Summary This paper describes a SlipChip to perform digital PCR in a very simple and inexpensive format. The fluidic path for introducing the sample combined with the PCR mixture was formed using elongated wells in the two plates of the SlipChip designed to overlap during sample loading. This fluidic path was broken up by simple slipping of the two plates that removed the overlap among wells and brought each well in contact with a reservoir preloaded with oil to generate 1,280 reaction compartments (2.6 nL each) simultaneously. After thermal cycling, end-point fluorescence intensity was used to detect the presence of nucleic acid. Digital PCR on the SlipChip was tested quantitatively by using Staphylococcus aureus genomic DNA. As the concentration of the template DNA in the reaction mixture was diluted, the fraction of positive wells decreased as expected from the statistical analysis. No cross contamination was observed during the experiments. At the extremes of the dynamic range of digital PCR the standard confidence interval determined using a normal approximation of the binomial distribution is not satisfactory. Therefore, statistical analysis based on the score method was used to establish these confidence intervals. The SlipChip provides a simple strategy to count nucleic acids by using PCR. It may find applications in research applications such as single cell analysis, prenatal diagnostics, and point-of-care diagnostics. SlipChip would become valuable for diagnostics, including applications in resource-limited areas after integration with isothermal nucleic acid amplification technologies and visual readout.
Frontotemporal dementia and amyotrophic lateral sclerosis are closely related clinical syndromes with overlapping molecular pathogenesis. Several families have been reported with members affected by frontotemporal dementia, amyotrophic lateral sclerosis or both, which show genetic linkage to a region on chromosome 9p21. Recently, two studies identified the FTD/ALS gene defect on chromosome 9p as an expanded GGGGCC hexanucleotide repeat in a non-coding region of the chromosome 9 open reading frame 72 gene (C9ORF72). In the present study, we provide detailed analysis of the clinical features and neuropathology for 16 unrelated families with frontotemporal dementia caused by the C9ORF72 mutation. All had an autosomal dominant pattern of inheritance. Eight families had a combination of frontotemporal dementia and amyotrophic lateral sclerosis while the other eight had a pure frontotemporal dementia phenotype. Clinical information was available for 30 affected members of the 16 families. There was wide variation in age of onset (mean = 54.3, range = 34-74 years) and disease duration (mean = 5.3, range = 1-16 years). Early diagnoses included behavioural variant frontotemporal dementia (n = 15), progressive non-fluent aphasia (n = 5), amyotrophic lateral sclerosis (n = 9) and progressive non-fluent aphasia-amyotrophic lateral sclerosis (n = 1). Heterogeneity in clinical presentation was also common within families. However, there was a tendency for the phenotypes to converge with disease progression; seven subjects had final clinical diagnoses of both frontotemporal dementia and amyotrophic lateral sclerosis and all of those with an initial progressive non-fluent aphasia diagnosis subsequently developed significant behavioural abnormalities. Twenty-one affected family members came to autopsy and all were found to have transactive response DNA binding protein with M(r) 43 kD (TDP-43) pathology in a wide neuroanatomical distribution. All had involvement of the extramotor neocortex and hippocampus (frontotemporal lobar degeneration-TDP) and all but one case (clinically pure frontotemporal dementia) had involvement of lower motor neurons, characteristic of amyotrophic lateral sclerosis. In addition, a consistent and relatively specific pathological finding was the presence of neuronal inclusions in the cerebellar cortex that were ubiquitin/p62-positive but TDP-43-negative. Our findings indicate that the C9ORF72 mutation is a major cause of familial frontotemporal dementia with TDP-43 pathology, that likely accounts for the majority of families with combined frontotemporal dementia/amyotrophic lateral sclerosis presentation, and further support the concept that frontotemporal dementia and amyotrophic lateral sclerosis represent a clinicopathological spectrum of disease with overlapping molecular pathogenesis.
Two studies recently identified a GGGGCC hexanucleotide repeat expansion in a non-coding region of the chromosome 9 open reading frame 72 gene (C9ORF72) as the cause of chromosome 9p-linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In a cohort of 231 probands with ALS, we identified the C9ORF72 mutation in 17 familial (27.4 %) and six sporadic (3.6%) cases. Patients with the mutation presented with typical motor features of ALS, although subjects with the C9ORF72 mutation had more frequent bulbar onset, compared to those without this mutation. Dementia was significantly more common in ALS patients and families with the C9ORF72 mutation and was usually early-onset FTD. There was striking clinical heterogeneity among the members of individual families with the mutation. The associated neuropathology was a combination of ALS with TDP-ir inclusions and FTLD-TDP. In addition to TDP-43-immunoreactive pathology, a consistent and specific feature of cases with the C9ORF72 mutation was the presence of ubiquitin-positive, TDP-43-negative inclusions in a variety of neuroanatomical regions, such as the cerebellar cortex. These findings support the C9ORF72 mutation as an important newly-recognized cause of ALS, provide a more detailed characterization of the associated clinical and pathological features and further demonstrate the clinical and molecular overlap between ALS and FTD.
The highly dynamic bundling activity of fission yeast α-actinin SpAin1 was biochemically characterized, and its importance for contractile ring formation in vivo was tested. Investigation of a mutant with higher bundling activity, SpAin1(216E), revealed that dynamic SpAin1-mediated bundling is crucial for proper ring assembly and constriction.
Objective-To assess the influence of rs5848 polymorphism in serum progranulin (PGRN) level in a cohort of subjects with Alzheimer and related dementias from a tertiary referral clinic.Background-Mutations in the GRN gene cause autosomal dominant frontotemporal dementia (FTD) with TDP-43 pathology (FTLD-TDP) through haploinsufficiency. It has recently been shown that homozygous carriers of the T-allele of rs5848 have an elevated risk developing FTD, and this polymorphism may play a role in the pathogenesis of other dementia by modifying progranulin level. We hypothesize that genotype of rs5848 may influence serum PGRN level in AD, FTD, and other dementias.
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