Supplementary Figure 2: Identification of a heterozygous deletion encompassing exon 4 of HCN1 in a female subject with ID and ASD, inherited from her asymptomatic father.
From: De novo mutations in HCN1 cause early infantile epileptic encephalopathy
(a) Cyto-12 SNP array (Illumina) profiles of the patient with HCN1 exon 4 deletion and details of the coding sequences included the deletion: the y axis indicates the B allele frequency (above) and the log R ratio (below), and the x axis indicates the position on chromosome 5. The deletion spans ~33 kb and theoretically leads to an in-frame deletion of 73 amino acids (p.Asn338_Lys410del) in the extracellular loop between the S5 and S6 domains containing the pore region. (b) Confirmation of the presence of the HCN1 heterozygous deletion in the proband and her father by quantitative PCR using primers located on exon 4 (forward: 5'-CCACCTGCTATGCCATGTTT-3'; reverse: 5'-ATACTGCCGCCTCGAAGsAAT-3'). RT-PCR experiments were performed using 10 ng of genomic DNA, 0.8 μM of each primer and 12.5 μl of SYBR Green PCR master mix (Applied Biosystems) in a total volume of 25 μl. RNase P (RNAse P Control Assay, Applied Biosystems) was used as the reference amplicon. Each sample was run in triplicate on an ABI PRISM 7700 Detection System (Applied Biosystems), and three different experiments were used for final quantification. Relative ratios were calculated using the formula r = with ΔΔCt = (Ct mutation – Ct RNaseP) ind tested – (Ct mutation – Ct RNaseP) ind ref.
