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

Molecular Neurodegeneration

Pamela J Shaw⁴  Natalia Ninkina³  Olga D Razinskaya¹  Janine Kirby⁴  Adrian Higginbottom⁴  Natalie Connor-Robson²  Johnathan Cooper-Knock⁴  Vladimir L Buchman³ 

[1] Pirogov Russian National Research Medical University, Ostrovitianov str. 1, Moscow, 117997, Russian Federation;School of Biosciences, Cardiff University, Museum Avenue, Cardiff, CF10 3AX, UK;Institute of Physiologically Active Compounds of RAS, 1 Severniy Proezd, Chernogolovka, Moscow Region, 142432, Russian Federation;Sheffield Institute for Translational Neuroscience (SITraN), University of Sheffield, 385A Glossop Road, Sheffield, S10 2HQ, UK

关键词: Southern hybridisation;    Amyotrophic lateral sclerosis;    C9ORF72;   

Others  :  863273
DOI  :  10.1186/1750-1326-8-12

 received in 2013-02-06, accepted in 2013-03-22,  发布年份 2013

【 摘 要 】

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.

【 授权许可】

   
2013 Buchman et al.; licensee BioMed Central Ltd.

【 图 表 】

【 参考文献 】

• [1]DeJesus-Hernandez M, Mackenzie , Ian R, Boeve , Bradley F, Boxer , Adam L, Baker M, Rutherford , Nicola J, Nicholson , Alexandra M, Finch , NiCole A, Flynn H, Adamson J, Kouri N, Wojtas A, Sengdy P, Hsiung G, Yuek R, Karydas A, Seeley , William W, Josephs , Keith A, Coppola G, Geschwind , Daniel H, Wszolek , Zbigniew K, Feldman H, Knopman , David S, Petersen , Ronald C, Miller , Bruce L, Dickson , Dennis W, Boylan , Kevin B, Graff-Radford , Neill R, Rademakers R: Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 2011, 72:245-256.
• [2]Renton , Alan E, Majounie E, Waite A, Simón-Sánchez J, Rollinson S, Gibbs JR, Schymick , Jennifer C, Laaksovirta H, Van Swieten , John C, Myllykangas L, Kalimo H, Paetau A, Abramzon Y, Remes , Anne M, Kaganovich A, Scholz , Sonja W, Duckworth J, Ding J, Harmer , Daniel W, Hernandez , Dena G, Johnson , Janel O, Mok K, Ryten M, Trabzuni D, Guerreiro , Rita J, Orrell , Richard W, Neal J, Murray A, Pearson J, Jansen , Iris E: A Hexanucleotide Repeat Expansion in C9ORF72 Is the Cause of Chromosome 9p21-Linked ALS-FTD. Neuron 2011, 72:257-268.
• [3]Ash PE, Bieniek KF, Gendron TF, Caulfield T, Lin WL, DeJesus-Hernandez M, van Blitterswijk MM, Jansen-West K, Paul JW 3rd, Rademakers R, Boylan KB, Dickson DW, Petrucelli L: Unconventional Translation of C9ORF72 GGGGCC Expansion Generates Insoluble Polypeptides Specific to c9FTD/ALS. Neuron 2013, 77:639-646.
• [4]Mori K, Weng SM, Arzberger T, May S, Rentzsch K, Kremmer E, Schmid B, Kretzschmar HA, Cruts M, Van Broeckhoven C, Haass C, Edbauer D: The C9orf72 GGGGCC Repeat Is Translated into Aggregating Dipeptide-Repeat Proteins in FTLD/ALS. Science 2013. Published online 7 February 2013
• [5]Todd PK, Paulson HL: RNA-mediated neurodegeneration in repeat expansion disorders. Ann Neurol 2010, 67:291-300.
• [6]Gijselinck I, Van Langenhove T, van der Zee J, Sleegers K, Philtjens S, Kleinberger G, Janssens J, Bettens K, Van Cauwenberghe C, Pereson S, Engelborghs S, Sieben A, De Jonghe P, Vandenberghe R, Santens P, De Bleecker J, Maes G, Bäumer V, Dillen L, Joris G, Cuijt I, Corsmit E, Elinck E, Van Dongen J, Vermeulen S, Van den Broeck M, Vaerenberg C, Mattheijssens M, Peeters K, Robberecht W: A C9orf72 promoter repeat expansion in a Flanders-Belgian cohort with disorders of the frontotemporal lobar degeneration-amyotrophic lateral sclerosis spectrum: a gene identification study. Lancet Neurol 2012, 11:54-65.
• [7]Prior TW: Technical standards and guidelines for myotonic dystrophy type 1 testing. Genet Med 2009, 11:552-555.
• [8]Ishiura H, Takahashi Y, Mitsui J, Yoshida S, Kihira T, Kokubo Y, Kuzuhara S, Ranum L, Tamaoki T, Ichikawa Y, Date H, Goto J, Tsuji S: C9orf72 repeat expansion in amyotrophic lateral sclerosis in the kii peninsula of japan. Arch Neurol 2012, 69:1154-1158.
• [9]Takada L, Pimentel M, Dejesus-Hernandez M, Fong J, Yokoyama J, Karydas A, Thibodeau M, Rutherford N, Baker M, Lomen-Hoerth C, Rademakers R, Miller B: Frontotemporal dementia in a Brazilian kindred with the c9orf72 mutation. Arch Neurol 2012, 69:1149-1153.
• [10]Southern E: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975, 98:503-17.
• [11]Rigby PWJ, Dieckmann M, Rhodes C, Berg P: Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 1977, 113:237-251.