Multimodal characterisation of the SMN locus in SMA: copy number quantification and hybrid gene identification

Figures

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  Fig. 1. Representative results from the MLPA analysis. In the Coffalyser output, the lower X-axis represents the genomic region analysed, while the upper X-axis shows the corresponding probe name, which indicates its hybridisation site, along with the size of the resulting fragment. The Y-axis displays the final ratio obtained after normalisation. In the ratio chart, the 95% confidence interval over the reference samples is shown as a fixed coloured bar, blue for test probes and green for reference probes, whereas the 95% confidence interval for each sample is displayed as error bars surrounding the final probe ratio (dot). Yellow dots denote inconclusive results, red dots mark absence of signal, purple dots show that the arbitrary border has not been crossed, and blue dots indicate that the upper arbitrary border has been exceeded. Arbitrary borders (red and blue lines) are set at ±0.3 from the mean value of each probe in the reference samples. When a probe ratio in a sample crosses these borders, it may indicate a deletion or duplication, although not necessarily, as the variability of the probe in the reference samples may also cross them. A final ratio (dot) of 0 indicates a…

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  Fig. 2. Representation of the expected values according to the fitted model and the observed values according to read counts in long-read sequencing (LRS). a Patient 17, b patient 18, c patient 32, d patient 32, e patient 45, f patient 56, g patient 62, h patient 63, i patient 65, and j patient 66. Bar plots show the comparison between observed (blue) and expected (green) read counts for different SMN2 haplotypes. Magenta dots indicate the estimated copy number of SMN2 in SMA patients.

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  Fig. 3. Characterisation of SMN2 copy number and SMN hybrid structures detected by LRS. a Patient 17, b patient 18, c patient 32, d patient 32, e patient 45, f patient 56, g patient 62, h patient 63, i patient 65, and j patient 66. The distribution of paralogous sequence variants (PSVs) across haplotypes is shown: each row corresponds to a haplotype, while each column represents a PSV position. PSVs specific to SMN1 are indicated in dark blue, whereas those of SMN2 are shown in light blue.

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  Fig. 4. Paralogous sequence variants (PSVs) haplotypes for SMN hybrid structures detected by LRS. H1, H3 and H5 had been described previously17,47. The remaining six structures have not been reported to date. PSVs of SMN1 are indicated in dark blue, while those of SMN2 are shown in a lighter blue. The positions of these PSVs (intron 6, exon 7, intron 7, or exon 8) are indicated at the top of the figure by a curly brace.

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  Fig. 5. Visualisation of LRS results in patient 45 using integrative genomic viewer (IGV). A 6.3 kb deletion was identified, encompassing from chr5:70.072.862 to chr5:70.079.171 (GRCh38/hg38).