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Figure 4 | BMC Medical Genetics

Figure 4

From: Clinical and genetic analyses of three Korean families with hereditary hemorrhagic telangiectasia

Figure 4

Allele-specific expression analyses of the ENG c.1-127 C > T mutant allele. (A) Restriction fragment length polymorphism analysis. (Left panel) F1 and R1 primers were used for amplification of the ENG genomic DNA. BtsCI, which recognizes the mutant allele, digested of 313 bp PCR product from the mutant allele into 216 bp and 97 bp fragments (arrowheads). The mutant allele-specific BtsCI-digested PCR products were detected in affected family member (Patient II-3) but not in an unaffected family member. (Right panel) F2 and R2 primers were used to amplify ENG transcripts from ENG cDNA generated by reverse transcriptase using ENG-specific R3 primer. BtsCI digestion of RT-PCR product amplified from mutant allele is supposed to yield 284 bp and 97 bp fragments if the mutant transcripts are stably present. Unlike genomic PCR products, BtsCI-digested PCR fragments from RT-PCR products of affected individuals were barely detectable (arrowheads). (B) Direct sequencing of the PCR products amplified from genomic DNA of a patient II-4 and cDNAs of an unaffected family member and three patients (II-2, II-3, II-4). The ENG c.1-127 positions were indicated by arrows. (C) Real-time RT-PCR analysis of ENG transcripts. Total RNAs isolated from the blood of three unaffected persons and three patients (II-2, II-3, II-4) were used for first-strand cDNA synthesis. Mean values of ENG ΔΔCt ratios and standard deviations are shown as filled box and bar above each box, respectively. The relative amount of ENG transcripts in each sample was normalized with the amount of Cyclophilin in the sample. * p < 0.01, compared with the unaffected samples, as determined by Student's t test.

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