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Acute otitis media (OM) is a common disease which often develops through complex interactions between the host, the pathogen and environmental factors. We studied single nucleotide polymorphisms (SNPs) of genes involved in innate and adaptive immunity, and other host and environmental factors for their role in OM. Using Sequenom Massarray platform, 21 SNPs were studied in 653 children from prospective (n = 202) and retrospective (n = 451) cohorts. Data were analyzed for the relationship between SNPs and upper respiratory infection (URI) frequency, risk of acute OM during URI episodes, and proneness to recurrent OM. Increased risk for OM proneness was associated with CX3CR1 (Thr280Met) SNP and with a jointly interactive group of IL-10 (-1082) SNP, IL-1β (-511) wild type genotype and white race. Family history of OM proneness independently increased the risk for frequent URIs, OM occurrence during URI, and OM proneness. Additionally, IL-1β (-31) SNP was associated with increased risk for frequent URIs, but IL-10 (-592), IL-1β (-511), IL-5 (-746) and IL-8 (-251) SNPs were associated with decreased risk of URI. IL-1β (-31), CX3CR1 (Thr280Met), IL-10 (-1082) and IL-1β (-511) SNPs were associated with increased risk for frequent URIs or OM proneness.

Canavan disease (CD) is a fatal leukodystrophy caused by mutation of the aspartoacylase (ASPA) gene, which leads to deficiency in ASPA activity, accumulation of the substrate N‐acetyl‐L‐aspartate (NAA), demyelination, and spongy degeneration of the brain. There is neither a cure nor a standard treatment for this disease. In this study, human induced pluripotent stem cell (iPSC)‐based cell therapy is developed for CD. A functional ASPA gene is introduced into patient iPSC‐derived neural progenitor cells (iNPCs) or oligodendrocyte progenitor cells (iOPCs) via lentiviral transduction or TALEN‐mediated genetic engineering to generate ASPA iNPC or ASPA iOPC. After stereotactic transplantation into a CD (Nur7) mouse model, the engrafted cells are able to rescue major pathological features of CD, including deficient ASPA activity, elevated NAA levels, extensive vacuolation, defective myelination, and motor function deficits, in a robust and sustainable manner. Moreover, the transplanted mice exhibit much prolonged survival. These genetically engineered patient iPSC‐derived cellular products are promising cell therapies for CD. This study has the potential to bring effective cell therapies, for the first time, to Canavan disease children who have no treatment options. The approach established in this study can also benefit many other children who have deadly genetic diseases that have no cure. Canavan disease (CD) is a leukodystrophy caused by mutation of the aspartoacylase(ASPA) gene. A functional ASPA gene is introduced into patient induced pluripotent stem cell (iPSC)‐derived neural progenitor or oligodendrocyte progenitor cells. After transplantation into CD (Nur7) mice, the engrafted cells could rescue major pathological features of CD, including vacuolation, defective myelination, and motor function deficits, and prolonge survival.

Background Exon-targeted microarrays can detect small (<1000 bp) intragenic copy number variants (CNVs), including those that affect only a single exon. This genome-wide high-sensitivity approach increases the molecular diagnosis for conditions with known disease-associated genes, enables better genotype–phenotype correlations, and facilitates variant allele detection allowing novel disease gene discovery. Methods We retrospectively analyzed data from 63,127 patients referred for clinical chromosomal microarray analysis (CMA) at Baylor Genetics laboratories, including 46,755 individuals tested using exon-targeted arrays, from 2007 to 2017. Small CNVs harboring a single gene or two to five non-disease-associated genes were identified; the genes involved were evaluated for a potential disease association. Results In this clinical population, among rare CNVs involving any single gene reported in 7200 patients (11%), we identified 145 de novo autosomal CNVs (117 losses and 28 intragenic gains), 257 X-linked deletion CNVs in males, and 1049 inherited autosomal CNVs (878 losses and 171 intragenic gains); 111 known disease genes were potentially disrupted by de novo autosomal or X-linked (in males) single-gene CNVs. Ninety-one genes, either recently proposed as candidate disease genes or not yet associated with diseases, were disrupted by 147 single-gene CNVs, including 37 de novo deletions and ten de novo intragenic duplications on autosomes and 100 X-linked CNVs in males. Clinical features in individuals with de novo or X-linked CNVs encompassing at most five genes (224 bp to 1.6 Mb in size) were compared to those in individuals with larger-sized deletions (up to 5 Mb in size) in the internal CMA database or loss-of-function single nucleotide variants (SNVs) detected by clinical or research whole-exome sequencing (WES). This enabled the identification of recently published genes ( BPTF , NONO , PSMD12 , TANGO2 , and TRIP12 ), novel candidate disease genes ( ARGLU1 and STK3 ), and further confirmation of disease association for two recently proposed disease genes ( MEIS2 and PTCHD1 ). Notably, exon-targeted CMA detected several pathogenic single-exon CNVs missed by clinical WES analyses. Conclusions Together, these data document the efficacy of exon-targeted CMA for detection of genic and exonic CNVs, complementing and extending WES in clinical diagnostics, and the potential for discovery of novel disease genes by genome-wide assay.

Canavan's disease (CD) is a fatal, hereditary disorder of CNS development that has been linked to mutations in the gene for the enzyme aspartoacylase (ASPA) (EC 3.5.1.15). ASPA acts to hydrolyze N-acetylaspartate (NAA) into L-aspartate and acetate, but the connection between ASPA deficiency and the failure of proper CNS development is unclear. We hypothesize that one function of ASPA is to provide acetate for the increased lipid synthesis that occurs during postnatal CNS myelination. The gene encoding ASPA has been inactivated in the mouse model of CD, and here we show significant decreases in the synthesis of six classes of myelin-associated lipids, as well as reduced acetate levels, in the brains of these mice at the time of peak postnatal CNS myelination. Analysis of the lipid content of white matter from a human CD patient showed decreased cerebroside and sulfatide relative to normal white matter. These results demonstrate that myelin lipid synthesis is significantly compromised in CD and provide direct evidence that defective myelin synthesis, resulting from a deficiency of NAA-derived acetate, is involved in the pathogenesis of CD.

Phenylketonuria patients harboring a subset of phenylalanine hydroxylase (PAH) mutations have recently shown normalization of blood phenylalanine levels upon oral administration of the PAH cofactor tetrahydrobiopterin [(6R)-L-erythro-5,6,7,8-tetrahydrobiopterin ( BH4)]. Several hypotheses have been put forward to explain BH4responsiveness, but the molecular basis for the corrective effect(s) of BH4has not been understood. We have investigated the biochemical, kinetic, and structural changes associated with BH4-responsive mutations (F39L, I65T, R68S, H170D, E178G, V190A, R261Q, A300S, L308F, A313T, A373T, V388M, E390G, P407S, and Y414C). The biochemical and kinetic characterization of the 15 mutants studied points toward a multifactorial basis for the BH4responsiveness; the mutants show residual activity (>30% of WT) and display various kinetic defects, including increased Km( BH4) and reduced cooperativity of substrate binding, but no decoupling of cofactor ( BH4) oxidation. For some, BH4seems to function through stabilization and protection of the enzyme from inactivation and proteolytic degradation. In the crystal structures of a phenylketonuria mutant, A313T, minor changes were seen when compared with the WT PAH structures, consistent with the mild effects the mutant has upon activity of the enzyme both in vitro and in vivo. Truncations made in the A313T mutant PAH form revealed that the N and C termini of the enzyme influence active site binding. Of fundamental importance is the observation that BH4appears to increase Phe catabolism if at least one of the two heterozygous mutations has any residual activity remaining.

Interstitial deletions of the short arm of chromosome 6 are rare and have been associated with developmental delay, hypotonia, congenital anomalies, and dysmorphic features. We used array comparative genomic hybridization in a South Carolina Autism Project (SCAP) cohort of 97 subjects with autism spectrum disorders (ASDs) and identified an ~ 5.4 Mb deletion on chromosome 6p22.3-p23 in a 15-year-old patient with intellectual disability and ASDs. Subsequent database queries revealed five additional individuals with overlapping submicroscopic deletions and presenting with developmental and speech delay, seizures, behavioral abnormalities, heart defects, and dysmorphic features. The deletion found in the SCAP patient harbors ATXN1 , DTNBP1 , JARID2 , and NHLRC1 that we propose may be responsible for ASDs and developmental delay.

The role of N -acetylaspartate in the brain is unclear. Here we used specific antibodies against N -acetylaspartate and immunocytochemistry of carbodiimide-fixed adult rodent brain to show that, besides staining of neuronal cell bodies in the grey matter, N -acetylaspartate labelling was present in oligodendrocytes/myelin in white matter tracts. Immunoelectron microscopy of the rat hippocampus showed that N -acetylaspartate was concentrated in the myelin. Also neuronal cell bodies and axons contained significant amounts of N -acetylaspartate, while synaptic elements and astrocytes were low in N -acetylaspartate. Mitochondria in axons and neuronal cell bodies contained higher levels of N -acetylaspartate compared to the cytosol, compatible with synthesis of N -acetylaspartate in mitochondria. In aspartoacylase knockout mice, in which catabolism of N -acetylaspartate is blocked, the levels of N -acetylaspartate were largely increased in oligodendrocytes/myelin. In these mice, the highest myelin concentration of N -acetylaspartate was found in the cerebellum, a region showing overt dysmyelination. In organotypic cortical slice cultures there was no evidence for N -acetylaspartate-induced myelin toxicity, supporting the notion that myelin damage is induced by the lack of N -acetylaspartate for lipid production. Our findings also implicate that N -acetylaspartate signals on magnetic resonance spectroscopy reflect not only vital neurons but also vital oligodendrocytes/myelin.

Childhood ataxia with diffuse central nervous system hypomyelination syndrome (CACH) is a recently described leukodystrophy of unknown etiology. To characterize the neuropathological features and gain insight as to the pathogenesis of this disorder, we studied cerebral tissue from six patients with the CACH syndrome. Evaluation of toluidine blue-stained, semithin sections of white matter from CACH patients disclosed unusual cells with \"foamy\" cytoplasm, small round nuclei and fine chromatin. Electron microscopy (EM) revealed cells in the white matter with abundant cytoplasm containing many mitochondria and loosely clustered, membranous structures, but lacking the lysosomal structures seen in macrophages. Further analysis of tissue sections with antibodies and special stains demonstrated that the abnormal cells with abundant cytoplasm labeled with oligodendroglial markers, but did not react with macrophage or astrocytic markers. Double immunolabeling with macrophage and oligodendroglial markers clearly distinguished macrophages from the \"foamy\" oligodendroglial cells (FODCs). Proteolipid protein (PLP) mRNA in situ hybridization demonstrated PLP mRNA transcripts in a high proportion of oligodendrocytes in CACH patients compared to control patients, and PLP mRNA transcript signal in cells, morphologically consistent with FODCs. Normal and pathological brain control tissues did not contain FODCs. These neuropathological findings will be useful pathological identifiers of CACH, and may provide clues to the pathogenesis of this disorder.

Canavan’s disease (CD) is a fatal pediatric leukodystrophy caused by mutations in aspartoacylase (AspA) gene. Currently, there is no effective treatment for CD; however, gene therapy is an attractive approach to ameliorate the disease. Here, we studied progressive neuropathology and gene therapy in short-lived (≤1 month) AspA−/− mice, a bona-fide animal model for the severest form of CD. Single intravenous (IV) injections of several primate-derived recombinant adeno-associated viruses (rAAVs) as late as postnatal day 20 (P20) completely rescued their early lethality and alleviated the major disease symptoms, extending survival in P0-injected rAAV9 and rAAVrh8 groups to as long as 2 years thus far. We successfully used microRNA (miRNA)-mediated post-transcriptional detargeting for the first time to restrict therapeutic rAAV expression in the central nervous system (CNS) and minimize potentially deleterious effects of transgene overexpression in peripheral tissues. rAAV treatment globally improved CNS myelination, although some abnormalities persisted in the content and distribution of myelin-specific and -enriched lipids. We demonstrate that systemically delivered and CNS-restricted rAAVs can serve as efficacious and sustained gene therapeutics in a model of a severe neurodegenerative disorder even when administered as late as P20.