(A, B) Human dermal fibroblast cultures from 7 heterozygous cell cultures and 2 homozygous cell cultures. Estimated absolute copy numbers of the lamin A (A) and lamin C (B) transcripts were normalized to the endogenous control β-glucoronidase. The mean value from different batches of the same cell culture is presented. Mean value of AG11498 and AG11498A is presented as AG11498. Mean value of AG06917A and AG06917B is presented as AG06917. AG01972B and GM01972B correspond to different biopsies from the same patient. Quantification of the homozygous control samples (AG03258 and AG03512) showed only weak amplification with the C assay and no amplification with the T assay in the absence of the respective alleles. (C) Mean and standard deviation of the frequencies of lamin A and lamin C transcripts for each allele across the 7 heterozygous human dermal fibroblast cultures derived from 6 different individuals. Two-tailed unpaired t-test. Asterisks represent significant differences: ***P<0.0001.

(A) Schematic illustration of the location, in the 3′ end of the lamin A and lamin C cDNA, of the primers and probes used in the different assays. The rs4641 SNP is located in the last nucleotide of exon 10, which is present in lamin A and lamin C transcripts. The forward primer, 5′-CAACTCCACTGGGGAAGAAGTG-3′ (orange arrows), and the TaqMan MGB probe, 5′-ATGCGCAAGCTGGTG-3′ (black bars), were the same in all 4 assays. Reverse primers (blue and red arrows) make the assay specific for each allele and transcript by allele-specific 3′-end termination. The reverse primer in the lamin A allele C assay was 5′-GCTGCAGTGGGAGCCG-3′ in the lamin A allele T assay, 5′-TGCTGCAGTGGGAGCCA-3′ in the lamin C allele C assay, 5′-GGCGGCTACCACTCACG-3′, and in the lamin C allele T assay, 5′-CGGCGGCTACCACTCACA-3′. Amplification of the lamin A and lamin C transcripts also results in co-amplification of the lamin AΔ150 and lamin C2, respectively. (B) Target transcripts and sizes of the PCR products of the 4 designed TaqMan assays and the endogenous control. (C) Standard curves of plasmids containing the assay-specific amplicon, estimated absolute copy numbers of transcript lamin C allele C (left) and transcript lamin C allele T (right). A minimum of 5 standards (blue) ranging from 1.1×10¹ to 1.1×10⁷ estimated copies of plasmids were used. Standards and samples from 11 different human dermal fibroblast cultures (red). Cultures were derived from 4 heterozygote HGPS patients (N = 7), heterozygote controls (N = 2), and homozygote controls (N = 2). Ct: cycle threshold.