Dataset Information

Simultaneous and independent detection of C9ORF72 alleles with low and high number of GGGGCC repeats using an optimised protocol of Southern blot hybridisation.

ABSTRACT:

Background

Sizing of GGGGCC hexanucleotide repeat expansions within the C9ORF72 locus, which account for approximately 10% of all amyotrophic lateral sclerosis (ALS) cases, is urgently required to answer fundamental questions about mechanisms of pathogenesis in this important genetic variant. Currently employed PCR protocols are limited to discrimination between the presence and absence of a modified allele with more than 30 copies of the repeat, while Southern hybridisation-based methods are confounded by the somatic heterogeneity commonly present in blood samples, which might cause false-negative or ambiguous results.

Results

We describe an optimised Southern hybridisation-based protocol that allows confident detection of the presence of a C9ORF72 repeat expansion alongside independent assessment of its heterogeneity and the number of repeat units. The protocol can be used with either a radiolabeled or non-radiolabeled probe. Using this method we have successfully sized the C9ORF72 repeat expansion in lymphoblastoid cells, peripheral blood, and post-mortem central nervous system (CNS) tissue from ALS patients. It was also possible to confidently demonstrate the presence of repeat expansion, although of different magnitude, in both C9ORF72 alleles of the genome of one patient.

Conclusions

The suggested protocol has sufficient advantages to warrant adoption as a standard for Southern blot hybridisation analysis of GGGGCC repeat expansions in the C9ORF72 locus.

SUBMITTER: Buchman VL

PROVIDER: S-EPMC3626718 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Publications

Simultaneous and independent detection of C9ORF72 alleles with low and high number of GGGGCC repeats using an optimised protocol of Southern blot hybridisation.

Buchman Vladimir L VL Cooper-Knock Johnathan J Connor-Robson Natalie N Higginbottom Adrian A Kirby Janine J Razinskaya Olga D OD Ninkina Natalia N Shaw Pamela J PJ

Molecular neurodegeneration 20130408

<h4>Background</h4>Sizing of GGGGCC hexanucleotide repeat expansions within the C9ORF72 locus, which account for approximately 10% of all amyotrophic lateral sclerosis (ALS) cases, is urgently required to answer fundamental questions about mechanisms of pathogenesis in this important genetic variant. Currently employed PCR protocols are limited to discrimination between the presence and absence of a modified allele with more than 30 copies of the repeat, while Southern hybridisation-based method ...[more]

PMID: 23566336

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Project description:ObjectiveAn intronic GGGGCC-repeat expansion of C9ORF72 is the most common genetic variant of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. The mechanism of neurodegeneration is unknown, but a direct effect on RNA processing mediated by RNA foci transcribed from the repeat sequence has been proposed.MethodsGene expression profiling utilised total RNA extracted from motor neurons and lymphoblastoid cell lines derived from human ALS patients, including those with an expansion of C9ORF72, and controls. In lymphoblastoid cell lines, expansion length and the frequency of sense and antisense RNA foci was also examined.ResultsGene level analysis revealed a number of differentially expressed networks and both cell types exhibited dysregulation of a network functionally enriched for genes encoding 'RNA splicing' proteins. There was a significant overlap of these genes with an independently generated list of GGGGCC-repeat protein binding partners. At the exon level, in lymphoblastoid cells derived from C9ORF72-ALS patients splicing consistency was lower than in lines derived from non-C9ORF72 ALS patients or controls; furthermore splicing consistency was lower in samples derived from patients with faster disease progression. Frequency of sense RNA foci showed a trend towards being higher in lymphoblastoid cells derived from patients with shorter survival, but there was no detectable correlation between disease severity and DNA expansion length.SignificanceUp-regulation of genes encoding predicted binding partners of the C9ORF72 expansion is consistent with an attempted compensation for sequestration of these proteins. A number of studies have analysed changes in the transcriptome caused by C9ORF72 expansion, but to date findings have been inconsistent. As a potential explanation we suggest that dynamic sequestration of RNA processing proteins by RNA foci might lead to a loss of splicing consistency; indeed in our samples measurement of splicing consistency correlates with disease severity.

Dataset Information

Simultaneous and independent detection of C9ORF72 alleles with low and high number of GGGGCC repeats using an optimised protocol of Southern blot hybridisation.

Background

Results

Conclusions

Publications

Simultaneous and independent detection of C9ORF72 alleles with low and high number of GGGGCC repeats using an optimised protocol of Southern blot hybridisation.

Similar Datasets

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