case_images: PMC3626300_01
case_images: PMC3626300_02
case_images: PMC3626300_03
case_images: Sequence chromatogram of the Cys86X mutation in COL2A1 exon 2. The sequence chromatogram of COL2A1 exon 2 encompassing codon 86 demonstrates the c.258C>A mutation (GenBank NM_001844.4, +1 in cDNA numbering corresponding to the A of the methionine translation initiation codon) converting a cysteine codon to a stop codon in two affected individuals while the mutation is not present in two unaffected individuals. DNA analysis of all other affected family members cosegregated with this mutation while the mutation was not present in all unaffected family individuals.
case_images: mv-v19-759-f2.jpg
case_images: PMC3626300_03_mv-v19-759-f2.jpg
case_images: f2
case_images: The nonsense mutation was a C to A change (Figure 2) in exon 2 (c.258C>A; NM_001844.4), converting codon TGC for cysteine at position 86 to codon TGA for a premature stop codon (Cys86Stop; NP_001835.3). Subsequent sequencing of the remaining family members showed mutation cosegregation with the disease phenotype (Figure 2).
case_images: COL2A1 cDNA structure and COL2A1 cDNA primer design. Exon 2 undergoes tissue-dependent alternative splicing. The COL2A1 type IIA isoform (A), expressed in the eye vitreous and in embryonic chondroprogenitor cells, includes exon 2 (Ex 2) whereas this exon is spliced in the COL2A1 type IIB isoform (B), which is expressed by adult differentiated chondrocytes. Primers were designed to amplify both cDNA isoforms: The COL2A1 cDNA primers span 303 bp when amplifying COL2A1 type IIB cDNA (excluding exon 2), and 510 bp when amplified COL2A1 type IIA cDNA (including exon 2). Ex 1, Ex 2, Ex 3, and Ex 8 depict exons 1, 2, 3, and 8.
case_images: mv-v19-759-f3.jpg
case_images: PMC3626300_03_mv-v19-759-f3.jpg
case_images: f3
case_images: We examined COL2A1 expression across normal fetal eye tissues to verify the presence or absence of type IIA and/or type IIB isoforms (Figure 3).