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Figure 8 | Orphanet Journal of Rare Diseases

Figure 8

From: Molecular genetic studies and delineation of the oculocutaneous albinism phenotype in the Pakistani population

Figure 8

Functional analysis of c.1045-15 T > G mutation. A. Electropherograms of amplimers from genomic DNA templates illustrating homozygosity for the wild-type and c.1045-15 T > G substitution mutation found in the affected individuals of the five OCA2 families. The arrow indicates the site of the mutation. B. To determine the effect of the c.1045-15 T > G mutation on splicing, exon 10 with 200 bp of the flanking intron of OCA2 was introduced into the pSPL3 vector and analyzed through an in vitro splicing assay. The transfected empty pSPL3 vector produced the expected product of 177 bp, whereas the wild-type exon 10 splice site produced two bands with (249 bp) and without (177 bp) exon-10 splicing when amplified with vector primers, which might indicate the presence of weak splice junctions around exon 10. The construct with the c.1045-15 T > G mutation produced a band of 177 bp, which upon sequencing, revealed the skipping of exon 10. With the mutant construct, a weak band of ~249 bp was occasionally observed, but sequencing revealed an aberrant splice product. C. Human OCA2 isoforms with and without exon 10 are expressed in many tissues. D. Molecular genetic analysis of known OCA genes in a cohort of forty Pakistani families indicates that (a) OCA2 mutations are the most common cause of OCA, and (b) a significant number of families do not have mutations in the known OCA genes. E. Real-time quantitative RT–PCR analysis of TYR mRNAs level in human melanocytes, retina and testis cDNA libraries. CT is the observed threshold number of PCR cycles required for detection of the amplification product; ΔCT is the calculated difference in CT between the TYR gene and an internal control standard (GAPDH) measured in the same sample. ΔΔCT is the calculated difference in ΔCT between the experimental and exon 9–11 isoform in retina. Compared to the retina and testis, melanocytes have a relatively high expression of both exon 9–10 and exon 9–11 isoforms of TYR.

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