Identification of Variants in CNGA3 As Cause for Achromatopsia by Exome Sequencing of a Single Patient

Supplementary Online Content

Lam K, Guo H, Wilson GA, Kohl S, Wong F. Identification of variants in CNGA3 as cause for achromatopsia by exome sequencing of a single patient. Arch Ophthalmol. 2011;129(9):1212-1217.

eAppendix. Supplemental data: retinal disease genes in RetNet that were not captured by Agilent’s SureSelect human all exon kit version.

eFigure. Curve showing cumulative percent of bases covered (left axis) and histogram showing percent bases covered (right axis) for the range of read depths (horizontal axis).

eTable 1. Types of variants identified.

eTable 2. Potentially damaging changes (15) detected in 13 retinal disease genes based on predictions by PolyPhen-2.

This supplementary material has been provided by the authors to give readers additional information about their work.

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Retinal disease genes in RetNet that were not captured by Agilent’s SureSelect human all exon kit version 1. Of the 177 genes, 21 (11.86%) were not captured: AHI1, ARMS2, CC2D2A, CEP290, FSCN2, GPR98, GRK1, KSS, LHON, MT-ATP6, MT-TH, MT-TL1, MT-TP, MT-TS2, MYO7A, NPHP4, NR2E3, PROM1, RIMS1, RPGRIP1, SAG.

eFigure. Curve showing cumulative percent of bases covered (left axis) and histogram showing percent bases covered (right axis) for the range of read depths (horizontal axis).

120% 18.00%

16.00% 100% 14.00%

80% 12.00% % Bases 10.00% 60% 8.00%

Cumulave % Bases 40% 6.00%

4.00% 20% 2.00%

0% 0.00% 0 20 40 60 80 100 120 140 160 180 200 220 Read Depth

eTable 1. Types of variants identified.

Indels were identified by SAMtools after aligning SOLiD reads to hg18 using BWA (http://bio-bwa.sourceforge.net/). Exonic indels were annotated using the SeattleSeq database (http://gvs-p.gs.washington.edu/SeattleSeqAnnotation); none of the exonic indels were in the 177 retinal disease genes in RetNet. The 62,518 SNPs identified by SAMtools were submitted to SeattleSeq for sorting splice-site, missense and nonsense mutations. None of the splice-site variants coincided with the 177 retinal disease genes in RetNet.

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eTable 2. Potentially damaging changes (15) detected in 13 retinal disease genes based on predictions by PolyPhen-2.

cDNA Protein

Allelic Change Residue change Gene Name SNP ID Hom/Het Read Depth dbSNP 130 ALMS1 rs45501594 hom 45 Yes c.10631 C>G p.3544 T>S ALMS1 het 36 No c.1570 T>C p.524 S>P BBS7 het 27 No c.2037 T>C p.679 I>M C3 het 18 No c.701 A>G p.234 V>A CNGA3 het 34 No c.1580 T>G p.527 L>R CNGA3 rs104893620 het 38 Yes c.829 C>T p.277 R>C HMCN1 rs10798035 het 17 Yes c.8678 A>G p.2893 E>G LRP5 het 17 No c.473 T>C p.158 L>S PRPF8 het 22 No c.5618 T>C p.1873 E>G RHO rs104893770 het 49 Yes c.133 T>C p.45 F>L RP1L1 rs4840502 hom 32 Yes c.3436 C>T p.1146 R>W RPGRIP1L het 11 No c.181 T>C p.61 N>D TLR3 hom 8 No c.2635 T>C p.879 W>R USH2A rs696723 het 36 Yes c.2137 G>C p.713 G>R WFS1 rs734312 hom 21 Yes c.1832 G>A p.611 R>H

Based on a recessive model, all the heterozygous variants were eliminated except the CNGA3 variants and the ALMS1 variants. The common polymorphisms identified in ALMS1 (homozygous), RP1L1 and WFS1 were observed in normal genomes, including that of Venter; ALMS1 variant was in Watson as well. Elimination of the ALMS1 (homozygous) polymorphism also removed the ALMS1 (heterozygous) variant based on a recessive model. Furthermore, our patient has a non-syndromic retinal disease; hence, ALMS1 and WFS1 could have been eliminated on this basis as well. The list of candidate genes was thus reduced to two: CNGA3 with two heterozygous candidate mutations and TLR3 with one homozygous candidate mutation. Of those variants included in dbSNP130, c.829C>T p.R277C in CNGA3 is known to cause achromatopsia.

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