Characterization of the 2'-5'-oligoadenylate synthetase ubiquitin-like family

The interferon-induced 2′–5′-oligoadenylate synthetases (OAS) are important for the antiviral activity of interferons. The human and murine OAS gene families each contain four genes: OAS1, OAS2, OAS3 and OASL, all having one or more conserved OAS units composed of five translated exons. The OASL gene has both an OAS unit and a C-terminus of two ubiquitin-like repeats. In this study, we demonstrate that murine Oasl1 protein is inactive while murine Oasl2 is active as an OAS. Further more, murine Oasl2 requires double-stranded RNA as co-factor. The affinity of murine Oasl2 for the double-stranded RNA activator is higher than that of human OAS1 (p42 isoform). We propose a model for the evolutionary origin of the murine Oasl1 and Oasl2 genes. The identification of a human orthologue (hOASL2) to the murine Oasl2 gene establishes that the OASL gene was duplicated prior to the radiation of the rodent and primate groups. We suggest that murine Oasl2, which has both enzymatic activity and a ubiquitin-like domain, is a functional intermediate between the active OAS species and the inactive human OASL1/murine Oasl1 proteins. In addition, we propose that murine Oasl1 appears to have gained a hitherto uncharacterized function independent of 2′–5′-linked oligoadenylate synthesis.     

De novo MECP2 disomy in a Mexican male carrying a supernumerary marker chromosome and no typical Lubs syndrome features

Xq28 duplication, including the MECP2 gene, is among the most frequently identified Xq subtelomeric rearrangements. The resulting clinical phenotype is named Lubs syndrome and mainly consists of intellectual disability, congenital hypotonia, absent speech, recurrent infections, and seizures. Here we report a Mexican male patient carrying a supernumerary marker chromosome with de novo Xq28 gain. By MLPA, duplication of MECP2, GDI1, and SLC6A8 was found and a subsequent a-CGH analysis demonstrated that the gain spanned ~ 2.1 Mb. Despite gain of the MECP2 gene, the features of this patient do not evoke Lubs syndrome. Probably the mosaicism of the supernumerary marker chromosome is modifying the phenotype in this patient.     

Novel Mutation in the <Emphasis Type="Italic">MECP2</Emphasis> Gene Identified in a Group of Rett Syndrome Patients from Ukraine

Mutations in the MECP2 gene are known to cause Rett syndrome (RTT)—a neurodevelopmental disorder, one of the most common causes of intellectual disability in females, with an incidence of 1 in 10000–15000. We have investigated exons 3 and 4 of the MECP2 gene, that coding MBD and TRD domains of the MeCP2 protein, in 21 RTT patients from Ukraine by PCR-DGGE analysis followed by Sanger sequencing of PCR fragments with abnormal migration profiles. In 13 of 21 (61.9%) patients 7 different mutations were identified one nonsense mutation—c. NC_000023.11:g.154031326G>A (MECP2:c.502C>T) and 4 missense mutation NC_000023.11:g.154031409G>T (MECP2:c.419C>T), NC_000023.11:g.154031355G>A (MECP2:c.473C>T), NC_000023.11:g.154031354A>C (MECP2:c.472A>C), NC_000023.11:g.154031431G>A (MECP2:c.397C>T) located in exon 4, a rare RTT-causing splice site mutation NC_000023.10:g.153296903T>G (MECP2:c.378-2A>C) in intron 3 and deletion NC_000023.10:g.1532 96079_153296122del44 in exon 4. The novel mutation MECP2:c.472A>C identified in our study in patients withclassic RTT phenotype leds to T158P substitution. It is one more confirmation of crucial role that 158 codon in MECP2 protein function.     

A Multi-Exon-Skipping Detection Assay Reveals Surprising Diversity of Splice Isoforms of Spinal Muscular Atrophy Genes

Humans have two near identical copies of Survival Motor Neuron gene: SMN1 and SMN2. Loss of SMN1 coupled with the predominant skipping of SMN2 exon 7 causes spinal muscular atrophy (SMA), a neurodegenerative disease. SMA patient cells devoid of SMN1 provide a powerful system to examine splicing pattern of various SMN2 exons. Until now, similar system to examine splicing of SMN1 exons was unavailable. We have recently screened several patient cell lines derived from various diseases, including SMA, Alzheimer’s disease, Parkinson’s disease and Batten disease. Here we report a Batten disease cell line that lacks functional SMN2, as an ideal system to examine pre-mRNA splicing of SMN1. We employ a multiple-exon-skipping detection assay (MESDA) to capture simultaneously skipping of multiple exons. Our results show surprising diversity of splice isoforms and reveal novel splicing events that include skipping of exon 4 and co-skipping of three adjacent exons of SMN. Contrary to the general belief, MESDA captured oxidative-stress induced skipping of SMN1 exon 5 in several cell types, including non-neuronal cells. We further demonstrate that the predominant SMN2 exon 7 skipping induced by oxidative stress is modulated by a combinatorial control that includes promoter sequence, endogenous context, and the weak splice sites. We also show that an 8-mer antisense oligonucleotide blocking a recently described GC-rich sequence prevents SMN2 exon 7 skipping under the conditions of oxidative stress. Our findings bring new insight into splicing regulation of an essential housekeeping gene linked to neurodegeneration and infant mortality.     

Multiplex ligation-dependent probe amplification (MLPA) detects large deletions in the MECP2 gene of Swedish Rett syndrome patients

Mutations in the methyl-CpG-binding protein-2 (MECP2) gene on Xq28 have been found to be a cause of Rett syndrome (RS). In a previous mutation screening, we found MECP2 mutations in 81% of Swedish classical Rett women. In this study, we have analyzed 22 patients for MECP2 deletions using multiplex-ligation-dependent probe amplification (MLPA). Clinically, 11 of the patients who were classical Rett women, 3 were forme fruste, 1 was congenital RS, and 7 were Rett variants. As inclusion criteria, we used DNA from patients in whom previous sequencing results showed no mutations in coding portions of the MECP2 gene. MLPA is a method based on multiplex PCR. In one PCR, as many as 40 probes are amplified with the same primers. The specificity of the amplification products is determined by the site-specific hybridization of each probe construct, prior to amplification. Each PCR product has a unique length, which makes it possible to identify it by size separation. In 3 of 11 (27%) classical Rett women, we detected large deletions in MECP2 using MLPA. All these patients had deletions covering two exons; in 2 cases the deletion involved exons 3 and 4 and, in one case, exons 1 and 2 were missing. In the forme fruste, congenital and Rett-variant patients, we found no large deletions. We have found that MLPA is useful when it comes to finding large deletions compromising whole exons in MECP2. Used as a complementary method to DNA sequencing, it revealed new MECP2 mutations in classical RS patients.     

Replication of recently identified systemic lupus erythematosus genetic associations: a case–control study

Introduction

We aimed to replicate association of newly identified systemic lupus erythematosus (SLE) loci.

Methods

We selected the most associated SNP in 10 SLE loci. These 10 SNPs were analysed in 1,579 patients with SLE and 1,726 controls of European origin by single-base extension. Comparison of allele frequencies between cases and controls was done with the Mantel–Haenszel approach to account for heterogeneity between sample collections.

Results

A previously controversial association with a SNP in the TYK2 gene was replicated (odds ratio (OR) = 0.79, P = 2.5 × 10-⁵), as well as association with the X chromosome MECP2 gene (OR = 1.26, P = 0.00085 in women), which had only been reported in a single study, and association with four other loci, 1q25.1 (OR = 0.81, P = 0.0001), PXK (OR = 1.19, P = 0.0038), BANK1 (OR = 0.83, P = 0.006) and KIAA1542 (OR = 0.84, P = 0.001), which have been identified in a genome-wide association study, but not found in any other study. All these replications showed the same disease-associated allele as originally reported. No association was found with the LY9 SNP, which had been reported in a single study.

Conclusions

Our results confirm nine SLE loci. For six of them, TYK2, MECP2, 1q25.1, PXK, BANK1 and KIAA1542, this replication is important. The other three loci, ITGAM, STAT4 and C8orf13-BLK, were already clearly confirmed. Our results also suggest that MECP2 association has no influence in the sex bias of SLE, contrary to what has been proposed. In addition, none of the other associations seems important in this respect.     

Exercise therapy for the management of osteoarthritis of the hip joint: a systematic review

Introduction

We aimed to replicate association of newly identified systemic lupus erythematosus (SLE) loci.

Methods

We selected the most associated SNP in 10 SLE loci. These 10 SNPs were analysed in 1,579 patients with SLE and 1,726 controls of European origin by single-base extension. Comparison of allele frequencies between cases and controls was done with the Mantel–Haenszel approach to account for heterogeneity between sample collections.

Results

A previously controversial association with a SNP in the TYK2 gene was replicated (odds ratio (OR) = 0.79, P = 2.5 × 10-⁵), as well as association with the X chromosome MECP2 gene (OR = 1.26, P = 0.00085 in women), which had only been reported in a single study, and association with four other loci, 1q25.1 (OR = 0.81, P = 0.0001), PXK (OR = 1.19, P = 0.0038), BANK1 (OR = 0.83, P = 0.006) and KIAA1542 (OR = 0.84, P = 0.001), which have been identified in a genome-wide association study, but not found in any other study. All these replications showed the same disease-associated allele as originally reported. No association was found with the LY9 SNP, which had been reported in a single study.

Conclusions

Our results confirm nine SLE loci. For six of them, TYK2, MECP2, 1q25.1, PXK, BANK1 and KIAA1542, this replication is important. The other three loci, ITGAM, STAT4 and C8orf13-BLK, were already clearly confirmed. Our results also suggest that MECP2 association has no influence in the sex bias of SLE, contrary to what has been proposed. In addition, none of the other associations seems important in this respect.     

Species differences in cannabinoid receptor 2 (CNR2 gene): identification of novel human and rodent CB2 isoforms, differential tissue expression and regulation by cannabinoid receptor ligands

Cannabinoids, endocannabinoids and marijuana activate two well-characterized cannabinoid receptors (CB-Rs), CB1-Rs and CB2-Rs. The expression of CB1-Rs in the brain and periphery has been well studied, but neuronal CB2-Rs have received much less attention than CB1-Rs. Many studies have now identified and characterized functional glial and neuronal CB2-Rs in the central nervous system. However, many features of CB2-R gene structure, regulation and variation remain poorly characterized in comparison with the CB1-R. In this study, we report on the discovery of a novel human CB2 gene promoter transcribing testis (CB2A) isoform with starting exon located ca 45 kb upstream from the previously identified promoter transcribing the spleen isoform (CB2B). The 5' exons of both CB2 isoforms are untranslated 5'UTRs and alternatively spliced to the major protein coding exon of the CB2 gene. CB2A is expressed higher in testis and brain than CB2B that is expressed higher in other peripheral tissues than CB2A. Species comparison found that the CB2 gene of human, rat and mouse genomes deviated in their gene structures and isoform expression patterns. mCB2A expression was increased significantly in the cerebellum of mice treated with the CB-R mixed agonist, WIN55212-2. These results provide much improved information about CB2 gene structure and its human and rodent variants that should be considered in developing CB2-R-based therapeutic agents.     

An abundant evolutionarily conserved CSB-PiggyBac fusion protein expressed in Cockayne syndrome

Cockayne syndrome (CS) is a devastating progeria most often caused by mutations in the CSB gene encoding a SWI/SNF family chromatin remodeling protein. Although all CSB mutations that cause CS are recessive, the complete absence of CSB protein does not cause CS. In addition, most CSB mutations are located beyond exon 5 and are thought to generate only C-terminally truncated protein fragments. We now show that a domesticated PiggyBac-like transposon PGBD3, residing within intron 5 of the CSB gene, functions as an alternative 3′ terminal exon. The alternatively spliced mRNA encodes a novel chimeric protein in which CSB exons 1–5 are joined in frame to the PiggyBac transposase. The resulting CSB-transposase fusion protein is as abundant as CSB protein itself in a variety of human cell lines, and continues to be expressed by primary CS cells in which functional CSB is lost due to mutations beyond exon 5. The CSB-transposase fusion protein has been highly conserved for at least 43 Myr since the divergence of humans and marmoset, and appears to be subject to selective pressure. The human genome contains over 600 nonautonomous PGBD3-related MER85 elements that were dispersed when the PGBD3 transposase was last active at least 37 Mya. Many of these MER85 elements are associated with genes which are involved in neuronal development, and are known to be regulated by CSB. We speculate that the CSB-transposase fusion protein has been conserved for host antitransposon defense, or to modulate gene regulation by MER85 elements, but may cause CS in the absence of functional CSB protein.     

Different unequal cross-over events between NCF1 and its pseudogenes in autosomal p47phox-deficient chronic granulomatous disease

Chronic granulomatous disease (CGD) is a rare congenital disorder in which phagocytes cannot generate superoxide (O₂^?) and other microbicidal oxidants due to mutations in one of the five components of the O₂^?-generating NADPH oxidase complex. The most common autosomal subtype of CGD is caused by mutations in NCF1, encoding the NADPH subunit p47^phox. Usually, these mutations are the result of unequal exchange of chromatid between NCF1 and one of its two pseudogenes. We have now investigated in detail the breakpoints within or between these (pseudo) NCF1 genes in 43 families with p47^phox-deficient CGD by means of multiplex ligase-dependent probe amplification (MLPA). In 24 families the patients totally lacked NCF1 sequences, indicating that in these families the cross-over points are located between NCF1 and its pseudogenes. Six other families were compound heterozygous for a total NCF1 deletion and another mutation in NCF1 on the other allele. In 8 families, the patients lacked NCF1 exons 1–4 but had retained NCF1 exons 6–10, indicating that a cross-over point is located within NCF1 between exons 4 and 6. Similarly, in 4 families a cross-over point was located within NCF1 between exons 2 and 4. Similar cross-overs, in heterozygous form, were observed in family members of the patients. Several patients were compound heterozygous for total and partial NCF1 deletions. Thus, at least three different cross-over points exist within the NCF1 gene cluster, indicating that autosomal p47^phox-deficient CGD is genetically heterogeneous but can be dissected in detail by MLPA.