Mutations in UBQLN2 cause dominant X-linked juvenile and adult-onset ALS and ALS/dementia.

Amyotrophic lateral sclerosis (ALS) is a paralytic and usually fatal disorder caused by motor-neuron degeneration in the brain and spinal cord. Most cases of ALS are sporadic but about 5-10% are familial. Mutations in superoxide dismutase 1 (SOD1), TAR DNA-binding protein (TARDBP, also known as TDP43) and fused in sarcoma (FUS, also known as translocated in liposarcoma (TLS)) account for approximately 30% of classic familial ALS. Mutations in several other genes have also been reported as rare causes of ALS or ALS-like syndromes. The causes of the remaining cases of familial ALS and of the vast majority of sporadic ALS are unknown. Despite extensive studies of previously identified ALS-causing genes, the pathogenic mechanism underlying motor-neuron degeneration in ALS remains largely obscure. Dementia, usually of the frontotemporal lobar type, may occur in some ALS cases. It is unclear whether ALS and dementia share common aetiology and pathogenesis in ALS/dementia. Here we show that mutations in UBQLN2, which encodes the ubiquitin-like protein ubiquilin 2, cause dominantly inherited, chromosome-X-linked ALS and ALS/dementia. We describe novel ubiquilin 2 pathology in the spinal cords of ALS cases and in the brains of ALS/dementia cases with or without UBQLN2 mutations. Ubiquilin 2 is a member of the ubiquilin family, which regulates the degradation of ubiquitinated proteins. Functional analysis showed that mutations in UBQLN2 lead to an impairment of protein degradation. Therefore, our findings link abnormalities in ubiquilin 2 to defects in the protein degradation pathway, abnormal protein aggregation and neurodegeneration, indicating a common pathogenic mechanism that can be exploited for therapeutic intervention.

Molecular dissection of ALS-associated toxicity of SOD1 in transgenic mice using an exon-fusion approach.

Mutations in Cu,Zn superoxide dismutase (SOD1) are associated with amyotrophic lateral sclerosis (ALS). Among more than 100 ALS-associated SOD1 mutations, premature termination codon (PTC) mutations exclusively occur in exon 5, the last exon of SOD1. The molecular basis of ALS-associated toxicity of the mutant SOD1 is not fully understood. Here, we show that nonsense-mediated mRNA decay (NMD) underlies clearance of mutant mRNA with a PTC in the non-terminal exons. To further define the crucial ALS-associated SOD1 fragments, we designed and tested an exon-fusion approach using an artificial transgene SOD1(T116X) that harbors a PTC in exon 4. We found that the SOD1(T116X) transgene with a fused exon could escape NMD in cellular models. We generated a transgenic mouse model that overexpresses SOD1(T116X). This mouse model developed ALS-like phenotype and pathology. Thus, our data have demonstrated that a 'mini-SOD1' of only 115 amino acids is sufficient to cause ALS. This is the smallest ALS-causing SOD1 molecule currently defined. This proof of principle result suggests that the exon-fusion approach may have potential not only to further define a shorter ALS-associated SOD1 fragment, thus providing a molecular target for designing rational therapy, but also to dissect toxicities of other proteins encoded by genes of multiple exons through a 'gain of function' mechanism.

High fidelity PCR with an off/on switch mediated by proofreading polymerases combining with phosphorothioate-modified primer.

In the initial report, introducing a single phosphorothioate modification at the very 3' terminus of the oligodeoxynucleotide primer has been shown to effectively protect the oligodeoxynucleotide degradation due to the 3' exonuclease activity. In this study, we reported a novel finding that phosphorothioate modification at the 3' end of primers could not only effectively prevent the primer from degradation, but could also mediate an off-switch extension by Pfu polymerase when primers also carry single or multiple mismatched bases located in the first eight bases of the 3' terminus. This suggests that the combination of 3' phosphorothioate-modified primers with exo+ polymerases such as Pfu constituted an on/off switch, which allows perfectly matched primers to be extended but not mismatched primers. Furthermore, we found that polymerases with different fidelities showed different efficiencies in turning off mismatched-primer mediated extension. So we described here a SYBR green-based real-time quantitative PCR assay for the detection of abundance level of gene expression that did not require fluorescently labeled gene-specific probes or complicated primer combinations. The emergence of real-time quantitative RT-PCR technology is thus suited for a diverse application with a need for high-throughput methods to detect and quantify different gene expressions by way of simplicity, versatility, and accuracy, and thus could complement global microarray-based expression profiling strategies.