Largest GWAS of PTSD (N=20 070) yields genetic overlap with schizophrenia and sex differences in heritability.

The Psychiatric Genomics Consortium-Posttraumatic Stress Disorder group (PGC-PTSD) combined genome-wide case-control molecular genetic data across 11 multiethnic studies to quantify PTSD heritability, to examine potential shared genetic risk with schizophrenia, bipolar disorder, and major depressive disorder and to identify risk loci for PTSD. Examining 20 730 individuals, we report a molecular genetics-based heritability estimate (h2SNP) for European-American females of 29% that is similar to h2SNP for schizophrenia and is substantially higher than h2SNP in European-American males (estimate not distinguishable from zero). We found strong evidence of overlapping genetic risk between PTSD and schizophrenia along with more modest evidence of overlap with bipolar and major depressive disorder. No single-nucleotide polymorphisms (SNPs) exceeded genome-wide significance in the transethnic (overall) meta-analysis and we do not replicate previously reported associations. Still, SNP-level summary statistics made available here afford the best-available molecular genetic index of PTSD-for both European- and African-American individuals-and can be used in polygenic risk prediction and genetic correlation studies of diverse phenotypes. Publication of summary statistics for ∼10 000 African Americans contributes to the broader goal of increased ancestral diversity in genomic data resources. In sum, the results demonstrate genetic influences on the development of PTSD, identify shared genetic risk between PTSD and other psychiatric disorders and highlight the importance of multiethnic/racial samples. As has been the case with schizophrenia and other complex genetic disorders, larger sample sizes are needed to identify specific risk loci.

Exome sequencing detection of two untranslated GFPT1 mutations in a family with limb-girdle myasthenia.

The term 'limb-girdle myasthenia' (LGM) was first used to describe three siblings with proximal limb weakness without oculobulbar involvement, but with EMG decrement and responsiveness to anticholinesterase medication. We report here that exome sequencing in the proband of this family revealed several sequence variations in genes linked to proximal limb weakness. However, the only mutations that cosegregated with disease were an intronic IVS7-8A>G mutation and the previously reported 3'-UTR c.*22C>A mutation in GFPT1, a gene linked to LGM. A minigene assay showed that IVS7-8A>G activates an alternative splice acceptor that results in retention of the last seven nucleotides of intron 7 and a frameshift leading to a termination codon 13 nucleotides downstream from the new splice site. An anconeus muscle biopsy revealed mild reduction of the axon terminal size and postsynaptic fold simplification. The amplitudes of miniature endplate potentials and quantal release were also diminished. The DNA of the mildly affected father of the proband showed only the intronic mutation along with sequence variations in other genes potentially relevant to LGM. Thus, this study performed in the family originally described with LGM showed two GFPT1 untranslated mutations, which may cause disease by reducing GFPT1 expression and ultimately impairing protein glycosylation.

A recombination-based assay demonstrates that the fragile X sequence is transcribed widely during development.

To identify transcribed sequences rapidly and efficiently, we have developed a recombination-based assay to screen bacteriophage lambda libraries for sequences that share homology with a given probe. This strategy determines analytically whether a given probe is transcribed in a given tissue at a given time of development, and may also be used to isolate preparatively the transcribed sequence free of the screening probe. We illustrate this technology for the fragile X sequence, demonstrating that it is transcribed ubiquitously in an 11 week fetus, in a variety of 20 week human fetal tissues, including brain, spinal cord, eye, liver, kidney and skeletal muscle, and in adult jejunum.

Plasmids for recombination-based screening.

To facilitate recombination-based screening, we constructed the ColE1-based plasmid, pi G4, that confers chloramphenicol resistance, contains a polylinker with multiple unique restriction enzyme recognition sequences, and contains the genetic marker, supF. To facilitate recombination-based screening followed by rapid DNA sequencing, we inserted the selectable marker, supF, into each of 20 high-copy-number (hcn) pUC-derived NoC plasmids that were designed for multiplex DNA sequencing. To facilitate recombination-based screening of common cDNA libraries that often contain ColE1 sequences, we constructed a supF-carrying plasmid whose replication was driven from an R6K replicon that does not share sequence homology with ColE1. Furthermore, we incorporated a useful polylinker and increased the copy number of this plasmid to create the 4.4-kb hcn plasmid, pMAD1. Thus, these plasmids allow: (1) background-free transformation of cells by a supF plasmid carrying an antibiotic-resistance marker; (2) simultaneous performance of the recombination-based assay and DNA sequencing; and (3) screening bacteriophage cDNA libraries that contain ColE1 sequences by recombination with a supF plasmid that is not homologous to ColE1 derivatives.

Improved adenoviral vectors for gene therapy of Duchenne muscular dystrophy.

We have been exploring the feasibility of gene therapy for Duchenne muscular dystrophy by characterizing parameters important for the design of therapeutic protocols. These studies have used transgenic mice to analyze expression patterns of multiple dystrophin vectors, and have been accompanied by the development of viral vectors for gene transfer to dystrophic mdx mouse muscle. Analysis of transgenic mdx mice indicates that greater than 50% of the fibers in a muscle group must express dystrophin to prevent development of a significant dystrophy, and that low-level expression of truncated dystrophins can function very well. These results suggest that gene therapy of DMD will require methods to transduce the majority of fibers in critical muscle groups with vectors that express moderate levels of dystrophin proteins. Strategies for the development of viral vectors able to deliver dystrophin genes to muscle include the use of muscle specific regulatory sequences coupled with deletion of viral gene sequences to limit virus-induced immune rejection of transduced tissues. These strategies should enable production of adenoviral vectors expressing full-length dystrophin proteins in muscle.

Prevalence of myocilin mutations in adults with primary open-angle glaucoma in Ghana, West Africa.

PURPOSE: Investigators have noted that primary open-angle glaucoma (POAG) in West Africa has an earlier age of onset and appears to be more clinically severe than in the United States and Europe. Primary open-angle glaucoma patients with mutations in myocilin have a similar phenotype. Therefore, we investigated the role of mutations in myocilin in patients with POAG in a West African population. MATERIALS AND METHODS: Patients seen at the Emmanuel Eye Clinic in Accra, Ghana, were recruited for this study. Informed consent was obtained from all study patients. Glaucoma specialists from the sponsoring institution (PC, LWH, or RRA) ascertained all POAG and control patients. Age-matched unaffected controls were obtained in patients with an IOP < 22 mm Hg and normal-appearing optic nerves. PCR amplification of each of the three myocilin exons was performed. Denaturing high-performance liquid chromatography (Transgenomics Corp.) was used to detect allelic differences and samples demonstrating a mobility shift were sequenced in both directions. RESULTS: Ninety unrelated affecteds with POAG and 76 control patients were recruited. Four individuals with severe POAG were found to have novel missense mutations in exon 3. Two exhibit an Asp380Asn mutation and two an Arg342Lys mutation. These changes were not detected in 152 ethnically matched control chromosomes. Fourteen affected individuals and eight controls exhibit a translationally silent polymorphism in codon 325 (Thr325Thr). CONCLUSIONS: A total of 4.4% of patients with POAG have novel disease-associated mutations in myocilin. Mutations in myocilin appear to play a limited role in the pathogenesis of POAG in this region of West Africa.

Variation of neutrophil function with age in calves.

Neutrophil function was evaluated on 2 occasions in 5 calves from each of the following age groups: 4 to 5 weeks, 9 to 11 weeks, 16 to 19 weeks, and 12 to 14 months. Of the neutrophil functions examined, the iodination reaction, which evaluates the activity of the myeloperoxidase-hydrogen peroxide-halide antibacterial system of the neutrophil, reflected the most marked differences among age groups. The iodination values for the 2 youngest age groups were approximately 52% of the value for the oldest cattle. This difference could not be attributed to the amount of myeloperoxidase in the neutrophil granules. Antibody-dependent cell-mediated cytotoxicity also tended to be lower in neutrophils from the 3 younger age groups. Nitroblue tetrazolium reduction, a measure of superoxide anion generation by neutrophils, was lower in the youngest age group only. The capability of neutrophils to ingest Staphylococcus aureus was higher in the 3 youngest groups of calves than in the oldest group. The observed differences in neutrophil function in young vs older calves may be partially responsible for the increased susceptibility of young calves to infectious disease.

Vascular tone pathway polymorphisms in relation to primary open-angle glaucoma.

AIMS: Vascular perfusion may be impaired in primary open-angle glaucoma (POAG); thus, we evaluated a panel of markers in vascular tone-regulating genes in relation to POAG. METHODS: We used Illumina 660W-Quad array genotype data and pooled P-values from 3108 POAG cases and 3430 controls from the combined National Eye Institute Glaucoma Human Genetics Collaboration consortium and Glaucoma Genes and Environment studies. Using information from previous literature and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, we compiled single-nucleotide polymorphisms (SNPs) in 186 vascular tone-regulating genes. We used the 'Pathway Analysis by Randomization Incorporating Structure' analysis software, which performed 1000 permutations to compare the overall pathway and selected genes with comparable randomly generated pathways and genes in their association with POAG. RESULTS: The vascular tone pathway was not associated with POAG overall or POAG subtypes, defined by the type of visual field loss (early paracentral loss (n=224 cases) or only peripheral loss (n=993 cases)) (permuted P≥0.20). In gene-based analyses, eight were associated with POAG overall at permuted P<0.001: PRKAA1, CAV1, ITPR3, EDNRB, GNB2, DNM2, HFE, and MYL9. Notably, six of these eight (the first six listed) code for factors involved in the endothelial nitric oxide synthase activity, and three of these six (CAV1, ITPR3, and EDNRB) were also associated with early paracentral loss at P<0.001, whereas none of the six genes reached P<0.001 for peripheral loss only. DISCUSSION: Although the assembled vascular tone SNP set was not associated with POAG, genes that code for local factors involved in setting vascular tone were associated with POAG.

Site-specific integration of the Haemophilus influenzae bacteriophage HP1. Identification of the points of recombinational strand exchange and the limits of the host attachment site.

Isotopic transfer experiments and boundary replacement studies were used to define the size and cleavage points of the Haemophilus influenzae attB site for phage HP1 integration. The points of strand cleavage and transfer were separated by 5' extensions with a spacing or overlap region most probably 7 residues long. The complete HP1 attB site is included within an 18-base pair (bp) sequence surrounding the cleavage sites. The sequence of HP1 attB is remarkably symmetric. Two 8-bp inverted repeats surround the central residue of the 7-bp overlap sequence; this central residue is the second residue of the anticodon sequence of the H. influenzae tRNA(leu)(UUR) gene which contains attB, and this symmetric segment encodes the anticodon stem and loop.

Site-specific integration of the Haemophilus influenzae bacteriophage HP1: location of the boundaries of the phage attachment site.

Plasmids containing DNA segments from the attachment region of phage HP1 were constructed and tested for the ability to replace the phage attachment site substrate in site-specific recombination reactions. The distance separating the boundaries of the functional site was 418 bp. Replacements within the 11-residue segment 5'-GGCGGTTATCG at the left boundary or within the 12-residue segment 5'-GGATTTTTTGAA at the right boundary abolished substrate activity. A segment of the 418-residue sequence preserves the integrity of an operon of three Haemophilus influenzae tRNA genes after HP1 insertion within the coding sequence.

Production and characterization of improved adenovirus vectors with the E1, E2b, and E3 genes deleted.

Adenovirus (Ad)-based vectors have great potential for use in the gene therapy of multiple diseases, both genetic and nongenetic. While capable of transducing both dividing and quiescent cells efficiently, Ad vectors have been limited by a number of problems. Most Ad vectors are engineered such that a transgene replaces the Ad E1a, E1b, and E3 genes; subsequently the replication-defective vector can be propagated only in human 293 cells that supply the deleted E1 gene functions in trans. Unfortunately, the use of high titers of E1-deleted vectors has been repeatedly demonstrated to result in low-level expression of viral genes still resident in the vector. In addition, the generation of replication-competent Ad (RCA) by recombination events with the E1 sequences residing in 293 cells further limits the usefulness of E1-deleted Ad vectors. We addressed these problems by isolating new Ad vectors deleted for the E1, E3, and the E2b gene functions. The new vectors can be readily grown to high titers and have several improvements, including an increased carrying capacity and a theoretically decreased risk for generating RCA. We have also demonstrated that the further block to Ad vector replication afforded by the deletion of both the E1 and E2b genes significantly diminished Ad late gene expression in comparison to a conventional E1-deleted vector, without destabilization of the modified vector genome. The results suggested that these modified vectors may be very useful both for in vitro and in vivo gene therapy applications.

Expression of full-length and truncated dystrophin mini-genes in transgenic mdx mice.

Duchenne and Becker muscular dystrophy are caused by defects in the dystrophin gene, and are candidates for treatment by gene therapy. We have shown previously that overexpression of a full-length dystrophin cDNA prevents the development of dystrophic symptoms in mdx mice. We show here that this functional correction can be achieved by expressing the full-length muscle isoform at a lower level than is present in control animals. Gene therapy for DMD may necessitate the use of truncated dystrophin mini-genes to accommodate the limited cloning capacity of current-generation viral delivery vectors. We have constructed both murine and human mini-genes deleted for exons 17-48, and have demonstrated that expression of either mini-gene can almost completely prevent the development of dystrophic symptoms in transgenic mdx mice. These results suggest that viral-mediated expression of moderate levels of a truncated dystrophin could be an effective treatment for DMD.

Analysis of muscle creatine kinase regulatory elements in recombinant adenoviral vectors.

Adenoviral gene transfer holds promise for gene therapy, but effective transduction of a large and distributed tissue such as muscle will almost certainly require systemic delivery. In this context, the use of muscle-specific regulatory elements such as the muscle creatine kinase (MCK) promoter and enhancer will avoid potentially harmful ectopic expression of transgenes. We describe here the development and testing of adenoviral vectors containing small, striated muscle-specific, highly active MCK expression cassettes. One of these regulatory elements (CK6) is less than 600 bp in length and is 12% as active as the CMV promoter/enhancer in muscle. A recombinant adenoviral vector containing this regulatory element retains very high muscle specificity, expressing 600-fold higher levels of transgene in muscle than in liver. Muscle-specific regulatory elements may also increase persistence of transduced muscle cells. Adenoviral transduction of dendritic cells has been shown to stimulate cytotoxic T-lymphocyte (CTL) responses directed against transgene epitopes. We show that human dendritic cells infected in vitro with MCK-containing adenoviruses do not express significant levels of transgene. Furthermore, while adenoviral vectors containing nonspecific promoters are normally cleared from muscle tissue within 1 month, we show that MCK-containing vectors express significant levels of transgene 4 months after intramuscular injection.

Myotilin is mutated in limb girdle muscular dystrophy 1A.

We have identified a mutation in the myotilin gene in a large North American family of German descent expressing an autosomal dominant form of limb girdle muscular dystrophy (LGMD1A). We have previously mapped this gene to 5q31. Symptoms of this adult onset disease are progressive weakness of the hip and shoulder girdles, as well as a distinctive dysarthric pattern of speech. Muscle of affected individuals shows degeneration of myofibers, variations in fiber size, fiber splitting, centrally located myonuclei and a large number of autophagic vesicles. Affected muscle also exhibits disorganization and streaming of the Z-line similar to that seen in nemaline myopathy. We have identified a C450T missense mutation in the myotilin gene that is predicted to result in the conversion of residue 57 from threonine to isoleucine. This mutation has not been found in 396 control chromosomes. The mutant allele is transcribed and normal levels of correctly localized myotilin protein are seen in LGMD1A muscle. Myotilin is a sarcomeric protein that binds to alpha-actinin and is localized in the Z-line. The observed missense mutation does not disrupt binding to alpha-actinin.

Apolipoprotein E controls the risk and age at onset of Parkinson disease.

BACKGROUND: Similarities between Alzheimer disease (AD) and Parkinson disease (PD) suggest a possible role for apolipoprotein E (APOE) in PD. Most previous studies seeking to establish such a link used case-control datasets and results have been inconsistent. OBJECTIVE: To investigate APOE's role in PD using family-based association analyses. METHODS: APOE functional polymorphisms were genotyped for 658 PD affected families, including 282 multiplex and 376 singleton families. The pedigree disequilibrium test (PDT) and the genotype-PDT were used to test the risk effect of APOE. The Monks-Kaplan test was used to evaluate the effect of APOE on age at onset of PD. RESULTS: APOE was significantly associated with risk of developing PD. Stratified analysis revealed that APOE was most strongly associated with families with a positive PD family history (global p = 0.003). Like AD, the APOE-4 allele increases disease risk while the APOE-3 allele decreases risk. We detected a positive association of APOE-3 (p = 0.019) and a negative association of APOE-4 (p = 0.015) with age at onset in PD. CONCLUSIONS: The APOE-4 allele increases risk and decreases age at onset of PD, an association that may not be dependent upon cognitive impairment.

Association of single-nucleotide polymorphisms of the tau gene with late-onset Parkinson disease.

CONTEXT: The human tau gene, which promotes assembly of neuronal microtubules, has been associated with several rare neurologic diseases that clinically include parkinsonian features. We recently observed linkage in idiopathic Parkinson disease (PD) to a region on chromosome 17q21 that contains the tau gene. These factors make tau a good candidate for investigation as a susceptibility gene for idiopathic PD, the most common form of the disease. OBJECTIVE: To investigate whether the tau gene is involved in idiopathic PD. DESIGN, SETTING, AND PARTICIPANTS: Among a sample of 1056 individuals from 235 families selected from 13 clinical centers in the United States and Australia and from a family ascertainment core center, we tested 5 single-nucleotide polymorphisms (SNPs) within the tau gene for association with PD, using family-based tests of association. Both affected (n = 426) and unaffected (n = 579) family members were included; 51 individuals had unclear PD status. Analyses were conducted to test individual SNPs and SNP haplotypes within the tau gene. MAIN OUTCOME MEASURE: Family-based tests of association, calculated using asymptotic distributions. RESULTS: Analysis of association between the SNPs and PD yielded significant evidence of association for 3 of the 5 SNPs tested: SNP 3, P =.03; SNP 9i, P =.04; and SNP 11, P =.04. The 2 other SNPs did not show evidence of significant association (SNP 9ii, P =.11, and SNP 9iii, P =.87). Strong evidence of association was found with haplotype analysis, with a positive association with one haplotype (P =.009) and a negative association with another haplotype (P =.007). Substantial linkage disequilibrium (P<.001) was detected between 4 of the 5 SNPs (SNPs 3, 9i, 9ii, and 11). CONCLUSIONS: This integrated approach of genetic linkage and positional association analyses implicates tau as a susceptibility gene for idiopathic PD.