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Fig. 1 | Genome Medicine

Fig. 1

From: Genomic diagnosis for children with intellectual disability and/or developmental delay

Fig. 1

Intronic variants in ALG1 and MTOR disrupt splicing and introduce early stop codons. a Diagram showing the region of ALG1 surrounding the variant found in the proband and mother, an A > G transition three nucleotides downstream from the splicing donor site of intron 11. E = exon. b The ALG1 variant leads to increased retention of intron 11. cDNA from patient derived RNA extracted from blood was amplified using the PCR F/PCR R primer set (shown in panel 1A) to test for intron 11 retention. The control samples are cDNA derived from RNA extracted from blood of an unrelated individual as well as the father of the proband that did not harbor the variant. The proband, and mom, from which the variant was transmitted, both harbor the incorrectly spliced transcript retaining intron 11. Control reactions lacking RT were also performed and did not show the PCR product containing the fully retained intron (data not shown). c, d qPCR analysis shows that the variant leads to inclusion of the entire intron 11. Controls are two unrelated individuals and the father of the proband. The affected individuals are the proband and mother. e Diagram showing the region of MTOR surrounding the variant, an A > G transition two nucleotides upstream of the splicing acceptor site. E = exon. f The region surrounding intron 4 was amplified using PCR F and PCR R (position indicated in (e)), and shows partial retention of the intron. The retained partial intron was not detected in control reactions lacking RT (data not shown). g, h qPCR from blood RNA shows that the 5′ splice site is not affected by the variant, but that the 3′ acceptor site is, leading to partial retention (134 bp) of intron 4. Controls included unrelated individuals and the maternal half aunt of the proband. Affected individuals are the proband and half-sibling. For all qPCR analyses, RNA was extracted from blood and ΔΔCT values were calculated as a percent of affected individuals and normalized to GAPDH. The sequences of all oligos used are found in Additional file 3: Table S7

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