Table 1.
Primers used in Sanger sequencing for validation of identified HaloPlex/NGS variants in the GNAI3 gene.
Table 2.
Summary of GNAI3 gene exons, introns and 5’ and 3’ UTR sequences (ENST00000369851) in the 5' to 3' direction (Ensembl GRCh38/hg38).
Fig 1.
GNAI3 gene locus in human Chr1, Agilent Haloplex amplicons and Median coverage of GNAI3.
A.GNAI3 is located in the p arm of chromosome 1, band 13.3 (red line). B. Agilent Haloplex Amplicons designed to amplify the GNAI3 9 exons, introns and 100 bp of each 5’ and 3’ UTRs are depicted in green and show that all regions of the gene were effectively covered. The GNAI3 RefSeq is shown in black and was aligned against the GRCh37/hg19 human genome using the UCSC-BLAT browser. C. The median coverage level varied from 14 to 8,009 across the targeted GNAI3 region analyzed in the HaloPlex design; only 1.2% of the target GNAI3 region was not covered.
Table 3.
Summary of SNVs in the GNAI3 5' UTR of ocular albino patients.
Table 4.
Summary of variants in exons of GNAI3 with likely functional effects in patients diagnosed with ocular albinism.
Table 5.
Summary of SNVs in the first 2109 bp of the GNAI3 non-coding exon 9/3’ UTR in patients affected with ocular albinism.
Table 6.
Summary of deletions and insertions in the first 2109 bp of the GNAI3 non-coding exon 9/3’ UTR in patients affected with ocular albinism.
Fig 2.
Variants identified in the 5’UTR and non-coding exon 9/3’ UTR using the HaloPlex target enrichment system/IIlumina MiSeq sequencing and visualized with the Integrative Genomics Viewer (IGV).
A. SNVs found in the 5’ UTR of the GNAI3 gene, represented by the blue horizontal bar before the start codon encoding Methionine (green), at the beginning of exon 1. B. SNVs, insertions and deletions found in the GNAI3 2109 bp of non-coding exon 9/3’ UTR, represented by the blue horizontal bar. Variants are homozygous [red or blue (lower frequency)] and heterozygous (half blue and half red).
Table 7.
Summary of SNVs, deletions and insertions in GNAI3 introns of patients diagnosed with ocular albinism.
Fig 3.
Validation of HaloPlex SNVs in the 5’ UTR of GNAI3 by Sanger Sequencing.
A. Homozygous SNV (c.-72C>T) of Patient 2. At the top of the Figure the track annotation panel shows the Chr1 location of the C >T variant at position 109548649. Next, the forward sequences of the reference (R) and test sample (S) are shown, followed by the confidence score of the peak corresponding to the variant. From the bottom up, the reverse sequences of the reference and test sample peak profiles and the confidence score demonstrate the same variant. The red arrows point to the mutated nucleotide. B. Heterozygous SNV (c.-61C>T) of Patient 10. Same as A, except for the track annotation panel, which shows the C>T SNV at position 109548660 in Chr1.
Fig 4.
Validation of HaloPlex heterozygous insertion (c.*1994_*1995insA) of Patient 18 in the non-coding exon 9/3’ UTR of GNAI3 by Sanger Sequencing.
As in the previous figures, the track annotation panel depicts the Chr1 location of the insertion at position 109594317, followed by the forward sequences of the reference (R) and test sample (S), the confidence score peaks and reverse reference and test sample sequences. The red arrows point to the insertion in the sequences.
Fig 5.
Validation of HaloPlex homozygous deletion (c.*1379_*1380delAT) of Patient 22 in the non-coding exon 9/3’ UTR of GNAI3 by Sanger Sequencing.
In addition to the track annotation panel clearly indicating the deletion at positions 109593703–109593704, the figure shows the forward sequences of the reference (R) and test sample (S) and the reverse sequences. The red arrows point to the deleted nucleotides.
Fig 6.
Structural representation of heterotrimeric GNAI3.
A) In the Gαβγ model, two domains of Gα are depicted: the Gα-helical insertion domain (GαAH) is in cyan and the Ras-like GTPase domain (GαRas) is in gold; the Gβ subunit is in green, and the Gγ subunit in blue. Sites of observed amino-acid mutations are indicated with red spheres. B) Magnified representation of mutations D102E and V109F as well as of amino-acid R105. Wild-type side chain carbons are in cyan while mutated residues are in red. C) Magnified representation of the “T-shaped” π-stacking interaction between F223 and F250 in wild-type GNAI3 (gold) that is lacking in the F223V mutant (red). The distance from the ortho carbon on F250 to the centroid of F223 is 3.5 Å, shown with a dotted black line. D) Magnified representation depicting the “parallel displaced” π-stacking interaction between α-H213 (gold) and β-W332 (green) that is absent in the H213L mutant (red). The distance between the centroids of the aromatic rings is 4.8 Å, shown with a dotted black line.