A deep intronic mutation in an Ataxia Telangiectasia patient identified by genomic resequencing of the ATM region

Recent development of Next Generation Sequencing (NGS) techniques is changing the approach to search for mutations in human genetic diseases. We decided to apply NGS to study a patient affected by Ataxia Telangiectasia (A-T), in which one of the two expected mutations was not found after DHPLC, cDNA sequencing and MLPA screening. Linkage analysis showed the mutated unknown allele was maternally inherited. The entire ATM genomic region (160 Kb) was divided in 32 partially overlapping fragments of 4-6 kb and amplified by long-range PCR (Takara) in the patient and her mother. Fragments were quantified, pooled in equimolar amounts and sequenced by the Baseclear service using an IlluminaGAIIX platform. Raw data were analyzed by the CLC Genomics workbench software. Average coverage was 300X. Comparison between the mother and daughter sequences, allowed to identify six variants shared in heterozygosis by the two genomes and not reported in the SNP (ver 131) database. All were intronic, but only one c.1236-405C>T (coverage 670X), located in IVS11 was predicted to be pathogenic as it affected splicing (BDGP, www.fruitfly.org/seq). This mutation creates a novel donor splice site (score 0.22 wt to 1.00 mutated) in position -405 of intron 11 and as a result may activate an alternative acceptor splice site in position -619 of intron 11 (score 0.71). cDNA analysis in the patient using forward and reverse primers in exon 11 and 12, showed a normal band of 260 bp and a larger extra band of 472 bp. Sequence analysis showed that the larger band contained a “pseudoexon” of 212 bp, which used the alternative 5’ and 3’ splice sites at positions -619 and -405 of intron 11. This insertion results in a frameshift on the cDNA and premature stop codon. This approach based on resequencing of a genomic region is a powerful strategy to identify rare mutations that affect splicing, and it is suitable for a selected group of patients in which DHPLC or cDNA sequencing plus MLPA was unsuccessful. The identification of splicing variants is important for their possible therapeutic targeting using AMO (Antisense Morpholino Oligonucleotides).