PMC4746300_01
Overview of the phenotype and molecular findings of two patients with CdLS. (a) Phenotype of patient 1. (b) Phenotype of patient 2. (c) Localization of the mutations on NIPBL gene and their consequences upon transcript processing. Boxes mean exons and lines mean introns. Dark boxes represent the nontranslated region. The localization of each mutation and chromatograms on genomic DNA are shown above the gene. Agarose gel of the cDNA PCR products in each patient' pictures and chromatograms are shown underneath the gene. Patient 1 yielded normal product of 634 bp and an aberrant fragment of 535 bp corresponding to exon 28 skipping. Patient 2 showed the normal product of 363 bp and an additional band of 208 bp corresponding to exon 37 skipping (MW: molecular weight, WT: wild type, P1: patient 1, and P2: patient 2).
BMRI2016-8742939.001.jpg
PMC4746300_02_BMRI2016-8742939.001.jpg
fig1
She had small hands with brachymesophalangy V and restriction of elbow movements (Figure 1(b)). In patient 1 a transition (c.5329-15A>G in intron 27) and in patient 2 a deletion (c.6344del(-13)_(-8) in intron 36) were identified (Figure 1(c)). Sequencing analysis of the aberrantly spliced product revealed a loss of a 99 bp fragment representing the entire exon 28 region (Figure 1(c)). Subsequent sequencing analysis showed a deletion of 155 bp representing exon 37 of the NIPBL gene (Figure 1(c)).
PMC4746300_02
Overview of the phenotype and molecular findings of two patients with CdLS. (a) Phenotype of patient 1. (b) Phenotype of patient 2. (c) Localization of the mutations on NIPBL gene and their consequences upon transcript processing. Boxes mean exons and lines mean introns. Dark boxes represent the nontranslated region. The localization of each mutation and chromatograms on genomic DNA are shown above the gene. Agarose gel of the cDNA PCR products in each patient' pictures and chromatograms are shown underneath the gene. Patient 1 yielded normal product of 634 bp and an aberrant fragment of 535 bp corresponding to exon 28 skipping. Patient 2 showed the normal product of 363 bp and an additional band of 208 bp corresponding to exon 37 skipping (MW: molecular weight, WT: wild type, P1: patient 1, and P2: patient 2).
BMRI2016-8742939.001.jpg
PMC4746300_02_BMRI2016-8742939.001.jpg
fig1
She had small hands with brachymesophalangy V and restriction of elbow movements (Figure 1(b)). In patient 1 a transition (c.5329-15A>G in intron 27) and in patient 2 a deletion (c.6344del(-13)_(-8) in intron 36) were identified (Figure 1(c)). Sequencing analysis of the aberrantly spliced product revealed a loss of a 99 bp fragment representing the entire exon 28 region (Figure 1(c)). Subsequent sequencing analysis showed a deletion of 155 bp representing exon 37 of the NIPBL gene (Figure 1(c)).
Functional analyses of the intronic mutations c.5329-15A>G and c.6344del(-13)_(-8). (a) Minigene constructs were generated by site-directed mutagenesis. Deletion of c.6344del(-13)_(-8) causes an exon skipping that was also found by a transversion of the five deleted purines into pyrimidines (PUR). (b) Quantitative real-time PCR analysis of total NIPBL mRNA from leukocytes of patients with CdLS and controls. NIPBL transcript levels were normalized by GAPDH expression. The values presented are the medians of triplicate determinations +- SD. The mean ratio of total NIPBL mRNA in controls was assigned as 100% (CT: controls, P1: patient 1, P2: patient 2, and ***: P < 0.001 versus control).
BMRI2016-8742939.002.jpg
PMC4746300_02_BMRI2016-8742939.002.jpg
fig2
Transfection using the exon 37 WT construct generated two fragments, the main one of 423 bp containing exon 37 and another of 268 bp without this exon (Figure 2(a)). The presence of the mutation c.6344del(-13)_(-8) leads to a rise of the transcript with exon 37 deletion while the normal transcript disappeared (Figure 2(a)). The disruption of the polypyrimidine tract of exon 37 (PUR) produced a similar effect to mutation c.6344del(-13)_(-8) (Figure 2(a)).