WO2017037539A2

WO2017037539A2 - Modulation of the phospholipase a2-activating protein for treatment of myelin related diseases

Info

Publication number: WO2017037539A2
Application number: PCT/IB2016/001350
Authority: WO
Inventors: Tzipora C. FALIK ZACCAI
Original assignee: Health Corp Of Galilee Medical Center
Current assignee: Health Corp Of Galilee Medical Center
Priority date: 2015-09-02
Filing date: 2016-09-02
Publication date: 2017-03-09
Anticipated expiration: 2018-03-02
Also published as: WO2017037539A3

Abstract

The present invention provides a method of treating a subject afflicted with a white matter disorder comprising administering to the subject an agent that induces the activity of the Phospholipase A2-Activating Protein (PLAA) pathway in the subject.

Description

MODULATION OP THE PHOSPHOLIPASE A2-ACTIVATING PROTEIN FOR

TREATMENT OF MYELIN RELATED DISEASES

This application claims priority of U.S. Provisional Applications No. 62/213,339, filed September 2, 2015, the contents of each of which are hereby incorporated by reference in their entirety.

Throughout this application, various publications are referenced, including referenced in parenthesis. Full citations for publications referenced in parenthesis may be found listed at the end of the specification immediately preceding the claims. The disclosures of all referenced publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

BACKGROUND OF INVENTION Leukoencephalopathies are white matter disorders categorized by neuroradiological and pathophysiological criteria into: (a) Hypomyelinating diseases that are primary disturbances in myelin formation; (b) Dysmyelinating disorders with delayed and disturbed myelination; (c) Leukodystrophies involving progressive demyelination; (d) Disorders related to cystic degeneration and (e) Disorders secondary to axonal damage (van der Knaap MS, 2001) ( Di Rocco et al., 2004) .

Over the past two decades, several new inherited disorders with white matter abnormalities have been described. Common examples include: Pelizaeus-Merzbacher disease, Cockayne syndrome and

Trichothiodystrophy, SOXlO-associated disease, 18q deletion syndrome, metachromatic leukodystrophy and Krabbe ' s disease (Vanderver et al., 1993) . Nevertheless, approximately 50% of patients with leukoencephalopathies remain without specific diagnoses and treatments. SUMMARY OF THE INVENTION

The present invention also provides a method of determining if a subject is a carrier of leukoencephalopathy comprising:

a) obtaining a biological sample from a subject;

b) analyzing the genomic DNA of the biological sample for the presence of a 22540T mutation;

c) determining the presence of a 22540T mutation in the Plaa gene; and

d) determining the subject as a carrier of leukoencephalopathy if the presence of a 22540T mutation in the Plaa gene is detected.

The present invention also provides a method of determining whether a subject is afflicted with, or predisposed to leukoencephalopathy comprising :

a) obtaining a biological sample from a subject;

b) analyzing the genomic DNA of the biological sample for the presence of a 22540T mutation; and

c) determining the presence of a 22540T mutation in the

Plaa gene.

The present invention also provides a diagnostic kit for determining the presence of, or predisposition to leukoencephalopathy in a subject, comprising a forward primer and a reverse primer for amplifying at least a region of the Plaa gene from a biological sample according to a polymerase chain reaction.

The present invention also provides a diagnostic kit for determining the presence of, or predisposition to leukoencephalopathy in a subject, comprising a specific probe for the 22540T mutation, and/or PCR primers designed to specifically amplify the 22540T mutation .

The present invention also provides a method of treating a subject suffering from neuroinflammation comprising administering to the subject a PLAA inhibitor.

The present invention also provides a transgenic mouse comprising a homozygous Leu752Phe mutation of the Plaa gene.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Pedigree of the investigated families.

A. family I: 6 affected individuals (filled shapes), 6 obligatory carriers (parents of affected, dotted shapes) and 15 unaffected individuals (asterisked white shapes) available for genetic analysis.

B. family II, containing another affected individual. A high rate of consanguinity and an autosomal recessive pattern of inheritance are evident .

Figure 2. Patients ' brain MRI . A and B. Tl brain MRI of patient IV-2 (family II), at age of 1 year old shows white matter atrophy. Corpus callosum is complete but thin.

C (T2 MRI Imaging) and D (Tl MRI Imaging) . MRI of brain of patient VI-3 (family I), at age of 14 year old shows moderate white matter atrophy and severe corpus callosum thinning. E (T2 MRI Imaging) and F (Tl MRI Imaging) . MRI of brain of patient V-4 (family I), at age 32 years shows severe general atrophy. The cortex is usually preserved but very thin, corpus callosum is complete but also very thin. Basal ganglia appear normal.

Figure 3. Analysis of the G.22540T mutation in exon 14 of PLAA. Sequence analysis is shown for an unaffected individual, an obligatory carrier, and an affected individual.

Figure 4. Sequence alignment of human PLAA to orthologues in the mutation area.

The Leucine at position 752 (boxed) in this protein is highly conserved throughout evolution.

Figure 5. Effect of L752F (Leu-^»Phe) substitution on PLAA structure.

Shown is a ribbon diagram of the PUL domain of PLAA (PDB ID 3EBB) which adopts a banana like shaped Armadillo domain. The conserved residues of PLAA (homo sapiens, mus musculus, rattus norvegicus, xenopus laevis, and S. cerevisae) are displayed in stick- representation and form the putative binding site of PLAA. Mutation of Leu752 shown in ball-representation disrupts the rigid leucine network that tightly holds together the Armadillo domain. Figure 6. PGE2 levels and CPLA2 activity are lower in the patients ' cells .

A. Levels of prostaglandin E₂ in cell culture media after 24 hour stimulation with LPS or cholera toxin. nPlaa denotes cells with a wild type Plaa, while mPlaa denotes cells homozygous for the p.Leu752Phe mutation in Plaa. Levels of PGE₂ were normalized against protein concentration in the supernatants . All cells were primary human fibroblasts except RAW264.7 cells which are murine macrophage like cells used as a positive control.

B. Activity of cytoplasmic phospholipase A2 ( C PLA2 ) in the membrane fractions of fibroblasts and RAW264.7 macrophages. Cells were stimulated with or without LPS for 24 hours before harvesting and purification of the membrane fractions. C PLA2 activity was normalized to amount of protein added to the assay.

Figure 7. PGE2 levels and CPLA2 activity are lower in the patients . A. PGE2 levels in the cell culture media after transfection with CMV promoter-based pIRES2-DsRed2 plasmid containing the native plaa gene and a fluorescent marker of transfection. Cells were treated as follows: ctl = no transfection; Vec = transfection with empty vector; PLAA = transfection with plasmid vector containing the wild type PLAA.

B. C PLA2 activity from membrane fractions of fibrobalsts after transfection with a plasmid containing the nPLAA in a CMV promoter- based vector system and a fluorescent marker of transfection.

Figure 8. Linkage analysis . A. Haplotypes for each family member were constructed for 11 microsatellite markers spanning the neurodegenerative interval. Markers analyzed are given in the left, according to their physical order. Haplotyes are represented by bars, with the disease- associated haplotype shaded in gray. Reduction in the affected linked region to 1.9Mb was due to healthy individuals Vic and IV₄ who bear fraction of the affected haplotype in a homozygous manner. B. Physical location of genes and predicted transcripts in the chromosome 9 linked interval. Asterisk denotes genes not approved by the HUGO Gene Nomenclature Committee (HGNC) . "Strand" refers to transcription orientation. Bold names indicate gene analyzed by direct sequencing. Physical location obtained from UCSC Human Genome Browser Gateway (hgl9 assembly) .

Figure 9. luRNA levels for PLAA and confocal microscopy of fibroblasts for the presence of PLAA protein.

A. Presence of full length transcript for PLAA from fibroblasts of affected patients and the control subject based on PCR. B. RT-qPCR for the detection of PLAA transcript from fibroblasts of a patient versus the healthy control normalized to four housekeeping genes coding for human 18S RNA, GAPDH, PolB, and L19 ribosomal protein. Arithmetic means ± standard deviations from three biological replicates performed in triplicate were shown. C. Fibroblasts (nPLAA or mPLAA) were counterstained with DAPI (blue) for the nucleus and with fluorophore conjugated phalloidin (red) for actin. Cells were fixed, subjected to immunofluorescence staining for PLAA (green) , and observed by confocal microscopy.

D. Mean fluorescence intensity of regions of interest corresponding to the cytoplasm and nucleus of imaged cells (Image J processing program, NIH) . Figure represents results from 3 sets of images and error bars represent standard deviations.

Figure 10. Fold changes in transcripts for IL-6, IL-8, and MIF based on RT-qPCR. Fold changes in transcripts for IL-6 (A) , IL-8 (B) , and MIF (C) based on RT-qPCR. Arithmetic means ± standard deviations from three independent experiments performed in triplicate were plotted and the data were analyzed using one way ANOVA with Tukey post-hoc correction .

Figure 11. Evaluation of NF-κΒ translocation and Wnt signaling.

A. Samples were counterstained with DAPI (blue) for the nucleus and with fluorophore conjugated phalloidin (red) for actin. Cells were stimulated with or without LPS for 1 hour, then fixed and subjected to immunofluorescence staining for p65 of NF-κΒ (green) .

B. Mean fluorescence intensity of regions of interest (Image J processing program) corresponding to the nucleus and cytoplasm of cells stimulated with and without LPS. Figure represents results from 3 set of images and error bars indicate standard deviations.

C. The whole cell lysates from nPLAA and mPLAA fibroblasts with and without LPS treatment were subjected to Western blot analysis and probed with non-phospho (active) β-catenin antibodies. Antibodies to β-tubulin were used as a loading control. Three independent experiments were performed and fold changes in the level of β- catenin (based on densitometer scanning of the bands) and normalized to the internal control with and without LPS treatment are shown.

Figure 12. Plaa gene targeting . Exon-2-7 region of the mouse Plaa gene was replaced with a neo cassette flanked by loxP sites. The cassette was subsequently removed via cre-loxP recombination. The Plaa mutant allele with neo cassette and the one without the cassette resulted in the same phenotype . A. Maps of the Plaa wild-type (WT) allele, knockout (KO) vector, KO^ne° allele, and KO allele. Exons 1-14 are shown as boxes. Blue, pink, yellow, and red triangles indicate genotyping primers: Plaa T, PlaaM+F, PlaaM-F, and PlaaCMR, respectively. A, AspllQ; BI, Bgll; BII, Bgll I; H, Hindi I I; and S, Sail. B. Southern blot analysis of genomic DNA isolated from three plaa KO"^e°/+ mouse embryonic stem cell clones and a random integration clone (R) . The 3' flanking probe hybridized to a 22.6-kb Bgll DNA fragment from WT allele, and a 14.7-kb Bgll DNA fragment from KO"^eo allele. The neo probe also hybridized to the 14.7-kb Bgll DNA fragment. Three ES cell clones were identified that incorporated the Plaa mutation by Southern blot analysis. The clones #27 and 75 were derived from Tel ES cells; the clone #52 was derived from G4 ES cells. The clones #27 and 52 transmitted the mutation to the germ line of the mouse.

C. PCR genotyping of the mouse. The upper panel shows genotyping results for E14.5 embryos produced by Plaa-KO^neo heterozygous intercrosses; the lower panel shows genotyping results for Plaa-KO lines .

Figure 13. PGE2 levels in embryonic mouse tissues .

Embryos of WT, Plaa ^+/~, Plaa ^~'^~ genotype were sacrificed at E18.5 and organs were isolated and prostaglandin levels determined for the lung (A) , brain (B) , liver (C) , and heart (D) . Data represent arithmetic means + standard deviations from tissues representing 3 WT, 3 Plaa ^+/~, and 4 Plaa ^"/"embryos and obtained from three independent littermates . Significance was determined by one way ANOVA with Tukey post-hoc. * denotes p<0.05 *** denotes p<0.001. Figure 14. Photographs of patient VI5 (pedigree A)

Photographs of patient VI5 (pedigree A) illustrating: coarse facial features (A) pectus carinatum, dystonic posturing, rigidity/freezing and shortening of tendons (B, C) , and rocker bottom feet (D) .

Figure 15. Hxstopathology of embryonic mouse tissues . Histopathology of embryonic mouse tissues. Lungs (A), brain cerebral cortex (B) , and skin (C) were H&E stained and analyzed in a blinded fashion. Tissues representing 2 WT, 2 Plaa^+>'^~, and 4 Plaa^''^' embryos were analyzed. Multiple fields for each tissue were visualized and typical representations are shown with magnifications. DETAILED DESCRIPTION OF THE INVENTION

In some embodiments, inducing the activity of the Phospholipase A2- Activating Protein (PLAA) pathway comprises inducing the activity of the Phospholipase A2-Activating Protein (PLAA), increasing prostaglandin levels, increasing phospholipase A2 (PLA₂) levels, increasing phospholipase A2 (PLA;) activity or a combination thereof. In some embodiments, inducing the activity of the Phospholipase A2- Activating Protein (PLAA) pathway comprises inducing the activity of the Phospholipase A2-Activating Protein (PLAA) .

In some embodiments, inducing the activity of the Phospholipase A2- Activating Protein (PLAA) pathway comprises increasing prostaglandin levels . In some embodiments, inducing the activity of the Phospholipase A2- Activating Protein (PLAA) pathway comprises increasing Phospholipase A2 (PLA;:) protein levels.

In some embodiments, inducing the activity of the Phospholipase A2- Activating Protein (PLAA) pathway comprises increasing Phospholipase A2 (PLA₂) activity.

In some embodiments, the phospholipase A? is cytosolic phospholipase A₂ (cPLA₂)

In some embodiments, the cytosolic phospholipase A₂ (cPLA₂) activity is measured in the membrane fraction of fibroblast cells.

In some embodiments, the agent is a gene therapy agent.

In some embodiments, at least one copy of Plaa gene of the subject has at least one mutation. In some embodiments, the subject has reduced level of PLAA activity relative to a subject not afflicted with a white matter disorder.

In some embodiments, the subject has reduced level of cytoplasmic Phospholipase A2 (PLA;) activity relative to a subject not afflicted with a white matter disorder.

In some embodiments, the subject has reduced level of at least one endogenous prostaglandin relative to a subject not afflicted with a white matter disorder.

In some embodiments, the at least one endogenous prostaglandin is Prostaglandin 2 (PGE₂) .

In some embodiments, the subject has about 50% or less endogenous PGE2 relative to a subject not afflicted with a white matter disorder

In some embodiments, the Plaa gene of the subject encodes a PLAA protein comprising a mutation at position Leu752.

In some embodiments, the mutation is a Leu752Phe mutation.

In some embodiments, the Plaa gene of the subject comprises a 22540T mutation.

In some embodiments, the white matter disorder is hereditary.

In some embodiments, the disorder is a leukoencephalopathy .

In some embodiments, the white matter disorder is a leukoencephalopathy .

In some embodiments, the subject has a dysmyelinating disorder.

In some embodiments, the disorder is a late onset white matter disease . In some embodiments, the disorder is a demyelinating disease.

a) obtaining a biological sample from a subject;

c) determining the presence of a 22540T mutation in the Plaa gene; and

In some embodiments, analyzing the genomic DNA of the biological sample for the presence of a 22540T mutation comprises sequencing the genomic DNA of the biological sample comprising a segment of the Plaa gene, using a specific probe for the 22540T mutation, or using PCR primers designed to specifically amplify the 22540T mutation. The present invention also provides a method of determining whether a subject is afflicted with, or predisposed to leukoencephalopathy comprising :

a) obtaining a biological sample from a subject;

c) determining the presence of a 22540T mutation in the Plaa gene.

In some embodiments, analyzing the genomic DNA of the biological sample for the presence of a 22540T mutation comprises sequencing the genomic DNA of the biological sample comprising a segment of the Plaa gene, using a specific probe for the 22540T mutation, or using PCR primers designed to specifically amplify the 22540T mutation. In some embodiments, the method further comprises determining the presence of the wild type allele of the Plaa gene. In some embodiments, if the wild type allele of the Plaa gene is not detected then the subject is determined to be afflicted with or predisposed to leukoencephalopathy . In some embodiments, the method further comprises the steps of;

a) analyzing the genomic DNA of the biological sample for the presence of a wild type Plaa allele; and

b) determining the subject as afflicted with or predisposed to leukoencephalopathy if the wild-type Plaa allele is not detected.

In some embodiments, the biological sample is an embryonic sample.

In some embodiments, the embryo is an embryo at the eight-cell stage or from a blastocyst

In some embodiments, the biological sample is a fetal sample.

In some embodiments, the fetal sample is taken from amniotic fluid.

In some embodiments, the region of the Plaa gene encodes a portion of the PUL domain of the PLAA protein. In some embodiments, the biologic sample is taken from an embryo or fetus

In some embodiments, the region of the Plaa gene encodes a portion of the PLAA protein comprising L752.

The present invention also provides a diagnostic kit for determining the presence of, or predisposition to leukoencephalopathy in a subject, comprising a specific probe for the 22540T mutation, and/or PCR primers designed to specifically amplify the 22540T mutation . The present invention also provides a method of treating a subject suffering from neuroinflammation comprising administering to the subject a PLAA inhibitor.

In some embodiments, the subject is diagnosed as suffering from a neurodegenerative disease.

The present invention also provides a transgenic mouse comprising a homozygous Leu752Phe mutation of the Plaa gene. Definition

For the purposes of the present disclosure, the term "agent" when used in the phrase "agent that induces the activity of the Phospholipase A2-Activating Protein (PLAA) pathway" is an agent that is exemplified by an oligonucleotide, a polynucleotide, a polypeptide, a protein, an antibody or a chemical compound. An "agent that induces the activity of the Phospholipase A2 -Activating Protein (PLAA) pathway" as used herein may be a gene therapy agent.

As used herein, "inducing the activity of the Phospholipase A2- Activating Protein (PLAA) pathway" as used herein means increasing the level and/or activity of non-mutant (wild-type) PLAA protein, increasing the level and/or activity of CPLA2 and/or increasing the level and/or activity of PGE_?„, or a combination thereof, resulting for example in increased levels or activity of cPLA₂ and/or PGE₂. In addition, "inducing the activity of the Phospholipase A2 -Activating Protein (PLAA) pathway" as used herein, includes inducing a change in the level and/activity of at least one molecule in the downstream signaling pathway of PLAA, a change which is effective to treat a white matter disease, including for example leukoencephalopathy.

As used herein, "inducing the activity of the Phospholipase A2- Activating Protein (PLAA)" means increasing the level and/or activity of the non-mutant PLAA protein, resulting for example in increased levels or activity of cPLA₂ and/or PGE2. In an embodiment, "inducing the activity of the Phospholipase A2-Activating Protein (PLAA)" restores the levels or activity of the non-mutant PLAA protein to a level that is sufficient to increase levels or activity of cPLA? and/or PGE;, for example to levels of a non-affected subject. In an embodiment, "inducing the activity of the Phospholipase A2- Activating Protein (PLAA)" restores the levels or activity of the non-mutant PLAA protein to a level that is similar to the activity in a healthy subject. As used herein, a "PLAA inhibitor" is an agent that reduces the level and/or activity of the non-mutant (wild-type) Phospholipase A2-Activating Protein (PLAA) in a cell, tissue or a living organism. The agent can be for example an oligonucleotide, a polynucleotide, a polypeptide, a protein, an antibody or a chemical compound. The agent may, in addition, be a gene therapy agent. A PLAA inhibitor may, for example, reduce the number or concentration of non-mutant PLAA molecules in a certain cell, group of cells or tissue, and/or inhibit, reduce or abrogate the activity of the non-mutant PLAA protein. In an embodiment, the PLAA inhibitor inhibits or reduces the biogenesis of the non-mutant PLAA protein. In an embodiment, the PLAA inhibitor is a mutant PLAA protein, or a fragment thereof.

As used herein a "gene therapy agent" is an agent that alters the seguence of a nucleic acid in a cell, group or cells, tissue or tissues, ex vivo or in vivo, for example the sequence of a gene, a DNA, an RNA or a non-encoding sequence, alters the expression pattern or expression level of a gene, and/or an agent which supports the productions of a specific protein, proteins, protein segment or peptide in a patient, exemplified by a vector comprising a gene or a gene segment, or cells treated to produce the protein, proteins, protein segment or peptide. In an embodiment, the gene therapy agent is a vector comprising the PLAA gene or a segment thereof. In an embodiment, the gene therapy agent is cells inserted with a vector comprising the PLAA gene or a segment thereof. In an embodiment, the gene therapy agent is a vector comprising the PLA2 gene or a segment thereof. In an embodiment, the gene therapy agent is cells inserted with a vector comprising the PLA2 gene or a segment thereof. In an embodiment, the gene therapy agent is a vector comprising the PGE; gene or a segment thereof. In an embodiment, the gene therapy agent is cells inserted with a vector comprising the PGE; gene or a segment thereof. In an embodiment, the vector is a viral vector.

As used herein "altered PLAA activity" means activity which is different from the activity of the PLAA protein in a healthy subject, for example, reduced or absent activity. Such altered activity can result for example in reduced levels of cPLA? and/or PGE? . As used herein, the term "effective amount" refers to the quantity of a component that is sufficient to treat a subject i.e. a therapeutically effective amount. The specific effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of subject being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the composition or its derivatives.

As used herein, the term "white matter disorder" refers to a white matter abnormality, including but not limited to genetic, demyelinative, infectious, inflammatory, toxic, metabolic, vascular, traumatic, neoplastic, and hydrocephalic. Clinical characteristics of patients with white matter abnormalities may include white matter atrophy or reduced mass of white matter. One subset a white matter disorder is a brain white matter disorder, a specific subclass of which is referred to as leukoencephalopathy . Leukoencephalopathies can be hereditary disorders or acquired disorders . Examples of leukoencephalopathies include progressive multifocal leukoencephalopathy, toxic leukoencephalopathy, leukoencephalopathy with vanishing white matter, leukoencephalopathy with neuroaxonal spheroids, reversible posterior leukoencephalopathy syndrome, and Megalencephalic leukoencephalopathy with subcortical cysts. Other examples of white matter disorders include white matter abnormalities such as: Pelizaeus-Merzbacher disease, Cockayne syndrome and Trichothiodystrophy, SOXlO-associated disease, 18q deletion syndrome, metachromatic leukodystrophy and Krabbe ' s disease (Vanderver et al . , 1993).

The term "subject" herein refers to any live entity amenable to the methods described herein. In certain non-limiting embodiments the subject is a human. In certain non-limiting embodiments the subject is a mammal. In certain non-limiting embodiments the subject is an embryo. In certain non-limiting embodiments the subject is a fetus.

The term "increasing the level" in the context of molecules as used herein refers to increasing the number of molecules per se, increasing the concentration of the molecules at a certain tissues, or the combination thereof.

The term "reducing the level" in the context of molecules as used herein refers to reducing the number of molecules per se, reducing the concentration of the molecules at a certain tissues, or the combination thereof.

To "treat" a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

The term "mutation" used herein with respect to DNA refers to a change of the nucleotide sequence. The term "mutation" used herein with respect to polypeptide or protein refers to a change of the amino acid sequence.

The term "deletion" is a mutation in which a part of a chromosome or a sequence of DNA is missing. Any number of nucleotides can be deleted, from a single base to a piece of chromosome.

The term "endogenous" refers to developing or originating within the organism or arising from causes within the organism. The term "wild-type" refers to the normal, non-mutated version of a gene or protein common in nature.

"Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting there from. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

As used herein, "PCR primers designed to specifically amplify the 2254C>T mutation" are primers which are designed by the person skilled in the art to amplify a nucleic acid fragment of the PLAA 22540T mutated allele, but not of the wild-type PLAA allele. The term "protein analog" refers to a protein which has a covalent attachment of moieties .

"About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of +20% or +10%, more preferably ±5%, even more preferably ±1%, and still more preferably +0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

All publications and other references mentioned herein are incorporated by reference in their entirety, as if each individual publication or reference were specifically and individually indicated to be incorporated by reference. Publications and references cited herein are not admitted to be prior art.

This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as defined in the claims which follow thereafter .

Experimental Details

Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only .

Example

Seven individuals from two consanguineous Arab families presenting a unique phenotype of severe spastic quadriparesis , progressive microcephaly, thin corpus callosum, significant startle response and severe global developmental delay were observed. Genetic investigation revealed a novel missense variant in the phospholipase A-2-Activating Protein (PLAA) gene and disclosed a new mechanism required for the normal development and maintenance of CNS white matter .

Methods

Patients

The Ethics Committee for genetic experiments of the Israeli Ministry of Health approved the proposed studies. Seven affected and twenty- three healthy individuals from two consanguineous families were enrolled in the study; they or their legal guardians gave written, informed consent. Clinical investigations included medical procedures, imaging and electrophysiological studies, and muscle biopsies. Skin biopsy was performed as part of the research protocol.

In Table 1, data were collected regarding medical history, metabolic measurements, imaging, electrophysiological studies and muscle biopsy. Complete physical, neurological and developmental examinations were performed on the seven affected patients. The disease phenotype in all patients was similarly severe. In Table 1, NA represents Not Available; MRI represents Magnetic Resonance Imaging; and SSEP represents Somatosensory Evoked Potentials. Table 1. Clinical characteristics of patients.

Patient A(Vh) A(VL) A(VIs) A(V₆) A(Vl ,o) B(1V₂) A(Vli)

Sex F F M M M M M

Age (y) 15 1 1 16 34 5 -> 2

4 ->

AO (m) 4 4 j j

FTT +++ + +++ ++ +++

Progressive

+++

microcepha *+ + ₊₊₊ + ly

Pyramidal

++ +++

Signs Babinski

(plegia) Babinski Babinski Babinski +++ Lower sign,

Babinski sign sign sign

extremities Clonus

sign

Pyramidal

Signs ++ ++ ₊₊ ++ ₊₊₊ Upper

extremities

Extra¬

++ +++ +++ ++ f++ +++ pyramidal

Signs

GMFCS

V V V V V V V

(Level)

Cognitive

and

Language Severe Severe Severe Severe Severe Severe Severe Developme

nt Delay

Exaggerate

d Startle + + ♦ NA + + + Response

Seizures

- - + + - + - Rl/CT of Reduce Enlargem White Severe White Delayed Delayed Patient A(V1₃) A(VI₄) A(VIs) A(V₆) A(VI io) B(IV₂) A(VI i ) brain mass of ent of matter general matter myelinatio myelinatio white ventricul atrophy and atrophy, n. Thin n matter. ar system with especially periventric Corpus (at age

Thin Thin periventric white ular Enlargeme 9m)

Corpus Corpus ular matter and nt of

Callosum Callosum lesions atrophy subcortica ventricular

Delayed ormal resemble and thin 1 lesions system

Myelinati MRI of PVL Corpus Thin (at age 2y) on spinal Thin Callosum Corpus

(especially cord (at Corpus (at age Callosum

along age l y) Callosum 30y Enlargeme

Cortico(at age 2y) nt of

spinal ventricular

tract and system

posterior (at age 1 y

Genu of 9m)

Capsula

Interna (at

age 13m)

Worsenin

g of brain

atrophy at

the age of

3 2/12.

Sparing of

basal

ganglia.

Kyphosis/

Pectus

Carinatum +-/+++ +/ +.+ +/+++ _+++/+++ +/+^■+ -/++

Hypertrichosis + + NA + + - -

Small

joints

Hyper- + + ++ - ++ + ₊ Patient A(Vh) ^Γ A(V1₄) ^~ A(VIs) A(V₆) A(VI io) B(1V₂) A(VI .) flexibility

+++

Large +++ Rocker

joints Rocker bottom

Contracture bottom feet

s feet +++ ₊₊₊ +++ ++ +

Intensive

sweating of

palms and

feet ₊ + - + + + +

Moderate/

severe

hearing

impairme Muscle

nt biopsy:

SSEP: mild

central reduction

SSEP: Occasiona

bilateral of

central 1

disturbanc cytochrom

bilateral horizontal

e in e C

disturban nystagmus

central oxidase

ce in . Retinal

tract activity.

Miscellane central atrophy

conductio Normal

ous conducti with

n above Immunohi

on abnormal

brainstem. stochemic

above VEP'S and

Muscle al

brainste ERG

biopsy staining.

m responses

revealed Normal

sediment muscle

of cells

glycogen structure .

like

material

(PAS

positive). Patient A(VI₃) A(VI₄) A(VIs) A(V₆) A(Vl ,o) B(IV₂) A(VI ,)

EM

examinati

on

confirmed

accumulat

ion of

glycogen.

Normal

respiratory

chain

Molecular studies

Genetic Linkage Analyses Linkage and haplotype analyses were performed as previously described ( Zivony-Elboum et al., 2012).

An analysis of 2050 polymorphic markers, spread across the genome at approximately 2cM intervals was performed for 9 family members. Statistical analysis of the logarithm of the odds (LOD) score was performed using the Pedtool-superlink tool. Areas with high LOD score were further examined using Linkage Mapping Set v2.5 HD5 kit and v2.5 MD10 (Applied Biosystems, Grand Island, NY) on 24 family members, according to the manufacturer's protocol.

Molecular Inversion Probes and Massively Parallel Sequencing Molecular Inversion Probes were designed as described in Teer et al., ( Teer et al . , 2010) to cover the 2Mb of the candidate region (LC Sciences, Houston, TX) . A total of 6498 amplimers had an average length of 433bp (+/-22bp) . The amplimers covered 97% of the candidate region. DNA capture, library preparation, GAIIx sequencing (Illumina, San Diego, CA) , and data analysis were performed as described in (Teer et al . , 2010) . Potential variants were filtered and visualized with VarSifter (Teer et al., 2010). Sanger Sequencing

For dideoxy sequencing, primers were designed to cover the candidate seguence variations . Direct sequencing of the polymerase chain reaction (PGR) amplification products was performed using BigDye 3.1 Terminator chemistry (Applied Biosystems Grand Island, NY) and separated on an ABI 3130x1 genetic analyzer (Applied Biosystems) . Data were evaluated using Sequencher v5.0 software (Gene Codes Corporation, Ann Arbor, MI) .

Molecular modeling Molecular modeling of the PLAA protein and assessment of the sequence variation impact was performed using the PyMOL Molecular Graphics System ( Schrodinger, New York, NY) ( Baugh et al., 2011) .

Expression analyses

Reverse Transcription of Full-Length PLAA Transcript Primary fibroblasts [obtained from the Skin biopsy from healthy controls (nPLAA) and patients (mPLAA) ] were harvested from one near- confluent 25 cm² flask and RNA extracted using the RNeasy mini kit (Qiagen, Valencia, CA) . RNA samples were quantified using a Nanodrop Spectrophotometer (Nanodrop Technologies, Wilmington, DE) and qualified by analysis on an RNA NanoChip using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) . Synthesis of complimentary DNA (cDNA) was performed using the Taqman Reverse Transcription Reagents Kit (Applied Biosystems) . The reaction conditions were as follow: 10 min at 25°C; 30 min at 48°C; and 5 min at 95°C. PCR amplifications of cDNA were performed using FailSafe buffer C (Epicenter Biotechnologies, Madison, WI) with PLAA primers 5 ' CGAGCGGCGCAACCAGGTACC3 ' and 5 ' GCATTCACTTACTTTAGCTGGTTCTG3 ' at a final concentration of 1 μΜ . Thermal conditions for 40 cycles of PCR were as follow: 94°C for 30 sec, 60°C for 30 sec, and 68°C for 7 min. Real Time (RT) -quantitative (q) PCR

One g of RNA extracted from fibroblasts from healthy controls and patients was subjected to cDNA synthesis followed by RT-qPCR using the iTaq Universal SYBR Green mix ( Bio-Rad, Hercules, CA) . The final concentration of the PLAA primers (5'GACT TGGGAATCCC AGCTTTTC3 ' and 5 ' TTCCCATACTTGCAGAACCTG3 ' ; Accession # N _001031689) was 300 nM . RT- qPCR assays were performed with human 18S RNA, glyceraldehyde 3- phosphate dehydrogenase (GAPDH) , L19 ribosomal protein, and polymerase beta (PolB) as housekeeping protein encoding genes to normalize PLAA transcript levels. Absolute analysis was performed using known amounts of a synthetic transcript of the gene of interest All RT-qPCR assays were run on the ABI Prism 7500 Sequence Detection System and the conditions were as follow: 50°C for 2 min, 95°C for 10 min, and then 40 cycles of 95°C for 15 sec and 60°C for 1 min. The results shown were the averages and standard deviations from three independent experiments performed in triplicate.

The pro-inflammatory gene expression in fibroblasts with nPLAA or the PLAA gene was carried out using the appropriate assays-on-demand™ gene expression assay mix consisting of a 20X mix of unlabeled PCR primers and TaqMan® MGB probe, FA TM dye-labeled (Life Science Technology Inc, CA) . Human GADPH, β-actin, and 18S RNA encoding genes were used to normalize transcripts for various cytokines. The reactions were carried out according to the manufacturer's instruction using a Bio-Rad Q5 RT-qPCR machine. The results shown were the averages and standard deviations from three independent experiments performed in triplicate.

Western blot analysis β-catenin. Briefly, 70 pg of protein samples from healthy controls' and patients' fibroblasts with or without lipopolysaccharide [LPS; 10 g/mL] stimulation) were electrophoresed on 4-20% Mini-Protean TGX Pre-cast Tris/Glycine gels (Bio-Rad) and then transferred to nitrocellulose membranes. The membranes were probed with non-phospho (active) β-catenin (Cell Signaling Technology, Danvers, MA) and β- Tubulin (Santa Cruz) antibodies as described by the manufacturer. An anti-rabbit horseradish peroxidase conjugated secondary antibody (Southern Biotech, Birmingham, AL) was then added, and proteins detected by using enhanced chemiluminescence with Super Signal West Femto Maximum Sensitivity substrate (Thermo Scientific) . The membranes were then imaged with GE ImageQuant LAS 4 000 (General Electric, Fairfield, CT) .

Ubiquitin. Western blotting was performed with a SDS-PAGE Electrophoresis System as described previously (Khayat e t al., 2008 ) . Briefly, 30 \ig protein samples from healthy controls' and patients' fibroblasts were prepared in a reducing sample buffer, and then electrophoresed on a 7 . 5 % Tris gel with Tris running buffer; blotted to nitrocellulose membrane; and probed with mouse anti-ubiquitin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) antibodies. A horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Santa Cruz) was then added, and proteins detected by autoradiography using enhanced chemiluminescence substrate (Pierce ECL kit, Thermo Scientific, Grand Island, NY) .

Biochemical studies Measurement of Prostaglandin E2 (PGE₂)

Primary human fibroblasts from healthy controls and patients were grown in Dulbecco modified essential medium (DMEM) with 15 % fetal bovine serum (FBS) at 37°C and 5 % CO; . Fibroblasts from patients and control subjects were treated with 10 g/mL LPS or cholera toxin for 24 h and the cell culture supernatants were collected. PGE; levels were determined using enzyme immunoassay kit (Cayman Chemicals, Ann Arbor, MI) . To examine PGE₂ levels in mouse tissues, samples were subjected to solid phase extraction on Cie columns (Cayman Chemicals) prior to measurements. Preparation of membrane fractions from fibroblasts

Membrane fractions from healthy controls and patients' unstimulated and LPS-stimulated fibroblasts (with native PLAA [nPLAA] or mutated PLAA [mPLAA] ) were isolated by using established procedures (refs) . Protein concentrations in the membrane fractions were determined using the Bradford Protein Reagent (Bio-Rad, Hercules, CA) . Measurement of cytosolic PLA₂ (cPLA₂) activity

The CPLA2 activity in membrane fractions of fibroblasts from patients and healthy controls was determined using PLA₂ activity kit (Cayman Chemicals) . The enzymatic activity was normalized to protein concentration for each sample. Bee Venom PLA: was provided in the kit and used as a positive control.

Complemen ation studies

Fibroblasts from patients (mPLAA) or healthy controls (nPLAA) were grown and electroporated with the recombinant plasmid or the vector alone using Lonza Nucleofector and Human Dermal Fibroblast kit (Lonza, Basel, Switzerland) . The mPLAA fibroblasts were electroporated with either CMV promoter-based pIRES2-DsRed2-nplaa for complementation or pIRES2-DsRed2 vector (Clontech, Moutain View, CA) alone as a control. nPLAA Fibroblasts were also electroporated with the vector alone to serve as an additional control.

Mouse model

All animal experiments were performed at the University of Texas Medical Branch. Animals were housed in a specific-pathogen free facility at a constant temperature (68-79°F) and humidity (30-70%) on a 12 hours light-dark cycle. Autoclaved water and irradiated feed were given to the animals ad libitum. All procedures were performed in accordance with the protocol reviewed and approved by University of Texas Medical Branch Institutional Animal Care and Use Committee, and in compliance with the institutional policies/guidelines and the Guide for the Care and Use of Laboratory Animals, 8^th edition. Euthanasia methods used in the procedures were consistent with the American Veterinary Medical Association Guidelines for the Euthanasia of Animals, 2013 edition. Generation of Plaa-null mice (Plaa gene targeting) and genotyping of the mice have been described in Supplementary Materials and Methods section.

Preparation of Mouse Tissue Samples for PGE₂ Measurements

In brief, mouse tissues were suspended in homogeni zation buffer (0.1M disodium phosphate buffer, pH 7.4 , 1 mM EDTA, 10 μΜ indomethacin) and sonicated. Samples were normalized by measuring protein concentrations using the Bradford Protein Reagent (Bio-Rad) . After homogenization, 4 volumes of ethanol were added and samples centrifuged at 3000 x g for 10 min at 4°C. Supernatants were collected and ethanol removed by vacuum centri fugation before acidification of the samples with 1M acetate buffer. The samples were then loaded on pre-washed Cie cartridges, washed with ¾0, and eluted with ethyl acetate and 1% methanol (99:1 v/v) . Ethyl acetate was removed by vacuum centrifugation and samples reconstituted in PGE₂ assay buffer for measuring PGE₂.

Histopathology

Sections (5 μπι) representing skin, lungs, and the brain cerebral cortex from embryonic day (E) 18.5 mouse embryos were fixed in 10% neutral buffered formalin. The tissue sections were mounted on slides and stained with hematoxylin and eosin. The histopathological evaluation of the tissue sections was performed in a blinded fashion.

Statistical analysis

Where appropriate, at least three independent experiments were performed in triplicate and the data analyzed using one-way ANOVA with Turkey or Turkey post-hoc correction.

Yeast Experiments

Wild type DOAl gene expressing plasmid and DOAl deleted yeast strain {Saccharomyces cerevisiae) were kindly provided by Prof. Tzachi Pilpel and Dr. Orna Dahan (Weizmann Institute of Science, Israel). Mutant p.Leu677Phe (Leucine^→ Phenylalanine) DOAl was produced using the Quikchange site-directed mutagenesis. Wild type or mutant DOAl expressing plasmid, or a mock plasmid, was transformed into the indicated yeast strain by standard lithium acetate method. The yeast was grown on a regular synthetic medium at 30°C, 37°C or at 30°C supplemented with 0.5 pg/ml cycloheximide . After three days, the growth of the yeast was assessed. Cell Biology Studies

Immunofluorescence Confocal Microscopy

Confocal microscopy was performed on fixed fibroblasts by established procedures with slight modifications. Briefly, nPLAA and mPLAA were treated with 10 pg/mL LPS for 60 min, with LPS untreated cells serving as controls, before fixation with 4% paraformaldehyde. Subsequently, fibroblasts were permeabilized with 0.5% Triton X-100. Goat anti-p65 C-20 antibody (Santa Cruz) or rabbit anti-PLAA antibody (GenWay Biotech, Inc., San Diego, CA) , and Alexa Fluor 488 donkey anti-goat antibody (Molecular Probes, Carlsbad, CA) or Alexa Fluor 488 donkey anti-rabbit antibody, were used as primary and secondary antibodies, respectively, as appropriate. Samples stained with secondary antibody alone were used as negative controls. Fibroblasts were then counterstained with DAPI (Life Technologies, Grand Island, NY) and Alexa Fluor 594 phalloidin (Life Technologies) to stain the nucleus and actin, respectively. Fibroblasts were subsequently visualized using a Zeiss LSM510 confocal scanning microscope.

Plaa Gene Targeting

A mouse genomic library, ES129SvJ, was screened for clones containing the Plaa gene. Two overlapping pUC18 clones, 10-1-lA carrying a -17- kb Plaa upstream-intron 2 region and 7-2-1A carrying a ~12-kb Plaa intron 2-intron 10 region, were validated for the organization of the gene by DNA restriction enzyme digestion and sequence analysis, and used for knockout vector construction. A 3.1-kb Hindlll-Bglll fragment (intron 1, 5' homology arm), and a 6.0-kb Sa2I-Asp718 (located in pUC18) fragment containing exons 8-10 (3' homology arm) were subcloned into a plasmid vector, pBluescript II KS (-) (Agilent Technologies) . A positive selection marker PGKneobpA (Soriano et al., 1991) flanked by loxP sites was inserted between two homology arms, and a negative selection marker HSVtkpA (Mansour et al., 1988) was inserted between pBluescript and the 5' homology arm (Figure 12A) .

The vector was linearized at the end of the 3' homology arm by Asp718 and electroporated into 129S inbred Tel (George et al . , 2007) kindly provided by Phillip Leder, Harvard Medical School, Boston, MA) and B6129Fi hybrid G4 (George et al . , 2007) kindly provided by Andras Nagy, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, NY) mouse embryonic stem (ES) cells.

A 3' -flanking probe was used in Southern blot for the identification of targeted clones (Figure 12A) . Mutant ES cell clones were injected into C57BL/6J blastocysts. We crossed chimeric mice and C57BL/6J to produce animals heterozygous for the Plaa knockout (KO)^neo allele. To establish mutant lines with Plaa exon-2-7 deletion (KO) allele, we crossed K0^neo/+ mice to a germ-line ere deleter strain, Zp3-cre transgenic mouse, on a C57BL/6J background (Lewandoski et al., 1997) C57BL/6J-Tg (Zp3-cre) 93Knw/J, The Jackson Laboratory, Stock Number: 003651) . The mice were maintained by backcrossing to C57BL/6J

Genotyping

We genotyped mice initially by Southern blot analysis using the 3' probe, and subsequently by PCR (Figures 12B and 12C) . The 3' probe was generated by PCR with primers: XB-X-Plaa, 5'- CTTCTCGAGTTCCTGAATGTCTGGGAAAA-3 ' ; and XB-B-P2aa, 5'-

ACGAGATCTAGTGATGTGGCAATGCCTTT-3 ' ; and mouse genomic DNA as the template. The PCR product was treated with Klenow DNA polymerase, digested with Bgill, and subcloned into pBluescript II SK (-) at -EcoRV and Bamtil sites (pPlaa 3' XB) . The digestion of the plasmid pPlaa 3' XB by Xhol and Xjbal restriction enzymes released a ~450-bp DNA fragment corresponding to Plaa exon 13 and its flanking region. Genotyping PCR was performed with the following primers: PlaaWTF, 5'- GGGGGTGGCGTTCCATGTGT-3 ' ; PlaaM+F, 5 ' -GTGGGGCAGGACAGCAAG-3 ' ; PlaaM-F, 5 ' -CCATTTGGCTTTTTGGTCTT-3 ' ; and PlaaCMR, 5 ' -CCACCTCCCGTCACTAACACTCCA- 3' .

Results

Patients Seven individuals from two families, all were products of consanguineous marriages and uneventful pregnancies, presented with progressive leukoencephalopathy (Figure 1A and IB), defined as dysmyelinating according to the known categories of Van der Knaap and his colleagues (van der Knaap, 2001). Affected individuals were normal at birth, with onset of neurological symptoms at age 2-4 months (Table 1) . Symptoms included spasticity of the lower limbs rapidly progressing to the upper extremities, resulting in severe quadriparesis with symptoms of corticospinal tract impairment and posture deformation. Involvement of extrapyramidal system function included dystonic posturing, rigidity/freezing and hypomimia/amimia. All patients suffered from severe mental and language developmental delay. The motor functions were also prominently impaired (level V, according to Gross Motor Function Classification System (Palisano et al . , 1997). Abnormally exaggerated startle reflex to an auditory stimulus was observed in 6 patients, and seizures developed in three.

Head circumferences, normal at birth, decreased to more than 2 standard deviation below the mean in the ensuing years. In two patients, we observed an unexplained gradual increase in Head circumferences up to 75% after the age of approximately 5 years. Weight and height, also normal at birth, fell to 3-4 SD below mean in 5 patients but returned to 50-75% in two out of five patients. Progressive chest deformities (kyphosis/pectus carinatum) were observed in all patients. Additional phenotypic characteristics include contractures of large joints, hyperextensibility of small ones, rocker bottom feet, hypertrichosis, and hyperhidrosis of the palms and feet (Figures 14A-D) .

Brain MRI demonstrated radiological signs of periventricular and subcortical damage including delayed myelination and atrophy, which worsened with age as a result of enlargement of ventricular system.

Thin corpus callosum was a prominent feature in all of the patients.

In one case, periventricular lesions were observed (Figures 2A-F) .A muscle biopsy in patient VI3 showed normal oxidative phosphorylation and increased aggregation of collagen. Molecular Analysis

Molecular Studies

Linkage analysis, performed on 24 individuals from family I, identified a 1.9 Mb region between markers D9S265 and rsl330920 with maximal LOD score of 3.24 at D9S1121 (Figures 8A and 8B) ; the region contained 11 genes. Seven samples were sequenced (two affected, 2 obligate carriers, and 3 unaffected individuals from the same village) with average coverage of 82 + 2% (all coding regions were covered) . Haplotype analysis (D9S259-D9S169) supported a common ancestral haplotype in families A and B (Table 2) .

Table 2. Common ancestral haplotypes in families A and B.

Haplotypes for each corresponding family member were constructed from 6 microsatellite markers spanning the neurodegenerative interval. In Figure 8A, markers analyzed are given in the left, according to their physical order. The disease-associated haplotype is shaded in gray. The shared interval between the two families is narrowed to 1.4 Mb, surrounding the gene PLAA.

Next Generation Sequencing

A total of 4289 variants were identified, but only 4 of them affected protein sequences (Table 3) . Out of these four variants, only one was present in a homozygous state in the affected individuals and in a heterozygous state in the obligate carriers. The three others were identified in the samples of unaffected individuals. The missense variant, NM_001031689.2 : c.22540T (p . Leu752 Phe ) , was confirmed by Sanger sequencing (Figure 3) in all affected individuals and obligate carriers. All seven affected individuals were homozygous for this sequence variation; all their parents were heterozygous.

Table 3. Genotypes of all the variants identified by molecular inverted probes in the coding regions of 4 family members ( V6 , VIS, V14 and V13) and 3 healthy controls from the same village (CI, C2, C3)

rsID: dbSNP identifier (ncbi.nlm.nih.gov/SNP); ND: Not Determined; Not Available.

The above PLAA variant (p . Leu752Phe) was neither observed in the Exome Aggregation Consortium (60.706 unrelated individuals, Exome Aggregation Consortium [ExAC] , Cambridge, MA, exac.broadinstitute.org, accessed February 2016) nor in the NHLBI database (6,500 unrelated individuals, Exome Variant Server, NHLBI GO Exome Sequencing Project, Seattle, A, evs . gs . Washington . edu/EVS , accessed February 2016). Population screening of 92 healthy village residents revealed 3 carriers of this sequence variation (prevalence 3.3%).

The leucine at position 752 in PLAA is highly conserved through Saccharomyces . cerevisiae (Figure 4), with the exclusion of Zebrafish (threonine) and Caenorhabditis . elegans (valine). This amino acid substitution is predicted to be deleterious by SIFT (Score: 0.04) (sift.jcvi.org/www/SIFT_aligned_seqs_submit.html) and probably damaging by PolyPhen-2 (Score of 0.983) (genetics.bwh.harvard.edu/pph2/index.shtml) .

Structural effects of p. Leu752Phe substitution in PLAA

The structure of the PUL domain of PLAA, in which Leu752 resides, was recently determined with atomic resolution (Qiu et al., 2010). PUL domain consists of 15 tightly packed α-helices that forming a 6-mer Armadillo domain. This protein fold consists of tightly packed helices in a single rigid structure found in several proteins such as importin-a, β-catenins, and Hsp70 binding protein (Hatzfeld, 1999) . The Armadillo domain of PLAA is held together mainly through conserved leucine residues that zip together adjacent a-helices. On average, leucine is present every 3 or 4 residues, corresponding to one turn of the helical wheel. Such Armadillo repeats form banana- shaped domains that generate good binding surfaces, particularly on the inside curvature (Hatzfeld, 1999) .

In PLAA, the putative binding site is also paved with conserved residues (Sievers et al . , 2011). Based on these data, the p.Leu752Phe mutation appears to disrupt the tightly packed leucine network of the PLAA PUL domain, and deform the banana-like binding surface (Figure 5) . Expression Studies

PLAA mRNA Expression

At the transcriptional level, fibroblasts from patients were capable of expressing full length PLAA transcript similar to that of nPLAA fibroblasts (Figure 9A) . Furthermore, based on RT-qPCR, no difference in the levels of PLAA transcript was noted between nPLAA versus PLAA fibroblasts (Figure 9B) . As amino acid changes can have unexpected effects on protein stability, we sought to confirm production of PLAA protein in fibroblasts with and without the defined mutation in the PLAA gene. Confocal microscopy was performed to localize PLAA in normal and patient fibroblasts . All patient fibroblasts tested showed some localization of PLAA in the nucleus and majority of PLAA in the cytoplasm, which were similar in levels found in the fibroblasts of healthy controls (Figures 9C and 9D) . Functional effects of p . Leu752Phe

Previous studies showed that PLAA loss causes severe ubiquitin depletion, accumulation of misfolded proteins and impaired cellular survival in S. cerevisiae (Mullally et al., 2006; Qiu et al., 2010). However, these effects were neither shown in the growth of S. cerevisiae and its &DOA1 (an ortholog of human PLAA in yeast) mutant nor on ubiquitin depletion in fibroblasts from healthy controls versus patients. While the ΔΩΟΑ1 strain displayed abrogated growth, transformation of the &DOA1 strain with a plasmid expressing either the native DOA1 gene or its p.Leu677Phe variant, which corresponds to Leu752 in human PLAA, completely rescued the growth phenotype of S. cerevisiae ADOAl mutant strain. Likewise, no evidence of effect of p.Leu752Phe variation of PLAA on ubiquitin depletion in fibroblasts from healthy controls versus patients based on Western blot analysis was found. We examined other known functions of the PLAA protein. PLAA induces PGE2 production by increasing levels of PLA2 and cyclooxygenase (COX)- 2 proteins, two major regulators of prostaglandins (Calignano et al., 1991; Zhang et al., 2008). To investigate this function, PGE 2 levels were measured in patients' fibroblasts. Healthy, un-stimulated cells expressing nPLAA exhibited ~2-fold higher levels of PGE2 in comparison to patients' fibroblasts (Figure 6A , black bars), and this difference became much more prominent after lipopolysaccharide (LPS) and cholera toxin treatments of the cultured cells nPLAA cells (~5000-fold (light gray bars) and -1000-fold (dark gray bars), respectively). LPS treatment also induced cPLA; activity in normal fibroblasts, but was unable to elicit a similar response in the patients' cells (Figure 6B) . These results suggest that the p.Leu752Phe mutation in PLAA abrogates its ability to induce prostaglandin biogenesis and elicit the proper response to related stresses. Finally, transfection with a plasmid expressing nPLAA rescued PGE 2 levels and C PLA2 activity in both untreated and LPS- stimulated patient fibroblasts (Figure 7A and 7B) . It was previously observed that PLAA regulates NF- KB-mediated inflammatory responses, and in particular inducible interleukin (IL)- 6 (Zhang et al., 2008). p.Leu752Phe variation in PLAA abrogated expression of LPS induced IL-6, IL-8, and macrophage migration inhibitory factor (MIF) expression in patient fibroblasts when compared to fibroblasts from a representative healthy control based on RT-qPCR (Figures lOA-C) .

Cell Biology studies

NF-κΒ Recruitment to the Nucleus is Unaffected by p . Leu752Phe in the PLAA. NF-κΒ signaling pathway is known to be regulated by PLAA (Zhang et al. , 2008) .

As the secretion of PGE2 as well as the activity of cPLA₂ was decreased in mPLAA fibroblasts, whether NF-κΒ signaling was intact was investigated. nPLAA and mPLAA cells were stimulated with 10 pg/mL LPS for 60 min and compared nuclear localization of p65 by confocal microscopy. Unstimulated nPLAA and mPLAA fibroblasts exhibited comparable fluorescent staining primarily in the cytoplasm with little to no nuclear staining (Figure 11A and 11B) . Upon stimulation with LPS, both nPLAA and mPLAA fibroblasts showed localized NF - KB staining primarily in the nucleus, with no significant differences between cell types (nPLAA versus mPLAA fibroblasts) . These data suggested that the NF- Β signaling pathway remained intact and that differences in phenotype observed for mPLAA fibroblasts could be localized downstream of NF-κΒ signaling. β-catenin wingless integration (Wnt) Signal ing is Not Affected by p.Leu752Phe PLAA

Wnt signaling was investigated by examining levels of non-phospho (active) β-catenin in nPLAA versus mPLAA fibroblasts with and without LPS stimulation. As shown in Figure 11C, the levels of active β- catenin were increased after LPS stimulation to a similar extent in both types of fibroblasts. β-catenin mediated wingless integration (Wnt) signaling has been shown to play an important role in early embryonic development, neurogenesis, central nervous system morphogenesis, hirsutism, sweat gland morphology, short tendons, and kyphosis (Haara et al., 2011; Joksimovic and Awatramani, 2014; Toribio et al., 2010; Zhang et al., 2014; Chenn, 2008) . Han and collegues have shown this pathway to be regulated by cPLAaa (Han et al., 2008) . Therefore, Wnt signaling was investigated by examining levels of non-phospho (active) β-catenin in nPLAA versus mPLAA fibroblasts with and without LPS stimulation. As shown in Figure 11C, the levels of active β-catenin were increased after LPS stimulation to a similar extent in both types of fibroblasts. These data indicated that the variant PLAA in the patients did not alter canonical Wnt signaling pathway.

Mouse Model

Inactivation of Plaa gene results in perinatal lethality of mice

Plaa null mice using gene targeting technology were generated (Figure 12A) . While heterozygous (Plaa*''^") mutants were viable and fertile, the homozygous (Plaa ^') mutants exhibited perinatal lethality.

Initially 66 pups derived from heterozygous intercrosses, typically on postnatal day 4-9, were generated. Twenty-eight pups were wild- type, thirty-eight were Plaa^+/", and there was no Plaa -'^' mutant. To determine when the Plaa-null mice died, timed heterozygous intercross

mating was set up and embryos were examined at different time points (Table 4) .

Table . Embryos and neonates from heterozygous intercrosses .

DOB, day of birth; E, embryonic day; Het, heterozygous; Homo, homozygous; P, postnatal day; WT, wild-type At E14.5, live, overtly normal Plaa^~/' embryos were recovered, which were indistinguishable from Plaa^+/" or wild-type littermates. At E18.5, live (with a beating heart) were mostly found but some dead Plaa^~/~ embryos were also found. Plaa^"-'^'" embryos were grossly normal but smaller than Plaa^+/"" or wild-type littermates. The average body weights of live Plaa^"'^', Plaa*^/", and wild-type embryos were 0.83 ± 0.11 g (n=14), 1.03 + 0.17 g (n=32), and 1.34 ± 0.14 (n=9) at E18.5, respectively. While differences in weights between wild-type and Plaa^+/" were not statisitcally significant, weight differences between wild-type and Plaa^"/" (<0.0001) and Plaa^"''^" and Plaa^^" (<0.001) were significant by one way ANOVA with Tukey post hoc correction.

Gross examination revealed that all of the near-term Plaa^'/~ embryos had abnormal or underdeveloped spleens, which were transparent/pale and smaller. Interestingly, one embryo was found with exencephaly/microcephaly among a total of 41 Plaa^"''^'" embryos examined. Resuscitate was attempted on some of the E18.5 embryos, but Plaa"^'" embryos could not be resuscitated. While Plaa*'^" and wild-type embryos reacted to the pinch and started gasping for air, Plaa ' embryos did not make any voluntary or involuntary movement. Subsequently, five Plaa^"''^' neonates were found to be naturally delivered, but they were all found dead. Notably, one of the dead Plaa^''^' neonates had posterior truncation. The dead Plaa ^■'^" neonates appeared to have no air in their lungs. To date, no live Plaa^"'^' neonate has been found. These observations suggest that Plaa- null mice likely died shortly before or after birth.

A Tissue-Specific PGE₂ Reduction and Perinatal Lethality of Plaa - Null Mice

To validate the results observed for the human fibroblasts carrying mPLAA, the levels of PGE₂ in wild-type, Plaa''^", and Plaa^"'^" embryos were evaluated. Lungs, brain, liver, and heart tissues were isolated from E18.5 embryos, and PGE2 levels were determined for each organ individually (Figures 13A-D) . In the brain, there was a gene copy dependent reduction of PGE? with significant reduction in Plaa*'^' embryos compared to wild-type (p<0.001) as well as a significant decreased level of PGE₂ in Plaa^~'^~ compared to Plaa*'^' (p<0.05) embryos (Figure 13B) . PGE2 levels were significantly decreased in Plaa^"'^" lungs, and Plaa*'^" and Plaa^"'^' hearts, but not in the liver (Figures 13A, 13C, and 13D) . Notably, this is the first report of an effect of PLAA on PGE2 levels in the brain.

Histopathological Analysis of Skin, Lungs, and Brain Cerebral Cortex of Plaa-Null Mice

As shown in Figure 15A, lungs from WT mice embryos exhibited the presence of organized alveolar spaces and thin alveolar walls. However, embryos from Plaa*'^" and Plaa^"'^"mice showed progressively unorganized alveolar spaces and thickening of the alveolar walls, suggesting underdeveloped or immature lungs.

In the brain cerebral cortex of wild type embryos, the neurons showed large nuclei and were fully matured, with no indication of degeneration. No signs of apoptotic bodies were noted. There were a few round dark cells that represented either oligodendroglia or granular immature neurons (Figure 15B) . The Plaa'^:' embryos had smaller neuronal nuclei and about the same density of the round dark cells. On the contrary, Plaa^"-' embryos had a vast area of neurons with smaller dark-stained round nuclei that could be described generally as "more granular" in type, an indication of less maturity and differentiation. No significant differences were observed in the skin of wild type versus mutant mouse embryos (Figure 15C) . Typical tissue sections representing multiple fields and from 2-4 embryos are shown.

Diagnosis

Diagnosis of c.2254C>T (p . Leu752Phe ) in patients, carrier, and as pre-gestational and pre-natal diagnosis

In order to test if a subject is a carrier of the missense variant, a sample, for example blood or skin can be taken from the subject and analyzed for the presence of the c.2254C>T (p . Leu752 Phe ) mutation.

Alternatively, the missense variant could be tested in embryonic or fetal tissues or cells. For example, for use in pre-implantation genetic diagnosis, a cell or cells could be taken from an embryo, for example an embryo in the eight-cell stage or from a blastocyst and analyzed for the presence of the c.2254C>T (p .Leu752Phe) mutation. In case the embryo is homozygous for c.2254C>T (p . Leu752 Phe ) , it could be decided that the embryo would not be implanted. In addition, prenatal testing could be performed in fetal cells or tissues. Fetal cells could be obtained during pregnancy, for example from amniotic fluid or by Chorionic villus sampling (CVS) , to test whether the fetus is homozygous for the c.2254C>T (p . Leu752Phe ) mutation.

A patient suffering from neurological symptoms manifesting leukoencephalopathy can be tested and diagnosed as a patient suffering from leukoencephalopathy based on their genotype having homozygosity for the missense variant. The patient can also be diagnosed as suffering from PLAA-related leukoencephalopathy if the patient is homozygous for the c.2254C>T (p . Leu752Phe) mutation. Specifically, the the c.2254C>T (p . Leu752Phe ) mutationcan be tested using adequate probes, polymorphic markers, primers or sequencing, which a person skilled in the art could design based on the wild-type sequence (NCBI reference sequence NM_001031689.2) , and the knowledge that the missense variant has a 22540T mutation. The presence of the 22540T mutation can be tested using known methods in the art, e.g. dynamic allele-specific hybridization (DASH) , molecular beacons, restriction fragment length polymorphism (RFLP) analysis, Polymerase Chain Reaction (PCR) , primer extension, TaqMan assay, oligonucleotide ligation assay, single strand conformation polymorphism, temperature gradient gel electrophoresis, denaturing high performance liquid chromatography, microarray, or sequencing, or a combination thereof. For sequencing, the genome or a region in the genome containing the 22540T mutation can be sequenced using known methods in the art including but not limited to Sanger sequencing, massively parallel signature sequencing, polony sequencing, pyrosequencing, sequencing by synthesis, sequencing by ligation, single molecule real time sequencing or nanopore DNA sequencing, etc.

Discussion PLAA is a regulatory molecule implicated in modulating production of phospholipases (e.g., phospholipase A₂ [PLA₂]) in the host cell (Clark et al., 1991; Ribardo et al . , 2002). The induction of PLA₂ is highly regulated by mitogen-activated protein kinases and nuclear factor- kappa B (NF-κΒ) ( Zhang et al . , 2008). PLA; hydrolyzes membrane phospholipids to produce arachidonic acid (AA) , which is used as a substrate to produce prostaglandins and leukotrienes ( eicosanoids ) through the cyclooxygenase and lipoxygenase pathways, respectively (Ribardo et al., 2002).

Seven patients from two families with severe unique progressive leukoencephalopathy were examined. Based on the clinical and radiological findings, these patients could be categorized into the group of primary delay in myelin formation and disturbed myelination (van der Knaap, 2001) . Brain biopsies were not performed and histopathological data are not available to confirm or rule out our clinical impression. All patients are homozygous for a founder sequence variant (p. Leu752Phe) in PLAA that did not lead to the production of unstable transcript or protein. The PLAA protein is composed of three major domains. The N ' terminal, multi-protein complex assembly domain contains 7 WD40 repeats. The central PFU domain includes a ubiquitin binding region and an SH3 region, and the C terminal PUL domain consists of 6 Armadillo repeats and binds to valosin-containing protein (VCP) , also known as Cdc48 and p97 (Qiu et al . , 2010). Results herein suggest that the p.Leu752Phe sequence variant disrupts the protein's Armadillo domain, possibly impairing the ability of cells to induce production of prostaglandins through a non-NF- κΒ signaling pathway.

Armadillo folds, such as those found in PLAA, importin-a, and β- catenins, play a role in the development of the central nervous system in Drosophila. Specifically, disruption of the cell-cell adhesion function of Armadillo results in defects in the construction of the axonal scaffold (Loureiro and Peifer, 1998) .

A recent study suggested that phopholipase A2a regulates the Wnt/β- catenin pathway (Han et al., 2008), which is implicated in neurogenesis, morphogenesis of the central nervous system, hirsutism, sweat gland morphology, short tendons, and kyphosis (Haara et al., 2011 ; Joksimovic and Awatramani, 2014; Toribio et al . , 2010; Zhang et al . , 2014). In this pathway, β-catenins transduce WNT signals during embryonic development. It is likely that PLAA, which activates phospholipase A2 , indirectly regulates the Wnt/p-catenin pathway, accounting for the pathology seen in the patients. However, the data indicate that the p.Leu752Phe in PLAA found in our patients did not alter Wnt signaling.

Experimentally, activation of PLAA was recently shown to occur via la,25(OH)₂D3 binding to a specific membrane-associated receptor, Pdia3, in caveolae, regulating growth zone chondrocytes (Doroudi et al., 2014) . These findings might explain the non-neurological features of progressive chest deformities (kyphosis/pectus carinatum) present in affected individuals. Complex phospholipid defects involving the central nervous system have received much attention of late (Lamari et al . , 2013), providing insights into the late onset neurodegenerative disease pathophysiology, such as gene PLA2G6 encoding PLA₂, underlying AR infantile neuroaxonal dystrophy, neurodegeneration associated with brain iron accumulation, and early-onset dystonia/parkinsonism (Gregory et al., 2008; Khateeb et al . , 2006).

Furthermore, PGE2 plays a dual role, both neurotoxic and neuroprotective, in the brain and nervous system, a role modulated by its four receptors (Milatovic et al . , 2011). Different binding affinities, varying cellular expression profiles, and attenuation of secondary messengers of these receptors lead to intricate, and sometimes opposing, signal transduction. While in Alzheimer's disease and amyotrophic lateral sclerosis it plays a neurotoxic role (Bazan et al . , 2002), in excitotoxicity and cerebral ischemia scenarios, PGE₂ is neuroprotective (Gregory et al . , 2008). Thus, modulation of PGE2 appears critical for neurological function. When PGE2 levels are reduced by deficiency of synthetic enzyme PLA2 (Gregory et al . , 2008), COX-1 and COX-2 (FitzGerald, 2003) or PGE2 receptor (EP1-4), neurological impairment might occur.

Mohri et al . have described prostaglandins as neuroinflammatory molecules that heighten the pathological response to demyelination in twitcher mice (Mohri et al . , 2006). Similarly, the arachidonic acid (AA) pathway was shown to be modulated during cuprizone neurotoxin induced-demyelination and remyelination processes (Palumbo et al., 2011) . These data support a causative relationship between abnormal PLAA activity and the severe leukoencephalopathy seen in the patients.

Of special interest is the prominent feature of exaggerated startle response seen in six patients, previously linked to dysmyelination/hypomyelination disorders, such as multiple sclerosis (MS) . In 1978 Mertin and Stackpoole showed that treatment with essential fatty acids, including arachidonic acid, suppresses experimental autoimmune encephalomyelitis in rats, and is abolished by inhibition of prostoglandin biosynthesis (Mertin and Stackpoole, 1978). In addition, decreased inhibition of startle generator structure was reported to be associated with MS (Ruprecht et al . , 2002). The calcium-independent PLA₂ inhibitor was also linked to reduced pre-pulse inhibition of the acoustic startle reflex in two other studies (Lee et al., 2009). Multiple studies link the PGE2 and AA pathways to neurodegenerative disorders, but their exact roles in causing white matter disorders remains unclear. The prominent dys-inhibition of the startle reflex in the patients may be related to brainstem lesions as a part of diffuse axonal and myelin damage. Additional interesting observation was that p.Leu752Phe substitution in PLAA results inability of the patient fibroblasts to induce IL-6, IL-8, and MIF in response to the NF-κΒ activating molecule LPS. While the pathophysiological relevance of this observation is yet to be determined, an association between neurodegenerative disorders and inflammatory cytokine responses has been suggested recently (Schmitz et al., 2015) . These cytokines are known for their pleiotropic function and are implicated in activation of microglia, proliferation, migration, and homing of different immune and non-immune cells. In addition, PGE; has been shown to be important leading to increased production of IL-6 and IL-8 (Cho et al., 2014). Thus, taken together, the pathogenesis of the leukoencephalopathy linked to the p.Leu752Phe substitution in PLAA implicates inability to mount appropriate inflammatory responses during pre-/post-neonatal development.

The phenotype of the patients, who are homozygous for p.Leu752Phe in PLAA with reduced C PLA2 activity and PGE₂ levels, adds new insights into this axis and its role in the pathogenesis of leukoencephalopathic disorders' pathogenesis.

Knockout mouse data provide a clue into potential disease mechanisms seen in the patients described herein. The disturbance in prostaglandin signaling results in a variety of pathological conditions. PLAA-deficient mice showed some phenotypes, specifically perinatal death, immature lungs with reduced PGE2 , and reduced body weight similar to that of Ptgsl (Cox-1 ) -Ptgs2 (Cox-2 ) double knockouts (Yu et al., 2006) and Ptgs3 knockouts (Nakatani et al., 2007) . The double mutants, as well as Ptgs4 knockouts (Nguyen et al., 1997) died perinatally due to patent ductus arteriosus, Ptgs3 knockouts showed perinatal death, possessed immature lungs, and PGE₂ levels were markedly decreased in the organ. The mutants also exhibited decreased body weight and skin morphological and physiological defects. Additionally, prostaglandin signaling is implicated for its roles in a range of physiological processes such as cell fate decision (Nissim et al . , 2014), cell differentiation (Li et al., 2000), and ciliogenesis (Jin et al., 2014). The inability of Plaa-null mice to survive, which may stem from impaired neuronal development in the brain, together with the findings that PGE? levels were significantly reduced in the brain and the lung of Plaa-null mouse embryos, raise the possibility that the pathogenesis of the condition observed in PLAA-deficient mice and in patients with a nonfunctional PLAA could be a developmental defect caused at least partly by ineffective prostaglandin signaling.

Clearly, the Plaa null mouse model provides an important tool to study the role of PLAA in CNS development and maintenance. Creating PJaa-conditional knockout mice or Plaa knock-in mice carrying the p.Leu752Phe sequence variant would enable researchers to perform more detailed histopathological studies of the brain and thus to learn what type of leukoencephalopathy is caused by the PLAA sequence variant described herein or by PLAA deficiency. Such an animal model would further contribute to the understanding of the significant role of arachidonic acid and PGE₂ pathway in the normal development and maintenance of the brain.

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Claims

What is claimed is:

1. A method of treating a subject afflicted with a white matter disorder comprising administering to the subject an agent that induces the activity of the Phospholipase A2-Acti ating Protein (PLAA) pathway in the subject.

2. The method of claim 1, wherein inducing the activity of the Phospholipase A2-Activating Protein (PLAA) pathway comprises inducing the activity of the Phospholipase A2-Activating Protein (PLAA) , increasing prostaglandin levels, increasing phospholipase A2 (PLA2) levels, increasing phospholipase A2

(PLA;) activity or a combination thereof.

3. The method of claim 1, wherein inducing the activity of the Phospholipase A2-Activating Protein (PLAA) pathway comprises inducing the activity of the Phospholipase A2-Activating Protein

(PLAA) .

4. The method of claim 1, wherein inducing the activity of the Phospholipase A2-Activating Protein (PLAA) pathway comprises increasing prostaglandin levels.

5. The method of claim 1, wherein inducing the activity of the Phospholipase A2-Activating Protein (PLAA) pathway comprises increasing Phospholipase A2 (PLA;) protein levels.

6. The method of claim 1, wherein inducing the activity of the Phospholipase A2-Activating Protein (PLAA) pathway comprises increasing Phospholipase A2 (PLA?) activity. 7. The method of claim 6, wherein the phospholipase A2 is cytosolic phospholipase A₂ (CPLA2)

The method of claim 7, wherein the cytosolic phospholipase A? (cPLA;) activity is measured in the membrane fraction of fibroblast cells. The method of any one of claims 1-8 wherein the agent is a gene therapy agent.

The method of any one of claims 1-9, wherein at least one copy of Plaa gene of the subject has at least one mutation.

The method of any one of claims 1-10, wherein the subject has reduced level of PLAA activity relative to a subject not afflicted with a white matter disorder.

The method of any one of claims 1-11, wherein the subject has reduced level of cytoplasmic Phosphol ipase A2 (PLA₂) activity relative to a subject not afflicted with a white matter disorder.

The method of any one of claims 1-12, wherein the subject has reduced level of at least one endogenous prostaglandin relative to a subject not afflicted with a white matter disorder.

The method of claim 13, wherein the at least one endogenous prostaglandin is Prostaglandin 2 (PGE₂) .

The method of any one of claims 1-14, wherein the subject has about 50% or less endogenous PGE; relative to a subject not afflicted with a white matter disorder.

The method of any one of claims 1-15, wherein the Plaa gene of the subject encodes a PLAA protein comprising a mutation at position Leu752.

The method of claim 16, wherein the mutation is a Leu752Phe mutation .

The method of claim 16 or 17, wherein the Plaa gene of the subject comprises a 22540T mutation.

The method of any one of claims 1-18 wherein the white matter disorder is hereditary.

20. The method of claim 19 wherein the disorder is a leukoencephalopathy .

21. The method of claim 20, wherein the subject has a dysmyelinating disorder

22. The method of any one of claims 1-18 wherein the disorder is a late onset white matter disease.

23. The method of claim 22 wherein the disorder is a demyelinating disease .

24. A method of determining if a subject is a carrier of leukoencephalopathy comprising: a) obtaining a biological sample from a subject; b) analyzing the genomic DNA of the biological sample for the presence of a 22540T mutation; c) determining the presence of a 22540T mutation in the Plaa gene; and d) determining the subject as a carrier of leukoencephalopathy if the presence of a 22540T mutation in the Plaa gene is detected.

25. The method of claim 24 wherein analyzing the genomic DNA of the biological sample for the presence of a 22540T mutation comprises sequencing the genomic DNA of the biological sample comprising a segment of the Plaa gene, using a specific probe for the 22540T mutation, or using PCR primers designed to specifically amplify the 22540T mutation.

26. A method of determining whether a subject is afflicted with, or predisposed to leukoencephalopathy comprising: a) obtaining a biological sample from a subject; b) analyzing the genomic DNA of the biological sample for the presence of a 22540T mutation; and c) determining the presence of a 22540T mutation in the Plaa gene . The method of claim 26, wherein analyzing the genomic DNA of the biological sample for the presence of a 22540T mutation comprises sequencing the genomic DNA of the biological sample comprising a segment of the Plaa gene, using a specific probe for the 22540T mutation, or using PCR primers designed to specifically amplify the 22540T mutation.

The method of any one of claims 24-27, wherein the method further comprises determining the presence of the wild type allele of the Plaa gene.

The method of claim 28, wherein if the wild type allele of the Plaa gene is not detected then the subject is determined to be afflicted with or predisposed to leukoencephalopathy.

The method of claim 26 or 27, further comprising the steps of; d) analyzing the genomic DNA of the biological sample for the presence of a wild type Plaa allele; and e) determining the subject as afflicted with or predisposed to leukoencephalopathy if the wild-type Plaa allele is not detected .

The method of any one of claims 24-30, wherein the biological sample is an embryonic sample.

The method of claim 31, wherein the embryo is an embryo at the eight-cell stage or from a blastocyst

The method of any one of claims 24-30, wherein the biological sample is a fetal sample.

The method of claim 33, wherein the fetal sample is taken from amniotic fluid.

A diagnostic kit for determining the presence of, or predisposition to leukoencephalopathy in a subject, comprising a forward primer and a reverse primer for amplifying at least a region of the Plaa gene from a biological sample according to a polymerase chain reaction.

36. The diagnostic kit of claim 35, wherein the region of the Plaa gene encodes a portion of the PUL domain of the PLAA protein.

37. The diagnostic kit of claim 35-36, wherein the biologic sample is taken from an embryo or fetus

38. The diagnostic kit of any one of claims 35-37, wherein the region of the Plaa gene encodes a portion of the PLAA protein comprising L752.

39. A diagnostic kit for determining the presence of, or predisposition to leukoencephalopathy in a subject, comprising a specific probe for the 22540T mutation, and/or PCR primers designed to specifically amplify the 22540T mutation.

40. A method of treating a subject suffering from neuroinflammation comprising administering to the subject a PLAA inhibitor.

41. The method of claim 40, wherein the subject is diagnosed as suffering from a neurodegenerative disease.

42. A transgenic mouse comprising a homozygous Leu752Phe mutation of the Plaa gene.