means of RNA or minigene studies and were subsequently confirmed in the case of p.Leu146Val. Peripheral neuropathy was noted in four patients harbouring intragenic mutations that altered RNA processing, but was absent from all PLP1-duplication patients. Unprecedentedly, family studies revealed the de novo occurrence of the PLP1 duplication at a frequency of 20%.

Pelizaeus-Merzbacher-like Disease Caused by AIMP1/p43 Homozygous Mutation

Pelizaeus-Merzbacher disease is an X-linked hypomyelinating leukodystrophy caused by PLP1 mutations. A similar autosomal-recessive phenotype, Pelizaeus-Merzbacher-like disease (PMLD), has been shown to be caused by homozygous mutations in GJC2 or HSPD1. We report a consanguineous Israeli Bedouin kindred with clinical and radiological findings compatible with PMLD in which linkage to PLP1, GJC2, and HSPD1 was excluded. Through genome-wide homozygosity mapping and mutation analysis, we demonstrated in all affected individuals a homozygous frameshift mutation that fully abrogates the main active domain of AIMP1, encoding ARS-interacting multifunctional protein 1. The mutation fully segregates with the disease-associated phenotype and was not found in 250 Bedouin controls. Our findings are in line with the previously demonstrated inability of mutant mice lacking the AIMP1/p43 ortholog to maintain axon integrity in the central and peripheral neural system.

Clinical neurophysiology for differentiation between Pelizaeus-Merzbacher Disease and Pelizaeus-Merzbacher-like Disease

Background: Hypomyelinating leukoencephalopathies constitute a large subgroup of white matter disorders with onset in childhood. Whereas a few conditions among this category are identifiable by typical clinical or neuroradiological features, Pelizaeus-Merzbacher disease (PMD) and Pelizaeus-Merzbacher-like disease (PMLD) show almost indistinguishable clinical and neuroradiological features. PMD is associated with X-linked PLP1 mutations. PMLD is genetically heterogeneous, and in about 8% of patients autosomal recessive mutations of the gap junction protein α12 (GJA12/GJC2) gene on chromosome 1q41–42 were detected. If a patient's pedigree is consistent with either an X-linked recessive or an autosomal recessive inheritance or a de novo mutation, the decision which genetic testing, PLP1 or a GJA12/GJC2, should be performed first is arbitrary. This study aimed at separating PMD from GJA12/GJC2-associated PMLD based on neurophysiologic investigations.

Clinical neurophysiology in GJA12 -related hypomyelination vs Pelizaeus-Merzbacher disease

Among the hypomyelinating leukoencephalopathies with onset in childhood, Pelizaeus-Merzbacher disease (PMD) and Pelizaeus-Merzbacher-like disease (PMLD) constitute a large subgroup with almost indistinguishable clinical and neuroradiologic features. Whereas PMD is due to X-linked PLP1 mutations, PMLD is genetically heterogeneous, with about 8% of patients carrying autosomal recessive GJA12/GJC2 mutations. The aim of this study was to evaluate whether neurophysiologic testing may separate PMD from GJA12/GJC2-associated PMLD. Retrospective data collection study with reevaluation of evoked potentials (EP) and nerve conduction studies (NCS) in 10 patients from 7 families with PMLD due to various GJA12/GJC2 mutations and 8 boys from 7 families with classic PMD caused by a PLP1 duplication or missense mutation. In brainstem auditory EP, waves III-V were absent in all patients with PMD, but clearly recordable in 11 of 13 investigations in 8 patients with PMLD. Visual evoked potentials and somatosensory evoked potentials revealed conduction delay in both PMD and PMLD, without significant difference. NCS were normal in all patients with PMD and indicated mild peripheral neuropathy in only 2 of 10 patients with PMLD. In a patient with clinical and neuroradiologic features of Pelizaeus-Merzbacher disease/Pelizaeus-Merzbacher-like disease and a pedigree consistent with both conditions, brainstem auditory evoked potentials provide good selectivity between these disorders and point in the right direction for identifying the primary genetic defect.

Hereditary spastic paraplegia caused by the PLP1 ‘rumpshaker mutation’

BackgroundHereditary spastic paraplegia (HSP) is a group of clinically and genetically heterogeneous neurodegenerative disorders characterised by progressive spasticity and weakness in the lower limbs. Mutations in PLP1 on the X...

Genomewide Association Study of Movement-Related Adverse Antipsychotic Effects

Understanding individual differences in the development of extrapyramidal side effects (EPS) as a response to antipsychotic therapy is essential to individualize treatment. We performed genomewide association studies to search for genetic susceptibility to EPS. Our sample consisted of 738 schizophrenia patients, genotyped for 492K single nucleotide polymorphisms (SNPs). We studied three quantitative measures of antipsychotic adverse drug reactions-the Simpson-Angus Scale (SAS) for Parkinsonism, the Barnes Akathisia Rating Scale, and the Abnormal Involuntary Movement Scale (AIMS)-as well as a clinical diagnosis of probable tardive dyskinesia. Two SNPs for SAS, rs17022444 and rs2126709 with p = 1.2 x 10(-10) and p = 3.8 x 10(-7), respectively, and one for AIMS, rs7669317 with p = 7.7 x 10(-8), reached genomewide significance (Q value < .1). rs17022444 and rs7669317 were located in intergenic regions and rs2126709 was located in ZNF202 on 11q24. Fourteen additional signals were potentially interesting (Q value < .5). The ZNF202 is a transcriptional repressor controlling, among other genes, PLP1, which is the major protein in myelin. Mutations in PLP1 cause Pelizaeus-Merzbacher disease, which has Parkinsonism as an occurring symptom. Altered mRNA expression of PLP1 is associated with schizophrenia. Although our findings require replication and validation, this study demonstrates the potential of genomewide association studies to discover genes and pathways that mediate adverse effects of antipsychotics.

Differences in endoplasmic-reticulum quality control determine the cellular response to disease-associated mutants of proteolipid protein

Missense mutations in human PLP1, the gene encoding myelin proteolipid protein (PLP), cause dysmyelinating Pelizaeus-Merzbacher disease of varying severity. Although disease pathology has been linked to retention of misfolded PLP in the endoplasmic reticulum (ER) and induction of the unfolded protein response (UPR), the molecular mechanisms that govern phenotypic heterogeneity remain poorly understood. To address this issue, we examined the cellular response to missense mutants of PLP that are associated with distinct disease phenotypes. We found that the mild-disease-associated mutants, W162L and G245A, were cleared from the ER comparatively quickly via proteasomal degradation and/or ER exit. By contrast, the more ;aggressive' A242V mutant, which causes severe disease, was significantly more stable, accumulated at the ER and resulted in a specific activation of the UPR. On the basis of these findings, we propose that the rate at which mutant PLP proteins are cleared from the ER modulates disease severity by determining the extent to which the UPR is activated.

Different Proteolipid Protein Mutants Exhibit Unique Metabolic Defects

PMD (Pelizaeus–Merzbacher disease), a CNS (central nervous system) disease characterized by shortened lifespan and severe neural dysfunction, is caused by mutations of the PLP1 (X-linked myelin proteolipid protein) gene. The majority of human PLP1 mutations are caused by duplications; almost all others are caused by missense mutations. The cellular events leading to the phenotype are unknown. The same mutations in non-humans make them ideal models to study the mechanisms that cause neurological sequelae. In the present study we show that mice with Plp1 duplications (Plp1tg) have major mitochondrial deficits with a 50% reduction in ATP, a drastically reduced mitochondrial membrane potential and increased numbers of mitochondria. In contrast, the jp (jimpy) mouse with a Plp1 missense mutation exhibits normal mitochondrial function. We show that PLP in the Plp1tg mice and in Plp1-transfected cells is targeted to mitochondria. PLP has motifs permissive for insertion into mitochondria and deletions near its N-terminus prevent its co-localization to mitochondria. These novel data show that Plp1 missense mutations and duplications of the native Plp1 gene initiate uniquely different cellular responses.

Comprehensive genetic analyses of PLP1 in patients with Pelizaeus–Merzbacher disease applied by array-CGH and fiber-FISH analyses identified new mutations and variable sizes of duplications

Pelizaeus–Merzbacher disease (PMD; MIM#312080) is a rare X-linked recessive neurodegenerative disorder. The main cause of PMD is alterations in the proteolipid protein 1 gene ( PLP1) on chromosome Xq22.2. Duplications and point mutations of PLP1 have been found in 70% and 10–25% of all patients with PMD, respectively, with a wide clinical spectrum. Since the underlining genomic abnormalities are heterogeneous in patients with PMD, clarification of the genotype-phenotype correlation is the object of this study. Comprehensive genetic analyses using microarray-based comparative genomic hybridization (aCGH) analysis and genomic sequencing were applied to fifteen unrelated male patients with a clinical diagnosis of PMD. Duplicated regions were further analyzed by fiber-fluorescence in situ hybridization (FISH) analysis. Four novel and one known nucleotide alterations were identified in five patients. Five microduplications including PLP1 were identified by aCGH analysis with the sizes ranging from 374 to 951-kb. The directions of five PLP1 duplications were further investigated by fiber-FISH analysis, and all showed tandem duplications. The common manifestations of the disease in patients with PLP1 mutations or duplications in this study were nystagmus in early infancy, dysmyelination revealed by magnetic resonance imaging (MRI), and auditory brain response abnormalities. Although the grades of dysmyelination estimated by MRI findings were well correlated to the clinical phenotypes of the patients, there is no correlation between the size of the duplications and the phenotypic severity.

Variable Expression of a Novel PLP1 Mutation in Members of a Family With Pelizaeus-Merzbacher Disease

Pelizaeus-Merzbacher disease is a rare X-linked disorder caused by mutations of the proteolipid protein 1 gene that encodes a structural component of myelin. It is characterized by progressive psychomotor delay, nystagmus, spastic quadriplegia, and cerebellar ataxia. Variable clinical expression was seen in 5 members of a family bearing a novel missense mutation in proteolipid protein 1, c.619T>C. Symptomatic patients included a 6-year-old girl, her younger brother, and their maternal uncle, a 29-year-old college graduate initially diagnosed with cerebral palsy; their brain magnetic resonance imaging studies showed diffuse dysmyelination. The mother had a history of delayed walking, achieved independently by age 3; she and the maternal grandmother were asymptomatic on presentation. Review of clinical information and family history led to consideration of Pelizaeus-Merzbacher disease. Subsequent identification of the causal mutation enabled preimplantation genetic diagnosis and the birth of an unaffected child.

Mitochondrial Hsp60 Chaperonopathy Causes an Autosomal-Recessive Neurodegenerative Disorder Linked to Brain Hypomyelination and Leukodystrophy

Hypomyelinating leukodystrophies (HMLs) are disorders involving aberrant myelin formation. The prototype of primary HMLs is the X-linked Pelizaeus-Merzbacher disease (PMD) caused by mutations in PLP1. Recently, homozygous mutations in GJA12 encoding connexin 47 were found in patients with autosomal-recessive Pelizaeus-Merzbacher-like disease (PMLD). However, many patients of both genders with PMLD carry neither PLP1 nor GJA12 mutations. We report a consanguineous Israeli Bedouin kindred with clinical and radiological findings compatible with PMLD, in which linkage to PLP1 and GJA12 was excluded. Using homozygosity mapping and mutation analysis, we have identified a homozygous missense mutation (D29G) not previously described in HSPD1, encoding the mitochondrial heat-shock protein 60 (Hsp60) in all affected individuals. The D29G mutation completely segregates with the disease-associated phenotype. The pathogenic effect of D29G on Hsp60-chaperonin activity was verified by an in vivo E. coli complementation assay, which demonstrated compromised ability of the D29G-Hsp60 mutant protein to support E. coli survival, especially at high temperatures. The disorder, which we have termed MitCHAP-60 disease, can be distinguished from spastic paraplegia 13 (SPG13), another Hsp60-associated autosomal-dominant neurodegenerative disorder, by its autosomal-recessive inheritance pattern, as well as by its early-onset, profound cerebral involvement and lethality. Our findings suggest that Hsp60 defects can cause neurodegenerative pathologies of varying severity, not previously suspected on the basis of the SPG13 phenotype. These findings should help to clarify the important role of Hsp60 in myelinogenesis and neurodegeneration.

Pelizaeus-Merzbacher disease: Genetic and cellular pathogenesis.

Pelizaeus-Merzbacher disease (PMD) and the allelic spastic paraplegia type 2 (SPG2) arise from mutations in the X-linked gene encoding myelin proteolipid protein (PLP). Analysis of mutations affecting PLP, the major protein in central nervous system myelin, has revealed previously unsuspected roles for myelinating glia in maintaining the integrity of the nervous system. The disease spectrum for PMD and SPG2 is extraordinarily broad and can be best understood by accounting not only for the wide range of mutations that can occur but also for the effects of PLP1 mutations on both cell autonomous and non-cell autonomous processes in myelinating cells. Appreciating the wide range of genetic and cellular effects of PLP1 mutations is important for patient and family counseling, understanding disease pathogenesis, and, ultimately, for developing future disease-specific therapies.

A novel deletion in the GJA12 gene causes Pelizaeus–Merzbacher-like disease

Pelizaeus-Merzbacher disease (PMD) and Pelizaeus-Merzbacher-like disease (PMLD) are hypomyelinating disorders of the central nervous system with a very similar phenotype. PMD is an X-linked disorder caused by mutations in PLP1. PMLD is an autosomal recessive condition caused by mutations in GJA12. We report a 5-year-old girl with a complex neurological syndrome and severe hypomyelination on brain magnetic resonance imaging. She harbored a homozygous 34-bp deletion in the coding region of GJA12. There are no distinctive features for the differential diagnosis of PMD/PMLD. GJA12 should be analyzed in all patients without PLP1 mutations but should also be considered the initial genetic test in women and in patients with consanguineous parents.

Golli‐MBP Copy Number Analysis by FISH, QMPSF and MAPH in 195 Patients with Hypomyelinating Leukodystrophies

The inherited disorders of CNS myelin formation represent a heterogeneous group of leukodystrophies. The proteolipoprotein (PLP1) gene has been implicated in two X-linked forms, Pelizaeus-Merzbacher disease (PMD) and spastic paraplegia type 2, and the gap junction protein alpha12 (GJA12) gene in a recessive form of PMD. The myelin basic protein (MBP) gene, which encodes the second most abundant CNS myelin protein after PLP1, presents rearrangements in hypomyelinating murine mutants and is always included in the minimal region deleted in 18q- patients with an abnormal hypomyelination pattern on cerebral MRI. In this study, we looked at the genomic copy number at the Golli-MBP locus in 195 patients with cerebral MRI suggesting a myelin defect, who do not have PLP1 mutation. Although preliminary results obtained by FISH suggested the duplication of Golli-MBP in 3 out of 10 patients, no abnormal gene quantification was found using Quantitative Multiplex PCR of Short Fluorescent fragments (QMPSF), Multiplex Amplifiable Probe Hybridization (MAPH), or another FISH protocol using directly-labelled probes. Pitfalls and interest in these different techniques to detect duplication events are emphasised. Finally, the study of this large cohort of patients suggests that Golli-MBP deletion or duplication is rarely involved in inherited defects of myelin formation.