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

Because the target XbaI-EcoRI fragments are quite similar in size (thus, no effect of the transfer efficiency during capillary blotting) but the ratio of probe length for detecting each fragment is approximately 1:2, the ratio of intensity of the two bands in the absence of an expanded allele, should be approximately 1:2. Indeed DNA samples from peripheral blood (Figure 1B, lanes 2, 4, 6) or lymphoblastoid cell lines (Figure 1C, lanes 2, 23 and Additional file 1: Table S1) from ALS patients without the repeat expansion show this pattern.

A swing of the intensity ratio of the bands to approximately 1:1 is an indicator of heterozygosity in the C9ORF72 locus. If the difference in the number of repeats in the two alleles is small (i.e. no pathological repeat expansion in either allele) two close and equally weighted bands are present on Southern blots even when whole blood cell DNA is analysed, as illustrated in Figure 1B lane 7, using a DNA sample from a patient carrying one allele with a single copy of the GGGGCC sequence and another allele with 15 copies.

In cases with a pathological repeat expansion in one allele, a band of larger size is detected in DNA samples extracted from patients’ lymphoblastoid cell lines. Examples of these can be found in Figure 1C lanes 10 and 20 (~6 kb band corresponds to an allele with ~750 repeats), 15 (~7.5 kb band corresponds to an allele with ~1000 repeats), 16 and 22 (~8.5 kb band corresponds to an allele with ~1200 repeats), 6 (~10 kb band corresponds to an allele with ~1500 repeats), 3, 7 and 13 (~12 kb band corresponds to an allele with ~1800 repeats), 5, 8, 17 (>12 kb band corresponds to an allele with >2000 repeats). For accuracy of size estimates see Methods section. The presence in the sample of more than one of these larger bands (for example, lanes 4, 8, 14, 18 in Figure 1C, lanes 4, 5 in Figure 2) might indicate instability of the GGGGCC repeat region in the cultured lymphoblastoid cells. Alternatively it might reflect instability and somatic heterogeneity of the repeat region in ALS patients’ lymphoid cells, coupled with non-monoclonal origin of the analysed lymphoblastoid cell lines. In whole blood DNA such somatic heterogeneity is clearly evident in some samples, leading to the appearance of multiple bands that coalesce into an ambiguous smear on Southern blots (Figure 1B, lanes 1, 5). For two other samples in the same panel (Figure 1B, lanes 2, 6) lower quality of DNA caused appearance of similar smears but the ratio of 1.05 kb and 1.33 kb band intensities in these cases are close to 1:2 suggesting that both alleles of the C9ORF72 locus have small number of GGGGCC repeats. Therefore, even in those cases with an ambiguous smear, the presence of a pathological allele could be immediately predicted from the ~1:1 ratio of 1.05 kb and 1.33 kb band intensities.

The described protocol readily identifies patients with repeat expansions within both C9ORF72 loci. These cases are characterised by the absence of a 1.33 kb band on Southern blots, as illustrated in Figure 2, lane 1 for a patient carrying one allele with ~50 and another allele with >2000 GGGGCC repeats in the C9ORF72 locus.

When our Southern hybridisation protocol was used for analysis of genomic DNA extracted from peripheral blood, cerebellum, cortex and a lymphoblastoid cell line of patients with GGGGCC repeat expansion, a different pattern of high molecular mass fragments has been detected for each DNA sample of the same patient, as illustrated in Figure 3 and Additional file 2: Figure S1. This observation strongly suggests that an expanded repeat region in the C9ORF72 locus is unstable not only in nucleated blood cells but also in other somatic cells, including cells of the central nervous system.

Although a ³²P-radiolabeled hybridisation probe remains the best option for detection of GGGGCC repeat expansions in the C9ORF72 locus with the generation of clearer and more quantitative results compared to those obtained using DIG-labeled probes, in a pilot experiments we successfully used the latter detection method with our protocol (data not shown).

Whole blood samples were obtained from the Sheffield MND Blood DNA Biobank and CNS tissue was obtained from the Sheffield Brain Tissue Bank. The South Sheffield Research Ethics Committee approved the study, and informed consent was obtained for all samples. Lymphoblastoid cell lines were obtained from patients with ALS from the Wellcome Trust/Motor Neurone Disease Association-funded ALS/MND DNA bank and associated lymphoblastoid cell line repository in the UK.

Before lysis with a digest buffer (50 mM Tris–HCl pH 8.0; 100 mM NaCl; 5 mM EDTA; 1% SDS; 2 mg/ml proteinase K) frozen CNS tissues were grinded in liquid nitrogen. Following overnight digestion at 55°C genomic DNA was extracted twice with phenol and once with phenol/chloroform and ethanol precipitated.

A genomic fragment was amplified from a human DNA using oligonucleotide primers AGTTCCAGAGCTTGCTACAG and GAACAGTAGGAAAAGGGTCTG and cloned into the pCR-BluntII-TOPO vector (Invitrogen) to produce a pCh9.1 plasmid carrying an insert used as the hybridisation probe (Additional file 3: Figure S2). Well-established and widely used methods of Southern transfer, preparation of the probe and hybridisation procedure were used. In brief, approximately 20 μg of genomic DNA was digested with EcoRI and XbaI and the resulting fragments separated in 1% TAE agarose gel. After incubating the gel in 0.25 N HCl for 20 min, 0.5 N NaOH; 1.5 M NaCl for 40 min and 0.5 M Tris–HCl pH7.2; 3 M NaCl for 40 min at room temperature, DNA was transferred to a nylon membrane (Hybond N+, GE Healthcare) by capillary blotting [10]. An eukaryotic insert of the plasmid pCh9.1 was excised using KpnI and XbaI, separated from the plasmid backbone in agarose gel, purified using Qiagen kit and used for preparing hybridisation probes. The probe was labelled with ³²P in a nick-translation reaction [11]. Blots were prehybridised in HB (4×SSC; 0.5% SDS; 5×Denhardt’s solution; 100 μg/ml denatured salmon testis DNA) at 67°C for 4 h. Labelled DNA was denatured by incubation at 100°C for 5 min followed by immediate mixing with ice–cold HB. Hybridisation was carried out at 67°C for 16–20 h. Blots were washed 3 times in in 2×SSC; 0.2% SDS at 67°C and exposed to X-ray film. The detailed protocol as well as the pCh9.1 plasmid carrying an insert used as the hybridisation probe can be obtained by sending a request to Vladimir Buchman (buchmanvl@cf.ac.uk).

DNA extracted from 32 lymphoblastoid cell lines obtained from the MND National Biobank was used for Southern hybridisation analysis. This included 29 cases with a C9ORF72 expansion identified by repeat-PCR and 3 cases with non-C9ORF72 ALS. For certain cases DNA was also extracted from peripheral blood and CNS tissue available from the Sheffield Brain Tissue and DNA bank.

When at least one normal (without repeat expansion) DNA sample is present on the blot as a reference, the difference in relative intensity of 1.33 kb and 1.05 kb bands in this sample and in samples with repeat expansion can be easily recognised by eye. However, this difference can be quantified by scanning X-ray films, measuring bands using a software available for every gel documentation system and calculating the ratio of pixels in 1.33 kb and 1.05 bands for each sample. Even when for any technical reasons, the ratio of bands in samples with repeat expansion is higher than conventional 1:1, this is accompanied by corresponding increase of the ratio in a normal sample(s) present on the same Southern blot, which still allows discrimination of repeat-bearing and normal cases by eye. An example of this is shown in Figure 3.

The number of repeats within the expanded allele is estimated based on the size of corresponding fragment in base pairs minus the size of the non-expanded fragment (1330 base pairs) and divided by the size of the repeat unit (6 base pairs). For example, if the size of a fragment was 3 kb, estimated number of repeats in the corresponding locus is (3000 – 1330)/6 = 278. As with any method based on agarose gel electrophoresis, an accuracy of the repeat number estimate using the described protocol varies with the size of detected fragments. In the range of the repeat-bearing fragment size between 1.5 kb (which corresponds to an upper limit for a “normal” allele, i.e. less than 30 repeats = 0.18 kb + 1.33 kb -> 1.51 kb) and 3 kb (~300 repeats) the accuracy of the estimate might be around 10 repeats; between 3 kb and 6 kb (~750 repeats) it drops to ~50 repeats and between 6 kb and 10 kb (~1500 repeats) – to 200 repeats. Any bands above 12 kb should be considered as >2000 repeats.