De novo mutations of KIAA2022 in females cause intellectual disability and intractable epilepsy.

BACKGROUND: Mutations in the KIAA2022 gene have been reported in male patients with X-linked intellectual disability, and related female carriers were unaffected. Here, we report 14 female patients who carry a heterozygous de novo KIAA2022 mutation and share a phenotype characterised by intellectual disability and epilepsy. METHODS: Reported females were selected for genetic testing because of substantial developmental problems and/or epilepsy. X-inactivation and expression studies were performed when possible. RESULTS: All mutations were predicted to result in a frameshift or premature stop. 12 out of 14 patients had intractable epilepsy with myoclonic and/or absence seizures, and generalised in 11. Thirteen patients had mild to severe intellectual disability. This female phenotype partially overlaps with the reported male phenotype which consists of more severe intellectual disability, microcephaly, growth retardation, facial dysmorphisms and, less frequently, epilepsy. One female patient showed completely skewed X-inactivation, complete absence of RNA expression in blood and a phenotype similar to male patients. In the six other tested patients, X-inactivation was random, confirmed by a non-significant twofold to threefold decrease of RNA expression in blood, consistent with the expected mosaicism between cells expressing mutant or normal KIAA2022 alleles. CONCLUSIONS: Heterozygous loss of KIAA2022 expression is a cause of intellectual disability in females. Compared with its hemizygous male counterpart, the heterozygous female disease has less severe intellectual disability, but is more often associated with a severe and intractable myoclonic epilepsy.

Neuronal ceroid lipofuscinosis with DNAJC5/CSPα mutation has PPT1 pathology and exhibit aberrant protein palmitoylation.

Neuronal ceroid lipofuscinoses (NCL) are a group of inherited neurodegenerative disorders with lysosomal pathology (CLN1-14). Recently, mutations in the DNAJC5/CLN4 gene, which encodes the presynaptic co-chaperone CSPα were shown to cause autosomal-dominant NCL. Although 14 NCL genes have been identified, it is unknown if they act in common disease pathways. Here we show that two disease-associated proteins, CSPα and the depalmitoylating enzyme palmitoyl-protein thioesterase 1 (PPT1/CLN1) are biochemically linked. We find that in DNAJC5/CLN4 patient brains, PPT1 is massively increased and mis-localized. Surprisingly, the specific enzymatic activity of PPT1 is dramatically reduced. Notably, we demonstrate that CSPα is depalmitoylated by PPT1 and hence its substrate. To determine the consequences of PPT1 accumulation, we compared the palmitomes from control and DNAJC5/CLN4 patient brains by quantitative proteomics. We discovered global changes in protein palmitoylation, mainly involving lysosomal and synaptic proteins. Our findings establish a functional link between two forms of NCL and serve as a springboard for investigations of NCL disease pathways.

Efficient Delivery of FMR1 across the Blood Brain Barrier Using AAVphp Construct in Adult FMR1 KO Mice Suggests the Feasibility of Gene Therapy for Fragile X Syndrome.

Background Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability and autism. Gene therapy may offer an efficient method to ameliorate the symptoms of this disorder. Methods An AAVphp.eb-hSyn-mFMR1IOS7 vector and an empty control were injected into the tail vein of adult Fmr1 knockout (KO) mice and wildtype (WT) controls. The KO mice were injected with 2 × 1013 vg/kg of the construct. The control KO and WT mice were injected with an empty vector. Four weeks following treatment, the animals underwent a battery of tests: open field, marble burying, rotarod, and fear conditioning. The mouse brains were studied for levels of the Fmr1 product FMRP. Results: No significant levels of FMRP were found outside the CNS in the treated animals. The gene delivery was highly efficient, and it exceeded the control FMRP levels in all tested brain regions. There was also improved performance in the rotarod test and partial improvements in the other tests in the treated KO animals. Conclusion: These experiments demonstrate efficient, brain-specific delivery of Fmr1 via peripheral administration in adult mice. The gene delivery led to partial alleviation of the Fmr1 KO phenotypical behaviors. FMRP oversupply may explain why not all behaviors were significantly affected. Since AAV.php vectors are less efficient in humans than in the mice used in the current experiment, studies to determine the optimal dose using human-suitable vectors will be necessary to further demonstrate feasibility.

On BC1 RNA and the fragile X mental retardation protein.

The fragile X mental retardation protein (FMRP), the functional absence of which causes fragile X syndrome, is an RNA-binding protein that has been implicated in the regulation of local protein synthesis at the synapse. The mechanism of FMRP's interaction with its target mRNAs, however, has remained controversial. In one model, it has been proposed that BC1 RNA, a small non-protein-coding RNA that localizes to synaptodendritic domains, operates as a requisite adaptor by specifically binding to both FMRP and, via direct base-pairing, to FMRP target mRNAs. Other models posit that FMRP interacts with its target mRNAs directly, i.e., in a BC1-independent manner. Here five laboratories independently set out to test the BC1-FMRP model. We report that specific BC1-FMRP interactions could be documented neither in vitro nor in vivo. Interactions between BC1 RNA and FMRP target mRNAs were determined to be of a nonspecific nature. Significantly, the association of FMRP with bona fide target mRNAs was independent of the presence of BC1 RNA in vivo. The combined experimental evidence is discordant with a proposed scenario in which BC1 RNA acts as a bridge between FMRP and its target mRNAs and rather supports a model in which BC1 RNA and FMRP are translational repressors that operate independently.

Aggregation of mutant cysteine string protein-α via Fe-S cluster binding is mitigated by iron chelators.

Point mutations in cysteine string protein-α (CSPα) cause dominantly inherited adult-onset neuronal ceroid lipofuscinosis (ANCL), a rapidly progressing and lethal neurodegenerative disease with no treatment. ANCL mutations are proposed to trigger CSPα aggregation/oligomerization, but the mechanism of oligomer formation remains unclear. Here we use purified proteins, mouse primary neurons and patient-derived induced neurons to show that the normally palmitoylated cysteine string region of CSPα loses palmitoylation in ANCL mutants. This allows oligomerization of mutant CSPα via ectopic binding of iron-sulfur (Fe-S) clusters. The resulting oligomerization of mutant CSPα causes its mislocalization and consequent loss of its synaptic SNARE-chaperoning function. We then find that pharmacological iron chelation mitigates the oligomerization of mutant CSPα, accompanied by partial rescue of the downstream SNARE defects and the pathological hallmark of lipofuscin accumulation. Thus, the iron chelators deferiprone (L1) and deferoxamine (Dfx), which are already used to treat iron overload in humans, offer a new approach for treating ANCL.

Tissue and developmental regulation of fragile X mental retardation 1 exon 12 and 15 isoforms.

The pre-mRNA of the fragile X mental retardation 1 gene (FMR1) is subject to exon skipping and alternative splice site selection, which can generate up to 12 isoforms. The expression and function of these variants in vivo has not yet been fully explored. In the present study, we investigated the distribution of Fmr1 exon 12 and exon 15 isoforms. Exon 12 encodes an extension of KH(2) domain, one of the RNA binding domains in the FMR1 gene product (FMRP) and we show that exon 12 variant proteins differentially interact with kissing complex RNA. Alternative splicing at exon 15 produces FMRPs differing in RNA binding ability and each is distinguished by unique post-translational modifications. Using semiquantitative RT-PCR and Northern blotting, we found that particular Fmr1 exon 12 and exon 15 isoforms change during neuronal differentiation. Interestingly, Fmr1 exon 12 variants display tissue-specific and developmental differences, while exon 15-containing transcripts vary less. Altogether, the spatio-temporal plasticity of FMR1 mRNA is consistent with complex RNA processing that is mis-regulated in fragile X syndrome.

Chemical and structural probing of the N-terminal residues encoded by FMR1 exon 15 and their effect on downstream arginine methylation.

Exon 15 of the fragile X mental retardation protein gene (FMR1) is alternatively spliced into three variants. The amino acids encoded by the 5' end of the exon contain several regulatory determinants including phosphorylation sites and a potential conformational switch. Residues encoded by the 3' end of the exon specify FMRP's RGG box, an RNA binding domain that interacts with G-quartet motifs. Previous studies demonstrated that the exon 15-encoded N-terminal residues influence the extent of arginine methylation, independent of S 500 phosphorylation. In the present study we focus on the role the putative conformational switch plays in arginine methylation. Chemical and structural probing of Ex15 alternatively spliced variant proteins and several mutants leads to the following conclusions: Ex15c resides largely in a conformation that is refractory toward methylation; however, it can be methylated by supplementing extracts with recombinant PRMT1 or PRMT3. Protein modeling studies reveal that the RG-rich region is part of a three to four strand antiparallel beta-sheet, which in other RNA binding proteins functions as a platform for nucleic acid interactions. In the Ex15c variant the first strand of this sheet is truncated, and this significantly perturbs the side-chain conformations of the arginine residues in the RG-rich region. Mutating R 507 in the conformational switch to K also truncates the first strand of the beta-sheet, and corresponding decreases in in vitro methylation were found for this and R 507/R 544 and R 507/R 546 double mutants. These effects are not due to the loss of R 507 methylation as a conformational switch-containing peptide reacted under substrate excess and in methyl donor excess was not significantly methylated. Consistent with this, similar changes in beta-sheet structure and decreases in in vitro methylation were observed with a W 513-K mutant. These data support a novel model for FMRP arginine methylation and a role for conformational switch residues in arginine modification.

Oxidative stress reveals heterogeneity of FMRP granules in PC12 cell neurites.

PC12 cells are a well-known model of parasympathetic neurons. They have also been used to study the dynamics of heterologously expressed fragile X mental retardation (FMRP) granule trafficking down neurites. Here, we demonstrate that undifferentiated and differentiated PC12 cells harbor endogenous FMRP-containing granules. These granules are not stress granules because they do not associate with an authentic stress granule marker protein T-cell internal antigen 1 (TIA-1). Treatment with sodium arsenite induces stress granule formation in undifferentiated and differentiated PC12 cells. In NGF-treated cells, FMRP-containing stress granules are observed in the soma, neurites and growth cones by co-immunostaining with anti-TIA-1 antibody. These data demonstrate that all three microdomains respond similarly to oxidative stress. Nevertheless, we find significantly less co-localization of FMRP and TIA-1 and FMRP and its homologs in the neurites of differentiated PC12 cells treated with sodium arsenite than in the soma or growth cones. The heterogeneity of these granules suggests that FMRP has multiple roles in neurites.

Increased Expression of the Large Conductance, Calcium-Activated K+ (BK) Channel in Adult-Onset Neuronal Ceroid Lipofuscinosis.

Cysteine string protein (CSPα) is a presynaptic J protein co-chaperone that opposes neurodegeneration. Mutations in CSPα (i.e., Leu115 to Arg substitution or deletion (Δ) of Leu116) cause adult neuronal ceroid lipofuscinosis (ANCL), a dominantly inherited neurodegenerative disease. We have previously demonstrated that CSPα limits the expression of large conductance, calcium-activated K+ (BK) channels in neurons, which may impact synaptic excitability and neurotransmission. Here we show by western blot analysis that expression of the pore-forming BKα subunit is elevated ~2.5 fold in the post-mortem cortex of a 36-year-old patient with the Leu116∆ CSPα mutation. Moreover, we find that the increase in BKα subunit level is selective for ANCL and not a general feature of neurodegenerative conditions. While reduced levels of CSPα are found in some postmortem cortex specimens from Alzheimer's disease patients, we find no concomitant increase in BKα subunit expression in Alzheimer's specimens. Both CSPα monomer and oligomer expression are reduced in synaptosomes prepared from ANCL cortex compared with control. In a cultured neuronal cell model, CSPα oligomers are short lived. The results of this study indicate that the Leu116∆ mutation leads to elevated BKα subunit levels in human cortex and extend our initial work in rodent models demonstrating the modulation of BKα subunit levels by the same CSPα mutation. While the precise sequence of pathogenic events still remains to be elucidated, our findings suggest that dysregulation of BK channels may contribute to neurodegeneration in ANCL.

A novel p.Leu(381)Phe mutation in presenilin 1 is associated with very early onset and unusually fast progressing dementia as well as lysosomal inclusions typically seen in Kufs disease.

Whole exome sequencing in a family with suspected dominant Kufs disease identified a novel Presenilin 1 mutation p.Leu(381)Phe in three brothers who, along with their father, developed progressive dementia and motor deficits in their early 30 s. All affected relatives had unusually rapid disease progression (on average 3.6 years from disease onset to death). In silico analysis of mutation p.Leu(381)Phe predicted more detrimental effects when compared to the common Presenilin 1 mutation p.Glu(280)Ala. Electron microscopy study of peripheral fibroblast cells of the proband showed lysosomal inclusions typical for Kufs disease. However, brain autopsy demonstrated typical changes of Alzheimer's disease.

Dysmorphic features, cognitive disability, chronic inflammation, and predisposition to vascular disease in two sisters: a new autosomal recessive disorder?

A 20-year-old woman presented with mental retardation and a history of stroke related to moyamoya disease at the age of 8 years. She had cognitive impairment which became more pronounced after the stroke. This patient's parents were first cousins and six close family relatives had strokes in their 60s or 70s. The patient's 16-year-old sister had learning disability, chronic muscle pain, and an ECG suggestive of previous hypoxemic heart injury. The two sisters had similar dysmorphic facial appearance including a prominent philtrum, bulbous nose, and severe acne. They both had increased subcutaneous tissue in their faces, whereas their bodies were slim. Both sisters were found to have elevated levels of rheumatoid factor, C-reactive protein, and erythrocyte sedimentation rate on repeat measurements. Partial autoimmunity screening in one of the patients was negative. Chromosome analysis and array comparative genomic hybridization analyses were also normal. Nerve conduction findings in the younger sister were consistent with distal, predominantly motor, demyelinating neuropathy localized to the lower extremities. We propose that these two sisters suffer from a new autosomal recessive syndrome. Carrier status for this condition may predispose to later onset stroke.

Mutations in the gene DNAJC5 cause autosomal dominant Kufs disease in a proportion of cases: study of the Parry family and 8 other families.

BACKGROUND: The Neuronal Ceroid Lipofuscinoses (NCL) comprise at least nine progressive neurodegenerative genetic disorders. Kufs disease, an adult-onset form of NCL may be recessively or dominantly inherited. Our study aimed to identify genetic mutations associated with autosomal dominant Kufs disease (ADKD). METHODOLOGY AND PRINCIPAL FINDINGS: We have studied the family first reported with this phenotype in the 1970s, the Parry family. The proband had progressive psychiatric manifestations, seizures and cognitive decline starting in her mid 20 s. Similarly affected relatives were observed in seven generations. Several of the affected individuals had post-mortem neuropathological brain study confirmatory for NCL disease. We conducted whole exome sequencing of three affected family members and identified a pLeu116del mutation in the gene DNAJC5, which segregated with the disease phenotype. An additional eight unrelated affected individuals with documented autosomal dominant or sporadic inheritance were studied. All had diagnostic confirmation with neuropathological studies of brain tissue. Among them we identified an additional individual with a p.Leu115Arg mutation in DNAJC5. In addition, a pAsn477Ser change in the neighboring gene PRPF6, a gene previously found to be associated with retinitis pigmentosa, segregated with the ADKD phenotype. Interestingly, two individuals of the Parry family did report visual impairment. CONCLUSIONS: Our study confirmed the recently reported association of DNAJC5 mutations with ADKD in two out of nine well-defined families. Sequence changes in PRPF6 have not been identified in other unrelated cases. The association of vision impairment with the expected PRPF6 dysfunction remains possible but would need further clinical studies in order to confirm the co-segregation of the visual impairment with this sequence change.

Clavicular pseudoarthrosis, anomalous coronary artery and extra crease of the fifth finger-previously unreported features in individuals with class II 1q21.1 microdeletions.

Submicroscopic deletions of two adjacent regions within chromosomal band 1q21.1 were recently associated with two distinct phenotypes: A deleted region of 200-500 kb was found in individuals with Thrombocytopenia Absent Radius syndrome (TAR). Deletion in another region of about 1.25 Mb that is located just telomeric of the TAR region, referred to as distal 1q21.1 region, was found to be associated with a phenotype of cognitive impairment, congenital heart defects and other variable manifestations. A significant proportion of individuals with either of the two deletions did not have phenotypic abnormalities. More than 40 individuals with distal 1q21.1 deletions have been reported to-date. A proportion of them (9 reported individuals) had larger (>2 Mb) deletions involving both the TAR and the distal 1q21.1 regions, referred to as class II deletions. We describe here four additional individuals from two families with such class II deletions, who presented with previously unreported manifestations: clavicular pseudoarthrosis and anomalous origin of the coronary artery in the proband of the first family; an extra transverse crease of the fifth finger, segregating in two of the three deletion carriers in the second family. Previously reported features, associated with such microdeletions - absolute or relative microcephaly, cognitive impairment and short stature, were variably observed in the reported individuals. This report expands the phenotypic spectrum associated with class II 1q21.1 deletions, and demonstrates striking phenotypic variability even within the same family.

Mitochondrial T9098C sequence change in the MTATP6 gene and development of severe mitochondrial disease after in utero antiretroviral prophylaxis.

Mitochondrial toxicity is a well-recognized adverse effect of nucleoside reverse transcriptase inhibitor therapy for human immunodeficiency virus (HIV) infection. Transient lactic acidosis is often observed in children born after in utero antiretroviral prophylaxis against mother-to-child transmission of HIV. However, the extent and the mechanism of in utero adverse effects are largely unknown. We describe a 4-year-old girl who presented with manifestations of severe mitochondrial disease, specifically, developmental and growth delay, hypotonia, lactic acidosis, congenital cataracts, and pancreatitis. Her HIV-positive mother was receiving lamivudine, zidovudine, and nelfinavir mesylate during her pregnancy. The child tested HIV negative after birth. She was found to have a homoplastic T9098C sequence change in the mitochondrial gene coding for adenosine 5'-triphosphate synthase 6 (MTATP6) that was previously reported as a mitochondrial polymorphism. This polymorphism is in the MTATP6 gene-coding sequence and leads to the replacement of a nonpolar amino acid with a polar amino acid. Because of the typical clinical manifestations of mitochondrial disorder and because of the nature of the mitochondrial sequence change, the observed polymorphism likely predisposed this patient to develop severe antiretroviral-associated mitochondrial disease. Mitochondrial sequence alterations may be important factors in mitochondrial toxicity associated with antiretroviral treatment. Mitochondrial sequencing may be warranted in cases of persistent lactic acidosis after antiretroviral prophylaxis to further study this association.

A novel 1.4 Mb de novo microdeletion of chromosome 1q21.3 in a child with microcephaly, dysmorphic features and mental retardation.

A 2.5 years old girl presented with moderate mental retardation, microcephaly, arching eyebrows, low set ears, long eyelashes, persistent fetal pads and clinodactyly. About 1 Mb deletion in the chromosomal region 1q21.3 was identified using BAC array CGH analysis. The parental follow up FISH analysis was normal. Further study of the deletion using a 244K oligo-array of Agilent Technologies Inc., Santa Clara, CA, USA defined the deleted region to span about 1.4 Mb with approximate genomic location chr1:152,511,593-153,993,103 (NCBI genome build 36). This is a novel deletion, not reported to-date. Larger proximal 1q deletions that were previously reported typically included microcephaly, mental retardation and multiple congenital anomalies. The deleted region reported here includes at least 30 coding genes. Among them of interest is a three-gene cluster of the ephrin gene family (EFNA1, EFNA3 and EFNA4). This is a group of receptor protein-tyrosine kinase type genes with presumed, but not completely characterized function in nervous system development.

De-novo 2.15 Mb terminal Xq duplication involving MECP2 but not L1CAM gene in a male patient with mental retardation.

Distal Xq disomy in males results in characteristic phenotypes that typically include mental retardation, microcephaly, prominent hypotonia and hypogonadism. The 8-year-old male patient reported here presented with mental retardation, prominent ears, abnormally wide and unstable gait and flat occiput. He did not have microcephaly or hypogonadism. Subtelomeric multi-fluorescence in-situ hybridization analysis identified a duplicated terminal portion of chromosome Xq/Yq located distally on Yp. Further analysis of the duplicated region using additional FISH probes, specific for the distal Xq and Yp chromosomal regions, and array comparative genomic hybridization analysis using the 244 K oligo-array of Agilent, showed that it spans approximately 2.15 Mb of the terminal Xq region and includes MECP2 but not L1CAM gene. This is the smallest well-characterized terminal Xq duplication reported to date. Genes proximal to MECP2 that are not duplicated in our patient are likely responsible for additional clinical manifestations including characteristic facial dysmorphic features, microcephaly, hypogonadism and more severe hypotonia, as noted in patients with larger distal Xq duplications. Our patient's features are similar to previously reported MECP2 gene duplication cases, thus suggesting minor or no contribution of duplicated genes distal of MECP2 to the reported phenotype.

Prosegment of tripeptidyl peptidase I is a potent, slow-binding inhibitor of its cognate enzyme.

Tripeptidyl peptidase I (TPP I) is the first mammalian representative of a family of pepstatin-insensitive serine-carboxyl proteases, or sedolisins. The enzyme acts in lysosomes, where it sequentially removes tripeptides from the unmodified N terminus of small, unstructured polypeptides. Naturally occurring mutations in TPP I underlie a neurodegenerative disorder of childhood, classic late infantile neuronal ceroid lipofuscinosis (CLN2). Generation of mature TPP I is associated with removal of a long prosegment of 176 amino acid residues from the zymogen. Here we investigated the inhibitory properties of TPP I prosegment expressed and isolated from Escherichia coli toward its cognate protease. We show that the TPP I prosegment is a potent, slow-binding inhibitor of its parent enzyme, with an overall inhibition constant in the low nanomolar range. We also demonstrate the protective effect of the prosegment on alkaline pH-induced inactivation of the enzyme. Interestingly, the inhibitory properties of TPP I prosegment with the introduced classic late infantile neuronal ceroid lipofuscinosis disease-associated mutation, G77R, significantly differed from those revealed by wild-type prosegment in both the mechanism of interaction and the inhibitory rate. This is the first characterization of the inhibitory action of the sedolisin prosegment.

Alternative splicing modulates protein arginine methyltransferase-dependent methylation of fragile X syndrome mental retardation protein.

The fragile X mental retardation protein (FMRP) is an RNA binding protein that is methylated by an endogenous methyltransferase in rabbit reticulocyte lysates. We mapped the region of methylation to the C-terminal arginine-glycine-rich residues encoded by FMR1 exon 15. We additionally demonstrated that mutation of R(544) to K reduced the endogenous methylation by more than 80%, while a comparable mutant R(546)-K reduced the endogenous methylation by 20%. These mutations had no effect on the subcellular distribution of FMRP, recapitulating previous results using the methyltransferase inhibitor adenosine-2',3'-dialdehyde. Using purified recombinant protein arginine methyltransferases (PRMTs), we showed that the C-terminal domain could be methylated by PRMT1, PRMT3, and PRMT4 in vitro and that both the R(544)-K mutant and the R(546)-K mutant were refractory toward these enzymes. We also report that truncating the N-terminal 12 residues encoded by FMR1 exon 15, which occurs naturally via alternative splicing, had no effect on FMRP methylation, demonstrating conclusively that phosphorylation of serine residue 500 (S(500)), one of the 12 residues, was not required for methylation. Nevertheless, truncating 13 additional amino acids, as occurs in the smallest alternatively spliced variant of FMR1 exon 15, reduced methylation by more than 85%. This suggests that differential expression and methylation of the FMRP exon 15 variants may be an important means of regulating target mRNA translation, which is consonant with recently demonstrated functional effects mediated by inhibiting FMRP methylation in cultured cells.

Methylation regulates the intracellular protein-protein and protein-RNA interactions of FMRP.

FMRP, the fragile X mental retardation protein, is an RNA-binding protein that interacts with approximately 4% of fetal brain mRNA. We have recently shown that a methyltransferase (MT) co-translationally methylates FMRP in vitro and that methylation modulates the ability of FMRP to bind mRNA. Here, we recapitulate these in vitro data in vivo, demonstrating that methylation of FMRP affects its ability to bind to FXR1P and regulate the translation of FMRP target mRNAs. Additionally, using double-label fluorescence confocal microscopy, we identified a subpopulation of FMRP-containing small cytoplasmic granules that are distinguishable from larger stress granules. Using the oxidative-stress induced accumulation of abortive pre-initiation complexes as a measure of the association of FMRP with translational components, we have demonstrated that FMRP associates with ribosomes during initiation and, more importantly, that methylation regulates this process by influencing the ratio of FMRP-homodimer-containing mRNPs to FMRP-FXR1P-heterodimer-containing mRNPs. These data suggest a vital role for methylation in normal FMRP functioning.