Abstract
Tuberous sclerosis (TSC) is an autosomal dominant disorder caused by mutations in either of two genes, TSC1 and TSC2. Point mutations and small indels account for most TSC1 and TSC2 mutations. We examined 261 TSC DNA samples (209 small-mutation-negative and 52 unscreened) for large deletion/duplication mutations using multiplex ligation-dependent probe amplification (MLPA) probe sets designed to permit interrogation of all TSC1/2 exons, as well as 15–50 kb of flanking sequence. Large deletion/duplication mutations in TSC1 and TSC2 were identified in 54 patients, of which 50 were in TSC2, and 4 were in TSC1. All but two mutations were deletions. Only 13 deletions were intragenic in TSC2, and one in TSC1, so that 39 (73%) deletions extended beyond the 5′, 3′ or both ends of TSC1 or TSC2. Mutations were identified in 24% of small-mutation-negative and 8% of unscreened samples. Eight of 54 (15%) mutations were mosaic, affecting 34–62% of cells. All intragenic mutations were confirmed by LR-PCR. Genotype/phenotype analysis showed that all (21 of 21) patients with TSC2 deletions extending 3′ into the PKD1 gene had kidney cysts. Breakpoints of intragenic deletions were randomly distributed along the TSC2 sequence, and did not preferentially involve repeat sequence elements. Our own 20-plex probe sets gave more robust performance than the 40-plex probe sets from MRC-Holland. We conclude that large deletions in TSC1 and TSC2 account for about 0.5 and 6% of mutations seen in TSC patients, respectively, and MLPA is a highly sensitive and accurate detection method, including for mosaicism.
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Acknowledgments
We thank all of the patients and clinicians who contributed DNA samples and clinical information; Mei Lin and Dawn Ciulla for assistance with capillary electrophoresis and peak height determinations; NIH NS31535 for support.
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Fig5
Supplementary Figure 1. Electropherograms of MRC-Holland and homemade MLPA probe sets. Control samples are shown. The location of each probe within the gene is shown (e – exons, 5′ and 3′ – flanking sequences), while for control probes the size of the amplified product is shown (in bp). The size of amplified probes are 136-418 and 130-481 for the MRC-Holland probe sets, and 90-148 and 90-156 for the homemade probe sets. The asterisk indicates a probe whose signal was variable due to polymorphism and was not used for analysis. The view windows of the electropherograms were adjusted to permit better display of these patterns, which is why the width of peaks is different on the different electropherograms. Note the relative high range of peak size (4.7-5.2-fold) for the MRC-Holland probe sets in comparison to our homemade probe sets (2.0-fold). (TIF 1,385 kb)
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Kozlowski, P., Roberts, P., Dabora, S. et al. Identification of 54 large deletions/duplications in TSC1 and TSC2 using MLPA, and genotype-phenotype correlations. Hum Genet 121, 389–400 (2007). https://doi.org/10.1007/s00439-006-0308-9
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DOI: https://doi.org/10.1007/s00439-006-0308-9