Table S1. in Silico Predictions for Missense Mutations Discussed in This Study

Table S1. In silico predictions for missense mutations discussed in this study.

Gene / cDNA / Protein / PhyloP / AA cons / Grantham distance / Region
Domain / Align GVGD / SIFT
(score) / PolyPhen-2
(score)
LRAT / c.326G>T / p.Arg109Leu / 6.02 / Up to Tetraodon / 102 / Topological domain (cytoplasmatic) / C65 / Deleterious
(0) / Probably damaging
(1)
RDH12 / c.464C>T
(rs121434337) / p.Thr155Ile / 6.18 / Up to Fruitfly / 89 / Short-chain dehydrogenase/reductase protein domain / C65 / Deleterious
(0) / Probably damaging
(1)
RDH12 / c.803G>T / p.Cys268Phe / 5.77 / Up to Tetraodon / 205 / NA / C0 / Tolerated
(0.05) / Probably damaging
(0.999)
USH2A / c.10721G>T / p.Gly3574Val / 3.68 / Up to Chicken / 109 / 20th fibronectin type III repeat / C65 / Deleterious
(0) / Benign
(0.246)
USH2A / c.12574C>T / p.Arg4192Cys / 3.03 / Up to Cow / 180 / 27th fibronectin type III repeat / C45 / Deleterious
(0) / Probably damaging
(0.999)
NMNAT1 / c.205A>G / p.Met69Val / 4.81 / Up to Baker's yeast / 21 / NA / C15 / Deleterious
(0) / Probably damaging
(1)
NMNAT1 / c.769G>A
(rs150726175) / p.Glu257Lys / 3.84 / Moderately conserved / 56 / NA / C0 / Tolerated
(0.24) / Benign
(0.089)
MERTK / c.2180G>A / p.Arg727Gln / 6.02 / Up to Opossum / 43 / Protein kinase domain / C35 / Deleterious
(0) / Probably damaging
(1)

Novel mutations are indicated in bold. Transcripts used for mutation nomenclature and evaluation are: NM_004744.3 (LRAT), NM_152443.2 (RDH12), NM_206933.2 (USH2A), NM_022787.3 (NMNAT1), NM_006343.2 (MERTK). Domains/regions are derived from UniProtKB using the following accession numbers: O95237 (LRAT), Q96NR8 (RDH12), O75445 (USH2A), Q12866 (MERTK), Q9HAN9 (NMNAT1). Abbreviations used: AA cons: amino acid conservation; NA: not applicable.

Table S2. Exome sequencing mapping statistics.

Exome enrichment kit / Sample / Alignment / Coverage depth
Total no of reads / Mapped reads / Mapped reads following duplicate read removal / Average coverage / % of regions 10x / % of regions 20x / % of regions 40x
TruSeq / F13 / 119,917,434 / 105,678,111 (88%) / 92,350,315 (77%) / 78.6 / 92.9 / 88.6 / 76.5
F14 / 127,021,034 / 112,292,265 (88%) / 96,087,831 (76%) / 85 / 93.6 / 90.1 / 80.6
F15 / 116,498,066 / 102,706,013 (88%) / 91,818,537 (79%) / 78.5 / 92.9 / 88.6 / 76.8
F16 / 89,384,650 / 79,891,736 (89%) / 60,341,347 (68%) / 60.0 / 86.7 / 79.2 / 62.1
F18 / 134,471,164 / 117,351,206 (87%) / 109,405,319 (81%) / 97.7 / 93.5 / 89.9 / 81.3
F20 / 136,004,204 / 120,479,337 (89%) / 106,893,783 (79%) / 92.9 / 93.2 / 89.5 / 80.3
F21 / 140,925,274 / 124,935,282 (89%) / 111,955,654 (79%) / 97.9 / 93.5 / 90.1 / 81.5
Nextera / F12 / 129,208,884 / 112,697,415 (87%) / 94,310,056 (73%) / 87.0 / 91.5 / 86.5 / 74.3
F17 / 128,039,454 / 112,600,321 (88%) / 94,065,168 (73%) / 86.7 / 91.2 / 86.2 / 74.2
F19 / 128,579,140 / 111,967,859 (87%) / 93,329,248 (73%) / 85.4 / 91.1 / 85.8 / 73.2

Coverage depth is calculated for all regions enriched by the TruSeq Exome Enrichment kit or the Nextera Rapid Capture Expanded Exome kit (both Illumina).

Table S3. Largest IBD regions of the families used for filtering of whole exome data.

Family / No of individuals / Par cons / Chr / Start (hg19)
(bp) / Stop (hg19)
(bp) / Length
(Mb) / No of SNPs / Used to filter exome data
F12 / 1 aff / NA / 14 / 20,942,451 / 24,311,085 / 3.4 / 351 / x
2 / 194,169,329 / 197,149,497 / 3.0 / 263 / x
4 / 43,281,240 / 45,786,807 / 2.5 / 239 / x
3 / 82,976,735 / 85,820,181 / 2.8 / 218 / x
15 / 96,817,467 / 98,880,510 / 2.1 / 180 / x
1 / 72,508,296 / 74,259,955 / 1.8 / 167
F13 / 1 aff / NA / 15 / 72,030,066 / 81,671,975 / 9.6 / 1,336 / x
2 / 20,695,707 / 29,638,906 / 8.9 / 558 / x
8 / 43,383,815 / 51,964,292 / 8.6 / 428 / x
2 / 88,541,437 / 97,549,668 / 9.0 / 400 / x
10 / 37,845,751 / 43,429,929 / 5.6 / 199
F14 / 1 aff
2 unaff / FC / 16 / 20,364,588 / 65,795,203 / 45.4 / 4,000 / x
21 / 21,903,798 / 44,589,215 / 22.7 / 3,175 / x
2 / 219,000,966 / 243,029,573 / 24.0 / 2,638 / x
10 / 87,685,741 / 115,373,277 / 27.7 / 2,519 / x
6 / 44,503,829 / 65,986,503 / 21.5 / 2,413 / x
10 / 34,415,490 / 55,293,890 / 20.9 / 1,698 / x
9 / 113,300,835 / 130,985,525 / 17.7 / 1,683 / x
5 / 109,204,024 / 124,500,651 / 15.3 / 1,501 / x
18 / 35,445,641 / 55,190,626 / 19.7 / 1,453 / x
5 / 149,650,925 / 155,810,954 / 6.2 / 481
F15 / 1 aff / NA / 6 / 100,539,396 / 107,252,119 / 6.7 / 864 / x
7 / 56,668,557 / 64,221,817 / 7.6 / 335 / x
3 / 87,371,723 / 88,418,911 / 1.1 / 237
F16 / 1 aff
3 unaff / FC / 2 / 109,804,521 / 147,023,441 / 37.2 / 3,354 / x
11 / 6,082,903 / 16,844,924 / 10.8 / 1,135
F17 / 1 aff / FC / 2 / 16,205,386 / 48,592,202 / 32.4 / 3,221 / x
3 / 107,202,656 / 141,641,015 / 34.4 / 2,994 / x
18 / 35,558,983 / 55,645,543 / 20.1 / 2,157 / x
15 / 62,648,611 / 91,801,266 / 29.2 / 2,078 / x
1 / 202,442,082 / 223,484,727 / 21.0 / 2,059 / x
21 / 25,071,454 / 38,042,412 / 13.0 / 1,612 / x
6 / 136,209,748 / 151,252,985 / 15.0 / 1,409 / x
16 / 91,010 / 10,423,631 / 10.3 / 1,171 / x
15 / 39,162,244 / 52,816,195 / 13.7 / 1,079 / x
2 / 60,750,560 / 71,848,412 / 11.1 / 952 / x
18 / 220,071 / 5,452,357 / 5.2 / 643
F18 / 1 aff / NA / 4 / 139,599,898 / 170,935,245 / 31.34 / 3,094 / x
8 / 29,226,940 / 65,419,106 / 36.19 / 2,973 / x
1 / 234,273,318 / 249,143,646 / 14.87 / 1,613 / x
5 / 66,251,173 / 82,123,527 / 15.87 / 1,342 / x
4 / 41,841,547 / 57,457,918 / 15.62 / 1,077 / x
10 / 122,175,979 / 131,398,005 / 9.22 / 1,070 / x
11 / 78,821,945 / 86,962,378 / 8.14 / 990 / x
2 / 71,756,645 / 80,010,375 / 8.25 / 738 / x
13 / 76,144,233 / 80,924,165 / 4.78 / 569 / x
6 / 100,522,097 / 105,710,719 / 5.19 / 523 / x
4 / 29,099 / 6,757,184 / 6.73 / 519 / x
20 / 69,408 / 4,625,558 / 4.56 / 410 / x
22 / 17,171,377 / 24,136,542 / 6.97 / 393 / x
22 / 25,088,629 / 27,825,443 / 2.74 / 305
F19 / 1 aff / NA / 2 / 43,151,102 / 140,799,875 / 97.6 / 8,239 / x
12 / 79,311,580 / 124,043,296 / 44.7 / 4,213 / x
9 / 82,794,762 / 115,594,108 / 32.8 / 3,491 / x
4 / 17,261,697 / 38,285,407 / 21.0 / 2,342 / x
10 / 120,709,474 / 135,422,505 / 14.7 / 1,569 / x
11 / 4,325,792 / 17,833,727 / 13.5 / 1,551 / x
3 / 73,603 / 8,611,070 / 8.5 / 1,416 / x
6 / 159,779,440 / 170,874,759 / 11.1 / 1,187 / x
5 / 56,273,488 / 66,511,535 / 10.2 / 1,081 / x
2 / 11,731,846 / 20,797,088 / 9.1 / 1,013 / x
3 / 188,568,652 / 197,833,758 / 9.3 / 788 / x
1 / 178,686,247 / 185,977,870 / 7.3 / 640
11 / 69,848,710 / 77,195,482 / 7.3 / 438
F20 / 2 aff
1 unaff / FC / 7 / 99,747,378 / 113,769,768 / 14.0 / 1,126 / x
10 / 36,921,500 / 44,200,970 / 7.3 / 286 / x
13 / 22,526,325 / 23,893,657 / 1.4 / 201
F21 / 1 aff / NA / 7 / 63,249,780 / 64,844,720 / 1.6 / 260
12 / 111,350,999 / 113,125,249 / 1.8 / 255
1 / 145,550,922 / 147,020,456 / 1.5 / 242

IBD regions are sorted according to the number of consecutive homozygous SNPs. Abbreviations used: no: number; par cons: parental consanguinity; chr: chromosome; bp: base pairs; Mb: mega bases; aff: affected individual; unaff: unaffected individual; FC: first cousins; NA: not available; x: region included in exome variant filtering.

Table S4. Exome sequencing variant analysis.

Patient / IBD regions (VAF ≥70%) / F12 / F14 / F16 / F17 / F18 / 209 RetNet genes (VAF ≥20%) / F13 / F15 / F19 / F20 / F21
Initial diagnosis / LCA / EORD / ARRP / ARRP / ARRP / LCA / EORD / ARRP / ARCRD / LCA + ID
IBD
mapping / Total size of IBD regions (Mb) /

13.8

215

37

200

168

/ NA /

NA

/ NA /

No of genes in IBD regions /

300

2,389

348

2,623

1,885

/ NA /

Variant filtering / 1. Coverage ≥ 5x /

342

9,954

1,478

7,701

7,576

2,652

2,489

2,136

2,476

2,585

2. Coding variants

507

548

405

187

158

170

163

Nonsense/frameshift

1/0

4/7

1/0

0/21

5/16

0/2

2/2

0/2

2/4

1/3

In-frame deletion/insertion /

Missense / Total /

491

522

379

/ 184 /

182

/ 155 /

162

158

Novel or rare (MAF≤ 0.05) /

/ 53 /

/ 44
Predicted pathogenic /

/ 16 /

/ 11
3. Splice site effect / Total /

389

Novel or rare (MAF≤ 0.05) /

140

Nearest splice site +/- 20bp / 3
Causal mutation(s) / Gene /

RPGRIP1

CLN3

MERTK

C2orf71

PDE6B

/ NMNAT1 / USH2A /

CERKL

IQCB1

Mutation / HoZ

c.2758C>T

p.Gln920* / HoZ
c.784A>T

p.Lys262*

HoZ

c.2180G>A p.Arg727Gln

/ HoZ

c.1764del

p.Glu589fs / HoZ
c.121_125delins15

p.Pro41fs

/ Comp HeZ
c.205A>G
p.Met69Val
c.769G>A
p.Glu257Lys / HoZ
c.10721G>T
p.Gly3574Val / HoZ
c.847C>T

p.Arg283*

/ HoZ
c.1241_1242del

p.Leu414fs

VAF /

100

/ 54/45 / 100 /

100

93.5

Coverage /

131

127

/ 74/89 / 89 /

154

Numbers indicated in bold refer to variant groups in which the causal mutation has been identified. Because of this, further filter steps have not been performed in these patients (grey filling).

Variant analysis was performed either against IBD regions (F12, F14, F16-F18) or against 209 RetNet genes (F13, F15, F19-F21). The variant allele frequency was set on minimum 70% and 20%, respectively, in order to identify homozygous variants in the IBD regions, and both homozygous and heterozygous variants in the RetNet genes. Following selection of all variants with a coverage equal to or above 5x, coding variants altering the amino acid sequence were divided into three groups (nonsense/frameshift, in-frame deletion/insertion, missense) based on CLC variant annotation. Subsequently, missense mutations were analyzed with Alamut HT, providing minor allele frequencies (MAF) from dbSNP and the NHLBI GO Exome Sequencing Project, and detailed in silico predictions. Following the exclusion of known variants with a dbSNP global MAF above 5%, missense variants were considered possibly pathogenic if at least 2/4 prediction programs suggest a pathogenic effect (SIFT: deleterious, Grantham score ≥ 75, AGVGD: C45, C55 or C65, MAPP: bad). In parallel with the assessment of coding variants, variants with a local splice site effect were selected from Alamut HT starting from all variants with a coverage equal to or above 5x. A local splice site effect is predicted when at least two of MaxEntScan, NNSPLICE, or HSF predictions for the variant vicinity are significant. Of these, rare variants and variants located 20 bp up- or downstream of an exon boundary were selected.

Abbreviations used: EORD: early-onset retinal dystrophy; ARRP: autosomal recessive retinitis pigmentosa; LCA: Leber Congenital Amaurosis; ARCRD: autosomal recessive cone-rod dystrophy; ID: intellectual disability; IBD: identity-by-descent; Mb: megabases; MAF: minor allele frequency (dbSNP137); bp: base pairs; NA: not applicable; HoZ: homozygous; Comp HeZ: compound heterozygous.

Table S5. Benefit of IBD-guided WES variant filtering.

Patient / IBD regions, VAF ≥70% / F12 / F14 / F16 / F17 / F18 / Whole exome, VAF ≥70% / F12 / F14 / F16 / F17 / F18
Initial diagnosis / LCA / EORD / ARRP / ARRP / ARRP / LCA / EORD / ARRP / ARRP / ARRP
IBD
mapping / Total size of IBD regions (Mb) /

13.8

215

200

168

/ NA / NA / NA / NA / NA
No of genes in IBD regions /

300

2,389

348

2,623

1,885

/ NA / NA / NA / NA / NA
Variant filtering / 1. Coverage ≥ 5x /

342

9,954

1,478

7,701

7,576

76,255

89,255

61,656

78,840

88,354

2. Coding variants

507

548

405

4,385

4,846

4,644

4,698

4,621

Nonsense/frameshift

1/0

4/7

1/0

0/21

5/16

25/139

36/134

26/116

19/151

26/131

In-frame deletion/insertion /

Missense /

491

522

379

/ 4,176 /

4,631

/ 4,452 /

4,476

4,420

Comparison between the number of variants obtained when selecting 1) all variants located in major IBD regions with a variant allele frequency equal to or above 70%, and 2) all variants in the whole exome with a variant allele frequency equal to or above 70%.

Abbreviations used: EORD: early-onset retinal dystrophy; ARRP: autosomal recessive retinitis pigmentosa; LCA: Leber Congenital Amaurosis; IBD: identity-by-descent; Mb: megabases; NA: not applicable.